BP2016 - Vol.01

British Pharmacopoeia 2016
Volume I
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: bpcom@mhra.gsi.gov.uk
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: bpcrs@mhra.gsi.gov.uk
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
I-vii
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.
I-ix
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 Professor Kevin M G Taylor BPharm PhD FRPharmS
Professor of Clinical Pharmaceutics, UCL School of Pharmacy
Vice-Chair 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 Dr Samantha Atkinson BSc MSc PhD MRSC
Director Visiting Fellow, University of Reading
I-xvi
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 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
BIO: Biological and L Tsang (Chair), P Varley (Vice-Chair), L Bisset*, A F Bristow*, C Bums,
Biotechnological D H Calam, K Chidwick*, A Cook*, J Cook*, L Findlay*, S Gill,
Products 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)
HCM: Herbal and E Williamson (Chair), L A Anderson (Vice-Chair), P Anderson, A Bligh,
Complementary T Chapman, A Charvill, S Gibbons, K Helliwell, P Hylands, C Leon, R
Medicines Middleton, A C Moffat, B Moore, M Pires, M Rowan, K Strohfeldt-
Venables, J Sumal*, P Viner, C Welham, C Wright, K Zhao
(Corresponding members SS Handa, A Krauss, Z-T Wang)
MCI: Medicinal A G Davidson (Chair), D Cairns (Vice-Chair), M Ahmed, J C Berridge,
Chemicals M Broughton, A J Caws, P Fleming, A James, V Loh, W J Lough,
D Malpas
MC2: Medicinal G Cook (Chair), C T Goddard (Vice-Chair), M Cole, J Cowie, D Edwards,
Chemicals A Gibson, J Lim, J Miller, P Murray, J Qiu, A Ruggiero, M Turgoose,
N Wynne
(Corresponding members M Brits, W Sherwin)
MC3: Medicinal V Fenton-May (Chair), E Williamson (Vice-Chair), M Almond, S Arkle,
Chemicals 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 Granell-
Villen, 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 M G Lee (Chair), V Fenton-May (Vice-Chair), G Bennett, S Branch,
Medicines 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 K Chidwick, A R Hubbard, J More, P Varley
Products
CX: Excipients 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 C T Goddard (Chair), M Almond, S Atherton, S Boland, A C Cartwright,
General Chemicals 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 J Ballinger, J Brain, D Graham, S R Hesslewood, G Inwards, P Maltby, R
Materials D Pickett, R Smith, S Waters
VET: Veterinary E Williamson (Chair), A Coulson (Vice-Chair), A Cairns, S Cockbill,
Medicines D Evans, E Flahive, P Lees, B Ward
VIP: Veterinary A M Brady, R Banks, K Redhead, J Salt, P W Wells, R Woodland
Immunological
Products
WORKING PARTIES
AQbD: Analytical G Cook (Chair), S Brown, S Ellison, M Hanna-Brown, S Jones,
Quality by Design D Makohon, P Nethercote, E Razzano
(Corresponding member K Barnett)
DNA: Identification K Helliwell (Chair), J Hawkins, E Mee, A Slater, E Williamson
Techniques
MCS: Microscopy E Williamson (Chair), R Arroo, R Reck, K Helliwell, K MacLellan Gibson
I-xviii
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 C Howard, C Lockie-Williams
Administrative 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
2016 Introduction I-xxiii
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-commission-
triennial-review.
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.
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
Surgical Materials
Volume V Infrared Reference Spectra
Appendices
Supplementary Chapters
Index
Volume VI British Pharmacopoeia (Veterinary') 2016
Effective Date 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.
2016 Introduction I-xxv
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 As with the previous edition, the reference spectra are placed in alphabetical
Spectra 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 One new Supplementary Chapter to harmonise with the European

Chapters Pharmacopoeia was first published in the British Pharmacopoeia 2015
electronic updates. It has been consolidated in the new edition as follows.
2016 Introduction I-xxvii
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 Co-operation Agreement

Pharmacopoeia
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 It should be noted that any article intended for medicinal use which is
Requirements 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 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).
Websites 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.
2016 Introduction I-xxix
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 Therapeutic Goods A dm inistration, A ustralia The British

Collaboration 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
I-xxx Introduction 2016
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 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.
Acknowledgements 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
2016 Introduction I-xxxi
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
I-xxxii Introduction 2016
Tolterodine Tartrate*
Zanamivir Hydrate*
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*
2016 Introduction I-xxxiii
Normal Immunoglobulin for Subcutaneous Administration*
Influenza Vaccine (Live, Nasal)*
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
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

• This formulated preparation is now controlled by the monograph for Codeine Phosphate
Oral Solution.
I-xxxiv Introduction 2016

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

Abacavir Tablets Identification; Related substances
Adapalene Cream Related substances
Adapalene Gel Related substances
Alendronic Acid Tablets Dissolution; Related substances —>
Phosphate and Phosphite; Assay
Alfuzosin Tablets Identification; Related substances
Prolonged-release Alfuzosin Tablets Identification; Related substances
Aminophylline Injection Content of theophylline
Aminophylline Tablets Content of ethylenediamine
Prolonged-release Aminophylline Content of ethylenediamine; Labelling
Tablets
Carbimazole Tablets Identification (colour test deleted);
Thiamazole and other related
substances
Chloral Hydrate Oral Solution Definition
Clobazam Oral Suspension Removal of unlicensed status; Non
interchangeability statement added;
Dissolution (deleted)
Clobazam Tablets Non-interchangeability statement
added
Clonazepam Tablets Non-interchangeability statement
added
Clonidine Tablets Assay
Clotrimazole Pessaries Definition; Identification test A
Codeine Phosphate Oral Solution Definition; Extemporaneous
Preparation (deleted); Identification;
Assay
Desmopressin Tablets Uniformity of content
Dexamethasone Sodium Phosphate Content of dexamethasone phosphate
Injection -* Content of dexamethasone;
Identification; Free dexamethasone -*■
Related substances; Assay; Labelling
Prolonged-release Dipyridamole Related substances
Capsules
Dipyridamole Infusion Related substances
2 Monograph suppressed by European Pharmacopoeia Commission on-1 April 2015.

2016 Introduction I-xxxv
Doxorubicin Injection Related substances

Econazole Pessaries Definition; Identification test A;
Related substances
Enoxaparin Sodium Injection Sodium
Ergocalciferol Injection Definition; Content of ergocalciferol
Ethinylestradiol Tablets Uniformity of content
Paediatric Ferrous Sulfate Oral Extemporaneous Preparation (deleted)
Solution
Glimepiride Tablets Dissolution
Goserelin Implants Drug release; Related substances tests
A , B and C
Heparin Injection Identification tests A and B; Assay
Hydroxychloroquine Tablets Disintegration; Related substances
Hydroxyzine Oral Solution Identification test A; Related
substances; Impurities
Kaolin Mixture Extemporaneous Preparation (deleted)
Kaolin and Morphine Mixture Definition; Extemporaneous
Preparation (deleted)
Lamotrigine Tablets Non-interchangeability statement
added
Dispersible Lamotrigine Tablets Non-interchangeability statement
added
Levonorgestrel Tablets Identification test A
Lisinopril Oral Solution Related substances
Lymecycline Capsules Light-absorbing impurities (deleted);
Related substances; Water
Aromatic Magnesium Carbonate Extemporaneous Preparation (deleted)
Mixture
Magnesium Hydroxide Mixture Extemporaneous Preparation (deleted)
Magnesium Sulfate Mixture Extemporaneous Preparation (deleted)
Magnesium Trisilicate Mixture Extemporaneous Preparation (deleted)
Melphalan Injection Related substances test B
Methotrexate Injection Related substances '
Methotrexate Oral Solution Identification
Morphine Sulfate Injection Assay
Naloxone Injection Identification test A; Related
substances
Omeprazole Oral Suspension Alkalinity (deleted); Dissolution
Oxymetholone Tablets Reference to unlicensed status added
Liquid Paraffin Oral Emulsion Definition; Extemporaneous
Preparation (deleted)
Liquid Paraffin and Magnesium Definition; Extemporaneous
Hydroxide Oral Emulsion Preparation (deleted)
Paroxetine Tablets Related substances; Impurities
Phénobarbital Elixir Non-interchangeability statement
added
Paediatric Phénobarbital Oral Patient monitoring statement added
Solution
Phénobarbital Tablets Non-interchangeability statement
added
Phénobarbital Sodium Tablets Non-interchangeabUity statement
added
Phentolâmine Injection Related substances
I-xxxvi Introduction 2016
Phenytoin Capsules Non-interchangeability statement

added; Related substances
Phenytoin Oral Suspension Non-interchangeability statement
added
Phenytoin Tablets Non-interchangeability statement
added
Potassium Citrate Mixture Extemporaneous Preparation (deleted)
Prednisolone Oral Solution Related substances
Primidone Oral Suspension Non-interchangeability statement
added
Primidone Tablets Non-interchangeability statement
added
Ramipril Capsules Related substances
Ramipril Tablets Related substances
Simvastatin Oral Suspension Acidity or alkalinity (deleted)
Sodium Bicarbonate Oral Solution Labelling
Compound Sodium Chloride Extemporaneous Preparation (deleted)
Mouthwash
Sodium Chloride Oral Solution Definition; Labelling
Sodium Valproate Oral Solution Non-interchangeabUity statement
added; Identification; Related
substances
Sodium Valproate Tablets Non-interchangeability statement
added; Related substances
Gastro-resistant Sodium Valproate Non-interchangeability statement
Tablets added; Dissolution; Related substances
Sumatriptan Injection Content of sumatriptan succinate ->
Content of sumatriptan;
Identification; Impurities A and H;
Related substances; Assay
Sumatriptan Nasal Spray Impurities A and H; Related
substances; Assay
Sumatriptan Tablets Content of sumatriptan succinate —►
Content of sumatriptan;
Identification; Dissolution; Impurities
A and H; Related substances; Assay
Vinblastine Injection Labelling
Vincristine Injection Requirements for powder for re
constitution replaced by requirements
for ready-to-use solution; Labelling
Zidovudine Capsules Related substances
Zidovudine Tablets Related substances
Zidovudine and Lamivudine Tablets Related substances
Zuclopenthixol Decanoate Injection Identification

Products
Acid Gentian Mixture Extemporaneous Preparation (deleted)
Alkaline Gentian Mixture Extemporaneous Preparation (deleted)
2016 Introduction I-xxxvii
Changes in Tide 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 BRITISH PHARM A CO PO EIA

2015 2016
Medicinal and Pharm aceutical Substances

Noscapine Hydrochloride Noscapine Hydrochloride Hydrate
Sodium Alendronate Sodium Alendronate Trihydrate
Normal Immunoglobulin Normal Immunoglobulin for
Intramuscular Administration
Herbal Drugs, H erbal D rug Preparations and Herbal Medicinal

Products
Extracts Herbal Drug Extracts
2016 General Notices 1-1
General Notices
1-2 General Notices 2016
CONTENTS OF THE GENERAL NOTICES
Part I Implementation of Pharmacopoeial Methods

Italic introduction Conventional Terms
European Pharmacopoeia Interchangeable Methods
References to Regulatory Documents
Part II
Italic introduction 1.2 Other Provisions Applying to General
Official Standards Chapters and Monographs
Definition of Terms Quantities
Expression of Standards Apparatus and Procedures
Temperature Water-bath
Weights and Measures Drying and Ignition to Constant Mass
Atomic Weights Reagents
Constant Weight Solvents
Expression of Concentrations Expression of Content
Water Bath Temperature
Reagents 1.3 General Chapters
Indicators Containers
Caution Statements 1.4 Monographs
Titles Tides
Chemical Formulae Relative Atomic and Molecular Masses
Definition Chemical Abstracts Service (CAS) Registry
Production Number
Manufacture of Formulated Preparations Definition
Freshly and Recently Prepared Limits of Content
Methods of Sterilisation Herbal Drugs
Water Production
Excipients Choice of Vaccine Strain^ Choice of
Colouring Agents Vaccine Composition
Antimicrobial Preservatives Potential Adulteration
Characteristics Characters
Solubility Solubility
Identification Identification
Reference Spectra Scope
Assays and Tests First and Second Identifications
Biological Assays and Tests Powdered Herbal Drugs
Reference Substances and Reference Preparations Tests and Assays
Chemical Reference Substances Scope
Biological Reference Preparations Calculation
Storage Limits
Labelling Indication of Permitted Limit of Impurities
Action and Use Herbal Drugs
Crude Drugs; Traditional Herbal and Equivalents
Complementary Medicines Culture Media
Monograph Tide Storage
Definition Labelling
Characteristics Warnings
Control Methods Impurities
Homoeopathic Medicines Functionality-related Characteristics of
Unlicensed Medicines Excipients
Reference Standards
Part HI
Italic introduction 1.5 Abbreviations and Symbols
General Notices of the European Pharmacopoeia Abbreviations used in the Monographs on
1.1 General Statements Immunoglobulins, Immunosera and
Quality Systems Vaccines
Alternative Methods Collections of Micro-organisms
Demonstration of Compliance with the 1.6 Units of the International System (SI) used
Pharmacopoeia in the Pharmacopoeia and Equivalence
Grade of Materials with other Units
General Monographs International System of Units (SI)
Validation of Pharmacopoeial Methods Notes
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.
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
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.
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 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).
Constant Weight 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.
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 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.
Reagents 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
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 -C H 3 Bu* -CH(CH 3)CH 2CH 3

Et - c h 2c h 3 Bu" - c h 2c h 2c h 2c h 3
Pr! -CH(CH 3)2 Bur -C(CH 3)3
Pr” - c h 2c h 2c h 3 Ph - c 6h 5
Bul -C H 2CH(CH 3)2 Ac -c o c h 3
Definition 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.
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 Attention is drawn to the need to observe adequate hygienic precautions in

Formulated the preparation and dispensing of pharmaceutical formulations. The
Preparations 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:
I-10 General Notices 2016
( 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 The direction, given under the heading Extemporaneous Preparation, that a
Recently Prepared 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 The methods of sterilisation used in preparing the sterile materials

Sterilisation 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
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 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.
Colouring Agents 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 When the term ‘suitable antimicrobial preservative’ is used it is implied that
Preservatives 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 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 10,000
practically insoluble more than 10,000
The term ‘partly soluble’ is used to describe a mixture of which only

some of the components dissolve.
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
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
I
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 Methods of assay described as Suggested methods are not obligatory, but
and Tests 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 Certain monographs require the use of a reference substance, a reference

Substances and preparation or a reference spectrum. These are chosen with regard to their
Reference intended use as prescribed in the monographs of the Pharmacopoeia and
Preparations 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.
Storage 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
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.
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; Herbal and complementary medicines are classed as medicines under European
T raditional H erbal Directive 2001/83/EC as amended. It is emphasised that, although requirements
and C om plem entary for the quality of the material are provided in the monograph to assist the
M edicines 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.
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 Homoeopathic medicines are classed as medicines under European Directive

Medicines 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.
Unlicensed The General Monograph for Unlicensed Medicines applies to those

Medicines 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.
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
I
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 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.
Alternative methods 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
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
Demonstration of (1) An article is not of Pharmacopoeia quality unless it complies with all
compliance with the the requirements stated in the monograph. This does not imply that
Pharmacopoeia 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 Substances and preparations that are the subject of an individual

monographs 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.
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 The test methods given in monographs and general chapters have been
pharmac opoeial validated in accordance with accepted scientific practice and current
methods recommendations on analytical validation. Unless otherwise stated in the
monograph or general chapter, validation of the test methods by the analyst
is not required.
Implementation o f When implementing a pharmacopoeial method, the user must assess

pharmac opoeial whether and to what extent the suitability of the method under the actual
methods conditions of use needs to be demonstrated according to relevant
monographs, general chapters and quality systems.
Conventional terms 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.
Interchangeable Certain general chapters contain a statement that the text in question is
methods 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
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 Monographs and general chapters may contain references to documents

regulatory issued by regulatory authorities for medicines, for example directives and
documents 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 Volumetric glassware complies with Class A requirements of the appropriate
procedures 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.
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 The terms ‘dried to constant mass’ and ‘ignited to constant mass’ mean
to constant mass 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 In defining content, the expression ‘per cent’ is used according to

content 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.
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 Monograph titles are in English and French in the respective versions and
there is a Latin subtitle.
Relative Atomic and The relative atomic mass (A r) or the relative molecular mass (Mr) is shown,
Molecular Masses 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.
Chemical Abstracts CAS registry numbers are included for information in monographs, where
Service (CAS) applicable, to provide convenient access to useful information for users.
Registry Number 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
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 Due to the increasing number of fraudulent activities and cases of

Adulteration 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

per gram of solute
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 10 000
Practically insoluble more than 10 000

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
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 non-
compliance 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
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.
Functionality- Monographs on excipients may have a section on functionality-related

Related characteristics. The characteristics, any test methods for determination and
Characteristics of any tolerances are not mandatory requirements; they may nevertheless be
Excipients relevant for use of the excipient and are given for information (see also
section 1.1. General statements).
Reference Certain monographs require the use of reference standards (chemical

Standards 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.
1.5. ABBREVIATIONS AND SYMBOLS
A Absorbance mp M elting point

*1 per cent Specific absorbance r,20 Refractive index
A \ cm
At Relative atom ic mass Ph. Eur. U. European Pharm acopoeia U nit
Md Specific optical rotation PPb Parts per billion (micrograms per kilogram)
bp Boiling point ppm Parts per million (milligrams p er kilogram)
BRP Biological Reference Preparation R Substance or solution defined under
CRS Chemical Reference Substance 4. Reagents
j20 Rf Retardation factor (see chapter 2.2.46)
“20 Relative density
X W avelength R:t U sed in chrom atography to indicate the ratio
o f the distance travelled by a substance to the
HRS H erbal reference standard distance travelled by a reference substance
IU International U nit RV Substance used as a prim ary standard in
M M olarity volumetric analysis (chapter 4.2.1)
Mr Relative m olecular mass
Abbreviations used in the monographs on imm unoglobulins, immunosera and vaccines
L D 50 T h e statistically determ ined quantity o f a Lo/10 dose T h e largest quantity of a toxin that, in the
substance that, when administered by the conditions of the test, when mixed with 0.1 IU
specified route, may be expected to cause the o f antitoxin and adm inistered by the specified
death o f 50 p er cent of the test animals within route, does not cause symptoms of toxicity in
a given period 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
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 CCIDso T h e statistically determ ined quantity o f virus
of antitoxin and administered by the specified th at may be expected to infect 50 p er cent of
route, causes th e death of the test anim als the cell cultures to which it is added
within a given period
EID50 T h e statistically determ ined quantity o f virus
L + dose T he smallest quantity o f a toxin that, in the th at may be expected to infect 50 per cent of
conditions of th e test, when mixed with 1 IU fertilised eggs into which it is inoculated
of antitoxin an d adm inistered by the specified ID 50 T h e statistically determ ined quantity of a virus
route, causes the death of the test animals that may be expected to infect 50 per cent of
within a given period the animals into which it is inoculated
lr/100 dose T he smallest quantity o f a toxin that, in the T h e statistically determ ined dose of a vaccine
P D 50
conditions o f the test, when mixed with that, in the conditions of the test, may be
0.01 IU of antitoxin and injected expected to protect 50 per cent o f the animals
intracutaneously causes a characteristic against a challenge dose of the micro
reaction at the site o f injection within a organisms or toxins against which it is active
given period
EDsn T h e statistically determ ined dose of a vaccine
L p/10 dose T h e smallest quantity o f toxin that, in the that, in the conditions of the test, may be
conditions o f the test, when mixed w ith 0.1 IU expected to induce specific antibodies in
of antitoxin an d adm inistered by the specified 50 per cent o f the animals for the relevant
route, causes paralysis in the test anim als vaccine antigens
within a given period
PFU Pock-forming units or plaque-form ing units
SPF Specified-pathogen-free.
Collections of micro-organisms
A TCC American Type Culture Collection NCTC N ational Collection of Type Cultures
10801 University Boulevard C entral Public Health Laboratory
Manassas, Virginia 20110-2209, USA Colindale Avenue
London NW9 5HT, Great Britain
C.I.P. Collection de Bactéries de l’Institut Pasteur
B.P. 52, 25 rue du D octeur Roux NCYC N ational Collection of Yeast C ultures
75724 Paris Cedex 15, France A FRC Food Research Institute
Colney Lane
IM I International Mycological Institute
Norwich NR4 7UA, Great Britain
Bakeham Lane
Surrey T W 20 9TY, Great Britain N IT E Biological Resource Center
D epartm ent of Biotechnology
I.P . Collection Nationale de Culture de
N ational Institute o f Technology and
Microorganismes (C.N .C.M .)
Evaluation
Institut Pasteur
2-5-8 Kazusakamatari, Kisarazu-shi, Chiba,
25, rue du D octeur Roux
292-0818
75724 Paris Cedex 15, France
Japan
N C IM B N ational Collection of Industrial and M arine
S.S.I. Statens Serum Institut
Bacteria Ltd
80 Amager Boulevard, Copenhagen, D enm ark
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
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.
Notes 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.
The second is the duration of 9 192 631 770 periods of the radiation corresponding
Time t second s 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 T kelvin K The kelvin is the fraction 1/273.16 of the thermodynamic temperature of the triple
temperature 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.
Table 1.6.-2. - SI units used in the European Pharmacopoeia and equivalence with other units
Quantity Unit
Name Name Symbol Expression b SI Expression in other Conversion of other units into SI units
Symbol
base units SI units
Wave number V one per metre 1/m m '1
pm
3° 3"
Wavelength X micrometre
o o
nanometre nm
Area A, S square metre m2 mJ
Volume V cubic metre m3 m3 1 mL = 1 cm3 = 10'6 m3
Frequency V hertz Hz s '1
Density P kilogram per cubic kg/m3 kg-m"3 1 g/mL = 1 g/cm3 =■ 103 kg-m'3
metre
Velocity V metre per second m/s m-s'1
Force F newton N m-kg-s"5 1 dyne = 1 g-cm-s'3 = 10*s N

1 kp = 9.806 65 N
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 V pascal second Pa-s m'^kg-s'1 N-s-m'2 1 P - 10'1Pa-s - 10'1N-s-m'1
viscosity 1 cP =* 1 mPa-s
Kinematic V square metre per m2/ s m2-s*‘ Pa-s-n^-kg-1 1 St = 1 cm2-s'1* 10'* m2-s'’
viscosity second N-m-s-kg"1
Energy W joule J m2-kg-s"2 N-m 1 erg - 1 cm2-g-s‘2 = 1 dyne-cm = 10'7J
l e a l» 4.1868 J
Power P watt W m2-kg-s'3 N-m-s"1 1 e r g / s - 1 dyne-cm-s'1=
Radiant flux J-s"1 10'7 W = 10'7 N-m-s'1* 10-7J-s'1
Absorbed dose D gray Gy m^s"2 /•kg-1 1 rad » lO'2 Gy
(of radiant
energy)
Electric U volt V m2- kg-s'3-A*‘ W-A*1
potential,
electromotive
force
Electric R ohm Q m2- kg-s'3-A'2 V-A-*
resistance
Quantity of Q coulomb c A-s
electricity
Activity of a A becquerel Bq s '1 1 Ci - 37-109 Bq = 37-109 s '1
radionuclide
Concentration c mole per cubic mol/m3 mol-m'3 1 m o l/L * 1 M * 1 mol/dm5 - 103 mol-m'3
(of amount of metre
substance),
molar
concentration
Mass P kilogram per cubic kg/m3 kg-m'3 1 g/L = 1 g/dm3» 1 kg-m'3
concentration metre
Table 1.6.-3. - Units used with the International System

Quantity Unit Value in SI units
Name Symbol
Time 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
Mass tonne t 1 t » 103 kg
Rotational revolution r/m in 1 r/min =* (1/60) s*1

frequency per minute
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
106 mega M 10-9 nano n
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
2016 General Monographs 1-37
T h e manufacture o f active substances m ust take place under

MEDICINAL AND PHARMACEUTICAL conditions o f good manufacturing practice.
SUBSTANCES 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:
Substances ★ ★ — is a recom binant protein or another substance obtained as
★ ★ a direct gene product based on genetic modification,
for Pharmaceutical Use ***** where applicable, the substance also complies with the
(Ph. Eur. monograph 2034) requirements o f the general monograph Products of
recombinant DNA technology (0784)’,
PhEur------ -----------------------------------------------------------------------------—
— is obtained from animals susceptible to transmissible
D E F IN IT IO N spongiform encephalopathies other than by experimental
Substances for pharmaceutical use are any organic or challenge, where applicable, the substance also complies
inorganic substances that are used as active substances or with the requirements of the general monograph Products
excipients for the production of medicinal products for with risk of transmitting agents of animal spongiform
hum an or veterinary use. They may be obtained from natural encephalopathies (1483)',
sources or produced by extraction from raw materials, — is a substance derived from a fermentation process,
fermentation or synthesis. whether or n o t the micro-organisms involved are modified
T his general m onograph does not apply to herbal drugs, by traditional procedures or recom binant D N A (rDNA)
herbal drugs for homoeopathic preparations, herbal drug technology, where applicable, the substance also complies
preparations, extracts, or m other tinctures for homoeopathic with the requirements o f the general monograph Products
preparations, which are the subject of separate general offermentation (1468).
monographs (Herbal drugs (1433), Herbal drugs for I f solvents are used during production, they are of suitable
homoeopathic preparations (2045), Herbal drug quality. In addition, their toxidty and their residual level are
preparations (1434), Extracts (0765), Mother tinctures for taken into consideration (5.4). If water is used during
homoeopathic preparations (2029)). It does not apply to raw production, it is o f suitable quality.
materials for homoeopathic preparations, except where there I f substances are produced or processed to yield a certain
is an individual m onograph for the substance in the non- form or grade, that specific form or grade of the substance
homoeopathic p art of the Pharmacopoeia. complies with the requirements of the monograph. Certain
Where a substance for pharmaceutical use not described in functionality-related tests may be described to control
an individual monograph o f the Pharmacopoeia is used in a properties that may influence the suitability of the substance
medicinal product prepared for the special needs of and subsequently the properties o f dosage forms prepared
individual patients, the need for compliance with the present from it.
general monograph is decided in the light o f a risk Powdered substances May be processed to obtain a certain
assessment that takes account o f the available quality o f the degree of fineness (2.9.35).
substance and its intended use.
Compacted substances Are processed to increase the particle
W here medicinal products are manufactured using size or to obtain particles o f a specific form and/or to obtain
substances for pharmaceutical use of hum an or animal origin, a substance with a higher bulk density.
the requirements o f chapter 5.1.7. Viral safety apply.
Coated active substances Consist o f particles of the active
Substances for pharmaceutical use may be used as such or as substance coated with one or more suitable excipients.
starting materials for subsequent formulation to prepare
Granulated active substances Are particles of a specified size
medicinal products. Depending on the formulation, certain
and/or form produced from the active substance by
substances may be used either as active substances or as
granulation directly or with one or more suitable excipients.
excipients. Solid substances may be compacted, coated,
granulated, pow dered to a certain fineness, or processed in I f substances are processed with excipients, these excipients
other ways. A m onograph is applicable to a substance comply with the requirements of the relevant monograph or,
processed with an excipient only where such processing is where no such monograph exists, the approved specification.
mentioned in the definition section of the monograph. W here active substances have been processed with excipients
Substance for pharmaceutical use of special grade Unless to produce, for example, coated or granulated substances, the
otherwise indicated or restricted in the individual processing is carried out under conditions of good
monographs, a substance for pharmaceutical use is intended manufacturing practice and the processed substances are
for hum an and veterinary use, and is of appropriate quality regarded as intermediates in the manufacture of a medicinal
for the m anufacture o f all dosage forms in which it can be p ro d u ct
used. CHARACTERS
Polymorphism Individual monographs do not usually specify T h e statements under the heading Characters
crystalline or am orphous forms, unless bioavailability is (e.g. statements about the solubility or a decomposition
affected. All forms of a substance for pharmaceutical use point) are n o t to be interpreted in a strict sense and are not
comply with the requirements of the monograph, unless requirements. T hey are given for information.
otherwise indicated. W here a substance may show polymorphism, this may be
P R O D U C T IO N stated under Characters in order to draw this to the attention
Substances for pharm aceutical use are manufactured by o f the user who may have to take this characteristic into
procedures that are designed to ensure a consistent quality consideration during formulation o f a preparation.
and comply with the requirements of the individual
m onograph or approved specification.
1-38 General Monographs 2016
ID E N T IF IC A T IO N T he requirem ents above do n o t apply to biological and

W here under Identification an individual monograph biotechnological products, oligonucleotides,
contains subdivisions entitled ‘First identification’ and radiopharmaceuticals, products of ferm entation and semi
‘Second identification’, the test or tests th a t constitute the synthetic products derived therefrom, to crude products of
‘First identification’ may be used in all circumstances. animal or plant origin or herbal products.
T he test or tests that constitute the ‘Second identification’ F o r active substances in a new application for a medicinal
may be used in pharmacies provided it can be dem onstrated p roduct for hum an use, the requirem ents o f the guideline on
that the substance or preparation is fully traceable to a batch the limits o f genotoxic impurities and the corresponding
certified to comply with all the other requirem ents o f the questions and answers docum ents published on the website
monograph. o f the European Medicines Agency (or similar evaluation
C ertain monographs give two o r m ore sets of tests for the principles for non-E uropean U nion m em ber states) m ust be
purpose of the first identification, which are equivalent and followed.
may be used independently. O ne o r m ore o f these sets R e sid u a l so lv en ts
usually contain a cross-reference to a test prescribed in the are limited according to the principles defined in chapter 5.4,
Tests section o f the m onograph. It may be used to simplify using general m ethod 2.4.24 or another suitable method.
the work of the analyst carrying out the identification and the W here a quantitative determ ination o f a residual solvent is
prescribed tests. For example, one identification set cross- carried out and a test for loss on drying is n o t carried out,
refers to a test for enantiomeric purity while the other set the content o f residual solvent is taken into account for
gives a test for specific optical rotation: the intended purpose calculation o f the assay content o f the substance, the specific
of the two is the same, that is, verification that the correct optical rotation and the specific absorbance.
enantiom er is present.
M ic ro b io lo g ic a l q u a lity
TESTS Individual monographs give accqptance criteria for
P o ly m o rp h is m (5.9) microbiological quality wherever such control is necessary.
If the nature o f a crystalline or am orphous form imposes T able 5.1.4.-2. - Acceptance criteria for microbiological quality of
restrictions on its use in preparations, the nature o f the rum-sterile substances for pharmaceutical use in chapter 5.1.4.
specific crystalline or am orphous form is identified, its Microbiological quality of non-sterUe pharmaceutical preparations
morphology is adequately controlled and its identity is stated and substances for pharmaceutical use gives recom mendations
on the label. on microbiological quality th a t are o f general relevance for
R e la te d su b s ta n c e s substances subject to microbial contamination. D epending on
Unless otherwise prescribed or justified and authorised, the nature of the substance and its intended use, different
organic impurities in active substances are to be reported, acceptance criteria may be justified.
identified wherever possible, and qualified as indicated in S te rility (2. 6 .I)
T able 2034.-1 or in Table 2034.-2 for peptides obtained by I f intended for use in the m anufacture o f sterile dosage forms
chemical synthesis. w ithout a further appropriate sterilisation procedure, o r if
offered as sterile grade, the substance for pharmaceutical use
Table 2034.-1. - Reporting, identification and qualification of complies with the test for sterility.
organic impurities in active substances B a c te ria l e n d o to x in s ('2.6.14)
Use Maximum Report Identification Qualification I f offered as bacterial endotoxin-free grade, the substance for
daily ing threshold threshold pharm aceutical use complies with the test for bacterial
dose threshold
endotoxins. T h e limit and test m ethod (if n o t gelation
Human £ 2 g/day > 0.05 per > 0.10 per > 0.15 per
use or cent cent or a cent or a m ethod A) are stated in the individual monograph. T h e limit
human daily intake daily intake is calculated in accordance with the recom mendations in
and of > 1.0 mg of > 1.0 mg general chapter 5.1.10. Guidelines for using the test for bacterial
veterinary (whichever is (whichever is
use the lower) the lower) endotoxins, unless a lower limit is justified from results from
Human > 2 g/day > 0.03 per > 0.05 per cent > 0.05 per production batches or is required by the com petent authority.
use or cent cent W here a test for bacterial endotoxins is prescribed, a test for
human
and pyrogens is n o t required.
veterinary
use
P y ro g e n s (2.6.8)
Veterinary Not > 0.10 per > 0.20 per cent > 0.50 per I f the test for pyrogens is justified rather than the test for
use only applicable cent cent 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
Table 2034.-2. - Reporting, identification and qualification of individual m onograph or approved by the competent
organic impurities in peptides obtained by chemical synthesis authority. Based on appropriate test validation for bacterial
endotoxins and pyrogens, the test for bacterial endotoxins
Reporting Identification Qualification
may replace the test for pyrogens.
threshold threshold threshold
> 0.1 per cent > 0.5 per cent > 1.0 per cent 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
Specific thresholds m ay be applied for impurities known to individual m anufacturing processes or formulations. Grades
be unusually potent or to produce toxic o r unexpected (such as sterile, endotoxin-free, pyrogen-free) may be
pharmacological effects. produced with a view to m anufacture o f preparations for
If the individual m onograph does n o t provide suitable control parenteral administration or other dosage forms and
for a new im purity, a suitable test for control must be
developed and included in the specification for the substance.
2016 Abacavir Sulfate 1-39
appropriate requirements may be specified in an individual Content

monograph. 99.0 per cent to 101.0 per cent (anhydrous substance).
ASSAY CHARACTERS
Unless justified and authorised, contents of substances for Appearance
pharmaceutical use are determined. Suitable methods are W hite or almost white powder.
used. Solubility
L A B E L L IN G Soluble in water, practically insoluble in ethanol
In general, labelling is subject to supranational and national (96 per cent) and in methylene chloride.
regulation and to international agreements. T h e statements IDENTIFICATION
under the heading Labelling therefore are n o t comprehensive A. Infrared absorption spectrophotometry (2.2.24).
and, moreover, for the purposes of the Pharmacopoeia only
Comparison abacavir sulfate CRS.
those statements that are necessary to demonstrate
compliance or non-compliance with the monograph are B. Enantiomeric purity (see Tests).
mandatory. Any other labelling statements are included as C. Solution S (see Tests) gives reaction (a) of sulfates
recommendations. W hen the term ‘label' is used in the (2.3.1).
Pharmacopoeia, the labelling statements may appear on the TESTS
container, the package, a leaflet accompanying the package or Solution S
a certificate of analysis accompanying the article, as decided Dissolve 0.250 g in water R and dilute to 25.0 m L with the
by the com petent authority. same solvent
Where appropriate, the label states that the substance is:
Enantiomeric purity
— intended for a specific use;
Liquid chromatography (2.2.29).
— of a distinct crystalline form;
— of a specific degree of fineness;
Solution A Mix 0.5 m L of trifluoroacenc acid R and 100 mT. of
— compacted;
methanol R.
— coated; Solution B Mix 30 volumes of methanol R, 30 volumes of 2-
— granulated; propanol R and 40 volumes of heptane R.
— sterile; Test solution Dissolve 40 mg of the substance to be examined
— free from bacterial endotoxins; in 30 m L of solution A. Sonicate until dissolution is
— free from pyrogens; complete. Add 30 m L of 2-propanol R and dilute to
— containing gliding agents. 100.0 m L with heptane R.
W here applicable, the label states: Reference solution (a) Dissolve 2 m g of abacavirfor system
— the degree o f hydration; suitability CRS (containing impurities A and D) in 1.5 m L of
— the nam e and concentration of any excipient. solution A. Sonicate until dissolution is complete.
__________________________________________________________ Ph Eur 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 Column:
*. * — size: I = 0.25 m , 0 = 4.6 mm;
(Ph Eur monograph 2589) *
— 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)\
Time Mobile phase A Mobile phase B

(min) (per cent V/V) (per cent V/V)
C28H 38N 120 6S 671 188062-50-2 0 -2 5 100 0
A ctio n a n d u se 25 - 27 100 -»0 0 100

Nucleoside reverse transcriptase inhibitor; antiviral (HIV). 27 - 37 0 100
P re p a r a tio n s
Abacavir Oral Solution
Flow rate 1.0 mL/min.
Abacavir Tablets
Detection Spectrophotometer at 286 nm.
Abacavir, Zidovudine and Lamivudine Tablets
Injection 20 nL.
Abacavir and Lamivudine Tablets
Identification of impurities Use the chromatogram supplied
PhEir __________________________________________________________ with abacavir for system suitability CRS and the chromatogram
obtained with reference solution (a) to identify the peaks due
D E F IN IT IO N
to impurities A and D.
Bis[[(l5,4R)-4-[2-amino-6-(cyclopropylamiiio)-9i/-purin-9-
yl]cyclopent-2-enyl]methanol] sulfate.
1-40 Abacavir Sulfate 2016
Relative retention W ith reference to abacavir (retention — total: n o t more than 5 times the area o f the principal peak
time = about 17 min): im purity D = about 0.8; in the chrom atogram obtained with reference solution (b)
im purity A = about 0.9. (0.5 per cent);
System suitability: reference solution (a): — disregard limit. 0.5 times the area o f the principal peak in
— resolution: minim um 1.5 betw een the peaks due to the chrom atogram obtained with reference solution (b)
impurities D and A; m inim um 1.5 between the peaks due (0.05 per cent).
to impurity A and abacavir. H eav y m e ta ls (2.4.8)
Limit. M aximum 20 ppm.
— impurity A: not m ore than 3 times the area o f the 12 m L of solution S complies with test A. Prepare the
principal peak in the chrom atogram obtained with reference solution using 2 m L of lead standard solution
reference solution (b) (0.3 p er cent). (1 ppm Pb) R.
Related substances W a te r (2.5.32)
Liquid chromatography (2.2.29). Prepare the solutions M aximum 0.5 p er cent, determined on 60.0 mg.
immediately before use and transfer them to low-adsorption, inert
S u lfa te d a s h (2.4.14)
glass vials.
M aximum 0.2 per cent, determined on 1.0 g.
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. A SSAY
Sonicate until dissolution is complete. Dissolve 0.300 g in 50 m L of water R. T itrate with 0.1 M
Reference solution (a) Dissolve 2.5 mg o f abacavir for peak sodium hydroxide, determining the end-point
potentiometrically (2 . 2 . 2 0 ).
identification CRS (containing impurities B and D ) in
10.0 m L of water R. 1 m L of 0.1 M sodium hydroxide is equivalent to 33.54 mg of
Reference solution (b) D ilute 1.0 m L of the test solution to C 28H 38N 12O 6S.
100.0 m L with water R. D ilute 1.0 m L o f this solution to IM P U R IT IE S
10.0 m L w ith water R. Specified impurities: A , B.
Column: Other detectable impurities (the following substances would, if
— size. I = 0.15 m , 0 = 3.9 m m ; present at a sufficient level, be detected by one or other o f
— stationary phase: end-capped octadecylsQyl silica gelfor the tests in the monograph. They are limited by the general
chromatography R (5 pm); acceptance criterion for other/unspecified impurities and/or
— temperature: 30 °C. by the general m onograph Substances for pharmaceutical use
Mobile phase: (2034). It is therefore n o t necessary to identify these
— mobile phase A: dilute 0.5 m L of trifluoroacedc acid R in impurities for dem onstration o f compliance. See also 5.10.
1000 m L o f water R ; Control of impurities in substances for pharmaceutical use): C, D,
— mobile phase B: water R, methanol R (15:85 V!V)\ E, F.

(min) (per cent V7V) (per cent V/V)
0 -5 95 5
5 - 25 95 + 70 5 + 30
25 -4 0 70->10 30 + 90

Detection Spectrophotom eter at 254 nm . A. [(li?,45)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-
yl] cydopent-2-enyl] methanol,
Irqection 20 pL.
with abacavir for peak identification CRS and the
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 nh 2
Hv = height above the baseline o f the lowest point o f the
curve separating this peak from the peak due to abacavir. B. 6-(cyclopropylamino)-9-[(li?,4S)-4-[[(2,5-diam ino-6-
Limits: chloropyrimidin-4-yi)oxy]methyI] cyclopent-2-enyl] -9H-
— impurity B: not m ore than twice the area o f the principal purine-2-am ine,
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);
2016 Acacia 1-41
NH2 CHARACTERS
Acacia is almost completely b u t very slowly soluble, after
HjNr x >N 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).
C. [(15j4R )-4-(2,6-diarnino-9/f-purin-9-yl)cyclopent-2-
IDENTIFICATION
enyl] m ethanol,
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.
^ nI^ " >
h2na n In the centre o f an unbroken tear there is sometimes a small
cavity.
H
"Oc OH
B. Reduce to a powder (355) (2.9.12). T he pow der is white
or yellowish-white. Examine under a microscope using a
D. [(1 i?34i?)-4 -[2-amino-6-(cyclopropylainino)-9/f-purin-9- 50 per cent V/V solution of glycerol R. T h e pow der shows the
yl] cyclopent-2-enyl] methanol, 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
^N H 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.
H—r ^ \ ^ H N o other im portant zocies are visible, particularly in the
upper p art o f the chromatogram.
D . Dissolve 1 g o f the powdered herbal drug (355) (2.9.12)
E. [(li?,35)-3-[2-amino-6-(cyclopropylamino)-9/f-purin-9- in 2 m L of water R by stirring frequently for 2 h. Add 2 m L
yl] cyclopentyl] methanol, 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.
NH TESTS
Solution S
H2NJ¿C> N 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.
"O<>x 0H
h í‘ h 3c ch3
Insoluble matter
M aximum 0.5 per cent.
T o 5.0 g o f the powdered herbal drug (355) (2.9.12) add
F. 6-(cyclopropylamino)-9- [(li?,45)-4- [ [(1,1 - 100 m L o f water R and 14 m L of dilute hydrochloric add R,
dimethylethyl)oxy] methyl] cyclopent-2-enyl] -9jFí-purine-2- boil gently for 15 min, shaking frequently and filter while hot
amine. through a tared sintered-glass filter (2.1.2). W ash with hot
PhEur water R and dry at 100-105 °C. The residue weighs a
maximum o f 25 mg.
Glucose and fructose
Thin-layer chromatography (2.2.27).
Acacia ★★+ ★★ 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
(Ph. Ever, monograph 0307) ***** 100 g/L solution of trifluoroacetic acid R, shake vigorously to
dissolve the forming gel, stopper the tube and heat the
Action and use mixture at 120 °C for 1 h. Centrifuge the hydrolysate,
Bulk-forming laxative; excipient transfer the clear supernatant carefully into a 50 m L flask,
W hen Powdered Acacia is prescribed or demanded, material add 10 m L of water R and evaporate the solution to dryness
complying with the requirements below with the exception of under reduced pressure. To the resulting clear film add
Identification test A shall be dispensed or supplied. 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
PhEur. necessary, to 1 m L with methanol R.
DEFINITION Reference solution Dissolve 10 mg of arabinose R, 10 mg of
Air-hardened, gummy exudate flowing naturally from or galactose R, 10 mg of glucose R, 10 mg o f rhamnose R and
obtained by incision of the trunk and branches o f Acacia 10 mg of xylose R in 1 m L of water R and dilute to 10 m L
Senegal L. Willd. (syn. Senegalia Senegal (L.) Britton), other with methanol R.
species of Acacia of African origin and Acacia seyal Delile.
1-42 Acacia 2016
Plate TUG silica gel plate R. part of the monograph since they also represent mandatory quality
Mobile phase 16 g/L solution of sodium dihydrogen phosphate R, criteria. In such cases, a cross-reference to the tests described in the
butanol R , acetone R (10:40:50 VIV/V). mandatory part is included in the Functionality-related
Application 10 ^L as bands. characteristics section. Control of the characteristics can contribute
to the quality of a medicinal product by improving the consistency
Development A Over a path o f 10 cm. of the manufacturing process and the performance of the medicinal
Drying A In a current o f warm air for a few minutes. product during use. Where control methods are cited, they are
Development B Over a path o f 15 cm using the same mobile recognised as being suitable for the purpose, but other methods can
phase. also be used. Wherever results for a particular characteristic are
Drying B A t 110 °C for 10 m in. reported, the control method must be indicated.
Detection treat with amsaldehyde solution R and heat at 110 °C The following characteristic may be relevant for acacia used as a
for 10 min. viscosity-increasing agent andlor suspending agent in aqueous
Results T he chromatogram obtained with the reference preparations.
solution shows 5 clearly separated coloured zones due to Apparent viscosity
galactose (greyish-green or green), glucose (grey), arabinose D eterm ine the dynamic viscosity using a capillary viscometer
(yellowish-green), xylose (greenish-grey or yellowish-grey) (2.2.9) or a rotating viscometer (2.2.10) on a 100 g/L
and rham nose (yellowish-green), in order of increasing RF solution o f acacia (dried substance).
value. T he chrom atogram obtained with the test solution ___________________________________________________________ PhEur
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 Spray-dried Acacia ******
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 (Ph Eur monograph 0308) *
develops. PhEur___________________________________________________________
S te rc u lia g u m D E F IN IT IO N
A. Place 0.2 g of the powdered herbal drug (355) (2.9.12) in Spray-dried acacia is obtained from a solution of acacia.
a 10 m L ground-glass-stoppered cylinder graduated in-
CHARACTERS
0.1 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 mT- It dissolves completely and rapidly, after about 20 m in, in
twice its mass of water. T h e liquid obtained is colourless or
B. T o 1.0 g of the powdered herbal drug (355) (2.9.12) add
yellowish, dense, viscous, adhesive, translucent and weakly
100 m L of water R and shake. Add 0.1 m L of methyl red
a d d to blue litmus paper. Spray-dried acada is practically
solution R. N o t m ore than 5.0 m L o f 0.01 M sodium hydroxide
insoluble in ethanol (96 p er cent).
is required to change the colour o f the indicator.
ID E N T IF IC A T IO N
T a n n in s
T o 10 m L o f solution S add 0.1 m L o f ferric chloride A. Examined under a microscope, in ethanol (96 per cent) R,
solution R l. A gelatinous precipitate is formed, bu t neither the the powder is seen to consist predom inantly o f spheroidal
precipitate nor the liquid are dark blue. particles about 4-40 (jm in diameter, w ith a central cavity
containing 1 or several air-bubbles; a few m inute flat
T ra g a c a n th
fragments are p resen t Only traces o f starch granules are
Examine the chromatograms obtained in the test for glucose visible. N o vegetable tissue is seen.
and fructose.
B. Examine the chromatograms obtained in the test for
Results T he chrom atogram obtained with the test solution glucose and fructose.
shows no greenish-grey or yellowish-grey zone corresponding
to the zone of xylose in the chrom atogram obtained with the
Results T h e chrom atogram obtained with the test solution
shows 3 zones due to galactose, arabinose and rham nose.
reference solution.
N o other im portant zones are visible, particularly in the
L oss o n d ry in g (2.2.32) upper p art o f the chromatogram.
M axim um 15.0 p er cent, determ ined on 1.000 g o f the
C. Dissolve 1 g of the drug to be examined in 2 m L of
pow dered herbal drug (355) (2.9.12) by drying in an oven at
water R by stirring frequently for 20 min. A dd 2 m L of
105 °C.
ethanol (96 per cent) R. After shaking a white gelatinous
T o ta l a sh (2.4.16) m udlage is formed which becomes fluid on adding 10 m L of
M axim um 4.0 per cent. water R.
M ic ro b ia l c o n ta m in a tio n TESTS
T A M C : acceptance criterion 104 C FU /g (2.6.12).
S o lu tio n S
TY M C : acceptance criterion 104 C FU /g (2.6.12). Dissolve 3.0 g o f th e drug to be examined in 25 m L of
Absence o f Escherichia cod (2.6.13). water R by stirring for 10 min. Allow to stand for 20 m in and
Absence o f Salmonella (2.6.13). dilute to 30 m L with water R.
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 G lu co se a n d fru c to se
This section provides information on characteristics that are
recognised as being relevant control parameters for one or more Test solution T o 0.100 g in a thick-walled centrifuge tube add
functions of the substance when used as an excipient (see chapter 2 m L o f a 100 g/L solution o f trifluoroacetic acid R, shake
5.15). Some of the characteristics described in the Functionality- vigorously to dissolve the forming gel, stopper the tube and
related characteristics section may also be present in the mandatory h eat the mixture at 120 °C for 1 h. Centrifuge the
hydrolysate, transfer the clear supernatant carefully, into a
2016 Acamprosate Calcium 1-43
50 m L flask, add 10 m L o f water R and evaporate to dryness Absence of Salmonella (2.6.13).

under reduced pressure. T o the resulting clear film add 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
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
recognised as being relevant control parameters for one or more
necessary, to 1 m L with methanol R.
functions of the substance when used as an excipient (see chapter
Reference solution Dissolve 10 mg o f arabinose R, 10 m g of 5.15). This section is a non-mandatory part of the monograph
galactose R, 10 m g o f glucose R, 10 m g of rhamnose R and and it is not necessary to verify the characteristics to demonstrate
10 mg o f xylose R in 1 m l. of water R and dilute to 10 m L compliance. Control of these characteristics can however contribute
with methanol R. to the quality of a medicinal product by improving the consistency
Plate TLC silica gel plate R. of the manufacturing process and the performance of the medicinal
Mobile phase 16 g/L solution o f sodium dihydrogen phosphate R, product during use. Where control methods are cited, they are
butanol R , acetone R (10:40:50 V/V/V). recognised as being suitable for the purpose, but other methods can
Application 10 pL as bands. also be used. Wherever results for-a particular characteristic are
reported, the control method must be indicated.
Development A Over a path o f 10 cm.
The following characteristic may be relevantfor spray-dried acacia
Drying A In a current of warm air for a few minutes. used as a viscosity-increasing agent and/or suspending agent in
Development B Over a path of 15 cm using the same mobile aqueous preparations.
phase.
A p p a re n t v isco sity
Drying B At 110 °C for 10 min. Determine the dynamic viscosity using a capillary viscometer
Detection Spray with ardsaldehyde solution R and heat at (2.2.9) or a rotating viscometer (2.2.10) on a 100 g/L
110 °C for 10 min. solution of spray-dried acacia (dried substance).
Results T h e chromatogram obtained with the reference PhEur
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 Acamprosate Calcium ★ ★
shows n o grey zone and no greyish-green zone between the *****
(Ph Eur monograph 1585)
zones corresponding to galactose and arabinose in the
chrom atogram obtained w ith the reference solution.
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 Ca2+
0.1 m L of 0.05 M iodine. N o blue or reddish-brown colour J2
develops.
Sterculia gum C io H ^ C a N ^ A ^ 400.5 77337-73-6
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 A ctio n a n d u se
(60 per cent VIV) R and shake. Any gel formed occupies not Treatm ent o f alcoholism.
more than 1.5 mL.
P re p a r a tio n
B. T o 1.0 g add 100 m L o f water R and shake. Add 0.1 m L Gastro-resistant Acamprosate Tablets
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 PhEur__________________________________
the indicator. D E F IN IT IO N
T a n n in s Calcium bis[3-(acetylamino)propane-l-sulfonate].
T o 10 m L of solution S add 0.1 m L o f ferric chloride C o n te n t
solution R l. A gelatinous precipitate is formed, b u t neither the 98.0 per cent to 102.0 per cent (dried substance).
precipitate nor the liquid shows a dark blue colour.
CHARACTERS
Tragacanth
A p p e a ra n c e
Examine the chromatograms obtained in the test for Glucose
White or almost white powder.
and fructose.
Results T h e chromatogram obtained with the test solution Solubility
shows no greenish-grey o r yellowish-grey zone corresponding Freely soluble in water, practically insoluble in ethanol
to the zone of xylose in the chromatogram obtained with the (96 per cent) and in methylene chloride.
reference solution. ID E N T IF IC A T IO N
L oss o n d ry in g (2.2.32) A. Infrared absorption spectrophotometry (2.2.24).
M axim um 10.0 per cent, determined on 1.000 g by drying in Comparison Ph. Eur. reference spectrum of acamprosate calcium.
an oven at 105 °C. B. It gives reaction (a) o f calcium (2.3.1).
T o ta l a s h C2.4.16) TESTS
M axim um 4.0 per c e n t
S o lu tio n S
M ic ro b ia l c o n ta m in a tio n Dissolve 5.0 g in carbon dioxide-free water R and dilute to
T A M C : acceptance criterion 104 CFU/g (2.6.12). 100 m L with the same solvent.
T Y M C : acceptance criterion 102 C FU/g (2.6.12). A p p e a ra n c e o f so lu tio n
Absence of Escherichia colt (2.6.13). Solution S is clear (2.2.1) and colourless (2 .2 .2 , Method II).
1-44 Acarbose 2016
p H (2.2.3) 0.1 M sodium hydroxide, determining the end-point

