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British Pharmacopoeia 2016 Volume I

British Pharmacopoeia 2016 Volume I The British Pharmacopoeia Commission has caused this British Pharmacopoeia 2016 to be prepared under regulation 317(1) of the Human Medicines Regulations 2012 and, in accordance with regulation 317(4), the Ministers have arranged for it to be published. It has been notified in draft to the European Commission in accordance with Directive 98/34/EEC. The monographs of the Eighth Edition of the European Pharmacopoeia (2013), as amended by Supplements 8.1 to 8.5, published by the Council of Europe are reproduced either in this edition of the British Pharmacopoeia or in the associated edition of the British Pharmacopoeia (Veterinary). See General Notices

Effective date: 1 January 2016 see Notices

London: The Stationery Office

In respect of Great Britain: THE DEPARTMENT OF HEALTH In respect of Northern Ireland: THE DEPARTMENT OF HEALTH, SOCIAL SERVICES AND PUBLIC SAFETY © Crown Copyright 2015 Published by The Stationery Office on behalf of the Medicines and Healthcare products Regulatory Agency (MHRA) except that: European Pharmacopoeia monographs are reproduced with the permission of the Council of Europe and are not Crown Copyright. These are identified in the publication by a chaplet of stars. This publication is a ‘value added’ product. If you wish to re-use the Crown Copyright material from this publication, applications must be made in writing, clearly stating the material requested for re-use, and the purpose for which it is required. Applications should be sent to: Dr S Atkinson, MHRA, 5th Floor, 151 Buckingham Palace Road, London SW1W 9SZ. First Published 2015 ISBN 978 Oil 3230 006 British Pharmacopoeia Commission Office: MHRA 5th Floor 151 Buckingham Palace Road London SW1W 9SZ Telephone: +44 (0)20 3080 6561 E-mail: [email protected] Web site: http://www.pharmacopoeia.com Laboratory: British Pharmacopoeia Commission Laboratory Queen’s Road Teddington Middlesex TW11 OLY Telephone: +44 (0)20 8943 8960 E-mail: [email protected] Web site: http://www.pharmacopoeia.com

FOREWORD The British Pharmacopoeia (BP), after 150 years of publication, continues to help ensure the quality of medicinal substances globally. One of its key attributes has been to take advantage of novel science and this edition of the BP is no different. Authentication of botanical constituents, to ensure the safety and quality of Traditional Herbal Medicines, provides many challenges. With advances in molecular genetics, however, reliable methods of identifying herbs are now available. This has enabled the BP and the National Institute for Biological Standards and Control (NIBSC) to work collaboratively and successfully on a pilot project to determine the deoxyribonucleic acid (DNA) profile of Ocimum tenuiflorum. The BP 2016 introduces the species specific sequence of the selected barcode region in a new BP Appendix as an identification method for Ocimum tenuiflorum. General guidance on how to conduct DNA-based identification methods for herbal drugs is also included in this new Appendix. In addition to this innovation, the BP 2016 publishes new and revised monographs for substances and products used in a wide range of medicines. The collegial partnerships between the staff, the appointed experts of the British Pharmacopoeia Commission and international partners have enabled these important quality standards to be made publicly available. The significant progress made jointly by the BP and NIBSC facilitates the reliable authentication of herbal drugs and will enhance the protection of public health, the primary objective of the Medicines and Healthcare products Regulatory Agency.

Professor Sir Michael Rawlins Chairman Medicines and Healthcare products Regulatory Agency

Contents Contents of Volume I FOREWORD NOTICES PREFACE BRITISH PHARMACOPOEIA COMMISSION EXPERT ADVISORY GROUPS, PANELS OF EXPERTS AND WORKING PARTIES CODE OF PRACTICE MEMBERSHIP BP Commission, Expert Advisory Groups, Panels of Experts, Working Parties STAFF British Pharmacopoeia, BP Laboratory, Publisher INTRODUCTION Additions, Omissions, Technical Changes, Changes in Tide GENERAL NOTICES MONOGRAPHS Medicinal and Pharmaceutical Substances (A - 1) Contents of Volume II NOTICES GENERAL NOTICES MONOGRAPHS Medicinal and Pharmaceutical Substances Q - Z) Contents of Volume m NOTICES GENERAL NOTICES MONOGRAPHS Formulated Preparations: General Monographs Formulated Preparations: Specific Monographs

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Contents of Volume IV NOTICES GENERAL NOTICES MONOGRAPHS Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal Products Materials for use in the Manufacture of Homoeopathic Preparations Blood-related Products Immunological Products Radiopharmaceutical Preparations Surgical Materials Contents of Volume V NOTICES GENERAL NOTICES INFRARED REFERENCE SPECTRA APPENDICES SUPPLEMENTARY CHAPTERS INDEX

Notices Monographs of the European Pharmacopoeia are distinguished by a chaplet of stars against the title. The term European Pharmacopoeia, used without qualification, means the eighth edition of the European Pharmacopoeia comprising, unless otherwise stated, the main volume, published in 2013, as amended by any subsequent supplements and revisions. Patents

In this Pharmacopoeia certain drugs and preparations have been included notwithstanding the existence of actual or potential patent rights. In so far as such substances are protected by Letters Patent theừ inclusion in this Pharmacopoeia neither conveySj nor implies, licence to manufacture.

Effective dates

New and revised monographs of national origin enter into force on 1 January 2016. The monographs are brought into effect under regulation 320(2) of the Human Medicines Regulations 2012. Monographs of the European Pharmacopoeia have previously been published by the European Dừectorate for the Quality of Medicines & Healthcare in accordance with the Convention on the Elaboration of a European Pharmacopoeia and have been brought into effect under European Dừectives 2001/82/ECj 2001/83/EC and 2003/63/ECj as amended, on medicines for human and veterinary use.

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Preface The British Pharmacopoeia Commission has caused this British Pharmacopoeia 2016 to be prepared under regulation 317(1) of the Human Medicines Regulations 2012 and, in accordance with regulation 317(4), the Ministers have arranged for it to be published. The British Pharmacopoeia 2016 contributes significantly to the quality control of medicinal products for human use. It contains publicly available, legally enforceable standards that provide an authoritative statement of the quality that a product, material or article is expected to meet at any time during its period of use. The Pharmacopoeial standards are designed to complement and assist the licensing and inspection processes and are part of the overall system for safeguarding purchasers and users of medicinal products in the U K The British Pharmacopoeia Commission wishes to record its appreciation of the services of all those who have contributed to this important work.

British Pharmacopoeia Commission The British Pharmacopoeia Commission is appointed, on behalf of the Secretaiy of State for Health, by the Department of Health’s Appointments Team who are responsible for appointments to all of the Advisory Bodies appointed under the Human Medicines Regulations 2012. Under the terms of the Human Medicines Regulations 2012 , the duties of the British Pharmacopoeia Commission are as follows: (a)

the preparation and publication of any new edition of the British Pharmacopoeia [regulations 317(1) and 317(4)];

(b)

the preparation and publication of any compendium containing information relating to substances and articles which are or may be used in the practice of veterinary medicine or veterinary surgery [regulations 317(3)(b) and 317(4)];

(c)

the preparation and publication of a list of names to be used as the headings to monographs in the British Pharmacopoeia [regulations 318(1) and 318(2)];

(d)

the preparation of any amendments to the above publications [regulation 317(5)(a)].

Members of the British Pharmacopoeia Commission are appointed for a renewable term of 4 years and, under the requirements laid down by the Office of the Commissioner for Public Appointments, can serve for a maximum of 10 years. In order to ensure that the British Pharmacopoeia Commission fulfils its duties under the Human Medicines Regulations 2012, the members also have the following duties: ( 1)

to frame clear and unequivocal technical advice in order to discharge the Commission’s responsibilities both for the British Pharmacopoeia, the British Pharmacopoeia (Veterinary) and British Approved Names and as the national pharmacopoeial authority with respect to the European Pharmacopoeia;

(2)

to develop clear policies for the preparation and publication of the British Pharmacopoeia and its related publications;

(3)

to serve on one or more Expert Advisory Groups or Panels of Experts of the BP Commission, usually in the position of Chair or Vice-Chair;

(4)

to approve new and revised text for inclusion in new editions of the British Pharmacopoeia and British Pharmacopoeia (Veterinary);

(5)

to approve new and revised names for inclusion in new editions of British Approved Names and its annual supplements. I-xi

In addition to the duties listed above, the Chair of the British Pharmacopoeia Commission has the following additional duties: (1)

To chair all scheduled and unscheduled meetings;

(2)

To carry out members appraisals in accordance with Department of Health policies and timelines;

(3)

To participate in the process to appoint/re-appoint members of the British Pharmacopoeia Commission.

Expert Advisory Groups, Panels o f Experts and Working Parties Members of Expert Advisory Groups, Panels of Experts and Working Parties are appointed by the British Pharmacopoeia Commission. The duties of the members are as follows: (a)

to collaborate in the preparation and revision of Monographs, Appendices and Supplementary Chapters for inclusion in the British Pharmacopoeia and British Pharmacopoeia (Veterinary);

(b)

to collaborate in the preparation and revision of Monographs, Methods and General Chapters of the European Pharmacopoeia;

(c)

to review reports from the British Pharmacopoeia Laboratory in terms of technical content and, where possible, provide independent experimental data to assist in decision making;

(d)

to collaborate in the preparation and revision of the list of names to be used as titles for monographs of the British Pharmacopoeia and British Pharmacopoeia (Veterinary).

Members of Expert Advisory Groups, Panels of Experts and Working Parties are usually appointed for a renewable term of 4 years.

I-xiii

Code of Practice Members of the British Pharmacopoeia Commission and its supporting Expert Advisory Groups, Panels of Experts and Working Parties are required to comply with a Code of Practice on Declaration of Interests in the Pharmaceutical Industry. British Pharmacopoeia Commission Chairs and members of the British Pharmacopoeia Commission are required to make a full declaration of interests on appointment and annually thereafter. They must also inform the BP Secretariat promptly of any changes to these interests during the year. These interests are published in the Medicines Advisory Bodies Annual Reports. Relevant interests must be declared at meetings and are recorded in the Minutes. Expert Advisory Groups, Panels of Experts and Working Parties Chairs and members are required to make a full declaration of interests on appointment and to update the Secretariat if these interests change during their term of office. A record is kept of those experts who have declared specific interests, but these are not published. Relevant interests must be declared at meetings and are recorded in the Minutes.

Membership of the British Pharmacopoeia Commission The list below includes those members who served during the period 2014 to 2015. Chair Vice-Chair

Professor Kevin M G Taylor BPharm PhD FRPharmS Professor of Clinical Pharmaceutics, UCL School of Pharmacy Professor Alastair Davidson BSc PhD FRPharmS Visiting Professor of Pharmaceutical Sciences, University of Strathclyde Professor Donald Cairns BSc PhD MRPharmS CSci CChem FRSC Head: School of Pharmacy and Life Sciences, Robert Gordon University, Aberdeen Mr Barry Capon CBE MA DL {Lay representative) Former Non-executive Director, Norfolk and Suffolk NHS Foundation Trust Dr Graham D Cook BPharm PhD MRPharmS Senior Director, Process Knowledge!Quality by Design, Pfizer Mr Andrew Coulson BVetMed MSc MRCVS Member of the Royal College of Veterinary Surgeons; former Superintending Inspector, Science & Research Group, The Home Office Mr Christopher Goddard BSc DIS CSci EurChem CChem FRSC Quality Control Technical Manager, Recipharm Limited Dr Keith Helliwell BPharm PhD Senior Technical Adviser, William Ransom & Son PLC Dr Rodney L Horder BPharm PhD MRPharmS Former Divisional Vice President, European Quality and Regulatory Strategy, Abbott Dr Gerard Lee BPharm PhD FRPharmS MRSC CChem Former Group Manager, British Pharmacopoeia and Laboratory Services (MHRA); former Secretary & Scientific Director of the British Pharmacopoeia Commission Dr Brian R Matthews BPharm PhD FRPharmS FTOPRA MRI Consultant on pharmaceutical and medical device regulatory affairs; former Senior Director, EC Registration, Alcon Laboratories Professor John Miller MSc PhD MRSC CChem Visiting Professor, Strathclyde Institute of Pharmacy and Biomedical Sciences; former Head of the EDQM Laboratory Dr Ronald Torano BSc PhD MRSC CChem Pharmacopoeial Intelligence and Advisory Specialist; GlaxoSmithKline

I-xv

Dr Lincoln Tsang BPharm LLB PhD FRSC FIBiol FRSA FRPharmS Solicitor Life Sciences Lawyer; Partner, Arnold & Porter LLP Mrs Josephine Turnbull T-T R {Lay representative) Former Chair of Tees, Esk and Wear Valley NHS Foundation Trust Dr Paul Varley BSc PhD Vice President of Biopharmaceutical Development, Medimmune Limited Professor Elizabeth Williamson BPharm PhD MRPharmS Former Professor of Pharmacy, University of Reading Secretary and Scientific Director

I-xvi

Dr Samantha Atkinson BSc MSc PhD MRSC Visiting Fellow, University of Reading

Membership of Expert Advisory Groups, Panels of Experts and Working Parties The Commission appointed the following Expert Advisory Groups, Panels of Experts and Working Parties to advise it in carrying out its duties. Membership has changed from time to time; the lists below include all who have served during the period 2014 to 2015.

EXPERT ADVISORY GROUPS ABS: Antibiotics

BIO: Biological and Biotechnological Products

HCM: Herbal and Complementary Medicines

R L Horder (Chair), G Cook (Vice-Chair), P Ellis, E Flahive, A Gibson, V Jaitely, A Livingstone, W Mann, J Miller, N Thomas, B White, I R Williams L Tsang (Chair), P Varley (Vice-Chair), L Bisset*, A F Bristow*, C Bums, D H Calam, K Chidwick*, A Cook*, J Cook*, L Findlay*, S Gill, E Griffiths, C Jones*, A Kippen*, B Patel, A M Pickett*, T Pronce, L Randon, I Rees*, S Schepelmann*, D Sesardic, P Sheppard, P Stickings*, W J Tarbit, A H Thomas, R Thorpe, M Wadhwa* (Corresponding members A Onadipe, J N A Tettey) E Williamson (Chair), L A Anderson (Vice-Chair), P Anderson, A Bligh, T Chapman, A Charvill, S Gibbons, K Helliwell, P Hylands, C Leon, R Middleton, A C Moffat, B Moore, M Pires, M Rowan, K StrohfeldtVenables, J Sumal*, P Viner, C Welham, C Wright, K Zhao (Corresponding members SS Handa, A Krauss, Z-T Wang)

MCI: Medicinal Chemicals

A G Davidson (Chair), D Cairns (Vice-Chair), M Ahmed, J C Berridge, M Broughton, A J Caws, P Fleming, A James, V Loh, W J Lough, D Malpas

MC2: Medicinal Chemicals

G Cook (Chair), C T Goddard (Vice-Chair), M Cole, J Cowie, D Edwards, A Gibson, J Lim, J Miller, P Murray, J Qiu, A Ruggiero, M Turgoose, N Wynne (Corresponding members M Brits, W Sherwin)

MC3: Medicinal Chemicals

V Fenton-May (Chair), E Williamson (Vice-Chair), M Almond, S Arkle, J Beach, J Beaman, C T Goddard, P Hampshire, W K L Pugh, B Rackstraw, R Torano, M Tubby, I R Williams

NOM: Nomenclature

J K Aronson (Chair), L Tsang (Vice-Chair), M Ahmed, D Mehta, G P Moss, R Thorpe (Corresponding members R G Balocco Mattavelli, E M Cortes Montejano, J S Robertson)

PCY: Pharmacy

R L Horder (Chair), B R Matthews (Vice-Chair), M Ahmed*, M Aulton, E Baker, J Beach, N Broad, G Davison, G Eccleston, D Elder, B GranellVillen, J Lim*, R Lowe, J MacDonald, J F McGuire, T Purewal, L Randon, K Taylor (Corresponding member J Churchill)

Denotes a specialist member.

I-xvii

ULM: Unlicensed Medicines

M G Lee (Chair), V Fenton-May (Vice-Chair), G Bennett, S Branch, D Caulfield, A Charvill, W Goddard, N Hussain, S Jones, M A Oldcome, N J Precious, J Rothwell, M Santdllo, J Smith, A Sully, P Weir

PANELS OF EXPERTS BLP: Blood Products CX: Excipients

K Chidwick, A R Hubbard, J More, P Varley B R Matthews (Chair), C Mroz (Vice-Chair), E Anno, C Cable, R Cawthome, W Cook, D Deutsch, N Hussain, M I Robertson, K Slevin

IGC: Inorganic and General Chemicals

C T Goddard (Chair), M Almond, S Atherton, S Boland, A C Cartwright, D Caulfield, P Henrys, G Lay, D Malpas, C Mroz, D Riches

MIC: Microbiology

V Fenton-May (Chair), S Denyer, D P Hargreaves, B R Matthews, P Newby

RAD: Radioactive Materials VET: Veterinary Medicines VIP: Veterinary Immunological Products

J Ballinger, J Brain, D Graham, S R Hesslewood, G Inwards, P Maltby, R D Pickett, R Smith, S Waters E Williamson (Chair), A Coulson (Vice-Chair), A Cairns, S Cockbill, D Evans, E Flahive, P Lees, B Ward A M Brady, R Banks, K Redhead, J Salt, P W Wells, R Woodland

WORKING PARTIES AQbD: Analytical Quality by Design DNA: Identification Techniques MCS: Microscopy

I-xviii

G Cook (Chair), S Brown, S Ellison, M Hanna-Brown, S Jones, D Makohon, P Nethercote, E Razzano (Corresponding member K Barnett) K Helliwell (Chair), J Hawkins, E Mee, A Slater, E Williamson E Williamson (Chair), R Arroo, R Reck, K Helliwell, K MacLellan Gibson

Current British Pharmacopoeia Staff Secretariat

M VaUender (Editor-in-Chief) S Young (.Head of Analytical Science) M Barrett, H Corns, P Crowley, A Evans, J Francomb, A Gibb, P Holland, G Li-Ship, R A Pask-Hughes, C Pitt, J Pound, F J Swanson, M Whaley

NIBSC-based Staff Administrative

C Howard, C Lockie-Williams B F Delahunty, W Jeffries, J Paine

ISO 9001 FS 27268

Current British Pharmacopoeia Laboratory Staff K Courtney (Laboratory Manager) A Ciesluk, D Colmer, J Elliot, C Galdino, P Gallagher, S Humphries, R Mannan, C Marcolan, A Murphy, M Nanasi, A Panchal, E Sanderson, N Vadukal, A Vasilaki, M Wallis, S Wilson

ISO 9001 FS 27613

I-xx

Current Staff of the Publisher of the British Pharmacopoeia J Hook (MD Public Sector EMEA) A Prince (Client Services Director) N Billington (Client Services Manager) A Hughes (Account Manager) C Hackett (.Project Manager) P Allard, A Dampier, M Grant, A Hood, S Page, M Parka, N Pope, M Rainbird, A Ray, P Relfe, C Spary, J Stoker, I Webb, K Williams

ISO 9001 FS 22428

I-xxi

Introduction I-xxiii

2016

Introduction Triennial Review of the British Pharmacopoeia Commission The Department of Health conducted a Triennial Review of the British Pharmacopoeia Commission (BPC) to provide assurance to the Department and the public that the functions of the Commission are required and that it is operating effectively. The review of the BPC was undertaken in two stages, the first examined the functions and form of the BPC and the second examined the efficiency, governance and performance of the BPC. The recommendation from the review of form and function is that “the BP Commission should continue to deliver its existing functions as an Advisory Non-Departmental Public Body” . This model facilitates the BP Commission to be in a position close to the Medicines and Healthcare products Regulatory Agency whilst being independent of it and facilitates harnessing potential information, communication, laboratory and expertise synergies. The evidence gathered from the review of efficiency, governance and performance suggested that “the BP commission operates efficiently, is mostly compliant with the principles of good corporate governance and is considered to be a leading pharmacopoeia. In particular, stakeholders highlighted the BP Commission’s innovative work, the dedication and expertise of members and the Secretariat, industry engagement and transparency” . The Triennial Review report, published in March 2015, makes a number of m inor recommendations which will be taken forward by the BPC Secretariat. The full report of the Review can be found on the website www.gov.uk/government/consultations/british-pharmacopoeia-commissiontriennial-review. British Pharmacopoeia 2016 The British Pharmacopoeia 2016 supersedes the British Pharmacopoeia 2015. It has been prepared by the British Pharmacopoeia Commission, with the collaboration and support of its Expert Advisory Groups, Panels of Experts and Working Parties and contains almost 4000 monographs for substances, preparations and articles used in the practice of medicine. Some of these monographs are of national origin and have been elaborated or revised under the auspices of the British Pharmacopoeia Commission whilst others (indicated to users by a chaplet of stars) have been elaborated, or revised, under the auspices of the European Pharmacopoeia Commission, supported by its Groups of Experts and Working Parties, and are reproduced from the European Pharmacopoeia. This edition, together with its companion volume, the British Pharmacopoeia (Veterinary) 2016, incorporates all the monographs of the 8th Edition of the European Pharmacopoeia, as amended by Supplements 8.1 to 8.5. The user of the British Pharmacopoeia thereby benefits by finding within this comprehensively indexed compendium all current United Kingdom pharmacopoeial standards for medicines for human use.

I-xxiv Introduction

2016

The BP 2016 comprises six volumes as follows.

Effective Date

Volumes I and II

Medicinal Substances

Volume III

Formulated Preparations: General Monographs Formulated Preparations: Specific Monographs

Volume IV

Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal Products Materials for use in the Manufacture of Homoeopathic Preparations Blood-related Products Immunological Products Radiopharmaceutical Preparations Surgical Materials

Volume V

Infrared Reference Spectra Appendices Supplementary Chapters Index

Volume VI

British Pharmacopoeia (Veterinary') 2016

The effective date for British Pharmacopoeia monographs in this edition is 1 January 2016. National monographs omitted from this or earlier editions of the British Pharmacopoeia remain effective in accordance with Regulation 252(2) (c) of the Human Medicines Regulations 2012. Implementation dates regarding European Pharmacopoeia publications are provided in Supplementary Chapter IV B: Dates of Implementation. European Pharmacopoeia monographs are identified by a chaplet of stars alongside the title.

Additions

A list of monographs included for the first time in the British Pharmacopoeia 2016 is given at the end of this introduction. It includes 37 new monographs of national origin and 25 new monographs reproduced from the 8th Edition of the European Pharmacopoeia as amended by Supplements 8.1 to 8.5. Traditional H erbal M edicines; Homoeopathic Preparations Work is continuing on the development of monographs for herbs used in traditional herbal medicines and homoeopathic medicines. The Latin scientific names cited in BP monographs for herbal drugs are consistent with the advice provided by the Medicinal Plant Names Services at the Royal Botanic Gardens, Kew. As stated in previous editions, the requirements for the quality of the material are provided in the monograph to set the standards for Traditional Herbal Medicines in the UK and to assist the UK Traditional Herbal Medicines Registration Scheme. The British Pharmacopoeia Commission, however, has not assessed the safety and efficacy of the materials in traditional use.

Introduction I-xxv

2016

Likewise, the British Pharmacopoeia Commission has not assessed the safety and efficacy of materials for use in homoeopathic preparations for which monographs are published. This edition sees the first publication of a DNA-based identification method for Holy Basil. The user benefits from modem technology for the identification of herbal drugs, providing a direct measure of the species in use (see under Appendices). Unlicensed Medicines With this new edition, a further 5 monographs for unlicensed formulations have been added. These individual monographs are characterised by a statement that they are not currently licensed in the United Kingdom. The general and individual monographs are intended to apply to all types of Unlicensed Medicines, that is, those formulations prepared under a Manufacturer’s ‘Specials’ Licence and those prepared extemporaneously under the supervision of a pharmacist. Revisions

A significant number (141 comprising 87 technical revisions and 54 editorial revisions) of national monographs have been amended by means of this edition. Of these monographs, those with major technical revisions are listed at the end of this Introduction. For the benefit of the reader this list indicates the section, or sections, of each monograph which has/have been revised. The list of revisions appended to this Introduction is as comprehensive as practicable. However, to ensure that the reader uses the current standard, it is essential to refer to the full text of each individual monograph. For those texts reproduced from the European Pharmacopoeia, the European Directorate for the Quality of Medicines & Healthcare (EDQM) database (see below, under Websites) provides information on revisions of the monographs or other texts on a historical basis, beginning from the 5th Edition of the European Pharmacopoeia. Title Changes Four monograph titles have been amended in this edition. The list of changes is appended at the end of this Introduction. Anti-epiletic Drugs (AEDs) The Commission on Human Medicines (CHM) reviewed reports of spontaneous adverse reactions received by the Medicines and Healthcare products Regulatory Agency (MHRA) and publications that reported potential harm arising from switching of oral preparations of AEDs in patients previously stabilised on manufacturer’s product. Following this review, CHM concluded that reports of loss of seizure control and/or worsening of side effects around the time of switching between products could be explained as chance associations, but that a causal role of switching could not be ruled out in all cases. This includes switching between branded original and generic products, and between different generic products of a particular drug.

I-xxvi Introduction

2016

As a consequence of the conclusion by CHM, the Medicines and Healthcare products Regulatory Agency circulated guidance to stakeholders on the non-interchangeability of anti-epileptic drugs. The guidance can be found on the website www.gov.uk/drug-safety-update/antiepileptic-drugs. In considering the published monographs for AEDs, the BP Commission recommended that statements would be included in monographs for orally administered category 1 AEDs, that is, those where products were not interchangeable and category 2 AEDs, that is, products that were changed based on clinical judgement. Statements that category 1 products “are not interchangeable” and category 2 products “may not be interchangeable” have been included in AEDs monographs in this edition. A suitable statement has also been added to one monograph for antiepileptic unlicensed medicine to reflect that different formulations may vary in bioavailability and patients should be adequately monitored. Inhaled Products A programme of revision to BP monographs for inhaled products has been established and the changes will be introduced in future editions of the British Pharmacopoeia. Omissions

Thirty five monographs have been omitted from the British Pharmacopoeia 2016. The list of omissions is appended at the end of this Introduction. In line with recommendations from the Commission on Human Medicines and the British Pharmacopoeia Commission, reference to chloroform as an ingredient in licensed medicines has been removed from all affected monographs in this publication. This has been through either removing the monograph from the publication or deleting the formula and/or method of preparation.

Infrared Reference Spectra

As with the previous edition, the reference spectra are placed in alphabetical order within this edition. Two new spectra have been added to the collection.

Appendices

Two new Appendices to harmonise with the European Pharmacopoeia were first published in the British Pharmacopoeia 2015 electronic updates. These have been consolidated in the new edition as follows. Appendix VUIX: Methyl, Ethyl and Isopropyl Toluenesulfonate in Active Substances (Ph. Eur. method 2.5.40) Appendix XV K: Carrier Proteins for the Production of Conjugated Polysaccharide Vaccines for Human Use (Ph. Eur. method 5.2.11) A new BP method, Appendix XI V: Deoxyribonucleic Acid Based Identification Techniques for Herbal Drugs, is introduced in this edition and is accompanied by a worked example for Holy Basil.

Supplementary Chapters

One new Supplementary Chapter to harmonise with the European Pharmacopoeia was first published in the British Pharmacopoeia 2015 electronic updates. It has been consolidated in the new edition as follows.

Introduction I-xxvii

2016

Supplementary Chapter VII C: Monographs on Herbal Drugs (Ph. Eur. General Chapter 5.23) Supplementary Chapter I O: Inhaled Products This Supplementary Chapter has been reintroduced in this edition to reflect the revised BP policy on monographs for inhaled products. Supplementary Chapter I E: Dissolution Testing o f Solid Oral Dosage Forms This Supplementary Chapter has been updated to reflect the monographs that are omitted from this edition. Supplementary Chapter V: Unlicensed Medicines This Supplementary Chapter has been amended to recommend that use of chloroform as an ingredient in unlicensed medicines should be avoided. European Pharmacopoeia

Co-operation Agreement As a consequence of the Co-operation Agreement with the EDQM of the Council of Europe, the British Pharmacopoeia Commission is pleased to note the integration of European Pharmacopoeia texts for the British Pharmacopoeia 2015 in-year online updates and for this edition of the British Pharmacopoeia. In accordance with previous practice, all monographs and requirements of the European Pharmacopoeia are reproduced in this edition of the British Pharmacopoeia or, where appropriate, within its companion edition, the British Pharmacopoeia (Veterinary) 2016. Where a monograph has been reproduced from the European Pharmacopoeia, this is signified by the presence of a chaplet of stars alongside its title. Additionally, reference to the European Pharmacopoeia monograph number is included immediately below the title in italics in the form lPh. Eur. monograph xxxx\ Where the title in the British Pharmacopoeia is different from that in the European Pharmacopoeia, an approved synonym has been created (see Appendix XXI B) and the European Pharmacopoeia title is included before the monograph number. The entire European Pharmacopoeia text is delineated by two horizontal lines bearing the symbol (Ph. Eur.\ The European Pharmacopoeia texts have been reproduced in their entirety but, where deemed appropriate, additional statements of relevance to UK usage have been added (e.g. action and use statement, a list of British Pharmacopoeia preparations). It should be noted, however, that in the event of doubt of interpretation in any text of the European Pharmacopoeia, the text published in English under the direction of the Council of Europe should be consulted. Correspondence between the general methods of the European Pharmacopoeia and the appendices of the British Pharmacopoeia is indicated in each appendix and by inclusion of a list at the beginning of the appendices section.

Pharmacopoeial Requirements

It should be noted that any article intended for medicinal use which is described by a name at the head of a monograph in the current edition of the Pharmacopoeia must comply with that monograph *whether or not it is referred to as BP.

I-xxviii Introduction

2016

It is also important to note that no requirement of the Pharmacopoeia can be taken in isolation. A valid interpretation of any particular requirement depends upon it being read in the context of (i) the monograph as a whole, (ii) the specified method of analysis, (iii) the relevant General Notices and (iv) where appropriate, the relevant General Monograph(s). Familiarity with the General Notices of the Pharmacopoeia will facilitate the correct application of the requirements. Additional guidance and information on the basis of phaimacopoeial requirements is provided in Supplementary Chapter I. This non-mandatory text describes the general underlying philosophy and current approaches to particular aspects of pharmacopoeial control. Expert Advisory G roups; Panels of Experts; Working Parties The British Pharmacopoeia Commission has reviewed the membership of all of its Expert Advisory Groups, Panels of Experts and Working Parties. The tenure of appointed members runs for a period of 4 years from 1 January 2015 and is renewable. Criteria for the appointment of experts are available on the consolidated website (www.pharmacopoeia.com) . Panels of Experts The British Pharmacopoeia Commission has changed the status of the former Working Party on Excipients to a Panel of Experts based on its current workload. Working Parties The British Pharmacopoeia Commission has established 3 new Working Parties as follows. Two of the Working Parties, DNA: Identification Techniques and MCS: Microscopy, relate to the programme of work on herbal analysis. The third Working Party, AQbD: Analytical Quality by Design, has been established to support the work of the joint MHRA-BP Analytical Quality by Design project. The joint project examines the application of Quality by Design concepts to analytical methods and includes representation from the MHRA Licensing Division, the Good Manufacturing Practice Inspectorate and industry. Code of Practice

Websites

Members of the British Pharmacopoeia Commission and its supporting Expert Advisory Groups, Panels of Experts and Working Parties are required to comply with a Code of Practice on Declaration of Interests in • the pharmaceutical industry. Details of the Code are published on the website (www.pharmacopoeia.com). B ritish Pharm acopoeia Websites The British Pharmacopoeia websites, www.pharmacopoeia.co.uk and www.pharmacopoeia.com, have been consolidated and the new website, https://www.pharmacopoeia.com, is now available, containing information relating to the British Pharmacopoeia and allowing subscribers to access the British Pharmacopoeia 2016 and British Pharmacopoeia (Veterinary) 2016 online and British Approved Names publications. The consolidated website provides new and improved functionality together with greater access to improved BP-supporting resources. These resources include freely available example chromatograms, herbal micrographs and omitted surgical materials monographs for all users. Draft new and revised texts of the BP will continue to be placed on the website in an improved and more accessible way.

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Subscribers to the BP online will find that these resources will also be linked with relevant texts and directly accessible from the BP online content. Access to previous editions of the BP will be available as a BP archive product for purchase by new and existing BP online subscribers. The content of the archive will start from the BP 2014 onwards and will grow year-on-year as superseded editions are added to the archive. Improvements and new features have also been made to the BPCRS catalogue and ordering facility. Users will be able to receive notifications on the status of out-of-stock items and view order histories. BPCRS products will also be linked with relevant BP monographs and subscribers to the BP online will be able to purchase these directly from the BP online content. BPCRS customers will continue to be able to make purchases through invoice or credit card orders. An email subscription feature will allow users to keep abreast with BP news and BPCRS updates. In line with a policy of continuous improvement, users of the newly consolidated www.pharmacopoeia.com website are invited to provide the Secretariat with feedback on their experience. European Pharm acopoeia Websites httpss://extranet.edqm.eu/publications/recherches_sw.shtml For those texts reproduced from the European Pharmacopoeia, the EDQM website provides access to a database (the Knowledge database) containing information of various sorts related to monographs and intended to facilitate their proper use. Information is provided on chromatographic columns used in monograph development, suppliers of reagents and equipment that may be difficult to find for some users, the status of monographs (in development, adopted, published, under revision), revisions of the monographs on a historical basis, beginning from the 5th Edition of the European Pharmacopoeia as well as other useful information. https://pharmeuropa.edqm.eu/home The European Pharmacopoeia Forum, Pharmeuropa, is published quarterly as an aid for the elaboration of monographs and as a vehicle for information on pharmacopoeial and related matters. Pharmeuropa is available as a free on-line publication. International Collaboration

Therapeutic Goods A dm inistration, A ustralia The British Pharmacopoeia Commission is pleased to continue its long-standing co­ operation with the Australian Department of Health Therapeutic Goods Adm inistration (TGA). The TGA continues to provide advice to British Pharmacopoeia Commission Expert Advisory Groups, participate in inter­ laboratory evaluation of British Pharmacopoeia monographs and review data jointly. This collaboration has enabled the production of robust, high quality monographs for users. Chinese Pharm acopoeia The British Pharmacopoeia Commission is pleased to continue its collaboration with the Chinese Pharmacopoeia on the development of monographs and mutually agreed projects. Three Joint Working Groups have been established to cover Traditional Herbal

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Medicines, Biological Products and Active Pharmaceutical Ingredients and Excipients and associated formulated products. The Croatian Agency for M edicinal Products and M edical Devices (“ HALMED” ) Following the signing of a Collaboration Agreement in January 2015, the Medicines and Healthcare products Regulatory Agency has granted HALMED a licence to use the information in tile British Pharmacopoeia on unlicensed medicines. State Pharm acopoeia of the Republic o f Kazakhstan Following the signing of a Collaboration Agreement in April 2014, die Medicines and Healthcare products Regulatory Agency has granted die Committee on Surveillance of Medical and Pharmaceutical Activities of tile Ministry of Health of the Republic of Kazakhstan a licence to use relevant contents of the British Pharmacopoeia in the State Pharmacopoeia of the Republic of Kazakhstan. U nited States Pharm acopeia The informal harmonisation between the British Pharmacopoeia and die United States Pharmacopeia continues with a projected work programme. Both Pharmacopoeias are committed to harmonising analytical procedures for such monographs wherever possible. W orld Health O rganization Following the renewal of a Collaboration Agreement in April 2014, the British Pharmacopoeia Commission is pleased to exchange information with the International Pharmacopoeia and to collaborate on the development of monographs for formulated preparations. Forward Look

Acknowledgements

Electronic Updates The British Pharmacopoeia 2016 online updates will be published on tile website, www.pharmacopoeia.comj to enable users to keep up to date with monographs published in tile European Pharmacopoeia. These updates will be integrated annually with the publication of the main edition of the British Pharmacopoeia. The British Pharmacopoeia Commission is greatly indebted to die members of its Expert Advisory Groups, Panels of Experts and Working Parties for theừ dedicated enthusiasm and assistance in the preparation of this edition. Close co-operation has continued with many organisations at home and overseas. These include the Medicines and Healthcare products Regulatory Agency, the Veterinary Medicines Dừectorate, the Royal Pharmaceutical Society, the Association of the British Pharmaceutical Industry, the British Association of Homoeopathic Manufacturers, die United Kingdom Herbal Forum, The China Food and Drug Administration, the Chinese Pharmacopoeia Commission, tile European Pharmacopoeia Commission and the European Dừectorate for the Quality of Medicines & Healthcare, tile Therapeutic Goods Adminisưation (Australia), the Health Products and Food Branch of Health Canada, the United States Pharmacopeia, tile Quality Assurance and Safety: Medicines Department of the World Health Organization (WHO), the Health Sciences Authority of Singapore and the Royal Botanic Gardens, Kew. The British Pharmacopoeia Commission wishes to thank the European Dừectorate for the Quality of Medicines & Healthcare for theừ support and assistance in tile reproduction of the European Pharmacopoeia texts and monographs. The British Pharmacopoeia Commission acknowledges

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the importance of the work of the European Pharmacopoeia (Ph. Eur.) Commission and its Groups of Experts and Working Parties. The British Pharmacopoeia Commission is also grateful for the generous contribution by the UK experts to the work of the Groups of Experts and Working Parties of the European Pharmacopoeia Commission. The British Pharmacopoeia is especially indebted to the Chinese Pharmacopoeia for facilitating the training of two members of staff at the Institute of Chinese Materia Medica on molecular methods of identification for herbal drugs. The Commission appreciates the training provided by Professor Chen Shilin, Professor Xu Jiang and their colleagues. The British Pharmacopoeia Commission is grateful for the contribution of Dr Ann Tough, Dr Graeme Kay and Ernie John Janus and Pamela Methven, from the Robert Gordon University for their collaboration and advice in the practical evaluation of monographs for this edition. The British Pharmacopoeia is also grateful for the contribution of Ms Harjit Jagpal for administrative and events management, Mr S Maddox, formerly of the BP Laboratory, and for the contribution of Rebecca Hendry, Marta Hemandez-Rueda and Walyd Mohammed of the Medicines and Healthcare products Regulatory Agency Laboratory for their collaboration and practical evaluation of monographs in this edition. The British Pharmacopoeia Commission acknowledges the contribution of Professor Frederick A Senese, Department of Chemistry, Frostburg State University, USA, for his kind permission to reproduce the indicator colour chart. The British Pharmacopoeia Commission also acknowledges and appreciates the advice of the publishing team at The Stationery Office, in particular, Ms Nichola Billington, Mr Colin Hackett, Mr Paul Allard, Mr Paul Relfe and Mr Ian Webb, in the production of this edition. The British Pharmacopoeia Commission is grateful for the commitment and contribution of the website team at the Stationery Office, in particular, Mr Terry Blake, Mr Andrew Hood and Mr Vinod Sathyamoorthy, in the consolidation of the two websites. Additions

The following monographs of the British Pharmacopoeia 2016 were not included in the British Pharmacopoeia 2015. Medicinal and Pharm aceutical Substances Eplerenone* Glucosamine Sulfate Potassium Chloride* Imatinib Mesilate* High-molecular-mass Macrogols* Macrogol Isotridecyl Ether* Meldonium Dihydrate* Methane* Permethrin* Polyoxypropylene Stearyl Ether* Pullulan* Rosuvastatin Calcium* Sulfadimethoxine* Tizanidine Hydrochloride* * denotes a monograph of the European Pharmacopoeia

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Tolterodine Tartrate* Zanamivir Hydrate* Formulated Preparations: Specific Monographs Abacavir and Lamivudine Tablets Abacavir, Zidovudine and Lamivudine Tablets Gastro-resistant Acamprosate Tablets Bendroflumethiazide Oral Suspension Carvedilol Tablets Ceftazidime Eye Drops Clotrimazole Eye Drops Clotrimazole Vaginal Tablets Diamorphine Tablets Estradiol Vaginal Tablets Etynodiol Tablets Fluconazole Capsules Fluconazole Infusion Fluconazole Oral Suspension Flumetasone and Clioquinol Ear Drops Flupentixol Tablets Fluticasone and Salmeterol Pressurised Inhalation Powder, pre-dispensed Fluticasone and Salmeterol Pressurised Inhalation, Suspension Interferon Beta-la Injection Ketoconazole Cream Ketoconazole Shampoo Lorazepam Oral Solution Miconazole Eye Drops Olanzapine Tablets Orodispersible Olanzapine Tablets Olmesartan Tablets Soluble Prednisolone Tablets Rizatriptan Tablets Orodispersible Rizatriptan Tablets Prolonged-release Sodium Valproate Capsules Prolonged-release Sodium Valproate Tablets Terbinafine Tablets Vecuronium Bromide for Injection Zidovudine Infusion Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal Products Agnus Castus Fruit Dry Extract* Barbary Wolfberry Fruit* Holy Basil Leaf Nettle Root* Phellodendron Amurense Bark Phellodendron Chínense Bark Materials for use in the Manufacture of Homoeopathic Preparations Coated Homoeopathic Pillules* Agaricus Phalloides for Homoeopathic Preparations* Ignatia for Homoeopathic Preparations* Nux-vomica for Homoeopathic Preparations* * denotes a monograph of the European Pharmacopoeia

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Blood-related Products Normal Immunoglobulin for Subcutaneous Administration* Immunological Products Influenza Vaccine (Live, Nasal)* Radiopharmaceutical Preparations Fluoroethyl-L-tyrosine (18F) Injection* Omissions

The following monographs of the British Pharmacopoeia 2015 are not included in the British Pharmacopoeia 2016. Medicinal and Pharmaceutical Substances Amitriptyline Embonate Carbenoxolone Sodium Chloroform Fosfestrol Sodium Halibut-liver Oil Nandrolone Phenylpropionate Formulated Preparations: Specific Monographs Aminoglutethimide Tablets Chloroform Spirit Chloroform and Morphine Tincture Double Strength Chloroform Water Codeine Linctus1 Paediatric Codeine Linctus1 Codergocrine Tablets Desipramine Tablets Diethylstilbestrol Pessaries Dydrogesterone Tablets Ergometrine Tablets Fosfestrol Injection Fosfestrol Tablets Fructose Infusion Glucose Irrigation Solution Halibut-liver Oil Capsules Isoconazole Pessaries Menadiol Phosphate Injection Methylcellulose Granules Morphine and Atropine Injection Neonatal Naloxone Injection Nandrolone Phenylpropionate Injection Orciprenaline Oral Solution Orciprenaline Tablets Paraffin Ointment Protriptyline Tablets Tretinoin Solution

* denotes a monograph of the European Pharmacopoeia • This formulated preparation is now controlled by the monograph for Codeine Phosphate Oral Solution.

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Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal Products Standardised Liquorice Ethanolic Liquid Extract2 Concentrated Peppermint Emulsion Technical Changes

The following monographs in the British Pharmacopoeia 2016 have been technically amended since the publication of the British Pharmacopoeia 2015, or have had a significant editorial change. This list does not include revised monographs of the European Pharmacopoeia. An indication of the nature of the change or the section of the monograph that has been changed is given in italic type in the right hand column. Medicinal and Pharmaceutical Substances Nicorandil Loss on drying —> Water; Assay Phenelzine Sulfate Assay Formulated Preparations: Specific Monographs Identification; Related substances Abacavir Tablets Related substances Adapalene Cream Related substances Adapalene Gel Dissolution; Related substances —> Alendronic Acid Tablets Phosphate and Phosphite; Assay Identification; Related substances Alfuzosin Tablets Prolonged-release Alfuzosin Tablets Identification; Related substances Content of theophylline Aminophylline Injection Content of ethylenediamine Aminophylline Tablets Content of ethylenediamine; Labelling Prolonged-release Aminophylline Tablets Identification (colour test deleted); Carbimazole Tablets Thiamazole and other related substances Definition Chloral Hydrate Oral Solution Removal of unlicensed status; Non­ Clobazam Oral Suspension interchangeability statement added; Dissolution (deleted) Non-interchangeability statement Clobazam Tablets added Non-interchangeability statement Clonazepam Tablets added Assay Clonidine Tablets Definition; Identification test A Clotrimazole Pessaries Definition; Extemporaneous Codeine Phosphate Oral Solution Preparation (deleted); Identification; Assay Uniformity of content Desmopressin Tablets Dexamethasone Sodium Phosphate Content of dexamethasone phosphate -* Content of dexamethasone; Injection Identification; Free dexamethasone -*■ Related substances; Assay; Labelling Related substances Prolonged-release Dipyridamole Capsules Related substances Dipyridamole Infusion 2 Monograph suppressed by European Pharmacopoeia Commission on-1 April 2015.

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Doxorubicin Injection Econazole Pessaries Enoxaparin Sodium Injection Ergocalciferol Injection Ethinylestradiol Tablets Paediatric Ferrous Sulfate Oral Solution Glimepiride Tablets Goserelin Implants Heparin Injection Hydroxychloroquine Tablets Hydroxyzine Oral Solution Kaolin Mixture Kaolin and Morphine Mixture Lamotrigine Tablets Dispersible Lamotrigine Tablets Levonorgestrel Tablets Lisinopril Oral Solution Lymecycline Capsules Aromatic Magnesium Carbonate Mixture Magnesium Hydroxide Mixture Magnesium Sulfate Mixture Magnesium Trisilicate Mixture Melphalan Injection Methotrexate Injection Methotrexate Oral Solution Morphine Sulfate Injection Naloxone Injection Omeprazole Oral Suspension Oxymetholone Tablets Liquid Paraffin Oral Emulsion Liquid Paraffin and Magnesium Hydroxide Oral Emulsion Paroxetine Tablets Phénobarbital Elixir Paediatric Phénobarbital Oral Solution Phénobarbital Tablets Phénobarbital Sodium Tablets Phentolâmine Injection

Related substances Definition; Identification test A; Related substances Sodium Definition; Content of ergocalciferol Uniformity of content Extemporaneous Preparation (deleted) Dissolution Drug release; Related substances tests A , B and C Identification tests A and B; Assay Disintegration; Related substances Identification test A; Related substances; Impurities Extemporaneous Preparation (deleted) Definition; Extemporaneous Preparation (deleted) Non-interchangeability statement added Non-interchangeability statement added Identification test A Related substances Light-absorbing impurities (deleted); Related substances; Water Extemporaneous Preparation (deleted) Extemporaneous Preparation (deleted) Extemporaneous Preparation (deleted) Extemporaneous Preparation (deleted) Related substances test B Related substances ' Identification Assay Identification test A; Related substances Alkalinity (deleted); Dissolution Reference to unlicensed status added Definition; Extemporaneous Preparation (deleted) Definition; Extemporaneous Preparation (deleted) Related substances; Impurities Non-interchangeability statement added Patient monitoring statement added Non-interchangeability statement added Non-interchangeabUity statement added Related substances

2016

I-xxxvi Introduction

Phenytoin Capsules Phenytoin Oral Suspension Phenytoin Tablets Potassium Citrate Mixture Prednisolone Oral Solution Primidone Oral Suspension Primidone Tablets Ramipril Capsules Ramipril Tablets Simvastatin Oral Suspension Sodium Bicarbonate Oral Solution Compound Sodium Chloride Mouthwash Sodium Chloride Oral Solution Sodium Valproate Oral Solution

Non-interchangeability statement added; Related substances Non-interchangeability statement added Non-interchangeability statement added Extemporaneous Preparation (deleted) Related substances Non-interchangeability statement added Non-interchangeability statement added Related substances Related substances Acidity or alkalinity (deleted) Labelling Extemporaneous Preparation (deleted)

Definition; Labelling Non-interchangeabUity statement added; Identification; Related substances Non-interchangeability statement Sodium Valproate Tablets added; Related substances Gastro-resistant Sodium Valproate Non-interchangeability statement added; Dissolution; Related substances Tablets Content of sumatriptan succinate -> Sumatriptan Injection Content of sumatriptan; Identification; Impurities A and H; Related substances; Assay Impurities A and H; Related Sumatriptan Nasal Spray substances; Assay Content of sumatriptan succinate —► Sumatriptan Tablets Content of sumatriptan; Identification; Dissolution; Impurities A and H; Related substances; Assay Labelling Vinblastine Injection Requirements for powder for re­ Vincristine Injection constitution replaced by requirements for ready-to-use solution; Labelling Related substances Zidovudine Capsules Related substances Zidovudine Tablets Zidovudine and Lamivudine Tablets Related substances Zuclopenthixol Decanoate Injection Identification Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal Products Acid Gentian Mixture Extemporaneous Preparation (deleted) Alkaline Gentian Mixture Extemporaneous Preparation (deleted)

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The following list gives the alterations in the titles of monographs of the British Pharmacopoeia 2015 that have been retained in the British Pharmacopoeia 2016. BRITISH PH A RM A CO PO EIA 2015

BRITISH PHARM A CO PO EIA 2016

Medicinal and Pharm aceutical Substances Noscapine Hydrochloride Noscapine Hydrochloride Hydrate Sodium Alendronate Sodium Alendronate Trihydrate Blood-related Products Normal Immunoglobulin

Normal Immunoglobulin for Intramuscular Administration

Herbal Drugs, H erbal D rug Preparations and Herbal Medicinal Products Extracts Herbal Drug Extracts

General Notices 1-1

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General Notices

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CONTENTS OF THE GENERAL NOTICES Part I Italic introduction European Pharmacopoeia Part II Italic introduction Official Standards Definition of Terms Expression of Standards Temperature Weights and Measures Atomic Weights Constant Weight Expression of Concentrations Water Bath Reagents Indicators Caution Statements Titles Chemical Formulae Definition Production Manufacture of Formulated Preparations Freshly and Recently Prepared Methods of Sterilisation Water Excipients Colouring Agents Antimicrobial Preservatives Characteristics Solubility Identification Reference Spectra Assays and Tests Biological Assays and Tests Reference Substances and Reference Preparations Chemical Reference Substances Biological Reference Preparations Storage Labelling Action and Use Crude Drugs; Traditional Herbal and Complementary Medicines Monograph Tide Definition Characteristics Control Methods Homoeopathic Medicines Unlicensed Medicines Part HI Italic introduction General Notices of the European Pharmacopoeia 1.1 General Statements Quality Systems Alternative Methods Demonstration of Compliance with the Pharmacopoeia Grade of Materials General Monographs Validation of Pharmacopoeial Methods

Implementation of Pharmacopoeial Methods Conventional Terms Interchangeable Methods References to Regulatory Documents 1.2 Other Provisions Applying to General Chapters and Monographs Quantities Apparatus and Procedures Water-bath Drying and Ignition to Constant Mass Reagents Solvents Expression of Content Temperature 1.3 General Chapters Containers 1.4 Monographs Tides Relative Atomic and Molecular Masses Chemical Abstracts Service (CAS) Registry Number Definition Limits of Content Herbal Drugs Production Choice of Vaccine Strain^ Choice of Vaccine Composition Potential Adulteration Characters Solubility Identification Scope First and Second Identifications Powdered Herbal Drugs Tests and Assays Scope Calculation Limits Indication of Permitted Limit of Impurities Herbal Drugs Equivalents Culture Media Storage Labelling Warnings Impurities Functionality-related Characteristics of Excipients Reference Standards 1.5 Abbreviations and Symbols Abbreviations used in the Monographs on Immunoglobulins, Immunosera and Vaccines Collections of Micro-organisms 1.6 Units of the International System (SI) used in the Pharmacopoeia and Equivalence with other Units International System of Units (SI) Notes

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General Notices

Part I The British Pharmacopoeia comprises the entire text within this publication. The word ‘official3is used in the Pharmacopoeia to signify (of the Pharmacopoeia\ It applies to any title, substance, preparation} method or statement included in the general notices, monographs and appendices of the Pharmacopoeia. The abbreviation for British Pharmacopoeia is BP. European Monographs of the European Pharmacopoeia are reproduced in this edition Pharmacopoeia of the British Pharmacopoeia by incorporation of the text published under the direction of the Council of Europe (Partial Agreement) in accordance with the Convention on the Elaboration of a European Pharmacopoeia (Treaty Series No. 32 (1974) CMND 5763) as amended by the Protocol to the Convention (Treaty Series No. MISC16 (1990) CMND 1133). They are included for the convenience of users of the British Pharmacopoeia. In cases of doubt or dispute reference should be made to the Council of Europe text. Monographs of the European Pharmacopoeia are distinguished by a V * chaplet of stars against the title and by reference to the European * Pharmacopoeia monograph number included immediately below the title in italics. The beginning and end of text from the European Pharmacopoeia are denoted by means of horizontal lines with the symbol (Ph Eur* ranged leftand right, respectively. The general provisions of the European Pharmacopoeia relating to different types of dosage form are included in the appropriate general monograph in that section of the British Pharmacopoeia entided Monographs: Formulated Preparations. These general provisions apply to all dosage forms of the type defined, whether or not an individual monograph is included in the British Pharmacopoeia. In addition, the provisions of the European Pharmacopoeia General Monograph for Pharmaceutical Preparations apply to all dosage forms, whether or not an individual monograph is included in the British Pharmacopoeia. Texts of the European Pharmacopoeia are governed by the General Notices of the European Pharmacopoeia. These are reproduced as Part HE of these notices.

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Part II The following général notices apply to the statements made in the monographs of the British Pharmacopoeia other than those reproduced from the European Pharmacopoeia and to the statements made in the Appendices of the British Pharmacopoeia other than when a method, test or other matter described in an appendix is invoked in a monograph reproduced from the European Pharmacopoeia. Official Standards

The requirements stated in the monographs of the Pharmacopoeia apply to articles that are intended for medicinal use but not necessarily to articles that may be sold under the same name for other purposes. An article intended for medicinal use that is described by means of an official title must comply with the requirements of the relevant monograph. A formulated preparation must comply throughout its assigned shelf-life (period of validity). The subject of any other monograph must comply throughout its period of use. A monograph is to be construed in accordance with any general monograph or notice or any appendix, note or other explanatory material that is contained in this edition and that is applicable to that monograph. All statements contained in the monographs, except where a specific general notice indicates otherwise and with the exceptions given below, constitute standards for the official articles. An article is not of pharmacopoeial quality unless it complies with all of the requirements stated. This does not imply that a manufacturer is obliged to perform all the tests in a monograph in order to assess compliance with the Pharmacopoeia before release of a product. The manufacturer may assure himself that a product is of pharmacopoeial quality by other means, for example, from data derived from validation studies of the manufacturing process, from in-process controls or from a combination of the two. Parametric release in appropriate circumstances is thus not precluded by the need to comply with the Pharmacopoeia. The general notice on Assays and Tests indicates that analytical methods other than those described in the Pharmacopoeia may be employed for routine purposes. Requirements in monographs have been framed to provide appropriate limitation of potential impurities rather than to provide against all possible impurities. Material found to contain an impurity not detectable by means of the prescribed tests is not of pharmacopoeial quality if the nature or amount of the impurity found is incompatible with good pharmaceutical practice. The status of any statement given under the headings Definition, Production, Characteristics, Storage, Labelling or Action and use is defined within the general notice relating to the relevant heading. In addition to any exceptions indicated by one of the general notices referred to above, the following parts of a monograph do not constitute standards: (a) a graphic or molecular formula given at the beginning of a monograph; (b) a molecular weight; (c) a Chemical Abstracts Service Registry Number; (d) any information given at the end of a monograph concerning impurities known to be limited by that monograph; (e) information in any annex to a

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monograph. Any statement containing the word ‘should5 constitutes non-mandatory advice or recommendation. The expression ‘unless otherwise justified and authorised’ means that the requirement in question has to be met, unless a competent authority authorises a modification or exemption where justified in a particular case. The term ‘competent authority5 means the national, supranational or international body or organisation vested with the authority for making decisions concerning the issue in question. It may, for example, be a licensing authority or an official control laboratory. For a formulated preparation that is the subject of monograph in the British Pharmacopoeia any justified and authorised modification to, or exemption from, the requirements of the relevant general monograph of the European Pharmacopoeia is stated in the individual monograph. For example, the general monograph for Tablets requires that Uncoated Tablets, except for chewable tablets, disintegrate within 15 minutes; for Calcium Lactate Tablets a time of 30 minutes is permitted. Many of the general monographs for formulated preparations include statements and requirements additional to those of the European Pharmacopoeia that are applicable to the individual monographs of the British Pharmacopoeia. Such statements and requirements apply to all monographs for that dosage form included in the Pharmacopoeia unless otherwise indicated in the individual monograph. Where a monograph on a biological substance or preparation refers to a strain, a test, a method, a substance, etc., using the qualifications ‘suitable’ or ‘appropriate’ without further definition in the text, the choice of such strain, test, method, substance, etc., is made in accordance with any international agreements or national regulations affecting the subject concerned. Definition of Terms

Where the term ‘about’ is included in a monograph or test it should be taken to mean approximately (fairly correct or accurate; near to the actual value). Where the term ‘corresponds’ is included in a monograph or test it should be taken to mean similar or equivalent in character or quantity. Where the term ‘similar’ is included in a monograph or test it should be taken to mean alike though not necessarily identical. Further qualifiers (such as numerical acceptance criteria) for the above terms are not included in the BP. The acceptance criteria for any individual case is set based on the range of results obtained from known reference samples, the level of precision of the equipment or apparatus used and the level of accuracy required for the particular application. The user should determine the variability seen in his/her own laboratory and set in-house acceptance criteria that he/she judges to be appropriate based on the local operating conditions.

Expression of Where the standard for the content of a substance described in a Standards monograph is expressed in terms of the chemical formula for that substance an upper limit exceeding 100% may be stated. Such an upper limit applies to the result of the assay calculated in terms of the equivalent content of the specified chemical formula. For example, the statement ‘contains not less than 99.0% and not more than 101.0% of C 20H 24N 2O2JHCI’ implies that the result of the assay is not less than 99.0% and not more than 101.0%, calculated in terms of the equivalent content of C20H 24N 2O2JHCI.

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Where the result of an assay or test is reqmred to be calculated with . .. . reference to die dried, anhydrous or ignited substance, the substance free from a specified solvent or to the peptide content, thệ determination of loss on drying, water content, loss on ignition, content of the specified solvent or peptide content is carried Gut by the method prescribed in the relevant test in the monograph. Temperature The Celsius thermometric scale is used in expressing temperatures. Weights and The metric system of weights and measures is employed; SI Units have Measures generally been adopted. Metric measures are required to have been graduated at 20 ° and all measurements involved in the analytical operations of die Pharmacopoeia are intended, unless otherwise stated, to be made at that temperature. Graduated glass apparatus used in analytical operations should comply with Class A requirements of the appropriate International Standard issued by the International Organization for Standardization. The abbreviation for litre is ‘L’ throughout die Pharmacopoeia. In line with European Dừeetive 80/181/EEC, the abbreviation T is also permitted for use. Atomic Weights

Constant Weight

The atomic weights adopted are the values given in the Table of Relative Atomic Weights 2001 published by the International Union of Pure and Applied Chemistry (Appendix XXV). The term ‘constant weight’, used in relation to the process of drying or the process of ignition, means that two consecutive weighings do not differ by more than 0.5 mg, the second weighing being made after an additional period of drying or ignition under the specified conditions appropriate to die nature and quantity of the residue (1 hour is usually suitable).

Expression of The term ‘per cent’ or more usually the symbol .*.%■is used with one of four Concentrations different meanings in the expression of concenưations according to cừcumstances. In order that the meaning to be attached to the expression in each instance is clear, the following notation is used: Per cent w/w (% w/w) (percentage weight in weight) expresses the number of grams of solute in 100 g of product. Per cent w/v (% w/v) (percentage weight in volume) expresses the number of grams of solute in 100 mL of product. Per cent v/v (% v/v) (percentage volume in volume) expresses the number of millilitres of solute in 100 mL of product. Per cent v/w (% v/w) (percentage volume in weight) expresses the number OĨ millilitres of solute in 100 g of product. Usually the sưength of solutions of solids in liquids is expressed as percentage weight in volume, of liquids in liquids as percentage volume in volume and of gases in liquids as percentage weight in weight. When the concentration of a solution is expressed as parts per million (ppm), it means weight in weight, unless otherwise specified. When the concentration of a solution is expressed as parts of dissolved substance in parts of the solution, it means parts by weight (g) of a solid in parts by volume (mL) of the final solution; or parts by volume (mL) of a liquid in pans by volume (mL) of the final solution; or parts by weight (g) of a gas in parts by weight (g) of the final solution.

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When the concentration of a solution is expressed in molarity designated by the symbol m preceded by a number, it denotes the number of moles of the stated solute contained in sufficient Purified Water (unless otherwise stated) to produce 1 litre of solution. Water Bath

Reagents

The term ‘water bath’ means a bath of boiling water, unless water at some other temperature is indicated in the text. An alternative form of heating may be employed providing that the required temperature is approximately maintained but not exceeded. The reagents required for the assays and tests of the Pharmacopoeia are defined in appendices. The descriptions set out in the appendices do not imply that the materials are suitable for use in medicine.

Indicators

Indicators, the colours of which change over approximately the same range of pH, may be substituted for one another but in the event of doubt or dispute as to the equivalence of indicators for a particular purpose, the indicator specified in the text is alone authoritative. The quantity of an indicator solution appropriate for use in acid-base titrations described in assays or tests is 0.1 mL unless otherwise stated in the text. Any solvent required in an assay or test in which an indicator is specified is previously neutralised to the indicator, unless a blank test is prescribed.

Caution Statements

A number of materials described in the monographs and some of the reagents specified for use in the assays and tests of the Pharmacopoeia may be injurious to health unless adequate precautions are taken. The principles of good laboratory practice and the provisions of any appropriate regulations such as those issued in the United Kingdom in accordance with the Health and Safety at Work etc. Act 1974 should be observed at all times in carrying out the assays and tests of the Pharmacopoeia. Attention is drawn to particular hazards in certain monographs by means of an italicised statement; the absence of such a statement should not however be taken to mean that no hazard exists.

Titles

Subsidiary tides, where included, have the same significance as the main tides. An abbreviated title constructed in accordance with the directions given in Appendix XXI A has the same significance as the main title. Titles that are derived by the suitable inversion of words of a main or subsidiary tide, with the addition of a preposition if appropriate, are also official titles. Thus, the following are all official tides: Aspirin Tablets, Tablets of Aspirin; Atropine Injection, Injection of Atropine. A title of a formulated preparation that includes the full nonproprietary name of the active ingredient or ingredients, where this is not included in ^ the title of the monograph, is also an official tide. For example, the title Promethazine Hydrochloride Oral Solution has the same significance as Promethazine Oral Solution and the title Brompheniramine Maleate Tablets has the same significance as Brompheniramine Tablets. Where the English tide at the head of a monograph in the European Pharmacopoeia is different from that at the head of the text incorporated into the British Pharmacopoeia, an Approved Synonym has been created on the recommendation of the British Pharmacopoeia Commission. Approved Synonyms have the same significance as the main title and are thus official

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titles. A cumulative list of such Approved Synonyms is provided in Appendix XXI B. Where the names of pharmacopoeial substances, preparations and other materials occur in the text they are printed with capital initial letters and this indicates that materials of Pharmacopoeial quality must be used. Words in the text that name a reagent or other material, a physical characteristic or a process that is described or defined in an appendix are printed in italic type, for example, methanol, absorbance, gas chromatography, and these imply compliance with the requirements specified in the appropriate appendix. Chemical Formulae

When the chemical composition of an official substance is known or generally accepted, the graphic and molecular formulae, the molecular weight and the Chemical Abstracts Service Registry Number are normally given at the beginning of the monograph for information. This information refers to the chemically pure substance and is not to be regarded as an indication of the purity of the official material. Elsewhere, in statements of standards of purity and strength and in descriptions of processes of assay, it is evident from the context that the formulae denote the chemically pure substances. Where the absolute stereochemical configuration is specified, the International Union of Pure and Applied Chemistry (IUPAC) R/S and E/Z systems of designation have been used. If the substance is an enantiomer of unknown absolute stereochemistry the sign of the optical rotation, as determined in the solvent and under the conditions specified in the monograph, has been attached to the systematic name. An indication of sign of rotation has also been given where this is incorporated in a trivial name that appears on an IUPAC preferred list. All amino acids, except glycine, have the L-configuration unless otherwise indicated. The three-letter and one-letter symbols used for amino acids in peptide and protein sequences are those recommended by the Joint Commission on Biochemical Nomenclature of the International Union of Pure and Applied Chemistry and the International Union of Biochemistry and Molecular Biology. In the graphic formulae the following abbreviations are used: Me Et Pr! Pr” Bul

Definition

-C H 3 - c h 2c h 3 -CH(CH 3)2 - c h 2c h 2c h 3 -C H 2CH(CH 3)2

Bu* Bu" Bur Ph Ac

-CH(CH 3)CH 2CH 3 - c h 2c h 2c h 2c h 3 -C(CH 3)3 - c 6h 5 -c o c h 3

Statements given under the heading Definition constitute an official definition of the substance, preparation or other article that is the subject of the monograph. They constitute instructions or requirements and are mandatory in nature. Certain medicinal or pharmaceutical substances and other articles are defined by reference to a particular method of manufacture. A statement that a substance or article is prepared or obtained by a certain method constitutes part of the official definition and implies that other methods are not permitted. A statement that a substance may be prepared or obtained by a certain method, however, indicates that this is one possible method and does not imply that other methods are proscribed.

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Additional statements concerning the definition of formulated preparations are given in the general notice on Manufacture of Formulated Preparations. Production

Statements given under the heading Production draw attention to particular aspects of the manufacturing process but are not necessarily comprehensive. They constitute mandatory instructions to manufacturers. They may relate, for example, to source materials, to the manufacturing process itself and its validation and control, to in-process testing or to testing that is to be carried out by the manufacturer on the final product (bulk material or dosage form) either on selected batches or on each batch prior to release. These statements cannot necessarily be verified on a sample of the final product by an independent analyst. The competent authority may establish that the instructions have been followed, for example, by examination of data received from the manufacturer, by inspection or by testing appropriate samples. The absence of a section on Production does not imply that attention to features such as those referred to above is not required. A substance, preparation or article described in a monograph of the Pharmacopoeia is to be manufactured in accordance with the principles of good manufacturing practice and in accordance with relevant international agreements and supranational and national regulations governing medicinal products. Where in the section under the heading Production a monograph on a vaccine defines the characteristics of the vaccine strain to be used, any test methods given for confirming these characteristics are provided as examples of suitable methods. The use of these methods is not mandatory. Additional statements concerning the production of formulated preparations are given in the general notice on Manufacture of Formulated Preparations.

Manufacture of Formulated Preparations

Attention is drawn to the need to observe adequate hygienic precautions in the preparation and dispensing of pharmaceutical formulations. The principles of good pharmaceutical manufacturing practice should be observed. The Definition in certain monographs for pharmaceutical preparations is given in terms of the principal ingredients only. Any ingredient, other than those included in the Definition, must comply with the general notice on Excipients and the product must conform with the Pharmacopoeial requirements. The Definition in other monographs for pharmaceutical preparations is presented as a full formula. No deviation from the stated formula is permitted except those allowed by the general notices on Colouring Agents and Antimicrobial Preservatives. Where additionally directions are given under the heading Extemporaneous Preparation these are intended for the extemporaneous preparation of relatively small quantities for short-term supply and use. When so prepared, no deviation from the stated directions is permitted. If, however, such a pharmaceutical preparation is manufactured on a larger scale with the intention that it may be stored, deviations from the stated directions are permitted provided that the final product meets the following criteria:

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( 1) compliance with all of the requirements stated in the monograph; (2) retention of the essential characteristics of the preparation made strictly in accordance with the directions of the Pharmacopoeia. Monographs for yet other pharmaceutical preparations include both a Definition in terms of the principal ingredients and, under the side-heading Extemporaneous Preparation, a full formula together with, in some cases, directions for their preparation. Such full formulae and directions are intended for the extemporaneous preparation of relatively small quantities for short-term supply and use. When so prepared, no deviation from the stated formula and directions is permitted. If, however, such a pharmaceutical preparation is manufactured on a larger scale with the intention that it may be stored, deviations from the formula and directions stated under the heading Extemporaneous Preparation are permitted provided that any ingredient, other than those included in the Definition, complies with the general notice on Excipients and that the final product meets the following criteria: ( 1) accordance with the Definition stated in the monograph; (2) compliance with all of the requirements stated in the monograph; (3) retention of the essential characteristics of the preparation made strictly in accordance with the formula and directions of the Pharmacopoeia. In the manufacture of any official preparation on a large scale with the intention that it should be stored, in addition to following any instruction under the heading Production, it is necessary to ascertain that the product is satisfactory with respect to its physical and chemical stability and its state of preservation over the claimed shelf-life. This applies irrespective of whether the formula of the Pharmacopoeia and any instructions given under the heading Extemporaneous Preparation are followed precisely or modified. Provided that the preparation has been shown to be stable in other respects, deterioration due to microbial contamination may be inhibited by the incorporation of a suitable antimicrobial preservative. In such circumstances the label states appropriate storage conditions, the date after which the product should not be used and the identity and concentration of the antimicrobial preservative. Freshly and Recently Prepared

The direction, given under the heading Extemporaneous Preparation, that a preparation must be freshly prepared indicates that it must be made not more than 24 hours before it is issued for use. The direction that a preparation should be recendy prepared indicates that deterioration is likely if the preparation is stored for longer than about 4 weeks at 15° to 25°.

Methods of Sterilisation

The methods of sterilisation used in preparing the sterile materials described in the Pharmacopoeia are given in Appendix XVIIL For aqueous preparations, steam sterilisation (heating in an autoclave) is the method of choice wherever it is known to be suitable. Any method of sterilisation must be validated with respect to both the assurance of sterility and the integrity of the product and to ensure that the final product complies with the requirements of the monograph.

Water

The term water used without qualification in formulae for formulated preparations means either potable water freshly drawn direct from the public supply and suitable for drinking or freshly boiled and cooled Purified

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Water. The latter should be used if the public supply is from a local storage tank or if the potable water is unsuitable for a particular preparation. Excipients

Colouring Agents

Where an excipient for which there is a pharmacopoeial monograph is used in preparing an official preparation it shall comply with that monograph. Any substance added in preparing an official preparation shall be innocuous, shall have no adverse influence on the therapeutic efficacy of the active ingredients and shall not interfere with the assays and tests of the Pharmacopoeia. Particular care should be taken to ensure that such substances are free from harmful organisms. If in a monograph for a formulated preparation defined by means of a full formula a specific colouring agent or agents is prescribed, suitable alternatives approved in the country concerned may be substituted.

Antimicrobial Preservatives

When the term ‘suitable antimicrobial preservative’ is used it is implied that the preparation concerned will be effectively preserved according to the appropriate criteria applied and interpreted as described in the test for efficacy of antimicrobial preservation (Appendix XVI C). In certain monographs for formulated preparations defined by means of a full formula, a specific antimicrobial agent or agents may be prescribed; suitable alternatives may be substituted provided that their identity and concentration are stated on the label.

Characteristics

Statements given under the heading Characteristics are not to be interpreted in a strict sense and are not to be regarded as official requirements. Statements on taste are provided only in cases where this property is a guide to the acceptability of the material (for example, a material used primarily for flavouring). The status of statements on solubility is given in the general notice on Solubility. Solubility Statements on solubility given under the heading Characteristics are intended as information on the approximate solubility at a temperature between 15° and 25°, unless otherwise stated, and are not to be considered as official requirements. Statements given under headings such as Solubility in ethanol express exact requirements and constitute part of the standards for the substances under which they occur. The following table indicates the meanings of the terms used in statements of approximate solubilities. Descriptive term

Approximate volume of solvent in millilitres per gram of solute

very soluble freely soluble soluble sparingly soluble slightly soluble very slightly soluble practically insoluble

less than 1 from 1 to 10 from 10 to 30 from 30 to 100 from 100 to 1000 from 1000 to 10,000 more than 10,000

The term ‘partly soluble’ is used to describe a mixture of which only some of the components dissolve.

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Identification

The tests described or referred to under the heading Identification are not necessarily sufficient to establish absolute proof of identity. They provide a means of verifying that the identity of the material being examined is in accordance with the label on the container. Unless otherwise prescribed, identification tests are carried out at a temperature between 15° and 25°. Reference spectra Where a monograph refers to an infrared reference spectrum, this spectrum is provided in a separate section of the Pharmacopoeia. A sample spectrum is considered to be concordant with a reference spectrum if the transmission minima (absorption maxima) of the principal bands in the sample correspond in position, relative intensities and shape to those of the reference. Instrumentation software may be used to calculate concordance with a previously recorded reference spectrum. When tests for infrared absorption are applied to material extracted from formulated preparations, strict concordance with the specified reference spectrum may not always be possible, but nevertheless a close resemblance between the spectrum of the extracted material and the specified reference spectrum should be achieved.

Assays an d Tests

The assays and tests described are the official methods upon which the standards of the Pharmacopoeia depend. The analyst is not precluded from employing alternative methods, including jnethods of micro-analysis, in any assay or test if it is known that the method used will give a result of equivalent accuracy. Local reference materials may be used for routine analysis, provided that these are calibrated against the official reference materials. In the event of doubt or dispute, the methods of analysis, the reference materials and the reference spectra of the Pharmacopoeia are alone authoritative. Where the solvent used for a solution is not named, the solvent is Purified Water. Unless otherwise prescribed, the assays and tests are carried out at a temperature between 15° and 25°. A temperature in a test for Loss on drying, where no temperature range is given, implies a range of ± 2 ° about the stated value. Visual com parative tests, unless otherwise prescribed, are carried out

using identical tubes of colourless, transparent, neutral glass with a flat base. The volumes of liquid prescribed are for use with tubes 16 mm in internal diameter; tubes with a larger internal diameter may be used but the volume of liquid examined must be increased so that the depth of liquid in the tubes is not less than that obtained when the prescribed volume of liquid and tubes 16 mm in internal diameter are used. Equal volumes of the liquids to be compared are examined down the vertical axis of the tubes against a white background or, if necessary, against a black background. The examination is carried out in diffuse light. Where a direction is given that an analytical operation is to be carried out ‘in subdued light’, precautions should be taken to avoid exposure to direct sunlight or other strong light. Where a direction is given that an analytical operation is to be carried out ‘protected from light’, precautions should be taken to exclude actinic light by the use of low-actinic glassware, working in a dark room or similar procedures. For preparations other than those of fixed strength, the quantity to be taken for an assay or test is usually expressed in terms of the active ingredient. This means that the quantity of the active ingredient expected to

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be present and the quantity of the preparation to be taken are calculated from the strength stated on the label. In assays the approximate quantity to be taken for examination is indicated but the quantity actually used must not deviate by more than 10% from that stated. The quantity taken is accurately weighed or measured and the result of the assay is calculated from this exact quantity. Reagents are measured and the procedures are carried out with an accuracy commensurate with the degree of precision implied by the standard stated for the assay. In tests the stated quantity to be taken for examination must be used unless any divergence can be taken into account in conducting the test and calculating the result. The quantity taken is accurately weighed or measured with the degree of precision implied by the standard or, where the standard is not stated numerically (for example, in tests for Clarity and colour of solution), with the degree of precision implied by the number of significant figures stated. Reagents are measured and the procedures are carried out with an accuracy commensurate with this degree of precision. The limits stated in monographs are based on data obtained in normal analytical practice; they take account of normal analytical errors, of acceptable variations in manufacture and of deterioration to an extent considered acceptable. No further tolerances are to be applied to the limits prescribed to determine whether the article being examined complies with the requirements of the monograph. In determining compliance with a numerical limit, the calculated result of a test or assay is first rounded to the number of significant figures stated, unless otherwise prescribed. The last figure is increased by 1 when the part rejected is equal to or exceeds one half-unit, whereas it is not modified when the pan rejected is less than a half-unit. In certain tests, the concentration of impurity is given in parentheses either as a percentage or in parts per million by weight (ppm). In chromatographic tests such concentrations are stated as a percentage irrespective of the limit. In other tests they are usually stated in ppm unless the limit exceeds 500 ppm. In those chromatographic tests in which a secondary spot or peak in a chromatogram obtained with a solution of the substance being examined is described as corresponding to a named impurity and is compared with a spot or peak in a chromatogram obtained with a reference solution of the same impurity, the percentage given in parentheses indicates the limit for that impurity. In those chromatographic tests in which a spot or peak in a chromatogram obtained with a solution of the substance being examined is described in terms other than as corresponding to a named impurity (commonly, for example, as any (other) secondary spot or peak) but is compared with a spot or peak in a chromatogram obtained with a reference solution of a named impurity, the percentage given in parentheses indicates an impurity limit expressed in terms of a nominal concentration of the named impurity. In chromatographic tests in which a comparison is made between spots or peaks in chromatograms obtained with solutions of different concentrations of the substance being examined, the percentage given in parentheses indicates an impurity limit expressed in terms of a nominal concentration of the medicinal substance itself. In some monographs, in particular those for certain formulated preparations, the impurity limit is expressed in terms of a nominal concentration of the active moiety rather than of the medicinal

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substance itself. Where necessary for clarification the terms in which the limit is expressed are stated within the monograph. In all cases where an impurity limit is given in parentheses, the figures given are approximations for information only; conformity with the requirements is determined on the basis of compliance or otherwise with the stated test. The use of a proprietary designation to identify a material used in an assay or test does not imply that another equally suitable material may not be used. Biological Assays and Tests

Methods of assay described as Suggested methods are not obligatory, but when another method is used its precision must be not less than that required for the Suggested method. For those antibiotics for which the monograph specifies a microbiological assay the potency requirement is expressed in the monograph in International Units (IU) per milligram. The material is not of pharmacopoeial quality if the upper fiducial limit of error is less than the stated potency. For such antibiotics the required precision of the assay is stated in the monograph in terms of the fiducial limits of error about the estimated potency. For other substances and preparations for which the monograph specifies a biological assay, unless otherwise stated, the precision of the assay is such that the fiducial limits of error, expressed as a percentage of the estimated potency, are within a range not wider than that obtained by multiplying by a factor of 10 the square roots of the limits given in the monograph for the fiducial limits of error about the stated potency. In all cases fiducial limits of error are based on a probability of 95% (P = 0.95). Where the biological assay is being used to ascertain the purity of the material, the stated potency means the potency stated on the label in terms of International Units (IU) or other Units per gram, per milligram or per millilitre. When no such statement appears on the label, the stated potency means the fixed or minimum potency required in the monograph. This interpretation of stated potency applies in all cases except where the m onograph specifically directs otherwise.

Where the biological assay is being used to determine the total activity in the container, the stated potency means the total number of International Units (IU) or other Units stated on the label or, if no such statement appears, the total activity calculated in accordance with the instructions in the monograph. Wherever possible the primary standard used in an assay or test is the respective International Standard or Reference Preparation established by the World Health Organization for international use and the biological activity is expressed in International Units (IU). In other cases, where Units are referred to in an assay or test, the Unit for a particular substance or preparation is, for the United Kingdom, the specific biological activity contained in such an amount of the respective primary standard as the appropriate international or national organisation indicates. The necessary information is provided with the primary standard. Unless otherwise directed, animals used in an assay or a test are healthy animals, drawn from a uniform stock, that have not previously been treated with any material that will interfere with the assay or test. Unless otherwise stated, guinea-pigs weigh not less than 250 g or, when used in systemic

General Notices 1-15

toxicity tests, not less than 350 g. When used in skin tests they are white or light coloured. Unless otherwise stated, mice weigh not less than 17 g and not more than 22 g. Certain of the biological assays and tests of the Pharmacopoeia are such that in the United Kingdom they may be carried out only in accordance with the Animals (Scientific Procedures) Act 1986. Instructions included in such assays and tests in the Pharmacopoeia, with respect to the handling of animals, are therefore confined to those concerned with the accuracy and reproducibility of the assay or test. Reference Substances and Reference Preparations

Storage

Certain monographs require the use of a reference substance, a reference preparation or a reference spectrum. These are chosen with regard to their intended use as prescribed in the monographs of the Pharmacopoeia and are not necessarily suitable in other circumstances. Any information necessary for proper use of the reference substance or reference preparation is given on the label or in the accompanying leaflet or brochure. Where no drying conditions are stated in the leaflet or on the label, the substance is to be used as received. No certificate of analysis or other data not relevant to the prescribed use of the product are provided. The products are guaranteed to be suitable for use for a period of three months from dispatch when stored under the appropriate conditions. The stability of the contents of opened containers cannot be guaranteed. The current lot is listed in the BP Laboratory website catalogue. Additional information is provided in Supplementary Chapter III E. Chemical Reference Substances The abbreviation BPCRS indicates a Chemical Reference Substance established by the British Pharmacopoeia Commission. The abbreviation CRS or EPCRS indicates a Chemical Reference Substance established by the European Pharmacopoeia Commission. Some Chemical Reference Substances are used for the microbiological assay of antibiotics and their activity is stated, in International Units, on the label or on the accompanying leaflet and defined in the same manner as for Biological Reference Preparations. Biological Reference Preparations The majority of the primary biological reference preparations referred to are the appropriate International Standards and Reference Preparations established by the World Health Organisation. Because these reference materials are usually available only in limited quantities, the European Pharmacopoeia has established Biological Reference Preparations (indicated by the abbreviation BRP or EPBRP) where appropriate. Where applicable, the potency of the Biological Reference Preparations is expressed in International Units. For some Biological Reference Preparations, where an international standard or reference preparation does not exist, the potency is expressed in European Pharmacopoeia Units. Statements under the side-heading Storage constitute non-mandatory advice. The substances and preparations described in the Pharmacopoeia are to be stored under conditions that prevent contamination and, as far as possible, deterioration. Unless otherwise stated in the monograph, the substances and preparations described in the Pharmacopoeia are kept in well-closed containers and stored at a temperature not exceeding 25°. Precautions that should be taken in relation to the effects of the atmosphere, moisture, heat and light are indicated, where appropriate, in

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the monographs. Further precautions may be necessary when some materials are stored in tropical climates or under other severe conditions. The expression ‘protected from moisture’ means that the product is to be stored in an airtight container. Care is to be taken when the container is opened in a damp atmosphere. A low moisture content may be maintained, if necessary, by the use of a desiccant in the container provided that direct contact with the product is avoided. The expression ‘protected from light’ means that the product is to be stored either in a container made of a material that absorbs actinic light sufficiently to protect the contents from change induced by such light or in a container enclosed in an outer cover that provides such protection or stored in a place from which all such light is excluded. The expression ‘tamper-evident container3 means a closed container fitted with a device that reveals irreversibly whether the container has been opened, whereas, the expression ‘tamper-proof container’ means a closed container in which access to the contents is prevented under normal conditions of use. The two terms are considered to be synonymous by the European Pharmacopoeia Commission. Labelling

The labelling requirements of the Pharmacopoeia are not comprehensive, and the provisions of regulations issued in accordance with the requirements of the territory in which the medicinal product is to be used should be met. Licensed medicines intended for use within the United Kingdom must comply with the requirements of The Human Medicines Regulations 2012 and European Directive 2001/83/EC, Title V (as amended) in respect of their labelling and package leaflets, together with those regulations for the labelling of hazardous materials. Best practice guidance on the labelling and packaging of medicines for use in the United Kingdom advises that certain items of information are deemed critical for the safe use of the medicine (see “Best Practice Guidance on the Labelling and Packaging of Medicines” issued by the MHRA, 2012) . Further information and guidance on the labelling of medicinal products can be found in Supplementary Chapter I G. Such matters as the exact form of wording to be used and whether a particular item of information should appear on the primary label and additionally, or alternatively, on the package or exceptionally in a leaflet are, in general, outside the scope of the Pharmacopoeia. When the term ‘label’ is used in Labelling statements of the Pharmacopoeia, decisions as to where the particular statement should appear should therefore be made in accordance with relevant legislation. The label of every official formulated preparation other than those of fixed strength also states the content of the active ingredient or ingredients expressed in the terms required by the monograph. Where the content of active ingredient is required to be expressed in terms other than the weight of the official medicinal substance used in making the formulation, this is specifically stated under the heading Labelling. Unless otherwise stated in the monograph, the content of the active ingredient is expressed in terms of the official medicinal substance used in making the formulation. These requirements do not necessarily apply to unlicensed preparations supplied in accordance with a prescription. For requirements for unlicensed medicines see the general monograph on Unlicensed Medicines.

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Action an d Use

The statements given under this heading in monographs are intended only as information on the principal pharmacological actions or the uses of the materials in medicine or pharmacy. It should not be assumed that the substance has no other action or use. The statements are not intended to be binding on prescribers or to limit their discretion.

C rude D rugs; T raditional H erbal and C om plem entary M edicines

Herbal and complementary medicines are classed as medicines under European Directive 2001/83/EC as amended. It is emphasised that, although requirements for the quality of the material are provided in the monograph to assist the registration scheme by the UK Licensing Authority, the British Pharmacopoeia Commission has not assessed the safety or efficacy of the material in traditional use. Monograph Title For traditional herbal medicines, the monograph title is a combination of the binomial name together with a description of use. Monographs for the material that has not been processed (the herbal drug) and the processed material (the herbal drug preparation) are published where possible. To distinguish between the two, the word ‘Processed’ is included in the relevant monograph title. Definition Under the heading Definition, the botanical name together with any synonym is given. Where appropriate, for material that has not been processed, information on the collection/harvesting and/or treatment/ drying of the whole herbal drug may be given. For processed materials, the method of processing, where appropriate, will normally be given in a separate section. Characteristics References to odour are included only where this is highly characteristic. References to taste are not included. Control methods Where applicable, the control methods to be used in monographs are: (a) macroscopical and microscopical descriptions and chemical/ chromatographic tests for identification (b) tests for absence of any related species (c) microbial test to assure microbial quality (d) tests for inorganic impurities and non-specific purity tests, including extractive tests, Sulfated ash and Heavy metals where appropriate (e) test for Loss on drying or Water (f) wherever possible, a method for assaying the active constituent(s) or suitable marker constituent (s). The macroscopical characteristics include those features that can be seen by the unaided eye or by the use of a hand lens. When two species/ subspecies of the same plant are included in the Definition, individual differences between the two are indicated where possible. The description of the microscopical characteristics of the powdered drug includes information on the dominant or the most specific characters. Where it is considered to be an aid to identification, illustrations of the powdered drug may be provided. The following aspects are controlled by the general monograph for Herbal Drugs: they are required to be free from moulds, insects, decay, animal matter and animal excreta. Unless otherwise prescribed the amount of foreign matter is not more than 2 % w/w. Microbial contamination should be minimal.

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In determining the content of the active constituents or the suitable marker substances measurements are made with reference to the dried or anhydrous herbal drug. In the tests for Acid-insoluble ash. Ash, Extractive soluble in ethanol. Loss on drying, Sulfated ash, Water, Water-soluble ash and Water-soluble extractive of herbal drugs, the calculations are made with reference to the herbal drug that has not been specifically dried unless otherwise prescribed in the monograph. Homoeopathic Medicines

Unlicensed Medicines

Homoeopathic medicines are classed as medicines under European Directive 2001/83IEC as amended. It is emphasised that, although requirements for the quality of the material are provided in the relevant monograph in order to assist the simplified registration scheme by the UK Licensing Authority, the British Pharmacopoeia Commission has not assessed the safety or efficacy of the material in use. All materials used for the production of homoeopathic medicines, including excipients, must comply with European Pharmacopoeia or British Pharmacopoeia monographs for those materials. Where such European Pharmacopoeia or British Pharmacopoeia monographs do not exist, each material used for the production of homoeopathic medicines must comply with an official national pharmacopoeia of a Member State. British Pharmacopoeia monographs for homoeopathic medicines apply to homoeopathic stocks and mother tinctures only, but may be prefaced by a section which details the quality requirements applicable to the principle component where there is no European Pharmacopoeia or British Pharmacopoeia monograph for the material. These monographs also include either general statements on the methods of preparation or refer to specific methods of preparation given in the European Pharmacopoeia. Homoeopathic stocks and mother tinctures undergo the further process referred to as potentisation. Potentisation is a term specific to homoeopathic medicine and is a process of dilution of stocks and mother tinctures to produce the final product. Identification tests are established for the components in homoeopathic stocks and usually relate to those applied to the materials used in the production of the homoeopathic stocks. An assay is included for the principal component(s) where possible. For mother tinctures, an identification test, usually chromatographic, is established and, where applicable, an assay for the principle component(s); where appropriate, other tests, related to the solvent, dry matter or known adulterants, are included. Specifications have not been set for final homoeopathic products due to the high dilution used in their preparation and the subsequent difficulty in applying analytical methodology. Statements under Crude Drugs; Traditional Herbal and Complementary Medicines also apply to homoeopathic stocks and mother tinctures, when appropriate. The General Monograph for Unlicensed Medicines applies to those formulations used in human medicine that are prepared under a Manufacturer’s ‘Specials’ Licence or prepared extemporaneously under the supervision of a pharmacist, whether or not there is a published monograph for the specific dosage form. An article intended for medicinal use that is described by means of an official title must comply with the requirements of the relevant monograph.

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A formulated preparation must comply throughout its assigned shelf-life (period of validity). The subject of any other monograph must comply throughout its period of use. Unlicensed medicines that are prepared under a Manufacturer’s ‘Specials’ Licence comply with the requirements of the General Monograph for Pharmaceutical Preparations, the requirements of the General Monograph for Unlicensed Medicines and, where applicable, the requirements of the individual monograph for the specific dosage form. Unlicensed medicines prepared extemporaneously under the supervision of a pharmacist comply with the requirements of the General Monograph for Pharmaceutical Preparations, the requirements of the General Monograph for Unlicensed Medicines and, where applicable, the requirements of the individual monograph for the specific dosage form. While it is expected that extemporaneous preparations will demonstrate pharmacopoeial compliance when tested, it is recognised that it might not be practicable to carry out the pharmacopoeial tests routinely on such formulations. In the event of doubt or dispute, the methods of analysis, the reference materials and the reference spectra of the Pharmacopoeia are alone authoritative.

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Part III Monographs and other texts of the European Pharmacopoeia that are incorporated in thừ edition of the British Pharmacopoeia are governed by the general notices of the European Pharmacopoeia; these are reproduced below. GENERAL NOTICES OF THE EUROPEAN PHARMACOPOEIA 1.1. GENERAL STATEMENTS The General Notices apply to all monographs and other texts of the European Pharmacopoeia. The official texts of the European Pharmacopoeia are published in English and French. Translations in other languages may be prepared by die signatory States of die European Pharmacopoeia Convention. In case of doubt or dispute, die English and French versions are alone authoritative. In the texts of the European Pharmacopoeia, the word ‘Pharmacopoeia5 without qualification means the European Pharmacopoeia. The official abbreviation Ph. Eur. may be used to indicate the European Pharmacopoeia. The use of the title or the subtitle of a monograph implies that the article complies with die requirements of die relevant monograph. Such references to monographs in die texts of the Pharmacopoeia are shown using die monograph title and reference number in italics. A preparation must comply throughout its period of validity; a distinct period of validity and/or specifications for opened or broached containers may be decided by the competent authority. The subject of any other monograph must comply throughout its period of use. The period of validity that is assigned to any given article and the time from which that period is to be calculated are decided by the competent authority in light of experimental results of stability studies. Unless otherwise indicated in the General Notices or in the monographs, statements in monographs constitute mandatory reqmrements. General chapters become mandatory when referred to in a monograph, unless such reference is made in a way that indicates that it is not die intention to make the text referred to mandatory but rather to cite it for information. The active substances, excipients, pharmaceutical preparations and other articles described in the monographs are intended for human and veterinary use (unless explicitly restricted to one of these uses). Quality systems

Alternative methods

The quality standards represented by monographs are valid only where the articles in question are produced within the framework of a suitable quality system. The quality system must assure that the articles consistently meet die requirements of die Pharmacopoeia. The tests and assays described are the official methods upon which the standards of die Pharmacopoeia are based. With the agreement of die competent authority, alternative methods of analysis may be used for conưol purposes, provided that the methods used enable an unequivocal decision to be made as to whether compliance with the standards of the

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monographs would be achieved if the official methods were used. In the event of doubt or dispute, the methods of analysis of the Pharmacopoeia are alone authoritative. Demonstration of compliance with the Pharmacopoeia

(1) An article is not of Pharmacopoeia quality unless it complies with all the requirements stated in the monograph. This does not imply that performance of all the tests in a monograph is necessarily a prerequisite for a manufacturer in assessing compliance with the Pharmacopoeia before release of a product. The manufacturer may obtain assurance that a product is of Pharmacopoeia quality on the basis of its design, together with its control strategy and data derived, for example, from validation studies of the manufacturing process. (2) An enhanced approach to quality control could utilise process analytical technology (PAT) and/or real-time release testing (including parametric release) strategies as alternatives to end-product testing alone. Real-time release testing in circumstances deemed appropriate by the competent authority is thus not precluded by the need to comply with the Pharmacopoeia. (3) Reduction of animal testing: the European Pharmacopoeia is dedicated to phasing out the use of animals for test purposes, in accordance with the 3Rs (Replacement, Reduction, Refinement) set out in the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes. In demonstrating compliance with the Pharmacopoeia as indicated above ( 1), manufacturers may consider establishing additional systems to monitor consistency of production. With the agreement of the competent authority, the choice of tests performed to assess compliance with the Pharmacopoeia when animal tests are prescribed is established in such a way that animal usage is minimised as much as possible.

Grade of materials

Certain materials that are the subject of a pharmacopoeial monograph may exist in different grades suitable for different purposes. Unless otherwise indicated in the monograph, the requirements apply to all grades of the material. In some monographs, particularly those on excipients, a list of functionality-related characteristics that are relevant to the use of the substance may be appended to the monograph for information. Test methods for determination of one or m ore of these characteristics may be given, also for information.

General monographs

Substances and preparations that are the subject of an individual monograph are also required to comply with relevant, applicable general monographs. Cross-references to applicable general monographs are not normally given in individual monographs. General monographs apply to all substances and preparations within the scope of the Definition section of the general monograph, except where a preamble limits the application, for example to substances and preparations that are the subject of a monograph of the Pharmacopoeia. General monographs on dosage forms apply to all preparations of the type defined. The requirements are not necessarily comprehensive for a given specific preparation and requirements additional to those prescribed in the general monograph may be imposed by the competent authority.

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General monographs and individual monographs are complementary. If the provisions of a general monograph do not apply to a particular product, this is expressly stated in the individual monograph. Validation of pharmac opoeial methods

Implementation o f pharmac opoeial methods

Conventional terms

Interchangeable methods

The test methods given in monographs and general chapters have been validated in accordance with accepted scientific practice and current recommendations on analytical validation. Unless otherwise stated in the monograph or general chapter, validation of the test methods by the analyst is not required. When implementing a pharmacopoeial method, the user must assess whether and to what extent the suitability of the method under the actual conditions of use needs to be demonstrated according to relevant monographs, general chapters and quality systems. The term ‘competent authority’ means the national, supranational or international body or organisation vested with the authority for making decisions concerning the issue in question. It may, for example, be a national pharmacopoeia authority, a licensing authority or an official control laboratory. The expression ‘unless otherwise justified and authorised’ means that the requirements have to be met, unless the competent authority authorises a modification or an exemption where justified in a particular case. Statements containing the word ‘should’ are informative or advisory. In certain monographs or other texts, the terms ‘suitable’ and ‘appropriate’ are used to describe a reagent, micro-organism, test method etc.; if criteria for suitability are not described in the monograph, suitability is demonstrated to the satisfaction of the competent authority. Medicinal product (a) Any substance or combination of substances presented as having properties for treating or preventing disease in human beings and/or animals; or (b) any substance or combination of substances that may be used in or administered to human beings and/or animals with a view either to restoring, correcting or modifying physiological functions by exerting a pharmacological, immunological or metabolic action, or to making a medical diagnosis. Herbal m edicinal product Any medicinal product, exclusively containing as active ingredients one or more herbal drugs or one or more herbal drug preparations, or one or more such herbal drugs in combination with one or more such herbal drug preparations. Active substance Any substance intended to be used in the manufacture of a medicinal product and that, when so used, becomes an active ingredient of the medicinal product. Such substances are intended to furnish a pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or to affect the structure and function of the body. Excipient (auxiliary substance). Any constituent of a medicinal product that is not an active substance. Adjuvants, stabilisers, antimicrobial preservatives, diluents, antioxidants, for example, are excipients. Certain general chapters contain a statement that the text in question is harmonised with the corresponding text of the Japanese Pharmacopoeia and/or the United States Pharmacopeia and that these texts are interchangeable. This implies that if a substance or preparation is found to

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comply with a requirement using an interchangeable method from one of these pharmacopoeias it complies with the requirements of the European Pharmacopoeia. In the event of doubt or dispute, the text of the European Pharmacopoeia is alone authoritative. References to regulatory documents

Monographs and general chapters may contain references to documents issued by regulatory authorities for medicines, for example directives and notes for guidance of the European Union. These references are provided for information for users for the Pharmacopoeia. Inclusion of such a reference does not modify the status of the documents referred to, which may be mandatory or for guidance. 1.2. OTHER PROVISIONS APPLYING TO GENERAL CHAPTERS AND MONOGRAPHS

Quantities

In tests with numerical limits and assays, the quantity stated to be taken for examination is approximate. The amount actually used, which may deviate by not more than 10 per cent from that stated, is accurately weighed or measured and the result is calculated from this exact quantity. In tests where the limit is not numerical, but usually depends upon comparison with the behaviour of a reference substance in the same conditions, the stated quantity is taken for examination. Reagents are used in the prescribed amounts. Quantities are weighed or measured with an accuracy commensurate with the indicated degree of precision. For weighings, the precision corresponds to plus or minus 5 units after the last figure stated (for example, 0.25 g is to be interpreted as 0.245 g to 0.255 g). For the measurement of volumes, if the figure after the decimal point is a zero or ends in a zero (for example, 10.0 mL or 0.50 mL), the volume is measured using a pipette, a volumetric flask or a burette, as appropriate; otherwise, a graduated measuring cylinder or a graduated pipette may be used. Volumes stated in microlitres are measured using a micropipette or micro syringe. It is recognised, however, that in certain cases the precision with which quantities are stated does not correspond to the number of significant figures stated in a specified numerical limit. The weighings and measurements are then carried out with a sufficiently improved accuracy.

Apparatus and procedures

Volumetric glassware complies with Class A requirements of the appropriate International Standard issued by the International Organisation for Standardisation. Unless otherwise prescribed, analytical procedures are carried out at a temperature between 15 °C and 25 °C. Unless otherwise prescribed, comparative tests are carried out using identical tubes of colourless, transparent, neutral glass with a flat base; the volumes of liquid prescribed are for use with tubes having an internal diameter of 16 mm, but tubes with a larger internal diameter may be used provided the volume of liquid used is adjusted (2.1.5). Equal volumes of the liquids to be compared are examined down the vertical axis of the tubes against a white background, or if necessary against a black background. The exam ination is carried out in diffuse light. Any solvent required in a test or assay in which an indicator is to be used is previously neutralised to the indicator, unless a blank test is prescribed.

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Water-bath

The term ‘water-bath’ means a bath of boiling water unless water at another temperature is indicated. Other methods of heating may be substituted provided the temperature is near to but not higher than 100 °C or the indicated temperature.

Drying and ignition to constant mass

The terms ‘dried to constant mass’ and ‘ignited to constant mass’ mean that 2 consecutive weighings do not differ by more than 0.5 mg, the 2nd weighing following an additional period of drying or of ignition respectively appropriate to the nature and quantity of the residue. Where drying is prescribed using one of the expressions ‘in a desiccator’ or ‘in vacuo’, it is carried out using the conditions described in chapter 2.2.32. Loss on drying.

Reagents

The proper conduct of the analytical procedures described in the Pharmacopoeia and the reliability of the results depend, in part, upon the quality of the reagents used. The reagents are described in general chapter 4. It is assumed that reagents of analytical grade are used; for some reagents, tests to determine suitability are included in the specifications.

Solvents

Where the name of the solvent is not stated, the term ‘solution’ implies a solution in water. Where the use of water is specified or implied in the analytical procedures described in the Pharmacopoeia or for the preparation of reagents, water complying with the requirements of the monograph Purified water (0008) is used, except that for many purposes the requirements for bacterial endotoxins (Purified water in bulk) and microbial contamination (Purified water in containers) are not relevant. The term ‘distilled water’ indicates purified water prepared by distillation. The term ‘ethanol’ without qualification means anhydrous ethanol. The term ‘alcohol’ without qualification means ethanol (96 per cent). Other dilutions of ethanol are indicated by the term ‘ethanol’ or ‘alcohol’ followed by a statement of the percentage by volume of ethanol (C2H 60 ) required.

Expression of content

In defining content, the expression ‘per cent’ is used according to circumstances with one of 2 meanings: — per cent m/m (percentage, mass in mass) expresses the number of grams of substance in 100 g of final product; — per cent V/V (percentage, volume in volume) expresses the number of millilitres of substance in 100 mL of final product. The expression ‘parts per million’ (or ppm) refers to mass in mass, unless otherwise specified.

Temperature

Where an analytical procedure describes temperature without a figure, the general terms used have the following meaning: — in a deep-freeze: below -1 5 °C; — in a refrigerator: 2 °C to 8 °C; — cold or cool: 8 °C to 15 °C; — room temperature: 15 °C to 25 °C.

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1.3. GENERAL CHAPTERS Containers

Materials used for containers are described in general chapter 3.1. General names used for materials, particularly plastic materials, each cover a range of products varying not only in the properties of the principal constituent but also in the additives used. The test methods and limits for materials depend on the formulation and are therefore applicable only for materials whose formulation is covered by the preamble to the specification. The use of materials with different formulations, and the test methods and limits applied to them, are subject to agreement by the competent authority. The specifications for containers in general chapter 3.2 have been developed for general application to containers of the stated category, but in view of the wide variety of containers available and possible new developments, the publication of a specification does not exclude the use, in justified circumstances, of containers that comply with other specifications, subject to agreement by the competent authority. Reference may be made within the monographs of the Pharmacopoeia to the definitions and specifications for containers provided in chapter 3.2. Containers. The general monographs for pharmaceutical dosage forms may, under the heading Definition/Production, require the use of certain types of container; certain other monographs may, under the heading Storage, indicate the type of container that is recommended for use. 1.4. MONOGRAPHS

Titles

Relative Atomic and Molecular Masses

Chemical Abstracts Service (CAS) Registry Number

Monograph titles are in English and French in the respective versions and there is a Latin subtitle. The relative atomic mass (A r) or the relative molecular mass (Mr) is shown, as and where appropriate, at the beginning of each monograph. The relative atomic and molecular masses and the molecular and graphic formulae do not constitute analytical standards for the substances described. CAS registry numbers are included for information in monographs, where applicable, to provide convenient access to useful information for users. CAS Registry Number^ is a registered trademark of the American Chemical Society.

Definition

Statements under the heading Definition constitute an official definition of the substance, preparation or other article that is the subject of the monograph. Limits of content Where limits of content are prescribed, they are those determined by the method described under Assay. Herbal drugs In monographs on herbal drugs, the definition indicates whether the subject of the monograph is, for example, the whole drug or the drug in powdered form. Where a monograph applies to the drug in several states, for example both to the whole drug and the drug in powdered form, the definition states this.

Production

Statements under the heading Production draw attention to particular aspects of the manufacturing process but are not necessarily comprehensive. They constitute mandatory requirements for manufacturers, unless otherwise stated. They may relate, for example, to source materials; to the manufacturing process itself and its validation and control; to in-process

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testing] or to testing that is to be carried out by the manufacturer on the final article, either on selected batches or on each batch prior to release. These statements cannot necessarily be verified on a sample of the final article by an independent analyst. The competent authority may establish that the instructions have been followed, for example, by examination of data received from the manufacturer, by inspection of manufacture or by testing appropriate samples. The absence of a Production section does not imply that attention to features such as those referred to above is not required. Choice of vaccine strain, Choice of vaccine composition The Production section of a monograph may define the characteristics of a vaccine strain or vaccine composition. Unless otherwise stated, test methods given for verification of these characteristics are provided for information as examples of suitable methods. Subject to approval by the competent authority, other test methods may be used without validation against the method shown in the monograph. Potential Adulteration

Due to the increasing number of fraudulent activities and cases of adulteration, information may be made available to Ph. Eur. users to help detect adulterated materials (i.e. active substances, excipients, intermediate products, bulk products and finished products). To this purpose, a method for the detection of potential adulterants and relevant limits, together with a reminder that all stages of production and sourcing are subjected to a suitable quality system, may be included in this section of monographs on substances for which an incident has occurred or that present a risk of deliberate contamination. The frequency of testing by manufacturers or by users (e.g. manufacturers of intermediate products, bulk products and finished products, where relevant) depends on a risk assessment, taking into account the level of knowledge of the whole supply chain and national requirements. This section constitutes requirements for the whole supply chain, from manufacturers to users (e.g. manufacturers of intermediate products, bulk products and finished products, where relevant). The absence of this section does not imply that attention to features such as those referred to above is not required.

Characters

The statements under the heading Characters are not to be interpreted in a strict sense and are not requirements. Solubility In statements of solubility in the Characters section, the terms used have the following significance, referred to a temperature between 15 °C and 25 °C. Descriptive terra

Approximate volume of solvent in millilitres

Very soluble

less than

1

Freely soluble

from

1

to

10

Soluble

from

10

to

30

Sparingly soluble

from

30

to

100

Slightly soluble

from

100

to

1000

Very slightly soluble

from

1000

to

Practically insoluble

more than

per gram of solute

10 000 10 000

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The term ‘partly soluble’ is used to describe a mixture where only some of the components dissolve. The term ‘miscible’ is used to describe a liquid that is miscible in all proportions with the stated solvent. Identification

Scope The tests given in the Identification section are not designed to give a full confirmation of the chemical structure or composition of the product; they are intended to give confirmation, with an acceptable degree of assurance, that the article conforms to the description on the label. First and second identifications Certain monographs have subdivisions entitled ‘First identification’ and ‘Second identification’. The test or tests that constitute the ‘First identification’ may be used in all circumstances. The test or tests that constitute the ‘Second identification’ may be used in pharmacies provided it can be demonstrated that the substance or preparation is fully traceable to a batch certified to comply with all the other requirements of the monograph. Certain monographs give two or more sets of tests for the purpose of the first identification, which are equivalent and may be used independently. One or more of these sets usually contain a cross-reference to a test prescribed in the Tests section of the monograph. It may be used to simplify the work of the analyst carrying out the identification and the prescribed tests. For example, one identification set cross-refers to a test for enantiomeric purity while the other set gives a test for specific optical rotation: the intended purpose of the two is the same, that is, verification that the correct enantiomer is present. Powdered herbal drugs Monographs on herbal drugs may contain schematic drawings of the powdered drug. These drawings complement the description given in the relevant identification test.

Tests and Assays

Scope The requirements are not framed to take account of all possible impurities. It is not to be presumed, for example, that an impurity that is not detectable by means of the prescribed tests is tolerated if common sense and good pharmaceutical practice require that it be absent. See also below under Impurities. Calculation Where the result of a test or assay is required to be calculated with reference to the dried or anhydrous substance or on some other specified basis, the determination of loss on drying, water content or other property is carried out by the method prescribed in the relevant test in the monograph. The words ‘dried substance’ or ‘anhydrous substance’ etc. appear in parentheses after the result. Where a quantitative determination of a residual solvent is carried out and a test for loss on drying is not carried out, the content of residual solvent is taken into account for the calculation of the assay content of the substance, the specific optical rotation and the specific absorbance. No further indication is given in the specific monograph. Limits The limits prescribed are based on data obtained in normal analytical practice; they take account of normal analytical errors, of acceptable variations in manufacture and compounding and of deterioration to an extent considered acceptable. No further tolerances are to be applied to the limits prescribed to determine whether the article being examined complies with the requirements of the monograph. In determining compliance with a numerical limit, the calculated result of a test or assay is first rounded to the number of significant figures stated, unless otherwise prescribed. The limits, regardless of whether the values are

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expressed as percentages or as absolute values, are considered significant to the last digit shown (for example 140 indicates 3 significant figures). The last figure of the result is increased by one when the part rejected is equal to or exceeds one half-unit, whereas it is not modified when the part rejected is less than a half-unit. Indication of permitted lim it of impurities The acceptance criteria for related substances are expressed in monographs either in terms of comparison of peak areas (comparative tests) or as numerical values. For comparative tests, the approximate content of impurity tolerated, or the sum of impurities, may be indicated in brackets for information only. Acceptance or rejection is determined on the basis of compliance or noncompliance with the stated test. If the use of a reference substance for the named impurity is not prescribed, this content may be expressed as a nominal concentration of the substance used to prepare the reference solution specified in the monograph, unless otherwise described. Herbal drugs For herbal drugs, the sulfated ash, total ash, water-soluble matter, alcohol-soluble matter, water content, content of essential oil and content of active principle are calculated with reference to the drug that has not been specially dried, unless otherwise prescribed in the monograph. Equivalents Where an equivalent is given, for the purposes of the Pharmacopoeia only the figures shown are to be used in applying the requirements of the monograph. Culture media The culture media described in monographs and general chapters have been found to be satisfactory for the intended purpose. However, the components of media, particularly those of biological origin, are of variable quality, and it may be necessary for optimal performance to modulate the concentration of some ingredients, notably: — peptones and meat or yeast extracts, with respect to their nutritive properties; — buffering substances; — bile salts, bile extract, deoxycholate, and colouring matter, depending on their selective properties; — antibiotics, with respect to their activity. Storage

The information and recommendations given under the heading Storage do not constitute a pharmacopoeial requirement but the competent authority may specify particular storage conditions that must be met. The articles described in the Pharmacopoeia are stored in such a way as to prevent contamination and, as far as possible, deterioration. Where special conditions of storage are recommended, including the type of container (see section 1.3. General chapters) and limits of temperature, they are stated in the monograph. The following expressions are used in monographs under Storage with the meaning shown. In an airtight container Means that the product is stored in an airtight container (3.2). Care is to be taken when the container is opened in a damp atmosphere. A low moisture content may be maintained, if necessary, by the use of a desiccant in the container provided that direct contact with the product is avoided. Protected fr o m light Means that the product is stored either in a container made of a material that absorbs actinic light sufficiently to protect the contents from change induced by such light, or in a container enclosed

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in an outer cover that provides such protection, or is stored in a place from which all such light is excluded. Labelling

In general, labelling of medicines is subject to supranational and national regulation and to international agreements. The statements under the heading Labelling are not therefore comprehensive and, moreover, for the purposes of the Pharmacopoeia only those statements that are necessary to demonstrate compliance or non-compliance with the monograph are mandatory. Any other labelling statements are included as recommendations. When the term ‘label’ is used in the Pharmacopoeia, the labelling statements may appear on the container, the package, a leaflet accompanying the package, or a certificate of analysis accompanying the article, as decided by the competent authority.