5.5 to 7.0 for solution S. potentiometrically (2.2.20).
Im p u rity A 1 m L of 0.1 M sodium hydroxide corresponds to 20.02 m g of
Liquid chromatography (2.2.29). CioH20CaN2O8S2.
Test solution Dissolve 0.40 g of the substance to be examined IM P U R IT IE S
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 H2N^ V / S O , H
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. A. 3-aminopropane- 1-sulfonic a d d (homotaurine).
Add 0.15 m L of a freshly prepared 5 g/L solution o f
__________________________________________________________ PhEur
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. Acarbose *****
Reference solution Dissolve 50 m g o f acamprosate *. *
impurity A CRS in distilled water R and dilute to 200.0 m L (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 OH OH OH OH
chromatography R (5 pm) with a specific surface area of
170 m 2/g and a pore size o f 12 nm .
C25H43N018 646 56180-94-4
Mobile phase acetomtrUe R, methanol R, 0.1 M phosphate buffer
solution pH 6.5 R (10:10:80 VIVIV). A ctio n a n d u se
Flow rate 1 mL/min. Alpha-glucosidase inhibitor; treatm ent o f diabetes mellitus.
Detection Spectrophotom eter at 261 nm . PhEur__________________________________________________________
Irgection 20 |iL.
D E F IN IT IO N
Run time 6 times the retention time o f impurity A 0-4,6-Dideoxy-4- [ [(1 S,4R,5S, 6 S)-4,5,6-trihydroxy-3-
Retention times Fluorescamine = about 4 min; (hydroxymethyl) cyclohex-2-enyl] amino] -a-D-glucopyrano syl-
im purity A = about 8 min; acamprosate is not detected by (1-+ 4)-0-a-D-glucopyranosyl-( 1-*4)-D-glucopyranose, which
this system. is produced by certain strains of Actinopkmes utahensis.
Limits: C o n te n t
— impurity A: not m ore than the area of the corresponding 95.0 per cent to 102.0 per cent (anhydrous substance).
peak in the chromatogram obtained with the reference
solution (0.05 per cent). CHA RACTERS
A p p e a ra n c e
H eav y m e ta ls (2.4.8)
W hite or yellowish, hygroscopic, amorphous powder.
M aximum 10 ppm.
Dissolve 2.0 g in distilled water R and dilute to 20 m L with S o lubility
the same solvent. 12 m L o f the solution complies with test A. Very soluble in water, soluble in methanol, practically
insoluble in methylene chloride.
Prepare the reference solution using 10 m L of lead standard
solution (1 ppm Pb) R. ID E N T IF IC A T IO N
L oss o n d ry in g ('2.2.32) A. Infrared absorption spectrophotometry (2.2.24).
M aximum 0.4 per cent, determ ined on 1.000 g by drying in Comparison acarbose for identification CRS.
an oven at 105 °C. B. Examine the chromatograms obtained in the assay.
A SSAY Results T he prindpal peak in the chromatogram obtained
T o 4 g of cation-exchange resin R (75-150 pm) add 20 m L of with the test solution is similar in retention time and size to
disnUed water R and stir magnetically for 10 min. Introduce the prindpal peak in the chromatogram obtained with
this suspension into a glass column, 45 cm long and 2.2 cm reference solution (a).
in internal diameter, equipped with a polytetrafluoroethylene TESTS
flow cap covered by a glass-wool plug. Allow a few millilitres
S o lu tio n S
of this solution to flow, then place a plug o f glass wool over
Dissolve 1.00 g in carbon dioxide-free water R and dilute to
the resin. Pass 50 m L o f 1 M hydrochloric acid through the
20.0 m L with the same solvent.
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 p H (2.2.3)
eluate at p H 6. Dissolve 0.100 g o f the substance to be 5.5 to 7.5 for solution S.
examined in 15 m L of distilled water R. Pass through the S pecific o p tic a l ro ta tio n (2.2.7)
colum n and wash with 3 quantities, each o f 25 m l , o f + 168 to + 183 (anhydrous substance).
distilled water R, collecting the eluate. Allow to elute until an D ilute 2.0 m L o f solution S to 10.0 m L with water R.
eluate at p H 6 is obtained. Titrate the solution obtained with
A b so rb a n c e (2.2.25)
M aximum 0.15 at 425 nm for solution S.
2016 Acarbose 1-45
Related substances chromatogram obtained with reference solution (c)

Liquid chrom atography (2.2.29). (0.3 per cent);
Test solution Dissolve 0.200 g of the substance to be — impurity E: n o t more than 0.2 times the area of the
examined in water R and dilute to 10.0 m L with the same principal peak in the chromatogram obtained with
solvent. reference solution (c) (0.2 per cent);
— arty other impurity: for each impurity, n o t more than
Reference solution (a) Dissolve the contents of a vial of
0.2 times the area o f the principal peak in the
acarbose CRS in 5.0 m L of water R.
chromatogram obtained with reference solution (c)
Reference solution (b) Dissolve the contents of a vial of (0.2 p er cent);
acarbose for peak identification CRS (acarbose containing — total: n o t more than 3 times the area o f the principal peak
impurities A, B, C , D , E, F and G) in 1 m L of water R. in the chromatogram obtained with reference solution (c)
Reference solution (c) Dilute 1.0 m L of the test solution to (3.0 p er cent);
100.0 m L with water R. — disregard limit. 0.1 times the area of the prindpal peak in
Column: the chromatogram obtained with reference solution (c)
— size: I = 0.25 m , 0 = 4 mm; (0.1 per cent).
— stationary phase: aminopropylsUyl silica gel for H eav y m e ta ls (2.4.8)
chromatography R (5 pm); M aximum 20 ppm.
Dissolve 1.5 g in water R and dilute to 15 m L with the same
Mobile phase M ix 750 volumes of acetomtrUe R1 and solvent. If the solution is not clear, cany out prefiltration and
250 volumes of a solution containing 0.60 g/L of potassium use the filtrate. 10 m L complies with test E. Prepare the
dihydrogen phosphate R and 0.35 g/L o f disodium hydrogen reference solution using 20 m L o f lead standard solution
phosphate dihydrate R. (1 ppm Pb) R.
Flow rate 2.0 mL/min. W a te r (2.5.12)
Detection Spectrophotom eter at 210 nm. M aximum 4.0 per cent, determined on 0.300 g.
Injection 10 pL o f the test solution and reference solutions (b) S u lfa te d a s h (2.4.14)
and (c). M aximum 0.2 per cent, determined on 1.0 g.
Run time 2.5 times the retention time of acarbose.
A SSAY
Identification of impurities Use the chromatogram supplied Liquid chromatography (2.2.29) as described in the test for
with acarbose for peak identification CRS and the related substances with the following modification.
chromatogram obtained with reference solution (b) to
Injection T est solution and reference solution (a).
identify the peaks due to impurities A, B, C , D, E, F and G.
Calculate the percentage content o f C 25H 43N O i 8 taking into
Relative retention W ith reference to acarbose (retention
account the assigned content o f acarbose CRS.
time = about 16 min): impurity D = about 0.5;
impurity B = about 0.8; impurity A = about 0.9; STORA GE
impurity C = about 1.2; impurity E = about 1.7; In an airtight container.
impurity F = about 1.9; impurity G = about 2.2.
IM P U R IT IE S
System suitability, reference solution (b): Specified impurities A, B, C, D , E, F , G
— the chromatogram obtained is similar to the
chromatogram supplied with acarbose for peak
Other detectable impurities (the following substances would, if
present at a sufficient levd, be detected by one or other of
identification CRS;
— peak-to-vaRey ratio: minimum 1.2, where Hp — height the tests in the monograph. T hey are limited by the general
acceptance criterion for other/unspecified impurities. It is
above the baseline of the peak due to impurity A and
therefore n o t necessary to identify these impurities for
demonstration of compliance. See also 5.10. Control of
curve separating this peak from the peak due to acarbose.
impurities in substances for pharmaceutical use): H.
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) A. 0-4,6-dideoxy-4-[[(15,4i?,5«S,66)-4,5,6-trihydroxy-3-
(1.0 per cent); (hydroxymethyl) cyclohex-2 -enyl] amino] -a-r>-
— impurity A: n o t m ore than 0.6 times the area of the glucopyranosyl-(l -►4)-0-a-D-glucopyranosyl-( 1 -> 4 )-d -
principal peak in the chromatogram obtained with arafono-hex-2 -ulopyranose,
— impurity B: not m ore than 0.5 times the area o f the
— impurities F, G: for each impurity, not more than
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-2-
en yl]am in o]-a-D -gIu cop yran osyl-(l -> 4 )-0 -a -D -
glucopyranosyl-(l ->4)-0 -a-D-glucopyranoside (a -D -
glucopyranosyl a-acarboside).
C. a-D-glucopyranosyl 4-0-[4,6-dideoxy-4-[[(15,4R,55,65)- H . 0 -4,6-dideoxy-4-[[(15,4i?,55,65)-4,5,6-trihydroxy-3-

4,5,6-trihydroxy-3-(hydroxymethyi)cydohex-2- (hydroxymethyl)cyclohex-2-enyl] amino] - a - D -
enyl] am ino]-a-D -glu cop yran osyl]-a-D -glucopyranosid e, glucopyranosyl-( 1-*■4)-0 -6-deoxy-a-D-glucopyranosyl-
(1 ->4)-D-glucopyranose.
ho ch HO PhEur
Acebutolol Hydrochloride ★★*★★

OH OH 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-D-
glucopyranosyl] -D -glucopyranose,
G )T 0H
lo \ — * OH
and enantiomer
Ci{1H29CIN2O4 3 72.9 34381-68-5

O X"—f Ö x —- f °
OH OH OH A ctio n a n d u se
Beta-adrenoceptor antagonist.
E. 0 -4 ,6-dideoxy-4- [[(15,4i?,55, 65)-4,5,6-trihydroxy-3- P re p a ra tio n s
(hydroxymethyi)cyclohex-2-enyI] amino] -a-D - Acebutolol Capsules
glucopyranosyl-(l -►4)-0-a-D-giucopyranosyl-(l -> 4 )-0 -a- Acebutolol Tablets
D-glucopyranosyl-(l ->4)-D-anzÄmo-hex-2-ulopyranose
PhEur.
(4-0-a-acarbosyl-D-fhictopyranose),
DEFINITION
HO HO. HO HO N- [3-Acetyl-4- [(2J?5)-2-hydroxy-3-
[(1 -methyletbyl) amino] propoxy] phenyl] butanamide
hydrochloride.
OH
C o n te n t
99.0 per cent to 101.0 per cent (dried substance).
OH OH OH OH OH
CHARACTERS
F. 0 -4,6-dideoxy-4- [[(15,4i?,55,65)-4,5,6-trihydroxy-3- A p p e a ra n c e
(hydroxymethyl) cyclohex-2-enyl] amino] -om> W hite or alm ost white, crystalline powder.
glucopyranosyl-(l -»4)-0 -a-D-glucopyranosyl-(l ->4)-0-a- S olu b ility
D -g lu co p y ra n o sy l-(l -*■4)-D-glucopyranose (4-0 -a- Freely soluble in w ater and in ethanol (96 per cent), very
acarb osyl-D -glu cop yran ose), slightly soluble in acetone and in-methylene chloride.
2016 Acebutolol Hydrochloride 1-47
mp mobile phase A Dilute 0.5 m L o f this solution to 50.0 m L

A bout 143 °C. with mobile phase A.
ID E N T IF IC A T IO N Reference solution (b) Dissolve the contents of a vial of
First identification B, D. acebutolol impurity I CRS in 1.0 m L of mobile phase A.
Second identification A , C, D. Reference solution (c) M ix 2.0 m L o f reference solution (a)
and 1.0 m L of reference solution (b) and dilute to 10.0 m L
A. Ultraviolet and visible absorption spectrophotometry
with mobile phase A.
(2.2.25).
Reference solution (d) Dissolve 5.0 m g o f acebutolol
Test solution Dissolve 20.0 mg in a 0.1 per cent V/V solution
impurity C CRS in 10 m L of acetomtrûe R and dilute to
of hydrochloric acid R and dilute to 100.0 m L with the same
25.0 m L with mobile phase A. D ilute 0.5 m L of this solution
acid solution. Dilute 5.0 m L of this solution to 100.0 m L
to 50.0 m L with mobile phase A.
with a 0.1 per cent V/V solution of hydrochloric add R.
Reference solution (e) Dissolve 5.0 m g o f acebutolol
Spectral range 220-350 nm.
impurity B CRS in 10.0 m L of acetonitrile R and dilute to
Absorption maxima At 233 nm and 322 nm. 25.0 m L with mobile phase A. D ilute 1.0 m L of this solution
Specific absorbance at the absorption maximum 555 to 605 at 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,
Preparation Discs. — stationary phase:, end-capped octadecylsüyl silica gel for
Comparison acebutold hydrochloride CRS. chromatography R (5 pm),
C. Thin-layer chromatography (2.2.27).
Mobile phase:
Test solution Dissolve 20 mg of die substance to be examined — mobile phase A: mix 2.0 m L of phosphoric add R, and
in methanol R and dilute to 20 m L with the same solvent. 3.0 m L of triethylamine R and dilute to 1000 m L with
Reference solution (a) Dissolve 20 mg of acebutolol water R;
hydrochloride CRS in methanol R and dilute to 20 m L with the — 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 Mobile phase B
Mobile phase A
T o 1 m L o f this solution add 1 m L of reference solution (a). (min) (per cent V/V) (per cent V/V)
Plate TLC silica gel F 254 plate R. 0-2 98 2
Mobile phase perchloric add R, methanol R, water R 2 - 30.5 98-» 10 2 -> 90
(5:395:600 V/V/V).
30.5 - 41 10 90
Application 10 pL.
Development Over 3/4 of the plate.
Drying In air. Flow rate 1.2 mlVmin.
Detection Examine in ultraviolet light at 254 nm. Detection Spectrophotom eter at 240 nm .
System suitability T he chromatogram obtained with reference Injection 25 pL.
solution (b) shows 2 clearly separated principal spots. System suitability: reference solution (c):
Results T h e principal spot in the chromatogram obtained with — resolution: minim um 7.0 between the peaks due to
the test solution is similar in position and size to the principal im purity I and acebutolol.
spot in th e chromatogram obtained with reference Limits:
solution (a). — impurity B: n o t m ore than the area of the principal peak
D . It gives reaction (a) of chlorides (2.3.1). in the chromatogram obtained with reference solution (e)
(0.2 per cent);
TESTS — impurity C: not m ore than the area o f the principal peak
A p p e a ra n c e o f so lu tio n in the chromatogram obtained with reference solution (d)
T he solution is not more opalescent than reference (0.1 per cent);
suspension II (2.2.1) and not more intensely coloured than — impurity I: not m ore than twice the area of the principal
reference solution BY 5 (2.2.2, Method II). peak in the chromatogram obtained with reference
Dissolve 0.5 g in water R and dilute to 10 m L with the same solution (a) (0.2 p er cent);
solvent. — any other impurity: for each impurity, not more than the
p H (2.2.3) area of the principal peak in the chromatogram obtained
5.0 to 7.0. 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)
20 m L w ith the same solvent.
(0.5 per cent);
R e la te d su b s ta n c e s — disregard limit. 0.5 times the area of the principal peak in
Liquid chromatography (2.2.29). the chromatogram obtained with reference solution (a)
Test solution Dissolve 0.100 g of die substance to be (0.05 per cent).
examined in mobile phase A and dilute to 50.0 m L with H eav y m e ta ls (2.4.8)
mobile phase A. M axim um 20 ppm.
Reference solution (a) Dissolve 20.0 m g of the substance to be Dissolve 0.50 g in 20.0 m L of water R. T h e solution
examined in mobile phase A and dilute to 100.0 m L with 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) F. R = OH: N- [3-acetyl-4- [(2&S)-2,3-
M aximum 0.1 per cent, determined on 1.0 g. dihydroxypropoxy]phenyl]butanamide,
A SSAY I. R = NH-CH2-CH3: N - [3-acetyl-4- [(2J?S)-3-(ethylamino)-
Dissolve 0.300 g in 50 m L o f ethanol (96 per cent) R and add 2-hydroxypropoxy]phenyl]butanamide,
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 H3C C H 3 H3C
OH T OH
volume added between the 2 points o f inflexion.
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 G . NyN'-[[( 1-methylethyl)imino]bis[(2-hydroxypropane-l,3-
Specified impurities A, B, C, D , E, F, G , H , I, J, K. diyl) oxy(3-acetyl-1,4-phenylene)]] dibutanam ide (biamine),
O and enantiomer
A N - [3-acetyl-4- [(2i?3)-oxiran-2-
ylmethoxy]phenyl]butanamide, H . NyN'-[(2-hydroxypropane-l,3-diyl)bis[oxy(3-acetyl-1,4-
phenylene)]]dibutanamide.
R2 H OH PhEur
° ^ X ^ N CH3
j and enantiomer
R1. 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 (Ph Eicr monograph 1281) *****
(diacetolol),
o co 2h
D . R I = H , R2 = CO-CH3: 1- [5-amino-2-[(2&S)-2-
hydroxy-3-[( 1-methyiethyl)amino]propoxy]phenyI] ethanone,
E. R I = C O -C H 2-C H 2-C H 33 R2 = H : N-[4-[(2i?5)-2-
o a
hydroxy-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- C16H13a 2N04 354.2 89796-99-6
[( 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- A ctio n a n d u se
[(2i?5)-2-hydroxy-3- Cyclo-oxygenase inhibitor; analgesic; anti-inflammatory.
[( 1-methylethyl) amino] propoxy]phenyI]butanamide,
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).
CHARACTERS
C. N-(3-acetyl-4-hydroxyphenyI)butanamide, 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).
First identification B.
2016 Aceclofenac 1-49
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
(2.2.25).
Test solution Dissolve 50.0 mg in methanol R and dilute to 25 - 30 50 -» 20 50 -» 80
100.0 m L with the same solvent. Dilute 2.0 m L o f the 30 - 50 20 80
solution to 50.0 m L with methanol R.
Spectral range 220-370 nm. Flow rate 1.0 mL/min.
Absorption maximum At 275 nm. Detection Spectrophotom eter at 275 nm.
Specific absorbance at the absorption maximum 320 to 350. Injection 10 jiL o f the test solution and reference
solutions (c), (e), (f) and (g).
B. Infrared absorption spectrophotometry (2.2.24).
Comparison: Ph. Eur. reference spectrum of aceclofenac.
with aceclofenac for peak identification CRS and the
C . Dissolve about 10 mg in 10 m L of ethanol (96 per cent) R. chromatogram obtained witinreference solution (g) to
T o 1 m L o f the solution, add 0.2 m L of a mixture, prepared identify the peaks due to impurities B, C, D , E and G.
immediately before use, o f equal volumes o f a 6 g/L solution
Relative retention W ith reference to aceclofenac (retention
o f potassium ferricyanide R and a 9 g/L solution of ferric
time = about 11 min): impurity A = about 0.8;
chloride R. Allow to stand protected from light for 5 min.
impurity G = about 1.3; impurity H = about 1.5;
A dd 3 m l, of a 10.0 g/L solution of hydrochloric acid R. Allow
impurity I = about 2.3; impurity D = about 3.1;
to stand protected from light for 15 min. A blue colour
impurity B = about 3.2; impurity E = about 3.3;
develops and a precipitate is formed.
impurity C = about 3.5; impurity F = about 3.7.
TESTS System suitability: reference solution (c):
Related substances — resolution: minim um 5.0 between the peaks due to
Liquid chromatography (2.2.29). Prepare the solutions impurity A and aceclofenac.
immediately before use. Limits:
Solvent mixture M obile phase A, mobile phase B (30:70 V/V). — impurity A: n o t more than the area o f the corresponding
Test solution Dissolve 50.0 mg o f the substance to be peak in the chromatogram obtained with reference
examined in the solvent mixture and dilute to 25.0 m L with solution (c) (0.2 per cent);
the solvent mixture. — impurities B, C, D, E, G: for each impurity, no t more than
Reference solution (a) Dissolve 21.6 mg of diclofenac the area of the peak due to aceclofenac in the
sodium CRS (impurity A) in the solvent mixture and dilute to chrom atogram obtained with reference solution (e)
50.0 m L with the solvent mixture. (0.2 per cent);
— impurity F: n o t more than the area o f the corresponding
peak in the chromatogram obtained with reference
10.0 m L with the solvent mixture.
solution (e) (0.2 per cent);
Reference solution (c) Mix 1.0 m L o f reference solution (a) — impurity H: n o t more than the area o f the corresponding
and 1.0 m L o f reference solution (b) and dilute to 100.0 m L peak in the chromatogram obtained with reference
w ith the solvent mixture. solution (e) (0.1 per cent);
Reference solution (d) Dissolve 4.0 mg of aceclofenac — impurity I: no t more than the area of the corresponding
impurity F CRS and 2.0 mg of aceclofenac impurity H CRS in peak in the chromatogram obtained with reference
the solvent mixture, then dilute to 10.0 m L with the solvent solution (f) (0.1 per cent);
mixture. — unspecified impurities: not more than 0.5 times die area of
Reference solution (e) Mix 1.0 m L of reference solution (b) the peak due to aceclofenac in the chromatogram
and 1.0 m L o f reference solution (d) and dilute to 100.0 m L obtained with reference solution (e) (0.10 per cent);
with the solvent mixture. — total: n o t m ore than 0.7 per cent;
Reference solution (j) Dissolve the contents of a vial of — disregard Ixmitr. 0.1 times the area of the peak due to
diclofenac impurity A CRS (aceclofenac impurity I) in 1.0 m L aceclofenac in the chromatogram obtained with reference
o f the solvent mixture, add 1.5 m L of the solvent mixture solution (e) (0.02 per cent).
and mix, Heavy m etals ( 2.4.8)
Reference solution (g) Dissolve 4 mg of aceclofenac for peak M aximum 10 ppm .
identification CRS (containing impurities B, C, D , E and G) T o 2.0 g in a silica crucible, add 2 m L of sulfuric acid R to
in 2.0 m L o f the solvent mixture. wet die substance. H eat progressively to ignition and
Column: continue heating until an almost white or at m ost a greyish
— size: I = 0.25 m , 0 = 4.6 mm; residue is obtained. Carry out the ignition at a tem perature
— stationary phase: spherical end-capped octadecylsüyl silica gel not exceeding 800 °C. Allow to cool. Add 3 m L of
for chromatography R (5 pm) with a pore size o f 10 n m hydrochloric acid R and 1 m L o f nitric acid R. H eat and
and a carbon loading of 19 per cent; evaporate slowly to dryness. Cool and add 1 m L of a 100 g/L
— temperature'. 40 °C. solution o f hydrochloric acid R and 10.0 m L o f cUsaHed
Mobile phase: water R. N eutralise with a 1.0 g/L solution of ammonia R
— mobile phase A: 1.12 g/L solution of phosphoric acid R using 0.1 m L o f phenolphthalein solution R as indicator.
adjusted to p H 7.0 with a 42 g/L solution of sodium Add 2.0 m L of a 60 g/L solution of anhydrous acetic acid R
hydroxide R, and dilute to 20 m L with distilled water R. 12 m L of the
— mobile phase B: water R, acetonitrüe R (10:90 ViV)\ solution complies with test A. Prepare the reference solution
using lead standard solution (1 ppm Pb) R.
1-50 Aceclofenac 2016
o
M aximum 0.5 per cent, determ ined on 1.000 g by drying in
an oven at 105 °C.
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 E. ethyl [[[2-[(2,6-
potentiometrically (2 . 2 . 2 0 ). dichlorophenyl)amino]phenyl] acetyl] oxy] acetate (ethyl ester
1 m L of 0.1 M sodium hydroxide is equivalent to 35.42 mg o f o f aceclofenac),
C 16H 13CI2N O 4.
STORAGE o
IM P U R IT IE S
Specified impurities A, B, C, D , E, F , G, H , I
F. benzyl [[[2-[(2,6-
dichlorophenyl)amino]phenyI]acetyl]oxy]acetate (benzyl ester
o f aceclofenac),
O
A [2-[(2,6-dichlorophenyi)amino]phenyI] acetic acid
(diclofenac).
G. [[[[[2-[(2,6-
dichlorophenyl)amino]phenyl]acetyI] oxy] acetyl] oxy]acetic
acid (acetic aceclofenac),
B. methyl [2-[(2j6-dichlorophenyl)amino]phenyl]acetate
(methyl ester of diclofenac),
o
^ Y ^ r 0' ^ o / v 0 ^ ' c ° 2H
H . [ [[ [[[[2-[(2,6-dichlorophenyl)amino]phenyl] acetyl] oxy]

C. ethyl [2-[(2,6-dichlorophenyl)amino]phenyl]acetate (ethyl
acetyl]oxy]acetyl]oxy]acetic a d d (diacetic acedofenac),
ester o f diclofenac),
• OCT^
1. 1-(2 ,6-dichlorophenyl)-1,3-dihydro-2/f-indol-2-one.
PhEur
D . methyl [[[2-[(2,6-
dichlorophenyl)amino]phenyl]acetyl]oxy]acetate (methyl ester
of aceclofenac),
2016 Acemetacin 1-51
★ ★ — temperature: 40 °C.
Acemetacin ★ ★ Mobile phase:
(Ph Eitr monograph 1686) ***** — 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]

(min) (percent V/V) (per cent V/V)
0-5 95 5
C jxH wCINO î 415.8 53164-05-9 5 -9 95->65 5 -»35

9 - 16 65 35
A c tio n a n d u se
16-28 65 -»20 35 -»80
Cyclo-oxygenase inhibitor; analgesic; anti-inflammatory.
28 - 34 20 80
PhEur_______________________________ ___________________________
DEFINITION
[ [[ l-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3- Flow rate 1.0 mTVmin.
yl] acetyl] oxy] acetic ad d . Detection Spectrophotom eter at 235 nm.
C o n te n t Injection 20 pL.
99.0 per cent to 101.0 per cent (dried substance). Identification of impurities:
CHARACTERS — use the chromatogram supplied with acemetacin
A p p e a ra n c e impurity mixture CRS and the chromatogram obtained
Yellow or greenish-yellow, crystalline powder. with reference solution (e) to identify the peaks due to
impurities C, D , E and F;
Solubility — use the chromatogram obtained with reference
Practically insoluble in water, soluble in acetone, slightly solution (b) to identify the peak due to impurity B.
soluble in anhydrous ethanol.
Relative retention W ith reference to acemetacin (retention
It shows polymorphism (5.9). tim e = about 15 min): impurity A = about 0.7;
IDENTIFICATION impurity B = about 0.9; impurity F = about 1.2;
Infrared absorption spectrophotometry (2.2.24). impurity C = about 1.3; impurity D = about 1.5;
Comparison acemetacin CRS. impurity E = about 2.2.
I f the spectra obtained in the solid state show differences, System suitability Reference solution (d):
dissolve the substance to be examined and the reference — peak-to-valley ratio: minimum 15, where Hp = htight
substance separately in acetone R, evaporate to dryness and above the baseline of the peak due to impurity B and
record new spectra using the residues. Hv = height above the baseline of the lowest point o f the
curve separating this peak from the peak due to
TESTS acemetacin.
R e la te d su b sta n c e s Limits:
Liquid chromatography (2.2.29). — correction factors: for the calculation of content, multiply
Test sohaion Dissolve 0.100 g of the substance to be the peak areas o f the following impurities by the
examined in acetonitrUe for chromatography R and dilute to corresponding correction factor: impurity C = 1.3;
20.0 m L with the same solvent. impurity D = 1.4; impurity F = 1.3;
Reference solution (a) Dilute 5.0 mT. of the test solution to — impurity E: no t more than 3 times the area of the
50.0 m L with acetonitrUe for chromatography R. Dilute 1.0 m L p rindpal peak in the chromatogram obtained with
o f this solution to 100.0 m L with acetonitrUe for reference solution (a) (0.3 per cent);
chromatography R. — impurity B: not more than the area of the mi-responding
Reference sohaion (b) Dissolve 5.0 mg of acemetacin peak in the chromatogram obtained with reference
impurity A CRS and 10.0 m g o f indometacin CRS solution (c) (0.2 per cent);
(impurity B) in acetonitrUe for chromatography R, and dilute to — impurity A: not more than the area of the corresponding
50.0 m L with the same solvent. peak in the chromatogram obtained with reference
solution (c) (0.1 per cent);
Reference solution (c) Dilute 1.0 m L o f reference solution (b)
— impurities C, D, F: for each impurity, not more than the
to 20.0 m L with acetomtrUe for chromatography R.
area o f the prindpal peak in th e chromatogram obtained
Reference sohaion (d) T o 1 m L of reference solution (b), add with reference solution (a) (0.1 per cent);
10 m L o f the test solution and dilute to 20 m L with — unspecified impurities: for each impurity, not more than the
acetomtrUefor chromatography R. area o f the prindpal peak in the chromatogram obtained
Reference sohaion (e) Dissolve the contents o f a vial of with reference solution (a) (0.10 per cent);
acemetacin impurity mixture CRS (containing impurities C, D , — total: n o t more than 4 times the area of the principal peak
E and F) in 1.0 m L o f the test solution. in the chromatogram obtained with reference solution (a)
Column: (0.4 per cent);
— size: I = 0.25 m , 0 = 4 mm; — disregard limit. 0.5 times the area of the prindpal peak in
— stationary phase: spherical end-capped octadecylsUyl siHca gel the chromatogram obtained w ith reference solution (a)
for chromatography R (5 pm); (0.05 p er cent).
1-52 Acenocoumarol 2016
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.
an oven at 105 °C.
M aximum 0.1 per cent, determined on 1.0 g. and enantiomer
A SSAY
Dissolve 0.350 g in 20 m L o f acetone R and add 10 m L of c 19h 15n o 6 353.3 152-72-7
water R. T itrate with 0.1 M sodium hydroxide, determining the
end-point potentiometrically (2 . 2 . 2 0 ). A ctio n a n d use
Vitamin K epoxide reductase inhibitor; oral anticoagulant.
1 m L o f 0.1 M sodium hydroxide is equivalent to 41.58 mg
o f C 2iH i8C 1N 06. P re p a r a tio n
Acenocoumarol Tablets
STORA GE
Protected from light. D E F IN IT IO N
IM P U R IT IE S Acenocoumarol is (R.S)-4-hydroxy-3-( 1-p-nitrophenyl-3-
Specified impurities A, B, C, D , E, F oxobutyl)coumarin. It contains not less than 98.5% and not
more than 100.5% o f C 19H15N 0 6, calculated with reference
COjH to the dried substance.
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,
T he infrared absorption spectrum, Appendix II A, is concordant
with the reference spectrum of acenocoumarol (RS 001). If the
R3
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.
B. R1 = R2 = R3 = H : [l-(4-chlorobenzoyl)-5-methoxy-2- Prepare a new spectrum o f the residue.
methylindol-3-yl]acetic acid (indom etadn),
TESTS
C. R1 = Cl, R2 = H , R3 = C H 2 -C 0 2H: [[[l-(3,4-
C la rity a n d c o lo u r o f so lu tio n
dichlorobenzoyl)-5-methoxy-2-methyl-l.H'-indol-3- A. A 2.0% w/v solution in acetone is clear, Appendix IV A.
yl] acetyl] oxy] acetic acid,
B. T he absorbance of a 4-cm layer of a 2.0% w/v solution in
D. R1 = H , R2 = C (C H 3)3, R3 = C H 2 -C 0 2H : [[[1-
acetone at 460 nm is not more than 0.12, Appendix II B.
(4-chlorobenzoyl)-6-( 1,1 -dimethylethyl)-5-methoxy-2-methyl-
1H-indol-3-yl] acetyl] oxy] acetic ad d , C. A 2.0% w/v solution in 0.1m sodium hydroxide is dear,
Appendix IV A, and yellow.
E. R1 = R2 = H , R3 = C H 2 -C 0 -0 -C (C H 3) 3:
1,1 -dimethylethyl [[[1 -(4-chlorobenzoyl)-5-methoxy-2- L ig h t a b so rp tio n
methyl- l/i-indol-3-yl] acetyl] oxy]acetate, 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
F. R1 = R2 = H , R3 = C H 2- C 0 -0 - C H 2- C 0 2H: [[[[[1-
the maximum at 306 nm, 0.50 to 0.54, calculated with
(4-chlorobenzoyl)-5-methoxy-2-methjd-l/i-indol-3-
reference to the dried substance, Appendix II B.
yl] acetyl] oxy] acetyl] oxy] acetic ad d .
R e la te d su b sta n c e s
__________________________________________________________ PhEur
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.
2016 Acesulfame Potassium 1-53
(e) After removal o f the plate, allow it to dry in air and Reference solution (a) Dissolve 5 mg o f acesulfame
im m ediately examine under ultraviolet light (254 nm). potassium CRS in water R and dilute to 5 m L with the same
solvent.
M O BILE PHASE
20 volumes of glacial acetic acid, 50 volumes of cychhexane Reference solution (b) Dissolve 5 mg o f acesulfame
and 50 volumes o f cHchloromethane.
potassium CRS and 5 m g o f saccharin sodium R in water R and
dilute to 5 m L with the same solvent.
LIMITS
Plate cellulose for chromatography R as the coating substance.
Any secondary spot in the chromatogram obtained with
Mobile phase concentrated ammonia R, acetone R, ethyl acetate R
solution (1) is n o t more intense than the spot in the
(10:60:60 V/V/V).
chrom atogram obtained with solution (2) (0.1%).
Application 5 pL as bands.
L oss o n d ry in g
W hen dried to constant weight at 105°, loses not more than Development Twice over 2/3 of the plate.
0.5% o f its weight. U se 1 g. Drying In a current o f warm air.
S u lfa te d ash Detection Examine in ultraviolet light at 254 nm.
N o t m ore than 0.1% , Appendix IX A. System suitability: reference solution (b):
— the chromatogram shows 2 clearly separated zones.
A SSA Y
Dissolve 0 .6 g in 5 0 m L of acetone and titrate with
Results T he principal zone in the chromatogram obtained
with the test solution is similar in position and size to the
0 .1 m sodium hydroxide VS using bromothymol blue solution R3
prindpal zone in the chromatogram obtained with reference
as indicator. Repeat the operation without the substance
solution (a).
being examined. T h e difference between the titrations
represents the am ount of sodium hydroxide required. Each C. 0.5 m L o f solution S (see Tests) gives reaction (b) of
mT. o f 0 .1 m sodium hydroxide KS is equivalent to 3 5 .3 3 m g of potassium (2.3.1).
C 19H 15N CV TESTS
S o lu tio n S
50 m L with the same solvent.
Acesulfame Potassium ****** 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).
Test solution Dissolve 0.80 g of the substance to be examined
C4H4KNO4S 201.2 55589-62-3
in water R and dilute to 10 m L with the same solvent
A c tio n a n d use Reference solution (a) Dissolve 50 m g o f acetylacetamide R
Sweetening agent. (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
PhEur________________________________________________________ __ dilute to 100 m L with methanol R.
D E F IN IT IO N Reference solution (b) T o 10 m L of reference solution (a) add
Potassium 6-methyl-1,2,3-oxathiazin-4-olate 2,2-dioxide. 1 m L o f the test solution and dilute to 20 m L with
C o n te n t methanol R.
99.0 per cent to 101.0 per cent (dried substance). Plate TLC silica gel plate R.
CHARACTERS Mobile phase water R, ethanol (96 per cent) R, ethyl acetate R
(2:15:74 V/VIV).
A p p e a ra n c e
W hite or almost white, crystalline powder or colourless Application 5 piL.
crystals. Development Over 2/3 o f the plate.
S o lu b ility Drying In air until the solvents are completely removed.
Soluble in water, very slightly soluble in acetone and in Detection Spray with phosphoric vanillin solution R and heat at
ethanol (96 per cent). 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. solution (a) shows a clearly visible spot and the
chromatogram obtained with reference solution (b) shows
Second identification B, C.
2 clearly separated spots.
A. Infrared absorption spectrophotometry (2.2.24).
Limit.
Comparison acesulfame potassium CRS. — impurity A: any spot due to im purity A is not more
B. Thin-layer chromatography (2.2.27). intense than the spot in the chromatogram obtained with
Test solution Dissolve 5 mg of the substance to be examined reference solution (a) (0.125 per cent).
in water R and dilute to 5 m L with the same solvent. I m p u rity B
1-54 Acetazolamide 2016
Test solution Dissolve 0.100 g o f die substance to be IMPURITIES

examined in water R and dilute to 10.0 m L with the same Specified impurities: A , B.
solvent.
Reference solution (a) Dissolve 4.0 m g o f acesulfame potassium H3C\ ^ 0
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
A. 3-oxobutanamide (acetylacetamide),
examined in reference solution (a) and dilute to 10.0 m L
with the same solution.
h 3c . .0 ^ »
Column: V s=o
— sizer. I = 0.25 m, 0 = 4.6 mm; ,NH
— stationary phase: octadecylsQyl silica gel for chromatography R ° A r ’
(3 nm).
Mobile phase M ix 40 volumes o f acetomtrUe R and 60 volumes B. 5-chloro-6-methyi-1,2,3-oxathiazm -4(3/f)-one 2,2-dioxide.
of a 3.3 g/L solution o f tetrabuiylammomwn hydrogen sulfate R.
__________________________________________________________ PhEur
Flow rate 1 mL/min.
Detection Spectrophotom eter at 234 nm .
Injection 20 nL.
Run time Twice the retention time o f acesulfame. Acetazolamide ★★* ★★
★ ★
Relative retention W ith reference to acesulfame (retention * * * * *
time = about 5.3 min): im purity B = about 1.6.
System suitability: o\w/o H
— signal-to-noise ratio: minimum 10 for the peak due to ,s. N^CHs
impurity B in the chromatogram obtained with reference HjN' 'Y Y T
N -N
solution (a);
— peak-to-vaUey ratio: minimum 1.2, where Hp = height
above the baseline o f the peak due to impurity B and 222.2 59-66-5
Hv = height above the baseline o f the lowest point o f the Action and use
curve separating this peak from the peak due to Carbonic anhydrase inhibitor; diuretic; treatm ent of
acesulfame, in the chromatogram obtained with reference glaucoma and ocular hypertension; treatm ent o f m o untain
solution (b). sickness.
Limit: Preparation
— impurity B: not m ore than the area o f the principal peak Acetazolamide Tablets
PhEur__________________________________________________________
(20 ppm ).
D E F IN IT IO N
Fluorides
Maximum 3 ppm. 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-ionic- CHARACTERS
strength-adjustment buffer R 1 and dilute to 50.0 m L with Appearance
distilled water R. White or almost white, crystalline powder.
Reference solutions T o 0.5 mT, 1.0 mT, 1.5 m L and 3.0 m L Solubility
of fluoride standard solution (10 ppm F) R add 15.0 m L of Very slighdy soluble in water, slighdy soluble in ethanol
total-ionic-strerigth-adjustment buffer R1 and dilute to 50.0 mL (96 per cent). It dissolves in dilute solutions o f alkali
with distilled water R. hydroxides.
Indicator electrode Fluoride-selective. It shows polymorphism (5.9).
Reference electrode Silver-silver chloride. IDENTIFICATION
H eav y m e ta ls (2.4.8) First identification A, B
M aximum 5 ppm . Second identification A , C, D
12 m L of solution S complies with test A. Prepare die A Ultraviolet and visible absoiption spectrophotometry
reference solution using lead standard solution (1 ppm Pb) R (2.2.25).
L oss o n d ry in g (2.2.32) Solution A Dissolve 30.0 m g in 0.01 M sodium hydroxide and
M aximum 1.0 per cent, determined on 1.000 g by drying in dilute to 100.0 m L with die same solvent. D ilute 10.0 m L of
an oven at 105 °C for 3 h. the solution to 100.0 m L with 0.01 M sodium hydroxide.
A SSA Y Solution B Dilute 25.0 m L o f solution A to 100.0 m L with
Dissolve 0.150 g in 50 m L o f anhydrous acetic add R. Titrate 0.01 M sodium hydroxide.
with 0.1 M perchloric add, determining the end-point Spectral range 230-260 nm for solution A; 260-350 n m for
potentiometrically (2 .2 . 2 0 ). solution B.
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.
2016 Acetazolamide 1-55
Specific absorbance at the absorption maximum 162 to 176 for — impurities A , B, C, D, E, F: for each impurity, not more
solution A; 570 to 620 for solution B. than 1.5 times the area of the principal peak in the
B. Infrared absorption spectrophotometry (2.2.24). chromatogram obtained with reference solution (a)
(0.15 per cent);
Comparison acetazolamide CRS.
— unspecified impurities: for each impurity, not more than the
If the spectra obtained in the solid state show differences, area o f the principal peak in th e chromatogram obtained
dissolve the substance to be examined and the reference with reference solution (a) (0.10 per cent);
substance separately in ethanol (96 per cent) R, evaporate to
— total: not m ore than 6 times the area o f the principal peak
dryness and record new spectra using the residues. in the chromatogram obtained with reference solution (a)
C. Introduce about 20 mg into a test-tube and add 4 m L of (0.6 per cent);
dilute hydrochloric add R and 0.2 g o f zinc powder R — disregard limit. 0.5 times the area of the principal peak in
Im mediately place a piece of lead acetate paper R over the the chromatogram obtained w ith reference solution (a)
m outh o f the tube. T he paper shows a brownish-black (0.05 per cent).
colour.
S u lfates (2.413)
D . Dissolve about 25 mg in a mixture o f 0.1 m L o f dilute M axim um 500 ppm.
sodium hydroxide sdurion R and 5 m L of water R Add 0.1 m L
T o 0.4 g add 20 m L o f distilled water R and dissolve by
o f copper sulfate solution R. A greenish-blue precipitate is
heating to boiling. Allow to cool w ith frequent shaking and
formed.
filter.
TESTS H eav y m e ta ls (2.48)
Appearance of solution M aximum 20 ppm.
T he solution is no t more opalescent than reference
1.0 g complies with test C. Prepare the reference solution
suspension II (2.2.1) and not more intensely coloured than
using 2 m L o f lead standard solution (10 ppm Pb) R.
reference solution Y 5 or BY5 (2.2.2, Method II).
Dissolve 1.0 g in 10 m L of 1 M sodium hydroxide.
Related substances an oven at 105 °C.
S u lfa te d a sh (2.414)
Test solution Dissolve 40 mg o f die substance to be examined M aximum 0.1 per cent, determined on 1.0 g.
in the mobile phase and dilute to 100.0 m L with the mobile
phase. A SSAY
Reference solution (a) Dilute 1.0 m L o f the test solution to Dissolve 0.200 g in 25 m L of dimethylforrnamide R. T itrate
100.0 m L with the mobile phase. Dilute 1.0 m L o f this with 0.1 M ethanolic sodium hydroxide, determining the
end-point potentiometrically (2 . 2 . 2 0 ).
solution to 10.0 m L with the mobile phase.
Reference solution (b) Dissolve the contents o f a vial o f 1 m L of 0.1 M ethanolic sodium hydroxide is equivalent to
acetazolamide for system suitability CRS (containing 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 IM P U R IT IE S
phase. Specified impurities A, B, C, D , E, F
Cdumn: Other detectable impurities (the following substances would, if
— size: I = 0.15 m , 0 = 4.6 mm; present at a sufficient level, be detected by one o r other of
— stationary phase: end-capped propoxybenzene silica gel for the tests in the monograph. They are limited by the general
chromatography R (4 (im). acceptance criterion for other/unspecified impurities and/or
Mobile phase acetonitrüe for chromatography R, 6.8 g/L solution by the general monograph Substances for pharmaceutical use
of potassium dihydrogen phosphate R (10:90 V!V). (2034). It is therefore not necessary to identify these
Flow rate 1.0 mL/min. impurities for demonstration of compliance. See also 5.10.
Detection Spectrophotom eter at 265 nm. Control of impurities in substancesfor pharmaceutical use): G.
Injection 25 jil. H
Run time 3.5 times the retention time of acetazolamide.
\
N -N
Y R1
with acetazolamide for system suitability CRS and the
chromatogram obtained with reference solution (b) to A. R1 = C O -C H 3, R2 = Cl: N -(5-chloro-l,3,4-thiadiazol-2-
identify the peaks due to impurities A, B, C, D , E and F. yl)acetamide,
Relative retention W ith reference to acetazolamide (retention B. R1 = C O -C H 3, R2 = H: 2V-(l,3,4-thiadiazol-2-
time = about 8 min): impurity E = about 0.3; yl)acetamide,
impurity D = about 0.4; impurity B = about 0.6;
C. R1 = C O -C H 3, R2 = SH: N-(5-sulfanyl-1,3,4-thiadiazol-
impurity C = about 1.4; impurity A = about 2.1;
2-yl)acetamide,
impurity F = about 2.6.
D . R 1 = H , R2 = S 0 2-N H 2: 5-am ino-1,3,4-thiadiazole-2-
System suitability: reference solution (b):
sulfonamide,
— resolution: minim um 2.0 between the peaks due to
impurities E and D . E. R1 = C O -C H 3, R2 = S 0 2-0 H : 5-acetamido-1,3,4-
thiadiazole-2-sulfonic ad d ,
Limits:
— correction factory, for the calculation o f content, multiply
corresponding correction factor impurity B = 2.3;
impurity C = 2 . 6; impurity D = 1.6;
1-56 Acetic Acid 2016
Dissolve the residue obtained in the test for residue on

"’V V rY x V V "
O N -N N -N 0
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
F. N- [5- [(5-acetam ido-l 33,4-thiadiazol-2- reference solution using lead standard solution (2 ppm Pb) R.
yl) sulfonyl] sulfamoyl-1,3,4-thiadiazol-2-yI] acetamide, R esid u e o n e v a p o ra tio n
H
Sx sy N
H
N -N
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.
G. 5-am ino-1,3,4-thiadiazole-2-thioL A SSA Y

Weigh accurately a conical flask with a ground-glass stopper
PhEur
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
***** hydroxide.
Glacial Acetic Acid ★ ★ 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.
STO R A G E
0
In an airtight container.
I
H3C OH PhEur
C2H 4O 2 60.1 64-19-7

PhEur__________________________________________________________
D E F IN IT IO N Acetic Acid (6 per cent)

C o n te n t Dilute Acetic A d d
99.0 per cent mlm to 100.5 per cent mlm.
D E F IN IT IO N
CHARACTERS Acetic A d d (6 per cent) contains not less than 5.7% and not
A p p e a ra n c e more than 6.3% w/w of acetic ad d , C 2H 4O 2. It may be
Crystalline mass or clear, colourless, volatile liquid. prepared by mnring 182 g of Acetic A d d (3 3 per cent) with
S o lu b ility 818 g of Purified Water.
Miscible with water, with ethanol (96 per cent) and with IDENTIFICATION
methylene chloride.
A. Strongly addic.
ID E N T IF IC A T IO N B. W hen neutralised, yields the reactions characteristic of
A. A 100 g/L solution is strongly acid (2.2.4). acetates, Appendix VI.
B. T o 0.03 m L add 3 m L o f water R and neutralise with
TESTS
dilute sodium hydroxide solution R T he solution gives W eig h t p e r m L
reaction (b) of acetates (2.3.1).
About 1.005 g, Appendix V G.
TESTS H eav y m e ta ls
S o lu tio n S Evaporate 20.0 m l, to dryness and add 20 m L of water.
Dilute 20 m L to 100 m L with distilled water R 12 m L o f the resulting solution complies with limit test A for
A p p e a ra n c e heavy metals, Appendix VII. Use lead standard solution
T he substance to be examined is clear (2.2.1) and colourless ( 1 ppm Pb) to prepare the standard (1 ppm).
(2.2.2, Method II). C h lo rid e

F re e z in g p o in t (2.2.18) Dilute 5.0 m l, with suffident water to produce 100 m L
M inim um 14.8 °C. 15 m L o f the resulting solution complies with the limit test for
R e d u c in g su b s ta n c e s chlorides, Appendix VII (70 ppm).
Dilute 2.0 m L to 10.0 m L with water R Add 0.1 m L of S u lfate
0.02 M potassium permanganate. H eat on a water-bath for 12.5 m L o f the solution used in the test for Chloride, diluted
1 min, the colour remains pink. to 15 m l . with water, complies with the limit test for sulfates,
C h lo rid e s (2.4.4) Appendix VII (240 ppm ).
Maximum 25 mg/L. A ldehydes
Dilute 10 m L of solution S to 15 m L with water R. 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
S u lfates (2.4.13)
with 5m sodium hydroxide, allow to stand for 5 minutes and
M aximum 50 mg/L, determined on solution S.
addify with 1m sulfuric acid. T h e solution shows not more
Iro n (2.4.9) than a faint turbidity.
M aximum 5 ppm.
F o rm ic a c id a n d o x id isab le im p u ritie s
Dilute 5.0 m L of solution A obtained in the test for heavy Mix 5 m l . with 6 m l , of sulfuric acid and cool to 20°.
metals to 10.0 m L with water R. Add 0 .4 m L o f 0 .0 1 6 7 m potassium dichromate VS, allow to
H eav y m e ta ls (2.4.8) stand for 1 minute, add 25 m l, o f water and 1 m L o f freshly
M aximum 5 ppm. prepared dilute potassium iodide solution and titrate the
2016 Acetone 1-57
liberated iodine with 0.1m sodium tkiosidfate F 5 using starch mucilage as indicator. N ot less than 1.0 m L of 0.1m sodium
mucilage as indicator. N ot less than 0 .2 m L o f 0.1m sodium tkiosidfate VS is required.
thiosulfate F 5 is required. R ead ily o x id isa b le im p u ritie s
R ead ily o x id isab 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
T o 2 5 m L add 0 .2 m L of 0.02m potassium permanganate FiS 0.02m potassium permanganate KS and allow to stand for
and allow to stand for 1 minute. T he pink colour is not 1 minute. T h e pink colour is not entirely discharged.
entirely discharged. N o n -v o la tile m a t te r
N o n -v o latile m a t te r W hen evaporated to dryness and dried at 105°, leaves not
W hen evaporated to dryness and dried at 105°, leaves not more th an 0 .01 % w/w of residue.
more than 0 .01 % w/w o f residue.
ASSAY
A SSAY Weigh 5 g into a stopper flask containing 5 0 m L o f water and
A dd 30 m L of water to 20 g in a stopper flask and titrate titrate w ith 1m sodium hydroxide F 5 using phenolphthalein
with 1m sodium hydroxide F 5 using phenolphthalein solution R1 solution R1 as indicator. Each mL o f 1m sodium hydroxide F S
as indicator. E ach m L of 1m sodium hydroxide F 5 is is equivalent to 6 0 .0 5 mg o f C2H 4 0 2.
equivalent to 60.05 mg of C 2H 4O 2.
Acetone ★ ★
★ ★
Acetic Acid (33 per cent) *****
Acetic A d d
P r e p a r a tio n 0
Acetic A d d (6 p er cent) X , CH;
H3C
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. CjHeO 58.08 67-64-1
PhEir___
A clear, colourless liquid. D E F IN IT IO N
M isdble with water, with ethanol (96%) and with glycerol. Propanone.
ID E N T IF IC A T IO N CHARACTERS
A. Strongly addic, even when diluted fredy. A p p e a ra n c e
Volatile, clear, colourless liquid.
B. W hen neutralised, yields the reactions characteristic of
acetates, A ppendix VI. S o lu b ility
M isdble w ith w ater and with ethanol (96 per cent).
TESTS
W eig h t p e r m l . T h e vapour is flammable.
1.040 to 1.042 g, Appendix V G. ID E N T IF IC A T IO N
H eav y m e ta ls A. Relative density (see Tests).
Evaporate 10.0 m L to dryness and add 20 m L o f water. B. T o 1 m L, add 3 m L o f dilute sodium hydroxide solution R
12 m L of the resulting solution complies with limit test A for and 0.3 m L of a 25 g/L solution o f sodium nitroprusside R.
heavy metals, Appendix VII. Use lead standard solution An intense red colour is produced which becomes violet with
(1 ppm Pb) to prepare the standard (2 ppm). the addition of 3.5 m L o f acetic acid R.
Chloride C. T o 10 m L of a 0.1 per cent VIV solution o f the substance
Dilute 5.0 m L w ith suffident water to produce 100 m L to be examined in ethanol (50 per cent VIV) R, add 1 m L o f a
15 m L of the resulting solution complies with the limit test for 10 g/L solution o f nitrobenzaldehyde R in ethanol
chlorides, Appendix VII (70 ppm). (50 per cent VIV) R and 0.5 mL o f strong sodium hydroxide
S u lfate solution R. Allow to stand for about 2 min and addify with
12.5 m L of the solution used in the test for Chloride, diluted
acetic acid R. A greenish-blue colour is produced.
to 15 m L with water, complies with the limit test for sulfates, TESTS
Appendix VII (240 ppm). A p p e a ra n c e o f so lu tio n
A ldehydes T o 10 m L add 10 m L o f water R. T he solution is clear
Distil 15 m L T o the first 5 m L of the distillate add 10 m L (2.2.1) and colourless (2.2.2, Method II).
of a 5% w/v solution of mercury(n) chloride, make alkaline A c id ity o r a lk a lin ity
w ith 5m sodium hydroxide, allow to stand for 5 minutes and T o 5 m L add 5 m L of carbon dioxide-free water R, 0.15 m L of
make addic w ith 1m sulfuric acid. The solution shows not phenolphthalein solution R and 0.5 m L of 0.01 M sodium
m ore than a faint turbidity. hydroxide. T h e solution is pink. A dd 0.7 m L o f 0.01 M
F o rm ic a c id a n d o x id isab le im p u ritie s hydrochloric acid and 0.05 m L of methyl red solution R.
M ix 5 m L with 6 m L o f sulfuric acid and cool to 20°. T h e solution is red or orange.
A dd 2 m L of 0 .0 1 6 7 m potassium dickromate F5, allow to R e lativ e d e n sity (2.2.5)
stand for 1 m inute, add 25 m L of water and 1 m L of freshly 0.790 to 0.793.
prepared dilute potassium iodide solution and titrate the
liberated iodine w ith 0.1m sodium thiosulfate VS using starch
1-58 Acetylcholine Chloride 2016
R e d u c in g su b sta n c e s R esid u e o n e v a p o ra tio n