Warnings

Materials described in monographs and reagents specified for use in the Pharmacopoeia may be injurious to health unless adequate precautions are taken. The principles of good quality control laboratory practice and the provisions of any appropriate regulations are to be observed at all times. Attention is drawn to particular hazards in certain monographs by means of a warning statement; absence of such a statement is not to be taken to mean that no hazard exists.

Impurities

A list of all known and potential impurities that have been shown to be detected by the tests in a monograph may be given. See also chapter 5.10. Control of impurities in substances for pharmaceutical use. The impurities are designated by a letter or letters of the alphabet. Where a letter appears to be missing, the impurity designated by this letter has been deleted from the list during monograph development prior to publication or during monograph revision.

FunctionalityRelated Characteristics of Excipients

Reference Standards

Monographs on excipients may have a section on functionality-related characteristics. The characteristics, any test methods for determination and any tolerances are not mandatory requirements; they may nevertheless be relevant for use of the excipient and are given for information (see also section 1.1. General statements). Certain monographs require the use of reference standards (chemical reference substances, herbal reference standards, biological reference preparations, reference spectra). See also chapter 5.12. Reference standards. The European Pharmacopoeia Commission establishes the official reference standards, which are alone authoritative in case of arbitration. These reference standards are available from the European Directorate for the Quality of Medicines & HealthCare (EDQM). Information on the available reference standards and a batch validity statement can be obtained via the EDQM website.

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1.5. ABBREVIATIONS AND SYMBOLS A *1 per cent A \ cm At

Absorbance

mp

M elting point

Specific absorbance

r,20

Refractive index

Relative atom ic mass

Ph. Eur. U.

European Pharm acopoeia U nit

Md bp

Specific optical rotation

PPb

Parts per billion (micrograms per kilogram)

Boiling point

ppm

Parts per million (milligrams p er kilogram)

BRP

Biological Reference Preparation

R

Substance or solution defined under

CRS j20 “20

Chemical Reference Substance Relative density

X

W avelength

HRS

H erbal reference standard

IU

International U nit

M

M olarity

Mr

Relative m olecular mass

4. Reagents Rf

Retardation factor (see chapter 2.2.46)

R:t

U sed in chrom atography to indicate the ratio o f the distance travelled by a substance to the distance travelled by a reference substance

RV

Substance used as a prim ary standard in volumetric analysis (chapter 4.2.1)

Abbreviations used in the monographs on imm unoglobulins, immunosera and vaccines L D 50

T h e statistically determ ined quantity o f a substance that, when administered by the specified route, may be expected to cause the death o f 50 p er cent of the test animals within a given period

Lo/10 dose

T h e largest quantity of a toxin that, in the conditions of the test, when mixed with 0.1 IU o f antitoxin and adm inistered by the specified route, does not cause symptoms of toxicity in the test animals within a given period

M LD

M inim um lethal dose

L f dose

T h e quantity of toxin or toxoid that flocculates in the shortest time with 1 IU o f antitoxin

CCIDso

T h e statistically determ ined quantity o f virus th at may be expected to infect 50 p er cent of the cell cultures to which it is added

EID50

T h e statistically determ ined quantity o f virus th at may be expected to infect 50 per cent of fertilised eggs into which it is inoculated

ID 50

T h e statistically determ ined quantity of a virus that may be expected to infect 50 per cent of the animals into which it is inoculated

P D 50

T h e statistically determ ined dose of a vaccine that, in the conditions of the test, may be expected to protect 50 per cent o f the animals against a challenge dose of the micro­ organisms or toxins against which it is active

EDsn

T h e statistically determ ined dose of a vaccine that, in the conditions of the test, may be expected to induce specific antibodies in 50 per cent o f the animals for the relevant vaccine antigens

PFU

Pock-forming units or plaque-form ing units

SPF

Specified-pathogen-free.

L + /1 0 dose T h e smallest quantity of a toxin that, in the conditions of the test, when mixed with 0.1 IU of antitoxin and administered by the specified route, causes th e death of the test anim als within a given period L + dose

T he smallest quantity o f a toxin that, in the conditions of th e test, when mixed with 1 IU of antitoxin an d adm inistered by the specified route, causes the death of the test animals within a given period

lr/100 dose

T he smallest quantity o f a toxin that, in the conditions o f the test, when mixed with 0.01 IU of antitoxin and injected intracutaneously causes a characteristic reaction at the site o f injection within a given period

L p/10 dose

T h e smallest quantity o f toxin that, in the conditions o f the test, when mixed w ith 0.1 IU of antitoxin an d adm inistered by the specified route, causes paralysis in the test anim als within a given period

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Collections of micro-organisms A TCC

American Type Culture Collection 10801 University Boulevard Manassas, Virginia 20110-2209, USA

C.I.P.

Collection de Bactéries de l’Institut Pasteur B.P. 52, 25 rue du D octeur Roux 75724 Paris Cedex 15, France

IM I

International Mycological Institute Bakeham Lane Surrey T W 20 9TY, Great Britain

I.P .

Collection Nationale de Culture de Microorganismes (C.N .C.M .) Institut Pasteur 25, rue du D octeur Roux 75724 Paris Cedex 15, France

N C IM B

N ational Collection of Industrial and M arine Bacteria Ltd 23 St Machar Drive Aberdeen AB2 1RY, Great Britain

N CPF

N ational Collection of Pathogenic Fungi London School of Hygiene and Tropical Medicine Keppel Street London W C1E 7 H T , Great Britain

NCTC

N ational Collection of Type Cultures C entral Public Health Laboratory Colindale Avenue London NW9 5HT, Great Britain

NCYC

N ational Collection of Yeast C ultures A FRC Food Research Institute Colney Lane Norwich NR4 7UA, Great Britain

N IT E

Biological Resource Center D epartm ent of Biotechnology N ational Institute o f Technology and Evaluation 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, 292-0818 Japan

S.S.I.

Statens Serum Institut 80 Amager Boulevard, Copenhagen, D enm ark

1.6. UNITS OF THE INTERNATIONAL SYSTEM (SI) USED IN THE PHARMACOPOEIA AND EQUIVALENCE WITH OTHER UNITS International The International System of Units comprises 3 classes of units, namely base System O f Units units, derived units and supplementary units1. The base units and their (SI) definitions are set out in Table 1.6-1. The derived units may be formed by combining the base units according to the algebraic relationships linking the corresponding quantities. Some of these derived units have special names and symbols. The SI units used in the Pharmacopoeia are shown in Table 1.6-2. Some important and widely used units outside the International System are shown in Table 1.6-3. The prefixes shown in Table 1.6-4 are used to form the names and symbols of the decimal multiples and submultiples of SI units.

1 The definitions of the units used in the International System are given in the booklet "Le Systeme International d’Unites (SI)” published by the Bureau International des Poids et Mesures, Pavillion de Breteuil, F-92310 Sevres.

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1. In the Pharmacopoeia, the Celsius temperature is used (symbol r). This is defined by the following equation: t = T-To

where T0 = 213.15 K by definition. The Celsius or centigrade temperature is expressed in degree Celsius (symbol °C). The unit ‘degree Celsius’ is equal to the unit ‘kelvin’. 2. The practical expressions of concentrations used in the Pharmacopoeia are defined in the General Notices. 3. The radian is the plane angle between two radii of a circle that cut off on the circumference an arc equal in length to the radius. 4. In the Pharmacopoeia, conditions of centrifugation are defined by reference to the acceleration due to gravity (g): g = 9.806 65 m • s ~

2

5. Certain quantities without dimensions are used in the Pharmacopoeia: relative density (2.2.5), absorbance (2.2.25), specific absorbance (2.2.25) and refractive index (2.2.6). 6. The microkatal is defined as the enzymic activity that, under defined conditions, produces the transformation (e.g. hydrolysis) of 1 micromole of the substrate per second.

Table 1.6.-1. - S I base units Quantity

Unit

Definition

Name

Symbol

Name

Symbol

Length

I

metre

m

The metre is the length of the path travelled by light in a vacuum during a time interval of 1/299 792 458 of a second.

Mass

m

kilogram

kg

The kilogram is equal to the mass of the international prototype of the kilogram.

Time

t

second

s

The second is the duration of 9 192 631 770 periods of the radiation corresponding to the transition between the two hyperfine levels of the ground state of the caesium-133 atom.

Electric current

I

ampere

A

The ampere is that constant current which, maintained in two straight parallel conductors of infinite length, of negligible circular cross-section and placed 1 metre apart in vacuum would produce between these conductors a force equal to 2 x 10'7 newton per metre of length.

Thermodynamic temperature

T

kelvin

K

The kelvin is the fraction 1/273.16 of the thermodynamic temperature of the triple point of water.

Amount of substance

n

mole

mol

The mole is the amount of substance of a system containing as many elementary entities as there are atoms in 0.012 kilogram of carbon-12*.

Luminous intensity

I,

candela

cd

The candela is the luminous intensity in a given direction of a source emitting monochromatic radiation with a frequency of 540 x 10IJ hertz and whose energy intensity in that direction is 1/683 watt per steradian.

* When the mole is used, the elementary entities must be specified and may be atoms, molecules, ions, electrons, other particles or specified groups of such particles.

General Notices 1-33

2016

Table 1.6.-2. - SI units used in the European Pharmacopoeia and equivalence with other units Unit Conversion of other units into SI units

Name

Symbol

Name

Symbol

Expression b SI base units

Wave number

V

one per metre

1/m

m '1

Wavelength

X

micrometre nanometre

pm nm

o o 3° 3"

Quantity

Area

A, S

square metre

m2

mJ

Volume

V

cubic metre

m3

m3

Frequency

V

hertz

Hz

s '1

Density

P

kilogram per cubic metre

kg/m3

kg-m"3

Velocity

V

metre per second

m/s

m-s'1

Force

F

newton

N

m-kg-s"5

Pressure

P

pascal

Pa

m‘ Lkg-s'2

N-m'5

1 dyne/cm2 = 10*1 Pa = 10'1N-m"2 1 atm = 101 325 Pa =* 101.325 kPa 1 bar - 10s Pa = 0.1 MPa 1 mm Hg =* 133.322 387 Pa lT o rr= 133.322 368 Pa 1 psi = 6.894 757 kPa

Dynamic viscosity

V

pascal second

Pa-s

m'^kg-s'1

N-s-m'2

1 P - 10'1Pa-s - 10'1N-s-m'1 1 cP =* 1 mPa-s

Kinematic viscosity

V

square metre per second

m2/ s

m2-s*‘

Pa-s-n^-kg-1 N-m-s-kg"1

1 St = 1 cm2-s'1* 10'* m2-s'’

Energy

W

joule

J

m2-kg-s"2

N-m

Power Radiant flux

P

watt

W

m2-kg-s'3

N-m-s"1 J-s"1

1 e r g / s - 1 dyne-cm-s'1= 10'7 W = 10'7 N-m-s'1* 10-7J-s'1

Absorbed dose (of radiant energy)

D

gray

Gy

m^s"2

/•kg-1

1 rad

Electric potential, electromotive force

U

volt

V

m2- kg-s'3-A*‘

W-A*1

Electric resistance

R

ohm

Q

m2- kg-s'3-A'2

V-A-*

Quantity of electricity

Q

coulomb

c

A-s

Activity of a radionuclide

A

becquerel

Bq

s '1

Concentration (of amount of substance), molar concentration

c

mole per cubic metre

mol/m3

mol-m'3

1 m o l/L * 1 M * 1 mol/dm5 - 103 mol-m'3

Mass concentration

P

kilogram per cubic metre

kg/m3

kg-m'3

1 g/L = 1 g/dm3» 1 kg-m'3

Expression in other SI units

1 mL = 1 cm3 = 10'6 m3

1 g/mL = 1 g/cm3 =■ 103 kg-m'3

1 dyne = 1 g-cm-s'3 = 10*s N 1 kp = 9.806 65 N

1 erg - 1 cm2-g-s‘2 = 1 dyne-cm = 10'7J l e a l» 4.1868 J

»

lO'2 Gy

1 Ci - 37-109 Bq = 37-109 s '1

1-34 General Notices

2016

Table 1.6.-3. - Units used with the International System Quantity

Unit

Value in SI units

Name

Symbol

minute

min

1 min = 60 s

hour

h

1 h = 60 mũi = 3600 s

day

d

1 d = 24 h - 86 400 s

Plane angle

degree

»

1° = (n/180) rad

Volume

liừe

L

1 L “ 1 dm1 ” 10"3 m3

Time

Mass

tonne

t

1 t » 103 kg

Rotational frequency

revolution per minute

r/m in

1 r/min =* (1/60) s*1

Table 1.6.4. - Decimal multiples and sub-multiples o f units Factor

Prefix

Symbol

Factor

Prefix

Symbol

1018

exa

E

10-1

deci

d

10>5

peta

p

10-*

centi

c

10“

tera

T

10-3

milli

m

109

giga

G

10-*

micro

ụ

n

106

mega

M

10-9

nano

103

kilo

k

10-«

pico

p

102

hecto

h

10-“

fern to

f

10'

deca

da

10-18

atto

a

Monographs

Medicinal and Pharmaceutical Substances A to I

General Monographs 1-37

2016

MEDICINAL AND PHARMACEUTICAL SUBSTANCES

Substances for Pharmaceutical Use

★ ★ ★ ★ *****

(Ph. Eur. monograph 2034) PhEur------ -----------------------------------------------------------------------------—

D E F IN IT IO N Substances for pharmaceutical use are any organic or inorganic substances that are used as active substances or excipients for the production of medicinal products for hum an or veterinary use. They may be obtained from natural sources or produced by extraction from raw materials, fermentation or synthesis. T his general m onograph does not apply to herbal drugs, herbal drugs for homoeopathic preparations, herbal drug preparations, extracts, or m other tinctures for homoeopathic preparations, which are the subject of separate general monographs (Herbal drugs (1433), Herbal drugs for

homoeopathic preparations (2045), Herbal drug preparations (1434), Extracts (0765), Mother tinctures for homoeopathic preparations (2029)). It does not apply to raw materials for homoeopathic preparations, except where there is an individual m onograph for the substance in the nonhomoeopathic p art of the Pharmacopoeia. Where a substance for pharmaceutical use not described in an individual monograph o f the Pharmacopoeia is used in a medicinal product prepared for the special needs of individual patients, the need for compliance with the present general monograph is decided in the light o f a risk assessment that takes account o f the available quality o f the substance and its intended use. W here medicinal products are manufactured using substances for pharmaceutical use of hum an or animal origin, the requirements o f chapter 5.1.7. Viral safety apply. Substances for pharmaceutical use may be used as such or as starting materials for subsequent formulation to prepare medicinal products. Depending on the formulation, certain substances may be used either as active substances or as excipients. Solid substances may be compacted, coated, granulated, pow dered to a certain fineness, or processed in other ways. A m onograph is applicable to a substance processed with an excipient only where such processing is mentioned in the definition section of the monograph.

Substance for pharmaceutical use of special grade Unless otherwise indicated or restricted in the individual monographs, a substance for pharmaceutical use is intended for hum an and veterinary use, and is of appropriate quality for the m anufacture o f all dosage forms in which it can be used.

Polymorphism Individual monographs do not usually specify crystalline or am orphous forms, unless bioavailability is affected. All forms of a substance for pharmaceutical use comply with the requirements of the monograph, unless otherwise indicated. P R O D U C T IO N Substances for pharm aceutical use are manufactured by procedures that are designed to ensure a consistent quality and comply with the requirements of the individual m onograph or approved specification.

T h e manufacture o f active substances m ust take place under conditions o f good manufacturing practice. T h e provisions of general chapter 5.10 apply to the control of impurities in substances for pharmaceutical use. W hether or no t it is specifically stated in the individual monograph th at the substance for pharmaceutical me: — is a recom binant protein or another substance obtained as a direct gene product based on genetic modification, where applicable, the substance also complies with the requirements o f the general monograph Products of

recombinant DNA technology (0784)’, — is obtained from animals susceptible to transmissible spongiform encephalopathies other than by experimental challenge, where applicable, the substance also complies with the requirements of the general monograph Products

with risk of transmitting agents of animal spongiform encephalopathies (1483)', — is a substance derived from a fermentation process, whether or n o t the micro-organisms involved are modified by traditional procedures or recom binant D N A (rDNA) technology, where applicable, the substance also complies with the requirements o f the general monograph Products

offermentation (1468). I f solvents are used during production, they are of suitable quality. In addition, their toxidty and their residual level are taken into consideration (5.4). If water is used during production, it is o f suitable quality. I f substances are produced or processed to yield a certain form or grade, that specific form or grade of the substance complies with the requirements of the monograph. Certain functionality-related tests may be described to control properties that may influence the suitability of the substance and subsequently the properties o f dosage forms prepared from it.

Powdered substances May be processed to obtain a certain degree of fineness (2.9.35). Compacted substances Are processed to increase the particle size or to obtain particles o f a specific form and/or to obtain a substance with a higher bulk density.

Coated active substances Consist o f particles of the active substance coated with one or more suitable excipients. Granulated active substances Are particles of a specified size and/or form produced from the active substance by granulation directly or with one or more suitable excipients. I f substances are processed with excipients, these excipients comply with the requirements of the relevant monograph or, where no such monograph exists, the approved specification. W here active substances have been processed with excipients to produce, for example, coated or granulated substances, the processing is carried out under conditions of good manufacturing practice and the processed substances are regarded as intermediates in the manufacture of a medicinal p ro d u ct CHARACTERS T h e statements under the heading Characters (e.g. statements about the solubility or a decomposition point) are n o t to be interpreted in a strict sense and are not requirements. T hey are given for information. W here a substance may show polymorphism, this may be stated under Characters in order to draw this to the attention o f the user who may have to take this characteristic into consideration during formulation o f a preparation.

1-38 General Monographs

2016

ID E N T IF IC A T IO N W here under Identification an individual monograph contains subdivisions entitled ‘First identification’ and ‘Second identification’, the test or tests th a t constitute the ‘First identification’ may be used in all circumstances. T he test or tests that constitute the ‘Second identification’ may be used in pharmacies provided it can be dem onstrated that the substance or preparation is fully traceable to a batch certified to comply with all the other requirem ents o f the monograph. C ertain monographs give two o r m ore sets of tests for the purpose of the first identification, which are equivalent and may be used independently. O ne o r m ore o f these sets usually contain a cross-reference to a test prescribed in the Tests section o f the m onograph. It may be used to simplify the work of the analyst carrying out the identification and the prescribed tests. For example, one identification set crossrefers to a test for enantiomeric purity while the other set gives a test for specific optical rotation: the intended purpose of the two is the same, that is, verification that the correct enantiom er is present. TESTS P o ly m o rp h is m (5.9) If the nature o f a crystalline or am orphous form imposes restrictions on its use in preparations, the nature o f the specific crystalline or am orphous form is identified, its morphology is adequately controlled and its identity is stated on the label. R e la te d su b s ta n c e s Unless otherwise prescribed or justified and authorised, organic impurities in active substances are to be reported, identified wherever possible, and qualified as indicated in T able 2034.-1 or in Table 2034.-2 for peptides obtained by chemical synthesis. Table 2034.-1. - Reporting, identification and qualification of

organic impurities in active substances Use

Human use or human and veterinary use Human use or human and veterinary use Veterinary use only

Maximum daily dose £ 2 g/day

Report­ ing threshold > 0.05 per cent

> 2 g/day

> 0.03 per cent

Not applicable

> 0.10 per cent

Identification threshold

Qualification threshold

> 0.10 per cent or a daily intake of > 1.0 mg (whichever is the lower) > 0.05 per cent

> 0.15 per cent or a daily intake of > 1.0 mg (whichever is the lower) > 0.05 per cent

> 0.20 per cent

> 0.50 per cent

Table 2034.-2. - Reporting, identification and qualification of

organic impurities in peptides obtained by chemical synthesis Reporting threshold > 0.1 per cent

Identification threshold > 0.5 per cent

Qualification threshold > 1.0 per cent

Specific thresholds m ay be applied for impurities known to be unusually potent or to produce toxic o r unexpected pharmacological effects. If the individual m onograph does n o t provide suitable control for a new im purity, a suitable test for control must be developed and included in the specification for the substance.

T he requirem ents above do n o t apply to biological and biotechnological products, oligonucleotides, radiopharmaceuticals, products of ferm entation and semi­ synthetic products derived therefrom, to crude products of animal or plant origin or herbal products. F o r active substances in a new application for a medicinal p roduct for hum an use, the requirem ents o f the guideline on the limits o f genotoxic impurities and the corresponding questions and answers docum ents published on the website o f the European Medicines Agency (or similar evaluation principles for non-E uropean U nion m em ber states) m ust be followed. R e sid u a l so lv en ts are limited according to the principles defined in chapter 5.4, using general m ethod 2.4.24 or another suitable method. W here a quantitative determ ination o f a residual solvent is carried out and a test for loss on drying is n o t carried out, the content o f residual solvent is taken into account for calculation o f the assay content o f the substance, the specific optical rotation and the specific absorbance. M ic ro b io lo g ic a l q u a lity Individual monographs give accqptance criteria for microbiological quality wherever such control is necessary. T able 5.1.4.-2. - Acceptance criteria for microbiological quality of rum-sterile substances for pharmaceutical use in chapter 5.1.4.

Microbiological quality of non-sterUe pharmaceutical preparations and substances for pharmaceutical use gives recom mendations on microbiological quality th a t are o f general relevance for substances subject to microbial contamination. D epending on the nature of the substance and its intended use, different acceptance criteria may be justified. S te rility (2. 6 .I) I f intended for use in the m anufacture o f sterile dosage forms w ithout a further appropriate sterilisation procedure, o r if offered as sterile grade, the substance for pharmaceutical use complies with the test for sterility. B a c te ria l e n d o to x in s ('2.6.14) I f offered as bacterial endotoxin-free grade, the substance for pharm aceutical use complies with the test for bacterial endotoxins. T h e limit and test m ethod (if n o t gelation m ethod A) are stated in the individual monograph. T h e limit is calculated in accordance with the recom mendations in general chapter 5.1.10. Guidelines for using the test for bacterial endotoxins, unless a lower limit is justified from results from production batches or is required by the com petent authority. W here a test for bacterial endotoxins is prescribed, a test for pyrogens is n o t required. P y ro g e n s (2.6.8) I f the test for pyrogens is justified rather than the test for bacterial endotoxins and if a pyrogen-free grade is offered, the substance for pharm aceutical use complies with the test for pyrogens. T h e limit and test m ethod are stated in die individual m onograph or approved by the competent authority. Based on appropriate test validation for bacterial endotoxins and pyrogens, the test for bacterial endotoxins may replace the test for pyrogens. A d d itio n a l p ro p e rtie s Control o f additional properties (e.g. physical characteristics, functionality-related characteristics) m ay be necessary for individual m anufacturing processes or formulations. Grades (such as sterile, endotoxin-free, pyrogen-free) may be produced with a view to m anufacture o f preparations for parenteral administration or other dosage forms and

Abacavir Sulfate 1-39

2016

appropriate requirements may be specified in an individual monograph.

Content

ASSAY Unless justified and authorised, contents of substances for pharmaceutical use are determined. Suitable methods are used.

CHARACTERS Appearance

99.0 per cent to 101.0 per cent (anhydrous substance).

W hite or almost white powder.

Solubility

L A B E L L IN G In general, labelling is subject to supranational and national regulation and to international agreements. T h e statements under the heading Labelling therefore are n o t comprehensive and, moreover, for the purposes of the Pharmacopoeia only those statements that are necessary to demonstrate compliance or non-compliance with the monograph are mandatory. Any other labelling statements are included as recommendations. W hen the term ‘label' is used in the Pharmacopoeia, the labelling statements may appear on the container, the package, a leaflet accompanying the package or a certificate of analysis accompanying the article, as decided by the com petent authority. Where appropriate, the label states that the substance is: — intended for a specific use; — of a distinct crystalline form; — of a specific degree of fineness; — compacted; — coated; — granulated; — sterile; — free from bacterial endotoxins; — free from pyrogens; — containing gliding agents.

Soluble in water, practically insoluble in ethanol (96 per cent) and in methylene chloride.

IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24).

Comparison abacavir sulfate CRS. B. Enantiomeric purity (see Tests). C. Solution S (see Tests) gives reaction (a) of sulfates

(2.3.1). TESTS Solution S Dissolve 0.250 g in water R and dilute to 25.0 m L with the same solvent

Enantiomeric purity Liquid chromatography (2.2.29).

Solution A Mix 0.5 m L of trifluoroacenc acid R and 100 mT. of methanol R. Solution B Mix 30 volumes of methanol R, 30 volumes of 2propanol R and 40 volumes of heptane R. Test solution Dissolve 40 mg of the substance to be examined in 30 m L of solution A. Sonicate until dissolution is complete. Add 30 m L of 2-propanol R and dilute to 100.0 m L with heptane R.

Reference solution (a) Dissolve 2 m g of abacavirfor system suitability CRS (containing impurities A and D) in 1.5 m L of

W here applicable, the label states: — the degree o f hydration; — the nam e and concentration of any excipient. __________________________________________________________ Ph Eur

solution A. Sonicate until dissolution is complete. Add 1.5 m L of 2-propanol R and dilute to 5.0 m L with

heptane R. Reference solution (b) Dilute 1.0 m L of the test solution to 100.0 m L with solution B. Dilute 1.0 m L of this solution to 10.0 m L with solution B.

Abacavir Sulfate

*.

(Ph Eur monograph 2589)

*

*

Column: — size: I = 0.25 m , 0 = 4.6 mm; — stationary phase: amylose derivative of silica gel for chiral separation R (10 |im); . — temperature: 30 °C.

Mobile phase: — mobile phase A: diethylamine R, 2-propanol R, heptane R (0.1:15:85 V/V/V); — mobile phase B: heptane R, 2-propanol R (50:50 V!V)\

C28H 38N 120 6S

671

188062-50-2

A ctio n a n d u se Nucleoside reverse transcriptase inhibitor; antiviral (HIV). P re p a r a tio n s Abacavir Oral Solution Abacavir Tablets Abacavir, Zidovudine and Lamivudine Tablets Abacavir and Lamivudine Tablets PhEir __________________________________________________________

D E F IN IT IO N Bis[[(l5,4R)-4-[2-amino-6-(cyclopropylamiiio)-9i/-purin-9yl]cyclopent-2-enyl]methanol] sulfate.

Time (min) 0 -2 5

Mobile phase A (per cent V/V) 100

25 - 27

100 -»0

27 - 37

0

Mobile phase B (per cent V/V) 0 0

100 100

Flow rate 1.0 mL/min. Detection Spectrophotometer at 286 nm. Injection 20 nL. Identification of impurities Use the chromatogram supplied with abacavir for system suitability CRS and the chromatogram obtained with reference solution (a) to identify the peaks due to impurities A and D.

1-40 Abacavir Sulfate

2016

Relative retention W ith reference to abacavir (retention time = about 17 min): im purity D = about 0.8; im purity A = about 0.9. System suitability: reference solution (a): — resolution: minim um 1.5 betw een the peaks due to impurities D and A; m inim um 1.5 between the peaks due to impurity A and abacavir.

Limit. — impurity A: not m ore than 3 times the area o f the principal peak in the chrom atogram obtained with reference solution (b) (0.3 p er cent).

Related substances Liquid chromatography (2.2.29). Prepare the solutions

immediately before use and transfer them to low-adsorption, inert glass vials. Test solution Dissolve 25 mg o f the substance to be examined in water R and dilute to 100.0 m l. with the same solvent. Sonicate until dissolution is complete.

Reference solution (a) Dissolve 2.5 mg o f abacavir for peak identification CRS (containing impurities B and D ) in 10.0 m L of water R. Reference solution (b) D ilute 1.0 m L of the test solution to 100.0 m L with water R. D ilute 1.0 m L o f this solution to 10.0 m L w ith water R. Column: — size. I = 0.15 m , 0 = 3.9 m m ; — stationary phase: end-capped octadecylsQyl silica gelfor chromatography R (5 pm); — temperature: 30 °C. Mobile phase: — mobile phase A: dilute 0.5 m L of trifluoroacedc acid R in 1000 m L o f water R ; — mobile phase B: water R, methanol R (15:85 V!V)\ Time (min) 0 -5

Mobile phase A (per cent V7V) 95

Mobile phase B (per cent V/V) 5

5 - 25

95 + 70

5 + 30

25 -4 0

70->10

30 + 90

Flow rate 1.0 mL/min. Detection Spectrophotom eter at 254 nm . Irqection 20 pL. Identification of impurities Use the chromatogram supplied with abacavir for peak identification CRS and the

— total: n o t more than 5 times the area o f the principal peak in the chrom atogram obtained with reference solution (b) (0.5 per cent); — disregard limit. 0.5 times the area o f the principal peak in the chrom atogram obtained with reference solution (b) (0.05 per cent). H eav y m e ta ls (2.4.8) M aximum 20 ppm. 12 m L of solution S complies with test A. Prepare the reference solution using 2 m L of lead standard solution

(1 ppm Pb) R. W a te r (2.5.32) M aximum 0.5 p er cent, determined on 60.0 mg. S u lfa te d a s h (2.4.14) M aximum 0.2 per cent, determined on 1.0 g. A SSAY Dissolve 0.300 g in 50 m L of water R. T itrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2 . 2 . 2 0 ). 1 m L of 0.1 M sodium hydroxide is equivalent to 33.54 mg of C 28H 38N 12O 6S. IM P U R IT IE S

Specified impurities: A , B. Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other o f the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general m onograph Substances for pharmaceutical use (2034). It is therefore n o t necessary to identify these impurities for dem onstration o f compliance. See also 5.10.

Control of impurities in substances for pharmaceutical use): C, D, E, F.

A. [(li?,45)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9yl] cydopent-2-enyl] methanol,

chrom atogram obtained with reference solution (a) to identify the peaks due to impurities B and D.

Relative retention W ith reference to abacavir (retention time = about 22 min): im purity D = about 1.04; impurity B = about 1.3.

System suitability: reference solution (a): — peak-to-vaEey ratio: m inim um 3.0, where Hp = height above the baseline o f the peak due to impurity D and Hv = height above the baseline o f the lowest point o f the curve separating this peak from the peak due to abacavir. Limits: — impurity B: not m ore than twice the area o f the principal peak in the chrom atogram obtained with reference solution (b) (0.2 p er cent); — unspecified impurities: for each impurity, not m ore than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.10 per cent);

nh 2

B. 6-(cyclopropylamino)-9-[(li?,4S)-4-[[(2,5-diam ino-6chloropyrimidin-4-yi)oxy]methyI] cyclopent-2-enyl] -9Hpurine-2-am ine,

Acacia 1-41

2016 NH2 HjNr

CHARACTERS Acacia is almost completely b u t very slowly soluble, after about 2 h, in twice its mass of water leaving only a very small residue of vegetable particles; the liquid obtained is colourless or yellowish, dense, viscous, adhesive, translucent and weakly a d d to blue litmus paper. Acada is practically insoluble in ethanol (96 per cent).

x >N

C. [(15j4R )-4-(2,6-diarnino-9/f-purin-9-yl)cyclopent-2enyl] m ethanol,

IDENTIFICATION A. A cada occurs as yellowish-white, yellow or pale amber, sometimes with a pinkish tint, friable, opaque, spheroidal, oval or reniform pieces (tears) o f a diameter from about 1-3 cm, frequently with a cracked surface, easily broken into irregular, whitish or slightly yellowish angular fragments with conchoidal fracture and a glassy and transparent appearance. In the centre o f an unbroken tear there is sometimes a small cavity.

h2na ^ nI^ " > n

H "Oc

OH

D. [(1 i?34i?)-4 -[2-amino-6-(cyclopropylainino)-9/f-purin-9yl] cyclopent-2-enyl] methanol,

^N H

B. Reduce to a powder (355) (2.9.12). T he pow der is white or yellowish-white. Examine under a microscope using a 50 per cent V/V solution of glycerol R. T h e pow der shows the following diagnostic characters: angular, irregular, colourless, transparent fragments. Only traces of starch or vegetable tissues are visible. N o stratified membrane, is ap p arent C. Examine the chromatograms obtained in the test for glucose and fructose.

Results T h e chromatogram obtained with the test solution h2n^ n^

n

shows 3 zones due to galactose, arabinose and rhamnose. N o other im portant zocies are visible, particularly in the upper p art o f the chromatogram.

H—r ^ \ ^ H

E. [(li?,35)-3-[2-amino-6-(cyclopropylamino)-9/f-purin-9yl] cyclopentyl] methanol,

D . Dissolve 1 g o f the powdered herbal drug (355) (2.9.12) in 2 m L of water R by stirring frequently for 2 h. Add 2 m L o f ethanol (96 per cent) R. After shaking, a white, gelatinous mucilage is formed which becomes fluid on adding 10 m L of

water R. TESTS Solution S

NH

J¿C>

H2N

N

Dissolve 3.0 g o f the powdered herbal drug (355) (2.9.12) in 25 m L o f water R by stirring for 30 min. Allow to stand for 30 min and dilute to 30 m L with water R.

N

h "Ox 0H í‘ h 3c

Insoluble matter

ch3

M aximum 0.5 per cent.

F. 6-(cyclopropylamino)-9- [(li?,45)-4- [ [(1,1 dimethylethyl)oxy] methyl] cyclopent-2-enyl] -9jFí-purine-2amine. PhEur

T o 5.0 g o f the powdered herbal drug (355) (2.9.12) add 100 m L o f water R and 14 m L of dilute hydrochloric add R, boil gently for 15 min, shaking frequently and filter while hot through a tared sintered-glass filter (2.1.2). W ash with hot water R and dry at 100-105 °C. The residue weighs a maximum o f 25 mg.

Glucose and fructose

Acacia (Ph. Ever, monograph 0307)

★★+ ★★ ★ ★ *****

Action and use Bulk-forming laxative; excipient W hen Powdered Acacia is prescribed or demanded, material complying with the requirements below with the exception of Identification test A shall be dispensed or supplied. PhEur.

DEFINITION Air-hardened, gummy exudate flowing naturally from or obtained by incision of the trunk and branches o f Acacia Senegal L. Willd. (syn. Senegalia Senegal (L.) Britton), other species of Acacia of African origin and Acacia seyal Delile.

Thin-layer chromatography (2.2.27).

Test solution T o 0.100 g of the powdered herbal drug (355) (2.9.12) in a thick-walled centrifuge tube add 2 m L of a 100 g/L solution of trifluoroacetic acid R, shake vigorously to dissolve the forming gel, stopper the tube and heat the mixture at 120 °C for 1 h. Centrifuge the hydrolysate, transfer the clear supernatant carefully into a 50 m L flask, add 10 m L of water R and evaporate the solution to dryness under reduced pressure. To the resulting clear film add 0.1 m L o f water R and 0.9 m L of methanol R. Centrifuge to separate the amorphous predpitate. Dilute the supernatant, if necessary, to 1 m L with methanol R.

Reference solution Dissolve 10 mg of arabinose R, 10 mg of galactose R, 10 mg of glucose R, 10 mg o f rhamnose R and 10 mg of xylose R in 1 m L of water R and dilute to 10 m L with methanol R.

1-42 Acacia

Plate TUG silica gel plate R. Mobile phase 16 g/L solution of sodium dihydrogen phosphate R, butanol R , acetone R (10:40:50 VIV/V). Application 10 ^L as bands. Development A Over a path o f 10 cm. Drying A In a current o f warm air for a few minutes. Development B Over a path o f 15 cm using the same mobile phase.

Drying B A t 110 °C for 10 m in. Detection treat with amsaldehyde solution R and heat at 110 °C for 10 min.

Results T he chromatogram obtained with the reference solution shows 5 clearly separated coloured zones due to galactose (greyish-green or green), glucose (grey), arabinose (yellowish-green), xylose (greenish-grey or yellowish-grey) and rham nose (yellowish-green), in order of increasing RF value. T he chrom atogram obtained with the test solution shows no grey zone and no greyish-green zone between the zones corresponding to galactose and arabinose in the chrom atogram obtained with the reference solution. S ta r c h , d e x trin a n d a g a r T o 10 m L o f solution S previously boiled and cooled add 0.1 m L of 0.05 M iodine. N o blue or reddish-brown colour develops. S te rc u lia g u m A. Place 0.2 g of the powdered herbal drug (355) (2.9.12) in a 10 m L ground-glass-stoppered cylinder graduated in0.1 m L . Add 10 m L o f ethanol (60 per cent VIV) R and shake. Any gel form ed occupies a m aximum o f 1.5 mTB. T o 1.0 g of the powdered herbal drug (355) (2.9.12) add 100 m L of water R and shake. Add 0.1 m L of methyl red solution R. N o t m ore than 5.0 m L o f 0.01 M sodium hydroxide is required to change the colour o f the indicator. T a n n in s T o 10 m L o f solution S add 0.1 m L o f ferric chloride solution R l. A gelatinous precipitate is formed, bu t neither the precipitate nor the liquid are dark blue. T ra g a c a n th Examine the chromatograms obtained in the test for glucose and fructose. Results T he chrom atogram obtained with the test solution shows no greenish-grey or yellowish-grey zone corresponding to the zone of xylose in the chrom atogram obtained with the reference solution. L oss o n d ry in g (2.2.32) M axim um 15.0 p er cent, determ ined on 1.000 g o f the pow dered herbal drug (355) (2.9.12) by drying in an oven at 105 °C.

2016

part of the monograph since they also represent mandatory quality criteria. In such cases, a cross-reference to the tests described in the mandatory part is included in the Functionality-related characteristics section. Control of the characteristics can contribute to the quality of a medicinal product by improving the consistency of the manufacturing process and the performance of the medicinal product during use. Where control methods are cited, they are recognised as being suitable for the purpose, but other methods can also be used. Wherever results for a particular characteristic are reported, the control method must be indicated. The following characteristic may be relevant for acacia used as a viscosity-increasing agent andlor suspending agent in aqueous preparations. Apparent viscosity D eterm ine the dynamic viscosity using a capillary viscometer (2.2.9) or a rotating viscometer (2.2.10) on a 100 g/L solution o f acacia (dried substance). ___________________________________________________________ PhEur

Spray-dried Acacia

******

(Ph Eur monograph 0308) D E F IN IT IO N Spray-dried acacia is obtained from a solution of acacia. CHARACTERS It dissolves completely and rapidly, after about 20 m in, in twice its mass of water. T h e liquid obtained is colourless or yellowish, dense, viscous, adhesive, translucent and weakly a d d to blue litmus paper. Spray-dried acada is practically insoluble in ethanol (96 p er cent). ID E N T IF IC A T IO N A. Examined under a microscope, in ethanol (96 per cent) R, the powder is seen to consist predom inantly o f spheroidal particles about 4-40 (jm in diameter, w ith a central cavity containing 1 or several air-bubbles; a few m inute flat fragments are p resen t Only traces o f starch granules are visible. N o vegetable tissue is seen. B. Examine the chromatograms obtained in the test for glucose and fructose.

Results T h e chrom atogram obtained with the test solution shows 3 zones due to galactose, arabinose and rham nose. N o other im portant zones are visible, particularly in the upper p art o f the chromatogram. C. Dissolve 1 g of the drug to be examined in 2 m L of

water R by stirring frequently for 20 min. A dd 2 m L of ethanol (96 per cent) R. After shaking a white gelatinous

T o ta l a sh (2.4.16) M axim um 4.0 per cent.

m udlage is formed which becomes fluid on adding 10 m L of

M ic ro b ia l c o n ta m in a tio n T A M C : acceptance criterion 104 C FU /g (2.6.12).

TESTS S o lu tio n S Dissolve 3.0 g o f th e drug to be examined in 25 m L of water R by stirring for 10 min. Allow to stand for 20 m in and dilute to 30 m L with water R.

TY M C : acceptance criterion 104 C FU /g (2.6.12). Absence o f Escherichia cod (2.6.13). Absence o f Salmonella (2.6.13). F U N C T IO N A L IT Y -R E L A T E D C H A R A C T E R IS T IC S

This section provides information on characteristics that are recognised as being relevant control parameters for one or more functions of the substance when used as an excipient (see chapter 5.15). Some of the characteristics described in the Functionalityrelated characteristics section may also be present in the mandatory

*

PhEur___________________________________________________________

water R.

G lu co se a n d fru c to se Thin-layer chromatography (2.2.27).

Test solution T o 0.100 g in a thick-walled centrifuge tube add 2 m L o f a 100 g/L solution o f trifluoroacetic acid R, shake vigorously to dissolve the forming gel, stopper the tube and h eat the mixture at 120 °C for 1 h. Centrifuge the hydrolysate, transfer the clear supernatant carefully, into a

Acamprosate Calcium 1-43

2016 50 m L flask, add 10 m L o f water R and evaporate to dryness under reduced pressure. T o the resulting clear film add 0.1 m L o f water R and 0.9 m L of methanol R. Centrifuge to separate the amorphous precipitate. Dilute the supernatant, if necessary, to 1 m L with methanol R.

Reference solution Dissolve 10 mg o f arabinose R, 10 m g of galactose R, 10 m g o f glucose R, 10 m g of rhamnose R and 10 mg o f xylose R in 1 m l. of water R and dilute to 10 m L with methanol R. Plate TLC silica gel plate R. Mobile phase 16 g/L solution o f sodium dihydrogen phosphate R, butanol R , acetone R (10:40:50 V/V/V). Application 10 pL as bands. Development A Over a path o f 10 cm. Drying A In a current of warm air for a few minutes. Development B Over a path of 15 cm using the same mobile phase.

Drying B At 110 °C for 10 min. Detection Spray with ardsaldehyde solution R and heat at 110 °C for 10 min.

Absence of Salmonella (2.6.13). FU N C T IO N A L IT Y -R E L A T E D C H A R A C T E R IS T IC S

This section provides information on characteristics that are recognised as being relevant control parameters for one or more functions of the substance when used as an excipient (see chapter 5.15). This section is a non-mandatory part of the monograph and it is not necessary to verify the characteristics to demonstrate compliance. Control of these characteristics can however contribute to the quality of a medicinal product by improving the consistency of the manufacturing process and the performance of the medicinal product during use. Where control methods are cited, they are recognised as being suitable for the purpose, but other methods can also be used. Wherever results for-a particular characteristic are reported, the control method must be indicated. The following characteristic may be relevantfor spray-dried acacia used as a viscosity-increasing agent and/or suspending agent in aqueous preparations. A p p a re n t v isco sity Determine the dynamic viscosity using a capillary viscometer (2.2.9) or a rotating viscometer (2.2.10) on a 100 g/L solution of spray-dried acacia (dried substance).

Results T h e chromatogram obtained with the reference solution shows 5 clearly separated coloured zones due to galactose (greyish-green or green), glucose (grey), arabinose (yellowish-green), xylose (greenish-grey or yellowish-grey) and rham nose (yellowish-green), in order of increasing Rp value. T he chromatogram obtained with the test solution shows n o grey zone and no greyish-green zone between the zones corresponding to galactose and arabinose in the chrom atogram obtained w ith the reference solution.

PhEur

(Ph Eur monograph 1585)

S ta r c h , d e x trin a n d a g a r T o 10 m l. of solution S previously boiled and cooled add 0.1 m L of 0.05 M iodine. N o blue or reddish-brown colour develops.

Ca2+ J2

Sterculia gum

C io H ^ C a N ^ A ^

A. Place 0.2 g in a 10 m L ground-glass-stoppered cylinder graduated in 0.1 mL. Add 10 m L of ethanol (60 per cent VIV) R and shake. Any gel formed occupies not more than 1.5 mL.

A ctio n a n d u se Treatm ent o f alcoholism.

B. T o 1.0 g add 100 m L o f water R and shake. Add 0.1 m L o f methyl red solution R. N o t more than 5.0 m L of 0.01 M sodium hydroxide is required to change the colour of the indicator. T a n n in s T o 10 m L of solution S add 0.1 m L o f ferric chloride solution R l. A gelatinous precipitate is formed, b u t neither the precipitate nor the liquid shows a dark blue colour.

Tragacanth Examine the chromatograms obtained in the test for Glucose and fructose.

Results T h e chromatogram obtained with the test solution shows no greenish-grey o r yellowish-grey zone corresponding to the zone of xylose in the chromatogram obtained with the reference solution. L oss o n d ry in g (2.2.32) M axim um 10.0 per cent, determined on 1.000 g by drying in an oven at 105 °C. T o ta l a s h C2.4.16) M axim um 4.0 per c e n t M ic ro b ia l c o n ta m in a tio n T A M C : acceptance criterion 104 CFU/g (2.6.12). T Y M C : acceptance criterion 102 C FU/g (2.6.12). Absence of Escherichia colt (2.6.13).

★★*★★ ★ ★ *****

Acamprosate Calcium

400.5

77337-73-6

P re p a r a tio n Gastro-resistant Acamprosate Tablets PhEur__________________________________

D E F IN IT IO N Calcium bis[3-(acetylamino)propane-l-sulfonate]. C o n te n t 98.0 per cent to 102.0 per cent (dried substance). CHARACTERS A p p e a ra n c e White or almost white powder. Solubility Freely soluble in water, practically insoluble in ethanol (96 per cent) and in methylene chloride. ID E N T IF IC A T IO N A. Infrared absorption spectrophotometry (2.2.24).

Comparison Ph. Eur. reference spectrum of acamprosate calcium. B. It gives reaction (a) o f calcium (2.3.1). TESTS S o lu tio n S Dissolve 5.0 g in carbon dioxide-free water R and dilute to 100 m L with the same solvent. A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and colourless (2 .2 .2 , Method II).

1-44 Acarbose

2016

p H (2.2.3) 5.5 to 7.0 for solution S.

0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20).

Im p u rity A Liquid chromatography (2.2.29).

1 m L of 0.1 M sodium hydroxide corresponds to 20.02 m g of CioH20CaN2O8S2.

Test solution Dissolve 0.40 g of the substance to be examined in distilled water R and dilute to 20.0 m L with the same solvent. D ilute 10.0 m L of this solution to 100.0 m L with borate buffer solution pH 10.4 R. Place 3.0 m L of the solution obtained in a 25 m L ground-glass-stoppered tube. Add 0.15 m L of a freshly prepared 5 g/L solution o f fluorescamine R in acetomtrUe R. Shake immediately and vigorously for 30 s. Place in a water-bath at 50 °C for 30 min. Cool under a stream o f cold water. Centrifuge and filter the supernatant through a suitable mem brane filter (nominal pore size 0.45 pm), 25 mm in diameter.

IM P U R IT IE S

Reference solution Dissolve 50 m g o f acamprosate impurity A CRS in distilled water R and dilute to 200.0 m L

H2N^ V / S O , H A. 3-aminopropane- 1-sulfonic a d d (homotaurine). __________________________________________________________ PhEur

Acarbose

***** *. *

(Ph. Eur. monograph 2089)

*

with the same solvent. Dilute 0.4 m L o f the solution to 100.0 m L with borate buffer solution pH 10.4 R. T reat 3.0 m L of this solution in the same way as the test solution

Column: — size. I = 0.15 m , 0 = 4.6 mm; — stationary phase, spherical octadecylsilyl silica gel for chromatography R (5 pm) with a specific surface area of 170 m 2/g and a pore size o f 12 nm .

Mobile phase acetomtrUe R, methanol R, 0.1 M phosphate buffer solution pH 6.5 R (10:10:80 VIVIV). Flow rate 1 mL/min. Detection Spectrophotom eter at 261 nm . Irgection 20 |iL. Run time 6 times the retention time o f impurity A Retention times Fluorescamine = about 4 min; im purity A = about 8 min; acamprosate is not detected by this system.

Limits: — impurity A: not m ore than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.05 per cent). H eav y m e ta ls (2.4.8) M aximum 10 ppm.

OH

C25H43N018

OH

OH

OH

646

56180-94-4

A ctio n a n d u se Alpha-glucosidase inhibitor; treatm ent o f diabetes mellitus. PhEur__________________________________________________________

D E F IN IT IO N 0-4,6-Dideoxy-4- [ [(1 S,4R,5S, 6 S)-4,5,6-trihydroxy-3(hydroxymethyl) cyclohex-2-enyl] amino] -a-D-glucopyrano syl(1-+ 4)-0-a-D-glucopyranosyl-( 1-*4)-D-glucopyranose, which is produced by certain strains of Actinopkmes utahensis. C o n te n t 95.0 per cent to 102.0 per cent (anhydrous substance). CHA RACTERS A p p e a ra n c e W hite or yellowish, hygroscopic, amorphous powder.

Dissolve 2.0 g in distilled water R and dilute to 20 m L with the same solvent. 12 m L o f the solution complies with test A. Prepare the reference solution using 10 m L of lead standard

S o lubility Very soluble in water, soluble in methanol, practically insoluble in methylene chloride.

solution (1 ppm Pb) R. L oss o n d ry in g ('2.2.32)

ID E N T IF IC A T IO N A. Infrared absorption spectrophotometry (2.2.24).

M aximum 0.4 per cent, determ ined on 1.000 g by drying in an oven at 105 °C.

B. Examine the chromatograms obtained in the assay.

A SSAY T o 4 g of cation-exchange resin R (75-150 pm) add 20 m L of disnUed water R and stir magnetically for 10 min. Introduce this suspension into a glass column, 45 cm long and 2.2 cm in internal diameter, equipped with a polytetrafluoroethylene flow cap covered by a glass-wool plug. Allow a few millilitres of this solution to flow, then place a plug o f glass wool over the resin. Pass 50 m L o f 1 M hydrochloric acid through the column. T he p H of the eluate is close to 1. W ash with 3 quantities, each of 200 m L, o f distilled water R to obtain an eluate at p H 6. Dissolve 0.100 g o f the substance to be examined in 15 m L of distilled water R. Pass through the colum n and wash with 3 quantities, each o f 25 m l , o f distilled water R, collecting the eluate. Allow to elute until an eluate at p H 6 is obtained. Titrate the solution obtained with

Comparison acarbose for identification CRS. Results T he prindpal peak in the chromatogram obtained with the test solution is similar in retention time and size to the prindpal peak in the chromatogram obtained with reference solution (a). TESTS S o lu tio n S Dissolve 1.00 g in carbon dioxide-free water R and dilute to 20.0 m L with the same solvent. p H (2.2.3) 5.5 to 7.5 for solution S. S pecific o p tic a l ro ta tio n (2.2.7) + 168 to + 183 (anhydrous substance). D ilute 2.0 m L o f solution S to 10.0 m L with water R. A b so rb a n c e (2.2.25) M aximum 0.15 at 425 nm for solution S.

Acarbose 1-45

2016 Related substances Liquid chrom atography (2.2.29).

Test solution Dissolve 0.200 g of the substance to be examined in water R and dilute to 10.0 m L with the same solvent. Reference solution (a) Dissolve the contents of a vial of acarbose CRS in 5.0 m L of water R. Reference solution (b) Dissolve the contents of a vial of acarbose for peak identification CRS (acarbose containing impurities A, B, C , D , E, F and G) in 1 m L of water R. Reference solution (c) Dilute 1.0 m L of the test solution to 100.0 m L with water R. Column: — size: I = 0.25 m , 0 = 4 mm; — stationary phase: aminopropylsUyl silica gel for chromatography R (5 pm); — temperature: 35 °C. Mobile phase M ix 750 volumes of acetomtrUe R1 and 250 volumes of a solution containing 0.60 g/L of potassium dihydrogen phosphate R and 0.35 g/L o f disodium hydrogen phosphate dihydrate R. Flow rate 2.0 mL/min. Detection Spectrophotom eter at 210 nm. Injection 10 pL o f the test solution and reference solutions (b)

—

—

—

—

chromatogram obtained with reference solution (c) (0.3 per cent); impurity E: n o t more than 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.2 per cent); arty other impurity: for each impurity, n o t more than 0.2 times the area o f the principal peak in the chromatogram obtained with reference solution (c) (0.2 p er cent); total: n o t more than 3 times the area o f the principal peak in the chromatogram obtained with reference solution (c) (3.0 p er cent); disregard limit. 0.1 times the area of the prindpal peak in the chromatogram obtained with reference solution (c) (0.1 per cent).

H eav y m e ta ls (2.4.8) M aximum 20 ppm. Dissolve 1.5 g in water R and dilute to 15 m L with the same solvent. If the solution is not clear, cany out prefiltration and use the filtrate. 10 m L complies with test E. Prepare the reference solution using 20 m L o f lead standard solution

(1 ppm Pb) R. W a te r (2.5.12) M aximum 4.0 per cent, determined on 0.300 g.

and (c).

S u lfa te d a s h (2.4.14) M aximum 0.2 per cent, determined on 1.0 g.

Run time 2.5 times the retention time of acarbose. Identification of impurities Use the chromatogram supplied with acarbose for peak identification CRS and the

A SSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modification.

chromatogram obtained with reference solution (b) to identify the peaks due to impurities A, B, C , D, E, F and G.

Injection T est solution and reference solution (a).

Relative retention W ith reference to acarbose (retention

Calculate the percentage content o f C 25H 43N O i 8 taking into account the assigned content o f acarbose CRS.

time = about impurity B = impurity C = impurity F =

16 min): impurity D = about 0.5; about 0.8; impurity A = about 0.9; about 1.2; impurity E = about 1.7; about 1.9; impurity G = about 2.2. System suitability, reference solution (b): — the chromatogram obtained is similar to the chromatogram supplied with acarbose for peak identification CRS; — peak-to-vaRey ratio: minimum 1.2, where Hp — height above the baseline of the peak due to impurity A and Hv = height above the baseline o f the lowest point o f the curve separating this peak from the peak due to acarbose.

Limits: — correction factors: for the calculation of content, multiply the peak areas o f the following impurities by the corresponding correction factor impurity B = 0.63; im purity D = 0.75; impurity E = 1.25; impurity F = 1.25; impurity G = 1.25; — impurity C: not m ore than 1.5 times the area o f the principal peak in the chromatogram obtained with reference solution (c) (1.5 per cent); — impurity D: n o t m ore than the area of the principal peak in the chromatogram obtained with reference solution (c) (1.0 per cent); — impurity A: n o t m ore than 0.6 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.6 per cent); — impurity B: not m ore than 0.5 times the area o f the principal peak in the chromatogram obtained with reference solution (c) (0.5 per cent); — impurities F, G: for each impurity, not more than 0.3 times the area o f the principal peak in the

STORA GE In an airtight container. IM P U R IT IE S

Specified impurities A, B, C, D , E, F , G Other detectable impurities (the following substances would, if present at a sufficient levd, be detected by one or other of the tests in the monograph. T hey are limited by the general acceptance criterion for other/unspecified impurities. It is therefore n o t necessary to identify these impurities for demonstration of compliance. See also 5.10. Control of

impurities in substances for pharmaceutical use): H.

A. 0-4,6-dideoxy-4-[[(15,4i?,5«S,66)-4,5,6-trihydroxy-3(hydroxymethyl) cyclohex-2 -enyl] amino] -a-r>glucopyranosyl-(l -►4)-0-a-D-glucopyranosyl-( 1 -> 4 )-d arafono-hex-2 -ulopyranose,

1-46 Acebutolol Hydrochloride

2016

B. (li?,4i?,55,6iQ-4,5,6-trihydroxy-2(hydroxymethyl)cydohex-2-enyl 4-0-[4,6-dideoxy-4[ [(1 S,4uR,55, 65)-4,5,6-trihydroxy-3(hydroxymethyl)cyclohex-2-enyI] am ino]-a-D glucopyranosyl] -^-D -glucopyranoside,

G. a-D-glucopyranosyl 0 -4,6-dideoxy-4-[[(15,4ß ,55,65)-

4j5j6-trihydroxy-3-(hydroxymethyl)cyclohex-2en yl]am in o]-a-D -gIu cop yran osyl-(l -> 4 )-0 -a -D -

glucopyranosyl-(l ->4)-0 -a-D-glucopyranoside glucopyranosyl a-acarboside).

C. a-D-glucopyranosyl 4-0-[4,6-dideoxy-4-[[(15,4R,55,65)4,5,6-trihydroxy-3-(hydroxymethyi)cydohex-2enyl] am ino]-a-D -glu cop yran osyl]-a-D -glucopyranosid e, ho

H . 0 -4,6-dideoxy-4-[[(15,4i?,55,65)-4,5,6-trihydroxy-3(hydroxymethyl)cyclohex-2-enyl] amino] - a - D -

glucopyranosyl-( 1-*■4)-0 -6-deoxy-a-D-glucopyranosyl(1 ->4)-D-glucopyranose.

HO

ch

OH

(a -D -

OH

PhEur

★★*★★ ★ ★

Acebutolol Hydrochloride

OH

* * * * *

(Ph Eur monograph 0871) D . 4 -0 - [4,6-dideoxy-4- [[(15,4i?,55, 65)-4,5,6-trihydroxy-3(hydroxymethyl)cyclohex-2-enyl] amino] -a-Dglucopyranosyl] -D -glucopyranose,

G )T 0H

lo \ — *

and enantiomer

OH

Ci{1H29CIN2O4 O X"—f OH

Ö x —- f

OH

°

3 72.9

34381-68-5

A ctio n a n d u se Beta-adrenoceptor antagonist.

OH

P re p a ra tio n s Acebutolol Capsules

E. 0 -4 ,6-dideoxy-4- [[(15,4i?,55, 65)-4,5,6-trihydroxy-3(hydroxymethyi)cyclohex-2-enyI] amino] -a-D glucopyranosyl-(l -►4)-0-a-D-giucopyranosyl-(l -> 4 )-0 -aD-glucopyranosyl-(l ->4)-D-anzÄmo-hex-2-ulopyranose (4-0-a-acarbosyl-D-fhictopyranose),

Acebutolol Tablets

HO

N- [3-Acetyl-4- [(2J?5)-2-hydroxy-3-

PhEur.

DEFINITION HO.

HO

HO

[(1 -methyletbyl) amino] propoxy] phenyl] butanamide hydrochloride. OH OH

OH

OH

OH

OH

F. 0 -4,6-dideoxy-4- [[(15,4i?,55,65)-4,5,6-trihydroxy-3-

(hydroxymethyl) cyclohex-2-enyl] amino] -om> glucopyranosyl-(l -»4)-0 -a-D-glucopyranosyl-(l ->4)-0-aD -g lu co p y ra n o sy l-(l -*■4)-D-glucopyranose (4-0 -aacarb osyl-D -glu cop yran ose),

C o n te n t 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS A p p e a ra n c e W hite or alm ost white, crystalline powder. S olu b ility Freely soluble in w ater and in ethanol (96 per cent), very slightly soluble in acetone and in-methylene chloride.

Acebutolol Hydrochloride 1-47

2016

mp A bout 143 °C.

mobile phase A Dilute 0.5 m L o f this solution to 50.0 m L with mobile phase A.

ID E N T IF IC A T IO N

Reference solution (b) Dissolve the contents of a vial of acebutolol impurity I CRS in 1.0 m L of mobile phase A. Reference solution (c) M ix 2.0 m L o f reference solution (a)

First identification B, D. Second identification A , C, D. A. Ultraviolet and visible absorption spectrophotometry

(2.2.25). Test solution Dissolve 20.0 mg in a 0.1 per cent V/V solution of hydrochloric acid R and dilute to 100.0 m L with the same acid solution. Dilute 5.0 m L of this solution to 100.0 m L with a 0.1 per cent V/V solution of hydrochloric add R.

Spectral range 220-350 nm. Absorption maxima At 233 nm and 322 nm. Specific absorbance at the absorption maximum 555 to 605 at

and 1.0 m L of reference solution (b) and dilute to 10.0 m L with mobile phase A.

Reference solution (d) Dissolve 5.0 m g o f acebutolol impurity C CRS in 10 m L of acetomtrûe R and dilute to 25.0 m L with mobile phase A. D ilute 0.5 m L of this solution to 50.0 m L with mobile phase A.

Reference solution (e) Dissolve 5.0 m g o f acebutolol impurity B CRS in 10.0 m L of acetonitrile R and dilute to 25.0 m L with mobile phase A. D ilute 1.0 m L of this solution to 50.0 m L with mobile phase A.

233 nm .

Column:

B. Infrared absorption spectrophotometry (2.2.24).

— sizer. I = 0.125 m, 0 = 4 mm, — stationary phase:, end-capped octadecylsüyl silica gel for chromatography R (5 pm), — temperature: 40 °C.

Preparation Discs. Comparison acebutold hydrochloride CRS. C. Thin-layer chromatography (2.2.27). Test solution Dissolve 20 mg of die substance to be examined in methanol R and dilute to 20 m L with the same solvent. Reference solution (a) Dissolve 20 mg of acebutolol hydrochloride CRS in methanol R and dilute to 20 m L with the

Mobile phase: — mobile phase A: mix 2.0 m L of phosphoric add R, and 3.0 m L of triethylamine R and dilute to 1000 m L with water R; — mobile phase B: mix equal volumes o f acetonitrile R and

same solvent.

mobile phase A;

Reference solution (b) Dissolve 20 mg o f pindolol CRS in methanol R and dilute to 20 m L with the same solvent.

Time (min)

T o 1 m L o f this solution add 1 m L of reference solution (a).

Plate TLC silica gel F 254 plate R. Mobile phase perchloric add R, methanol R, water R (5:395:600 V/V/V). Application 10 pL. Development Over 3/4 of the plate. Drying In air. Detection Examine in ultraviolet light at 254 nm. System suitability T he chromatogram obtained with reference

0-2

Mobile phase A (per cent V/V) 98

Mobile phase B (per cent V/V) 2

2 - 30.5

98-» 10

2 -> 90

30.5 - 41

10

90

Results T h e principal spot in the chromatogram obtained with

Flow rate 1.2 mlVmin. Detection Spectrophotom eter at 240 nm . Injection 25 pL. System suitability: reference solution (c): — resolution: minim um 7.0 between the peaks due to

the test solution is similar in position and size to the principal spot in th e chromatogram obtained with reference solution (a).

Limits: — impurity B: n o t m ore than the area of the principal peak

solution (b) shows 2 clearly separated principal spots.

im purity I and acebutolol.

D . It gives reaction (a) of chlorides (2.3.1).

TESTS A p p e a ra n c e o f so lu tio n T he solution is not more opalescent than reference suspension II (2.2.1) and not more intensely coloured than reference solution BY 5 (2.2.2, Method II). Dissolve 0.5 g in water R and dilute to 10 m L with the same solvent.

—

—

—

p H (2.2.3) 5.0 to 7.0. Dissolve 0.20 g in carbon dioxide-free water R and dilute to 20 m L w ith the same solvent.

—

R e la te d su b s ta n c e s Liquid chromatography (2.2.29).

—

Test solution Dissolve 0.100 g of die substance to be examined in mobile phase A and dilute to 50.0 m L with mobile phase A. Reference solution (a) Dissolve 20.0 m g of the substance to be examined in mobile phase A and dilute to 100.0 m L with

in the chromatogram obtained with reference solution (e) (0.2 per cent); impurity C: not m ore than the area o f the principal peak in the chromatogram obtained with reference solution (d) (0.1 per cent); impurity I: not m ore than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 p er cent); any other impurity: for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent); total: no t more th an 5 times the area o f the principal peak in the chrom atogram obtained with reference solution (a) (0.5 per cent); disregard limit. 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent).

H eav y m e ta ls (2.4.8) M axim um 20 ppm. Dissolve 0.50 g in 20.0 m L of water R. T h e solution complies with test E. Prepare the .reference solution by

1-48 Aceclofenac

2016

diluting 10.0 m L o f lead standard solution (1 ppm Pb) R to 20.0 m L w ith water R. and enantiomer

L oss o n d ry in g (2.2.32) M aximum 0.5 per cent, determined on 1.000 g by drying in an oven at 105 °C for 3 h. S u lfated a s h (2.4.14) M aximum 0.1 per cent, determined on 1.0 g.

F. R = OH: N- [3-acetyl-4- [(2&S)-2,3dihydroxypropoxy]phenyl]butanamide,

A SSAY Dissolve 0.300 g in 50 m L o f ethanol (96 per cent) R and add 1 m L of 0.1 M hydrochloric acid. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the 2 points o f inflexion.

I. R = NH-CH2-CH3: N - [3-acetyl-4- [(2J?S)-3-(ethylamino)2-hydroxypropoxy]phenyl]butanamide, H3C C H 3 OH T OH

H3C

1 m L of 0.1 M sodium hydroxide is equivalent to 37.29 m g of C 18H 29CIN 2O 4. STORAGE Protected from light. IM P U R IT IE S

Specified impurities A, B, C, D , E, F, G , H , I, J, K.

G . NyN'-[[( 1-methylethyl)imino]bis[(2-hydroxypropane-l,3diyl) oxy(3-acetyl-1,4-phenylene)]] dibutanam ide (biamine),

O and enantiomer

A N - [3-acetyl-4- [(2i?3)-oxiran-2ylmethoxy]phenyl]butanamide, R2

H . NyN'-[(2-hydroxypropane-l,3-diyl)bis[oxy(3-acetyl-1,4phenylene)]]dibutanamide.

H OH ° ^ X ^ N

PhEur

CH3 j

R1.

and enantiomer

CH3

★ ★ ★ ★ *****

Aceclofenac B. R I = R2 = C O -C H 3: N - [3-acetyl-4-[(2i?5)-2-hydroxy-3[( 1-methylethyl) amino] propoxy]phenyl] acetamide (diacetolol),

(Ph Eicr monograph 1281) o

D . R I = H , R2 = CO-CH3: 1- [5-amino-2-[(2&S)-2hydroxy-3-[( 1-methyiethyl)amino]propoxy]phenyI] ethanone,

o a

E. R I = C O -C H 2-C H 2-C H 33 R2 = H : N-[4-[(2i?5)-2hydroxy-3[( 1-methylethyl) amino] propoxy]phenyI] butanamide, J. R I = CO-CH2-CH3, R2 = CO-CH3: Ar-[3-acetyl-4[(2i?5)-2-hydroxy-3[( 1-methylethyl) amino] propoxy] phenyl] propanamide, K R I = R2 = C O -C H 2-C H 2-C H 3: N-[3-butanoyl-4[(2i?5)-2-hydroxy-3[( 1-methylethyl) amino] propoxy]phenyI]butanamide,

co 2h

C16H13a 2N04

354.2

89796-99-6

A ctio n a n d u se Cyclo-oxygenase inhibitor; analgesic; anti-inflammatory. PhEur_______________________________________________________

DEFINITION [[[2-[(2,6-Dichlorophenyl)amino]phenyl]acetyl]oxy]acetic ad d . C o n te n t 99.0 per cent to 101.0 p er cent (dried substance). C. N-(3-acetyl-4-hydroxyphenyI)butanamide,

CHARACTERS A p p e a ra n c e W hite or almost white, crystalline powder. S o lu b ility Practically insoluble in water, freely soluble in acetone, soluble in ethanol (96 p er cent). ID E N T IF IC A T IO N

First identification B.

Aceclofenac 1-49

2016 Second identification A, C.

Time Mobile phase A Mobile phase B (min)____________ (per cent V/V)________ (per cent V/V) 0 - 25 70 -» 50 30 -» 50

A. Ultraviolet and visible absorption spectrophotometry (2.2.25). Test solution Dissolve 50.0 mg in methanol R and dilute to 100.0 m L with the same solvent. Dilute 2.0 m L o f the solution to 50.0 m L with methanol R.

Spectral range 220-370 nm. Absorption maximum At 275 nm. Specific absorbance at the absorption maximum 320 to 350. B. Infrared absorption spectrophotometry (2.2.24).

Comparison: Ph. Eur. reference spectrum of aceclofenac. C . Dissolve about 10 mg in 10 m L of ethanol (96 per cent) R. T o 1 m L o f the solution, add 0.2 m L of a mixture, prepared immediately before use, o f equal volumes o f a 6 g/L solution o f potassium ferricyanide R and a 9 g/L solution of ferric chloride R. Allow to stand protected from light for 5 min. A dd 3 m l, of a 10.0 g/L solution of hydrochloric acid R. Allow to stand protected from light for 15 min. A blue colour develops and a precipitate is formed. TESTS

Related substances Liquid chromatography (2.2.29). Prepare the solutions

immediately before use. Solvent mixture M obile phase A, mobile phase B (30:70 V/V). Test solution Dissolve 50.0 mg o f the substance to be examined in the solvent mixture and dilute to 25.0 m L with the solvent mixture.

Reference solution (a) Dissolve 21.6 mg of diclofenac sodium CRS (impurity A) in the solvent mixture and dilute to 50.0 m L with the solvent mixture.

Reference solution (b) Dilute 2.0 m L of the test solution to 10.0 m L with the solvent mixture.

Reference solution (c) Mix 1.0 m L o f reference solution (a) and 1.0 m L o f reference solution (b) and dilute to 100.0 m L w ith the solvent mixture.

Reference solution (d) Dissolve 4.0 mg of aceclofenac impurity F CRS and 2.0 mg of aceclofenac impurity H CRS in the solvent mixture, then dilute to 10.0 m L with the solvent mixture.

Reference solution (e) Mix 1.0 m L of reference solution (b) and 1.0 m L o f reference solution (d) and dilute to 100.0 m L with the solvent mixture.

Reference solution (j) Dissolve the contents of a vial of diclofenac impurity A CRS (aceclofenac impurity I) in 1.0 m L o f the solvent mixture, add 1.5 m L of the solvent mixture and mix,

Reference solution (g) Dissolve 4 mg of aceclofenac for peak identification CRS (containing impurities B, C, D , E and G) in 2.0 m L o f the solvent mixture.

Column: — size: I = 0.25 m , 0 = 4.6 mm; — stationary phase: spherical end-capped octadecylsüyl silica gel for chromatography R (5 pm) with a pore size o f 10 n m and a carbon loading of 19 per cent; — temperature'. 40 °C. Mobile phase: — mobile phase A: 1.12 g/L solution of phosphoric acid R adjusted to p H 7.0 with a 42 g/L solution of sodium

hydroxide R, — mobile phase B: water R, acetonitrüe R (10:90 ViV)\

25 - 30

50 -» 20

50 -» 80

30 - 50

20

80

Flow rate 1.0 mL/min. Detection Spectrophotom eter at 275 nm. Injection 10 jiL o f the test solution and reference solutions (c), (e), (f) and (g).

Identification of impurities Use the chromatogram supplied with aceclofenac for peak identification CRS and the chromatogram obtained witinreference solution (g) to identify the peaks due to impurities B, C, D , E and G.

Relative retention W ith reference to aceclofenac (retention time = about 11 min): impurity A = about 0.8; impurity G = about 1.3; impurity H = about 1.5; impurity I = about 2.3; impurity D = about 3.1; impurity B = about 3.2; impurity E = about 3.3; impurity C = about 3.5; impurity F = about 3.7. System suitability: reference solution (c): — resolution: minim um 5.0 between the peaks due to impurity A and aceclofenac.

Limits: — impurity A: n o t more than the area o f the corresponding peak in the chromatogram obtained with reference solution (c) (0.2 per cent); — impurities B, C, D, E, G: for each impurity, no t more than the area of the peak due to aceclofenac in the chrom atogram obtained with reference solution (e) (0.2 per cent); — impurity F: n o t more than the area o f the corresponding peak in the chromatogram obtained with reference solution (e) (0.2 per cent); — impurity H: n o t more than the area o f the corresponding peak in the chromatogram obtained with reference solution (e) (0.1 per cent); — impurity I: no t more than the area of the corresponding peak in the chromatogram obtained with reference solution (f) (0.1 per cent); — unspecified impurities: not more than 0.5 times die area of the peak due to aceclofenac in the chromatogram obtained with reference solution (e) (0.10 per cent); — total: n o t m ore than 0.7 per cent; — disregard Ixmitr. 0.1 times the area of the peak due to aceclofenac in the chromatogram obtained with reference solution (e) (0.02 per cent).

Heavy m etals ( 2.4.8) M aximum 10 ppm . T o 2.0 g in a silica crucible, add 2 m L of sulfuric acid R to wet die substance. H eat progressively to ignition and continue heating until an almost white or at m ost a greyish residue is obtained. Carry out the ignition at a tem perature not exceeding 800 °C. Allow to cool. Add 3 m L of hydrochloric acid R and 1 m L o f nitric acid R. H eat and evaporate slowly to dryness. Cool and add 1 m L of a 100 g/L solution o f hydrochloric acid R and 10.0 m L o f cUsaHed water R. N eutralise with a 1.0 g/L solution of ammonia R using 0.1 m L o f phenolphthalein solution R as indicator. Add 2.0 m L of a 60 g/L solution of anhydrous acetic acid R and dilute to 20 m L with distilled water R. 12 m L of the solution complies with test A. Prepare the reference solution using lead standard solution (1 ppm Pb) R.

1-50 Aceclofenac

2016 o

L oss o n d ry in g (2.2.32) M aximum 0.5 per cent, determ ined on 1.000 g by drying in an oven at 105 °C. S u lfa te d a s h (2.4.14) M aximum 0.1 per cent, determ ined on 1.0 g. A SSA Y Dissolve 0.300 g in 40 m L o f methanol R. T itrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2 . 2 . 2 0 ). 1 m L of 0.1 M sodium hydroxide is equivalent to 35.42 mg o f C 16H 13CI2N O 4.

E. ethyl [[[2-[(2,6dichlorophenyl)amino]phenyl] acetyl] oxy] acetate (ethyl ester o f aceclofenac),

o

STORAGE Protected from light. IM P U R IT IE S

Specified impurities A, B, C, D , E, F , G, H , I

F. benzyl [[[2-[(2,6dichlorophenyl)amino]phenyI]acetyl]oxy]acetate (benzyl ester o f aceclofenac),

O

A [2-[(2,6-dichlorophenyi)amino]phenyI] acetic acid (diclofenac).

B. methyl [2-[(2j6-dichlorophenyl)amino]phenyl]acetate (methyl ester of diclofenac),

G. [[[[[2-[(2,6dichlorophenyl)amino]phenyl]acetyI] oxy] acetyl] oxy]acetic acid (acetic aceclofenac),

o ^ Y

C. ethyl [2-[(2,6-dichlorophenyl)amino]phenyl]acetate (ethyl ester o f diclofenac),

•

^ r 0' ^ o

/ v

0 ^ ' c ° 2H

H . [ [[ [[[[2-[(2,6-dichlorophenyl)amino]phenyl] acetyl] oxy] acetyl]oxy]acetyl]oxy]acetic a d d (diacetic acedofenac),

OCT^ 1. 1-(2 ,6-dichlorophenyl)-1,3-dihydro-2/f-indol-2-one. PhEur

D . methyl [[[2-[(2,6dichlorophenyl)amino]phenyl]acetyl]oxy]acetate (methyl ester of aceclofenac),

2016

Acemetacin 1-51

★ ★ ★ ★ *****

Acemetacin (Ph Eitr monograph 1686)

C jxH wCINO î

415.8

— temperature: 40 °C.

Mobile phase: — mobile phase A: dissolve 1.0 g of potassium dihydrogen phosphate R in 900 m L of water R, adjust to p H 6.5 with 1 M sodium hydroxide and dilute to 1000 m L with water R, — mobile phase B: acetonitrUefor chromatography R]

53164-05-9

A c tio n a n d u se Cyclo-oxygenase inhibitor; analgesic; anti-inflammatory.

Time (min) 0-5

Mobile phase A (percent V/V) 95

Mobile phase B (per cent V/V) 5

5 -9

95->65

5 -»35

9 - 16

65

35

16-28

65 -»20

35 -»80

28 - 34

20

80

PhEur_______________________________ ___________________________

DEFINITION [ [[ l-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3yl] acetyl] oxy] acetic ad d . C o n te n t 99.0 per cent to 101.0 per cent (dried substance).

CHARACTERS A p p e a ra n c e Yellow or greenish-yellow, crystalline powder.

Solubility Practically insoluble in water, soluble in acetone, slightly soluble in anhydrous ethanol. It shows polymorphism (5.9).

IDENTIFICATION Infrared absorption spectrophotometry (2.2.24).

Comparison acemetacin CRS. I f the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in acetone R, evaporate to dryness and record new spectra using the residues.

TESTS R e la te d su b sta n c e s Liquid chromatography (2.2.29).

Test sohaion Dissolve 0.100 g of the substance to be examined in acetonitrUe for chromatography R and dilute to 20.0 m L with the same solvent. Reference solution (a) Dilute 5.0 mT. of the test solution to 50.0 m L with acetonitrUe for chromatography R. Dilute 1.0 m L o f this solution to 100.0 m L with acetonitrUe for chromatography R. Reference sohaion (b) Dissolve 5.0 mg of acemetacin impurity A CRS and 10.0 m g o f indometacin CRS (impurity B) in acetonitrUe for chromatography R, and dilute to 50.0 m L with the same solvent.

Reference solution (c) Dilute 1.0 m L o f reference solution (b) to 20.0 m L with acetomtrUe for chromatography R. Reference sohaion (d) T o 1 m L of reference solution (b), add 10 m L o f the test solution and dilute to 20 m L with

acetomtrUefor chromatography R. Reference sohaion (e) Dissolve the contents o f a vial of acemetacin impurity mixture CRS (containing impurities C, D , E and F) in 1.0 m L o f the test solution.

Column: — size: I = 0.25 m , 0 = 4 mm; — stationary phase: spherical end-capped octadecylsUyl siHca gel for chromatography R (5 pm);

Flow rate 1.0 mTVmin. Detection Spectrophotom eter at 235 nm. Injection 20 pL. Identification of impurities: — use the chromatogram supplied with acemetacin impurity mixture CRS and the chromatogram obtained with reference solution (e) to identify the peaks due to impurities C, D , E and F; — use the chromatogram obtained with reference solution (b) to identify the peak due to impurity B.

Relative retention W ith reference to acemetacin (retention tim e = about impurity B = impurity C = impurity E =

15 min): impurity A = about 0.7; about 0.9; impurity F = about 1.2; about 1.3; impurity D = about 1.5; about 2.2. System suitability Reference solution (d): — peak-to-valley ratio: minimum 15, where Hp = htight above the baseline of the peak due to impurity B and Hv = height above the baseline of the lowest point o f the curve separating this peak from the peak due to acemetacin.

Limits: — correction factors: for the calculation of content, multiply the peak areas o f the following impurities by the corresponding correction factor: impurity C = 1.3; impurity D = 1.4; impurity F = 1.3; — impurity E: no t more than 3 times the area of the p rindpal peak in the chromatogram obtained with reference solution (a) (0.3 per cent); — impurity B: not more than the area of the mi-responding peak in the chromatogram obtained with reference solution (c) (0.2 per cent); — impurity A: not more than the area of the corresponding peak in the chromatogram obtained with reference solution (c) (0.1 per cent); — impurities C, D, F: for each impurity, not more than the area o f the prindpal peak in th e chromatogram obtained with reference solution (a) (0.1 per cent); — unspecified impurities: for each impurity, not more than the area o f the prindpal peak in the chromatogram obtained with reference solution (a) (0.10 per cent); — total: n o t more than 4 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.4 per cent); — disregard limit. 0.5 times the area of the prindpal peak in the chromatogram obtained w ith reference solution (a) (0.05 p er cent).

1-52 Acenocoumarol

2016

H eav y m e ta ls (2.4.8) M aximum 20 ppm.

Acenocoumarol

Solvent mixture methanol R, acetone R (10:90 V!V). 0.250 g complies with test H . Prepare the reference solution using 0.5 m L o f lead standard sohaion (10 ppm Pb) R. L oss o n d ry in g (2.2.32) M aximum 0.5 per cent, determ ined on 1.000 g by drying in an oven at 105 °C. S u lfa te d a s h (2.4.14) M aximum 0.1 per cent, determined on 1.0 g. A SSAY Dissolve 0.350 g in 20 m L o f acetone R and add 10 m L of water R. T itrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2 . 2 . 2 0 ). 1 m L o f 0.1 M sodium hydroxide is equivalent to 41.58 mg o f C 2iH i8C 1N 06. STORA GE Protected from light.

and enantiomer

c

19h 15n o 6

353.3

152-72-7

A ctio n a n d use Vitamin K epoxide reductase inhibitor; oral anticoagulant. P re p a r a tio n Acenocoumarol Tablets D E F IN IT IO N Acenocoumarol is (R.S)-4-hydroxy-3-( 1-p-nitrophenyl-3oxobutyl)coumarin. It contains not less than 98.5% and not more than 100.5% o f C 19H15N 0 6, calculated with reference to the dried substance.

IM P U R IT IE S

Specified impurities A, B, C, D , E, F COjH

C H A R A C T E R IS T IC S An almost white to buff powder. Practically insoluble in water and in ether, slightly soluble in ethanol (96%). It dissolves in aqueous solutions of the alkali hydroxides. It exhibits polymorphism.

A. 4-chlorobenzoic acid,

R3

B. R1 = R2 = R3 = H : [l-(4-chlorobenzoyl)-5-methoxy-2methylindol-3-yl]acetic acid (indom etadn), C. R1 = Cl, R2 = H , R3 = C H 2 -C 0 2H: [[[l-(3,4dichlorobenzoyl)-5-methoxy-2-methyl-l.H'-indol-3yl] acetyl] oxy] acetic acid, D. R1 = H , R2 = C (C H 3)3, R3 = C H 2 -C 0 2H : [[[1(4-chlorobenzoyl)-6-( 1,1 -dimethylethyl)-5-methoxy-2-methyl1H-indol-3-yl] acetyl] oxy] acetic ad d , E. R1 = R2 = H , R3 = C H 2 -C 0 -0 -C (C H 3) 3: 1,1 -dimethylethyl [[[1 -(4-chlorobenzoyl)-5-methoxy-2methyl- l/i-indol-3-yl] acetyl] oxy]acetate, F. R1 = R2 = H , R3 = C H 2- C 0 -0 - C H 2- C 0 2H: [[[[[1(4-chlorobenzoyl)-5-methoxy-2-methjd-l/i-indol-3yl] acetyl] oxy] acetyl] oxy] acetic ad d . __________________________________________________________ PhEur

ID E N T IF IC A T IO N T he infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of acenocoumarol (RS 001). If the spectra are n o t concordant, dissolve 0.1 g o f the substance being examined in 10 m L o f acetone and add water drop wise until the solution becomes turbid. H eat on a w ater bath until the solution is clear and allow to stand. Filter, wash the crystals with a mixture o f equal volumes of acetone and water and dry at 100° at a pressure o f 2 kPa for 30 minutes. Prepare a new spectrum o f the residue. TESTS C la rity a n d c o lo u r o f so lu tio n A. A 2.0% w/v solution in acetone is clear, Appendix IV A. B. T he absorbance of a 4-cm layer of a 2.0% w/v solution in acetone at 460 nm is not more than 0.12, Appendix II B. C. A 2.0% w/v solution in 0.1m sodium hydroxide is dear, Appendix IV A, and yellow. L ig h t a b so rp tio n

Absorbance of a 0.001% w/v solution in a mixture of 1 volume o f 1m hydrochloric add and 9 volumes o f methanol at the maximum at 306 nm, 0.50 to 0.54, calculated with reference to the dried substance, Appendix II B. R e la te d su b sta n c e s Carry out the m ethod for thin-layer chromatography, Appendix HI A, using the following solutions in acetone. (1) 2.0% w/v of the substance being examined. (2) 0.0020% w/v o f the substance being examined. CHROMATOGRAPHIC CONDITIONS (a) U se as the coating silica gd GF2 s4 (b) Use die mobile phase as described below. (c) Apply 20 nL of each solution. (d) Develop the plate to 15 cm.

Acesulfame Potassium 1-53

2016

Reference solution (a) Dissolve 5 mg o f acesulfame potassium CRS in water R and dilute to 5 m L with the same

(e) After removal o f the plate, allow it to dry in air and im m ediately examine under ultraviolet light (254 nm).

solvent.

M O BILE PHASE

20 volumes of glacial acetic acid, 50 volumes of cychhexane and 50 volumes o f cHchloromethane.

Reference solution (b) Dissolve 5 mg o f acesulfame potassium CRS and 5 m g o f saccharin sodium R in water R and

LIMITS

Plate cellulose for chromatography R as the coating substance. Mobile phase concentrated ammonia R, acetone R, ethyl acetate R (10:60:60 V/V/V). Application 5 pL as bands. Development Twice over 2/3 of the plate. Drying In a current o f warm air. Detection Examine in ultraviolet light at 254 nm. System suitability: reference solution (b):

dilute to 5 m L with the same solvent.

Any secondary spot in the chromatogram obtained with solution (1) is n o t more intense than the spot in the chrom atogram obtained with solution (2) (0.1%). L oss o n d ry in g W hen dried to constant weight at 105°, loses not more than 0.5% o f its weight. U se 1 g. S u lfa te d ash N o t m ore than 0.1% , Appendix IX A. A SSA Y Dissolve 0 .6 g in 5 0 m L of acetone and titrate with 0 .1 m sodium hydroxide VS using bromothymol blue solution R3 as indicator. Repeat the operation without the substance being examined. T h e difference between the titrations represents the am ount of sodium hydroxide required. Each mT. o f 0 .1 m sodium hydroxide KS is equivalent to 3 5 .3 3 m g of C 19H 15N CV

Acesulfame Potassium

******

*+ +*

(Ph Eur monograph 1282)

*

—

the chromatogram shows 2 clearly separated zones.

Results T he principal zone in the chromatogram obtained with the test solution is similar in position and size to the prindpal zone in the chromatogram obtained with reference solution (a). C. 0.5 m L o f solution S (see Tests) gives reaction (b) of potassium (2.3.1). TESTS S o lu tio n S Dissolve 10.0 g in carbon dioxide-free water R and dilute to 50 m L with the same solvent. A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and colourless (2.2.2, Method IT). A cid ity o r alk a lin ity T o 20 m L of solution S add 0.1 m L of bromothymol blue solution R l. N o t m ore than 0.2 m L o f 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is required to change the colour of the indicator. Im p u rity A Thin-layer chrom atography (2.2.27).

C4H4KNO4S

201.2

55589-62-3

A c tio n a n d use Sweetening agent. PhEur________________________________________________________ __

D E F IN IT IO N Potassium 6-methyl-1,2,3-oxathiazin-4-olate 2,2-dioxide. C o n te n t 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS A p p e a ra n c e W hite or almost white, crystalline powder or colourless crystals. S o lu b ility Soluble in water, very slightly soluble in acetone and in ethanol (96 per cent).

Test solution Dissolve 0.80 g of the substance to be examined in water R and dilute to 10 m L with the same solvent Reference solution (a) Dissolve 50 m g o f acetylacetamide R (impurity A) in water R and dilute to 25 m L with the same solvent. T o 5 m L o f the solution add 45 m L of water R and dilute to 100 m L with methanol R. Reference solution (b) T o 10 m L of reference solution (a) add 1 m L o f the test solution and dilute to 20 m L with

methanol R. Plate TLC silica gel plate R. Mobile phase water R, ethanol (96 per cent) R, ethyl acetate R (2:15:74 V/VIV). Application 5 piL. Development Over 2/3 o f the plate. Drying In air until the solvents are completely removed. Detection Spray with phosphoric vanillin solution R and heat at 120 °C for about 10 min; examine in daylight

ID E N T IF IC A T IO N

System suitability T h e chromatogram obtained with reference

First identification A , C. Second identification B, C.

solution (a) shows a clearly visible spot and the chromatogram obtained with reference solution (b) shows 2 clearly separated spots.

A. Infrared absorption spectrophotometry (2.2.24).

Comparison acesulfame potassium CRS. B. Thin-layer chromatography (2.2.27). Test solution Dissolve 5 mg of the substance to be examined in water R and dilute to 5 m L with the same solvent.

Limit. — impurity A: any spot due to im purity A is not more intense than the spot in the chromatogram obtained with reference solution (a) (0.125 per cent). I m p u rity B Liquid chromatography (2.2.29).

1-54 Acetazolamide

Test solution Dissolve 0.100 g o f die substance to be examined in water R and dilute to 10.0 m L with the same solvent.

2016

IMPURITIES Specified impurities: A , B.

H3C\ ^ 0

Reference solution (a) Dissolve 4.0 m g o f acesulfame potassium impurity B CRS in water R and dilute to 100.0 m L with the same solvent. Dilute 1.0 m L o f the solution to 200.0 m L with water R.

Reference solution (b) Dissolve 0.100 g o f the substance to be examined in reference solution (a) and dilute to 10.0 m L with the same solution.

A. 3-oxobutanamide (acetylacetamide), h 3c .

Column: — sizer. I = 0.25 m, 0 = 4.6 mm; — stationary phase: octadecylsQyl silica gel for chromatography R

V

.0 ^ »

s=o

,NH

° A

r ’

(3 nm).

Mobile phase M ix 40 volumes o f acetomtrUe R and 60 volumes of a 3.3 g/L solution o f tetrabuiylammomwn hydrogen sulfate R. Flow rate 1 mL/min. Detection Spectrophotom eter at 234 nm . Injection 20 nL. Run time Twice the retention time o f acesulfame. Relative retention W ith reference to acesulfame (retention time = about 5.3 min): im purity B = about 1.6.

B. 5-chloro-6-methyi-1,2,3-oxathiazm -4(3/f)-one 2,2-dioxide. __________________________________________________________ PhEur

* * * * *

(Ph Eur monograph 0454)

System suitability: — signal-to-noise ratio: minimum 10 for the peak due to impurity B in the chromatogram obtained with reference solution (a); — peak-to-vaUey ratio: minimum 1.2, where Hp = height above the baseline o f the peak due to impurity B and Hv = height above the baseline o f the lowest point o f the curve separating this peak from the peak due to acesulfame, in the chromatogram obtained with reference solution (b).

★★* ★★ ★ ★

Acetazolamide o\w/o ,s. HjN' 'Y

H

N^CHs

Y T N -N

222.2

Action and use Carbonic anhydrase inhibitor; diuretic; treatm ent of glaucoma and ocular hypertension; treatm ent o f m o untain sickness.

Limit:

Preparation

— impurity B: not m ore than the area o f the principal peak in the chrom atogram obtained with reference solution (a) (20 ppm ).

Acetazolamide Tablets

Fluorides Maximum 3 ppm.

D E F IN IT IO N 2V-(5-Sulfamoyi-1,3,4-thiadiazol-2-yI) acetamide.

Potentiom etry (2.2.36, Method I).

Content

Test solution Dissolve 3.000 g o f the substance to be examined in distilled water R, add 15.0 m l. of total-ionicstrength-adjustment buffer R 1 and dilute to 50.0 m L with distilled water R. Reference solutions T o 0.5 mT, 1.0 mT, 1.5 m L and 3.0 m L of fluoride standard solution (10 ppm F) R add 15.0 m L of total-ionic-strerigth-adjustment buffer R1 and dilute to 50.0 mL with distilled water R. Indicator electrode Fluoride-selective. Reference electrode Silver-silver chloride. H eav y m e ta ls (2.4.8) M aximum 5 ppm . 12 m L of solution S complies with test A. Prepare die reference solution using lead standard solution (1 ppm Pb) R L oss o n d ry in g (2.2.32) M aximum 1.0 per cent, determined on 1.000 g by drying in an oven at 105 °C for 3 h.

59-66-5

PhEur__________________________________________________________

98.5 per cent to 101.0 per cent (dried substance).

CHARACTERS Appearance White or almost white, crystalline powder.

Solubility Very slighdy soluble in water, slighdy soluble in ethanol (96 per cent). It dissolves in dilute solutions o f alkali hydroxides. It shows polymorphism (5.9).

IDENTIFICATION First identification A, B Second identification A , C, D A Ultraviolet and visible absoiption spectrophotometry

(2.2.25). Solution A Dissolve 30.0 m g in 0.01 M sodium hydroxide and dilute to 100.0 m L with die same solvent. D ilute 10.0 m L of the solution to 100.0 m L with 0.01 M sodium hydroxide.

A SSA Y Dissolve 0.150 g in 50 m L o f anhydrous acetic add R. Titrate with 0.1 M perchloric add, determining the end-point potentiometrically (2 .2 . 2 0 ).

Solution B Dilute 25.0 m L o f solution A to 100.0 m L with 0.01 M sodium hydroxide. Spectral range 230-260 nm for solution A; 260-350 n m for

1 m L o f 0.1 M perchloric add is equivalent to 20.12 mg

Absorption maximum A t 240 nm for solution A; at 292 nm for

Of

C4H4KNO4S.

solution B. solution B.

2016 Specific absorbance at the absorption maximum 162 to 176 for solution A; 570 to 620 for solution B. B. Infrared absorption spectrophotometry (2.2.24).

Comparison acetazolamide CRS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in ethanol (96 per cent) R, evaporate to dryness and record new spectra using the residues. C. Introduce about 20 mg into a test-tube and add 4 m L of

dilute hydrochloric add R and 0.2 g o f zinc powder R Im mediately place a piece of lead acetate paper R over the m outh o f the tube. T he paper shows a brownish-black colour. D . Dissolve about 25 mg in a mixture o f 0.1 m L o f dilute sodium hydroxide sdurion R and 5 m L of water R Add 0.1 m L o f copper sulfate solution R. A greenish-blue precipitate is formed.

Acetazolamide 1-55 — impurities A , B, C, D, E, F: for each impurity, not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.15 per cent); — unspecified impurities: for each impurity, not more than the area o f the principal peak in th e chromatogram obtained with reference solution (a) (0.10 per cent); — total: not m ore than 6 times the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.6 per cent); — disregard limit. 0.5 times the area of the principal peak in the chromatogram obtained w ith reference solution (a) (0.05 per cent). S u lfates (2.413) M axim um 500 ppm. T o 0.4 g add 20 m L o f distilled water R and dissolve by heating to boiling. Allow to cool w ith frequent shaking and filter.

TESTS Appearance of solution

H eav y m e ta ls (2.48) M aximum 20 ppm.

T he solution is no t more opalescent than reference suspension II (2.2.1) and not more intensely coloured than reference solution Y 5 or BY5 (2.2.2, Method II).

1.0 g complies with test C. Prepare the reference solution using 2 m L o f lead standard solution (10 ppm Pb) R.

Dissolve 1.0 g in 10 m L of 1 M sodium hydroxide.

Related substances Liquid chromatography (2.2.29).

Test solution Dissolve 40 mg o f die substance to be examined in the mobile phase and dilute to 100.0 m L with the mobile phase.

L oss o n d ry in g (2.2.32) M aximum 0.5 per cent, determined on 1.000 g by drying in an oven at 105 °C. S u lfa te d a sh (2.414) M aximum 0.1 per cent, determined on 1.0 g.

100.0 m L with the mobile phase. Dilute 1.0 m L o f this solution to 10.0 m L with the mobile phase.

A SSAY Dissolve 0.200 g in 25 m L of dimethylforrnamide R. T itrate with 0.1 M ethanolic sodium hydroxide, determining the end-point potentiometrically (2 . 2 . 2 0 ).

Reference solution (b) Dissolve the contents o f a vial o f acetazolamide for system suitability CRS (containing

1 m L of 0.1 M ethanolic sodium hydroxide is equivalent to 22.22 m g of C 4H 6N 4O 3S2.

impurities A, B, C , D , E and F) in 1.0 m L o f the mobile phase.

IM P U R IT IE S

Reference solution (a) Dilute 1.0 m L o f the test solution to

Cdumn: — size: I = 0.15 m , 0 = 4.6 mm; — stationary phase: end-capped propoxybenzene silica gel for chromatography R (4 (im). Mobile phase acetonitrüe for chromatography R, 6.8 g/L solution of potassium dihydrogen phosphate R (10:90 V!V). Flow rate 1.0 mL/min. Detection Spectrophotom eter at 265 nm. Injection 25 jil. Run time 3.5 times the retention time of acetazolamide. Identification of impurities Use the chromatogram supplied with acetazolamide for system suitability CRS and the

Specified impurities A, B, C, D , E, F Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one o r other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these impurities for demonstration of compliance. See also 5.10.

Control of impurities in substancesfor pharmaceutical use): G. H

\

Y

N -N

R1

chromatogram obtained with reference solution (b) to identify the peaks due to impurities A, B, C, D , E and F.

A. R1 = C O -C H 3, R2 = Cl: N -(5-chloro-l,3,4-thiadiazol-2yl)acetamide,

Relative retention W ith reference to acetazolamide (retention

B. R1 = C O -C H 3, R2 = H: 2V-(l,3,4-thiadiazol-2yl)acetamide,

time = about 8 min): impurity E = about 0.3; impurity D = about 0.4; impurity B = about 0.6; impurity C = about 1.4; impurity A = about 2.1; impurity F = about 2.6. System suitability: reference solution (b): — resolution: minim um 2.0 between the peaks due to impurities E and D .

Limits: — correction factory, for the calculation o f content, multiply the peak areas o f the following impurities by the corresponding correction factor impurity B = 2.3; impurity C = 2 . 6; impurity D = 1.6;

C. R1 = C O -C H 3, R2 = SH: N-(5-sulfanyl-1,3,4-thiadiazol-

2-yl)acetamide, D . R 1 = H , R2 = S 0 2-N H 2: 5-am ino-1,3,4-thiadiazole-2sulfonamide, E. R1 = C O -C H 3, R2 = S 0 2-0 H : 5-acetamido-1,3,4thiadiazole-2-sulfonic ad d ,

1-56 Acetic Acid

2016

Dissolve the residue obtained in the test for residue on evaporation by h eating with 2 quantities, each of 15 m L, of water R and dilute to 50.0 m L with water R (solution A). 12 m l. o f solution A complies with test A. Prepare the reference solution using lead standard solution (2 ppm Pb) R.

"’V V rY x V V " O

N -N

N -N

0

F. N- [5- [(5-acetam ido-l 33,4-thiadiazol-2yl) sulfonyl] sulfamoyl-1,3,4-thiadiazol-2-yI] acetamide,

R esid u e o n e v a p o ra tio n M aximum 0.01 per cent.

H Sx sy N H 2

Evaporate 20 g to dryness on a water-bath and dry at 100-105 °C. T h e residue weighs a maximum of 2.0 mg.

N -N

G. 5-am ino-1,3,4-thiadiazole-2-thioL PhEur

A SSA Y Weigh accurately a conical flask with a ground-glass stopper containing 25 m l, o f water R A dd 1.0 m L o f the substance to be examined and weigh again accurately. A dd 0.5 m L of phenolpkthalein solution R and titrate with 1 M sodium

Glacial Acetic Acid

***** ★ ★

hydroxide. 1 m i, o f 1 M sodium hydroxide is equivalent to 60.1 mg

(Ph Eitr monograph 0590)

*****

o f C 2H 4O 2.

H3C

C2H 4O 2

0 I

STO R A G E In an airtight container. OH

60.1

PhEur

64-19-7

PhEur__________________________________________________________

D E F IN IT IO N C o n te n t 99.0 per cent mlm to 100.5 per cent mlm. CHARACTERS A p p e a ra n c e Crystalline mass or clear, colourless, volatile liquid. S o lu b ility Miscible with water, with ethanol (96 per cent) and with methylene chloride. ID E N T IF IC A T IO N A. A 100 g/L solution is strongly acid (2.2.4). B. T o 0.03 m L add 3 m L o f water R and neutralise with dilute sodium hydroxide solution R T he solution gives reaction (b) of acetates (2.3.1). TESTS S o lu tio n S Dilute 20 m L to 100 m L with distilled water R A p p e a ra n c e T he substance to be examined is clear (2.2.1) and colourless

(2.2.2, Method II). F re e z in g p o in t (2.2.18) M inim um 14.8 °C. R e d u c in g su b s ta n c e s Dilute 2.0 m L to 10.0 m L with water R Add 0.1 m L of 0.02 M potassium permanganate. H eat on a water-bath for 1 min, the colour remains pink. C h lo rid e s (2.4.4) Maximum 25 mg/L. Dilute 10 m L of solution S to 15 m L with water R. S u lfates (2.4.13) M aximum 50 mg/L, determined on solution S. Iro n (2.4.9) M aximum 5 ppm. Dilute 5.0 m L of solution A obtained in the test for heavy metals to 10.0 m L with water R. H eav y m e ta ls (2.4.8) M aximum 5 ppm.

Acetic Acid (6 per cent) Dilute Acetic A d d D E F IN IT IO N Acetic A d d (6 per cent) contains not less than 5.7% and not more than 6.3% w/w of acetic ad d , C 2H 4O 2. It may be prepared by mnring 182 g of Acetic A d d (3 3 per cent) with 818 g of Purified Water.

IDENTIFICATION A. Strongly addic. B. W hen neutralised, yields the reactions characteristic of acetates, Appendix VI. TESTS W eig h t p e r m L About 1.005 g, Appendix V G. H eav y m e ta ls Evaporate 20.0 m l, to dryness and add 20 m L of water. 12 m L o f the resulting solution complies with limit test A for heavy metals, Appendix VII. Use lead standard solution ( 1 ppm Pb) to prepare the standard (1 ppm). C h lo rid e Dilute 5.0 m l, with suffident water to produce 100 m L 15 m L o f the resulting solution complies with the limit test for chlorides, Appendix VII (70 ppm). S u lfate 12.5 m L o f the solution used in the test for Chloride, diluted to 15 m l . with water, complies with the limit test for sulfates, Appendix VII (240 ppm ). A ldehydes Distil 7 5 m l^ T o the first 5 m L of the distillate add 10 m L o f a 5 % w/v solution o f mercuTy(n) chloride, make alkaline with 5m sodium hydroxide, allow to stand for 5 minutes and addify with 1m sulfuric acid. T h e solution shows not more than a faint turbidity. F o rm ic a c id a n d o x id isab le im p u ritie s Mix 5 m l . with 6 m l , of sulfuric acid and cool to 20°. Add 0 .4 m L o f 0 .0 1 6 7 m potassium dichromate VS, allow to stand for 1 minute, add 25 m l, o f water and 1 m L o f freshly prepared dilute potassium iodide solution and titrate the

Acetone 1-57

2016 liberated iodine with 0.1m sodium tkiosidfate F 5 using starch mucilage as indicator. N ot less than 0 .2 m L o f 0.1m sodium thiosulfate F 5 is required. R ead ily o x id isab le im p u ritie s T o 2 5 m L add 0 .2 m L of 0.02m potassium permanganate FiS and allow to stand for 1 minute. T he pink colour is not entirely discharged. N o n -v o latile m a t te r W hen evaporated to dryness and dried at 105°, leaves not more than 0 .01 % w/w o f residue. A SSAY A dd 30 m L of water to 20 g in a stopper flask and titrate with 1m sodium hydroxide F 5 using phenolphthalein solution R1 as indicator. E ach m L of 1m sodium hydroxide F 5 is equivalent to 60.05 mg of C 2H 4O 2.

Acetic Acid (33 per cent) Acetic A d d

mucilage as indicator. N ot less than tkiosidfate VS is required.

C H A R A C T E R IS T IC S A clear, colourless liquid. M isdble with water, with ethanol (96%) and with glycerol. ID E N T IF IC A T IO N A. Strongly addic, even when diluted fredy. B. W hen neutralised, yields the reactions characteristic of acetates, A ppendix VI. TESTS W eig h t p e r m l . 1.040 to 1.042 g, Appendix V G. H eav y m e ta ls Evaporate 10.0 m L to dryness and add 20 m L o f water. 12 m L of the resulting solution complies with limit test A for heavy metals, Appendix VII. Use lead standard solution (1 ppm Pb) to prepare the standard (2 ppm).

Chloride Dilute 5.0 m L w ith suffident water to produce 100 m L 15 m L of the resulting solution complies with the limit test for chlorides, Appendix VII (70 ppm). S u lfate 12.5 m L of the solution used in the test for Chloride, diluted to 15 m L with water, complies with the limit test for sulfates, Appendix VII (240 ppm). A ldehydes Distil 15 m L T o the first 5 m L of the distillate add 10 m L of a 5% w/v solution of mercury(n) chloride, make alkaline w ith 5m sodium hydroxide, allow to stand for 5 minutes and make addic w ith 1m sulfuric acid. The solution shows not m ore than a faint turbidity. F o rm ic a c id a n d o x id isab le im p u ritie s M ix 5 m L with 6 m L o f sulfuric acid and cool to 20°. A dd 2 m L of 0 .0 1 6 7 m potassium dickromate F5, allow to stand for 1 m inute, add 25 m L of water and 1 m L of freshly prepared dilute potassium iodide solution and titrate the liberated iodine w ith 0.1m sodium thiosulfate VS using starch

sodium

R ead ily o x id isa b le im p u ritie s T o 5 .0 m L add 2 0 m L o f water and 0 .2 m L of 0.02m potassium permanganate KS and allow to stand for 1 minute. T h e pink colour is not entirely discharged. N o n -v o la tile m a t te r W hen evaporated to dryness and dried at 105°, leaves not more th an 0 .01 % w/w of residue. ASSAY Weigh 5 g into a stopper flask containing 5 0 m L o f water and titrate w ith 1m sodium hydroxide F 5 using phenolphthalein solution R1 as indicator. Each mL o f 1m sodium hydroxide F S is equivalent to 6 0 .0 5 mg o f C2H 4 0 2.

★ ★ ★ ★ *****

Acetone (Ph Eur monograph 0872)

P r e p a r a tio n Acetic A d d (6 p er cent) D E F IN IT IO N Acetic A d d (33 p er cent) contains not less than 32.5% and n o t m ore than 33.5% w/w of acetic a d d , C 2H 4O 2.

1.0 m L of 0.1m

0 X , CH; H3C

CjHeO

58.08

67-64-1

PhEir___

D E F IN IT IO N Propanone. CHARACTERS A p p e a ra n c e Volatile, clear, colourless liquid. S o lu b ility M isdble w ith w ater and with ethanol (96 per cent). T h e vapour is flammable. ID E N T IF IC A T IO N A. Relative density (see Tests). B. T o 1 m L, add 3 m L o f dilute sodium hydroxide solution R and 0.3 m L of a 25 g/L solution o f sodium nitroprusside R. An intense red colour is produced which becomes violet with the addition of 3.5 m L o f acetic acid R. C. T o 10 m L of a 0.1 per cent VIV solution o f the substance to be examined in ethanol (50 per cent VIV) R, add 1 m L o f a 10 g/L solution o f nitrobenzaldehyde R in ethanol (50 per cent VIV) R and 0.5 mL o f strong sodium hydroxide solution R. Allow to stand for about 2 min and addify with acetic acid R. A greenish-blue colour is produced. TESTS A p p e a ra n c e o f so lu tio n T o 10 m L add 10 m L o f water R. T he solution is clear (2.2.1) and colourless (2.2.2, Method II). A c id ity o r a lk a lin ity T o 5 m L add 5 m L of carbon dioxide-free water R, 0.15 m L of phenolphthalein solution R and 0.5 m L of 0.01 M sodium hydroxide. T h e solution is pink. A dd 0.7 m L o f 0.01 M hydrochloric acid and 0.05 m L of methyl red solution R. T h e solution is red or orange. R e lativ e d e n sity (2.2.5) 0.790 to 0.793.

2016

1-58 Acetylcholine Chloride

R e d u c in g su b sta n c e s T o 30 m l. add 0.1 m l. o f 0.02 M potassium permanganate and allow to stand in the dark for 2 h. T he mixture is not completely decolourised

R esid u e o n e v a p o ra tio n M aximum 50 ppm.

R e la te d su b sta n c e s G as chromatography (2.2.28).

W a te r (.2.5.12) Maximum 3 g/L, determined on 10.0 m L. U se 20 m L of anhydrous pyridine R as solvent.