T o 30 m l. add 0.1 m l. o f 0.02 M potassium permanganate M aximum 50 ppm.
and allow to stand in the dark for 2 h. T he mixture is not Evaporate 20.0 g to dryness on a w ater-bath and dry at
completely decolourised 100-105 °C. T h e residue weighs a maximum o f 1 mg.
R e la te d su b sta n c e s W a te r (.2.5.12)
G as chromatography (2.2.28). Maximum 3 g/L, determined on 10.0 m L. U se 20 m L of
Test solution T he substance to be examined. anhydrous pyridine R as solvent.
Reference solution (a) T o 0.5 m L o f methanol R add 0.5 m L of STORAGE
2-propanol R and dilute to 100.0 m L with the test solution. Protected from light.
D ilute 1.0 m L of this solution to 10.0 m L with the test
IM P U R IT IE S
solution.
Specified impurities: A, B, C.
Reference solution (b) Dilute 100 pL o f benzene R to 100.0 m L
w ith the test solution. Dilute 0.20 m L o f this solution to A. C H 3-OH: m ethanol,
100.0 m L with the test solution. B. C H 3-C H O H -C H 3: propan-2-ol (isopropanol),
Column: C. O H ^ : benzene.
— material: fused silica, PhEur
— 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. Acetylcholine Chloride ★★*★*
★ ★
Split ratio 1:50. *****
(Ph Ever monograph 1485)
Temperature:
H3C + CH3
Time Temperature .N^ Cl
CH3
(min) (°C)
Column 0 -1 1 45 + 100
C7H16o n o 2 181.7 60-31-1
11-20 100
Injection port 150 A c tio n a n d u se

Cholinoceptor agonist.
Detector 250
PhEur__________________
Detection Flam e ionisation. D E F IN IT IO N

2-(Acetyloxy)-iV>W,N-trimethylethanaminium chloride.
Injection 1 pL.
Retention time Impurity C = about 7.5 min. C o n te n t
System suitability:
— resolution: minimum 5.0 between the peak due to CHARACTERS
impurity A (2nd peak) and the peak due to impurity B A p p e a ra n c e
(3rd peak) in the chromatogram obtained with reference W hite or almost white crystalline powder or colourless
solution (a), crystals, very hygroscopic.
— signal-to-notse ratio: minim um 5 for the peak due to S o lu b ility
impurity C in the chromatogram obtained with reference Very soluble in water, freely soluble in alcohol, slightly
solution (b). soluble in methylene chloride.
Limits:
— impurities A , B: for each impurity, n o t m ore than the
difference between the areas of the corresponding peaks First identification B, E.
in the chromatogram obtained with reference solution (a) Second identification A , C, D, E.
and the areas o f the corresponding peaks in the A. Melting point (2.2.14): 149 °C to 152 °C.
chromatogram obtained with the test solution Introduce the substance to be examined into a capillary tube.
(0.05 per cent VIV), D ry in an oven at 100-105 °C for 3 h. Seal the tube and
— impurity C: not more than die difference between the area determine the melting point.
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 Comparison acetylcholine chloride CRS.
the test solution (2 ppm VIV), C. Examine the chromatograms obtained in the test for
— any other impurity, for each impurity, not m ore than the related substances.
difference between the area o f the peak due to impurity A Results T h e principal zone in the chromatogram obtained
in the chromatogram obtained with reference solution (a) with test solution (b) is similar in position, colour and size to
and the area o f the corresponding peak in die the principal zone in the chromatogram obtained with
chromatogram obtained with the test solution reference solution (b).
(0.05 p er cent VIV). D . T o 15 m g add 10 m L of dilute sodium hydroxide solution R,
M a tte r in so lu b le in w a te r 2 m l. o f 0.02 M potassium permanganate and heat.
D ilute 1.0 m L to 20 m L w ith water R. T he solution is clear T h e vapours formed change the colour o f red litmus paper R
(2.2.1). to blue.
2016 Acetylcysteine 1-59
E. 0.5 m L of solution S (see Tests) gives reaction (a) o f A SSAY

chlorides (2.3.1). Dissolve 0.200 g in 20 m L of carbon dioxide-free water R.
TESTS Neutralise with 0.01 M sodium hydroxide using 0.15 m l. of
S o lu tio n S
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
A p p e a ra n c e o f so lu tio n
C 7H 16C IN 0 2.
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). STO R A G E
A cid ity In ampoules, protected from light.
D ilute 1 m L o f solution S to 10 m L with carbon dioxide-free IM P U R IT IE S
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 H3C + CH3
change the colour of the indicator to pink. \ N^ Cf
ch 3
R e la te d su b s ta n c e s
Thin-layer chromatography (2.2.27). Prepare the solutions A. 2- chloride
immediately before use. (choline chloride),
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
solvent.
Test solution (b) D ilute 1 m L of test solution (a) to 10 m L
with methanol R.
B. 2-(acetyloxy)-N>N-dimethylethanaminium chloride,
Reference solution (a) Dilute 1 m L of test solution (a) to
100 m L with methanol R. ch 3
Reference solution (b) Dissolve 20.0 mg of acetylcholine
chloride CRS in methanol R and dilute to 2.0 m L with the H3C ch 3
same solvent.
Reference solution (c) Dissolve 20 mg of choline chloride R in C. NjN-dimethylmethanamine.
methanol R, add 0.4 m L of test solution (a) and dilute to PhEur
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 Acetylcysteine ★* * ★★
60 volumes of acetonârUe R. ★ ★
Application 5 nL as bands o f 10 mm by 2 mm. (Ph. Eur. monograph 0967) *****
Development Over 2/3 o f the plate.
ch 3
Detection Spray with potassium iodobismuthate solution R3.
System suitability T h e chromatogram obtained with reference
0={
H NH
solution (c) shows 2 clearly separated zones.
co 2h
Limits:
— any impurity: any zones in the chromatogram obtained
with test solution (a), apart from the principal zone, are C 5H 9N 03 S 163.2 616-91-1
n o t more intense than the principal zone in the
Action and use
chrom atogram obtained with reference solution (a)
Sulfydryl donor; antidote to paracetamol poisoning;
(1 per cent).
mucolytic.
T rim e th y la m in e
P r e p a r a tio n
Dissolve 0.1 g in 10 m L of sodium carbonate solution R and
Acetylcysteine eye drops
heat to boiling. N o vapours appear which turn red litmus
paper R blue. Acetylcysteine Injection
H eav y m e ta ls (2.4.8) PhEur.

M axim um 10 ppm .
D E F IN IT IO N
12 m L o f solution S complies with test A. Prepare the (2i?)-2-(Acetylamino)-3-sulfanylpropanoic add.
reference solution using lead standard solution (1 ppm Pb) R.
C o n te n t
M axim um 1.0 p er cent, determined on 1.000 g by drying in
an oven at 105 °C for 3 h. CHARACTERS
A p p e a ra n c e
S u lfa te d a sh (2.4.14)
W hite or almost white, crystalline powder or colourless
M axim um 0.1 p er cent, determined on the residue obtained
crystals.
in the test for loss on drying.
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 Injection 20 (iL, 3 times; inject 0.01 M hydrochloric acid as a

First identification A , C. blank.
Second identification A , B, D, E. Run time 5 times the retention time o f acetylcysteine (about
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 =
about 2.4 min; 2-methyl-2-thiazoline-4-carboxylic acid,
C. Infrared absorption spectrophotometry (2.2.24).
originating in test solution (c) = about 3.3 min;
Preparation Discs of potassium bromide R. acetylcysteine = about 6.4 min; im purity C = about 12 min;
Comparison acetylcysteine CRS. impurity D = about 14 min.
D . Examine the chromatograms obtained in the test for System suitability: reference solution (a):
related substances. — resolution: minimum 1.5 between the peaks due to
Results T he principal peak in the chromatogram obtained impurities A and B and minim um 2.0 between the peaks
with test solution (b) is similar in retention time and size to due to impurities C and D .
the principal peak in the chromatogram obtained with
reference solution (b). A \ x m 2 x 100
E. T o 0.5 m L of solution S (see Tests) add 0.05 m L of a A-----------
11 = ----~ x mi
50 g/L solution of sodium nitroprusside R and 0.05 m L of
_ A 3 x m 3 x 100
concentrated ammonia R. A dark violet colour develops. 12 = ----------------
A* x m i
TESTS
Solution S
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);
Appearance of solution A 2 = peak area o f the corresponding individual im purity
Solution S is clear (2 . 2 . 1 ) and colourless (2.2.2, Method II). (impurity A, im purity B, im purity C and impurity
pH (2.2.3) D ) in the chrom atogram obtained with reference
2.0 to 2.8. solution (a);
T o 2 m L o f solution S add 8 m L of carbon dioxide-free A 3 = peak area of unknown impurity in the chromatogram
water R and mix. obtained with test solution (a);
A 4 = peak area o f acetylcysteine in the chromatogram
Specific optical rotation (2.2.7)
obtained with reference solution (b);
+ 21.0 to + 27.0 (dried substance).
mi = mass o f the substance to be examined in test
In a 25 m L volumetric flask, mix 1.25 g with 1 m L of a solution (a);
10 g/L solution of sodium edetate R. A dd 7.5 m L o f a 40 g/L m2 = mass of the individual im purity in reference solution
solution o f sodium hydroxide R, mix and dissolve. Dilute to (a);
25.0 m L with phosphate buffer solution pH 7.0 R2. m3 = mass of acetylcysteine in reference solution (b).
Related substances Limits:
Liquid chrom atography (2.2.29). Except where otherwise — impurities A , B, C, D: for each im purity, maximum
prescribed, prepare the solutions immediately before use. 0.5 per cent;
Test solution (a) Suspend 0.80 g of the substance to be — arty other impurity: for each impurity, maximum
examined in 1 m L o f 1 M hydrochloric acid and dilute to 0.5 per cent;
100.0 m L with water R. — total: maximum 0.5 p er cent;
Test solution (b) Dilute 5.0 m L of test solution (a) to — disregard Umar. 0.1 times the area o f the principal peak in
100.0 m L with water R. Dilute 5.0 m l. of this solution to the chromatogram obtained with reference solution (b)
50.0 m L w ith water R. (0.05 per cent); disregard any peak w ith a retention time
Test solution (c) Use test solution (a) after storage for at least of about 3.3 m in due to 2-methyl-2-thiazoline-4-
1 h. carboxylic ad d .
Reference solution (a) Suspend 4.0 m g o f acetylcysteine CRS, H eav y m e ta ls (2.4.8)
4.0 mg of L-cystme R (impurity A), 4.0 mg o f l -cysteine R M aximum 10 ppm .
(impurity B), 4.0 m g of acetylcysteine impurity C CRS and 2.0 g complies with test C. Prepare the reference solution
4.0 mg of acetylcysteine impurity D CRS in 1 m L o f 1 M using 2 m L o f lead standard solution (10 ppm Pb) R.
hydrochloric add and dilute to 100.0 m L with water R. Z in c
Reference solution (b) Suspend 4.0 mg o f acetylcysteine CRS in Maximum 10 ppm.
1 m L of 1 M hydrochloric add and dilute to 100.0 m l. with Atomic absorption spectrometry (2.2.23, Method II).
water R.
Test solution Dissolve 1.00 g in 0.001 M hydrochloric add and
Column: dilute to 50.0 m L with the same ad d .
— sizer. I = 0.25 m, 0 = 4 mm;
Reference solutions Prepare the reference solutions using zinc
— stationary phase: octadecylsifyl silica gel for chromatography R
(5 pm).
standard solution (5 mglmL Zn) R, diluting with 0.001 M
hydrochloric add.
Mobile phase Stir 3 volumes of acetonitrüe R and 97 volumes
Source Zinc hollow-cathode lamp.
o f water R in a beaker; adjust to pH 3.0 with phosphoric
add R. Wavelength 213.8 nm .
Flow rate 1.0 mL/min. Atomisation device Air-acetylene flame.
Detection Spectrophotom eter at 220 nm . U se a correction procedure for non-specific absorption.
2016 Acetyldigoxin 1-61
Loss o n d ry in g (2.2.52)
Acetyldigoxin ★■k+ic★
M aximum 1.0 per cent, determined on 1.000 g by drying in ★ ★
★ ★
an oven in vacuo at 70 °C for 3 h. 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
IMPURITIES
Specified impurities A, B, C, D
H NH,
HOjC
COjH
H NH2 C43H660 15 823 5355-48-6
A. 3,3 '-disulfenediylbis [(2J?)-2-aminopropanoic acid] Action and use

(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-D-
B. (2i^-2-am ino-3-sulfanylpropanoic a d d (L-cysteine), n&o-hexopyranosyl)oxy]-l 2P, 14-dihydroxy-5P-card-20(22)-
enolide.
ch 3
Content
0=\ 97.0 p er cent to 102.0 p er cent (dried substance).
H NH
ho 2c CHARACTERS
> r ^ S ' Sx— X 'COjH
Appearance
H NH
W hite or alm ost white powder.
°= <
ch 3 Solubility
Practically insoluble in water, sparingly soluble in methylene
C. (2i?,2/i?)-3>3 /-disulfanediylbis[2-(acetylamino)propanoic chloride, slightly soluble in ethanol (96 per cent).
add] (N ^'-diacetyl-L-cystine), IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
ch 3 Comparison ^-acetyldigoxin CRS.
0=< TESTS
H NH
h3C^ S p ecific o p tic a l ro ta tio n (2.2.7)
Yo COjH
Dissolve 0.50 g in a mixture of equal volumes o f methanol R
and methylene chloride R and dilute to 25.0 m L with the same
D. (2i?)-2-(acetylamino)-3-(acetylsulfenyl)propanoic a d d m ixture o f solvents.
(2V,.S-diacetyl-L-cysteine).
Related substances
PhEur
L iquid chrom atography (2.2.29). Prepare the solutions
immediately before use.
Solvent mixture M ix equal volumes of methanol R2 and
acetomtrUe for chromatography R.
Test solution Dissolve 50.0 mg of the substance to be
examined in the solvent mixture and dilute to 100.0 m L w ith
the solvent m ixture.
Reference solution (a) Dissolve 10.0 m g o f /?-acetyldigoxin CRS
in the solvent m ixture and dilute to 20.0 m L with the solvent
mixture.
20.0 m L w ith th e solvent mixture. D ilute 1.0 m L of this
solution to 10.0 m L with the solvent mixture.
1-62 Acetyldigoxin 2016
Reference solution (c) Dissolve 5 m g o f gitoxin CRS Loss o n d ry in g (2.2.32)

(impurity D ) in the solvent mixture and dilute to 100.0 m L M aximum 1.5 per cent, determ ined on 1.000 g by drying in
with the solvent mixture. T o 5.0 m L o f this solution, add an oven at 105 °C.
0.5 m L of reference solution (a) and dilute to 100.0 m L with S u lfa te d a s h (2.4.14)
the solvent mixture. Maximum 0.1 per cent, determ ined on the residue obtained
Reference solution (d) Dissolve 5.0 m g o f (¡-acetyldigoxin for in the test for loss on drying.
peak identification CRS (containing impurities A and B) in ASSAY
10.0 m L o f the solvent mixture. Liquid chromatography (2.2.29) as described in the test for
Column: related substances with the following modification.
— sizer. I = 0.125 m , 0 = 4.0 mm; Injection T est solution and reference solution (a).
— stationary phase: octadecylsQyl silica gelfor chromatography R Calculate the percentage content o f C 43H 660 15 from the
(4 nm).
declared content of fi-acetyldigoxin CRS.
Mobile phase:
STO RA G E
— mobile phase A: water for chromatography R',
— mobile phase B: acetonitrUe for chromatography R\ Protected from light.
IM P U R IT IE S
Specified impurities A, B, D.
(min) (per cent V/V) (percent V/V) Other detectable impurities (the following substances would, if
0 - 10 70 30 present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by the general
1 0 -2 0 70->35 30-» 65
20 - 20.1 35-» 70 65-» 30 therefore not necessary to identify these impurities for
20.1 - 25 70 30 demonstration o f compliance. See also 5.10. Control of
impurities in substances for pharmaceutical use): C, E, F, G, H.
Flow rate 1.5 mlVmin.

Detection Spectrophotom eter at 2 2 5 nm .
Injection 10 pL o f the test solution and reference
solutions (b), (c) and (d).
Identification of impurities Use the chromatograms obtained
with reference solutions (c) and (d) to identify the peaks due
to impurities A, B and D.
Relative retention W ith reference to ^-acetyldigoxin (retention
time = about 9 min): im purity B = about 0.3;
impurity A = about 0 .7 ; impurity D = about 1.2.
System suitability: reference solution (c):
P-acetyldigoxin and impurity D;
— symmetry factor, maximum 2 .5 for the peak due to
^-acetyldigoxin.
CH3
Limits:
— impurities A , B: for each impurity, not more than the area A. 3|3-[(3-O-acetyl-2,6-dideoxy-p-D-rt&0-hexopyranosyl-
o f the principal peak in the chromatogram obtained with (1 -> 4)-2, 6-dideoxy-P-D-ribo-hexopyranosyl- (1 -*•4)-2, 6-
reference solution (b) (0 .5 per cent); dideoxy-P-D-n&o-hexopyranosyl)oxy]-12(3,14-dihydroxy-5 P-
— impurity D: not m ore than 0 .6 times the area of the card-20 (22)-enolide (a-acetyldigoxin),
reference solution (b) (0 .3 per cent);
— any other impurity: for each impurity, not m ore than
0 .4 times the area o f the principal peak in the
chromatogram obtained with reference solution (b)
(0.2 per cent);
— sum of impurities other than A , B and D~. not m ore than
1 .2 times the area of the principal peak in the
(0.6 per cent);
in the chromatogram obtained with reference solution (b)
(1 .5 per cent);
— disregard limit. 0.1 times the area o f the principal peak in
the chromatogram obtained with reference solution (b)
(0 .0 5 per cent).
T he thresholds indicated under Related substances B. 3 P- [(2, 6-dideoxy-P-D-n&o-hexopyranosyl-( 1-*-4)-2,6-
(Table 2 0 3 4 .-1 ) in the general m onograph Substances for dideoxy-P-D-n&o-hexopyranosyi-( 1->4)-2, 6-dideoxy-P-D-nJo-
pharmaceutical use (2034) do n o t apply. hexopyranosyl)oxy]-12j},14-dihydroxy-5(3-card-20(22)-enolide
(digoxin),
2016 Acetyldigoxin 1-63
C. 3ß, 12ßjl4-trihydroxy-5ß-card-20(22)-enolide
(digoxigenin),
CH3
F . 3 ß- [(3,4-0-diacetyl-2,6-dideoxy-ß-i>nk>-hexopyranosyl-
( 1-^4)-2,6-dideoxy-ß-D-rÄö-hexopyranosyl-(l ->4)-2j6-
dideoxy-ß-D-rÄo-hexopyranosyl) oxy] -12 ßj 14-dihydroxy-5 ß-
card-20 (22)-enolide (diacetyldigoxin),
D . 3ß-[(2j6-dideoxy-ß-D-rÄo-hexopyranosyl-(l^4)-2j6-
dideoxy-ß-D-rÄo-hexopyranosyl-(l-^4)-2j6-dideoxy-ß-D-rÄo-
hexopyranosyl)oxy]-14jl6ß-dihydroxy-5ß-card-20(22)-enolide
(gitoxin),
ch3
G . 3 ß- [(3-0-acetyl-2,6-dideoxy-ß-i>nZ>o-hexopyranosyl-
(1 -+4)-2j6-*dideoxy-ß-D-r&o-hexopyranosyl-(l -+4)-2,6-
dideoxy-ß-D-rÄo-hexopyranosyl) oxy] -14-hydroxy-5 ß-caid-
20 (22)-enolide (a-acetyldigitoxin)j
E. 3ß- [(2,6-dideoxy-ß-i>rjfcö-hexopyranosyl-(l -+4)-2,6-

dideoxy-ß-D-r&o-hexopyranosyl-(l-+4)-2j6-dideoxy-ß-D-ri&o-
hexopyranosyl)oxy]- 14-hydroxy-5 ß-card-20(22)-enolide
(digitoxin).
H . 3ß-[(4-0-acetyl-2j6-dideoxy-ß-i>nZ>o-hexopyranosyl-
(1 ^4)-2,6-dideoxy-ß-D-rÄo-hexopyranosyl-(l -+4)-2,6-
dideoxy-ß-D-r&o-hexopyranosyl) oxy] -14-hydroxy-5 ß-card-
20(22)-enolide (ß-acetyldigitoxm).
___________________ :______________________________________ PhEur
1-64 Acetyltryptophan 2016
E. It gives the reaction o f acetyl (2.3.1). Proceed as described

Acetyltryptophan ***** for substances hydrolysable only with difficulty.
(N-Acetyltryptophan, Ph Eur monograph 1383) * TESTS
o T he solution is clear (2.2. T) and n o t m ore intensely coloured
CH and enanttomer. than reference solution or GY 7 (2.2.2, Method II).
COjH Dissolve 1.0 g in a 40 g/L solution o f sodium hydroxide R and
dilute to 100 m L with the same alkaline solution.
O p tic a l ro ta tio n (2.2.7)
C 13H 14N203 246.3 87-32-1
- 0 . 1° to + 0 . 1°.
PhEtr___________________________________________________________
Dissolve 2.50 g in a 40 g/L solution o f sodium hydroxide R
D E F IN IT IO N and dilute to 25.0 m L with the same alkaline solution.
(i?5)-2-Acetylamino-3-( 1#-indol-3-yl)propanoic add. R e la te d su b s ta n c e s
C o n te n t Liquid chromatography (2.2.29). Prepare the test and reference
99.0 p er cent to 101.0 per cent (dried substance). solutions immediately before use.
P R O D U C T IO N Buffer solution pH 2.3 Dissolve 3.90 g o f sodium dihydrogen
T ryptophan used for the production o f N-acetyltiyptophan
phosphate R in 1000 m L o f water R. A dd about 700 m L o f a
2.9 g/L solution o f phosphoric add R and adjust to p H 2.3
complies with the test for impurity A and other related
with the same a d d solution.
substances in the monograph on Tryptophan (1272).
Solvent mixture acetonitrile R, water R (10:90 VIV).
CHARACTERS
Test solution Dissolve 0.10 g o f the substance to be examined
A p p e a ra n c e
in a mixture o f 50 volumes o f acetonitrile R and 50 volumes
W hite or almost white, crystalline powder, or colourless
o f water R and dilute to 20.0 m L with the same mixture of
crystals.
solvents.
S o lu b ility
Reference solution (a) Dilute 1.0 m L o f the test solution to
Slightly soluble in water, very soluble in ethanol
100.0 m L w ith the solvent mixture.
(96 p er cent). It dissolves in dilute solutions of alkali
hydroxides. Reference solution (b) Dilute 4.0 m L o f reference solution (a)
to 100.0 m L with the solvent mixture.
mp
Reference solution (c) Dissolve the contents o f a vial o f 1,1'-
A bout 205 °C.
ethyUdenebisttyptophan CRS in 1 m L o f reference solution (b).
ID E N T IF IC A T IO N Column:
First ideritification A , B. — sizer. I = 0.25 m , 0 = 4.6 mm;
Second identification A , C, D, E. — stationary phase: octadecylsUyl silica gel for chromatography R
A Optical rotation (see Tests). (5 nm);
B. Infrared absorption spectrophotom etry (2.2.24).
Mobile phase:
Comparison N-acetyltryptophan CRS.
— mobile phase A: acetonitrile R, buffer solution p H 2.3
C. Thin-layer chromatography (2.2.27). (115:885 VIV);
Test solution Dissolve 50 mg o f the substance to be examined — mobile phase B: acetonitrile R, buffer solution p H 2.3
in 0.2 m L of concentrated ammonia R and dilute to 10 m L (350:650 VIV)’,
with footer R.
Reference solution (a) Dissolve 50 mg o f N- Mobile phase A Mobile phase B
Time
acetyltryptophan CRS in 0.2 m L o f concentrated ammonia R (min) (percent V/V) (per cent V/V)
and dilute to 10 m L with water R. 0 -1 0 100 0
Reference solution (b) Dissolve 10 m g o f tryptophan R in the 0+100
10-45 100 -> 0
test solution and dilute to 2 m l. with the test solution.
4 5 -6 5 0 100
Plate TLC silica gel F 254 plate R.
Mobile phase glacial acetic add R, water R, butanol R
(25:25:40 VIVJV). Flow rate 0.7 mL/min.
Application 2 |iL. Detection Spectrophotom eter at 220 nm .
Development Over a path of 10 cm. Injection 20 p L o f the test solution and reference solutions (a)
Drying In an oven at 100-105 °C for 15 min. and (c).
Detection Examine in ultraviolet light at 254 nm. Retention time N-acetyltryptophan = about 29 min;
System suitability, reference solution (b): 1,1 '-ethylidenebis(tryptophan) = about 34 min.
— the chrom atogram shows 2 clearly separated spots. System suitability, reference solution (c):
Results T he principal spot in the chrom atogram obtained with — resolution: minimum 8.0 between the peaks due to
the test solution is similar in position and size to the prindpal N -acetyltryptophan and 1,1 '-ethylidenebis(tryptophan);
spot in the chrom atogram obtained w ith reference if necessary, adjust the time program m e for die elution
solution (a). gradient (an increase in the duration o f elution with
mobile phase A produces longer retention times and a
D . Dissolve about 2 m g in 2 m L o f water R Add 2 m L o f
b etter resolution);
dimethylaminobenzcddekyde solution R 6 . H eat on a water-bath.
A blue or greenish-blue colour develops.
2016 Acetyltryptophan 1-65
— 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 C. R = H : (S)-2-amino-4-(2-aminophenyI)-4-oxobutanoic
principal peak in the chromatogram obtained with a d d (kynurenine),
reference solution (a) (0.25 per cent); E. R = C H O : (5)- 2-amino-4-[2- (formylamino)phenyl]-4-
— total: not m ore than 0.5 times the area of the principal oxobutanoic a d d (AT-fonnylkynurenine) 3
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) HO
Maximum 200 ppm , determined on 0.10 g.
Prepare the standard using 0.2 m L o f ammonium standard D . (S)- 2 -amino-3-(5-hydroxy - 1H-indol-3-yl)propanoic a d d
solution ( 1 0 0 ppm N H J R. (5-hydroxytryptophan),
Ir o n (2.4.9)
M aximum 10 ppm . h Hv Nh2
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 F . (S)-2-amino-3-(phenylamino)propanoic a d d
(3-phenylaminoalanine),
aqueous layer.
H eavy m e ta ls (2.4.8)
M aximum 10 ppm .
M axim um 0.5 per cent, determined on 1.000 g by drying in G. 0S)-2-amino-3-(2-hydroxy-lH-indol-3-yl)propanoic a d d
an oven at 105 °C. (2-hydroxytryptophan),
M aximum 0.1 per cent, determined on 1.0 g. 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 ).
H . R = H : (3i?5)-l,2,3,4-tetrahydro-9/f-P-carboline-3-
C 13H14N20 3 .
carboxylic ad d ,
STORA GE I. R = C H 3: 1-m ethyl-1,2,3,4-tetrahydro-9/f-P-carboline-3-
Protected from light. carboxylic 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), 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)-1H-
and epimer at C*
indol-3-yl] propanoic ad d .
CO2H
B. (S)-2-amino-3-[(3i?S)-3-hydroxy-2-oxo-2,3-dihydro-li/-
indol-3-yl]propanoic a d d (dioxyindolylalanine),
1-66 Acetyltyrosine 2016
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.
L. 1-(lH-indol-3-yimet±iyl)-li2j3,4-tetxahydro-9f/-ß- Solution S is d e a r (2.2.1) and colourless (2.2.2, Method II).
carboline-3-carboxylic ad d . S p ecific o p tic a l r o ta tio n (2.2.7)
___________________________________________________________PhEur + 46 to + 49 (dried substance).
Dilute 10.0 m L o f solution S to 25.0 m L with water R.
Liquid chromatography (2.2.29). Cany out the test protected
from light.
Acetyltyrosine ? **%
** * Test solution Dissolve 50.0 m g of die substance to be
(N-Acetyltyrosine, Ph Eier monograph 1384) ** examined in mobile phase A and dilute to 50.0 m L with
mobile phase A.
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.
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
Cn H 13N 0 4 223.2 537-55-3 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.
PhEir ___________________________________________________________
D E F IN IT IO N to 10.0 m L with mobile phase A.
(2S)-2-(Acetylamino)-3-(4-hydroxyphenyI)propanoic ad d .
Reference solution (d) Dilute 1.0 m L o f reference solution (b)
C o n te n t to 20.0 m L with the test solution.
98.5 p er cent to 101.0 p er cent (dried substance). Column:
CHARACTERS — size. I = 0.15 m , 0 = 3 mm;
A p p e a ra n c e — stationary phase, spherical octadecylsUyl silica gel for
W hite or almost white, crystalline powder or colourless chromatography R (3 pm);
crystals. — temperature. 40 °C.
S o lu b ility
Mobile phase:
Fredy soluble in water, practically insoluble in cyclohexane. — mobile phase A: mix 1.0 m L of phosphoric add R and
1000 m L of water for chromatography R;
ID E N T IF IC A T IO N — mobile phase B: acetonitrile Rl;
First ideruification A , B
Second identification A , C, D Time Mobile phase A Mobile phase B
A Specific optical rotation (see Tests). (min) (per cent V/V) (per cent V/V)
B. Infrared absorption spectrophotom etry (2.2.24). 0 -2 97 3
Comparison N-acetyltyrosine CRS. 2 - 15 97 -» 62 3 -» 38
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 Detection Spectrophotom eter at 219 nm .
to 10 m L with the same mixture of solvents. Iiyection 2 [iL o f the test solution and reference solutions (a),
Reference solution Dissolve 80 m g o f N-acetyltyrosine CRS in a (c) and (d).
m ixture of 3 volumes o f glacial acetic acid R, 3 volumes of Relative retention W ith reference to N-acetyltyrosine (retention
water R and 94 volumes o f anhydrous ethanol R , and dilute to time = about 6 min): impurity A = about 0.5.
10 m L with the same mixture o f solvents. System suitability Reference solution (d):
Plate TLC silica gel F 254 plate R. — resolution: m inim um 5.0 between the principal peak and
the peak due to im purity A.
Mobile phase water R, glacial acetic add R} ethyl acetate R
(10:15:75 V/VIV). Limits:
— impurity A: n o t m ore than 0.8 times the area o f the
Application 5 [iL.
corresponding peak in the chrom atogram obtained with
Development Over 2/3 o f the plate. reference solution (c) ( 0.8 per cent);
Drying In air. — unspecified impurities: for each impurity, n o t m ore than the
Detection Examine in ultraviolet light at 254 nm. area o f the principal peak in the chrom atogram obtained
Results T he p rin d p al spot in the chrom atogram obtained with with reference solution (a) (0.10 per cent);
the test solution is similar in position and size to. the p rindpal — 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).
COjH
C h lo rid e s (2.4.4)
M aximum 200 ppm .
B. (25)-2-(acetylamino)-3-[4-(acetoxy)phenyl]propanoic a d d
Dilute 10 m L o f solution S to 15 m L with water R.
(diacetyltyrosine).
S u lfates C2.4.13)
PhEur
M axim um 200 ppm .
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)
Aciclovir ★+** ★
Maximum 200 ppm , determined on 0.100 g. ★ ★
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)
"¿0
M axim um 20 ppm .
OH
In a separating funnel, dissolve 0.5 g in 10 m L of dilute
h 2n
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
C 8H 11N 5O 3 225.2 592 7 7 -8 9 -3
shake for 3 m in. T h e aqueous layer complies with the test.
H eav y m e ta ls (2.4.8) A c tio n a n d u se
Maximum 10 ppm . Purine nucleoside analogue; antiviral (herpesviruses).
Dissolve 2.0 g in water R and dilute to 20 m L with the same P re p a r a tio n s
solvent. 12 m L o f the solution complies with test A. Prepare Aciclovir C ream
the reference solution using lead standard solution Aciclovir Eye O intm ent
(1 ppm Pb) R.
Acidovir Infusion
A d d o v ir Oral Suspension
M aximum 0.5 p er cent, determined on 1.000 g by drying in
an oven at 105 °C. Adclovir Tablets
Dispersible A dd o v ir Tablets
M aximum 0.1 per cent, determined on 1.0 g. P h E u r __________________________________________________________________________________________
B a c te ria l e n d o to x in s (2.6.14)
D E F IN IT IO N
Less than 25 IU /g, if intended for use in the manufacture of
2-Amino-9- [(2-hydroxyethoxy) methyl] -1,9-dihydro-6//-purin-
parenteral preparations without a further appropriate
6-one.
procedure for the removal of bacterial endotoxins.
C o n te n t
A SSAY 98.5 per cent to 101.0 per cent (anhydrous substance).
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 CHARACTERS
end-point potentiometrically (2 . 2 . 2 0 ). A p p e a ra n c e ^
C n H 13N 0 4. S o lu b ility
Slightly soluble in water, very slightly soluble in ethanol
STORA GE
(96 per cent), practically insoluble in heptane. It dissolves in
Protected from light. If the substance is sterile, store in a dilute solutions o f mineral adds and alkali hydroxides.
sterile, airtight, tam per-proof container.
IM P U R IT IE S
Specified impurities A
Comparison aciclovir CRS.
present at a sufficient level, be detected by one or other of TESTS
the tests in the monograph. They are limited by the general A p p e a ra n c e o f so lu tio n
acceptance criterion for other/unspecified impurities and/or T he solution is clear (2.2.1) and n o t more intensely coloured
by the general monograph Substances for pharmaceutical use than reference solution Y7 (2.2.2, Method IT).
(2034). It is therefore not necessary to identify these Dissolve 0.25 g in a 4 g/L solution of sodium hydroxide R and
impurities for demonstration o f compliance. See also 5.10. dilute to 25 m L with the same solvent.
Control of impurities in substancesfor pharmaceutical use): B. . R e la te d su b sta n c e s
Liquid chromatography (2.2.29). Prepare the solutions
Solvent mixture dimethyl sulfoxide R, water R (20:80 VIV).
Phosphate buffer solution pH 2.5 Dissolve 3.48 g of dipotassium
A. (26)-2-amino-3-(4-hydroxyphenyl)propanoic acid hydrogen phosphate R in 1000 m L of water R and adjust to
(tyrosine), p H 2.5 with phosphoric add R.
1-68 Aciclovir 2016
Phosphate btcffer solution pH 3.1 Dissolve 3.48 g of dipotassium Limits:

hydrogen phosphate R in 1000 m L o f water R and adjust to — correction factor, for the calculation o f content, multiply the
p H 3.1 with phosphoric acid R. peak area o f im purity I by 1.5;
Test solution Dissolve 25 m g o f the substance to be examined — impurity B: not more than 7 times the area o f the
in 5.0 mT- o f dimethyl sulfoxide R and dilute to 25.0 m L with principal peak in the chrom atogram obtained with
water R. reference solution (b) (0.7 per cent);
— sum of impurities O and Q: n o t m ore than 3 times the area
Reference solution (a) Dissolve 5 mg o f addovir for system
o f the principal peak in the chrom atogram obtained with
suitability CRS (containing impurities A, B, J, K , N , O and
reference solution (b) (0.3 per cent);
P) in 1 m L o f dimethyl sulfoxide R and dilute to 5.0 m L with
— sum cf impurities K and R: n o t m ore than twice the area of
water R.
the principal peak in the chrom atogram obtained with
Reference solution (b) Dilute 1.0 m L o f the test solution to reference solution (b) (0.2 per cent);
100.0 m L with the solvent mixture. Dilute 1.0 mT. o f this — impurities A , G, J, N, F. for each impurity, n o t m ore than
solution to 10.0 m L with the solvent mixture. twice the area o f the principal peak in the chromatogram
Reference solution (c) Dissolve the contents of a vial o f aciclovir obtained with reference solution (b) (0.2 per cent);
for peak identification 1 CRS (containing impurities C and I) — impurities C, F, I: for each impurity, not m ore than the
in 200 pL o f dimethyl sulfoxide R and dilute to 1.0 m L with area o f the principal peak in the chrom atogram obtained
water R. with reference solution (b) ( 0.1 per cent);
Reference solution (d) Dissolve the contents o f a vial of — unspecified impurities: for each impurity, n o t m ore than
aciclovir for peak identification 2 CRS (containing impurities F 0.5 times the area o f the principal peak in the
and G ) in 1.0 m L of reference solution (a). chrom atogram obtained with reference solution (b)
Column’. (0.05 per cent);
— size: I = 0.25 m , 0 = 4.6 mm; — total: n o t m ore than 15 times the area o f the principal
— stationary phase: end-capped octadecylsüyl silica gel for peak in the chromatogram obtained with reference
chromatography R (5 pm). solution (b) (1.5 per cent);
Mobile phase: — disregard limit. 0.3 times the area o f the p rin d p al peak in
— mobile phase A: acetonitrile R, phosphate buffer solution the chrom atogram obtained with reference solution (b)
p H 3.1 (1:99 VIV); (0.03 per cent).
— mobile phase B: acetonitrile R, phosphate buffer solution W a te r (2.5.12)
p H 2.5 (50:50 VIV)’, M aximum 6.0 per cent, determ ined on 0.500 g.
Time Mobile phase A Mobile phase B M aximum 0.1 per cent, determ ined on 1.0 g.
(min) (per cent V/V) (per cent V/V) B a c te ria l e n d o to x in s (2.6.14, Method D)
0-5 100 0 Less than 0.50 IU/m g, if intended for use in th e manufacture
5 - 27 100 -» 80 0 -» 20 o f parenteral preparations without a further appropriate
procedure for the removal o f bacterial endotoxins.
27 - 40 80 20
A SSA Y
Dissolve 0.150 g in 60 m L o f anhydrous acetic add R. T itrate
Flow rate 1.0 mL/min. with 0.1 M perchloric add, determining the end-point
Detection Spectrophotom eter at 254 nm . potentiometrically (2.2.20). C an y out a blank titration.
Injection 10 pL o f the test solution and reference 1 m L o f 0.1 Mperchloric add is equivalent to 22.52 m g
solutions (b), (c) and (d). 0f C 8H u N 5O 3.
Identification of impurities Use the chrom atogram supplied IM P U R IT IE S
with aciclovir for peak identification 1 CRS and the Specified impurities A, B, C, F, G , I, J, K, N , O , P, Q, R
chrom atogram obtained with reference solution (c) to identify Other detectable impurities (the following substances would, if
the peaks due to impurities C and I; use the chrom atogram
present at a suffirient levd, be detected by one or other o f
supplied with addovir for peak identification 2 CRS and the
the tests in the monograph. T hey are limited by the general
chrom atogram obtained with reference solution (d) to
acceptance criterion for other/unspecified impurities and/or
identify the peaks due to impurities A, B, F, G, J, K, N , O
by the general monograph Substances for pharm aceutical use
and P.
(2034). It is therefore not necessary to identify these
Relative retention W ith reference to aciclovir (retention impurities for demonstration o f compliance. See also 5.10.
time = about 13 min): impurity B = about 0.4; Control of impurities in substances for pharmaceutical use): L, M.
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; O
im purity I = about 1.57; im purity J = about 1.62;
N O
im purity F = about 1.7; im purity A = about 1.8;
impurities K and R = about 2.5; impurity G = about 2.6. > p
N / -' CH3
System suitability: ^—o
— resolution: minimum 1.5 between the peaks due to
im purity C and aciclovir in the chrom atogram obtained A. 2 -[( 2 -am ino- 6-oxo- l,6-dihydro-9/i-purin-9-
with reference solution (c); m inim um 1.5 between the yl)methoxy] ethyl acetate,
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).
2016 Aciclovir 1-69
H2N
i V>
h N N
1 aV>
h3c ^ n^ n^ n
B. 2-amincHl ,7-dihydro-6i7-purin-6-one (guanine),

h y o
ch 3
L. N - (9-acetyl-6-oxo-6,9-dihydro-1.ff-purin-2-yl) acetamide
o -O OH (2^9-diacetylguanine),
1 1 / VJ
H2N N N 0
/^°x P ~ich3
C. 2-am ino-7- [(2-hydroxyethoxy)methyl]-l ,7-dihydro-6/i-
purin-6-onej
h3c^X ^JL^JL^
n n n
H
o
M . 2- [ [2-(acetylamino)-6-oxo-1,6-dihydro-7H-puiin-7-
yl]methoxy] ethyl acetate,
h3c ^ X a JCn> ;
" nA
H
^ n
\ — 0'
N . unknown structure,
O. unknown structure,
F. N-[9-[(2-hydroxyethoxy)methyl]-6-oxo-6,9-dihydro-l/i- 0
HNX .
purin-2-yl] acetamide,
o h2n ^ lX > n^ n^ ^ oh
O
i iY> ^
/
h3c ^ n ^ n ^ - n j—f ( 3
ch
P. 2-amino-9-(2-hydroxyethyl)-1,9-dihydro-6/f-purin-6-onej
H \— o
0
G. 2- [ [2-(acetylamino)-6-oxo-1,6-dihydro-9H-purin-9-
yl]methoxy] ethyl acetate.
x YN > N OH
V
HN JLn O
+
i xN >
H2N
x V>
h -O
/ \
OH
N NH2
I. 2-am ino-7-[ [2- [(2-amino-6-oxo-1,6-dihydro9H -purin-9- Q. mixture o f 2-amino-9-[[2-

yl)methoxy]ethoxy]methyl]-l,7-dihydro-6i/-purin-6-one, (hydroxymethoxy) ethoxy] methyl] -1 j9-dihydro-6//-purin-6-
one and 2-amino-9-[[2-(hydroxyethoxy)methoxy]methyl]-l,9-
dihydro-6//-purin-6-one,
H2N
a N
I> N / -----\
ccSl
N N NH2
'—O O—’
J. 9 ,9 '- [ethylenebis(oxymethylene)]bis(2-amino-l,9-dihydro-
6//-purin-6-one),
R. 9 ,9 '-
[methylenebis(oxyethyleneoxymethylene)]bis(2-amino-l,9-
dihydro-6//-purin-6-one).
PhEur
K. 2,2'-(methylenediimino)bis[9-[(2-hydroxyethoxy)methyl]-
1,9-dihydro-6/i-purin-6-one],
1-70 Acitretin 2016
TESTS
Acitretin ******
Related substances
(Ph Eur monograph 1385) * Liquid chrom atography (2.2.29). Maintain the sampler at
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
Cj i H ^ O s 326.4 55079-83-9 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
A ctio n a n d u se solution to 2 5 .0 m l. with anhydrous ethanol R
Vitamin A analogue (retinoid); treatm ent o f psoriasis; Reference solution (b) Dissolve 1 .0 m g of tretinoin CRS in
ichthyosis; D arier’s disease. anhydrous ethanol R and dilute to 2 0 .0 m L with the same
P re p a r a tio n solvent. M ix 5 .0 m L o f this solution with 2 .5 m L o f
A dtretin Capsules reference solution (a) and dilute to 1 0 0 .0 m L with anhydrous
ethanol R
PhEtr___________________________________________________________
Reference solution (c) Dilute 2 .5 m L o f reference solution (a)
D E F IN IT IO N to 5 0 .0 m L with anhydrous ethanol R. Dilute 3 .0 m L o f this
(aU-£)-9-(4-Methoxy-2,3, 6-trimethylphenyl)-3,7 - solution to 2 0 .0 m l. with anhydrous ethanol R
dimethylnona-2j4j6j8-tetraenoic ad d . Column’.
C o n te n t — size I = 0 .2 5 m , 0 = 4 mm;
98.0 per cent to 102.0 per cent (dried substance). — stationary phase: microparticulate octadecylsQyl silica gel for
chromatography R (5 pm) with a specific surface area of
CHARACTERS 2 0 0 m 2/g, a pore size o f 15 n m and a carbon loading o f
A p p e a ra n c e 20 per cent;
Yellow or greenish-yellow, crystalline powder. — temperature: 25 °C.
S o lubility Mobile phase A 0 .3 per cent V/V solution o f glacial acetic
Practically insoluble in water, sparingly soluble in acid R in a mixture o f 8 volumes o f water R and 9 2 volumes
tetrahydrofuran, slightly soluble in acetone and in ethanol o f anhydrous ethanol R.
(96 per cent), very slightly soluble in cyclohexane. Flow rate 0 .6 mL/min.
It is sensitive to air, heat and light, espedally in solution. Detection Spectrophotom eter at 3 6 0 nm .
It shows polymorphism. Injection 10 pL o f test solution (a) and reference solutions (b)
Cany ota all operations as rapidly as possible and avoid exposure and (c).
to actinic light; use freshly prepared solutions. Run time 2 .5 times the retention time o f adtretin.
ID E N T IF IC A T IO N Retention time Impurity A = about 4 .8 min; tretinoin = about
First identification B 5 .2 m in; adtretin = about 6 .2 min; impurity B = about
Second identification A , C 10.2 min.
A. Ultraviolet and visible absorption spectrophotom etry System suitability: reference solution (b):
(2.2.25). — resolution: minim um 2.0 betw een the peaks due to
Test solution Dissolve 15.0 m g in 10 m L o f tetrahydrofuran R adtretin and tretinoin; if necessary, adjust the
and dilute immediately to 100.0 m L with the same solvent. concentration o f anhydrous ethanol R
D ilute 2.5 m L o f this solution to 100.0 m L with Limits:
tetrahydrofuran R — 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);
Absorption maximum A t 358 nm . — total: n o t more than the area o f the peak due to ad tretin
Specific absorbance at the absorption maximum 1350 to 1475. in the chrom atogram obtained with reference solution (b)
B. Infrared absorption spectrophotom etry (2.2.24). (1.0 per cent);
Preparation Discs. — disregard limit. 0.1 times the area o f the principal peak in
the chrom atogram obtained with reference solution (c).
Comparison acitretin CRS.
If the spectra obtained in the solid state show differences, Palladium
dissolve die substance to be examined and the reference M axim um 10 ppm.
substance separately in 2-propanol R heating under reflux, A tomic absorption spectrometry (2.2.23, Method I).
filter, evaporate to dryness and record new spectra using the Test solution Introduce 2 .0 g into a quartz beaker and add
residues. 3 m l. o f magnesium nitrate solution R. H eat in a muffle
C. Examine the chrom atograms obtained in the assay. furnace to 3 5 0 °C at a rate o f 4 0 °C/min to incinerate the
Results T he prindpal peak in the chrom atogram obtained 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
with test solution (b) is similar in retention time to the
m ixture o f 0 .7 5 m L o f hydrochloric add R and 0 .2 5 m L o f
principal peak in the chrom atogram obtained with reference
solution (a).
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 (Ph. Eur. monograph 2445) *
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
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 A ctio n a n d u se
vising 2 m l. o f lead standard solution (10 ppm Pb) R. Vitamin A analogue (retinoid); treatm ent of acne.
L oss o n d ry in g (2.2.32) P re p a r a tio n s
M aximum 0.5 per cent, determined on 1.000 g by drying Adapalene Cream
in vacuo at 100 °C for 4 h. Adapalene Gel
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 6-(4-Methoxy-3-tricyclo [3.3.1.13,7] dec-1-
flasks and prepare the solutions immediately before use. ylphenyI)naphthalene-2-carboxylic acid.
Liquid chromatography (2.2.29) as described in the test for C o n te n t
related substances with the following modifications. 98.0 per cent to 102.0 per cent (dried substance).
Injection T est solution (b) and reference solution (a). CHARACTERS
System suitability: A p p e a ra n c e
— repeatability: maximum relative standard deviation of White or almost white powder.
1.0 per cent after 6 injections o f reference solution (a); Solubility
if necessary, adjust the integration parameters.
Practically insoluble in water, sparingly soluble in
Calculate the percentage content of C 21H 26O 3 from the tetrahydrofuran, practically insoluble in ethanol
declared content of acitrean CRS. (96 per cent).
STORAGE ID E N T IF IC A T IO N
In an airtight container, protected from light, at a Infrared absorption spectrophotometry (2.2.24).
tem perature o f 2 °C to 8 °C. Comparison adapalene CRS.
I t is recom mended that the contents o f an opened container
TESTS
be used as soon as possible and any unused part be protected
by an atmosphere o f inert gas. 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
IM P U R IT IE S than reference solution BY6 (2.2.2, Method II).
Specified impurities A, B. Dissolve 0.2 g in tetrahydrofuran R and dilute to 20 m l. with
the same solvent.
ch 3 ch3 ch 3
h3co
Solvent mixture tetrahydrofuran R, acetomtrUe R, water R
(20:37:43 V/V/V).
ch 3
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
A. (2Zj4£,6£’,8£)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-
solvent mixture and dilute to 20.0 m L with tetrahydrofuran R.
dim ethylnona-2,4,6,8-tetraenoic add.
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)-3,7-
Reference solution (b) Dissolve 2.4 m g of adapalene
dim ethylnona-2,4,6,8-tetraenoate.
impurity C CRS in 2 m L of tetrahydrofuran R and dilute to
—-________________________________________________________ PhEur 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.
1-72 Adapalene 2016
Reference solution (c) Dissolve the contents of a vial of — disregard Omit: 0.5 times the area of the principal peak in
adapalene for peak identification CRS (containing impurities A, the chrom atogram obtained w ith reference solution (a)
C and D ) in 0.5 m L o f tetrahydrqfuran R and dilute to (0.05 per cent).
1.0 m L with the solvent mixture. H eav y m e ta ls (2.4.8)
Reference solution (d) Dissolve 20.0 m g o f adapalene CRS in M axim um 20 ppm .
50 m L of tetrahydrqfuran R> add 35 m L o f the solvent 0.250 g complies with test G . Prepare the reference solution
m ixture and dilute to 100.0 m L with tetrahydrofuran R. u sin g 0.5 m L of lead standard solution (10 ppm Pb) R.
D ilute 5.0 m L of the solution to 50.0 m L with die solvent
mixture.
M axim um 0.5 p er cent, determ ined on 1.000 g by drying in
Column: an oven at 105 °C for 4 h.
— size. I = 0.25 m , 0 = 4.6 mm;
— stationary phase: end-capped phenylsQyl silica gel for S u lfa te d a s h ( 2.4.14)
chromatography R (5 tun) with a carbon loading of M axim um 0.1 per cent, determ ined on 1.0 g.
7.5 per cent; A SSA Y
— temperature: 30 °C. Liquid chromatography (2.2.29) as described in the test for
Mobile phase: related substances w ith the following modification.
— mobile phase A: glacial acetic add R, water R (0.1:100 VIV)\ Irtjection T est solution (b) and reference solution (d).
— mobile phase B: tetrahydrofuran R, acetomtrUe R
Calculate the percentage content o f adapalene from the
(35:65 VIV)\
declared content o f adapalene CRS.
IM P U R IT IE S
(min)
Specified impurities A, C , D
(percent V/V) (per cent V/V)
0 -2 .5 50 50 Other detectable impurities (the following substances would, if
present at a sufficient level, be detected by one or other o f
23 - 40 50-» 28 50*72
the tests in the m onograph. T hey are limited by die general
40 - 42 28 72 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
Flow rate 1 .2 mL/min. impurities for dem onstration o f compliance. See also 5.10.
Detection Spectrophotom eter at 2 7 0 nm . Control of impurities in substances for pharmaceutical use): B.
Injection 2 5 |iL of test solution (a) and reference solutions (a),
(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 ; A. 2 ,2 /-binaphthalene- 6,6 '-dicarboxylic acid,
impurity C = about 0 .9 ; im purity D = about 1.9.
— 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.
Limits: COjH
the peak areas o f the following impurities by the B. 6-[3-(3-hydroxytricydo[3.3.1.1 3,7]dec-l-yl)-4-
corresponding correction facto r im purity A = 0 .7 ; methoxyphenyl] naphthalene- 2-carboxylic ad d ,
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
C. l-(2-m ethoxyphenyi)tricydo[3.3.1. l 3,7]decane,
— 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) D . 1, 1 '- [4,4 '-bis(methoxy)biphenyl-3,3
(0 .5 per cent); diyl]bis(tricyclo [3.3.1.13’7] decane).
______________________________________________■ PhEut
2016 Adenine 1-73
Adenine ★★* ★★ Test solution (b) Dilute 1 m L of test solution (a) to 10 m L