Test solution T he substance to be examined. Reference solution (a) T o 0.5 m L o f methanol R add 0.5 m L of 2-propanol R and dilute to 100.0 m L with the test solution. D ilute 1.0 m L of this solution to 10.0 m L with the test solution.

Reference solution (b) Dilute 100 pL o f benzene R to 100.0 m L

Evaporate 20.0 g to dryness on a w ater-bath and dry at 100-105 °C. T h e residue weighs a maximum o f 1 mg.

STORAGE Protected from light. IM P U R IT IE S

Specified impurities: A, B, C.

w ith the test solution. Dilute 0.20 m L o f this solution to 100.0 m L with the test solution.

A. C H 3-OH: m ethanol, B. C H 3-C H O H -C H 3: propan-2-ol (isopropanol),

Column:

C. O H ^ : benzene.

— material: fused silica, — size. I = 50 m, 0 = 0.3 mm, — stationary phase: macrogol 20 000 R (film thickness 1 (im).

Carrier gas helium for chromatography R. Linear velocity 21 cm/s. Split ratio 1:50. Temperature:

Column

PhEur

Acetylcholine Chloride (Ph Ever monograph 1485)

Time (min) 0 -1 1

Temperature (°C) 45 + 100

11-20

100

H3C + CH3 .N^ Cl CH3

C7H16o n o 2

Injection port

150

Detector

250

★★*★* ★ ★ *****

181.7

60-31-1

A c tio n a n d u se Cholinoceptor agonist. PhEur__________________

Detection Flam e ionisation. Injection 1 pL. Retention time Impurity C = about 7.5 min. System suitability: — resolution: minimum 5.0 between the peak due to impurity A (2nd peak) and the peak due to impurity B (3rd peak) in the chromatogram obtained with reference solution (a), — signal-to-notse ratio: minim um 5 for the peak due to impurity C in the chromatogram obtained with reference solution (b).

Limits: — impurities A , B: for each impurity, n o t m ore than the difference between the areas of the corresponding peaks in the chromatogram obtained with reference solution (a) and the areas o f the corresponding peaks in the chromatogram obtained with the test solution (0.05 per cent VIV), — impurity C: not more than die difference between the area o f die peak due to impurity C in the chromatogram obtained with reference solution (b) and the area of the corresponding peak in the chromatogram obtained with the test solution (2 ppm VIV), — any other impurity, for each impurity, not m ore than the difference between the area o f the peak due to impurity A in the chromatogram obtained with reference solution (a) and the area o f the corresponding peak in die chromatogram obtained with the test solution (0.05 p er cent VIV). M a tte r in so lu b le in w a te r D ilute 1.0 m L to 20 m L w ith water R. T he solution is clear (2.2.1).

D E F IN IT IO N 2-(Acetyloxy)-iV>W,N-trimethylethanaminium chloride. C o n te n t 98.5 per cent to 101.5 per cent (dried substance). CHARACTERS A p p e a ra n c e W hite or almost white crystalline powder or colourless crystals, very hygroscopic. S o lu b ility Very soluble in water, freely soluble in alcohol, slightly soluble in methylene chloride. ID E N T IF IC A T IO N

First identification B, E. Second identification A , C, D, E. A. Melting point (2.2.14): 149 °C to 152 °C. Introduce the substance to be examined into a capillary tube. D ry in an oven at 100-105 °C for 3 h. Seal the tube and determine the melting point. B. Infrared absorption spectrophotometry (2.2.24).

Comparison acetylcholine chloride CRS. C. Examine the chromatograms obtained in the test for related substances.

Results T h e principal zone in the chromatogram obtained with test solution (b) is similar in position, colour and size to the principal zone in the chromatogram obtained with reference solution (b). D . T o 15 m g add 10 m L of dilute sodium hydroxide solution R, 2 m l. o f 0.02 M potassium permanganate and heat. T h e vapours formed change the colour o f red litmus paper R to blue.

Acetylcysteine 1-59

2016

E. 0.5 m L of solution S (see Tests) gives reaction (a) o f chlorides (2.3.1). TESTS S o lu tio n S Dissolve 5.0 g in carbon dioxide-free water R and dilute to 50 m L with the same solvent. A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y 6 o r BY6 (2.2.2, Method II). A cid ity D ilute 1 m L o f solution S to 10 m L with carbon dioxide-free water R. A dd 0.05 m L of phenolphthalein solution R. N o t more than 0.4 m L o f 0.01 M sodium hydroxide is required to change the colour of the indicator to pink. R e la te d su b s ta n c e s Thin-layer chromatography (2.2.27). Prepare the solutions

immediately before use. Test solution (a) Dissolve 0.30 g of the substance to be examined in methanol R and dilute to 3.0 m L with the same

A SSAY Dissolve 0.200 g in 20 m L of carbon dioxide-free water R. Neutralise with 0.01 M sodium hydroxide using 0.15 m l. of phenolphthalein solution R as indicator. Add 20.0 m L o f 0.1 M sodium hydroxide and allow to stand for 30 min. T itrate with

0.1 M hydrochloric acid. 1 m L of 0 . 1 M sodium ftydroxide is equivalent to 18.17 mg of C 7H 16C IN 0 2. STO R A G E In ampoules, protected from light. IM P U R IT IE S

\

H3C + CH3 N^

Cf

ch 3

A. 2(choline chloride),

chloride

solvent.

Test solution (b) D ilute 1 m L of test solution (a) to 10 m L with methanol R. Reference solution (a) Dilute 1 m L of test solution (a) to 100 m L with methanol R. Reference solution (b) Dissolve 20.0 mg of acetylcholine chloride CRS in methanol R and dilute to 2.0 m L with the

B. 2-(acetyloxy)-N>N-dimethylethanaminium chloride, ch 3

H3C

ch 3

same solvent.

Reference solution (c) Dissolve 20 mg of choline chloride R in methanol R, add 0.4 m L of test solution (a) and dilute to 2.0 m L with methanol R. Plate TLC silica gel plate R. Mobile phase Mix 20 volumes of a 40 g/L solution o f ammonium nitrate R, 20 volumes of methanol R and 60 volumes of acetonârUe R. Application 5 nL as bands o f 10 mm by 2 mm. Development Over 2/3 o f the plate. Detection Spray with potassium iodobismuthate solution R3. System suitability T h e chromatogram obtained with reference

C. NjN-dimethylmethanamine. PhEur

★* * ★★ ★ ★ *****

Acetylcysteine (Ph. Eur. monograph 0967) ch 3

0={

H NH

solution (c) shows 2 clearly separated zones.

Limits: — any impurity: any zones in the chromatogram obtained with test solution (a), apart from the principal zone, are n o t more intense than the principal zone in the chrom atogram obtained with reference solution (a) (1 per cent). T rim e th y la m in e Dissolve 0.1 g in 10 m L of sodium carbonate solution R and heat to boiling. N o vapours appear which turn red litmus paper R blue. H eav y m e ta ls (2.4.8) M axim um 10 ppm . 12 m L o f solution S complies with test A. Prepare the reference solution using lead standard solution (1 ppm Pb) R. L oss o n d ry in g (2.2.32) M axim um 1.0 p er cent, determined on 1.000 g by drying in an oven at 105 °C for 3 h. S u lfa te d a sh (2.4.14) M axim um 0.1 p er cent, determined on the residue obtained in the test for loss on drying.

co 2h

C 5H 9N 03 S

163.2

616-91-1

Action and use Sulfydryl donor; antidote to paracetamol poisoning; mucolytic. P r e p a r a tio n Acetylcysteine eye drops Acetylcysteine Injection PhEur.

D E F IN IT IO N (2i?)-2-(Acetylamino)-3-sulfanylpropanoic add. C o n te n t 98.0 per cent to 101.0 per cent (dried substance). CHARACTERS A p p e a ra n c e W hite or almost white, crystalline powder or colourless crystals. S o lu b ility Freely soluble in water and in ethanol (96 per cent), practically insoluble in methylene chloride.

1-60 Acetylcysteine

2016

IDENTIFICATION First identification A , C. Second identification A , B, D, E.

Injection 20 (iL, 3 times; inject 0.01 M hydrochloric acid as a

A. Specific optical rotation (see Tests).

30 min).

B. Melting point (2.2.14): 104 °C to 110 °C.

Retention time Im purity A = about 2.2 m in; impurity B =

C. Infrared absorption spectrophotometry (2.2.24).

Preparation Discs of potassium bromide R. Comparison acetylcysteine CRS. D . Examine the chromatograms obtained in the test for related substances.

Results T he principal peak in the chromatogram obtained with test solution (b) is similar in retention time and size to the principal peak in the chromatogram obtained with reference solution (b).

blank.

Run time 5 times the retention time o f acetylcysteine (about

about 2.4 min; 2-methyl-2-thiazoline-4-carboxylic acid, originating in test solution (c) = about 3.3 min; acetylcysteine = about 6.4 min; im purity C = about 12 min; impurity D = about 14 min. System suitability: reference solution (a): — resolution: minimum 1.5 between the peaks due to impurities A and B and minim um 2.0 between the peaks due to impurities C and D .

A \ x m 2 x 100

A----------11 = ----~ x mi

E. T o 0.5 m L of solution S (see Tests) add 0.05 m L of a 50 g/L solution of sodium nitroprusside R and 0.05 m L of concentrated ammonia R. A dark violet colour develops.

TESTS Solution S Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 m L with the same solvent.

Appearance of solution Solution S is clear (2 . 2 . 1 ) and colourless (2.2.2, Method II). pH (2.2.3) 2.0 to 2.8. T o 2 m L o f solution S add 8 m L of carbon dioxide-free water R and mix.

Specific optical rotation (2.2.7) + 21.0 to + 27.0 (dried substance). In a 25 m L volumetric flask, mix 1.25 g with 1 m L of a 10 g/L solution of sodium edetate R. A dd 7.5 m L o f a 40 g/L solution o f sodium hydroxide R, mix and dissolve. Dilute to 25.0 m L with phosphate buffer solution pH 7.0 R2.

Related substances Liquid chrom atography (2.2.29). Except where otherwise

prescribed, prepare the solutions immediately before use. Test solution (a) Suspend 0.80 g of the substance to be examined in 1 m L o f 1 M hydrochloric acid and dilute to 100.0 m L with water R. Test solution (b) Dilute 5.0 m L of test solution (a) to 100.0 m L with water R. Dilute 5.0 m l. of this solution to 50.0 m L w ith water R. Test solution (c) Use test solution (a) after storage for at least 1 h.

Reference solution (a) Suspend 4.0 m g o f acetylcysteine CRS, 4.0 mg of L-cystme R (impurity A), 4.0 mg o f l -cysteine R (impurity B), 4.0 m g of acetylcysteine impurity C CRS and 4.0 mg of acetylcysteine impurity D CRS in 1 m L o f 1 M hydrochloric add and dilute to 100.0 m L with water R. Reference solution (b) Suspend 4.0 mg o f acetylcysteine CRS in 1 m L of 1 M hydrochloric add and dilute to 100.0 m l. with water R. Column: — sizer. I = 0.25 m, 0 = 4 mm; — stationary phase: octadecylsifyl silica gel for chromatography R (5 pm).

Mobile phase Stir 3 volumes of acetonitrüe R and 97 volumes o f water R in a beaker; adjust to pH 3.0 with phosphoric add R. Flow rate 1.0 mL/min. Detection Spectrophotom eter at 220 nm .

_

A 3 x m 3 x 100 A* x m i

12 = ----------------

A1

= peak area o f individual im purity (impurity A, impurity B, impurity C and im purity D) in the chromatogram obtained with test solution (a); A 2 = peak area o f the corresponding individual im purity (impurity A, im purity B, im purity C and impurity D ) in the chrom atogram obtained with reference solution (a); A 3 = peak area of unknown impurity in the chromatogram obtained with test solution (a); A 4 = peak area o f acetylcysteine in the chromatogram obtained with reference solution (b); mi = mass o f the substance to be examined in test solution (a); m2 = mass of the individual im purity in reference solution (a); m3 = mass of acetylcysteine in reference solution (b).

Limits: — impurities A , B, C, D: for each im purity, maximum 0.5 per cent; — arty other impurity: for each impurity, maximum 0.5 per cent; — total: maximum 0.5 p er cent; — disregard Umar. 0.1 times the area o f the principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent); disregard any peak w ith a retention time of about 3.3 m in due to 2-methyl-2-thiazoline-4carboxylic ad d . H eav y m e ta ls (2.4.8) M aximum 10 ppm . 2.0 g complies with test C. Prepare the reference solution using 2 m L o f lead standard solution (10 ppm Pb) R. Z in c Maximum 10 ppm. Atomic absorption spectrometry (2.2.23, Method II).

Test solution Dissolve 1.00 g in 0.001 M hydrochloric add and dilute to 50.0 m L with the same ad d . Reference solutions Prepare the reference solutions using zinc standard solution (5 mglmL Zn) R, diluting with 0.001 M hydrochloric add. Source Zinc hollow-cathode lamp. Wavelength 213.8 nm . Atomisation device Air-acetylene flame. U se a correction procedure for non-specific absorption.

Acetyldigoxin 1-61

2016

Loss o n d ry in g (2.2.52) M aximum 1.0 per cent, determined on 1.000 g by drying in an oven in vacuo at 70 °C for 3 h.

★■k+ic★ ★ ★ ★ ★

Acetyldigoxin fl}~Acetyldigoxin, Ph Eur monograph 2168)

S u lfated a s h (2.4.14) Maximum 0.2 per cent, determined on 1.0 g.

ASSAY Dissolve 0.140 g in 60 m L o f water R and add 10 m L of dilute hydrochloric acid R. After cooling in iced water, add 10 m L of potassium iodide solution R and titrate with 0.05 M iodine, using 1 m L of starch solution R as indicator. 1 m L of 0.05 M iodine is equivalent to 16.32 m g of C5H 9 N 0 3S.

STORAGE Protected from light.

IMPURITIES Specified impurities A, B, C, D H NH, HOjC

COjH

C43H660 15

H NH2

823

5355-48-6

Action and use

A. 3,3 '-disulfenediylbis [(2J?)-2-aminopropanoic acid] (L-cystine),

Cardiac Glycoside. PhEur_______________

H NH,

DEFINITION

co 2h

3 P- [(4- O-Acetyl-2,6-dideoxy-P-D-n&o-hexopyranosyl- (1 -> 4)2,6-dideoxy-P-D-nZ>o-hexopyranosyl-(l->4)-2,6-dideoxy-P-Dn&o-hexopyranosyl)oxy]-l 2P, 14-dihydroxy-5P-card-20(22)enolide.

B. (2i^-2-am ino-3-sulfanylpropanoic a d d (L-cysteine), ch 3

Content

0=\

97.0 p er cent to 102.0 p er cent (dried substance).

H NH

CHARACTERS Appearance

ho 2c

> r ^ S ' Sx— X 'COjH H NH

°=
3 /-disulfanediylbis[2-(acetylamino)propanoic add] (N ^'-diacetyl-L-cystine),

Practically insoluble in water, sparingly soluble in methylene chloride, slightly soluble in ethanol (96 per cent).

IDENTIFICATION Infrared absorption spectrophotometry (2.2.24).

ch 3

Comparison ^-acetyldigoxin CRS.

0=
0

0+100

4 5 -6 5

0

100

Flow rate 0.7 mL/min. Detection Spectrophotom eter at 220 nm . Injection 20 p L o f the test solution and reference solutions (a) and (c).

Retention time N-acetyltryptophan = about 29 min; 1,1 '-ethylidenebis(tryptophan) = about 34 min.

System suitability, reference solution (c): — resolution: minimum 8.0 between the peaks due to N -acetyltryptophan and 1,1 '-ethylidenebis(tryptophan); if necessary, adjust the time program m e for die elution gradient (an increase in the duration o f elution with mobile phase A produces longer retention times and a b etter resolution);

Acetyltryptophan 1-65

2016 — symmetry factor, maximum 3.5 for the peak due to ljl'-ethylidenebistcyptophan in the chromatogram obtained with reference solution (c).

Limits: — impurities A , B, C, D, E, F, G, H, I, J, K, L: for each impurity, n o t more than 0.25 times the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.25 per cent); — total: not m ore than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent); — disregard limit: 0.01 times the area of the principal peak the chrom atogram obtained with reference solution (a) (0.01 per cent). A m m o n iu m (2.4.1, Method B) Maximum 200 ppm , determined on 0.10 g. Prepare the standard using 0.2 m L o f ammonium standard

solution ( 1 0 0 ppm N H J R. Ir o n (2.4.9) M aximum 10 ppm .

C. R = H : (S)-2-amino-4-(2-aminophenyI)-4-oxobutanoic a d d (kynurenine), E. R = C H O : (5)- 2-amino-4-[2- (formylamino)phenyl]-4oxobutanoic a d d (AT-fonnylkynurenine) 3

HO

D . (S)- 2 -amino-3-(5-hydroxy - 1H-indol-3-yl)propanoic a d d (5-hydroxytryptophan),

h

Dissolve 1.0 g in 50 m L o f hydrochloric acid R1, with heating at 50 °C. Allow to cool. In a separating funnel, shake with 3 quantities, each of 10 m L, of methyl isobutyl ketone R1, shaking for 3 min each time. T o the combined organic layers add 10 m L o f water R and shake for 3 min. Examine the aqueous layer.

Hv Nh2

F . (S)-2-amino-3-(phenylamino)propanoic a d d (3-phenylaminoalanine),

H eavy m e ta ls (2.4.8) M aximum 10 ppm . 2.0 g complies with test C. Prepare the reference solution using 2 m L o f lead standard solution (10 ppm Pb) R. L oss o n d ry in g (2.2.32) M axim um 0.5 per cent, determined on 1.000 g by drying in an oven at 105 °C. S u lfa te d a sh (2.4.14) M aximum 0.1 per cent, determined on 1.0 g.

G. 0S)-2-amino-3-(2-hydroxy-lH-indol-3-yl)propanoic a d d (2-hydroxytryptophan),

R

A SSAY Dissolve 0.200 g in 5 m L o f methanol R. Add 50 m L of anhydrous ethanol R Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2 .2 . 2 0 ). 1 m L of 0.1 M sodium hydroxide is equivalent to 24.63 m g of

C 13H14N20 3 .

H . R = H : (3i?5)-l,2,3,4-tetrahydro-9/f-P-carboline-3carboxylic ad d ,

STORA GE Protected from light.

I. R = C H 3: 1-m ethyl-1,2,3,4-tetrahydro-9/f-P-carboline-3carboxylic ad d ,

IM P U R IT IE S

Specified impurities A, B, C , D , E, F, G, H , I, J, K , L

c o 2h

A. (S)-2-amino-3-(lH-indol-3-yl)propanoic acid (tryptophan),

CO2H

and epimer at C*

B. (S)-2-amino-3-[(3i?S)-3-hydroxy-2-oxo-2,3-dihydro-li/indol-3-yl]propanoic a d d (dioxyindolylalanine),

J. R = C H O H -C H 2-O H: (5)-2-amino-3-[2- [2,3-dihydroxy-1(l/f-indol-3-yl) propyl] - lH-indol-3-yl]propanoic add, K. R = H : (5)-2-amino-3-[2-(1 //-indol-3-ylmethyl)-1Hindol-3-yl] propanoic ad d .

2016

1-66 Acetyltyrosine

spot in the chrom atogram obtained with th e reference solution. D. Solution S (see Tests) is strongly a d d (2.2.4). TESTS S o lu tio n S Dissolve 2.50 g in water R and dilute to 100.0 m L with the same solvent. A p p e a ra n c e o f so lu tio n Solution S is d e a r (2.2.1) and colourless (2.2.2, Method II).

L. 1-(lH-indol-3-yimet±iyl)-li2j3,4-tetxahydro-9f/-ßcarboline-3-carboxylic ad d . ___________________________________________________________PhEur

S p ecific o p tic a l r o ta tio n (2.2.7) + 46 to + 49 (dried substance). Dilute 10.0 m L o f solution S to 25.0 m L with water R. R e la te d su b s ta n c e s Liquid chromatography (2.2.29). Cany out the test protected

Acetyltyrosine

? **%

(N-Acetyltyrosine, Ph Eier monograph 1384)

** * **

from light. Test solution Dissolve 50.0 m g of die substance to be examined in mobile phase A and dilute to 50.0 m L with mobile phase A.

Reference solution (a) Dilute 1.0 m L o f the test solution to 100.0 m L with mobile phase A. D ilute 1.0 m l. o f this solution to 10.0 m L with mobile phase A.

Cn H 13N 0 4

223.2

537-55-3

PhEir ___________________________________________________________

D E F IN IT IO N (2S)-2-(Acetylamino)-3-(4-hydroxyphenyI)propanoic ad d .

Reference solution (b) Dissolve 20.0 m g o f tyrosine CRS (impurity A) in 2 m L of a 40 g/L solution o f sodium hydroxide R and dilute to 20.0 m L w ith water R. Dilute 1.0 m l. o f this solution to 10.0 m L with water R. Reference solution (c) Dilute 1.0 m L o f reference solution (b) to 10.0 m L with mobile phase A.

Reference solution (d) Dilute 1.0 m L o f reference solution (b)

C o n te n t 98.5 p er cent to 101.0 p er cent (dried substance).

to 20.0 m L with the test solution.

CHARACTERS A p p e a ra n c e W hite or almost white, crystalline powder or colourless crystals.

— size. I = 0.15 m , 0 = 3 mm; — stationary phase, spherical octadecylsUyl silica gel for chromatography R (3 pm); — temperature. 40 °C.

S o lu b ility Fredy soluble in water, practically insoluble in cyclohexane. ID E N T IF IC A T IO N

First ideruification A , B Second identification A , C, D

Column:

Mobile phase: — mobile phase A: mix 1.0 m L of phosphoric add R and 1000 m L of water for chromatography R; — mobile phase B: acetonitrile Rl;

B. Infrared absorption spectrophotom etry (2.2.24).

Time (min) 0 -2

Mobile phase A (per cent V/V) 97

Mobile phase B (per cent V/V) 3

Comparison N-acetyltyrosine CRS.

2 - 15

97 -» 62

3 -» 38

A Specific optical rotation (see Tests).

C. Thin-layer chrom atography (2.2.27).

Test solution Dissolve 80 mg of the substance to be examined in a mixture o f 3 volumes of glacial acetic acid R, 3 volumes of water R and 94 volumes o f anhydrous ethanol R, and dilute to 10 m L with the same mixture of solvents. Reference solution Dissolve 80 m g o f N-acetyltyrosine CRS in a m ixture of 3 volumes o f glacial acetic acid R, 3 volumes of water R and 94 volumes o f anhydrous ethanol R , and dilute to 10 m L with the same mixture o f solvents. Plate TLC silica gel F 254 plate R. Mobile phase water R, glacial acetic add R} ethyl acetate R (10:15:75 V/VIV). Application 5 [iL. Development Over 2/3 o f the plate. Drying In air. Detection Examine in ultraviolet light at 254 nm. Results T he p rin d p al spot in the chrom atogram obtained with the test solution is similar in position and size to. the p rindpal

Flow rate 0.7 mL/min. Detection Spectrophotom eter at 219 nm . Iiyection 2 [iL o f the test solution and reference solutions (a), (c) and (d).

Relative retention W ith reference to N-acetyltyrosine (retention time = about 6 min): impurity A = about 0.5.

System suitability Reference solution (d): — resolution: m inim um 5.0 between the principal peak and the peak due to im purity A.

Limits: — impurity A: n o t m ore than 0.8 times the area o f the corresponding peak in the chrom atogram obtained with reference solution (c) ( 0.8 per cent); — unspecified impurities: for each impurity, n o t m ore than the area o f the principal peak in the chrom atogram obtained with reference solution (a) (0.10 per cent); — total: m axim um 1.0 p er cent;

2016

Aciclovir 1-67

— disregard, limit. 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). C h lo rid e s (2.4.4) M aximum 200 ppm . Dilute 10 m L o f solution S to 15 m L with water R.

COjH

B. (25)-2-(acetylamino)-3-[4-(acetoxy)phenyl]propanoic a d d (diacetyltyrosine).

S u lfates C2.4.13) M axim um 200 ppm .

PhEur

Dissolve 1.0 g in distilled water R and dilute to 20 m L with the same solvent. A m m o n iu m (2.4.1, Method B) Maximum 200 ppm , determined on 0.100 g.

Aciclovir

Prepare the standard using 0.2 m L of ammonium standard

(Ph Eur monograph 0968)

★+** ★ ★ ★ *****

solution (100 ppm N H J R. Ir o n (2.4.9) M axim um 20 ppm . In a separating funnel, dissolve 0.5 g in 10 m L of dilute hydrochloric add R. Shake with 3 quantities, each o f 10 m L, of methyl isobutyl ketone R l, shaking for 3 m in each time. T o the combined organic layers add 10 m L o f water R and shake for 3 m in. T h e aqueous layer complies with the test.

h 2n

"¿0

C 8H 11N 5O 3

OH

225.2

592 7 7 -8 9 -3

H eav y m e ta ls (2.4.8) Maximum 10 ppm .

A c tio n a n d u se Purine nucleoside analogue; antiviral (herpesviruses).

Dissolve 2.0 g in water R and dilute to 20 m L with the same solvent. 12 m L o f the solution complies with test A. Prepare the reference solution using lead standard solution

P re p a r a tio n s Aciclovir C ream

(1 ppm Pb) R. L oss o n d ry in g (2.2.32) M aximum 0.5 p er cent, determined on 1.000 g by drying in an oven at 105 °C. S u lfa te d a sh (2.4.14) M aximum 0.1 per cent, determined on 1.0 g. B a c te ria l e n d o to x in s (2.6.14) Less than 25 IU /g, if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal of bacterial endotoxins. A SSAY Dissolve 0.180 g in 50 m L o f carbon dioxide-free water R. T itrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2 . 2 . 2 0 ). 1 m L of 0.1 M sodium hydroxide is equivalent to 22.32 m g of C n H 13N 0 4. STORA GE Protected from light. If the substance is sterile, store in a sterile, airtight, tam per-proof container. IM P U R IT IE S

Specified impurities A Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these impurities for demonstration o f compliance. See also 5.10.

Control of impurities in substancesfor pharmaceutical use): B. .

A. (26)-2-amino-3-(4-hydroxyphenyl)propanoic acid (tyrosine),

Aciclovir Eye O intm ent Acidovir Infusion A d d o v ir Oral Suspension Adclovir Tablets Dispersible A dd o v ir Tablets P h E u r __________________________________________________________________________________________

D E F IN IT IO N 2-Amino-9- [(2-hydroxyethoxy) methyl] -1,9-dihydro-6//-purin6-one. C o n te n t 98.5 per cent to 101.0 per cent (anhydrous substance). CHARACTERS A p p e a ra n c e ^ W hite or almost white, crystalline powder. S o lu b ility Slightly soluble in water, very slightly soluble in ethanol (96 per cent), practically insoluble in heptane. It dissolves in dilute solutions o f mineral adds and alkali hydroxides. ID E N T IF IC A T IO N Infrared absorption spectrophotometry (2.2.24).

Comparison aciclovir CRS. TESTS A p p e a ra n c e o f so lu tio n T he solution is clear (2.2.1) and n o t more intensely coloured than reference solution Y7 (2.2.2, Method IT). Dissolve 0.25 g in a 4 g/L solution of sodium hydroxide R and dilute to 25 m L with the same solvent. R e la te d su b sta n c e s Liquid chromatography (2.2.29). Prepare the solutions

immediately before use. Solvent mixture dimethyl sulfoxide R, water R (20:80 VIV). Phosphate buffer solution pH 2.5 Dissolve 3.48 g of dipotassium hydrogen phosphate R in 1000 m L of water R and adjust to p H 2.5 with phosphoric add R.

2016

1-68 Aciclovir

Phosphate btcffer solution pH 3.1 Dissolve 3.48 g of dipotassium hydrogen phosphate R in 1000 m L o f water R and adjust to p H 3.1 with phosphoric acid R. Test solution Dissolve 25 m g o f the substance to be examined in 5.0 mT- o f dimethyl sulfoxide R and dilute to 25.0 m L with water R. Reference solution (a) Dissolve 5 mg o f addovir for system suitability CRS (containing impurities A, B, J, K , N , O and P) in 1 m L o f dimethyl sulfoxide R and dilute to 5.0 m L with water R. Reference solution (b) Dilute 1.0 m L o f the test solution to 100.0 m L with the solvent mixture. Dilute 1.0 mT. o f this solution to 10.0 m L with the solvent mixture.

Reference solution (c) Dissolve the contents of a vial o f aciclovir for peak identification 1 CRS (containing impurities C and I) in 200 pL o f dimethyl sulfoxide R and dilute to 1.0 m L with water R. Reference solution (d) Dissolve the contents o f a vial of aciclovir for peak identification 2 CRS (containing impurities F and G ) in 1.0 m L of reference solution (a).

Column’. — size: I = 0.25 m , 0 = 4.6 mm; — stationary phase: end-capped octadecylsüyl silica gel for chromatography R (5 pm). Mobile phase: — mobile phase A: acetonitrile R, phosphate buffer solution p H 3.1 (1:99 VIV); — mobile phase B: acetonitrile R, phosphate buffer solution p H 2.5 (50:50 VIV)’, Time (min) 0-5

Mobile phase A (per cent V/V) 100

Mobile phase B (per cent V/V) 0

5 - 27

100 -» 80

0 -» 20

27 - 40

80

20

Flow rate 1.0 mL/min. Detection Spectrophotom eter at 254 nm . Injection 10 pL o f the test solution and reference solutions (b), (c) and (d).

Identification of impurities Use the chrom atogram supplied with aciclovir for peak identification 1 CRS and the chrom atogram obtained with reference solution (c) to identify the peaks due to impurities C and I; use the chrom atogram supplied with addovir for peak identification 2 CRS and the chrom atogram obtained with reference solution (d) to identify the peaks due to impurities A, B, F, G, J, K, N , O and P. Relative retention W ith reference to aciclovir (retention time = about 13 min): impurity B = about 0.4; im purity P = about 0.7; im purity C = about 0.9; im purity N = about 1.37; impurities O and Q = about 1.42; im purity I = about 1.57; im purity J = about 1.62; im purity F = about 1.7; im purity A = about 1.8; impurities K and R = about 2.5; impurity G = about 2.6. System suitability: — resolution: minimum 1.5 between the peaks due to im purity C and aciclovir in the chrom atogram obtained with reference solution (c); m inim um 1.5 between the peaks due to impurities F and A and minimum 1.5 between the peaks due to impurities K and G in the chrom atogram obtained with reference solution (d).

Limits: — correction factor, for the calculation o f content, multiply the peak area o f im purity I by 1.5; — impurity B: not more than 7 times the area o f the principal peak in the chrom atogram obtained with reference solution (b) (0.7 per cent); — sum of impurities O and Q: n o t m ore than 3 times the area o f the principal peak in the chrom atogram obtained with reference solution (b) (0.3 per cent); — sum cf impurities K and R: n o t m ore than twice the area of the principal peak in the chrom atogram obtained with reference solution (b) (0.2 per cent); — impurities A , G, J, N, F. for each impurity, n o t m ore than twice the area o f the principal peak in the chromatogram obtained with reference solution (b) (0.2 per cent); — impurities C, F, I: for each impurity, not m ore than the area o f the principal peak in the chrom atogram obtained with reference solution (b) ( 0.1 per cent); — unspecified impurities: for each impurity, n o t m ore than 0.5 times the area o f the principal peak in the chrom atogram obtained with reference solution (b) (0.05 per cent); — total: n o t m ore than 15 times the area o f the principal peak in the chromatogram obtained with reference solution (b) (1.5 per cent); — disregard limit. 0.3 times the area o f the p rin d p al peak in the chrom atogram obtained with reference solution (b) (0.03 per cent). W a te r (2.5.12) M aximum 6.0 per cent, determ ined on 0.500 g. S u lfa te d a s h (2.4.14) M aximum 0.1 per cent, determ ined on 1.0 g. B a c te ria l e n d o to x in s (2.6.14, Method D) Less than 0.50 IU/m g, if intended for use in th e manufacture o f parenteral preparations without a further appropriate procedure for the removal o f bacterial endotoxins. A SSA Y Dissolve 0.150 g in 60 m L o f anhydrous acetic add R. T itrate with 0.1 M perchloric add, determining the end-point potentiometrically (2.2.20). C an y out a blank titration.

1 m L o f 0.1 Mperchloric add is equivalent to 22.52 m g 0f C 8H u N 5O 3. IM P U R IT IE S

Specified impurities A, B, C, F, G , I, J, K, N , O , P, Q, R Other detectable impurities (the following substances would, if present at a suffirient levd, be detected by one or other o f the tests in the monograph. T hey are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharm aceutical use (2034). It is therefore not necessary to identify these impurities for demonstration o f compliance. See also 5.10.

Control of impurities in substances for pharmaceutical use): L, M.

O N

> ^—o

N

O

p

/ -'

A. 2 -[( 2 -am ino- 6-oxo- l,6-dihydro-9/i-purin-9yl)methoxy] ethyl acetate,

CH3

2016

Aciclovir 1-69 0

h i V>

H2N

N

1 aV> h y

h3c ^

N

n^

n^

n

B. 2-amincHl ,7-dihydro-6i7-purin-6-one (guanine),

o

-O

L. N - (9-acetyl-6-oxo-6,9-dihydro-1.ff-purin-2-yl) acetamide (2^9-diacetylguanine),

OH

1 1 / H2N

N

o

^

\ — 0'

X ^JL^JL^

h3c^

n

n

n

H

O. unknown structure,

F. N-[9-[(2-hydroxyethoxy)methyl]-6-oxo-6,9-dihydro-l/ipurin-2-yl] acetamide,

0 HNX

.

lX >

o

h2n ^

H

n^

n^

^ oh

O /

i iY> ^ j—f

h3c ^ n ^ n ^ - n

P ~ich3

N . unknown structure,

n

H

/^°x

M . 2- [ [2-(acetylamino)-6-oxo-1,6-dihydro-7H-puiin-7yl]methoxy] ethyl acetate,

X a JCn> ; " nA

VJ

0

N

C. 2-am ino-7- [(2-hydroxyethoxy)methyl]-l ,7-dihydro-6/ipurin-6-onej

h3c ^

ch 3

o

( 3 ch

P. 2-amino-9-(2-hydroxyethyl)-1,9-dihydro-6/f-purin-6-onej

\— o

0

G. 2- [ [2-(acetylamino)-6-oxo-1,6-dihydro-9H-purin-9yl]methoxy] ethyl acetate.

x YN > N

OH

V

HN

JLn

O

i xN >

h x V>

H2N

N

a

N

I> N

/ -----\

'—O

/

-O

\

OH

NH2

I. 2-am ino-7-[ [2- [(2-amino-6-oxo-1,6-dihydro9H -purin-9yl)methoxy]ethoxy]methyl]-l,7-dihydro-6i/-purin-6-one,

H2N

+

Q. mixture o f 2-amino-9-[[2(hydroxymethoxy) ethoxy] methyl] -1 j9-dihydro-6//-purin-6one and 2-amino-9-[[2-(hydroxyethoxy)methoxy]methyl]-l,9dihydro-6//-purin-6-one,

ccSl N

O—’

N

NH2

J. 9 ,9 '- [ethylenebis(oxymethylene)]bis(2-amino-l,9-dihydro6//-purin-6-one), R. 9 ,9 '[methylenebis(oxyethyleneoxymethylene)]bis(2-amino-l,9dihydro-6//-purin-6-one). PhEur

K. 2,2'-(methylenediimino)bis[9-[(2-hydroxyethoxy)methyl]1,9-dihydro-6/i-purin-6-one],

2016

1-70 Acitretin

Acitretin

******

(Ph Eur monograph 1385)

*

TESTS

Related substances Liquid chrom atography (2.2.29). Maintain the sampler at

A ctio n a n d u se Vitamin A analogue (retinoid); treatm ent o f psoriasis; ichthyosis; D arier’s disease.

4 °C. Test solution (a) Dissolve 2 5 .0 m g o f the substance to be examined in 5 m L of tetrahydrofuran R and dilute immediately to 1 0 0 .0 m L with anhydrous ethanol R. Test solution (b) Dilute 1 0 .0 m L o f test solution (a) to 2 5 .0 m l. with anhydrous ethanol R. Reference solution (a) Dissolve 2 5 .0 m g o f acitretin CRS in 5 m L of tetrahydrofuran R and dilute immediately to 100.0 m L with anhydrous ethanol R. D ilute 10.0 m L o f this solution to 2 5 .0 m l. with anhydrous ethanol R Reference solution (b) Dissolve 1 .0 m g of tretinoin CRS in anhydrous ethanol R and dilute to 2 0 .0 m L with the same

P re p a r a tio n A dtretin Capsules

solvent. M ix 5 .0 m L o f this solution with 2 .5 m L o f reference solution (a) and dilute to 1 0 0 .0 m L with anhydrous

Cj i H ^ O s

326.4

55079-83-9

PhEtr___________________________________________________________

D E F IN IT IO N (aU-£)-9-(4-Methoxy-2,3, 6-trimethylphenyl)-3,7 dimethylnona-2j4j6j8-tetraenoic ad d . C o n te n t 98.0 per cent to 102.0 per cent (dried substance). CHARACTERS A p p e a ra n c e Yellow or greenish-yellow, crystalline powder. S o lubility Practically insoluble in water, sparingly soluble in tetrahydrofuran, slightly soluble in acetone and in ethanol (96 per cent), very slightly soluble in cyclohexane. It is sensitive to air, heat and light, espedally in solution. It shows polymorphism.

Cany ota all operations as rapidly as possible and avoid exposure to actinic light; use freshly prepared solutions. ID E N T IF IC A T IO N

ethanol R Reference solution (c) Dilute 2 .5 m L o f reference solution (a) to 5 0 .0 m L with anhydrous ethanol R. Dilute 3 .0 m L o f this solution to 2 0 .0 m l. with anhydrous ethanol R Column’. — size I = 0 .2 5 m , 0 = 4 mm; — stationary phase: microparticulate octadecylsQyl silica gel for chromatography R (5 pm) with a specific surface area of 2 0 0 m 2/g, a pore size o f 15 n m and a carbon loading o f

20 per cent; — temperature: 25 °C.

Mobile phase A 0 .3 per cent V/V solution o f glacial acetic acid R in a mixture o f 8 volumes o f water R and 9 2 volumes o f anhydrous ethanol R. Flow rate 0 .6 mL/min. Detection Spectrophotom eter at 3 6 0 nm . Injection 10 pL o f test solution (a) and reference solutions (b) and (c).

Run time 2 .5 times the retention time o f adtretin. Retention time Impurity A = about 4 .8 min; tretinoin = about

First identification B Second identification A , C

5 .2 m in; adtretin = about 6 .2 min; impurity B = about

A. Ultraviolet and visible absorption spectrophotom etry (2.2.25). Test solution Dissolve 15.0 m g in 10 m L o f tetrahydrofuran R and dilute immediately to 100.0 m L with the same solvent. D ilute 2.5 m L o f this solution to 100.0 m L with

System suitability: reference solution (b): — resolution: minim um 2.0 betw een the peaks due to

tetrahydrofuran R Spectral range 300-400 nm. Absorption maximum A t 358 nm . Specific absorbance at the absorption maximum 1350 to 1475. B. Infrared absorption spectrophotom etry (2.2.24). Preparation Discs. Comparison acitretin CRS. If the spectra obtained in the solid state show differences, dissolve die substance to be examined and the reference substance separately in 2-propanol R heating under reflux, filter, evaporate to dryness and record new spectra using the residues. C. Examine the chrom atograms obtained in the assay.

Results T he prindpal peak in the chrom atogram obtained with test solution (b) is similar in retention time to the principal peak in the chrom atogram obtained with reference solution (a).

10.2 min.

adtretin and tretinoin; if necessary, adjust the concentration o f anhydrous ethanol R

Limits: — impurities A , B: for each im purity, n o t more than the area o f the peak due to adtretin in the chrom atogram obtained w ith reference solution (c) (0 .3 p er cent); — total: n o t more than the area o f the peak due to ad tretin in the chrom atogram obtained with reference solution (b) (1.0 per cent); — disregard limit. 0.1 times the area o f the principal peak in the chrom atogram obtained with reference solution (c).

Palladium M axim um 10 ppm. A tomic absorption spectrometry (2.2.23, Method I).

Test solution Introduce 2 .0 g into a quartz beaker and add m l. o f magnesium nitrate solution R. H eat in a muffle

3

furnace to 3 5 0 °C at a rate o f 4 0 °C/min to incinerate the content. Ignite at about 4 5 0 °C for 8 h and then at 5 5 0 ± 5 0 °C for a further hour. Dissolve the residue in a m ixture o f 0 .7 5 m L o f hydrochloric add R and 0 .2 5 m L o f nitric acid R, warming gently. Cool, then transfer the solution

2016

Adapalene 1-71

into a volumetric flask contain in g water R and dilute to 50.0 m L w ith the same solvent.

Adapalene

Reference solution Dissolve 0.163 g o f heavy magnesium oxide R in a mixture o f 0.5 m L of nitric acid R, 1.5 m L o f hydrochloric acid R and 50 m L o f water R, add 2.0 m L o f palladium standard solution (20 ppm Pd) R and dilute to 100.0 m L with

(Ph. Eur. monograph 2445)

water R. Source Palladium hollow-cathode lamp. Wavelength 247.6 nm. Atomisation device Air-acetylene flame. H eavy m e ta ls {2.4.8)

*****

*

M aximum 20 ppm.

C ss H a A

412.5

J 06685-40-9

2.0 g complies with test C. Prepare the reference solution vising 2 m l. o f lead standard solution (10 ppm Pb) R.

A ctio n a n d u se Vitamin A analogue (retinoid); treatm ent of acne.

L oss o n d ry in g (2.2.32) M aximum 0.5 per cent, determined on 1.000 g by drying in vacuo at 100 °C for 4 h.

P re p a r a tio n s Adapalene Cream Adapalene Gel

S u lfated a s h (2.4.14) M aximum 0.1 per cent, determined on 1.0 g.

Ph Eur____________ _____________________________________________

ASSAY

DEFINITION

Cany out the assay protected from light, use amber volumetric flasks and prepare the solutions immediately before use. Liquid chromatography (2.2.29) as described in the test for

6-(4-Methoxy-3-tricyclo [3.3.1.13,7] dec-1ylphenyI)naphthalene-2-carboxylic acid.

related substances with the following modifications.

C o n te n t 98.0 per cent to 102.0 per cent (dried substance).

Injection T est solution (b) and reference solution (a). System suitability: — repeatability: maximum relative standard deviation of

CHARACTERS A p p e a ra n c e White or almost white powder.

1.0 per cent after 6 injections o f reference solution (a); if necessary, adjust the integration parameters.

Solubility

Calculate the percentage content of C 21H 26O 3 from the declared content of acitrean CRS.

Practically insoluble in water, sparingly soluble in tetrahydrofuran, practically insoluble in ethanol (96 per cent).

STORAGE In an airtight container, protected from light, at a tem perature o f 2 °C to 8 °C.

ID E N T IF IC A T IO N Infrared absorption spectrophotometry (2.2.24).

I t is recom mended that the contents o f an opened container be used as soon as possible and any unused part be protected by an atmosphere o f inert gas. IM P U R IT IE S

Specified impurities A, B. ch 3

Comparison adapalene CRS. TESTS A p p e a ra n c e o f solution T h e solution is clear (2.2.1) and not more intensely coloured than reference solution BY6 (2.2.2, Method II). Dissolve 0.2 g in tetrahydrofuran R and dilute to 20 m l. with the same solvent.

ch3

ch 3

h3co ch 3

A. (2Zj4£,6£’,8£)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7dim ethylnona-2,4,6,8-tetraenoic add.

R e la te d su b sta n c e s Liquid chromatography (2.2.29).

Solvent mixture tetrahydrofuran R, acetomtrUe R, water R (20:37:43 V/V/V). Test solution (a) Dissolve 40.0 mg o f the substance to be examined in 10 m L o f tetrahydrofuran R, add 7 m L of the solvent mixture and dilute to 20.0 m L with tetrahydrofuran R. Test solution (b) Dissolve 20.0 m g o f the substance to be examined in 50 m L of tetrahydrofuran R, add 35 m l. of the solvent mixture and dilute to 100.0 m L with tetrahydrofuran R. Dilute 5.0 m L o f the solution to 50.0 m L with the solvent mixture.

Reference solution (a) Dilute 1.0 m L o f test solution (a) to 10.0 m L with tetrahydrofuran R. D ilute 1.0 m l. o f this solution to 100.0 m l, with the solvent mixture. B. ethyl (all-£)-9-(4-methoxy-2,336-trimethylphenyl)-3,7dim ethylnona-2,4,6,8-tetraenoate. —-________________________________________________________ PhEur

Reference solution (b) Dissolve 2.4 m g of adapalene impurity C CRS in 2 m L of tetrahydrofuran R and dilute to 20.0 m L with the same solvent. Dilute 2.0 m L o f the solution to 20.0 m L with the solvent mixture. T o 2.0 m L of this solution add 2.0 m L of reference solution (a) and dilute to 20.0 m l. with the solvent mixture.

2016

1-72 Adapalene

Reference solution (c) Dissolve the contents of a vial of adapalene for peak identification CRS (containing impurities A, C and D ) in 0.5 m L o f tetrahydrqfuran R and dilute to

— disregard Omit: 0.5 times the area of the principal peak in the chrom atogram obtained w ith reference solution (a) (0.05 per cent).

1.0 m L with the solvent mixture.

H eav y m e ta ls (2.4.8) M axim um 20 ppm .

Reference solution (d) Dissolve 20.0 m g o f adapalene CRS in 50 m L of tetrahydrqfuran R> add 35 m L o f the solvent m ixture and dilute to 100.0 m L with tetrahydrofuran R. D ilute 5.0 m L of the solution to 50.0 m L with die solvent mixture.

Column: — size. I = 0.25 m , 0 = 4.6 mm; — stationary phase: end-capped phenylsQyl silica gel for chromatography R (5 tun) with a carbon loading of 7.5 per cent; — temperature: 30 °C.

Mobile phase: — mobile phase A: glacial acetic add R, water R (0.1:100 VIV)\ — mobile phase B: tetrahydrofuran R, acetomtrUe R (35:65 VIV)\

0.250 g complies with test G . Prepare the reference solution u sin g 0.5 m L of lead standard solution

(10 ppm Pb) R.

L oss o n d ry in g (2.2.32) M axim um 0.5 p er cent, determ ined on 1.000 g by drying in an oven at 105 °C for 4 h. S u lfa te d a s h ( 2.4.14) M axim um 0.1 per cent, determ ined on 1.0 g. A SSA Y Liquid chromatography (2.2.29) as described in the test for related substances w ith the following modification.

Irtjection T est solution (b) and reference solution (d). Calculate the percentage content o f adapalene from the declared content o f adapalene CRS. IM P U R IT IE S

Time (min) 0 -2 .5

Mobile phase A (percent V/V) 50

Mobile phase B (per cent V/V) 50

23 - 40

50-» 28

50*72

40 - 42

28

72

Flow rate 1 .2 mL/min. Detection Spectrophotom eter at 2 7 0 nm . Injection 2 5 |iL of test solution (a) and reference solutions (a),

Specified impurities A, C , D Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other o f the tests in the m onograph. T hey are limited by die general acceptance criterion for other/unspecified impurities and/or by the general m onograph Substances for pharmaceutical use (2034). It is therefore n o t necessary to identify these impurities for dem onstration o f compliance. See also 5.10.

Control of impurities in substances for pharmaceutical use): B.

(b) and (c).

Identification of impurities Use the chrom atogram supplied with adapalene for peak identification CRS and the chrom atogram obtained with reference solution (c) to identify the peaks due to impurities A, C and D.

Relative retention W ith reference to adapalene (retention tim e = about 2 0 min): impurity A = about 0 .3 ; impurity C = about 0 .9 ; im purity D = about 1.9. System suitability: reference solution (b): — resolution: m in im u m 4 .5 between the peaks due to impurity C and adapalene; — signal-to-noise ratio: minimum 1 0 for the peak due to im purity C.

A. 2 ,2 /-binaphthalene- 6,6 '-dicarboxylic acid,

COjH

Limits:

— correction factors: for the calculation of content, multiply the peak areas o f the following impurities by the corresponding correction facto r im purity A = 0 .7 ; impurity C = 7; impurity D = 1.4; - — impurity A: not m ore than 3 times die area o f the principal peak in the chrom atogram obtained with reference solution (a) (0 .3 p er cent); — impurity D: n o t m ore than twice die area o f the principal peak in the chrom atogram obtained with reference solution (a) (0.2 per cent); — impurity C: n o t m ore than 1.5 times the area o f the principal peak in die chrom atogram obtained with reference solution (a) (0 .1 5 p er cent); — unspecified impurities: for each impurity, n o t m ore than the area o f the principal peak in the chrom atogram obtained w ith reference solution (a) (0.10 per cent); — total: n o t m ore th an 5 times the area o f the principal peak in the chrom atogram obtained with reference solution (a) (0 .5 per cent);

B. 6-[3-(3-hydroxytricydo[3.3.1.1 3,7]dec-l-yl)-4methoxyphenyl] naphthalene- 2-carboxylic ad d ,

C. l-(2-m ethoxyphenyi)tricydo[3.3.1. l 3,7]decane,

D . 1, 1 '- [4,4 '-bis(methoxy)biphenyl-3,3 diyl]bis(tricyclo [3.3.1.13’7] decane). ______________________________________________■

PhEut

Adenine 1-73

2016 ★★* ★★ ★ ★ *****

Adenine (Ph Eur monograph 0800) nh 2

nV

I

C5H5N5

^ v I /)

135.1

73-24-5

Action and use C onstituent of anticoagulant and preservative solutions for -blood. PhEir.

DEFINITION Adenine contains not less than 98.5 p er cent and not more than the equivalent of 101.0 per cent o f 7H-purin-6-amine, calculated w ith reference to the dried substance.

CHARACTERS A white or almost white powder, very slightly soluble in water and in alcohol. It dissolves in dilute m in eral a d d s and in dilute solutions of alkali hydroxides.

IDENTIFICATION First identification A. Second identification B, C. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with adenine CRS. Examine the substances prepared as discs. B. Examine the chromatograms obtained in the test for related substances. The principal spot in the chromatogram obtained with test solution (b) is similar in position and size to the prindp al spot in the chromatogram obtained with reference solution (a). C. T o 1 g add 3.5 m L of propionic anhydride R and boil for 15 min with stirring. Cool. T o the resulting crystalline mass add 15 m L o f light petroleum R and heat to boiling with vigorous stirring. Cool and filter. W ash the predpitate with two quantities, each o f 5 mT, of light petroleum R. Dissolve the predpitate in 10 m L of water R and boil for 1 min. Filter the mixture at 30 °C to 40 °C. Allow to cool. Filter, and dry the predpitate at 100 °C to 105 °C for 1 h. T he melting point (2.2.14) o f the predpitate is 237 °C to 241 °C.

TESTS Solution S Suspend 2.5 g in 50 m L o f distilled water R and boil for 3 m in. Cool and dilute to 50 m L with distilled water R. Filter. Use the filtrate as solution S.

Appearance o f solution Dissolve 0.5 g in dilute hydrochloric acid R and dilute to 50 m L with the same ad d . The solution is clear (2.2.1) and colourless (2.2.2, Method II). Acidity or alkalinity T o 10 m L o f solution S add 0.1 mL o f bromothymol blue solution R1 and 0.2 m L of 0.01 M sodium hydroxide. The solution is blue. Add 0.4 m L of 0.01 M hydrochloric acid. T he solution is yellow.

Related substances Examine by thin-layer chromatography (2.2.27), using silica gel GF2 5 4 R as the coating substance. Test solution (a) Dissolve 0.10 g of the substance to be examined in dilute acetic acid R, with heating if necessary, and dilute to 10 m L with the same add.

Test solution (b) Dilute 1 m L of test solution (a) to 10 m L with dilute acetic add R. Reference solution (a) Dissolve 10 m g of adenine CRS in dilute acetic add R, with heating if necessary, and dilute to 10 m L with the same ad d . Reference solution (b) Dilute 1 m L o f test solution (b) to 20 m L with dilute acetic add R. Reference solution (c) Dissolve 10 m g of adenine CRS and 10 mg o f adenosine R in dilute acetic add R, with heating if necessary, and dilute to 10 mL w ith the same add. Apply to the plate 5 jiL o f each solution. Develop over a path of 12 cm using a mixture of 20 volumes of concentrated ammonia R, 40 volumes o f ethyl acetate R and 40 volumes of propanol R. Dry the plate in a current of warm air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with test solution (a), apart from the prindpal spot, is not m ore intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). T h e test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly separated spots. Chlorides (2.4.4) T o 10 m L o f solution S add 1 m L of concentrated ammonia R and 3 m L o f silver nitrate solution R2. Filter. Wash the predpitate with a little water R and dilute the filtrate to 15 m L with water R. T h e solution complies with the limit test for chlorides (100 ppm). W hen carrying out the test, add 2 m L of dilute nitric add R instead o f 1 m L of dilute nitric

add R. Sulfates (2.4.13) Dilute 10 m L o f solution S to 15 m L with distilled water R. T he solution complies with the limit test for sulfates (300 ppm). A m m on iu m

Prepare a cell consisting of two watch-glasses 60 mm in diameter placed edge to edge. T o the inner wall of the upper watch-glass stick a piece o f red litmus paper R 5 mm square and wetted with a few drops of water R. Finely powder the substance to be examined, place 0.5 g in the lower watchglass and suspend in 0.5 mL of water J?. T o the suspension add 0.30 g of heavy magnesium oxide R. Briefly triturate with a glass rod. Immediately close the cell by putting the two watch-glasses together. H eat at 40 °C for 15 min. T he litmus paper is n o t more intensdy blue coloured than a standard prepared at the same time and in the same manner using 0.05 m L o f ammonium standard solution (100 ppm NH 4) R, 0.5 m L o f water R and 0.30 g of heavy magnesium oxide R (10 ppm).

Heavy metals (2.4.8) 1.0 g complies with test C for heavy metals (20 ppm). Prepare the reference solution using 2 m L of lead standard

solution (10 ppm Pb) R. Loss on drying (2.2.32) N o t more than 0.5 per cent, determined on 1.000 g by drying in an oven at 105 °C.

Sulfated ash (2.4.14) N ot more than 0.1 per cent, determined on 1.0 g.

ASSAY Dissolve 0.100 g in a mixture o f 20 m L of acetic anhydride R and 30 m L of anhydrous acetic acid R. T itrate with 0.1 M perchloric add, determining the end-point potentiometrically (2.2.20). 1 m L of 0 . 1 M perchloric add is equivalent to 13.51 m g of

CsHsNs. __________________________________________________________ PhEur

2016

1-74 Adenosine

★ ★ ★ ★ *****

Adenosine (Ph. Eur. monograph 1486)

Reference solution (a) Dilute 1.0 m L o f the test solution to 100.0 m L with the mobile phase. Dilute 1.0 m L o f this solution to 10.0 m L with the mobile phase.

Reference solution (b) Dissolve 5 m g o f adenine R (impurity A) and 5 m g o f inosine R (impurity G) in the mobile phase and

NH2

dilute to 50 m L with the mobile phase. D ilute 4 m L o f this solution to 100 m L with the mobile phase.

Column: — size: I = 0.25 m , 0 = 4.6 mm; — stationary phase: end-capped octadecykHyl silica gel for chromatography R (5 (im). OH OH

C 10H 13N 5O4

267.2

58-61-7

Action and use Antiarrhythmic. P h E tr ____________________

DEFINITION 9-P-D-Ribofuranosyl-9ii-purin-6-araine.

Content 99.0 per cent to 101.0 per cent (dried substance).

CHARACTERS Appearance W hite or alm ost white, crystalline powder.

Solubility Slightly soluble in water, soluble in h o t water, practically insoluble in ethanol (96 per cent) and in methylene chloride. It dissolves in dilute mineral adds.

mp A bout 234 °C.

IDENTIFICATION Infrared absorption spectrophotom etry (2.2.24).

Comparison adenosine CRS. TESTS Solution S Suspend 5.0 g in 100 m L o f distilled water R and heat to boiling. Allow to cool, filter with the aid o f vacuum and dilute to 100 m L w ith distilled water R.

Appearance o f solution Solution S is colourless (2.2.2, Method II). Acidity or alkalinity T o 10 m L o f solution S, add 0.1 mT. o f bromocresolpurple solution R and 0.1 m L o f 0.01 M hydrochloric add. T he solution is yellow. Add 0.4 m L o f 0.01 M sodium hydroxide. T h e solution is violet-blue. Specific optical rotation (2.2.7) - 4 5 to - 4 9 (dried substance). Dissolve 1.25 g in / M hydrochloric acid and dilute to 50.0 m L with the same ad d . Examine within 10 min o f preparing the solution.

Related substances Liquid chrom atography (2.2.29).

Solvent mixture Dissolve 6.8 g o f potassium hydrogen sulfate R and 3.4 g o f tetrabutylammonium hydrogen sulfate R in water R, adjust to p H 6.5 with a 60 g/L solution o f potassium hydroxide R and dilute to 1000 m L with the same solvent. U se a freshly prepared solvent mixture.

Test solution Dissolve 20 m g o f the substance to be examined in the mobile phase and dilute to 20 m L with the mobile phase.

Mobile phase water R, solvent mixture (40:60 VIV). Flow rate 1.5 mlVmin. Detection Spectrophotom eter at 254 nm. Injection 20 pL. Run time 1.5 times the retention time o f adenosine. Relative retention W ith reference to adenosine (retention time = about 13 min): impurity A = about 0.3; im purity G = about 0.4. System suitability, reference solution (b): — resolution: minim um 1.5 between the peaks due to impurities A and G.

Limits: — correction factors: for the calculation o f content, multiply the peak areas o f the following impurities by the corresponding correction facto r impurity A = 0.6; im purity G = 1.4; — impurity A: n o t m ore than twice the area o f the prindpal peak in the chromatogram obtained with reference solution (a) ( 0.2 per cent); — impurity G: n o t more than the area o f the prin d p al peak in the chrom atogram obtained with reference solution (a) (0.1 p er cent); — unspecified impurities: for each impurity, no t m ore than the area o f the prin d p al peak in the chrom atogram obtained with reference solution (a) (0.10 per cent); — total: n o t m ore than 5 times the area o f the prin d p al peak in the chrom atogram obtained with reference solution (a) (0.5 p er cent); — disregard ltmir. 0.5 times the area of the prin d p al peak in the chrom atogram obtained with reference solution (a) (0.05 p er cent). C h lo rid e s (2.4.4) M axim um 100 ppm . D ilute 10 m L o f solution S to 15 m L with water R. S u lfa te s (2.4.13) M axim um 200 ppm , determined on solution S. A m m o n iu m (2.4.1, Method B) M axim um 10 ppm , determined on 0.5 g. Prepare the standard using 5 m L o f ammonium standard

solution (1 ppm NH 4 ) R. L o ss o n d ry in g (2.2.32) Maximum 0.5 per cent, determined on 1.000 g by drying in an oven a t 105 °C. S u lfa te d a s h (2.4.14) M axim um 0.1 p er cent, determined on 1.0 g. A SSA Y Dissolve 0.200 g, warming slightly if necessary, in a mixture o f 20 m L o f acetic anhydride R and 30 m L o f anhydrous acetic add R. 1111316 w ith 0.1 M perchloric add, determ ining the end-point potentiometrically (2 . 2 . 2 0 ).

2016

Adipic A dd 1-75

1 m L of 0 . 1 M perchloric acid is equivalent to 26.72 m g o f C 10H 13N 5O 4.

Adipic Acid

IM P U R IT IE S

(Fh Eur monograph 1586)

Specified impitrities A, G. Other detectable impurities (the following substances would, if present at a sufficient levd, be detected by one or other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these impurities for demonstration of compliance. See also 5.10.

Control of impurities in substances for pharmaceutical use): F, H.

HO2C' C6H10O4

146.1

124-04-9

A c tio n a n d u se Exdpient. PhEur.

D E F IN IT IO N Hexanedioic ad d .

nh2

C o n te n t 99.0 per cent to 101.0 per cent (dried substance).

CT> N N

★ ★ ★ ★ * .★ *★*

N H

CHARACTERS A p p e a ra n c e W hite or alm ost white, crystalline powder.

A. 7H-purin-6-amine (adenine).

S o lu b ility Sparingly soluble in water, soluble in boiling water, fredy soluble in ethanol (96 per cent) and in methanol, soluble in acetone. ID E N T IF IC A T IO N A. M d tin g point {2.2.14): 151 °C to 154 °C. B. Infrared absorption spectrophotometry {2.2.24).

OH OH

Comparison adipic acid CRS. TESTS S o lu tio n S Dissolve 5.0 g with heating in distilled water R and dilute to 50 m L with the same solvent. Allow to cool and to crystallise. Filter through a sintered-glass filter (40) {2.1.2). W ash the filter with distilled water R. Collect the filtrate and the washings until a volume of 50 m L is obtained.

F. l-^i>ribofuranosylpyrim idine-2j4(l//j3/i)-dione (uridine),

HN

A p p e a ra n c e o f so lu tio n T he solution is clear {2.2.1) and colourless (2.2.2,

HO

Method II). Dissolve 1.0 g in methanol R and dilute to 20 m l. with the same solvent.

OH OH

G . 9-P-D-ribofuranosyl-l,9-dihydro-6H-purin-6-one (inosine),

R e la te d su b sta n c e s Liquid chromatography (2.2.29).

Test solution Dissolve 0.20 g of the substance to be examined in the mobile phase and dilute to 10.0 m L with the mobile phase.

0

Reference solution (a) Dissolve 20 m g of ghitaric acid R in

h2n

1.0 m L o f the test solution and dilute to 10.0 m L with the mobile phase.

ho

V? OH

Reference solution (b) Dilute 1.0 m L o f the test solution to 100.0 m L with the mobile phase, dilute 1.0 m L o f the solution to 10.0 m L with the mobile phase.

oh

Column: H . 2-amino-9-P-D-ribofuranosyl-1,9-dihydro-6H-purin-6-one (guanosine). PhEur

— size. I = 0.125 m , 0 = 4.0 mm, — stationary phase, spherical octadecylstiyl silica gel for chromatography R (5 Jim) with a specific surface area o f 350 m 2/g and a pore size of 10 nm, — temperature: 30 °C.

Mobile phase M ix 3 volumes of acetonitrüe R and 97 volumes o f a 24.5 g/L solution o f dilute phosphoric acid R. Flow rate 1 m L/m in. Detection Spectrophotometer at 209 nm. Injection 20 (iL.

2016

1-76 Adrenaline

Run time 3 times the retention time o f adipic ad d . System suitability: reference solution (a): — resolution: minimum 9.0 between the peaks due to glutaric

IM P U R IT IE S HO2C

acid and adipic a d d .

Limits'.

A R = C H 2- C 0 2H: pentanedioic a d d (glutaric ad d ),

— any impurity: not m o re than the area o f the p rindpal peak in the chrom atogram obtained with reference solution (b) (0.1 per cent), — total: not m ore th an 5 times the area o f the prindpal peak in the chrom atogram obtained with reference solution (b) (0.5 per cent), — disregard limit: 0.5 times the area o f th e prindpal peak in the chrom atogram obtained with reference solution (b) (0.05 per cent).

B. R = C 0 2H : butanedioic a d d (succinic a d d ),

C h lo rid e s (2.4.4) M axim um 200 ppm .

C. R = [ C H J 3-C O 2H : heptanedioic a d d (pimelic ad d ). ________________________________:___________________________ PhEur

Adrenaline / Epinephrine

** * ***

(Ph Eur monograph 2303)

** ** *

D ilute 2.5 m L o f solution S to 15 m L with water R N itra te s M aximum 30 ppm . T o 1 m L of solution S add 2 m L of concentrated ammonia R, 0.5 m L of a 10 g/L solution o f manganese sulfate R, 1 m L of a 10 g/L solution o f sulfanilamide R and dilute to 20 m L with water R. Add 0.10 g o f zinc powder R and cool in iced water for 30 min; shake from time to time. Filter and cool 10 m L of the filtrate in iced water. A dd 2.5 m L o f hydrochloric add R1 and 1 m L o f a 10 g/L solution of naphthylethylenediamine dihydrochloride R. Allow to stand at room tem perature. A fter 15 m in the mixture is no t more intensely coloured th a n a standard prepared at the sam e time and in the sam e m anner, using 1.5 m L o f nitrate standard solution ( 2 ppm NO j) R instead o f 1 m L o f solution S. T he test is invalid if a blank solution prepared at the same time and in the same m anner, using 1 m L o f water R instead of 1 m L of solution S, is more intensely coloured than a 2 m g/L solution o f potassium permanganate R. S u lfates (2.4.13) Maximum 500 ppm. D ilute 3 m L o f solution S to 15 m L w ith distilled water R. Ir o n (2.4.9) Maximum 10 ppm , determ ined on solution S.

C9H 13N 0 3

183.2

51-43-4

A c tio n a n d u se Adrenoceptor agonist P re p a r a tio n s Adrenaline Eye Drops/Epinephrine Eye D rops D ilute Adrenaline Injection (I in 10,000)/Dilute Epinephrine Injection (1 in 10,000) Ph Eur

---------------------------------------------------------------------------------

D E F IN IT IO N 4- [(1R) - 1-Hydroxy-2-(methyiamino) ethyl] benzene-1,2-diol. Synthetic p ro d u c t C o n te n t

99.0

per cent to 101.0 per cent (dried substance).

CHARACTERS A p p e a ra n c e White or alm ost white crystalline powder, becoming coloured on exposure to air and lig h t

H eav y m e ta ls (2.4.8) M aximum 10 ppm .

S o lu b ility Practically insoluble in water, in ethanol (96 per cent) and in methylene chloride. It dissolves in hydrochloric ad d .

12 m L of solution S complies with test A. Prepare the reference solution using lead standard solution (1 ppm Pb) R.

ID E N T IF IC A T IO N A Infrared absorption spectrophotom etry (2.2.24).

L oss o n d ry in g (2.2.32) M aximum 0.2 per cent, determined on 1.000 g by drying in an oven at 105 °C. S u lfa te d a s h (2.4.14) M axim um 0.1 per c e n t M d t 1.0 g completely over a gas burner, then ignite the m d te d substance with the burner. After ignition, lower or remove the flame in order to prevent th e substance from boiling and keep it burning until completely carbonised. Carry out the test for sulfated ash using the residue. A SSA Y Dissolve 60.0 m g in 50 m L o f water R. A dd 0.2 m L o f phenolphthalein solution R and titrate w ith 0.1 M sodium

hydroxide. 1 m L of 0.1 M sodium hydroxide is equivalent to 7.31 m g of C6H 10O4.

Comparison adrenaline CRS. B. Spedfic optical rotation (see Tests). TESTS S o lu tio n S Dissolve 1.000 g in a 25.75 g/L solution o f hydrochloric add R and dilute to 50.0 m L with the same solvent Examine the solution immediately. A p p e a ra n c e o f so lu tio n Solution S is not more opalescent than reference suspension II (2.2.1) and n o t m ore intensely coloured than reference solution BY5 (2.2.2, Method II). S pecific o p tic a l ro ta tio n (2.2.7) - 5 0 .0 to - 5 4 .0 (dried substance), determined on solution S. R e la te d su b s ta n c e s Liquid chromatography (2.2.29). Prepare the solutions protected

from light. Solvent mixture A Dissolve 5.0 g o f potassium dihydrogen phosphate R and 2.6 g o f sodium octanesulfonate R in waterfor

2016

Adrenaline 1-77

chromatography R and dilute to 1000 m L with the same solvent (it is usually necessary to stir for at least 30 min to achieve complete dissolution). Adjust to p H 2.8 with

phosphoric acid R. Solvent mixture B acetomtrUe R l, solvent mixture A (13:87 VIV). Test solution Dissolve 40 mg of the substance to be examined in 5 m L o f 0.1 M hydrochloric add and dilute to 50.0 m L with solvent mixture B.

—

—

—

Reference solution (a) Dilute 1.0 m L of the test solution to 100.0 m L with solvent mixture B. Dilute 1.0 m L o f this solution to 10.0 m L with solvent mixture B.

—

Reference solution (b) Dissolve 1.5 mg of noradrenaline tartrate CRS (impurity B) and 1.5 m g of adrendone hydrochloride R (impurity C) in solvent mixture B, add

—

1.0 m L of the test solution and dilute to 100 m L with solvent mixture B.

Reference solution (c) Dissolve the contents o f a vial o f adrenaline impurity mixture CRS (containing impurities D and E) in 1.0 m L o f the blank solution.

Reference solution (d) Dissolve 4 m g o f adrenaline with impurity F CRS in 0.5 m L of 0.1 M hydrochloric add and dilute to 5 m L with solvent mixture B.

Blank solution 0.1 M hydrochloric acid, solvent mixture B (1:9 VIV). Column: — size. I = 0.10 m , 0 = 4.6 mm; — stationary phase, end-capped octadecylsOyl silica gel for chromatography R (3 |xm); — temperature: 50 °C. Mobile phase: — mobile phase A: acetomtrUe R l, solvent mixture A (5:95 VIV)-, — mobile phase B: acetonitrUe R l, solvent mixture A (45:55 VIV)-,

corresponding correction factor: impurity D = 0.7; impurity E = 0.6; impurities B, C, F: for each impurity, not more than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent); impurities D, E: for each impurity, not more than the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.1 p er cent); unspedfied impurities: for each impurity, not more than the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.10 per cent); total: not m ore than 5 times the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent); disregard limit. 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent).

Loss on drying (2.2.32) M axim um 0.5 per cent, determined on 1.000 g by drying over diphosphorus pentoxide R at a pressure not exceeding 0.7 kPa for 18 h.

Sulfated ash (2.4.14) M axim um 0.1 per cent, determined on 1.0 g.

ASSAY Dissolve 0.150 g in 50 m L of anhydrous acetic acid R. T itrate with 0 . 1 M perchloric add, determining the end-point potentiometrically (2 . 2 .2 0 ). 1 m L of 0.1 M perchloric add is equivalent to 18.32 mg o f C 9H 13N 0 3.

STORAGE U nder nitrogen, protected from light. IM P U R IT IE S

Specified impurities B, C , D , E, F

HO

Time (min) 0 - 15

Mobile phase A (per cent V7V) 92*50

Mobile phase B (per cent V/V) 8*50

15-20

50*92

50*8

2 0 -2 5

92

8

HO

B. (1 i?)-2-am ino-1-(3,4-dihydroxyphenyl)ethanol (noradrenaline), O

Flow rate 2.0 mL/min. Detection Spectrophotom eter at 210 nm. Injection 20 (lL. Identification of impurities Use the chromatogram supplied with adrenaline impurity mixture CRS and the chromatogram obtained with reference solution (c) to identify the peaks due to impurities D and E; use the chromatogram supplied with adrenaline with impurity F CRS and the chromatogram obtained with reference solution (d) to identify the peak due to im purity F.

C . 1-(3,4-dihydroxyphenyl)-2-(methylamino) ethanone (adrenalone),

Relative retendon W ith reference to adrenaline (retention time = about 4 min): impurity F = about 0.2; impurity B = about 0.8; impurity C = about 1.3; impurity D = about 3.3; impurity E = about 3.7. System suitability: reference solution (b): — resolution: m inim um 3.0 between the peaks due to im purity B and adrenaline.

Limits: — correction factors: for the calculation of content, multiply the peak areas o f the following impurities by the

D . 4-[(li?)-2-(benzyImethylamino)-l-hydroxyethyl]benzene1,2-diol,

2016

1-78 Adrenaline Acid Tartrate

(2.2.7) of the residue (adrenaline base) is —53.5 to —50, determ ined using a 20.0 g/L solution in 0.5 M hydrochloric

add. B. Infrared absorption spectrophotom etry (2.2.24).

Preparation Discs o f adrenaline base prepared as described under identification test A. E. 2-(beri2ylmethylamino)-l-(3,4-dihydroxyphenyl)ethanonej H O ,s

h

H

C. 0.2 m L o f the filtrate obtained in identification test A gives reaction (b) of tartrates (2.3.1).

F. (li?)-l-(3,4-dihydroxyphenyl)-2(methylamino)ethanesulfonic ad d .

TESTS PhEur

Adrenaline Acid Tartrate / Epinephrine Acid Tartrate

* * * * * ★ ★ *****

OH

H

OH COjH

HOjC H

C 13H 19NO 9

OH

333.3

Appearance of solution T he solution is not m ore opalescent than reference suspension II (2 . 2 . 1 ) and n o t m ore intensely coloured than reference solution BY5 (2.2.2, Method II). Dissolve 0.5 g in water R and dilute to 10 m L w ith the same solvent. Examine the solution im m ediatdy.

Related substances Liquid chromatography (2.2.29). Prepare the solutions protected from light. Solvent mixture A Dissolve 5.0 g o f potassium dihydrogen phosphate R and then 2.6 g o f sodium octanesidfonate R in water for chromatography R, and dilute to 1000 m L with the

(Adrenaline Tartrate, Ph Eur monograph 0254) H

Comparison U se adrenaline base prepared as described under identification test A from 50 m g o f adrenaline tartrate CRS dissolved in 5 m L o f a 5 g/L solution o f sodium metabisulfite R. Keep the mixture at room tem perature for at least 30 m in . Filter through a sintered-giass filter (2.1.2).

same solvent (it is usually necessary to stir for at least 30 min to achieve complete dissolution). Adjust to p H 2.8 with 51-42-3

Action and use Adrenoceptor agonist.

Preparations

phosphoric acid R. Solvent mixture B acetonitrUe R l, solvent m ixture A (130:870 V/V). Test sdution Dissolve 75 m g of the substance to be examined in 5 m L o f 0.1 M hydrochloric add and dilute to 50 m L with

Adrenaline Injection/Epinephrine Injection

solvent mixture B.

D ilute Adrenaline Injection (1 in 10,000)/Dilute Epinephrine Injection (1 in 10,000)

Reference solution (a) Dilute 1.0 m L o f the test solution to

Adrenaline Solution/Epinephrine Solution Adrenaline and Cocaine Intranasal Solution Bupivacaine and Adrenaline Injection/Bupivacaine and Epinephrine Injection Lidocaine and Adrenaline Injection/Lidocaine and Epinephrine Injection PhEur___________________________________________________________

D E F IN IT IO N (li?)-l-(3j4-Dihydroxyphenyl)-2-(m ethylam ino)ethanol hydrogen (2i?,3i?)-2,3-dihydroxybutanedioate.

Content 98.5 per cent to 101.0 per cent (dried substance).

CHARACTERS Appearance W hite or greyish-white, crystalline powder.

Solubility F red y soluble in water, slightly soluble in ethanol (96 per cent). ID E N T IF IC A T IO N A. Dissolve 5 g in 50 m L of a 5 g/L solution of sodium metabisidfite R and m ake alkaline by addition of ammonia R. Keep, the mixture at room tem perature for a t least 15 min and filter. Reserve the filtrate for identification test C . W ash the predpitate with 3 quantities, each o f 10 mL, of methanol R. D ry at 80 °C. T h e specific optical rotation

100.0 m L with solvent mixture B. Dilute 1.0 m L o f this solution to 10.0 m L with solvent mixture B.

Reference solution (b) Dissolve 1.5 mg of noradrenaline tartrate CRS (impurity B) and 1.5 m g of adrenalone hydrochloride R (impurity C) in solvent mixture B, add 1.0 m L o f the test solution and dilute to 100.0 m L with solvent m ixture B.

Reference solution (c) Dissolve the contents o f a vial of adrenaline impurity mixture CRS (impurities D and E) in 0.1 m L o f 0.1 M hydrochloric add and 0.9 m L o f solvent mixture B.

Reference solution (d) Dissolve 7.5 mg o f adrenaline tartrate with impurity A CRS in 0.5 m L o f 0.1 M hydrochloric add and dilute to 5.0 m L with solvent mixture B.

Blank solution 0.1 M hydrochloric add, solvent mixture B (1:9 VIV). Column: — size: I = 0.10 m , 0 = 4.6 mm; — stationary phase: end-capped octadecylsilyl silica gel for chromatography R (3 pm); — temperature: 50 °C. Mobile phase: — mobile phase A: acetomtrUe R l, solvent mixture A (5:95 V/V); — mobile phase B: acetomtrUe R l, solvent mixture A (45:55 V/V);

I

2016

Agar 1-79

Tune (min) 0 - 15

Mobile phase A (per cent V/V) 92->50

Mobile phase B (per cent V/V) 8 -> 50

15 -2 0

50->92

50 -> 8

20 - 25

92

8

Flow rate 2.0 mL/min. Detection Spectrophotom eter at 210 nm. Injection 20 |xL. Identification of impurities Use the chromatogram supplied with adrenaline impurity mixture CRS and the chromatogram obtained w ith reference solution (c) to identify the peaks due to impurities D and E; use the chromatogram supplied with adrenaline tartrate with impurity A CRS and the chromatogram obtained w ith reference solution (d) to identify the peak due to impurity A. ,

Relative retention W ith reference to adrenaline (retention

IM P U R IT IE S

Specified impurities A, B, C , D , E A. unknown structure, H

OH

B. ( 1i?)-2-amino-1-(3,4-dihydroxyphenyl)ethanol (noradrenaline),

C. 1-(3,4-dihydroxyphenyl)-2-(methyiamino)ethanone (adrenalone),

time = about 4 min): impurity B = about 0.8; impurity C = about 1.3; impurity A = about 3.2; impurity D = about 3.3; impurity E = about 3.7. System suitability: reference solution (b): — resolution: minimum 3.0 between the peaks due to impurity B and adrenaline.

Limits: — correction factors: for the calculation o f content, multiply the peak areas o f the following impurities by the corresponding correction factor impurity D = 0.7; im purity E = 0.6; — impurity A: not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.3 per cent); — impurities B, C: for each impurity, no t more than twice the area o f the principal peak in the chromatogram obtained w ith reference solution (a) ( 0.2 per cent); — impurities D, E: for each impurity, n o t more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent); — unspecified impurities: for each impurity, not more than the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.10 per cent); — total: n o t more than 6 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.6 per cent); — disregard limit. 0.5 times the area o f the principal peak in the chrom atogram obtained with reference solution (a) (0.05 p er cent).

Loss on drying ('2.2.32) M aximum 0.5 per cent, determined on 1.000 g by drying in vacuo for 18 h.

Sulfated ash (2.4.14) Maximum 0.1 per cent, determined on 1.0 g.

ASSAY Dissolve 0.300 g in 50 m L of anhydrous acetic acid R, heating gently if necessary. Titrate with 0.1 M perchloric acid until a bluish-green colour is obtained, using 0.1 m L of crystal violet solution R as indicator.

1 m L of 0.1 M perchloric acid is equivalent to 33.33 mg of C 13H 19N O 9.

D . 4-[(li?)-2-(benzylmethylamino)-l-hydroxyethyl]benzene1, 2-diol,

E. 2-(benzyimethylamino)-l-(3,4-dihydroxyphenyl)ethanone. — ___ ____________________________________________________ PhEtr

Agar

★ ★

★ ★

(Ph. Eur. monograph 0310)

*****

Action and use Excipient. Ph Eur_______________________

DEFINITION Polysaccharides from various species of Rhodophyceae mainly belonging to the genus Gelidium. It is prepared by treating the algae with boiling water; the extract is filtered whilst hot, concentrated and dried.

CHARACTERS Appearance Powder or crumpled strips 2-5 m m wide or sometimes flakes, colourless or pale yellow, translucent, somewhat tough and difficult to break, becoming more britde on drying. Mucilaginous taste.

STORAGE

IDENTIFICATION

In an airtight container, or preferably in a sealed tube under vacuum or u n d er an inert gas, protected from light.

A. Examine under a microscope. W hen m ounted in 0.005 M iodine, the strips or flakes are partly stained brownish-violet. Magnified 100 times, they show the following diagnostic characters: numerous m inute, colourless, ovoid or rounded

2016

1-80 Air

grains on an am orphous background; occasional brown, round or ovoid spores with a reticulated surface, measuring up to 60 nm, may be present. Reduce to a powder, if necessary. T h e pow der is yellowish-white. Examine un d er a microscope using 0.005 M iodine. T h e powder presents angular fragments w ith num erous grains similar to those seen in the strips and flakes; some o f the fragments are stained brownish-violet. B. Dissolve 0.1 g with heating in 50 m L of vocaer R. Cool. T o 1 m L of the mucilage carefully add 3 m L of water R so as to form 2 separate layers. A dd 0.1 m L o f 0.05 M iodine. A dark brownish-violet colour appears at the interface. Mix. T he liquid becomes pale yellow. C. H eat 5 m L o f the mucilage prepared for identification test B on a w ater-bath with 0.5 m L o f hydrochloric acid R for 30 min. A dd 1 m L o f barium chloride solution R l. A white turbidity develops within 30 min. D. H eat 0.5 g with 50 m L o f water R on a water-bath until dissolved. Only a few fragments rem ain insoluble. D uring cooling, the solution gels between 35 °C and 30 °C. H eat the gel thus obtained on a water-bath; it does not liquefy below 80 °C. TESTS Sw elling in d e x (2.8.4) M inim um 10 and within 10 p er cent o f the value stated on the label, determ ined on the pow dered herbal drug (355)

(2.9.12). In so lu b le m a t te r M aximum 1.0 per cent.

solution add 5 m L o f picric acid solution R. N o turbidity appears within 10 min. L o ss o n d ry in g (2.2.32) M axim um 20.0 per cent, determ ined on 1.000 g o f the pow dered herbal drug (355) (2.9.12) by drying in an oven at 105 °C. T o ta l a s h (2.4.16) M axim um 5.0 per c e n t M ic ro b ia l c o n ta m in a tio n T A M C : acceptance criterion 103 C FU /g (2.6.12). T Y M C : acceptance criterion 102 C FU /g (2.6.12). Absence o f Escherichia coli (2.6.13). Absence o f Salmonella (2.6.13). L A B E L L IN G T h e label states th e swelling index. ___________________________________________________________ PhEur

Medical Air

*****

(Medicinal Air\ Ph Eur monograph 1238) When Medical Air is intended for use in a room in which magnetic resonance imaging (MRJ) is being performed, the cylinder and fittings should be made from suitable nonferromagnetic materials and labelled accordingly.

*

PhEur___________________________________________________________

T o 5.00 g o f the powdered herbal drug (355) (2.9.12) add 100 m i. of water R and 14 m L o f dilute hydrochloric add R. Boil gendy for 15 m in with frequent stirring. Filter the hot liquid through a tared, sintered-glass filter (160) (2 . 1 . 2 ), rinse the filter with hot water R and dry at 100-105 °C. T h e residue weighs a maximum o f 50 mg. G elatin T o 1.00 g add 100 m L of water R and heat on a water-bath until dissolved. Allow to cool to 50 °C. T o 5 m L o f this

D E F IN IT IO N Compressed am bient air. C o n te n t 20.4 per cent VIV to 21.4 per cent VIV o f oxygen (O 2). CHARACTERS A p p e a ra n c e Colourless gas. S o lu b ility A t 20 °C at a pressure of 101 kPa, 1 volume dissolves in about 50 volumes of water.

t

Photomultiplier

Figure 1238.-1. - UV fluorescence analyser

t

Amplifier

2016

Air 1-81

PRODUCTION Carbon dioxide

than 0.05 ppm V/V of nitrogen monoxide and nitrogen dioxide.

M axim um 500 p pm V/V, determined using an infrared analyser (2.5.24).

Reference gas (b) U se a mixture o f 2 ppm V/V o f nitrogen monoxide R in nitrogen R l.

Gas to be examined Filter the substance to be examined to avoid stray light phenomena.

Calibrate the apparatus and set the sensitivity using reference gases (a) and (b). Measure the content o f nitrogen monoxide and nitrogen dioxide in the gas to be examined.

Reference gas (a) Use a mixture of 21 per cent V/V o f oxygen R and 79 per cent VIV of nitrogen R l, containing less than 1 ppm V/V o f carbon dioxide R l. Reference gas (b) Use a mixture of 21 per cent VIV of oxygen R and 79 per cent ViV of nitrogen R l, containing 500 ppm VIV o f carbon dioxide R l.

Water

Calibrate the apparatus and set the sensitivity using reference gases (a) and (b). M easure the content of carbon dioxide in the gas to be examined.

Maximum 67 ppm V/V, determined using an electrolytic hygrometer (2.5.28), except where the com petent authority decides that the following limit applies to medicinal air generated on-site and distributed in pipe-line systems operating at a pressure not greater than 10 bars and a tem perature not less than 5 °C: m axim um 870 ppm V/V, determined using an electrolytic hygrometer (2.5.28).

Carbon monoxide

Assay

M axim um 5 p p m VIV, determined using an infrared analyser

Determ ine the concentration of oxygen in air using a paramagnetic analyser (2.5.27).

(2.5.25). Gas to be examined Filter the substance to be examined to avoid stray light phenomena.

Reference gas (a) U se a mixture of 21 per cent V/V o f oxygen R and 79 per cent VIV of nitrogen R l, containing less than 1 ppm VIV o f carbon monoxide R. Reference gas (b) U se a mixture of 21 per cent VIV of oxygen R and 79 per cent VIV of nitrogen R l, containing 5 ppm V/V o f carbon monoxide R. Calibrate the apparatus and set the sensitivity using reference gases (a) and (b). M easure the content of carbon monoxide in the gas to be examined.

Sulfur dioxide M axim um 1 ppm V/V, determined using an ultraviolet fluorescence analyser (Figure 1238.-1). T h e apparatus consists o f the following: — a system generating ultraviolet radiation with a wavelength o f 210 nm , made up o f an ultraviolet lamp, a collimator, and a selective filter; the beam is blocked periodically by a chopper rotating at high speeds; — a reaction chamber, through which flows the gas to be examined; — a system that detects radiation emitted at a wavelength of 350 nm , m ade up o f a selective filter, a photomultiplier tube and an amplifier.

Gas to be examined Filter the substance to be examined. Reference gas (a) U se a mixture of 21 per cent V/V of oxygen R and 79 per cent V/V of nitrogen R l. Reference gas (b) U se a mixture of 21 per cent V/V of oxygen R and 79 per cent VIV of nitrogen R l, containing 0.5 ppm VIV to 2 ppm V/V of sulfur dioxide R l. Calibrate the apparatus and set the sensitivity using reference gases (a) and (b). M easure the content of sulfur dioxide in the gas to be examined

Oil M axim um 0.1 m g/m 3, determined using an oil detector tube (2. 1 . 6 ), when an oil-lubricated compressor is used for the production.

Nitrogen m onoxide and nitrogen dioxide M axim um 2 p pm V/V in total, determined using a chemiluminescence analyser (2.5.26). Gas to be examined T h e substance to be examined. Reference gas (a) U se a mixture of 21 per cent V/V of oxygen R and 79 per cent V/V o f nitrogen R l, containing less

Figure 1238.-2. - Gas burette

2016

1-82 Air

ID E N T IF IC A T IO N

First identification C. Second identification A , B. A. In a conical flask containing the substance to be examined, place a glowing wood splinter. T he splinter rem ains glowing. B. U se a gas burette (Figure 1238.-2) o f 25 m L capacity in the form of a cham ber in the middle o f which is a tube graduated in 0.2 per cent between 19.0 per cent and 23.0 per cent, and isolated at each end by a tap with a conical barrel. T he lower tap is joined to a tube with an olive-shaped nozzle and is used to introduce the gas into the apparatus. A cylindrical funnel above the upper tap is used to introduce die absorbent solution. W ash the burette with water R and dry. O pen the 2 taps. C onnect the nozzle to the source of the gas to be examined and set the flow rate to 1 L/m in. Flush the burette by passing the gas to be examined through it for 1 min. Close the lower tap of the burette and immediately afterwards the upper tap. Rapidly disconnect the burette from the source o f the gas to be examined. Rapidly give a half turn to the upper tap to eliminate any excess pressure in the burette. Keeping the burette vertical, fill the funnel with a freshly prepared mixture of 21 m L of a 560 g/L solution of potassium hydroxide R and 130 m L o f a 200 g/L Irolution o f sodium dithionite R. O pen the upper tap slowly. T he solution absorbs the oxygen and enters the burette. Allow to stand for 10 min w ithout shaking. Read the level of the liquid meniscus on the graduated part of the burette. This figure represents the percentage V/V of oxygen. T he value read is 20.4 to 21.4. C. It complies with the limits o f the assay. TESTS C a rb o n dioxide M axim um 500 ppm V/V, determ ined using a carbon dioxide detector tube (2. 1 . 6 ). S u lfu r dioxide M aximum 1 ppm V/V, determ ined using a sulfur dioxide detector tube (2. 1 . 6). O il M axim um 0.1 mg/m3, determ ined using an oil detector tube (2 . 1 . 6 ), when an oil-lubricated compressor is used for the production. N itro g e n m o n o x id e a n d n itro g e n diox id e M axim um 2 ppm V/V, determ ined using a nitrogen monoxide and nitrogen dioxide detector tube (2 . 1 . 6 ). C a rb o n m o n o x id e M axim um 5 ppm V/V, determ ined using a carbon monoxide detector tube (2 . 1 . 6 ). W a te r'v a p o u r M axim um 67 ppm V/V, determ ined using a water vapour detector tube (2 . 1 . 6 ), except where the competent authority decides that the following limit applies to medicinal air generated on-site and distributed in pipe-line systems operating at a pressure not greater than 10 bars and a tem perature n o t less than 5 °C: maximum 870 ppm V/V, determined using a w ater vapour detector tube (2 . 1 . 6 ). STORAGE As a gas, in suitable containers complying with the legal regulations or as a gas supplied by a pipe network. L A B E L L IN G W here applicable, the label states the production m ethod, as regards to the use o f an oil - lubricated compression.

IM P U R IT IE S A. C O 2: carbon dioxide, B. S 0 2: sulfur dioxide, C. N O : nitrogen monoxide, D . N 0 2: nitrogen dioxide, E. oil, F. CO: carbon monoxide, G . H 20 : water. Ph Eur

Synthetic Air

★ ★ ★ ★ *★ ★*

(Synthetic Medicinal Air, Ph Eur monograph 1684) When Synthetic Air is intended for use in a room in which magnetic resonance imaging (MBI) is being performed, the cylinder and fittings should be made from suitable non­ ferromagnetic materials and labelled accordingly.

*

P h E u r ___________________________________________________________________________________________

D E F IN IT IO N M ixture of Nitrogen (1247) an d Oxygen (0417). C o n te n t 95.0 per cent to 105.0 per cent o f the nominal value which is between 21.0 per cent V/V to 22.5 per cent V/V o f oxygen

(Oa). CHARACTERS Colourless and odourless gas. S o lu b ility A t a tem perature of 20 °C and a pressure o f 101 kPa, 1 volume dissolves in about 50 volumes o f water. P R O D U C T IO N W a te r (2.5.28) M axim um 67 ppm V/V. A ssay (2.5.27) Carry out the determination o f oxygen in gases. ID E N T IF IC A T IO N

First identification C Second identification A, B A. In a conical flask containing the substance to be examined, place a glowing splinter o f wood. T h e splinter remains glowing. B. U se a gas burette (Figure 1684.-1) o f 25 m L capacity in the form o f a chamber, in the middle o f which is a tube graduated in 0.2 per cent between 19.0 per cent and 23.0 per cent, and isolated at each end by a tap with a conical barrel. T h e lower tap is joined to a tube with an olive-shaped nozzle and is used to introduce the gas into the apparatus. A cylindrical funnel above the upper tap is used to introduce the absorbent solution. W ash the burette with water R and dry. O pen both taps. C onnect the nozzle to the source of the substance to be examined and set the flow rate to 1 L/min. Flush the burette by passing the substance to be examined through it for 1 m in. Close the lower tap o f the burette and immediately afterwards the upper tap. Rapidly disconnect the burette from the source of the substance to be examined. Rapidly give a h alf tu rn o f the upper tap to eliminate any excess pressure in the burette. Keeping the b urette vertical, fill the funnel with a freshly prepared mixture of 2 1 m L o f a 560 g/L solution o f potassium hydroxide R and 130 m L o f a 200 g/L solution o f sodium dithionite R. O pen the upper tap slowly. T he solution absorbs the oxygen and

Alanine 1-83

2016

STORAGE As a compressed gas in suitable containers complying w ith the legal regulations or as a compressed gas supplied by a pipe network, after mixing of the components. L A B E L L IN G T he label states the nominal content of 0 2 in per cent VIV.

IMPURITIES A. H 20 : water. __________________________________________________________ PhEur

Alanine

★★*★★ ★ ★

(Ph Eitr monograph 0752)

***** H

NH,

V HjC

C3H7N 0 2

COjH

89.1

5 6 -4 1 -7

A ctio n a n d u se Amino ad d . PhEur

D E F IN IT IO N (2S)-2-Aminopropanoic add. C o n te n t 98.5 per cent to 101.0 per cent (dried substance). CHARACTERS A p p e a ra n c e W hite or almost white, crystalline powder or colourless crystals. S o lu b ility F red y soluble in water, very slightly soluble in ethanol (96 per cent). ID E N T IF IC A T IO N

First identification A , B Second identification A , C, D A. Specific optical rotation (see Tests). B. Infrared absorption spectrophotometry (2.2.24).

Comparison alanine CRS. C. Thin-layer chromatography (2.2.27).

Test solution Dissolve 10 mg of the substance to be examined in water R and dilute to 50 m L w ith the same solvent. Reference solution. Dissolve 10 mg of alanine CRS in water R and dilute to 50 m L with the same solvent. Figure 1684.-1,- Gas burette

enters the burette. Allow to stand for 10 min without shaking. Read the level of the liquid meniscus on the graduated part o f the burette. T his figure represents the percentage VIV o f oxygen. T h e value read is 95.0 per cent to 105.0 p er cent o f the nominal value. C. It complies w ith the limits of the assay. TESTS W a te r v a p o u r M axim um 67 p p m VIV, determined using a water vapour detector tube (2 . 1 . 6 ).

Plate TLC silica gel plate R. Mobile phase glacial acetic add R, water R, butanol R (20:20:60 V/V/V). Application 5 |xL. Development Over 2/3 of the plate. Drying In air. Detection Spray with ninhydrin solution R and heat at 105 °C for 15 min.

Results T h e p rindpal spot in the chromatogram obtained with the T est solution is similar in position, colour and size to the prindpal spot in the chromatogram obtained with the reference solution.

2016

1-84 Alanine

D . Dissolve 0.5 g in a mixture o f 0.25 m L o f hydrochloric add R l, 0.5 m L of a 100 g/L solution of sodium nitrite R and 1 m L of water R. Shake; gas is given off, Add 2 m L o f dilute sodium hydroxide solution R, followed by 0.25 m L o f iodinaxed potassium iodide solution R. After about 30 min, a yellow precipitate is formed. TESTS S o lu tio n S Dissolve 2.5 g in distilled water R and dilute to 50 m L with the same solvent. A p p e a ra n c e o f so lu tio n T he solution is clear ('2.2.1) and not more intensely coloured than reference solution BY 6 (2.2.2, Method II). Dilute 10 m L of solution S to 20 m L with water R. S pecific o p tic a l r o ta tio n (2.2.7) + 13.5 to + 15.5 (dried substance). Dissolve 2.50 g in hydrochloric add R l and dilute to 25.0 m L with the same acid. N in h y d rin -p o sitiv e su b sta n c e s Amino a d d analysis (2.2.56). F or analysis, use M ethod 1. T he concentrations o f the test solution and the reference solutions may be adapted according to the sensitivity of the equipm ent used. T h e concentrations o f all solutions are adjusted so that the system suitability requirements described in general chapter 2.2.46 are fulfilled, keeping the ratios of concentrations between all solutions as described.

Solution A dilute hydrochloric acid R l or a sample preparation buffer suitable for the apparatus used. Test solution Dissolve 30.0 m g o f the substance to be examined in solution A and dilute to 50.0 m L with solution A. Reference solution (a) D ilute 1.0 m L of the test solution to 100.0 m L with solution A Dilute 2.0 m L of this solution to 10.0 m L with solution A.

Reference solution (b) Dissolve 30.0 mg of proUne R in solution A and dilute to 100.0 m L with solution A. Dilute 1.0 m L of the solution to 250.0 m L with solution A.

Reference solution (c) Dilute 6.0 m L o f ammonium standard solution (100 ppm NH 4 ) R to 50.0 m L with solution A. D ilute 1.0 m L of this solution to 100.0 m L with solution A. Reference solution (d) Dissolve 30 m g o f isoleucine R and 30 m g of leudne R in solution A and dilute to 50.0 m L with solution A. Dilute 1.0 m L o f the solution to 200.0 m L with solution A.

Blank solution Solution A. Inject suitable, equal am ounts of the test, blank and reference solutions into the amino a d d analyser. Run a program suitable for the determination of physiological amino adds. System suitability Reference solution (d): — resolution: minimum 1.5 between the peaks due to isoleucine and leucine.

Calculation of percentage contents: — for any ninhydrin-positive substance detected at 570 nm , use the concentration of alanine in reference solution (a); — for any ninhydrin-positive substance detected at 440 nm , use the concentration o f proline in reference solution (b); if a peak is above the reporting threshold at both wavelengths, use the result obtained at 570 nm for quantification.

Limits: — any ninhydrin-positive substance: for each impurity, maximum 0.10 p er cent;

— total: maximum 0.5 per cent; — reporting threshold: 0.05 per cent. C h lo rid e s (2.4.4) M aximum 200 ppm. Dilute 5 m L of solution S to 15 m L with water R. S u lfates (2.4.13) M aximum 300 ppm. Dilute 10 m L of solution S to 15 m L with dxstHled water R. A m m onium

Amino a d d analysis (2.2.56) as described in the test for ninhydrin-positive substances with the following modifications.

Injection T est solution, reference solution (c) and blank solution.

Limit: — ammonium at 570 ran: not more than the area of the corresponding peak in the chrom atogram obtained with reference solution (c) (0.02 per cent), taking into account the peak due to ammonium in the chrom atogram obtained with the blank solution. Ir o n (2.4.9) M aximum 10 ppm. In a separating funnd, dissolve 1.0 g in 10 m L of dilute hydrochloric add R. Shake with 3 quantities, each of 10 mL, of methyl isobutyl ketone R l, shaking for 3 m in each time. T o the combined organic layers add 10 m L of water R and shake for 3 min. Use the aqueous layer. H eav y m e ta ls (2.4.8) M aximum 10 ppm. Dissolve 2.0 g in water R and dilute to 20 m L with the same solvent. 12 m L of the solution complies with test A. Prepare the reference solution using lead standard solution

(1 ppm Pb) R. L oss o n d ry in g (2.2.32) Maximum 0.5 per cent, determined on 1.000 g by drying in an oven at 105 °C. S u lfa te d a s h (2.4.14) Maximum 0.1 per cent, determined on 1.0 g. A SSAY Dissolve 80.0 mg in 3 mL o f anhydrous formic add R. Add 30 m l. of anhydrous acetic add R. T itrate with 0.1 M perchloric add, determining the end-point potentiometrically (2.2.20). 1 m L of 0.1 M perchloric acid is equivalent to 8.91 m g of c 3h 7n o 2. STO RA G E Protected from light. IM P U R IT IE S

Other detectable impurities (the following substances would, if present at a suffident levd, be detected by one or other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore no t necessary to identify these impurities for demonstration of compliance. See also 5.10. Control of impurities in substances for pharmaceutical use): A , B. H HO!° ' ~ X

nh 2 c0 !h

A. (25)-2-aminobutanedioic acid (aspartic ad d ),

Albendazole 1-85

2016 Column: h o 2c '

B. (25)-2-am inopentanedioic ad d (glutamic ad d ). PhEur

Albendazole

★ ★

★ ★

(Ph Eur monograph 1386)

***** ch 3

-NH

C 12H 15N 3O 2S

265.3

54965-21-8

A ctio n a n d u se Benzimidazole antihelminthic. P re p a r a tio n s Albendazole Oral Suspension Albendazole Oral Suspension with Minerals PhEir.

D E F IN IT IO N M ethyl [5-(propylsulfanyl)-1 /f-benzimidazol-2 -yl] carbamate. C o n te n t 98.0 per cent to 102.0 per cent (dried substance). CHARACTERS A p p e a ra n c e W hite or slightly yellowish powder. S o lu b ility Practically insoluble in water, freely soluble in anhydrous formic a d d , very slightly soluble in methylene chloride, practically insoluble in ethanol (96 p er cent). ID E N T IF IC A T IO N Infrared absorption spectrophotometry (2.2.24).

Preparation Discs. Comparison albendazole CRS. TESTS A p p e a ra n c e o f so lu tio n T he solution is clear (2.2.1) and not m ore intensely coloured than reference solution BY 6 (2.2.2, Method II). Dissolve 0.10 g in a mixture of 1 volume o f anhydrous formic add R and 9 volumes o f methylene chloride R and dilute to 10 m L w ith the same mixture of solvents. R e la te d s u b s ta n c e s liq u id chrom atography (2.2.29).

Test solution Dissolve 25.0 m g o f die substance to be examined in 5 m L o f methanol R containing 1 per cent VIV o f sulfuric add R and dilute to 50.0 m L with the mobile phase.

— size. I = 0.25 m , 0 = 4.6 mm; — stationary phase’, spherical end-capped octadecylsUyl silica gel for chromatography R (5 pm) with a pore size of 10 nm and a carbon loading o f 19 p er cent.

Mobile phase Mix 300 volumes of a 1.67 g/L solution of ammonium dihydrogen phosphate R and 700 volumes of methanol R. Flow rate 0.7 mL/min. Detection Spectrophotom eter at 254 nm. Injection 20 |iL. Run time 1.5 times the retention time of albendazole. Relative retention W ith reference to albendazole: impurity D = about 0.40; impurities B and C = about 0.43; impurity E = about 0.47; impurity F = about 0.57; impurity A = about 0.80. System suitability", reference solution (b): — resolution', m inim um 3.0 between the peaks due to albendazole and oxibendazole.

Limits: — impurities A, B, C, D, E, F: for each impurity, not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.75 p er cent); — total: n o t more than 3 times the area o f the prindpal peak in the chrom atogram obtained with reference solution (a) (1.5 p er cent); — disregard limit: 0.1 times the area of the prindpal peak in the chromatogram obtained with reference solution (a) (0.05 p er cent).

Loss on drying (2.2.32) M aximum 0.5 p er cent, determined on 1.000 g by drying in an oven at 105 °C for 4 h.

Sulfated ash (2.4.14) M aximum 0.2 p er cent, determ ined on 1.0 g.

ASSAY In order to avoid overheating during the titration, mix thoroughly throughout and stop the titration immediately after the end-point has been reached. Dissolve 0.250 g in 3 m L of anhydrous formic acid R and add 40 m L o f anhydrous acetic add R. Titrate with 0.1 M perchloric add, determining the end-point potentiometrically (2.2.20). 1 m L of 0.1 M perchloric add is equivalent to 26.53 mg Ci2Hj5N302S.

Of

STORAGE Protected from light.

IMPURITIES Specified impurities A, B, C , D , E, F.

-NH,

Reference solution (a) Dissolve 10.0 m g o f the substance to be examined in 10 m L of methanol R containing 1 p er cent VIV o f sulfuric acid R and dilute to 100.0 m L with the mobile

A. R = S-CH2-CH2-CH3: 5-(propylsulfanyl)-IH benzimidazol- 2-am ine,

phase. D ilute 0.5 m L of this solution to 20.0 m L with the mobile phase.

D . R = SO 2-C H 2-C H 2-C H 3: 5-(propyisulfonyI)-liibenzimidazol- 2-amine,

Reference solution (b) Dissolve 50.0 m g o f the substance to be examined and 50 m g o f oxibendazole CRS in 5 m L o f methanol R c o n taining l per cent VIV o f sulfuric add R and dilute to 100.0 m L with the mobile phase.

1-86 Alcuronium Chloride O

1

2016 CH,

y -o '

> -N H

B. R = SO -C H 2-C H 2-C H 3: methyi [5-(propylsuLfinyl)~ÎHbenzimidazol-2-yl]carfaamate, C. R = SO 2-C H 2-C H 2-C H 3: methyl [5-(propylsulfonyl)-1Hbenzimida 7ol- 2-yl] carbamate, E. R = H: methyl ( l.ff-benzimidazol-2-yl) carbamate, F. R = S-C H 3: methyl [5-(methylsulfanyl)-1/i-benziinidazol2-yl] carbamate. __________________________________________________________ PhEur

Reference solution Dissolve 10 m g o f alcuronium chloride CRS in methanol R and dilute to 10 m l. with the same solvent. Plate TLC silica gel plate R. Mobile phase Mix 15 volumes o f a 58.4 g/L solution of sodium chloride R, 35 volumes of dilute ammonia R2 and 50 volumes of methanol R. Application 10 |iL. Development Over a path of 15 cm. Drying In air for 10 min. Detection Spray with 0.1 M ammonium and cerium nitrate. Results T he principal spot in the chrom atogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. C. It gives reaction (a) of chlorides (2.3.1).

★*★ ★ ★ ★ ★ *****

Alcuronium Chloride (Ph Eur monograph 1285)

TESTS S o lu tio n S Dissolve 0.250 g in carbon dioxide-free water R and dilute to 25.0 m L with the same solvent. A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and n o t more intensely coloured than reference solution Y6, BY 6 or B 6 (2.2.2, Method I).

2 Cl

C44H 50C I2N 4O2

738

15180-03-7

A ctio n a n d u se Non-depolarizing neuromuscular blocker. PhEur______________________________________

D E F IN IT IO N (lfl,3aS,10S,llaS,12J?,14aS,19aS,20bS,21S,22aS,23.E,26£)23,26-bis(2-Hydroxyethylidene)-l, 12-bis(prop-2-enyl)2,3,11,1 la , 13,14,22,22a-octahydro-10tf,21/f-l, 21:10,12diethano-19aH ,20b/f- [1,5]diazocino [ 1,2,3-Zm: 5,6,7l'm '] dipyrrolo[2',3 '-d:2'',3":d ']dicaibazolediium dichloride (4 ,4 /-didesmethyl-4,4/-bis(prop-2-enyl)toxiferm I dichloride). C o n te n t 98.0 per cent to 102.0 per cent (anhydrous substance). CHARACTERS A p p e a ra n c e W hite or slightly greyish-white, crystalline powder. S olubility Freely soluble in water and in methanol, soluble in ethanol (96 per cent), practically insoluble in cyclohexane.

Carry out the identification, tests and assay as rapidly as possible avoiding exposure to actinic light

A cid ity o r a lk alin ity T o 10 m L of solution S add 0.1 m L of methyl red solution R and 0.2 m L of 0.01 M hydrochloric add T he solution is red. Add 0.4 m L of 0.01 M sodium hydroxide. T he solution is yellow. Specific o p tic a l ro ta tio n (2.2.7) —430 to —451 (anhydrous substance), determined on solution S. P ro p a n -2 -o l (2.4.24, System A) Maximum 1.0 per cent. R e la te d su b s ta n c e s Liquid chromatography (2.2.29).

Solvent mixture M ix 100 m L of methanol R, 200 m L o f acetonitrile R and 200 m L of a 6.82 g/L solution of potassium dihydrogen phosphate R. Dissolve 1.09 g o f sodium laurylsulfonate for chromatography R in the mixture and adjust the apparent p H to 8.0 with a 100 g/L solution of sodium hydroxide R Test solution Dissolve 0.20 g o f the substance to be examined in the solvent mixture and dilute to 100.0 m L with the solvent mixture.

Reference solution (a) Dilute 0.5 m L o f the test solution to 100.0 m L with the solvent mixture.

Reference solution (b) Dilute 4.0 m l, of reference solution (a) to 10.0 m L with the solvent mixture.

Reference solution (c) Dilute 1.0 m L o f reference solution (a) to 10.0 m L with the solvent mixture.

Reference solution (d) T o 5.0 m L o f the test solution add 5.0 mg of aUylstrychnine bromide CRS, dissolve in the solvent mixture and dilute to 100.0 m L with the solvent mixture.

ID E N T IF IC A T IO N

Column:

First identification A , C Second identification B, C

— size. I = 0.25 m, 0 = 4 mm; — stationary phase: octylsQyl silica gel for chromatography R (5 jim).

A. Infrared absorption spectrophotometry (2.2.24).

Comparison alcuronium chloride CRS. B. Thin-layer chromatography (2.2.27). Test solution Dissolve 10 m g o f the substance to be exam ined in methanol R and dilute to 10 m L with the same solvent.

Mobile phase Mix 200 m L o f methanol R, 400 m L of acetonitrile R and 400 m L o f a 6.82 g/L solution o f potassium dihydrogen phosphate R Dissolve 2.18 g o f sodium laurylsulfonate for chromatography R in the mixture and adjust

Alfacalcidol 1-87

2016 the apparent p H to 5.4 with a 100 g/L solution oiphosphoric

add R. Flow rate 1.2 mL/min. Detection Spectrophotom eter at 254 nm. Irgecdon 10 jxL~ Run time Twice the retention time o f alcuronium. System suitability Reference solution (d): — resolution: m inim um 4.0 between the peaks due to N ’-allylstrychnine and alcuronium.