★ ★ with dilute acetic add R.
(Ph Eur monograph 0800) ***** 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
nh 2
with the same ad d .
nV ^ v Reference solution (b) Dilute 1 m L o f test solution (b) to
I I /) 20 m L with dilute acetic add R.
Reference solution (c) Dissolve 10 m g of adenine CRS and
C5H5N5 135.1 73-24-5
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.
Action and use Apply to the plate 5 jiL o f each solution. Develop over a
C onstituent of anticoagulant and preservative solutions for path of 12 cm using a mixture of 20 volumes of concentrated
-blood. 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
PhEir. examine in ultraviolet light at 254 nm. Any spot in the
DEFINITION chromatogram obtained with test solution (a), apart from the
Adenine contains not less than 98.5 p er cent and not more prindpal spot, is not m ore intense than the spot in the
than the equivalent of 101.0 per cent o f 7H-purin-6-amine, chromatogram obtained with reference solution (b)
calculated w ith reference to the dried substance. (0.5 per cent). T h e test is not valid unless the chromatogram
obtained with reference solution (c) shows two clearly
CHARACTERS separated spots.
A white or almost white powder, very slightly soluble in
Chlorides (2.4.4)
water and in alcohol. It dissolves in dilute m in eral a d d s and
T o 10 m L o f solution S add 1 m L of concentrated ammonia R
in dilute solutions of alkali hydroxides.
and 3 m L o f silver nitrate solution R2. Filter. Wash the
IDENTIFICATION predpitate with a little water R and dilute the filtrate to
First identification A. 15 m L with water R. T h e solution complies with the limit
Second identification B, C. 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
A. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with add R.
adenine CRS. Examine the substances prepared as discs. 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
related substances. The principal spot in the chromatogram
(300 ppm).
obtained with test solution (b) is similar in position and size
A m m on iu m
to the prindp al spot in the chromatogram obtained with
reference solution (a). Prepare a cell consisting of two watch-glasses 60 mm in
diameter placed edge to edge. T o the inner wall of the upper
C. T o 1 g add 3.5 m L of propionic anhydride R and boil for
watch-glass stick a piece o f red litmus paper R 5 mm square
15 min with stirring. Cool. T o the resulting crystalline mass
and wetted with a few drops of water R. Finely powder the
add 15 m L o f light petroleum R and heat to boiling with
substance to be examined, place 0.5 g in the lower watch-
vigorous stirring. Cool and filter. W ash the predpitate with
glass and suspend in 0.5 mL of water J?. T o the suspension
two quantities, each o f 5 mT, of light petroleum R. Dissolve
add 0.30 g of heavy magnesium oxide R. Briefly triturate with
the predpitate in 10 m L of water R and boil for 1 min. Filter
a glass rod. Immediately close the cell by putting the two
the mixture at 30 °C to 40 °C. Allow to cool. Filter, and dry
watch-glasses together. H eat at 40 °C for 15 min. T he litmus
the predpitate at 100 °C to 105 °C for 1 h. T he melting
paper is n o t more intensdy blue coloured than a standard
point (2.2.14) o f the predpitate is 237 °C to 241 °C.
prepared at the same time and in the same manner using
TESTS 0.05 m L o f ammonium standard solution (100 ppm NH 4) R,
Solution S 0.5 m L o f water R and 0.30 g of heavy magnesium oxide R
Suspend 2.5 g in 50 m L o f distilled water R and boil for (10 ppm).
3 m in. Cool and dilute to 50 m L with distilled water R. Filter. Heavy metals (2.4.8)
Use the filtrate as solution S. 1.0 g complies with test C for heavy metals (20 ppm).
Appearance o f solution Prepare the reference solution using 2 m L of lead standard
Dissolve 0.5 g in dilute hydrochloric acid R and dilute to solution (10 ppm Pb) R.
50 m L with the same ad d . The solution is clear (2.2.1) and Loss on drying (2.2.32)
colourless (2.2.2, Method II). N o t more than 0.5 per cent, determined on 1.000 g by
Acidity or alkalinity drying in an oven at 105 °C.
T o 10 m L o f solution S add 0.1 mL o f bromothymol blue Sulfated ash (2.4.14)
solution R1 and 0.2 m L of 0.01 M sodium hydroxide. N ot more than 0.1 per cent, determined on 1.0 g.
The solution is blue. Add 0.4 m L of 0.01 M hydrochloric acid. ASSAY
T he solution is yellow.
Dissolve 0.100 g in a mixture o f 20 m L of acetic anhydride R
Related substances and 30 m L of anhydrous acetic acid R. T itrate with 0.1 M
Examine by thin-layer chromatography (2.2.27), using silica perchloric add, determining the end-point potentiometrically
gel GF2 5 4 R as the coating substance. (2.2.20).
Test solution (a) Dissolve 0.10 g of the substance to be 1 m L of 0 . 1 M perchloric add is equivalent to 13.51 m g of
examined in dilute acetic acid R, with heating if necessary, and CsHsNs.
dilute to 10 m L with the same add. __________________________________________________________ PhEur
1-74 Adenosine 2016
★ ★ Reference solution (a) Dilute 1.0 m L o f the test solution to

Adenosine ★ ★ 100.0 m L with the mobile phase. Dilute 1.0 m L o f this
(Ph. Eur. monograph 1486) ***** solution to 10.0 m L with the mobile phase.
Reference solution (b) Dissolve 5 m g o f adenine R (impurity A)
NH2 and 5 m g o f inosine R (impurity G) in the mobile phase and
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
Mobile phase water R, solvent mixture (40:60 VIV).
C 10H 13N 5O4 267.2 58-61-7
Detection Spectrophotom eter at 254 nm.
Action and use Injection 20 pL.
Antiarrhythmic. Run time 1.5 times the retention time o f adenosine.
P h E tr ____________________
Relative retention W ith reference to adenosine (retention
DEFINITION im purity G = about 0.4.
9-P-D-Ribofuranosyl-9ii-purin-6-araine. System suitability, reference solution (b):
Content — resolution: minim um 1.5 between the peaks due to
99.0 per cent to 101.0 per cent (dried substance). impurities A and G.
CHARACTERS Limits:
Appearance — correction factors: for the calculation o f content, multiply
W hite or alm ost white, crystalline powder. the peak areas o f the following impurities by the
corresponding correction facto r impurity A = 0.6;
Solubility
im purity G = 1.4;
Slightly soluble in water, soluble in h o t water, practically
— impurity A: n o t m ore than twice the area o f the prindpal
insoluble in ethanol (96 per cent) and in methylene chloride.
It dissolves in dilute mineral adds.
solution (a) ( 0.2 per cent);
mp — impurity G: n o t more than the area o f the prin d p al peak
A bout 234 °C. in the chrom atogram obtained with reference solution (a)
IDENTIFICATION (0.1 p er cent);
Infrared absorption spectrophotom etry (2.2.24). — 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
Comparison adenosine CRS.
with reference solution (a) (0.10 per cent);
TESTS — total: n o t m ore than 5 times the area o f the prin d p al peak
Solution S in the chrom atogram obtained with reference solution (a)
Suspend 5.0 g in 100 m L o f distilled water R and heat to (0.5 p er cent);
boiling. Allow to cool, filter with the aid o f vacuum and — disregard ltmir. 0.5 times the area of the prin d p al peak in
dilute to 100 m L w ith distilled water R. the chrom atogram obtained with reference solution (a)
Appearance o f solution (0.05 p er cent).
Solution S is colourless (2.2.2, Method II). C h lo rid e s (2.4.4)
Acidity or alkalinity M axim um 100 ppm .
T o 10 m L o f solution S, add 0.1 mT. o f bromocresolpurple D ilute 10 m L o f solution S to 15 m L with water R.
solution R and 0.1 m L o f 0.01 M hydrochloric add. S u lfa te s (2.4.13)
T he solution is yellow. Add 0.4 m L o f 0.01 M sodium M axim um 200 ppm , determined on solution S.
hydroxide. T h e solution is violet-blue.
Specific optical rotation (2.2.7) M axim um 10 ppm , determined on 0.5 g.
- 4 5 to - 4 9 (dried substance).
Prepare the standard using 5 m L o f ammonium standard
Dissolve 1.25 g in / M hydrochloric acid and dilute to solution (1 ppm NH 4 ) R.
50.0 m L with the same ad d . Examine within 10 min o f
L o ss o n d ry in g (2.2.32)
preparing the solution.
Maximum 0.5 per cent, determined on 1.000 g by drying in
Related substances an oven a t 105 °C.
Liquid chrom atography (2.2.29).
Solvent mixture Dissolve 6.8 g o f potassium hydrogen sulfate R M axim um 0.1 p er cent, determined on 1.0 g.
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 A SSA Y
hydroxide R and dilute to 1000 m L with the same solvent. Dissolve 0.200 g, warming slightly if necessary, in a mixture
U se a freshly prepared solvent mixture. o f 20 m L o f acetic anhydride R and 30 m L o f anhydrous acetic
Test solution Dissolve 20 m g o f the substance to be examined add R. 1111316 w ith 0.1 M perchloric add, determ ining the
end-point potentiometrically (2 . 2 . 2 0 ).
in the mobile phase and dilute to 20 m L with the mobile
phase.
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 ★ ★
* .★
(Fh Eur monograph 1586) *★*
IM P U R IT IE S
Specified impitrities A, G.
Other detectable impurities (the following substances would, if HO2C'
present at a sufficient levd, be detected by one or other of
C6H10O4 146.1 124-04-9
by the general monograph Substances for pharmaceutical use A c tio n a n d u se
(2034). It is therefore not necessary to identify these Exdpient.
impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use): F, H. PhEur.
D E F IN IT IO N
nh2
Hexanedioic ad d .
C o n te n t
CT> N
N N
H
CHARACTERS
A p p e a ra n c e
A. 7H-purin-6-amine (adenine).
W hite or alm ost white, crystalline powder.
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.
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.
F. l-î>ribofuranosylpyrim idine-2j4(l//j3/i)-dione TESTS
(uridine), 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).
HN W ash the filter with distilled water R. Collect the filtrate and
the washings until a volume of 50 m L is obtained.
HO 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 II).
Dissolve 1.0 g in methanol R and dilute to 20 m l. with the
OH OH
same solvent.
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

0 Test solution Dissolve 0.20 g of the substance to be examined
phase.
h2n
Reference solution (a) Dissolve 20 m g of ghitaric acid R in
ho 1.0 m L o f the test solution and dilute to 10.0 m L with the
mobile phase.
V? OH 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
Column:
H . 2-amino-9-P-D-ribofuranosyl-1,9-dihydro-6H-purin-6-one — size. I = 0.125 m , 0 = 4.0 mm,
(guanosine). — stationary phase, spherical octadecylstiyl silica gel for
PhEur chromatography R (5 Jim) with a specific surface area o f
350 m 2/g and a pore size of 10 nm,
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.
Injection 20 (iL.
1-76 Adrenaline 2016
Run time 3 times the retention time o f adipic ad d . IM P U R IT IE S

— resolution: minimum 9.0 between the peaks due to glutaric 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 B. R = C 0 2H : butanedioic a d d (succinic a d d ),
in the chrom atogram obtained with reference solution (b) C. R = [ C H J 3-C O 2H : heptanedioic a d d (pimelic ad d ).
(0.1 per cent),
________________________________:___________________________ PhEur
— 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) Adrenaline / Epinephrine ** * ***
(0.05 per cent). ** **
M axim um 200 ppm .
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,
C9H 13N 0 3 183.2 51-43-4
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 A c tio n a n d u se
water R. Add 0.10 g o f zinc powder R and cool in iced water Adrenoceptor agonist
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
Adrenaline Eye Drops/Epinephrine Eye D rops
add R1 and 1 m L o f a 10 g/L solution of
naphthylethylenediamine dihydrochloride R. Allow to stand at D ilute Adrenaline Injection (I in 10,000)/Dilute Epinephrine
room tem perature. A fter 15 m in the mixture is no t more Injection (1 in 10,000)
intensely coloured th a n a standard prepared at the sam e time
Ph Eur ---------------------------------------------------------------------------------
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. D E F IN IT IO N
T he test is invalid if a blank solution prepared at the same 4- [(1R) - 1-Hydroxy-2-(methyiamino) ethyl] benzene-1,2-diol.
time and in the same m anner, using 1 m L o f water R instead Synthetic p ro d u c t
of 1 m L of solution S, is more intensely coloured than a
C o n te n t
2 m g/L solution o f potassium permanganate R.
S u lfates (2.4.13)
CHARACTERS
Maximum 500 ppm.
A p p e a ra n c e
D ilute 3 m L o f solution S to 15 m L w ith distilled water R. White or alm ost white crystalline powder, becoming coloured
Ir o n (2.4.9) on exposure to air and lig h t
Maximum 10 ppm , determ ined on solution S.
S o lu b ility
H eav y m e ta ls (2.4.8) Practically insoluble in water, in ethanol (96 per cent) and in
M aximum 10 ppm . methylene chloride. It dissolves in hydrochloric ad d .
12 m L of solution S complies with test A. Prepare the ID E N T IF IC A T IO N
reference solution using lead standard solution (1 ppm Pb) R. A Infrared absorption spectrophotom etry (2.2.24).
L oss o n d ry in g (2.2.32) Comparison adrenaline CRS.
B. Spedfic optical rotation (see Tests).
an oven at 105 °C.
S u lfa te d a s h (2.4.14) TESTS
M axim um 0.1 per c e n t S o lu tio n S
Dissolve 1.000 g in a 25.75 g/L solution o f hydrochloric add R
M d t 1.0 g completely over a gas burner, then ignite the
and dilute to 50.0 m L with the same solvent Examine the
m d te d substance with the burner. After ignition, lower or
solution immediately.
remove the flame in order to prevent th e substance from
boiling and keep it burning until completely carbonised. A p p e a ra n c e o f so lu tio n
Carry out the test for sulfated ash using the residue. Solution S is not more opalescent than reference
suspension II (2.2.1) and n o t m ore intensely coloured than
A SSA Y reference solution BY5 (2.2.2, Method II).
Dissolve 60.0 m g in 50 m L o f water R. A dd 0.2 m L o f
S pecific o p tic a l ro ta tio n (2.2.7)
phenolphthalein solution R and titrate w ith 0.1 M sodium
- 5 0 .0 to - 5 4 .0 (dried substance), determined on solution S.
hydroxide.
1 m L of 0.1 M sodium hydroxide is equivalent to 7.31 m g of R e la te d su b s ta n c e s
Liquid chromatography (2.2.29). Prepare the solutions protected
C6H 10O4.
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 corresponding correction factor: impurity D = 0.7;
solvent (it is usually necessary to stir for at least 30 min to impurity E = 0.6;
achieve complete dissolution). Adjust to p H 2.8 with — impurities B, C, F: for each impurity, not more than twice
phosphoric acid R. the area of the principal peak in the chromatogram
Solvent mixture B acetomtrUe R l, solvent mixture A obtained with reference solution (a) (0.2 per cent);
(13:87 VIV). — impurities D, E: for each impurity, not more than the area
Test solution Dissolve 40 mg of the substance to be examined o f the principal peak in the chromatogram obtained with
reference solution (a) (0.1 p er cent);
in 5 m L o f 0.1 M hydrochloric add and dilute to 50.0 m L
with solvent mixture B.
— unspedfied impurities: for each impurity, not more than the
area o f the principal peak in the chromatogram obtained
Reference solution (a) Dilute 1.0 m L of the test solution to with reference solution (a) (0.10 per cent);
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.
in the chromatogram obtained with reference solution (a)
Reference solution (b) Dissolve 1.5 mg of noradrenaline (0.5 per cent);
tartrate CRS (impurity B) and 1.5 m g of adrendone — disregard limit. 0.5 times the area of the principal peak in
hydrochloride R (impurity C) in solvent mixture B, add the chromatogram obtained with reference solution (a)
1.0 m L of the test solution and dilute to 100 m L with (0.05 per cent).
solvent mixture B.
Loss on drying (2.2.32)
Reference solution (c) Dissolve the contents o f a vial o f M axim um 0.5 per cent, determined on 1.000 g by drying
adrenaline impurity mixture CRS (containing impurities D over diphosphorus pentoxide R at a pressure not exceeding
and E) in 1.0 m L o f the blank solution. 0.7 kPa for 18 h.
Reference solution (d) Dissolve 4 m g o f adrenaline with Sulfated ash (2.4.14)
impurity F CRS in 0.5 m L of 0.1 M hydrochloric add and M axim um 0.1 per cent, determined on 1.0 g.
dilute to 5 m L with solvent mixture B.
Blank solution 0.1 M hydrochloric acid, solvent mixture B ASSAY
(1:9 VIV). 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
Column:
potentiometrically (2 . 2 .2 0 ).
— size. I = 0.10 m , 0 = 4.6 mm;
— stationary phase, end-capped octadecylsOyl silica gel for 1 m L of 0.1 M perchloric add is equivalent to 18.32 mg
chromatography R (3 |xm); o f C 9H 13N 0 3.
— temperature: 50 °C. STORAGE
Mobile phase: U nder nitrogen, protected from light.
— mobile phase A: acetomtrUe R l, solvent mixture A
IM P U R IT IE S
(5:95 VIV)-,
Specified impurities B, C , D , E, F
— mobile phase B: acetonitrUe R l, solvent mixture A
(45:55 VIV)-,
HO
(min) (per cent V7V) (per cent V/V) HO
0 - 15 92*50 8*50
15-20 50*92 50*8 B. (1 i?)-2-am ino-1-(3,4-dihydroxyphenyl)ethanol

2 0 -2 5 92 8 (noradrenaline),
O
Injection 20 (lL.
with adrenaline impurity mixture CRS and the chromatogram C . 1-(3,4-dihydroxyphenyl)-2-(methylamino) ethanone
obtained with reference solution (c) to identify the peaks due (adrenalone),
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.
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): D . 4-[(li?)-2-(benzyImethylamino)-l-hydroxyethyl]benzene-
— resolution: m inim um 3.0 between the peaks due to 1,2-diol,
im purity B and adrenaline.
Limits:
1-78 Adrenaline Acid Tartrate 2016
(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.
Preparation Discs o f adrenaline base prepared as described
under identification test A.
E. 2-(beri2ylmethylamino)-l-(3,4-dihydroxyphenyl)ethanonej Comparison U se adrenaline base prepared as described under
identification test A from 50 m g o f adrenaline tartrate CRS
H O ,s h H 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).
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
Appearance of solution
PhEur
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
Adrenaline Acid Tartrate / * * * * *
★ ★ solvent. Examine the solution im m ediatdy.
Epinephrine Acid Tartrate ***** Related substances

Liquid chromatography (2.2.29). Prepare the solutions protected
(Adrenaline Tartrate, Ph Eur monograph 0254)
from light.
H OH
H OH
Solvent mixture A Dissolve 5.0 g o f potassium dihydrogen
COjH
phosphate R and then 2.6 g o f sodium octanesidfonate R in
HOjC water for chromatography R, and dilute to 1000 m L with the
H OH same solvent (it is usually necessary to stir for at least 30 min
to achieve complete dissolution). Adjust to p H 2.8 with
C 13H 19NO 9 333.3 51-42-3 phosphoric acid R.
Solvent mixture B acetonitrUe R l, solvent m ixture A
Action and use (130:870 V/V).
Adrenoceptor agonist.
Test sdution Dissolve 75 m g of the substance to be examined
Preparations 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 Reference solution (a) Dilute 1.0 m L o f the test solution to
Injection (1 in 10,000) 100.0 m L with solvent mixture B. Dilute 1.0 m L o f this
Adrenaline Solution/Epinephrine Solution solution to 10.0 m L with solvent mixture B.
Adrenaline and Cocaine Intranasal Solution Reference solution (b) Dissolve 1.5 mg of noradrenaline
Bupivacaine and Adrenaline Injection/Bupivacaine and tartrate CRS (impurity B) and 1.5 m g of adrenalone
Epinephrine Injection 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
Lidocaine and Adrenaline Injection/Lidocaine and
solvent m ixture B.
Epinephrine Injection
Reference solution (c) Dissolve the contents o f a vial of
PhEur___________________________________________________________ adrenaline impurity mixture CRS (impurities D and E) in
D E F IN IT IO N 0.1 m L o f 0.1 M hydrochloric add and 0.9 m L o f solvent
mixture B.
(li?)-l-(3j4-Dihydroxyphenyl)-2-(m ethylam ino)ethanol
hydrogen (2i?,3i?)-2,3-dihydroxybutanedioate. 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
Content
dilute to 5.0 m L with solvent mixture B.
Blank solution 0.1 M hydrochloric add, solvent mixture B
CHARACTERS (1:9 VIV).
Appearance Column:
W hite or greyish-white, crystalline powder. — size: I = 0.10 m , 0 = 4.6 mm;
Solubility — stationary phase: end-capped octadecylsilyl silica gel for
F red y soluble in water, slightly soluble in ethanol chromatography R (3 pm);
(96 per cent). — temperature: 50 °C.
ID E N T IF IC A T IO N Mobile phase:
A. Dissolve 5 g in 50 m L of a 5 g/L solution of sodium — mobile phase A: acetomtrUe R l, solvent mixture A
metabisidfite R and m ake alkaline by addition of ammonia R. (5:95 V/V);
Keep, the mixture at room tem perature for a t least 15 min — mobile phase B: acetomtrUe R l, solvent mixture A
and filter. Reserve the filtrate for identification test C . W ash (45:55 V/V);
the predpitate with 3 quantities, each o f 10 mL, of
methanol R. D ry at 80 °C. T h e specific optical rotation
I
2016 Agar 1-79
Tune Mobile phase A Mobile phase B IM P U R IT IE S

(min) (per cent V/V) (per cent V/V) Specified impurities A, B, C , D , E
0 - 15 92->50 8 -> 50
A. unknown structure,
15 -2 0 50->92 50 -> 8
H OH
20 - 25 92 8

Detection Spectrophotom eter at 210 nm. B. ( 1i?)-2-amino-1-(3,4-dihydroxyphenyl)ethanol
Injection 20 |xL. (noradrenaline),
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. , C. 1-(3,4-dihydroxyphenyl)-2-(methyiamino)ethanone
(adrenalone),
Relative retention W ith reference to adrenaline (retention
time = about 4 min): impurity B = about 0.8;
impurity B and adrenaline.
Limits:
— correction factors: for the calculation o f content, multiply D . 4-[(li?)-2-(benzylmethylamino)-l-hydroxyethyl]benzene-
the peak areas o f the following impurities by the 1, 2-diol,
corresponding correction factor impurity D = 0.7;
im purity E = 0.6;
— impurity A: not more than 3 times the area of the
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 E. 2-(benzyimethylamino)-l-(3,4-dihydroxyphenyl)ethanone.
of the principal peak in the chromatogram obtained with — ___ ____________________________________________________ PhEtr
with reference solution (a) (0.10 per cent); ★ ★
— total: n o t more than 6 times the area of the principal peak Agar ★ ★
in the chromatogram obtained with reference solution (a) (Ph. Eur. monograph 0310) *****
(0.6 per cent);
— disregard limit. 0.5 times the area o f the principal peak in Action and use
the chrom atogram obtained with reference solution (a) Excipient.
(0.05 p er cent).
Ph Eur_______________________
Loss on drying ('2.2.32)
M aximum 0.5 per cent, determined on 1.000 g by drying DEFINITION
in vacuo for 18 h. Polysaccharides from various species of Rhodophyceae
Sulfated ash (2.4.14) mainly belonging to the genus Gelidium. It is prepared by
Maximum 0.1 per cent, determined on 1.0 g. treating the algae with boiling water; the extract is filtered
whilst hot, concentrated and dried.
ASSAY
Dissolve 0.300 g in 50 m L of anhydrous acetic acid R, heating CHARACTERS
gently if necessary. Titrate with 0.1 M perchloric acid until a Appearance
bluish-green colour is obtained, using 0.1 m L of crystal violet Powder or crumpled strips 2-5 m m wide or sometimes flakes,
solution R as indicator. colourless or pale yellow, translucent, somewhat tough and
difficult to break, becoming more britde on drying.
1 m L of 0.1 M perchloric acid is equivalent to 33.33 mg
of C 13H 19N O 9. Mucilaginous taste.
STORAGE IDENTIFICATION
In an airtight container, or preferably in a sealed tube under A. Examine under a microscope. W hen m ounted in 0.005 M
vacuum or u n d er an inert gas, protected from light. 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
1-80 Air 2016
grains on an am orphous background; occasional brown, solution add 5 m L o f picric acid solution R. N o turbidity
round or ovoid spores with a reticulated surface, measuring appears within 10 min.
up to 60 nm, may be present. Reduce to a powder, if L o ss o n d ry in g (2.2.32)
necessary. T h e pow der is yellowish-white. Examine un d er a M axim um 20.0 per cent, determ ined on 1.000 g o f the
microscope using 0.005 M iodine. T h e powder presents pow dered herbal drug (355) (2.9.12) by drying in an oven at
angular fragments w ith num erous grains similar to those seen 105 °C.
in the strips and flakes; some o f the fragments are stained
T o ta l a s h (2.4.16)
brownish-violet.
B. Dissolve 0.1 g with heating in 50 m L of vocaer R. Cool.
M ic ro b ia l c o n ta m in a tio n
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 Y M C : acceptance criterion 102 C FU /g (2.6.12).
T he liquid becomes pale yellow. Absence o f Escherichia coli (2.6.13).
C. H eat 5 m L o f the mucilage prepared for identification Absence o f Salmonella (2.6.13).
test B on a w ater-bath with 0.5 m L o f hydrochloric acid R for
L A B E L L IN G
30 min. A dd 1 m L o f barium chloride solution R l. A white
T h e label states th e swelling index.
turbidity develops within 30 min.
___________________________________________________________ PhEur
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. Medical Air *****
TESTS
(Medicinal Air\ Ph Eur monograph 1238) *
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
When Medical Air is intended for use in a room in which
magnetic resonance imaging (MRJ) is being performed, the
the label, determ ined on the pow dered herbal drug (355)
cylinder and fittings should be made from suitable non-
(2.9.12).
ferromagnetic materials and labelled accordingly.
In so lu b le m a t te r
PhEur___________________________________________________________
T o 5.00 g o f the powdered herbal drug (355) (2.9.12) add D E F IN IT IO N
100 m i. of water R and 14 m L o f dilute hydrochloric add R. Compressed am bient air.
Boil gendy for 15 m in with frequent stirring. Filter the hot C o n te n t
liquid through a tared, sintered-glass filter (160) (2 . 1 . 2 ), rinse 20.4 per cent VIV to 21.4 per cent VIV o f oxygen (O 2).
the filter with hot water R and dry at 100-105 °C.
CHARACTERS
T h e residue weighs a maximum o f 50 mg.
A p p e a ra n c e
G elatin Colourless gas.
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 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
t
Amplifier
Figure 1238.-1. - UV fluorescence analyser

2016 Air 1-81
PRODUCTION than 0.05 ppm V/V of nitrogen monoxide and nitrogen

Carbon dioxide dioxide.
M axim um 500 p pm V/V, determined using an infrared Reference gas (b) U se a mixture o f 2 ppm V/V o f nitrogen
analyser (2.5.24). monoxide R in nitrogen R l.
Gas to be examined Filter the substance to be examined to Calibrate the apparatus and set the sensitivity using reference
avoid stray light phenomena. gases (a) and (b). Measure the content o f nitrogen monoxide
Reference gas (a) Use a mixture of 21 per cent V/V o f and nitrogen dioxide in the gas to be examined.
oxygen R and 79 per cent VIV of nitrogen R l, containing less Water
than 1 ppm V/V o f carbon dioxide R l. Maximum 67 ppm V/V, determined using an electrolytic
Reference gas (b) Use a mixture of 21 per cent VIV of hygrometer (2.5.28), except where the com petent authority
oxygen R and 79 per cent ViV of nitrogen R l, containing decides that the following limit applies to medicinal air
500 ppm VIV o f carbon dioxide R l. generated on-site and distributed in pipe-line systems
Calibrate the apparatus and set the sensitivity using reference operating at a pressure not greater than 10 bars and a
gases (a) and (b). M easure the content of carbon dioxide in tem perature not less than 5 °C: m axim um 870 ppm V/V,
the gas to be examined. 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
(2.5.25). paramagnetic analyser (2.5.27).
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.
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
Figure 1238.-2. - Gas burette
oxygen R and 79 per cent V/V o f nitrogen R l, containing less
1-82 Air 2016
ID E N T IF IC A T IO N IM P U R IT IE S
First identification C. A. C O 2: carbon dioxide,
Second identification A , B. B. S 0 2: sulfur dioxide,
A. In a conical flask containing the substance to be C. N O : nitrogen monoxide,
examined, place a glowing wood splinter. T he splinter D . N 0 2: nitrogen dioxide,
rem ains glowing. E. oil,
B. U se a gas burette (Figure 1238.-2) o f 25 m L capacity in F. CO: carbon monoxide,
the form of a cham ber in the middle o f which is a tube
G . H 20 : water.
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 Ph Eur
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 Synthetic Air ★ ★
water R and dry. O pen the 2 taps. C onnect the nozzle to the *★ ★*
(Synthetic Medicinal Air, Ph Eur monograph 1684) *
source of the gas to be examined and set the flow rate to
When Synthetic Air is intended for use in a room in which
1 L/m in. Flush the burette by passing the gas to be examined
magnetic resonance imaging (MBI) is being performed, the
through it for 1 min. Close the lower tap of the burette and
cylinder and fittings should be made from suitable non
immediately afterwards the upper tap. Rapidly disconnect the
ferromagnetic materials and labelled accordingly.
burette from the source o f the gas to be examined. Rapidly
give a half turn to the upper tap to eliminate any excess P h E u r ___________________________________________________________________________________________
pressure in the burette. Keeping the burette vertical, fill the D E F IN IT IO N

funnel with a freshly prepared mixture of 21 m L of a 560 g/L M ixture of Nitrogen (1247) an d Oxygen (0417).
solution of potassium hydroxide R and 130 m L o f a 200 g/L
C o n te n t
Irolution o f sodium dithionite R. O pen the upper tap slowly.
95.0 per cent to 105.0 per cent o f the nominal value which is
T he solution absorbs the oxygen and enters the burette.
between 21.0 per cent V/V to 22.5 per cent V/V o f oxygen
Allow to stand for 10 min w ithout shaking. Read the level of
the liquid meniscus on the graduated part of the burette. (Oa).
This figure represents the percentage V/V of oxygen. CHARACTERS
T he value read is 20.4 to 21.4. Colourless and odourless gas.
C. It complies with the limits o f the assay. S o lu b ility
TESTS A t a tem perature of 20 °C and a pressure o f 101 kPa,
C a rb o n dioxide 1 volume dissolves in about 50 volumes o f water.
M axim um 500 ppm V/V, determ ined using a carbon dioxide P R O D U C T IO N
detector tube (2. 1 . 6 ). W a te r (2.5.28)
S u lfu r dioxide M axim um 67 ppm V/V.
M aximum 1 ppm V/V, determ ined using a sulfur dioxide A ssay (2.5.27)
detector tube (2. 1 . 6). Carry out the determination o f oxygen in gases.
O il ID E N T IF IC A T IO N
M axim um 0.1 mg/m3, determ ined using an oil detector tube First identification C
(2 . 1 . 6 ), when an oil-lubricated compressor is used for the
Second identification A, B
production.
A. In a conical flask containing the substance to be
N itro g e n m o n o x id e a n d n itro g e n diox id e
examined, place a glowing splinter o f wood. T h e splinter
M axim um 2 ppm V/V, determ ined using a nitrogen
remains glowing.
monoxide and nitrogen dioxide detector tube (2 . 1 . 6 ).
B. U se a gas burette (Figure 1684.-1) o f 25 m L capacity in
C a rb o n m o n o x id e the form o f a chamber, in the middle o f which is a tube
M axim um 5 ppm V/V, determ ined using a carbon monoxide graduated in 0.2 per cent between 19.0 per cent and
detector tube (2 . 1 . 6 ). 23.0 per cent, and isolated at each end by a tap with a
W a te r'v a p o u r conical barrel. T h e lower tap is joined to a tube with an
M axim um 67 ppm V/V, determ ined using a water vapour olive-shaped nozzle and is used to introduce the gas into the
detector tube (2 . 1 . 6 ), except where the competent authority apparatus. A cylindrical funnel above the upper tap is used to
decides that the following limit applies to medicinal air introduce the absorbent solution. W ash the burette with
generated on-site and distributed in pipe-line systems water R and dry. O pen both taps. C onnect the nozzle to the
operating at a pressure not greater than 10 bars and a source of the substance to be examined and set the flow rate
tem perature n o t less than 5 °C: maximum 870 ppm V/V, to 1 L/min. Flush the burette by passing the substance to be
determined using a w ater vapour detector tube (2 . 1 . 6 ). examined through it for 1 m in. Close the lower tap o f the
STORAGE burette and immediately afterwards the upper tap. Rapidly
disconnect the burette from the source of the substance to be
As a gas, in suitable containers complying with the legal
examined. Rapidly give a h alf tu rn o f the upper tap to
regulations or as a gas supplied by a pipe network.
eliminate any excess pressure in the burette. Keeping the
L A B E L L IN G b urette vertical, fill the funnel with a freshly prepared mixture
W here applicable, the label states the production m ethod, as of 2 1 m L o f a 560 g/L solution o f potassium hydroxide R and
regards to the use o f an oil - lubricated compression. 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
2016 Alanine 1-83
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 COjH
C3H7N 0 2 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
CHARACTERS
A p p e a ra n c e
crystals.
S o lu b ility
F red y soluble in water, very slightly soluble in ethanol
(96 per cent).
First identification A , B
Second identification A , C, D
A. Specific optical rotation (see Tests).
Comparison alanine CRS.
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
(20:20:60 V/V/V).
enters the burette. Allow to stand for 10 min without Application 5 |xL.
shaking. Read the level of the liquid meniscus on the Development Over 2/3 of the plate.
graduated part o f the burette. T his figure represents the
Drying In air.
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. Detection Spray with ninhydrin solution R and heat at 105 °C
for 15 min.
C. It complies w ith the limits of the assay.
Results T h e p rindpal spot in the chromatogram obtained with
TESTS the T est solution is similar in position, colour and size to the
W a te r v a p o u r prindpal spot in the chromatogram obtained with the
M axim um 67 p p m VIV, determined using a water vapour reference solution.
detector tube (2 . 1 . 6 ).
1-84 Alanine 2016
D . Dissolve 0.5 g in a mixture o f 0.25 m L o f hydrochloric — total: maximum 0.5 per cent;
add R l, 0.5 m L of a 100 g/L solution of sodium nitrite R and — reporting threshold: 0.05 per cent.
1 m L of water R. Shake; gas is given off, Add 2 m L o f dilute C h lo rid e s (2.4.4)
sodium hydroxide solution R, followed by 0.25 m L o f iodinaxed M aximum 200 ppm.
potassium iodide solution R. After about 30 min, a yellow
Dilute 5 m L of solution S to 15 m L with water R.
precipitate is formed.
S u lfates (2.4.13)
TESTS M aximum 300 ppm.
S o lu tio n S
Dilute 10 m L of solution S to 15 m L with dxstHled water R.
Dissolve 2.5 g in distilled water R and dilute to 50 m L with
the same solvent. A m m onium
Amino a d d analysis (2.2.56) as described in the test for
ninhydrin-positive substances with the following
T he solution is clear ('2.2.1) and not more intensely coloured
modifications.
than reference solution BY 6 (2.2.2, Method II).
Injection T est solution, reference solution (c) and blank
solution.
S pecific o p tic a l r o ta tio n (2.2.7) Limit:
+ 13.5 to + 15.5 (dried substance). — ammonium at 570 ran: not more than the area of the
Dissolve 2.50 g in hydrochloric add R l and dilute to 25.0 m L corresponding peak in the chrom atogram obtained with
with the same acid. reference solution (c) (0.02 per cent), taking into account
N in h y d rin -p o sitiv e su b sta n c e s the peak due to ammonium in the chrom atogram
Amino a d d analysis (2.2.56). F or analysis, use M ethod 1. obtained with the blank solution.
T he concentrations o f the test solution and the reference Ir o n (2.4.9)
solutions may be adapted according to the sensitivity of the M aximum 10 ppm.
equipm ent used. T h e concentrations o f all solutions are In a separating funnd, dissolve 1.0 g in 10 m L of dilute
adjusted so that the system suitability requirements described hydrochloric add R. Shake with 3 quantities, each of 10 mL,
in general chapter 2.2.46 are fulfilled, keeping the ratios of of methyl isobutyl ketone R l, shaking for 3 m in each time.
concentrations between all solutions as described. T o the combined organic layers add 10 m L of water R and
Solution A dilute hydrochloric acid R l or a sample preparation shake for 3 min. Use the aqueous layer.
buffer suitable for the apparatus used. H eav y m e ta ls (2.4.8)
Test solution Dissolve 30.0 m g o f the substance to be M aximum 10 ppm.
examined in solution A and dilute to 50.0 m L with Dissolve 2.0 g in water R and dilute to 20 m L with the same
solution A. solvent. 12 m L of the solution complies with test A. Prepare
Reference solution (a) D ilute 1.0 m L of the test solution to the reference solution using lead standard solution
100.0 m L with solution A Dilute 2.0 m L of this solution to (1 ppm Pb) R.
10.0 m L with solution A. L oss o n d ry in g (2.2.32)
Reference solution (b) Dissolve 30.0 mg of proUne R in Maximum 0.5 per cent, determined on 1.000 g by drying in
solution A and dilute to 100.0 m L with solution A. Dilute an oven at 105 °C.
1.0 m L of the solution to 250.0 m L with solution A. S u lfa te d a s h (2.4.14)
Reference solution (c) Dilute 6.0 m L o f ammonium standard Maximum 0.1 per cent, determined on 1.0 g.
solution (100 ppm NH 4 ) R to 50.0 m L with solution A. D ilute
A SSAY
1.0 m L of this solution to 100.0 m L with solution A.
Dissolve 80.0 mg in 3 mL o f anhydrous formic add R.
Reference solution (d) Dissolve 30 m g o f isoleucine R and Add 30 m l. of anhydrous acetic add R. T itrate with 0.1 M
30 m g of leudne R in solution A and dilute to 50.0 m L with
perchloric add, determining the end-point potentiometrically
solution A. Dilute 1.0 m L o f the solution to 200.0 m L with
(2.2.20).
solution A.
1 m L of 0.1 M perchloric acid is equivalent to 8.91 m g of
Blank solution Solution A.
c 3h 7n o 2.
Inject suitable, equal am ounts of the test, blank and reference
solutions into the amino a d d analyser. Run a program STO RA G E
suitable for the determination of physiological amino adds. Protected from light.
System suitability Reference solution (d): IM P U R IT IE S
— resolution: minimum 1.5 between the peaks due to Other detectable impurities (the following substances would, if
isoleucine and leucine. present at a suffident levd, be detected by one or other of
Calculation of percentage contents: the tests in the monograph. They are limited by the general
— for any ninhydrin-positive substance detected at 570 nm , acceptance criterion for other/unspecified impurities and/or
use the concentration of alanine in reference solution (a); by the general monograph Substances for pharmaceutical use
— for any ninhydrin-positive substance detected at 440 nm , (2034). It is therefore no t necessary to identify these
use the concentration o f proline in reference solution (b); impurities for demonstration of compliance. See also 5.10.
if a peak is above the reporting threshold at both Control of impurities in substances for pharmaceutical use): A , B.
wavelengths, use the result obtained at 570 nm for
quantification. H nh 2
Limits: HO!° ' ~ X c0 !h

— any ninhydrin-positive substance: for each impurity,
maximum 0.10 p er cent;
A. (25)-2-aminobutanedioic acid (aspartic ad d ),
2016 Albendazole 1-85
Column:
h o 2c ' — size. I = 0.25 m , 0 = 4.6 mm;
— stationary phase’, spherical end-capped octadecylsUyl silica gel
B. (25)-2-am inopentanedioic ad d (glutamic ad d ). for chromatography R (5 pm) with a pore size of 10 nm
PhEur 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.
Albendazole ★ ★
(Ph Eur monograph 1386) *****
Injection 20 |iL.
ch 3 Run time 1.5 times the retention time of albendazole.
Relative retention W ith reference to albendazole:
-NH 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.
C 12H 15N 3O 2S 265.3 54965-21-8 System suitability", reference solution (b):
— resolution', m inim um 3.0 between the peaks due to
A ctio n a n d u se
albendazole and oxibendazole.
Benzimidazole antihelminthic.
Limits:
P re p a r a tio n s — impurities A, B, C, D, E, F: for each impurity, not more
Albendazole Oral Suspension than 1.5 times the area of the principal peak in the
Albendazole Oral Suspension with Minerals chromatogram obtained with reference solution (a)
(0.75 p er cent);
PhEir.
— total: n o t more than 3 times the area o f the prindpal peak
D E F IN IT IO N in the chrom atogram obtained with reference solution (a)
M ethyl [5-(propylsulfanyl)-1 /f-benzimidazol-2 -yl] carbamate. (1.5 p er cent);
C o n te n t — disregard limit: 0.1 times the area of the prindpal peak in
98.0 per cent to 102.0 per cent (dried substance). the chromatogram obtained with reference solution (a)
(0.05 p er cent).
CHARACTERS
A p p e a ra n c e
W hite or slightly yellowish powder.
S o lu b ility
Sulfated ash (2.4.14)
Practically insoluble in water, freely soluble in anhydrous
M aximum 0.2 p er cent, determ ined on 1.0 g.
formic a d d , very slightly soluble in methylene chloride,
practically insoluble in ethanol (96 p er cent). ASSAY
In order to avoid overheating during the titration, mix thoroughly
throughout and stop the titration immediately after the end-point
has been reached.
Preparation Discs.
Dissolve 0.250 g in 3 m L of anhydrous formic acid R and add
Comparison albendazole CRS. 40 m L o f anhydrous acetic add R. Titrate with 0.1 M
TESTS perchloric add, determining the end-point potentiometrically
A p p e a ra n c e o f so lu tio n (2.2.20).
T he solution is clear (2.2.1) and not m ore intensely coloured 1 m L of 0.1 M perchloric add is equivalent to 26.53 mg
than reference solution BY 6 (2.2.2, Method II). OfCi2Hj5N302S.
Dissolve 0.10 g in a mixture of 1 volume o f anhydrous formic STORAGE
add R and 9 volumes o f methylene chloride R and dilute to Protected from light.
10 m L w ith the same mixture of solvents.
IMPURITIES
R e la te d s u b s ta n c e s
Specified impurities A, B, C , D , E, F.
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
-NH,
o f sulfuric add R and dilute to 50.0 m L with the mobile
phase.
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 A. R = S-CH2-CH2-CH3: 5-(propylsulfanyl)-IH -
o f sulfuric acid R and dilute to 100.0 m L with the mobile benzimidazol- 2-am ine,
phase. D ilute 0.5 m L of this solution to 20.0 m L with the D . R = SO 2-C H 2-C H 2-C H 3: 5-(propyisulfonyI)-lii-
mobile phase. benzimidazol- 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 2016
O CH,
Reference solution Dissolve 10 m g o f alcuronium chloride CRS
1 y -o '
in methanol R and dilute to 10 m l. with the same solvent.
> -N H
Mobile phase Mix 15 volumes o f a 58.4 g/L solution of sodium
B. R = SO -C H 2-C H 2-C H 3: methyi [5-(propylsuLfinyl)~ÎH- chloride R, 35 volumes of dilute ammonia R2 and 50 volumes
benzimidazol-2-yl]carfaamate, of methanol R.
C. R = SO 2-C H 2-C H 2-C H 3: methyl [5-(propylsulfonyl)-1H- Application 10 |iL.
benzimida 7ol- 2-yl] carbamate, Development Over a path of 15 cm.
E. R = H: methyl ( l.ff-benzimidazol-2-yl) carbamate, Drying In air for 10 min.
F. R = S-C H 3: methyl [5-(methylsulfanyl)-1/i-benziinidazol- Detection Spray with 0.1 M ammonium and cerium nitrate.
2-yl] carbamate. Results T he principal spot in the chrom atogram obtained with
__________________________________________________________ PhEur 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 ★ ★ TESTS
★ ★
S o lu tio n S
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).
A cid ity o r a lk alin ity
2 Cl 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.
C44H 50C I2N 4O2 738 15180-03-7
P ro p a n -2 -o l (2.4.24, System A)
A ctio n a n d u se Maximum 1.0 per cent.
Non-depolarizing neuromuscular blocker. R e la te d su b s ta n c e s
PhEur______________________________________
Solvent mixture M ix 100 m L of methanol R, 200 m L o f
D E F IN IT IO N acetonitrile R and 200 m L of a 6.82 g/L solution of potassium
(lfl,3aS,10S,llaS,12J?,14aS,19aS,20bS,21S,22aS,23.E,26£)- dihydrogen phosphate R. Dissolve 1.09 g o f sodium
23,26-bis(2-Hydroxyethylidene)-l, 12-bis(prop-2-enyl)- laurylsulfonate for chromatography R in the mixture and adjust
2,3,11,1 la , 13,14,22,22a-octahydro-10tf,21/f-l, 21:10,12- the apparent p H to 8.0 with a 100 g/L solution of sodium
diethano-19aH ,20b/f- [1,5]diazocino [ 1,2,3-Zm: 5,6,7- hydroxide R
l'm '] dipyrrolo[2',3 '-d:2'',3":d ']dicaibazolediium dichloride
(4 ,4 /-didesmethyl-4,4/-bis(prop-2-enyl)toxiferm I dichloride).
in the solvent mixture and dilute to 100.0 m L with the
C o n te n t solvent mixture.
98.0 per cent to 102.0 per cent (anhydrous substance).
CHARACTERS 100.0 m L with the solvent mixture.
A p p e a ra n c e Reference solution (b) Dilute 4.0 m l, of reference solution (a)
W hite or slightly greyish-white, crystalline powder. to 10.0 m L with the solvent mixture.
S olubility Reference solution (c) Dilute 1.0 m L o f reference solution (a)
Freely soluble in water and in methanol, soluble in ethanol to 10.0 m L with the solvent mixture.
(96 per cent), practically insoluble in cyclohexane. Reference solution (d) T o 5.0 m L o f the test solution add
Carry out the identification, tests and assay as rapidly as possible 5.0 mg of aUylstrychnine bromide CRS, dissolve in the solvent
avoiding exposure to actinic light mixture and dilute to 100.0 m L with the solvent mixture.
First identification A , C — size. I = 0.25 m, 0 = 4 mm;
— stationary phase: octylsQyl silica gel for chromatography R
Second identification B, C
(5 jim).
Mobile phase Mix 200 m L o f methanol R, 400 m L of
Comparison alcuronium chloride CRS. acetonitrile R and 400 m L o f a 6.82 g/L solution o f potassium
B. Thin-layer chromatography (2.2.27). dihydrogen phosphate R Dissolve 2.18 g o f sodium
Test solution Dissolve 10 m g o f the substance to be exam ined laurylsulfonate for chromatography R in the mixture and adjust
in methanol R and dilute to 10 m L with the same solvent.
2016 Alfacalcidol 1-87
the apparent p H to 5.4 with a 100 g/L solution oiphosphoric

ch2
add R.
Flow rate 1.2 mL/min. ci'
Irgecdon 10 jxL~
Run time Twice the retention time o f alcuronium.
System suitability Reference solution (d):
B. (4bS, 1R,1 aS, 8 ai?, 13i?, 13ai?, 13 bS )-l 3-hydroxy-7-(prop-2-
— resolution: m inim um 4.0 between the peaks due to
enyI)-5,6,7 a, 8, 8a, 11,13,13a, 13b, 14-decahydro-7,9-methano-
N ’-allylstrychnine and alcuronium.
7H-oxepino [3,4-a] pyrrolo [2,3-d] carbazolium chloride
Limits: ((4 R, 17i?)-4-allyl-l 7,18-epoxy-17 -hydroxy-19,2 0-
— impurities A , B: for each impurity, not m ore than the area didehydrocuranium chloride).
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
reference solution (b) ( 0.2 per cent);
★
— total: not m ore than twice the area of the principal peak in ★ ★
Alfacalcidol ★ ★
*****
(1 per cent); (Ph Eur monograph 1286)
— disregard limit: th e area o f the principal peak in the
chromatogram obtained w ith reference solution (c)
(0.05 per cent).
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.
C 27H 44O 2 400.6 41294-56-8
1 m L of 0.1 M perchloric add is equivalent to 36.9 m g
Of C44H5()Cl2N402. A ctio n a n d u se
STORA GE Vitamin D analogue.
In an airtight container under nitrogen, protected from light,
Ph Eur__________________________________________________________
at a tem perature o f 2 °C to 8 °C.
D E F IN IT IO N
IM P U R IT IE S
(5Z ,7£)-9,10-Secocholesta-5,7,10(19)-triene-l 0,3 P-diol.
Specified impurities A, B
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.
S o lu b ility
Practically insoluble in water, freely soluble in ethanol
(96 per cent), soluble in fatty oils.
A. (l£,3aS,9Æ,9a£,10Æ,llaS,12Æ,14aS,19aS,20Æ,20aÆ,
20bS,21i?,22aS)-l, 12-bis(prop-2-enyl)- It is sensitive to air, heat and light.
2,3,9a,11,1 la,13,14,19a,20a,21,22,22a-dodecahydro- ID E N T IF IC A T IO N
10tf,2 0 b H -l,2 3 :12,27-dim ethano-9,10:20,21- A. Infrared absorption spectrophotometry (2.2.24).
bis (epoxyprop [2] eno)-9H,2 OH- [1,5] diazocino [ 1,2,3-lm: 5,6,7-
Comparison Ph. Eur. reference spectrum of aÿacalddol.
l'm '] dipyirolo[2 ',3 '-dr.2 " ,3 " :d']dicarbazolediium dichloride
(4,4 '-diallylcaracurin V dichloride),
related substances.
Results T he principal peak in the chromatogram obtained
with the test solution is similar in retention time and size to
reference solution (a).
1-88 Alfacalcidol 2016
TESTS T h e contents of an opened container are to be used