Limits: — impurities A , B: for each impurity, not m ore than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent) and not m ore than one of the peaks has an area greater than the area of the principal peak in the chromatogram obtained with reference solution (b) ( 0.2 per cent); — total: not m ore than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (1 per cent); — disregard limit: th e area o f the principal peak in the chromatogram obtained w ith reference solution (c) (0.05 per cent).

ch2 ci'

B. (4bS, 1R,1 aS, 8 ai?, 13i?, 13ai?, 13 bS )-l 3-hydroxy-7-(prop-2enyI)-5,6,7 a, 8, 8a, 11,13,13a, 13b, 14-decahydro-7,9-methano7H-oxepino [3,4-a] pyrrolo [2,3-d] carbazolium chloride ((4 R, 17i?)-4-allyl-l 7,18-epoxy-17 -hydroxy-19,2 0didehydrocuranium chloride).

★ ★ ★ ★ ★ *****

Alfacalcidol (Ph Eur monograph 1286)

W a te r (2.5.12) Maximum 5.0 p er cent, determined on 0.500 g. S u lfa te d ash (2.4.14) M aximum 0.1 p er cent, determ ined on 1.0 g. A SSA Y Dissolve 0.300 g by stirring in 70 m L of acetic anhydride R for 1 min. T itrate with 0.1 M perchloric add until the colour changes from violet-blue to greenish-blue, using 0.1 m L of crystal violet solution R as indicator. 1 m L of 0.1 M perchloric add is equivalent to 36.9 m g Of C44H5()Cl2N402. STORA GE In an airtight container under nitrogen, protected from light, at a tem perature o f 2 °C to 8 °C. IM P U R IT IE S

Specified impurities A, B

C 27H 44O 2

400.6

41294-56-8

A ctio n a n d u se Vitamin D analogue. Ph Eur__________________________________________________________

D E F IN IT IO N (5Z ,7£)-9,10-Secocholesta-5,7,10(19)-triene-l 0,3 P-diol. C o n te n t 97.0 per cent to 102.0 per cent. A reversible isomérisation to pre-alfacalcidol takes place in solution, depending on temperature and time. T he activity is due to b oth compounds (see Assay). CHARACTERS A p p e a ra n c e W hite or almost white crystals.

A. (l£,3aS,9Æ,9a£,10Æ,llaS,12Æ,14aS,19aS,20Æ,20aÆ, 20bS,21i?,22aS)-l, 12-bis(prop-2-enyl)2,3,9a,11,1 la,13,14,19a,20a,21,22,22a-dodecahydro10tf,2 0 b H -l,2 3 :12,27-dim ethano-9,10:20,21bis (epoxyprop [2] eno)-9H,2 OH- [1,5] diazocino [ 1,2,3-lm: 5,6,7l'm '] dipyirolo[2 ',3 '-dr.2 " ,3 " :d']dicarbazolediium dichloride (4,4 '-diallylcaracurin V dichloride),

S o lu b ility Practically insoluble in water, freely soluble in ethanol (96 per cent), soluble in fatty oils. It is sensitive to air, heat and light. ID E N T IF IC A T IO N A. Infrared absorption spectrophotometry (2.2.24).

Comparison Ph. Eur. reference spectrum of aÿacalddol. B. Examine the chromatograms obtained in the test for related substances.

Results T he principal peak in the chromatogram obtained with the test solution is similar in retention time and size to the principal peak in the chromatogram obtained with reference solution (a).

1-88 Alfacalcidol

2016

TESTS Related substances

T h e contents of an opened container are to be used immediately.

Liquid chromatography (2.2.29): use the normalisation procedure. Cany out the test as rapidfy as possible, avoiding

IMPURITIES Specified impurities A, B. Other detectable impurities (die following substances would, if

exposure to light and air. Test solution Dissolve 1.0 m g o f the substance to be examined without heating in 10.0 m L o f the mobile phase.

Reference solution (a) Dissolve 1.0 mg o f alfacalcidol CRS without heating in 10.0 m L o f the mobile phase.

Reference solution (b) Dilute 1.0 m L of reference solution (a) to 100.0 m L w ith the mobile phase. Dilute 1.0 m L o f this solution to 20.0 m L with the mobile phase.

present at a suffident levd, be detected by one or other o f the tests in the monograph. T hey are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these impurities for demonstration o f compliance. See also 5.10.

Control of impurities m substances for pharmaceutical use): C.

Reference solution (c) In order to prepare pre-alfacalddol in situ, dissolve the contents o f a vial o f alfacalcidolfor system suitability CRS (containing impurities A and B) in 25 m L of the mobile phase, heat in a water-bath at 80 °C under a reflux condenser for 2 h and cool.

Column: — sizer. I = 0.25 m , 0 = 4.6 mm; — stationary phase: end-capped octadecylsUyl silica gel for chromatography R (5 |im).

Mobile phase ammonia R, water R, acetomtrUe R (1:200:800 V/V/V).^ Flow rate 2.6 mL/min. Detection Spectrophotom eter at 265 nm . Injection 100 (iL o f the test solution and reference

A. (51%7E)-9,10-secocholesta-5,7,10(19)-triene-1a,3{$-diol (zrans-alfacalddol),

solutions (b) and (c).

Run time Twice the retention time o f alfacaladol. Identification of impurities Use the chromatogram supplied with alfacalcidol for system suitability CRS and the chromatogram obtained with reference solution (c) to identify the peaks due to impurities A and B.

Relative retention W ith reference to alfacaladol (retention time = about 21 min): pre-alfacalddol = about 0 .88; impurity A = about 0.93; impurity B = about 1.1. System suitability: reference solution (c): — resolution: m inim um 1.5 between the peaks due to prealfacalddol and impurity A and minimum l .5 between the peaks due to im purity A and alfacaladol.

B. (5Z ,7£)-9,10-secocholesta-5,7,10(19)-triene-l P,3fi-diol ( 10-calddol),

Limitsr. — impurities A , B: for each impurity, maximum 0.5 p er cent; — unspecified impurities: for each impurity, maximum 0.10 per cent; — total: maximum 1.0 per cent; — disregard limit: the area o f the prindpal peak in the chromatogram obtained with reference solution (b) (0.05 per cent); disregard the peak due to pre-alfacalddol.

ASSAY Liquid chromatography (2.2.29) as described in the test for related substances w ith the following modifications.

Injection T est solution and reference solutions (a) and (c). System suitability: reference solution (c): — repeatability: maximum relative standard deviation o f 1 per cent for the peak due to alfacaladol after 6 injections. Calculate the percentage content of C 27H 44O 2 taking into account the assigned content o f alfacaladol CRS and, if necessary, the peak due to pre-alfacalddol.

STORAGE U nder nitrogen, in an airtight container, protected from light, at a tem perature of 2 °C to 8 °C.

C. 6^-[(3S,5i?)-3,5-dihydroxy-2-methylcydohex-l-en-l-yl]-2phenyl-2,5,10-triaza-4,19-dinor-9^-cholest-7-ene-1,3-dione. P b E tr

Alfadex 1-89

2016

Alfadex

f * \

temperature. Add 10 m L of ammonium molybdate reagent R1 and allow to stand for 15 min.

A lphacydodextrin

*★ ★* *

Reference solution Prepare a reference solution at the same time and in the same manner as the test solution, using 1 m L of a 0.02 g/L solution o f glucose R.

(Ph. Eur. monograph 1487)

Measure the absorbance (2.2.25) o f the test solution and the reference solution at the absorption maximum at 740 nm using water R as the compensation liquid. T he absorbance of the test solution is not greater than that of the reference solution.

Light-absorbing impurities Examine solution S between 230 nm and 750 nm . Between 230 nm and 350 nm, the absorbance (2.2.25) is not greater than 0.10. Between 350 nm and 750 nm, the absorbance (2.2.25) is n o t greater than 0.05.

Related substances Liquid chromatography (2.2.29). [C6H 10O5]6

973

10016-20-3

A ctio n a n d u se Cyclodextran; carrier molecule for drug delivery systems. PhEur__________________________________________________________

D E F IN IT IO N Cyclohexakis-(l -> 4)- (a-D-glucopyranosyl) (cyclom altohexaose

Test solution (a) Dissolve 0.25 g o f the substance to be examined in water R with heating, cool and dilute to 25.0 m L with the same solvent. Test solution (b) Dilute 5.0 m L o f test solution (a) to 50.0 m L with water R. Reference solution (a) Dissolve 25.0 m g of betadex CRS (impurity A), 25.0 mg of gammacydodextrin CRS (impurity B) and 50.0 mg of alfadex CRS in water R, then

o r a-cyclodextrin).

dilute to 50.0 m L with the same solvent.

C o n te n t 97.0 per cent to 102.0 per cent (dried substance).

Reference solution (b) Dilute 5.0 m L o f reference solution (a) to 50.0 m L w ith water R. Reference solution (c) Dissolve 25.0 m g of alfadex CRS in water R and dilute to 25.0 m L with the same solvent.

CHA RACTERS A p p e a ra n c e White or almost white, amorphous or crystalline powder. S o lu b ility Freely soluble in water, slightly soluble in propylene glycol, practically insoluble in anhydrous ethanol and in methylene chloride. ID E N T IF IC A T IO N A. Specific optical rotation (see Tests). B. Examine the chromatograms obtained in the assay.

Results T he principal peak in the chromatogram obtained with test solution (b) is similar in retention time and size to the principal peak in the chromatogram obtained with reference solution (c). C. Dissolve 0.2 g in 2 m L of iodine solution R4 by warming on a water-bath, and allow to stand at room temperature; a yellowish-brown precipitate is formed. TESTS S o lu tio n S Dissolve 1.000 g in carbon dioxide-free water R and dilute to 100.0 m L with the same solvent A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1). p H (2.2.3) 5.0 to 8.0. Mix 1 m L o f a 223.6 g/L solution o f potassium chloride R and 30 m L o f solution S. S p ecific o p tic a l ro ta tio n (2.2.7) + 147 to + 152 (dried substance), determined on solution S. R e d u c in g s u g a rs M aximum 0.2 p e r cent.

Test solution T o 1 m L o f solution S add 1 m L o f cupri-tartaric solution R4. H eat on a water-bath for 10 min, cool to room

Column: — sizer. I = 0.25 m, 0 = 4.6 mm; — stationary phase:, octadecylsUyl silica gel for chromatography R (10 |im).

Mobile phase methanol R, water R (10:90 VIV). Flow rate 1.5 mL/min. Detection Differential refractometer. Equilibration W ith the mobile phase for about 3 h. Injection 50 |JL of test solution (a) and reference solutions (a) and (b).

Run time 3.5 times the retention time of alfadex. Relative retention W ith reference to alfadex (retention time = about 10 min): impurity B = about 0.7; impurity A = about 2.2. System suitability: reference solution (a): — resolution: minimum 1.5 between the peaks due to impurity B and alfadex; if necessary, adjust the concentration of methanol in the mobile phase.

Limits: — impurities A , B: for each impurity, not more than 0.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (b) (0.25 per cent); — sum of impurities other than A and B: not more than 0.5 times the area of the peak due to alfadex in the chromatogram obtained with reference solution (b) (0.5 per cent).

Heavy metals (2.4.8) Maximum 10 ppm. 2.0 g complies with test C. Prepare the reference solution using 2 m L o f lead standard solution (10 ppm Pb) R.

1-90 Alfentanil Hydrochloride

2016

L oss o n d ry in g (2.2.32) M axim um 11 per cent, determ ined on 1.000 g by drying in an oven at 120 °C for 2 h.

★ .★

Alfentanil Hydrochloride

★ ★

*****

(Ph. Eur. monograph 1062)

S u lfa te d a s h (2.4.14) M axim um 0.1 per cent, determ ined on 1.0 g. n « nv

A SSA Y Liquid chromatography (2.2.29) as described in the test for related substances with the following modifications.

Injection T est solution (b) and reference solutions (a) and (c). System suitability: — repeatability: maximum relative standard deviation o f 2.0 per cent for the peak due to alfadex after 5 injections o f reference solution (a). Calculate the percentage content o f [ O H i o C ^ from the assigned content of alfadex CRS.

J.

ch3

N -V HCI

ch3

C21H33CIN6O3

453.0

69049-06-5

A c tio n a n d u se Opioid receptor agonist; analgesic. PhEur_______________________________

STO R A G E In an airtight container.

D E F IN IT IO N N -[l- [2-(4-Ethyl-4,5-dihydro-5-oxo-1H -tetrazol-1-yl) ethyl] -4(methoxymethyl)piperidin-4-yl]-N-phenylpropanamide hydrochloride.

IM P U R IT IE S

Specified impurities A, B OH

C o n te n t 98.5 per cent to 101.5 per cent (anhydrous substance).

HO

CHARACTERS A p p e a ra n c e W hite or alm ost white powder. S o lu b ility F red y soluble in water, in ethanol (96 p er cent) and in methanol. mp: about 140 °C, with decomposition. ID E N T IF IC A T IO N A. Infrared absorption spectrophotom etry (2.2.24).

A. cycloheptakis-(l-»4)-(a-D-glucopyranosyl) (betadex or

cyclomaltoheptaose or ß-cyclodextrin),

Comparison Ph. Eur. reference spectrum of alfentanil hydrochloride. B. Dissolve 50 mg in a mixture of 0.4 m L o f ammonia R and 2 m L of water R. Mix, allow to stand for 5 min and filter. A ddify the filtrate with dilute nitric add R. It gives reaction (a) of chlorides (2.3.1). TESTS A p p e a ra n c e o f so lu tio n T he solution is clear (2.2.1) and colourless (2.2.2,

Method IT). Dissolve 0.2 g in water R and dilute to 20 m L with the same solvent. R e la te d su b s ta n c e s Liquid chrom atography (2.2.29).

Test solution Dissolve 0.100 g of the substance to be examined in methanol R and dilute to 10.0 m L with die same solvent. Reference solution (a) In order to produce impurity E in situ,

B. cyclooctakis-(l -*4)-(a-D-glucopyranosyl)

(cydomaltooctaose or y-cydodextrin). PhEur

dissolve 10 mg of the substance to be examined in 10.0 m L o f dilute hydrochloric add R. H eat on a water-bath under a reflux condenser for 4 h. Neutralise with 10.0 m L o f dilute sodium hydroxide solution R. Evaporate to dryness on a waterbath. Cool and take up the residue in 10 m L o f methanol R. Filter.

Reference solution (b) Dilute 1.0 m L o f the test solution to 100.0 m L with methanol R. Dilute 5.0 m L o f this solution to 20.0 m L w ith methanol R. Column: — size. I = 0.1 m , 0 = 4.6 mm;

Alfentanil Hydrochloride 1-91

2016 — stationary phase: octadecybüyl silica gel for chromatography R (3 \im).

h2

Ar- =

R-

Mobile phase. — mobile phase A: 5 g/L solution o f ammonium carbonate R in a mixture o f 10 volumes of tetrahydrqfuran R and 90 volumes o f water R; — mobile phase B: acetonitrüe R] Time (min) 0 -15

Mobile phase A (per cent V/V) 90->40

Mobile phase B (percent V/V) 10 -»60

1 5 -2 0

40

60

2 0 -2 5

40 ->90

60-» 10

I f

V xe”3

o

v

A. cis-N- [ 1- [2-(4-ethyl-4,5-dihydro-5-oxo-1//-tetrazol-1yI)ethyl]-4-(methoxymethyl)piperidin-4-yl]-iVphenylpropanamide N-oxide, r

Flow rate 1.5 mL/min. Detection Spectrophotom eter at 220 nm. Equilibration W ith acetomtrSe R for at least 30 min and then with the mobile phase at the initial composition for at least 5 min.

Irqection 10 jiL; inject methanol R as a blank. Retention time Im purity E = about 6 min; alfentanil = about 7

i^

x î r

o

^C H 3 V e

0

B . trans-N- [ 1- [2-(4-ethyl-4,5-dihydro-5-oxo- lH -tetrazol-1yl)ethyl]-4-(methoxymethyl)piperidin-4-yl]-iVphenylpropanamide N-oxide, Ar / x I f NH

min.

Identification of impurities Use the chromatogram obtained with reference solution (a) to identify the peak due to impurity E; disregard any other peak. System suitability: reference solution (a): — resolution: m in im u m 4.0 between the peaks due to alfentanil and impurity E; if necessary, adjust the concentration o f acetonitrile in the mobile phase or adjust the time programme for the linear-gradient elution.

o -R N

Ar

o

*^0 - CH3

C. N - [4-(methoxymethyl)piperidm-4-yl]-Nphenylpropanamide,

Limits: — impurities A , B, C, D, E, F, G, H: for each impurity, not m ore than the area of the principal peak in the chrom atogram obtained with reference solution (b) (0.25 p er cent); — total: not more than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent); — disregard limit. 0.2 times the area of the principal peak in the chrom atogram obtained with reference solution (b) (0.05 p er cent); disregard any peak due to the blank. W a te r (2.5.12) 3.0 per cent to 4.0 p er cent, determined on 0.500 g. A SSA Y Dissolve 0.350 g in 50 m L of a m ixture o f 1 volume of ethanol (96 per cent) R and 4 volumes o f water R and add 5.0 m L o f 0.01 M hydrochloric add. T itrate with 0.1 M sodium hydroxide, d eterm ining the end-point potentiometrically (2.2.20). Read the volume added between the 2 points of inflexion. 1 m L o f 0.1 M sodium hydroxide is equivalent to 45.30 mg o f

C21H33C 1N 60 3 .

D . N -[l- [2-(4-ethyl-4,5-dihydro-5-oxo-1//-tetrazol-1yl)ethyI]-4-(methoxymethyl)piperidm-4-yl]-Nphenylacetamide,

E. 1-ethyl-1,4-dihydro-4- [2-[ [4-(methoxymethyl)-4phenylamino] piperidin-1-yl] ethyl]-5/f-tetrazol-5-one, Ar / s , 1

hO

I

N

t n^

o

k 0 .CH3

F. N- [ 1-(2-hydroxyethyl)-4-(methoxymethyl)piperidin-4-yl] iV-phenylpropanamide,

STO RA G E Protected from light.

IMPURITIES Specified impurities A, B, C, D , E, F, G, H

G. N -[l- [2-(4-ethyl-4,5-dihydro-5-oxo-1 //-tetrazo l-1yl) ethyl] -4-(propanoyloxymethyl)piperidin-4-yI] -Nphenylpropanamide,

2016

1-92 Alfuzosin Hydrochloride

Reference solution (b) Dissolve 4 m g o f alfuzosin for system suitability CRS (containing impurities A and D ) in the mobile phase and dilute to 10 m L with the mobile phase.

Column: — sizer. I = 0.15 m , 0 = 4.6 mm ; — stationary phase: end-capped octadecylsUyl silica gel for chromatography R (5 Jim).

H . N -[l-[2-(4-ethyl-4j5-dihydro-5-oxo-li/-tetrazol-1yl) ethyl] -4-(methoxymethyl)piperidin-4-yi] -Nphenylbutanamide. PhEur

Alfuzosin Hydrochloride

★ ★

★ ★

(Ph Eur monograph 1287)

*****

HzC0' y ^ Y ' h3c o - ^ Y

C 19H 28Q N 5O 4

obtained with reference solution (b) to identify the peaks due to impurities A and D.

n nh2

and enantiomer

425.9

Mobile phase Mix 1 volume of tetrahydrcfuran R, 20 volumes o f acetonitrile R and 80 volumes o f a solution prepared as follows: dilute 5.0 m L o f perchloric add R in 900 m L o f water R, adjust to p H 3.5 with dilute sodium hydroxide solution R and dilute to 1000 m L with water R. Flow rate 1.5 mL/min. Detection Spectrophotom eter at 254 nm. Injection 10 jiL. Run time Twice the retention tim e o f alfuzosin. Identification of impurities Use the chrom atogram supplied with alfuzosin for system suitability CRS and the chromatogram

Relative retention W ith reference to alfuzosin (retention 81403-68-1

A ctio n a n d u se Alpha ^adrenoceptor antagonist. P re p a r a tio n s Alfuzosin Tablets Prolonged-release Alfuzosin Tablets PhEir ________________________________

D E F IN IT IO N (2RS)-N- [3- [(4-Amino-6j7 -dimethoxyquinazolm- 2-yl) methylaminojpropyl] tetrahydrofuran- 2-carboxamide hydrochloride. C o n te n t 99.0 per cent to 101.0 per cent (anhydrous substance). CHARACTERS A p p e a ra n c e W hite or alm ost white, crystalline powder, slightly hygroscopic. S o lu b ility Freely soluble in water, sparingly soluble in ethanol (96 per cent), practically insoluble in methylene chloride. ID E N T IF IC A T IO N A Infrared absorption spectrophotometry (2.2.24).

Comparison alfuzosin hydrochloride CRS. It gives reaction (a) of chlorides (2.3.1).

B.

TESTS p H (2.2.3) 4.0 to 5.5. Dissolve 0.500 g in carbon dioxide-free water R and dilute to 25.0 m L with the same solvent. Use a freshly prepared solution. R e la te d su b s ta n c e s Liquid chromatography (2.2.29).

Test solution Dissolve 40 mg o f the substance to be examined in the mobile phase and dilute to 100.0 m L with the mobile phase. Reference solution (a) Dilute 1.0 m L o f the test solution to 100.0 m L with the mobile phase. Dilute 1.0 m L o f this solution to 10.0 m L with the mobile phase.

tim e = about 8 min): impurity D = about 0.4; impurity A = about 1.2. System suitability: reference solution (b): — peak-to-valUy ratio: minimum 5.0, where Hp = height above the baseline o f the peak due to im purity A and Hv = height above the baseline o f the lowest point of the curve separating this peak from the peak due to alfuzosin.

Limits: — impurity D: not more than twice the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent); — unspecified impurities: for each impurity, n o t more than the area o f die principal peak in the chromatogram obtained with reference solution (a) ( 0.10 per cent); — total: n o t more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.3 p e r cent); — disregard limit: 0.5 times the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.05 p er cent). W a te r (2.5.12) M axim um 0.5 per cent, determ ined on 1.000 g. S u lfa te d a sh (2.4.14) M axim um 0.1 per cent, determ ined on 1.0 g. A SSA Y Dissolve 0.300 g in a mixture o f 40 m L o f anhydrous acetic add R and 40 m L o f acetic anhydride R T itrate with 0.1 M perchloric add, determining the end-point potentiometrically (2.2.20). 1 m l, o f 0.1 Mperchloric add is equivalent to 42.59 m g o f C 19H 28CIN 5O 4. STO R A G E In an airtight container, protected from light. IM P U R IT IE S

Specified impurities D . Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other of the tests in the monograph. T hey are limited by die general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these

Alginic Acid 1-93

2016 impurities for dem onstration o f compliance. See also 5.10.

IDENTIFICATION

Control of impurities in substances for pharmaceutical use): A , B, C, E.

A. T o 0.2 g add 20 m L o f water R and 0.5 m L o f sodium carbonate solution R. Shake and filter. T o 5 m L of the filtrate add 1 m L of calcium chloride solution R. A voluminous gelatinous mass is formed. B. T o 5 m L o f the filtrate obtained in identification test A add 0.5 m L o f a 123 g/L solution of magnesium sulfate R. N o voluminous gelatinous mass is formed. C . T o 5 m g add 5 m L of water R, 1 m L of a freshly prepared 10 g/L solution of 1,3-dihydroxynaphthalene R in ethanol (96 per cent) R and 5 m L of hydrochloric add R. Boil gently for 3 min, cool, add 5 m L o f water R, and shake with 15 m L of di-isopropyl ether R. Carry out a blank test. T h e upper layer obtained with the substance to be examined exhibits a deeper bluish-red colour than that obtained with the blank.

nh2

A. N-[3-[(4-amino-6,7-dimethoxyquinazolin-2-yl) methylamino] propyl] furan-2-carboxamide3

TESTS Chlorides

nh2

M axim um 1.0 p er cent.

B. R = Cl: 2N,2-trimethyl-3-(5-oxido-l OH-phenothiazin-10yl) propan-1-am ine,

bath. A dark blue colour develops, which becomes red after cooling and pouring into about 10 m L of water R. D . H eat about 0.5 g. Ammonia vapour is evolved, which turns red litmus paper R blue.

3

and

TESTS S o lu tio n S Dissolve 0.5 g in carbon dioxide-free water R, with heating if necessary, and dilute to 100 m L with the same solvent.

enantiomer

B . (2i?S}-ATj2-dimethyl-3-( 1OH-phenothiazin-10-yl)propan-1amine,

A cid ity o r a lk a lin ity T o 5 m L o f solution S add 5 m L of carbon dioxide-free water R, 0.1 m L o f methyl red solution R and 0.2 m L of 0.01 M sodium hydroxide. T he solution is yellow. Add 0.4 m L o f 0.01 M hydrochloric acid The solution is red. O p tic a l ro ta tio n (2.2.7) T h e angle of optical rotation, determined on solution S, is -0 .1 0 ° to + 0.10°.

C. 1OH-phenothiazine. Ph Bur

★ ★

Allantoin

★ ★

* * * * *

(Ph. Eur. monograph 1288) o

f

H. H-

. y ' NH

and enantiomer

H,N

€4 ^ 403

158.1

97-59-6

A ctio n a n d u se Astringent; keratolytic. PhEir ___________________

D E F IN IT IO N Allantoin contains n o t less than 98.5 per cent and not more than the equivalent o f 101.0 per cent of (RS) -(2 j 5-dioxoimidazolidin-4-yl)urea. CHARACTERS A white or alm ost white, crystalline powder, slightly soluble in water, very slightly soluble in alcohol.

R ed u c in g su b sta n c e s Shake 1.0 g with 10 m L o f water R for 2 min. Filter. Add 1.5 m L o f 0.02 Mpotassium permanganate. T he solution m ust rem ain violet for at least 10 min. R e la te d su b sta n c e s Examine by thin-layer chromatography (2.2.27), using a suitable cellulose for chromatography R as the coating substance.

Test solution (a) Dissolve 0.10 g o f the substance to be examined in 5.0 m L of water R with heating. Allow to cool. Dilute to 10 m l, with methanol R. Use the solution immediately after preparation. Test solution (b) Dilute 1 m L of test solution (a) to 10 m L with a mixture o f 1 volume of methanol R and 1 volume o f water R. Reference solution (a) Dissolve 10 m g of allantoin CRS in a mixture o f 1 volume o f methanol R and 1 volume of water R and dilute to 10 m L with the same mixture of solvents.

Reference solution (b) Dissolve 10 m g of urea R in 10 m L o f water R. Dilute 1 m L o f this solution to 10 m L with methanol R Reference solution (c) M ix 1 m L o f reference solution (a) and 1 m L of reference solution (b). Apply to the plate 10 jiL o f test solution (a) and 5 |iL each o f test solution (b), reference solution (a), reference solution (b) and reference solution (c). Develop over a path o f 10 cm using a mixture of 15 volumes of glacial acetic add R, 25 volumes o f water R and 60 volumes of butanol R.

1-96 Allergen products

2016

Allow the plate to dry in air. Spray the plate with a 5 g/L solution of dimethylaminobenzaldehyde R in a mixture of 1 volume of hydrochloric add R and 3 volumes of methanol R. Dry the plate in a current o f h o t air. Examine in daylight after 30 min. Any spot in the chromatogram obtained with test solution (a), apart from th e principal spot, is not more intense than the spot in the chrom atogram obtained with reference solution (b) (0.5 p er cent). T h e test is not valid unless the chrom atogram obtained with reference solution (c) shows two clearly separated principal spots. L oss o n d ry in g ( 2.2.32) N ot more than 0.1 p e r cent, determined on 1.000 g by drying in an oven at 105 °C. S u lfa te d a s h (2.4.14) N ot more than 0.1 p e r cent, determined on 1.0 g.

1 m L of 0.1 M sodium hydroxide is equivalent to 15.81 m g of C 4H 6N 4O 3. IM P U R IT IE S

To

CO2H

T h e source materials are defined by their origin, nature, m ethod o f collection or production and pretreatm en t Control m ethods and acceptance criteria relating to identity and purity are established. T h e acceptance criteria m ust ensure the consistency o f the allergenic source material from a qualitative and quantitative point o f view. T h e source materials are stored under controlled conditions justified by stability data.

T h e content of the relevant residual solvents, heavy metals and pesticides is determ ined on a num ber o f batches according to a justified sampling plan. Residual solvents and pesticides are limited according to the principles defined in general chapter 2.4.24. Identification and control of residual solvents and 2.8.13. Pesticide residues respectively.

o

B. carbamide (urea). _____________________________PhEur

Allergen Products

*****

(Ph Eur. monograph 1063)

*

Ph Fur

P R O D U C T IO N S O U R C E M A T E R IA L S Source materials for the preparation of allergen products are products o f animal o r vegetable origin, mostly pollens, moulds, mites, animal epithelia and outgrowths (such as hair and feathers) and/or dander, hymenoptera venoms, insects and certain foods.

T h e collection or production, as well as the handling o f the source materials are such that uniform composition is ensured as far as possible from batch to batch.

A. glyoxylic acid,

--------------------------------- — .—

F or specific immunotherapy, allergen products may be either unmodified extracts or extracts modified chemically and/or by adsorption onto different carriers (for example, aluminium hydroxide, calcium phosphate or tyrosine).

W here allergen products are m anufactured using materials of hum an or animal origin, the requirements o f chapter 5.1.7. Viral safety apply.

ASSAY Dissolve 120.0 mg in 40 m L o f water R. T itrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20).

H

prepared by dilution of aqueous or glycerinated extracts, or by reconstitution o f unmodified freeze-dried extracts.

_________________________________________

This monograph does not apply to: chemicals that are used solely for diagnosis of contact dermatitis; chemically synthesised products; allergens derived by recombinant DNA technology. It does not necessarily apply to allergen products for veterinary use. D E F IN IT IO N Allergen products are pharm aceutical preparations derived from extracts of naturally occurring source materials containing allergens, which are substances that lead to and/or provoke allergic reactions. T h e allergenic components are most often o f a proteinaceous nature. Allergen products are intended for in vivo diagnosis o r treatm ent o f allergic diseases attributed to these allergens.

P o lle n s Potential chemical contam inants, such as pesticides, heavy metals and solvents, m ust be minimised. T h e content of foreign pollen m ust be limited to 1 per cent o f total mixed pollens and 0.5 per cent o f any individual pollen as determined by a microscopic particle count. D etectable m ould spores m ust not exceed 1 per c e n t T h e contam ination with particles o f plant origin other than pollen m ust be kept to a minim um. T he m axim u m allowed contam ination m ust be justified. M o u ld s Biologically active contam inants such as mycotoxins in moulds m ust be m in im ised and any presence justified. Appropriate measures have to be im plem ented to avoid c o n tam inatio n by foreign m ould strains. C are m ust be taken to minimise any allergenic constituents o f the media vised for the cultivation of moulds as source materials. Culture media th at contain substances of hum an or anim al origin m ust be justified and, when required, m ust be suitably treated to ensure the inactivation or elimination of possible transmissible agents of disease.

Allergen products are available as finished products, and as finished products used on a nam ed-patient basis. Allergen products are generally presented as parenteral preparations, eye preparations, preparations for inhalation, preparations for oral use, sublingual preparations or preparations for skin tests.

T h e production m ethod is validated to demonstrate that allergen products obtained from moulds and intended for parenteral administration, if tested* would comply with the test for abnormal toxicity for immunosera and vaccines for hum an use (2.6.9).

For in vivo diagnostic use, allergen products are usually prepared as unmodified extracts in a 50 per cent VIV solution of glycerol for skin testing. F o r intradermal diagnosis or for provocation tests by nasal, ocular or bronchial administration, suitable dilutions o f allergen products may be

M ite s Appropriate measures have to be implem ented to avoid co n tam inatio n by foreign m ite strains. Care m ust be taken to m in im ise any allergenic constituents o f the media used for the cultivation of mites as source materials. Culture media

Allergen products 1-97

2016

th at contain substances of hum an or animal origin m ust be justified and, when required, must be suitably treated to ensure the inactivation or elimination of possible transmissible agents of disease.

of suitable reagents, as well as the intended use. The characterised IHRP is used as the reference in the batch control of active substances and intermediates and, if possible, in the batch control offinished products.

Anim al epithelia and outgrowths and/or dander

T he IH R P is characterised by the protein content determination and a protein profile using appropriate methods (such as isoelectric focusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Immunoelectrophoresis, capillary electrophoresis, chromatographic techniques and mass spectrometry).

T hey are obtained from healthy animals selected to avoid possible transmissible agents of disease.

Hymenoptera venoms T h e species of hymenoptera from which the venom is extracted is identified and specified. T h e m ethods of insect collection and venom extraction are described and m ust ensure that the source material is of proper quality.

Food T h e scientific nam e (species, variety, strain etc.) o f the an im al or vegetable species is indicated and the p art used is stated, if applicable. Foods must be o f a quality suitable for hum an consum ption. T he origin o f the food stuff as well as its processing stage is stated.

MANUFACTURING PROCESS Allergen products are generally obtained by extraction, and m ay be purified, from the source materials using appropriate m ethods shown to preserve the allergenic properties o f the com ponents. Allergens for which there are not enough patients to determine the total allergenic activity in vivo or in vitro, the extraction ratio indicating the relative proportions (mlV) o f allergenic source materials and solvents is a minimum requirem ent. Allergen products presented as parenteral preparations, eye preparations, preparations for inhalation and preparations for skin testing are m anufactured u nder aseptic conditions. In the manufacture, packaging, storage and distribution o f allergen products intended for administration by other routes, suitable measures are taken to ensure their microbial quality; recom m endations on this aspect are provided in chapter

5.1.4. Microbial quality of non-sterUe pharmaceutical preparations and substances for pharmaceutical use. All allergen preparations are m anufactured under conditions designed to minimise exogenous and endogenous enzymatic degradation. Any purification procedure is designed to minimise the content o f any potential irritant low molecular mass com ponents and non-allergenic components. Allergen products m ay contain suitable antimicrobial preservatives, the nature and concentration of which have to be justified.

Allergenic components may be detected by appropriate methods (for example, immunoblotting or crossed radioim munoelecupphoresis). Characterisation of the allergenic components may include identification o f relevant allergens based on serological or other techniques using pooled or individual sera from allergic patients, or allergen-specific polyclonal or monoclonal antibodies. Determ ination o f the content of relevant allergens is performed wherever possible. T his determination may be m ade using individual allergen-specific reference standards, w hen available. T h e choice of the relevant allergen components subjected to the determination m ust be justified. Individual allergens are identified and named according to internationally established nomenclature wherever possible. T h e biological potency o f the first IH R P is determined in patients by in vivo techniques such as skin testing, and expressed in units of biological activity except when not enough patients are available. In this case, the potency of the first IH R P is determ ined by an m vitro method. Subsequently, the biological activity of future EHRPs is demonstrated by m vitro methods by comparison with the results obtained with the first IH R P. T he in vitro potency may be measured by a suitable immunoassay (for example, an assay based on the inhibition o f the binding capacity of specific immunoglobulin E antibodies).

IDENTIFICATION T h e tests for identification are performed as late as possible in the manufacturing process. In the case of products used on a nam ed-patient basis, the control is performed on the active substance and/or at the intermediate stage between the active substance and the finished product. Identity is confirmed by comparison with the IH R P using protein profiling by appropriate methods (for example, isoelectric focusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Immunoelectrophoresis, immunoblotting, liquid chromatography o r mass spectrometry).

T h e manufacturing process comprises various stages: — source material; — active substance: it is generally a modified or an unm odified allergen extract; where applicable it is stored under conditions ensuring its stability, for example freezedried; — finished product.

In exceptional cases, if no IH R P is available, a representative batch may be used to confirm identity.

All other stages of the manufacturing process are considered as intermediates.

T h e tests are perform ed as late as possible in the manufacturing process. In the case o f products used on a named-patient basis, th e control is performed on the active substance and/or at the intermediate stage between the active substance and the finished product.

IN-HOUSE REFERENCE PREPARATION An appropriate representative preparation is selected as the in-house reference preparation (IHRP), characterised and used to verify batch-to-batch consistency. T he IH R P is stored in suitably sized aliquots under conditions ensuring its stability, for example freeze-dried.

Characterisation o f the in-house reference preparation The extent of characterisation of the IHRP depends on the source material, knowledge of the aUergejric components and availability

Identity may also be confirmed by comparison with individual allergen-specific reference standards, when available.

TESTS

Various biochemical and immunological tests have been developed in order to characterise allergens qualitatively and quantitatively. In those cases where such m ethods cannot be applied, particularly for the determination o f allergenic activity and allergen and/or protein profile, justification m ust be provided.

1-98 Allopurinol

W a te r (2.5.12) or (2 J J 2 ) M aximum 5 per cent for freeze-dried products. In the case o f oral lyophilisates, the w ater content may be higher than 5 per cent, where justified and authorised. S te rility (2.6.1) Allergen products presented as parenteral preparations, eye preparations, preparations for inhalation or preparations for skin testing comply with the test for sterility. M icro b ial c o n ta m in a tio n For non-sterile allergen products, recommendations are provided in 5.1.4. Microbial quality of non-sterile pharmaceutical

preparations and substances for pharmaceutical use. P ro te in c o n te n t (2.5.33) 80 per cent to 120 p er cent o f die stated content, unless otherwise justified and authorised. If the biological potency can be determined then the test for protein content is performed as a batch-to-batch consistency test and the protein content is within 50 per cent to 150 per cent o f the stated content. W hen the finished product contains proteinaceous excipients, the test for protein content is performed as late as possible during production before addition o f the proteinaceous excipient. P ro te in p ro file T he protein profile determined by suitable methods corresponds to that o f the IH R P. T h e presence o f relevant allergen components is verified, where possible. T he choice of relevant allergen components to be tested for m ust be justified.

Various additional tests, some with increasing selectivity, depending on the allergen product concerned can be applied, but in any casefor allergen products intended for therapeutic use, a validated test measuring the potency (total allergenic activity, determination of individual allergens or any otherjustified tests) must be applied. A lu m in iu m (2.5.13) 80 per cent to 120 per cent o f the stated amount bu t in any case not m ore than 1.25 m g per hum an dose unless otherwise justified and authorised, w hen aluminium hydroxide or aluminium phosphate is used as adsorbent. C a lc iu m (2.5.14) 80 per cent to 120 p er cent o f the stated amount when calcium phosphate is used as adsorbent. A llergen p ro file Relevant allergenic components are identified by means of suitable techniques using allergen-specific hum an or animal antibodies. T o ta l allerg en ic a c tiv ity 50 per cent to 150 per cent o f the stated am ount as assayed by inhibition o f the binding capacity o f specific im munoglobulin E antibodies or a suitable equivalent in vitro method. In d iv id u a l allerg en s 50 per cent to 200 per cent o f the stated amount o f each relevant allergen com ponent, determined by a suitable method. STO RA G E Adsorbed allergen products are not to be frozen, unless otherwise justified and authorised. L A B E L L IN G

The label states: — the nam e o f the allergen product;

2016

the biological potency and/or the protein content and/or the extraction concentration; the route o f administration and die intended use; the storage conditions; where applicable, the name and am ount of added antimicrobial preservative; where applicable, for freeze-dried preparations: — the name, composition and volume o f the reconstituting liquid to be added; — the period o f tim e within which the preparation is to be used after reconstitution; where applicable, that the preparation is sterile; where applicable, the nam e and am ount of adsorbent. PhEur

★*★ ★ ★ ★ ★

Allopurinol

* * * * *

(Ph. Eur. monograph 0576)

NH

J C5H4N4O

136.1

315-30-0

A c tio n a n d u se Xanthine oxidase inhibitor; treatm ent o f gout and hyperuricaemia. P re p a r a tio n s Allopurinol Oral Suspension Allopurinol Tablets PhEur__________________________________________________________

D E F IN IT IO N 1,5-Dihydro-4ii-pyrazolo [3,4 -d]pyrimidin-4-one. C o n te n t 97.0 per cent to 102.0 per cent (dried substance). CHARACTERS A p p e a ra n c e W hite o r alm ost white powder. S o lu b ility Very slighdy soluble in water and in ethanol (96 p er cent). It dissolves in dilute solutions o f alkali hydroxides. ID E N T IF IC A T IO N

First identification B Second identification A , C, D A. Ultraviolet and visible absorption spectrophotometry

(2.2.25). Test solution Dissolve 10 mg in 1 m L o f a 4 g/L solution of sodium hydroxide R and dilute to 100.0 m L with a 10.3 g/L solution o f hydrochloric acid R. Dilute 10.0 m L o f this solution to 100.0 m L with a 10.3 g/L solution o f hydrochloric acid R. Spectral range 220-350 nm . Absorption maximum A t 250 nm . Absorption minimum At 231 nm. Absorbance ratio ^ 231^250 = 0-52 to 0.62. B. Infrared absorption spectrophotom etry (2.2.24). Comparison aOopurinol CRS.

Allopurinol 1-99

2016 C. Dissolve 0.3 g in 2.5 m L of düute sodium hydroxide solution R and add 50 m L o f water R. Add slowly and with shaking 5 m L o f silver nitrate solution R l. A white precipitate is formed which does not dissolve on the addition of 5 m L of

ammonia R. D . Thin-layer chromatography (2.2.27).

Test solution Dissolve 20 m g of the substance to be examined in concentrated ammonia R and dilute to 10 m L with the same solvent. Reference solution Dissolve 20 mg of aUopurinol CRS in concentrated ammonia R and dilute to 10 m L with the same solvent.

Plate TLC silica gel F2 5 4 plate R. Mobile phase anhydrous ethanol R, methylene chloride R (40:60 VIV). Application 10 |iL. Development Over 2/3 o f the plate. Drying In air. Detection Examine in ultraviolet light at 254 nm . Results T h e principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.

Limits: — impurity A: n o t more than twice the area of th e principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent); — impurity B: n o t more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent); — impurity C: n o t more than the area of the corresponding peak in the chromatogram obtained with reference solution (b) (0.1 per cent); — unspecified impurities: for each impurity, not m ore than the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.10 per cent); — sum of impurities other than A , B and C: not m ore than 3 times the area of the principal peak in the chrom atogram obtained with reference solution (a) (0.3 p er cent); — disregard limit. 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 p er cent).

Impurities D and E Liquid chromatography (2.2.29). Use freshfy prepared solutions.

Related substances

Store and inject them at 8 °C} using a cooled autosampler. Solution A 1.25 g/L solution of potassium dihydrogen phosphate R. Test solution Dissolve 50.0 mg of the substance to be examined in 5.0 m L of a 4 g/L solution o f sodium hydroxide R

Liquid chromatography (2.2.29). Use freshly prepared solutions.

and dilute immediately to 100.0 m L with solution A.

Store and inject them at 8 °C, using a cooled autosampler. Test solution (a) Dissolve 25.0 mg of the substance to be examined in 2.5 m L o f a 4 g/L solution of sodium hydroxide R

Reference solution Dissolve 5.0 m g o f aUopurinol impurity D CRS and 5.0 m g of aUopurinol impurity E CRS in 5.0 m L o f a 4 g/L solution of sodium hydroxide R and dilute

and dilute immediately to 50.0 m L with the mobile phase.

immediately to 100.0 m L with solution A. Dilute 1.0 m L of this solution to 100.0 m L with solution A.

TESTS

Test solution (b) Dissolve 20.0 mg o f the substance to be examined in 5.0 m L o f a 4 g/L solution of sodium hydroxide R and dilute immediately to 250.0 m L with the mobile phase.

Reference solution (a) Dilute 2.0 m L o f test solution (a) to 100.0 m L with the mobile phase. Dilute 5.0 m L of this solution to 100.0 m L with the mobile phase.

Reference solution (b) Dissolve 5 mg o f aUopurinol impurity A CRS, 5 m g of aUopurinol impurity B CRS and 5.0 mg o f aUopurinol impurity C CRS in 5.0 m L o f a 4 g/L solution o f sodium hydroxide R and dilute immediately to 100.0 m L with the mobile phase. Dilute 1.0 m L o f this solution to 100.0 m L with the mobile phase.

Reference solution (c) Dissolve 20.0 m g o f aUopurinol CRS in 5.0 m L o f a 4 g/L solution o f sodium hydroxide R and dilute immediately to 250.0 m L with the mobile phase.

Column: — size: I = 0.25 m , 0 = 4.6 mm; — stationary phase: octadecylsSyl silica gel for chromatography R (5 nm).

Mobile phase 1.25 g/L solution of potassium dihydrogen phosphate R. Flow rate 1.4 mL/min. Detection Spectrophotom eter at 230 nm . Injection 20 |iL of test solution (a) and reference solutions (a) and (b).

Run time Twice the retention time of aUopurinol. Elution order Im purity A, impurity B, impurity C , aUopurinol. Retention time AUopurinol = about 10 min. System suitability: reference solution (b): — resolution: minim um 1.1 between the peaks due to impurities B and C.

Column: — size: I = 0.05 m , 0 = 4.6 mm; — stationary phase: base-deactivated octadecylsUyl silica gel for chromatography R (3 (am).

Mobile phase methanol R> 1.25 g/L solution of potassium dihydrogen phosphate R (10:90 VIV). Flow rate 2 mL/min. Detection Spectrophotom eter at 230 nm. Irtjection 20 |iL. Run time 1.5 times the retention time o f impurity E. Retention times Im purity D = about 3.6 min; im purity E = about 4.5 min. System suitability: reference solution: — resolution: minim um 2.0 between the peaks due to impurities D and E.

Limits: — impurity D: n o t more than the area of the corresponding peak in the chromatogram obtained with the reference solution (0.1 p er cent); — impurity E: n o t more than the area o f the corresponding peak in the chromatogram obtained with the reference solution (0.1 p er cent).

Impurity F Liquid chromatography (2.2.29). U nder the following conditions, any hydrazine in the sample reacts with benzaldehyde to give benzaldehyde azine.

Solvent mixture M ix equal volumes o f dilute sodium hydroxide solution R and methanol R.

2016

I- 100 Almagate

Solution A Dissolve 2.0 g of benzaldehyde R in the solvent m ixture and dilute to 50.0 m L with the solvent mixture. Prepare immediately before use. N‘ H

Test solution Dissolve 250.0 m g of the substance to be examined in 5 m L o f the solvent mixture. Add 4 m L of solution A, mix and allow to stand for 2.5 h at room tem perature. Add 5.0 m L o f hexane R and shake for 1 min. Allow the layers to separate and use the upper layer.

Reference solution Dissolve 10.0 mg o f hydrazine sulfate R in the solvent mixture by sonicating for about 2 min and dilute to 50.0 m L with the solvent mixture. Dilute 1.0 m L to 20.0 m L with the solvent mixture. D ilute 1.0 m L o f this solution to 20.0 m L with the solvent mixture. T o 5.0 m L of the solution obtained, add 4 m L o f solution A, mix and allow to stand for 2.5 h at room temperature. Add 5.0 m L o f hexane R and shake for 1 m in. Allow the layers to separate and use the upper layer.

mN

A. R1 = N H 23 R2 = H : 5-am ino-li/-pyrazole-4carboxamide, B. R1 = N H 2, R2 = C H O : 5-(fonnylam ino)-lH-pyrazole-4carboxamide, D . R1 = 0 - C 2H 5, R2 = H: ethyl 5-am ino-lH-pyrazole-4carboxylate, E. R1 = O -C 2H 5, R2 = C HO : ethyl 5-(form ylam ino)-l//pyrazole-4-caiboxylate,

nh2

Blank solution T o 5 m L of the solvent mixture add 4 m L of solution A, mix and allow to stand for 2.5 h at room tem perature. Add 5.0 m L o f hexane R and shake for 1 min. Allow the layers to separate and use the upper layer. Column: — sizer. I = 0.25 m ,0 = 4.0 mm; — stationary phase', cyanosifyl silica gelfor chromatography R (5 Jim) with a pore size o f 10 nm; — temperature'. 30 °C.

Mobile phase 2-propanol R, hexane R (5:95 VIV). Flow rate 1.5 mL/min. Detection Spectrophotom eter at 310 nm . Injection 20 pL. Relative retention W ith reference to benzaldehyde (retention tim e = about 2.8 min): benzaldehyde azine = about 0.8.

System suitability: reference solution: — resolution: minimum 2 between the peaks due to benzaldehyde azine and benzaldehyde; — signal-to-noise ratio: minimum 20 for the peak due to benzaldehyde azine.

Limit. — impurity F: the area of the peak due to benzaldehyde azine in the chromatogram obtained with the test solution is not m ore than the area o f the corresponding peak in the chromatogram obtained with the reference solution (10 ppm of hydrazine sulfate equivalent to 2.5 ppm of hydrazine). H eav y m e ta ls (2.4.8) Maximum 20 ppm. 1.0 g complies with test C. Prepare the reference solution using 2 m L of lead standard solution (10 ppm Pb) R. L o ss o n d ry in g (2.2.32) M axim um 0.5 per cent, determined on 1.000 g by drying in an oven at 105 °C. S u lfa te d a s h (2.4.14) M axim um 0.1 per cent, determined on 1.0 g. A SSA Y Liquid chromatography ( 2.2.29) as described in the test for related substances with the following modification.

Injection T est solution (b) and reference solution (c). Calculate the percentage content o f C5H4N4O from the declared content of aUopurinol CRS. IM P U R IT IE S

Specified impurities: A, B, C, D , E, F.

L

/N

C. 5-(4H-l,2,4-triazol-4-yl)-lii-pyrazole-4-carboxam ide, F. H 2N -N H 2: diazane (hydrazine). PhEur

★*★ * •* ★ ★

Almagate

* * * * *

(Ph Eur monograph 2010)

AhMftCsDaoHi^fflaO

630

66827-12-1

A c tio n a n d u se Antacid. P h & i _____________________

DEFINmON H ydrated aluminium magnesium hydroxycarbonate. C o n te n t — aluminium: 15.0 per cent to 17.0 per cent (calculated as A120 3), — magnesium: 36.0 per cent to 40.0 per cent (calculated as M gO ), — carbonic acid: 12.5 p er cent to 14.5 per cent (calculated as C O 2). CHARACTERS A p p e a ra n c e W hite or almost white, fine, crystalline powder. S o lu b ility Practically insoluble in water, in ethanol (96 per cent) and in methylene chloride. It dissolves with effervescence and heating in dilute mineral adds.

IDENTIFICATION A. Infrared absorption spectrophotom etry (2.2.24).

Comparison Ph. Eur. reference spectrum of almagate. B. Dissolve 0.15 g in dilute hydrochloric add R and dilute to 20 m L with the same ad d . 2 m L o f the solution gives the reaction o f aluminium (2.3.1).

C. 2 m L o f the solution prepared under identification test B gives the reaction o f magnesium (2.3.1). TESTS p H (2.2.3) 9.1 to 9.7.

2016 Disperse 4.0 g in 100 m L of carbon dioxide-free water R, stir for 2 m in and filter. N e u tra lisin g cap a c ity

Carry out the testât 37 °C. Disperse 0.5 g in 100 m L of water R, heat, add 100.0 m L o f 0.1 M hydrochloric add, previously heated and stir continuously; the p H (2.2.3) o f the solution between 5 m in and 20 min is not less than 3.0 and not greater than 4.5. Add 10.0 m L o f 0.5 M hydrochloric add, previously heated, stir continuously for 1 h and titrate with 0.1 M sodium hydroxide to p H 3.5; n o t m ore than 20.0 m L of 0 . 1 M sodium hydroxide is required.

Almagate 1-101 Magnesium Introduce 10.0 m L of solution A prepared in the assay of alum inium into a 500 m L conical flask, add 200 m L of water R, 20 m L o f triethanolamine R with shaking, 10 m l. of ammonium chloride buffer solution pH 10.0 R and 50 mg of mordant black 11 triturate R Titrate with 0.05 M sodium edetate until the colour changes from violet to pure blue. 1 m L of 0.05 M sodium edetate is equivalent to 2.015 mg o f MgO. C a rb o n ic a c id 12.5 per cent to 14.5 per cen t

C h lo rid e s (2.4.4) Maximum 0.1 per c e n t

Test sample Place 7.00 m g of the substance to be examined in a tin capsule. Seal the capsule.

Dissolve 0.33 g in 5 m L of dilute nitric acid R and dilute to 100 m L with water R. Prepare simultaneously the standard by diluting 0.7 m L o f dilute nitric add R to 5 m L with water R and adding 10 m L o f chloride standard solution (5 ppm

Reference sample Place 7.00 mg o f ahnagate CRS in a tin

Cl) R. S u lfates (2.4.13) M aximum 0.4 per cent. Dissolve 0.25 g in 5 m L o f dilute hydrochloric add R and dilute to 100 m L with distilled water R. Prepare simultaneously the standard by adding 0.8 m L o f dilute hydrochloric add R to 15 m L of sulfate standard solution

(10 ppm SO 4) R Sodium M aximum 150 ppm. Atomic absorption spectrometry (2.2.23, Method I).

Test solution Dissolve 0.25 g in 50 m L of a 103 g/L solution o f hydrochloric acid R. Reference solutions Prepare the reference solutions using sodium standard solution (200 ppm Na) R, diluted as necessary with a 103 g/L solution of hydrochloric add R. H eavy m e ta ls (2.4.8) Maximum 20 ppm. Dissolve 1.0 g in dilute hydrochloric acid R and dilute to 20.0 m L w ith the same acid. 12 m L o f the solution complies with test A. Prepare the reference solution using lead standard

solution (1 ppm Pb) R. L oss o n ig n itio n 43.0 per cent to 49.0 per cent, determined on 1.000 g by ignition at 900 ± 50 °C.

capsule. Seal the capsule. Introduce separately the test sample and the reference sample into a combustion chamber of a C H N analyser purged with helium for chromatography R and maintained at a tem perature o f 1020 °C. Simultaneously, introduce oxygen R at a pressure o f 40 kPa and a flow rate of 20 m I7m in and allow complete combustion o f the sample. Sweep the combustion gases through a reduction reactor and separate the gases form ed by gas chromatography (2.2.28).

Column: — sizer. I = 2 m , 0 = 4 mm; — stationary phase: ethylvinylbenzene-dxvinylbenzene

copolymer R l. Carrier gas heHum for chromatography R. Flow rate 100 mL/min. Temperature: — column: 65 °C; — detector. 190 °C. Detection Therm al conductivity. Run time 16 m in . System suitability: — average percentage o f carbon in 5 reference samples m ust be within ± 0.2 per cent of the value assigned to the CRS; the difference between the upper and the lower values o f the percentage of carbon in these samples m ust be below 0.2 per cent. Calculate the percentage content o f carbonic acid in the test sample according to the following formula:

M ic ro b ia l c o n ta m in a tio n TA M C: acceptance criterion 103 C FU /g (2.6.12).

CxKx — m

TY M C: acceptance criterion 102 C FU /g (2.6.12). Absence o f Escherichia colt (2.6.13).

C

Absence o f Pseudomonas aeruginosa (2.6.13). A SSAY A lu m in iu m Dissolve 1.000 g in 5 m L o f hydrochloric acid R, heating if necessary. Allow to cool to room tem perature and dilute to 100.0 m L with water R (solution A). Introduce 10.0 m L of solution A into a 250 m L conical flask, add 25.0 m L of 0.05 M sodium edetate, 20 m L of buffer solution pH 3.5 R, 40 m L o f ethanol R and 2 m L of a freshly prepared 0.25 g/L solution o f dithizone R in ethanol R. T itrate the excess of sodium edetate with 0.05 M zinc sulfate until the colour changes from greenish-violet to pink. 1 m L of 0.05 M sodium edetate is equivalent to 2.549 mg of AI2O 3.

K

A

percentage content o f carbonic a d d in the reference sample; m ean value for the 5 reference samples of the ratio o f the mass in milligrams to the area o f the peak due to carbonic add; area o f the peak due to carbonic a d d in the chrom atogram obtained with the test sample; sample mass, in milligrams.

STORAGE In an airtight container. PhEur

1-102 Almond Oil

Virgin Almond Oil Almond Oil

2016 ★* * ★ ★ ★ ★ *****

(Ph. Eur. monograph 0261) P re p a r a tio n Almond Oil E ar Drops PhEur_______________________

D E F IN IT IO N Fatty oil obtained by cold expression from the ripe seeds of Prunus dulcis (Mill.) D A .W ebb var. dulcis or Prunus dulds (Mill.) D A .W ebb var. amara (DC.) Buchheim or a mixture of both varieties. CHARACTERS A p p e a ra n c e Yellow, d ear liquid.

— erucic add: maximum 0.1 per cent. S tero ls (2.4.23)

Composition of sterolfraction of the oil: cholesterol: maximum 0.7 per cent, campesterol: maximum 4.0 per cent, sugmasteroh maximum 3.0 per cent, f}-sitosterol: 73.0 per cent to 87.0 per cent, A5-avenasterol: minimum 10.0 per cent, A7-stigmastenol: maximum 3.0 per cent, A7-avenasterol: maximum 3.0 per cent, brassicasterol: maximum 0.3 per cent. W a te r (2.5.32)

— — — — — — — —

Maximum 0.1 per cent, determ ined on 1.00 g. STORA GE In a well-filled container, protected from light. PhEur

S o lu b ility Slightly soluble in ethanol (96 per cent), m isdble with light petroleum. R elative d e n sity About 0.916.

Refined Almond Oil

***** *+ +* *

It solidifies at about - 1 8 °C.

(Ph. Eur. monograph 1064)

ID E N T IF IC A T IO N

PhEur__________________________________________________________

First identification A, C. Second identification A, B. A Absorbance (see Tests). B. Identification of fatty oils by thin-layer chromatography

(2.3.2). Results T he chromatogram obtained is similar to the corresponding chromatogram shown in Figure 2.3.2.-1. C. Composition of fatty adds (see Tests). TESTS Specific a b so rb a n c e (2.2.25) M aximum 0.2, determined at the absorption maximum at 270 nm . T he ratio o f the absorbance measured at 232 nm to that measured at 270 nm is greater than 7. T o 0.100 g add cyclohexane R and dilute to 10.0 m L with the same solvent. Adapt the concentration of the solution so that the absorbance lies between 0.5 and 1.5, measured in a 1 cm cell.

D E F IN IT IO N Fatty oil obtained from the ripe seeds of Prunus dulds (Mill.) D A . Webb var. dulcis or Prunus dulcis (Mill.) D A . W ebb var. amara (DC.) Buchheim or a mixture o f both varieties by cold expression. It is then refined. A suitable antioxidant may be added. CHA RACTERS A p p e a ra n c e Pale yellow, d ear liquid. S o lubility Slightly soluble in ethanol (96 per cent), m isdble with light petroleum. R elativ e d en sity A bout 0.916. It solidifies at about - 1 8 °C. ID E N T IF IC A T IO N A Identification o f fatty oils by thin-layer chromatography

A d d valu e (2.5.1) M aximum 2.0, determined on 5.0 g.

(2.3.2). Results T he chromatogram obtained is similar to the

P e ro x id e v alu e (2.5.5, Method A) Maximum 15.0.

B. Composition of fatty a d d s (see Tests).

U n sap o n ifiab le m a tte r (2.5.7) Maximum 0.9 per cent, determined on 5.0 g. C o m p o sitio n o f fa tty acid s (2.4.22, Method A) Use the mixture of calibrating substances in Table 2.4.22.-3.

Composition of the fatty-acid fraction of the oil: — saturated fatty acids of chain length less than Cj^. maximum 0.1 per cent, — palmitic acid: 4.0 per cent to 9.0 per cent, — palrmtoleic add: maximum 0.8 per cent, — margaric add', maximum 0.2 per cent, — stearic add: maximum 3.0 p er cent, — oleic add: 62.0 per cent to 86.0 per cent, — linoleic add: 20.0 per cent to 30.0 per cent, — linolenic acid: maximum 0.4 per cent, — arackidic add: maximum 0.2 per cent, — eicosenoic acid: maximum 0.3 per cent, — behenic add: maximum 0.2 per cent,

corresponding chromatogram shown in Figure 2.3.2.-1. TESTS Specific a b so rb a n c e (2.2.25) 0.2 to 6.0, determined at the absorption maximum at 270 nm. T o 0.100 g add cyclohexane R and dilute to 10.0 m L with the same solvent. Adapt the concentration o f the solution so that the absorbance lies between 0.5 and 1.5, measured in a 1 cm cell. A cid valu e (2.5.1) M aximum 0.5, determined on 5.0 g. P e ro x id e v alu e (2.5.5, Method A) Maximum 5.0. U n sap o n ifiab le m a tte r (2.5.7) M aximum 0.9 per cent, determined on 5.0 g. C o m p o sitio n o f fa tty a c id s (2.4.22, Method A) Use the mixture of calibrating substances in Table 2.4.22.-3.

2016 Composition of the fatty-acid fraction of the oü: — saturated fatty adds of chain length less than Ci£ maximum 0.1 per cent; — palmitic add: 4.0 per cent to 9.0 per cent; — paltmtoleic add: maximum 0.8 per cent; — margaric acid: maximum 0.2. per cent; — stearic add: maximum 3.0 per cent; — oleic acid: 62.0 per cent to 86.0 per cent; — linoleic acid: 20.0 per cent to 30.0 per cent; — Imolenic add: maximum 0.4 per cent; — arachidic acid: maximum 0.2 per cent; — eicosenoic acid: maximum 0.3 per cent; — behenic acid, maximum 0.2 per cent; — erucic acid: maximum 0.1 per cent. S tero ls (2.4.25)

Composition of the sterolfraction of the oü: cholesterol: maximum 0.7 per cent; campesterol: maximum 5.0 per cent; stigmasterol: maximum 4.0 per cent; ^-sitosterol: 73.0 per cent to 87.0 per cent; A5-avenasterol: minim um 5.0 per cent; A 7-sngmastenol: maximum 3.0 per cent; A 7-avenasterol: maximum 3.0 per cent; brassicasterol: maximum 0.3 per cent. W a te r (2.5.32)

— — — — — — — —

M aximum 0.1 per cent, determined on 1.00 g. STORA GE In a well-filled container, protected from light. __________________________________________________________________________________________ P h E tr

Aloxiprin 9014-67-9 A ctio n a n d u se Salicylate; non-selective cyclo-oxygenase inhibitor; antipyretiq analgesic; anti-inflammatory. P re p a ra tio n Aloxiprin Tablets D E F IN IT IO N Aloxiprin is a polymeric condensation product of aluminium oxide and O-acetylsalicylic add. It contains n o t less than 7.5% and not m ore than 8.5% o f aluminium, Al, and not less than 79.0% and n o t more than 87.4% o f total salicylates, calculated as O-acetylsalicylic ad d , CgHsOi, both calculated with reference to the dried substance. C H A R A C T E R IS T IC S A fine, white or slightly pink powder. Practically insoluble in water, practically insoluble in ethanol (96%) and in ether. ID E N T IF IC A T IO N A. Boil 1 g with 2 0 m L o f 2 m hydrochloric add, cool, filter and reserve the filtrate. Dissolve the residue in 10 m L of 0 .1 m sodium hydroxide and neutralise with 1m acetic add. 1 m L of the resulting solution yidds reaction A characteristic o f salicylates, Appendix VI. B. T h e filtrate reserved in test A yields the reaction characteristic o f aluminium salts, Appendix VI.

Aloxiprin 1-103

TESTS H eav y m e ta ls Carefully ignite 2 .0 g at a low tem perature until completely charred, cool, add 2 m L of nitric acid and 0 .25 m L of sulfuric add, heat cautiously until white fumes are evolved and ignite at 5 00° to 60 0 °. Cool, add 2 m L of hydrochloric acid, evaporate to dryness on a water bath and carry out the procedure for limit test C for heavy metals, Appendix VH, beginning at the words Dissolve the residue...’. Use 2 m L of lead standard solution (10 ppm Pb) to prepare die standard (10 ppm). F re e acetylsalicylic a cid T o a quantity containing the equivalent o f 1.0 g of total salicylates add 5 0 m L of dry ether and shake for 3 0 minutes. Filter quickly through fluted filter paper, wash the paper with several portions o f dry ether and dilute the combined filtrate and washings to 1 0 0 m L with dry ether. T he absorbance o f the solution at the maximum at 2 7 8 nm is n o t more than 0 .3 6 , Appendix I I B (0.5% , calculated with reference to the content o f total salicylates). S alicylic a c id T he absorbance o f the solution used in the test for Free acetylsalicylic a d d at the maximum at 3 0 8 nm is not more than 0 .5 0 , Appendix II B (0.15% , calculated with reference to the content o f total salicylates). C o m b in e d salicy late N o t more than 9.5% , calculated as salicylic ad d , C7H 60 3, with reference to the content o f total salicylates calculated as O-acetylsalicylic a d d when determined in the following manner. T o 0.1 g add 4 0 m L o f a 0.5% w/v solution of sodium fluoride in 0.1m hydrochloric add and shake for 5 minutes. Allow the solution to stand for 10 minutes, shaking at frequent intervals. Extract with six 2 0 m L quantities of dichloromethane, filter the combined extracts through a layer o f anhydrous sodium sulfate, wash with 3 0 m l . o f dichloromethane and dilute the combined filtrate and washings to 2 0 0 m L with dichloromethane. Dilute 2 0 mT. o f the solution to 5 0 m L with dichloromethane and measure the absorbance of the resulting solution at the maximum at 3 0 8 nm , Appendix IIB . Calculate the content of taking 2 9 3 as the value of A (l% , 1 cm) at the maximum at 3 0 8 nm . L oss on d ry in g W hen dried to constant weight over phosphorus pentoxide at a pressure not exceeding 0 .7 kPa, loses n o t more than 2.0% of its w dght. Use 1 g. ASSAY

For ahimm mm Ignite 2 g in a tared silica crudble, heat gently until the organic m atter is destroyed and then ignite to constant w dght at 1000°. Each g of residue is equivalent to 0 .5 2 9 2 g of Al.

For total salicylates T o 0 .2 5 g add 5 0 m L of 1m sodium hydroxide and boil gendy until dissolved. Cool, add 50 m L of water, adjust the pH to between 2 .4 0 and 2 .5 0 with 1m hydrochloric acid and dilute to 5 0 0 m L with water. T o 5 m L add 4 m L of iron(m) chloride solution, allow to stand for 3 0 minutes, dilute to 5 0 m L w ith water and measure the absorbance of the resulting solution at the maximum at 5 3 0 nm , Appendix II B, using in the reference cell a solution prepared by diluting 4 m L of iron(m) chloride solution to 5 0 m L with water. Calculate the content of total salicylates as C ^ H ^ from the absorbance obtained by repeating the procedure using 4 m L of a 0.05% w/v solution

2016

1-104 Alprazolam

of salicyUc add in place o f the solution being examined and b eg in n in g at the words ‘add 4 m L o f

ừon(w) chloride

Reference solution (b) Dissolve 10 m g o f alprazolam CRS and 10 m g o f midazolam CRS in methanol R and dilute to 10 m L

solution..’. Each g o f salicylic a d d is equivalent to 1.305 g of

with the same solvent.

C 9Ỉ Ỉ 8O 4.

Plate TLC silica gel GF2 5 4 plate R. Mobile phase glacial acetic acid R, water R, methanol R, ethyl acetate R (2:15:20:80 VIV/VIV). Application 5 |iL. Development Over a path o f 12 cm. Drying In. air. Detection Examine in ultraviolet light at 254 nm. System suitability: reference solution (b):

★ ★ ★ ★ *****

Alprazolam (Ph. Eur. monograph 1065) H3C ^

w

—

the chromatogram shows 2 clearly separately spots.

Results T h e principal spot in the chrom atogram obtained with the test solution is similar in position and size to the principa] spot in the chromatogram obtained with reference solution (a).

TESTS Related substances

PhEir __________________________________________________________

Liquid chromatography (2.2.29). Buffer solution Dissolve 7.7 g of ammonium acetate R in 1000 m L o f water R and adjust to p H 4.2 with glacial acetic add R. Test solution Dissolve 0.100 g o f the substance to be examined in dimethylformamide R and dilute to 10.0 m L with

DEFINITION

the same solvent.

8-Chloro-1-m ethyl-6-phenyl-4/i- [ 1,2,4] triazolo [4,3-a] [1,4] benzodiazepine.

Reference solution (a) Dissolve 2 m g o f alprazolam CRS and 2 m g o f triazolam CRS in dimethylformamide R and dilute to

C17H13aN4

308.8

28981-97-7

Action and use Benzodiazepine.

100.0 m L with the same solvent.

Content 99.0

per cent to 101.0 per cent (dried substance).

CHARACTERS Appearance W hite or almost white, crystallin e powder.

Solubility Practically insoluble in water, freely soluble in methylene chloride, sparingly soluble in acetone and in ethanol (96 per cent). It shows polymorphism (5.9).

IDENTIFICATION First identification B. Second identification A , C. A. Dissolve the substance to be examined in the smallest necessary quantity o f ethyl acetate R and evaporate to dryness on a water-bath. Thoroughly mix 5.0 m g o f the substance to be examined with 5.0 mg of alprazolam CRS. T he melting point (2.2.14) o f the mixture does n o t differ by more than 2 °C from the melting point o f the substance to be examined. B. Infrared absorption spectrophotometry (2.2.24).

Preparation Discs. Comparison alprazolam CRS.

Reference solution (b) Dilute 5.0 m L o f the test solution to 100.0 m l. with dimethylformamide R. Dilute 0.5 m L o f this solution to 10.0 m L with dimethylformamide R. Cdumn: — size. I = 0.25 m, 0 = 4.6 mm; — stationary phase: phenylsüyl silica gelfor chromatography R1 (5 nm).

Mobile phase: — mobile phase A: buffer solution, methanol R (44:56 VIV)\ — mobile phase B: buffer solution, methanol R (5:95 V!V)\ — temperature:. 40 °C; lim e (min) 0 -1 5

Mobile phaae A (percent V/V) 98

Mobile phase B (ptT cent V7V) 2

15 -3 5

98 -> 1

2 + 99

3 5 -4 0

1

99

Flow rate 2 mL/min. Detection Spectrophotom eter at 254 nm. Irgecdon 10 |iL; inject dtmethylformamide R a sa blank. Retention time Triazolam = about 9 min;

If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in the m in im u m volume of ethyl acetate R, evaporate to dryness on a water-bath and record new spectra using the residues.

alprazolam = about 10 min. System suitability: reference solution (a): — resolution: minim um 1.5 between the peaks due to triazolam and alprazolam.

C. Thin-layer chromatography (2.2.27).

— totah not more than the area o f the principal peak in the chrom atogram obtained with reference solution (b) (0.25 per cent); — disregard lima: 0.2 times the area o f the principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent).

Test solution Dissolve 10 m g o f the substance to be exam in ed in methanol R and dilute to 10 m L w ith the same solvent. Reference solution (a) Dissolve 10 m g o f alprazolam CRS in methanol R and dilute to 10 m L with the same solvent.

Limits:

2016

Alprazolam 1-105

L oss o n d ry in g (2.2.32) M axim um 0.5 per cent, determined on 1.000 g by drying in an oven at 105 °C. S u lfa te d a s h (2.4.14) Maximum 0.1 per cent, determined on 1.0 g. A SSA Y Dissolve 0.140 g in 50 m L of a mixture o f 2 volumes of acetic anhydride R and 3 volumes of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). Titrate to the 2nd point of inflexion.

G. 7-chloro- l-methyl-5-phenyl[ 1,2,4]triazolo [4,3-a] quinolin4-amine,

1 m L of 0.1 M perchloric add is equivalent to 15.44 mg o f C 17H 13CIN 4. STO R A G E Protected from light. IM P U R IT IE S N y CH3 and enantiomer

H . bis[[4-(2-ben2oyl-4-chlorophenyl)-5-methyl-4H'-lj2}4triazol-3-yl] methyl] amine.

OH

A. (4R5)-3-amino-6-chloro-2-methyl-4-phenyl-3,4dihydroquinazolin-4-olj and enantiomer

B. R = C H 2O H : [5-chloro-2-[3-(hydroxymethyl)-5-methyl4 H - 1j2j4-triazol-4-yl] phenyl] phenyimethanone,

I. [5-chloro-2-[3- [[(6i?5)-8-chloro- 6-hydroxy-1-methyl-6 phenyl-4H- [ 1,2,4] triazolo [4,3-a] [ 1,4]benzodiazepin-5 (6H)-yI] methyl] -5-m ethyl-4ii-1,2,4-triazol-4-yl] phenyl] phenylmethanone,

C. R = H : [5-chloro-2-[3-methyl-4/f-1,2,4-triazol-4-yl] phenyl] phenylmethanone, F. R = C H 2C1: [5-chloro-2-[3-(chloromethyl)-5-methyl-4H1,2 }4-triazol-4-yl] phenyl] phenylmethanone,

J. 2,17-dichloro-6,13-dimethyl-l 8b, 19a-diphenyl8b, 19adihydro-1OH, 18b H- [ 1,2,4] triazolo [4 " ',3 '" : 1 ",2 "] quinolo [3 ",4 " :4 ',5 '] oxazolo [3 ',2 '-d\-1,2,4-triazolo [4,3-a] [ 1,4] benzodiazepine. PhEur

D . 8-chloro-1-ethenyl-6-phenyl-4/i- [1,2,4] triazolo [4,3-a] [ 1,4] benzodiazepine,

E. (2 -amino-5-chlorophenyl)phenylmethanone,

2016

1-106 Alprenolol Hydrochloride

Alprenolol Hydrochloride

***\

(Ph Eur monograph 0876)

*

Test solution (a) Dissolve 0.50 g o f die substance to be examined in methanol R and dilute to 10 m L w ith die same solvent. Test solution (b) D ilute 1 m L o f test solution (a) to 50 m L with methanol R. Reference solution (a) Dissolve 10 m g o f alprenolol hydrochloride CRS in methanol R and dilute to 10 m L with the same solvent.

C 15H 24C1N02

285.8

13707-88-5

Action and use Beta-adrenoceptor antagonist. PhEur___________________________________________________________

DEFINITION (2RS)-1 -[(1 -M ethylethyl)amino]-3- [2-(prop-2-enyI)phenoxy] propan-2-ol hydrochloride.

Content 99.0

per cent to 101.0 per cent (dried substance).

CHARACTERS Appearance W hite or alm ost white, crystalline pow der o r colourless crystals.

Solubility Very soluble in water, freely soluble in ethanol (96 p er cent) and in methylene chloride.

IDENTIFICATION First identification B, D. Second identification A , C, D. A. M elting point (2.2. 14): 108 °C to 112 °C. B. Infrared absorption spectrophotom etry (2.2.24). Comparison alprenolol hydrochloride CRS. C. Examine the chrom atograms obtained in the test for im purity D.

Detection Examine in daylight, after exposure to iodine vapour for 30 min. Results T he principal spot in the chrom atogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chrom atogram obtained with reference solution (a). D . It gives reaction (a) of chlorides (2.3.1).

TESTS Solution S Dissolve 1.0 g in carbon dioxide-free water R and dilute to 50 m L with the same solvent.

Appearance of solution Solution S is clear (2.2.1) and n o t m ore intensely coloured than reference solution B9 (2.2.2, Method II). Acidity or alkalinity T o 10 m L o f solution S add 0.2 mT. o f methyl red solution R and 0.2 m L o f 0.01 M hydrochloric acid; the solution is red. A dd 0.4 m L o f 0.01 M sodium hydroxide; the solution is yellow.

Impurity C M axim um 0.1 per c e n t Dissolve 0.25 g in ethanol (96 per cent) R and dilute to 25 m L with the same solvent. T h e absorbance (2.2.25) measured at 297 nm is n o t greater than 0.20.

Impurity D Thin-layer chrom atography (2.2.27).

Reference solution (b) Dissolve 10 m g o f alprenolol hydrochloride CRS and 10 m g o f oxprenold hydrochloride CRS in methanol R and dilute to 10 m L with the same solvent. Reference solution (c) Dilute 5 m L o f test solution (b) to 50 mT. with methanolR Plate TLC silica gel G {date R. Mobile phase Place 2 beakers each containing 30 m L of ammonia R at die bottom o f die tank containing a mixture of 5 volumes o f methanol R and 95 volumes o f ethyl acetate R. Application 5 |iL. Development Over a p ath o f 15 cm in a tank saturated for at least 1 h.

Drying Ax. 100 °C for 15 min. Detection Expose to iodine vapour for up to 6 h. System suitability: reference solution (b): — the chrom atogram shows 2 clearly separated spots.

Limits: test solution (a): — impurity D-. any spot with an Rp value greater than th at of the principal spot is not more intense than die principal spot in the chrom atogram obtained with reference solution (c) (0.2 per cent). Related substances Liquid chrom atography (2.2.29).

Test solution Dissolve 20.0 m g of die substance to be exam ine d in the mobile phase and dilute to 10.0 m L with

the mobile phase.

Reference solution (a) Dissolve 4.0 m g o f alprenolol hydrochloride CRS and 0.8 m g of 4-isopropylphenol R in the mobile phase and dilute to 100.0 m L with the mobile phase.

Reference solution (b) D ilute 4.0 m L of the test solution to 100.0 m L with die mobile phase. D ilute 1.0 m L o f this solution to 10.0 m L with the mobile phase.

Column: — size. I = 0.15 m , 0 = 4 mm; — stationary phase, octylsüyl silica gel for chromatography R (5 pm). Mobile phase M ix 0.656 g o f sodium octanesidfonate R with 150 mT. o f acetomtrüe R and dilute to 500 m L with phosphate buffer p H 2.8 prepared as follows: mix 1.78 g of phosphoric add R and 15.6 g o f sodium dihydrogen phosphate R and dilute to 2000 m L w ith water R

Flow rate 1 mlVmin. Detection Spectrophotom eter at 280 nm . Equilibration W ith the mobile phase for about 1 h. Injection 20 nL. Run time Twice the retention time of alprenolol. Retention time Alprenolol = about 11 min; 4-isopropylphenol = about 18 min.

System suitability: reference solution (a): — resolution: m inim um 5 between the peaks due to alprenolol and 4-isopropylphenol; if necessary, adjust the concentration o f sodium octanesulfonate and/or acetonitrile in the mobile phase (increase the

2016

Alprostadil 1-107

concentration o f sodium octanesulfonate to increase the retention time of alprenolol and increase the concentration of acetonitrile to decrease the retention times o f both compounds).

Limits: — unspecified impurities: for each impurity, not more than 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.10 per cent); — total: not more than the area of the principal peak in the chromatogram obtained with reference solution (b) (0.4 per cent); — disregard limit: 0.1 times the area o f the principal peak in the chromatogram obtained with reference solution (b) (0.04 per cent).

D . 1,1 '-[(l-methylethyl)imino]bis[3-[2-(prop-2-enyl) phenoxy]propan- 2 -ol]. ------------------------------------------------------------------------------------------------------------------------------------------------------ P hE ir

H eav y m e ta ls (2.4.8) M aximum 10 ppm. Dissolve 2.0 g in 20 m L o f water R. 12 m L o f the solution complies with test A. Prepare the reference solution using

lead standard solution (1 ppm Pb) R. L oss o n d ry in g (2.2.31) M aximum 0.5 per cent, determined on 1.000 g by drying over diphosphorus pentoxide R at a pressure n ot exceeding 2.7 kPa. S u lfa te d a sh (2.4.14) M aximum 0.1 per cent, determined on 1.0 g. A SSA Y Dissolve 0.400 g in 25 m L o f a m ixture of equal volumes o f anhydrous ethanol R and water R. A dd 10 m L of 0.01 M hydrochloric add. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the 2 points of inflexion.

1 m L of 0.1 M sodium hydroxide is equivalent to 28.58 mg of C 15H 24CINO 2. STORA GE Protected from light. IM P U R IT IE S

Specified impurities C, D Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these impurities for demonstration of compliance. See also 5.10.

Control of impurities in substances for pharmaceutical use): A, B.

354.5

745-65-3

A ctio n a n d u se Prostaglandin E x (PG Ei) P h E ir __________________________________________________________________________________________

D E F IN IT IO N 7- [( 1 2R,3R) -3-Hydroxy-2-[( 1£,35) -3-hydroxyoa - 1-enyl] 5-oxocyclopentyl]heptanoic acid. C o n te n t 95.0 per cent to 102.5 p er cent (anhydrous substance). CHARACTERS A p p e a ra n c e W hite or slighdy yellowish, crystalline powder. S olu b ility Practically insoluble in water, freely soluble in ethanol (96 p er cent), soluble in acetone, slightly soluble in ethyl acetate. ID E N T IF IC A T IO N A Specific optical rotation (2.2.7): -7 0 to —60 (anhydrous substance). Immediately before use, dissolve 50 m g in ethanol (96per cent) R and dilute to 10.0 m l . with the same solvent. B. Infrared absorption spectrophotometry (2.2.24).

Comparison alprostadil CRS. C. Examine the chromatograms obtained in the assay.

H OH

0.

C20H 34O 5

.R1

and enantiomer

R2

A. R1 = O H , R 2 = C H 2-C H = C H 2: (2ftS)-3-[2-(prop-2enyl) phenoxy] propan- 1, 2-diol, C. R1 = N H -C H (C H 3) 2, R2 = C H = C H -C H 3: (2RS) -1 - [(1 -methylethyl)amino] -3- [2 -(prop-1-enyl) phenoxy] propan- 2-ol,

Results T he principal peak in the chromatogram obtained with the test solution is similar in retention time and size to the principal peak in the chromatogram obtained with the reference solution. TESTS R e la te d su b stan c e s Liquid chromatography (2.2.29). Prepare the solutions protected

from light. Test solution Dissolve 10.0 mg o f the substance to be exam in ed in a mixture o f equal volumes of acetonitrile R1 and water R and dilute to 10.0 mL with the same mixture of solvents.

ch2

B. 2-(prop-2-enyl)phenol,

Reference solution (a) Dilute 100 (xL o f the test solution to 20.0 m L with a mixture o f equal volumes of acetonitrile R1 and water R.

2016

1-108 Alprostadil

Reference solution (b) Dissolve 1.0 m g o f dinoprostone impurity C CRS (alprostadil impurity H ) and 1.0 m g o f the

Time Mobile phase A Mobile phase B (min)____________ (percent V7V)________ (per cent V7V) 0 100 0 -5 0

substance to be examined in a mixture o f equal volumes of acetomtrile R1 and water R and dilute to 20.0 m L with the sam e mixture o f solvents.

50-51

Reference solution (c) In order to prepare impurities A and B in situ, dissolve 1 mg o f the substance to be examined in 100 pL o f 1 M sodium hydroxide (the solution becomes

51 -61

0

100

61 -62

0+100

100 + 0

6 2 -7 2

100

0

brownish-red), wait for 3 min and add 100 |iL o f a 112 g/L solution o f phosphoric add R (yellowish-white opalescent solution); dilute to 5.0 m L with a mixture of equal volumes o f acetomtrUe R1 and water R. S y ste m A

100 + 0

0+100

Relative retention W ith reference to alprostadil (retention time = about 7 min): impurity A = about 2.4; impurity B = about 2.6.

System suitability:

Column: — size: I = 0.25 m , 0 = 4.0 mm; — stationary phase: base-deactivated octylsUyl silica gel for chromatography R (4 pm) with a pore size of 6 nm ; — temperature: 35 °C.

— resolution: m in im u m 1 .5 between the peaks due to impurity A and impurity B in the chrom atogram obtained w ith reference solution (c).

Mobile phase: — mobile phase A: dissolve 3.9 g of sodium dihydrogen phosphate R in water R and dilute to 1.0 L with the same

Limits: — correction factors: for the calculation of content, multiply

solvent; adjust to p H 2.5 with a 2.9 g/L solution of phosphoric add R (approximately 600 m L is required); to 740 m L o f the buffer solution add 260 m L o f acetomtrUe R1; — mobile phase B: dissolve 3.9 g o f sodium dihydrogen phosphate R in water R and dilute to 1.0 L with the same solvent; adjust to p H 2.5 with a 2.9 g/L solution o f phosphoric add R (approximately 600 m L is required); to 200 m L o f the buffer solution add 800 m L of acetomtrUe R1;

C arry out the test according to system A and B.

the peak areas o f the impurities listed in Table 1488.-1 by the corresponding correction factor; Table 1488.-1. Imparity impurity G

Relative retention Relative retention Correction factor (system A) (system B) 0.7 0.80 0.88

-

impurity D

0.90

-

1.0

impurity H

0.96

-

0.7

impurity E

1.10

-

0.7

impurity F

0.8

Time (min) 0 - 75

Mobile phase A (per cent V7V) 100

Mobile phase B (per cent V7V) 0

impurity C

1.36

1.9

impurity K

1.85

0.06

75 - 76

100-»0

0+100

impurity A

232

0.7

2.45

1.5

7 6 -8 6

0

100

impurity B

8 6 -8 7

0+100

100 + 0

impurity I

4.00

1.0

0

impurity J

5.89

1.0

87 - 102

100

Flow rate 1 mL/m in. Detection Spectrophotom eter at 200 nm . Injection 20 |iL. Retention time Alprostadil = about 63 m in . System suitability: — resolution: m in im u m 1.5 between the peaks due to im purity H and alprostadil in the chromatogram obtained with reference solution (b). S y ste m B U se the same conditions as for system A with the following mobile phase and elution programme: — mobile phase A: dissolve 3.9 g of sodium dihydrogen phosphate R in water R and dilute to 1.0 L with the same solvent; adjust to p H 2.5 with a 2.9 g/L solution o f phosphoric add R (approximately 600 m L is required); to 600 m L o f the buffer solution add 400 m L of

acetomtrUe R l; — mobile phase B: use mobile phase B as described under system A;

— impurity A: n o t more than 3 times the area of the principal peak in the chrom atogram obtained with reference solution (a) (1.5 per cent); — impurity B: n o t more than the area o f the principal peak in the chrom atogram obtained with reference solution (a) (0.5 per cent); — any other impurity: n o t m ore than 1.8 times the area o f the principal peak in the chrom atogram obtained with reference solution (a) (0.9 per cent), and n o t more than 1 such peak has an area greater than the area o f the principal peak in the chrom atogram obtained with reference solution (a) (0.5 per cent). Evaluate impurities appearing at relative retentions less than 1.2 by system A and impurities appearing at relative retentions greater than 1.2 by system B; — total: not more than 3 times the area o f the principal peak in the chromatogram obtained with reference solution (a) (1.5 per cent); — disregard limit. 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). W a te r ( 2.5.32) M axim um 0.5 per cent, determ ined on 50 mg.

2016

Alprostadil 1-109

ASSAY Liquid chromatography (2.2.29) as described in the test for related substances, system A. Prepare the solutions protected

from Ught. Test solution Dissolve 10.0 m g of the substance to be examined in a mixture of equal volumes of acetonitrile R1 and water R and dilute to 25.0 m L with the same mixture of solvents. Dilute 3.0 m L of the solution to 20.0 m L with a mixture o f equal volumes of acetonitrile R1 and water R.

H OH

E. 7-[(li?,2J?,35)-3-hydroxy-2-[(l£,35)-3-hydroxyoct-lenyl]-5-oxocyclopentyl]heptanoic acid (11-epiprostaglandin E i),

Reference solution Dissolve 5.0 mg of alprostadil CRS in a mixture of equal volumes of acetonitrile R1 and water R and dilute to 25.0 m L with the same mixture o f solvents. Dilute 6.0 m L o f the solution to 20.0 m L with a mixture of equal volumes o f acetonitrile R1 and water R. HO"

Injection 20 ^L.

H OH

Calculate the percentage content of C 20H 34O 5 taking into account the assigned content of alprostadil CRS. ST O R A G E At a tem perature o f 2 °C to 8 °C.

F . 7-[(l S,2R,3R)-3-hydroxy-2-[(1 £ ,3 6 )-3-hydroxyoct-1enyI]-5-oxocyclopentyl]heptanoic a d d ( 8-epiprostaglandin E i),

IM P U R IT IE S

H ’oh H OH

A 7- [(1 R,2S)-2- [(1 £,3.S)-3-hydroxyoct-l -enyI]-5oxocyclopent-3-enyI]heptanoic acid (prostaglandin A 0 ,

G . (5Z)-7- [(1 i?,2i?3^)-3-hydroxy-2-[(l£,35)-3-hydroxyoctl-enyI]-5-oxocydopentyl]hept-5-enoic a d d (dinoprostone),

O CO2H CH3 H

OH

B. 7- [2- [(1£,3.S)-3-hydroxyoct-1-enyl] -5-oxocydopent-1enyl]heptanoic a d d (prostaglandin B i),

H OH

H . (5£)-7-[(l.R,22?,3.R)-3-hydroxy-2-[(l£,3.S)-3-hydroxyoctl-enyI]-5-oxocydopentyl]hept-5-enoic ad d ((5£)-prostaglandin E 2),

H OH

C. 7-[(li?,2i?,3i?)-3-hydroxy-2-[(l£)-3-oxooct-l-enyI]-5oxocydopentyl]heptanoic a d d (15-oxoprostaglandin E j),

I. ethyl 7-[(1 i?,2i?,3i?)-3-hydroxy-2- [(1 £ ,3 5 )-3-hydroxyoctl-enyI]-5-oxocydopentyi]heptanoate (prostaglandin E i, ethyl ester),

H OH

D . 7-[(li?,2i?,3i?)-3-hydroxy-2-[(l£*3.R)-3-hydroxyoct-lenyl]-5-oxocyclopentyl]heptanoic a d d (15-epiprostagJandin E i),

H OH

J. 1-methylethyl 7-[(li?,2i?,3i?)-3-hydroxy-2-[(l£,36)-3hydroxyoct-l-enyl]-5-oxocyclopentyl]heptanoate (prostaglandin E i, isopropyl ester),

2016

1-110 Alteplase for Injection

If alteplase is stored in bulk form, stability (m aintenance o f potency) in the intended storage conditions m ust be demonstrated. T he production, purification and product consistency are checked by a num ber of analytical m ethods described below, carried out routinely as in-process controls. K. triphenylphosphine oxide. PhEur

Alteplase for Injection

★ ★

(Ph E ut monograph 1170)

*****

SYQVICRDEK

TQMIYQQHQS

WLRPVLRSNR

★ ★

VEYCWCNSGR

I------------- 1 = i ------------------- ' AQCHSVPVKS CSEPRCFNGG TCQQALYFSD FVCQCPEGFA ,____________ 1 " i_l GKCCEIDTRA

TCYEDQGISY

LAQKPYSGRR

PDAIRLGLGN

HNYCRNPDRD

SKPWCYVFKA

GKYSSEFCST

PACSEGNSDC

YFGNGSAYRG

THSLTESGAS

CLPWNSMILI

GKVYTAQNPS ?sj^AAQALGLGKHN

YCRNPDGDAK

LTWEYCDVPS

CSTCGLRQYS

QPQFR

AAIFAKHRRS

PGERFLCGGI

LISSC W ILSA

.PWCHVLKNRR 1—i

RGTWSTAESG

'------------------------------

r

AECTNWNSSA

'“ I

IKGGL FADIASHPWQ 1 AHCFQERFPP

HHLTVILGRT

YRWPGEEEQ

KFEVEKYIVH

KEFDDDTYDN

DIALLQLK5D

SSRCAQESSV

VRTVCLPPAD

LQLPDWTECE

LSGYGKHEAL

SPFYSERLKE

AHVRLYPSSR

CTSQHLLNRT

VTDNMLCAGD

TRSGGPQANL

HDACQGDSGG ■

PLVCLNDGRM

TLVGIISWGL

GCGQKDVPGV

YTKVTNYLDW

C 2736H 4174N 91 4O 824S 45

(non-glycosylated protein) 105857-23-6

A c tio n a n d u se Tissue-type plasm inogen activator; fibrinolytic. PhEur___________________________________________________________

D E F IN IT IO N Alteplase for injection is a sterile, fieeze-dried preparation o f alteplase, a tissue plasminogen activator produced by recom binant D N A technology. It has a potency of n o t less than 500 000 IU per milligram of protein. Tissue plasminogen activator binds to fibrin clots and activates plasminogen, leading to die generation of plasmin and to the degradation of fibrin clots or blood coagulates. Alteplase consists of 527 amino acids with a calculated relative molecular mass of 59 050 w ithout consideration o f the carbohydrate moieties attached at positions Asn 117, Asn 184 and Asn 448. T he total relative molecular mass is approximately 65 000. Alteplase is cleaved by plasmin betw een amino-acids 275 and 276 into a two-chain form (A chain and B chain) that are connected by a disulfide bridge betw een Cys 264 and Cys 395. T he single-chain form and the two-chain form show comparable fibrinolytic activity in

vitro. P R O D U C T IO N Alteplase is produced by recom binant D NA synthesis in cell culture; the fermentation takes place in serum-free m edium. T he purification process is designed to remove efficiendy potential impurities, such as antibiotics, D N A and protein contam inants derived both from the h ost cell and from the production m edium , and potential viral contaminants.

P ro te in c o n te n t T he protein concentration of alteplase solutions is determ ined by measuring the absorbance (2.2.25) o f the protein solution at 280 nm and at 320 nm , using formulation buffer as the compensation liquid. If dilution o f alteplase samples is necessary, the samples are diluted in formulation buffer. F or the calculation of the alteplase concentration, the absorbance value (A2so —-^320) is divided by the specific absorption coefficient for alteplase o f 1.9. P o te n c y T h e potency of alteplase is determ ined in an in vitro clot-lysis assay as described under Assay. T he specific activity o f bulk alteplase is approximately 580 000 IU per milligram o f alteplase. iV -te rm in a l se q u e n ce TV-terminal sequencing is applied to determ ine the correct N-terminal sequence and to determine semiquantitatively additional cleavage sites in the alteplase molecule, for example at position AA 275-276 o r at position AA 27-28. T he AT-terminal sequence m ust conform with the sequence o f hum an tissue plasminogen activator. Is o e le c tric fo cu sin g T he consistency in the microheterogeneity o f gjycosylation of the alteplase molecule can be dem onstrated by isoelectric focusing (IEF). A complex banding pattern with 10 major and several minor bands in the p H range 6.5-8.5 is observed. Denaturing conditions are applied to achieve a good separation o f differently charged variants o f alteplase. T he broad charge distribution observed is due to a population o f molecules, which differ in the fine structure o f biantenary and triantenary complex-type carbohydrate residues, with different degrees o f substitution with sialic acids. T h e banding pattern o f alteplase test samples m ust be consistent w ith the pattern of alteplase reference standard. S in g le -c h a in a lte p la se c o n te n t T h e alteplase produced by C H O (Chinese ham ster ovary) cells in serum-free medium is predom inantly single-chain alteplase. T h e single-chain form can be separated from the two-chain form by gel-permeation liquid chromatography under reducing conditions as described under Single-chain content (see Tests). T h e single-chain alteplase content in bulk samples m ust be higher than 60 p er cent. T ry p tic -p e p tid e m a p p in g T he primary structure o f the alteplase molecule is verified by tryptic-peptide mapping as described under Identification B. T h e reduced and carboxymethylated molecule is cleaved by trypsin into about 50 peptides, which are separated by reverse-phase liquid chromatography. A characteristic chrom atogram (fingerprint) is obtained. T h e identity of the tryptic-peptide map o f a given alteplase sample with the profile o f a well-characterised reference standard is an indirect confirmation of the amino-acid sequence, becam e even single amino-acid exchanges in individual peptides can be detected by this sensitive technique. In addition, complex peaks o f the glycopeptides can be isolated from the trypticpeptide m ap and separated in a second dimension, either by reverse-phase liquid chrom atography under modified conditions o r by capillary electrophoresis. By this two­

Alteplase for Injection 1-111

2016

dimensional separation of glycopeptide variants, lot-to-lot consistency of the microheterogeneity o f glycosylation can be demonstrated. T he tryptic-peptide map o f alteplase samples must be consistent with the tryptic-peptide map of alteplase reference standard.

Monomer content T h e m onom er content of alteplase is m easured by gelperm eation liquid chromatography under non-reduced conditions as described under M onomer content (see Tests). T he m onom er content of alteplase bulk samples must be higher than 95 per cent

Type I/Type II alteplase content C H O cells produce 2 glycosylation variants o f alteplase. Type I alteplase contains 1 polymannose-type glycosylation at position Asn 117 and 2 complex-type glycosylation sites at positions Asn 184 and Asn 448. Type II alteplase is only glycosylated at positions Asn 117 and Asn 448. T he ratio o f Type I/Type II alteplase is constant in the range o f 45 to 65 per cent of Type I and 35 to 55 per cent of Type II. T h e content of alteplase Type I and Type II can be determ ined by a densitometric scan o f SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel. Plasmin-treated samples of alteplase, which are reduced and carboxymethylated before loading on the gel, are separated into 3 bands: Type I alteplase A-chain (AA 1-275), Type II alteplase A-chain (AA 1-275) and alteplase B-chain (AA 276-527). T h e ratio o f Type I/Type II alteplase is determ ined from a calibration curve, which is obtained by a densitometric scan of defined mixtures o f purified Type I alteplase and T ype II alteplase standards.

SDS-PAGE SD S-PAG E (silver staining) is used to demonstrate purity of the alteplase bulk material and the integrity o f the alteplase molecule. F or alteplase bulk samples, no additional protein bands compared to reference standard or degradation products m ust occur in SDS-PAGE gels at a loading am ount o f 2.5 |xg alteplase protein per lane and a limit of detection of 5 ng per protein (BSA) band.

Bacterial endotoxins (2.6.14) Less than 1 IU p er milligram of alteplase.

Sialic adds Proceed using a suitable validated m ethod devdoped according to general chapter 2.2.59. Glycan analysis of glycoproteins. T h e sialic ad d s content for the test samples m ust be in the range of 70 to 130 per cent compared to alteplase reference standard, which contains about 3 moles of sialic a d d s per mole of alteplase.

Neutral sugars Dilute alteplase samples and the reference standard in the assay buffer, containing 34.8 g/L of arginine R, 0.1 g/L of polysorbate 80 R and adjusted to pH 7.4 with phosphoric acid R, to a protein concentration of 50 [ig/m L Prepare the following concentrations o f mannose in the same assay buffer for a calibration curve: 20, 30, 40, 50 and 60 (ig/mL. Pipette 2 m L of alteplase samples and reference standard, as well as 2 m L o f each mannose concentration in duplicate in reagent tubes. A dd 50 jaL of phenol R, followed by 5 m L of sulfuric acid R, in each reagent tube. Incubate the mixture for 30 m in at room tem perature. M easure the absorbance at 492 nm for each tube. Read the content of neutral sugars from the mannose calibration curve. T he neutral sugar content is expressed in moles of neutral sugar per mole of alteplase, taking into account the dilution factor for alteplase samples

and reference standard and using a relative molecular mass of 180.2 for mannose and a relative molecular mass of 59 050 for the alteplase protein moiety. T h e neutral sugar content of the alteplase samples m ust be in the range o f 70 to 130 per cent compared to alteplase reference standard, which contains about 12 moles of neutral sugar per mole of alteplase.

CHARACTERS W hite or slightly yellow powder or solid friable mass.

Reconstitute the preparation as stated on the label immediately before carrying out the Identification, Tests (except those for solubility and water) and Assay. IDENTIFICATION A. T he assay serves also to identify the preparation. B. Tryptic-peptide mapping. Examine by liquid chromatography (2.2.29).

Test solution Dilute the preparation to be examined with water R to obtain a solution containing about 1 mg of alteplase per millilitre. Dialyse about 2.5 m L o f the solution for at least 12 h into a solution containing 480 g/L of urea R, 44 g/L o f tris(hydroxymethyl) aminomethane R and 1.5 g/L of sodium edetate R and adjusted to p H 8.6, using a mem brane with a cut-oflf point corresponding to a relative molecular mass o f 10 000 for globular proteins. Measure the volume of the solution, transfer it to a clean test-tube and add per millilitre 10 |iL o f a 156 g/L solution of dithiothreitol R. Allow to stand for 4 h, cool in iced water and add per millilitre of solution 25 pL o f a freshly prepared 190 g/L solution of iodoacetic add R. Allow to stand in the dark for 30 min. A dd per millilitre 50 |oL of dithiothreitol solution to stop the reaction. Dialyse for 24 h against an 8 g/L solution of ammonium hydrogen carbonate R. Add 1 part of trypsin for peptide mapping R to 100 parts o f the protein and allow to stand for 6 h to 8 h. Repeat the addition of trypsin and allow to stand for a total of 24 h.

Reference solution Prepare as for the test solution using a suitable reference standard instead of the preparation to be examined. T he chromatographic procedure may be carried out using: — a column 0.1 m long and 4.6 mm in internal diameter packed with octadecylsüyl silica gelfor chromatography R (5 jim to 10 pm); Mobile phase A 8 g/L solution of sodium dikydrogen phosphate R , adjusted to p H 2.85 with phosphoric acid R, filtered and degassed; Mobile phase B 75 per cent V/V solution of acetomtrUe R in mobile phase A; — as detector a spectrophotometer set at 210 nm. Equilibrate the system with mobile phase A at a flow rate of 1 mL/min. After injection of the solution, increase the proportion of mobile phase B at a rate of 0.44 per cent per m inute until the ratio of mobile phase A to mobile phase B is 60:40, then increase the proportion of mobile phase B at a rate o f 1.33 per cent per minute until the ratio o f mobile phase A to mobile phase B is 20:80 and then continue elution with this mixture for a further 10 min. Record the chromatogram for the reference solution: the test is not valid unless the resolution of peaks 6 (peptides 268-275) and 7 (peptides 1-7) is at least 1.5; whf and are n o t more than 0.4 min. Inject about 100 |iL o f the test solution and record the chromatogram. Verify the identity of the peaks by comparison with the chromatograms of the reference solution. There should n o t be any additional significant peaks o r shoulders, a significant peak or shoulder being defined as

1-112 Alteplase for Injection

one with an area response equal to or greater than 5 p er cent of peak 19 (peptides 278-296); no significant peak is missing. A type chromatogram for identification of the peaks cited is shown in Figure 1170.-1. TESTS A p p e a ra n c e o f so lu tio n T he reconstituted preparation is clear (2.2. I) and n o t more intensely coloured than reference solution Y7 (2.2.2,

Method II). p H (2.2.3) 7.1 to 7.5. S o lu b ility A dd the volume o f the liquid stated on the label. T h e preparation dissolves completely within 2 m in at 20 °C to 25 °C. P r o te in c o n te n t Prepare a solution of the substance to be examined with an accurately known concentration of about 1 g/L. Using a 34.8 g/L solution of arginine R adjusted to p H 7.3 with phosphoric acid R, dilute an accurately measured volume o f the solution o f the substance to be examined so that the absorbance measured at the maximum at about 280 nm is 0.5 to 1.0 (test solution). M easure the absorbance (2.2.25) o f the solution at the maximum at about 280 nm and at 320 nm using the arginine solution as the compensation liquid. Calculate the protein content in the portion o f alteplase taken from the following expression:

V (-^280 ~ -glucopyranosyl)-l-N-[(25)-4-amino-2hydroxybutanoyl] -2-deoxy-D-strq)tamine sulfate. Antimicrobial substance obtained from kanamycin A. G . 6 -0 - (3-amino-3-deoxy-a-D-glucopyranosyl)-4-0(6-amino-6-deoxy-a-D-glucopyranosyl)-l-N-[(2R)-4-amino-2hydroxybutanoyl] -2-deoxy-D-streptamine,

Semi-synthetic product derived from a fermentation product. C o n te n t 96.5 per cent to 102.0 per cent (dried substance).

CHARACTERS A p p e a ra n c e White or almost white powder.

Solubility Freely soluble in water, practically insoluble in acetone and in ethanol (96 p er cent). ID E N T IF IC A T IO N A. Infrared absorption spectrophotometry (2.2.24).

Comparison amikacin sulfate CRS. B. Thin-layer chromatography (2.2.27).

1-136 Amikacin Sulfate

2016

Test solution Dissolve 25 mg o f the substance to be examined in water R and dilute to 10 m L with the same solvent. Reference solution (a) Dissolve 25 m g of amikacin sulfate CRS in water R and dilute to 10 m l. with the same solvent. Reference solution (b) Dissolve 5 mg o f kanamycin monosulfate CRS in 1 m L o f the test solution and dilute to 10 m L with water R. Plate TLC silica gel plate R Mobile phase methylene chloride R, ammonia R, methanol R (25:30:40 V/V/V). Application 5 |iL. Development Over 3/4 of the plate. Drying In air. Detection Spray with mnhydrin solution R1 and heat at 110 °C for 5 min.

System suitability: reference solution (b): —

the chromatogram shows 2 clearly separated spots.

Results The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). C. It gives reaction (a) of sulfates (2.3.1). TESTS p H (2.2.3) 2.0 to 4.0. Dissolve 0.1 g in carbon dioxide-free water R and dilute to 10 m L with the same solvent. S pecific o p tic a l ro ta tio n (2.2.7) + 76 to + 84 (dried substance). Dissolve 0.50 g in water R and dilute to 25.0 m L with the sam e solvent. R e la te d su b s ta n c e s Liquid chrom atography (2.2.29).

Test solution Dissolve 33 m g o f the substance to be examined in mobile phase A and dilute to 50.0 m L with mobile phase A Reference solution (a) Dilute 1.0 m L o f the test solution to 100.0 m L w ith mobile phase A. Reference solution (b) Dilute 1.0 m L o f reference solution (a) to 10.0 m L with mobile phase A.

Reference solution (c) Dissolve 5 mg o f amikacin for system suitability CRS (containing impurities A, B, F and H ) in mobile phase A and dilute to 10 m l. with mobile phase A.

Reference solution (d) Dissolve 6.6 m g o f amikacin impurity I CRS in mobile phase A and dilute to 20.0 m L with mobile phase A. Dilute 1.0 m l. o f the solution to 100.0 m L w ith mobile phase A.

Column: — size. I = 0.25 m, 0 = 4.6 mm; — stationary phase: end-capped octadecyhüyl silica gel for chromatography R (5 pm); — temperature: 40 °C. Mobile phase: — mobile phase A: a mixture prepared with carbon dioxide-free water R, containing 1.8 g/L o f sodium octanesulfonate R, 20 g/L o f anhydrous sodium sulfate R l, 1.4 per cent V/V o f tetrahydrofuran R, and 5 per cent V/V of 0.2 M potassium dihydrogen phosphate R previously adjusted to p H 3.0 with dilute phosphoric acid R; degas; — mobile phase B: a mixture prepared with carbon dioxide-free water R, containing 1.8 g/L o f sodium octanesulfonate R, 28 g/L o f anhydrous sodium sulfate R l , 1.4 per cent V/V of

tetrahydrqfuran R, and 5 per cent V/V o f 0.2 M potassium dihydrogen phosphate R previously adjusted to p H 3.0 with dilute phosphoric add R; degas; Time (min) 0-3

Mobile phase A (per cent V/V) 100

Mobile phase B (per cent V/V) 0

3 - 38.0

1 00*30

0*70

38.0 - 38.1

30* 0

7 0 * 100

38.1 - 68

0

100

Flow rate 1.0 mL/min. Post-column solution M ixture o f 1 volume of carbonate-free sodium hydroxide solution R and 24 volumes o f previously degassed carbon dioxide-free water R, which is added in a pulseless m anner to the colum n effluent using a 375 pL polymeric mixing coil.

Flow rate of post-column solution 0.3 mL/min. Detection Pulsed amperometric detector or equivalent with a gold indicator electrode, a silver-silver chloride reference electrode, and a stainless steel auxiliary electrode which is the cell body, held at respectively + 0.05 V detection, + 0.75 V oxidation and — 0.15 V reduction potentials, with pulse durations according to the instrum ent used.

Injection 20 |iL. Identification of impurities Use the chromatogram supplied with armkadn for system suitability CRS and the chromatogram obtained with reference solution (c) to identify the peaks due to impurities A, B, F and H ; use the chromatogram obtained with reference solution (d) to identify the peak due to im purity I.

Relative retention W ith reference to amikacin (retention time = about 28 min): impurity I = about 0.13; impurity F = about 0.92; impurity B = about 0.95; impurity A = about 1.62; im purity H = about 1.95. System suitability: reference solution (c): — peak-to-vaUey ratio: minimum 5, where Hp = height above the baseline of the peak due to im purity B and Hv = height above the baseline o f the lowest point o f the curve separating this peak from the peak due to amikacin; if necessary, adjust the volume o f tetrahydrofuran in the mobile phase.

Calculation of percentage contents: — for impurity I, use the concentration o f im purity I in reference solution (d); — for impurities other than I, use the concentration of amikacin sulfate in reference solution (a).

Limits. — impurities A , B, F, H, I: for each impurity, maximum 0.5 p er cent; — arty other impurity: for each impurity, m axim um 0.5 p er cent; — total: maximum 1.5 p er cent; — reporting threshold: 0.1 p er cent.

Sulfate 23.3

per cent to 25.8 per cent (dried substance).

Dissolve 0.250 g in 100 m L o f water R and adjust the solution to p H 11 using concentrated ammonia R. A dd 10.0 m L o f 0.1 M barium chloride and about 0.5 m g of phthalein purple R Titrate with 0.1 M sodium edetate adding 50 m l. of ethanol (96 per cent) R when the colour o f the solution begins to change and continue the titration until the violet-blue colour disappears.

Amikacin Sulfate 1-137

2016 1 m l. o f 0.1 M barium chloride is equivalent to 9.606 mg of sulfate (SO 4). L oss o n d ry in g (2.2.32) M aximum 13.0 p e r cent, determined on 0.500 g by drying in an oven at 105 °C at a pressure not exceeding 0.7 kPa for 3 h. P y ro g e n s (2.6.8) If intended for use in the manufacture of parenteral preparations w ithout a further appropriate procedure for the removal of pyrogens, it complies with the test for pyrogens. Inject per kilogram o f the rabbit’s mass 5 m L o f a solution containing 25 m g o f the substance to be examined in water

for injections R.

A. 4-0-(3-amino-3-deoxy-a-D-glucopyranosyl)-6-0-(6-amino6 -deoxy-a-D-ghicopyranosyl)-1-N-[(2S) -4-amino-2 hydroxybutanoyl] - 2-deoxy-L-streptamine,

A SSAY Liquid chrom atography (2.2.29).

Test solution Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 10.0 m L with the mobile phase. Reference solution Dissolve 50.0 mg o f amikacin sulfate CRS in the mobile phase and dilute to 10.0 m L with the mobile phase.

Column'. — sizer. I — 0.25 m , 0 = 4.6 mm; — stationary phase', end-capped octadecylsUyl silica gel for chromatography R (5 |am); — temperature: 40 °C.

Mobile phase A mixture prepared with carbon dioxide-free water R, containing 1.8 g/L o f sodium octanesulfonate R, 20 g/L o f anhydrous sodium sulfate R l, 5.8 p er cent V/V of acetonitrile R l, and 5 per cent VIV o f 0.2 M potassium dihydrogen phosphate R previously adjusted to p H 3.0 with dilute phosphoric acid R; degas. Flow rate 1.0 m l7min. Detection Spectrophotom eter at 200 nm. Injection 20 |iL. Rim time 1.3 times the retention time of amikacin. Retention time Amikacin = about 30 min. System suitability: reference solution: — symmetry factor, maximum 1.5 for the peak due to amikacin; if necessary, adjust the am ount o f acetonitrile R l in the mobile phase; peak splitting may be observed when the retention tim e becomes too short; — repeatability: m axim um relative standard deviation of 1.5 per cent after 6 injections.

B. 4-0-(3-amino-3-deoxy-a-D-ghicopyranosyl)-6-0-(6-amino6-deoxy-a-D-glucopyranosyl)-1,3-N-bis [(25)-4-amino-2hydroxybutanoyl] -2-deoxy-L-streptamine,

C. 4-0-(6-amino-6-deoxy-a-D-glucopyranosyl)-6-0-[3-[[(25)-

4-amino-2-hydroxybutanoyl] amino]-3-deoxy-a-Dglucopyranosyl] -2-deoxy-D-streptamine,

Calculate the percentage content o f C 22H 47N 5O 21S 2 taking into account the assigned content o f amikacin sulfate CRS. STO R A G E If the substance is sterile, store in a sterile, airtight, tam per­ proof container. IM P U R IT IE S

Specified impurities A, B, F, H , I Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other of the tests in the m onograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by die general m onograph Substances for pharmaceutical use (2034). It is therefore n o t necessary to identify these impurities for dem onstration o f compliance. See also 5.10. Control of impurities in substances for pharmaceutical use): C, D,

E, G.

D . 6-0-(3-amino-3-deoxy-a-D-ghicopyranosyl)-4-0-

(6-amino-6-deoxy-a-D-glucopyranosyl)-2-deoxy-D-streptamine (kanamycin),

1-138 Amiloride Hydrochloride

2016

Amiloride Hydrochloride (Ph. Eur. monograph 0651)

* NH

O

CK

E. 4-0-(3-amino-3-deoxy-a-D-giucopyranosyl)-6-0-[6- [ [(25)-

4-amino-2-hydroxybutanoyI]amino]-6-deoxy-a-Dglucopyranosyfl-2-deoxy-L-streptamine,

JLN J k NH,

XX H Y

H2N

.N. N

C6HgQ2N70j2H20

, HCI, 2 H ,0 .

NH2

302.1

17440-83^1

Action and use Sodium channel blocker; potassium-sparing diuretic.

Preparations Amiloride Tablets Co-amilofruse Tablets Co-amilozide Oral Solution Co-amilozide Tablets PhEur__________________________________________________________

DEFINITION F. 6-0-(3-amino-3-deoxy-a-D-glucopyranosyl)-4-0- [6- [(25)-

4-amino-2-hydroxybutanoyl] amino-6-deoxy-a-DglucopyranosyQ-l-N- [(25)-4-amino-2-hydroxybutanoyi]-2deoxy-D -streptam ine}

3,5-Diarnino-Ar-carbamirnidoyl-6-chloropyrazine-2carboxamide hydrochloride dihydrate.

Content 98.0

per cent to 101.0 per cent (anhydrous substance).

CHARACTERS Appearance Pale yellow or greenish-yellow powder.

Solubility Slightly soluble in water and in anhydrous ethanol.

IDENTIFICATION First identification A, D. Second identification B, C, D. A. Infrared absorption spectrophotom etry (2.2.24). G. 6-0-(3-amino-3-deoxy-a-D-glucopyranosyl)-4-0-

(6-araino-6-deoxy-a-D-glucopyranosyl)-l-7V-[(2i?l)-4-ainino-2hydroxybutanoyl]-2-deoxy-D-streptamine,

H . 6-0-(3-amino-3-deoxy-a-D-gJucopyranosyJ)-l-N-[(25)-4amino-2-hydroxybutanoyI] -4-0- (2,6-diamino-2, 6-dideoxy-a-

D-gJucopyranosyl)-2-deoxy-D-streptaminej H° 2 C ^ ^ , N H 2 H OH

I. (25)-4-amino-2-hydroxybutanoic add. ___________________________________________________________ PhEur

Comparison amiloride hydrochloride CRS. B. Thin-layer chromatography (2.2.27).

Test solution Dissolve 40 m g o f the substance to be examined in methanol R and dilute to 10 m L w ith the same solvent. Reference solution Dissolve 40 m g o f amiloride hydrochloride CRS in methanol R and dilute to 10 m L with the same solvent. Plate TLC silica gel plate R. Mobile phase dilute ammonia R l, water R, dioxan R (6:6:88 VIV/V); freshly prepared mixture. Application 5 |iL. Development Over 2/3 of the plate. Drying In air. Detection Examine in ultraviolet light at 365 nm. Results T h e principal spot in the chrom atogram obtained with the test solution is similar in position, fluorescence and size to the principal spot in the chrom atogram obtained w ith the reference solution. C. Dissolve about 10 m g in 10 m L o f water R. Add 10 m L o f a 200 g/L solution of cetrimide R} 0.25 m L of dilute sodium hydroxide solution R and 1 m L o f bromine water R. A greenishyellow colour is produced. Add 2 m L o f dilute hydrochloric acid R. T h e solution becomes deep yellow and shows blue fluorescence in ultraviolet light at 365 nm. D. It gives reaction (b) o f chlorides (2.3.1).

Aminobenzoic Acid 1-139

2016

TESTS

Free acid Dissolve 1.0 g in a mixture o f 50 m L o f methanol R and 50 m L o f water R and titrate with 0.1 M sodium hydroxide, determ ining the end-point potentiometrically (2.2.20). N o t m ore than 0.3 m L o f 0.1 M sodium hydroxide is required to reach the end-point.

Related substances Liquid chrom atography (2.2.29).

Test solution Dissolve 20.0 m g o f the substance to be examined in a mixture of 1 volume o f acetonitrile R and 3 volumes o f water R and dilute to 10.0 m L with the same mixture o f solvents.

Reference solution (a) Dilute 1.0 m L o f the test solution to 100.0 m L with a mixture o f 1 volume o f acetomtrUe R and 3 volumes o f water R. Reference solution (b) Dilute 1.0 m L o f reference solution (a) to 10.0 m l, with a mixture o f 1 volume o f acetonitrUe R and 3 volumes of water R. Reference solution (c) Dissolve 5.0 m g o f amHoride impurity A CRS in a mixture of 1 volume o f acetonitrUe R and 3 volumes of water R and dilute to 5.0 m L with die same

ASSAY Dissolve 0.200 g in a mixture o f 5.0 m L o f 0.01 M hydrochloric add and 50 m L of ethanol (96 per cent) R. Carry out a potentiom etric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the 2 points o f inflexion. 1 m L of 0.1 M sodium hydroxide is equivalent to 26.61 m g Of C6H 9CI2N 7O. STORAGE Protected from light. IM P U R IT IE S

Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one o r other o f the tests in the monograph. T hey are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these impurities for demonstration o f compliance. See also 5.10. Control of impurities in substances for pharmaceutical use): A .

,c h 3

mixture o f solvents. Dilute 1.0 m L o f this solution to 100.0 m L with a mixture of 1 volume o f acetonitrUe R and 3 volumes o f water R.

Column: — sizer. I = 0.25 m , 0 = 4.6 mm; — stationary phase: octadecylsHyl silica gel for chromatography R (5 pm ).

Mobile phase M ix 5 volumes o f tetramethylammoraum hydroxide solution R, 250 volumes o f acetonitrUe R and 745 volumes of water R ; adjust to p H 7.0 with a mixture o f 1 volume of phosphoric add R and 9 volumes o f water R. Adjust the concentration o f acetonitrile in the mobile phase so that the retention time o f im purity A is 5-6 min (an increase in the concentration o f acetonitrile results in a shorter retention time). A djust die concentration o f tetramethylammonium hydroxide and o f phosphoric a d d keeping the p H at 7.0 so that the retention time of amiloride is 9-12 min (an increase in the concentration results in a shorter retention time for amiloride).

Flow rate 1 mL/min. Detection Spectrophotom eter at 254 run. Irqection 20 pL. Run time 5 times the retention time o f amiloride. System suitability: reference solution (b): — signal-to-noise ratio: m inim u m 5.0 for the peak due to amiloride.

Limits: — unspecified impurities: for each impurity, n o t more than 0.2 tim es the area of the peak due to impurity A in the chrom atogram obtained with reference solution (c) (0.10 per cent); — total: not m ore than the area o f the peak due to im purity A in the chromatogram obtained with reference solution (c) (0.5 per cent); — disregard limit. 0.1 times the area of the peak due to im purity A in the chromatogram obtained with reference solution (c) (0.05 per cent).

Water (2.5.12) 11.0 per cent to 13.0 per cent, determ ined on 0.200 g.

Sulfated ash (2.4.14) M axim um 0.1 p er cent, determined on 1.0. g.

HjN

N

NH2

A. methyl 3,5-diamino-6-chloropyrazine-2-carboxylate. PhEur

Aminobenzoic Acid (4-Aminobenzoic Add, Ph Eur monograph 1687)

h 2n

CzHyNOa

*

★

* * *★

★

* * * * *

XT 137.1

150-13-0

A ctio n a n d u se Skin protective. PhEtr__________ __

D E F IN IT IO N 4-Aminobenzoic acid. C o n te n t 99.0 p er cent to 101.0 per cent (anhydrous substance). CHARACTERS A p p e a ra n c e W hite o r slightly yellow, crystalline powder. S o lu b ility Slightly soluble in water, freely soluble in alcohol. It dissolves in dilute solutions o f alkali hydroxides. ID E N T IF IC A T IO N

First identification B Second identification A , C A. M elting point (2.2.14): 186 °C to 189 °C. B. Infrared absorption spectrophotometry (2.2.24). Comparison 4-aminobenzoic add CRS. C. Thin-layer chromatography (2.2.27).

1-140 Aminobenzoic Acid

Test solution Dissolve 20 mg o f the substance to be examined in methanol R and dilute to 20 m L w ith the same solvent. Reference solution (a) Dissolve 20 m g o f 4-aminobenzoic add CRS in methanol R and dilute to 20 m L with the same solvent.

Reference solution (b) Dissolve 10 m g o f 4-nitrobenzoic acid R in 10 m L o f reference solution (a).

Plate Suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm as the coatin g substance.

Mobile phase glacial acetic add R, hexane R, methylene ' chloride R (5:20:75 VIVIV). Application 1 |iL. Development Over a path of 10 cm. Drying In air. Detection Examine in ultraviolet light at 254 nm. System suitability T he chromatogram obtained with reference solution (b) shows 2 clearly separated spots.

Results T he principal spot in the chrom atogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a). TESTS A p p e a ra n c e o f so lu tio n The solution is clear (2.2.1) and no t m ore intensely coloured than reference solution B5 (2.2.2, Method II). Dissolve 1.0 g in alcohol R and dilute to 20 m L with the same solvent.

Related substances Liquid chromatography (2.2.29).

Test solution Dissolve 25.0 mg o f the substance to be examined in the mobile phase and dilute to 100.0 m L with the mobile phase.

Reference solution Dissolve 25.0 m g o f 4-nitrobenzoic add R and 25.0 m g o f benzocaine R in methanol R and dilute to 100.0 m L with the same solvent. D ilute 1.0 m L to 50.0 m L with the mobile phase. Dilute 1.0 m L o f this solution to 10.0 m L with the mobile phase.

Column: — size. I — 0.12 m , 0 = 4.0 m m , — stationary phase: octylsUyl silica gel for chromatography R (5 nm).

Mobile phase Mix 20 volumes o f a mixture o f 70 volumes of acetomtrUe R and 80 volumes o f methanol R, and 80 volumes of a solution containing 1.5 g/L o f potassium dihydrogen phosphate R and 2.5 g/L of sodium octanesulfoncae R adjusted to p H 2.2 with phosphoric add R. Flow rate 1.0 mlVmin. Detection Spectrophotom eter at 270 nm . Injection 20 |iL. Run time 11 times the retention time o f 4-aminobenzoic add. Relative retention W ith reference to 4-aminobenzoic a d d (retention tim e = about 3 min): impurity A = about 4; impurity B = about 9.

2016

— any other impurity: not more than 0.5 times the area o f the peak due to im purity A in the chrom atogram obtained with the reference solution (0.1 p er cent), — total: n o t more than 2.5 times the area o f the peak due to impurity A in the chrom atogram obtained with the reference solution (0.5 p er cent), — disregard limit: 0.1 times the area o f the peak due to impurity A in the chrom atogram obtained with the reference solution (0.02 per cent).

Impurity C and im purity D Gas chromatography (2.2.28). Internal standard solution Dissolve 20.0 m g o f lauric add R in methylene chloride R and dilute to 100.0 m L with the same solvent.

Test solution Dissolve 1.000 g o f the substance to be examined in 10.0 m L o f an 84 g/L solution o f sodium hydroxide R and extract with 2 quantities, each o f 10 mT, of methylene chloride R. Combine and wash with 5 m L of water R; filter through anhydrous sodium sulfate R. W ash the filter with methylene chloride R. Evaporate in a water-bath at 50-60 °C to obtain a volume o f about 1-5 mL. A dd 1.0 m L o f the internal standard solution and dilute to 10.0 m L with methylene chloride R. Reference solution (a) Dissolve 20.0 m g o f aniline R in methylene chloride R and dilute to 100.0 m L w ith the same solvent.

Reference solution (b) Dissolve 20.0 m g o f p-tohddine R in methylene chloride R and dilute to 100.0 m L with the same solvent.

Reference solution (c) Dilute 0.50 m L o f reference solution (a), 0.50 m L o f reference solution (b) and 10.0 m L o f the internal standard solution to 100.0 m L with methylene

chloride R. Column: — material: fused silica, — size. I = 30 m , 0 = 0.32 m m , — stationary phase. poly[methyl(95)phenyl(5)JsUoxane R (film thickness 0.5 nm).

Carrier gas helium for chromatography R. Flow rate 1.0 mL/min. Split ratio 1:10. Temperature:

Column

H um (min) 0 -4

Temperature (°C) 130

4-6.5

130->180

6.5 -11.5

180

Injection port

280

Detector

300

Detection Flame ionisation. Injection 2 nL; inject the test solution and reference

Limits:

solution (c).

— impurity A: not m ore than the area o f the corresponding peak in the chromatogram obtained with the reference solution (0.2 per cent), — impurity B: n o t more than the area o f the corresponding peak in the chromatogram obtained with the reference solution (0.2 per cent),

Retention time Internal standard = about 9.5 min. Limits: — impurity C: calculate th e ratio (K) o f the area o f the peak due to impurity C to the area of the peak due to the internal standard from the chromatogram obtained with reference solution (c); calculate the ratio o f the area o f the peak due to impurity C to the area o f the peak due to the internal standard from the chrom atogram obtained with

Aminocaproic Acid 1-141

2016 the test solution: this ratio is not greater than R (10 ppm ), — impurity D: calculate the ratio (R) of the area o f the peak due to impurity D to the area of the peak due to the internal standard from the chromatogram obtained with reference solution (c); calculate the ratio o f the area o f the peak due to impurity D to the area o f the peak due to the internal standard from the chromatogram obtained with the test solution: this ratio is not greater than R (10 ppm ). Ir o n (2.4.9) M axim um 40 ppm .

CHARACTERS A white or almost white, crystalline pow der or colourless crystals, fredy soluble in water, slightly soluble in alcohol. It melts at about 205 °C with decomposition. ID E N T IF IC A T IO N

First identification A. Second identification B, C, D. A. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with aminocaproic acid CRS. Examine the substances prepared as discs. B. Examine the chromatograms obtained in the test for ninhydrin-positive substances. T h e p rindpal spot in the chromatogram obtained with the test solution (b) is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a).

Dissolve 0.250 g in 3 m L of alcohol R and dilute to 10.0 m L w ith water R. H e a v y m e ta ls (2.4.8) M axim um 20 ppm . 1.0 g complies with test C . Prepare the reference solution using 2 m L o f lead standard solution (10 ppm Pb) R.

C. Dissolve 0.5 g in 4 m L of a mixture o f equal volumes of

dilute hydrochloric acid R and water R Evaporate to dryness by

W a te r (2.5.12) M axim um 0.2 p er cent, determined on 1.00 g.

heating on a water-bath. Dry the residue in a desiccator. Dissolve the residue in about 2 m L o f boiling ethanol R. Allow to cool and maintain at 4 °C to 8 °C for 3 h. Filter under reduced pressure. The residue washed with about 10 m L o f acetone R and dried at 60 °C for 30 min, m dts (2.2.14) at 131 °C to 133 °C.

S u lfa te d a s h (2.4.14) M axim um 0.1 per cent, determined on 1.0 g. A SSA Y Dissolve 0.100 g with heating in 50 m L o f carbon dioxide-free water R. T itrate with 0.1 M sodium hydroxide determining the end-point potentiometrically (2.2.20).

D . Dissolve about 5 mg in 0.5 m L o f distilled water R. A dd 3 m L o f dimetkylformarmde R and 2 m L of ascorbic acid solution R. H eat on a water-bath. An orange colour develops.

1 m L o f 0.1 M sodium hydroxide is equivalent to 13.71 m g o f C 7H 7N 0 2.

TESTS S o lu tio n S Dissolve 10.0 g in carbon dioxide-free water R and dilute to 50.0 m L w ith the same solvent.

STORA GE Protected from light. IM P U R IT IE S

A p p e a ra n c e o f so lu tio n Solution S is colourless (2.2.2, Method II) and remains clear (2.2.1) on standing for 24 h. p H (2.2.3) T h e p H o f solution S is 7.5 to 8.0. A b so rb a n c e (2.2.25) A. T h e absorbance o f solution S at 287 nm is not more th an 0.10 and at 450 nm is not more th an 0.03.

A. R = C 0 2H , R ' = N 0 2: 4-nitrobenzoic ad d , B. R = C 0 - 0 - C 2H 5, R ' = N H 2: ethyl 4-aminobenzoate (benzocaine), C. R = H , R ' = N H 2: aniline, D . R = C H 3, R ' = N H 2: 4-methylaniline (p-toluidine). __________________________________________________________ PhEur

Aminocaproic Acid

i***% *+

(Ph Eur monograph 0874)

*

B. Place 2.0 g in an even layer in a shallow dish 9 cm in diameter, cover and allow to stand at 98 °C to 102 °C for 72 h. Dissolve in water R and dilute to 10.0 m L with the same solvent. T h e absorbance o f the solution at 287 nm is no t more than 0.15 and at 450 nm is n o t more than 0.03. N in h y d rin -p o sitiv e su b stan c es Examine by thin-layer chromatography (2.2.27), using a suitable silica gel as the coating substance.

Test solution (a) Dissolve 0.10 g of the substance to be examined in water R and dilute to 10 m L with the same solvent. Test solution (b) Dilute 1 m L o f test solution (a) to 50 m L w ith water R.

Q H J3N 02

131.2

60-32-2

A c tio n a n d u s e Antifibrinolytic. PhEur__________________________________________________________

D E F IN IT IO N Aminocaproic a d d contains not less than 98.5 p er cent and not more th an the equivalent of 101.0 per cent of 6-am inohexanoic ad d , calculated with reference to the dried substance.

Reference solution (a) Dissolve 10 mg o f aminocaproic acid CRS in water R and dilute to 50 m L with the same solvent.

Reference solution (b) Dilute 5 m L o f test solution (b) to 20 m L with water R. Reference solution (c) Dissolve 10 mg o f aminocaproic acid CRS and 10 mg o f leucine CRS in water R an d dilute to 25 m L with the same solvent. Apply separately to the plate 5 (iL o f each solution. Allow the plate to dry in air. D evdop over a p ath of 15 cm using a

2016

1-142 Aminoglutethimide

mixture of 20 volumes o f glacial acetic acid R, 20 volumes of water R and 60 volumes o f butanol R. Dry the plate in a current of w arm air. Spray with mnhydrin solution R and heat at 100 °C to 105 °C for 15 min. Any spot in the chromatogram obtained with the test solution (a), apart from the principal spot, is n o t more intense than the spot in the chromatogram obtained with reference solution (b) (0.5 per cent). T he test is not valid unless the chromatogram obtained with reference solution (c) shows two clearly separated principal spots. H eav y m e ta ls (2.4.8) 12 m L of solution S complies with test A for heavy metals (10 ppm ). Prepare the reference solution using lead standard

solution (2 ppm Pb) R. L oss o n d ry in g (2.2.32) N ot more than 0.5 per cent, determined on 1.000 g by drying in an oven at 105 °C. S u lfa te d a sh (2.4.14) N ot more than 0.1 per cent, determined on 1.0 g.

C. Thin-layer chromatography (2.2.27).

Test solution Dissolve 25 m g of the substance to be examined in acetone R and dilute to 5 m L with the same solvent. Reference solution (a) Dissolve 25 m g of aminoglutethimide CRS in acetone R and dilute to 5 m L with the same solvent.

Reference solution (b) Dissolve 25 mg of aminoglutethimide CRS and 25 mg o f glutethimide CRS in acetone R and dilute to 5 m L with the same solvent. Plate TLC stHca gel F 254 plate R. Mobile phase glacial acetic add R, methanol R, ethyl acetate R (0.5:15:85 VIVIV). Application 5 |oL. Development Over 3/4 of the plate. Drying In air. Detection Examine in ultraviolet light at 254 nm. System suitability: reference solution (b): —

the chromatogram shows 2 clearly separed spots.

Results T h e principal spot in the chromatogram obtained with

A SSA Y Dissolve 0.100 g in 20 m L o f anhydrous acetic acid R. Using 0.1 m L of crystal violet solution R as indicator, titrate with 0.1 M perchloric add until the colour changes from bluishviolet to bluish-green.

the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a).

1 m L o f 0.1 M perchloric add is equivalent to 13.12 m g o f C sH jaN O * __________________________________________________________ PhEur

TESTS S o lu tio n S Dissolve 1.0 g in methanol R and dilute to 20.0 m L with the same solvent. A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y7 (2.2.2, Method II).

Aminoglutethimide

***** ★.

(Ph. Eur. monograph 1291)

* *

R e la te d su b sta n c e s Liquid chromatography (2.2.29).

H

O ^N ^O I

L J

\

Solvent mixture methanol R, acetate buffer solution pH 5.0 R (50:50 VIV). Test solution Dissolve 0.100 g of the substance to be

NH2 and enantiomer

H3< /

C i 3H16N 20 2

O p tic a l ro ta tio n (2.2.7) —0.10° to + 0.10°, determined on solution S.

examined in the solvent mixture and dilute to 50.0 m L with the solvent mixture. 232.3

125-84-8

A c tio n a n d u se Inhibitor of adrenal corticosteroid synthesis; used in chemical adrenalectomy.

Reference solution (a) Dissolve 5.0 m g o f aminoglutethimide impurity A CRS in the solvent mixture and dilute to 25.0 m L with the solvent mixture.

Reference solution (b) Dilute 1.0 m L of reference solution (a) to 10.0 m L with the solvent mixture.

PhEur__________________________________________________________

D E F IN IT IO N (3i?5)-3-(4-Aminophenyl)-3-ethylpiperidine-2j6-dione. C o n te n t 98.0 per cent to 101.5 per cent (dried substance). CHARACTERS A p p e a ra n c e W hite or slightly yellow, crystalline powder. S olubility Practically insoluble in water, freely soluble in acetone, soluble in methanol. ID E N T IF IC A T IO N

First identification B Second identification A, C A. Melting point (2.2.14): 150 °C to 154 °C. B. Infrared absorption spectrophotometry (2.2.24). Comparison aminoglutethimide CRS.

Reference solution (c) Dilute 1.0 m L o f the test solution to 100.0 m L with the solvent mixture.

Reference solution (d) Dilute 1.0 m L o f the test solution to 10.0 m L with reference solution (a). -

Column: — size. I = 0.15 m, 0 = 3.9 mm; — stationary phase, octadecylsüyl silica gel for chromatography R (4 Jim); — temperature: 40 °C.

Mobile phase Mix 27 volumes of methanol R and 73 volumes o f acetate buffer solution pH 5.0 R Flow rate 1.3 mL/min. Detection Spectrophotometer at 240 nm. Injection 10 pL of the test solution and reference solutions (b), (c) and (d).

Run time 4 times the retention time o f aminoglutethimide.

Aminoglutethimide 1-143

2016 Identification of impurities Use the chromatogram obtained with reference solution (b) to identify die peak due to impurity A.

Relative retention W ith reference to aminoglutethimide (retention time = about 9 min): impurity A = about 1.3.

System suitability Reference solution (d): — resolution: m inim u m 2.0 between the peaks due to am in oglu teth im id e and impurity A.

Limits: — impurity A: not m ore than twice die area o f the principal

—

—

— —

peak in the chromatogram obtained w ith reference solution (b) (2.0 p er cent); unspecified impurities: for each impurity, n o t more than 0.1 times the area o f the principal peak in die chromatogram obtained with reference solution (c) (0.10 per cent); sum of impurities other than A: not more than the area o f the principal peak in die chromatogram obtained with reference solution (c) (1.0 per cent); total: maximum 2.0 per cent for the sum of die contents o f all impurities; disregard limit: 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.05 per cent).

Impurity D Liquid chromatography (2.2.29). Carry out die test protected from light Use shaking, not sordcadon or heat, to dissolve the reference substance and the substance to be examined. Test solution Dissolve 0.100 g of the substance to be examined in dimethyl sulfoxide R and dilute to 100.0 m L with

Prepare the reference solution vising lead standard solution (1 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture o f 5 m L o f water R and 15 m L o f acetone R L oss o n d ry in g (2.2.32) M axim um 0.5 p er cent, determined on 1.000 g by drying in an oven at 105 °C. S u lfa te d a sh (2.4.14) M axim um 0.1 per cent, determined on 1.0 g. A SSA Y Dissolve 0.180 g in 50 m L of anhydrous acetic acid R and titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 m L of 0.1 M perchloric acid is equivalent to 23.23 mg Of C 13H 16N 2O 2. IM P U R IT IE S

Specified impurities A, D . Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one o r other o f the tests in the monograph. T hey are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these impurities for demonstration o f compliance. See also 5.10.

Control of impurities in substances for pharmaceutical use): B, C.

R4

and enantiomer

the same solvent.

Reference solution Dissolve 3.0 mg of aminoglutethimide impurity D CRS in dimethyl sulfoxide R and dilute to 100.0 m L with the same solvent. Dilute 1.0 m L of this solution to 100.0 m l, with dimethyl sulfoxide R.

A. R3 = N H 2) R4 = H: (3RS)-3-(3-aminophenyl)-3ethylpiperidine-2,6-dione (3-aminoglutethimide),

Column:

B. R3 = N O * R4 = H: (3i?S)-3-ethyl-3-(3-nitrophenyl)piperidine-2,6-dione,

— sizer. I = 0.12 m , 0 = 4 mm; — stationary phase: octadecylsUyl silica gel for chromatography R (5 pm).

Mobile phase Dissolve 0.285 g of sodium edetate R in water R, add 7.5 m L o f dilute acetic acid R and 50 m l, of 0.1 M potassium hydroxide and dilute to 1000 m L with water R; adjust to p H 5.0 with glacial acetic acid R’, mix 350 m L of this solution with 650 m L of methanol R. Flow rate 1.0 mL/min. Detection Spectrophotometer at 328 nm. Injection 10 |iL. System suitability T est solution: — number of theoretical plates: minimum 3300, calculated for the principal peak; — mass distribution ratio: 2.0 to 5.0 for the principal peak; — symmetry factor, m axim um 1.2 for the principal peak.

Limit: — impurity D: n o t m ore than the area of the principal peak in the chromatogram obtained with the reference solution (300 ppm).

Sulfates (2.4.13) M aximum 500 ppm . Dilute 6 m L o f solution S to 15 m L with distilled water R.

Heavy m etals (2.4.8) M aximum 10 ppm. Dissolve 2.0 g in 15 m L o f acetone R and dilute to 20 m L with water R. 12 m L of the solution complies with test B.

C. R3 = H , R4 = N 0 2: (3i?5)-3-ethyl-3-(4-nitrophenyl)piperidine-2,6-dione,

D . 3,3'-[diazenediylbis(4,l-phenylene)]bis(3-ethylpiperidine2, 6-dione) (azoglutethimide). PhEur

1-144 Aminophylline

2016

Aminophylline

;***%

(TheophyUine-ethyknediamine, anhydrous, Ph Eur monograph 0300)

*★ ★* *

E. W ater (see Tests). F . T h e pred p itate gives the reaction o f xanthines (2.3.1).

TESTS Appearance o f solution T h e solution is n o t m ore opalescent than reference suspension II (2.2.1) and n o t m ore intensely coloured than reference solution GY6 (2.2.2, Method II). Dissolve 0.5 g with gentle warming in 10 m L o f carbon

dioxide-free water R. Related substances Liquid chrom atography (2.2.29). C 16H 24N 10O 4

420.4

317-34-0

A c tio n a n d u se N on-sdective phosphodiesterase inhibitor; treatm ent o f reversible airways obstruction. P re p a r a tio n s Aminophylline Injection Aminophylline Tablets Prolonged-release Aminophylline Tablets

Test solution Dissolve 47 m g o f the substance to be examined in the mobile phase and dilute to 20.0 m L w ith the mobile phase. Reference solution (a) D ilute 1.0 m L o f the test solution to 100.0 m L with the mobile phase. D ilute 1.0 m l . o f this solution to 10.0 m l . with the mobile phase.

Reference solution (b) Dissolve 10 m g o f theobromine R (impurity G ) in the mobile phase, add 5 m L o f the test solution and dilute to 100 m L with the mobile phase. Dilute 5 m L o f this solution to 50 m L with the mobile phase.

Ph Eur___________________________________________________________

Column:

D E F IN IT IO N C o n te n t — theophylline (C 7H&N4O 2; 180.2): 84.0 per cent to 87.4 per cent (anhydrous substance); — ethylenedianane (C 2HgN 2; 60.1): 13.5 p er cent to 15.0 per cent (anhydrous substance).

— sizer. I = 0.25 m , 0 = 4 mm; — stationary phase: octadecylsUyl silica gelfor chromatography R (7 nm).

CHARACTERS A p p e a ra n c e W hite or slightly yellowish powder, sometimes granular, hygroscopic. S o lu b ility Freely soluble in water (the solution becomes cloudy through absorption o f carbon dioxide), practically insoluble in anhydrous ethanol. ID E N T IF IC A T IO N

First identification B, C, E. Second identification A , C, D, E, F. Dissolve 1.0 g in 10 m L o f water R and add 2 m l . o f dilute hydrochloric acid R dropwise with shaking. Filter. U se the predpitate for identification tests A, B, D and F and the filtrate for identification test C. A. M d tin g point (2.2.14): 270 °C to 274 °C, determ ined after w ash in g the predpitate with water R and drying at 105 °C. B. Infrared absorption spectrophotom etry (2.2.24). Preparation P redpitate, washed with water R and dried at 105 °C.

Comparison theophylline CRS. C. T o the filtrate add 0.2 m L o f benzoyl chloride R, m ake alkaline with dilute sodium hydroxide solution R and shake vigorously. F ilter the predpitate, wash with 10 m L o f water R, dissolve in 5 m L o f h o t ethanol (96 per cent) R and add 5 m L o f water R. A predpitate is formed, which, when washed and dried at 105 °C, m d ts (2.2.14) at 248 °C to 252 °C. D. H eat about 10 m g o f the predpitate with 1.0 m L o f a 360 g/L solution o f potassium hydroxide R in a water-bath at 90 °C for 3 m in, then add 1.0 m L o f diazodsed sulfanUic acid solution R. A red colour slowly devdops. C an y out a blank te s t

Mobile phase M ix 7 volumes o f acetomtrUefor chromatography R and 93 volumes o f a 1.36 g/L solution of sodium acetate R containing 0.50 per cent V/V o f glacial acetic add R. Flow rate 2.0 mL/min. Detection Spectrophotom eter at 272 nm . Irqection 20 jjL . Run time 3.5 times the retention time o f theophylline. Relative retention W ith reference to theophylline (retention tim e = about 6 min): impurity G = about 0.6. System suitability: reference solution (b): — resolution: m in im u m 2.0 between the peaks due to impurity G and theophylline.

Limits: — unspecified impurities: for each impurity, n o t more than the area o f the prindpal peak in the chrom atogram obtained with reference solution (a) (0.10 per cent); — total: n o t m ore than the area o f the prin d p al peak in the chrom atogram obtained with reference solution (a) (0.1 p er cent); — disregard Umit. 0.5 times the area o f the principal peak in the chrom atogram obtained with reference solution (a) (0.05 per cent).

Heavy m etals (2.4.8) M aximum 20 ppm.

Solvent water R. 0.500 g complies with test H . Prepare the reference solution using 1 m L o f lead standard solution (10 ppm Pb) R. T h e substance predpitates after addition o f bitjfer solution pH 3.5 R. D ilute to 100 m L with water R, the substance re-dissolves completely.

Water (2.5.12) M axim um 1.5 per cent, determ ined on 0.50 g.

Sulfated ash (2.4.14) M aximum 0.1 per cent, determ ined on 1.0 g.

2016

Aminophylline Hydrate 1-145

A SSA Y

Etfaylenediamine

^ n^ v n

Dissolve 0.250 g in 30 m L of water R. Add 0.1 m L of bromocresol green solution R. T itrate with 0.1 M hydrochloric add until a green colour is obtained.

0

1 m L of 0.1 M hydrochloric add is equivalent to 3.005 m g of C2HgN2‘ T h eo p h y llin e H eat 0.200 g to constant mass in an oven at 135 °C. Dissolve the residue with heating in 100 m L o f water R} allow to cool, add 20 m L of 0.1 M silver nitrate and shake. Add 1 mT. of bromothymol blue solution R l. Titrate with 0.1 M

n>< ch3

E. l,3-dim ethyl-7,9-dihydro-lH-purine-2,6,8(3/i)-trione,

OH

o

X x> I

sodium hydroxide.. 1 m L of 0.1 M sodium hydroxide is equivalent to 18.02 m g o f C 7H 8N 4O 2. STO RA G E In an airtight container, protected from light

ch3

F. 7-(2-hydroxyethyl)-1,3-dimethyl-3,7 -dihydro-1H -purine2, 6-dione (etofylline),

IM P U R IT IE S

Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these impurities for demonstration of compliance. See also 5.10.

H

1

X

0

pi

1

'

V> !

ch3

G. 3,7-dimethyl-3,7-dihydro-lH-purine-2,6-dione (theobromine).

Control of impurities in substances for pharmaceutical use): A, B, C, D, E, F, G. 0

pt

1

'

PhEur

★ ★ ★ ★ *****

Aminophylline Hydrate

lx >

Çlheophyïïme-eàiylenediamine Hydrate, Ph Eur monograph 0301)

1

ch3

A. 1,3,7-trimethyl-3,7-dihydro-lH-purine-2,6-dione (caffeine), H2N'

1 1 >

, xH 20

I

ch3

HN

X

//>

L

C 16H 24N 10O 4PCH2O

CH,

420.4

72487-55-9

(anhydrous substance) B. 3-methyl-3 37-dîhydro-1H-purine-236-dione3

A J.

JjC.

O

N

CHO

NH2

CH,

A c tio n a n d u se Non-selective phosphodiesterase inhibitor; treatm ent of reversible airways obstruction. P re p a r a tio n Aminophylline Injection Aminophylline Tablets Prolonged-release AminopyQine Tablets

C. AT-(6-am ino-l^-dim ethyl-2,4-dioxo-l,2,3,4tetrahydropyrimidin-5-yl)formamide,

h3c x

»V> hn ^

n

CH,

D. N-methyl-5-(methylamino)- lH-imidazole-4-carboxamide,

PhEur.

D E F IN IT IO N C o n te n t — theophylline (C7H8N402; 180.2): 84.0 per cent to 87.4 per cent (anhydrous substance); — ethylenediamine (C 2H 8N 2; 60.1): 13.5 per cent to 15.0 per cent (anhydrous substance). CHARACTERS A p p e a ra n c e W hite or slightly yellowish powder, sometimes granular.

1-146 Aminophylline Hydrate

2016

S o lu b ility Freely soluble in water (the solution becomes cloudy through absorption o f carbon dioxide), practically insoluble in anhydrous ethanol.

Injection 20 pL. Run time 3.5 times the retention time o f theophylline. Relative retention W ith reference to theophylline (retention

IDENTIFICATION

System suitability: reference solution (b): — resolution: minimum 2.0 between the peaks due to

First identification B, C, E. Second identification A , C, D, E, F. Dissolve 1.0 g in 10 m L of water R and add 2 m L o f dilute hydrochloric acid R dropwise with shaking. Filter. Use the precipitate for identification tests A, B, D and F and the filtrate for identification test C. A. Melting point (2.2.14): 270 °C to 274 °C, determined after washing the precipitate with water R and drying at 105 °C. B. Tnfrared absorption spectrophotom etry (2.2.24).

Preparation Precipitate, washed with water R and dried at 105 °C.

Comparison theophylline CRS. C. T o the filtrate add 0.2 m L o f benzoyl chloride R, make alkaline with dilute sodium hydroxide solution R and shake vigorously. Filter the precipitate, wash with 10 m L o f water R, dissolve in 5 m L o f h o t ethanol (96 per cent) R and add 5 m L o f water R. A precipitate is formed, which, when washed and dried at 105 °C, melts (2.2.14) at 248 °C to 252 °C.

tim e = about 6 min): im purity G = about 0.6.

im purity G and theophylline.

Limits: — unspecified impurities: for each impurity, n o t m ore than the area of the principal peak in the chrom atogram obtained with reference solution (a) (0.10 per cent); — total: n o t more than the area o f the principal peak in the chrom atogram obtained with reference solution (a) (0.1 per cent); — disregard limit. 0.5 times the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). H e a v y m e ta ls (2.4.8) M aximum 20 ppm.

Solvent water R. 0.500 g complies with test H . Prepare the reference solution using 1 m L o f lead standard solution (10 ppm Pb) R. T h e substance precipitates after addition o f buffer solution pH 3.5 R. D ilute to 100 m L with water R; the substance re-dissolves completely. W a te r (2.5.12) 3.0 per cent to 8.0 per cent, determ ined on 0.50 g.

D . H eat about 10 m g o f the precipitate with 1.0 m L o f a 360 g/L solution of potassium hydroxide R in a water-bath at 90 °C for 3 m in, then add 1.0 m L o f diazotised stdfanUic acid solution R. A red colour slowly develops. Carry out a blank te s t

A SSA Y

E. W ater (see Tests).

Ethyienediamine

F. T he precipitate gives the reaction o f xanthines (2.3.1).

Dissolve 0.250 g in 30 m L o f water R. A dd 0.1 m L o f bromocresol green solution R. T itrate with 0.1 M hydrochloric acid until a green colour is obtained.

TESTS A p p e a ra n c e o f so lu tio n T he solution is not m ore opalescent than reference suspension II (2.2.T) and n o t m ore intensely coloured than reference solution GY6 (2.2.2, Method II). Dissolve 0.5 g with gentle warming in 10 m l. of carbon

dioxide-free water R R e la te d su b s ta n c e s Liquid chrom atography (2.2.29).

Test solution Dissolve 50 mg o f the substance to be examined in the mobile phase and dilute to 20.0 m L with the mobile phase.

Reference solution (a) D ilute 1.0 m l, o f the test solution to 100.0 m L w ith the mobile phase. D ilute 1.0 m l. o f this solution to 10.0 m L w ith the mobile phase.

Reference solution (b) Dissolve 10 m g o f theobromine R (impurity G ) in the mobile phase, add 5 m L o f the test solution and dilute to 100 m L with the mobile phase. D ilute 5 m L o f this solution to 50 m L with the mobile phase.

Column: — size. I — 0.25 m, 0 = 4 mm; — stationary phase: octadecylsifyl silica gel for chromatography R (7 pm).

Mobile phase M ix 7 volumes o f acetortitrile for chromatography R and 93 volumes o f a 1.36 gJL solution of sodium acetate R containing 0.50 per cent V/V of glacial acetic add R. Flow rate 2.0 mL/min. Detection Spectrophotometer at 272 nm.

S u lfa te d a s h (2.4.14) M aximum 0.1 per cent, determ ined on 1.0 g.

1 mT. o f 0.1 M hydrochloric acid is equivalent to 3.005 m g of C 2H8N2.

Theophylline H eat 0.200 g to constant mass in an oven at 135 °C. Dissolve the residue with heating in 100 m L o f water R, allow to cool, add 20 mT. o f 0.1 M stiver nitrate and shake. A dd 1 mT. o f bromothymol blue solution R l. T itrate with 0.1 M

sodium hydroxide. 1 mT. o f 0.1 M sodium hydroxide is equivalent to 18.02 m g of C rH sN ^ .

STORAGE In a well-filled, airtight container, protected from light.

IMPURITIES Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other of the tests in the m onograph. T hey are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharm aceutical use (2034). It is therefore no t necessary to identify these impurities for dem onstration o f compliance. See also 5.10.

Control of impurities in substances fin- pharmaceutical use): A , B, C, D, E, F, G.

2016

Amiodarone Hydrochloride 1-147

1Y>

o^

n-

*****

Amiodarone Hydrochloride

i ? Hs H,c' N' l V ' N

★

^ n

ch3

★

* * * * *

(Ph. Eur. monograph 0803) H,C

A. 1,3,7-trim ethyl-3,7-dihydro-lH-purine-2,6-dione (caffeine),

N. .CH,

C25H3oCn2N 0 3

lV > I

682

,

HQ

19774-82-4

A ctio n a n d u se Potassium channel blocker; class ID antiarrhythmic.

ch3

P re p a ra tio n s Amiodarone Intravenous Infusion

B. 3-methyl-3,7-dihydro-lH-purine-2,6-dione,

Amiodarone Oral Suspension h3c^ N

I B |

Amiodarone Tablets

. c

PhEir. O ^N

NH2

D E F IN IT IO N (2-Butylben2ofuran-3-yl)[4-[2-(diethylamino)ethoxy]-3j5diiodophenyljmethanone hydrochloride.

ch3

C. N -(6-am ino-l,3-dim ethyl-2,4-dioxo-l,2,3,4tetrahydropyrimidin-5-yl)formamide,

H,C

C o n te n t 98.5 per cent to 101.0 p er cent (dried substance). CHARACTERS A p p e a ra n c e W hite or almost white, fine, crystalline powder.

rV s H N N

I

CH3

D . N-methyl-5-(methylamino)-lH-imidazole-4-carboxamide,

S olu b ility Very slightly soluble in water, freely soluble in methylene chloride, soluble in methanol, sparingly soluble in ethanol (96 per cent). ID E N T IF IC A T IO N A. Infrared absorption spectrophotometry (2.2.24).

Comparison amiodarone hydrochloride CRS.

H*C'1n\ A[ ) ==0 o ^ n ^ n I

CH,

B. It gives reaction (b) o f chlorides (2.3.1).

M

TESTS A p p e a ra n c e o f so lu tio n T h e solution is clear (2.2.1) and n o t more intensely coloured than reference solution GY5 or BY5 (2.2.2, Method II).

E. 1,3-dim ethyl-7,9-dihydro-lii-purine-2,6,8(3H)-trione,

Dissolve 1.0 g in methanol R and dilute to 20 m L with the same solvent

OH

1 oIA

pH (2.2.3)

JL > HA >

3.2 to 3.8.

I

Dissolve 1.0 g in carbon dioxide-free water R, heating at 80 °C, cool and dilute to 20 m L with the same solvent

ch3

F . 7-(2-hydroxyethyl)-1,3-dimethyl-3,7-dihydro-1H -purine2,6-dione (etofylline),

Im p u rity H Thin-layer chromatography (2.2.27). Prepare the solutions

immediately before use and keep protectedfrom bright light. Test solution Dissolve 0.500 g o f the substance to be examined in methylene chloride R and dilute to 5.0 m L with

X "

ÍJC>

the same solvent.

Reference solution (a) Dissolve 10.0 m g of (2-chloroethyl)diethylamine hydrochloride R (impurity H) in methylene chloride R and dilute to .50.0 m L with the same

I

ch3

solvent. Dilute 2.0 m L o f the solution to 20.0 m L with

G . 3,7-dim ethyl-3,7-dihydro-lii-purine-2,6-dione (theobromine). PhEur

methylene chloride R. Reference solution (b) Mix 2.0 m L o f the test solution and 2.0 m L o f reference solution (a).

Plate TLC silica gel F2 5 4 plate R.

1-148 Amiodarone Hydrochloride

2016

Mobile phase anhydrous formic add R, methanol R, methylene chloride R (5:10:85 VIVIV). Application 50 (iL o f the test solution and reference

— disregard limit. 0.25 times the area o f the peak due to amiodarone in the chrom atogram obtained with the reference solution (0.05 per cent).

solution (a); 100 pL o f reference solution (b).

Io d id e s M axim um 150 ppm.

Development Over 2/3 o f the plate. Drying In a current o f cold air. Detection Spray with potassium iodobismuthate solution R l and then with dilute hydrogen peroxide solution R', exam in e immediately in daylight. System suitability: reference solution (b): — the spot due to im purity H is clearly visible.

Limit: — impurity H: any spot with the same Rp as the spot due to im purity H in the chromatogram obtained with reference solution (b) is n o t m ore intense th an the spot in the chrom atogram obtained w ith reference solution (a) (0.02 per cent).

Prepare the test and reference solutions simultaneously. Solution A A dd 1.50 g o f the substance to be examined to 40 m L o f water R at 80 °C and shake until completely dissolved. Cool and dilute to 50.0 m L w ith water R. Test solution T o 15.0 m L o f solution A add 1.0 m L o f 0.1 M hydrochloric acid and 1.0 mT. o f 0.05 Mpotassium iodate. Dilute to 20.0 m L with water R. Allow to stand protected from light for 4 h.

Reference solution T o 15.0 m L o f solution A add 1.0 m L of 0.1 M hydrochloric add, 1.0 m L of an 88.2 mg/L solution o f potassium iodide R and 1.0 m L o f 0.05 Mpotassium iodate. D ilute to 20.0 m L with water R. Allow to stand protected

Related substances

from light for 4 h.

Liquid chromatography (2.2.29).

M easure the absorbances (2.2.25) o f the solutions at 420 nm , using a mixture o f 15.0 m L o f solution A and 1.0 m L o f 0.1 M hydrochloric acid diluted to 20.0 m L with water R as the compensation liquid. T h e absorbance o f the test solution is n o t greater than half the absorbance o f the reference solution.

Buffer solution pH 49 T o 800 mT. o f water R add 3.0 m L o f glacial acetic add R, adjust to p H 4.9 with dilute ammonia R l and dilute to 1000 m L with water R. Test solution Dissolve 0.125 g o f the substance to be ex a m in ed in a mixture of equal volumes of acetonitrUe R and water R and dilute to 25.0 m L with the same mixture of solvents.

Reference solution Dissolve 5 m g o f amiodarone impurity D CRS, 5 m g of amiodarone impurity E CRS and 5.0 m g o f amiodarone hydrochloride CRS in methanol R and dilute to 25.0 m L w ith the sam e solvent. Dilute 1.0 m L o f the solution to 20.0 m L with a mixture o f equal volumes o f acetonitrile R and water R.

Column: — size: I = 0.15 m , 0 = 4.6 mm; — stationary phase, end-capped octadecylsUyl silica gel for chromatography R (5 pm); — temperature: 30 °C.

Mobile phase Buffer solution p H 4.9, methanol R, acetonitrile R (30:30:40 VIVIV). Flow rate 1 mL/min. Detection Spectrophotom eter at 240 nm . Injection 10 (iL. Run time Twice the retention time o f am iodarone. Relative retention W ith reference to amiodarone (retention time = about im purity D = impurity B = im purity G =

24 m in): im purity A = about 0.26; about 0.29; im purity E = about 0.37; about 0.49; im purity C = about 0.55; about 0.62; im purity F = about 0.69. System suitability: reference solution: — resolution: m inim um 3.5 between the peaks due to impurities D and E.

H e a v y m e ta ls (2.4.8) M axim um 20 ppm . 1.0 g complies with test C. Prepare the reference solution using 2 m L o f lead standard solution (10 ppm Pb) R. L o ss o n d ry in g (2.2.32) Maximum 0.5 per cent, determ ined on 1.000 g by drying at 50 °C at a pressure not exceeding 0.3 kPa for 4 h. S u lfa te d a s h (2.4.14) M aximum 0.1 per cent, determ ined on 1.0 g. A SSA Y Dissolve 0.600 g in a mixture o f 5.0 m L o f 0.01 M hydrochloric add and 75 m L o f ethanol (96 per cent) R. Carry out a potentiom etric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between the 2 points o f inflexion. 1 mT. o f 0.1 M sodium hydroxide is equivalent to 68.18 m g of C 25H 3oCU2N 0 3 . STORAGE Protected from light, at a tem perature n o t exceeding 30 °C. IM P U R IT IE S

Specified impurities A, B, C , D , E, F , G, H

Limits: — impurities A , B, C, D, E, F, G: for each impurity, not m ore than the area o f the peak due to amiodarone in the chrom atogram obtained w ith the reference solution (0.2 per cent); — unspecified impurities: for each impurity, n o t more than 0.5 times the area o f the peak due to amiodarone in the chrom atogram obtained w ith the reference solution (0.10 per cent); — total: not m ore th an 2.5 times the area of the peak due to amiodarone in the chrom atogram obtained with the reference solution (0.5 per cent);

A. (2-butyIbenzofuran-3-yl) [4-[2-(diethylamino)ethoxy] phenyl] methanone,

Amisulpride 1-149

2016 and enantiomer

★* * ★ ★ ★ ★ *****

Amisulpride (Ph Eur monograph 1490) o\w/o

h 3c .

B. (2-butylbenzofuran-3-yI) [4-[2-(ethyiamino)ethoxy]-3,5diiodophenyl] methanone,

N H u^ ' O N h2n

och3

and enantiomer.

k CH3

and enantiomer C 17H 27N 3O 4S

369.5

71675-85-9

Action and use D opamine receptor antagonist; neuroleptic.

Preparations Amisulpride Oral Solution C. (2-butylbenzofuran-3-yl) [4-[2-(diethyiamino)ethoxy]-3iodophenyl] methanone,

Amisulpride Tablets P h E tr ______________________________________________________________

DEFINITION 4-Amino-N- [ [(2i?5)-1-ethylpyrrolidin-2-yl] methyl] -5(ethylsulfonyl)-2-methoxybenzamide.

Content 99.0 per cent to 101.0 per cent (dried substance). D . (2-butyIbenzofuran-3-yl)(4-hydroxy-3,5diiodophenyl)methanone,

CHARACTERS Appearance W hite or almost white, crystalline powder.

Solubility

H,c

Practically insoluble in water, freely soluble in methylene chloride, sparingly soluble in anhydrous ethanol.

mp About 126 °C.

IDENTIFICATION E. (2-butyIbenzofuran-3-yl) (4-hydroxyphenyl)methanone,

F. (2-butylbenzofuran-3-yl) (4-hydroxy-3-iodophenyl) methanone,

Infrared absorption spectrophotometry (2.2.24).

Comparison amisulpride CRS. TESTS Appearance o f solution T h e solution is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). Dissolve 1.0 g in 3 m L of a mixture of 1 volume of acetic acid R and 4 volumes of water Rs and dilute to 20 m L with water R. Impurity A Thin-layer chromatography (2.2.27).

Test solution Dissolve 0.20 g of th e substance to be examined in methanol R and dilute to 10 m L with the same solvent. Reference solution (a) Dissolve 5 mg of sulpiride impurity A CRS (amisulpride impurity A) in methanol R and dilute to 25 mT. with the same solvent. Dilute 2 m L of the solution to 20 m L with methanol R. Reference solution (b) Dilute 1 m L of the test solution to 10 m L with reference solution (a). G. [4-[2-(diethylamino)ethoxy]-3,5-diiodophenyl] [2-[(1 jR5)1-methoxybutyl] benzofuran-3-yl] methanone, ,c h 3

r

,N_XH3

H . 2-chloro-N,N-diethylethanamine (2-chlorotriethylamine, (2-chloroethyl)diethyIamine).

Plate TLC silica gel G plate R. Mobile phase 50 per cent VIV solution of concentrated ammonia R, anhydrous ethanol R, di-isopropyl ether R (10:25:65 VIVIV); use the upper layer obtained after shaking the mixture.

Application 10 (iL. Development Over 2/3 of the plate. Drying In air.

2016

1-150 Amisulpride

Detection Spray with ninhydrin solution R and heat at

— reporting threshold: 0.05 per cent.

100-105 °C for 15 min.

H ea v y m e ta ls (2.4.8) M axim um 10 ppm.

Retardation factors Im purity A = about 0.2; amisulpride = about 0.5.

System suitability T he chrom atogram obtained with reference solution (b) shows 2 clearly separated spots.

Limit. — impurity A: any spot due to impurity A is not more intense than the corresponding spot in the chromatogram obtained with reference solution (a) (0.1 per cent). R e la te d su b s ta n c e s liq u id chromatography (2.2.29).

dilute to 100.0 m L with mobile phase A.

Reference solution (a) Dilute 1.0 m L o f the test solution to 100.0 m L with the solvent mixture. Dilute 1.0 m L o f this solution to 10.0 m L with the solvent mixture.

Reference solution (b) Dissolve the contents o f a vial of amisulpride for system suitabUity CRS (containing impurity B) in 1.0 m L of the solvent mixture.

Column: — size: I = 0.25 m , 0 = 4.6 mm ; — stationary phase:, base-deactivated oaylsüyl sUica gel for chromatography R (5 nm); — temperature: 40 °C.

Mobile phase: — mobile phase A: dissolve 0.7 g o f sodium octanesulfonate R in 930 m L o f water R , add 45.0 m l. o f a 5 per cent V/V solution o f dilute sulfuric acid R, adjust to p H 2.3 with a 5 per cent V/V solution o f dilute sulfuric add R, and dilute to 1000 m L with water R; — mobile phase B: methanol R2; — mobile phase C: acetomtrUe R l :;

18 - 35

Mobile phase A (per cent V/V/V) 72 72

Mobile phase B (per cent V/V/V) 16

50

L o ss o n d ry in g (2.2.32) Maximum 0.5 per cent, determ ined on 1.000 g by drying in an oven at 105 °C for 3 h. S u lfa te d a s h (2.4.14) M axim um 0.1 per cent, determ ined on 1.0 g.

Solvent mixture acetomtrUe R l, methanol R2, mobile phase A (12:16:72 V/V/V). Test solution Dissolve 0.10 g o f the substance to be examined in 16 m L o f methanol R2, add 12 m L of acetomtrUe R l and

Time (min) 0 -1 8

Dissolve 4.0 g by gently heating in 5 m L o f dilute acetic add R. Allow to cool and dilute to 20 m L w ith water R. 12 m L o f the solution complies with test A. Prepare the reference solution using lead standard solution (2 ppm Pb) R.

16

38

Mobile phase C (percent V/V/V) 12

A SSA Y Dissolve 0.300 g with shaking in a mixture o f 5 m L o f acetic anhydride R and 50 m L o f anhydrous acetic acid R. T itrate w ith 0.1 M perchloric add, determining the end-point potentiometrically (2.2.20). 1 m L o f 0.1 M perchloric add is equivalent to 36.95 m g o f C 17H 2 7N 3O 4 S .

IM P U R IT IE S

Spedfied impurities A Other detectable impurities (the following substances w ould, if present at a sufficient level, be detected by one or other o f the tests in the monograph. They are lim ited by th e general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these impurities for demonstration o f compliance. See also 5.10.

Control of impurities in substances for pharmaceutical use): B, C, D, E, F, G3 H.

h2n

H

N

and enantiomer

CH3

A. [(1RS) -1 -ethylpyrrolidin-2-yI] m ethanam ine, o

\

12

Flow rate 1.5 mL/min. Detection Spectrophotom eter a t 225 nm . Injection 10 ¿iL. Identification of impurities U se the chromatogram obtained

and enantiomer

B . 4 - am in o - A/-[[(2i?«S)-1 -ethylpyrrolidin-2-yl] methyl]-5-

(ethylsulfonyl)-2-hydroxybenzamide,

with reference solution (b) to identify the peak due to im purity B. and enantiomer

Relative retention W ith reference to amisulpride (retention tim e = about 17 min): im purity B = about 1.1. System suitability: reference solution (b): — peak-to-vaUey ratio: minimum 2.0, where Hp = height above the baseline o f the peak due to impurity B and Hv = height above the baseline o f the lowest point o f the curve separating this peak from the peak due to amisulpride.

Calculation of percentage contents Use the concentration of amisulpride in reference solution (a).

Limits: — unspecified impurities: for each impurity, maximum 0.10 per cent; — total: m aximum 0.3 per cent;

H,N

C . 4 - am ino - iV- [[(2ÆS)-1 -ethylpyirolidin-2-yl]methyl] -5-iodo2-methoxybenzamide,

Amitriptyline Hydrochloride 1-151

2016 o o *o

Content

h 3c

N -^ O H H N

99.0 per cent to 101.0 per cent (dried substance).

and enantiomer

CHARACTERS Appearance

och3

h2n

CH3

W hite or almost white powder or colourless crystals. D . 4-am ino-N - [ [ (2RS)-1 -ethylpyrrolidin-2-yl] methyl] -2methoxy-5-(methylsulfonyl)benzamide,

Solubility Freely soluble in water, in ethanol (96 per cent) and in methylene chloride.

o o

IDENTIFICATION

.XX.

H2N

^

A. Infrared absorption spectrophotometry (2.2.24).

OCH3

Comparison amitriptyline hydrochloride CRS. B. 20 m g gives reaction (a) of chlorides (2.3.1).

E. 4-amino-5-(ethylsulfonyl)-2-methoxybenzoic acid.

TESTS

na H

h2n

QN

Appearance of solution T he solution is clear (2.2.1) and not more intensely coloured than reference solution B7 (2.2.2, Method II). Dissolve 1.25 g in water R and dilute to 25 m L with the

and enantiomer

I '0

0CH3

I

ch3

same solvent

Acidity or alkalinity Dissolve 0.20 g in carbon dioxide-free water R and dilute to 10 m L with the same solvent. Add 0.1 m L of methyl red solution R and 0.2 m L o f 0.01 M sodium hydroxide. T he solution is yellow. Add 0.4 m L of 0.01 M hydrochloric acid. T he solution is red.

F. 4-am ino-N- [[(2i?S)-1-ethyl-1-oxidopyrrolidin-2-yl] methyl]-5-(ethylsulfonyl)-2-methoxybenzamide, o o ❖'!

h 3c

s

H,N

^CH3

N H H

and enantiomer

Related substances

0CH3

Liquid chromatography (2.2.29).

G . 4-amincHN-[(3i?S)-l-ethylpiperidin-3-yl]-5-(ethylsulfonyl)2-methoxybenzamide,

Test solution Dissolve 50.0 mg o f the substance to be examined in the mobile phase and dilute to 50.0 m L with the mobile phase. Reference solution (a) Dissolve 5.0 m g of dibenzosuberone CRS (impurity A) and 5.0 m g of cydobenzaprine hydrochloride CRS

and enantiomer

H!N ' ^ A o c g3H>H

(impurity B) in 5.0 m L o f the test solution and dilute to 100.0 m L with the mobile phase.

^ CH,

Reference solution (b) Dilute 1.0 m L of reference solution (a) to 50.0 m L with the mobile phase.

H . 4-am ino-N - [ [(2i?S)-l -ethylpyrrolidin-2-yl] methyl] -5(ethylsulfonyl)-2-methoxy-AT-methylbenzamide.

Column: PhEur

Amitriptyline Hydrochloride

★ ★

(Ph Eur monograph 0464)

*****

N

I

★ ★

CH3 HCI

CH3

C 20H 24Q N

549-18-8

A c tio n a n d u se M onoam ine reuptake inhibitor; tricyclic antidepressant.

Preparation Amitriptyline Tablets PhEur.

D E F IN IT IO N 3-(10jl 1-Dihydro-5//-dibenzo [a,d\ [7] annulen-5-ylidene) iVJV-dimethylpropan-1-amine hydrochloride.

— sizer. I = 0.15 m, 0 = 4.6 mm; — stationary phase: end-capped polar-embedded octadecylsUyl amorphous organosUica polymer R (5 pm); — temperature: 40 °C.

Mobile phase Mix 35 volumes of acetonitrile R and 65 volumes o f a 5.23 g/L solution o f dipotassium hydrogen phosphate R previously adjusted to p H 7.0 with phosphoric add R. Flow rate 1.2 mL/min. Detection Spectrophotometer at 220 nm. Injection 10 |iL. Run time 3 times the retention time of amitriptyline. Relative retention W ith reference to amitriptyline (retention time = about 14 min): impurity B = about 0.9; im purity A = about 2.2. System suitability: reference solution (a): — resolution: minimum 2.0 between the peaks due to impurity B and amitriptyline.

Limits: — impurity B: no t more than the area of the corresponding peak in the chromatogram obtained with reference solution (b) (0.1 p er cent); — impurity A: n o t more than 0.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (b) (0.05 per cent);

1-152 Amlodipine Besilate

2016

— unspecified impurities: for each impurity, not more than the area o f the peak due to amitriptyline in the chromatogram obtained with reference solution (b) (0.10 per cent); — total: not more than 3 times the area o f the peak due to amitriptyline in the chromatogram obtained with reference solution (b) (0.3 per cent); — disregard limit. 0.5 times the area o f the peak due to amitriptyline in the chromatogram obtained with reference solution (b) (0.05 per cent). H e a v y m e ta ls (2.4.8) M axim um 20 ppm. 1.0 g complies with test F. Prepare the reference solution using 2 m L o f lead standard solution (10 ppm Pb) R.

D . R = C H 2-C H 2-C H 2-N (C H 3)2: 5-[3-(dimethylamino)propyl]-10,l l-d ihydro-5//dibenzo [a,d\ [7] annulen-5-ol, G . R = H: 10,1 l-dihydro-5H-dibenzo[a,if] [7]annulen-5-ol (dibenzosuberol), ch3

L oss o n d ry in g (2.2.32) M axim um 0.5 per cent, d eterm ined on 1.000 g by drying in an oven at 105 °C for 2 h.

N.

CH3

S u lfa te d a s h (2.4.14) M axim um 0.1 per cent, determined on 1.0 g. A SSA Y Dissolve 0.250 g in 30 m L of ethanol (96 per cent) R. Titrate with 0.1 M sodium hydroxide, determ ining the end-point potentiometrically (2.2.20).

E. NyN-àimexhÿ{-3-( 1,2,3,4,4a, 10,11,11 a-octahydro-5H dibenzo[a,4I [7] annul en-5-ylidene)propan-l-am ine,

1 m L o f 0.1 M sodium hydroxide is equivalent to 31.39 mg of C 20H 24C1N. STORA GE Protected from light. IM P U R IT IE S

Specified impurities A, B Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these impurities for demonstration o f compliance. See also 5.10.

Control of impurities in substances for pharmaceutical use): C, D, E, F, G. A

F . (5£Z, 10£S>5-[3-(dim ethylam ino)propylidene]-10,11dihydro-5/f-dibenzo [a^d] [7]annulen-10-ol. PhEur

★ ★ ★ ★ *****

Amlodipine Besilate (Ph Eur monograph 1491) h3c

,0 .

n^

Jl 1

^ o ^ s^ .0 ,

H *c Yo t CT ^ : o A. 10,11 -dihydro-5H-dibenzo [a,d\ [7] annulen-5-one (dibenzosuberone),

CH3

nh2

.c h 3

SOjH

o ci

and enantiomer

Q eH a j O N ^ g S

567.1

111470-99-6

n

i

ch3

A c tio n a n d u se C alcium channel blocker. PhEur--------------------------------------------------------------------------------

B. 3-(5if-dibenzo[a,d] [7]annulen-5-ylidene)-isyVdimethylpropan-1-amine (cydobenzaprine),

.C H ,

D E F IN IT IO N 3-Ethyl 5-methyl (4R^)-2-[(2-aminoethoxy)methyl]-4(2-chlorophenyl)-6-methyl-l,4-dihydropyridine-3,5dicarboxylate benzenesulfonate. C o n te n t 97.0 per cent to 102.0 per cent (anhydrous substance).

C. 3-(10,l l-dihydro-5H-dibenzo[a,d] [7]annulen-5-ylidene)N -m ethylpropan-1-am ine (nortriptyline),

CHARACTERS A p p e a ra n c e W hite or almost white powder.

Amlodipine Besilate 1-153

2016 Solubility Slightly soluble in water, freely soluble in methanol, sparingly soluble in anhydrous ethanol, slightly soluble in 2-propanol. ID E N T IF IC A T IO N Infrared absorption spectrophotometry (2.2.24).

Comparison amlodipine besilate CRS. TESTS O p tic a l ro ta tio n (2.2.7) — 0.10° to + 0.10°. Dissolve 0.250 g in methanol R and dilute to 25.0 m L with the same solvent.

Related substances Liquid chromatography (2.2.29). Carry out the test protected front light. Test solution (a) Dissolve 50.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 m L with the mobile phase.

Test solution (b) Dilute 5.0 m L of test solution (a) to 100.0 m L with the mobile phase.

Reference solution (a) Dilute 1.0 m L o f test solution (a) to 10.0 m L with the mobile phase. Dilute 1.0 m L o f this solution to 100.0 m L with the mobile phase.

Reference solution (b) Dissolve 5 mg of amlodipine impurity B CRS and 5 mg o f amlodipine impurity G CRS in the mobile phase and dilute to 50.0 m L with the mobile phase. Dilute 1.0 m L o f the solution to 10.0 m L with the mobile phase.

Reference solution (c) Dissolve 5 mg o f amlodipine for peak identification CRS (containing impurities D , E and F) in 10 m L of the mobile phase.

Reference solution (d) Dissolve 5.0 mg of amlodipine impurity A CRS in acetomtrUe R and dilute to 5.0 m L with the same solvent. Dilute 1.0 m L o f the solution to 100.0 m L with the mobile phase. Dilute 1.0 m L o f this solution to 10.0 m L with the mobile phase.

System suitability: reference solution (b): — resolution: minimum 2.0 between the peaks due to impurities G and B. Limits: — correction factors: for the calculation o f content, multiply the peak areas o f the following impurities by the corresponding correction factor: impurity D = 1.7; impurity F = 0.7; — impurity D: not more than 3 times the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.3 per cent); — impurity A: n o t more than 1.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (d) (0.15 p er cent); — impurities E, F: for each impurity, not more than 1.5 times the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.15 per cent); — unspecified impurities: for each impurity, not more than the area o f the prindpal peak in th e chromatogram obtained w ith reference solution (a) (0.10 per cent); — total: maximum 0.8 per cent; — disregard limit. 0.5 times the area of the principal peak in the chromatogram obtained w ith reference solution (a) (0.05 per cent); disregard any peak due to benzene sulfonate (relative retention = about 0.14). W a te r (2.5.12) M aximum 0.5 p er cent, determined on 1.000 g. S u lfa te d a s h (2.4.14) M aximum 0.2 per cent, determined on 1.0 g. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modification.

Injection T est solution (b), reference solution (e). Calculate the percentage content o f C26H31C1N208S from the declared content o f amlodipine besilate CRS.

Reference solution (e) Dissolve 50.0 mg o f amlodipine besilate CRS in the mobile phase and dilute to 50.0 m L with

STO RA G E In an airtight container, protected from light.

the mobile phase. Dilute 5.0 m L of the solution to 100.0 m L with the mobile phase.

IM P U R IT IE S

Column: — size: I = 0.25 m , 0 = 4.0 mm; — stationary phase: octadecylsüyl silica gel for chromatography R (5 |im ); — temperature: 30 °C.

Mobile phase 2.3 g/L solution of ammonium acetate R, methanol R (30:70 VIV). Flow rate 1.5 mlVmin. Detection Spectrophotom eter at 237 nm. Injection 20 |iL o f test solution (a) and reference solutions (a),

Specified impurities A, D , E, F Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other o f the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general m onograph Substances for pharmaceutical use (2034). It is therefore n o t necessary to identify these impurities for demonstration of compliance. See also 5.10.

Control of impurities in substances for pharmaceutical use): B, G, H.

(b), (c) and (d).

Run time Twice the retention time o f amlodipine. Identification of impurities Use the chromatogram supplied with amlodipine for peak identification CRS and the chromatogram obtained with reference solution (c) to identify the peaks due to impurities D , E and F; use the chromatogram obtained with reference solution (d) to identify the peak due to impurity A.

Relative retention W ith reference to amlodipine (retention time = about 20 min): impurity G = about 0.21; impurity B = about 0.25; impurity D = about 0.5; impurity F = about 0.8; impurity E = about 1.3.

A. 3-ethyl 5-methyl (4&S)-4-(2-chlorophenyl)-2-[[2-(l,3dioxo-1,3-dihydro-2//-isoindol-2-yl) ethoxy] methyl]-6m ethyl-1,4-dihydropyridine-3,5-dicarboxylate,

1-154 Ammonia

2016 h3c x

NH

a and enantiomer

B. 3-ethyl 5-methyl (4&S)-4-(2-chlorophenyi)-6-methyl-2[ [2-[[2-(methylcarbamoyl)benzoyl] amino] ethoxy] methyl] -1,4dihydropyridine-3,5-dicarboxylate,

H . 2- [ [2- [[(4&S)-4-(2-chlorophenyl)-3 -(ethoxycarbonyl)-S(m ethoxycaibonyl)-6-methyl-1,4-dihydropyridin-2-yI] methoxy] ethyl] carbamoyl]benzoic acid. ___________________________________________________________ PhEur

Strong Ammonia Solution (Ammonia Solution, Concentrated, Ph Eur monograph 0877) NH3

***** *★ ** *

17.03

Preparation D. 3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4(2-chlorophenyl)-6-methylpyridine-3,5-dicarboxyiate,

D ilute Ammonia Solution PhEur___________________________________________________________

DEFINITION Content 25.0

per cent m/m to 30.0 p er cent mtm.

CHARACTERS Appearance Clear, colourless liquid, very caustic.

Solubility Miscible with water and with ethanol (96 per cent). E. diethyl (4/25)-2-[(2-aminoethoxy)methyl]-4(2-chlorophenyl)-6-methyl-l,4-dihydropyridine-3,5dicarboxylate,

IDENTIFICATION A. Relative density (2.2.5): 0.892 to 0.910. B. It is strongly alkaline (2.2.4). C. T o 0.5 m L add 5 m L o f water R. Bubble air through die solution and lead the gaseous mixture obtained over the surface o f a solution containing 1 m L o f 0.1 M hydrochloric acid and 0.05 m L o f methyl red solution R. T h e colour changes from red to yellow. Add 1 m L o f sodium cobabtmtrite solution R. A yellow precipitate is formed.

TESTS Solution S F. dimethyl (4RS)-2-[(2-aminoethoxy)methyi]-4(2-chlorophenyl) -6-m ethyl-134-dihydropyridme-3,5dkarboxylate,

Evaporate 220 m L almost to dryness on a water-bath. Cool, add 1 m L o f dilute acetic add R and dilute to 20 m L with

distilled water R. Appearance of solution T h e solution is clear (2.2.1) and colourless (2.2.2, Method II). T o 2 m L add 8 m L of water R. Oxidisable substances Cautiously add, whilst cooling, 8.8 m L to 100 m L of dilute

sulfuric add R. A dd 0.75 m L o f 0.002 Mpotassium permanganate. Allow to stand for 5 m in. T h e solution rem ains faintly pink. G. dimethyl 4-(2-chlorophenyl)-2j 6-dimethyl-1,4dihydropyridine-3,5-dicarboxylate,

Pyridine and related substances Maximum 2 ppm , calculated as pyridine. M easure the absorbance (2.2.25) at 252 n m using water R as the. compensation liquid. T h e absorbance is n o t greater th an 0.06.

Ammonio Methacrylate Copolymer 1-155

2016 Carbonates

Ammonio Methacrylate Copolymer (Type A)

★ ★

★ ★

*****

(Ph Eur monograph 2081) 0

-

X

«0 O \ O

X

sodium carbonate R.

J k 0 / * \ ch3

o

H2C ^ 2

r

° 1

M aximum 60 ppm. T o 10 m L in a test-tube with a ground-glass neck add 10 m l, o f calcium hydroxide solution R. Stopper immediately and mix. Any opalescence in the solution is not more intense than that in a standard prepared at the same time and in the same manner using 10 m L o f a 0.1 g/L solution of anhydrous

ch3

C h lo rid e s (2.4.4) M aximum 1 ppm. Dilute 5 m L o f solution S to 15 m L with water R.

cr

S u lfates (2.4.13) Maximum 5 ppm. Dilute 3 m L o f solution S to 15 m L with distilled water R. Iro n (2.4.9) M aximum 0.25 ppm. Dilute 4 m L o f solution S to 10 m L with water R. H eavy m e ta ls (2.4.8) M aximum 1 ppm. Dilute 4 m L o f solution S to 20 m L with water R. 12 m L o f the solution complies with test A. Prepare the reference solution using lead standard solution (2 ppm Pb) R. R esid u e o n e v a p o ra tio n Maximum 20 m g/L Evaporate 50 m L to dryness on a water-bath and dry at 100-105 °C for 1 h. T he residue weighs a maximum of 1 mg. ASSAY Weigh accurately a flask with a ground-glass neck containing 50.0 m L o f 1 M hydrochloric add. A dd 2 m L of the substance to be examined and re-weigh. Add 0.1 m L of methyl red solution R as indicator. T itrate with 1 M sodium hydroxide until the colour changes from red to yellow.

A ctio n a n d u se Excipient. PhEir.

D E F IN IT IO N Poly(ethyl propenoate-co-methyl 2-methylpropenoate-co-2(trimethylammonio)ethyl 2-methylpropenoate) chloride having a m ean relative molecular mass of about 150 000. T h e ratio o f ethyl propenoate groups to methyl 2-methylpropenoate groups to 2-(trimethylammonio)ethyl 2-methylpropenoate groups is about 1:2:0.2. C o n te n t o f a m m o n io m e th a c ry la te g ro u p s 8.9 per cent to 12.3 per cent (dried substance). CHARACTERS A p p e a ra n c e Colourless to white or almost white granules or powder.

1 m L of 1 M hydrochloric acid is equivalent to 17.03 m g of N H 3.

S o lu b ility Practically insoluble in water, freely soluble in anhydrous ethanol and in methylene chloride giving clear to cloudy solutions. D ue to the polymeric nature o f the substance, a stirring time of u p to 5 h may be necessary.

STORAGE Protected from air, at a temperature not exceeding 20 °C.

ID E N T IF IC A T IO N A. Infrared absorption spectrophotometry (2.2.24).

__________________________________________________________ PhEur

Comparison Ph Eur. reference spectrum of ammonio methacrylate copolymer (type A ). B. Viscosity (see Tests). C. It complies with the limits o f the assay. TESTS S o lu tio n S Dissolve a quantity of the substance to be examined corresponding to 12.5 g of the dried substance in a mixture o f 35.0 g o f acetone R and 52.5 g of 2-propanol R. V iscosity (2.2.10) M aximum 15 mPa-s, determined on solution S.

Apparatus Rotating viscometer. Dimensionr. — spindle: diameter = 25.15 mm ; height = 90.74 mm ; shaft diam eter = 4.0 mm; — ctfinder. diameter = 27.62 mm ; height = 0.135 m .

Stirring speed 30 r/min. Volume of solution 16 m L of solution S. Temperature 20 °C. A p p e a ra n c e o f a film Spread 2 m L of solution S evenly on a glass plate. U pon drying a clear film is formed.

1-156 Ammonio Methacrylate Copolymer

2016

M o n o m e rs L iquid chrom atography (2.2.29). Solution A Dissolve 3.5 g o f sodium perchlorate R in water for chromatograpky R and dilute to 100 m L with the same solvent. Test solution Dissolve 5.00 g of the substance to be examined in methanol R and dilute to 50.0 m L with the same solvent. T o 10.0 m L o f this solution add 5.0 m L o f solution A, dropwise, while continuously stirring. Remove the precipitated polymer by centrifugation. U se the clear supernatant solution. Reference solution Dissolve 50.0 m g o f ethyl acrylate R and 10.0 m g o f methyl methacrylate R in methanol R and dilute to 50.0 m L with the sam e solvent. Dilute 1.0 m L o f the solution to 100.0 m L with methanol R. A dd 10 m L o f this solution to 5 m L of solution A.

Ammonio Methacrylate Copolymer (Type B)

Column: — sizer. I = 0.12 m , 0 = 4.6 mm; — stationary phase: octadecylsUyl silica gel for chromatography R

A c tio n a n d u se Excipient

(7 Jim).

Mobile phase Dilute phosphoric add R with water for chromatography R to obtain a solution at p H 2.0; mix 800 m L o f this solution and 200 m L o f methanol R, filter and degas.

* * * * *

★

★

* * * * *

(Ph Eur monograph 2082) o o

ch3

HjC ^ A ^ C H s ch3

9 H*0

H,C CH3

-"•'C H ,

cr

PhEur_____________

D E F IN IT IO N Poly(ethyl propenoate-co-m ethyl 2-m ethylpropenoate-co-2(trimethylammpnio)ethyl 2-m ethylpropenoate) chloride having a m ean relative m olecular mass o f about 150 000. T h e ratio o f ethyl propenoate groups to methyl 2-methylpropenoate groups to 2-(trimethylam monio)ethyl 2-methylpropenoate groups is about 1:2 :0 . 1.

Flow rate 2.0 mL/min. Detection Spectrophotom eter at 202 nm . Injection 50 pL. System suitability: reference solution: — resolution: minimum 1.5 between the peaks due to

C o n te n t o f a m m o n io m e th a c ry la te g ro u p s 4.5 per cent to 7.0 per cent (dried substance).

impurity A and im purity B.

Limits: — impurity A: not m ore than the area of the corresponding

CHARACTERS A p p e a ra n c e Colourless to white or almost white granules or powder.

peak in the chrom atogram obtained w ith the reference solution (100 ppm ); — impurity B: not m ore than 2.5 times the area of the corresponding peak in the chrom atogram obtained with the reference solution (50 ppm).

S o lu b ility Practically insoluble in water, freely soluble in anhydrous ethanol and in methylene chloride giving clear to cloudy solutions. D ue to the polymeric nature o f die substance, a stirring time o f up to 5 h may be necessary.

M e th a n o l (2.4.24, System A) M axim um 1.5 per c e n t H e a v y m e ta ls (2.4.8) M axim um 20 ppm. 1.0 g complies with test C. Prepare the reference solution using 2.0 m L of lead standard solution (10 ppm Pb) R. L o ss o n d ry in g (2.2.32) M axim um 3.0 per cent, determ ined on 1.000 g by drying in vacuo at 80 °C for 5 h. A SSA Y Dissolve 1.000 g in a mixture o f 3 mL o f anhydrous formic add R and 30 m L o f anhydrous acetic add R and heat to dissolve. A dd 20 m L o f acetic anhydride R. T itrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 m L of 0.1 M perchloric add is equivalent to 20.77 mg o f C 9H 1802 N C 1 (ammonio methacrylate groups). IM P U R IT IE S

ID E N T IF IC A T IO N A. Infrared absorption spectrophotom etry (2.2.24).

Comparison Ph. Eur. reference spectrum of ammonio methacrylate copolymer (type B). B. Viscosity (see Tests). C. It complies w ith the limits of the assay. TESTS S o lu tio n S Dissolve a quantity o f die substance to be examined corresponding to 12.5 g o f the dried substance in a m ixture of 35.0 g of acetone R and 52.5 g o f 2-propanol R. V iscosity (2.2.10) M axim um 15 mPa-s, determ ined on solution S.

Apparatus Rotating viscometer. Dimensions: — spindle: diam eter = 25.15 m m ; height = 90.74 m m ; shaft diam eter = 4.0 mm; — cylinder, diam eter = 27.62 m m ; height = 0.135 m.

Specified impurities A, B

Stirring speed 30 r/min. Volume of sdution 16 m L of solution S. Temperature 20 °C.

HjC

A p p e a ra n c e o f a film Spread 2 m L o f solution S evenly on a glass plate. U p o n drying a clear film is formed.

A. R = H , R ; = C 2H 5: ethyl propenoate (ethyl acrylate), B. R = R ' = C H 3: methyl 2-m ethyipropenoate (methyl methacrylate). PhEur

Ammonium Bicarbonate 1-157

2016

M o n o m e rs L iquid chromatography (2.2.29). Solution A Dissolve 3.5 g of sodium perchlorate R in water for chromatography R and dilute to 100 m L with the same solvent. Test solution Dissolve 5.00 g of the substance to be examined in methanol R and dilute to 50.0 m L with the same solvent. T o 10.0 m L of this solution add 5.0 m L o f solution A, dropwise, while continuously stirring. Remove the precipitated polymer by centrifugation. Use the clear supernatant solution. Reference solution Dissolve 50.0 mg of ethyl acrylate R and 10.0 m g o f methyl methacrylate R in methanol R and dilute to 50.0 m L with the same solvent. Dilute 1.0 m L of the solution to 100.0 m L with methanol R. Add 10 m L o f this solution to 5 m L o f solution A.

Column: — sizer. I = 0.12 m , 0 = 4.6 mm; — stationary phase: octadecylsSyl silica gel for chromatography R (7 pm).

Mobile phase Dilute phosphoric acid R with water for chromatography R to obtain a solution at p H 2.0; mix 800 m L of this solution and 200 m L o f methanol R, filter and degas. Flow rate 2.0 mL/min. Detection Spectrophotometer at 202 nm. Injection 50 (iL. System suitability: reference solution: — resolution: minimum 1.5 between the peaks due to impurity A and impurity B.

Limits: — impurity A: n o t more than the area o f the corresponding peak in the chromatogram obtained with the reference solution (100 ppm); — impurity B: not more than 2.5 times the area o f the corresponding peak in the chromatogram obtained with the reference solution (50 ppm). M e th a n o l (2.4.24, System A) M aximum 1.5 per c e n t H eav y m e ta ls (2.4.8) M axim um 20 ppm. 1.0 g complies with test C. Prepare the reference solution using 2.0 m L of lead standard solution (10 ppm Pb) R. L oss o n d ry in g (2.2.32) M axim um 3.0 p er cent, determined on 1.000 g by drying in vacuo at 80 °C for 5 h. A SSAY Dissolve 2.000 g in a mixture of 3 m L of anhydrous formic acid R and 30 m L o f anhydrous acetic add R and h eat to dissolve. Add 20 m L o f acetic anhydride R. Titrate with 0.1 M perchloric add, determining the end-point potentiometrically ( 2.2.20). 1 m L of 0.1 M perchloric add is equivalent to 20.77 mg o f C 9H i80 2N C 1 (ammonio methacrylate groups). IM P U R IT IE S

Specified impurities A, B o

HiCY ^ 0' R R

Ammonium Bicarbonate

?**%

(Ammonium Hydrogen Carbonate, Ph Eur monograph 1390)

*★ ★* *

NHtHCOa

79.1

1066-33-7

A ctio n a n d u se Expectorant P re p a ra tio n s Aromatic Ammonia Solution Strong Ammonium Acetate Solution Aromatic Ammonia Spirit PhEur__________________________________________________________

D E F IN IT IO N C o n ten t 98.0 per cent to 101.0 per cent. CHA RACTERS A p p e a ra n c e Fine, white or almost white, crystalline powder or white or almost white crystals, slightly hygroscopic. Solubility Freely soluble in water, practically insoluble in ethanol (96 per cent). It volatilises rapidly at 60 °C. T h e volatilisation takes place slowly at ambient temperatures if the substance is slightly moist. It is in a state of equilibrium with ammonium carbamate. ID E N T IF IC A T IO N A. It gives the reaction o f carbonates and bicarbonates

(2.3.1). B. Dissolve 50 m g in 2 m L of water R. T he solution gives the reaction o f amm onium salts (2.3.1). TESTS S o lu tio n S . Dissolve 14.0 g in 100 m L of distilled water R. Boil, to remove the ammonia, allow to cool and dilute to 100.0 m L with

distilled water R. C h lo rid es (2.4.4) Maximum 70 ppm . Dilute 5 m L of solution S to 15 m L with water R. S u lfates (2.4.IS) Maximum 70 ppm , determined on solution S. Iro n (2.4.9) Maximum 40 ppm . Dilute 1.8'm L o f solution S to 10 m L with water R. H eav y m e ta ls (2.4.8) Maximum 10 ppm . Dissolve cautiously 2.5 g in 25 m L o f 1 M hydrochloric acid. 12 m L of the solution complies with test A. Prepare the reference solution using lead standard solution (1 ppm Pb) R. ASSAY Dissolve cautiously 1.0 g in 20.0 m L o f 0.5 M sulfuric acid and dilute to 50 m L with water R. Boil, cool and titrate the excess of a d d w ith 1 M sodium hydroxide, using 0.1 m L o f methyl red solution R as indicator.

A. R = H , R ; = C 2H 5: ethyl propenoate (ethyl acrylate),

1 m L o f 0.5 M sulfuric acid is equivalent to 79.1 mg o fN H tH C O s.

B. R = R ' = C H 3: methyl 2-methylpropenoate (methyl methacrylate).

ST O R A G E In an airtight container.

________________________________ !___________ ;______________ PhEur

_________________________________________________________ PhEur

1-158 Ammonium Bromide

2016

Ammonium Bromide

***** +* *

(Ph. Eur. monograph 1389) N H iB r

97.9

12124-97-9

PhEur___________________________________________________________

D E F IN IT IO N C o n te n t 98.5 per cent to 101.0 per cent (dried substance). CHARACTERS A p p e a ra n c e W hite or almost white, crystalline pow der or colourless crystals, hygroscopic. S o lu b ility Freely soluble in water, sparingly soluble in ethanol (96 p er cent). It becomes yellow w hen exposed to light or air. ID E N T IF IC A T IO N A. It gives reaction (a) o f bromides (2.3.1). B. 10 m L of solution S (see Tests) gives the reaction o f amm onium salts (2.3.1). TESTS S o lu tio n S Dissolve 10.0 g in carbon dioxide-free water R and dilute to 100 m L with the sam e solvent. A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and colourless (2.2.2, Method II). A c id ity o r a lk a lin ity T o 10 m L o f solution S add 0.05 m l. o f methyl red solution R. N ot more than 0.5 m L o f 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is required to change the colour o f the indicator. B ro m a te s T o 10 m L o f solution S add 1 m L o f starch solution R, 0.1 m L of a 100 g/L solution o f potassium iodide R and 0.25 m L of 0.5 M sulfuric add and allow to stand protected from light for 5 min. N o blue or violet colour develops.

Flow rate 0.4 m U m in. Detection Conductivity detector equipped with a suitable ion suppressor.

Irqection 50 pL o f test solution (b), reference solutions (a) and (b) and the blank solution.

Run time 2.5 times the retention time o f bromide. Retention tone C hloride = about 5 min; brom ide = about 8

m in; sulfate = about 16 min.

System suitability: reference solution (b): — resolution: m inim um 8.0 betw een the peaks due to chloride and brom ide.

Calculation of percentage contents'. — for chlorides, use the concentration of chloride in reference solution (a); correct the area o f the peak due to chloride in the chrom atogram obtained w ith reference solution (a) by subtracting the area of the peak due to chloride in the chrom atogram obtained with test solution (b); — for sulfates, use the concentration o f sulfate in reference solution (a); correct the area o f the peak due to sulfate in the chrom atogram obtained with reference solution (a) by subtracting the area o f the peak due to sulfate in the chrom atogram obtained w ith test solution (b).

Limits: — chlorides: maximum 0.6 p er cent; — sulfates: m axim um 0.01 p er cent.

Iodides T o 5 m L o f solution S add 0.15 m L o f ferric chloride solution R1 and 2 m L o f methylene chloride R. Shake and allow to separate. T h e lower layer is colourless (2.2.2, Method I).

Iron (2.4.9) Maximum 20 ppm . D ilute 5 m L o f solution S to 10 m L with water R.

M agnesium and alkaline-earth m etals (2.4.7) Maximum 200 ppm , calculated as Ca. 10.0 g complies w ith the test for magnesium and alkalineearth metals. T h e volume o f 0.01 M sodium edetate used does n o t exceed 5.0 m L

C h lo rid e s a n d su lfa te s L iquid chrom atography (2.2.29).

Heavy m etals (2.4.8)

Test solution (a) Dissolve 0.400 g o f the substance to be examined in 50 m L o f water for chromatography R and dilute

12 m L o f solution S complies with test A. Prepare the reference solution using lead standard solution (1 ppm Pb) R.

to 100.0 m L w ith the same solvent.

Test solution (b) D ilute 25.0 m L o f test solution (a) to 50.0 m L with water for chromatography R. Reference solution (a) T o 25.0 m L o f test solution (a) add 1.0 m L o f sulfate standard solution (10 ppm SO 4) R and 12.0 m L of chloride standard solution (50 ppm Cl) R and dilute to 50.0 m L with water for chromatography R. Reference solution (b) D ilute 10.0 m L o f test solution (a) to 100.0 m L with water for chromatography R. T o 2.0 m l, of this solution add 8.0 m L o f chloride standard solution (50 ppm Cl) R and dilute to 20.0 m L w ith water for chromatography R. Blank solution water for chromatography R. Column: — size. I — 0.25 m , 0 = 2 m m ; — stationary phase, strongly basic anion-exchange resin for chromatography R (13 pm). Mobile phase Dissolve 0.600 g o f potassium hydroxide R in water for chromatography R and dilute to 1000.0 m L with the same solvent.

Maximum 10 ppm .

Loss on drying (2.2.32) Maximum 1.0 per cent, determ ined on 1.000 g by drying in an oven at 105 °C.

Sulfated ash (2.4.14) Maximum 0.1 p er cent, determ ined on 1.0 g. A SSA Y Dissolve 80.0 m g in water R , add 5 m L o f dilute nitric add R and dilute to 50 m L with water R. T itrate with 0.1 M silver nitratey determ ining the end-point potentiometrically (2.2.20). 1 m L o f 0.1 M silver nitrate is equivalent to 9.794 m g of N H iB r. Calculate the percentage content of N H ^B r using the following expression: o - 2.763 b

a b

= percentage content o f N H jB r and NH4CI obtained in the assay and calculated as NH*Br; = percentage content o f Cl obtained in the test for chlorides.

Ammonium Glycyrrhizinate 1-159

2016

STO RA G E In an airtight container, protected from light. __________________________________________________________ PhEur

L oss o n d ry in g (2.2.32) M aximum 1.0 per cent, determined on 1.00 g by drying in an oven at 105 °C for 2 h. S u lfa ted a s h (2.4.14) M aximum 0.1 per cent, determined on 2.0 g.

Ammonium Chloride *+ +*

(Ph Eur monograph 0007) NH 4CI

*

53.49

12125-02-9

A ction a n d use Used for the acidification of urine and to correct metabolic alkalosis.

ASSAY Dissolve 1.000 g in 20 m L of water R and add a mixture of 5 m L of formaldehyde solution R, previously neutralised to phenolphthalein solution R, and 20 m l. o f water R. After 1-2 min, titrate slowly with 1 M sodium hydroxide, using a further 0.2 m L of the same indicator. 1 m L of 1 M sodium hydroxide is equivalent to 53.49 mg o f N H 4CI. __________________________________________________________PhEur

P re p a r a tio n Ammonium Chloride Mixture PhEtr----------------------------------------------------------------------------------------

D E F IN IT IO N C o n ten t 99.0 per cent to 100.5 per cent (dried substance).

Ammonium Glycyrrhizinate

*****

(Ammonium Gfycyrrhizate, Ph Eur monograph 1772)

*★ ★* *

CHARACTERS A p p e a ra n c e White o r almost white, crystalline powder or colourless crystals. S o lu b ility Freely soluble in water. ID E N T IF IC A T IO N A It gives the reactions of chlorides (2.3.1). B. 10 m L of solution S (see Tests) gives the reaction of amm onium salts (2.3.1). TESTS S o lu tio n S Dissolve 10.0 g in carbon dioxide-free water R prepared from distilled water R an d dilute to 100 m L with the same solvent. A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and colourless (2.2.2, Method II). A cidity o r alk a lin ity T o 10 m L of solution S add 0.05 m L of methyl red solution R. N ot m ore than 0.5 m l. of 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is required to change the colour of the indicator.

Bromides and iodides T o 10 m l. of solution S add 0.1 m L of dilute hydrochloric acid R and 0.05 m l. of chloramme solution R. After 1 min, add 2 m l. o f chloroform R and shake vigorously. T h e chloroform layer remains colourless (2.2.2, Method I). S u lfates (2.4.13) M aximum 150 ppm . Dilute 10 m L o f solution S to 15 m L with distilled water R. C a lc iu m (2.4.3) M axim um 200 ppm .

GKHssNOje

840

53956-04-0

PhEtr__________________________________________________________

D E F IN IT IO N M ixture o f amm onium 18a- and 18^-glycyrrhizate (ammonium salt of (20P)-3^-[[2-0-(^- dglucopyranosyhironic acid)-a-D-glucopyranosyluronic acid]oxy]-ll-oxoolean-12-en-29-oic ad d ), the 18|3-isomer being the m ain com ponent C o n te n t 98.0 per cent to 102.0 per cent (anhydrous substance). CHARACTERS A p p e a ra n c e W hite or yellowish-white, hygroscopic powder. S olu b ility Slightly soluble in water, very slightly soluble in anhydrous ethanol, practically insoluble in acetone. It dissolves in dilute solutions of adds and of alkali hydroxides. ID E N T IF IC A T IO N A Infrared absorption spectrophotometry (2.2.24).

Dilute 5 m L of solution S to 15 m L with distilled water R.

Comparison ammonium gfycyrrkizate CRS. Dissolve 0.1 g in 20 m L of water R, add 2 m L o f dilute sodium hydroxide solution R and heat cautiously. O n heating,

Iro n (2.4.9) M aximum 20 ppm .

the solution gives off vapours th at may be identified by the alkaline reaction o f wet litmus paper (2.3.1).

Dilute 5 m L of solution S to 10 m L with water R.

TESTS A p p e a ra n c e o f so lu tio n T he solution is clear (2.2.1) and n o t more intensely coloured than reference solution BY7 (2.2.2, Method I).

H eavy m e ta ls ( 2.4.8) M aximum 10 ppm . 12 m L o f solution S complies with test A. Prepare the reference solution using lead standard solution (1 ppm Pb) R.

B.

Dissolve 1.0 g in ethanol (20 per cent V/V) R and dilute to 100.0 m L with the same solvent.

2016

1-160 Amobarbital

Specific optical rotation (2.2.7) + 49.0 to + 54.0 (anhydrous substance). Dissolve 0.5 g in ethanol (50 per cent V/V) R and dilute to 50.0 m L w ith the same solvent.

STO RA G E In an airtight container. IM P U R IT IE S

Related substances Liquid chromatography (2.2.29).

Test solution Dissolve 0.100 g o f the substance to be examined in the mobile phase and dilute to 100.0 m L with the mobile phase. Reference solution (a) D ilute 1.0 m L o f the test solution to 20.0 m L with the mobile phase.

Reference solution (b) Dissolve 50 m g o f ammonium gfycyrrhizate CRS in the mobile phase and dilute to 50.0 m L with the mobile phase. Dilute 1.0 m L o f the solution to 20.0 m L with the mobile phase.

Column: — size: I = 0.25 m , 0 = 4.0 mm, — stationary phase: octadecykifyl silica gelfor chromatography R (5-10 pm).

A. (4(3,20P)-3fH[2-0-({^-i>giucopyranosyluronic add)-a-Dglucopyranosyluronic add]oxy]-23-hydroxy-l l-oxoolean-12en-29-oic a d d (24-hydroxyglycyrrhizinic a d d ).

Mobile phase glacial acetic acid R, acetomtrUe R, water R (6:380:614 V/V/V). Flow rate 1.2 mlVmin. Detection Spectrophotom eter at 254 nm . Injection 10 pL. Run time 3 times the retention time o f 18fi-glycyrrhizic ad d . Relative retention W ith reference to 18{i-glycyrrhizic a d d

___________________________________________________________ PhEur

Amobarbital *

(Ph Eur monograph 0594)

*

*

(retention tim e = about 8 min): impurity A = about 0.8; 18a-glycyrrhizic a d d = about 1.2 . System suitability: reference solution (b): — resolution: minim um 2.0 between the peaks due to 18fi-glycynhizic a d d and 18a-glycyrrhizic ad d .

Limits: — 18a-glycyrrhizic acid: n o t m ore than twice the sum o f the areas of the peaks in the chrom atogram obtained with reference solution (a) ( 10.0 per cent), — impurity A: not m ore than the sum o f the areas o f the peaks in the chrom atogram obtained with reference solution (a) (5.0 p e r cent), — any other impurity: for each impurity, not more than 0.4 times the sum o f the areas o f the peaks in the chromatogram obtained with reference solution (a) (2.0 per cent), — sum of other impurities: not more than 1.4 times the sum o f the areas o f the peaks in the chromatogram obtained with reference solution (a) (7.0 per cent), — disregard limit: 0.04 times the sum o f the areas o f the peaks in the chrom atogram obtained with reference solution (a) (0.2 p er cent).

Heavy m etals (2.4.8) Maximum 20 ppm .

1.0 g complies with limit test C. Prepare the reference solution vising 2 m L o f lead standard solution (10 ppm Pb) R.

Water (2.5.12) M aximum 6.0 per cent, determ ined on 0.250 g.

Sulfated ash (2.4.14) M axim vim 0.2 per cent, determ ined on 1.0 g.

ASSAY Dissolve 0.600 g in 60 m L o f anhydrous acetic acid R heating at 80 °C if necessary. Cool. T itrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 m L o f 0.1 M perchloric add is equivalent to 84.0 m g Of C 42H 65N O 16.

C n H 18N 20 3

226.3

57-43-2

A c tio n a n d u se Barbiturate. PhEur__________________________________________________________

D E F IN IT IO N Amobarbital contains n o t less than 99.0 p er cent and not more than the equivalent of 101.0 p er cent o f 5-ethyl-5(3-methylbutyI)pyrimidin-2,4,6 (1 H,3H,5H) -trione, calculated with reference to die dried substance. CHARACTERS A white or alm ost white, crystalline powder, very slightly soluble in water, freely soluble in alcohol, soluble in methylene chloride. It forms water-soluble compounds with alkali hydroxides and carbonates and with ammonia. ID E N T IF IC A T IO N

First identification A , B. Second identification A , C, D. A. D eterm ine the m d tin g point (2.2.14) o f the substance to be ex a m in ed . Mix equal parts o f the substance to be exam in e d and amobarbital CRS and determine the m d tin g point o f the mixture. T h e difference between the m d tin g points (which are about 157 °C) is n o t greater than 2 °C. B. Examine by infrared absorption spectrophotom etry (2.2.24), comparing with the spectrum obtained with

amobarbital CRS. C. Examine by thin-layer chromatography (2.2.27), using

silica gel GF2 5 4 R as the coating substance.

Amobarbital Sodium 1-161

2016 Test solution Dissolve 0.1 g of the substance to be examined in alcohol R and dilute to 100 m L with the same solvent.

Amobarbital Sodium

*****

Reference solution Dissolve 0.1 g o f amobarbital CRS in alcohol R and dilute to 100 m L with the same solvent.

(Ph Eur monograph 0166)

*+ +* *

Apply separately to the plate 10 tiL o f each solution. Develop over a path of 18 cm using the lower layer from a mixture of 5 volumes o f concentrated ammonia R, 15 volumes o f alcohol R and 80 volumes of chloroform R. Examine immediately in ultraviolet light at 254 nm. The principal spot in die chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. D . It gives the reaction of non-nitrogen substituted barbiturates (2.3.1). TESTS A p p e a ra n c e o f so lu tio n Dissolve 1.0 g in a mixture of 4 m L o f dilute sodium hydroxide solution R and 6 m L o f water R. T he solution is clear (2.2.1) and not more intensely coloured than reference solution Y 6

(2.2.2, Method II). A cid ity o r alk alin ity T o 1.0 g add 50 m L o f water R and boil for 2 m in. Allow to cool and filter. T o 10 m L of the filtrate add 0.15 m L of methyl red solution R and 0.1 m L of 0.01 M sodium hydroxide. T he solution is yellow. Add 0.2 m L of 0.01 M hydrochloric add. T h e solution is red. R e la te d su b sta n c e s Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance.

Test solution Dissolve 1.0 g of the substance to be examined in alcohol R and dilute to 100 m L with the same solvent. Reference solution D ilute 0.5 m L of the test solution to 100 m L with alcohol R. Apply separately to the plate 20 |jL o f each solution. Develop over a p ath of 15 cm using the lower layer from a mixture of 5 volumes of concentrated ammonia R, 15 volumes o f alcohol R and 80 volumes o f chloroform R. Examine the plate immediately in ultraviolet light at 254 nm . Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is n o t more intense than the spot in the chromatogram obtained with the reference solution. Spray with diphenylcarbazone mercuric reagent R. Allow die plate to dry in air and spray with freshly prepared alcoholic potassium hydroxide solution R diluted 1 in 5 with aldehyde-free alcohol R. Heat at 100 °C to 105 °C for 5 min and examine immediately. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is n o t more intense than the spot in the chromatogram obtained with the reference solution (0.5 per cent). L oss o n d ry in g (2.2.32) N ot m ore than 0.5 per cent, determined on 1.000 g by drying in an oven at 105 °C. S u lfa te d a sh (2.4.14) N ot m ore than 0.1 per cent, determined on 1.0 g. A SSAY Dissolve 0.100 g in 5 m L of pyridine R. A dd 0.5 m L of thymolphthalein solution R and 10 m L o f silver nitrate solution in pyridine R. Titrate with 0.1 M ethanolic sodium hydroxide until a pure blue colour is obtained. Carry out a blank titration. 1 m L o f 0.1 M ethanolic sodium hydroxide is equivalent to 11.31 m g o f C 11H 18N 2O 3. ____________________________________________ _____________ Ph Eur

C 1iH 17N aN a03

248.3

6443-7

A ctio n a n d u se Barbiturate. PhEur__________________________________________________________

D E F IN IT IO N Amobarbital sodium contains not less than 98.5 per cent and not more than the equivalent of 102.0 per cent o f sodium derivative o f 5-ethyl-5-(3-methylbutyi)pyrimidin2,4,6(lH ,3H ,5ii)-trione, calculated with reference to the dried substance. CHARACTERS A white or almost white, granular powder, hygroscopic, very soluble in carbon dioxide-free water (a small fraction may be insoluble), freely soluble in alcohol. ID E N T IF IC A T IO N

First identification A , B, E Second identification A , C, D, E. A. Acidify 10 m L of solution S (see Tests) with dilute hydrochloric add R and shake with 20 m L o f ether R. Separate the ether layer, wash w ith 10 m L of water R, dry over anhydrous sodium sulfate R and filter. Evaporate the filtrate to dryness and dry the residue at 100 °C to 105 °C (test residue). Repeat the operations using 0.1 g of amobarbital sodium CRS (reference residue). Determine the melting point (2.2.14) of the test residue. Mix equal parts of the test residue and the reference residue and determine the melting point of the mixture. T h e difference between the melting points (which are about 157 °C) is not greater th an 2 °C. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing the spectrum obtained with the reference residue prepared from amobarbital sodium CRS with that obtained with the test residue (see identification test A). C. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance. Test solution Dissolve 0.1 g of the substance to be examined in alcohol R and dilute to 100 m l, with the same solvent.

Reference solution Dissolve 0.1 g of amobarbital sodium CRS in alcohol R and dilute to 100 m L with the same solvent. Apply separately to the plate 10 nL of each solution. Develop over a path of 18 cm using the lower layer of a mixture of 5 volumes o f concentrated ammonia R, 15 volumes o f alcohol R and 80 volumes of chloroform R. Examine immediately in ultraviolet light at 254 nm . T he principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. D . It gives the reaction o f non-nitrogen substituted barbiturates (2.3.1). E. It gives reaction (a) o f sodium (2.3.1).

1-162 Amoxicillin Sodium

2016

TESTS S o lu tio n S Dissolve 5.0 g in alcohol (50 per cent V/V) R and dilute to 50 m L with the same solvent.

(Ph. Eur. monograph 0577)

A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and not m ore intensely coloured than reference solution Y7 (2.2.2, Method II). p H (2.2.3) Dissolve 5.0 g in carbon dioxide-free water R and dilute to 50 m L with the same solvent. Disregard any slight residue. T h e p H of the solution is n o t m ore than 11.0.

! COzHa

C 16H18N 3N a 0 5S

R e la te d su b s ta n c e s Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating substance.

A c tio n a n d u se Penicillin antibacterial.

Test solution Dissolve 1.0 g o f the substance to be examined in alcohol R and dilute to 100 m L w ith the same solvent.

P re p a r a tio n s Amoxicillin Injection

Reference solution D ilute 0.5 m L of the test solution to 100 m L with alcohol R.

Co-amoxiclav Injection

Apply separately to the plate 20 jiL o f each solution. Develop over a path o f 15 cm using the lower layer of a mixture of 5 volumes o f concentrated ammonia R, 15 volumes o f alcohol R and 80 volumes of chloroform R. Examine the plate immediately in ultraviolet light at 254 nm . Spray with diphenylcarbazone mercuric reagent R. Allow the plate to dry in air and spray with freshly prepared alcoholic potassium hydroxide, solution R diluted 1 in 5 with aldehyde-free alcohol R. H eat at 100 °C to 105 °C for 5 m in and exam in e immediately. W hen examined in ultraviolet light and after spraying, any spot in the chromatogram obtained with the test solution, apart from the principal spot, is no t more intense than the spot in the chromatogram obtained with the reference solution (0.5 per cent). Disregard any spot at the point o f application. L oss o n d ry in g (2.2.32) N o t m ore than 3.0 per cent, determined on 0.50 g by drying in an oven at 130 °C. A SSA Y Dissolve 0.200 g in 5 m L o f ethanol R. Add 0.5 m L o f thymolphthalein solution R and 10 m L o f silver nitrate solution in pyridine R. T itrate with 0.1 M ethanolic sodium hydroxide until a pure blue colour is obtained. Carry out a blank titration. 1 m L oi 0.1 M ethanolic sodium hydroxide is equivalent to 24.83 m g o f C n H jy ^ N a C ^ .

*** ★ * ★ ★ *****

Amoxicillin Sodium

387.4

34642-77-8

PhEur.