Related substances immediately.
Liquid chromatography (2.2.29): use the normalisation IMPURITIES
procedure. Cany out the test as rapidfy as possible, avoiding Specified impurities A, B.
exposure to light and air.
Other detectable impurities (die following substances would, if
Test solution Dissolve 1.0 m g o f the substance to be examined present at a suffident levd, be detected by one or other o f
without heating in 10.0 m L o f the mobile phase.
Reference solution (a) Dissolve 1.0 mg o f alfacalcidol CRS acceptance criterion for other/unspecified impurities and/or
without heating in 10.0 m L o f the mobile phase. by the general monograph Substances for pharmaceutical use
Reference solution (b) Dilute 1.0 m L of reference solution (a) (2034). It is therefore not necessary to identify these
to 100.0 m L w ith the mobile phase. Dilute 1.0 m L o f this impurities for demonstration o f compliance. See also 5.10.
solution to 20.0 m L with the mobile phase. 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).^
A. (51%7E)-9,10-secocholesta-5,7,10(19)-triene-1a,3{$-diol
(zrans-alfacalddol),
Injection 100 (iL o f the test solution and reference
solutions (b) and (c).
Run time Twice the retention time o f alfacaladol.
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.
— resolution: m inim um 1.5 between the peaks due to pre-
alfacalddol and impurity A and minimum l .5 between B. (5Z ,7£)-9,10-secocholesta-5,7,10(19)-triene-l P,3fi-diol
the peaks due to im purity A and alfacaladol. ( 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
(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): C. 6^-[(3S,5i?)-3,5-dihydroxy-2-methylcydohex-l-en-l-yl]-2-
— repeatability: maximum relative standard deviation o f phenyl-2,5,10-triaza-4,19-dinor-9^-cholest-7-ene-1,3-dione.
1 per cent for the peak due to alfacaladol after P b E tr
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.
2016 Alfadex 1-89
temperature. Add 10 m L of ammonium molybdate reagent R1

Alfadex f * \ and allow to stand for 15 min.
*★ ★*
A lphacydodextrin * Reference solution Prepare a reference solution at the same
(Ph. Eur. monograph 1487) 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.
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
Test solution (a) Dissolve 0.25 g o f the substance to be
[C6H 10O5]6 973 10016-20-3
examined in water R with heating, cool and dilute to
A ctio n a n d u se
Cyclodextran; carrier molecule for drug delivery systems. Test solution (b) Dilute 5.0 m L o f test solution (a) to
50.0 m L with water R.
PhEur__________________________________________________________
Reference solution (a) Dissolve 25.0 m g of betadex CRS
D E F IN IT IO N (impurity A), 25.0 mg of gammacydodextrin CRS
Cyclohexakis-(l -> 4)- (a-D-glucopyranosyl) (cyclom altohexaose (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 Reference solution (b) Dilute 5.0 m L o f reference solution (a)
97.0 per cent to 102.0 per cent (dried substance). to 50.0 m L w ith water R.
CHA RACTERS
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.
A p p e a ra n c e
Column:
White or almost white, amorphous or crystalline powder.
— sizer. I = 0.25 m, 0 = 4.6 mm;
S o lu b ility — stationary phase:, octadecylsUyl silica gel for chromatography R
Freely soluble in water, slightly soluble in propylene glycol, (10 |im).
practically insoluble in anhydrous ethanol and in methylene
Mobile phase methanol R, water R (10:90 VIV).
chloride.
Detection Differential refractometer.
Equilibration W ith the mobile phase for about 3 h.
B. Examine the chromatograms obtained in the assay.
Injection 50 |JL of test solution (a) and reference solutions (a)
and (b).
with test solution (b) is similar in retention time and size to
the principal peak in the chromatogram obtained with Run time 3.5 times the retention time of alfadex.
reference solution (c). Relative retention W ith reference to alfadex (retention
C. Dissolve 0.2 g in 2 m L of iodine solution R4 by warming time = about 10 min): impurity B = about 0.7;
on a water-bath, and allow to stand at room temperature; impurity A = about 2.2.
a yellowish-brown precipitate is formed. System suitability: reference solution (a):
TESTS
impurity B and alfadex; if necessary, adjust the
S o lu tio n S concentration of methanol in the mobile phase.
Limits:
100.0 m L with the same solvent
— impurities A , B: for each impurity, not more than
A p p e a ra n c e o f so lu tio n 0.5 times the area of the corresponding peak in the
Solution S is clear (2.2.1). chromatogram obtained with reference solution (b)
p H (2.2.3) (0.25 per cent);
5.0 to 8.0. — sum of impurities other than A and B: not more than
Mix 1 m L o f a 223.6 g/L solution o f potassium chloride R and 0.5 times the area of the peak due to alfadex in the
30 m L o f solution S. chromatogram obtained with reference solution (b)
(0.5 per cent).
S p ecific o p tic a l ro ta tio n (2.2.7)
+ 147 to + 152 (dried substance), determined on solution S. Heavy metals (2.4.8)
Maximum 10 ppm.
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
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
Alfentanil Hydrochloride .★ ★
an oven at 120 °C for 2 h. (Ph. Eur. monograph 1062) *****
M axim um 0.1 per cent, determ ined on 1.0 g.
n « nv ch3
A SSA Y J. N -V
Liquid chromatography (2.2.29) as described in the test for HCI
related substances with the following modifications.
Injection T est solution (b) and reference solutions (a) and (c). ch3
System suitability:
— repeatability: maximum relative standard deviation o f
2.0 per cent for the peak due to alfadex after 5 injections C21H33CIN6O3 453.0 69049-06-5
o f reference solution (a).
Calculate the percentage content o f [ O H i o C ^ from the Opioid receptor agonist; analgesic.
assigned content of alfadex CRS.
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-
IM P U R IT IE S
(methoxymethyl)piperidin-4-yl]-N-phenylpropanamide
Specified impurities A, B hydrochloride.
OH HO
C o n te n t
CHARACTERS
A p p e a ra n c e
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.
A. Infrared absorption spectrophotom etry (2.2.24).
Comparison Ph. Eur. reference spectrum of alfentanil
hydrochloride.
A. cycloheptakis-(l-»4)-(a-D-glucopyranosyl) (betadex or B. Dissolve 50 mg in a mixture of 0.4 m L o f ammonia R and
cyclomaltoheptaose or ß-cyclodextrin), 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
T he solution is clear (2.2.1) and colourless (2.2.2,
Method IT).
solvent.
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,
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
B. cyclooctakis-(l -*4)-(a-D-glucopyranosyl) reflux condenser for 4 h. Neutralise with 10.0 m L o f dilute
(cydomaltooctaose or y-cydodextrin). sodium hydroxide solution R. Evaporate to dryness on a water-
bath. Cool and take up the residue in 10 m L o f methanol R.
PhEur
Filter.
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;
2016 Alfentanil Hydrochloride 1-91
— stationary phase: octadecybüyl silica gel for chromatography R h2

(3 \im). 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; o
Ar -R
— mobile phase B: acetonitrüe R] I f N
Time Mobile phase A Mobile phase B o

V xe”3
v
(min) (per cent V/V) (percent V/V)
0 -15 90->40 10 -»60 A. cis-N- [ 1- [2-(4-ethyl-4,5-dihydro-5-oxo-1//-tetrazol-1-
1 5 -2 0 40 60 yI)ethyl]-4-(methoxymethyl)piperidin-4-yl]-iV-
phenylpropanamide N-oxide,
2 0 -2 5 40 ->90 60-» 10
r i^ x î r
Flow rate 1.5 mL/min. o
Detection Spectrophotom eter at 220 nm. ^C H 3
Equilibration W ith acetomtrSe R for at least 30 min and then
0 V e
with the mobile phase at the initial composition for at least
B . trans-N- [ 1- [2-(4-ethyl-4,5-dihydro-5-oxo- lH -tetrazol-1-
5 min.
yl)ethyl]-4-(methoxymethyl)piperidin-4-yl]-iV-
Irqection 10 jiL; inject methanol R as a blank. phenylpropanamide N-oxide,
Retention time Im purity E = about 6 min; alfentanil = about
7 min. Ar / x
I f NH
Identification of impurities Use the chromatogram obtained
with reference solution (a) to identify the peak due to
o *^0 - CH3
impurity E; disregard any other peak.
— resolution: m in im u m 4.0 between the peaks due to C. N - [4-(methoxymethyl)piperidm-4-yl]-N-
alfentanil and impurity E; if necessary, adjust the phenylpropanamide,
concentration o f acetonitrile in the mobile phase or adjust
the time programme for the linear-gradient elution.
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); D . N -[l- [2-(4-ethyl-4,5-dihydro-5-oxo-1//-tetrazol-1-
— total: not more than twice the area of the principal peak in yl)ethyI]-4-(methoxymethyl)piperidm-4-yl]-N-
the chromatogram obtained with reference solution (b) phenylacetamide,
(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.
E. 1-ethyl-1,4-dihydro-4- [2-[ [4-(methoxymethyl)-4-
A SSA Y phenylamino] piperidin-1-yl] ethyl]-5/f-tetrazol-5-one,
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 Ar / s ,
5.0 m L o f 0.01 M hydrochloric add. T itrate with 0.1 M 1 I N
sodium hydroxide, d eterm ining the end-point hO t n^

potentiometrically (2.2.20). Read the volume added between o k 0 .CH3
the 2 points of inflexion.
1 m L o f 0.1 M sodium hydroxide is equivalent to 45.30 mg o f F. N- [ 1-(2-hydroxyethyl)-4-(methoxymethyl)piperidin-4-yl] -
C21H33C 1N 60 3 . iV-phenylpropanamide,
STO RA G E
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-1-

yl) ethyl] -4-(propanoyloxymethyl)piperidin-4-yI] -N-
phenylpropanamide,
1-92 Alfuzosin Hydrochloride 2016
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 ;
H . N -[l-[2-(4-ethyl-4j5-dihydro-5-oxo-li/-tetrazol-1- — stationary phase: end-capped octadecylsUyl silica gel for
yl) ethyl] -4-(methoxymethyl)piperidin-4-yi] -N- chromatography R (5 Jim).
phenylbutanamide.
Mobile phase Mix 1 volume of tetrahydrcfuran R, 20 volumes
PhEur 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.
Alfuzosin Hydrochloride ★ ★
Injection 10 jiL.
Run time Twice the retention tim e o f alfuzosin.
HzC0' y ^ Y '
with alfuzosin for system suitability CRS and the chromatogram
h3c o - ^ Y n obtained with reference solution (b) to identify the peaks due
to impurities A and D.
nh2 and enantiomer
Relative retention W ith reference to alfuzosin (retention
tim e = about 8 min): impurity D = about 0.4;
C 19H 28Q N 5O 4 425.9 81403-68-1 impurity A = about 1.2.
A ctio n a n d u se System suitability: reference solution (b):
Alpha âdrenoceptor antagonist. — 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
Alfuzosin Tablets curve separating this peak from the peak due to alfuzosin.
Prolonged-release Alfuzosin Tablets Limits:
PhEir ________________________________
— impurity D: not more than twice the area o f the principal
D E F IN IT IO N solution (a) (0.2 per cent);
(2RS)-N- [3- [(4-Amino-6j7 -dimethoxyquinazolm- 2-yl) — unspecified impurities: for each impurity, n o t more than the
methylaminojpropyl] tetrahydrofuran- 2-carboxamide area o f die principal peak in the chromatogram obtained
hydrochloride. with reference solution (a) ( 0.10 per cent);
C o n te n t — total: n o t more than 3 times the area of the principal peak
99.0 per cent to 101.0 per cent (anhydrous substance). in the chromatogram obtained with reference solution (a)
(0.3 p e r cent);
CHARACTERS
— disregard limit: 0.5 times the area o f the principal peak in
A p p e a ra n c e
W hite or alm ost white, crystalline powder, slightly
(0.05 p er cent).
hygroscopic.
W a te r (2.5.12)
S o lu b ility
Freely soluble in water, sparingly soluble in ethanol
(96 per cent), practically insoluble in methylene chloride. S u lfa te d a sh (2.4.14)
A Infrared absorption spectrophotometry (2.2.24). A SSA Y
Dissolve 0.300 g in a mixture o f 40 m L o f anhydrous acetic
Comparison alfuzosin hydrochloride CRS.
add R and 40 m L o f acetic anhydride R T itrate with 0.1 M
B. It gives reaction (a) of chlorides (2.3.1). perchloric add, determining the end-point potentiometrically
TESTS (2.2.20).
p H (2.2.3) 1 m l, o f 0.1 Mperchloric add is equivalent to 42.59 m g
4.0 to 5.5. o f C 19H 28CIN 5O 4.
Dissolve 0.500 g in carbon dioxide-free water R and dilute to STO R A G E
25.0 m L with the same solvent. Use a freshly prepared In an airtight container, protected from light.
solution.
IM P U R IT IE S
Specified impurities D .
present at a sufficient level, be detected by one or other of
the tests in the monograph. T hey are limited by die general
phase.
Reference solution (a) Dilute 1.0 m L o f the test solution to by the general monograph Substances for pharmaceutical use
100.0 m L with the mobile phase. Dilute 1.0 m L o f this (2034). It is therefore not necessary to identify these
2016 Alginic Acid 1-93
impurities for dem onstration o f compliance. See also 5.10. IDENTIFICATION

Control of impurities in substances for pharmaceutical use): A , B, A. T o 0.2 g add 20 m L o f water R and 0.5 m L o f sodium
C, E. 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.
nh2 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
A. N-[3-[(4-amino-6,7-dimethoxyquinazolin-2-yl) ethanol (96 per cent) R and 5 m L of hydrochloric add R. Boil
methylamino] propyl] furan-2-carboxamide3 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 TESTS
Chlorides
B. R = Cl: 2<hloro-637-dimethoxyquina2olin-4-amine3 M axim um 1.0 p er cent.
D . R = N (C H 3)-[C H 2]3-N H 2: N-(4-amino-637- T o 2.50 g add 50 m L of dilute nitric acid R, shake for 1 h
dimethoxyquinazolin-2-yl) -N-methylpropane-1j3-diamine3 and dilute to 100.0 m L with dilute nitric add R. Filter.
T o 50.0 m L of the filtrate add 10.0 m L of 0.1 M silver nitrate
E. R = N (C H 3)-[C H 2]3-N H -CO -H : AT-[3-[(4-amino-6,7-
and 5 m L of toluene R. Titrate with 0.1 M ammonium
dimethoxyquinazolin-2-yl)methylamino]propyi]formamide,
thiocyanate, using 2 m L o f ferric ammonium sulfate solution R2
as indicator and shaking vigorously towards the end-point.
H H. / - , 1 m L of 0.1 M silver nitrate is equivalent to 3.545 mg o f Cl.
H’ °Y ^Y V N'v ^ NY b J Heavy m etals (2.4.8)
h 3c o
M axim um 20 ppm .
NH2 and enantiomer 1.0 g complies w ith test F . Prepare the reference solution
C . (2RS)-N-[3-[(4-amino-6,7-dimethoxyquinazolin-2-yl) Loss on drying (2.2.32)
amino] propyl] -N-meiiiyltetrahydrofuran-2-carfaoxam ide. M axim um 15.0 p er cent, determined on 0.1000 g by drying
in an oven at 105 °C for 4 h.
PhEur
Maximum 8.0 p er cent (dried substance), determined on
0.100 g.
M icrobial contamination
Alginic Acid ?**% T A M C : acceptance criterion 102 C FU /g (2.6.12).
(Ph Eur monograph 0591) * Absence o f Escherichia coli (2.6.13).
Absence o f Salmonella (2.6.13).
Action and use
T reatm ent o f gastro-oesophageal reflux disease; excipient; ASSAY
thickening agent. T o 0.2500 g add 25 m L of footer R, 25.0 m L o f 0.1 M
sodium hydroxide and 0.2 m L of phenolphthalein solution R.
PhEur__________________________________________________________ T itrate with 0.1 M hydrochloric acid.
DEFINITION 1 m L of 0.1 M sodium hydroxide is equivalent to 4.502 m g of
M ixture of polyuronic acids [(C6H80 6) J composed of carboxyl groups (-C 0 2H ).
residues of p-m annuronic and L-guluronic acids, obtained FUNCTIONALITY-RELATED CHARACTERISTICS
mainly from algae belonging to the Phaeophyceae. A small This section provides information on characteristics that are
proportion o f the carboxyl groups may be neutralised. recognised as being relevant control parameters for one or more
Content functions of the substance when used as an excipient (see chapter
19.0 per cent to 25.0 p er cent of carboxyl groups (-C 0 2H) 5.15). This section is a non-mandatory part of the monograph
(dried substance). and it is not necessary to verify the characteristics to demonstrate
CHARACTERS compliance. Control of these characteristics can however contribute
Appearance
of the manufacturing process and the performance of the medicinal
W hite or pale yellowish-brown, crystalline or amorphous
product during use. Where control methods are cited, they are
powder.
recognised as bang suitable for the purpose, but other methods can
Solubility also be used. Wherever results for a particular characteristic are
Very slightly soluble o r practically insoluble in ethanol reported, the control method must be indicated
(96 per cent), practically insoluble in organic solvents.
The following characteristics may be relevant for alginic add used
It swells in w ater b u t does not dissolve; it dissolves in
as disintegrant and/or binder.
solutions of alkali hydroxides.
1-94 Alimemazine 2016
P a rtic le -siz e d is trib u tio n (2.9.31 or 2.9.38). Dissolve 1.0 g in water R and dilute to 10 m L with the same
S e ttlin g v o lu m e solvent.
Place 75 m L o f water R in a 100 m L graduated cylinder and p H (2.2.3)
add 1.5 g of die substance to be examined in 0.5 g portions, 5.0 to 6.5. Carry out the test protected from light and use a
shaking vigorously after each addition. Dilute to 100.0 m L freshly prepared solution.
with water R and shake again until the substance is Dissolve 1.0 g in carbon dioxide-free water R and dilute to
homogeneously distributed. Allow to stand for 4 h and 50 m L with the same solvent.
determine the volume o f the settled mass.
Related substances
The following characteristic may be relevant for alginic acid used Liquid chromatography (2.2.29). Carry out the test protected
as gelling agent or viscosity-increasing agent. from light and use freshly prepared solutions.
A p p a re n t viscosity Solvent mixture acetomtrüe R, water R (20:80 VIV).
D eterm ine the dynamic viscosity using a rotating viscometer
Test solution Dissolve 35 m g o f the substance to be examined
(2.2.1Q).
in the solvent mixture and dilute to 100.0 m L with the
Prepare a 20 g/L suspension o f alginic a d d (dried substance) solvent mixture.
and add 0.1 M sodium hydroxide until a solution is obtained.
Reference solution (a) D ilute 1.0 m L o f the test solution to
__________________________________________________________ PhEur 100.0 m L with the solvent mixture. D ilute 1.0 m L of this
Reference solution (b) Dissolve 3.5 m g o f alimemazine for
system suitability CRS (containing impurities A, B and C) in
***** the solvent mixture and dilute to 10.0 m L with the solvent
Alimemazine Tartrate ★ ★ mixture.
(Alimemazine Hemitartrate Ph. Bur. monograph ***** Column:
2650) — size. I = 0.15 m , 0 = 4.6 mm;
— stationary phase, base-deactivated end-capped octadecylsüyl
silica gelfor chromatography R (3 |im );
H OH — temperature. 40 °C.
n' ^ v ^ ' n ' CH3 COjH Mobile phase acetomtrüe R, methanol R , 3.854 g/L solution o f
I I H i i and dnantiomer , HOî C
CH3 c h 3 ammonium acetate R (10:40:50 VÍVIV).
H OH
Injection 20 jiL.
C20H 25N 2O3S 373.5 4330-99-8
Run time Twice the retention time o f alimemazine.
A ctio n a n d u se Identification of impurities Use the chrom atogram supplied
Histamine H i, receptor antagonist; sedative with alimemazine for system suitability CRS and the
P re p a ra tio n s chromatogram obtained with reference solution (b) to
Paediatric Alimemazine Oral Solution identify the peaks due to impurities A, B and C.
Strong Paediatric Alimemazine Oral Solution Relative retention W ith reference to alimemazine (retention
Alimemazine Tablets time = about 27 min): impurity A = about 0.1;
impurity B = about 0.5; impurity C = about 1.4.
PhEur__________________________________________ System suitability: reference solution (b):
D E F IN IT IO N — resolution: minimum 5.0 between the peaks due to
(2RS)-N,N, 2-Trimethyi-3-( 1OH-phenothiazin-10-yl)propan- alimemazine and im purity C.
1-am ine hemi [(2Æ,3iQ-2,3-dihydroxybutanedioate]. Calculation of percentage contents:
C o n te n t — correction factors: multiply the peak areas o f the following
99.0 per cent to 101.0 per cent (dried substance). impurities by the corresponding correction facto r
impurity A = 4.4; impurity C = 0.4;
CHARACTERS — for each impurity, use the concentration o f alimemazine
A p p e a ra n c e in reference solution (a).
W hite or very slightly yellowish powder. Limits:
S o lu b ility — impurity B: máximum 0.3 per cent;
F red y soluble in water, sparingly soluble in ethanol — impurities A, C: for each impurity, maximum
(96 per cent), practically insoluble in toluene. 0.15 p er cent;
It deteriorates when exposed to air and light. — unspecified impurities: for each impurity, maximum
0.10 p er cent;
— total: maximum 0.5 p er cent;
Infrared absorption spectrophotom etry (2.2.24). — reporting threshold: 0.05 per cent.
Comparison alimemazine hemitartrate CRS. Loss on drying (2.2.32)
TESTS M axim um 0.5 per cent, determined on 1.000 g by drying in
A p p e a ra n c e o f so lu tio n an oven at 105 °C for 3 h.
T he solution is not m ore opalescent than reference Sulfated ash (2.4.14)
suspension II (2.2.1) and n o t more intensely coloured than M axim um 0.1 per cent, determ ined on 1.0 g.
reference solution BY5 (2.2.2, Method IT).
2016 Allantoin 1-95
ASSAY It melts at about 225 °C, with decomposition.

D isso lv e 0.300 g in 50 m l , o f anhydrous acetic acid R. Titrate ID E N T IF IC A T IO N
with 0.1 M perchloric acid, determining the end-point First identification A.
potentiometrically (2.2.20).
Second identification B, C, D.
1 m L of 0.1 M perchloric acid is equivalent to 37.35 mg of
C 20H 25N 2O 3S .
(2.2.24), comparing with the spectrum obtained with
STORAGE allantoin CRS.
In an airtight container, protected from light. B. Examine the chromatograms obtained in the test for
IM P U R IT IE S related substances. T h e principal spot in the chrom atogram
Specified impurities A, B, C obtained with test solution (b) is similar in position, colour
and size to the principal spot in the chromatogram obtained
with reference solution (a).
C. Boil 20 m g w ith a mixture of 1 m L of dilute sodium
enantiomer hydroxide solution R and 1 m L of water R. Allow to cool.
s A CH3 CH3 Add 1 m L o f dilute hydrochloric add R. T o 0.1 m L of the
solution add 0.1 m L o f a 100 g/L solution of potassium
u bromide R, 0.1 m L o f a 20 g/L solution o f resorcmd R and
3 m L o f sulfuric add R. H eat for 5 min to 10 min on a water-
A. (2 i^ -iS />N,2-trimethyl-3-(5-oxido-l OH-phenothiazin-10- bath. A dark blue colour develops, which becomes red after
yl) propan-1-am ine, 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.
enantiomer TESTS
3 and
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.
A cid ity o r a lk a lin ity
B . (2i?S}-ATj2-dimethyl-3-( 1OH-phenothiazin-10-yl)propan-1-
T o 5 m L o f solution S add 5 m L of carbon dioxide-free
amine,
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.
T h e angle of optical rotation, determined on solution S, is
-0 .1 0 ° to + 0.10°.
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.
C. 1OH-phenothiazine. Add 1.5 m L o f 0.02 Mpotassium permanganate. T he solution
Ph Bur m ust rem ain violet for at least 10 min.
Examine by thin-layer chromatography (2.2.27), using a
suitable cellulose for chromatography R as the coating
★ ★ substance.
Allantoin ★ ★
(Ph. Eur. monograph 1288) * * * * * 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
o after preparation.
f H-
H. . y ' NH and enantiomer Test solution (b) Dilute 1 m L of test solution (a) to 10 m L
H,N 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
€4 ^ 403 158.1 97-59-6 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.
A ctio n a n d u se
Astringent; keratolytic. 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
PhEir ___________________ methanol R
D E F IN IT IO N Reference solution (c) M ix 1 m L o f reference solution (a) and
Allantoin contains n o t less than 98.5 per cent and not more 1 m L of reference solution (b).
than the equivalent o f 101.0 per cent of Apply to the plate 10 jiL o f test solution (a) and 5 |iL each
(RS) -(2 j 5-dioxoimidazolidin-4-yl)urea. o f test solution (b), reference solution (a), reference
solution (b) and reference solution (c). Develop over a path
CHARACTERS
o f 10 cm using a mixture of 15 volumes of glacial acetic
A white or alm ost white, crystalline powder, slightly soluble
add R, 25 volumes o f water R and 60 volumes of butanol R.
in water, very slightly soluble in alcohol.
1-96 Allergen products 2016
Allow the plate to dry in air. Spray the plate with a 5 g/L prepared by dilution of aqueous or glycerinated extracts, or
solution of dimethylaminobenzaldehyde R in a mixture of by reconstitution o f unmodified freeze-dried extracts.
1 volume of hydrochloric add R and 3 volumes of methanol R. F or specific immunotherapy, allergen products may be either
Dry the plate in a current o f h o t air. Examine in daylight unmodified extracts or extracts modified chemically and/or
after 30 min. Any spot in the chromatogram obtained with by adsorption onto different carriers (for example, aluminium
test solution (a), apart from th e principal spot, is not more hydroxide, calcium phosphate or tyrosine).
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 P R O D U C T IO N
unless the chrom atogram obtained with reference solution (c) S O U R C E M A T E R IA L S
shows two clearly separated principal spots. Source materials for the preparation of allergen products are
products o f animal o r vegetable origin, mostly pollens,
L oss o n d ry in g ( 2.2.32)
moulds, mites, animal epithelia and outgrowths (such as hair
N ot more than 0.1 p e r cent, determined on 1.000 g by
and feathers) and/or dander, hymenoptera venoms, insects
drying in an oven at 105 °C.
and certain foods.
W here allergen products are m anufactured using materials of
N ot more than 0.1 p e r cent, determined on 1.0 g.
hum an or animal origin, the requirements o f chapter 5.1.7.
ASSAY Viral safety apply.
Dissolve 120.0 mg in 40 m L o f water R. T itrate with 0.1 M T h e source materials are defined by their origin, nature,
sodium hydroxide, determining the end-point m ethod o f collection or production and pretreatm en t
potentiometrically (2.2.20). Control m ethods and acceptance criteria relating to identity
1 m L of 0.1 M sodium hydroxide is equivalent to 15.81 m g of and purity are established. T h e acceptance criteria m ust
C 4H 6N 4O 3. ensure the consistency o f the allergenic source material from
a qualitative and quantitative point o f view. T h e source
IM P U R IT IE S
materials are stored under controlled conditions justified by
H CO2H stability data.
To T h e collection or production, as well as the handling o f the
source materials are such that uniform composition is
A. glyoxylic acid, ensured as far as possible from batch to batch.
T h e content of the relevant residual solvents, heavy metals
o 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
B. carbamide (urea).
solvents and 2.8.13. Pesticide residues respectively.
--------------------------------- — .— _____________________________PhEur
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
Allergen Products ***** pollens and 0.5 per cent o f any individual pollen as
determined by a microscopic particle count. D etectable
(Ph Eur. monograph 1063) * m ould spores m ust not exceed 1 per c e n t
Ph Fur _________________________________________ T h e contam ination with particles o f plant origin other than
This monograph does not apply to: chemicals that are used solely pollen m ust be kept to a minim um. T he m axim u m allowed
for diagnosis of contact dermatitis; chemically synthesised products; contam ination m ust be justified.
allergens derived by recombinant DNA technology. It does not M o u ld s
necessarily apply to allergen products for veterinary use. Biologically active contam inants such as mycotoxins in
D E F IN IT IO N moulds m ust be m in im ised and any presence justified.
Appropriate measures have to be im plem ented to avoid
Allergen products are pharm aceutical preparations derived
c o n tam inatio n by foreign m ould strains. C are m ust be taken
from extracts of naturally occurring source materials
to minimise any allergenic constituents o f the media vised for
containing allergens, which are substances that lead to and/or
provoke allergic reactions. T h e allergenic components are the cultivation of moulds as source materials. Culture media
th at contain substances of hum an or anim al origin m ust be
most often o f a proteinaceous nature. Allergen products are
intended for in vivo diagnosis o r treatm ent o f allergic diseases justified and, when required, m ust be suitably treated to
attributed to these allergens. 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 T h e production m ethod is validated to demonstrate that
products are generally presented as parenteral preparations, allergen products obtained from moulds and intended for
parenteral administration, if tested* would comply with the
eye preparations, preparations for inhalation, preparations for
test for abnormal toxicity for immunosera and vaccines for
oral use, sublingual preparations or preparations for skin
tests. hum an use (2.6.9).
For in vivo diagnostic use, allergen products are usually M ite s

prepared as unmodified extracts in a 50 per cent VIV Appropriate measures have to be implem ented to avoid
solution of glycerol for skin testing. F o r intradermal diagnosis co n tam inatio n by foreign m ite strains. Care m ust be taken to
or for provocation tests by nasal, ocular or bronchial m in im ise any allergenic constituents o f the media used for
administration, suitable dilutions o f allergen products may be the cultivation of mites as source materials. Culture media
2016 Allergen products 1-97
th at contain substances of hum an or animal origin m ust be of suitable reagents, as well as the intended use. The characterised
justified and, when required, must be suitably treated to IHRP is used as the reference in the batch control of active
ensure the inactivation or elimination of possible substances and intermediates and, if possible, in the batch control
transmissible agents of disease. offinished products.
Anim al epithelia and outgrowths and/or dander T he IH R P is characterised by the protein content
T hey are obtained from healthy animals selected to avoid determination and a protein profile using appropriate
possible transmissible agents of disease. methods (such as isoelectric focusing, sodium dodecyl sulfate
Hymenoptera venoms polyacrylamide gel electrophoresis, Immunoelectrophoresis,
T h e species of hymenoptera from which the venom is capillary electrophoresis, chromatographic techniques and
mass spectrometry).
extracted is identified and specified. T h e m ethods of insect
collection and venom extraction are described and m ust Allergenic components may be detected by appropriate
ensure that the source material is of proper quality. methods (for example, immunoblotting or crossed radio-
im munoelecupphoresis). Characterisation of the allergenic
Food
components may include identification o f relevant allergens
T h e scientific nam e (species, variety, strain etc.) o f the
based on serological or other techniques using pooled or
an im al or vegetable species is indicated and the p art used is
individual sera from allergic patients, or allergen-specific
stated, if applicable. Foods must be o f a quality suitable for
polyclonal or monoclonal antibodies.
hum an consum ption. T he origin o f the food stuff as well as
its processing stage is stated. Determ ination o f the content of relevant allergens is
performed wherever possible. T his determination may be
MANUFACTURING PROCESS m ade using individual allergen-specific reference standards,
Allergen products are generally obtained by extraction, and w hen available. T h e choice of the relevant allergen
m ay be purified, from the source materials using appropriate components subjected to the determination m ust be justified.
m ethods shown to preserve the allergenic properties o f the Individual allergens are identified and named according to
com ponents. Allergens for which there are not enough internationally established nomenclature wherever possible.
patients to determine the total allergenic activity in vivo or in
T h e biological potency o f the first IH R P is determined in
vitro, the extraction ratio indicating the relative proportions
patients by in vivo techniques such as skin testing, and
(mlV) o f allergenic source materials and solvents is a
expressed in units of biological activity except when not
minimum requirem ent. Allergen products presented as
enough patients are available. In this case, the potency of the
parenteral preparations, eye preparations, preparations for
first IH R P is determ ined by an m vitro method.
inhalation and preparations for skin testing are m anufactured
Subsequently, the biological activity of future EHRPs is
u nder aseptic conditions.
demonstrated by m vitro methods by comparison with the
In the manufacture, packaging, storage and distribution o f results obtained with the first IH R P. T he in vitro potency
allergen products intended for administration by other routes, may be measured by a suitable immunoassay (for example,
suitable measures are taken to ensure their microbial quality; an assay based on the inhibition o f the binding capacity of
recom m endations on this aspect are provided in chapter specific immunoglobulin E antibodies).
5.1.4. Microbial quality of non-sterUe pharmaceutical preparations
and substances for pharmaceutical use. IDENTIFICATION
T h e tests for identification are performed as late as possible
All allergen preparations are m anufactured under conditions
in the manufacturing process. In the case of products used
designed to minimise exogenous and endogenous enzymatic
on a nam ed-patient basis, the control is performed on the
degradation.
active substance and/or at the intermediate stage between the
Any purification procedure is designed to minimise the active substance and the finished product.
content o f any potential irritant low molecular mass
Identity is confirmed by comparison with the IH R P using
com ponents and non-allergenic components.
protein profiling by appropriate methods (for example,
Allergen products m ay contain suitable antimicrobial isoelectric focusing, sodium dodecyl sulfate polyacrylamide
preservatives, the nature and concentration of which have to gel electrophoresis, Immunoelectrophoresis, immunoblotting,
be justified. liquid chromatography o r mass spectrometry).
T h e manufacturing process comprises various stages: In exceptional cases, if no IH R P is available, a representative
— source material; batch may be used to confirm identity.
— active substance: it is generally a modified or an
Identity may also be confirmed by comparison with
unm odified allergen extract; where applicable it is stored
individual allergen-specific reference standards, when
under conditions ensuring its stability, for example freeze-
available.
dried;
— finished product. TESTS
All other stages of the manufacturing process are considered T h e tests are perform ed as late as possible in the
as intermediates. manufacturing process. In the case o f products used on a
named-patient basis, th e control is performed on the active
IN-HOUSE REFERENCE PREPARATION substance and/or at the intermediate stage between the active
An appropriate representative preparation is selected as the substance and the finished product.
in-house reference preparation (IHRP), characterised and
Various biochemical and immunological tests have been
used to verify batch-to-batch consistency. T he IH R P is
developed in order to characterise allergens qualitatively and
stored in suitably sized aliquots under conditions ensuring its
quantitatively. In those cases where such m ethods cannot be
stability, for example freeze-dried.
applied, particularly for the determination o f allergenic
Characterisation o f the in-house reference preparation activity and allergen and/or protein profile, justification m ust
The extent of characterisation of the IHRP depends on the source be provided.
material, knowledge of the aUergejric components and availability
1-98 Allopurinol 2016
W a te r (2.5.12) or (2 J J 2 ) the biological potency and/or the protein content and/or

M aximum 5 per cent for freeze-dried products. the extraction concentration;
In the case o f oral lyophilisates, the w ater content may be the route o f administration and die intended use;
higher than 5 per cent, where justified and authorised. the storage conditions;
where applicable, the name and am ount of added
S te rility (2.6.1)
antimicrobial preservative;
Allergen products presented as parenteral preparations, eye
where applicable, for freeze-dried preparations:
preparations, preparations for inhalation or preparations for
— the name, composition and volume o f the
skin testing comply with the test for sterility.
reconstituting liquid to be added;
M icro b ial c o n ta m in a tio n — the period o f tim e within which the preparation is to
For non-sterile allergen products, recommendations are be used after reconstitution;
provided in 5.1.4. Microbial quality of non-sterile pharmaceutical where applicable, that the preparation is sterile;
preparations and substances for pharmaceutical use. where applicable, the nam e and am ount of adsorbent.
P ro te in c o n te n t (2.5.33) PhEur
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 Allopurinol ★ ★
stated content. W hen the finished product contains
(Ph. Eur. monograph 0576) * * * * *
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 NH
T he protein profile determined by suitable methods J
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 C5H4N4O 136.1 315-30-0
justified.
Various additional tests, some with increasing selectivity, Xanthine oxidase inhibitor; treatm ent o f gout and
depending on the allergen product concerned can be applied, but in hyperuricaemia.
any casefor allergen products intended for therapeutic use, a
validated test measuring the potency (total allergenic activity,
Allopurinol Oral Suspension
determination of individual allergens or any otherjustified tests)
must be applied. Allopurinol Tablets
A lu m in iu m (2.5.13) PhEur__________________________________________________________
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 D E F IN IT IO N
otherwise justified and authorised, w hen aluminium 1,5-Dihydro-4ii-pyrazolo [3,4 -d]pyrimidin-4-one.
hydroxide or aluminium phosphate is used as adsorbent. C o n te n t
C a lc iu m (2.5.14) 97.0 per cent to 102.0 per cent (dried substance).
80 per cent to 120 p er cent o f the stated amount when CHARACTERS
calcium phosphate is used as adsorbent. A p p e a ra n c e
A llergen p ro file W hite o r alm ost white powder.
Relevant allergenic components are identified by means of S o lu b ility
suitable techniques using allergen-specific hum an or animal Very slighdy soluble in water and in ethanol (96 p er cent).
antibodies. It dissolves in dilute solutions o f alkali hydroxides.
T o ta l allerg en ic a c tiv ity ID E N T IF IC A T IO N
50 per cent to 150 per cent o f the stated am ount as assayed First identification B
by inhibition o f the binding capacity o f specific
im munoglobulin E antibodies or a suitable equivalent in vitro
method. A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
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 Test solution Dissolve 10 mg in 1 m L o f a 4 g/L solution of
relevant allergen com ponent, determined by a suitable sodium hydroxide R and dilute to 100.0 m L with a 10.3 g/L
method. 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
STO RA G E acid R.
Adsorbed allergen products are not to be frozen, unless
Spectral range 220-350 nm .
otherwise justified and authorised.
Absorption maximum A t 250 nm .
L A B E L L IN G
Absorption minimum At 231 nm.
The label states:
Absorbance ratio ^ 231^250 = 0-52 to 0.62.
— the nam e o f the allergen product;
Comparison aOopurinol CRS.
2016 Allopurinol 1-99
C. Dissolve 0.3 g in 2.5 m L of düute sodium hydroxide Limits:

solution R and add 50 m L o f water R. Add slowly and with — impurity A: n o t more than twice the area of th e principal
shaking 5 m L o f silver nitrate solution R l. A white precipitate peak in the chromatogram obtained with reference
is formed which does not dissolve on the addition of 5 m L of solution (a) (0.2 per cent);
ammonia R. — impurity B: n o t more than the area of the principal peak
D . Thin-layer chromatography (2.2.27). in the chromatogram obtained with reference solution (a)
(0.1 per cent);
Test solution Dissolve 20 m g of the substance to be examined
— impurity C: n o t more than the area of the corresponding
in concentrated ammonia R and dilute to 10 m L with the same
solvent.
solution (b) (0.1 per cent);
Reference solution Dissolve 20 mg of aUopurinol CRS in — unspecified impurities: for each impurity, not m ore than the
concentrated ammonia R and dilute to 10 m L with the same area o f the principal peak in the chromatogram obtained
solvent. with reference solution (a) (0.10 per cent);
Plate TLC silica gel F2 5 4 plate R. — sum of impurities other than A , B and C: not m ore than
Mobile phase anhydrous ethanol R, methylene chloride R 3 times the area of the principal peak in the
(40:60 VIV). chrom atogram obtained with reference solution (a)
Application 10 |iL. (0.3 p er cent);
Drying In air. (0.05 p er cent).
Detection Examine in ultraviolet light at 254 nm . Impurities D and E
Results T h e principal spot in the chromatogram obtained with Liquid chromatography (2.2.29). Use freshfy prepared solutions.
the test solution is similar in position and size to the principal Store and inject them at 8 °C} using a cooled autosampler.
spot in the chromatogram obtained with the reference
Solution A 1.25 g/L solution of potassium dihydrogen
solution.
phosphate R.
TESTS Test solution Dissolve 50.0 mg of the substance to be
Related substances 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. Reference solution Dissolve 5.0 m g o f aUopurinol
Test solution (a) Dissolve 25.0 mg of the substance to be impurity D CRS and 5.0 m g of aUopurinol impurity E CRS in
examined in 2.5 m L o f a 4 g/L solution of sodium hydroxide R 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
Test solution (b) Dissolve 20.0 mg o f the substance to be this solution to 100.0 m L with solution A.
examined in 5.0 m L o f a 4 g/L solution of sodium hydroxide R Column:
and dilute immediately to 250.0 m L with the mobile phase. — size: I = 0.05 m , 0 = 4.6 mm;
Reference solution (a) Dilute 2.0 m L o f test solution (a) to — stationary phase: base-deactivated octadecylsUyl silica gel for
100.0 m L with the mobile phase. Dilute 5.0 m L of this chromatography R (3 (am).
solution to 100.0 m L with the mobile phase. Mobile phase methanol R> 1.25 g/L solution of potassium
Reference solution (b) Dissolve 5 mg o f aUopurinol dihydrogen phosphate R (10:90 VIV).
impurity A CRS, 5 m g of aUopurinol impurity B CRS and Flow rate 2 mL/min.
5.0 mg o f aUopurinol impurity C CRS in 5.0 m L o f a 4 g/L Detection Spectrophotom eter at 230 nm.
solution o f sodium hydroxide R and dilute immediately to
Irtjection 20 |iL.
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. Run time 1.5 times the retention time o f impurity E.
Reference solution (c) Dissolve 20.0 m g o f aUopurinol CRS in Retention times Im purity D = about 3.6 min;
5.0 m L o f a 4 g/L solution o f sodium hydroxide R and dilute im purity E = about 4.5 min.
immediately to 250.0 m L with the mobile phase. System suitability: reference solution:
Column: — resolution: minim um 2.0 between the peaks due to
— size: I = 0.25 m , 0 = 4.6 mm; impurities D and E.
— stationary phase: octadecylsSyl silica gel for chromatography R Limits:
(5 nm). — impurity D: n o t more than the area of the corresponding
Mobile phase 1.25 g/L solution of potassium dihydrogen peak in the chromatogram obtained with the reference
phosphate R. solution (0.1 p er cent);
— impurity E: n o t more than the area o f the corresponding
Detection Spectrophotom eter at 230 nm . solution (0.1 p er cent).
Injection 20 |iL of test solution (a) and reference solutions (a) Impurity F
and (b). Liquid chromatography (2.2.29).
Run time Twice the retention time of aUopurinol. U nder the following conditions, any hydrazine in the sample
Elution order Im purity A, impurity B, impurity C , aUopurinol. reacts with benzaldehyde to give benzaldehyde azine.
Retention time AUopurinol = about 10 min. Solvent mixture M ix equal volumes o f dilute sodium hydroxide
System suitability: reference solution (b): solution R and methanol R.
impurities B and C.
I- 100 Almagate 2016
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‘ m-
Test solution Dissolve 250.0 m g of the substance to be H N
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 A. R1 = N H 23 R2 = H : 5-am ino-li/-pyrazole-4-
tem perature. Add 5.0 m L o f hexane R and shake for 1 min. carboxamide,
Allow the layers to separate and use the upper layer. B. R1 = N H 2, R2 = C H O : 5-(fonnylam ino)-lH-pyrazole-4-
Reference solution Dissolve 10.0 mg o f hydrazine sulfate R in carboxamide,
the solvent mixture by sonicating for about 2 min and dilute D . R1 = 0 - C 2H 5, R2 = H: ethyl 5-am ino-lH-pyrazole-4-
to 50.0 m L with the solvent mixture. Dilute 1.0 m L to carboxylate,
20.0 m L with the solvent mixture. D ilute 1.0 m L o f this
E. R1 = O -C 2H 5, R2 = C HO : ethyl 5-(form ylam ino)-l//-
solution to 20.0 m L with the solvent mixture. T o 5.0 m L of
pyrazole-4-caiboxylate,
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. 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 L /N
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.
C. 5-(4H-l,2,4-triazol-4-yl)-lii-pyrazole-4-carboxam ide,
Column:
— sizer. I = 0.25 m ,0 = 4.0 mm; F. H 2N -N H 2: diazane (hydrazine).
— stationary phase', cyanosifyl silica gelfor chromatography R PhEur
(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. Almagate * •*
★ ★
Detection Spectrophotom eter at 310 nm . * * * * *
Injection 20 pL.
Relative retention W ith reference to benzaldehyde (retention AhMftCsDaoHi^fflaO 630 66827-12-1
tim e = about 2.8 min): benzaldehyde azine = about 0.8. A c tio n a n d u se
System suitability: reference solution: Antacid.
— resolution: minimum 2 between the peaks due to
benzaldehyde azine and benzaldehyde; P h & i _____________________
— signal-to-noise ratio: minimum 20 for the peak due to DEFINmON
benzaldehyde azine.
H ydrated aluminium magnesium hydroxycarbonate.
Limit.
C o n te n t
— impurity F: the area of the peak due to benzaldehyde
— aluminium: 15.0 per cent to 17.0 per cent (calculated
azine in the chromatogram obtained with the test solution
as A120 3),
is not m ore than the area o f the corresponding peak in
— magnesium: 36.0 per cent to 40.0 per cent (calculated
the chromatogram obtained with the reference solution
as M gO ),
(10 ppm of hydrazine sulfate equivalent to 2.5 ppm of
— carbonic acid: 12.5 p er cent to 14.5 per cent (calculated
hydrazine).
as C O 2).
Maximum 20 ppm. CHARACTERS
A p p e a ra n c e
W hite or almost white, fine, crystalline powder.
using 2 m L of lead standard solution (10 ppm Pb) R.
S o lu b ility
Practically insoluble in water, in ethanol (96 per cent) and in
M axim um 0.5 per cent, determined on 1.000 g by drying in
methylene chloride. It dissolves with effervescence and
an oven at 105 °C.
heating in dilute mineral adds.
M axim um 0.1 per cent, determined on 1.0 g. IDENTIFICATION
A SSA Y
Comparison Ph. Eur. reference spectrum of almagate.
Liquid chromatography ( 2.2.29) as described in the test for
related substances with the following modification. 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
Injection T est solution (b) and reference solution (c).
reaction o f aluminium (2.3.1).
Calculate the percentage content o f C5H4N4O from the
C. 2 m L o f the solution prepared under identification test B
declared content of aUopurinol CRS.
gives the reaction o f magnesium (2.3.1).
IM P U R IT IE S
TESTS
Specified impurities: A, B, C, D , E, F.
p H (2.2.3)
9.1 to 9.7.
2016 Almagate 1-101
Disperse 4.0 g in 100 m L of carbon dioxide-free water R, stir Magnesium

for 2 m in and filter. Introduce 10.0 m L of solution A prepared in the assay of
N e u tra lisin g cap a c ity alum inium into a 500 m L conical flask, add 200 m L of
Carry out the testât 37 °C. Disperse 0.5 g in 100 m L of water R, 20 m L o f triethanolamine R with shaking, 10 m l. of
water R, heat, add 100.0 m L o f 0.1 M hydrochloric add, ammonium chloride buffer solution pH 10.0 R and 50 mg of
previously heated and stir continuously; the p H (2.2.3) o f the mordant black 11 triturate R Titrate with 0.05 M sodium
solution between 5 m in and 20 min is not less than 3.0 and edetate until the colour changes from violet to pure blue.
not greater than 4.5. Add 10.0 m L o f 0.5 M hydrochloric add, 1 m L of 0.05 M sodium edetate is equivalent to 2.015 mg
previously heated, stir continuously for 1 h and titrate with o f MgO.
0.1 M sodium hydroxide to p H 3.5; n o t m ore than 20.0 m L of C a rb o n ic a c id
0 . 1 M sodium hydroxide is required. 12.5 per cent to 14.5 per cen t
C h lo rid e s (2.4.4) Test sample Place 7.00 m g of the substance to be examined in
Maximum 0.1 per c e n t a tin capsule. Seal the capsule.
Dissolve 0.33 g in 5 m L of dilute nitric acid R and dilute to Reference sample Place 7.00 mg o f ahnagate CRS in a tin
100 m L with water R. Prepare simultaneously the standard capsule. Seal the capsule.
by diluting 0.7 m L o f dilute nitric add R to 5 m L with Introduce separately the test sample and the reference sample
water R and adding 10 m L o f chloride standard solution (5 ppm into a combustion chamber of a C H N analyser purged with
Cl) R. helium for chromatography R and maintained at a tem perature
S u lfates (2.4.13) o f 1020 °C. Simultaneously, introduce oxygen R at a pressure
M aximum 0.4 per cent. o f 40 kPa and a flow rate of 20 m I7m in and allow complete
Dissolve 0.25 g in 5 m L o f dilute hydrochloric add R and combustion o f the sample. Sweep the combustion gases
dilute to 100 m L with distilled water R. Prepare through a reduction reactor and separate the gases form ed by
simultaneously the standard by adding 0.8 m L o f dilute gas chromatography (2.2.28).
hydrochloric add R to 15 m L of sulfate standard solution Column:
(10 ppm SO 4) R — sizer. I = 2 m , 0 = 4 mm;
Sodium — stationary phase: ethylvinylbenzene-dxvinylbenzene
M aximum 150 ppm. copolymer R l.
Atomic absorption spectrometry (2.2.23, Method I). Carrier gas heHum for chromatography R.
Test solution Dissolve 0.25 g in 50 m L of a 103 g/L solution Flow rate 100 mL/min.
o f hydrochloric acid R. Temperature:
— column: 65 °C;
Reference solutions Prepare the reference solutions using sodium
— detector. 190 °C.
standard solution (200 ppm Na) R, diluted as necessary with a
103 g/L solution of hydrochloric add R. Detection Therm al conductivity.
H eavy m e ta ls (2.4.8) Run time 16 m in .
Maximum 20 ppm. System suitability:
Dissolve 1.0 g in dilute hydrochloric acid R and dilute to — average percentage o f carbon in 5 reference samples m ust
20.0 m L w ith the same acid. 12 m L o f the solution complies be within ± 0.2 per cent of the value assigned to
with test A. Prepare the reference solution using lead standard the CRS; the difference between the upper and the lower
solution (1 ppm Pb) R. values o f the percentage of carbon in these samples m ust
be below 0.2 per cent.
L oss o n ig n itio n
Calculate the percentage content o f carbonic acid in the test
43.0 per cent to 49.0 per cent, determined on 1.000 g by
sample according to the following formula:
ignition at 900 ± 50 °C.
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 percentage content o f carbonic a d d in the reference
Absence o f Pseudomonas aeruginosa (2.6.13). sample;
A SSAY K m ean value for the 5 reference samples of the ratio
A lu m in iu m o f the mass in milligrams to the area o f the peak
Dissolve 1.000 g in 5 m L o f hydrochloric acid R, heating if due to carbonic add;
necessary. Allow to cool to room tem perature and dilute to A area o f the peak due to carbonic a d d in the
100.0 m L with water R (solution A). Introduce 10.0 m L of chrom atogram obtained with the test sample;
solution A into a 250 m L conical flask, add 25.0 m L of sample mass, in milligrams.
0.05 M sodium edetate, 20 m L of buffer solution pH 3.5 R, STORAGE
40 m L o f ethanol R and 2 m L of a freshly prepared 0.25 g/L In an airtight container.
solution o f dithizone R in ethanol R. T itrate the excess of
PhEur
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.
1-102 Almond Oil 2016
★* * — erucic add: maximum 0.1 per cent.