D E F IN IT IO N Sodium (2S,5R,6R)-6-[[(2i?)-2-amino-2-(4-hydroxyphenyl) acetyl] amino] -3,3-dimethyl-7 -oxo-4-thia-1 azabicydo[3.2.0]heptane-2-carboxylate. Semi-synthetic product derived from a fermentation product. C o n te n t 89.0 per cent to 102.0 per cent (anhydrous substance). CHARACTERS A p p e a ra n c e W hite or almost white, very hygroscopic, powder. S o lu b ility Very soluble in water, sparingly soluble in anhydrous ethanol, very slightly soluble in acetone. ID E N T IF IC A T IO N

First identification A , D. Second identification B, C, D. A. Infrared absorption spectrophotom etry (2.2.24). Preparation Dissolve 0.250 g in 5 m L o f water R, add 0.5 m L o f dilute acetic add R, swirl and allow to stand for 10 m in in iced water. Filter die crystals and wash with 2-3 m L o f a mixture o f 1 volume o f water R and 9 volumes o f acetone R, then dry in an oven at 60 °C for 30 min.

Comparison amoxicillin trihydrate CRS. B. Thin-layer chromatography (2.2.27).

STORAGE Store in an airtight container.

Test solution Dissolve 25 m g o f die substance to be examined in 10 m L o f sodium hydrogen carbonate solution R. PhEur

Reference solution (a) Dissolve 25 m g o f amoxicillin trihydrate CRS in 10 m L o f sodium hydrogen carbonate solution R. Reference solution (b) Dissolve 25 m g o f amoxicillin trihydrate CRS and 25 mg o f ampidHin trihydrate CRS in 10 m L o f sodium hydrogen carbonate solution R. Plate TLC sUamsed silica gel plate R. Mobile phase M ix 10 volumes o f acetone R and 90 volumes of a 154 g/L solution o f ammonium acetate R previously adjusted to p H 5.0 with glacial acetic acid R. Application 1 |jL. Development Over a path o f 15 cm. Drying In air. Detection Expose to iodine vapour until the spots appear and examine in daylight-

Amoxicillin Sodium 1-163

2016 System suitability: reference solution (b): —

the chromatogram shows 2 clearly separated spots.

Results T h e principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). C. Place about 2 m g in a test-tube about 150 m m long and about 15 mm in diameter. M oisten with 0.05 m L o f water R and add 2 m L o f sulfuric acid-formaldehyde reagent R. M ix the contents o f the tube by swirling; the solution is practically colourless. Place the test-tube in a water-bath for 1 min; a dark yellow colour develops. D . It gives reaction (a) o f sodium (2.3.1). TESTS A p p e a ra n c e o f so lu tio n T h e solution is n o t more opalescent than reference suspension II (2.2. 1), it may show an initial, b u t transient, pink colour, and after 5 min, its absorbance (2.2.25) at 430 nm is not greater than 0.20. Dissolve 1.0 g in water R and dilute to 10.0 m L with the same solvent Examine immediately after dissolution. p H (2.2.3) 8.0 to 10.0. Dissolve 2.0 g in carbon dioxide-free water R and dilute to 20 m L with the same solvent. S p ecific o p tic a l ro ta tio n (2.2.7) + 240 to + 290 (anhydrous substance). Dissolve 62.5 m g in a 4 g/L solution of potassium hydrogen phthalate R and dilute to 25.0 m L with the same solution. R e la te d su b sta n c e s Liquid chromatography (2.2.29).

Test solution (a) Dissolve 30.0 mg o f the substance to be examined in mobile phase A and dilute to 50.0 m L with mobile phase A. Test solution (b) Dissolve 30.0 m g o f the substance to be examined in mobile phase A and dilute to 20.0 m L with mobile phase A. Prepare immediately before use. Reference solution (a) Dissolve 30.0 m g o f amoxicillin trihydrate CRS in mobile phase A and dilute to 50.0 m L with mobile phase A.

Reference solution (b) Dissolve 4.0 m g of cefadroxü CRS in mobile phase A and dilute to 50 m L with mobile phase A. T o 5.0 m L o f this solution add 5.0 m L of reference solution (a) and dilute to 100 m L with mobile phase A.

Reference solution (c) Dilute 2.0 m L o f reference solution (a) to 20.0 m L with mobile phase A. Dilute 5.0 m L of this solution to 20.0 m L with mobile phase A.

Reference solution (d) T o 0.20 g of amoxicillin trihydrate R add 1.0 m L o f water R. Shake and add dropwise dilute sodium hydroxide solution R to obtain a solution. T he p H of the solution is about 8.5. Store the solution at room tem perature for 4 h. Dilute 0.5 m L of this solution to 50.0 m L with mobile phase A.

Column: — sizer. I = 0.25 m , 0 = 4.6 mm; — stationary phase: octadecylsüyl silica gel for chromatography R (5 pm ).

Mobile phase: — mobile phase A: mix 1 volume o f acetonitrüe R and 99 volumes o f a 25 per cent V/V solution o f 0.2 M potassium dihydrogen phosphate R adjusted to p H 5.0 with dilute sodium hydroxide solution R]

— mobile phase B: mix 20 volumes of acetonitrüe R and 80 volumes o f a 25 per cent V/V solution o f 0.2 M potassium dihydrogen phosphate R adjusted to p H 5.0 with

dilute sodium hydroxide solution R\ Time (min) 0 -t,

Mobile phase A (per cent V/V) 92

Mobile phase B (per cent V/V) 8

f ,- ( f , + 25)

92 -» 0

8 4 100

(Í, + 25) - (Í, + 40)

0

100

(t^ + íO M ^ + ss)

92

8

t, = retention time of amoxicillin determined with reference solution (c)

I f the mobile phase has been adjusted to achieve the required resolution, the adjusted composition will apply at time zero in the gradient and in the assay.

Flow rate 1.0 mL/min. Detection Spectrophotom eter at 254 nm. Injection 50 |iL o f reference solutions (b) and (c) with isocratic elution at the initial mobile phase composition and 50 |iL o f test solution (b) and reference solution (d) according to the elution gradient described under Mobile phase; inject mobile phase A as a blank according to the elution gradient described under Mobile phase.

Identification of impurities Use the chromatogram obtained w ith reference solution (d) to identify the 3 principal peaks eluted after the m ain peak corresponding to impurity C, amoxicillin dim er (impurity J; n = 1) and amoxicillin trim er (impurity J; n = 2).

Relative retention W ith reference to amoxicillin: im purity C = about 3.4; impurity J (n = 1) = about 4.1; impurity J (n = 2) = about 4.5. System suitability, reference solution (b): — resolution: m inim um 2.0 between the peaks due to amoxicillin and cefadroxil; if necessary, adjust the ratio A:B o f the mobile phase.

Limits: — impurity J (n = 1): not more than 3 times the area o f the principal peak in the chromatogram obtained with reference solution (c) (3 per cent); — any other impurity: for each impurity, n o t more than twice the area o f th e principal peak in the chromatogram obtained w ith reference solution (c) (2 per cent); — total: not m ore than 9 times the area o f the principal peak in the chromatogram obtained with reference solution (c) (9 per cent); — disregard limit. 0.1 times the area of the principal peak in the chromatogram obtained w ith reference solution (c) (0.1 per cent). iV ^V -D im ethylaniline (2.4.26, Method A or B) M aximum 20 ppm. 2 -E th y lh ex a n o ic a c id (2.4.28) Maximum 0.8 per cent m/m. H e a v y m e ta ls (2.4.8) M aximum 20 ppm. 1.0 g complies with test C. Prepare the reference solution using 2 m L o f lead standard solution (10 ppm Pb) R. W a te r (2.5.12) M axim um 3.0 p er cent, determined on 0.400 g.

1-164 Amoxicillin Sodium

2016

B a c te ria l e n d o to x in s (2.6.14) Less than 0.25 IU/mg, if intended for use in the manufacture o f parenteral preparations w ithout a further appropriate procedure for the removal of bacterial endotoxins. A SSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modifications.

Mobile phase Initial composition o f the mixture of mobile phases A and B, adjusted where applicable.

H NH*

H\jP O2H HN ^CH , j î ^ ^ / ^ c H j 3nd epimer a tC *.

I 1

I O

H

E. (2RS,4S)-2-[ [[(2Æ)-2-amino-2-(4-hydroxyphenyl) acetyl] amino]methyI] -5,5-dimethylthiazolidine-4-carboxylic a d d (penilloic adds of amoxidllin),

Injection T est solution (a) and reference solution (a). System suitability: reference solution (a): — repeatability: maximum relative standard deviation of 1.0 per cent after 6 injections. Calculate the percentage content of amoxicillin sodium by multiplying the percentage content o f amoxicillin by 1.060. STORAGE In an airtight container. If the substance is sterile, store in a sterile, airtight, tam per-proof container.

F. 3-(4-hydroxyphenyl)pyrazin-2-ol,

IM P U R IT IE S o V

V ^ H ^ h,

nA

H2n - -|— k -S H H

CHa

A. (2S,5/?,6i?)-6-amino-3,3-dimethyl-7-oxo-4-thia-lazabicyclo[3.2.0]heptane-2-carboxylic a d d (6-aminopenidllanic acid),

G. (2S,5R,6R)-6 - [[(2R)-2-[[(2Æ)-2-amino-2(4-hydroxyphenyl) acetyl] amino] 2(4-hydroxyphenyl) acetyl] amino]-3,3-dim ethyl-7-oxo-4-thia-1azabicydo[3.2.0]heptane2-carboxylic a d d

(D-(4-hydroxyphenyl)glycylamoxidllm), h3c

0

HjCJ__ff h-î /

h

!NH C02H

B. (2S,52?,6Æ)-6-[[(2S)-2-amino-2-(4-hydroxyphenyl)acetyl] amino] -3,3-dime thyl-7-oxo-4-thia-1azabicyclo[3.2.0]heptane-2-caiboxylic acid (L-amoxicillin),

H . (2i?)-2-[(2,2-dimethylpropanoyl)amino]-2(4-hydroxyphenyl) acetic ad d ,

I. (2R) -2-amino-2-(4-hydroxyphenyl) acetic a d d ,

C. (4S)-2- [5-(4-hydroxyphenyl)-3,6-dioxopiperazin-2-yl] -5,5dimethylthiazolidine-4-carboxylic acid (amoxicillin diketopiperazines), *1 COzH H NH2 u

H N -A .C H 3 CO2H

D . (45)-2-[ [[(2Æ)-2-amino-2-(4-hydroxyphenyl)acetyI] amino] carboxymethyl]-5,5-dimethylthiazolidine-4-carboxylic a d d (penidlloic ad d s o f amoxicillin),

J. co-oligomers of amoxidllin and penidlloic ad d s of amoxicillin,

Amoxicillin Trihydrate 1-165

2016

K. oligomers o f penicilloic ad d s of amoxicillin. ____ ______________________________________________________ PhEur

Reference solution (b) Dissolve 25 m g of anumcSlin trihydrate CRS and 25 mg of ampidHin trihydrate CRS in 10 m L of sodium hydrogen carbonate solution R Plate TLC sUamsed silica gel plate R. Mobile phase Mix 10 volumes of acetone R and 90 volumes o f a 154 g/L solution of ammonium acetate R previously adjusted to pH 5.0 with glacial acetic acid R. Application 1 pL. Development Over a path of 15 cm. Drying la air. Detection Expose to iodine vapour until the spots appear and examine in daylight. System suitability: reference solution (b): — the chromatogram shows 2 clearly separated spots.

Amoxicillin Trihydrate

Results T he prindpal spot in the chromatogram obtained w ith

(Ph Eur monograph 0260)

*

the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a). C. Place about 2 m g in a test-tube about 150 m m long and about 15 m m in diameter. M oisten with 0.05 m L of water R and add 2 m L o f sulfuric acid-formaldehyde reagent R. Mix the contents o f the tube by swirling; the solution is practically colourless. Place the test-tube in a water-bath for 1 min; a dark yellow colour devdops.

C 16H 19N 30 5S,3H20

419.4

61336-70-7

A ctio n a n d u se Penicillin antibacterial. P re p a r a tio n s Amoxicillin Capsules Amoxicillin Oral Suspension Co-amoxiclav Oral Suspension Co-amoxiclav T ablets Dispersible Co-amoxiclav Tablets PhEur------------------------------------------------------------------------------------------

D E F IN IT IO N (25j5i?j6i?)-6-[[(2R)-2-Amino-2-(4-hydroxyphenyl)acetyl] amino]-3,3-dimethyl-7 -oxo-4-thia-1 azabicyclo [3.2.0]h ep tane-2-carboxylic a d d trihydrate. Semi-synthetic product derived from a fermentation product. C o n te n t 95.0 per cent to 102.0 per cent (anhydrous substance). CHARACTERS A p p e a ra n c e White or almost white, crystalline powder.

Solubility Slightly soluble in w ater, very slightly soluble in ethanol (96 per cent), practically insoluble in fatty oils. It dissolves in dilute a d d s and dilute solutions o f alkali hydroxides. ID E N T IF IC A T IO N

First identification A Second identification B, C A. Infrared absorption spectrophotometry (2.2.24).

Comparison amoxicillin trihydrate CRS. B. Thin-layer chrom atography (2.2.27).

TESTS S o lu tio n S With the aid of ultrasound or gentle heating, dissolve 0.100 g in carbon dioxide-free water R and dilute to 50.0 m L with the same solvent. p H (2.2.3) 3.5 to 5.5 for solution S. S pecific o p tic a l ro ta tio n (2.2.7) + 290 to + 315 (anhydrous substance), determined on solution S. R e la te d su b sta n c e s Liquid chromatography (2.2.29).

Buffer solution pH 50 T o 250 m L o f 0.2 M potassium dihydrogen phosphate R add dilute sodium hydroxide solution R to p H 5.0 and dilute to 1000.0 m L with water R Test solution (a) Dissolve 30.0 m g of the substance to be examined in mobile phase A and dilute to 50.0 m L with mobile phase A.

Test solution (b) Dissolve 30.0 m g of the substance to be examined in mobile phase A and dilute to 20.0 m l. with mobile phase A Prepare immediately before use. Reference solution (a) Dissolve 30.0 m g o f amoxicillin trihydrate CRS in mobile phase A and dilute to 50.0 m L with mobile phase A

Reference solution (b) Dissolve 4.0 m g of cefadroxU CRS in mobile phase A and dilute to 50 m L with mobile phase A. T o 5.0 m L o f this solution add 5.0 m L o f reference solution (a) and dilute to 100 m L with mobile phase A

Reference solution (c) Dilute 2.0 m L o f reference solution (a) to 20.0 m L with mobile phase A. D ilute 5.0 m L o f this solution to 20.0 m L with mobile phase A.

Column:

Test solution Dissolve 25 m g o f the substance to be examined in 10 m l, o f sodium hydrogen carbonate solution R.

— size: I = 0.25 m , 0 = 4.6 mm; — stationary phase: octadecylsHyl silica gel for chromatography R (5 pm).

Reference solution (a) Dissolve 25 mg of amoxicillin trihydrate CRS in 10 m L o f sodium hydrogen carbonate solution R.

Mobile phase: — mobile phase A: acetonitrüe R, buffer solution p H 5.0 (1:99 V!V)\

1-166 Amoxicillin Trihydrate

2016

mobile phase B: acetonitrUe R, buffer solution p H 5.0 (20:80 V!V)\ Time (min) 0-h

Mobile phase A (per cent V/V) 92

Mobile phase B (per cent V/V) 8

f, * (i* + 25)

92 -> 0

8 -> 100

(f* + 25) - (/a + 40)

0

100

(fÄ+ 4 0 )-(f, + 55)

92

8

B. (25, 5R,6R)-6-[[(25)-2-amino-2-(4-hydroxyphenyl)acetyl] amino] -3,3-dimethyl-7 -oxo-4-thia-1 azabicyclo[3.2.0]heptane-2-carboxylic a d d (L-amoxicillin),

tR= retention time of amoxicillin determined with reference solution (c) If the mobile phase composition has been adjusted to achieve the required resolution, the adjusted composition will apply at time zero in the gradient and in the assay.

Flow rate 1.0 mL/min. Detection Spectrophotom eter at 254 nm . Injection 50 (iL of reference solutions (b) and (c) with isocratic elution at the initial mobile phase composition and 50 jiL of test solution (b) according to the elution gradient described under Mobile phase; inject mobile phase A as a blank according to the elution gradient described under M obile phase. System suitability: reference solution (b): — resolution: minim um 2.0 between the peaks due to amoxicillin and cefadroxil; if necessary, adjust the ratio A:B o f the mobile phase. ;

Limit. — arty impurity: for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (c) (1 per cent).

C. (45)-2-[5-(4-hydroxyphenyl)-3,6-dioxopiperazin-2-yl]-5,5dimethylthiazolidine-4-carboxylic a d d (amoxicillin diketopiperazines), h

H N *h

CH* C02H D . (45)-2-[[[ (2R)-2-amino-2-(4-hydroxyphenyl)acetyl] amino] carboxymethyl] -5,5-dimethylthiazolidine-4-carboxylic a d d (penicilloic ad d s o f amoxicillin),

N ,N -D im e th y la n ilin e (2.4.26, Method A orB) M aximum 20 ppm. W a te r (2.5.12) 11.5 per cent to 14.5 per cent, determined on 0.100 g.

c o 2h

CO2H h

nh2

h n ^ c h 3 k g ^ X H s and epimer at C*

S u lfa te d a s h (2.4.14) M aximum 1.0 per cent, determined on 1.0 g. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances w ith the following modifications. Mobile phase Initial composition o f the mixture o f mobile phases A and B, adjusted where applicable.

E. (2RS,45)-2-[[[ (2R)-2-amino-2-(4-hydroxyphenyl)acetyl] amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic a d d (penilloic ad d s o f amoxicillin),

Injection T e st solution (a) and reference solution (a). System suitability: reference solution (a): — repeatability: maximum relative standard deviation of 1.0 per cent after 6 injections. Calculate the percentage content of C i6H 19N 30 5S taking into account the assigned content o f amoxicillin

F . 3-(4-hydroxyphenyl)pyrazin-2-ol,

trihydrate CRS. STORAGE In an airtight container. IM P U R IT IE S O

> C°2H

V i

> 0

15+ 100

(f„ + 30) - (f, + 45)

0

100

(f* + 45) - (f, + 60)

85

15

f, = retention time of ampicillin determined with reference solution (c)

If the mobile phase composition has been adjusted to achieve the required resolution, the adjusted composition will apply at time zero in the gradient and in the assay.

Flow rate 1.0 mL/min. Detection Spectrophotom eter at 254 nm . Irgecdon 50 pL o f reference solutions (b) and (c) with isocratic elution at the initial mobile phase composition and 50 pL of test solution (b) according to the elution gradient described under Mobile phase; inject mobile phase A as a blank according to the elution gradient described under M obile phase. System suitability: reference solution (b): — resolution: minimum 3.0 between the peaks due to ampicillin and cefradin; if necessary, adjust the ratio A:B o f die mobile phase.

Lima:

Dissolve 0.1 g in carbon dioxide-free water R and dilute to 40 m L with the same solvent.

— any impurity, for each impurity, n o t m ore than the area of the principal peak in the chrom atogram obtained with reference solution (c) (1.0 per cent).

S p ecific o p tic a l ro ta tio n (2.2.7) + 280 to + 305 (anhydrous substance).

N jA f-D im ethylaniline (2.4.26, Method B) M axim um 20 ppm .

Dissolve 62.5 m g in water R and dilute to 25.0 m L with the sam e solvent.

W a te r (2.5.12) M axim um 2.0 per cent, determ ined on 0.300 g.

R e la te d su b s ta n c e s L iquid chromatography (2.2.29).

S u lfa te d a s h (2.4.14) M axim um 0.5 per cent, determ ined on 1.0 g.

Test solution (a) Dissolve 27.0 m g o f the substance to be examined in mobile phase A and dilute to 50.0 m L with m obile phase A.

A SSA Y liq u id chromatography (2.2.29) as described in the test for related substances with the following modifications.

Test solution (b) Dissolve 27.0 mg o f the substance to be examined in mobile phase A and dilute to 10.0 m L with mobile phase A. Prepare immediately before use.

Mobile phase Initial composition o f the mixture o f mobile

Reference solution (a) Dissolve 27.0 mg pf anhydrous ampicillin CRS in mobile phase A and dilute to 50.0 m L with mobile phase A.

Reference solution (b) Dissolve 2.0 mg of cefradine CRS in mobile phase A and dilute to 50 m L with mobile phase A. T o 5.0 m L o f this solution add 5.0 m l, o f reference solution (a).

Reference solution (c) Dilute 1.0 m L of reference solution (a) to 20.0 m L w ith mobile phase A.

phases A and B, adjusted where applicable.

Irgecdon T est solution (a) and reference solution (a). System suitability: reference solution (a): — repeatability: maximum relative standard deviation of 1.0 p er cent after 6 injections. Calculate die percentage content o f C ie H ig ^ C ^ S from die declared content o f anhydrous ampicillin CRS. STORAGE In an airtight container, at a tem perature n o t exceeding 30 °C.

Ampicillin 1-171

2016

im p u r it ie s

0

V t

> C°2H >< CH3

^ " r r s

CHj

H H

A. (2S,5R, 6i?)-6-amino-3j3-dimethyl-7-oxo-4-thia-1azabicydo [3.2.0] heptane-2-carboxyHc a d d (6-am inopenidllanic ad d ),

H . 3-phenylpyrazin-2-ol, h

nh 2

° 0

J > C°2H

H NH H V n A ^ C H 3 H H

B. (2S,5R,6K)-6-[ [(2.S)-2-amino-2-phenylacetyl] amino]-3,3dim ethyl-7-oxo-4-thia-l -azabicydo [3.2.0] heptane-2carboxylic a d d (L-ampicillin),

I. (2S,5R,6R)-6- [ [(2R)-2- [ [(2R)-2-amino-2-phenylacetyl] amino]-2-phenyiacetyl] amino]-3,3-dimethyl-7 -oxo-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylic a d d (D-phenylglycylampicillin),

° v _ N*- V\ *^CH3 CH, II

0

H H

J. (2S,5i?j6i?)-6-[(2,2-dimethylpropanoyl) amino] -3,3dimethy 1-7-oxo-4-thia-1-azabicydo [3.2.0] heptane- 2carboxylic ad d , C. (4S)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5dimethylthiazolidine-4-caiboxylic a d d (diketopiperazines o f am pidtlin),

CH3 0 H3C H NH

^ . c o 2h H NH2 h

CO2H

H N ''A C°2H xh3

nA

H H

CHa

A. (2S,5R, 6/?)-6-amino-3,3-dimethyl-7 -oxo-4-thia-1 azabicydo[3.2.0]heptane-2-carboxylic a d d (6-aminopenidllanic a d d ),

Test solution (a) Dissolve 1.0 g o f the substance to be examined in water R and dilute to 10.0 m L with the same solvent. Test solution (b) Dissolve 1.0 g o f the substance to be examined in water R, add 1.0 m L o f the internal standard solution and dilute to 10.0 m L with water R. Reference solution Dissolve 1.0 m L o f methylene chloride R in water R and dilute to 500.0 m L w ith the same solvent. T o 1.0 m L o f this solution add 1.0 m L o f the internal standard solution and dilute to 10.0 m L with water R.

Column: — material: glass; — size. / = 1.5 m , 0 = 4 mm;

B. (25,5i?,6i?)-6-[[(25)-2-amino-2-phenylacetyI]amino]-3,3dimethyl-7 -oxo-4-thia-1 -azabicyclo [3.2.0] heptane-2carboxylic a d d (L-ampicillin),

1-174 Ampicillin Sodium

2016

C. (45)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5dimethylthiazolidme-4-caiboxylic a d d (diketopiperazines of ampidllin),

J. (2S,5i?,6.R)-6-[(2,2-dimethylpropanoyl)amino]-3,3dim ethyl-7-oxo-4-thia-l-azabicydo[3.2.0]heptane-2carboxylic ad d ,

CH3 o H3C^ _ ^

COjH

H ^

H N - \ .C H 3

X

^ Nn î A

I

0

S

H3C H NH CO2H

CH3

R

D . R = C 0 2H : (4 68

15 -* 32

32-37

68

32

A (6aJ?)-l 0-methoxy-6-methyl-5,6,6a,7-tetrahydro-4ifdibenzo [deg\quinolin-1 l-o l (apocodeine),

Aprotinin 1-183

2016 CHARACTERS Appearance Almost white hygroscopic powder.

Solubility Soluble in water and in isotonic solutions, practically insoluble in organic solvents. B. 7,8-didehydro-4,5a-epoxy-17-methyimorphinan-3,6a-diol (m orphine).

IDENTIFICATION A. Thin-layer chromatography (2.2.27).

Test solution Solution S (see Tests). Reference solution D ilute aprotinin solution BRP in water R to obtain a concentration of 15 Ph. Eur. UVmL.

C. (6aR)-9-[7,8-didehydro-4,5a-epoxy-3-hydroxy-17m ethylm orphinan- 6a-yl] -6-methyl-5,6 , 6 a, 7-tetrahydr o-4Hdibenzo [de,g\ quinoline- 10, 11-diol (m orphine-apomorphine dimer). __________________________________________________________ PhEi*

Plate TLC silica gel G plate R. Mobile phase water R, glacial acetic acid R (80:100 V/V) co n taining 100 g/L o f sodium acetate R. Application 10 |iL. Development Over a path o f 12 cm. Drying In air. Detection Spray with a solution of 0.1 g of ninhydrin R in a mixture o f 6 m L o f a 10 g/L solution of cupric chloride R, 21 m L of glacial acetic acid R and 70 m L o f anhydrous ethanol R. Dry the plate at 60 °C. Results T he principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution.

Aprotinin

*****

(Ph Eur monograph 0580)

*+ ** *

H - A rg-Pro - Asp - P h e -C y s -L e u -G lu -P r o -P r o -T y r -------------------------------------- . t(J T h r-G ly -P ro -C y s -L y s -A )a -A rg — lie— lie—Arg-

I______________________20 Tyr - Phe - Tyr - Asn - Ala - Lys - Ala - Gly - Leu - Cys -

_____________________________ m

G ln -T h r-P h e -V a l-T y r -G ly -G ly -C y s -A rg -A la — I_____________ i 2_____

Lys - Arg - Asn - Asn - Phe - Lys - Ser - Ala - Glu - Asp -

_I Cys - Met - Arg - Thr - Cys - Gly ----------------------C 284H 432N 84O79S7

50

Gly - Ala - OH

58

6511

A ctio n a n d u se Antifibrinolytic. PhEtr__________________________________________________________

D E F IN IT IO N Aprotinin is a polypeptide consisting of a chain of 58 amino acids. It inhibits stoichiometrically the activity of several proteolytic enzymes such as chymotrypsin, kallikrein, plasmin and trypsin. It contains not less than 3.0 Ph. Eur. U . of aprotinin activity per milligram, calculated with reference to the dried substance. P R O D U C T IO N T he animals from which aprotinin is derived must fulfil the requirem ents for the health of animals suitable for hum an consumption. T he m ethod o f manufacture is validated to demonstrate that the product, if tested, would comply with the following tests. A b n o rm a l to x ic ity (2.6.9) Inject into each mouse a quantity o f the substance to be examined containing 2 Ph. Eur. U. dissolved in a sufficient quantity o f waterfor injections R to give a volume o f 0.5 mL. H is ta m in e (2.6.10) M axim um 0.2 |ig o f histamine base p er 3 Phi Eur. U .

B. Determine the ability of the substance to be examined to inhibit trypsin activity using the m ethod described below.

Test solution Dilute 1 m L o f solution S to 50 m L w ith buffer solution pH 7.2 R. Trypsin solution Dissolve 10 mg of trypsin BRP in 0.002 M hydrochloric add and dilute to 100 m L with the same acid. Casein solution Dissolve 0.2 g of casein R in buffer solution pH 7.2 R and dilute to 100 m L w ith the same buffer solution.

Predpitating solution gladd acetic add R, water R, anhydrous ethanol R (1:49:50 V/V/V). Mix 1 m L o f the test solution with 1 m L o f the trypsin solution. Allow to stand for 10 m in and add 1 m L o f the casein solution. Incubate at 35 °C for 30 m in. Cool in iced water and add 0.5 m L o f the precipitating solution. Shake and allow to stand at room temperature for 15 min. T h e solution is cloudy. Carry out a blank test under the same conditions using buffer solution pH 7.2 R instead of the test solution. T he solution is not cloudy.

TESTS Solution S Prepare a solution o f the substance to be examined containing 15 Ph. Eur. UVmL, calculated from the activity stated on the label.

Appearance of solution Solution S is clear (2.2.1). Absorbance (2.2.25) Maximum 0.80 by measuring at the absorption maximum at 277 nm. Prepare a solution o f the substance to be examined containing 3.0 Ph. Eur. U ./ mL.

Des-Ala-aprotinin and des-Ala-des-Gly-aprotinin Capillary zone electrophoresis (2.2.47): Use the normalisation procedure.

2016

1-184 Aprotinin

Test solution Prepare a solution o f the substance to be water R containing n o t less than

ex am in ed in

1

dihydrate R and 66.07 g o f ammonium sulfate R in 1000 m L of water; filter and degas;

Ph. E ur. U ./m L.

Reference solution D ilute aprotinin solution BRP in water R to

Time (min) 0 -2 1

obtain the same concentration as the test solution.

Capillary: — material: uncoated fused silica; — sizer, effective length = 45-60 cm, 0 = 75 Jim.

Temperature 25 °C. CZE buffer Dissolve 8.21 g o f potassium ¿¡hydrogen phosphate R in 400 m L o f water R, adjust to p H 3.0 with phosphoric add R, dilute to 500.0 m L with water R and filter through a m em brane filter (nominal pore size 0.45 Jim).

Detection Spectrophotom eter a t 214 nm . Between-run rinsing Rinse the capillary for at least 1 min with 0.1 M sodium hydroxide filtered through a m em brane filter (nominal pore size 0.45 Jim) and for 2 min with the C ZE buffer.

Irgection U nder pressure or vacuum (for example, 3 s a t a differential pressure o f 3.5 kPa).

Migration Apply a field strength o f 0.2 kV/cm, using the C ZE buffer as the electrolyte in b oth buffer reservoirs. Run time 30 min. Identification of impurities Use the electropherogram supplied with aprotinin solution BRP and the electropherogram obtained with the reference solution to identify the peaks due to impurities A and B.

Relative migration W ith reference to aprotinin (migration tim e = about 22 m in): impurity A = about 0.98; im purity B = about 0.99.

System suitability Reference solution after at least 6 injections: — migration time: aprotinin = 19.0 m in to 25.0 m in ; — resolution: m inim um 0.8 between the peaks due to impurities A and B; minimum 0.5 between the peaks due to im purity B and aprotinin; — peak distribution: the electrophoregram obtained is qualitatively and quantitatively sim ilar to the electropherogram supplied with aprotinin solution BRP, — height of the prindpal peak: at least 1000 times the height o f the baseline noise. If necessary, adjust the sample load to give peaks o f sufficient height. Limits: — impurity A: maximum 8.0 per cent; — impurity B: m axim um 7.5 per cent.

Pyroghitamyl-aprotinin and related compounds L iquid chrom atography (2.2.29): use the normalisation procedure.

Test solution Prepare a solution of the substance to be e x a m in ed in mobile phase A, containing about

5 Ph. E ur. UVmL.

Reference sdudon Dissolve the contents of a vial o f aprotinin for system suitability CRS in 2.0 m L o f mobile phase A. Column: — size. I = 0.075 m , 0 = 7.5 mm; — stationary phase, strong cation-exchange silica gelfor chromatography R (10 pm); — temperature. 40 °C. Mobile phase: — mobile phase A: dissolve 3.52 g o f potassium dihydrogen phosphate R and 7.26 g o f disodium hydrogen phosphate dihydrate R in 1000 m L o f water; filter and degas; — mobile phase B: dissolve 3.52 g o f potassium dihydrogen phosphate R, 7.26 g o f disodium hydrogen phosphate

21 -3 0

Mobile phase A (percent V/V) 92 64 64

0

Mobile phase B (percent V/V) 8*36 3 6 * 100

Flow rate 1.0 mlVmin. Detection Spectrophotom eter a t 210 nm . Irgection 40 |xL. Relative retention W ith reference to aprotinin (retention tim e = 17.0 min to 20.0 min): impurity C = about 0.9. System suitability: reference solution: — resolution: minimum 1.5 betw een the peaks due to impurity C and aprotinin; —■symmetry factor, maximum 1.3 for the peak due to aprotinin.

Limits: — impurity C: m aximum 1.0 p er cent; — any other impurity: m axim um 0.5 p er cent; — sum of impurities other than C: m aximum 1.0 p er c e n t Aprotinin oligomers Size-exclusion chromatography (2.2.30): U se the norm alisation procedure.

Test solution Prepare a solution o f the substance to be examined in water R containing about 5 Ph. Eur. UVmL. Reference solution T reat the substance to be examined to obtain about 2 per cent aprotinin oligomers. F or example, heat freeze-dried aprotinin at about 110 °C for about 4 h. T h en dissolve in water R to obtain a concentration o f about 5 Ph. Eur. UVmL.

Column 3 columns coupled in series: — size. I = 0.30 m , 0 = 7.8 m m ; — stationary phase, hydrophilic silica gelfor chromatography R o f a grade suitable for fractionation o f globular proteins in the relative molecular mass range o f 20 000 to 10 000 000 (8 jam). Mobile phase acetomtrUe R, glacial acetic add R, water R (2:2:6 V/VIV)-, filter and degas. Flow rate 1.0 mlVmin. Detection Spectrophotom eter at 277 nm . Irgection 100 |iL. Run lime 40 min. Relative retention W ith reference to aprotinin m onom er (retention time = 24.5 min to 25.5 min): aprotinin dim er = about 0.9. System suitability: reference solution: — resolution: minimum 1.3 betw een the peaks due to aprotinin dim er and monom er; — symmetry factor, maximum 2.5 for the peak due to aprotinin m onom er.

Limit: — total: maxim um 1.0 p er cent.

Loss on drying (2.2.32) Maximum 6.0 per cent, determ ined on 0.100 g by drying

in vacuo. B a c te r ia l e n d o to x in s (2.6.14) Less than 0.14 IU per European Pharm acopoeia U nit o f aprotinin, if intended for use in the m anufacture of parenteral preparations w ithout a further appropriate procedure for the removal o f bacterial endotoxins.

Aprotinin 1-185

2016

ASSAY T he activity of aprotinin is determined by measuring its inhibitory action on a solution of trypsin o f known activity. T he inhibiting activity of the aprotinin is calculated from the difference between the initial activity and the residual activity of die trypsin.

L A B E L L IN G

T he inhibiting activity of aprotinin is expressed in European Pharmacopoeia Units. 1 Ph. Eur. U . inhibits 50 p er cent of the enzymatic activity of 2 micro katals of trypsin.

IM P U R IT IE S

Use a reaction vessel with a capacity of about 30 m L, provided with: — a device that will maintain a temperature of 25 ± 0.1 ° Q — a stirring device, such as a magnetic stirrer; — a lid with 5 holes for accommodating the electrodes, the tip of a burette, a tube for the admission of nitrogen and the introduction of the reagents. An automatic o r manual titration apparatus may be used. In the latter case the burette is graduated in 0.05 m L and the pH -m eter is provided with a wide reading scale and glass and calomel or glass-silver-silver chloride electrodes.

Test solution Prepare a solution of the substance to be examined in 0.0015 M borate buffer solution pH 8.0 R expected to contain 1.67 Ph. Eur. UVmL (about 0.6 mg (m mg) per millilitre).

Trypsin solution Prepare a solution o f trypsin BRP containing about 0.8 micro katals per millilitre (about 1 mg/mL), using 0.001 M hydrochloric acid as the solvent. Use a freshly prepared solution and keep in iced water. Trypsin and aprotinin solution T o 4.0 m L o f the trypsin solution add 1.0 m L of the test solution. Dilute immediately to 40.0 m L with 0.0015 M borate buffer solution pH 8.0 R Allow to stand a t room tem perature for 10 min and then keep in iced water. Use within 6 h o f preparation.

Dilute trypsin solution Dilute 0.5 m L of the trypsin solution to 10.0 m L with 0.0015 M borate buffer solution pH 8.0 R Allow to stand at room tem perature for 10 min and then keep in iced water. M aintain an atm osphere o f nitrogen in the reaction flask and stir continuously; introduce 9.0 m L o f 0.0015 M borate buffer solution pH 8.0 R and 1.0 m L of a freshly prepared 6.9 g/L solution of benzqylarginine ethyl ester hydrochloride R. Adjust to p H 8.0 with 0.1 M sodium hydroxide. W hen the tem perature has reached equilibrium at 25 ± 0.1 °C, add 1.0 m L of die trypsin and aprotinin solution and start a timer. M aintain at p H 8.0 by the addition of 0.1 M sodium hydroxide and note the volume added every 30 s. Continue the reaction for 6 m in . D eterm ine the num ber of m illilitres of 0.1 M sodium hydroxide used p er second (nx mL). Carry out, under the same conditions, a titration using 1 .0 m L of the dilute trypsin solution. Determ ine the num ber o f millilitres of 0.1 M sodium hydroxide used per second (n2 mL). Calculate the aprotinin activity in European Pharmacopoeia Units per milligram using the following expression:

4000 (2na — ni)

m T he estimated activity is n o t less than 90 per cent and not more than 110 per cent o f the activity stated on the label. ST O R A G E In an airtight, tam per-proof container, protected from light.

The label states: — the num ber o f European Pharmacopoeia Units of aprotinin activity per milligram; — where applicable, that the substance is suitable for use in the m anufacture o f parenteral preparations.

Ra - Arg - Pro - A s p -P h e -C y s -L e u -G lu -P ro -P ro - Tyr -

___ !_________________ |

to

T h r-G ly -P ro -C y s -L y s -A la -A rg — lie— lie—Arg -

I

«

Tyr - Phe - Tyr - Asn - Ala - Lys - Ala - Gly - Leu - Cys -

_____________________________ m

Gin - Thr - Ptie - Val - Tyr - Gly - Gly - Cys - Arg - Ala-

w

I

Lys - Arg - Asn - Asn - Phe - Lys - Ser - Ala - Glu - Asp -

—i Cys - M e t-A rg -T h r-C y s -G ly -R b ----------------------------- «

30

A. Ra = H , Rb = OH: aprotinin-(1-56)-peptide, B. Ra = H , Rb = Gly-OH: aprotinin-(l-57)-peptide, C. Ra = Glp, Rb = Gly-Ala-OH: (5-oxoprolyl)aprotinin (pyroglutamylaprotinin). __________________________________________________ PhEur

Aprotinin Concentrated Solution (Ph Eur monograph 0579)

*

H - Arg - Pro - Asp - Phe - Cys - Leu - Glu - Pro - Pro - TyrT h r-G ly -P ro -C y s -L y s -A la -A rg — lie— lie—Arg-

|_________________

20

T y r -P h e -T y r-A s n -A la -L y s -A la -G ly -L e u -C y s -

________________________________________ 221 Gin - Thr - Phe - Val - Tyr - Gly - Gly - Cys - Arg - Ala -

Lys - Arg - Asn - Asn - Phe - Lys - Ser - Ala - Glu - Asp -

—i

Cys - Met - Arg- Thr - Cys - Gly - Gly - Ala - OH

C 284H 432N 84O79S7

30

6511

A c tio n a n d u se Antifibrinolytic. PhEur_________________________________________________________

D E F IN IT IO N Aprotinin concentrated solution is a solution o f aprotinin, a polypeptide consisting of a chain of 58 amino adds, which inhibits stoichiometrically the activity o f several proteolytic enzymes such as chymotrypsin, kallikrem, plasmin and trypsin. It contains not less than 15.0 Ph. Eur. U. o f aprotinin activity per millilitre. P R O D U C T IO N T h e animals from which aprotinin is derived m ust fulfil the requirem ents for the health of anim als suitable for hu m an consumption. T h e m ethod of manufacture is validated to demonstrate that the product, if tested, would comply with the following tests. A b n o rm a l to x icity (2.6.9) Inject into each mouse a quantity of the preparation to be examined containing 2 Ph. Eur. U . diluted with a suffident quantity of water for injections R to give a volume of 0.5 mL. H ista m in e (2.6.10) M aximum 0.2 |ig o f histamine base per 3 Ph. Eur. U.

1-186 Aprotinin

CHARACTERS A p p e a ra n c e Clear, colourless liquid. ID E N T IF IC A T IO N A. Thin-layer chromatography (2.2.27).

Test solution Solution S (see Tests). Reference solution D ilute aprotinin solution BRP in water R to obtain a concentration o f 15 Ph. Eur. U ./m L.

Plate TLC silica gel G plate R. Mobile phase water R, glacial acetic acid R (80:100 V/V) containing 100 g/L o f sodium acetate R. Application 10 jiL. Development Over a path of 12 cm. Drying In air. Detection Spray with a solution of 0.1 g o f mnhydrin R in a m ixture o f 6 m L o f a 10 g/L solution o f cupric chloride R, 21 m L o f glacial acetic acid R and 70 m L o f anhydrous ethanol R. D ry the plate at 60 °C. Results T he principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chrom atogram obtained with the reference solution. B. D eterm ine the ability o f the preparation to be examined to inhibit trypsin activity using the m ethod described below.

Test solution D ilute 1 m L o f solution S to 50 m L with buffer solution pH 7.2 R Trypsin solution Dissolve 10 m g of trypsin BRP in 0.002 M hydrochloric add and dilute to 100 m L with the same acid. Casein solution Dissolve 0.2 g o f casein R in buffer solution pH 7.2 R and dilute to 100 m L with the same buffer solution.

Predpitating solution glacial acetic add R, water R, anhydrous ethanol R (1:49:50 VfVIV). M ix 1 m L o f the test solution with 1 m L o f the trypsin solution. Allow to stand for 10 min and add 1 m L of the casein solution. Incubate at 35 °C for 30 min. Cool in iced w ater and add 0.5 m L of the precipitating solution. Shake and allow to stand at room tem perature for 15 min. T h e solution is cloudy. Carry out a blank test under the sam e conditions using buffer solution pH 7.2 R instead o f the test solution. T h e solution is n o t cloudy. TESTS S o lu tio n S Prepare a solution c o n tain in g 15 Ph. Eur. U./m L, if necessary by dilution, on the basis o f the activity stated on the label. A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1). A b so rb a n c e (2.2.25) M axim um 0.80 by measuring at the absorption m m m n m at 277 nm . Prepare a solution containing 3.0 Ph. Eur. IJ /mT.. D e s -A la -a p ro tm in a n d d e s-A la -d e s-G ly -a p ro tin m Capillary zone electrophoresis (2.2.47) U se the normalisation procedure.

2016 — size, effective length = 45-60 cm , 0 = 75 pm.

Temperature 25 °C. CZE buffer Dissolve 8.21 g o f potassium dihydrogen phosphate R in 400 m L o f water R, adjust to p H 3.0 with phosphoric add R, dilute to 500.0 m L w ith water R and filter through a m em brane filter (nom inal pore size 0.45 pm).

Detection Spectrophotom eter at 214 nm . Between-run rinsing Rinse the capillary for at least 1 min with 0.1 M sodium hydroxide filtered through a m em brane filter (n o m in al pore size 0.45 Jim) and for 2 m in with the C ZE

buffer.

Injection U nder pressure or vacuum (ibr example, 3 s at a differential pressure o f 3.5 kPa).

Migration Apply a field strength o f 0.2 kV/cm, using the C ZE buffer as the electrolyte in b oth buffer reservoirs.

Run time 30 min. Identification of impurities Use the electropherogram supplied with aprotinin solution BRP and die electropherogram obtained with the reference solution to identify the peaks due to impurities A and B.

Relative migration W ith reference to aprotinin (migration tim e = about 22 m in): impurity A = about 0.98; im purity B = about 0.99. System suitability Reference solution after at least 6 injections: — migration time, aprotinin = 19.0 m in to 25.0 min; — resolution: m in im u m 0.8 between the peaks due to impurities A and B; minim um 0.5 between the peaks due to im purity B and aprotinin; — peak distribution: the electrophoregram obtained is qualitatively and quantitatively similar to the electropherogram supplied with aprotinin solution BRP, — height of the principal peak: at least 1000 times the height o f the baseline noise. I f necessary, adjust the sample load to give peaks o f a sufficient height.

Limits. — impurity A: m axim um 8.0 per cent; — impurity B: maximum 7.5 p er cent.

Pyroglutamyl-aprotinin and related compounds Liquid chrom atography (2.2.29): use die normalisation procedure.

Test solution D ilute th e preparation to be examined in mobile phase A to a concentration o f about 5 Ph. Eur. UVmL. Reference solution Dissolve the contents o f a vial o f aprotinin for system suitability CRS in 2.0 m L o f mobile phase A. Column: — size. I = 0.075 m , 0 = 7.5 mm ; — stationary phase, strong cation-exchange silica gel for chromatography R (10 pm); — temperature. 40 °C. Mobile phase. — mobile phase A: dissolve 3.52 g o f potassium dihydrogen phosphate R and 7.26 g o f disodium hydrogen phosphate dihydrate R in 1000 m L o f water; filter and degas; — mobile phase B: dissolve 3.52 g o í potassium dihydrogen phosphate R, 7.26 g o f disodium hydrogen phosphate dihydrate R and 66.07 g of ammonium sulfate R in 1000 m L o f water; filter and degas;

Test solution D ilute the preparation to be examined in water R to obtain a concentration o f n o t less than 1 P h Eur. U./m L. obtain the same concentration as the test solution.

Time (min) 0 -2 1

Mobile phase A (percent V/V) 92 -»64

Mobile phase B (percent V/V) 8*36

Capillary:

2 1 -3 0

64*0

3 6 * 100

Reference solution D ilute aprotinin solution BRP in water R to

— material: u nfoated fused silica;

Aprotinin 1-187

2016

Flow rate 1.0 mL/min. Detection Spectrophotom eter at 210 nm. Irgection 40 (iL. Relative retention W ith reference to aprotinin (retention time = 17.0 m in to 20.0 m in); impurity C = about 0.9. System suitability: reference solution: — resolution: minim um 1.5 between the peaks due to impurity C and aprotinin; — symmetry factor, maximum 1.3 for the peak due to aprotinin.

Limits: — impurity C: maximum 1.0 per cent; — any other impurity, maximum 0.5 per cent; — sum of impurities other than C: maximum 1.0 per cent.

Aprotinin oligomers Size-exclusion chromatography (2.2.30) Use the normalisation procedure.

T he inhibiting activity of the aprotinin is calculated from the difference between the initial activity and the residual activity o f the trypsin. T h e inhibiting activity of aprotinin is expressed in European Pharmacopoeia Units. 1 Ph. Eur. U . inhibits 50 per cent o f the enzymatic activity o f 2 microkatals of trypsin. U se a reaction vessel with a capacity o f about 30 mL, provided with: — a device th at will maintain a tem perature of 25 + 0.1 °C; — a stirring device, such as a magnetic stirrer; — a lid with 5 holes for accommodating the electrodes, the tip o f a burette, a tube for the admission of nitrogen and the introduction o f the reagents. A n autom atic o r manual titration apparatus may be used. In the latter case the burette is graduated in 0.05 m L and the pH -m eter is provided with a wide reading scale and glass and calomel or glass-silver-silver chloride electrodes.

Reference solution T reat the substance to be examined to

Test solution W ith 0.0015 M borate buffer solution pH 8.0 R prepare an appropriate dilution (D) of the aprotinin concentrated solution expected, on the basis of the stated potency, to contain 1.67 Ph. Eur. IJ VmT -

obtain about 2 p er cent aprotinin oligomers. F or example, heat freeze-dried aprotinin at about 110 °C for about 4 h. T hen dissolve in water R to obtain a concentration o f about 5 Ph. E ur. U./mL.

Trypsin solution Prepare a solution o f trypsin BRP containing about 0.8 microkatals per millilitre (about 1 mg/mL), using 0.001 M hydrochloric acid as the solvent. Use a freshly prepared solution and keep in iced water.

Column 3 columns coupled in series: — sizer. I = 0.30 m , 0 = 7.8 mm; — stationary phase: hydrophilic silica gel for chromatography R o f a grade suitable for fractionation of globular proteins in the relative molecular mass range of 20 000 to 10 000 000 (8 nm).

Trypsin and aprotinin solution T o 4.0 m L of the trypsin solution add 1.0 m L o f the test solution. Dilute immediately to 40.0 m L with 0.0015 M borate buffer solution pH 8.0 R. Allow to stand at room tem perature for 10 min and then keep in iced water. U se within 6 h of preparation.

Test solution Dilute the preparation to be examined in water R to obtain a concentration of about 5 Ph. E ur. UVmL.

Mobile phase acetordtrUe R, glacial acetic add R, water R (2:2:6 VIV/V); filter and degas. Flow rate 1.0 mL/min. Detection Spectrophotom eter at 277 nm. Irgection 100 (iL. Run time 40 min. Relative retention W ith reference to aprotinin m onom er (retention time = 24.5 min to 25.5 min): aprotinin dimer = about 0.9. System suitability, reference solution: — resolution: m in im u m 1.3 between the peaks due to aprotinin dim er and monomer; — symmetry factor, m axim u m 2.5 for the peak due to aprotinin m onom er.

Limit: — total: maxim um 1.0 per cen t

Specific activity o f the dry residue Minimum 3.0 Ph. Eur. U. o f aprotinin activity per milligram of dry residue. Evaporate 25.0 m l, to dryness in a water-bath, dry the residue at 110 °C for 15 h and weigh- From the mass of the residue and the activity determined as described below, calculate the nu m b er o f European Pharmacopoeia U nits per milligram of dry residue.

Bacterial endotoxins (2.6.14) Less than 0.14 IU per European Pharmacopoeia U nit of aprotinin, if intended for use in the manufacture of parenteral preparations without a further appropriate procedure for the removal o f bacterial endotoxins.

ASSAY T he activity of aprotinin is determined by measuring its inhibitory action on a solution of trypsin of.known activity.

Dilute trypsin solution Dilute 0.5 m L of the trypsin solution to 10.0 m L with 0.0015 M borate buffer solution pH 8.0 R. Allow to stand at room temperature for 10 min and then keep in iced water. Maintain an atmosphere of nitrogen in the reaction flask and stir continuously; introduce 9.0 m L of 0.0015 M borate buffer solution pH 8.0 R and 1.0 m L of a freshly prepared 6.9 g/L solution of benzoylarginine ethyl ester hydrochloride R.'A djust to p H 8.0 with 0.1 M sodium hydroxide. W hen the tem perature has reached equilibrium at 25 ± 0.1 °C, add 1.0 m L of the trypsin and aprotinin solution and start a timer. M aintain at p H 8.0 by the addition of 0.1 M sodium hydroxide and note the volume added every 30 s. Continue the reaction for 6 min. Determine the num ber o f millilitres of 0.1 M sodium hydroxide used per second ( ^ m L). Carry out, under the same conditions, a titration using 1.0 mL of the dilute trypsin solution. Determine the num ber of millilitres of 0.1 M sodium hydroxide used per second (n2 mL). Calculate the aprotinin activity in European Pharmacopoeia U nits per millilitre vising the following expression: 4000 (2ri2 — n i )

m D

= dilution factor of the aprotinin concentrated solution to be examined in order to obtain a solution containing 1.67 Ph. Eur. UVmL.

T h e estimated activity is not less than 90 per cent and not m ore than 110 per cent of the activity stated on the label. STO RA G E In an airtight, tam per-proof container, protected from light.

1-188 Arachis Oil

2016

L A B E L L IN G

Composition of the fatty-acid fraction of the oil:

The label states:

— saturated fatty acids of chain length less than C /* m axim u m 0.4 per cent; — palmitic acid: 5.0 per cent to 14.0 per cent; — stearic add. 1.3 p er cent to 6.5 per cent; — oleic add: 35.0 p er cent to 76.0 p er cent; — linoleic add. 8.0 per cent to 43.0 p er cent; — linolenic add: maximum 0.6 per cent; — arachidic add: 0.5 per cent to 3.0 p er cent; — eicosenoic acid: 0.5 per cent to 3.0 per cent; — behenic acid: 1.0 per cent to 5.0 p er cent; — erucic add: maximum 0.5 p er cent; — Ugnoceric acid: 0.5 per cent to 3.0 p er c e n t

— the num ber of European Pharmacopoeia U nits o f aprotinin activity p er millilitre; — where applicable, th at the substance is suitable for use in the m anufacture o f parenteral preparations. IM P U R IT IE S r g -—rPro r o - Asp A s p - Phe r n e - uCys y s - Leu Leu - Glu o i u - pPro r o - Pro P ro - Tyr Ra - *Arg

_!_________________ I

10

T h r-G ly -P ro -C y s -L y s -A la -A r g — Ile— Ile—ArgI I_____________________________________________20 _________________________________________20

Tyr - Phe - Tyr - Asn - Ala - Lys - Ala - Gly - Leu - Cys-

JOJ

G in -T h r-P h e -V a l—T y r -G ly -G ly -C y s -A r g -A la —

W a te r (2.5.32) M aximum 0.1 per cent, determ ined on 1.00 g.

Lys - Arg - Asn - Asn - Phe - Lys - Ser - Ala - Glu - Asp -

—I

Cys - Met - Arg - Thr - Cys - Gly - Rb

50

STO RA G E In a well-filled container, protected from light.

A. R a = H , Rb = OH: aprotinin-(l-56)-peptide,

PhEur

B. R a = H , R b = Gly-OH: aprotinin-(l-57)-peptide, C. R a = Glp, Rb = Gly-Ala-OH: (5-oxoprolyl)aprotinin (pyrogjutamylaprotinin). PhEur

Hydrogenated Arachis Oil Hydrogenated P eanut Oil

Arachis Oil P eanut Oil

★* * * ★ ★ ★ *****

(Refined Arachis OH, Ph. Eur. monograph 0263) P r e p a r a tio n Arachis Oil Enem a Ph Fir

_______________________________________

D E F IN IT IO N T h e refined fatty oil obtained from the shelled seeds o f Arachis hypogaea L. A suitable antioxidant may be added. CHARACTERS A p p e a ra n c e C lear, yellowish, viscous liquid. S o lu b ility Very slightly soluble in ethanol (96 per cent), miscible with light petroleum.

** ** *

(Ph. Eur. monograph 1171) PhEtr------------------------------------------------------------------------------------------

D E F IN IT IO N Oil obtained by refining, bleaching, hydrogenating and d eod orisin g oil obtained from the shelled seeds o f Arachis hypogaea L. Each type o f hydrogenated arachis oil is characterised by its nom inal drop point. CHARACTERS A p p e a ra n c e W hite or faintly yellowish, soft mass which m elts to a clear, pale yellow liquid w hen heated. S o lu b ility Practically insoluble in water, freely soluble in methylene chloride and in light petroleum (bp: 65-70 °C ), very slightly soluble in ethanol (96 per cent). ID E N T IF IC A T IO N

R e lativ e d e n sity A bout 0.915.

First identification A , B Second identification A , C A. D rop point (see Tests).

It solidifies at about 2 °C.

B. Identification of fatty oils by thin-layer chrom atography

ID E N T IF IC A T IO N Identification o f fatty oils by thin-layer chromatography

(2.3.2). Results T h e chromatogram obtained is similar to the

(2.3.2). Results T he chrom atogram obtained is similar to the

chrom atogram for arachis oil shown in Figure 2.3.2.-1.

corresponding chrom atogram shown in Figure 2.3.2.-1.

TESTS D ro p p o in t (2.2.17) 32 °C to 43 °C, and within 3 °C o f th e nom inal value.

TESTS A c id v alu e (2.5.1) M axim um 0.5, determ ined on 10.0 g. P e ro x id e v a lu e (2.5.5, Method A) M axim um 5.0. U n sa p o n ifia b le m a t te r (2.5.7) M axim um 1.0 per cent, determ ined on 5.0 g. A lk alin e im p u ritie s (2.4.19) It complies with the test. C o m p o sitio n o f fa tty a d d s (2.4.22, Method A). Use the mixture o f calibratin g substances in T able 2.4.22.-3.

C. Composition o f fatty ad d s (see Tests).

A c id v a lu e (2.5.1) M aximum 0.5. Dissolve 10.0 g in 50 m L o f the prescribed solvent by heating on a water-bath. P e ro x id e v a lu e (2.5.5, Method A) M axim um 5.0. Dissolve 5.0 g in 30 m L o f the prescribed solvent by heating on a water-bath. U n sa p o n ifia b le m a t te r (2.5.7) M axim um 1.0 per c e n t

Arginine 1-189

2016

A lkaline im p u ritie s (2.4.19) It complies with the test.

Arginine

C o m p o sitio n o f fa tty acid s (2.4.22, Method A) U se the mixture o f calibrating substances in Table 2A.22.-3.

(Ph. Eur. monograph 0806)

Column:

H H2N . .N . Y

— material: fused silica; — size: I = 25 m , 0 = 0.25 mm; — stationary phase: poly(cyanopropyl)sUoxane R (film thickness

0.2 |im). Carrier gas helium for chromatography R. Flow rate 0.7 mL/min. Split ratio 1:100. Temperature: — column: 180 °C for 20 min; — injection port and detector. 250 °C. Detection Flame ionisation. Composition of the fatty-acid fraction of the oil: — saturated fatty adds of chain length less than Crf. maximum 0.5 per cent;

myrisac add: maximum 0.5 per cent; palmitic add: 7.0 per cent to 16.0 per cent; stearic acid. 3.0 per cent to 19.0 per cent; oleic acid and isomers: 54.0 per cent to 78.0 per cent; Imoleic add and isomers: maximum 10.0 per cent; arachidic acid: 1.0 per cent to 3.0 per cent; eicosenoic acids: maximum 2.1 p er cent; behenic add: 1.0 per cent to 5.0 per cent; erucic acid and isomers: maximum 0.5 per cent; lignoceric add\ 0.5 per cent to 3.0 per cent. Nickel

***** *

H* -NHj X .

co 2h

NH

CfiH iiN^A

174.2

74-79-3

A c tio n a n d u se Amino acid; nutrient. PhEur__________________________________________________________

D E F IN IT IO N (2S)-2-Amino-5-guanidinopentanoic add. Ferm entation product, extract or hydrolysate of protein. C o n te n t 98.5 per cent to 101.0 p er cent (dried substance).

— — — — — — — — — —

CHARACTERS A p p e a ra n c e W hite o r almost white, crystalline powder or colourless crystals, hygroscopic. S o lu b ility Freely soluble in water, very slightly soluble in ethanol (96 per cent). ID E N T IF IC A T IO N

First identification A , C Second identification A , B, D, E

M aximum 1 ppm.

A. Specific optical rotation (see Tests).

Atomic absorption spectrometry (2.2.23, Method IT).

B. Solution S (see Tests) is strongly alkaline (2.2.4).

Test solution Into a platinum or silica crucible previously tared

C . Infrared absorption spectrophotometry (2.2.24).

after ignition introduce 5.0 g. Cautiously heat and introduce into the substance a wick formed from twisted ashless filter paper. Ignite the wick. W hen the substance has ignited stop heating. After combustion, ignite in a muffle furnace at about 600 ± 50 °C. Continue ignition until white ash is obtained. After cooling, take up the residue with 2 quantities, each of 2 mT, o f dilute hydrochloric acid R and transfer into a 25 m L graduated flask. Add 0.3 m L of nitric add R and dilute to 25.0 m L with water R.

Reference solutions Prepare 3 reference solutions by adding 1.0 mT, 2.0 m L and 4.0 m L o f nickel standard solution (0.2 ppm Ni) R to 2.0 m L of the test solution and diluting to 10.0 m L with water R. Source Nickel hollow-cathode lamp. Wavelength 232 nm. Atomisation device Graphite furnace. Carrier gas argon R.

★ *

Comparison arginine CRS. I f the spectra obtained show differences, dry the substance to be examined and the reference substance in an oven, at 105 °C and record new spectra. D . Thin-layer chromatography (2.2.27).

Test solution Dissolve 10 m g of the substance to be examined in a 10.3 g/L solution o f hydrochloric acid R and dilute to 50 m L with the same solution. Reference solution. Dissolve 10 mg of arginine CRS in a 10.3 g/L solution o f hydrochloric acid R and dilute to 50 m l, with the same solution.

STO RA G E Protected from light.

Hate TLC silica gel plate R. Mobile phase concentrated ammonia R, 2-propanol R (30:70 VIV). Application 5 |iL. Development Over 2/3 of the plate. Drying At 105 °C until the ammonia disappears completely. Detection Spray with mnhydrin solution R and heat at 105 °C

L A B E L L IN G T he label states the nominal drop point.

Results T he principal spot in the chromatogram obtained with

__________________________________________________________ PhEur

for 15 min. the T est solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution. E . Dissolve about 25 mg in 2 m L o f water R. Add 1 m L of a -naphthol solution R and 2 m L of a mixture of equal volumes o f strong sodium hypochlorite solution R and water R. A red colour develops.

1-190 Arginine

2016

TESTS S o lu tio n S Dissolve 2.5 g in distilled water R and dilute to 50 m L with the same solvent.

T o 5 m L o f solution S add 0.5 m L o f dilute nitric add R and dilute to 15 m L w ith water R.

A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and not m ore intensely coloured than reference solution BY6 (2.2.2, Method II).

T o 10 m L o f solution S, add 1.7 m L o f dilute hydrochloric add R and dilute to 15 m L with distilled water R.

Specific o p tic a l ro ta tio n (2.2.7) + 25.5 to + 28.5 (dried substance).

Amino a d d analysis (2.2.56) as described in the test for ninhydrin-positive substances with the following modifications.

Dissolve 2.00 g in hydrochloric add R1 and dilute to 25.0 m L with the same acid. N in h y d rin -p o sitiv e su b s ta n c e s Amino acid analysis (2.2.56). F or analysis, use M ethod 1. T he concentrations o f the test solution and the reference solutions may be adapted according to the sensitivity o f the equipm ent used. T he concentrations o f all solutions are adjusted so that the system suitability requirements described in general chapter 2.2.46 are fulfilled, keeping the ratios of concentrations between all solutions as described.

Solution A water R

ot

a sample preparation buffer suitable for

the apparatus used.

Test solution Dissolve 30.0 m g of the substance to be examined in solution A and dilute to 50.0 m L with solution A.

S u lfa tes (2.4.13) Maximum 300 ppm .

Amm onium

Irgection T est solution, reference solution (c) and blank solution.

Limit: — ammonium at 570 mn: not m ore than the area of the corresponding peak in the chrom atogram obtained with reference solution (c) (0.02 per cent), taking into account the peak due to am m onium in the chromatogram obtained with the blank solution. Ir o n (2.4.9) M axim um 10 ppm. In a separating funnel, dissolve 1.0 g in 10 m L o f dilute hydrochloric add R. Shake with 3 quantities, each o f 10 mL, o f methyl isobutyl ketone R l, shaking for 3 m in each time. T o the combined organic layers add 10 m L o f water R and

Reference solution (a) D ilute 1.0 m L o f the test solution to

shake for 3 m in . U se the aqueous layer.

100.0 m L with solution A. D ilute 2.0 m L o f this solution to 10.0 m L with solution A.

H eav y m e ta ls (2.4.8) M axim um 10 ppm.

Reference solution (b) Dissolve 30.0 m g o f proline R in

Dissolve 2.0 g in water R and dilute to 20 m L with the same solvent. 12 m L o f the solution complies with test A. Prepare the reference solution using lead standard solution

solution A and dilute to 100.0 mT. w ith solution A. Dilute 1.0 m L o f the solution to 250.0 m L with solution A.

Reference solution (c) D ilute 6.0 mT. o f ammonium standard solution (100 ppm N H J R to 50.0 m L with solution A. Dilute 1.0 m L of this solution to 100.0 m l. with solution A.

Reference solution (d) Dissolve 30 m g o f isoleucine R and 30 m g o f leudne R in solution A and dilute to 50.0 m l. with solution A. Dilute 1.0 m L o f the solution to 200.0 m L with solution A.

Blank solution Solution A. Inject suitable, equal am ounts of the test, blank and reference solutions into the amino acid analyser. R un a program suitable for the determination o f physiological amino adds. System suitability Reference solution (d): — resolution: minim um 1.5 between the peaks due to isoleucine and leudne.

Calculation ofpercentage contents:

(1 ppm Pb) R. L oss o n d ry in g (2.2.32) M axim um 0.5 per cent, determined on 1.000 g by drying in an oven at 105 °C. S u lfa te d a s h (2.4.14) M axim um 0.1 per cent, determined on 1.0 g. A SSA Y Dissolve 0.150 g in 50 m L of water R. T itrate with 0.1 M hydrochloric add, determining the end-point potentiometrically (2.2.20).

1 mT. o f 0.1 M hydrochloric add is equivalent to 17.42 mg of C 6HX4N 4O 2. STORAGE In an airtight container, protected from light.

— for any ninhydrin-positive substance detected at 570 nm , use the concentration of arginine in reference solution (a); — for any ninhydrin-positive substance detected at 440 nm , use the concentration of proline in reference solution (b); if a peak is above the reporting threshold at both w avdengths, use the result obtained at 570 n m for quantification.

Other detectable impurities (the following substances would, if present at a suffident levd, be detected by one or other of the tests in the m onograph. They are limited by the general acceptance criterion for other/unspecified impurities. It is therefore not necessary to identify these impurities for dem onstration of compliance. See also 5.10. Control of

Limits:

impurities in substances for pharmaceutical use): A, B, C.

— any ninhydrin-positive substance, for each impurity, maximum 0.2 per cent; — total: maximum 0.5 per cent; — reporting threshold: 0.05 p er c e n t T he thresholds indicated u n d er R d a te d substances (Table 2034.-1) in the general m onograph Substances for pharmaceutical use (2034) do not apply. C h lo rid e s (2.4.4) M axim um 200 ppm.

IM P U R IT IE S

h Hj N ^ ' s ^

x

nh2

- ^ S‘C02H

A. (25)-2,6-diaminohexanoic a d d Qysine),

Arginine Aspartate 1-191

2016

Dissolve 2.50 g in dilute hydrochloric acid R and dilute to 25.0 m L with the same ad d . N in h y d rin -p o sitiv e su b stan ces Thin-layer chromatography (2.2.27). B. (2S)-2-amino-5-(caibamoylamino)pentanoic a d d (dtrulline),

Test solution (a) Dissolve 0.20 g of the substance to be examined in water R and dilute to 10 m L with the same solvent. Test solution (b) Dilute 1 m L of test solution (a) to 10 m L with water R.

Reference solution (a) Dissolve 25 m g o f arginine R and 25 mg of aspartic acid R in water R and dilute to 25 m L with the

C. (2S)-2,5-diaminopentanoic a d d (ornithine). PhEur

★ ★ ★ ★ *****

Arginine Aspartate (Ph. Eur. monograph 2096) H N n h2 ho 2c

C ioH21N50 6

C02H

307.3

7675-83-4

A ctio n a n d u se Amino add; n u trien t PhEur_________________

D E F IN IT IO N (2S)-2-Amino-5-guanidinopentanoic a d d (2S)-2aminobutanedioate. C o n te n t 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS A p p e a ra n c e W hite or almost white granules or powder. S olubility Very soluble in water, practically insoluble in alcohol and in methylene chloride. ID E N T IF IC A T IO N A. Specific optical rotation (see Tests). B. Infrared absorption spectrophotometry (2.2.24).

Comparison arginine aspartate CRS. C . Examine the chromatograms obtained in the test for ninhydrin-positive substances.

Resubs T he 2 principal spots in the chromatogram obtained w ith test solution (b) are sim ilar in position, colour and size to the 2 prindpal spots in the chromatogram obtained with reference solution (a). TESTS S o lu tio n S Dissolve 5.0 g in carbon dioxide-free water R and dilute to 50 m L with the same solvent. A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y 7 (2.2.2, Method II). p H (2.2.3) 6.0 to 7.0 for solution S. Specific o p tic a l ro ta tio n (2.2.7) + 25 to + 27 (dried substance).

same solvent.

Reference solution (b) Dilute 2 m L o f reference solution (a) to 50 m L with water R. Plate TLC silica gel G plate R. Mobile phase ammonia R, propanol R (36:64 VIV). Application 5 pL. Development Over 2/3 of the plate. Drying At 100-105 °C for 10 min. Detection Spray with ninhydrm solution R and heat at 100-105 °C for 10 min.

System suitability: reference solution (b): — the chromatogram shows 2 clearly separated prindpal spots. Limit, test solution (a): — any impurity: any spots, apart from the 2 principal spots, are no t more intense than each of the 2 prindpal spots in the chromatogram obtained with reference solution (b) (0.2 p er cent). C h lo rid e s (2.4.4) M aximum 200 ppm. Dilute 2.5 m L of solution S to 15 m L with water R. S u lfates (2.4.13) M aximum 300 ppm. T o 0.5 g add 2.5 m L of dilute hydrochloric acid R and dilute to 15 m L with distilled water R. Examine after 30 min. A m m o n iu m (2.4.1) M aximum 100 ppm, determined on 100 mg. H eav y m e ta ls (2.4.8) M aximum 20 ppm. 12 m L of solution S complies with test A. Prepare the reference solution using lead standard solution (2 ppm Pb) R. L oss o n d ry in g (2.2.32) M aximum 0.5 per cent, determined on 1.000 g by drying in an oven at 60 °C for 24 h. S u lfa te d a s h (2.4.14) M aximum 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 80.0 mg in 2 m L of anhydrous formic add R. A dd 50 m L o f anhydrous acetic add R. T itrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2 .20). 1 m L of 0.1 M perchloric add is equivalent to 10.24 mg o f C 10H 21N 5O 6. __________________________________________________________PhEur

2016

1-192 Arginine Hydrochloride

Arginine Hydrochloride

}***%

(Ph Eur. monograph 0805) H

*

A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and not m ore intensely coloured than reference solution BY6 (2.2.2, Method II).

H* -NH2 • HCI

NH

C eH uC m A

210.7

TESTS S o lu tio n S Dissolve 2.5 g in distilled water R and dilute to 50 m L with the same solvent.

1119-34-2

A c tio n a n d u se Amino add; nutrient. P r e p a r a tio n Arginine Hydrochloride Infusion Arginine Hydrochloride Oral Suspension Sterile Arginine Hydrochloride C oncentrate PhEur___________________________________________________________

S p ecific o p tic a l ro ta tio n (2.2.7) + 21.0 to + 23.5 (dried substance). Dissolve 2.00 g in hydrochloric add R1 and dilute to 25.0 m L with the same ad d . N in h y d rin -p o sitiv e su b s ta n c e s Amino a d d analysis (2.2.56). F or analysis, use M ethod 1. T h e concentrations o f the test solution and die reference solutions may be adapted according to the sensitivity o f the equipm ent used. T h e concentrations o f all solutions are adjusted so that the system suitability requirem ents described in general chapter 2.2.46 are fulfilled, keeping the ratios o f concentrations betw een all solutions as described.

D E F IN IT IO N (25)-2-Amino-5-guanidinopentanoic a d d hydrochloride.

Solution A water R ot a sample preparation buffer suitable for

Ferm entation product, extract or hydrolysate o f protein.

the apparatus used.

C o n te n t 98.5 per cent to 101.0 per cent (dried substance).

Test solution Dissolve 30.0 m g o f the substance to be examined in solution A and dilute to 50.0 m L with solution A.

CHARACTERS A p p e a ra n c e W hite or alm ost white, crystalline pow der o r colourless crystals. S o lu b ility Freely soluble in w ater, very slightly soluble in ethanol (96 per cent). ID E N T IF IC A T IO N

First identification A , B, E Second identification A , C, D} E A. Specific optical rotation (see Tests). B. Infrared absorption spectrophotom etry (2.2.24).

Comparison arginine hydrochloride CRS. C . Thin-layer chromatography (2.2.27).

Reference solution (a) D ilute 1.0 m L o f the test solution to 100.0 m L with solution A. D ilute 2.0 m L of this solution to 10.0 m L with solution A.

Reference solution (b) Dissolve 30.0 m g o f proUne R in solution A and dilute to 100.0 m L with solution A. Dilute 1.0 m L o f the solution to 250.0 m L with solution A.

Reference solution (c) Dilute 6.0 m L o f ammonium standard solution (100 ppm N H J R to 50.0 m L with solution A. D ilute 1.0 m L o f this solution to 100.0 m L with solution A.

Reference solution (d) Dissolve 30 m g of isoleucine R and 30 m g o f leucine R in solution A and dilute to 50.0 m L with solution A. Dilute 1.0 m L o f the solution to 200.0 m L with solution A.

Blank solution Solution A.

sam e solvent.

Inject suitable, equal amounts o f the test, blank and reference solutions into die amino add analyser. Rim a program suitable for the determ ination o f physiological amino adds. System suitability Reference solution (d): — résolution: minimum 1.5 between die peaks due to isoleucine and leucine.

Plate TLC silica gel plate R. Mobile phase concentrated ammonia R, 2-propanol R (30:70 VIV). Application 5 fiL. Development Over 2/3 of the plate. Drying At 105 °C until the am m onia disappears completely. Detection Spray with mnhydrin solution R and heat at 105 °C

— for any ninhydrin-positive substance detected at 570 nm , use die concentration o f arginine in reference solution (a); — for any ninhydrin-positive substance detected at 440 nm , use d ie concentration o f proline in reference solution (b); if a peak is above the reporting threshold at both wavelengths, use the result obtained at 570 n m for quantification.

Test solution Dissolve 10 m g o f the substance to be examined in water R and dilute to 50 m L with the same solvent. Reference solution. Dissolve 10 m g o f arginine hydrochloride CRS in water R and dilute to 50 m L with the

Calculation of percentage contents:

for 15 min.

Limits:

Results T he p rindpal spot in the chrom atogram obtained with the T est solution is similar in position, colour and size to the p rindpal spot in the chrom atogram obtained with the reference solution.

— any ninhydrin-positive substance: for each impurity, m axim um 0.2 p er cent; — total: maximum 0.5 per cent; — reporting threshold: 0.05 p er cent.

D . Dissolve about 25 m g in 2 m L o f water R. Add 1 m L of a-naphthol solution R and 2 m L o f a mixture o f equal volumes o f strong sodium hypochlorite solution R and water R. A red

T h e thresholds indicated under Related substances (Table 2034.-1) in the general m onograph Substances for pharmaceutical use (2034) do n o t apply.

colour develops. E. I t gives reaction (a) o f chlorides (2.3.1).

Argon 1-193

2016

S u lfates (.2.4.13) M axim um 300 ppm. Dilute 10 m L o f solution S to 15 m L w ith distilled water R. A m m onium A m ino acid analysis (2.2.56) as described in the test for

ninhydrin-positive substances with the following modifications.

B. (2S)-2-amino-5-(carbamoylamino)pentanoic a d d (dtrulline),

Irgection T est solution, reference solution (c) and blank

H,N,

solution.

Limit. — ammonium at 570 nm: not more than the area of the corresponding peak in the chromatogram obtained with reference solution (c) (0.02 per cent), taking into account the peak due to am m onium in the chromatogram obtained w ith the blank solution.

C. (2S)-2,5-diaminopentanoic a d d (ornithine).

I r o n (2.4.9) M aximum 10 ppm.

Argon

★

(Ph Eur monograph 2407)

*****

In a separating funnel, dissolve 1.0 g in 10 m L of dilute hydrochloric acid R. Shake with 3 quantities, each of 10 mL, of methyl tsobutyl ketone R l, shaking for 3 min each time. T o the combined organic layers add 10 m L o f water R and shake for 3 min. U se the aqueous layer. H eav y m e ta ls (2.4.8) M axim u m 10 ppm. Dissolve 2.0 g in water R and dilute to 20 m L with the same solvent. 12 m L o f the solution complies with test A. Prepare the reference solution using lead standard solution

PhEur

Ar

*****

39.95

★

7440-37-1

PhEur_____________________________

D E F IN IT IO N Gas obtained by fractional distillation of ambient air. C o n te n t M inim um 99.995 per cent V/V o f Ar, calculated by deduction of the sum of impurities found when performing the test for impurities and the water content.

(1 ppm Pb) R.

T his monograph applies to argon for medicinal use.

L oss o n d ry in g (2.2.32) M axim um 0.5 per cent, determined on 1.000 g by drying in an oven at 105 °C.

CHARACTERS A p p e a ra n c e Colourless gas.

S u lfa te d a s h (2.4.14) M axim um 0.1 per cent, determined on 1.0 g.

S o lu b ility A t 20 °C and at a pressure of 101 kPa, 1 volume dissolves in about 29 volumes of water.

A SSA Y Dissolve 0.180 g in 3 m L of anhydrous formic acid R. A dd 30 m L o f anhydrous acetic acid R. T itrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2. 2. 20). 1 m L of 0.1 M perchloric acid is equivalent to 21.07 mg of C e H is C lN ^ . STORA GE Protected from light. IM P U R IT IE S

Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities. It is therefore not necessary to identify these impurities for dem onstration o f compliance. See also 5.10. Control of impurities in substances for pharmaceutical use): A , B, C.

H,N

A. (2S)-2,6-diaminohexanoic a d d (lysine),

ID E N T IF IC A T IO N A. Verify that the gas is not oxygen using a paramagnetic analyser (2.5.27). B. Gas chromatography (2.2.28).

Gas to be examined T he substance to be examined. Reference gas Use the following mixture of gases in argon R l: methane R l (5 ppm V/V), nitrogen R l (5 ppm V/V), oxygen R (5 ppm V/V). Column: — material: stainless steel; — sizer. I = 2 m, 0 = 3 mm; — stationary phase: molecular sieve for chromatography R (particle size 150-180 nm, pore size 0.5 nm).

Carrier gas helium for chromatography R. Flow rate 10 mL/min. Temperature: — column: 50 °C; — detector. 150 °C. Detection Thermal conductivity. Irgection 25 JiL. System suitability. Reference gas: — resolution: minim um 3.0 between the peaks due to argon/oxygen and nitrogen and minimum 2.0 between the peaks due to nitrogen and methane.

Results T h e prindpal peak in the chromatogram obtained with the gas to be examined is similar in retention time to the prindpal peak in the chromatogram obtained with the reference gas.

1-194 Aripiprazole

2016

TESTS Im p u ritie s Gas chromatography (2.2.28).

if* it

if

★

Aripiprazole

★

(Ph. Eur. monograph 2617)

*****

★

Gas to be examined T h e substance to be exam in ed. Reference gas U se the following mixture o f gases in argon Rl: methane R l (5 ppm VIV), nitrogen R l (5 ppm VIV), oxygen R (5 ppm VIV). Column• — material: stainless steel; — size. I = 4 m , 0 = 4 m m ; — stationary phase, molecular sieve for chromatography R (particle size 150-180 pm , pore size 0.5 nm ).

C23H27CI2N3O2

Carrier gas argon R l. Flow rate 70 mL/min. Temperature. — column: 80 °C; — detector. 40 °C. Detection Discharge ionisation. Injection 1 m lSample rate 100 mL/min. Relative retention W ith reference to im purity C (retention

448.4

129722-12-9

A c tio n a n d u se D opam ine D 2 receptor antagonist; neuroleptic PhEur___________________________________________________________

D E F IN IT IO N 7-[4- [4- (2,3-Dichlorophenyl)piperazin-1-yl] butoxy]-3,4dihydroquinolin-2(lii)-one. C o n te n t 98.0 per cent to 102.0 per cent (dried substance).

tim e = about 4.7 min): im purity A = about 0.4; impurity B = about 0.7. System suitability: Reference gas: — resolution: m in im u m 3.0 between the peaks due to impurities A and B and m in im u m 2.0 between the peaks due to impurities B and C.

Limits:

CHARACTERS A p p e a ra n c e W hite or alm ost white crystals o r crystalline powder. S o lu b ility Practically insoluble in water, soluble in methylene chloride, very slighty soluble in ethanol (96 per cent). It shows polym orphism (5.9).

— impurity A: not m ore than the area of the corresponding peak in the chrom atogram obtained with the reference gas (5.0 p pm V!V)\ — total: m axim um 0.0040 p er cent o f the sum of the areas o f all the peaks (40.0 p pm VIV).

ID E N T IF IC A T IO N Infrared absorption spectrophotom etry (2.2.24).

W a te r (2.5.28) M axim um 10.0 ppm VIV, d eterm ined u sin g an electrolytic hygrometer.

If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in methylene chloride R, evaporate to dryness and record new spectra using the residues.

STORAGE In gaseous or liquid state, in suitable con tainers, complying w ith the legal regulations. IM P U R IT IE S

Specified impurities A, D Other detectable impurities B, C.

TESTS A p p e a ra n c e o f so lu tio n I f intended for use in the m anufacture o f parenteral preparations, the solution is clear (2.2.1) and n o t more intensely coloured than reference solution GY 5 (2.2.2,

Method II). Dissolve 0.5 g in a mixture o f 10 volumes o f acetic acid R and 90 volumes o f anhydrous ethanol R and dilute to 20 m L with the same mixture of solvents. Sonicate for about 15 m in, shaking occasionally, until dissolution is complete.

A. oxygen, B. nitrogen, C. m ethane, D . water. ------ -----------------------------------------------------------------------------

Comparison aripiprazole CRS.

PhEur

R e la te d su b s ta n c e s L iquid chrom atography (2.2.29). Protect the solutions from

light. Solvent mixture acetic add R, methanol R, acetonitrile R, water R (1:10:30:60 VIVIVIV). Test solution Dissolve 50.0 m g o f the substance to be exam in ed in the solvent mixture and dilute to 50.0 m L with the solvent mixture. D ilute 5.0 m L o f the solution to 50.0 m L with the solvent mixture.

Reference solution (a) Dilute 1.0 m L o f the test solution to 100.0 m L with the solvent mixture. D ilute 1.0 m L o f this solution to 10.0 m L with the solvent mixture.

Reference solution (b) Dissolve 5 m g o f the substance to be exam in e d and 5 m g of aripiprazole impurity F CRS in the solvent m ixture and dilute to 100 m L with the solvent

Aripiprazole 1-195

2016

mixture. Dilute 1 m L of the solution to 50 m L with the solvent mixture.

Reference solution (c) Dissolve 50.0 mg of aripiprazole CRS in the solvent mixture and dilute to 50.0 m L with the solvent mixture. Dilute 5.0 m L of the solution to 50.0 m L with the solvent mixture.

Column: — sizer. I = 0.10 m , 0 = 4.6 mm; — stationary phase: end-capped. octadecylsUyl silica gel for chromatography R (3 pm).

Mobile phase: — mobile phase A: acetonitrile R, 0.05 per cent V/V solution o f trifluoroacetic add R (10:90 VfV)‘, — mobile phase B: 0.05 per cent V/V solution o f trifluoroacetic add R, acetonitrile R (10:90 V/V)', Time ( o i l) 0 -2

Mobile phase A (per cent V/V) 80

Mobile phase B (per cent V/V) 20

2 - 10

80 ->65

20-»35

10-20

65-» 10

35-»90

2 0 -2 5

10

90

STO RA G E Protected from light. If the substance is sterile, store in a sterile, airtight, tam per-proof container. L A B E L L IN G T h e label states, where applicable, that the substance is suitable for use in the manufacture o f parenteral preparations. IM P U R IT IE S

Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general m onograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these impurities for demonstration of compliance. See also 5.10. Control of impurities in substances for pharmaceutical use): A, B, C, D} E, F, G.

A. 7-hydroxy-3,4-dihydroquinolirt-2(lfi)-one,

Flow rate 1.2 m l/m in . Detection Spectrophotometer at 254 nm. Injection 20 |xL o f the test solution and reference solutions (a) and (b).

Relative retention W ith reference to aripiprazole (retention time — about 11 min): impurity F = about 1.1. System suitability: reference solution (b): — resolution: minimum 2.0 between the peaks due to aripiprazole and impurity F.

a B. l-(2,3-dichlorophenyl)piperazine,

Calculation of percentage contents: — for each impurity, use the concentration of aripiprazole in reference solution (a).

Limits: — unspecified impurities', for each impurity, maximum 0.10 per cent; — total: maximum 0.2 per cent; — reporting threshold: 0.05 per cent.

C . 7- [4- [4-(2-chlorophenyl)piperazin-1-yl] butoxy] -3,4dihydroquinolin-2( 1H)-ons,

L oss o n d ry in g (2.2.32) M axim um 0.5 per cent, determined on 1.000 g by drying in an oven at 105 °C for 3 h. S u lfa te d a s h (2.4.14) M axim um 0.1 p er cent, determined on 1.0 g. B a c te ria l en d o to x in s (2.6.14) Less than 5 IU/mg, if intended for use in the m anufacture o f parenteral preparations without a further appropriate procedure for the removal o f bacterial endotoxins.

ci D . 7-[4-[4-(3-chlorophenyl)piperazin-l-yl]butoxy]-3,4dihydroquinolin-2( 1H)-one,

Dissolve 1.0 mg o f the substance to be examined in 20 m L of a 5.17 g/L solution o f hydrochloric acid R. A SSA Y Liquid chromatography (2.2.29) as described in the test for related substances with the following modifications.

Injection T est solution and reference solution (c). System suitability: reference solution (c): — symmetry factor, maximum 2 .0 . Calculate the percentage content o f C 23H 27CI2N 3O 2 taking into account the assigned content o f aripiprazole CRS.

ci E. 7- [4- [4-(2,3-dichlorophenyl)piperazin-1-yl]butoxy] quinolin -2 (lii)-o n e ,

1-196 Articaine Hydrochloride

2016

Test solution Dissolve 0.1 g in 5 m L o f water R, add 3 m L of a saturated solution of sodium hydrogen carbonate R and shake twice with 2 m L o f methylene chloride R. C om bine the m ethylene chloride layers, dilute to 5.0 m L with methylene chloride R and dry over anhydrous sodium sulfate R. Comparison articaine hydrochloride CRS. C. Thin-layer chrom atography (2.2.27). Test solution Dissolve 20 m g o f the substance to be exam in ed in 5 m L o f ethanol (96 per cent) R. Reference solution Dissolve 20 m g o f articaine hydrochloride CRS in 5 m L o f ethanol (96 per cent) R Hate TLC silica gel F 254 plate R. Mobile phase triethykamne R, ethyl acetate R, heptane R (10:35:65 VIV/V). Application 5 jiL. Development Over a path of 15 cm. Drying In air. Detection Examine in ultraviolet light at 254 nm. Results T h e principal spot in the chrom atogram obtained with

F. 7-[4- [4-(2,3-dichlorophenyi)-1-oxidopiperazin-1-yl] butoxy] -3,4-dihydroquinolin-2 (l/f)-o n e ,

G. 7 , 7 [ethane-1,1 -diylbis [(2,3-dichlorobenzene-4,1 -diyl) piperazine-4, l-diyibutane-4, l-diyloxy]]bis[3,4dihydroquinolin-2 (1 if)-o n e]. PhEur

the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. D . It gives reaction (a) o f chlorides (2.3.1).

Articaine Hydrochloride

* * * * *

★

★

* * * * *

(Ph. Eur. monograph 1688)

A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and n o t more intensely coloured than reference solution BY 6 (2.2.2, Method I).

H CHS - HCI

p H (2.2.5) 4.2 to 5.2.

and enanb'omer

C13H21CIN2O3S

320.8

TESTS S o lu tio n S Dissolve 0.50 g in water R and dilute to 10 m L with the same solvent.

23964-57-0

A ctio n a n d u se Local anaesthetic.

Dissolve 0.20 g in carbon dioxide-free water R and dilute to 20.0 m L with the same solvent. R e la te d su b s ta n c e s Liquid chromatography (2.2.29).

Test solution Dissolve 10.0 m g o f the substance to be PhEur.

exam in ed in the mobile phase and dilute to 10.0 m L with

D E F IN IT IO N M ethyl 4-m ethyl-3- [[(2i?S)-2-(propyiamino)propanoyi] amino]thiophene- 2-carboxylate hydrochloride.

Reference solution (a) D ilute 1.0 m L o f the test solution to

C o n te n t 98.5 per cent to 101.0 per cent (dried substance). CHARACTERS A p p e a ra n c e W hite or alm ost white, crystalline powder. S o lu b ility Freely soluble in w ater and in ethanol (96 per cent). ID E N T IF IC A T IO N

First identification B, D Second identification A , C, D A. Dissolve 50.0 m g in a 1 g/L solution of hydrochloric add R and dilute to 100.0 m L with the same acid. Dilute 5.0 m L o f the solution to 100.0 m L with a 1 g/L solution o f hydrochloric add R. Exam ined between 200 n m and 350 nm (2.2.25), the solution shows an absorption m axim um at 272 nm . T he specific absorbance at the m axim u m is 290 to 320. B. Infrared absorption spectrophotom etry (2.2.24). Preparation Place dropwise 20 |iL o f the test solution on 300 mg discs.

the mobile phase. 100.0 m L with the mobile phase. D ilute 1.0 m L o f this solution to 10.0 m L with the mobile phase.

Reference solution (b) Dissolve 5.0 m g of articaine impurity A CRS and 2.5 m g o f articaine impurity E CRS in the mobile phase and dilute to 50.0 m L with the mobile phase. D ilute 1.0 m L o f the solution to 50.0 m L w ith the mobile phase. Column: — size: I = 0.25 m , 0 = 4.6 mm ; — stationary phase: spherical end-capped octadecylsUyl silica gel for chromatography R (5 pm); — temperature: 45 °C.

Mobile phase M ix 25 volumes o f acetomtrUe R and 75 volumes o f a solution prepared as follows: dissolve 2.02 g o f sodium heptanesulfonate R and 4.08 g o f potassium dihydrogen phosphate R in water R and dilute to 1000 m L with the same solvent. A djust to p H 2.0 with phosphoric acid R. Flow raze 1 mL/min. Detection Spectrophotom eter at 276 nm. Irgecdon 10 pL. Run time 5 times the retention tim e o f articaine.

Articaine Hydrochloride 1-197

2016 Relative retention W ith reference to articaine (retention tim e = about 9 min): impurity A = about 0.8; impurity E = about 0.86.

System suitability Reference solution (b): — resolution: m inim u m 1.2 between the peaks due to

HOzC

L1

H

CH CH3 and enantiomer

O CH3

B. 4-methyl-3-[[(22?S)-2-(propylamino)propanoyl]amino] thiophene-2-carboxylic acid (articaine add),

impurities A and £ .

Limits: — impurity A: not more than the area of the corresponding peak in the chromatogram obtained with reference solution (b) (0.2 per cent); — unspecified impurities: for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.10 per cent); — sum of impurites other than A: not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent); — disregard limit. 0.5 times the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.05 p er cent).

h

ch3

/ \ ^ ch3

C. 1-methylethyl 4-methyl-3-[ [(2RS)-2-(propjdamino) propanoyl]amino]thiophene-2-carboxylate (articaine isopropyl ester),

H

H eav y m e ta ls (2.4.8) M aximum 5 ppm.

CH3

Dissolve 4.0 g in 20.0 m L o f water R 12 m L of the solution complies with test A. Prepare the reference solution using

lead standard solution (1 ppm Pb) R. L oss on d ry in g (2.2.32) M aximum 0.5 per cent, determined on 1.000 g by drying in an oven at 105 °C for 5 h.

and enantiomer

and enantiomer

D . methyl 3-[[(2i?S)-2-(ethylamino)propanoyl]amino]-4methylthiophene-2-caiboxylate (ethylarticaine),

S u lfa te d a sh (2.4.14) M aximum 0.1 per cent, determined on 1.0 g. A SSA Y Dissolve 0.250 g in a mixture o f 5.0 m L o f 0.01 M hydrochloric add and 50 m L o f ethanol (96 per cent) R. C any out a potentiometric titration (2.2.20) using 0.1 M sodium hydroxide. Read the volume added between the 2 points of inflexion.

and enantiomer

E. methyl 4-methyl-3- [[(2ftS)-2-[( 1-methylethyl) amino] propanoyl] amino] thiophene-2-carboxylate (isopropylarticaine),

1 m L of 0.1 M sodium hydroxide is equivalent to 32.08 mg o f C 13H 21C1N20 3S. ST O R A G E Protected from light.

H

H- #CH3

o

IM P U R IT IE S

Specified impurities A Other detectable impurities (die following substances would, if present at a sufficient level, be detected by one or other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general m onograph Substances for pharmaceutical use (2034). It is therefore n o t necessary to identify these impurities for demonstration o f compliance. See also 5.10.

CHj

and enantiomer

F. 4-methyl-N-propyl-3- [ [(2RS)-2-(propylamino) propanoyl] amino] thiophene-2-carboxamide (articaine a d d propionamide),

Control of impurities in substances for pharmaceutical use): B, C, D ,E ,F ,G ,H ,I,J .

x\

^ ch3

G. methyl 3- [ [(2RS)-2-(butylamino)propanoyl] amino] -4methylthiophene-2-caiboxylate (butylarticaine),

A. methyl 4-methyl-3-[[2-(propylamino)acetyl] amino] thiophene-2-carboxylate (acetamidoarticaine), and enantiomer

H . methyl 3- [ [(2RS)-2-(dipropylamino)propanoyl] amino] -4methylthiophene-2-Carboxylate (dipropylarticaine),

1-198 Ascorbic Acid

2016

Second identification A , C, D A. Ultraviolet and visible absorption spectrophotom etry (2.2.25).

nh 2

Test solution Dissolve 0.10 g in water R and dilute immediately to 100.0 m L with the same solvent. Add 1.0 m L o f this solution to 10 m L o f 0.1 M hydrochloric add and dilute to 100.0 m L with water R

ch3

I.

methyl 3-am ino-4-methylthiophene-2-caiboxylate (3-aminoarticaine),

Absorption maximum At 243 nm , determ ined immediately after dissolution.

i Ch3

and enantiomer

CH3

J. methyl 3-[[(2i?S)-2-bromopropanoyl]amino]-4methylthiophene- 2 -carboxylate (bromo compound).

***** ★ ★

Ascorbic Acid

* * * * *

(Ph. Eur. monograph 0253)

Spedfic absorbance at the absorption maximum 545 to 585. B. Infrared absorption spectrophotom etry (2.2.24). Comparison ascorbic add CRS. C. p H (2.2.3): 2.1 to 2.6 for solution S (see Tests). D. T o 1 m L o f solution S add 0.2 m L o f dilute nitric acid R and 0.2 m L o f silver nitrate solution R2. A grey predpitate is formed. TESTS S o lu tio n S Dissolve 1.0 g in carbon dioxide-free water R and dilute to 20 m L with the same solvent. A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and n o t m ore intensdy coloured than reference solution BY 7 (2.2.2, Method II). S p ecific o p tic a l r o ta tio n (2.2.7) + 20.5 to + 21.5. Dissolve 2.50 g in water R and dilute to 25.0 m L with the same solvent.

HO

CsHgOg

OH

176.1

50-81-7

A c tio n a n d use Vitam in C. P re p a r a tio n s Ascorbic A d d Injection Ascorbic A d d Tablets Chewable Ascorbic A d d Tablets Paediatric Vitam ins A, C and D Oral Drops Potassium Ascorbate Eye D rops Vitamins B and C Injection W hen Vitamin C is prescribed or dem anded. Ascorbic A d d shall be dispensed or supplied. PhEur___________________________________________________________

D E F IN IT IO N (5i?)-5- [(1S) -1 ,2-Dihydroxyethyl] -3,4-dihydroxyfuran-2 (5H) one. C o n te n t 99.0 per cent to 100.5 per cent. CHARACTERS A p p e a ra n c e W hite or alm ost white, crystalline pow der or colourless crystals, becoming discoloured on exposure to air and moisture.

I m p u rity E M axim um 0.2 per cent.

Test solution Dissolve 0.25 g in 5 m L o f water R. Neutralise using dilute sodium hydroxide solution R and add 1 m L o f dilute acetic add R and 0.5 m L o f caldum chloride solution R. Reference solution Dissolve 70 m g o f oxalic add R in water R and dilute to 500 m L with the same solvent; to 5 m L o f this solution add 1 m L o f dilute acetic add R and 0.5 m L o f

caldum chloride solution R Allow the solutions to stand for 1 h. Any opalescence in the test solution is no t m ore intense than that in the reference solution. R e la te d su b s ta n c e s Liquid chrom atography (2.2.29). Prepare the solutions

immediately before use. Phosphate buffer solution Dissolve 6.8 g o f potassium dihydrogen phosphate R in water R and dilute to about 175 m L with the same solvent. Filter through a m em brane filter (nominal pore size 0.45 pm) and dilute to 1000 m L with water R.

Test solution Dissolve 0.500 g o f the substance to be examined in the mobile phase and dilute to 10.0 m L with the mobile phase. Reference solution (a) Dissolve 10.0 m g o f ascorbic add impurity C CRS in the mobile phase and dilute to 5.0 m L with the mobile phase.

Reference solution (b) Dissolve 5.0 m g o f ascorbic add impurity D CRS and 5.0 m g o f ascorbic add CRS in the mobile phase, add 2.5 m L o f reference solution (a) and dilute to 100.0 m L with the mobile phase.

S o lu b ility Freely soluble in water, sparingly soluble in ethanol (96 per cent).

Reference solution (c) D ilute 1.0 m L of the test solution to

mp A bout 190 °C, with decomposition.

200.0 m L with the mobile phase. M ix 1.0 m L of this solution with 1.0 m L of reference solution (a).

ID E N T IF IC A T IO N

— sizer. I — 0.25 m , 0 = 4.6 mm;

First identification: B , C.

Column:

Ascorbic Acid 1-199

2016

— stationary phase: aminopropylsifyl silica gel for chromatography R (5 nm)i — temperature: 45 °C.

Adjust the zero of the apparatus using 0.1 M nitric add.

Mobile phase Phosphate buffer solutionj acetonitrUe R1 (25:75 VIV). Flow rate 1.0 mlVmin. Detection Spectrophotom eter at 210 nm. Injection 20 |iL o f the test solution and reference solutions (b)

Dissolve 2.0 g in water R and dilute to 20 m L with the same solvent. 12 m L of the solution complies with test A. Prepare the reference solution using lead standard solution

and (c).

Run time 2.5 times the retention time of ascorbic acid. Identification of impttrities Use the chromatogram obtained with reference solution (b) to identify the peaks due to impurities C and D.

Relative retention W ith reference to ascorbic acid (retention time = about 11 min): impurity D = about 0.4; impurity C = about 1.7.

System suitability. — resolution: minim um 3.0 between the peaks due to ascorbic a d d and impurity C in the chromatogram obtained with reference solution (c); — signal-to-noise ratio: m in im u m 20 for the peak due to impurity C in the chromatogram obtained with reference solution (b).

Limits: — impurities C, D: for each impurity, n o t more than 1.5 times the area of the corresponding peak in the chrom atogram obtained with reference solution (b) (0.15 per cent); — unspecified impurities: for each impurity, n o t more than the area o f the peak due to ascorbic a d d in the chromatogram obtained with reference solution (b) (0.10 per cent); — total of impurities other than C and D: not more than twice the area of the peak due to ascorbic a d d in the chromatogram obtained with reference solution (b) (0.2 per cent); — disregard limic. 0.5 times the area of the peak due to ascorbic a d d in the chromatogram obtained with reference solution (b) (0.05 per cent). C opper M aximum 5 ppm .

H eav y m e ta ls (2.4.8) M aximum 10 ppm.

(1 ppm Pb) R. S u lfa te d a s h (2.4.14) M aximum 0.1 per cent, determined on 1.0 g. A SSAY Dissolve 0.150 g in a mixture of 10 m L of dilute sulfuric add R and 80 m L of carbon dioxide-free water R. Add 1 m L of starch solution R. Titrate with 0.05 M iodine until a persistent violet-blue colour is obtained. 1 m L of 0.05 M iodine is equivalent to 8.81 mg of CgHgOg. STORA GE In a non-metallic container, protected from light. IM P U R IT IE S

Specified impurities C, D , E Other detectable impurities (the following substances would, if present at a suffident level, be detected by one or other o f the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical vise (2034). It is therefore not necessary to identify these impurities for demonstration of compliance. See also 5.10.

Control of impurities in substances for pharmaceutical use): A , F, G, H. /° V -C H O

i

1

A. 2-furaldehyde, OH

oh

o

rV/™

OH OH O

C. D-xy/o-hex-2-ulosonic a d d (D-sorbosonic ad d ),

Atomic absorption spectrometry (2.2.25, Method I).

Test solution Dissolve 2.0 g in 0.1 M nitric add and dilute to 25.0 m L with the same ad d . Reference solutions Prepare the reference solutions (0.2 ppm , 0.4 ppm and 0.6 ppm ) by diluting copper standard solution (10 ppm Cu) R w ith 0.1 M nitric add.

OH OH o

OH

O

D . methyl D-xyfo-hex-2-ulosonate (methyl D-sorbosonate),

Source C opper hollow-cathode lamp. Wavelength 324.8 nm. Atomisation device Air-acetylene flame.

V"

Adjust the zero o f the apparatus using 0.1 M nitric add. Ir o n M aximum 2 ppm .

OH

o

E. oxalic ad d ,

Atomic absorption spectrometry {2.2.23, Method I).

Test solution Dissolve 5.0 g in 0.1 M nitric add and dilute to 25.0 m L with th e same ad d . Reference solutions Prepare the reference solutions (0.2 ppm, 0.4 ppm and 0.6 ppm ) by diluting iron standard solution (20 ppm Fe) R w ith 0.1 M nitric acid. Source Iron hollow-cathode lamp. Wavelength 248.3 nm. Atomisation device Air-acetylene flame.

HO

OH

F. (5R)-5-[(lR)-l,2-dihydroxyethyl]-3,4-dihydroxyfuran2 (5/i)-one,

1-200 Ascorbyl Palmitate

2016

S p ecific o p tic a l ro ta tio n (2.2.7) + 21 to + 24 (dried substance), determ ined on solution S.

.OH

HO

HO

OH

G. (2R)-2-[(2Æ)-3j4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl]2-hydroxyacetic acid.

R e la te d su b s ta n c e s T h e thresholds indicated under Related substances (Table 2034.-1) in the general m onograph Substances for pharmaceutical use (2034) do n o t apply. H e a v y m e ta ls (2.4.8) M axim um 10 ppm. 2.0 g complies with test C. Prepare the reference solution using 2 m L o f lead standard solution (10 ppm Pb) R.

HO

L o ss o n d ry in g (2.2.32) M axim um 1.0 p er cent, determ ined on 1.000 g by drying m vacuo at 60 °C for 5 h.

OH

S u lfa te d a s h (2.4.14) M aximum 0.1 per cent, determ ined on 1.0 g.

H . methyl (2i?)-2-[(22?)-3j4-dihydroxy-5-oxo-2j5dihydrofuran-2-yl]-2-hydroxyacetate. PhEur

*** ★

Ascorbyl Palmitate

* ★

(Ph. Eur. monograph 0807)

*****

★

A SSA Y Dissolve 0.200 g in 50 m L o f ethanol (96 per cent) R A dd 30 m L o f water R and titrate with 0. 05 M iodine until a yellow colour is obtained. 1 m L o f 0.05 M iodine is equivalent to 20.73 m g o f C 22H 38O 7. STORA GE In an airtight container, protected from lig h t ___________________________________________________________PhEur

***** ★ ★

Asparagine Monohydrate C22H 3807

414.5

137-66-6

* * * * *

E** monograph 2086)

A c tio n a n d u se Excipient.

X x™ 1

h2n '

, H20

^ ^ v co2h

PhEur.

C4H8N20 3iH 20 D E F IN IT IO N (25)-2-[(2i?)-3,4-Dihydroxy-5-oxo-2,5-dihydrofuraii-2-yl]-2hydroxyethyl hexadecanoate. C o n te n t 98.0 per cent to 100.5 per cent (dried substance). CHARACTERS A p p e a ra n c e W hite or yellowish-white powder. S o lu b ility Practically insoluble in water, freely soluble in ethanol (96 p er cent) and in m ethanol, practically insoluble in methylene chloride and in fatty oils. ID E N T IF IC A T IO N A. Specific optical rotation (see Tests). B. Infrared absorption spectrophotom etry (2.2.24).

Comparison ascorbyl palmitate CRS. C. Dissolve about 10 mg in 5 m L o f methanol R. T he solution decolourises dichlorophenolindophenol standard

solution R. TESTS S o lu tio n S Dissolve 2.50 g in methanol R and dilute to 25.0 m L with the same solvent A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) a n d n o t m ore intensely coloured than reference solution BY4 (2.2.2, Method I).

150.1

5794-13-8

A c tio n a n d u se Amino add. PhEur_____________

D E F IN IT IO N (2S)-2,4-Diam ino-4-oxobutanoic a d d m onohydrate. C o n te n t 99.0 per cent to 101.0 p er cent (dried substance). CHARACTERS A p p e a ra n c e W hite or almost white, crystalline pow der or colourless crystals. S o lu b ility Slightly soluble in water, practically insoluble in ethanol (96 per cent) and in methylene chloride. ID E N T IF IC A T IO N

First identification A , B Second identification A , C A. Specific optical rotation (see Tests). B. Infrared absorption spectrophotom etry (2.2.24).

Comparison asparagine monohydrate CRS. C. Examine the chrom atograms obtained in the test for ninhydrin-positive substances.

Results T h e principal spot in th e chrom atogram obtained with test solution (b) is similar in position, colour and size to the

2016

Aspartame 1-201

principal spot in the chromatogram obtained with reference solution (c).

organic phases w ith 10 m L of water R for 3 min. T h e aqueous phase complies with th e limit test for iron.

TESTS S o lu tio n S Dissolve with heating 2.0 g in carbon dioxide-free water R and dilute to 100 m L with the same solvent.

H eav y m e ta ls (2.4.8) M aximum 10 ppm .

A p p e a ra n c e o f so lu tio n Solution S is d ear (2.2.1) and colourless (2.2.2, Method II). p H (2.2.3) 4.0 to 6.0 for solution S. S p ecific o p tic a l ro ta tio n (2.2.7) + 33.7 to + 36.0 (dried substance). Dissolve 2.50 g in a 309.0 g/L solution o f hydrochloric acid R and dilute to 25.0 m L with the same acid. N in h y d rin -p o sitiv e su b sta n ce s Thin-layer chromatography (2.2.27).

Test solution (a) Dissolve 0.25 g of the substance to be examined in water R, heating to not more than 40 °Cj and dilute to 10 m L w ith the same solvent. Test solution (b) Dilute 1 m L of test solution (a) to 10 m L w ith water R. Reference solution (a) Dilute 1.0 m L o f test solution (a) to 200 m L with water R. Reference solution (b) Dissolve 25 mg o f glutamic acid R in water R, add 1 m L o f test solution (a) and dilute to 10 m L w ith water R. Reference solution (c) Dissolve 25 mg of asparagine monohydrate CRS in water R and dilute to 10 m L with the

Dissolve 2.0 g in a mixture of 3 m L of dilute hydrochloric acid R and 15 m L of water R with gentle warming if necessary. Dilute to 20 m L with water R. 12 m L o f the solution complies with test A. Prepare the reference solution using lead standard solution (1 ppm Pb) R. L oss o n d ry in g (2.2.32) 10.5 per cent to 12.5 p er cent, determined on 1.000 g by drying in an oven at 130 °C for 3 h. S u lfa te d a sh (2. 4.14) M aximum 0.1 per cent, determined on 1.0 g. A SSA Y Dissolve 0.110 g in 5 m L of anhydrous formic acid R. Add 50 m L of anhydrous acetic acid R. T itrate with 0.1 M perchloric acid, determining the end-point potentiometrically ( 2 .2.20). 1 m L of 0.1 M perchloric acid is equivalent to 13.21 mg o fC 4 H 8N 20 3. IM P U R IT IE S

Specified impurities: A, B. H nh2

A. (25)-2-aminobutanedioic a d d (aspartic ad d ),

sam e solvent.

Hate TLC silica gel G plate R. Mobile phase glacial acetic acid R, water R, butanol R (25:25:50 V/VIV). Application 5 pL. Development Over h alf of the plate. Drying A t 110 °C for 15 min. Detection Spray with ninhydrin solution R and heat at 110 °C

h

V

ho2c / S s ^

nh 2 n‘co2h

B. (2S)-2-aminopentanedioic a d d (glutamic ad d ). PhEur

for 10 min.

System suitability: reference solution (b): — the chromatogram shows 2 clearly separated principal spots.

★★* * ★ ★ ★ *****

Aspartame (Ph Eur monograph 0973)

Limit: test solution (a): — any impurity, any spot, apart from the principal spot, is not more intense than the principal spot in the chromatogram obtained with reference solution (a) (0.5 per cent). C h lo rid e s (2.4.4) M axim um 200 ppm . D ilute 12.5 m L o f solution S to 15 m L with water R. S u lfa te s (2.4.II) M axim um 200 ppm.

Q 4H 18N 2O 5

T o 0.75 g add 2.5 m L o f dilute hydrochloric acid R and dilute to 15 m L with distilled water R. Examine after 30 min.

A c tio n a n d u se Sweetening agent.

A m m o n iu m (2.4.1, Method B) M axim um 0.1 per cent, determined on 10 mg.

PhEur_____________

I r o n (2.4.9) M axim um 10 ppm. Dissolve 1.0 g in dilute hydrochloric acid R and dilute to 10 m L w ith the same add. Shake 3 times with 10 m L o f methyl isobutyl ketone R l for 3 min. Wash the combined

294.3

22839-47-0

D E F IN IT IO N (3S)-3-Amino-4- [ [(25) -1 -methoxy-1-oxo-3-phenylpropan-2yl] amino]-4-oxobutanoic a d d (methyl a-L-aspartyl-Lphenylalaninate). C o n te n t 98.0 per cent to 102.0 per cent (dried substance).