Virgin Almond Oil ★ ★
★ ★
S tero ls (2.4.23)
Almond Oil *****
Composition of sterolfraction of the oil:
(Ph. Eur. monograph 0261) — cholesterol: maximum 0.7 per cent,
P re p a r a tio n — campesterol: maximum 4.0 per cent,
Almond Oil E ar Drops — sugmasteroh maximum 3.0 per cent,
— f}-sitosterol: 73.0 per cent to 87.0 per cent,
PhEur_______________________
— A5-avenasterol: minimum 10.0 per cent,
D E F IN IT IO N — A7-stigmastenol: maximum 3.0 per cent,
Fatty oil obtained by cold expression from the ripe seeds of — A7-avenasterol: maximum 3.0 per cent,
Prunus dulcis (Mill.) D A .W ebb var. dulcis or Prunus dulds — brassicasterol: maximum 0.3 per cent.
(Mill.) D A .W ebb var. amara (DC.) Buchheim or a mixture W a te r (2.5.32)
of both varieties. Maximum 0.1 per cent, determ ined on 1.00 g.
CHARACTERS STORA GE
A p p e a ra n c e In a well-filled container, protected from light.
Yellow, d ear liquid.
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. D E F IN IT IO N
Second identification A, B. Fatty oil obtained from the ripe seeds of Prunus dulds
A Absorbance (see Tests). (Mill.) D A . Webb var. dulcis or Prunus dulcis (Mill.) D A .
B. Identification of fatty oils by thin-layer chromatography W ebb var. amara (DC.) Buchheim or a mixture o f both
(2.3.2). varieties by cold expression. It is then refined. A suitable
antioxidant may be added.
Results T he chromatogram obtained is similar to the
corresponding chromatogram shown in Figure 2.3.2.-1. CHA RACTERS
C. Composition of fatty adds (see Tests). A p p e a ra n c e
Pale yellow, d ear liquid.
TESTS
Specific a b so rb a n c e (2.2.25) S o lubility
M aximum 0.2, determined at the absorption maximum at Slightly soluble in ethanol (96 per cent), m isdble with light
270 nm . T he ratio o f the absorbance measured at 232 nm to petroleum.
that measured at 270 nm is greater than 7. R elativ e d en sity
T o 0.100 g add cyclohexane R and dilute to 10.0 m L with the A bout 0.916.
same solvent. Adapt the concentration of the solution so that It solidifies at about - 1 8 °C.
the absorbance lies between 0.5 and 1.5, measured in a 1 cm ID E N T IF IC A T IO N
cell.
A Identification o f fatty oils by thin-layer chromatography
A d d valu e (2.5.1) (2.3.2).
M aximum 2.0, determined on 5.0 g. Results T he chromatogram obtained is similar to the
P e ro x id e v alu e (2.5.5, Method A) corresponding chromatogram shown in Figure 2.3.2.-1.
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) TESTS
Specific a b so rb a n c e (2.2.25)
C o m p o sitio n o f fa tty acid s (2.4.22, Method A) Use the 0.2 to 6.0, determined at the absorption maximum at
mixture of calibrating substances in Table 2.4.22.-3. 270 nm.
Composition of the fatty-acid fraction of the oil: T o 0.100 g add cyclohexane R and dilute to 10.0 m L with the
— saturated fatty acids of chain length less than Cj^. maximum same solvent. Adapt the concentration o f the solution so that
0.1 per cent, the absorbance lies between 0.5 and 1.5, measured in a 1 cm
— palmitic acid: 4.0 per cent to 9.0 per cent, cell.
— palrmtoleic add: maximum 0.8 per cent,
A cid valu e (2.5.1)
— margaric add', maximum 0.2 per cent,
M aximum 0.5, determined on 5.0 g.
— stearic add: maximum 3.0 p er cent,
— oleic add: 62.0 per cent to 86.0 per cent, P e ro x id e v alu e (2.5.5, Method A)
— linoleic add: 20.0 per cent to 30.0 per cent, Maximum 5.0.
— linolenic acid: maximum 0.4 per cent, U n sap o n ifiab le m a tte r (2.5.7)
— arackidic add: maximum 0.2 per cent, M aximum 0.9 per cent, determined on 5.0 g.
— eicosenoic acid: maximum 0.3 per cent,
C o m p o sitio n o f fa tty a c id s (2.4.22, Method A)
— behenic add: maximum 0.2 per cent,
Use the mixture of calibrating substances in Table 2.4.22.-3.
2016 Aloxiprin 1-103
Composition of the fatty-acid fraction of the oü: TESTS

— saturated fatty adds of chain length less than Ci£ maximum H eav y m e ta ls
0.1 per cent; Carefully ignite 2 .0 g at a low tem perature until completely
— palmitic add: 4.0 per cent to 9.0 per cent; charred, cool, add 2 m L of nitric acid and 0 .25 m L of sulfuric
— paltmtoleic add: maximum 0.8 per cent; add, heat cautiously until white fumes are evolved and ignite
— margaric acid: maximum 0.2. per cent; at 5 00° to 60 0 °. Cool, add 2 m L of hydrochloric acid,
— stearic add: maximum 3.0 per cent; evaporate to dryness on a water bath and carry out the
— oleic acid: 62.0 per cent to 86.0 per cent; procedure for limit test C for heavy metals, Appendix VH,
— linoleic acid: 20.0 per cent to 30.0 per cent; beginning at the words Dissolve the residue...’. Use 2 m L of
— Imolenic add: maximum 0.4 per cent; lead standard solution (10 ppm Pb) to prepare die standard
— arachidic acid: maximum 0.2 per cent; (10 ppm).
— eicosenoic acid: maximum 0.3 per cent; F re e acetylsalicylic a cid
— behenic acid, maximum 0.2 per cent; T o a quantity containing the equivalent o f 1.0 g of total
— erucic acid: maximum 0.1 per cent.
salicylates add 5 0 m L of dry ether and shake for 3 0 minutes.
S tero ls (2.4.25) Filter quickly through fluted filter paper, wash the paper with
Composition of the sterolfraction of the oü: several portions o f dry ether and dilute the combined filtrate
— cholesterol: maximum 0.7 per cent; and washings to 1 0 0 m L with dry ether. T he absorbance o f the
— campesterol: maximum 5.0 per cent; solution at the maximum at 2 7 8 nm is n o t more than 0 .3 6 ,
— stigmasterol: maximum 4.0 per cent; Appendix I I B (0.5% , calculated with reference to the
— ^-sitosterol: 73.0 per cent to 87.0 per cent; content o f total salicylates).
— A5-avenasterol: minim um 5.0 per cent; S alicylic a c id
— A 7-sngmastenol: maximum 3.0 per cent; T he absorbance o f the solution used in the test for Free
— A 7-avenasterol: maximum 3.0 per cent; acetylsalicylic a d d at the maximum at 3 0 8 nm is not more
— brassicasterol: maximum 0.3 per cent. than 0 .5 0 , Appendix II B (0.15% , calculated with reference
W a te r (2.5.32) to the content o f total salicylates).
M aximum 0.1 per cent, determined on 1.00 g. C o m b in e d salicy late
STORA GE N o t more than 9.5% , calculated as salicylic ad d , C7H 60 3,
In a well-filled container, protected from light. with reference to the content o f total salicylates calculated as
__________________________________________________________________________________________ P h E tr
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
Aloxiprin 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
9014-67-9
washings to 2 0 0 m L with dichloromethane. Dilute 2 0 mT. o f
A ctio n a n d u se the solution to 5 0 m L with dichloromethane and measure the
Salicylate; non-selective cyclo-oxygenase inhibitor; absorbance of the resulting solution at the maximum at
antipyretiq analgesic; anti-inflammatory. 3 0 8 nm , Appendix IIB . Calculate the content of
taking 2 9 3 as the value of A (l% , 1 cm) at the
P re p a ra tio n
maximum at 3 0 8 nm .
Aloxiprin Tablets
L oss on d ry in g
D E F IN IT IO N W hen dried to constant weight over phosphorus pentoxide at a
Aloxiprin is a polymeric condensation product of aluminium pressure not exceeding 0 .7 kPa, loses n o t more than 2.0% of
oxide and O-acetylsalicylic add. It contains n o t less than its w dght. Use 1 g.
7.5% and not m ore than 8.5% o f aluminium, Al, and not ASSAY
less than 79.0% and n o t more than 87.4% o f total salicylates, For ahimm mm
calculated as O-acetylsalicylic ad d , CgHsOi, both calculated Ignite 2 g in a tared silica crudble, heat gently until the
with reference to the dried substance. organic m atter is destroyed and then ignite to constant
C H A R A C T E R IS T IC S w dght at 1000°. Each g of residue is equivalent to 0 .5 2 9 2 g
A fine, white or slightly pink powder. of Al.
Practically insoluble in water, practically insoluble in ethanol For total salicylates
(96%) and in ether. 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
A. Boil 1 g with 2 0 m L o f 2 m hydrochloric add, cool, filter
5 0 0 m L with water. T o 5 m L add 4 m L of iron(m) chloride
and reserve the filtrate. Dissolve the residue in 10 m L of
solution, allow to stand for 3 0 minutes, dilute to 5 0 m L w ith
0 .1 m sodium hydroxide and neutralise with 1m acetic add.
water and measure the absorbance of the resulting solution at
1 m L of the resulting solution yidds reaction A characteristic
the maximum at 5 3 0 nm , Appendix II B, using in the
o f salicylates, Appendix VI.
reference cell a solution prepared by diluting 4 m L of iron(m)
B. T h e filtrate reserved in test A yields the reaction chloride solution to 5 0 m L with water. Calculate the content of
characteristic o f aluminium salts, Appendix VI. total salicylates as C ^ H ^ from the absorbance obtained by
repeating the procedure using 4 m L of a 0.05% w/v solution
1-104 Alprazolam 2016
of salicyUc add in place o f the solution being examined and Reference solution (b) Dissolve 10 m g o f alprazolam CRS and
b eg in n in g at the words ‘add 4 m L o f ừon(w) chloride 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.
Alprazolam ★ ★ Development Over a path o f 12 cm.
★ ★
(Ph. Eur. monograph 1065) ***** Drying In. air.
Detection Examine in ultraviolet light at 254 nm.
H3C ^ w System suitability: reference solution (b):
— 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
C17H13aN4 308.8 28981-97-7
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
Action and use
Benzodiazepine. add R.
PhEir __________________________________________________________ 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] Reference solution (a) Dissolve 2 m g o f alprazolam CRS and
[1,4] benzodiazepine. 2 m g o f triazolam CRS in dimethylformamide R and dilute to
Content
99.0 per cent to 101.0 per cent (dried substance). 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
CHARACTERS solution to 10.0 m L with dimethylformamide R.
Appearance
Cdumn:
W hite or almost white, crystallin e powder.
— size. I = 0.25 m, 0 = 4.6 mm;
Solubility — stationary phase: phenylsüyl silica gelfor chromatography R1
Practically insoluble in water, freely soluble in methylene (5 nm).
chloride, sparingly soluble in acetone and in ethanol Mobile phase:
(96 per cent). — mobile phase A: buffer solution, methanol R (44:56 VIV)\
It shows polymorphism (5.9). — mobile phase B: buffer solution, methanol R (5:95 V!V)\
IDENTIFICATION — temperature:. 40 °C;
Second identification A , C. lim e Mobile phaae A Mobile phase B
(min) (percent V/V) (ptT cent V7V)
A. Dissolve the substance to be examined in the smallest
0 -1 5 98 2
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 15 -3 5 98 -> 1 2 + 99
be examined with 5.0 mg of alprazolam CRS. T he melting 3 5 -4 0 1 99
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. Flow rate 2 mL/min.
B. Infrared absorption spectrophotometry (2.2.24). Detection Spectrophotom eter at 254 nm.
Preparation Discs. Irgecdon 10 |iL; inject dtmethylformamide R a sa blank.
Comparison alprazolam CRS. Retention time Triazolam = about 9 min;
If the spectra obtained in the solid state show differences, alprazolam = about 10 min.
dissolve the substance to be examined and the reference System suitability: reference solution (a):
substance separately in the m in im u m volume of ethyl — resolution: minim um 1.5 between the peaks due to
acetate R, evaporate to dryness on a water-bath and record triazolam and alprazolam.
new spectra using the residues. Limits:
C. Thin-layer chromatography (2.2.27). — totah not more than the area o f the principal peak in the
Test solution Dissolve 10 m g o f the substance to be exam in ed chrom atogram obtained with reference solution (b)
in methanol R and dilute to 10 m L w ith the same solvent. (0.25 per cent);
— disregard lima: 0.2 times the area o f the principal peak in
Reference solution (a) Dissolve 10 m g o f alprazolam CRS in
methanol R and dilute to 10 m L with the same solvent.
(0.05 per cent).
2016 Alprazolam 1-105
an oven at 105 °C.
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 G. 7-chloro- l-methyl-5-phenyl[ 1,2,4]triazolo [4,3-a] quinolin-
potentiometrically (2.2.20). Titrate to the 2nd point of 4-amine,
inflexion.
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
IM P U R IT IE S
N y CH3
H . bis[[4-(2-ben2oyl-4-chlorophenyl)-5-methyl-4H'-lj2}4-
and enantiomer triazol-3-yl] methyl] amine.
OH
A. (4R5)-3-amino-6-chloro-2-methyl-4-phenyl-3,4-
dihydroquinazolin-4-olj
and enantiomer
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,
B. R = C H 2O H : [5-chloro-2-[3-(hydroxymethyl)-5-methyl-
4 H - 1j2j4-triazol-4-yl] phenyl] phenyimethanone,
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-4H-
1,2 }4-triazol-4-yl] phenyl] phenylmethanone,
J. 2,17-dichloro-6,13-dimethyl-l 8b, 19a-diphenyl-

8b, 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,
1-106 Alprenolol Hydrochloride 2016
Test solution (a) Dissolve 0.50 g o f die substance to be

Alprenolol Hydrochloride ***\ examined in methanol R and dilute to 10 m L w ith die same
(Ph Eur monograph 0876) * 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.
Reference solution (b) Dissolve 10 m g o f alprenolol
C 15H 24C1N02 285.8 13707-88-5 hydrochloride CRS and 10 m g o f oxprenold hydrochloride CRS
Action and use Reference solution (c) Dilute 5 m L o f test solution (b) to
Beta-adrenoceptor antagonist. 50 mT. with methanolR
PhEur___________________________________________________________ Plate TLC silica gel G {date R.
DEFINITION Mobile phase Place 2 beakers each containing 30 m L of
ammonia R at die bottom o f die tank containing a mixture of
(2RS)-1 -[(1 -M ethylethyl)amino]-3- [2-(prop-2-enyI)phenoxy]
5 volumes o f methanol R and 95 volumes o f ethyl acetate R.
propan-2-ol hydrochloride.
Application 5 |iL.
Content
99.0 per cent to 101.0 per cent (dried substance). Development Over a p ath o f 15 cm in a tank saturated for at
least 1 h.
CHARACTERS
Drying Ax. 100 °C for 15 min.
Appearance
W hite or alm ost white, crystalline pow der o r colourless
Detection Expose to iodine vapour for up to 6 h.
crystals. System suitability: reference solution (b):
— the chrom atogram shows 2 clearly separated spots.
Solubility
Very soluble in water, freely soluble in ethanol (96 p er cent) Limits: test solution (a):
— impurity D-. any spot with an Rp value greater than th at of
and in methylene chloride.
the principal spot is not more intense than die principal
IDENTIFICATION spot in the chrom atogram obtained with reference
First identification B, D. solution (c) (0.2 per cent).
Second identification A , C, D. Related substances
A. M elting point (2.2. 14): 108 °C to 112 °C. Liquid chrom atography (2.2.29).
B. Infrared absorption spectrophotom etry (2.2.24). Test solution Dissolve 20.0 m g of die substance to be
Comparison alprenolol hydrochloride CRS. exam ine d in the mobile phase and dilute to 10.0 m L with
C. Examine the chrom atograms obtained in the test for the mobile phase.
im purity D. Reference solution (a) Dissolve 4.0 m g o f alprenolol
Detection Examine in daylight, after exposure to iodine hydrochloride CRS and 0.8 m g of 4-isopropylphenol R in the
vapour for 30 min. mobile phase and dilute to 100.0 m L with the mobile phase.
Results T he principal spot in the chrom atogram obtained with 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
test solution (b) is similar in position, colour and size to the
principal spot in the chrom atogram obtained with reference solution to 10.0 m L with the mobile phase.
solution (a). Column:
— size. I = 0.15 m , 0 = 4 mm;
D . It gives reaction (a) of chlorides (2.3.1).
— stationary phase, octylsüyl silica gel for chromatography R
TESTS (5 pm).
Solution S Mobile phase M ix 0.656 g o f sodium octanesidfonate R with
Dissolve 1.0 g in carbon dioxide-free water R and dilute to 150 mT. o f acetomtrüe R and dilute to 500 m L with
50 m L with the same solvent. phosphate buffer p H 2.8 prepared as follows: mix 1.78 g of
Appearance of solution phosphoric add R and 15.6 g o f sodium dihydrogen phosphate R
Solution S is clear (2.2.1) and n o t m ore intensely coloured and dilute to 2000 m L w ith water R
than reference solution B9 (2.2.2, Method II). Flow rate 1 mlVmin.
Acidity or alkalinity Detection Spectrophotom eter at 280 nm .
T o 10 m L o f solution S add 0.2 mT. o f methyl red solution R Equilibration W ith the mobile phase for about 1 h.
and 0.2 m L o f 0.01 M hydrochloric acid; the solution is red.
Injection 20 nL.
A dd 0.4 m L o f 0.01 M sodium hydroxide; the solution is
yellow. Run time Twice the retention time of alprenolol.
Impurity C Retention time Alprenolol = about 11 min;
M axim um 0.1 per c e n t 4-isopropylphenol = about 18 min.
Dissolve 0.25 g in ethanol (96 per cent) R and dilute to

— resolution: m inim um 5 between the peaks due to
25 m L with the same solvent. T h e absorbance (2.2.25)
alprenolol and 4-isopropylphenol; if necessary, adjust the
measured at 297 nm is n o t greater than 0.20.
concentration o f sodium octanesulfonate and/or
Impurity D acetonitrile in the mobile phase (increase the
Thin-layer chrom atography (2.2.27).
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 D . 1,1 '-[(l-methylethyl)imino]bis[3-[2-(prop-2-enyl)
0.25 times the area of the principal peak in the phenoxy]propan- 2 -ol].
------------------------------------------------------------------------------------------------------------------------------------------------------ P hE ir
(0.10 per cent);
— total: not more than the area of the principal peak in the
(0.4 per cent);
(0.04 per cent).
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.
M aximum 0.5 per cent, determined on 1.000 g by drying
over diphosphorus pentoxide R at a pressure n ot exceeding C20H 34O 5 354.5 745-65-3
2.7 kPa.
A ctio n a n d u se
S u lfa te d a sh (2.4.14) Prostaglandin E x (PG Ei)
P h E ir __________________________________________________________________________________________
A SSA Y
Dissolve 0.400 g in 25 m L o f a m ixture of equal volumes o f D E F IN IT IO N
anhydrous ethanol R and water R. A dd 10 m L of 0.01 M 7- [( 1 2R,3R) -3-Hydroxy-2-[( 1£,35) -3-hydroxyoa - 1-enyl] -
hydrochloric add. Carry out a potentiometric titration (2.2.20), 5-oxocyclopentyl]heptanoic acid.
using 0.1 M sodium hydroxide. Read the volume added C o n te n t
between the 2 points of inflexion. 95.0 per cent to 102.5 p er cent (anhydrous substance).
1 m L of 0.1 M sodium hydroxide is equivalent to 28.58 mg CHARACTERS
of C 15H 24CINO 2. A p p e a ra n c e
STORA GE W hite or slighdy yellowish, crystalline powder.
Protected from light. S olu b ility
IM P U R IT IE S Practically insoluble in water, freely soluble in ethanol
Specified impurities C, D (96 p er cent), soluble in acetone, slightly soluble in ethyl
acetate.
present at a sufficient level, be detected by one or other of ID E N T IF IC A T IO N
the tests in the monograph. They are limited by the general A Specific optical rotation (2.2.7): -7 0 to —60 (anhydrous
acceptance criterion for other/unspecified impurities and/or substance).
by the general monograph Substances for pharmaceutical use Immediately before use, dissolve 50 m g in ethanol
(2034). It is therefore not necessary to identify these (96per cent) R and dilute to 10.0 m l . with the same solvent.
Control of impurities in substances for pharmaceutical use): A, B.
Comparison alprostadil CRS.
H OH C. Examine the chromatograms obtained in the assay.
0. .R1 Results T he principal peak in the chromatogram obtained
and enantiomer
R2 the principal peak in the chromatogram obtained with the
reference solution.
A. R1 = O H , R 2 = C H 2-C H = C H 2: (2ftS)-3-[2-(prop-2- TESTS
enyl) phenoxy] propan- 1, 2-diol, R e la te d su b stan c e s
C. R1 = N H -C H (C H 3) 2, R2 = C H = C H -C H 3: Liquid chromatography (2.2.29). Prepare the solutions protected
(2RS) -1 - [(1 -methylethyl)amino] -3- [2 -(prop-1-enyl) phenoxy] from light.
propan- 2-ol, 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 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
B. 2-(prop-2-enyl)phenol, and water R.
1-108 Alprostadil 2016
Reference solution (b) Dissolve 1.0 m g o f dinoprostone Time Mobile phase A Mobile phase B
impurity C CRS (alprostadil impurity H ) and 1.0 m g o f the (min)____________ (percent V7V)________ (per cent V7V)
substance to be examined in a mixture o f equal volumes of 0 -5 0 100 0
acetomtrile R1 and water R and dilute to 20.0 m L with the 50-51 100 + 0 0+100
sam e mixture o f solvents.
51 -61 0 100
Reference solution (c) In order to prepare impurities A and B
in situ, dissolve 1 mg o f the substance to be examined in 61 -62 0+100 100 + 0
100 pL o f 1 M sodium hydroxide (the solution becomes 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 Relative retention W ith reference to alprostadil (retention
o f acetomtrUe R1 and water R. time = about 7 min): impurity A = about 2.4;
S y ste m A impurity B = about 2.6.
Column: System suitability:
— size: I = 0.25 m , 0 = 4.0 mm; — resolution: m in im u m 1 .5 between the peaks due to
— stationary phase: base-deactivated octylsUyl silica gel for impurity A and impurity B in the chrom atogram obtained
chromatography R (4 pm) with a pore size of 6 nm ; w ith reference solution (c).
— temperature: 35 °C. C arry out the test according to system A and B.
Mobile phase: Limits:
— mobile phase A: dissolve 3.9 g of sodium dihydrogen — correction factors: for the calculation of content, multiply
phosphate R in water R and dilute to 1.0 L with the same the peak areas o f the impurities listed in Table 1488.-1 by
solvent; adjust to p H 2.5 with a 2.9 g/L solution of the corresponding correction factor;
phosphoric add R (approximately 600 m L is required);
to 740 m L o f the buffer solution add 260 m L o f Table 1488.-1.
acetomtrUe R1;
Imparity Relative retention Relative retention Correction factor
— mobile phase B: dissolve 3.9 g o f sodium dihydrogen (system A) (system B)
phosphate R in water R and dilute to 1.0 L with the same impurity G 0.80 - 0.7
solvent; adjust to p H 2.5 with a 2.9 g/L solution o f
impurity F 0.88 - 0.8
phosphoric add R (approximately 600 m L is required);
to 200 m L o f the buffer solution add 800 m L of impurity D 0.90 - 1.0
acetomtrUe R1; impurity H 0.96 - 0.7
impurity E 1.10 - 0.7

impurity C 1.36 1.9
(min) (per cent V7V) (per cent V7V)
0 - 75 100 0 impurity K 1.85 0.06
75 - 76 100-»0 0+100 impurity A 232 0.7
7 6 -8 6 0 100 impurity B 2.45 1.5
8 6 -8 7 0+100 100 + 0 impurity I 4.00 1.0
87 - 102 100 0 impurity J 5.89 1.0
Flow rate 1 mL/m in. — impurity A: n o t more than 3 times the area of the
Injection 20 |iL. — impurity B: n o t more than the area o f the principal peak
Retention time Alprostadil = about 63 m in . in the chrom atogram obtained with reference solution (a)
System suitability: (0.5 per cent);
— resolution: m in im u m 1.5 between the peaks due to — any other impurity: n o t m ore than 1.8 times the area o f the
im purity H and alprostadil in the chromatogram obtained principal peak in the chrom atogram obtained with
with reference solution (b). reference solution (a) (0.9 per cent), and n o t more than 1
S y ste m B such peak has an area greater than the area o f the
U se the same conditions as for system A with the following principal peak in the chrom atogram obtained with
mobile phase and elution programme: reference solution (a) (0.5 per cent). Evaluate impurities
— mobile phase A: dissolve 3.9 g of sodium dihydrogen appearing at relative retentions less than 1.2 by system A
phosphate R in water R and dilute to 1.0 L with the same and impurities appearing at relative retentions greater
solvent; adjust to p H 2.5 with a 2.9 g/L solution o f than 1.2 by system B;
phosphoric add R (approximately 600 m L is required); — total: not more than 3 times the area o f the principal peak
to 600 m L o f the buffer solution add 400 m L of in the chromatogram obtained with reference solution (a)
acetomtrUe R l; (1.5 per cent);
— mobile phase B: use mobile phase B as described under — disregard limit. 0.1 times the area of the principal peak in
system A; 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
related substances, system A. Prepare the solutions protected
from Ught.
Test solution Dissolve 10.0 m g of the substance to be H OH
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 E. 7-[(li?,2J?,35)-3-hydroxy-2-[(l£,35)-3-hydroxyoct-l-
solvents. Dilute 3.0 m L of the solution to 20.0 m L with a enyl]-5-oxocyclopentyl]heptanoic acid
mixture o f equal volumes of acetonitrile R1 and water R. (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
account the assigned content of alprostadil CRS.
F . 7-[(l S,2R,3R)-3-hydroxy-2-[(1 £ ,3 6 )-3-hydroxyoct-1-
ST O R A G E enyI]-5-oxocyclopentyl]heptanoic a d d
At a tem perature o f 2 °C to 8 °C. ( 8-epiprostaglandin E i),
IM P U R IT IE S
H ’oh
H OH
G . (5Z)-7- [(1 i?,2i?3^)-3-hydroxy-2-[(l£,35)-3-hydroxyoct-
A 7- [(1 R,2S)-2- [(1 £,3.S)-3-hydroxyoct-l -enyI]-5- l-enyI]-5-oxocydopentyl]hept-5-enoic a d d
oxocyclopent-3-enyI]heptanoic acid (prostaglandin A 0 , (dinoprostone),
O
CO2H
CH3
H OH H OH
B. 7- [2- [(1£,3.S)-3-hydroxyoct-1-enyl] -5-oxocydopent-1- H . (5£)-7-[(l.R,22?,3.R)-3-hydroxy-2-[(l£,3.S)-3-hydroxyoct-

enyl]heptanoic a d d (prostaglandin B i), l-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]-5-
oxocydopentyl]heptanoic a d d (15-oxoprostaglandin E j), I. ethyl 7-[(1 i?,2i?,3i?)-3-hydroxy-2- [(1 £ ,3 5 )-3-hydroxyoct-
l-enyI]-5-oxocydopentyi]heptanoate (prostaglandin E i,
ethyl ester),
H OH
D . 7-[(li?,2i?,3i?)-3-hydroxy-2-[(l£*3.R)-3-hydroxyoct-l- H OH
enyl]-5-oxocyclopentyl]heptanoic a d d
(15-epiprostagJandin E i),
J. 1-methylethyl 7-[(li?,2i?,3i?)-3-hydroxy-2-[(l£,36)-3-
hydroxyoct-l-enyl]-5-oxocyclopentyl]heptanoate
(prostaglandin E i, isopropyl ester),
1-110 Alteplase for Injection 2016
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. P ro te in c o n te n t
PhEur 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
★ ★
Alteplase for Injection ★ ★ buffer. F or the calculation of the alteplase concentration, the
***** absorbance value (A2so —-^320) is divided by the specific
(Ph E ut monograph 1170) absorption coefficient for alteplase o f 1.9.
P o te n c y
SYQVICRDEK TQMIYQQHQS WLRPVLRSNR VEYCWCNSGR
T h e potency of alteplase is determ ined in an in vitro clot-lysis
I------------- 1 = i ------------------- '
AQCHSVPVKS CSEPRCFNGG TCQQALYFSD FVCQCPEGFA assay as described under Assay. T he specific activity o f bulk
,____________ 1 " i_l
GKCCEIDTRA TCYEDQGISY RGTWSTAESG AECTNWNSSA alteplase is approximately 580 000 IU per milligram o f
'------------------------------ '“ I alteplase.
LAQKPYSGRR PDAIRLGLGN HNYCRNPDRD SKPWCYVFKA
GKYSSEFCST PACSEGNSDC YFGNGSAYRG THSLTESGAS iV -te rm in a l se q u e n ce

TV-terminal sequencing is applied to determ ine the correct
CLPWNSMILI ?sjÂAQALGLGKHN
GKVYTAQNPS YCRNPDGDAK
.PWCHVLKNRR
1—i r N-terminal sequence and to determine semiquantitatively
LTWEYCDVPS CSTCGLRQYS QPQFR
additional cleavage sites in the alteplase molecule, for
IKGGL example at position AA 275-276 o r at position AA 27-28.
FADIASHPWQ AAIFAKHRRS PGERFLCGGI LISSC W ILSA T he AT-terminal sequence m ust conform with the sequence o f
1
AHCFQERFPP HHLTVILGRT YRWPGEEEQ KFEVEKYIVH hum an tissue plasminogen activator.
KEFDDDTYDN DIALLQLK5D SSRCAQESSV VRTVCLPPAD Is o e le c tric fo cu sin g
LQLPDWTECE LSGYGKHEAL SPFYSERLKE AHVRLYPSSR T he consistency in the microheterogeneity o f gjycosylation of
VTDNMLCAGD TRSGGPQANL
the alteplase molecule can be dem onstrated by isoelectric
CTSQHLLNRT HDACQGDSGG
■ focusing (IEF). A complex banding pattern with 10 major
PLVCLNDGRM TLVGIISWGL GCGQKDVPGV YTKVTNYLDW
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.
C 2736H 4174N 91 4O 824S 45 (non-glycosylated protein) 105857-23-6 T he broad charge distribution observed is due to a
population o f molecules, which differ in the fine structure o f
Tissue-type plasm inogen activator; fibrinolytic. biantenary and triantenary complex-type carbohydrate
residues, with different degrees o f substitution with sialic
PhEur___________________________________________________________
acids. T h e banding pattern o f alteplase test samples m ust be
D E F IN IT IO N consistent w ith the pattern of alteplase reference standard.
Alteplase for injection is a sterile, fieeze-dried preparation o f S in g le -c h a in a lte p la se c o n te n t
alteplase, a tissue plasminogen activator produced by T h e alteplase produced by C H O (Chinese ham ster ovary)
recom binant D N A technology. It has a potency of n o t less cells in serum-free medium is predom inantly single-chain
than 500 000 IU per milligram of protein. alteplase. T h e single-chain form can be separated from the
Tissue plasminogen activator binds to fibrin clots and two-chain form by gel-permeation liquid chromatography
activates plasminogen, leading to die generation of plasmin under reducing conditions as described under Single-chain
and to the degradation of fibrin clots or blood coagulates. content (see Tests). T h e single-chain alteplase content in
Alteplase consists of 527 amino acids with a calculated bulk samples m ust be higher than 60 p er cent.
relative molecular mass of 59 050 w ithout consideration o f T ry p tic -p e p tid e m a p p in g
the carbohydrate moieties attached at positions Asn 117, T he primary structure o f the alteplase molecule is verified by
Asn 184 and Asn 448. T he total relative molecular mass is tryptic-peptide mapping as described under Identification B.
approximately 65 000. Alteplase is cleaved by plasmin T h e reduced and carboxymethylated molecule is cleaved by
betw een amino-acids 275 and 276 into a two-chain form trypsin into about 50 peptides, which are separated by
(A chain and B chain) that are connected by a disulfide bridge reverse-phase liquid chromatography. A characteristic
betw een Cys 264 and Cys 395. T he single-chain form and chrom atogram (fingerprint) is obtained. T h e identity of the
the two-chain form show comparable fibrinolytic activity in tryptic-peptide map o f a given alteplase sample with the
vitro. profile o f a well-characterised reference standard is an
P R O D U C T IO N indirect confirmation of the amino-acid sequence, becam e
even single amino-acid exchanges in individual peptides can
Alteplase is produced by recom binant D NA synthesis in cell
be detected by this sensitive technique. In addition, complex
culture; the fermentation takes place in serum-free m edium.
peaks o f the glycopeptides can be isolated from the tryptic-
T he purification process is designed to remove efficiendy peptide m ap and separated in a second dimension, either by
potential impurities, such as antibiotics, D N A and protein reverse-phase liquid chrom atography under modified
contam inants derived both from the h ost cell and from the conditions o r by capillary electrophoresis. By this two
production m edium , and potential viral contaminants.
2016 Alteplase for Injection 1-111
dimensional separation of glycopeptide variants, lot-to-lot and reference standard and using a relative molecular mass of
consistency of the microheterogeneity o f glycosylation can be 180.2 for mannose and a relative molecular mass of 59 050
demonstrated. for the alteplase protein moiety. T h e neutral sugar content of
T he tryptic-peptide map o f alteplase samples must be the alteplase samples m ust be in the range o f 70 to
consistent with the tryptic-peptide map of alteplase reference 130 per cent compared to alteplase reference standard, which
standard. contains about 12 moles of neutral sugar per mole of
alteplase.
Monomer content
T h e m onom er content of alteplase is m easured by gel- CHARACTERS
perm eation liquid chromatography under non-reduced W hite or slightly yellow powder or solid friable mass.
conditions as described under M onomer content (see Tests). Reconstitute the preparation as stated on the label immediately
T he m onom er content of alteplase bulk samples must be before carrying out the Identification, Tests (except those for
higher than 95 per cent solubility and water) and Assay.
Type I/Type II alteplase content IDENTIFICATION
C H O cells produce 2 glycosylation variants o f alteplase. A. T he assay serves also to identify the preparation.
Type I alteplase contains 1 polymannose-type glycosylation at
B. Tryptic-peptide mapping. Examine by liquid
position Asn 117 and 2 complex-type glycosylation sites at
chromatography (2.2.29).
positions Asn 184 and Asn 448. Type II alteplase is only
glycosylated at positions Asn 117 and Asn 448. Test solution Dilute the preparation to be examined with
water R to obtain a solution containing about 1 mg of
T he ratio o f Type I/Type II alteplase is constant in the range
alteplase per millilitre. Dialyse about 2.5 m L o f the solution
o f 45 to 65 per cent of Type I and 35 to 55 per cent of
for at least 12 h into a solution containing 480 g/L of urea R,
Type II. T h e content of alteplase Type I and Type II can be
44 g/L o f tris(hydroxymethyl) aminomethane R and 1.5 g/L of
determ ined by a densitometric scan o f SDS-PAGE (sodium
sodium edetate R and adjusted to p H 8.6, using a mem brane
dodecyl sulfate polyacrylamide gel electrophoresis) gel.
with a cut-oflf point corresponding to a relative molecular
Plasmin-treated samples of alteplase, which are reduced and
mass o f 10 000 for globular proteins. Measure the volume of
carboxymethylated before loading on the gel, are separated
the solution, transfer it to a clean test-tube and add per
into 3 bands: Type I alteplase A-chain (AA 1-275), Type II
millilitre 10 |iL o f a 156 g/L solution of dithiothreitol R. Allow
alteplase A-chain (AA 1-275) and alteplase B-chain
to stand for 4 h, cool in iced water and add per millilitre of
(AA 276-527). T h e ratio o f Type I/Type II alteplase is
solution 25 pL o f a freshly prepared 190 g/L solution of
determ ined from a calibration curve, which is obtained by a
iodoacetic add R. Allow to stand in the dark for 30 min.
densitometric scan of defined mixtures o f purified Type I
A dd per millilitre 50 |oL of dithiothreitol solution to stop the
alteplase and T ype II alteplase standards.
reaction. Dialyse for 24 h against an 8 g/L solution of
SDS-PAGE ammonium hydrogen carbonate R. Add 1 part of trypsin for
SD S-PAG E (silver staining) is used to demonstrate purity of peptide mapping R to 100 parts o f the protein and allow to
the alteplase bulk material and the integrity o f the alteplase stand for 6 h to 8 h. Repeat the addition of trypsin and allow
molecule. F or alteplase bulk samples, no additional protein to stand for a total of 24 h.
bands compared to reference standard or degradation
Reference solution Prepare as for the test solution using a
products m ust occur in SDS-PAGE gels at a loading am ount
suitable reference standard instead of the preparation to be
o f 2.5 |xg alteplase protein per lane and a limit of detection of
examined.
5 ng per protein (BSA) band.
T he chromatographic procedure may be carried out using:
Bacterial endotoxins (2.6.14) — a column 0.1 m long and 4.6 mm in internal diameter
Less than 1 IU p er milligram of alteplase. packed with octadecylsüyl silica gelfor chromatography R
Sialic adds (5 jim to 10 pm);
Proceed using a suitable validated m ethod devdoped Mobile phase A 8 g/L solution of sodium dikydrogen
according to general chapter 2.2.59. Glycan analysis of phosphate R , adjusted to p H 2.85 with phosphoric
glycoproteins. T h e sialic ad d s content for the test samples acid R, filtered and degassed;
m ust be in the range of 70 to 130 per cent compared to Mobile phase B 75 per cent V/V solution of
alteplase reference standard, which contains about 3 moles of acetomtrUe R in mobile phase A;
sialic a d d s per mole of alteplase. — as detector a spectrophotometer set at 210 nm.
Neutral sugars Equilibrate the system with mobile phase A at a flow rate of
Dilute alteplase samples and the reference standard in the 1 mL/min. After injection of the solution, increase the
assay buffer, containing 34.8 g/L of arginine R, 0.1 g/L of proportion of mobile phase B at a rate of 0.44 per cent per
polysorbate 80 R and adjusted to pH 7.4 with phosphoric m inute until the ratio of mobile phase A to mobile phase B is
acid R, to a protein concentration of 50 [ig/m L Prepare the 60:40, then increase the proportion of mobile phase B at a
following concentrations o f mannose in the same assay buffer rate o f 1.33 per cent per minute until the ratio o f mobile
for a calibration curve: 20, 30, 40, 50 and 60 (ig/mL. Pipette phase A to mobile phase B is 20:80 and then continue
2 m L of alteplase samples and reference standard, as well as elution with this mixture for a further 10 min. Record the
2 m L o f each mannose concentration in duplicate in reagent chromatogram for the reference solution: the test is not valid
tubes. A dd 50 jaL of phenol R, followed by 5 m L of sulfuric unless the resolution of peaks 6 (peptides 268-275) and 7
acid R, in each reagent tube. Incubate the mixture for 30 m in (peptides 1-7) is at least 1.5; whf and are n o t more than
at room tem perature. M easure the absorbance at 492 nm for 0.4 min. Inject about 100 |iL o f the test solution and record
each tube. Read the content of neutral sugars from the the chromatogram. Verify the identity of the peaks by
mannose calibration curve. T he neutral sugar content is comparison with the chromatograms of the reference
expressed in moles of neutral sugar per mole of alteplase, solution. There should n o t be any additional significant peaks
taking into account the dilution factor for alteplase samples o r shoulders, a significant peak or shoulder being defined as
1-112 Alteplase for Injection 2016
one with an area response equal to or greater than 5 p er cent — a colum n 0.6 m long and 7.5 m m in internal diam eter
of peak 19 (peptides 278-296); no significant peak is missing. packed with silica-based, rigid, hydrophilic gel with
A type chromatogram for identification of the peaks cited is spherical particles 10 pm to 13 pm in diam eter, suitable
shown in Figure 1170.-1. for size-exclusion chromatography;
— as mobile phase at a flow rate o f 0.5 m L/m in a solution
TESTS
c o n tainin g 30 g/L of sodium dihydrogen phosphate R and
1 g/L o f sodium dodecyl sulfate R, adjusted to p H 6.8 with
T he reconstituted preparation is clear (2.2. I) and n o t more
dilute sodium hydroxide solution R;
intensely coloured than reference solution Y7 (2.2.2,
— as detector a spectrophotom eter set at 214 nm .
Method II).
Inject about 50 pL o f the test solution and record the
p H (2.2.3)
chrom atogram. T he chrom atogram shows 2 m ajor peaks
7.1 to 7.5. corresponding to single-chain and two-chain alteplase.
S o lu b ility Calculate the relative am ount o f single-chain alteplase from
A dd the volume o f the liquid stated on the label. the peak area values.
T h e preparation dissolves completely within 2 m in at 20 °C T h e test is n o t valid unless: the num ber o f theoretical plates
to 25 °C. calculated on the basis o f the single-chain alteplase peak is at
P r o te in c o n te n t least 1000. T h e content of single-chain alteplase is n o t less
Prepare a solution of the substance to be examined with an than 60 per cent o f the total am ount o f alteplase-related
accurately known concentration of about 1 g/L. Using a substances found.
34.8 g/L solution of arginine R adjusted to p H 7.3 with M o n o m e r c o n te n t
phosphoric acid R, dilute an accurately measured volume o f Examine by liquid chromatography (2.2.29).
the solution o f the substance to be examined so that the
Test solution Reconstitute the preparation to be examined to
absorbance measured at the maximum at about 280 nm is
obtain a solution containing about 1 m g per millilitre.
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 T h e chromatographic procedure may be carried out using:
320 nm using the arginine solution as the compensation — a colum n 0.6 m long and 7.5 m m in internal diam eter
liquid. Calculate the protein content in the portion o f packed with silica-based rigid, hydrophilic gel with
alteplase taken from the following expression: spherical particles 10 pm to 13 pm in diameter, suitable
for size-exclusion chromatography;
— as mobile phase at a flow rate o f 0.5 m L/m in a solution
V (-^280 ~ -<4.320)
containing 30 g/L of sodium dihydrogen phosphate R and
1.9
1 g/L o f sodium dodecyl sulfate R, adjusted to p H 6.8 with
dilute sodium hydroxide solution R;
in which V is the volume o f th e test solution, A 2 S0 is the
— as detector a spectrophotom eter set at 214 nm .
absorbance at the maximum at about 280 nm and A 32q is the
absorbance at 320 nm. Inject the test solution and record the chromatogram.
T h e test is n o t valid unless the num ber o f theoretical plates
Single-chain c o n te n t
calculated for the alteplase m onom er peak is at least 1000.
Examine by liquid chrom atography (2.2.29).
M easure the response for all peaks, i.e. peaks corresponding
Test solution Dissolve the preparation to be examined in to alteplase species o f different molecular masses. Calculate
water R to obtain a solution containing about 1 m g o f the relative content of m onom er from the area values of these
alteplase per millilitre. Place about 1 m L o f the solution in a peaks. T h e m onom er content for alteplase m ust be at least
tube, add 3 m L of a 3 g/L solution o f dxthiotkreitd R in the 95 p er c e n t
mobile phase, place a cap on the tube and heat at about
80 °C for 3 m in to 5 min.
T h e chromatographic procedure may be carried out using:
2016 Altizide 1-113
W a te r (2.5.12) valid unless the correlation coefficient is —0.9900 to

N o t more than 4.0 per cent, determined by the semi-micro —1.0000. From the line equation and the clot-lysis time for
determ ination o f water. the test solution, calculate the logarithm o f the activity U&
B a c te ria l en d o to x in s (2.6.14) from the following equation:
Less than 1 IU per milligram of protein.
,o g u A = l O e g t A
S te rility (2. 6.1) 0
It complies with the test for sterility.
Calculate the alteplase activity in International U nits per
A SSA Y millilitre from the following expression:
T h e potency of alteplase is determined by comparing its
ability to activate plasminogen to form plasmin with the same D xU a
capacity o f a reference preparation calibrated in International
U nits. T he formation o f plasmin is measured by the in which D is the dilution factor for the test solution.
determ ination o f the lysis time of a fibrin clot in given Calculate the specific activity in the portion of the substance
conditions. to be examined from the following expression:
T h e International U nit is the activity of a stated quantity of
the International Standard o f alteplase. T he equivalence in Ua
International U nits of the International Standard is stated by P
the World H ealth Organization.
Solvent buffer A solution containing 1.38 g/L of sodium in which P is the concentration of protein obtained in the test
dihydrogen phosphate monohydrate R, 7.10 g/L of anhydrous for protein con ten t
disodium hydrogen phosphate R, 0.20 g/L o f sodium azide R and T h e estimated potency is not less than 90 per cent and not
0.10 g/L of polysorbate 80 R. m ore than 110 p er cent of the stated potency.
Human thrombin solution A solution o f human thrombin R STORA GE
containing 33 IU /m L in solvent buffer. Store in a colourless, glass container, under vacuum or under
Human fibrinogen solution A 2 g/L solution o ffibrinogen R in an inert gas, protected from light, at a tem perature of 2 °C to
solvent buffer. 30 °C.
Human plasminogen solution A 1 g/L solution of human LA B E LL IN G
plasminogen R in solvent buffer. The label states:
Test solutions U sing a solution of the substance to be — the num ber o f International Units per container;
examined containing 1 g/L, prepare serial dilutions using — the am ount o f protein per container;
solvent buffer, for example 1:5000, 1:10 000, 1:20 000. — the name and volume of the liquid to be used for
Reference solutions Using a solution of a suitable reference reconstitution.
standard having an accurately known concentration of about ---------------------------------------------------------------------- -------------- -- PhEur
1 g/L (580 000 IU o f alteplase per millilitre), prepare 5 serial
dilutions using water R to obtain reference solutions having
known concentrations in the range 9.0 IU /m L to 145 IU /m L.
T o each o f a set o f labelled glass test-tubes, add 0.5 m L of Altizide *****
hum an throm bin solution. Allocate each test and reference *, *
solution to a separate tube and add to each tube 0.5 m L o f (Ph Eur monograph 2185) *
the solution allocated to it. T o each o f a second set of
o o
labelled glass tubes, add 20 |iL of hum an plasminogen
solution, and 1 m L of hum an fibrinogen solution, mix and H’ N ' S y T S' r and enanUomer
store on ice. Beginning with the reference/thrombin mixture C l N ^s
containing the lowest num ber of International Units per H H
millilitre, record the time and separately add 200 pL o f each
o f the thrombin mixtures to the test tubes containing the C 11H 14QN30 4S3 383.9 5588-16-9
plasminogen-fibrinogen mixture. Using a vortex mixer,
intermittently mix the contents of each tube for a total of A c tio n a n d u se
15 s and carefully place in a rack in a circulating water-bath Thiazide diuretic.
at 37 °C. A visibly turbid clot forms within 30 s and bubbles
PhEur___ ______________________________________________________
subsequently form within die clot. Record the clot-lysis time
as the time between the first addition of alteplase solution D E F IN IT IO N
and the m om ent when the last bubble rises to the surface. (3RS) -6-Chloro-3- [(prop-2-enylsulfanyl) methyl] -3,4-dihydro-
U sing a least-squares fit, determine the equation o f the line 2/f-l,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide.
using the logarithms o f the concentrations of the reference C o n te n t
preparation in International Units per millilitre versus the 97.5 per cent to 102.0 per cent (anhydrous substance).
logarithms of the values of their clot-lysis times in seconds,
according to the following equation: CHA RACTERS
A p p e a ra n c e
logf = o + 6 (logl7s) W hite or almost white powder.
S o lu b ility
in which t is the clot-lysis time, Us the activity in Practically insoluble in water, soluble in methanol, practically
International U nits per millilitre of the reference preparation, insoluble in methylene chloride.
b is the slope and a the ^ in te rc ep t o f the line. T he test is not It shows polymorphism (5.9).
1-114 Altizide 2016
ID E N T IF IC A T IO N Detection Spectrophotom eter at 270 nm .

Infrared absorption spectrophotom etry (2.2.24). Injection 5 pL.
Comparison altizide CRS. Run time Twice the retention tim e o f altizide.
If the spectra obtained show differences, dissolve 50 m g of Relative retention W ith reference to altizide (retention
the substance to be examined and 50 m g of the reference time = about 25 min): im purity A = about 0.15;
substance separately in 2 m L o f acetone R and evaporate the furosemide = about 1.05.
solvent. Precipitate by adding 1 m L o f methylene chloride R. System suitability: reference solution (c):
Evaporate to dryness and record new spectra using the — resolution: minimum 1.0 between die peaks due to altizide
residues. and furosemide.
TESTS Limits:
Im p u rity B — impurity A: not more than 3 times die area o f the
Thin-layer chrom atography (2.2.27). principal peak in the chrom atogram obtained with
Test solution Dissolve 0.200 g o f the substance to be reference solution (a) (0.3 p er cent);
examined in acetone R and dilute to 2.0 m L with the same — unspecified impurities: for each impurity, n o t m ore than the
solvent. area o f the principal peak in the chrom atogram obtained
Reference solution (a) Dissolve 10.0 m g o f altizide
— total: not more than 5 times the area o f the principal peak
impurity B CRS in acetone R and dilute to 25.0 m L with the
same solvent.
(0.5 per cent);
Reference solution (b) T o 1.0 m L of reference solution (a) add — disregard limit. 0.5 times the area o f the principal peak in
1.0 m L o f the test solution. the chromatogram obtained w ith reference solution (a)
Reference solution (c) Dilute 5.0 mL o f reference solution (a) (0.05 per cent).
to 10.0 m L with acetone R. W a te r (2.5.32)
Plate TLC silica gel F2 5 4 plate R. Maximum 0.5 per cent, determ ined on 50.0 mg.
Mobile phase acetone R, methylene chloride R (25:75 VIV). S u lfa te d a s h (2.4.14)
Application 10 pL o f the test solution and reference M aximum 0.1 per cent, determ ined on 1.0 g.
ASSAY
Development Over 2/3 o f the plate. Liquid chromatography (2.2.29) as described in the test for
Drying In air. related substances, with the following modifications.
Detection Spray with a mixture o f equal volumes o f a 10 g/L Test solution Dissolve 25.0 m g o f the substance to be
solution of potassium permanganate R and a 50 g/L solution of examined in 2 mL o f acetomtrile R and dilute to 25.0 m L
sodium carbonate R, prepared immediately before use. Allow with the mobile phase.
to stand for 30 m in and examine in daylight. Reference sdution Dissolve 25.0 m g o f altizide CRS in 2 m L of
System suitability: reference solution (b): acetomtrile R and dilute to 25.0 m L with die mobile phase.
— the chrom atogram shows 2 clearly separated spots. Calculate the percentage content o f C iiH 14Q N 304 S 3 from
Limit Any spot due to im purity 6 is n o t more intense than the declared content o f altizide CRS.
the principal spot in the chrom atogram obtained with
IM P U R IT IE S
reference solution (c) (0.2 per cent).
liq u id chrom atography (2.2.29). Prepare the solutions
immediately before use, except reference sdution (b).
in 5 m L o f acetomtrUe R and dilute to 25 m L with the mobile
phase.
Reference solution (a) D ilute 1.0 m L o f the test solution to A. 4-am ino-6-chlorobenzene-l,3-disulfonam ide,
100.0 m L with the mobile phase. D ilute 1.0 m L of this
solution to 10.0 m L w ith the mobile phase. OCH,
Reference solution (b) In order to produce im purity A in situ,
dissolve 50 m g o f the substance to be examined in 5 m L o f
acetomtrile R and dilute to 25 m L with water R. Allow to
stand for 30 min. B. 3-[(2,2-dimethoxyethyl)sulfanyl]prop-1-ene.
Reference solution (c) Dissolve 4 mg o f furosemide CRS in ___________________________________________________________ PtiEur
2 m l . of acetomtrUe R, add 2 m L o f the test solution and
dilute to 100 m L with the mobile phase.
Column:
— size: I = 0.15 m , 0 = 3.9 mm;
— stationary phase: end-capped octadecylsüyl silica gelfor
chromatography R (5 pm);
Mobile phase acetomtrile R, water R previously adjusted to
p H 2.0 with perchloric add R (25:75 VIV).
Flow rate 0.7 mL/m in.
2016 Aluminium Chloride 1-115
Alum ***** Aluminium Chloride Hexahydrate ★ ★

★ ★
*★ ★* *****
Potash Alum; Aluminium Potassium Sulphate; * (Ph Eur monograph 0971)
Aluminium Potassium Sulfate A1C13,6H20 241.4 7784-13-6
A1K(S04)2j 12H20 474.4 7784-24-9
A stringent
A ction a n d u se P re p a r a tio n
Astringent. Aluminium Chloride Solution
PhEur__________________________________________________________ PhEtr.
D E F IN IT IO N D E F IN IT IO N
C o n ten t C o n te n t
99.0 per cent to 100.5 per cent of A IK C S O ^ 12H 20 . 95.0 p er cent to 101.0 per cent.
CHARACTERS CHARACTERS
A p p e a ra n c e A p p e a ra n c e
Granular powder or colourless, transparent, crystalline W hite or slightly yellow, crystalline powder or colourless
masses. crystals, deliquescent
S olubility S o lu b ility
Freely soluble in water, very soluble in boiling water, soluble Very soluble in water, freely soluble in ethanol (96 per cent),
in glycerol, practically insoluble in ethanol (96 per cent). soluble in glycerol.
ID E N T IF IC A T IO N ID E N T IF IC A T IO N
A. Solution S (see Tests) gives the reactions o f sulfates A. Dilute 0.1 m L of solution S2 (see Tests) to 2 m L with
(2.3.1). water R. T he solution gives reaction (a) o f chlorides (2.3.1).
B. Solution S gives the reaction of aluminium (2.3.1). B. Dilute 0.3 m L o f solution S2 to 2 m L with water R.
C. Shake 10 m L o f solution S with 0.5 g o f sodium hydrogen T he solution gives the reaction o f aluminium (2.3.1).
carbonate R and filter. T he filtrate gives reaction (a) o f TESTS
potassium (2.3.1). S o lu tio n S I
TESTS Dissolve 10.0 g in distilled water R and dilute to 100 m L w ith
S o lu tio n S the same solvent.
Dissolve 2.5 g in water R and dilute to 50 m L with the same S o lu tio n S 2
solvent Dilute 50 m L o f solution SI to 100 m L with water R.
A p p e a ra n c e o f so lu tio n 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). Solution S2 is clear (2.2.1) and no t more intensely coloured
p H (2.2.3) than reference solution B 7 (2.2.2, Method II).
3.0 to 3.5. S u lfates (2.4.13)
Dissolve 1.0 g in carbon dioxide-free water R and dilute to Maximum 100 ppm , determined on solution S I.
10 m L with die same solvent. Ir o n (2.4.9)
A m m o n iu m (2.4.1) Maximum 10 ppm , determined on solution S i.
Maximum 0.2 per cent. A lkali a n d alk alin e -e a rth m e ta ls
T o 1 m l, o f solution S add 4 m L of water R. Dilute 0.5 m L Maximum 0.5 per c e n t
o f this solution to 14 m L with water R. T o 20 m L o f solution S2 add 100 m L o f water R and heat to
Iron ( 2. 4. 9) boiling. T o the hot solution add 0.2 m l. of methyl red
Maximum 100 ppm. solution R. Add dilute ammonia R1 until the colour of the
Dilute 2 m L o f solution S to 10 m L with water R. U se in this indicator changes to yellow and dilute to 150 m L with
test 0.3 m L o f thiogfycoUic add R. water R. H eat to boiling and filter. Evaporate 75 m L of the
filtrate to dryness on a water-bath and ignite to constant
mass. T h e residue weighs a maximum of 2.5 mg.
M aximum 20 ppm.
12 m L o f solution S complies with test A. Prepare the
Maximum 20 ppm.
12 m L of solution SI complies w ith test A. Prepare the
ASSAY reference solution using lead standard solution (2 ppm Pb) R.
Dissolve 0.900 g in 20 m L of water R and carry out the
W a te r (2.5.12)
complexometric titration o f aluminium (2.5.11).
42.0 p e r cent to 48.0 per cent, determined on 50.0 mg.
ofA IK (S 04 ) 2, 12H 20 . A SSAY
Dissolve 0.500 g in 25.0 m L of water R. Carry out the
__________________________________________________________ PhEur
complexometric titration o f aluminium (2.5.11). Titrate with
0.1 M zinc sulfate until the colour o f the indicator changes
from greyish-green to pink. Carry out a blank titration.
o f A1C13,6H 20 .
1-116 Aluminium Glycinate 2016
STORAGE A ppendix VH. U s z lead standard solution (1 ppm Pb) to

In an airtight container. prepare the standard (20 ppm ).
PhEur Mercuric salts
Dissolve 2.0 g in 10 m L o f 1m sulfuric add, transfer to a
separating funnel with the aid o f water, dilute to about
50 m L w ith water and add 50 m L o f 0.5m sulfuric acid.
Add 100 m L o f water, 2 g o f hydroxylamine hydrochloride.,
Aluminium Glycinate 1 m L o f 0.05m disodium edetate and 1 m L o f glacial acetic
acid. A dd 5 m L o f chloroform, shake, allow to separate and
discard the chloroform layer. T itrate the aqueous layer with a
solution o f dithizone in chloroform containing 8 \ig per m L
,xH20 until the chloroform layer rem ain s green. After each addition,
shake vigorously, allow the layers to separate and discard the
chloroform layer. Repeat the operation using a solution
prepared by d ilu tin g 1 mT. o f mercury standard solution
C2H6A1N04}xH20 135.1 41354-48-7 (5 ppm Hg) to 100 m L with 0.5m sulfuric add and beginning
at the words ‘Add 100 m L of water . . . T h e volume o f the
Action and use dithizone solution required by the substance being examined
Antacid. does n o t exceed that required by the mercury standard
solution.
DEFINITION
Chloride
Aluminium Glycinate is a basic alu m in iu m monoglycinate,
Dissolve 1.0 g in 10 m L o f 2m nitric add and dilute to
partly hydrated. It contains n o t less than 34.5% and not
100 m L w ith water. 15 m L o f the resulting solution complies
more than 38.5% of AI2O 3 and not less than 9.9% and not
with the limit test for chlorides, Appendix VH (330 ppm).
more than 10.8% of N , both calculated with reference to the
dried substance. Loss on drying
W hen dried to constant w dght at 130°, loses n o t more than
CHARACTERISTICS 12.0% o f its w dght. U se 1 g.
A white or alm ost white powder.
ASSAY
Practically insoluble in water and in organic solvents.
It dissolves in dilute mineral a d d s and in aqueous solutions For Al203
Dissolve 0.25 g in a m ixture o f 3 m L o f 1m hydrochloric add
of the alkali hydroxides.
and 50 m L o f water, add 50 m L o f 0. 05m disodium edetate VS
IDENTIFICATION and neutralise with 1m sodium hydroxide using methyl red
A. A dd 0.1 g to 10 m L o f a solution prepared by dissolving solution as indicator. H eat the solution to boiling, allow to
0.84 g o f citric add in 8 m L o f 1m sodium hydroxide and stand for 10 m in u tes on a water bath, cool rapidly, add
diluting to 20 m L with water. Add 0.5 m L of a 0.1% w/v about 50 m g o f xylenol orange triturate and 5 g o f hexamine
solution of ninhydrin in methanol and warm. A purple colour and titrate the excess o f disodium edetate with 0. 05m lead
is produced. nitrate VS until the solution becomes red. Each m L o f 0. 05m
B. Suspend 1 g in 25 m L o f 0.5m hydrochloric add and heat disodium edetate VS is equivalent to 2.549 m g o f A120 3.
gently until a clear solution is produced. Reserve h alf o f the
solution. T o 2 m L o f the solution add 0.15 m L o f liquefied
phenol, shake and add carefully w ithout shaking 5 m L of
dilute sodium hypochlorite solution. A blue colour is produced. ★.* * * ★
C. T h e solution reserved in test B yields the reaction
Hydrated Aluminium Hydroxide tor ★ ★
characteristic o f aluminium salts, A ppendix VI. Adsorption *****
TESTS (Ph Eur monograph 1664)
Acidity or alkalinity [A10(0H)],nH20
p H o f a suspension o f 1 g in 25 m L o f carbon dioxide-free PhEur_____________
water, 6.5 to 7.5, Appendix V L.
DEFINITION
Neutralising capacity
Shake 0.2 g vigorously with 25 m L o f 0. 1m hydrochloric add
Content
90.0 p er cent to 110.0 per cent o f the content o f aluminium
for 5 m inutes and allow to stand for 5 minutes. T he p H o f
stated on the labd.
the mixture is greater than 3.0, Appendix V L.
NOTE: shake the gel vigorously for at least 30 s immediately
Arsenic
before examining.
Dissolve 2.0 g in 18 m L o f brominated hydrochloric acid and
32 m L of water. 25 m L o f the resulting solution complies CHARACTERS
with the limit test for arsenic, Appendix VII (1 ppm). Appearance
Heavy m etals W hite or alm ost white, translucent, viscous, colloidal g d .
Dissolve 1.5 g in 20 m L o f 2m hydrochloric add and 10 m L of A supernatant may be formed upon standing.
water, add 0.5 m L o f nitric acid and boil for about Solubility
30 seconds. Cool, add 2 g o f ammonium chloride and 2 g o f A clear or almost d e a r solution is obtained with alkali
ammonium tkiocyanate and extract w ith two 10 mT. q u an tities hydroxide solutions and mineral adds.
o f a mixture o f equal parts o f isoamyl alcohol and ether. IDENTIFICATION
T o the aqueous layer add 2 g o f citric acid. 12 m L o f the
Solution S (see Tests) gives the reaction o f aluminium.
resulting solution complies w ith limit test A for heavy metals,
2016 Aluminium Hydroxide 1-117
T o 10 m L o f solution S add about 0.5 m L o f dilute Place 5 g in a test-tube immersed in ice-water, add 0.4 m L
hydrochloric add R and about 0.5 m L of thioacetamide of a 100 g/L solution of potassium chloride R, 0.1 mT. of
reagent R. N o precipitate is formed. A dd dropwise 5 m L of diphenylandne solution R and, dropwise with sh a k in g , 5 m L of
dilute sodium hydroxide solution R. Allow to stand for 1 h. sulfuric acid R. Transfer the tube to a water-bath at 50 °C.
A gelatinous white precipitate is formed which dissolves upon After 15 min, any blue colour in the solution is no t more
addition o f 5 mT. of dilute sodium hydroxide solution R. intense than that in a standard prepared a t the same time
Gradually add 5 m L of ammonium chloride solution R and and in the same manner using 5 m L of nitrate standard
allow to stand for 30 min. T he gelatinous white precipitate is solution (100 ppm NO?) R.
re-formed. S u lfates (2.4.13)
TESTS M aximum 0.5 p er cent.
S o lu tio n S Dilute 2 m L o f solution S to 20 m L with water R.
A dd 1 g to 4 m L of hydrochloric acid R. H eat at 60 °C for A m m o n iu m (2. 4.1, Method B)
1 h, cool, dilute to 50 m L with distilled water R and filter if M aximum 50 ppm , determined on 1.0 g.
necessary.
Prepare the standard using 0.5 m L of ammonium standard
p H (2.2.5) solution (100 ppm NH 4) R.
5.5 to 8.5.
A rse n ic (2.4.2, Method A)
A d so rp tio n p o w e r M aximum 1 ppm , determined on 1 g.
Dilute the substance to be examined with distilled water R to
Ir o n (2.4.9)
obtain an aluminium concentration o f 5 mg/mL. Prepare
M aximum 15 ppm , determined on 0.67 g.
bovine albumin R solutions with the following concentrations
o f bovine albumin: 0.5 mg/mL, 1 mg/mL, 2 mg/mL, H eav y m e ta ls (2.4.8)
3 mg/mL, 5 m g/m L and 10 mg/mL. I f necessary, adjust the M aximum 20 ppm .
gel and the bovine albumin R solutions to p H 6.0 with dilute Dissolve 2.0 g in 10 m L o f dilute nitric acid R and dilute to
hydrochloric acid R or dilute sodium hydroxide solution R. 20 m L with water R. T h e solution complies with test A.
F or adsorption, mix 1 part o f the diluted gel with 4 parts o f Prepare the reference solution using lead standard solution
each of the solutions o f bovine albumin R and allow to stand (2 ppm Pb) R.
at room tem perature for 1 h. During this time shake the B a c te ria l en d o to x in s (2.6.14)
mixture vigorously at least 5 times. Centrifuge or filter Less than 5 IU of endotoxin per m illigram of alu m in iu m , if
through a non-protein-retaining filter. Immediately determine intended for use in the manufacture of an adsorbed product
the protein content (2.5.33, Method 2) o f either the w ithout a further appropriate procedure for the removal of
supernatant or the filtrate. bacterial endotoxins.
It complies with the test if no bovine alb u m in is detectable in A SSAY
the supernatant or filtrate of the 2 mg/mL bovine albumin R Dissolve 2.50 g in 10 m L o f hydrochloric acid R, heating for
solution (maximum level of adsorption) and in the 30 min at 100 °C on a water-bath. Cool and dilute to 20 m L
supernatant or filtrate o f bovine albumin R solutions o f lower with water R. T o 10 m L o f the solution, add concentrated
concentrations. Solutions containing 3 mg/mL, 5 m g/m L and ammonia R until a precipitate is obtained. Add the smallest
10 mg/mL bovine albumin R may show bovine albumin in the quantity o f hydrochloric acid R needed to dissolve the.
supernatant or filtrate, proportional to the am ount o f bovine precipitate and dilute to 20 m L with water R. Carry out the
albumin in the solutions. complexometric titration o f alu m in ium (2.5.11). Carry out a
S e d im e n ta tio n blank titration.
If necessary, adjust the substance to be examined to p H 6.0
STORA GE
using dilute hydrochloric add R or dilute sodium hydroxide
At a temperature not exceeding 30 °C. D o not allow to
solution R. D ilute with distilled water R to obtain an
freeze. If the substance is sterile, store in a sterile, airtight,
aluminium concentration of approximately 5 mg/mL. I f the
tam per-proof container.
aluminium content of the substance to be e x am in e d is lower
than 5 mg/m L, adjust to p H 6.0 and dilute with a 9 g/L LABELLING
solution o f sodium chloride R to obtain an aluminium T h e label states the declared content o f alu m in iu m .
concentration o f about 1 mg/mL. After shaking for at least -------------------:____________________________________________ PhEur
30 s, place 25 m L of the preparation in a 25 m L graduated
cylinder and allow to stand for 24 h.
It complies with the test if the volume o f the clear
supernatant is less than 5 m L for die gel with an aluminium
content o f about 5 mg/mL.
Dried Aluminium Hydroxide *****
*★ ★*
It complies with the test if the volume o f the clear (Hydrated Aluminium Oxide, *
supernatant is less than 20 m L for the gel with an aluminium Ph Eur monograph 0311)
content o f about 1 mg/mL.
A ctio n a n d u se
Antacid.
Maximum 0.33 per cent.
P re p a ra tio n s
Dissolve 0.5 g in 10 m L of dilute nitric add R and dilute to
Aluminium Hydroxide Oral Suspension
500 m L w ith water R.
Chewable Aluminium Hydroxide Tablets
N itra te s
Chewable Compound M agnesium Trisilicate Tablets
M axim um 100 ppm.
Co-magaldrox Oral Suspension
Co-magaldrox Tablets
1-118 Aluminium Magnesium Silicate 2016
PhEir ___________________________________ :_______________________ ASSAY

D E F IN IT IO N Dissolve 0.800 g in 10 m L of hydrochloric add R l, heating on
a water-bath. Cool and dilute to 50.0 m L w ith water R
C o n te n t
T o 10.0 m L o f the solution add dilute ammonia R1 until a
47.0 per cent to 60.0 per cent o f A120 3 (Mt 102.0).
predpitate begins to appear. Add the smallest quantity of
CHARACTERS dilute hydrochloric add R needed to dissolve the predpitate
A p p e a ra n c e and dilute to 20 m L with water R. Carry out the
W hite or almost white, amorphous powder. complexometric titration o f aluminium (2.5.11).
S o lu b ility 1 m ĩ, o f 0.1 M sodium edetate is equivalent to 5.098 mg
Practically insoluble in water. It dissolves in dilute mineral o f AI2O3.
adds and in solutions o f alkali hydroxides.
STORA GE
ID E N T IF IC A T IO N In an airtight container, at a tem perature not
Solution S (see Tests) gives the reaction o f aluminium exceeding 30 °c.
(2.3.1). 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
TESTS This section provides information on characteristics that are
S o lu tio n S recognised as bang relevant control parameters for one or more
Dissolve 2.5 g in 15 m L of hydrochloric add R, heating on a functions of the substance when used as an excipient (see chapter
water-bath. Dilute to 100 m L with distQled water R. 5.15). This section ừ a non-mandatory part of the monograph
A p p e a ra n c e o f so lu tio n and ừ ừ not necessary to verify the characteristics to demonstrate
Solution S is n o t more opalescent than reference compliance. Control of these characteristics can however contribute
suspension II (2.2.1) and not more intensely coloured than to the quality of a medicinal product by improving the consistency
reference solution GY6 (2.2.2, Method II). of the manufacturing process and the performance of the medicinal
A lkaline im p u ritie s
recognised as being suitable for the purpose, but other methods can
Shake 1.0 g with 20 m L of carbon dioxide-free water R for
also be used Wherever results for a particular characteristic are
1 m in and filter. T o 10 m L o f the filtrate add 0.1 m L of
reported, the control method must be indicated
phenolphthalein solution R. Any pink colour disappears on the
addition of 0.3 m L o f 0.1 M hydrochloric add.
The f(Mowing characteristics may be relevantfor hydrated
aluminium oxide used as adsorbent
N e u tra lisin g c a p a c ity
P a rtic le -siz e d is trib u tio n (2.9.31)
Cany out the test at 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, Specific su rfa c e a r e a (2.9.26)
previously heated, and stir continuously; the p H (2.2.3) of __________________________________________________________ Pi) Ell
the solution after 10 min, 15 min and 20 min is not less than
1.8, 2.3 and 3.0 respectively and is at no time greater than
4.5. Add 10.0 mL of 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 more than 35.0 m L o f 0.1 M Aluminium Magnesium Silicate *****
_ *. .*
sodium hydroxide is required. (Ph. Ear. monograph 1388) *
M axim um 1 p e r cent. A ctio n a n d u se
E xdpient.
Dissolve 0.1 g with heating in 10 m L o f dilute nitric add R
and dilute to 100 m L with water R. Dilute 5 m L of the PhEir___________________________________________________________
solution to 15 m L with water R.
D E F IN IT IO N
S u lfa te s (2.4.13)
M ixture of partides with colloidal particle size o f
M axim um 1 per cent.
montmorillonite and saponite, free from grit and non-
D ilute 4 m L o f solution S to 100 m L with distHkd water R. swellable ore.
A rse n ic (2.4.2, Method A) C o n te n t
M axim um 4 ppm , determ ined on 10 m L o f solution S. — aluminium (Al; A r 26.98): 95.0 p er cent to 105.0 per cent
H e a v y m e ta ls (2.4.8) o f the value stated on the labd;
M axim um 60 ppm. — magnesium (Mg; A T 24.30): 95.0 per cent to
Neutralise 20 m L o f solution S with concentrated ammonia R, 105.0 per cent o f the value stated on the lab d .
using metanU yellow solution R as an external indicator. Filter, CHARACTERS
if necessary, and dilute to 30 m L with water R. 12 m L o f the A p p e a ra n c e
solution complies with test A. Prepare the reference solution Almost white powder, granules o r plates.
using 10 m l- o f lead standard solution (1 ppm Pb) R.
S o lu b ility
M ic ro b ia l c o n ta m in a tio n Practically insoluble in water and in organic solvents.
It swells in water to produce a colloidal dispersion.
T Y M C : acceptance criterion 102 CFU/g (2.6.12).
Absence o f bile-tolerant gram-negative bacteria (2.6.13).
A. Fuse 1 g with 2 g of anhydrous sodium carbonate R. W arm
Absence o f Escherichia colt (2.6.13). the residue with water R and filter. Addify the filtrate with
hydrochloric add R and evaporate to dryness on a water-bath.
0.25 g o f the residue gives the reaction of silicates (2.3.1).
2016 Aluminium Magnesium Silicate 1-119
B. Dissolve the remainder o f the residue obtained in L oss o n d ry in g (2.2.32)

identification test A in a mixture o f 5 m L o f dilute hydrochloric M aximum 8.0 per cent, determined on 1.000 g by drying in
acid R and 10 m L o f water R. Filter and add ammonium an oven at 105 °C.
chloride buffer solution pH 10.0 R A white, gelatinous M ic ro b ia l c o n ta m in a tio n
precipitate is formed. Centrifuge and keep the supernatant TA M C: acceptance criterion 103 CFU/g (2.6.12).
for identification C. Dissolve the remaining precipitate in
TY M C: acceptance criterion 102 CFU/g (2.6.12).
dilute hydrochloric acid R T h e solution gives the reaction of
aluminium (2.3.T). Absence o f Escherichia coli (2.6. II).
C. T he supernatant obtained after centrifugation in A SSAY
identification test B gives the reaction of magnesium (2.3.1). A lu m in iu m
Atomic absorption spectrometry (2.2.23, Method I).
TESTS
p H (2.2.3) Test solution In a platinum crucible mix 0.200 g with 1.0 g of
9.0 to 10.0. lithium metaborate R. H eat slowly at first and ignite at
1000-1200 °C for 15 min. Allow to cool, then place the
Disperse 5.0 g in 100 m L o f carbon dioxide-free water R.
crucible in a 100 m L beaker containing 25 m L o f dilute nitric
A rsen ic (2.4.2, Method A) acid R and add an additional 50 m L of dilute nitric add R,
Maximum 3 ppm. filling and submerging the crucible. Place a
Transfer 16.6 g to a 250 m L beaker containing 100 m L of polytetrafluoroethylene-coated magnetic stirring b ar in the
dilute hydrochloric acid R Mix, cover with a watch glass and crucible and stir gently with a magnetic stirrer until
boil gently, with occasional stirring, for 15 min. Allow the dissolution is complete. Pour the contents into a 250 m L
insoluble m atter to settle and decant the supernatant through beaker and remove the crucible. W arm the solution and
a rapid-flow filter paper into a 250 m L volumetric flask, transfer through a rapid-flow filter paper into a 250 m L
retaining as m uch sediment as possible in the beaker. T o the volumetric flask, wash the filter and beaker with water R and
residue in the beaker add 25 m L o f hot dilute hydrochloric dilute to 250.0 m L with water R (solution A). T o 20.0 m L of
acid R, stir, heat to boiling, allow the insoluble m atter to solution A add 20 m L o f a 10 g/L solution of sodium
settle and decant the supernatant through the filter into the chloride R and dilute to 100.0 m L with water R.
volumetric flask. Repeat the extraction with 4 additional Reference solutions Dissolve, with gentle heating, 1.000 g of
quantities, each of 25 m l , of hot dilute hydrochloric acid R, aluminium R in a mixture of 10 m L of hydrochloric acid R and
decanting each supernatant through the filter into the 10 m L o f water R. Allow to cool, then dilute to 1000.0 m L
volumetric flask. A t the last extraction, transfer as m uch of with water R (1 mg of aluminium per millilitre). Into 3
the insoluble m atter as possible onto the filter. Allow the identical volumetric flasks, each containing 0.20 g o f sodium
combined filtrates to cool to room temperature and dilute to chloride R, introduce 2.0 mL, 5.0 m L and 10.0 m L of this
250.0 m L with dilute hydrochloric acid R. Dilute 5.0 m L of solution respectively, and dilute to 100.0 m L w ith water R.
this solution to 25.0 m L with dilute hydrochloric acid R.
Source Aluminium hollow-cathode lamp.
Lead Wavelength 309 nm.
Maximum 15 ppm.
Atomisation device Oxidising acetylene-nitrous oxide flame.
M a g n e siu m
Test solution Transfer 10.0 g to a 250 m L beaker containing Atomic absorption spectrometry (2.2.23, Method I).
100 m L of dilute hydrochloric acid R. Mix, cover with a watch
glass and boil for 15 min. Allow to cool to room tem perature Test solution Dilute 25.0 m L of solution A, prepared in the
assay for aluminium, to 50.0 m L with water R. T o 5.0 m L of
and allow the insoluble m atter to setde. D ecant the
supernatant through a rapid-flow filter paper into a 400 mL this solution add 20.0 m L of lanthanum nitrate solution R and
dilute to 100.0 m L with water R.
beaker. T o the insoluble m atter in the 250 m L beaker add
25 m L of hot water R. Stir, allow the insoluble m atter to Reference solutions Place 1.000 g of magnesium R in a 250 m L
setde and decant the supernatant through the filter into the beaker containing 20 m L o f water R and carefully add 20 m L
400 m L beaker. Repeat the extraction with 2 additional o f hydrochloric add R} warming if necessary to dissolve.
quantities, each o f 25 mL, o f water R, decanting each time Transfer the solution to a volumetric flask and dilute to
the supernatant through the filter into the 400 m L beaker. 1000.0 m L with water R (1 mg of magnesium p er millilitre).
Wash the filter with 25 m L o f hot water R, collecting this D ilute 5.0 m L of this solution to 250.0 m L with water R.
filtrate in the 400 m L beaker. Concentrate the combined Into 4 identical volumetric flasks, introduce 5.0 mL,
filtrates to about 20 m L by gently boiling. I f a precipitate 10.0 m L, 15.0 m L and 20.0 mL of the solution respectively.
appears, add about 0.1 m L o f nitric acid R, heat to boiling T o each flask add 20.0 m L of lanthanum nitrate solution R
and allow to cool to room temperature. Filter the and dilute to 100.0 m L with water R.
concentrated extracts through a rapid-flow filter paper into a Source Magnesium hollow-cathode lamp.
50 m L volumetric flask. Transfer the remaining contents of Wavelength 285 nm.
the 400 m L beaker through the filter paper and into the flask
Atomisation device Reducing air-acetylene flame.
w ith water R. D ilute this solution to 50.0 m L with water R
L A B E L L IN G
Reference solutions Prepare the reference solutions using lead
standard solution (10 ppm Pb) R, diluted as necessary with T h e label states the content of aluminium and magnesium.
water R. __________________________________________________________PhEur
Source Lead hollow-cathode lamp.
Wavelength 217 nm
Atomisation device Oxidising air-acetylene flame.
1-120 Aluminium Phosphate 2016
***** 15 min. M easure the absorbance (2.2.25) at 730 nm.

Dried Aluminium Phosphate ★ ★ Calculate the content of soluble phosphates from a
(Aluminium Phosphate, Hydrated, ***** calibration curve prepared using reference solutions (a),
Ph Eur monograph 1598) (b) and (c) after treatm en t
A1P04jxH20 122.0 7784-30-7 Sulfates (2.4.13)
M axim um 0.6 p er c e n t
(anhydrous substance)
D ilute 8 m L o f solution S to 100 m L with distilled water R.
A c tio n a n d u se A rse n ic (2.4.2)
Antacid. M aximum 1 ppm.
PhEur.
1.0 g complies with limit test A.
H e a v y m e ta ls (2.4.8)
D E F IN IT IO N
M axim um 20 ppm.
C o n te n t
94.0 per cent to 102.0 per cent of A1P0 4 (Mt 122.0) (ignited Dissolve 1.0 g in dilute hydrochloric add R and dilute to
substance). 20 m L with the same ad d . 12 m L o f the solution complies
with test A. Prepare the reference solution using lead standard
CHARACTERS solution (1 ppm Pb) R.
A p p e a ra n c e
L o ss o n ig n itio n
10.0 p er cent to 20.0 per cent, determ ined on 1.000 g at
S o lu b ility 800 ± 50 °C.
Very slightly soluble in water, practically insoluble in ethanol
N e u tra lisin g c a p a c ity
(96 per cent). It dissolves in dilute solutions o f mineral ad d s
A dd 0.50 g to 30 m L of 0.1 M hydrochloric add previously
and alkali hydroxides.
heated to 37 °C and maintain at this tem perature for 15 min
ID E N T IF IC A T IO N while stirring. T h e p H (2.2.3) o f the mixture after 15 m in at
A. Solution S (see Tests) gives reaction (b) of phosphates 37 °C is 2.0 to 2.5.
(2.3.1). A SSA Y
B. Solution S gives the reaction o f aluminium (2.3.1). Dissolve 0.400 g in 10 m L o f dilute hydrochloric add R and
TESTS dilute to 100.0 m L with water R. T o 10.0 m L o f the
S o lu tio n S solution, add 10.0 m L o f 0.1 M sodium edetate and 30 m L of
Dissolve 2.00 g in dilute hydrochloric acid R and dilute to a mixture o f equal volumes o f ammonium acetate solution R
100 m L with the same ad d . and dilute acetic add R Boil for 3 min, then cool. A dd 25 m L
o f ethanol (96 per cent) R and 1 m L o f a freshly prepared
0.25 g/L solution o f dithizone R in alcohol R. T itrate the
Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
excess of sodium edetate with 0.1 M zinc sulfate until the
p H (2.2.3) colour changes to pink.
5.5 to 7.2 1 m L o f 0.1 M sodium edetate is equivalent to 12.20 m g o f
Shake 4.0 g with carbon dioxide-free water R and dilute to AIPO 4.
STORA GE
PhEur
Dissolve 50.0 m g in 10 m L o f dilute nitric add R and dilute
to 200 m L with water R.
S o lu b le p h o sp h a te s
M axim um 1.0 per cent, calculated as PO 43-. ,* * * ★
★
Test solution Stir 5.0 g with 150 m L o f water R for 2 h. Filter Aluminium Phosphate Gel ★ ★
and wash the filter with 50 m L o f water R. Com bine the (Ph Eur monograph 2166) * * * * *
filtrate and the washings and dilute to 250.0 m L with
water R D ilute 10.0 m L o f this solution to 100.0 m L with A c tio n a n d u se
water R. A ntad d ; vacdne adjuvant
Reference solution (a) Dissolve 2.86 g o f potassium dihydrogen
PhEur-----------------------------------------------------------------------------------------
phosphate R in water R and dilute to 100 m L with th e same
solvent D E F IN IT IO N
Reference solution (b) Dilute 1 m L o f reference solution (a) to H ydrated A1P0 4 in g d form.
5 m L with water R. C o n te n t
Reference solution (c) Dilute 3 m L o f reference solution (a) to 19.0 per cent to 21.0 per cent o f AIPO 4.
5 m L with water R.
CHARACTERS
T reat each solution as follows. T o 5.0 m L add 4 m L o f dilute A p p e a ra n c e
sulfuric add R, 1 m L of ammonium mdybdate solution R, 5 m L Gel.
o f water R and 2 m L o f a solution containing 0.10 g of
S o lu b ility
4-methylaminophenol sulfate R, 0.5 g o f anhydrous sodium
Practically insoluble in water, in ethanol (96 per cent) and in
sulfite R and 20.0 g o f sodium metabisulfite R in 100 m L of
methylene chloride. It dissolves in dilute solutions o f mineral
water R Shake and allow to stand for 15 min. Dilute to
ad d s.
25.0 m L with water R and allow to stand for a further
2016 Aluminium Powder 1-121
ID E N T IF IC A T IO N Dissolve 4.0 g in dilute hydrochloric add R and dilute to

A. Solution S (see Tests) gives reaction (b) of phosphates 20 m L with the same ad d . 12 m L of the solution complies
(2.3.1). with test A. Prepare the reference solution using lead standard
B. Solution S gives the reaction of aluminium (2.3.1). solution (2 ppm Pb) R.
C. It complies with the assay. A cid n e u tra lisin g c a p acity
Add 2.0 g to 30 m L of 0.1 M hydrochloric acid heated to
TESTS 37 °C and maintain at 37 °C while shaking. Determine the
S o lu tio n S p H after 15 min. The p H (2.2.3) of the mixture is 2.0 to 2.5.
Dissolve 2.00 g in dilute hydrochloric acid R and dilute to
R e sid u e o n ig n itio n
100 m L with the same add.
19.0 per cent to 23.0 per c e n t
p H (2.2.3)
H eat 0.500 g at 50 °C for 5 hours, then ignite at
6.0 to 8.0.
500 ± 50 °C until constant mass.
. P e ro x id e s
M ic ro b ia l c o n tam in a tio n
M aximum 150 ppm , expressed as hydrogen peroxide.
TA M C: acceptance criterion 103 CFU/g (2.6.12).
Test solution Dissolve with heating 1,0 g of the substance to
TY M C: acceptance criterion 102 CFU/g (2.6.12).
be examined in 5 m L o f dilute hydrochloric acid R, then add
5 m L of water R and 2 m L of dmanadxum pentoxide solution in Absence o f bile-tolerant gram-negative bacteria (2.6.13).
sulfuric acid R. Absence of Escherichia coli (2.6.13).
Reference solution D ilute 1.0 m L of dilute hydrogen peroxide A SSAY
solution R to 200.0 m L with water R. To 1 m L of this Dissolve with heating 0.300 g in 5 m L of dilute hydrochloric
solution add 9 m L o f water R and 2 m L o f divanadium acid R. A dd 45 m L of water R, 10.0 mT, of 0.1 M sodium
pentoxide solution in sulfuric acid R. edetate and 30 m L of a mixture of equal volumes of
T he test solution is no t more intensely coloured than the ammonium acetate solution R and dilute acetic add R. H eat to
reference solution. boiling and maintain boiling for 3 min. Cool, then add
C h lo rid es (2.4.4) 25 m L o f ethanol (96 per cent) R. Titrate with 0.1 M zinc
M aximum 500 ppm . sulfate, determining the end-point potentiometrically (2.2.20).
Dissolve 1.3 g in 5 mT. o f dilute nitric acid R and dilute to 1 m L of 0.1 M zinc sulfate is equivalent to 12.2 m g of A1P04.
200 mT, w ith water R. STORAGE
Soluble p h o sp h a te s In an airtight container.
Maximum 0.5 per cent, expressed as P 0 4. _________________________________________________________ PhEur
Test solution Centrifuge 10.0 g until a clear supernatant is
obtained. T o 2.00 m L of the supernatant add 20.0 m L o f a
10.3 g/L solution of hydrochloric acid R and dilute to
100.0 m L with water R. T o 10.0 m L of this solution add
10.0 mT, o f nitro-mofybdovanadic reagent R and dilute to Aluminium Powder
50.0 m L with water R. Allow to stand protected from light A1 26.98 7429-90-5
for 15 min.
Reference solution Add 10.0 m L of nitro-mofybdovanadic A c tio n a n d u se
reagent R to 10.0 m L o f a 143 mg/L solution of potassium Topical protective.
dihydrogen phosphate R and dilute to 50.0 m L with water R. P re p a r a tio n
Allow to stand protected from light for 15 min. C om pound Aluminium Paste
Measure the absorbances (2.2.25) of the 2 solutions at
D E F IN IT IO N
400 nm . T h e absorbance of the test solution is not greater
than that o f the reference solution. Aluminium Powder consists mainly o f metallic aluminium in
the form o f very small flakes, usually with an appreciable
S u lfates (2.4.13) proportion of aluminium oxide; it is lubricated with stearic
Maximum 0.2 per cent. a d d to prevent oxidation. It contains not less than 86.0% of
Dilute 25 m L of solution S to 100 m L with distilled water R. Al, calculated with reference to the substance freed from
Soluble a lu m in iu m lubricant and volatile matter.
Maximum 50 ppm. C H A R A C T E R IS T IC S
T o 16.0 g add 50 m L o f water R. H eat to boiling for 5 min. A silvery grey powder.
Cool and centrifuge. Separate the supernatant W ash the Practically insoluble in water and in ethanol (96%).
residue with 20 m L of water R and centrifuge. Separate the It dissolves in dilute adds and in aqueous solutions of alkali
supernatant and add to the first supernatant. To the hydroxides, with the evolution of hydrogen.
combined supernatants add 5 m L of hydrochloric acid R and
20 m L o f water R. Introduce all o f this solution into a ID E N T IF IC A T IO N
500 m L conical flask and carry out the complexometric A solution in 2m hydrochloric add yields the reaction
titration o f aluminium (2.5.11) using 0.01 M sodium edetate. characteristic of aluminium salts, Appendix VI.
A rsen ic (2.4.2, Method A) TESTS
M aximum 1 ppm , determined on 1.0 g. S u rfa c e -c o v e rin g po w er
H eavy m e ta ls (2.4.8) N o t less than 4000 cm2 per g w hen determined by the
M aximum 10 ppm. following method. Fill with water a shallow trough measuring
approximately 60 cm x 12 cm x 1.5 cm, fitted with a
movable partition so constructed that it is a sliding fit and
1-122 Aluminium Sodium Silicate 2016
can be used to divide th e trough into two rectangular areas. A SSAY

Place the movable partition n ear one end and sprinkle 50 mg Transfer 0 .2 g, previously freed from lubricant by successive
o f the substance being examined on the surface o f the liquid washing with acetone and drying, to a three-necked 5 0 0 m L
confined in the smaller area. U sing a glass rod, spread the flask fitted with a 150 m L dropping funnel, an inlet tube
pow der evenly over the liquid surface until an unbroken film connected to a cylinder o f carbon dioxide and an outlet tube
covers die entire surface. M ove the partition so as to increase dipping into a water trap. Add 6 0 m L of water and disperse
the area confined and again spread the powder to cover the the substance being examined; replace the air by carbon
increased surface. Continue this process and d eterm in e the dioxide and add 100 m L o f a solution containing 56 g of
maximum unbroken surface area obtained. T h e surface- ammonium iron(m) sulfate and 7 .5 m L o f sulfuric add in water.
covering pow er is the area covered p er g of the pow der at the While m aintain in g an atm osphere o f carbon dioxide in the
breaking point of the film. flask, heat to boiling, boil for 5 minutes after the sample lias
Ir o n dissolved, cool rapidly to 2 0 ° and dilute to 2 5 0 m L with
Dissolve 10 m g in 20 m L of 2m hydrochloric acid and dilute to water. T o 5 0 mT. add 15 m L of orthophosphoric add and
100 m L with water. 10 m L o f the resulting solution complies titrate with 0 .0 2 m potassium permanganate KS. Each m L o f
with the limit test for iron, Appendix VII (1.0%). 0 .0 2 m potassium permanganate KS is equivalent to 0 .8 9 9 4 mg
o f Al.
Lead
Use two solutions prepared in the following manner.
For solution (1 ) boil 0 .4 0 g with 2 0 mT. of 2m hydrochloric
add and 10 m L of water until effervescence ceases, add
0 .5 m L o f nitric add, boil for 3 0 seconds and cool; add 2 g o f Aluminium Sodium Silicate *****
ammonium chloride and 2 g o f ammonium tfnocyanate, extract *+ +*
with three 10 m L quantities o f a m ix ture of equal volumes of (Ph Eur monograph 1676) *
amyl alcohol and ether, discard the extracts and add 2 g of PhEur___________________________________________________________
citric add. F o r solution (2) dissolve 2 g o f citric add in 10 m L
D E F IN IT IO N
of 2 m hydrochloric add and add 4 m L o f lead standard solution
(10 ppm Pb). Make solutions (1 ) and (2 ) alkaline with Silicic a d d aluminium sodium salt o f synthetic origin.
5 m ammonia and to each add 1 m L o f potassium cyanide solution C o n te n t
PbT. T he solutions should be not m ore than faintly — aluminium (Al; 26.98): 2.7 per cent to 7.9 per cent (dried
opalescent. If the colours o f the solutions differ, equalise by substance);
the addition o f about 0 .2 m L o f a highly diluted solution of — sodium (Na; 22.99): 3.7 per cent to 6.3 p er cent (dried
bu rn t sugar o r other non-reactive substance. Dilute each substance).
solution to 5 0 m L with water, add 0.1 m L of a 10% w/v CHARACTERS
solution o f sodium sulfide to each and mix thoroughly.
A p p e a ra n c e
T he colour produced in solution (1) is not more intense than
W hite o r alm ost white, fine, light, am orphous powder.
that produced in solution (2), when viewed against a white
background (100 ppm ). S o lu b ility
Practically insoluble in w ater and in organic solvents.
O th e r m e ta ls
Dissolve 2 g in 4 0 m L of 2 m hydrochloric add. D ilute 2 0 m L ID E N T IF IC A T IO N
o f the solution to 100 m L with water, make alkaline to litmus A. T ransfer 1.0 g to a 100 m L beaker and add 10 m L o f
paper by the addition o f 5 m ammonia, boil and filter. dilute hydrochloric acid R. Mix, cover with a w atch glass and
Evaporate the filtrate to dryness, add 0.05 m L o f sulfuric acid boil for 15 m in. Allow to cool to room tem perature, mix and
and ignite. T h e residue weighs not m ore than 2 mg. centrifuge the solution. 2 m L of the supernatant gives the
reaction o f aluminium {2.3.1).
L u b ric a n t
T o 2 g add 100 m L o f hot water, cover and add, drop wise, B. 2 m L o f the supernatant obtained in identification test A
sufficient o f a mixture o f equal volumes o f hydrochloric add gives reaction (a) of sodium {2.3.1).
and water to dissolve the metal alm ost completely. H eat to C. 0.2 g gives the reaction o f silicates {2.3.1).
complete dissolution, cool, filter through a hardened filter
TESTS
paper and wash the vessel and filter paper thoroughly with
p H {2.2.3)
water, dry b o th the vessel and paper at room tem perature.
9.5 to 11.5.
Extract the paper with three 100-mL quantities o f boiling,
freshly distilled acetone, using the original vessel to contain Disperse 5.0 g in 100 m L o f carbon dioxide-free water R.
the solvent and then wash th e paper with five 10-mL A rse n ic {2.4.2, Method A)
quantities o f freshly distilled acetone. Evaporate the combined M axim um 3 ppm.
filtrate and washings to dryness u sin g a rotary evaporator. Transfer 8.3 g to a 250 m L beaker containing 50 m L o f
T h e residue, after drying at 105° for 30 m inute s and allowing dilute hydrochloric acid R. M ix, cover with a watch glass and
to cool, weighs 10 to 60 mg. boil gently, with occasional stirring, for 15 min. Centrifuge,
W hen die basin containing the residue is floated in a beaker and decant the supernatant through a rapid-flow filter paper
of water suitably stirred and heated, the residue melts into a 250 m L volumetric flask. T o the residue in the beaker,
between 4 0 ° and 6 0 °. T h e residue is alm ost completely add 25 m L o f hot dilute hydrochloric acid R, stir, centrifuge,
soluble, w ith effervescence, in hot dilute sodium carbonate and decant the supernatant through the sam e filter into the
sdudon. volumetric flask. Repeat the extraction w ith 3 additional
V olatile m a t te r quantities, each o f 25 m L, o f ho t dilute hydrochloric add R,
filtering each supernatant through this filter into the
W hen heated to constant weight at 105°, loses no t m ore than
0.5% of its weight. U se 1 g. volumetric flask. Allow the combined filtrates to cool to room
tem perature and dilute to 250.0 m L with dilute hydrochloric
2016 Aluminium Stearate 1-123
add R. D ilute 10.0 m L of the solution to 25.0 m L with 1.0 m L of lanthanum chloride solution R and dilute to
water R. 50.0 m L with water R.
Lead Reference solutions Into 5 separate 50 m L volumetric flasks,
M axim um 5 ppm . introduce respectively 1.0 mL, 2.5 m l , 5.0 ml-, 7.5 m L and
10.0 m L o f aluminium standard solution (100 ppm Al) R, add
1 m L of lanthanum chloride solution R and 10 m L o f the blank
Test solution T ransfer 5.0 g to a 250 m L beaker containing solution, and dilute to 50.0 m L with water R.
50 m L o f dilute hydrochloric add R Mix, cover with a watch
glass an d boil for 15 min. Allow to cool to room Source Aluminium hollow-cathode lamp.
temperature. Centrifuge, and decant the supernatant through Wavelength 309.3 nm .
a rapid-flow filter paper into a 250 m L beaker. T o the Atomisation device Acetylene-nitrous oxide flame.
insoluble m atter add 25 m L of hot water R. Stir vigorously, Sodium
centrifuge, and decant the supernatant through the same Atomic emission spectrometry (2.2.22, Method i).
filter into the beaker. Repeat the extraction with 2 additional
Test solution T o 2.0 m L o f solution A, prepared in the assay
quantities, each o f 25 mL, o f hot water R, decanting each
o f aluminium, add 1 m L of a 12.5 g/L solution o f caesium
supernatant through the filter into the beaker. W ash the filter
chloride R and dilute to 20.0 m L with water R.
with 25 m L o f h o t water R, collecting die filtrate in the
beaker. C oncentrate the combined filtrates by gently boiling Reference solutions Into 5 separate 200 m L volumetric flasks,
to about 15 mT. Add about 0.05 m L o f heavy metal-free nitric each containing 10 m L o f a 12.5 g/L solution of caesium
add R, heat to boiling and allow to cool to room chloride R, introduce respectively 1.0 m l, 2.0 m l , 4.0 m l,
temperature. Filter the concentrated extracts through a rapid- 6.0 m L and 10.0 m L o f sodium standard solution
flow filter paper into a 25 m L volumetric flask. Transfer the (200 ppm Na) R and dilute to 200.0 m L with water R.
rem aining contents o f the beaker through the filter paper and Wavelettgth 589.0 nm .
into die volum etric flask with water R and dilute to 25.0 m L __________________________________________________________ PhEur
with the sam e solvent.
Reference solutions Into 4 separate 100 m L volumetric flasks,
introduce respectively 3.0 mL, 5.0 mL, 10.0 m L and
15.0 m L o f lead standard solution (10 ppm Pb) R, add
0.20 mT. o f heavy metal-free nitric add R and dilute to Aluminium Stearate *****
100.0 m L w ith water R.
Source L ead hollow-cathode lamp.
PhEur__________________________________________________________
D E F IN IT IO N
Aluminium salts o f a mixture of solid organic a d d s consisting
L oss o n d ry in g (2.2.32) mainly o f variable proportions o f aluminium stearate and
M axim um 8.0 per cent, determ ined on 1.000 g by drying in aluminium palmitate. T h e organic adds are obtained from
an oven at 105 °C for 4 h. sources o f vegetable o r animal origin.
L oss o n ig n itio n C o n te n t
5.0 per cent to 11.0 per cent (dried substance), determ ined — aluminium (Al; A t 26.98): 3.0 per cent to 9.0 per cent
on 1.000 g by ignition in a platinum crucible to constant (dried substance);
mass a t 1000 ± 25 °C. — stearic add in the fatty add fraction: m inim um
M ic ro b ia l c o n ta m in a tio n 40.0 per cent;
T A M C : acceptance criterion 103 CFU /g (2.6.12). — sum of stearic acid and palmitic add in the fatty add fraction:
T Y M C : acceptance criterion 102 C FU /g (2.6.12). m inim um 90.0 per cent.
Absence o f Escherichia coli (2.6.13). CHARACTERS