1-202 Aspartame

CHARACTERS A p p e a ra n c e W hite or alm ost white, slightly hygroscopic, crystalline powder. S o lu b ility Sparingly soluble or slightly soluble in water and in ethanol (96 per cent), practically insoluble in hexane and in methylene chloride. ID E N T IF IC A T IO N

First identification B. Second identification A , C, D. A. Ultraviolet and visible absorption spectrophotometry (2.2.25).

Test solution Dissolve 0.1 g in ethanol (96 per cent) R and dilute to 100 rnT. with the same solvent. Spectral range 230-300 nm. Absorption maxima A t 247 nm , 252 nm , 258 nm and 264 nm .

2016

Calculate the conductivity o f die solution o f the substance to be examined using the following expression:

C i - 0.992 C2

Specific optical rotation (2.2.7) + 14.5 to + 16.5 (dried substance). Dissolve 2.00 g in a 690 g/L solution of anhydrous formic add R and dilute to 50.0 m L with the same solution. M easure within 30 min o f preparation.

Related substances Liquid chromatography (2.2.29).

Test solution Dissolve 0.60 g o f the substance to be examined in a mixture o f 1.5 volumes o f glacial acetic add R and 98.5 volumes o f water R and dilute to 100.0 m L w ith the sam e mixture o f solvents. Reference solution (a) Dissolve 4.5 m g o f aspartame impurity A CRS in a mixture o f 1.5 volumes o f glacial acetic add R and 98.5 volumes o f water R and dilute to 50.0 m L

B. Infrared absorption spectrophotom etry (2.2.24).

w ith the sam e mixture o f solvents.

Preparation Discs. Comparison aspartame CRS. C. Thin-layer chrom atography (2.2.27).

Reference solution (b) Dissolve 30.0 m g o f phenylalanine R (impurity C) in a mixture o f 15 volumes o f glacial acetic add R and 85 volumes o f water R and dilute to 100.0 m L

Test solution Dissolve 15 m g o f the substance to be examined in 2.5 m L o f water R and dilute to 10 m L with acetic acid R

with the same mixture o f solvents. Dilute 1.0 m L o f this solution to 10.0 m L w ith water R.

Reference solution Dissolve 15 m g o f aspartame CRS in 2.5 m L of water R and dilute to 10 m L with acetic add R. Plate TLC silica gel G plate R. Mobile phase water R, anhydrous formic add R> methanol R, methylene chloride R (2:4:30:64 V/V/V/V). Application 20 |±L. Development O ver a p ath o f 15 cm. Drying In air. Detection Spray with nmhydrm solution R and heat at

Reference solution (c) D ilute 5.0 m L o f the test solution to 10.0 m L with water R D ilute 3.0 m L o f this solution to 100.0 m L with water R. Reference solution (d) Dissolve 30.0 m g o f L-aspartyl-Lphenylalanine R (impurity B) in a mixture o f 15 volumes of glacial acetic add R and 85 volumes o f water R and dilute to

100-105 °C for 15 min.

— sizer. I = 0.25 m , 0 = 4.0 mm ; — stationary phase: octadecylsQyl silica gel for chromatography R (5-10 nm).

Results T he spot in the chrom atogram obtained with the test solution is similar in position, colour and size to the spot in the chrom atogram obtained w ith the reference solution. D . Dissolve about 20 m g in 5 m L o f methanol R and add 1 m L o f alkaline hydroxylamme solution R l. H eat on a waterbath for 15 m in. Allow to cool and adjust to about p H 2 with dilute hydrochloric acid R. A dd 0.1 m L o f ferric chloride solution R l. A brownish-red colour is pro d u ced TESTS S o lu tio n S Dissolve 0.8 g in carbon dioxide-free water R and dilute to 100 m L with the sam e solvent. A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and not m ore intensely coloured than reference solution GY6 (2.2.2, Method II). C o n d u c tiv ity (2.2.38) M axim um 30 nS-cm-1 . Dissolve 0.80 g in carbon dioxide-free water R prepared from distilled water R and dilute to 100.0 m L with the same solvent. M easure the conductivity o f the solution (C x) and that o f the w ater used for preparing the solution (C 2). T he readings m ust be stable within 1 p er cent over a period o f 30 s.

100.0 m L with die same mixture o f solvents. D ilute 1.0 m L o f the solution to 10.0 m L with water R. M ix 1.0 m L o f this solution with 1.0 m L o f reference solution (b).

Column

Mobile phase M ix 10 volumes o f acetordtri1e R and 90 volumes o f a 6.8 g/L solution o f potassium dihydrogen phosphate R previously adjusted to p H 3.7 with phosphoric add R. Flow rate 1 mL/min. Detection Spectrophotom eter at 220 nm . Injection 20 |±L. Run time Twice the retention tim e o f aspartame. System suitability Reference solution (d): — resolution: minimum 3.5 betw een the peaks due to impurities B and C.

Limits: — impurity A: n o t m ore than die area o f the principal peak in the chrom atogram obtained with reference solution (a) (1.5 p er cent); — impurity C: n o t m ore than die area o f th e principal peak in the chrom atogram obtained with reference solution (b) (0.5 p er cent); — sum of impurities other than A and C: n o t more th an the area o f die principal peak in die chrom atogram obtained w ith reference solution (c) (1.5 per cent); — disregard limit: disregard any peak due to the solvent.

Heavy m etals (2.4.8) Maximum 10 ppm. 1.0 g complies with test C. Prepare die reference solution using 1 m L o f lead standard solution (10 ppm Pb) R.

Aspartic Acid 1-203

2016 L oss o n d ry in g (2.2.32) Maxim vim 4.5 per cent, determined on 1.000 g by drying in an oven at 105 °C.

★ ★ ★ ★ ★ ★ *★ *

Aspartic Acid (Ph. Eur. monograph 0797)

S u lfa te d a sh (2.4.14) M aximum 0.2 per cent, determined on 1.0 g.

H

ASSAY Dissolve 0.250 g in 1.5 m L o f anhydrous formic acid R and 60 m L o f anhydrous acetic add R T itrate immediately with 0.1 M perchloric add, determining the end-point potentiometrically (2.2.20).

1 m L of 0.1 M perchloric acid is equivalent to 29.43 mg Of C 14H 18N 2O 5.

C4H7N 0 4

NH 2

133.1

56-84-8

A c tio n a n d u se Amino a d d . P hB r_____________

STORAGE In an airtight container. IM P U R IT IE S

Specified impurities A, C Other detectable impurities (the following substances would, if

D E F IN IT IO N Aspartic a d d contains not less th an 98.5 per cent and not m ore than the equivalent of 101.5 p e r cent of (25)-2-am inobutanedioic add, calculated with reference to the dried substance.

present at a sufficient level, be detected by one or other of the tests in the m onograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general m onograph Substances for pharmaceutical use (2034). I t is therefore not necessary to identify these impurities for dem onstration of compliance. See also 5.10.

CHARACTERS A white o r almost white, crystalline powder or colourless crystals, slightly soluble in water, practically insoluble in alcohol. I t dissolves in dilute mineral adds and in dilute solutions o f alkali hydroxides.

Control of impurities in substances for pharmaceutical use): B.

ID E N T IF IC A T IO N

First identification A, C. Second identification A , B, D. A. Specific optical rotation (see T ests). B. A suspension of 1 g in 10 m L o f water R is strongly a d d

COzH

(2.2.4).

A. 2-[(25',53)-5-benzyl-3,6-dioxopiperazm-2-yl]acetic ad d .

C. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with aspartic add CRS. Examine the substances prepared as discs. D . Examine the chromatograms obtained in the test for ninhydrin-posTtive substances. T h e prindpal spot in the chrom atogram obtained with test solution (b) is similar in position, colour and size to the principal spot in the chrom atogram obtained with reference solution (a).

CO2H HOjC

B. (35)-3-amino-4- [[(13)-1 -carboxy-2-phenylethyI] amino] -4oxobutanoic a d d (a-L-aspartyl-L-phenylalanine),

C . (2S)-2-amino-3-phenylpropanoic a d d (L-phenylalanine). PhEir

TESTS A p p e a ra n c e o f so lu tio n Dissolve 0.5 g in 1 M hydrochloric acid and dilute to 10 m L w ith the same ad d . T he solution is clear (2.2.1) and not more intensely coloured than reference solution BY 6 (2.2.2,

Method II). S p ecific o p tic a l ro ta tio n (2.2.7) Dissolve 2.000 g in hydrochloric acid R l and dilute to 25.0 m L with the same add. T h e specific optical rotation is + 24.0 to + 26.0, calculated with reference to the dried substance. N in h y d rin -p o sitiv e su b stan ces Examine by thin-layer chromatography (2.2.27), using a

TLC silica gel plate R. Test solution (a) Dissolve 0.10 g o f th e substance to be exam in e d in 2 m L of ammonia R and dilute to 10 m L w ith water R. Test solution (b) Dilute 1 mL of test solution (a) to 50 m L with water R. Reference solution (a) Dissolve 10 m g of aspartic add CRS in 2 m L of dilute ammonia R l and dilute to 50 m L with water R. Reference solution (b) Dilute 5 m L o f test solution (b) to 20 m L w ith water R.

1-204 Aspirin

2016

Reference solution (c) Dissolve 10 m g o f aspartic add CRS and 10 m g of glutamic add CRS in 2 m L of dilute ammonia R1 and dilute to 25 m L with water R. Apply separately to the plate 5 pL of each solution. Allow die plate to dry in air. Develop over a p ath o f 15 cm using a m ixture of 20 volumes o f glacial acetic add R, 20 volumes of water R and 60 volumes o f butanol R. Allow the plate to dry in air, spray with nmhydrin solution R. H eat at 100-105 °C for 15 m in. Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is n o t more intense than the spot in die chromatogram obtained with reference solution (b) (0.5 per cent). T h e test is n o t valid unless the chromatogram obtained with reference solution (c) shows 2 clearly separated principal spots. C h lo rid e s (2.4.4) Dissolve 0.25 g in 3 m L o f dilute nitric add R and dilute to 15 m L with water R. T h e solution, to which 1 m L o f water R is added instead o f dilute nitric add R, complies with the limit test for chlorides (200 ppm). S u lfa te s (2.4.13) Dissolve 0.5 g in 4 m L of hydrochloric add R and dilute to 15 m L with distilled water R T h e solution complies with die limit test for sulfates (300 ppm ). Carry out the evaluation o f the test after 30 min. A m m o n iu m 2.4.1) 50 mg complies with limit test B (200 ppm ). Prepare the standard using 0.1 m L o f ammonium standard solution

(100 ppm N H J R. Ir o n (2.4.9) In a separating funnel, dissolve 1.0 g in 10 m L o f dilute hydrochloric add R. Shake with 3 quantities, each of 10 mT, of methyl isobutyl ketone R l, shaking for 3 min each time. T o d ie combined organic layers add 10 m L o f water R and shake for 3 min. T he aqueous layer complies with the limit test for iron (10 ppm ).

Aspirin (Acetylsalicylic Add, Ph Eur monograph 0309)

*. * * * ★ ★ ★ *****

C02H

. ch 3

C9H 804

180.2

50-78-2

A c tio n a n d u se Salicylate; non-selective cyclo-oxygenase inhibitor; antipyretic; analgesic; anti-inflammatory. P re p a ra tio n s Aspirin Tablets

a

Dispersible Aspirin Tablets

Effervescent Soluble Aspirin Tablets Gastro-resistant Aspirin Tablets Aspirin and Caffeine Tablets Co-codaprin Tablets Dispersible Co-codaprin Tablets PhEur.

D E F IN IT IO N 2-(Acetyloxy)benzoic acid. C o n te n t 99.5 per cent to 101.0 per cent (dried substance). CHARACTERS A p p e a ra n c e W hite or almost white, crystalline pow der or colourless crystals.

S o lu b ility H eav y m e ta ls (2.4.8) Slighdy soluble in water, freely soluble in ethanol 2.0 g complies with test D (10 ppm ). Prepare the reference (96 per cent). solution using 2 m L o f lead standard solution (10 ppm Pb) R. mp L oss o n d ry in g (2.2.32) About 143 °C (instantaneous method). N ot m ore than 0.5 per cent, determined on 1.000 g by ID E N T IF IC A T IO N drying in an oven at 105 °C.

First identification A, B Second identification B, C, D

Sulfa ted ash (2.4.14)

N ot m ore than 0.1 per cent, d eterm ined on 1.0 g. A SSA Y Dissolve 0.100 g in 50 m L o f carbon dioxide-free water R, with slight heating if necessary. Cool and add 0.1 m L o f bromothymol blue solution R l. T itrate with 0.1 M sodium hydroxide until the colour changes from yellow to blue. 1 m L o f 0.1 M sodium hydroxide is equivalent to 13.31 m g of C4H7N 0 4. STO RA G E Protected from light. PhEur

A Infrared absorption spectrophotometry (2.2.24).

Comparison acetylsalicylic add CRS. B. T o 0.2 g add 4 m L o f dilute sodium hydroxide solution R and boil for 3 min. Cool and add 5 mT. o f dilute sulfuric add R. A crystalline precipitate is formed. Filter, wash the precipitate and dry at 100-105 °C. T h e melting point (2.2.14) is 156 °C to 161 °C. C. In a test tube mix 0.1 g with 0.5 g o f caldum hydroxide R. H eat the mixture and expose to the fumes produced a piece o f filter paper impregnated with 0.05 m L o f nitrobenzaldehyde solution R. A greenish-blue o r greenish-yellow colour develops on the paper. M oisten the paper with dilute hydrochloric add R T h e colour becomes blue. D . Dissolve with heating about 20 m g o f the precipitate obtained in identification test B in 10 m L o f water R and cool. T h e solution gives reaction (a) o f salicylates (2.3.1). TESTS A p p e a ra n c e o f so lu tio n T h e solution is clear (2.2.1) and colourless (2.2.2,

Method 11).

Aspirin 1-205

2016 Dissolve 1.0 g in 9 m L o f ethanol (96 per cent) R. Related substances L iquid chromatography (2.2.29). Prepare the solutions

immediately before use. Test solution Dissolve 0.100 g of the substance to be exam ined in acetonitrile for chromatography R and dilute to 10.0

m L w ith the same solvent.

Reference solution (a) Dissolve 50.0 m g o f saUcyUc add R (im purity C) in th e mobile phase and dilute to 50.0 m L with the mobile phase. Dilute 1.0 m L of the solution to 100.0 m L w ith the mobile phase.

Reference solution (b) Dissolve 10 mg o f salicylic add R (impurity C) in th e mobile phase and dilute to 10.0 m L with the mobile phase. T o 1.0 m L of the solution add 0.2 m L of the test solution and dilute to 100.0 m L with the mobile phase.

Reference solution (c) Dissolve with the aid o f ultrasound the contents o f a vial of aceiylsalicylic add for peak identification CRS (containing impurities A, B, D , E and F) in 1.0 m L of acetonitrile R. Column: — size. I = 0.25 m , 0 = 4.6 mm; — stationary phase: octadecylsUyl silica gelfor chromatography R

Prepare the reference solution using lead standard solution (1 ppm Pb) obtained by diluting lead standard solution (100 ppm Pb) R with a mixture o f 6 volumes of water R and 9 volumes of acetone R. L oss o n d ry in g (2.2.32) M axim um 0.5 p er cent, determined on 1.000 g by drying

in vacuo. S u lfa te d a s h (2.4.14) M axim um 0.1 p e r cent, determined on 1.0 g. A SSAY In a flask with a ground-glass stopper, dissolve 1.000 g in 10 m L o f ethanol (96 per cent) R. Add 50.0 m L of 0.5 M sodium hydroxide. Close die flask and allow to stand for 1 h. U sing 0.2 m L o f phenolphthalan solution R as indicator, titrate with 0.5 M hydrochloric add. Carry out a blank titration. 1 m L of 0.5 M sodium hydroxide is equivalent to 45.04 m g o f C 9H 8O 4. STO R A G E In an airtight container. IM P U R IT IE S

Specified impurities A, B, C , D , E , F CO2H

(5 pm).

Mobile phase phosphoric add R, acetonitrile for chromatography R, water R (2:400:600 VIVfV). Flow rate 1 mL/min. Detection Spectrophotom eter at 237 nm . Injection 10 |iL. Run time 7 times the retention time o f acetylsalicylic ad d . Identification of impurities U se the chrom atogram obtained w ith reference solution (a) to identify the peak due to im purity C; use the chromatogram supplied with acetylsalicylic acid for peak identification CRS and the chrom atogram obtained with reference solution (c) to identify the peaks due to impurities A, B, D , E and F.

Relative retention W ith reference to acetylsalicylic a d d (retention tim e = about 5 min): impurity A = about 0.7; im purity B = about 0.8; im purity C = about 1.3; im purity D = about 2.3; impurity E = about 3.2; im purity F = about 6.0. System suitability: reference solution (b): — resolution: m in im u m 6.0 between the peaks due to acetylsalicylic a d d and impurity C .

HO

A. 4-hydroxybenzoic ad d , CO2H

h o 2c

OH

B. 4-hydroxybenzene-1,3-dicarboxylic a d d (4-hydroxyisophthalic ad d ), COjH

a

OH

C . 2-hydroxybenzenecarboxylic a d d (salicylic ad d ),

Limits: — impurities A , B, C, D, E, F: for each impurity, n o t m ore than 1.5 tim es the area of the principal peak in the chrom atogram obtained with reference solution (a) (0.15 p e r cent); — unspecified impurities', for each impurity, n o t more than 0.5 times the area of the principal peak in the chrom atogram obtained with reference solution (a) (0.05 p e r cent); — total: n o t m ore than 2.5 times the area o f the prindpal peak in the chrom atogram obtained with reference solution (a) (0.25 per cent); — disregard limit. 0.3 times the area o f the prindpal peak in die chrom atogram obtained with reference solution (a) (0.03 p er cent).

Heavy m etals (2.4.8) M axim um 20 ppm . Dissolve 1.0 g in 12 m L o f acetone R and dilute to 20 m L w ith water R. 12 m L of the solution complies with test B.

D . 2-[[2-(acetyloxy)benzoyl]oxy]benzoic a d d (acetylsalicylsalicylic ad d ), COjH

E. 2-[(2-hydroxybenzoyl)oxy]benzoic a d d (salsalate, salicylsalicylic a d d ),

1-206 Atenolol

2016

er

ch3

F. 2 -(acetyloxy)benzoic anhydride (acetylsalicylic anhydride). ___________________________________________________ PhEir

★★* * ★ ★ ★

Atenolol

* * * * *

(Ph Ettr. monograph 0703) H

OH

H N

CH3

T

and enant'omer

CH3

Reference solution Dissolve 10 m g o f atenolol CRS in 1 m L o f methanol R. Plate TLC sUamsed silica gel F 254 plate R. Mobile phase concentrated ammonia R l, methanol R (1:99 V!V). Application 10 (iL. Drying In air. Detection Examine in ultraviolet light at 254 nm. Results The principal spot in the chrom atogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution. TESTS S o lu tio n S Dissolve 0.10 g in water R and dilute to 10 m L with the same solvent. A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and n o t more intensely coloured than degree 6 of the range of reference solutions o f the m ost appropriate colour (2.2. 2, Method II). O p tic a l ro ta tio n (2.2.7) + 0.10° to —0.10°, determined on solution S.

C 14H 22N 2O 3

266.3

29122-68-7

A ctio n a n d u se Beta-adrenoceptor antagonist. P re p a r a tio n s Atenolol Injection Atenolol Oral Solution

R elated su b sta n c e s Liquid chromatography (2.2.29).

Test solution Dissolve 50 mg of the substance to be examined in 20 m L o f the mobile phase and dilute to 25.0 m L with the mobile phase. Reference solution (a) Dissolve 2 m g of atenololfor system suitability CRS (containing impurities B, F, G, I and J) in

Atenolol Tablets

1.0 m L of the mobile phase.

Co-tenidone Tablets

Reference solution (b) Dilute 1.0 m L o f the test solution to

PhEur__________________________

100.0 m L with the mobile phase. Dilute 1.0 m L o f this solution to 10.0 m L with the mobile phase.

D E F IN IT IO N 2- [4- [(2ftS)-2-Hydroxy-3- [(1 -methylethyl) amino] propoxy] phenyl] acetamide. C o n te n t 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS A p p e a ra n c e W hite or alm ost white powder. S o lu b ility Sparingly soluble in water, soluble in anhydrous ethanol, slightly soluble in methylene chloride. ID E N T IF IC A T IO N

First identification C. Second identification A , B, D. A. Melting point (2.2.14)? 152 °C to 155 °C.

Column: — size. I = 0.125 m , 0 = 4.0 m m ; — stationary phase, end-capped octadecylsilyl silica gel for

chromatography R (5 Jim). Mobile phase Dissolve 1.0 g of sodium octanesulfonate R and 0.4 g of tetrabutylammonium hydrogen sulfate R in 1 L of a mixture o f 20 volumes o f tetrahydrofuran R, 180 volumes of methanol R2, and 800 volumes o f a 3.4 g/L solution of potassium dihydrogen phosphate R; adjust the apparent p H to 3.0 with phosphoric add R Flow rate 0.6 mlVmin. Detection Spectrophotometer at 226 nm. Injection 10 jiL. Run time 5 times the retention time o f atenolol. Identification of impurities Use the chromatogram supplied with atenololfor system suitability CRS and the chromatogram

B. Ultraviolet and visible absorption spectrophotometry (2.2.25).

obtained with reference solution (a) to identify the peaks due to impurities B, F , G , I and J.

Test solution Dissolve 0.100 g in methanol R and dilute to 100 m L with the same solvent. Dilute 10.0 m L o f this solution to 100 m L with methanol R.

Relative retention W ith reference to atenolol (retention time = about 8 min): impurity B = about 0.3; impurity J = about 0.7; impurity I = about 0.8; impurity F = about 2.0 (pair o f peaks); impurity G = about 3.5. System suitability: reference solution (a): — resolution: minimum 1.4 between the peaks due to impurities J (unidentified impurity) and I.

Speared range 230-350 nm. Absorption maxima A t 275 nm and 282 nm. Absorbance ratio A?7s/A?8? = 1.15 to 1.20. C. Infrared absorption spectrophotometry (2.2.24). Comparison atenolol CRS. D. Thin-layer chromatography (2.2.27). Test solution Dissolve 10 mg o f the substance to be examined in 1 m L o f methanol R.

Limits: — correction factor, for the calculation o f content, multiply the peak area of impurity I by 1.5;

Atomoxetine Hydrochloride 1-207

2016 — impurity B: not m ore than twice the area of the principal peak in the chromatogram obtained with reference solution (b) (0.2 p er cent); — impurities F, G, I : for each impurity, not more than 1.5 times the area of the principal peak in the chrom atogram obtained with reference solution (b) (0.15 p er cent); — unspecified impurities, for each impurity, not more th an the area o f the principal peak in the chromatogram obtained with reference solution (b) (0.10 per cent); — totd: n o t more th an 5 limes the area of the principal peak in the chrom atogram obtained with reference solution (b) (0.5 per cent); — disregard limit. 0.5 times the area o f the principal peak in the chrom atogram obtained with reference solution (b) (0.05 per cent). C h lo rid e s (2.4.4) M axim um 0.1 per c e n t Dissolve 50 mg in a mixture o f 1 m L o f dilute nitric acid R and 15 m L o f water R. T he solution, without further addition of dilute nitric acid R, complies with the test.

OH

E. 2,2'- [(2-hydroxypropane-1,3-diyl)bis(oxy-4,1-phenylene)] diacetamide, h 3c

ch3

j

oh

F. 2,2'-[[(l-methylethyl)imino]bis[(2-hydroxypropane-3,ldiyI)oxy-4,1-phenylene]] diacetamide, V

H H

HO2C

G . 2- [4- [(2i?5)-2-hydroxy-3- [(1 -methylethyl)amino] propoxy]phenyl] acetic a d d , H

OH

H N.

and enantiomer

H . 2-[4- [(2i?i)-2-hydroxy-3- [(1-methylethyl)amino] propoxy]phenyl] acetonitrile, H

1 m L o f 0.1 M perchloric acid is equivalent to 26.63 m g o f C 14H 22N 2O 3.

.C H 3 CH3

ASSAY Dissolve 0.200 g in 80 m L o f anhydrous acetic acid R. T itrate with 0.1 M perchloric add, d eterm ining the end-point potentiometrically (2.2.20).

and enantiomer

CH3

L oss o n d ry in g (2.2.32) M axim um 0.5 per cent, determined on 1.000 g by drying in an oven at 105 °C. S u lfa te d a s h (2.4.14) M axim um 0.1 per cent, determined on 1.0 g.

^ ^ ch3

OH N

CH3

and enantiomer

I. 2-[4- [(2i?5)-3-(ethylamino)-2-hydroxypropoxy] phenyl] acetamide. PhEur

IMPURITIES Specified impurities B, F , G, I Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other o f the tests in the monograph. T hey are limited by the general acceptance criterion for other/unspecified impurities and/or by the general m onograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these impurities for dem onstration o f compliance. See also 5.10.

Atomoxetine Hydrochloride

★ ★

(Ph. Ettr. monograph 2640)

*****

Control of impurities in substances for pharmaceutical use): A , D, E, H.

, HCI

C 17H 22CINO

291.8

82248-59-7

H,N

A. R-H : 2-(4-hydroxyphenyl)acetamide, H

OH and enantiomer

B. 2-[4- [(2R5)-23-dihydroxypropoxy]phenyl] acetamide, H

OH and enantiomer

D . 2-[4- [(2jR5)-3-chloro-2-hydroxypropoxy]phenyl] acetamide,

* ★

A ctio n a n d u se Noradrenaline reuptake inhibitor, treatm ent of attention defidt hyperactivity disorder (A DH D). PhEur.

D E F IN IT IO N (3i?)-N-Methyl-3-(2-methylphenoxy)-3-phenylpropan-lamine hydrochloride. C o n te n t 98.0 per cent to 102.0 p er cent (dried substance). CHARACTERS A p p e a ra n c e W hite or almost white powder. S o lu b ility Sparingly soluble in water, soluble in anhydrous ethanol, practically insoluble in heptane.

1-208 Atomoxetine Hydrochloride

It shows polymorphism (5.9).

IDENTIFICATION A. Infrared absorption spectrophotom etry (2.2.24).

Comparison atomoxetine hydrochloride CRS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in anhydrous ethanol R, evaporate to dryness and record new spectra using the residues. B. Isomeric purity (see Tests).

2016 acid R previously adjusted to p H 2.5 w ith a 280 g/L solution o f potassium hydroxide R Test solution (a) Dissolve 25 m g o f the substance to be examined in the mobile phase and dilute to 10.0 m L with the mobile phase. Test solution (b) Dissolve 25.0 m g o f the substance to be examined in the mobile phase and dilute to 100.0 m L with the mobile phase. Reference solution (a) Dilute 1.0 m L o f test solution (a) to

C. It gives reaction (a) o f chlorides (2.3.1).

100.0 m L with the mobile phase. D ilute 1.0 m L o f this solution to 10.0 m L with the mobile phase.

TESTS Isomeric purity

Reference solution (b) Dissolve 7.5 m g o f 3-(methylammo)-lphenylpropan-l-ol R (impurity H ) and 5 mg o f mandeHc add R

Liquid chromatography (2.2.29): use the normalisation procedure.

(impurity E) in test solution (b) and dilute to 50 m L with test solution (b).

Test solution Dissolve 35.0 m g of the substance to be examined in 2.5 m L o f anhydrous ethanol R, sonicate until dissolution is complete and dilute to 10.0 m L with heptane R.

Reference solution (c) Dissolve 5 m g o f atomoxetine for impurity A identification CRS in the mobile phase and dilute

Reference solution (a) Dissolve 3.5 mg o f atomoxetine impurity B CRS and 1 mg o f atomoxetine impurity D CRS in 5 m L of anhydrous ethanol R, sonicate until dissolution is complete and dilute to 20.0 m L with heptane R. Reference solution (b) Dissolve 35.0 m g of the substance to be examined in 2.5 m L o f anhydrous ethanol R. Add 1.0 m L of reference solution (a) and dilute to 10.0 m L with heptane R. Reference solution (c) Dilute 1.0 m L of reference solution (a) to 100.0 m L with heptane R. Column: — size. I — 0.25 m , 0 = 4.6 mm; — stationary phase. cellulose derivative of silica gelfor chiral separation R (5 pm). Mobile phase M ix 1.5 m L of diethylamine R, 2.0 m L of trifluoroacetic acid R and 150.0 m L o f 2-propanol R and dilute to 1000 m l. with heptane R. Flow rate 1.0 mL/min. Detection Spectrophotom eter at 273 nm . Irqecdon 10 pL of the test solution and reference solutions (b) and (c).

Run time 1.3 times the retention time o f atomoxetine. Identification of impurities U se the chromatogram obtained with reference solution (b) to identify the peaks due to impurities B and D.

Relative retention W ith reference to atomoxetine (retention

to 20 m L with the mobile phase.

Reference solution (d) Dissolve 25.0 mg of atomoxetine hydrochloride CRS in the mobile phase and dilute to 100.0 m L with the mobile phase.

Column: — size. I = 0.15 m , 0 = 4.6 mm ; — stationary phase: end-capped octylsUyl silica gel for chromatography R (3.5 pm); — temperature. 40 °C. Mobile phase propanol R, solution A (27:73 VIV). Flow rate 1.0 mL/min. Detection Spectrophotom eter at 215 nm . Injection 10 pL of test solution (a) and reference solutions (a), (b) and (c).

Run time 2.5 times the retention time o f atomoxetine. Identification of impurities Use the chromatogram obtained w ith reference solution (b) to identify the peaks due to impurities E and H ; use the chromatogram supplied with atomoxetine for impurity A identification CRS and the chrom atogram obtained with reference solution (c) to identify the peak due to impurity A.

Relative retention W ith reference to atomoxetine (retention tim e = about 10 min): impurity E = about 0.2; impurity H = about 0.3; impurity A = about 0.7. System suitability: reference solution (b): — resolution: m in im u m 5.0 between the peaks due to impurities E and H.

time = about 12 min): impurity B = about 0.5; im purity D = about 0.6. System suitability: reference solution (b): — resolution: minimum 1.8 between the peaks due to impurities B and D .

— for each impurity, use the concentration o f atomoxetine hydrochloride in reference solution (a).

Limits: — impurity B: maximum 0.5 per cent; — impurity D: maximum 0.15 per cent; — unspecified impurities', for each impurity, maximum

— impurity A: maximum 0.3 per cent; — unspecified impurities: for each impurity, maximum 0.10 per cent; — total: maximum 0.5 per cent; — reporting threshold: 0.05 per cent.

0.10 per cent; — disregard limit: the area o f the peak due to impurity B in the chromatogram obtained with reference solution (c) (0.05 per cent); disregard any peak with a relative retention with reference to atomoxetine o f about 0.7 (impurity A).

Related substances Liquid chromatography (2.2.29).

Solution A Dissolve 5.9 g of sodium octanesulfonate monohydrate R in 1000 m L o f a 2.9 g/L solution of phosphoric

Calculation ofpercentage contents:

Limits:

H e a v y m e ta ls (2.4.8) M axim um 10 ppm.

Solvent mixture water R, methanol R (20:80 VIV). 0.250 g complies with test H. Prepare the reference solution using 0.25 m l . of lead standard solution (10 ppm Pb) R. L o ss o n d ry in g (2.2.32) M axim um 0.5 per cent, determined on 1.000 g by drying in vacuo at 105 °C for 2 h.

Atorvastatin Calcium Trihydrate 1-209

2016 Sulfated ash (2.4.14) M axim um 0.1 per cent, determined on 1.0 g. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modification.

Injection T est solution (b) and reference solution (d). Calculate the percentage content o f C i 7H 22C lN O taking into account the assigned content of atomoxetine

F. (35)-3-(3-fluoro-2-methylphenoxy)-N-methyl-3pheny lpropan-1-amine.

hydrochloride CRS. IMPURITIES Specified impurities A, B, D Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general m onograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these impurities for dem onstration of compliance. See also 5.10. Control of impurities in substancesfor pharmaceutical use): C, E,

h3Cv

xCH3

G. 3 j3 /-[(2-methylbenzene-lj3-diyl)bis(oxy)]bis(N-methyl-3phenylpropan-1-amine),

F, G, H.

H . 3-(methylamino)-1 -phenylpropan-1-ol. PhEur

A. N-methyl-3-phenoxy-3-phenylpropan-1-amine,

o

Atorvastatin Calcium Trihydrate

★ ★

★ ★

(Ph Ettr. monograph 2191)

*****

B . (3S)-N-methyl-3-(2-methylphenoxy)-3-phenylpropan-1amine,

HaC

C66H 68CaF2N40 1(b3H20

1209

344423-98-9

Action and use C . (3i?)-iV-methyl-3-(4-methylphenoxy)-3-phenylpropan-1amine,

H M G Co-A reductase inhibitor; lipid-regulating drug. PhEur.

DEFINITION Calcium (3i?,5jR)-7-[2-(4-fluorophenyl)-5-(l-methyiethyl)-3phenyl-4-(phenylcarbamoyi)-1//-pyrrol- l-yQ-3,5dihydroxyheptanoate trihydrate.

H ,C

Content 97.0 D . (3R)-N-methyl-3-(3-methylphenoxy)-3-phenylpropan-1amine, HO

H COjH

p er cent to 102.0 p er cent (anhydrous substance).

CHARACTERS Appearance W hite o r almost white powder.

Solubility Very slightly soluble in water, slightly soluble in ethanol (96 p er cent), practically insoluble in methylene chloride. It shows polymorphism (5.9).

E. (2S)-2-hydroxy-2-phenylacetic acid (L-mandelic acid),

IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24).

Comparison atorvastatin calcium trihydrate CRS.

1-210 Atorvastatin Calcium Trihydrate

2016

If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in methanol R, evaporate to dryness and record new spectra using the residues.

impurity D CRS and 2.5 m g o f the substance to be examined in dimethylformamide R and dilute to 50.0 m L with the same solvent.

B. Enantiom eric purity (see Tests).

— size: I = 0.25 m , 0 = 4.6 mm; — stationary phase: octylsifyl silica gel for chromatography R (5 pm); — temperature: 35 °C.

C. W ater (see Tests). D. Ignite. T h e residue gives reaction (b) o f calcium (2.3.1). Filtration may be necessary in case the residue does not completely dissolve.

TESTS Enantiomeric purity Liquid chrom atography (2.2.29).

Solvent mixture anhydrous ethanol R, methanol R (50:50 V!V). Test solution Dissolve 10 mg o f the substance to be examined

Column:

Mobile phase: — mobile phase A: tetrahydrofuran R, acetonitrüe R, 3.9 g/L solution o f ammonium acetate R adjusted to p H 5.0 with glacial acetic acid R (12:21:67 VIVIV)', — mobile phase B: tetrahydrofuran R, 3.9 g/L solution o f ammonium acetate R adjusted to p H 5.0 with glacial acetic add R, acetonitrüe R (12:27:61 VIVIV)',

in 4 m L o f the solvent mixture and dilute to 10.0 m L with

hexane R. Reference solution (a) Dissolve 2 mg o f atorvastatin impurity E CRS in methanol R and dilute to 20.0 m L with the same solvent (solution A). Dissolve 10 mg of the substance to be examined in 1.25 m L of methanol R, add 0.75 m L of solution A and 2 m L o f anhydrous ethanol R and dilute to 10.0 m L w ith hexane R.

Reference solution (b) T o 2.0 m L o f the test solution add 40.0 m L of the solvent mixture and dilute to 100.0 m L with hexane R. T o 3.0 m L o f this solution add 5 m L of the solvent mixture and dilute to 20.0 m L with hexane R.

Column: — size: I = 0.25 m, 0 = 4.6 mm; — stationary phase: amylose derivative of silica gel for chromatography R (10 jun). Mobile phase trifluoroacetic acid R, anhydrous ethanol R, hexane R (0.1:6:94 VIVIV). Flow rate 1.0 mlVmin. Detection Spectrophotom eter at 244 nm . Injection 20 )lL. Run time 1.2 times the retention time o f atorvastatin. Relative retention W ith reference to atorvastatin (retention time = about 44 min): impurity E = about 0.8.

System suitability: reference solution (a): — resolution: m inim um 2.0 between the peaks due to im purity E and atorvastatin.

Limit. — impurity E: not m ore than the area o f the principal peak in the chrom atogram obtained with reference solution (b) (0.3 per cent).

Related substances Liquid chrom atography (2.2.29).

Test solution (a) Dissolve 40.0 m g o f the substance to be examined in dimethytformamide R and dilute to 100.0 mT. with the same solvent. Test solution (b) Dissolve 50 m g o f the substance to be examined in dimethylformamide R and dilute to 50.0 m L with the same solvent. Reference solution (a) Dissolve 40.0 m g o f atorvastatin calcium trihydrate CRS in dimethylformamide R and dilute to 100.0 m L with the same solvent.

Reference solution (b) D ilute 1.0 m L o f test solution (b) to 100.0 m L with dimethylformamide R. Dilute 1.0 m L o f this solution to 10.0 m L w ith dimethylformarnide R. Reference solution (c) Dissolve 2.5 mg o f atorvastatin impurity A CRS, 2.5 m g o f atorvastatin impurity B CRS, 2.5 m g o f atorvastatin impurity C CRS, 2.5 mg o f atorvastatin

Time (min) 0 • 40

Mobile phase A (per cent V7V) 100

Mobile phase B (per cent V/V) 0

40 - 70

100->20

0 + 80

7 0 -8 5

20

0

80 -» 100

Flow rate 1.5 mlVmin. Detection Spectrophotom eter at 244 nm . Injection 20 )iL o f test solution (b) and reference solutions (b) and (c).

Identification of impurities U se the chrom atogram obtained with reference solution (c) to identify the peaks due to impurities A, B, C and D .

Relative retention W ith reference to atorvastatin (retention time = about 33 min): impurity A = about 0.8; im purity B = about 0.9; impurity C = about 1.2; impurity D = about 2.1. If necessary5 adjust the mobile phase by increasing or decreasing the percentage o f acetonitrile o r the p H o f the am m onium acetate solution to achieve a retention time o f about 33 rnin for atorvastatin. For example, raising the p H would decrease die retention time o f atorvastatin. System suitability: reference solution (c): — resolution: m inim um 1.5 between the peaks due to im purity B and atorvastatin.

Limits: — impurities A , B: for each impurity, n o t m ore than 3 times the area o f the principal peak in the chrom atogram obtained with reference solution (b) (0.3 per cent); — impurities C, D: for each impurity, n o t m ore than 1.5 times die area o f the principal peak in the chrom atogram obtained with reference solution (b) (0.15 p er cent); — unspecified impurities: for each im purity, not m ore than the area o f the principal peak in the chrom atogram obtained with reference solution (b) (0.10 p er cent); — total: n o t m ore than 15 times the area o f the principal peak in die chrom atogram obtained with reference solution (b) (1.5 p er cent); — disregard limit. 0.5 tim es the area o f the principal peak in the chrom atogram obtained w ith reference solution (b) (0.05 p er cent); disregard the peak due to dimethylformamide.

Sodium M axim u m 0.4 per cent (anhydrous substance).

Atomic absorption spectrometry (2.2.23, Method I).

2016

Atorvastatin Calcium Trihydrate 1-211

Solvent mixture hydrochloric acid R, water R, methanol R (2:25:75 VIVIV). Test solution Dissolve 5.0 mg in the solvent mixture and dilute to 100.0 mT. with the solvent mixture.

Reference solutions Prepare the reference solutions using sodium standard solution (50 ppm Na) R, diluting with the solvent m ixture.

Source Sodium hollow-cathode lamp. Wavelength 589.0 nm . Atomisation device Air-acetylene flame. H eav y m e ta ls (2.4.8) M axim um 20 ppm .

Solvent mixture water R, methanol R (10:90 VIV).

C. (3Æj5Æ)-7-[2j3-bis(4-fluorophenyl)-5-(l-methylethyl)-4(phenylcarbam oyl)-l//-pyrrol-l-yl]-3j5-dihydroxyheptanoic a d d (fluoroatorvastatin), h 3c n__ ch 3

It complies with test H with the following modifications.

Test solution Dissolve 0.250 g of the substance to be examined in 30 m L o f the solvent mixture. -Reference solution Dilute 0.5 m L o f lead standard solution (10 ppm Pb) R to 30 m L with the solvent mixture. Blank solution 30 m L of the solvent mixture. W a te r (.2.5.12)

D . 3-[(4-fluorophenyl)carbonyl]-2-(2-methylpropanoyl)-2Vj3diphenyloxirane-2-carboxamidej

3.5 per cent to 5.5 per cent, determined on 0.130 g. A SSA Y Liquid chromatography (2.2.29) as described in the test for related substances with the following modification.

Irgecdon T est solution (a) and reference solution (a). Calculate the percentage content o f C 66H 68CaF 2N 4O 10 from the declared content of atorvastatin calcium trihydrate CRS. IM P U R IT IE S

Specified impurities A, B, C 3 D , E. Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these impurities for demonstration of compliance. See also 5.10.

E. (3S,5S)-7-[2-(4-fluorophenyI)-5-(l-methylethyl)-3-phenyl4-(phenylcarbam oyl)-l//-pyirol-l-yi]-3,5-dihydroxyheptanoic a d d (ônr-atorvastatin), HO H H OH

Control of impurities in substances for pharmaceutical use): F, G, H.

A. (3i?j5iR)-3j5-dihydroxy-7-[5-(l-methylethyl)-2j3-diphenyl4-(phenylcarbamoyI)-1//-pyrrol-1-yl]heptanoic a d d (desfluoroatorvastatin),

B . (3RS,5SK)-7- [2-(4-fluorophenyl) -5-( 1-methylethyl)-3phenyl-4-(phenylcarbamoyl)-1//-pyrrol-1-yl] -3,5dihydroxyheptanoic ad d .

F . (3i?,5/$-7-[[(3i?,5£)-7-[2-(4-fluorophenyl)-5( 1-methylethyl)-3-phenyl-4-(phenylcarbamoyl)-1//-pyrrol-1yl] -3,5-dihydroxyheptanoyI] amino]-3,5-dihydroxyheptanoic add,

G . (3R,5R)-7- [2-(4-fluorophenyl)-i>-( 1-methylethyl)-3phenyl-4- (phenylcarbamoyl)-1//-pyrrol-1-yl] -5-hydroxy-3m ethoxyheptanoic a d d (3-0-methylatorvastatin)j

2016

1-212 Atovaquone o

Test solution Dissolve 25.0 m g o f the substance to be examined in the solvent mixture and dilute to 100.0 m L with the solvent mixture. Reference solution (a) Dissolve 25.0 m g o f atovaquone CRS in the solvent mixture and dilute to 100.0 m L with the solvent mixture. Reference solution (b) Dissolve 2.5 m g o f atovaquone for system suitability CRS (containing impurities B and C) in the solvent mixture and dilute to 10.0 m L with the solvent mixture.

Reference solution (c) Dilute 1.0 m L o f the test solution to

H. (4R,6R)-6-[2-[2-(4-fluorophenyI)-5-( 1-methylethyI)-3phenyl-4-(phenylcarbam oyl)-lif-pyrrol-l-yI]ethyl]-4hydroxytetrahydro-2Jï-pyran-2-one.

100.0 m L with die solvent mixture. Dilute 1.0 m L o f this solution to 10.0 m L with the solvent mixture.

__________________________________________________________ PhEur

Column: — size. I — 0.25 m, 0 = 4.6 mm; — stationary phase, end-capped octadecylsifyl silica gelfor chromatography R (5 pm).

Atovaquone

******

(Ph E ut monograph 2192)

*

Mobile phase phosphoric add R, methanol R2, water for chromatography R, acetomtrUe R1 (0.5:17.5:30:52.5 VIVIVIV). Flow rate 2.5 mL/min. Detection Spectrophotometer at 220 nm. Injection 20 jiL of the test solution and reference solutions (b) and (c).

Run time Twice the retention time of atovaquone. Identification of impurities Use the chromatogram supplied with atovaquone for system suitability CRS and the

O C22H19C103

366.8

95233-18-4

Action and use Antiprotozoal (malaria). PhEur__________________________________________________________

DEFINITION 2- [trans-4-(4-ChlorophenyI) cyclohexyl]-3hydroxynaphthalene-1,4-dione.

Content 97.5 per cent to 102.0 per cent (anhydrous substance).

CHARACTERS Appearance Yellow, crystalline powder.

Solubility Practically insoluble in water, sparingly soluble in methylene chloride, very slightly soluble in methanol. It shows polymorphism (5.9).

IDENTIFICATION Infrared absorption spectrophotometry (2.2.24).

Comparison atovaquone CRS. If the spectra obtained show differences, dissolve 0.1 g o f the substance to be examined and 0.1 g o f the reference substance separately in 2.5 m L of a 50 g/L solution of potassium hydroxide R in methanol R. Filter the solutions and add each filtrate dropwise to a mixture o f 0.8 m L o f acetic add R and 1.5 m L o f methanol R, stirring continuously. Filter, wash the residues with methanol R and then with water R, and dry under vacuum at 55 °C. Record new spectra using the residues.

TESTS Related substances Liquid chromatography (2.2.29). Cany out the test protected from light. Solvent mixture water R, acetonitrüe R1 (20:80 ViV).

chromatogram obtained with reference solution (b) to identify the peaks due to impurities B and C.

Relative retention W ith reference to atovaquone (retention time = about 15 min): impurity B = about 0.85; impurity C = about 0.90. System suitability: reference solution (b): — resolution: minimum 2.0 between the peaks due to impurity C and atovaquone; — peak-to-vaUey ratio: minim um 1.5, where Hp = height above the baseline of the peak due to impurity C and Hv ~ height above the baseline o f the lowest point of the curve separating this peak from the peak due to impurity B.

Calculation of percentage contents: — for each impurity, use the concentration o f atovaquone in reference solution (c).

Limits: — impurity B: maximum 0.5 per cent; — impurity C: maximum 0.2 per cent; — unspecified impurities: for each impurity, maximum 0.10 per cent; — total: maximum 0.6 per cent; — reporting threshold: 0.05 per cent. W a te r (2.5.32) M axim um 0.3 per cent, determ ined on 0.100 g using the evaporation technique: — temperature. 160 °C; — heating time. 3 min; — flow rater. 50 mL/min. S u lfa te d a s h (2.4.14) Maximum 0.1 per cent, determ ined on 1.0 g. A SSA Y Liquid chromatography (2.2. 29) as described in the test for related substances with the following modification.

Injection T est solution and reference solution (a). Calculate the percentage content o f C 22H 19CIO 3 taking into account the assigned content o f atovaquone CRS.

Atracurram Besilate 1-213

2016

IM P U R IT IE S

Specified impurities B, C Other detectable impurities (the following substances would, if

Atracurium Besilate

***** ★ ★

(Ph Ettr monograph 1970)

*****

present at a sufficient level, be detected by one or other of th e tests in the monograph. They are limited by die general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these impurities for demonstration of compliance. See also 5.10.

Control of impurities in substances for pharmaceutical use): A , D.

Q 5H 82N 2O 18S 2

1243

64228-81-5

A ctio n a n d u se Non-depolarizing neuromuscular blocker. PhEir______________________________________

D E F IN IT IO N Mixture of the cis-cis, cis-trans and trans-trans isomers of 2 ,2 '- [pentane-1,5-diylbis [oxy(3-oxopropane-l ,3-diyl)]] bis [ 1(3,4-dimethoxybenzyl)-6,7-dimethoxy-2-methyl-l ,2,3,4tetrahydroisoquinolinium] dibenzenesulfonate.

B. 2 -[cw-4-(4-chlorophenyl)cyclohexyl]-3hydroxynaphthalene-l,4-dione,

C o n te n t 96.0 per cent to 102.0 per cent (anhydrous substance). OH

CHARACTERS A p p e a ra n c e White or yellowish-white, slightly hygroscopic powder.

and enantiomer

C . 2- [( l.RS)-4-(4-chlorophenyl)cyclohex-3-en- 1-yl] -3hydroxynaphthalene-l,4-dione,

S olubility Soluble in water, very soluble in acetonitrile, in ethanol (96 per cent) and in methylene chloride. ID E N T IF IC A T IO N A Infrared absorption spectrophotometry (2.2.24).

Comparison atracurium besilate CRS. B. Examine the chromatograms obtained in the assay.

Results T he 3 principal isomeric peaks in the chromatogram D . 2-[mms-4-(4-chlorophenyl)cyclohexyl]-3methoxynaphthalene-1,4-dione. PhEur

obtained with test solution (a) are similar in retention tim e to those in the chromatogram obtained with reference solution (a). TESTS S o lu tio n S Dissolve 1.00 g in water R and dilute to 100 m L with the same solvent. A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y 7 (2.2.2, Method II). R e la te d su b sta n c e s Liquid chromatography (2.2.29).

Test solution (a) Dissolve 50.0 m g of the substance to be examined in mobile phase A and dilute to 50.0 m L with mobile phase A. Test solution (b) Dissolve 0.100 g of the substance to be examined in mobile phase A and dilute to 10.0 m L with mobile phase A.

1-214 Atracurium Besilate

2016

Reference solution (a) Dissolve 50.0 m g o f atracurium besilate CRS in mobile phase A and dilute to 50.0 m L with

im purity B = about 1.15; im purity C l = ab o u t 1.2; impurity C2 (major isomer) = about 1.3.

mobile phase A.

System suitability:

Reference solution (b) Dilute 1.0 m L o f test solution (a) to

— resolution: minimum 1.5 betw een the peaks due to the atracurium trans-trans isomer an d the atracurium cis-trans isomer, and m inim um 1.5 betw een the peaks due to the atracurium cis-trans isom er and th e atracurium cis-ds isomer in the chrom atogram obtained with reference solution (a); — pedk-to-valley ratio: m inim um 1.2, w here HP — height above the baseline o f the peak due to im purity A l and Hv = heigjit above the baseline o f the lowest point o f the curve separating this peak from th e peak due to the atracurium cis-cis isomer in the chrom atogram obtained with reference solution (d).

100.0 m L with mobile phase A.

Reference solution (c) Dissolve 20.0 m g o f methyl benzenesulfonate R in aceîonitrüe R and dilute to 100.0 m l. with the same solvent. Dilute 50 nL o f the solution to 100.0 m L with mobile phase A.

Reference solution (d) Dissolve 2.0 m g o f atracurium for peak identification CRS (containing impurities A l, A2, B, C l , C2, D l, D 2, E, G and K) in 2.0 m L o f mobile phase A.

Reference solution (e) Dissolve 2.0 m g o f atracurium for impurity F identification CRS in 2.0 m L o f mobile phase A. Column: — size: I = 0.25 m , 0 = 4.6 mm; — stationary phase: base-deactivated end-capped octadecylsifyl silica gelfor chromatography R (5 |im ). Mobile phase: — mobile phase A: mix 5 volumes of methanol R, 20 volumes o f acetonitrüe R and 75 volumes o f a 10.2 g/L solution of potassium dihydrogen phosphate R previously adjusted to p H 3.1 with phosphoric add R; — mobile phase B: mix 20 volumes o f acetomtrüe R, 30 volumes of methanol R and 50 volumes o f a 10.2 g/L solution o f potassium dihydrogen phosphate R previously adjusted to p H 3.1 with phosphoric add R; Urne (min) 0 -5

Mobile phase A (per cent V/V) 80

Mobile phase B (percent V/V) 20

5 -15

80 ->40

20 ->60

15-25

40

60

2 5 -3 0

40 ->0

60-> 100

30 -4 5

0

100

Flow rate 1 mlVmin. Detection Spectrophotom eter at 280 run. Injection 20 pL o f test solution (a) and reference solutions (a), (b), (d) and (e).

Identification of impurities Use the chromatogram obtained with reference solution (d) and the chromatogram supplied with atracurium for peak identification CRS to identify the peaks due to impurities A l, A2, B, C l, C2, D l, D 2, E, G and K; use the chromatogram obtained with reference solution (e) and the chromatogram supplied with atracurium for impurity F identification CRS to identify the peak due to im purity F.

Relative retention W ith reference to the atracurium tis-ds isomer (retention time = about 30 min): impurity E = about 0.2; impurity F = about 0.25; im purity G = about 0.3; impurity D l = about 0.45; impurity D 2 = about 0.5; atracurium trans-trans isom er = about 0.8; atracurium cis-trans isomer = about 0.9; im purity A l = about 1.04; im purity II = about 1.07; impurity H I = about 1.07 (shoulder on the front o f peak A2); im purity A2 (major isomer) = about 1.08; impurity K1 = about 1.09 (shoulder on the tail o f peak A2); impurity 12 (major isomer) = about 1.12; impurity H 2 (major isomer) = about 1.12; impurity K 2 (major isomer) = about 1.12;

Limits:

— correction factor, for the calculation o f content, multiply the peak area of impurity G by 0.5; — impurity E: not more than 1.5 tim es the sum o f the areas of the peaks due to the atracurium cis-cis, trans-trans and cis-trans isomers in the chrom atogram obtained with reference solution (b) (1.5 per cent); — impurities A, D: for each im purity, for the sum o f the areas o f the 2 isomer peaks, n o t m ore than 1.5 times the sum o f the areas o f the peaks due to the atracurium cis-cis, trans-trans and cis-trans isomers in the chromatogram obtained with reference solution (b) (1.5 per cent); — impurity C: for the sum o f the areas o f the 2 isom er peaks, not m ore than the sum o f the areas o f the peaks due to the atracurium cis-cis, trans-trans and cis-trans isomers in the chrom atogram obtained w ith reference solution (b) (1.0 p er cent); — impurities F, G: for each im purity, n o t m ore than the sum o f the areas of the peaks due to th e atracurium cis-ds, trans-trans and cis-trans isomers in the chrom atogram obtained with reference solution (b) (1.0 p er cent); — impurities H, 1, K. for the sum o f the areas o f the isom er peaks o f these impurities, not m ore than th e sum o f the areas o f the peaks due to the atracurium cis-cis, trans-trans and cis-trans isomers in the chrom atogram obtained with reference solution (b) (1.0 per cent); — unspecified impurities: for each im purity, n o t m ore th an 0.1 times the sum o f the areas o f the peaks due to the atracurium cis-cis, trans-trans and cis-trans isomers in the chromatogram obtained with reference solution (b) (0.10 p er cent); — total: n o t more than 3.5 times th e sum o f the areas o f the peaks due to the atracurium cis-cis, trans-trans and cis-trans isomers in the chrom atogram obtained with reference solution (b) (3.5 per cent); — disregard Umit. 0.05 times the sum o f the areas of the peaks due to the atracurium cis-cis, trans-trans an d cis-trans isomers in the chrom atogram obtained with reference solution (b) (0.05 per cent).

Impurity J Liquid chromatography (2.2.29) as described in the test for related substances with the following modifications.

Atracurium Besilate 1-215

2016

Mobile phase: Time (min) 0 -5

Mobile phase A (per cent V7V) 80

Mobile phase B (per cent V/V) 20

5 - 15

8 0 -»75

20 ->25

15 - 25

75

25

2 5 -3 0

75

55

25 ->45

3 0 -3 8

55 -» 0

45-» 100

38 - 45

0

100

Detection Spectrophotom eter at 217 nm. Irtjection 100 jiL o f test solution (b) and reference

o

A . l-(3,4-dimethoxybenzyl)-2-[13-[l-(3,4-dimethoxybenzyl)6,7-dim ethoxy-3,4-dihydroisoquinolin-2(l/i)-yI]-3,ll-dioxo4 ,1 0-dioxatridecyI]-6,7-dimethoxy-2-inethyl-l,2,3,4tetrahydroisoquinolinium (A1 = cis-trans isomer, A2 — cis-cis isomer),

solution (c).

Retention time Im purity J = about 25 min; atracurium trans-trans isomer = about 38 min. Limit. — impurity J: n o t more than the area of the principal peak in the chromatogram obtained with reference solution (c) (10 ppm).

o

o r a

A

o q/

W

^

o^

r

B. pentane-l,5-diyl bis[3-[l-(3,4-dimethoxybenzyl)-6,7dimethoxy-3,4-dihydroisoquinolin-2( li/)-yl] prop anoate], o

o

Isomer composition Liquid chromatography (2.2.29) as described in the test for related substances with the following modifications. U se the normalisation procedure.

Irtjection T est solution (a). Limits: — atracurium cis-cis isomer. 55.0 per cent to 60.0 per cent, — atracurium cis-trans isomer. 34.5 per cent to 38.5 per cent, — atracurium trans-trans isomer. 5.0 per cent to 6.5 per cent. Water (2.5.12) M aximum 5.0 per cent, determined on 1.000 g.

Sulfated ash (2.4.14) M aximum 0.1 p er cent, determined on 1.0 g. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modification.

C . 1-(3,4-dimethoxybenzyl)-2-(3,11 -dioxo-4,10-dioxatridec12-enyl)-6,7-dimethoxy-2-methyl-1,2,3,4tetrahydroisoquinolinium (C l = trans isomer, C 2 = cis isomer), o

D . l-(3,4-dimethoxybenzyl)-2-[3-[(5-hydroxypentyl)oxy]-3oxopropyl] -6,7-dimethoxy-2-methyl-1,2,3,4tetrahydroisoquinolinium (D1 = trans isomer, D 2 = cis isomer), .

o

Irtjection T est solution (a) and reference solution (a). Calculate the percentage content o f Q s H ^ ^ O j a S j from the sum of the areas o f the peaks due to the 3 isomers.

E . 2-(2-carboxyethyl)- l-(3,4-dimethoxybenzyl)-6,7dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinolinium,

STORAGE In an airtight containerj protected from light, at a tem perature o f 2 °C to 8 °C.

F . R+-C H 3: l-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2,2dim ethyl-1,2,3,4-tetrahydroisoquinolinium,

IM P U R IT IE S

Specified impurities A, C, D , E, F, G , H , I, J, K Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other of the tests in the monograph. T hey are limited by die general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore n o t necessary to identify these impurities for dem onstration of compliance. See also 5.10.

Control of impurities in substances for pharmaceutical usé): B.

G . R -C H 3: l-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2m ethyl-1,2,3,4-tetrahydroisoquinoline, o

O H . 2,2 [hexane- 1,6-diylbis [oxy(3-oxopropane-1,3dfyl)]]bis[l-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2-methylI.2,3,4-tetrahydroisoquinolinium] (H I = cis-trans isomer, H 2 = cis-cis isomer),

I. 2,2 [(3-methylpentane-l ,5-diyl)bis [oxy(3-oxopropane-1,3diyl)] ]bis [ 1-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2-methyl1,2,3,4-tetrahydroisoquinolinitim] (II = cis-trans isomer, 12 = cis-cis isomer),

1-216 Atropine

2016 o o

Detecdon After cooling, spray with dilute potassium iodobismuthate solution R. Results T h e principal spot in the chrom atogram obtained with

’sV CHs

the test solution is similar in position, colour and size to the principal spot in the chrom atogram obtained with the reference solution.

J. methyl benzenesulfonate,

D . Place about 3 m g in a porcelain crudble and add 0.2 m L of fuming nitric acid R. Evaporate to dryness on a water-bath. Dissolve the residue in 0.5 m L o f a 30 g/L solution of potassium hydroxide R in methanol R; a violet colour develops.

K. 2 ,2 '-[hexane-1j5-diylbis[oxy(3-oxopropane-l,3-diyl)]] bis [ 1-(3 j4-dimethoxybenzyi)-6j 7-dimethoxy-2-methyl1,2,3,4-tetrahydroisoquinolinium].

E. Optical rotation (see Tests).

PhEur

TESTS O p tic a l ro ta tio n (2.2.7) —0.70° to + 0.05° (measured in a 2 dm tube). Dissolve 1.25 g in ethanol (96 per cent) R and dilute to 25.0 m L with the same solvent.

★ ★ ★ ★ *****

Atropine (Ph Eter monograph 2056)

R e la te d su b s ta n c e s Liquid chromatography (2.2.29).

Test solution Dissolve 24 m g o f the substance to be examined in mobile phase A and dilute to 100.0 m L with mobile phase A. Reference solution (a) D ilute 1.0 m L of the test solution to

and enanttomer

100.0 m L with mobile phase A. Dilute 1.0 m L o f this solution to 10.0 m L with mobile phase A

Reference solution (b) Dissolve 5 mg o f atropine impurity B CRS in the test solution and dilute to 20.0 m L C j7H23N03

289.4

51-55-8

A ctio n a n d use Anticholinergic. PhEur.

D E F IN IT IO N (1 i?,3i?,53)-8-Methyl-8-azabicyclo[3.2. l]oct-3-yl (2RS)-3hydroxy-2-phenylpropanoate. C o n te n t 99.0 per cent to 101.0 p er cent (dried substance). CHARACTERS A p p e a ra n c e W hite or almost white, crystalline pow der or colourless crystals. S o lu b ility Very slightly soluble in water, freely soluble in ethanol (96 per cent) and in methylene chloride. ID E N T IF IC A T IO N

First identification: A, B, E. Second identification: A , C, D, E A. M elting point (2.2.14): 115 °C to 119 °C. B. Infrared absorption spectrophotom etry (2.2.24). Comparison atropine CRS. C. Thin-layer chrom atography (2.2.27). Test solution Dissolve 10 m g o f the substance to be examined in methanol R and dilute to 10 m L w ith die same solvent Reference solution Dissolve 10 m g o f atropine CRS in methanol R and dilute to 10 m L with the same solvent Plate TLC silica gel plate R. Mobile phase concentrated ammonia R, water R, acetone R (3:7:90 VIVIV). Application 10 jiL. Development Over half of the plate. Drying A t 100-105 °C for 15 min.

with the test solution. D ilute 5.0 m L o f this solution to 25.0 m L with mobile phase A.

Reference solution (c) Dissolve the contents o f a vial of atropine for peak identification CRS (containing impurities A, D , E, F, G and H ) in 1.0 m L o f mobile phase A

Reference solution (d) Dissolve 5 mg o f tropic acid R (impurity C) in mobile phase A and dilute to 10.0 m L with mobile phase A. Dilute 1.0 m L o f the solution to 100.0 m L with mobile phase A. D ilute 1.0 m L o f this solution to 10.0 m L with mobile phase A.

Column: — size. I = 0.10 m , 0 = 4.6 mm; — stationary phase: octadecylsUyl silica gel for chromatography R (3 nm).

Mobile phase: — mobile phase A: dissolve 3.5 g o f sodium dodecyl sulfate R in 606 m L o f a 7.0 g/L solution o f potassium dihydrogen phosphate R previously adjusted to p H 3.3 with a 5.8 g/L solution o f phosphoric add R, and mix with 320 m L o f acetomtrüe R1; — mobile phase B: acetomtrüe Rl; Time (min) 0 -2

Mobile phase A (per cent V7V) 95

Mobile phase B (per cent V/V) 5

2-20

95->70

5 -> 30

Flow rate 1 mL/min. Detection Spectrophotom eter at 210 nm. Injection 10 (iL. Identification of impurities U se the chrom atogram supplied with atropine for peak identification CRS and the chrom atogram obtained with reference solution (c) to identify the peaks due to impurities A, D , E , F , G and H ; use the chrom atogram obtained w ith reference solution (b) to identify the peak due to impurity B; use the chromatogram

Atropine 1-217

2016

obtained with reference solution (d) to identify the peak due to im purity C. Relative retention W ith reference to atropine (retention time = about 11 min): impurity C = about 0 .2; impurity E = about 0.67; impurity D = about 0.73; im purity F = about 0.8; impurity B = about 0.89; impurity H = about 0.93; impurity G = about 1.1; im purity A = about 1.7. System suitability: reference solution (b): — resolution: m in im u m 2.5 between the peaks due to im purity B and atropine.

CO2H

and enantiomer

C. (2i?S)-3-hydroxy-2-phenylpropanoic a d d (tropic add),

and epimer at C*

Limits: — correction factors: for the calculation of content, multiply the peak areas o f the following impurities by the corresponding correction facto r impurity A = 0.6; im purity C = 0.6; — impurities E, H: for each impurity, not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.3 per cent); — impurities A, B, C, D, F} &. for each impurity, n o t m ore than twice the area o f the principal peak in the chrom atogram obtained with reference solution (a) (0.2 per cent); — unspecified impurities: for each impurity, n ot m ore than the area o f the principal peak in the chromatogram obtained w ith reference solution (a) (0.10 per cent); — total: no t more than 5 times th e area o f the principal peak in the chrom atogram obtained w ith reference solution (a) (0.5 per cent); — disregard limit. 0.5 times the area o f the principal peak in the chrom atogram obtained w ith reference solution (a) (0.05 per cent).

Loss on drying (2.2.32) M axim um 0.2 per cent, determined on 1.000 g by drying in an oven at 105 °C for 2 h. A SSA Y Dissolve 0.250 g in 40 mT. of anhydrous acetic acid R, heating if necessary, and allow to cool. T itrate with 0.1 M perchloric acid, d e te rm in in g th e end-point potentiometrically (2.2.20).

D . (1 i?,3S,5i?,6RS)-6-hydroxy-8-methyl-8azabicyclo [3.2.1] oct-3-yl (23)-3-hydroxy-2-phenylpropanoate (6-hydroxyhyoscyamine),

and epimer at C*

E. (1 S,3i?,5Sj6i?5)-6-hydroxy-8-methjd-8azabicydo[3.2.l]oct-3-yl (25)-3-hydroxy-2-phenylpropanoate (7-hydroxyhyoscyamine),

X °* w v H OH

* H

F. (li?,2i?,4S,55,7j)-9-methyl-3-oxa-9azatricyclo[3.3.1 .O ^ n o n ^ - y i (26)-3-hydroxy-2phenylpropanoate (hyoscine),

1 m L o f 0.1 M perchloric acid is equivalent to 28.94 m g of C 17H 23N O 3.

and enantiomer

STORAGE Protected from light. IM P U R IT IE S

Specified impurities A, B, C , D , E, F , G, H .

G. (lÄ,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl (2RS)-2hydroxy-3-phenylpropanoate (littorine), H . unknown structure. PhEur

A. (12?,3r,55)-8-methyl-8-azabicydo[3.2.1]oct-3-yl 2-phenylpropenoate (apoatropine),

and enantiomer

B. (li?,3r,55)-8-azabicyclo[3.2.1] oct-3-yl (2RS)-3-hydroxy-2phenylpropanoate (noratropine),

1-218 Atropine Sulfate

2016

Atropine Sulfate

★* * ★ ★ ★ ★

Atropine Sulphate

*****

(Ph

Reference solution (a) Dilute 1.0 m L o f the test solution to

monograph 0068)

E ut.

Test solution Dissolve 24 m g of the substance to be examined in mobile phase A and dilute to 100.0 m L with mobile phase A. 100.0 m L with mobile phase A. Dilute 1.0 m L o f this solution to 10.0 m L with mobile phase A.

, h2s o 4 , h2o

Reference solution (b) Dissolve 5 m g o f atropine impurity B CRS in the test solution and dilute to 20 m L with the test solution. Dilute 5 m L o f this solution to 25 mT. with mobile phase A.

2

C34H 48N 201oS,H20

and enantiomer

695

5908-99-6

Action and use Anticholinergic.

Preparations Atropine Eye Drops

Reference solution (c) Dissolve the contents o f a vial o f atropine for peak identification CRS (containing impurities A, D , E, F, G and H ) in 1 m L of mobile phase A.

Reference solution (d) Dissolve 5 m g o f tropic add R (impurity C) in mobile phase A and dilute to 10 m L with mobile phase A. Dilute 1 m L o f the solution to 100 mT. with mobile phase A. Dilute 1 m L o f this solution to 10 m L with mobile phase A.

Atropine Eye O intm ent

Column:

Atropine Injection

— size. I = 0.10 m , 0 = 4.6 mm; — stationary phase: octadecylsifyl silica gel for chromatography R (3 nm).

Atropine Tablets PhEtr____________________

D E F IN IT IO N Bis [(li£j3r 35S)-8-methyl-8-azabicydo [3.2. 1] oct-3-yl (2RS)-3hydroxy-2-phenylpropanoate] sulfate monohydrate.

Content 99.0

per cent to 101.0 per cent (anhydrous substance).

Mobile phase: — mobile phase A: dissolve 3.5 g o f sodium dodecyl sulfate R in 606 m L o f a 7.0 g/L solution of potassium dihydrogen phosphate R previously adjusted to p H 3.3 with 0.05 M phosphoric add, and mix with 320 m L of acetomtrüe Rl; — mobile phase B: acetomtrüe R l ;

CHARACTERS Appearance W hite or alm ost white, crystalline pow der or colourless crystals.

Solubility Very soluble in water, freely soluble in ethanol (96 per cent).

Tune (min) 0 -2

Mobile phase A (per cent V7V) 95

Mobile phase B (per cent V/V) 5

2-20

95->70

5-> 30

ID E N T IF IC A T IO N

First identification A , B, E. Second identification C, D, E, F. A. Optical rotation (see Tests). B. Infrared absorption spectrophotom etry (2.2.24). Comparison atropine sulfate CRS. C. Dissolve about 50 m g in 5 m L o f water R and add 5 m L of picric, acid solution R. T he p redpitate, washed with water R and dried at 100-105 °C for 2 h, m d ts (2.2.14) at 174 °C to 179 °C. D. T o about 1 m g add 0.2 m L o f fuming nitric add R and evaporate to dryness in a water-bath. Dissolve the residue in 2 m L o f acetone R and add 0.1 m L o f a 30 g/L solution of potassium hydroxide R in methanol R!TA violet colour devdops. E. It gives the reactions of sulfates (2.3.1). F. It gives the reaction o f alkaloids (2.3.1). TESTS

pH (2.2.3) 4.5 to 6.2. Dissolve 0.6 g in carbon dioxide-free water R and dilute to 30 m L with the same solvent.

Optical rotation (2.2.7) -0 .5 0 ° to + 0.05° (measured in a 2 d m tube). Dissolve 2.50 g in water R and dilute to 25.0 m L with the same solvent.

Related substances Liquid chrom atography (2.2.29).

Flow rate 1 mL/m in. Detection Spectrophotom eter a t 210 nm. Injection 10 |xL. Identification of impurities U se the chromatogram supplied with atropine for peak identification CRS and the chrom atogram obtained with reference solution (c) to identify the peaks due to impurities A, D , E, F, G and H. U se the chrom atogram obtained with reference solution (b) to identify the peak due to impurity B, and use the chrom atogram obtained with reference solution (d) to identify the peak due to impurity C.

Relative retention W ith reference to atropine (retention 11 min): impurity C = about 0.2; about 0.67; impurity D = about 0.73; about 0.8; impurity B = about 0.89; about 0.93; impurity G = about 1.1; about 1.7. System suitability: reference solution (b): — resolution: minimnm 2.5 between the peaks due to im purity B and atropine. tim e = about im purity E = im purity F = impurity H = impurity A =

Limits: — correction factors: for the calculation o f content, multiply the peak areas o f the following impurities by the corresponding correction facto r impurity A = 0.6; impurity C = 0.6; — impurities E, H: for each impurity, not m ore than 3 times the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.3 per cent);

Attapulgite 1-219

2016 — impurities A, B, C, D, F, G: for each impurity, n o t more than twice the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent); — unspecified impurities: for each impurity, n o t m ore than the area of the principal peak in the chrom atogram obtained with reference solution (a) (0.10 per cent); — total: n o t m ore than 5 times the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent); — disregard limit. 0.5 times the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent).

and epimer at C*

E. (1 S,3R,5S,6i?3)-6-hydroxy-8-methyl-8azabicydo[3.2.1]oct-3-yl (25)-3-hydroxy-2-phenylpropanoate (7-hydroxyhyoscy amine),

W a te r (2.5.12) 2.0 per cent to 4.0 per cent, determined on 0.500 g. S u lfa te d a s h (2. 4.14) M aximum 0.1 per cent, determined on 1.0 g. A SSAY Dissolve 0.500 g in 30 m L o f anhydrous acetic add R, w anning if necessary. Cool the solution. T itrate w ith 0.1 M perchloric add, determining the end-point potentiometrically

F . (li?,2jR,4S,5S,7i)-9-methyl-3-oxa-9azatricyclo [3.3.1 .O ^non-T -yl (25)-3-hydroxy-2phenylpropanoate (hyoscine)j

(2.2.20).

and enantiomer

1 m L of 0.1 M perchloric acid is equivalent to 67.68 m g Of C 34H 48N 2O 10S. STO RA G E Protected from light.

G . (li?,3r,55)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl (2RS)-2~ hydroxy-3-phenylpropanoate (littorine).

IM P U R IT IE S

Spedfied impurities: A, B, C , D , E, F , G , H .

H . unknow n structure. PhEur

Attapulgite A. (IR ,3r,55)-8-methyl-8-azabicydo[3.2.l]oct-3-yl 2-phenylpropenoate (apoatropine)j

and enantiomer

A c tio n a n d u se Excipient. D E F IN IT IO N Attapulgite is a purified native hydrated magnesium alum inium silicate essentially consisting of the clay mineral palygorskite. C H A R A C T E R IS T IC S A light, cream or buffi, very fine powder, free or almost free from gritty particles.

B. (li?,3r,55)-8-azabicyclo[3.2.1]oct-3-yl (2RS)-3-hydroxy-2phenylpropanoate (noratropine),

CO2H

and enantiomer

C . (2RS)-3-hydroxy-2-phenylpropanoic a d d (tropic ad d )j H

« ^ /•4 — -o -\

, v

\

h

OH

r

H and epimer at C*

I

D . (IR,3S,5R,6RS)- 6-hydroxy-8-methyl-8 a2abicydo[ 3 .2 . l]oct-3-yl (25)-3-hydroxy-2-phenylpropanoate ( 6-hydiroxyhyoscyamine),

ID E N T IF IC A T IO N A. Ignite 0.5 g with 2 g of anhydrous sodium carbonate for 20 minutes, cool and extract with 25 m L o f boiling water. Cool, filter, wash the residue with water and add the washings to the filtrate. Reserve the residue for test B. Cautiously addify the combined filtrate and washings with hydrochloric add, evaporate to dryness, moisten the residue with 0.2 m l. of hydrochloric add, add 10 m l. of water and stir. A white, gelatinous predpitate is produced. B. W ash the residue reserved in test A with water and dissolve in 10 m L o f 2m hydrochloric add T o 2 m L o f the solution add a 10% whr solution o f ammonium thiocyanate. A n intense red colour is produced. C. T o 2 m L of the solution obtained in test B add 1 m L of

strong sodium hydroxide solution and filter. T o the filtrate add 3 m L of ammonium chloride solution. A gelatinous white predpitate is produced. D . T o 2 m L o f the solution obtained in test B add ammonium chloride and an excess o f 13.5m ammonia and

1-220 Attapulgite

filter. T o the filtrate add 0.15 m L o f magneson reagent and an excess o f 5 m sodium hydroxide. A blue precipitate is produced.

TESTS Acidity or alkalinity p H o f a 5% wN suspension in carbon dioxide-free water, after

2016 Loss on ignition W hen ignited at 600°, loses 15.0 to 27.0% o f its weight. Use 1 g.

shaking for 5 minutes, 7.0 to 9.5, Appendix V L.

Adsorptive capacity M oisture adsorption, 5 to 14% when determined by the following method. D ry in air and pow der a sufficient quantity o f th e substance being examined and pass through a sieve with a nominal m esh aperture of 150 jam. Spread 0.5 g as a thin layer on a previously weighed piece o f aluminium foil (60 m m x 50 mm ) o f nominal gauge 17.5 nm and transfer to a desiccator containing a dish o f sodium chloride crystals partially immersed in saturated brine at 25°. After 4 hours, remove from the desiccator and weigh immediately. D ry in an oven at 110° for 4 hours, allow to cool in a desiccator and weigh. T he moisture adsorption is the gain in weight o f the substance being examined expressed as a percentage o f its oven-dried w eight

Arsenic T o 0.13 g add 5 m L o f water, 2 m L o f sulfuric add and 10 m L of sulfur dioxide solution and evaporate on a water bath until the sulfur dioxide solution is removed and the volume reduced to about 2 mL. Transfer the solution to the generator flask with the aid o f 5 m L o f water. T he resulting solution complies with the limit test for arsenic, Appendix VII (8 ppm ).

Heavy metals A. N o t m ore than 2 0 ppm when determined by the following m ethod. Shake 6 .0 g with 4 0 m L o f 0 .5 m hydrochloric add at 3 7 ° for 3 0 minutes, cool and filter. W ash the residue with water and dilute the combined filtrate and washings to 5 0 m L with water. T o 2 0 m L add 2 g each o f ammonium chloride and ammonium tkiocyanate and dissolve. Shake the solution with 8 0 m L of a mixture o f equal volumes o f ether and isoamyl alcohol and separate, retaining the aqueous layer. Extract with a further 8 0 m L of the mixture. T o the aqueous layer add 2 g o f citric add, neutralise with 1 3 .5 m ammonia and dilute to 25 m L with water. 12 m L o f the resulting solution complies with limit test A for heavy metals, Appendix VII. Use lead standard solution (2 ppm Pb) to prepare the standard. B. N o t more than 10 ppm when determined by the following m ethod. Shake 6.0 g with 40 m L o f 0.5m sodium hydroxide at 37° for 30 minutes, cool and filter. W ash the residue with water and dilute the combined filtrate and washings to 50 mT. with water. Neutralise 20 m L o f the solution with hydrochloric add and dilute to 25 m L with water. 12 m L o f the resulting solution complies with limit test A for heavy metals, A ppendix VH. Use lead standard solution (1 ppm Pb) to prepare the standard.

Acid-soluble m atter Boil 2 g with 1 0 0 m L o f 0 .2 m hydrochloric add under a reflux condenser for 5 minutes, cool and filter. Evaporate 5 0 mT, o f the filtrate to dryness. T he residue, after ignition at about 6 0 0 ° for 3 0 minutes, weighs not m ore than 0 .2 5 g.

W ater-soluble matter Boil 10 g with 100 m L o f water under a reflux condenser for 5 m inutes, cool and filter. Evaporate 50 m L o f the filtrate to diyness. T h e residue, after ignition a t 600° for 30 minutes, weighs n o t m ore than 50 mg.

Loss on drying W hen dried to constant weight at 105°, loses n o t more than 17.0% o f its weight. Use 1 g.

Activated Attapulgite Action and use Antidiarrhoeal. '

DEFINITION Activated Attapulgite is a purified native hydrated magnesium alum inium silicate essentially consisting o f the clay mineral palygorskite that has been carefully heated to increase its adsorptive capacity.

CHARACTERISTICS A light, cream or buff, very fine powder, free or almost free from gritty particles.

IDENTIFICATION A. Ignite 0 .5 g with 2 g o f anhydrous sodium carbonate for 2 0 minutes, cool and extract with 2 5 m L o f boiling water. Cool, filter, wash the residue with water and add the washings to the filtrate. Reserve the residue for test B. Cautiously acidify the combined filtrate and washings with hydrochloric add, evaporate to dryness, m oisten the residue with 0 .2 m L o f hydrochloric add, add 10 m L of water and stir. A white, gelatinous precipitate is produced. B. W ash the residue reserved in test A with water and dissolve in 10 m L o f 2 m hydrochloric add. T o 2 m L of the solution add a 10% w/v solution of ammonium thiocyanate. A n intense red colour is produced. C. T o 2 m l, o f the solution obtained in test B add 1 m L of

strong sodium hydroxide solution and filter. T o the filtrate add 3 m L of ammonium chloride sdution. A gelatinous white precipitate is produced. D . T o 2 m L o f the solution obtained in test B add ammonium chloride and an excess o f 1 3 .5 m ammonia and filter. T o the filtrate add 0 .1 5 m L o f magneson reagent and an excess o f 5 m sodium hydroxide. A blue precipitate is produced.

TESTS Acidity or alkalinity p H o f a 5% w/v suspension in carbon dioxide-free water, after shaking for 5 minutes, 7 .0 to 9 .5 , Appendix V L.

Arsenic T o 0 .1 3 g add 5 mT, o f water, 2 m L o f sulfuric add and 10 mT, o f sulfur dioxide solution and evaporate on a w ater bath until the sulfur dioxide solution is removed and the volume reduced to about 2 mT. Transfer the solution to the generator flask with the aid o f 5 m L o f water. T h e resulting solution complies with the limit test for arsenic, Appendix VII (8 ppm).

Heavy m etals A. N o t m ore than 2 0 ppm when determ ined by the following m ethod. Shake 6 .0 g with 4 0 m L of 0.5m hydrochloric add at 3 7 ° for 3 0 minutes, cool and filter. W ash the residue with water and dilute die combined filtrate and washings to 5 0 m L with water. T o 2 0 mT, add 2 g each of ammonium chloride and ammonium thiocyanate and dissolve. Shake the solution with 8 0 m L o f a mixture o f equal volumes o f ether and isoamyl alcohol and separate, retaining the aqueous layer. Extract with a further 8 0 m L o f the mixture. T o the aqueous layer add 2 g o f citric add, neutralise with 13.5m ammonia and dilute to 2 5 m L w ith water. 12 m L o f the resulting solution complies

Azapropazone 1-221

2016 with limit test A for heavy metals, Appendix VII. Use lead standard solution (2 ppm Pb) to prepare die standard. B. N o t m ore than 10 ppm w hen determined by the following method. Shake 6.0 g with 40 m L o f 0.5m sodium hydroxide at 37° for 30 m in u tes, cool and filter. W ash die residue with water and dilute the combined filtrate and washings to 50 m L with water. Neutralise 20 m L o f the solution with hydrochloric acid and dilute to 25 m L with water. 12 m L of the resulting solution complies with limit test A for heavy metals, Appendix VII. U se lead standard solution (1 ppm Pb) to prepare the standard. A d d -s o lu b le m a t te r Boil 2 g with 100 m L o f 0.2m hydrochloric acid under a reflux condenser for 5 m inutes, cool and filter. Evaporate 50 m L of the filtrate to dryness. T h e residue, after ignition at about 600° for 30 minutes, weighs n o t more than 0.25 g. W ate r-so lu b le m a t te r Boil 10 g with 100 m L of water under a reflux condenser for 5 minutes, cool and filter. Evaporate 50 m L of the filtrate to dryness. T h e residue, after ignition at 600° for 30 minutes, weighs not more than 50 mg. A d so rp tiv e c a p a c ity In a stoppered bottle shake 1.0 g, in very fine powder, with 50 m L o f a 0.12% w/v solution of methylene blue for 5 minutes, allow to setde and centrifuge. T h e colour o f the clear supernatant solution is no t more intense than that of a 0.0012% w/v solution of methylene blue. L oss o n d ry in g W hen dried to constant weight at 105°, loses not more than 4.0% of its weight. U se 1 g. L oss o n ig n itio n W hen ignited at 600°, loses n o t more than 9.0% of its weight. U se 1 g.

IDENTIFICATION A. T he infrared absorption spectrum, Appendix II A, is concordant with die reference spectrum of azapropazone

(RS 016). B. T he light absorption, Appendix I I B , in the range 230 to 350 nm of a 0.0008% w/v solution in 0.1 m sodium hydroxide exhibits two maxima, at 255 nm and 325 nm . T he absorbances at 255 nm and 325 nm are about 0.86 and 0.18 respectively.

TESTS Acetic add N o t more than 0.2%, determined by the following m ethod. Dissolve 10 g in 25 m L o f methanol, add 75 m L of water and carry out a potendometric titration, Appendix V H IB , using 0.1 m sodium hydroxide KS as titrant to a p H o f 5.9. Each m L o f 0.1 m sodium hydroxide FS is equivalent to 6.005 mg of acetic ad d , C 2H 4O 2.

Related substances Carry out the following operations in subdued light using low-actinic glassware without delay. Carry out the method for liquid chromatography, Appendix HI D , using the following solutions in a mixture of 1 volume of phosphate buffer pH 4.0 and 3 volumes of methanol. ( 1) 0 . 10% w/v of die substance being examined. (2) 0.00010% w/v o f azapropazone impurity A BPCRS. (3) 0.00025% w/v of azapropazone impurity B BPCRS. (4) 0.00025% w/v of azapropazone impurity C BPCRS. (5) 0.0001% w/v o f the substance being examined. ( 6) 0.00005% w/v of the substance being examined. (7) 0.1% w/v o f azapropazone impurity standard BPCRS. CHROMATOGRAPHIC CONDITIONS

(a) U se a stainless steel column (30 cm x 3.9 mm) packed with octadecylsUyl silica gel for chromatography (10 nm) (jiBondapak C18 is suitable). (b) U se isocratic elution and the mobile phase described below.

Azapropazone

(c) U se a flow rate of 2.5 m L per minute. (d) U se ambient column temperature. (e) Use a detection wavelength of 254 nm. (f) Inject 20 |iL o f each solution. M OBILE PHASE

Ci6H 2oN40 2,2H20

336.4

13539-59-8 (anhydrous)

A ction a n d u se Cyclo-oxygenase inhibitor; analgesic; anti-inflammatory. P re p a r a tio n s Azapropazone Capsules Azapropazone Tablets D E F IN IT IO N Azapropazone is 5-dimethylamino-9-methyl-2propylpyrazolo [ 1,2-a] [1,2,4] benzotriazine-1,3 (2 H) -dione dihydrate. It contains not less than 99.0% and n o t m ore than 101.0% of C 16H 2oN402 , calculated with’reference to the anhydrous substance. C H A R A C T E R IS T IC S A white to pale yellow, crystalline powder. Very slightly soluble in water, soluble in ethanol (96%)-, it dissolves in solutions of alkali hydroxides..

1 volume o f glacial acetic acid, 36 volumes of methanol and 63 volumes o f a 0.068% w/v solution of sodium butanesulfonate in water. SYSTEM SUITABILITY

Inject solution (7) and continue the chromatography for 5 times the retention time of the principal peak. T h e test is n o t valid unless the chromatogram obtained with solution (7) closely resembles the reference chromatogram supplied with the azapropazone impurity standard. I f necessary adjust the proportion o f methanol in the mobile phase to give the required retention times. LIMITS

In the chromatogram obtained w ith solution (1): the area of any peak corresponding to azapropazone im purity A is n o t greater than the area o f the corresponding peak in the chromatogram obtained with solution (2) (0 . 1%); the area of any peak corresponding to azapropazone im purity B is n o t greater than the area of the corresponding

1-222 Azathioprine

2016

peak in the chromatogram obtained with solution (3) (0.25% ); Me

the area of any peak corresponding to azapropazone im purity C is not greater th an the area of the corresponding peak in the chromatogram obtained with solution (4) (0.25% ); the area o f any other secondary peak is not greater than the area of the peak in the chromatogram obtained with solution (5) (0.1%).

H eav y m e ta ls Dissolve 3.125 g in 20 m L o f water, boil for 10 minutes, dilute to 50 m L and filter (solution A). 12 m L o f solution A complies with limit test A for heavy metals, Appendix VII. Use 10 m L o f lead standard solution (1 ppm Pb) to prepare the standard (16 ppm). C h lo rid e A m ixture o f 10 m L o f solution A and 5 m L o f water complies with the limit test for chlorides, Appendix VII (80 ppm ).

to 11.5% w/w, Appendix IX C. Use 0.25 g.

S u lfa te d a s h N o t m ore than 0.1%, Appendix IX A

N'N

o

3. 5-hydroxy-9-methyl-2-propylpyrazolo [1,2-

HOOC

M e Y

Y

N

' N

Pr"

0

4. a-(3-dimethylamino-7-methyl-1,2-dihydro-1,2,4benzotriazin-2-ylcaibonyl)valeric a d d (impurity C).

.★ ** ★ ★ ★ ★ *****

Azathioprine (Ph. Eur. monograph 0369) n -—y

,n o 2

t~ C

rV

a] [1,2,4]benzotriazine-1,3 (2//)-dione (impurity B),

Calculate the content of impurities A, B and C using the respective reference solutions and the content of any unnam ed impurities using solution (5). T he total nominal content of impurities is not greater than 0.5%. Disregard any peak with an area less than the area o f the principal peak in the chromatogram obtained with solution (6) (0.05%).

V

S

cx> C9H7N70 2S

277.3

446-86-6

A ctio n a n d u se Immunosuppressant.

A SSA Y Carry out M ethod I for non-aqueous titration, A ppendix V III A, using 0.25 g and determining the end point potentiometrically. E ach m L o f 0 .1 m perchloric acid VS is equivalent to 30.04 mg o f C 16H 2oN40 2. IM P U R IT IE S

P re p a ra tio n s Azathioprine Tablets Azathioprine Oral Suspension PhEur__________________________

D E F IN IT IO N 6- [( l-M ethyl-4-nitro- lH-imidazol-5-yl)sulfenyl] -7H-purine. Me

N.

C o n te n t 98.5 per cent to 101.0 per cent (dried substance).

'N

N

X

NM62

1. 3-dimethylamino-7-methyi-l ,2,4-benzotriazine

(impurity A ), Bun Me

H

L

A NMe2 N 2. 3-dimethylamino- l,2-dihydro-7-methyl-2-valeryl-1,2,4benzotriazine,

CHARACTERS A p p e a ra n c e Pale-yellow powder. S o lubility Practically insoluble in water and in ethanol (96 p er cent). It is soluble in dilute solutions of alkali hydroxides and sparingly soluble in dilute mineral adds. ID E N T IF IC A T IO N Infrared absorption spectrophotom etry (2.2.24).

Comparison azathioprine CRS. TESTS R e la te d su b s ta n c e s Liquid chromatography (2.2.29).

Solution A 2.76 g/L solution o f sodium dihydrogen phosphate monohydrate R adjusted to p H 2.5 with phosphoric acid R. Test solution Dissolve 10 m g o f the substance to be examined in 35 m L o f a 0.8 g/L solution o f sodium hydroxide R and dilute to 100.0 m L with solution A.

Azathioprine 1-223

2016 Reference solution (a) Dissolve 5 mg o f azathioprine impurity A CRS and 5 m g o f mercaptopurine R (impurity B) in 8.75 m L o f a 0.8 g/L solution of sodium hydroxide R and dilute to 25.0 m L with solution A. T o 1.0 m L o f this solution, add 35 m L of a 0.8 g/L solution of sodium hydroxide R and dilute to 100.0 m L with solution A.

Reference solution (b) Dissolve 2.5 mg o f azathioprine impurity G CRS and 2.5 mg o f the substance to be examined in 8.8 m L of a 0.8 g/L solution o f sodium hydroxide R and

S u lfa te d a s h (2.4.14) M aximum 0.1 p er cent, determined on 1.0 g. ASSAY Dissolve 0.250 g in 25 m L of dimethylformamide R. Titrate with 0.1 M tetrabutylammonium hydroxide, d eterm in in g the end-point potentiometrically (2.2.20).

1 m L of 0.1 M tetrabutylammonium hydroxide is equivalent to 27.73 mg of C 9H 7N 7O 2S.

dilute to 25.0 m L with solution A. T o 1.0 m L o f this solution, add 17.5 m L of a 0.8 g/L solution of sodium hydroxide R and dilute to 50.0 m L with solution A.

STORAGE Protected from light.

Reference solution (c) Dilute 1.0 m L of die test solution to

Specified impurities A, B Other detectable impurities (the following substances would, if

100.0 m L with solution A. Dilute 1.0 m L o f this solution to 10.0 m L with solution A.

Column: — sizer. I = 0.15 m , 0 = 4.6 mm; — stationary phase: phenylsüyl silica gel for chromatography R (5 nm); — temperature: 30 °C.

Mobile phase'. — mobile phase A: methanol R, solution A (5:95 VIV)', — mobile phase B: solution A, methanol R (40:60 VIV)',

IM P U R IT IE S

present at a sufficient level, be detected by one or other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general m onograph Substances for pharmaceutical use (2034). It is therefore n o t necessary to identify these impurities for demonstration of compliance. See also 5.10.

Control of impurities in substances for pharmaceutical use): C, D, E, F, G. N- V

Time (min) 0 -5

Mobile phase A (per cent V7V) 100

Mobile phase B (per cent V/V) 0

5 - 15

100->0

0 -> 100

1 5 -20

0

100

ino 2

B. 7H-purine-6-thiol (mercaptopurine),

O, Q, R, S} U, V.

J. R1 = R2 = CO -C 2H 5: 9,lip-epoxy-16p-m ethyl-3,20dioxo-9 P-pregna-1,4-diene-17,21 -diyl dipropanoate, R. R1 = R2 = H : 9,11 p-epoxy-17,2 1-dihydroxy-l 6 P-methyl9P-pregna-l,4-diene-3,20-dione,

A. R1 = R3 = H , R2 = Cl, R4 = C O -Q H g: 9-chlorolip,17-dihydroxy-16p-m ethyl-3,20-dioxopregna-l,4-dien-21yl propanoate (beclometasone 21-propionate),

U . R1 = C O -C 2H 5, R2 = H: 9,HP-epoxy-21-hydroxy-16Pmethyl-3,20-dioxo-9p-pregna-l,4-dien-17-yl propanoate, V. R 1 = H , R2 = CO-C 2H 5: 9,llp-epoxy-17-hydroxy-16Pmethyl-3,20-dioxo-9P-pregna-l,4-dien-2 1-yl propanoate, o

B. R1 = H , R2 = Cl, R3 = CO-C2H 5, R4 = C O -C H 3: 2 l-(acetyloxy)-9-chloro-l 1P-hydroxy-16P-methyi-3,20dioxopregna-1,4-dien-17-yl propanoate (beclometasone 21-acetate 17-propionate), C. R1 = H , R2 = Cl, R3 = CO-C2H 5, R4 = C O -C H 2C H 2-C H 3: 9-chloro-l 1P-hydroxy-16P-methyl-3,20-dioxo-l 7(propanoyloxy)-pregna-1,4-dien-21-yl butanoate (beclometasone 21-butyrate 17-propionate), D . R1 = H , R2 = Br, R3 = R4 = C O -Q H g: 9 -b ro m o -llp hydroxy-16 P-methyl-3^0-dioxopregna-1,4-diene-17,21 -diyl dipropanoate,

L. 9-chloro-l 1P-hydroxy-16P-methyl-3,20-dioxopregn-4-ene17,21-diyl dipropanoate, o

F . R1 = Br, R2 = Cl, R3 = R4 = C O -Q H g: 6a-bromo-9chloro-11P-hydroxy-16P-methyl-3,20-dioxopregna-l,4-diene17,21-diyl dipropanoate,

M . 9-chloro-l 1P-hydroxy-16P-methyl-3,20-dioxopregna-4,6diene-17,21-diyl dipropanoate, o

E. R1 = Cl, R2 = CO -C 2H 5: 6a,9-dichloro-lip-hydroxy16p-methyl-3,20-dioxopregna-l ,4-diene-l 7,21 -diyl dipropanoate, H . R1 = R2 = H : 9-chloro-l ip ,2 1-dihydroxy-l 6 P-methyl3,20-dioxopregna-l,4-dien-17-yl propanoate (beclometasone 17-propionate), O

N . 2-bromo-9-chloro-l 1P-hydroxy-16P-methyl-3,20dioxopregna-1,4-diene-17,21-diyl dipropanoate, O 0 ^ y ° 0 'w-CH,

ChT H T

\-C H 3

H

I. 16P-methyi-3,20-diQxopregna-l,4,9(l l)-triene-17,21-diyl dipropanoate,

O. R1 = R2 = Cl: 9,liP-dichloro-16P-methyl-3,20dioxopregna-l,4-diene-17,21-diyl dipropanoate, Q. R1 = R2 = H : 16P-methyl-3,20-dioxopregna-l,4-diene17,21-diyl dipropanoate, S. R1 = O -C O -C 2H 5, R2 = Cl: 9-chloro-l6p-m ethyl-3,20dioxopregna-l,4-diene-l ip,17,21-triyl tripropanoate (beclometasone tripropionate). __________________________________________________________ PhEur

1-242 Beclometasone Dipropionate Monohydrate

Beclometasone Dipropionate Monohydrate

***** *****

(Ph. Eur. monograph 1709)

h° X ' s

Reference solution (c) Dissolve 5 m g o f beclometasone dipropionate for peak identification CRS (containing impurities B, C and L) in 3 m L o f mobile phase B and dilute to 5 m L w ith mobile phase A. U se 1 m L of this solution to dissolve die contents o f a vial o f beclometasone dipropionate

impurities F and N CRS. Reference solution (d) Dissolve 50.0 m g o f anhydrous beclometasone dipropionate CRS in 28 m L of mobile phase B

O

o

2016

fV lJ H i

and dilute to 50.0 m L with mobile phase A. D ilute 1.0 m L o f this solution to 50.0 m L w ith the solvent mixture.

5 v ° ' ^ CHi ■

ChX H 7 ^ W

Column: h

— sizer. I = 0.25 m , 0 = 4.6 mm ; — stationary phase: spherical difunctional bonded end-capped

octadecylsüyl silica gelfor chromatography R (5 Jim); C ^ H s tC IO ^ O

539.1

5534-09-8

Action and use Glucocorticoid.

Preparations Beclometasone Aqueous Nasal Spray

— temperature: 50 °C.

Mobile phase: — mobile phase A: 2.72 g/L solution o f potassium dihydrogen phosphate R adjusted to p H 2.35 w ith phosphoric add R', — mobile phase B: tetrahydrofuran R, acetomtrile R, methanol R (5:23:25 VIVIV)\

Beclometasone Inhalation Powder Beclometasone Inhalation Powder, pre-dispensed PhEur___________________________________________________________

D E F IN IT IO N 9-C hloro-l 1{5-hydroxy-16 P-methyi-3,20-dioxopregna-1,4diene-17,21-diyl dipropanoate monohydrate.

Content 97.0 per cent to 102.0 p er cent (dried substance).

CHARACTERS Appearance W hite or alm ost w hite powder.

Solubility Practically insoluble in water, freely soluble in acetone, sparingly soluble in ethanol (96 per cent). ID E N T IF IC A T IO N A. Infrared absorption spectrophotom etry (2.2.24).

Comparison beclometasone dipropionate monohydrate CRS. B. T reat 25 m g by th e oxygen-flask m ethod (2.5.10). U se a mixture of 1 m L o f 1 M sodium hydroxide and 20 m L o f water R to absorb the com bustion products. T h e solution gives reaction (a) o f chlorides (2.3.1). C. Loss on drying (see Tests). TESTS

Specific optical rotation (2.2.7) + 108 to + 115 (dried substance). Dissolve 0.100 g in ethanol (96 per cent) R and dilute to 10.0 m L with the sam e solvent.

Related substances Liquid chrom atography (2.2.29). Solvent mixture M obile phase A, mobile phase B (45:55 VIV). Test solution (a) Dissolve 50.0 m g o f the substance to be examined in 28 m L o f mobile phase B and dilute to 50.0 m L with mobile phase A.

Test solution (b) D ilute 1.0 m L o f test solution (a) to 50.0 m L with the solvent mixture. Reference solution (a) D ilute 5.0 m L o f test solution (b) to 100.0 m L with the solvent mixture.

Reference solution (b) Dissolve 5 m g o f beclometasone dipropionate for system suitability CRS (containing im purity D ) in 3 m L o f mobile phase B and dilute to 5 m L with mobile phase A.

Time (min) 0 -4

Mobile phase A (percent V/V) 40

4 -1 2

40-» 45

12 -5 9

45

Mobile phase B (per cent V/V) 60 60

55 55

Flow rate 1.4 mL/min. Detection Spectrophotom eter at 254 nm . Injection 20 pi o f test solution (a) and reference solutions (a), (b) and (c).

Identification of impurities U se the chrom atogram supplied with beclometasone dipropionate for peak identification CRS and the chrom atogram obtained with reference solution (c) to identify the peaks due to impurities B, C , F and L; use the chrom atogram supplied with beclometasone dipropionate for system suitability CRS and the chrom atogram obtained with reference solution (b) to identify the peak due to impurity D . Relative retention W ith reference to beclometasone dipropionate (retention time = about 25 min): im purity B = about 0.6; im purity D = about 1.1; impurity L = about 1.3; im purity C = about 1.8; im purity F = about 2.2. System suitability: reference solution (b): — peak-to-vaHey ratio: m inim um 1.5, where Hp = height above the baseline o f the peak due to im purity D and Hv — height above the baseline o f the lowest point o f the curve separating this peak from the peak due to beclom etasone dipropionate. Limits: — correction factor, for the calculation o f content, multiply the peak area o f im purity F by 1.3; — impurity B: n o t m ore than 5 times the area o f the principal peak in the chrom atogram obtained with reference solution (a) (0.5 per cent); — impurities C, F, L: for each impurity, not m ore than 1 .5 times the area o f the principal peak in the chrom atogram obtained w ith reference solution (a) (0.15 p er cent); — unspecified impurities: for each impurity, n o t more than the area of the principal peak in the chrom atogram obtained w ith reference solution (a) (0.10 per cent); — total: n o t m ore than 10 times the area of the principal peak in die chrom atogram obtained with reference solution (a) (1.0 per cent);

Beclometasone Dipropionate Monohydrate 1-243

2016 — disregard limit: 0.5 times the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). Loss o n d ry in g (2.2.32) 2.8 per cent to 3.8 per cent, determined on 1.000 g by drying in an oven at 105 °C for 3 h. A SSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modification.

Injection T est solution (b) and reference solution (d).

I. 16P-methyl-3,20-dioxopregna-l,4,9(l l)-trien e-17,21-diyl dipropanoate,

Calculate the percentage content of C 28H 37CIO 7 from the declared content of anhydrous beclometasone dipropionate CRS. IM P U R IT IE S

Specified impurities B, C, F, L Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). I t is therefore not necessary to identify these impurities for demonstration of compliance. See also 5.10.

Control of impurities in substances for pharmaceutical use): A, D, E, H, I, J, M, N, O, Q, R, S, U, V.

J. R I = R2 = C O -C 2H 5: 9,11 P-epoxy-16 P-methyl-3,20 dioxo-9 P-pregna-1,4-diene-17,21 -diyl dipropanoate, R. R I = R2 = H : 9,lip-epoxy-17,21-dihydroxy-16P-m ethyl9 P-pregna-l,4-diene-3,20-dione, U . R I = CO -C 2H 5, R 2 = H: 9,llp-epoxy-21-hydroxy-16Pmethyl-3,20-dioxo-9P~pregna-l,4-dien-l7-yl propanoate, V. R I = H , R 2 = C O -C 2H 5: 9,lip-epoxy-17-hydroxy-16Pmethyl-3,20-dioxo-9P-pregna-l,4-dien-2 1-yl propanoate,

on

°joA .\ -

A. R I = R3 = H , R2 = Cl, R4 = CO-Q-Hg: 9-chloro1 1P, 17 -dihydroxy-16 P-methyl-3 ,2 0-dioxopregna-1,4-dien-2 1yl propanoate (beclometasone 21-propionate), D. R I = H , R2 = Br, R3 = R4 = CO -C 2H 5: 9 -b ro m o -llp hydroxy-16 P-methyl-3,20-dioxopregna-1,4-diene-17,21 -diyl dipropanoate,

L. 9-chloro-liP-hydroxy-16P-methyl-3,20-dioxopregn-4-ene17,21-diyl dipropanoate,

E. R I = R2 = Cl, R3 = R4 = C O -C 2H 5: 6a,9-dichloro11 P-hydroxy-16P-methyI-3,20-dioxopregna-l ,4-diene-l 7,2 1diyl dipropanoate, H . R I = R4 = H , R2 = Cl, R3 = C O -C 2H 5: 9-chloro11 P,21-dihydroxy-16P-methyl-3,20-dioxopregna- 1,4-dien-l 7yl propanoate (beclometasone 17-propionate), -R 2

M . 9-chloro-l 1P-hydroxy-16P-methyl-3,20-dioxopregna-4,6diene-17 ,2 1-diyl dipropanoate,

H R1

B. R I = H , R2 = C O C H 3: 21-(acetyloxy)-9-chloro-liphydroxy-16 P-methyl-3,2 0-dioxopregna-1,4-dien-17-yl propanoate (beclometasone 21-acetate 17-propionate), C. R I = H , R2 = C O -C H 2-C H 2-C H 3: 9-chloro-l lp hydroxy-16P-methyl-3,20-dioxo-l 7-(propanoyloxy)-pregnal,4-dien-21-yl butanoate (beclometasone 21-butyrate 17-propionate), F. R I = Br, R2 = CO-C 2H 5: 6a-brom o-9-chloro-liphydroxy-16 P-methyl-3,20-dioxopregna- 1,4-diene-17,21 -diyl dipropanoate,

N . R I = Br, R2 = O H , R3 = Cl: 2-brom o-9-chloro-l 1Phydroxy-16P-methyl-3,20-dioxopregna-l,4-diene-l 7,2 1-diyl dipropanoate, O. R I = H , R2 = R3 = Cl: 9,11 P-dichloro-16P-methyl3,20-dioxopregna-l,4-diene-17,21-diyl dipropanoate,

1-244 Beeswax

2016

Q. R l = R2 = R3 = H : 16P-methyl-3,20-dioxopregna-l,4diene-17,21-diyl dipropanoate, S. R l = H , R2 = 0 - C 0 - C 2H 5, R3 = Cl: 9-chloro-16pmethyl-3,20-dioxopregna-l ,4 -d ien e-lip ,1 7 321-triyl tripropanoate (bedom etasone tripropionate). __________________________________________________________ PhEur

White Beeswax (Ph. Eur. monograph 0069)

of ethanol (96 per cent) R and xylene R and a few glass beads. H eat until the substance is dissolved. Add 25.0 m L o f 0.5 M alcoholic potassium hydroxide and heat under a reflux condenser for 3 h. Titrate the hot solution immediately with 0.5 M hydrochloric acid, using 1 m L o f phenolphthalein solution R l as indicator (nx mL). Reheat the solution to boiling several times during the course o f the titration. Carry out a blank test (n2 mL).

.* * * ★ ★ ★ ★ ★+ ★

Saponification value =

28.05 (na — m )

Ceresin, paraffins and certain other waxes Action and use Excipient PhEur_______________________

DEFINITION Wax obtained by bleaching yellow beeswax.

CHARACTERS Appearance White or yellowish-white pieces or platesj translucent when thin, with a fine-grained, m att and non-crystalline fracture; when w anned in the hand they become soft and malleable. It has an odour similar to that o f yellow beeswax, though fainter and never rancid. It is tasteless and does not stick to the teeth.

Solubility Practically insoluble in water, partially soluble in hot ethanol (90 p er cent V/V) and completely soluble in fatty and essential oils.

Relative density About 0.960.

TESTS Drop point (2.2.17) 61 °C to 66 °C. M elt the beeswax by heating on a water-bath, pour onto a glass plate and allow to cool to a semi-solid mass. Fill the metal cup by inserting the wider end into the beeswax and repeating the procedure until beeswax extrudes from the narrow opening. Remove the excess with a spatula and insert the therm om eter immediately. Remove the beeswax displaced. Allow to stand at room temperature for at least 12 h before determining the drop point.

Acid value 17.0

to 24.0.

To 2.00 g (m g), in a 250 m L conical flask fitted with a reflux condenser, add 40 m L of xylene R and a few glass beads. H eat until the substance is dissolved. Add 20 m L of ethanol (96 per cent) R and 0.5 m L o f phenolphthalein solution R l and titrate the hot solution with 0.5 M alcoholic potassium hydroxide until a red colour persists for at least 10 s («i mL). Carry out a blank test («2 mL).

Add value = 28-°^ (ni ~ ni) tn Ester value (2.5.2) 70 to 80.

Saponification value 87 to 104. To 2.00 g (m g), in a 250 m L conical flask fitted with a reflux condenser, add 30 m L o f a mixture o f equal volumes

T o 3.0 g, in a 100 m L round-bottom ed flask, add 30 m L of a 40 g/L solution o f potassium hydroxide R in aldehyde-free alcohol R and boil gently under a reflux condenser for 2 h. Remove the condenser and immediately insert a thermom eter. Place the flask in a water-bath at 80 °C and allow to cool, swirling the solution continuously. N o precipitate is formed until 65 °C, although the solution may be slightly opalescent. Beginning at 65 °C, the solution may become cloudy and precipitates may be formed. At 59 °C, the solution is cloudy.

Glycerol and other polyols M axim um 0.5 per cent m/m, calculated as glycerol. T o 0.20 g add 10 m L o f alcoholic potassium hydroxide solution R and heat on a water-bath under a reflux condenser for 30 min. Add 50 m L o f dilute sulfuric add R, cool and filter. Rinse the flask and the filter with dilute sulfuric add R. Combine the filtrate and washings and dilute to 100.0 m L w ith dilute sulfuric add R. Place 1.0 m L of the solution in a test-tube, add 0.5 m L o f a 10.7 g/L solution o f sodium periodate R, mix and allow to stand for 5 min. Add 1.0 m L of decolorisedfuchsin solution R and mix. Any precipitate disappears. Place the tube in a beaker containing water at 40 °C. D uring cooling observe for 10-15 min. Any violetblue colour in the solution is not more intense than that in a standard prepared at the same time and in the same m anner using 1.0 m L of a 10 m g/L solution o f glycerol R in dilute

sulfuric add R. __________________________________________________________ PhEur

Yellow Beeswax (Ph. Eur. monograph 0070)

***** ★ ★ *****

Action and use Excipient. PhEur.

DEFINITION Wax obtained by melting the walls o f the honeycomb made by the honey-bee, Apis meUifera L., with hot water and removing foreign matter.

CHARACTERS Appearance Yellow or light brown pieces or plates with a fine-grained, m att and non-crystalline fracture; when w anned in the hand they become soft and malleable. It has a faint odour, characteristic of honey. It is tasteless and does n o t stick to the teeth.

Benazepril Hydrochloride 1-245

2016

S olubility Practically insoluble in water, partially soluble in h o t ethanol (90 per cent V/V) and completely soluble in fatty and essential oils. R elativ e d e n sity A bout 0.960. TESTS D ro p p o in t (2.2.17) 61 °C to 66 °C. M elt the beeswax by heating on a water-bath, p our onto a glass plate and allow to cool to a semi-solid mass. Fill the metal cup by inserting the wider end into the beeswax and repeating the procedure until beeswax extrudes from the narrow opening. Remove the excess with a spatula and insert the therm om eter immediately. Remove the beeswax displaced. Allow to stand at room tem perature for at least 12 h before determining the drop point. A cid value 17.0 to 22.0.

filter. Rinse the flask and the filter with dilute sulfuric acid R. Combine the filtrate and washings and dilute to 100.0 m L with dilute sulfuric add R. Place 1.0 m l. o f the solution in a test-tube, add 0.5 m L of a 10.7 g/L solution of sodium periodate R, mix and allow to stand for 5 min. Add 1.0 m L of decolorisedfuchsm solution R and mix. Any precipitate disappears. Place the tube in a beaker containing water at 40 °C. D uring cooling observe for 10-15 min. Any violetblue colour in the solution is not more intense than that in a standard prepared at the same time and in the same m anner using 1.0 m L of a 10 mg/L solution o f glycerol R in dilute

sulfuric add R. __________________________________________________________ PhEur

Benazepril Hydrochloride

****% *.

(Ph. Eur. monograph 2388)

*

*

T o 2.00 g (m g), in a 250 m L conical flask fitted with a reflux condenser, add 40 m L of xylene R and a few glass beads. H eat until the substance is dissolved. Add 20 m L o f ethanol (96 per cent) R and 0.5 mL of phenolphxhalein solution R1 and titrate the hot solution with 0.5 M alcoholic potassium hydroxide until a red colour persists for at least 10 s (ni m L). Carry out a blank test («2 mL). A #J

,

28.05 ( n i - n 2)

A cid value = ---------------------

m

E s te r v alu e (2.5.2) 70 to 80.

461.0

86541-74-4

A c tio n a n d u se Angiotensin converting enzyme inhibitor. PhEir__________________________________________________________

S ap o n ific a tio n v alu e 87 to 102. T o 2.00 g (m g), in a 250 m L conical flask fitted with a reflux condenser, add 30 m L of a mixture o f equal volumes o f ethanol (96 per cent) R and xylene R and a few glass beads. H eat until the substance is dissolved. Add 25.0 m L o f 0.5 M alcoholic potassium hydroxide and heat under a reflux condenser for 3 h. T itrate the hot solution immediately with 0.5 M hydrochloric acid, using 1 m L o f phenolphthalein solution R1 as indicator («j mL). Reheat the solution to boiling several times during the course o f the titration. Carry out a blank test («2 mL)S ap o n ificatio n value =

C24H 29CIN 2O 5

m

C e re sin , p a ra ffin s a n d c e rta in o th e r w axes T o 3.0 g, in a 100 m L round-bottom ed flask, add 30 m L of a 40 g/L solution of potassium hydroxide R in aldehyde-free alcohol R and boil gently under a reflux condenser for 2 h. Remove the condenser and immediately insert a therm om eter. Place the flask in a water-bath at 80 °C and allow to cool, swirling the solution continuously. N o precipitate is formed until 65 °C, although the solution may be slightly opalescent. Beginning at 65 °C, the solution may become cloudy and precipitates may be formed. At 59 °C, the solution is cloudy. G lycerol a n d o th e r polyols M aximum 0.5 per cent m/m, calculated as glycerol. T o 0.20 g add 10 m L o f alcoholic potassium hydroxide solution R and heat on a water-bath under a reflux condenser for 30 min. A dd 50 m L o f dilute sulfuric acid R, cool and

D E F IN IT IO N [(3S)-3-[ [( 1S )-1-(Ethoxycarbonyl)-3-phenylpropyl] amino] - 2 oxo-2}3,4,5-tetrahydro- 1/f-l-benzazepin- 1-yl] acetic a d d hydrochloride. C o n te n t 97.5 per cent to 102.0 per cent (dried substance). CHARACTERS A p p e a ra n c e W hite or almost white, crystalline powder, hygroscopic. S o lu b ility Slightly soluble in water, freely soluble in anhydrous ethanol, very slightly soluble in ethyl acetate, practically insoluble in cyclohexane. It shows polymorphism (5.9). ID E N T IF IC A T IO N Carry out either tests A, B, D or tests B, C, D. A. Specific optical rotation (2.2.7): —141 to -1 3 6 (dried substance). Dissolve 1.000 g in anhydrous ethanol R and dilute to 50.0 m L with the same solvent. B. Infrared absorption spectrophotometry (2.2.24).

Comparison benazepril hydrochloride CRS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in methanol R, evaporate to dryness and record new spectra using the residues. C. Enantiomeric purity (see Tests). D . It gives reaction (a) o f chlorides (2.3.1).

1-246 Benazepril Hydrochloride

TESTS

Related substances Liquid chromatography (2.2.29).

Test solution (a) Dissolve 50.0 mg o f the substance to be examined in the mobile phase and dilute to 50.0 m L with the mobile phase. Test solution (b) Dilute 10.0 m L o f test solution (a) to 100.0 m L with the mobile phase.

Reference solution (a) Dissolve 50.0 mg o f benazepril hydrochloride CRS in the mobile phase and dilute to 50.0 m L with the mobile phase. Dilute 10.0 m L of this solution to 100.0 m L with the mobile phase.

Reference solution (b) Dissolve the contents of a vial of benazeprilfor system suitability CRS (containing impurities B, C, D , E, F and G) in 1.0 m L of test solution (a).

2016 — disregard limit. 0.25 times the area of the principal peak in the chromatogram obtained with reference solution (c) (0.05 per cent). E n a n tio m e ric p u rity Liquid chromatography (2.2.29).

Buffer solution pH 60 Dissolve 3.58 g o f disodium hydrogen phosphate R and 9.66 g o f potassium dihydrogen phosphate R in water R and dilute to 1000.0 m L with the same solvent Test solution Dissolve 50.0 mg o f the substance to be examined in the mobile phase and dilute to 50.0 m L with the mobile phase.

Reference solution (a) Dissolve 5.0 m g o f benazepril impurity A CRS in the mobile phase and dilute to 50.0 m L with the mobile phase.

Reference solution (b) Dilute 1.0 m L o f reference solution (a)

Reference solution (c) Dilute 1.0 m L of reference solution (a)

to 100.0 m L with the mobile phase.

to 50.0 m L with the mobile phase.

Reference solution (c) Dilute 1.0 m L of reference solution (a)

Column:

to 10.0 m L with the mobile phase. Dilute 1.0 m L o f this solution to 10.0 m L with the test solution.

— size: I = 0.30 m, 0 = 3.9 mm ; — stationary phase: end-capped octadecylsüyl silica gelfor chromatography R (10 pm).

Mobile phase Add 0.2 m L of glacial acetic acid R to 1000 m L o f a mixture o f 360 volumes o f water R and 640 volumes of methanol R2; add 0.81 g o f tetrabutylammonium bromide R and stir to dissolve.

Flow rate 1.0 mlVmin. Detection Spectrophotometer at 240 nm . Injection 25 pL of test solution (a) and reference solutions (b) and (c).

Run time 3 times the retention time o f benazepril. Relative retention W ith reference to benazepril (retention time = about impurity F = impurity B = impurity G =

6 min): impurity E = about 0.3; about 0.4; impurity C = about 0.5; about 1.8; impurity D = about 2.0; about 2.5.

Identification of impurities Use the chromatogram supplied with benazepril for system suitability CRS and the chrom atogram obtained with reference solution (b) to identify the peaks due to impurities B, C, D , E, F and G. System suitability.: reference solution (b): — resolution: m in im u m 2.5 between the peaks due to benazepril and impurity B and minimum 1.5 between the peaks due to impurities E and F.

Limits: — correction factors: for the calculation o f content, multiply the peak areas of the following impurities by the corresponding correction factor, impurity E = 0.5; im purity F = 0.7; — impurity B: n o tm o re than 2.5 times the area o f the principal peak in the chromatogram obtained with reference solution (c) (0.5 per cent); — impurity C: not m ore than 1.5 times die area of the principal peak in the chromatogram obtained with reference solution (c) (0.3 per cent); — impurities D, E, F, G: for each impurity, not more than the area o f the principal peak in the chromatogram obtained with reference solution (c) (0.2 per cent); — unspecified impurities: for each impurity, not more than 0.5 times the area o f the principal peak in the chrom atogram obtained with reference solution (c) (0.10 per cent); — total: not m ore than 10 times the area of the principal peak in the chromatogram obtained with reference solution (c) (2.0 per cent);

Column: — size: I = 0.10 m , 0 = 4.0 mm; — stationary phase: spherical silica gel AGP for chiral chromatography R (5 pm); — temperature: 30 °C.

Mobile phase methanol R2, buffer solution p H 6.0 (20:80 V/V). Flow rate 0.9 mL/min. Detection Spectrophotom eter at 240 nm . Injection 50 pL of the test solution and reference solutions (b) and (c).

Run time 3.5 times the retention tim e of benazepril. Relative retention W ith reference to benazepril (retention time — about 6 min): impurity A = about 1.9. System suitability: reference solution (c): — peak-to-valley ratio: minimum 2.5, where Hp = height above the baseline o f the peak due to impurity A and Hv = height above the baseline o f the lowest point o f the curve separating this peak from the peak due to benazepril.

Limit. — impurity A: n o t more than the area of the corresponding peak in the chromatogram obtained with reference solution (b) (0.1 per cent). H eav y m e ta ls (2.4.8) M aximum 20 ppm. 1.0 g complies with test C. Prepare the reference solution using 2 mT. o f lead standard solution (10 ppm Pb) R. L oss o n d ry in g (2.2.32) M aximum 1.5 per cent, determined on 1.000 g by drying in vacuo at 105 °C for 3 h. S u lfa te d a s h (2.4.14) M aximum 0.1 per cent, determined on 1.0 g. A SSA Y Liquid chromatography (2.2.29) as described in the test for related substances with the following modification.

Irqection T est solution (b) and reference solution (a). Calculate the percentage content o f C 24H 29CIN 2O 5 from the declared content o f benazepril hydrochloride CRS. STORAGE Protected from light, in an airtight container.

Bendroflumethiazide 1-247

2016

IM P U R IT IE S

Specified impurities A, B, C, D , E 3 F 3 G

Bendroflumethiazide

★

(Ph. Eitr. monograph 0370)

*****

O O * '/

,s H2n

C02h

f3c

A. [(3i?)-3- [ [(If ?)-1-(ethoxycarbonyl)-3-phenylpropyI] amino] 2 -o x o -2 ,3 34 35-tetrahydro- 1H- 1-benzazepin - 1-yl] acetic acid,

c o 2h

★

★

★

O 0 * // s.

‘NH

and enantiomer

A H H 421.4

73-48-3

A c tio n a n d u se Thiazide diuretic. P re p a r a tio n s Bendroflumethiazide Tablets

and enantiomer

Bendroflumethiazide Oral Suspension PhEir.

D E F IN IT IO N (3^5)-3-Benzyl-6-(trifluorom ethyl)-3,4-dihydro-2H-l,2,4benzothiadiazine-7-sulfonamide 1, 1-dioxide.

B. [(3i?S)-3-[[(1 SR)- 1-(ethoxycarbonyl)-3phenylpropyl]am ino]-2-oxo-2,3j4j5-tetrahydro-lii-lbenzazepin-l-yl] acetic acid.

C o n te n t 98.0 p er cent to 102.0 per cent (dried substance).

o

CHARACTERS A p p e a ra n c e W hite o r almost white, crystalline powder.

H H ||

S o lu b ility Practically insoluble in water, freely soluble in acetone, soluble in ethanol (96 per cent). C. R = H: (2S)-2-[[(3 * *★*

Benzalkonium Chloride

Benzaldehyde

(Ph. Eur. monograph 0372) CHO H3C CH3

CrHfiO

106.1

cr

8001-54-5

100-52-7

Action and use Flavour.

DEFINITION Benzaldehyde c o n tains not less than 98.0% w/w and not more than 100.5% w/w of G/H^O.

CHARACTERISTICS A clear, colourless liquid. Slightly soluble in water, miscible with ethanol (96%) and with ether.

TESTS Refractive index 1.544 to 1.546, Appendix V E.

Weight per mL 1.043 to 1.049 g, Appendix V G.

Free add N ot more than 1.0% w/v, calculated as benzoic ad d , CyHfiO^ w hen determ ined by the following method. T o 10 m L add 2 0 m L of ethanol (96%) previously neutralised to phenolphthalein solution R1 and titrate with 0 .1 m sodium hydroxide F 5 using phenolphthalein solution R1 as indicator. E ach m l , of 0 .1 m sodium hydroxide P'S is equivalent to 12.21 m g o f C 7H 60 2.

Chlorinated compounds N o t more than 0.05% w/v, calculated as Cl, when determined by the following method. T o 5 m L add 50 m L of isoamyl alcohol and 3 g o f sodium and boil under a reflux condenser for 1 hour. Cool, add 50 m L o f water and 15 m l, of nitric acid, cool, add 5 m L o f 0.1m silver nitrate FS, shake and titrate the excess silver nitrate with 0 . 1m ammonium thiocyanate VS using ammonium iron(III) sulfate solution R2 as indicator. Repeat the procedure without the substance being examined. T h e difference between die titrations represents the am ount o f silver nitrate required. Each m L o f 0 . 1m silver nitrate F51is equivalent to 3 .5 4 5 mg o f CL

ASSAY Carry out the m ethod for determination of aldehydes, Appendix X K, using 0.5 g. Each m L o f 0.5m potassium hydroxide in ethanol (60%) FiS is equivalent to 53.06 mg o f CrHfiO.

STORAGE Benzaldehyde should be kept in a well-filled container, protected from light and stored at a tem perature not exceeding 15°.

Action and use Antiseptic. PhEir____________

DEFINITION M ixture of alkylbenzyldrmethylaxnmonium chlorides, the alkyl groups mainly having chain lengths o f C 12, C J4 and

c16. Content 95.0 per cent to 104.0 p er cent of alkylbenzyldimethylammonium chlorides (anhydrous substance) calculated using the average relative molecular m ass (see Tests).

CHARACTERS Appearance W hite or yellowish-white powder or gelatinous, yellowishwhite fragments, hygroscopic. On heating it forms a d ear m olten mass.

Solubility Very soluble in water and in ethanol (96 p er cent). A n aqueous solution froths copiously when shaken.

IDENTIFICATION First identification B, E Second identification A, C, D, E A . Ultraviolet and visible absorption spectrophotometry (2.2.25). Test solution Dissolve 80 m g in water R and dilute to 100.0 m L with the same solvent.

Spectral range 220-350 nm . Absorption maxima At 257 nm, 263 nm and 269 nm. Shoulder A t about 250 nm . B. Examine the chromatograms obtained in the test for average rdative molecular mass and ratio o f alkyl components.

Results T he prindpal peaks in the chromatogram obtained w ith the test solution are similar in retention time to the p rin d p al peaks in the chromatogram obtained with the reference solution. C . T o 2 m L of solution S (see Tests) add 0.1 m L o f glacial acetic acid R and, dropwise, 1 m L o f sodium tetraphenylborate solution R. A white predpitate is formed. Filter. Dissolve the p redpitate in a mixture o f 1 m L o f acetone R and 5 m L o f ethanol (96 per cent) R, heating to not m ore than 70 °C. A dd water R dropwise to the warm solution until a slight opalescence forms. H eat gently until the solution is clear and allow to cool. W hite crystals separate. Filter, wash with 3 quantities, each of 10 m T, of water R and dry in vacuo over diphosphorus pentoxide R o r anhydrous silica gel R a t a tem perature not exceeding 50 °C. The crystals melt (2.2.14) at 127 °C to 133 °C. D . T o 5 m L o f dilute sodium hydroxide solution R add 0.1 m L o f bromophenol blue solution R1 and 5 m L o f methylene chloride R and shake. T he methylene chloride layer is

1-254 Benzalkonium Chloride

colourless. Add 0.1 m L of solution S and shake. T he methylene chloride layer becomes blue.

2016

Calculate the percentage of each homologue, using the following expression:

E. T o 2 m L o f solution S add 1 m L o f dilute nitric acid R. A white precipitate is formed which dissolves on the addition of 5 m L of ethanol (96 per cent) R. T h e solution gives reaction (a) of chlorides (2.3.1). TESTS S o lu tio n S Dissolve 1.0 g in carbon dioxide-free water R and dilute to 100 m L with the same solvent. A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y 6 (2.2.2, MethodII). A cid ity o r alk alin ity T o 50 m L of solution S add 0.1 m L o f bromocresolpurple solution R. N ot more than 0.1 m L of 0.1 M hydrochloric add or 0.1 M sodium hydroxide is required to change the colour of the indicator. A v erag e re la tiv e m o le c u la r m a s s a n d ra tio o f alkyl co m p o n e n ts L iquid chromatography (2.2.29).

Test solution Dissolve 0.400 g o f the substance to be examined in water R and dilute to 100.0 m L with the same solvent. Reference solution Dissolve the contents o f a vial of benzalkonium chloridefor system suitability CRS in 5.0 m L o f water R. Column: — sizer. I = 0.25 m , 0 = 4.6 mm; — stationary phase: end-capped nitrUe silica gelfor chromatography R (5 pm). Mobile phase Mix 45 volumes o f acetonitrUe R and 55 volumes of a 13.6 g/L solution o f sodium acetate R previously adjusted to p H 5.0 with glacial acetic add R. Flow rate 2.0 mL/min. Detection Spectrophotom eter at 254 nm. Injection 10 (iL. Identification of homologues Use the chromatogram supplied with benzalkonium chloride for system suitability CRS and the chromatogram obtained with die reference solution to identify the peaks due to Q jj, C 14 and C i6.

Relative retention W ith reference to C 12 homologue (retention time = about 6 min): C 14 homologue = about 1.3; C 16 homologue = about 1.7. System suitability: reference solution: — resolution: minim um 1.5 between the peaks due to the C i 2 and C 14 homologues. Calculate the average relative molecular mass o f the sample by summing the products for each homologue, using the following expression:

-(i)

100(I) C

= product of the relative molecular mass o f the given homologue and the area of the corresponding peak in the chromatogram obtained with the test solution; = sum o f the C values for all homologues quantified.

D Limits: — C1 2 homologue: minimum 40 per cent; — C14 homologue: minimum 20 p er cent; — sum of C 1 2 and C14 homologues: minim um 70 per cent. C 1 2 and C14 homologues Im p u ritie s A , B a n d C Liquid chromatography (2.2.29). Prepare the solutions

immediately before use. Test solution Dissolve 0.50 g o f the substance to be examined in methanol R1 and dilute to 10.0 m L with the same solvent. Reference solution (a) Dissolve 25.0 mg of benzyl alcohol CRS (impurity A) in methanol R1 and dilute to 100.0 m L with the same solvent.

Reference solution (b) Dissolve 75.0 m g of benzaldehyde CRS (impurity B) in methanol R1 and dilute to 100.0 m L with the same solvent. Dilute 1.0 m L o f this solution to 10.0 m L with

methanol R l. Reference solution (c) Dilute 1.0 m L o f reference solution (a) to 10.0 m L with methanol R l. Column: — size. I = 0.15 m, 0 = 4.6 mm; — stationary phase, end-capped octadecylsUyl silica gel for chromatography R (5 jam); — temperature: 30 °C. Mobile phase. — mobile phase A: dissolve 1.09 g o f sodium hexanesulfimate R and 6.9 g of sodium dihydrogen phosphate monohydrate R in water R; adjust to p H 3.5 with phosphoric add R and dilute to 1000.0 m L with the same solvent; — mobile phase B: m ethanol R l ; Time (min) 0-10

Mobile phase A (per cent V/V) 80

Nobile phase B (per cent V/V) 20

10-14

80 —> 50

2 0 -* 5 0

14-35

50

50

35 -3 6

50 —> 20

5 0 -» 8 0

3 6-55

20

80

Flow rate 1.0 mL/min. Detection Spectrophotom eter at 210 nm for impurities A and C, and at 257 n m for impurity B.

A B

W

= area o f the peak due to the given homologue in the chrom atogram obtained with the test solution; = sum of the areas o f the peaks due to all homologues in the chromatogram obtained with the test solution; = relative molecular mass for the given homologue: 340, 368 and 396 for the C J2, C 14 and C 16 homologues, respectively.

Injection 20 |iL. Relative retention W ith reference to impurity A (retention time = about 10 min): impurity B = about 1.3; impurity C = about 2.4. System suitability A t 210 nm: — signal-to-noise ratio: minimum 10 for the principal peak in the chromatogram obtained w ith reference solution (c);

Benzalkonium Chloride 1-255

2016 — symmetry factor, minimum 0.6 for the peak due to im purity A in the chromatogram obtained with reference solution (a).

Limits:.

OH

A. benzyl alcohol,

— correction factor, for the calculation of content, multiply the peak area of impurity C by 1.3; — impurity A: not more than die area of the corresponding peak in the chromatogram obtained with reference solution (a) (0.5 per cent); — impurity B: n o t more than the area of the corresponding peak in the chromatogram obtained with reference solution (b) (0.15 per cent); — impurity C: n o t more than 0.1 times the area o f the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent).

CHO

B. benzaldehyde,

C. (chloromethyl)benzene.

Amines and amine salts

PhEur

Dissolve 5.0 g with heating in 20 m L of a mixture of 3 volumes o f 1 M hydrochloric acid and 97 volumes of methanol R and add 100 m L o f 2-propanol R. Pass a stream of nitrogen R slowly through the solution. T itrate with up to 12.0 m L of 0.1 M tetrabutylammomum hydroodde and record the potentiometric titration curve (2.2.20). If the curve shows 2 points o f inflexion, the volume o f titrant added between the 2 points is not greater than 5.0 mL. If the curve shows no point o f inflexion, the substance to be examined does not comply with the te s t If the curve shows 1 point o f inflexion, repeat the test b u t add 3.0 m L of a 25.0 g/L solution of dimethyldecylamine R in 2-propanol R before the titration. If the titration curve after addition o f 12.0 m L o f the titrant shows only 1 point of inflexion, the substance to be examined does n o t comply with the test. W a te r (2.5.12) M axim um 10 per cent, determined on 0.300 g. S u lfa te d a sh ( 2.4.14) M axim um 0.1 per cent, determined on 1.0 g. A SSA Y Dissolve 2.00 g in water R and dilute to 100.0 m L with the same solvent. T ransfer 25.0 m L o f the solution to a separating funnel, add 25 m L of methylene chloride R, 10 m L of 0.1 M sodium hydroxide and 10.0 m L of a freshly prepared 50 g/L solution o f potassium iodide R. Shake well, allow to separate and discard the methylene chloride layer. Shake the aqueous layer with 3 quantities, each o f 10 m L, o f methylene chloride R and discard the methylene chloride layers. T o the aqueous layer add 40 m L o f hydrochloric acid R, allow to cool and titrate with 0.05 M potassium iodate until the deep-brown colour is almost discharged. Add 5 m L o f methylene chloride R and continue the titration, shaking vigorously, until the m ethylene chloride layer no longer changes colour. Carry out a blank titration on a mixture of 10.0 m L o f the freshly prepared 50 g/L solution o f potassium iodide R, 20 m L of water R and 40 m L o f hydrochloric acid R. 1 m L of 0.05 M potassium iodate is equivalent to

10

Benzalkonium Chloride Solution (Ph. Eur. monograph 0371)

***** *.

* *

A ctio n a n d u se Antiseptic. PhEur__________________________________________________________

D E F IN IT IO N Aqueous solution of a mixture of alkylbenzyldimethylammonium chlorides, the alkyl groups mainly having chain lengths of C 121 C 14 and C 16. C o n te n t 475 g/L to 525 g/L o f alkylbenzyldimethylammonium chlorides, calculated using the average relative molecular mass (see Tests). T h e solution may contain ethanol (96 per cent). CHARACTERS A p p e a ra n c e Clear, colourless or slightly yellowish liquid. S olu b ility Miscible with water and with ethanol (96 per cent). It froths copiously when shaken. ID E N T IF IC A T IO N

First identification B, E Second identification A , C, D, E A. Ultraviolet and visible absorption spectrophotometry

(2.2.25). Test solution D ilute 0.3 m L to 100.0 mL with water R. Spectral range 220-350 nm. Absorption maxima A t 257 nm, 263 nm and 269 nm. Shoulder A t about 250 nm . B. Examine the chromatograms obtained in the test for average relative molecular mass and ratio o f alkyl components.

Results T h e principal peaks in the chromatogram obtained m g of benzalkonium chloride where x is the average relative molecular mass o f the sample. STORA GE In an airtight container. IM P U R IT IE S

Specified impurities A, B, C.

with the test solution are similar in retention time to the principal peaks in the chromatogram obtained with the reference solution. C. T o 0.05 m L add 2 m L o f water R, 0.1 m L of glacial acetic acid R and, dropwise, 1 m L of sodium tetraphenylborate solution R. A white precipitate is formed. Filter. Dissolve the precipitate in a mixture o f 1 m L of acetone R and 5 m L of ethanol (96 per cent) R, heating to not m ore than 70 °C.

1-256 Benzalkonium Chloride

Add water R dropwise to the warm solution until a slight opalescence forms. H eat gently until the solution is clear and allow to cool. W hite crystals separate. Filter, wash with 3 quantities, each o f 10 mL, of water R and dry in vacuo over diphosphorus pentoxide R or anhydrous silica gel R a t a tem perature not exceeding 50 °C. T h e crystals melt (2.2.14) at 127 °C to 133 °C. D . T o 5 m L o f dilute sodium hydroxide solution R add 0.1 m L of bromophenol blue solution R1 and 5 m l. of methylene chloride R and shake. T he methylene chloride layer is colourless. A dd 0.05 m L o f the solution to be examined and shake. T he methylene chloride layer becomes blue. E. T o 0.05 m L add 1 m L o f dilute nitric add R. A white precipitate is form ed which dissolves on the addition of 5 m L o f ethanol (96 per cent) R. T h e solution gives reaction (a) of chlorides (2.3.1). TESTS S o lu tio n S Dilute 2.0 g to 100 m L with carbon dioxide-free water R. A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and not m ore intensely coloured than reference solution Y6 (2.2.2, Method IT). A cid ity o r alk a lin ity T o 50 m L o f solution S add 0.1 m L of bromocresol purple solution R. N o t m ore than 0.1 m L o f 0.1 M hydrochloric acid or 0.1 M sodium hydroxide is required to change the colour of the indicator. A verage re la tiv e m o le c u la r m a ss a n d ra tio o f alkyl c o m p o n e n ts Liquid chromatography (2.2.29).

Test solution D eterm ine the density (2.2.5) of the solution to be examined. D ilute a quantity of the solution to be examined equivalent to about 0.400 g o f benzalkonium chloride to 100.0 m L with water R Reference solution Dissolve the contents of a vial of benzalkonium chloride for system suitability CRS in 5.0 m l. of water R. Column: — size: I = 0.25 m , 0 = 4.6 mm; — stationary phase: end-capped nitrUe silica gel for chromatography R (5 |im). Mobile phase M ix 45 volumes o f acetomtrUe R and 55 volumes of a 13.6 g/L solution of sodium acetate R previously adjusted to p H 5.0 with glacial acetic acid R. Flow rate 2.0 mlVmin. Detection Spectrophotom eter at 254 nm . Injection 10 pL. Identification of homologues U se the chromatogram supplied with benzalkonium chloride for system suitability CRS and the chrom atogram obtained with the reference solution to identify the peaks due to homologues C 12s Q 4 and C 16.

Relative retention W ith reference to C 12 homologue (retention tim e = about 6 min): C 14 homologue = about 1.3; C i6 homologue = about 1.7.

2016

"(s ) A

= area o f the peak due to the given homologue in the chromatogram obtained with the test solution; — sum o f the areas of th e peaks due to all homologues in the chrom atogram obtained with the test solution; = relative molecular mass for the given homologue: 340, 368 and 396 for the C 12, C j4 and C i6 homologues, respectively.

B

W

Calculate the percentage o f each homologue, using the following expression:

100(§) C

= product o f the relative molecular mass o f the given homologue and the area o f the corresponding peak in the chromatogram obtained with the test solution; = sum o f the C values for all homologues quantified.

D Limits:

— C;2 homologue: minimum 40 per cent; — C14 homologue: m inim u m 20 per cent; — sum of C ] 2 and C14 homologues: minimum 70 per cent. Im p u ritie s A , B a n d C Liquid chromatography (2.2.29). Prepare the solutions

immediately before use. Test solution Determ ine the density (2.2.5) o f the solution to be examined. Dilute a quantity o f the solution to be examined equivalent to 2.5 g o f benzalkonium chloride to 50.0 m L with methanol R l.

Reference solution (a) Dissolve 25.0 mg o f benzyl alcohol CRS (impurity A) in methanol R l and dilute to 100.0 m L with the same solvent.

Reference solution (b) Dissolve 75.0 mg of benzaldehyde CRS (impurity B) in methanol R l and dilute to 100.0 m l. with the same solvent. D ilute 1.0 m L of this solution to 10.0 m L with

methanol R l. Reference solution (c) Dilute 1.0 m L of reference solution (a) to 10.0 m L with methanol R l. Column: — sizer. I = 0.15 m , 0 = 4.6 m m ; — stationary phase: end-capped octadecylsHyl silica gelfor chromatography R (5 pm); — temperature: 30 °C. Mobile phaser. — mobile phase A: dissolve 1.09 g o f sodium hexanesulfonate R and 6.9 g o f sodium dihydrogen phosphate monohydrate R in water R; adjust to p H 3.5 w ith phosphoric add R and dilute to 1000.0 m L with the same solvent; — mobile phase B: methanol Rl;

System suitability: reference solution: — resolution: minim um 1.5 between the peaks due to the C i2 and C 14 homologues.

Time Mobile phase A Mobile phase B (min)____________ (percent V/V)________ (percent V/V) 20 80 0 -10

Calculate the average relative molecular mass o f the sample by summing the products for each homologue, using the following expression:

10- 14

80-» 50

20-» 50

14-35

50

50

3 5 -3 6

50-» 20

50-»80

3 6 -5 5

20

80

Benzathine Benzylpenicillin 1-257

2016 Flow rate 1.0 mL/min. Detection Spectrophotom eter at 210 nm for impurities A and

L A B E L L IN G T he label states the content of ethanol (96 per cent), if any.

C, and at 257 nm for impurity B.

IM P U R IT IE S

Irqection 20 |iL. Relative retention W ith reference to impurity A (retention

Specified impurities: A, B, C.

time = about 10 min): impurity B = about 1.3; impurity C = about 2.4. System suitability A t 210 nm: — signal-to-noise ratio: m inim u m 10 for the principal peak in the chrom atogram obtained with reference solution (c); — symmetry factor, minimum 0.6 for the peak due to im purity A in the chromatogram obtained with reference solution (a).

Limits: — correction factor, for the calculation of contentj multiply the peak area of impurity C by 1.3; — impurity A: n o t m ore than the area of the corresponding peak in the chromatogram obtained with reference solution (a) (0.5 per cent); — impurity B: n o t more than the area of the corresponding peak in the chromatogram obtained with reference solution (b) (0.15 per cent); — impurity C: n o t more than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent). A m in es a n d a m in e salts M ix 10.0 gj while heating, with 20 m L of a mixture of 3 volumes of 1 M hydrochloric acid and 97 volumes o f methanol R and add 100 m L o f 2-propanol R. Pass a stream o f nitrogen R slowly through the solution. T itrate with up to 12.0 m L o f 0.1 M tetrabutylammonium hydroxide and record the potentiometric titration curve (2.2.20). If the curve shows 2 points o f inflexio n , the volume o f titrant added between the 2 points is not greater than 5.0 mL. If the curve shows no point of inflexio n , the solution to be exam ined does not comply with the te s t If the curve shows 1 point o f inflexion, repeat the test b u t add 3.0 m L of a 25.0 g/L solution o f dimethyldecylamme R in 2-propanol R before the titration. If the titration curve after the addition of 12.0 m L o f the titrant shows only 1 point of inflexion, the solution to be examined does n o t comply with the test. S u lfated a s h (2.4.14) M aximum 0.1 p er cent, determined on 1.0 g. ASSAY D eterm in e the density (2.2.5) of the solution to be examined. D ilute 4.00 g to 100.0 m L with water R. Transfer 25.0 m L of the solution to a separating funnel, add 25 m L of methylene chloride R, 10 m L of 0.1 M sodium hydroxide and 10.0 m L o f a freshly prepared 50 g/L solution of potassium iodide R. Shake well, allow to separate and discard the methylene chloride layer. Shake the aqueous layer with 3 quantities, each of 10 mT, of methylene chloride R and discard the methylene chloride layers. T o the aqueous layer add 40 m L o f hydrochloric acid R, allow to cool and titrate with 0.05 M potassium iodate until the deep-brown colour is almost discharged. Add 5 m L of methylene chloride R and continue the titration, shaking vigorously, until the methylene chloride layer no longer changes colour. Carry out a blank titration on a m ixture of 10.0 m L o f the freshly prepared 50 g/L solution o f potassium iodide R, 20 m L o f water R and 40 m L o f hydrochloric acid R.

1 m L of 0.05 M potassium iodate is equivalent to x 10 m g o f benzalkonium chloride where x is the average relative molecular mass o f the sample.

A. benzyl alcohol,

CHO

B. benzaldehyde,

C. (chloromethyl)benzene. PhEur

★ ★ ★ ★ ★ ★ *★ *

Benzathine Benzylpenicillin (Ph. Eur. monograph 0373) O

> C°2H

h iT x ™ 3 N- - r r s^ ch3 H H

C48H56N6O8S2

909

1538-09-6

A c tio n a n d use Penicillin antibacterial. PhEtr___________________

D E F IN IT IO N ATjN'-Dibenzylethane-1,2-diamine compound (1:2) with (2S,5i?,6i?)-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4thia- 1-azabicyclo [3.2.0]heptane-2-carboxylic add. Substance produced by the growth of certain strains of

PenicUUum notatum or related organisms, or obtained by any other means. C o n te n t — benzathine benzylpenicillin: 96.0 per cent to 102.0 per cent (anhydrous substance); — NyN'-dibenzylethylenedicunine (benzathine C 16H 20N 2; 240.3): 24.0 per cent to 27.0 p er cent (anhydrous substance). It contains a variable quantity o f water. Dispersing or suspending agents may be added. CHARACTERS A p p e a ra n c e W hite or almost white powder. S o lu b ility Very slightly soluble in water, freely soluble in dimethylformamide and in formamide, slightly soluble in ethanol (96 per cent).

1-258 Benzathine Benzylpenicillin

2016

ID E N T IF IC A T IO N

Reference solution (b) Dilute 1.0 m L o f reference solution (a)

First identification A. Second identification B, C, D.

to 100.0 m L with mobile phase A.

A. Infrared absorption spectrophotometry (2.2.24).

— size: I = 0.25 m , 0 = 4.0 mm; — stationary phase: end-capped octadecylsüyl silica gelfor chromatography R (5 ^m); — temperature: 40 °C.

Comparison benzathine benzylpenicillin CRS. B. Thin-layer chromatography (2.2.27). Test solution Dissolve 25 mg o f the substance to be examined in 5 m L o f methanol R. Reference solution Dissolve 25 m g o f benzathine benzylpenicillin CRS in 5 m L o f methanol R. Hate TLC sOamsed silica gel plate R. Mobile phase Mix 30 volumes of acetone R and 70 volumes of a 154 g/L solution o f ammonium acetate R adjusted to p H 7.0 with ammonia R. Application 1 [lL. Development Over a p ath o f 15 cm. Drying In air. Detection Expose to iodine vapour until the spots appear and

Column:

Mobile phase: — mobile phase A: mix 10 volumes o f a 34 g/L solution of potassium dihydrogen phosphate R adjusted to p H 3.5 with phosphoric add R, 30 volumes o f methanol R and 60 volumes o f water R; — mobile phase B: mix 10 volumes of a 34 g/L solution of potassium dihydrogen phosphate R adjusted to p H 3.5 with phosphoric acid R, 30 volumes o f water R and 60 volumes of methanol R;

examine in daylight

System suitability: reference solution:

Time (min) 0 - 10

Mobile phase A (per cent V/V) 75

Mobile phase B (per cent V/V) 25

10 - 20

7 5 ->0

25-» 100

the chromatogram shows 2 clearly separated spots.

20 - 55

0

100

Results T he 2 principal spots in the chromatogram obtained with the test solution are similar in position, colour and size to the 2 principal spots in the chromatogram obtained with the reference solution.

5 5 -7 0

75

25

—

C. Place about 2 mg in a test-tube about 150 mm long and 15 m m in diameter. M oisten with 0.05 m L of water R and add 2 m L o f sulfuric acid-formaldehyde reagent R. M ix the contents of the tube by swirling; the solution is practically colourless. Place the test-tube on a water-bath for 1 min; a reddish-brown colour develops. D . T o 0.1 g add 2 m l- of 1 M sodium hydroxide and shake for 2 m in. Shake the mixture with 2 quantities, each of 3 mT, of ether R. Evaporate the combined ether layers to dryness and dissolve the residue in 1 m L o f ethanol (50 per cent V/V) R. Add 5 m L o f picric acid solution R, heat at 90 °C for 5 min and allow to cool slowly. Separate the crystals and recrystallise from ethanol (25 per cent V/V) R containing 10 g/L of picric acid R. T he crystals melt (2.2.14) at about 214 °C. TESTS

Acidity or alkalinity T o 0.50 g add 100 m L of carbon dioxide-free water R and shake for 5 min. Filter through a sintered-glass filter (2.1.2). T o 20 m L o f the filtrate add 0.1 m L o f bromothymol blue solution R l. T he solution is green or yellow. N ot m ore than 0.2 m L of 0.02 M sodium hydroxide is required to change the colour of the indicator to blue. R e la te d su b stan ces Liquid chromatography (2.2.29). Prepare the solutions

immediately before use, using somcation (for about 2 min) to dissolve the samples. Avoid any overheating during the sample preparation. Test solution Dissolve 70.0 m g o f the substance to be examined in 25 m L o f methanol R and dilute to 50.0 m L with a solution containing 6.8 g/L o f potassium dihydrogen phosphate R and 1.02 g/L of disodium hydrogen phosphate R. Reference solution (a) Dissolve 70.0 m g o f benzathine benzylpemcillin CRS in 25 m L of methanol R and dilute to 50.0 m L with a solution containing 6.8 g/L o f potassium dihydrogen phosphate R and 1.02 g/L of disodium hydrogen phosphate R.

Flow rate 1 mlVmin. Detection Spectrophotom eter at 220 nm. Injection 20 |iL. System suitability: reference solution (a): — relative retention with reference to benzylpenicillin: benzathine = 0.3 to 0.4; impurity C = about 2.4; if necessary, adjust the concentration o f methanol in the mobile phase.

Limits: — impurity C: n o t more than twice the sum o f the areas of the 2 principal peaks in the chrom atogram obtained with reference solution (b) (2 per cent); — any other impurity, for each impurity, n o t more than the sum o f the areas of the 2 principal peaks in the chromatogram obtained with reference solution (b) (1 per cent); — disregard limit. 0.05 times the sum of the areas of the 2 principal peaks in the chromatogram obtained with reference solution (b) (0.05 p er cent). W a te r (2.5.12) 5.0 per cent to 8.0 per cent, determined on 0.300 g. B a c te ria l en d o to x in s (2.6.14, Method E) Less than 0.13 IU /m L, if intended for use in the manufacture of parenteral preparations w ithout a further appropriate procedure for the removal o f bacterial endotoxins. Suspend 20 mg in 20 m L o f a solution o f 0.1 M sodium hydroxide diluted 1 to 100, shake thoroughly and centrifuge. Examine the supernatant. A SSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modifications.

Mobile phase phosphate buffer solution pH 3.5 R, methanol R, water R (10:35:55 VIV/V). Injection T est solution and reference solution (a). Calculate the percentage contents o f benzathine and b enzathine benzylpenicillin. Calculate the percentage content

Benzatropine Mesilate 1-259

2016

I.C O 2H C , ch3 . . . { ^ / S h3

of benzathine benzylpenicillin by multiplying the percentage content o f benzylpenicillin by 1.36.

hn - ^

STORAGE In an airtight container. If the substance is sterile, store in a sterile, airtight, tam per-proof container. IM P U R IT IE S

Specified impurities C Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034) . It is therefore not necessary to identify these impurities for demonstration o f compliance. See also 5. 10. Control of impurities in substances for pharmaceutical use): A , B,

H and epimer at C*

F . (2RS,4S)-2 -[[(phenylacetyl)amino] methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penilloic a d d s of benzylpenicillin). PhEur

Benzatropine Mesilate Me

D, E} F. ^ ^ nh2

,CH3S 03 H

OCHPh2 A. monobenzylethylenediamine,

C 2 iH 2 5 N 0 ,C H 4 0 3S

403.5

132-17-2

Action and use

0 - C C H

Anticholinergic.

Preparations Benzatropine Injection B. phenylacetic ad d ,

Benzatropine Tablets D E F IN IT IO N Benzatropine Mesilate is (li?,3i?,55)-3-benzhydryloxytropane methanesulfonate. It contains not less than 98.0% and not m ore than 100.5% o f C 2iH 25N 0 ,C H 403S, calculated with reference to the dried substance. C H A R A C T E R IS T IC S A white, crystalline powder. It melts at about 144°. Very soluble in water, fredy soluble in ethanol (96%) \ practically insoluble in ether.

C. benzylpenicilloic ad d s benzathide,

,c o 2h ch 3 ch 3 H02C I H

n

D . (35,7i?,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7atetrahydroimidazo [5,1 -b]thiazole-3,7~dicarboxylic a d d (penillic a d d o f benzylpenicillin), I.COzH h n - A .c h 3

o

co 2h

E. (45)-2-[caiboxy[(phenylacetyl)amino]methyl]-5,5dimethylthiazoüdine-4-carboxylic a d d (penicilloic ad d s of benzylpenicillin),

ID E N T IF IC A T IO N A. D ry the substance at 105° for 3 hours. The infrared absorption spectrum, Appendix II A, is concordant with the reference spectrum of benzatropine mesilate (RS 026). B. T h e light absorption, Appendix II B, in the range 230 to 350 nm of a 0.1% w/v solution in 2m hydrochloric acid exhibits two maxima, at 253 and 258 nm . T he absorbance at 253 nm is about 0.96 and at 258 n m is about 1.1. C. Dissolve 10 m g in 2 m L of water, pour into 5 m L o f hot picric arid solution R1 and allow to c o o l T he melting point of the predpitate, after drying at 105°, is about 185°, Appendix V A. TESTS T ro p in e C arry out the m ethod for thin-layer chromatography, Appendix HI A, using silica gel G as the coating substance and a mixture of 75 volumes of ethanol (96%) and 15 volumes o f 13. 5m ammonia as the mobile phase. Apply separatdy to the plate 10 |iL of each o f two solutions in acetone containing (1) 4.0% w/v of the substance being examined and (2) 0.020% w/v o f tropine. After removal o f the plate, allow it to dry in air and spray with sodium iodobismuthate solution and then with a 0.4% w/v solution of sulfuric acid. Any spot corresponding to tropine in the

1-260 Benzbromarone

2016

chromatogram obtained with solution (1) is not more intense th an the spot in the chrom atogram obtained with solution (2).

Related substances Carry out the m ethod for liquid chromatography, Appendix DDE D , using the following solutions. For solution (1) mix with the aid o f ultrasound 50 m g of the substance being examined with 15 m L o f mobile phase A, dilute to 50 m L with the same solvent and filter. For solution (2) dilute 1 volume of solution (1) to 100 volumes with mobile phase A and further dilute 1 volume o f the resulting solution to 5 volumes with the same solvent. F o r solution (3) mix with the aid o f ultrasound 50. m g o f desmethyl benzatropine hydrochloride BPCRS with 15 m L o f mobile phase A, dilute to 100 m L and dilute 1 volume of the resulting solution to 100 volumes with the same solvent. Solution (4) contains 0.01% wfv each of benzatropine mesUate BPCRS and desmethyl benzatropine hydrochloride BPCRS in mobile phase A.

ASSAY Dissolve 0 .6 g in 2 5 m L of water, add 5 m L o f dilute sodium carbonate solution and extract with four 10 m L quantities of chloroform. Wash the combined extracts with 10 m L o f water, extract the washings with 5 m L of chloroform and add the chloroform to the com bined extracts. Filter and wash the filter with 5 m L o f chloroform. T o the combined filtrate and washings add 2 5 m L o f 1,4-dioxan and titrate with 0 .1 m perchloric add F51using 0 .1 5 m L of a 0.1% w fv solution o f methyl red in methanol as indicator. Each m L of 0 .1 m perchloric add P'S is equivalent to 4 0 .3 5 m g of

C2iH25N0,CH403S.

★ ★ ★ ★ *****

Benzbromarone (Ph. Eur. monograph 1393)

T he chromatographic procedure may be carried out using (a) a stainless steel column (25 cm x 4.6 mm) packed with phenylsQyl silica gdfor chromatography (5 jam) (Zorbax SB-Phenyl 5n is suitable). C an y out a linear gradient elution with a flow rate o f 1 m L p er minute using the following conditions. U se a detection wavelength o f 220 nm.

h3c

Mobile phase A

A mixture o f 5 volumes o f a 1m potassium phosphate buffer prepared as described for mobile phase B, 20 volumes o f acetonitrUe and 75 volumes o f water.

Mobile phase B A mixture o f 35 volumes of water, 6 0 volumes o f acetonitrUe and 5 volumes of a 1m potassium phosphate buffer prepared in the following m anner: dissolve 136.1 g o f potassium dihydrogen orthophosphate in 9 0 0 m L of water, add 5 m L of orthophosphoric acid (85%) and dilute to 1 0 0 0 mL.

Tima

Mobile phase A

M obile phase B

(M inutes)

(% v/v)

(% W v)

Comment

0-20

70->30

30-»70

linear gradient

20-30

30->0

70-+100

linear gradient

30-55

0

100

isocratic

55-65

70

30

isocratic

Inject 20 |iL of solution (4). T he test is not valid unless the resolution factor between the two principal peaks is at least 1. If necessary adjust the concentration o f acetonitrile or adjust the time program of the linear gradient elution. Inject separately 20 |iL o f mobile phase A as a blank and 20 jiL each o f solutions (1), (2) and (3). In the chromatogram obtained with solution (1) the area o f any peak corresponding to desmethyl benzatropine is n o t greater than the area o f the principal peak in the chromatogram obtained with solution (3) (0.5%), the area o f any other secondary peak is not greater that the area o f the principal peak in the chromatogram obtained with solution (2) (0.2%) and the sum o f the areas of any such peaks is not greater than 2.5 times the area o f the principal peak in the chrom atogram obtained with solution (2) (0.5%). In solution (1) disregard any peaks corresponding to the peaks in the chrom atogram obtained with the blank solution.

Loss on drying W hen dried to constant weight at 105°, loses not more than 5.0% o f its weight. U se 1 g.

Sulfated ash N ot more than 0.1%, Appendix IX A.

Ci7Hi2Br20 3

424.1

3562-84-3

Action and use Uricosuric; treatm ent of hyperuricaemia. Ph Eur_____________________________________

D E F IN IT IO N (3j5-Dibromo-4-hydroxyphenyl)(2-ethylbenzofuran-3yl)methanone.

Content 98.0 per cent to 101.0 per cent (dried substance).

CHARACTERS Appearance W hite or almost white, crystalline powder.

Solubility Practically insoluble in water, freely soluble in acetone and in methylene chloride, sparingly soluble in ethanol (96 per cent), mp: about 152 °C.

IDENTIFICATION A. Infrared absorption spectrophotometry (2.2.24).

Comparison benzbromarone CRS. B. By means of a copper wire, previously ignited, introduce a small am ount o f the substance to be examined into the nonluminous part o f a flame. T h e colour o f the flame becomes green.

TESTS Appearance o f solution T h e solution is clear (2.2. i) and n o t more intensely coloured than reference solution Y5 (2.2.2, Method U). Dissolve 1.25 g in dimethylformamide R and dilute to 25 m L with the same solvent.

Acidity or alkalinity Shake 0.5 g with 10 m L o f carbon dioxide-free water R for 1 m in and filter. T o 2.0 m L o f the filtrate add 0.1 m L of methyl red solution R and 0.1 m L o f 0.01 M hydrochloric add. T h e solution is red. Add 0.3 m L o f 0.01 M sodium hydroxide. T h e solution is yellow.

Benzethonium Chloride 1-261

2016 Related substances Liquid chromatography (2.2.29). Test solution Dissolve 0.125 g o f the substance to be examined in 30 m L of methanol R and dilute to 50.0 m L with the mobile phase.

Reference solution (a) Dilute 1.0 m L of the test solution to 100.0 m L with the mobile phase. Dilute 1.0 m L o f this solution to 10.0 m L with the mobile phase.

Reference solution (b) Dissolve 10 m g o f benzarone CRS (impurity C) in the mobile phase and dilute to 20 m L with the mobile phase. T o 5 m L of this solution add 1 m L of the test solution and dilute to 100 m L with the mobile phase.

Column:

L oss o n d ry in g (2.2.32) M aximum 0.5 per cent, determined on 1.000 g by drying in vacuo at 50 °C for 4 h. S u lfa te d a s h (2.4.14) M axim um 0.1 per cent, determined on 1.0 g. ASSAY Dissolve 0.300 g in 60 m L of methanol R. Stir until completely dissolved and add 10 m L of water R. T itrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20).

1 m L o f 0.1 M sodium hydroxide is equivalent to 42.41 mg o f C i 7H i 2Br 203 .

— sizer. I = 0.25 m , 0 = 4.6 mm; — stationary phase: octadecylsQyl silica gel far chromatography R (5 |im).

STORA GE Protected from light.

Mobile phase glacial acetic acid R, acetonitrUe R, water R, methanol R (5:25:300:990 VIVIVIV). Flow rate 1.5 mL/min. Detection Spectrophotom eter at 231 nm . Injection 20 |iL. Run time 2.5 times the retention time of benzbromarone. Relative retention W ith reference to benzbromarone:

Specified impurities A, B. Other detectable impurities (the following substances would, if

impurity A = about 0.6; impurity B = about 2. System suitability: reference solution (b): — resolution: m inim u m 10.0 between the peaks due to impurity C (1st peak) and benzbromarone (2nd peak).

IM P U R IT IE S

present at a sufficient level, be detected by one o r other o f the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore n o t necessary to identify these impurities for demonstration of compliance. See also 5.10.

Control of impurities in substances far pharmaceutical use): C.

Limits: — impurity A: n o t more than 4 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.4 per cent); — impurity B: n o t more than 10 times the area of the principal peak in the chromatogram obtained with reference solution (a) ( 1.0 per cent); — unspecified impurities: for each impurity, n o t more than the area of the principal peak in the chromatogram obtained with reference solution (a) ( 0.10 per cent); — sum of impurities other than A and B: no t more than twice the area of the principal peak in the chromatogram obtained with reference solution (a) ( 0.2 per cent); — disregard limit. 0.2 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.02 per cent). H a lid e s e x p ressed a s ch lo rid e s (2.4.4) M axim um 400 ppm.

A. R l = R2 = H , R3 = Bn (3-bromo-4hydroxyphenyl) (2-ethylbenzofuran-3-yl)methanone, B. R l = R2 = R3 = Br. (6-bromo-2-ethylbenzofuran-3yl) (3,5-dibromo-4-hydroxyphenyl)methanone, C. R l = R2 = R3 = H: (2-ethylbenzofuran-3yl)(4-hydroxyphenyl)methanone (benzarone). ____________________________ :_____________________________ PhEur

Benzethonium Chloride (Ph. Eur. monograph 0974)

*

Shake 1.25 g with a mixture o f 5 m L o f dilute nitric acid R and 15 m L o f water R. Filter. Rinse the filter with water R and dilute the filtrate to 25 m L with the same solvent. Dilute 2.5 m L o f this solution to 15 m L with water R. Ir o n (2.4.9) M axim um 125 ppm. M oisten the residue obtained in the test for sulfated ash with 2 m L o f hydrochloric add R and evaporate to dryness on a water-bath. Add 0.05 m L of hydrochloric add R and 10 m L of water R, heat to boiling and maintain boiling for 1 min. Allow to cool. Rinse the crucible with water R , collect the rinsings and dilute to 25 m L with water R. Dilute 2 m L of this solution to 10 m L with water R. H eav y m e ta ls (2.4.8) M axim um 20 ppm . 0.5 g complies with test C. Prepare the reference solution using 1 m L o f lead standard solution (10 ppm Pb) R.

C 27H 42O N O 2

448.1

121-54-0

A ctio n a n d u se Antiseptic. PhEur__________________________________________________________

D E F IN IT IO N N -B enzyl-N ^-dim ethyl-2- [2- [4-( 1,1,3,3tetramethylbutyl)phenoxy] ethoxy] ethanaminium chloride. C o n te n t 97.0 per cent to 103.0 per cent (dried substance).

1-262 Benzocaine

CHARACTERS A p p e a ra n c e W hite or yellowish-white powder. S o lubility Very soluble in water and in ethanol (96 per cent), freely soluble in methylene chloride. An aqueous solution froths copiously when shaken. ID E N T IF IC A T IO N A. Melting point (2.2.14): 158 °C to 164 °C, after drying at 105 °C for 4 h. B. Thin-layer chromatography (2.2.27).

Test solution Dissolve 25 mg o f the substance to be examined in water R and dilute to 5 m L with the same solvent. Reference solution Dissolve 25 m g of benzethonium chloride CRS in water R and dilute to 5 m L with the same solvent. Plate TLC silica gel F2 5 4 plate R. Mobile phase glacial acetic acid R, water R, methanol R (5:5:100 V/V/V). Application 20 pL. Development Over a path of 12 cm. Drying In a current of warm air. Detection Examine in ultraviolet light at 254 nm. Results T he principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution.

2016 ASSAY Dissolve 2.000 g in water R and dilute to 100.0 m L with the same solvent. Transfer 25.0 m L o f the solution to a separating funnel, add 10 m L of a 4 g/L solution o f sodium hydroxide R, 10.0 m L of a freshly prepared 50 g/L solution of potassium iodide R and 25 m L o f methylene chloride R. Shake vigorously, allow to separate and discard the lower layer. Shake the upper layer with 3 quantities, each of 10 mT, of methylene chloride R and discard the lower layers. T o the upper layer add 40 m L o f hydrochloric add R, allow to cool and titrate with 0.05 M potassium iodate until the deep brown colour is almost discharged. Add 4 m L o f methylene chloride R and continue the titration, shaking vigorously, until the lower layer is no longer brown. C any ou t a blank titration using a mixture of 10.0 m L o f a freshly prepared 50 g/L solution of potassium iodide R, 20 m L o f water R and 40 m L of

hydrochloric acid R. 1 m L of 0.05 Mpotassium iodate is equivalent to 44.81 m g of C 27H 42CINO 2.

STORAGE Protected from light. PhEur

(Ph. Eur. monograph 0011)

C. T o 5 m L of dilute sodium hydroxide solution R add 0.1 m L of bromophenol blue solution R l and 5 m L o f methylene chloride R and shake. T h e lower layer is colourless. Add 0.1 m L o f solution S (see Tests) and shake. A blue colour develops in the lower layer. D. T o 2 m L o f solution S add 1 m L of dilute nitric acid R. A white precipitate is formed which dissolves upon addition of 5 m L o f ethanol (96 per cent) R. T he solution gives reaction (a) o f chlorides (2.3.1). TESTS S o lu tio n S Dissolve 5.0 g in carbon dioxide-free water R and dilute to 50 m L with the same solvent.

Appearance o f solution Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II). Acidity or alkalinity T o 25 m L o f solution S add 0.1 m L of phenolphthalein solution R. T he solution is colourless. Add 0.3 m L o f 0.01 M sodium hydroxide. T h e solution is pink. Add 0.1 m L of methyl red solution R and 0.5 m L o f 0.01 M hydrochloric acid

★ ★ ★ ★ **★**

Benzocaine

'CH, h2n

CçHuNOa

165.2

A c tio n a n d u se Local anaesthetic. PhEur.

DEFINITION Ethyl 4-aminobenzoate.

Content 99.0 per cent to 101.0 per cent (dried substance).

CHARACTERS Appearance W hite or almost white, crystalline powder or colourless crystals.

T he solution is orange-red.

Solubility

V olatile b a se s a n d sa lts o f v o latile b ase s (2.4.1,

Very slightly soluble in water, freely soluble in ethanol (96 per cent).

Method B) M aximum 50 ppm , determined on 0.20 g.

It shows polymorphism (5.9).

Prepare the standard using 0.1 m L of ammonium standard solution (100 ppm N H J R. Replace heavy magnesium oxide by 2.0 m L o f strong sodium hydroxide solution R.

Infrared absorption spectrophotometry (2.2.24).

L oss o n d ry in g (2.2.32) M aximum 5.0 per cent, determined on 1.000 g by drying in an oven at 105 °C for 4 h. S u lfa te d a s h (2.4.14) Maximum 0.1 per cent, determined on 1.0 g.

94-09-7

IDENTIFICATION Comparison benzocaine CRS. If the spectra obtained show differences, dissolve the substance to be examined and the reference substance separately in anhydrous ethanol R, evaporate to dryness and record new spectra using the residues.

TESTS Related substances Liquid chromatography (2.2.29).

Benzocaine 1-263

2016 Solvent mixture acetonitrile R l, waterfor chromatography R (50:50 V/V). Test solution Dissolve 25.0 mg of the substance to be examined in 5 m L of acetonitrile R l and dilute to 50.0 m L with the solvent mixture.

Reference solution (a) Dilute 1.0 m L of the test solution to 100.0 m L with the solvent mixture. Dilute 1.0 m L of this solution to 10.0 m L with the solvent mixture.

Reference solution (b) Dissolve 5 mg of the substance to be examined and 5 mg of 4-rdtrobenzoic acid R (impurity E) in

IM P U R IT IE S

Other detectable impurities (the following substances would, if present at a sufficient level, be detected by one or other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore n o t necessary to identify these impurities for demonstration of compliance. See also 5.10. Control of impurities in substances for pharmaceutical use): A , B, C, D, E, F, G, H.

10.0 m L of the solvent mixture. Dilute 1.0 m L o f the solution to 50.0 m L with the solvent mixture.

Column: — sizer. I = 0.10 m, 0 = 4.6 mm; — stationary phase: end-capped octadecylsilyl silica gel for chromatography R (3 nm); — temperature: 35 °C.

Mobile phase: — mobile phase A: dilute 1 m L of perchloric acid R to 100 m L with water for chromatography R, dilute 1 m L of the solution to 100 m L with water for chromatography R; mix 9 volumes of this solution and 1 volume of acetonitrile R l ;

A. (4-aminophenyl)methanol,

B. (2-aminophenyl)methanol, o

— mobile phase B: acetonitrile Rl; Tune (min) 0 -2

Mobile phase A (per cent V/V) 100

2 - 15

100 -> 38.5

Mobile phase B (per cent V/V) 0 0

61.5

NHj C . ethyl 3-aminobenzoate, o

Flow rate 1.5 mL/min. Detection Spectrophotometer at 215 nm. Injection 10 (iL. Identification of impurities Use the chromatogram obtained with reference solution (b) to identify the peak due to im purity E.

D . ethyl 2-aminobenzoate,

Relative retention W ith reference to benzocaine (retention time = about 10 min): impurity E = about 0.9. System suitability-, reference solution (b): — resolution: minimum 5.0 between the peaks due to impurity E and benzocaine.

Calculation ofpercentage contents:

E . 4-nitrobenzoic ad d ,

— for each impurity, use the concentration of benzocaine in reference solution (a).

Limits: — unspecified impurities: for each impurity, maximum 0.10 per cent; — total: maximum 0.2 per cent; — reporting threshold: 0.05 per cent.

nh2

F . (3-aminophenyl)methanol,

L oss o n d ry in g (2.2.32) M aximum 0.5 per cent, determined on 1.000 g by drying

in vacuo. S u lfa te d a s h (2.4.14) M aximum 0.1 per cent, determined on 1.0 g.

G. 4-aminobenzoic acid,

A SSA Y Carry out the determination of primary aromatic aminonitrogen (2.5.5), using 0.400 g dissolved in a mixture of 25 m L of hydrochloric acid R and 50 m L o f water R.

o

1 m L o f 0.1 M sodium nitrite is equivalent to 16.52 mg o fG jH n N O z . ST O R A G E Protected from light.

H . methyl 4-aminobenzoate. __________________________________________________________PhEur

1-264 Benzoic Acid

2016 *** ★ ★ ★ ★ *****

Benzoic Acid (Ph. Eur. monograph 0066)

Ç C 7H 0O 2

T * 122.1

65-85-0

A c tio n a n d u se Antimicrobial preservative. P re p a r a tio n s C om pound Benzoic A d d O intm ent Benzoic Acid Solution PhEur.

D E F IN IT IO N Benzenecarboxyiic ad d . C o n te n t 99.0 per cent to 100.5 per c e n t CHARACTERS A p p e a ra n c e W hite or alm ost white, crystalline pow der or colourless crystals. S o lu b ility Slightly soluble in water, soluble in boiling water, freely soluble in ethanol (96 per cent) and in fatty oils. ID E N T IF IC A T IO N A. M dting point (2.2.14): 121 °C to 124 °C. B. Solution S (see Tests) gives reaction (a) of benzoates

(23.1).

, washings to 25.0 m L with water R. T his solution is used to preparer solution A.

Solution (b) In the same m anner, prepare a similar solution w ithout the substance to be examined. T his solution is used to prepare solution B. In four 25 m L volumetric flasks, place separatdy 10 m L o f solution (a), 10 m L o f solution (b), 10 m L o f chloride standard solution (8 ppm Cl) R (used to prepare solution C) and 10 m L o f water R. T o each flask add 5 m L o f ferric ammonium sulfate solution R5, mix and add dropwise and with swirling 2 m L o f nitric add R and 5 m L o f mercuric thiocyanate solution R. Shake. Dilute the contents o f each flask to 25.0 m L with water R and allow the solutions to stand in a water-bath at 20 °C for 15 min. M easure a t 460 n m the absorbance (2.2.25) o f solution A using solution B as the compensation liquid, and the absorbance o f solution C using the solution obtained with 10 m L o f water R as the compensation liquid. T h e absorbance o f solution A is n o t greater than that o f solution C. H e a v y m e ta ls (2.4.8) M axim um 10 ppm . 12 m L o f solution S complies with test B. Prepare the reference solution using a mixture o f 5 m L o f lead standard solution (1 ppm Pb) R and 5 m L of ethanol (96 per cent) R. S u lfa te d a s h (2.4.14) M aximum 0.1 p er cent, determined on 1.0 g. A SSA Y Dissolve 0.200 g in 20 m L of ethanol (96 per cent) R and titrate with 0.1 M sodium hydroxide, using 0.1 m L o f phenol red solution R as indicator, until the colour changes from yellow to violet-red. 1 m L of 0.1 M sodium hydroxide is equivalent to 12.21 m g of C 7H 6O 2.

TESTS S o lu tio n S Dissolve 5.0 g in ethanol (96 per cent) R and dilute to 100 m L with the same solvent.

___________________________________________________________PhEir

A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and colourless (2.2.2, Method II).

Hydrous Benzoyl Peroxide

C a rb o n is a b le su b sta n c e s Dissolve 0.5 g with shaking in 5 m L o f sulfuric add R . After 5 m in, the solution is not m ore intensely coloured than reference solution Y5 (2.2.2, Method I).

(Ph. Eur. monograph 0704)

O x id isab le su b sta n c e s Dissolve 0.2 g in 10 m L o f boiling water R. Cool, shake and filter. T o the filtrate add 1 m L of dihae sulfuric acid R and 0.2 m L of 0.02 M potassium permanganate. After 5 m in, the solution is still coloured pink. H a lo g e n a te d c o m p o u n d s a n d h a lid e s M axim um 300 ppm.

AU glassware used must be chloride-free and may be prepared by soaking overnight in a 500 g/L solution of nitric add R, rinsed with water R and storedfull of water R. It is recommended that glassware be reservedfor this test Solution (a) Dissolve 6.7 g in a mixture o f 40 m L o f 1 M sodium hydroxide and 50 m L o f ethanol (96 per cent) R and dilute to 100.0 m L with water R. T o 10.0 mT. o f this solution add 7.5 m L o f dilute sodium hydroxide solution R and 0.125 g o f nickel-ahaninium alloy R an d heat on a water-bath for 10 min. Allow to cool to room tem perature, filter into a 25 m L volumetric flask and wash w ith 3 quantities, each of 2 m L , o f ethanol (96 per cent) R. Dilute the filtrate and

★ ★

* * * * *

242.2

C 14H 10O4

★ ★

94-36-0

(anhydrous substance) A c tio n a n d u se U sed topically in the treatm ent of acne. P re p a r a tio n s Benzoyl Peroxide Cream Benzoyl Peroxide G d Benzoyl Peroxide Lotion Potassium Hydroxyquinoline Sulfate and Benzoyl Peroxide Cream PhEur.

D E F IN IT IO N C o n te n t 3j7,7atetrahydroimidazo [5,1 -è] thiazole-3,7-dicarboxylic a d d (penillic a d d of benzylpenidllin),

Untie. H

— any impurity: for each impurity, n o t more than die area of the principal peak in die chromatogram obtained with reference solution (c) (1 per cent).

2-Ethyihexanoic add (2.4.28)

h n -A

O

COjH

.c h 3

COaH

Maximum 0.5 per cent m/m. Loss o n d ry in g (2.2.32) Maximum 1.0 per cent, determined on 1.000 g by drying in an oven at 105 °C.

E. (45)-2- [carboxy[(phenylacetyl) amino]methyl]-5,5dimethyhhiazolidine-4-carboxylic a d d (penicOloic adds o f benzylpenidllin),

2016

1-274 Betacarotene H

H ç

r

r

C02H

TESTS Related substances

HN'^X.CHa

U / \ ru

and epimerat C*

Determine the absorbance (2.2.25) o f test solutions (b) and (a) used in Identification, at 455 nm an d at 340 nm respectively.

h

Absorbance ratio A 4 S5 / A ^ : minimum 1.5.

F. (2i?5,45)-2-[[(phenyIacetyl)amino]methyi]-5,5dimetiiylthiazoHdine-4-caiboxylic a d d (penifloic ad d s o f benzylpenidllin).

T h e thresholds indicated under Related substances (Table 2034.-1) in the general monograph Substances for pharmaceutical use (2034) do n o t apply. PhEur

★ ★

Betacarotene (Ph. Eur. monograph 1069)

★ ★ * •k*

Heavy metals (2.4.8) Maximum 10 ppm . 2.0 g complies with test D . Prepare the reference solution using 2 m L o f lead standard solution (10 ppm Pb) R

Loss on drying (2.2.32) M aximum 0.2 p er cent, determined on 1.000 g by drying in vacuo over diphosphorus pentoxide R at 40 °C for 4 h. Sulfated ash (2.4.14) M aximum 0.2 p er cent, determined on 1.0 g, moistened with a mixture o f 2 mT. o f dilute sulfuric add R and 5 m L o f ethanol (96 per cent) R ASSAY Measure the absorbance 4)-(a-D-giucopyranosyl) (cydomaltoheptaose or 0-cydodextrin).

7585-39-9

2016

CHARACTERS

Appearance White o r almost white, amorphous or crystalline powder. S o lu b ility Sparingly soluble in water and in propylene glycol, practically insoluble in anhydrous ethanol and in methylene chloride. ID E N T IF IC A T IO N A. Specific optical rotation (see Tests). B. Examine the chromatograms obtained in the assay.

Results T h e principal peak in the chromatogram obtained with test solution (b) is similar in retention time and size to the principal peak in the chromatogram obtained with reference solution (c). C. Dissolve 0.2 g in 2 m L of iodine solution R4 by wanning on a w ater-bath, and allow to stand at room temperature. A yellowish-brown precipitate is formed. TESTS S o lu tio n S Dissolve 1.000 g in carbon dioxide-free water R with heating, allow to cool and dilute to 100.0 m L with the same solvent A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1). p H (2.2.3) 5.0 to 8.0. T o 10 m L of solution S add 0.1 m L of a saturated solution of potassium chloride R. S pecific o p tic a l ro ta tio n (2.2.7) + 160 to 4- 164 (dried substance), determined on solution S. R e d u c in g su g a rs M aximum 0.2 per cent.

Test solution T o 1 m L o f solution S add 1 m L of cupri-tartaric solution R4. H eat on a water-bath for 10 min, cool to room tem perature. Add 10 m L of ammonium molybdate reagent R1

Betadex 1-275 Reference solution (c) Dissolve 25.0 mg of betadex CRS in water R and dilute to 25.0 m L with the same solvent. Column: — sizer. I — 0.25 m, 0 = 4.6 mm ; — stationary phase: octadecylsifyl silica gel for chromatography R (10 nm).

Mobile phase methanol R, water R (10:90 VIV). Flow rate 1.5 rnL/m in. Detection Differential refractometer. Equilibration W ith the mobile phase for about 3 h . Irtjection 50 |jL o f test solution (a) and reference solutions (a) and (b).

Run time 1.5 times the retention time of betadex. Relative retention W ith reference to betadex (retention time = about 10 min): impurity B = about 0.3; impurity A = about 0.45. System suitability: reference solution (a): — resolution: minim um 1.5 between the peaks d u e to impurities B and A; if necessary, adjust the concentration o f methanol in the mobile phase.

Limits: — impurities A, B: for each impurity, not more than 0.5 times the area of the corresponding peak in the chromatogram obtained with reference solution (b) (0.25 per cent); — sum of impurities other than A and B: not m ore than 0.5 times the area of the peak due to betadex in die chromatogram obtained with reference solution (b) (0.5 per cent).

Residual solvents Head-space gas chromatography (2.2.28) Use the standard additions method.

Reference solution Prepare a reference solution at the same

Internal standard ethylene chloride R. Test solutions In each o f 4 identical 20 m L flasks, dissolve 0.5 g o f the substance to be examined in water R and add 0.10 g of calcium chloride R and 30 |xL o f a-amylase solution R.

time and in the same m anner as the test solution, using 1 m L o f a 0.02 g/L solution of glucose R.

Add 1 m L of reference solutions (a), (b), (c) and (d), adding a different solution to each flask. Dilute to 10 m L with

M easure the absorbance (2.2.25) of the test solution and the reference solution at the absorption maximum at 740 nm using water R as the compensation liquid. T he absorbance of the test solution is not greater than that of the reference solution.

water R. Reference solutions Prepare a 10 \sUL solution o f ethylene chloride R (reference solution (a)). Prepare reference

and allow to stand for 15 min.

L ig h t-a b so rb in g im p u ritie s Examine solution S between 230 n m and 750 nm . Between 230 nm and 350 nm , the absorbance (2.2.25) is not greater than 0.10. Between 350 nm and 750 nm , the absorbance (2.2.2S) is not greater than 0.05. R e la te d su b sta n c e s Liquid chrom atography (2.2.29).

Test solution (a) Dissolve 0.25 g of the substance to be examined in water R with heating, cool and dilute to 25.0 m L with the same solvent. Test solution (b) Dilute 5.0 m L of test solution (a) to 50.0 m L with water R. Reference solution (a) Dissolve 25.0 mg o f alfadex CRS (impurity A), 25.0 mg o f gammacyclodextrin CRS (impurity B) and 50.0 mg of betadex CRS in water R, then dilute to 50.0 m L with the same solvent.

Reference solution (b) Dilute 5.0 m L o f reference solution (a) to 50.0 m L with water R.

solutions (b), (c) and (d) from reference solution (a) to contain respectively, per litre, 5 nL, 10 |iL and 15 |iL of both trichloroethylene R and toluene R.

Column: — material: fused silica; — sizer. I = 25 m , 0 = 0.32 mm ; — stationary phase: macrogol 20 000 R (film thickness 1 nm).

Carrier gas helium for chromatography R. Static head-space conditions which may be used: — equilibration temperature: 45 °C; — equilibration timer. 2 h. Temperature: — column: 50 °C; — infection port. 140 °C; — detector. 280 °C. Detection Flame ionisation. Irtjection 200 |jL of the head space, at least 3 times. Retention time Toluene = about 10 min.

1-276 Betahistine Dihydrochloride

2016

System suitability: — resolution: m in im u m 1.1 between the peaks due to trichloroethylene and toluene; minim um 1.1 between the peaks due to toluene and ethylene chloride; — repeatability: maximum relative standard deviations of the ratios o f the areas o f the peaks due to trichloroethylene and toluene to th at of the peak due to ethylene chloride o f 5 per cent. Calculate the content o f trichloroethylene and of toluene taking their relative densities to be 1.46 and 0.87, respectively.

Limits: — trichloroethylene: m axim um 10 ppm; — toluene, maximum 10 ppm . H eav y m e ta ls (2.4.8) M axim um 10 ppm. 1.0 g complies with test C. Prepare the reference solution using 1 m L o f lead standard solution (10 ppm Pb) R.

B. cyclooctakis-(l -+4)-(a-D-glucopyranosyl) (cyclomaltooctaose or y-cyclodextrin).

L oss o n d ry in g (2.2.32) M axim um 16.0 per cent, determ ined on 1.000 g by drying in an oven at 120 °C for 2 h. S u lfa te d a s h (2.4.14) M axim um 0.1 per cent, determ ined on 1.0 g.

Betahistine Dihydrochloride

area of the peak due to betadex of 2.0 per cent. Calculate the percentage content of [C 6H 10O 5]7 from the assigned content of betadex CRS. STORAGE In an airtight container. IM P U R IT IE S

Specified impurities A, B

* * * * *

(Ph. Eur. monograph 1665)

A SSA Y Liquid chromatography (2.2.29) as described in the test for related substances w ith the following modifications.

Injection T est solution (b) and reference solutions (a) and (c). System suitability: reference solution (a): — repeatability: maximum relative standard deviation of the

★* ★ ★ ★ ★ ★

R f

C8Hi4C12N 2

CH3 ,2HCI

209.1

5579-84-0

A c tio n a n d u se Histamine H ] receptor antagonist; antih istam in e. P r e p a r a tio n B etahistin e Dihydrochloride Tablets PhEur___________________________________________________________

D E F IN IT IO N N-M ethyl-2-(pyridin-2-yi)ethanamine dihydrochloride. C o n te n t 99.0 per cent to 101.0 per cent (dried substance). CHARACTERS A p p e a ra n c e W hite or slightly yellow powder, very hygroscopic. S o lu b ility Very soluble in water, soluble in ethanol (96 per cent), practically insoluble in 2 -propanol. ID E N T IF IC A T IO N

A. cyclohexakis-(l-*4)-(a-D-glucopyranosyl) (alfadex or

cyclomaltohexaose or a-cyclodextrin),

First identification B, D Second identification A , C, D A. M eltin g point (2.2.14): 150 °C to 154 °C. B. Infrared absorption spectrophotom etry (2.2.24). Comparison betahistine dihydrochloride CRS. C. Thin-layer chrom atography (2.2.27). Test solution Dissolve 10 m g of the substance to be examined in 2 m L o f ethanol (96 per cent) R. Reference solution Dissolve 10 m g o f betahistine dihydrochloride CRS in 2 m L o f ethanol (96 per cent) R. Plate TLC silica gel GF2s\ plate R. Mobile phase concentrated ammonia R, ethyl acetate R, methanol R (0.75:15:30 VIVIV). Application 2 |iL.

Betahistine Mesilate 1-277

2016 Development Over 2/3 of the plate. Drying At 110 °C for 10 min. Detection Examine in ultraviolet light at 254 nm. Results T he principal spot in the chromatogram obtained with the test solution is similar in position and size to the prindpal spot in the chromatogram obtained with the reference solution. D . It gives reaction (a) o f chlorides (2.3.1).

TESTS Solution S Dissolve 5.0 g in carbon dioxide-free water R, and dilute to 50 m L with the same solvent.

Appearance o f solution Solution S is clear (2.2.1) and not more intensely coloured than reference solution B 8 (2.2.2, Method II). pH (2.2.3) 2.0 to 3.0 for solution S. Related substances Liquid chromatography (2.2.29). Test solution Dissolve 25 mg o f die substance to be examined in the mobile phase and dilute to 25.0 m L with the mobile phase.

— total: not more than 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent); — disregard limit. 0.25 times the area o f the principal peak in the chromatogram obtained with reference solution (c) (0.05 p er cent). L oss o n d ry in g (2.2.32) M aximum 1.0 per cent, determined on 1.000 g by drying in an oven at 105 °C. S u lfa te d a sh (2.4.14) Maximum 0.1 p er cent, determined on 1.0 g. ASSAY Dissolve 80.0 m g in 50 m L of ethanol (96 per cent) R. T itrate with 0.1 M sodium hydroxide, determ in in g the end-point potentiometrically (2.2.20). Read the volume added to reach the second point of inflexion. 1 m L of 0.1 M sodium hydroxide is equivalent to 10.46 m g of C 8H 14C12N 2. STORA GE In an airtight container. IM P U R IT IE S

Specified impurities A, B, C.

Reference solution (a) Dissolve 10 m g of betahistine dihydrochloride CRS and 10 mg of 2-virtylpyridine R in the mobile phase and dilute to 50.0 m L with the mobile phase. Dilute 2.0 m L o f the solution to 50.0 m L with the mobile phase.

C

T

"

A. 2-ethenylpyridine (2-vinylpyridine),

Reference solution (b) Dilute 1.0 m L of the test solution to 100.0 m L with the mobile phase.

Reference solution (c) Dilute 2.0 m L of reference solution (b)

C

to 10.0 m L with the mobile phase.

Column: — sizer. I = 0.15 m , 0 = 3.0 mm; — stationary phase: base-deactivated end-capped octadecylsilyl silica gel for chromatography R (5 Jim). Mobile phase Dissolve 2.0 g o f sodium dodecyl sulfate R in a mixture of 15 m L of a 10 per cent V/V solution o f sulfuric acid R, 35 m L o f a 17 g/L solution of tetrabtaylammomum hydrogen sulfate R and 650 m L of water R; adjust to p H 3.3 using dilute sodium hydroxide solution R and mix w ith 300 m L of acetonitrile R. Flow rate 1 mlVmin. Detection Spectrophotom eter at 260 nm. Irtjection 20 (iL. Run time 4 times the retention time of betahistine. Relative retention W ith reference to betahistine (retention time = about 7 m in): impurity B = about 0.2; impurity A = about 0.3; impurity C = about 3. System suitability: reference solution (a): — resolution: minim um 3.5 between the peaks due to 2-vinylpyridine and betahistine.

P

'* '

B. 2-(pyridin-2-yl)ethanol, ch3 n.

O

T '- '^ O

C. N-methyl-2-(pyridin-2-yl)-N-[2-(pyridin-2yl)ethyl] ethanamine. PhEur

Betahistine Mesiiate

★ ★

★ ★

(Ph. Eur. monograph 1071)

* * * * *

CH3 , 2 H3C -S O 3H

Limits: — correction factor, for the calculation of content, multiply the peak area o f impurity B by 0.4; — impurities A , B, C: for each impurity, n o t more th an the area o f the principal peak in the chromatogram obtained with reference solution (c) (0.2 per cent); — unspecified impurities: for each impurity, not m ore than 0.5 times o f the area o f the principal peak in the chromatogram obtained with reference solution (c) (0.10 p er cent);

C 10H20N2O6S2

328.4

54856-23-4

A ctio n a n d u se Histamine H I receptor antagonist; antihistamine. PhEur_________________________________________________________

D E F IN IT IO N N-Methyl-2-(pyridin-2-yl)ethanamine bis(methanesulfonate). C o n te n t 98.0 per cent to 101.0 per cent (anhydrous substance).

2016

1-278 Betahistine Mesilate

P R O D U C T IO N It is considered that alkylsulfonate esters are genotoxic and are potential impurities in betahistine mesilate. T h e manufacturing process should be developed taking into consideration the principles o f quality risk management, together with considerations o f the quality of starting materials, process capability and validation. T he general m ethods 2.5.37. Methyl, ethyl and isopropyl methanesulfonate in

Test solution Dissolve 50 mg o f the substance to be examined in the mobile phase and dilute to 10.0 m L with the mobile phase.

methanesutfonic add, 2.5.38. Methyl, ethyl and isopropyl methanesulfonate in active substances and 2.5.39. Methanesulfonyl chloride in methanesulfonic acid are available to

Reference solution (b) Dilute 1.0 m L o f the test solution to

assist manufacturers. CHARACTERS A p p e a ra n c e W hite or alm ost white, crystalline powder, very hygroscopic. S o lu b ility Very soluble in water, freely soluble in ethanol (96 per cent), very slightly soluble in 2 -propanol. ID E N T IF IC A T IO N

First identification B. Second identification A, C, D. A. M elting point (2.2.14): 108 °C to 112 °C. B. Infrared absorption spectrophotom etry (2.2.24). Comparison betahistine mesilate CRS. C. Thin-layer chrom atography (2.2.27). Test solution Dissolve 10 m g o f the substance to be examined in ethanol (96 per cent) R and dilute to 2 m L with the same solvent.

Reference solution Dissolve 10 m g o f betahistine mesilate CRS in ethanol (96 per cent) R and dilute to 2 m L with the same solvent.

Plate TLC silica gel F2 5 4 plate R. Mobile phase concentrated ammonia R, ethyl acetate R, methanol R ( 0.75:15:30 VIV/V). Application 2 |iL. Development O ver 3/4 o f the plate. Drying At 110 °C for 10 min. Detection Examine in ultraviolet light at 254 nm. Results T he principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chrom atogram obtained with the reference solution.

Reference solution (a) Dissolve 10 m g of betahistine mesilate CRS and 10 m g o f 2-vinylpyridme R (impurity A) in the mobile phase and dilute to 50.0 m L with the mobile phase. Dilute 2.0 m L o f this solution to 50.0 m L with the mobile phase. 100.0 m L with the mobile phase.

Reference solution (c) Dilute 2.0 m L o f reference solution (b) to 10.0 m L with the mobile phase.

Column: — size: I = 0.25 m , 0 = 4.6 mm; — stationary phase: octadecylsUyl silica gel for chromatography R (5 jun).

Mobile phase Dissolve 2.0 g o f sodium dodecyl sulfate R in a mixture o f 15 volumes o f a 10 per cent V/V solution of sulfuric acid R, 35 volumes o f a 17 g/L solution of tetrabutylammonium hydrogen sulfate R and 650 volumes of water R; adjust to p H 3.3 using dilute sodium hydroxide solution R and mix with 300 volumes of acetonitrUe R. Flow rate 1 mlVmin. Detection Spectrophotom eter at 260 nm. Injection 20 jjL. Run time 3 times the retention tim e o f betahistine mesilate. Retention time Betahistine mesilate = about 8 min. System suitability: reference solution (a): — resolution: minimum 3.5 betw een the peaks due to im purity A and betahistine mesilate.

Limits: — impurity A: n o t more than the area o f the principal peak in the chromatogram obtained with reference solution (c) (0.2 per cent); — unspecified impurities: for each impurity, n o t more than 0.1 times the area of the principal peak in the chromatogram obtained with reference solution (b) ( 0.10 per cent); — total: not more than 0.5 times the area o f the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent); — disregard limit. 0.05 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.05 per cent).

D . T o 0.1 g add 5 m l. o f dilute hydrochloric acid R and shake for about 5 min. A dd 1 m L o f barium chloride solution R l. T he solution rem ains clear. T o a further 0.1 g add 0.5 g o f anhydrous sodium carbonate R, mix and ignite until a white residue is obtained. Allow to cool and dissolve the residue in 7 m L of water R. T he solution gives reaction (a) o f sulfates

2 -P ro p a n o l (2.424) M axim um 0.5 p er c e n t

(2.3.1).

S u lfa tes (2.413) M axim um 250 ppm.

C h lo rid e s (2.44) Maximum 35 ppm . T o 14 m L of solution S add 1 m L of water R.

TESTS S o lu tio n S Dissolve 5.0 g in carbon dioxide-free water R prepared from distilled water R, and dilute to 50 m L with the same solvent.

H ea v y m e ta ls (2.48) M axim um 20 ppm .

A p p e a ra n c e o f so lu tio n Solution S is clear (2.2.1) and colourless (2.2.2, Method II).

12 m L o f solution S complies with test A. Prepare the reference solution using lead standard solution (2 ppm Pb) R.

p H (2.2.3) 2.0 to 3.0 for solution S.

W a te r (2.5.12) Maximum 2.0 per cent, determ ined on 0.50 g.

R e la te d su b s ta n c e s Liquid chromatography (2.2.29).

A SSA Y Dissolve 0.140 g in 50 m L o f a mixture o f 1 volume o f anhydrous acetic acid R and 7 volumes of acetic anhydride R.

D ilute 6 m L o f solution S to 15 m L with distilled water R.

Betamethasone 1-279

2016 T itrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20).

chloride R, evaporate to dryness on a water-bath and record

1 m L of 0.1 M perchloric add is equivalent to 16.42 mg of C 10H 20N 2O 6S 2.

C. Thin-layer chromatography (2.2.27). Solvent mixture methanol R, methylene chloride R (1:9 V/V).

STORA GE In an airtight container.

Solvent mixture

new spectra using the residues.

Test solution Dissolve 10 mg of the substance to be examined in the solvent mixture and dilute to 10 m L with the solvent mixture.

IM P U R IT IE S

Specified impurities A

Reference solution (a) Dissolve 20 m g of betamethasone CRS in the solvent mixture and dilute to 20 m L with the solvent mixture.

C T "

Reference solution (b) Dissolve 10 m g of dexamethasone CRS in reference solution (a) and dilute to 10 m L with reference solution (a).

A. 2-ethenylpyridine (2-vinylpyridine). PhEur

★ ★ ★ ★ *****

Betamethasone (Ph. Eur. monograph 0312)

Plate TLC silica gel F 254 plate R. Mobile phase butanol R saturated with water R, toluene R, ether R (5:10:85 VIVIV). Application 5 |iL. Development Over a path of 15 cm. Drying In air. Detection A Examine in ultraviolet light at 254 nm. Results A T h e principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a).

Detection B Spray with alcoholic solution of sulfuric acid R. H eat C22H 29FO 5

392.5

378-44-9

at 120 °C for 10 m in or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at 365 nm .

Results T h e principal spot in the chromatogram obtained w ith A ctio n a n d u se Glucocorticoid. P re p a ra tio n Betamethasone T ablets PhEir ___________________

D E F IN IT IO N 9-Fluoro-l 1P, 17 j2 1-trihydroxy-16 ^-methylpr egna-1,4diene-3,20-dione. C o n te n t 97.0 per cent to 103.0 per cent (dried substance). CHARACTERS A p p earan ce White or almost white, crystalline powder. Solubility Practically insoluble in water, sparingly soluble in anhydrous ethanol, very slightly soluble in methylene chloride. ID E N T IF IC A T IO N

First identification B, C. Second identification A , C, D, E. A. Dissolve 10.0 m g in anhydrous ethanol R and dilute to 100.0 m L with the same solvent. Place 2.0 m L of this solution in a stoppered tube, add 10.0 m L o f phertylhydrazine-sidfuric acid solution R, mix and heat in a water-bath at 60 °C for 20 min. Cool immediately. The absorbance (2.2.25) measured at 419 n m is n o t greater than 0 . 10. B. Infrared absorption spectrophotometry (2.2.24).

Comparison betamethasone CRS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in the minimum volume of methylene

the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (a). System suitability: reference solution (b): — the chromatogram shows 2 spots which may, however, not be completely separated. D . Mix about 5 m g with 45 mg of heavy magnesium oxide R and ignite in a crucible until an almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 m L o f water R, 0.05 m L of phenolphthalein solution R1 and about 1 m L of dilute hydrochloric acid R to render the solution colourless. Filter. Add 1.0 m L of the filtrate to a freshly prepared mixture o f 0.1 m L of alizarin S solution R and 0.1 m L o f zirconyl nitrate solution R. Mix, allow to stand for 5 min and compare the colour of the solution with that o f a blank prepared in the same manner. T he test solution is yellow and the blank is red. E. Add about 2 m g to 2 m L of sidfuric acid R and shake to dissolve. W ithin 5 min, a deep reddish-brown colour develops. Add this solution to 10 m L of water R and mix. T h e colour is discharged and a clear solution remains. TESTS S p ecific o p tic a l ro ta tio n (2.2.7) + 118 to + 126 (dried substance). Dissolve 0.125 g in methanol R and dilute to 25.0 m L with the same solvent. R e la te d su b sta n c e s Liquid chromatography (2.2.29).

Test solution Dissolve 25.0 mg o f the substance to be examined in a mixture of equal volumes o f acetonitrHe R and methanol R and dilute to 10.0 m L with the same mixture o f solvents.

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1-280 Betamethasone

Reference solution (a) Dissolve 2 m g o f betamethasone CRS and 2 m g of methylprednisolone CRS in mobile phase A, then

Calculate the content of C 22H 29F O 5 taking the specific absorbance to be 395.

dilute to 100.0 m L with mobile phase A.

STO RA G E Protected from light.

Reference solution (b) Dilute 1.0 m L of the test solution to 100.0 m L with mobile phase A.

Column'. — size. I = 0.25 m , 0 = 4.6 mm; — stationary phase, octadecylsilyl silica gel for chromatography R

IM P U R IT IE S

Specified impurities A, B, C, D , E, F, G , H , I, J

(5 Jim); — temperature: 45 °C.

Mobile phase". — mobile phase A: in a 1000 m L volumetric flask mix 250 m l. o f acetomtrUe R w ith 700 m L of water R and allow to equilibrate; dilute to 1000 m L with water R and mix again; — mobile phase B: acetomtrUe R; Time (min) 0 - 15

Mobile phase A (per cent V/V) 100

Mobile phase B (per cent V/V) 0

15-40

100*0

0 * 100

4 0-41

0 * 100

10 0 * 0

4 1 -4 6

100

0

A. 9 -flu o ro -lip , 17,21 -trihydroxy-16a-m ethylpregna-1,4diene-3,20-dione (dexamethasone),

B. 21-chloro-9-fluoro-l ip,17-dihydroxy-16P-methylpregnal,4-diene-3,20-dione,

Flow rate 2.5 mlVmin. Detection Spectrophotom eter at 254 nm . Equilibration W ith mobile phase B for at least 30 min and then with mobile phase A for 5 min. F or subsequent chromatograms, use the conditions described from 40 min to 46 min.

Injection 20 pL; inject the mixture of equal volumes o f acetomtrUe R and methanol R asa, blank. Retention time M ethylprednisolone = about 11.5 min;

C. 17,21-dihydroxy-16P-methylpregna-l,4,9(l l)-triene-3,20dione,

betam ethasone = about 12.5 min. System suitability: reference solution (a): — resolution: minimum 1.5 between the peaks due to methylprednisolone and betamethasone; if necessary, adjust the concentration o f acetonitrfle in mobile phase A.

Limits: — impurities A , B, C, D, E, F, G, H, 1, J: for each impurity, no t more than the area o f the principal peak in the chrom atogram obtained w ith reference solution (b) ( 1.0 per cent), and not m ore than 1 such peak has an area greater than 0.5 times the area of the principal peak in the chrom atogram obtained with reference solution (b) (0.5 per cent); — total: not more than twice the area of the principal peak in ~ the chrom atogram obtained with reference solution (b) ( 2.0 per cent); — disregard limit. 0.05 times the area of the principal peak in the chrom atogram obtained with reference solution (b) (0.05 per cent). L oss o n d ry in g (2.2.32) M axim um 0.5 per cent, determ ined on 0.500 g by drying in an oven at 105 °C. A SSA Y Dissolve 0.100 g in ethanol (96 per cent) R and dilute to 100.0 m L w ith the same solvent. Dilute 2.0 m L of this solution to 100.0 m L with ethanol (96 per cent) R. M easure the absorbance (2.2.25) at the absorption maximum at 238.5 nm.

D . 9 -flu o ro -lip , 17-dihydroxy-16P-methyl-3,20-dioxopregnal,4-dien-21-yl ethoxycarboxylate,

E. 9,11 P-epoxy-17,21 -dihydroxy-16P-methyl-9 P-pregna-1,4diene-3,20-dione,

F . 17,21-dihydroxy-16P-methylpregna-l,4,l l-triene-3,20dione,

2016

Betamethasone Acetate 1-281

CHARACTERS A p p e a ra n c e W hite or almost white, crystalline powder. S olu b ility Practically insoluble in water, freely soluble in acetone, soluble in ethanol (96 per cent) and in methylene chloride. It shows polymorphism (5.9).

G . 1 la,17,21-trihydroxy-16ß-methylpregna-l,4-diene-3,20dione,

ID E N T IF IC A T IO N

First identification B, C Second identification A , C, D, E, F A. Dissolve 10.0 m g in anhydrous ethanol R and dilute to 100.0 m L with the same solvent. Place 2.0 m L o f this solution in a groimd-gjass-stoppered tube, add 10.0 m L o f phenyJhydrazme-sulfuric acid solution R, m ix and heat in a water-bath at 60 °C for 20 m in. Cool im m ediately. T he absorbance (2.2.25) measured at 419 nm is not greater than 0 . 10.

H . 14-fluoro-l lß,17,21-trihydroxy-16ß-methyl-8a,9ß,14ßpregna-1 ,4-diene-3,20-dione,

B. Infrared absorption spectrophotometry (2.2.24).

Comparison betamethasone acetate CRS. If the spectra obtained in the solid state show differences, dissolve the substance to be examined and the reference substance separately in the m inim um volume of methanol R, evaporate to dryness on a water-bath and record new spectra using the residues. C. Thin-layer chromatography (2.2.27). Solvent mixture methanol R, methylene chloride R (1:9 VIV). Test solution Dissolve 10 mg of the substance to be exam ined

I. 8-fluoro-llß,17,21-trihydroxy-16ß-methyl-8a,9ß-pregnal 34-diene-3,20-dione,

in the solvent mixture and dilute to 10 m L with the solvent mixture.

Reference solution (a) Dissolve 20 mg of betamethasone acetate CRS in the solvent mixture and dilute to 20 m L with the solvent mixture.

Reference solution (b) Dissolve 10 mg of prednisolone acetate CRS in reference solution (a) and dilute to 10 m L with reference solution (a). J. 17,21 -dihydroxy-16 (J-methylpregna-134-diene-3 ,2 0-dione. __________________________________________________________ PhEtr

Betamethasone Acetate

******

(Ph Eur monograph 0975)

*

Plate TLC silica gel F 254 plate R. Mobile phase Add a mixture of 1.2 volumes of water R and 8 volumes of methanol R to a mixture of 15 volumes of ether R and 77 volumes o f methylene chloride R. Application 5 pL. Development Over a path o f 15 cm. Drying In. air. Detection A Examine in ultraviolet light at 254 nm. Results A T h e principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with reference solution (a).

Detection B Spray with alcoholic solution of sulfuric acid R. H eat at 120 °C for 10 m in or until the spots appear. Allow to cool. Examine in daylight and in ultraviolet light at 365 n m .

Results B T h e principal spot in the chromatogram obtained C24H31F 0 6

434.5

987-24-6

A ctio n a n d u se Glucocorticoid. PhEur__________________________________________________________

D E F IN IT IO N 9-Fhioro-11(3,17-dihydroxy-16|}-inethyl-3,20-dioxopregnal,4-diene-21-yl acetate. C o n te n t 97.0 per cent to 103.0 per cent (anhydrous substance).

with the test solution is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with reference solution (a).

System suitability: reference solution (b): — the chromatogram shows 2 clearly separated spots. D . Add about 2 m g to 2 m L o f sulfuric acid R and shake to dissolve. W ithin 5 min, a deep reddish-brown colour develops. Add this solution to 10 m L of water R and mix. T h e colour is discharged and a clear solution rem ain s.

2016

1-282 Betamethasone Acetate

E. M ix about 5 mg with 45 m g of heavy magnesium oxide R and ignite in a crucible until an almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 m L of water R, 0.05 m L of phenolphthalein solution R1 and about 1 m L o f dilute hydrochloric acid R to render the solution colourless. Filter. T o a freshly prepared mixture o f 0.1 m L o f alizarin S solution R and 0.1 m L of zirconyl nitrate solution R, add 1.0 m L o f the filtrate. Mix, allow to stand for 5 min and compare the colour of the solution with that o f a blank prepared in the same m anner. T he test solution is yellow and the blank is red.

A SSAY Dissolve 0.100 g in ethanol (96 per cent) R and dilute to 100.0 m L with the same solvent. D ilute 2.0 m L of this solution to 100.0 m L with ethanol (96 per cent) R M easure the absorbance (2.2.25) at the absorption maximum at 240 nm .

F. A bout 10 m g gives the reaction o f acetyl (2.3.1).

IM P U R IT IE S

TESTS S p ecific o p tic a l ro ta tio n (2.2.7) + 120 to + 128 (anhydrous substance).

Calculate the content of C 24H 3iF 0 6 taking the specific absorbance to be 350. STO R A G E Protected from light.

Specified impurities A, B, C , D

Dissolve 0.250 g in dioxan R and dilute to 25.0 m L with the sam e solvent. R e la te d su b s ta n c e s Liquid chromatography (2.2.29).

Test solution Dissolve 25.0 m g of the substance to be examined in 4 m L of acetomtrUe R and dilute to 10.0 m L with the same solvent.

A. 9-fluoro-lip,17,21-trihydroxy-16|3-niethylpregna-l,4diene-3,20-dione (betamethasone),

Reference solution (a) Dissolve 2 mg o f betamethasone acetate CRS and 2 mg o f dexamethasone acetate CRS (impurity B) in the mobile phase, then dilute to 100.0 m L with the mobile phase.

Reference solution (b) D ilute 1.0 m L o f the test solution to 100.0 m L with the mobile phase.

Column: — size: I = 0.25 m , 0 = 4.6 mm; — stationary phase, octadecylsUyl silica gel for chromatography R (5 \un). Mobile phase In a 1000 m L volumetric flask mix 380 m l. of acetomtrUe R w ith 550 m L of water R and allow to equilibrate; dilute to 1000 m L with water R and mix again. Flow rate 1 mL/min. Detection Spectrophotom eter at 254 cm . Equilibration W ith the mobile phase for about 30 m in. Injection 20 jiL. Run time 2.5 times the retention time of betamethasone acetate.

Retention time Betam ethasone acetate = about 19 min;

B. 9-fluoro-l ip,l7-dihydroxy-16a-m ethyl-3,20-dioxopregnal,4-dien-21-yl acetate (dexamethasone acetate),

C . 9-fluoro-17-hydroxy-16 |3-methyi-3,20-dioxopregna-1,4d ie n e -lip , 21 -diyl diacetate (betamethasone 11, 21 -diacetate),

im purity B = about 22 m in. System suitability: reference solution (a): — resolution: minim um 3.3 between the peaks due to betam ethasone acetate and impurity B; if necessary, adjust slightly the concentration o f acetonitrile in the mobile phase.

Limits: — impurities A , B, C, D: for each impurity, not m ore than 0.5 times the area o f the principal peak in the chrom atogram obtained with reference solution (b) (0.5 per cent); — total: not m ore than 1.25 times the area of the principal peak in the chrom atogram obtained with reference solution (b) (1.25 per cent); — disregard limit. 0.05 times the area o f the principal peak in the chrom atogram obtained with reference solution (b) (0.05 per cent). W a te r (2.5.12) M axim um 4.0 per cent, determ ined on 0.100 g.

D . 9,11 (5-epoxy-17-hydroxy-l 6P-methyl-3,20-dioxo-9|3pregna-1,4-diene-21-yl acetate. __________________ ________________________________ __

PhEur

Betamethasone Dipropionate 1-283

2016

Betamethasone Dipropionate

******

(Ph. Eur. monograph 0809)

** +* * O

immediately pass a current of nitrogen R briskly through the solution for 5 m in. Stopper the tube. H eat in a water-bath at 45 °C, protected from light, for 2 h. Allow to cool.

Plate TLC silica gel F 254 plate R. Mobile phase Add a mixture of 1.2 volumes o f water R and 8 volumes of methanol R to a mixture of 15 volumes of ether R and 77 volumes o f methylene chloride R. Application 5 |iL. Development Over 3/4 of the plate. Drying In. air. Detection A Examine in ultraviolet light at 254 nm. Results A T he principal spot in each o f the chromatograms

Action and use

obtained with the test solutions is similar in position and size to the principal spot in the chromatogram obtained with the corresponding reference solution.

Glucocorticoid.

Detection B Spray with alcoholic solution of sulfuric acid R, heat

PhEtr -----------------------------------------------------------------------------------------

at 120 °C for 10 m in or until the spots appear, and allow to cool; examine in daylight and in ultraviolet light at 365 nm.

D E F IN IT IO N 9-Fluoro-l 1 P-hydroxy-16P-methyl-3}20-dioxopregna-l ,4diene-17j21-diyl dipropanoate.

Content 97.0 per cent to 102.0 per cent (dried substance). CHARACTERS

Appearance W hite or alm ost white, crystalline powder.

Solubility Practically insoluble in water, freely soluble in acetone and in methylene chloride, sparingly soluble in ethanol (96 per cent).

IDENTIFICATION First identification B. Second identification A , C, D, E. A. Dissolve 10.0 m g in anhydrous ethanol R and dilute to 100.0 m L with the same solvent Place 2.0 m L of the solution in a ground-glass-stoppered tube, add 10.0 m L of phenylhydrazine-sulfuric add solution R, mix and heat in a water-bath at 60 °C for 20 min. Cool immediately. T he absorbance (2.2.25) measured at 419 nm is not more than 0 . 10. B. Infrared absorption spectrophotometry (2.2.24).

Comparison betamethasone dipropionate CRS. C. Thin-layer chrom atography (2.2.27). Test solution (a) Dissolve 25 m g of the substance to be examined in methanol R with gende heating and dilute to 5 m l, with the same solvent (solution A). Dilute 2 m L of solution A to 10 m L with methylene chloride R. Test solution (b) Transfer 2 m L of solution A to a 15 m L glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 m L of saturated methanoUc potassium hydrogen carbonate solution R and immediately pass a current o f nitrogen R briskly through the solution for 5 min. Stopper the tube. H eat in a water-bath at 45 °C, protected from light, for 2 h. Allow to cool

Reference solution (a) Dissolve 25 mg of betamethasone dipropionate CRS in methanol R with gende heating and dilute to 5 m l, w ith the sam e solvent (solution B). Dilute 2 m L o f solution B to 10 m l. with methylene chloride R.

Reference solution (b) T ransfer 2 m L of solution B to a 15 m L glass tube with a ground-glass stopper or a polytetrafluoroethylene cap. Add 10 m L o f saturated methanoUc potassium hydrogen carbonate solution R and

Results B T he principal spot in each of the chromatograms obtained with the test solutions is similar in position, colour in daylight, fluorescence in ultraviolet light at 365 nm and size to the principal spot in the chromatogram obtained with the corresponding reference solution; the principal spot in each of the chromatograms obtained with test solution (b) and reference solution (b) has an Rp value distinctly lower than that o f the principal spot in each o f the chromatograms obtained with test solution (a) and reference solution (a). D . Add about 2 mg to 2 m L of sulfuric add R and shake to dissolve. W ithin 5 min, a deep reddish-brown colour develops. Add this solution to 10 m L o f water R and mix. T he colour is discharged and a clear solution remains. E. Mix about 5 mg with 45 mg o f heavy magnesium oxide R and ignite in a crucible until an alm ost white residue is obtained (usually less than 5 min). Allow to cool, add 1 m L o f water R, 0.05 m L of phenolphthalein solution R1 and about 1 m L of dilute hydrochloric add R to render the solution colourless. Filter. Add 1.0 m L o f the filtrate to a freshly prepared mixture o f 0.1 m L of alizarin S solution R and 0.1 m l , of zirconyl nitrate solution R. Mix, allow to stand for 5 min and compare the colour o f th e solution with that of a blank prepared in the same manner. T he test solution is yellow and the blank is red. TESTS S pecific o p tic a l ro ta tio n (2.2.7) + 84 to + 88 (dried substance). Dissolve 0.250 g in anhydrous ethanol R and dilute to 25.0 m L with the same solvent.

Related substances Liquid chromatography (2.2.29).

Test solution (a) Dissolve 60.0 m g o f the substance to be exam in e d in the mobile phase and dilute to 25.0 m l . with

the mobile phase.

Test solution (b) Dilute 1.0 m L o f test solution (a) to 10.0 m L with the mobile phase.

Reference solution (a) Dissolve 5 mg o f betamethasone dipropionate for system suitability CRS (containing impurities B, C, D , E and G) in the mobile phase and dilute to 2.0 m L with the mobile phase.

Reference solution (b) Dilute 1.0 m L o f test solution (a) to 100.0 m L with the mobile phase. Dilute 1.0 m L o f this solution to 10.0 m l. with the mobile phase.

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1-284 Betamethasone Dipropionate

Reference solution (c) Dissolve 60.0 mg o f betamethasone dipropionate CRS in the mobile phase and dilute to 25.0 m L w ith the mobile phase. Dilute 1.0 m L o f the solution to 10.0 m L with the mobile phase.

Reference solution (d) Dissolve 5 mg o f betamethasone dipropionate for peak identification CRS (containing impurity H) in the mobile phase and dilute to 2.0 m L with the mobile phase. Column: — size. I = 0.10 m , 0 = 2.0 mm; — stationary phase: octadecylsiJyl silica gel for chromatography R (2.5 pm); — temperature: 20 + 2 °C.

Mobile phase M ix 35 m L o f water R and 56 m L of acetomtrUe R and allow to equilibrate; dilute to 100 m L with water R and mix. Flow rate 0.2 mL/min. Detection Spectrophotom eter at 254 nm. Injection 5 jiL o f test solution (a) and reference solutions (a), (b) and (d).

Run time 3 times the retention time o f betamethasone dipropionate.

L oss o n d ry in g (2.2.32) M aximum 1.0 per cent, determ ined on 0.500 g by drying in an oven at 105 °C. ASSAY Liquid chromatography (2.2.29) as described in the test for related substances with the following modification.

Injection T est solution (b) and reference solution (c). Calculate the percentage content o f C 28H 37F O 7 from the declared content o f betamethasone dipropionate CRS. STO R A G E Protected from light. IM P U R IT IE S

Specified impurities B, C, D , E, G , H Other detectable impurities (die following substances would, if present at a sufficient level, be detected by one or other of the tests in the monograph. They are limited by the general acceptance criterion for other/unspecified impurities and/or by the general monograph Substances for pharmaceutical use (2034). It is therefore not necessary to identify these impurities for demonstration o f compliance. See also 5.10.

Control of impurities in substances for pharmaceutical use): A , F.

Identification of impurities Use the chromatogram supplied with betamethasone dipropionate for system suitabiliiy CRS and the chromatogram obtained with reference solution (a) to identify the peaks due to impurities B, C, D , E and G; use the chrom atogram supplied with betamethasone dipropionate for peak identification CRS and the chromatogram obtained with reference solution (d) to identify the peak due to impurity H. Relative retention W ith reference to betamethasone dipropionate (retention time = about 10 min): impurity B = about 0.4; impurity C = about 0.5; impurity D = about 0.7; impurity E = about 1.2; im purity H = about 1.7; impurity G = about 2 . 1. System suitability: reference solution (a): — peak-to-valley ratio: minimum 4.0, where Hp = height above the baseline of the peak due to impurity E and Hv = height above the baseline o f the lowest point of the curve separating this peak from the peak due to betamethasone dipropionate.

Limits: — correction factors: for the calculation o f content, multiply the peak areas o f the following impurities by the corresponding correction factor, im purity G = 1.3; impurity H = 1.4; — impurity C: not m ore than 5 times the area of the principal peak in the chromatogram obtained with reference solution (b) (0.5 per cent); — impurities B, H: for each impurity, not more than 3 times the area o f the principal peak in the chromatogram obtained with reference solution (b) (0.3 per cent); — impurities D, E,