ASSAY A p p e a ra n c e
Alum inium W hite or almost white, very fine, light powder.
Atomic absorption spectrometry (2.2.23, Method I). S olubility
A dd mixture A dd 50 m L o f nitric add R to 500 m L of Practically insoluble in water and in anhydrous ethanol.
water R. Dissolve in this solution 17 g of tartaric add R and ID E N T IF IC A T IO N
dilute to 1000 m L with water R First identification C, D
Blank solution Dissolve 1.4 g o f anhydrous lithium metaborate R Second identification A , B, D
in 60 m l. o f th e acid mixture and dilute to 200 m L with
A. Freezing point (2.2.18): minimum 53 °C, determined on
water R. the residue obtained in the preparation o f solution S
Test solution In a platinum crucible mix 0.200 g with 1.4 g o f (see Tests).
anhydrous lithium metaborate R. H eat slowly at first and ignite B. A d d value (2.5.1): 195 to 210.
at 1100 ± 25 °C for 15 m in. Cool, then place the crucible
Dissolve 0.200 g o f the residue obtained in the preparation of
in a 100 m L beaker containing 60 m L o f the a d d mixture.
solution S in 25 m L o f the prescribed mixture o f solvents.
Place a polytetrafluoroethylene-coated magnetic stirring bar
in the crucible and stir gently with a magnetic stirrer for C. Examine the chromatograms obtained in the assay of
16 h. T ran sfer the contents o f the crucible into a 200 m L stearic a d d and palmitic add.
volum etric flask. W ash the crucible, the magnetic stirring b ar Results T he 2 principal peaks in the chromatogram obtained
and the beaker with water R and dilute to 200.0 m l. with the with the test solution are similar in retention time to the
same solvent (solution A). T o 10.0 m L o f this solution, add 2 prindpal peaks in the chromatogram obtained with the
reference solution.
1-124 Aluminium Stearate 2016
D. 1 m L o f solution S gives the reaction of aluminium Reference solution Prepare a solution containing
(2.3.1). T he addition of 0.5 m L o f dilute hydrochloric add R 0.00165 ng/mL o f cadmium nitrate tetrahydrate R in the blank
described in the general m ethod is omitted. solution (equivalent to 0.006 pg/m L o f Cd).
TESTS D ilute 1.0 m L o f the test solution to 10.0 m L with the blank
S o lu tio n S solution. Prepare mixtures of this solution, the reference
T o 5.0 g add 50 m L o f peroxide-free ether R, 20 m L o f dilute solution and the blank solution in the following proportions:
nitric add R and 20 m L of distilled water R and heat gently (1.0:0:1.0 V/V/V), (1.0:0.25:0.75 V/V/V),
under a reflux condenser until dissolution is complete. Allow (1.0:0.5:0.5 V/V/V), (1.0:0.75:0.25 V/V/V). T o each mixture
to cool. In a separating funnel, separate the aqueous layer add 50 )iL o f the modifier solution and mix. These solutions
and shake the ether layer with 2 quantities, each o f 4 mT, o f contain respectively 0 jxg, 0.0015 jig, 0.0030 ng and
distilled water R. Com bine the aqueous layers, wash with 0.0045 ng o f cadmium per millilitre from the reference
15 m L of peroxide-free ether R and dilute to 50.0 m L with solution. K eep the remaining test solution for use in the test
distilled water R (solution S). Evaporate the ether layer to for lead and nickel.
dryness and dry the residue at 100-105 °C. Keep the residue Source Cadm ium hollow-cathode lamp.
for identification tests A and B. Wavelength 228.8 nm .
A cid ity o r alk a lin ity Atomisation device Furnace.
T o 1.0 g add 20 m L o f carbon dioxide-free water R and boil Platform Pyrolytically coated with integrated tube.
for 1 min with continuous sh ak ing. Cool and filter.
Operating conditions U se the tem perature programme
T o 10 m L o f the filtrate add 0.05 m L of bromothymol blue
recom m ended for cadmium by the GFAA manufacturer.
solution R4. N o t m ore than 0.05 m L o f 0.1 M hydrochloric A n example o f tem perature parameters for GFAA analysis of
add or 0.1 M sodium hydroxide is required to change the
cadm ium is shown below.
colour of the indicator.
C h lo rid es (2.4.4)
Stage Final temperature Ramp time Hold time
CC) (s) (s)
Dilute 0.5 m L o f solution S to 15 m L with water R. Drying 110 10 20
S u lfates (2.4.13)
Ashing 600 10 30
Atomisation 1800 0 5
Dilute 0.3 m L o f solution S to 15 m L with distilled water R.
C a d m iu m
M axim um 3 ppm . L ead
Atomic absorption spectrometry (2.2.23, Method II). M axim um 10 ppm .
For the preparation of all aqueous solutions and for the rinsing of Atomic absorption spectrometry (2.2.23, MethodII).
glassware before use, employ water that has been passed through a For the preparation of aU aqueous solutions and for the rinsing of
strong-acid, strong-base, mixed-bed ion-exchange resin before use. glassware before use, employ water that has been passed through a
Select all reagents to have as low a content of cadmium, lead and strong-acid, strong-base, mixed-bed ion-exchange resin before use.
nickel as practicable and store all reagent solutions in containers of Select all reagents to have as low a content of cadmium, lead and
borosUicate glass. Clean glassware before use by soaking in warm nickel as practicable and store aU reagent solutions in containers of
8 M nitric acid for 30 min and by rinsing with deionised water. borosUicate glass. Clean glassware before use by soaking in warm
Blank solution D ilute 25 m L o f cadmium- and lead-free nitric 8 M nitric add for 30 min and by rinsing with deionised water.
acid R to 100.0 m L w ith water R. Blank sdution U se the solution described in the test for
Modifier solution Dissolve 20 g o f ammonium dihydrogen cadmium.
phosphate R and 1 g o f magnesium nitrate R in water R and Modifier solution U se the solution described in the test for
dilute to 100 m L with the same solvent. Alternatively, use an cadmium.
appropriate matrix modifier as recommended by the graphite Test solution U se the solution described in the test for
furnace atom ic absorption (GFAA) spectrometer cadmium.
manufacturer.
Reference sdution Prepare a solution of 0.100 |ig/mL of Pb by
Test solution Place 0.100 g of the substance to be examined in suitable dilutions o f lead standard solution (100 ppm Pb) R
a polytetrafluoroethylene digestion bom b and add 2.5 m L o f with the blank solution.
cadmium- and lead-free nitric acid R. Close and seal th e bom b
Prepare mixtures o f the test solution, the reference solution
according to the m anufacturer's operating instructions. When
and the blank solution in the following proportions:
using a digestion bomb, be thoroughly familiar with the safety and
(1.0:0:1.0 V/V/V), (1.0:0.5:0.5 V/V/V), (1.0:1.0:0 V/V/V).
operating instructions. Carefully follow the bomb manufacturer's
T o each mixture add 50 pL o f the modifier solution and mix.
instructions regarding care and maintenance of these digestion
These solutions contain respectively 0 |ig5 0.025 |ig and
bombs. Do not use metal-jacketed bombs or liners that have been
0.05 jig o f lead p er millilitre from the reference solution.
used with hydrochloric acid due to contamination from corrosion of
the metal jacket by hydrochloric add. H eat the bom b in an oven Source L ead hollow-cathode lamp.
at 170 °C for 3 h. Cool the bom b slowly in air to room Wavelength 283.3 nm .
tem perature accord in g to the bom b m anufacturer's Atomisation device Furnace.
instructions. Place the bom b in a fume cupboard and open Platform Pyrolytically coated with integrated tube.
carefully as corrosive gases may be expelled. Dissolve die
Operating conditions Use the tem perature programme
residue in water R and dilute to 10.0 m L with the same
recom m ended for lead by the GFAA manufacturer.
solvent.
A n example of tem perature parameters for GFAA analysis of
lead is shown below.
2016 Aluminium Stearate 1-125
Stage Final temperature Ramp time Hold time solution for 30 m in under reflux on a w ater-bath, swirling
_________________CQ___________ &__________ isi__ frequently. Allow to cool. Add 100 m L o f water R and adjust
Drying 110 10 20 to about p H 1 by adding approximately 12 m L of dilute
Ashing 450 10 30 sodium hydroxide solution R. Add 20.0 m L o f 0.1 M sodium
edetate and adjust to between pH 5 and p H 6 by the addition
Atomisation 2000 0 5
of sodium acetate R. Add 70 mg of xylenol orange triturate R
and titrate immediately and quickly with 0.1 M zinc sulfate
until the colour changes from yellow to pinkish-violet.
Nickel
M axim um 5 ppm . 1 m L of 0.1 M sodium edetate is equivalent to 2.698 mg of
Atomic absorption spectrometry (2.2.23, Method II). Al.
For the preparation of all aqueous solutions and for the rinsing of S te a ric a c id a n d p a lm itic acid
glassware before me, employ water that has been passed through a Gas chromatography (2.2.28): use the normalisation
strong-acid, strong-base, mixed-bed ion-exchange resin before use. procedure.
Select al1 reagents to have as low a content of cadmium, lead and Test solution In a conical flask fitted with a reflux condenser,
nickel as practicable and store al1 reagent solutions in containers of dissolve 0.100 g o f the substance to be examined in 5 m L o f
borosUicate glass. Clean glassware before use by soaking in warm boron trifliioride-methanol solution R. Boil under a reflux
8 M nitric acid for 30 mm and by rinsing with deionised water. condenser for 10 min. Add 4 m L of heptane R through the
Blank solution U se the solution described in the test for condenser and boil again under a reflux condenser for
cadmium. 10 min. Allow to cool. A dd 20 m L of saturated sodium
chloride solution R. Shake and allow the layers to separate.
Modifier solution Dissolve 20 g of ammonium dihydrogen
D ry the organic layer over 0.1 g of anhydrous sodium sulfate R
phosphate R in water R and dilute to 100 m L with the same
previously washed with heptane R. D ilute 1.0 m L of the
solvent. Alternatively, use an appropriate matrix modifier as
solution to 10.0 m L with heptane R.
recom m ended by the GFAA spectrometer manufacturer.
Reference solution Prepare the reference solution in the same
Test solution U se the solution described in the test for
m anner as the test solution using 50.0 m g of palmitic
cadmium.
acid CRS and 50.0 mg of stearic acid CRS instead o f the
Reference solution Prepare a solution of 0.050 ng/mL of N i by substance to be examined.
suitable dilutions o f a 0.2477 |ig/mL solution of nickel nitrate
Column:
hexahydrate R with the blank solution.
— material: fused silica;
Prepare mixtures o f the test solution, the reference solution — size. I = 30 m , 0 = 0.32 mm;
and the blank solution in the following proportions: — stationary phase: macrogol 20 000 R (film thickness
(1.0:0:1.0 V/V/V), (1.0:0.5:0.5 VIVIV), (1.0:1.0:0 VIVIV). 0.5 tun).
T o each mixture add 50 (iL of the modifier solution and mix.
T hese solutions contain respectively 0 ng, 0.0125 fog and
0.025 ng o f nickel per millilitre from the reference solution. Flow rate 2.4 ml 7min.
Source Nickel hollow-cathode lamp. Temperature:
Time Temperature.
Atomisation device Furnace.
(min) rc)
Platform Pyrolytically coated with integrated tube. Column 0 -2 70
Operating conditions Use the temperature programme
2 -3 6 70 240
recom m ended for nickel by the GFAA manufacturer.
A n example o f tem perature parameters for GFAA analysis of 36-41 240
nickel is shown below. Injection port 220
Detector 260
Stage Final temperature Ramp time Hold time
CC) (») (s)
Drying 110 10 20 Detection Flam e ionisation.
Ashing 1000 20 30 Injection 1 (iL.
Atomisation 2300 0 5 Relative retention W ith reference to methyl stearate: methyl
palmitate = about 0.9.
System suitability: reference solution:
L oss o n d ry in g ( 2.2.32) — resolution: minimum 5.0 between the peaks due to methyl
M axim um 6.0 per cent, determined on 1.000 g by drying in palmitate and methyl stearate;
an oven at 105 °C. — repeatability: maximum relative standard deviation of
M ic ro b ia l c o n ta m in a tio n 3.0 per cent for the areas of the peaks due to methyl
T A M C : acceptance criterion 103 C FU/g (2.6.12). palmitate and methyl stearate after 6 injections; maximum
relative standard deviation of 1.0 per cent for the ratio of
T Y M C : acceptance criterion 102 CFU/g (2.6.12).
the areas of the peaks due to methyl palm itate to the
Absence of Escherichia colt (2.6.13). areas of the peaks due to methyl stearate after
Absence of Salmonella (2.6.13). 6 injections.
A SSA Y __________________________________________________________ PhEur
A lu m in iu m
T o 0.250 g in a 250 m L conical flask add 20 m L of
methanol R and, slowly, 2 m L o f sulfuric acid R. H eat the
1-126 Aluminium Sulfate 2016
★ ★ STO RA G E
Aluminium Sulfate ★ ★ In an airtight container.
Aluminium Sulphate *****
PhEur
A12(S04)3îxH20 342.1
★ ★
Alverine Citrate ★ ★
P re p a r a tio n *****
Aluminium Acetate E ar D rops
Ph E n _______________________________________ CO j H
ch3
D E F IN IT IO N CO2H
C o n te n t OH
51.0 per cent to 59.0 per cent of A12(S 04 )3. COjH
It contains a variable quantity of water of crystallisation.
CHARACTERS C 26H 35NO 7 473.6 5560-59-8
A p p e a ra n c e
Colourless, lustrous crystals or crystalline masses.
Sm ooth muscle relaxant; antispasmodic.
S o lu b ility
P re p a r a tio n
Soluble in cold water, freely soluble in hot water, practically
Alverine Capsules
insoluble in ethanol (96 per cent).
ID E N T IF IC A T IO N PhEur_____________________________________
A. Solution S (see Tests) gives reaction (a) o f sulfates (2.3.1). D E F IN IT IO N

B. Solution S gives the reaction of alu m in iu m (2.3.1). N-Ethyl-3-phenyl-N-(3-phenylpropyl)propan-1-amine
TESTS dihydrogen 2-hydroxypropane-l,2,3-tricarboxylate.
S o lu tio n S C o n te n t
Dissolve 2.5 g in water R and dilute to 50 m L with the same 99.0 per cent to 101.0 per cent (dried substance).
solvent. CHARACTERS
A p p e a ra n c e o f so lu tio n A p p e a ra n c e
Solution S is not m ore opalescent than reference W hite or almost white, crystalline powder.
suspension IU (2.2.1) and is colourless (2.2.2, Method II). S o lu b ility
p H (2.2.3) Slightly soluble in water and in methylene chloride, sparingly
2.5 to 4.0. soluble in ethanol (96 per cent).
Dissolve 0.5 g in carbon dioxide-free water R and dilute to mp
25 m L with the same solvent. A bout 104 °C.
A lk ali a n d a lk a lin e -e a rth m e ta ls ID E N T IF IC A T IO N
M axim um 0.4 per c e n t Infrared absorption spectrophotometry (2.2.24).
T o 20 m L of solution S add 100 m L of water R, heat and Comparison alverine citrate CRS.
add 0.1 m L of methyl red solution R. A dd dilute ammonia R l
until the colour o f the indicator changes to yellow. Dilute to TESTS
150 m L with water R, heat to boiling and filter. Evaporate p H (2.2.3)
75 m L of the filtrate to dryness on a water-bath and ignite. 3.5 to 4.5.
T he residue weighs a maximum o f 2 mg. Dissolve 0.250 g in carbon dioxide-free water R and dilute to
A m m onium (2.4.1) 50.0 m L with the same solvent.
M aximum 500 ppm. R e la te d su b sta n c e s
D ilute 0.4 m L of solution S to 14 m L with water R. G as chromatography (2.2.28): use the normalisation
procedure. Use freshly prepared solutions.
Ir o n (2.4.9)
M axim um 100 ppm. Test solution Dissolve 0.250 g of die substance to be
examined in water R and dilute to 20 m L w ith the same
D ilute 2 m L o f solution S to 10 m L with water R.
solvent A dd 2 m L of concentrated ammonia R and shake with
U se 0.3 m L o f thiogfycoUic add R in this test.
3 quantities, each of 15 m L, o f methylene chloride R. T o the
H eav y m e ta ls (2.4.8) com bined lower layers add anhydrous sodium sulfate R, shake,
M axim um 50 ppm . filter, and evaporate the filtrate at a tem perature not
D ilute 8 m L o f solution S to 20 m L with water R. 12 m L o f exceeding 30 °C, using a rotary evaporator. T ake up the
the solution complies with test A Prepare the reference residue with methylene chloride R and dilute to 10.0 m L with
solution using lead standard solution (1 ppm Pb) R. the same solvent.
A SSA Y Reference solution (a) Dissolve 5 m g o f alverine
Dissolve 0.500 g in 20 m L o f water R. Carry out the impurity D CRS (impurity D citrate) in 5 m L o f water R, add
complexometric titration o f aluminium (2.5.11). 1 m L o f concentrated ammonia R and shake with 3 quantities,
each of 5 m L, o f methylene chloride R. T o the combined lower
1 m L o f 0.1 M sodium edetate is equivalent to 17.11 mg
layers add anhydrous sodium sulfate R, shake, filter, and
ofAl2(S04)3. evaporate the filtrate at a tem perature no t exceeding 30 °C,
2016 Amantadine Hydrochloride 1-127
using a rotary evaporator. T ake up the residue with methylene ASSAY

chloride R, add 0.2 m L of the test solution and dilute to Dissolve 0.375 g in 50 m L of anhydrous acetic acid R. T itrate
2 m L with methylene chloride R. w ith 0.1 M perchloric acid, determ in in g the end-point
Reference solution (b) Dilute 1.0 m L o f the test solution to potentiometrically (2.2.20).
100.0 m l, with methylene chloride R. Dilute 1.0 m L of this 1 m L o f 0.1 M perchloric add is equivalent to 47.36 mg
solution to 20.0 m L with methylene chloride R. o f C 26H 35N O 7.
Reference solution (c) Dissolve the contents o f a vial of alverine STORAGE
for peak identification CRS (containing impurities C and E) in Protected from light.
1 m l, of methylene chloride R.
IMPURITIES
Column:
Specified impurities: A, B, C, D , E.
— sizer. I = 25 m , 0 = 0.32 mm;
— stationary phase: poly(dimethyl) (diphenyl) sUoxane R (film
thickness 0.45 (im).
Flow rate 2.2 mL/min. A. R = Cl: 1-chloro-3-phenylpropane,
Split ratio 1:11. B. R = O H : 3-phenylpropan-l-ol,
Temperature: C. R = N H -C 2H 5: N-ethyl-3-phenylpropan-1 -amine.
Time Temperature
(min) CC)
Column 0 -7 120
7 -1 3 120 -» 240 D . iV-(3-cyclohexylpropyl)-N-ethyl-3-phenylpropan-l-amine,

13-21 240
21-24 240 -» 290
24-39 290
Injection port 290
Detector 290
E. 3-phenyl-AT^ST-bis(3-phenylpropyl)propan-l-amine.
Detection Flame ionisation.
PtiEur
Irtjection 1 pL.
with alverine for peak identification CRS and the
the peaks due to impurities C and E. Amantadine Hydrochloride ***\
Relative retention W ith reference to alverine (retention
time = about 16 m in): impurity A = about 0.28; (Ph Em monograph 0463) *
impurity D and alverine.
C 10H 18CIN 187.7 665-66-7
Limits:
— impurities A , B: for each impurity, maximum 0.1 per cent; Action and use
— impurity C: maxim um 0.2 per cent; Viral replication inhibitor (influenza A); dopamine receptor
— impurities D, E: for each impurity, maximum 0.3 per cent; agonist; treatm ent of influenza and Parkinson’s disease.
0.10 per cent; Preparations
Amantadine Capsules
— disregard limit, the area o f the principal peak in the A m antadine Oral Solution
PhEir _______________________ :___________________________________
(0.05 per cent).
DEFINITION
Maximum 20 ppm . Tricyclo[3.3.1. l 3,7]decan-l-am ine hydrochloride.
0.5 g complies with test G. Prepare the reference solution Content

using 1 m L of lead standard solution (10 ppm Pb) R. 98.5 per cent to 101.0 per cent (anhydrous substance).
L oss o n d ry in g (2.2.32) CHARACTERS

Maximum 0.5 per cent, determined on 1.000 g by drying in Appearance
an oven at 80 °C for 2 h. W hite or almost white, crystalline powder.
S u lfa te d a sh (2.4.14) .Solubility
M axim um 0.1 per cent, determined on 1.0 g. Freely soluble in water and in ethanol (96 per cent).
1-128 Amantadine Hydrochloride 2016
It sublimes on hearing. Temperature:

IDENTIFICATION
First identification A, D. Time Temperature
(min) CC)
Column 0 -5 70
5 -2 3 70 ->250
Comparison amantadine hydrochloride CRS.
2 3 -4 0 250
B. T o 0.1 g add 1 m L o f pyridine R, mbc and add 0.1 m l. of
acetic anhydride R. H e a t to boiling for about 10 s. Pour the Injection port 220
hot solution into 10 m L o f dilute hydrochloric acid R, cool to Detector 300
5 °C and filter. T he precipitate, washed with water R and
dried m vacuo at 60 °C for 1 h, melts (2.2.14) at 147 °C to
151 °C. Detection Flam e ionisation.
C. Dissolve 0.2 g in 1 m L o f 0.1 M hydrochloric acid. Injection 1 [iL.
Add 1 m L o f a 500 g/L solution of sodium nitrite R A white Relative retention W ith reference to am antadine (retention
precipitate is formed. tim e = about 14 min): internal standard = about 0.8.
D . 1 m L of solution S (see Tests) gives reaction (a) of System suitability: reference solution:
chlorides (2.3.1). — resolution: minimum 5.0 between the peaks due to the
TESTS internal standard and amantadine.
S o lu tio n S Limits:
Dissolve 2.5 g in carbon dioxide-free water R and dilute to — unspecified impurities: calculate the ratio (R{) of the area of
25 m L with the same solvent. the peak due to amantadine to the area of the peak due
to the internal standard from the chrom atogram obtained
Appearance o f solution
with the reference solution; from the chromatogram
obtained with the test solution, calculate the ratio of the
than reference solution Y7 (2.2.2, Method II).
area o f any peak, apart from the principal peak and the
Acidity or alkalinity peak due to the internal standard, to the area of the peak
D ilute 2 m L of solution S to 10 m L with carbon dioxide-free due to the internal standard: this ratio is n o t greater than
water R. Add 0.1 m L o f methyl red solution R and 0.2 m l. of Ri (0.10 per cent);
0.01 M sodium hydroxide. T h e solution is yellow. Add 0.4 m L — total: calculate the ratio (R^) of 3 times the area of the
of 0.01 M hydrochloric add. T h e solution is red. peak due to amantadine to the area of the peak due to
Related substances the internal standard from the chrom atogram obtained
Gas chromatography (2.2.28). with the reference solution; from the chromatogram
Internal standard solution Dissolve 0.500 g o f adamantane R in obtained with the test solution, calculate the ratio of the
methylene chloride R and dilute to 10.0 m L with the same sum of the areas of any peaks, apart from the principal
solvent. peak and the peak due to the internal standard, to the
Test solution Weigh 0.5 g of the substance to be examined area o f the peak due to the internal standard: this ratio is
into a centrifuge tube. Add 9 m L of methylene chloride R and n o t greater than R2 (0.3 per cent);
— disregard limit calculate the ratio (R3) o f 0.5 times the
10 m L of a 210 g/L solution o f sodium hydroxide R. Shake for
area of the peak due to amantadine to the area of the
10 min. Discard the upper layer. Dry the lower layer over
peak due to the internal standard from the chromatogram
anhydrous sodium sulfate R. Filter and collect the filtrate in a
volu m etric flask. Add 0.1 m L o f the internal standard obtained with the reference solution; from the
chromatogram obtained with the test solution, calculate
solution and dilute to 10.0 m L with methylene chloride R.
the ratio o f the area of any peak, apart from the principal
Reference solution Weigh 5 m g o f amantadine peak and the peak due to the internal standard, to the
hydrochloride CRS into a centrifuge tube. Add 9 m L of area of the peak due to the internal standard: disregard
methylene chloride R and 10 m L of a 210 g/L solution o f any peak with a ratio less than R3 (0.05 p er cent).
sodium hydroxide R. Shake for 10 m in. Discard the upper
layer. Dry the lower layer over anhydrous sodium sulfate R. Heavy metals (2.4.8)
Filter and collect the filtrate in a volumetric flask. M axim um 20 ppm.
A dd 1.0 m L o f the internal standard solution and dilute to 12 m L o f solution S complies with test A Prepare the
100.0 m L with methylene chloride R. reference solution using lead standard solution (2 ppm Pb) R.
Column: Water (2.5.12)
— material: fused silica; M aximum 0.5 per cent, determ ined on 2.00 g.
— size. I = 30 m , 0 = 0.53 mm; Sulfated ash (2.4.14)
— stationary phase, base-deactivated M aximum 0.1 per cent, determ ined on 1.0 g.
pdy (dimethyl) (diphenyl)süoxane R (film thickness 1 Jim).
A SSA Y
Dissolve 0.150 g in a mixture o f 5.0 m L of 0.01 M
Flow rate 4 mL/min.
hydrochloric acid and 50 m L o f ethanol (96 per cent) R. Carry
Split ratio 1:50. out a potentiometric titration (2.2.20), vising 0.1 M sodium
hydroxide. Read the volume added betw een the 2 points of
inflexion.
1 m l. of 0.1 M sodium hydroxide is equivalent to 18.77 mg of
C 10H 18C1N.
2016 Ambroxol Hydrochloride 1-129
IMPURITIES B. Infrared absorption spectrophotom etry (2.2.24).

Other detectable impurities (the following substances would, if Comparison ambroxol hydrochloride CRS.
present at a sufficient level, be detected by one or other of C. Thin-layer chromatography (2.2.27).
by the general monograph Substances for pharmaceutical use
(2034). It is therefore n o t necessary to identify these Reference solution Dissolve 50 mg o f ambroxol
impurities for demonstration of compliance. See also 5.10. hydrochloride CRS in methanol R and dilute to 5 m L with the
Control of impurities in substances for pharmaceutical use): A , B. same solvent.
Hate TLC silica gel F 254 plate R.
a Mobile phase concentrated ammonia R, propanol R, ethyl
acetate R, hexane R (1:10:20:70 V/V/V/V).
Application 10 jjL.
A. l-chlorotricydo[3.3.1.13,7]decane, Development Over 2/3 of the plate.
Drying In air.
H Detection Examine in ultraviolet light at 254 nm .
N ^ /C H 3
T Results T h e prindpal spot in the chrom atogram obtained with
o the test solution is similar in position and size to the principal
B. N-(tricyclo[3.3.1 . l 3,7]dec-l-yl)acetamide. solution.
PhEur D . Dissolve 25 m g in 2.5 m L of water R, mix with 1.0 m L of
dilute ammonia R1 and allow to stand for 5 m in. Filter and
addify the filtrate with dilute nitric acid R. The filtrate gives
*****
Ambroxol Hydrochloride ★ ★ TESTS
*+ Solution S
(Ph. Eur. monograph 1489) *★ +*
*
Dissolve 0.75 g in methanol R and dilute to 15 m L with the
same solvent.
Br Solution S is clear (2.2.1) and n o t m ore intensely coloured
HCI
than reference solution Y 6 (2.2.2, MethodII).
NH2 pH (2.2.3)
Br 4.5 to 6.0.
Ci3H19Br2ClN20 414.6 23828-92-4
Action and use Related substances
M ucolytic expectorant. Liquid chromatography (2.2.29). Prepare the solutions
PhEir.
DEFINITION in water R and dilute to 50.0 m L with the same solvent.
trans-4- [(2-Amino-3,5-dibromobenzyl) amino] cydohexanol Reference solution (a) D ilute 1.0 m L o f the test solution to
hydrochloride. 100.0 m L with water R. Dilute 1.0 m L o f this solution to
Content 10.0 m L with the mobile phase.
99.0 per cent to 101.0 per cent (dried substance). Reference solution (b) In order to prepare impurity B in situ,
CHARACTERS dissolve 5 mg o f the substance to be examined in 0.2 m L of
Appearance methanol R, add 0.04 m l. of a mixture of 1 volum e of
W hite or yellowish, crystalline powder.
formaldehyde solution R and 99 volumes of water R. H eat at
60 °C for 5 min. Evaporate to dryness under a current of
Solubility nitrogen. Dissolve the residue in 5 m l. of water R and dilute
Sparingly soluble in water, soluble in methanol, practically to 20.0 m l, with the mobile phase.
Column:
IDENTIFICATION — sizer. I = 0.25 m , 0 = 4.0 mm ;
First identification By D. — stationary phase: octadecylstfyl silica gel for chromatography R
Second identification A j C, D. (5 pm).
A. U ltraviolet and visible absorption spectrophotometry Mobile phase A mixture o f equal volumes o f acetonitrik R and
(2.2.25). a solution prepared as follows: dissolve 1.32 g o f ammonium
Test solution Dissolve 20.0 m g in 0.05 M sulfuric acid and phosphate R in 900 m L o f water R, adjust to p H 7.0 with
dilute to 100.0 m L with the same ad d . Dilute 2.0 m L o f the
phosphoric acid R and dilute to 1000 m L with water R.
solution to 10.0 m L with 0.05 M sulfuric acid. Flow rate 1 mL/min.
Spectral range 200-350 nm. Detection Spectrophotom eter at 248 nm.
Absorption maxima At 245 nm and 310 nm . Injection 20 jiL.
Absorbance ratio ^ 245/^310 = 3.2 to 3.4. Run time 3 times the retention time o f ambroxol.
1-130 Amfetamine Sulfate 2016
.OH
Identification of impurities U se the chrom atogram obtained
with reference solution (b) to identify the peak due to
impurity B.
»-.X )
Relative retention W ith reference to ambroxol (retention C. trans-4- [[(£)-2-am ino-3,5-dibromobenzyliden] amino]
time = about 9 min): impurity B = about 0.6.
cyclohexanol.
— resolution: minimum 4.0 between the peaks due to .OH
impurity B and ambroxol.
Limits: . - „ O
H
D . os-4- [(2-amino-3,5-dibromobenzyl)amino] cyclohexanol,
with reference solution (a) (0.10 per cent),
— total: not m ore than 3 times the area of the principal peak E. A r-C H = 0 : 2-amino-3,5-dibromobenzaldehyde.
in the chromatogram obtained with reference solution (a) PhEtr
(0.3 per cent),
(0.05 per cent). ★*★
★ ★
H eavy m e ta ls (2.4.8) Amfetamine Sulfate ★ ★
M aximum 20 ppm. Amfetamine Sulphate * * * * *
1.0 g complies with test C. Prepare the reference solution (Ph Eur monograph 0368)
M aximum 0.5 per cent, determined on 1.000 g by drying in H2SO4 and enantiomer
an oven at 105 °C.
M aximum 0.1 per cent, determined on 1.0 g. C 18H 28N 2O 4S 368.5 60-13-9
ASSAY
Dissolve 0.300 g in 70 m L of ethanol (96 per cent) R and add
Releases dopamine; central nervous system stimulant.
5 m L of 0.01 M hydrochloric acid. C a n y out a potentiometric
titration (2.2.20), using 0.1 M sodium hydroxide. Read PhEur__________________________________________________________
the volume added between the 2 points o f inflexion.
D E F IN IT IO N
1 m L of 0.1 M sodium hydroxide is equivalent to 41.46 mg of
Bis [(2.&S)-1-phenylpropan-2-amine] sulfate.
C 13Hj9Br2C lN 20 .
C o n te n t
STORA GE
CHARACTERS
IM P U R IT IE S
A p p e a ra n c e
Other detectable impurities (the following substances would, if W hite or alm ost white powder.
the tests in the monograph. They are limited by the general S o lu b ility
acceptance criterion for other/unspecified impurities and/or Freely soluble in water, slightly soluble in ethanol
by the general monograph Substances for pharmaceutical use (96 per cent).
(2034). It is therefore no t necessary to identify these ID E N T IF IC A T IO N
impurities for demonstration o f compliance. See also 5.10. First identification A , B, E.
Control of impurities in substances for pharmaceutical use): A, B, Second identification A, C, D, E.
C, D, E.
A. Optical rotation (2.2.7): —0.04° to + 0.04° (measured in a
2 dm tube), determined on solution S (see Tests).
Ar- =
Preparation Mulls in liquid paraffin R.
Br Comparison Ph. Eur. reference spectrum of amfetamine sulfate.
C. T o 50 m L of solution S add 5 m L o f strong sodium
A. A r-C H 2O H : (2-amino-3,5-dibromophenyI)methanol, hydroxide solution R and 0.5 m l. o f benzoyl chloride R and
shake. Continue to add benzoyl chloride R in portions of
0.5 m L until no further precipitate is form ed Filter, wash
the precipitate with water R, reciystallise twice from a
mixture o f equal volumes o f ethanol (96 per cent) R and
w
water R, then dry at 100-105 °C. T h e crystals m elt (2.2.14)
at 131 °C to 135 °C.
Br D . T o about 2 m g add 1 m L o f sulfuric acid-formaldehyde
reagent R. A n orange colour develops and quickly becomes
B. trans-4-(6,8-dibrom o-1,4-dihydroquinazolin-3 (2H)-yl) dark-brown.
cyclohexanol, E. Solution S gives reaction (a) o f sulfates (2.3.1).
2016 Amidotrizoic Acid 1-131
TESTS B. Thin-layer chromatography (2.2.27).

S o lu tio n S Test solution Dissolve 25 m g of the substance to be examined
Dissolve 2.0 g in carbon dioxide-free water R and dilute to in a 3 p er cent V/V solution of ammonia R in methanol R and
100 m L with the same solvent. dilute to 5 m L with the same solution.
A p p e a ra n c e o f so lu tio n Reference solution Dissolve 25 m g of amidotrizoic add
Solution S is clear (2.2.1) and colourless (2 .2 .2 , Method II). dihydrate CRS in a 3 per cent V/V solution of ammonia R in
A cid ity o r alk a lin ity methanol R and dilute to 5 m L w ith the same solution.
T o 25 mL of solution S add 0.1 m L of methyl red solution R. Plate TLC silica gd GF2 5 4 plate R
N ot more than 0.1 mL o f 0.01 M hydrochloric add or 0.01 M Mobile phase anhydrous forme add R, methyl ethyl ketone R,
sodium hydroxide is required to change the colour o f the toluene R, (20:25:60 VIV/V).
indicator. Application 2 |iL.
L oss o n d ry in g (2.2.32) Development Over 2/3 o f the plate.
Drying In air until the solvents have evaporated.
an oven at 105 °C.
Detection In ultraviolet light at 254 nm .
S ulfa te d a s h (2.4.14)
Results T h e principal spot in the chromatogram obtained with
the test solution is similar in position and size to the principal
ASSAY spot in the chrom atogram obtained with the reference
Dissolve 0.300 g in 30 m L o f anhydrous acetic add R. T itrate solution.
with 0.1 M perchloric acid, determining the end-point C. H eat 50 m g gently in a small porcelain dish over a naked
potentiometrically (2 . 2 . 2 0 ). flame. Violet vapour is evolved.
TESTS
o f C 18H 2&N2O 4S .
STORAGE T h e solution is clear (2.2.1) and colourless (2.2.2,
Protected from light. Method II).
__________________________________________________________ PhEur Dissolve 1.0 g in dilute sodium hydroxide solution R and dilute
to 20 m L with the same solution.
★* ★
★ ★ Solvent mixture Dissolve 0.250 g o f sodium hydroxide R and
Amidotrizoic Acid Dihydrate ★ ★
0.860 g o f sodium dihydrogen phosphate R in 50 m L of water R
c o 2h
examined in 10.0 m L of the solvent mixture with the aid of
ultrasound.
2 HzO
Reference sdution (a) D ilute 1.0 m L o f the test solution to
100.0 m L with the solvent mixture. Dilute 1.0 m L of this
Reference solution (b) Dilute 1.0 m L o f reference solution (a)
C 11H 9l 3N 204J2H 20 650 50978-11-5 to 10.0 m L with the solvent mixture.
A ctio n a n d u se Reference solution (c) Dissolve the contents of a vial of

Iodinated contrast medium. amidotrizoic add for system suitability CRS (impurities A, B, C
and D) in 1.0 m L of the solvent mixture!
Preparation
Column:
Meglumine Amidotrizoate Injection
— size: I = 0.25 m , 0 = 4.6 mm;
PhEur________________________________ — stationary phase: end-capped octadecylstlyl silica gel for
chromatography R (5 jim).
D E F IN IT IO N
Mobile phase Dissolve 3.4 g of tetrabutylammonium hydrogen
3,5-Bis(acetylamino)-2,4,6-triiodobenzoic acid dihydrate.
sulfate R in a mixture o f 230 m L o f acetonitrile R and 770 m L
C o n te n t o f water R.
CHARACTERS Detection Spectrophotom eter at 236 nm.
A p p e a ra n c e Injection 20 (iL.
White or almost white, crystalline powder.
Run time 4 times the retention tim e o f amidotrizoic add.
S o lu b ility
Very slightly soluble in water and in ethanol (96 p er cent).
with amidotrizoic acid for system suitability CRS and the
It dissolves in dilute solutions of alkali hydroxides.
ID E N T IF IC A T IO N the peaks due to impurities A, B, C and D .
First identification A Relative retention W ith reference to amidotrizoic a d d
Second identification B, C (retention time = about 5 min): impurity B = about 0.8;
A. Infrared absorption spectrophotometry (2.2.24). impurity C = about 0.9; impurity A = about 1.4;
impurity D = about 1.8.
Comparison amidotrizoic acid dihydrate CRS.
1-132 Amidotrizoic Acid 2016
System suitability. through a sintered-glass filter (2.1.2) and wash the filter with
— resolution: m inim um 1.5 between the peaks due to several quantities o f water R. Collect the filtrate and
impurities B and C in the chromatogram obtained with washings. Add 40 m L o f dilute sulfuric acid R and titrate
reference solution (c); immediately with 0.1 M silver nitrate. D eterm ine the
— signal-to-noise ratio: minimum 25 for the principal peak in end-point potentiometrically (2.2.20), using a suitable
the chromatogram obtained with reference solution (b). electrode system such as silver/mercurous sulfate.
Limits: 1 m L of 0.1 M silver nitrate is equivalent to 20.47 m g of
— impurity B: not m ore than the area of the principal peak C 11H 9I 3N 2O 4.
STO RA G E
(0.1 per cent);
— impurities A , D: for each impurity, not more than the area
of the principal peak in the chromatogram obtained with IM P U R IT IE S
reference solution (b) (0.01 per cent); Specified impurities A, B, D
— unspecified impurities: for each impurity, not more than Other detectable impurities (the following substances would, if
0.5 times the area o f the principal peak in the present at a sufficient level, be detected by one or other o f
chrom atogram obtained with reference solution (a) the tests in the monograph. T hey are limited by the general
(0.05 per cent); acceptance criterion for other/unspecified impurities and/or
— total: not m ore than 1.5 times the area of the principal by the general m onograph Substances for pharmaceutical use
peak in the chromatogram obtained with reference (2034). It is therefore not necessary to identify these
solution (a) (0.15 per cent); impurities for dem onstration of compliance. See also 5.10.
— disregard limit. 0.3 times the area o f the principal peak in Control of impurities in substances for pharmaceutical use): C, E.
(0.03 per cent), except for the peaks due to impurities A CO2H
and D.
H a lid e s ex p ressed a s ch lo rid es (2.4.4)
M aximum 150 ppm . h2n
Dissolve 0.55 g in a mixture o f 4 m L o f dilute sodium
hydroxide solution R and 15 m L of water R. Add 6 m l. of
dilute nitric acid R and filter. A. 3-(acetylamino)-5-amino-2,4,6-triiodobenzoic acid,
F re e a ro m a tic a m in e s
Maintain the solutions and reagents in iced water, protected from c o 2h
bright light. T o 0.50 g in a 50 m L volumetric flask add

15 m L of water R. Shake and add 1 m L of dilute sodium
hydroxide solution R. Cool in iced water, add 5 m l. of a A XX1
freshly prepared 5 g/L solution o f sodium nitrite R and 12 m L I
of dilute hydrochloric acid R. Shake gently and allow to stand
for exactly 2 min after adding the hydrochloric acid. B. 3,5-bis(acetylamino)-2,4-diiodobenzoic ad d ,
Add 10 m L of a 20 g/L solution of ammonium stdfamate R.
Allow to stand for 5 min, shaking frequently, and add c o 2h
0.15 m L of a 100 g/L solution o f a-naphthol R in ethanol
(96 per cent) R. Shake and allow to stand for 5 min.
Add 3.5 m L o f buffer solution pH 10.9 R, mix and dilute to
50.0 m L with water R. The absorbance (2.2.25), measured
within 20 min at 485 nm using as the compensation liquid a
solution prepared at the same time and in the same m anner C. 3,5-bis(acetylamino)-2,6-diiodobenzoic a d d ,
but omitting the substance to be examined, is n o t greater
than 0.30.
Maximum 20 ppm.
Dissolve 2.0 g in 4 m L of dilute sodium hydroxide solution R
and dilute to 20 m L w ith water R. 12 m L of the solution
lead standard solution (2 ppm Pb) R. D . 3-(acetylamino)-5-[(iodoacetyl)amino]-2,4,6-
L oss o n d ry in g (2.2.32) triiodobenzoic ad d ,
4.5 p er cent to 7.0 per cent, determ ined on 0.500 g by
drying in an oven at 105 °C. CO2H
A SSA Y
T o 0.150 g in a 250 m L round-bottom ed flask add 5 m L of
strong sodium hydroxide solution R, 20 m l. o f water R, 1 g of
zinc powder R and a few glass beads. Boil under a reflux E. 3-(acetylamino)-5- (diacetylamino)-2,4,6-triiodobenzoic
condenser for 30 min. Allow to cool and rinse the condenser ad d .
with 20 m L o f water R, adding the rinsings to the flask. Filter
PhEur
2016 Amikacin 1-133

Amikacin 10 m L with the same solvent
(Ph Eur monograph 1289) * Specific o p tic a l ro ta tio n (2.2.7)
+ 97 to + 105 (anhydrous substance).
same solvent.
R elated su b sta n c e s
in mobile phase A and dilute to 50.0 m L w ith mobile
phase A.
Reference solution (a) Dilute 1.0 m L of the test solution to
100.0 m L with mobile phase A.
585.6 37517-28-5
Reference solution (b) Dilute 1.0 m L of reference solution (a)
A ctio n a n d u se
Aminoglycoside antibacterial. Reference solution (c) Dissolve 5 m g of amikacin for system
suitability CRS (containing impurities A, B, F and H ) in
PhEut______ ._____ ______________________________________________ mobile phase A and dilute to 10 m L with mobile phase A.
D E F IN IT IO N Reference solution (d) Dissolve 5.0 mg of amikacin
6-0-(3-Amino-3-deoxy-oc-D-glucopyranosyl)-4-0-(6-amino-6- impurity I CRS in mobile phase A and dilute to 20.0 m L with
deoxy-a-D-glucopyranosyl)-1-N- [(25)-4-amino-2- mobile phase A. Dilute 1.0 m L of the solution to 100.0 m L
hydroxybutanoyl] -2-deoxy-D-streptamine. with mobile phase A.
Antimicrobial substance obtained from kanamycin A. Column:
— sizer. I = 0.25 m , 0 = 4.6 mm;
Semi-synthetic product derived from a fermentation p ro d u ct
— stationary phase: end-capped octadecylsüyl silica gel for
C o n te n t chromatography R (5 pm);
96.5 per cent to 102.0 per cent (anhydrous substance). — temperature: 40 °C.
CHARACTERS Mobile phase:
A p p e a ra n c e — mobile phase A: a mixture prepared with carbon dioxide-free
White or almost white powder. water R, containing 1.8 g/L o f sodium octanesulfonate R,
S o lu b ility 20 g/L of anhydrous sodium sulfate R1, 1.4 per cent V/V of
Sparingly soluble in water, slightly soluble in methanol,
tetrahydrqfuran R, and 5 per cent V/V o f 0.2 M potassium
practically insoluble in acetone and in ethanol (96 per cent).
dihydrogen phosphate R previously adjusted to p H 3.0 with
dilute phosphoric acid R', degas;
ID E N T IF IC A T IO N — mobile phase B: a mixture prepared with carbon dioxide-free
A. Infrared absorption spectrophotometry (2.2.24). water R, containing 1.8 g/L o f sodium octanesulfonate R,
Comparison amikacin CRS. 28 g/L of anhydrous sodium sulfate R1, 1.4 per cent V!V of
B. Thin-layer chromatography (2.2.27). tetrahydrqfuran R, and 5 per cent V/V o f 0.2 M potassium
dilute phosphoric acid R ; degas;
in water R and dilute to 10 m L with the same solvent
Reference solution (a) Dissolve 25 m g o f amikacin CRS in
water R and dilute to 10 m L with the same solvent. Tune Mobile phase A Mobile phase B
Reference solution (b) Dissolve 5 mg o f kanamycin 0 -3 100 0
monosulfate CRS in 1 m L of the test solution and dilute to
10 m L with water R. 3 - 38.0 10030 0*70
Plate TLC silica gel plate R. 38.0 - 38.1 30 0 70-» 100

Mobile phase methylene chloride R, ammonia R, methanol R 38.1 - 68 0 100
(25:30:40 VfV/V).
Application 5 pL.
Post-column solution Mixture of 1 volume o f carbonate-free
Drying Luair. sodium hydroxide solution R and 24 volumes o f previously
Detection Spray w ith mnhydrin solution R1 and heat at 110 °C degassed carbon dioxide-free water R, which is added in a
for 5 min. pulseless m aim er to the column effluent using a 375 jiL
System suitability: reference solution (b): polymeric mixing coil.
— the chromatogram shows 2 clearly separated spots. Flow rate of post-column solution 0.3 mL/min.
Results T h e principal spot in the chromatogram obtained with Detection Pulsed amperometric detector or equivalent with a
the test solution is similar in position, colour and size to the gold indicator electrode, a silver-silver chloride reference
principal spot in the chromatogram obtained with reference electrode, and a stainless steel auxiliary electrode which is the
solution (a). cell body, held at respectively + 0.05 V detection, + 0.75 V
TESTS oxidation and — 0.15 V reduction potentials, with pulse
p H (2.2.3) durations according to the instrument used.
9.5 to 11.5. byection 20 pL.
1-134 Amikacin 2016
Identification of impurities Use the chromatogram supplied Calculate the percentage content o f C 22H 43N 5O 13 taking into
with amikacin for system suitability CRS and the account the assigned content o f armkadn CRS.
chromatogram obtained with reference solution (c) to identify IM P U R IT IE S
the peaks due to impurities A, B, F and H ; use the
Spedfied impurities A, B, F, H , I
chromatogram obtained with reference solution (d) to
identify the peak due to impurity I. Other detectable impurities (the following substances would, if
present at a sufficient level, be detected by one o r other of
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;
by the general monograph Substances for pharm aceutical use
impurity A = about 1.62; impurity H = about 1.95.
System suitability: reference solution (c): impurities for demonstration o f compliance. See also 5.10.
— peak-tchvalley ratio: minimum 5, where Hp = height above Control of impurities in substances for pharmaceutical use): C, D,
the baseline of the peak due to im purity B and E, G.
curve separating this peak from the peak due to amikacin;
if necessary, adjust the volume of tetrahydrofuran in the
mobile phase.
Calculation ofpercentage contents:
— for im purity I, use the concentration of impurity I in
reference solution (d);
— for impurities other than I, use the concentration o f
amikacin in reference solution (a).
Limits:
— impurities A , B, F, H} I: for each impurity, maximum
0.5 per cent;
— any other impurity: for each impurity, maximum
0.5 per cent; A. 4-0-(3-amino-3-deoxy-q-D-glucopyranosyl)-6-0-(6-amino-
— total: maximum 1.5 per cent; 6 -deoxy-a-D-glucopyranosyi)-1-N- [(2S)-4-amino-2-
— reporting threshold: 0.1 per cent. hydroxybutanoyl] -2-deoxy-L-streptamine,
W a te r (2.5.12)
A SSAY
Test solution Dissolve 50.0 m 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 Dissolve 50.0 mg o f amikacin CRS in the
mobile phase and dilute to 10.0 m L with the mobile phase.
Column: B. 4-0-(3-amino-3-deoxy-ce-D-glucopyranosyl)-6-0-(6-amino-
— size. I = 0.25 m , 0 = 4.6 mm; 6-deoxy-a-D-glucopyranosyl)-1,3-N-bis [(2S)-4-amino-2-
— stationary phase, end-capped octadecylsüyl silica gelfor hydroxybutanoyl] -2-deoxy-L-streptamine,
chromatography R (5 nm);
Mobile phase A mixture prepared with carbon dioxide-free
water R, containing 1.8 g/L o f sodium octanesulfonate R,
20 g/L of anhydrous sodium sulfate R1, 5.8 per cent V/V of
acetomtrüe R l, and 5 p er cent V/V o f 0.2 M potassium
dilute phosphoric add R; degas.
Detection Spectrophotom eter at 200 nm . *
Irtjection 20 |1L. C . 4-0-(6-amino-6-deoxy-cc-D-glucopyranosyl)-6-0-[3-[[(2S)-
Run time 1.3 times the retention time o f amikacin. 4-amino-2-hydroxybutanoyi] amino] -3-deoxy-a-D-
Retention time Amikacin = about 30 min. glucopyranosyl] -2-deoxy-D-streptamine,
amikacin; if necessary, adjust the am ount o f acetomtrüe R l
in the mobile phase; peak splitting may be observed w hen
the retention time becomes too short;
— repeatability: maximum relative standard deviation of
1.5 per cent after 6 injections.
2016 Amikacin Sulfate 1-135
D . 6-0-(3-amino-3-deoxy-a-D-glucopyranosyl)-4-0- H . 6-0-(3-amino-3-deoxy-a-D-glucopyranosyI) -1 -N- [(2S) -4-

(6-amino-6-deoxy-a-D-glucopyranosyI)-2-deoxy-D-streptamine amino-2-hydroxybutanoyl]-4-0-(2j6-diamino-2j6-dideoxy-a-
(kanamycin), D-glucopyranosyl)-2-deoxy-D-streptamine3
HOjC NH,
H OH
I. (25)-4-amino-2-hydroxybutanoic acid.
PhEur
★ ★
E. 4-0-(3-amino-3-deoxy-a-D-glucopyranosyl)-6-0- [6-[[(25)- Amikacin Sulfate ★ ★
4-amino-2-hydroxybutanoyl] amino] -6-deoxy-a-D- *****
Amikacin Sulphate
glucopyranosyl] -2-deoxy-L-streptamine3
(Ph. Eur. monograph 1290)
F. 6 -0 - (3-amino-3-deoxy-a-D-giucopyranosyl)-4-0- [6- [(25)-

4-amino-2-hydroxybutanoyl] amino-6-deoxy-a-D- 022^ 47^ 502 x82 782 39831-55-5
glucopyranosyl] -1 -N - [(25)-4-amino-2-hydroxybutanoyl] -2-
deoxy-p-stxeptamine,
Aminoglycoside antibacterial.
P re p a r a tio n
Amikacin Injection
PhEtr_____________________________
D E F IN IT IO N
6-0-(3-Amino-3-deoxy-a-D-glucopyranosyl)-4-0-(6-amino-6-
deoxy-a-i>glucopyranosyl)-l-N-[(25)-4-amino-2-
hydroxybutanoyl] -2-deoxy-D-strq)tamine sulfate.
Antimicrobial substance obtained from kanamycin A.
Semi-synthetic product derived from a fermentation product.
G . 6 -0 - (3-amino-3-deoxy-a-D-glucopyranosyl)-4-0-
(6-amino-6-deoxy-a-D-glucopyranosyl)-l-N-[(2R)-4-amino-2- C o n te n t
hydroxybutanoyl] -2-deoxy-D-streptamine, 96.5 per cent to 102.0 per cent (dried substance).
CHARACTERS
A p p e a ra n c e
Solubility
Freely soluble in water, practically insoluble in acetone and
in ethanol (96 p er cent).
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 tetrahydrqfuran R, and 5 per cent V/V o f 0.2 M potassium
in water R and dilute to 10 m L with the same solvent. dihydrogen phosphate R previously adjusted to p H 3.0 with
Reference solution (a) Dissolve 25 m g of amikacin sulfate CRS dilute phosphoric add R; degas;
in water R and dilute to 10 m l. with the same solvent.
Reference solution (b) Dissolve 5 mg o f kanamycin Time Mobile phase A Mobile phase B
monosulfate CRS in 1 m L o f the test solution and dilute to (min) (per cent V/V) (per cent V/V)
10 m L with water R. 0-3 100 0
Plate TLC silica gel plate R 3 - 38.0 1 00*30 0*70

Mobile phase methylene chloride R, ammonia R, methanol R 38.0 - 38.1 30* 0 7 0 * 100
(25:30:40 V/V/V).
38.1 - 68 0 100
Application 5 |iL.
Drying In air.
Post-column solution M ixture o f 1 volume of carbonate-free
Detection Spray with mnhydrin solution R1 and heat at 110 °C sodium hydroxide solution R and 24 volumes o f previously
for 5 min.
degassed carbon dioxide-free water R, which is added in a
System suitability: reference solution (b): pulseless m anner to the colum n effluent using a 375 pL
— the chromatogram shows 2 clearly separated spots. polymeric mixing coil.
Results The principal spot in the chromatogram obtained with Flow rate of post-column solution 0.3 mL/min.
the test solution is similar in position, colour and size to the
Detection Pulsed amperometric detector or equivalent with a
principal spot in the chromatogram obtained with reference
gold indicator electrode, a silver-silver chloride reference
solution (a).
electrode, and a stainless steel auxiliary electrode which is the
C. It gives reaction (a) of sulfates (2.3.1). cell body, held at respectively + 0.05 V detection, + 0.75 V
TESTS oxidation and — 0.15 V reduction potentials, with pulse
p H (2.2.3) durations according to the instrum ent used.
2.0 to 4.0. Injection 20 |iL.
Dissolve 0.1 g in carbon dioxide-free water R and dilute to Identification of impurities Use the chromatogram supplied
10 m L with the same solvent. with armkadn for system suitability CRS and the
S pecific o p tic a l ro ta tio n (2.2.7) chromatogram obtained with reference solution (c) to identify
+ 76 to + 84 (dried substance). the peaks due to impurities A, B, F and H ; use the
identify the peak due to im purity I.
sam e solvent.
Relative retention W ith reference to amikacin (retention
time = about 28 min): impurity I = about 0.13;
Test solution Dissolve 33 m g o f the substance to be examined impurity A = about 1.62; im purity H = about 1.95.
in mobile phase A and dilute to 50.0 m L with mobile System suitability: reference solution (c):
phase A — peak-to-vaUey ratio: minimum 5, where Hp = height above
Reference solution (a) Dilute 1.0 m L o f the test solution to the baseline of the peak due to im purity B and
100.0 m L w ith mobile phase A. Hv = height above the baseline o f the lowest point o f the
Reference solution (b) Dilute 1.0 m L o f reference solution (a) curve separating this peak from the peak due to amikacin;
to 10.0 m L with mobile phase A. if necessary, adjust the volume o f tetrahydrofuran in the
Reference solution (c) Dissolve 5 mg o f amikacin for system mobile phase.
suitability CRS (containing impurities A, B, F and H ) in Calculation of percentage contents:
mobile phase A and dilute to 10 m l. with mobile phase A. — for impurity I, use the concentration o f im purity I in
Reference solution (d) Dissolve 6.6 m g o f amikacin reference solution (d);
impurity I CRS in mobile phase A and dilute to 20.0 m L with — for impurities other than I, use the concentration of
mobile phase A. Dilute 1.0 m l. o f the solution to 100.0 m L amikacin sulfate in reference solution (a).
w ith mobile phase A. Limits.
Column: — impurities A , B, F, H, I: for each impurity, maximum
— size. I = 0.25 m, 0 = 4.6 mm; 0.5 p er cent;
— stationary phase: end-capped octadecyhüyl silica gel for — arty other impurity: for each impurity, m axim um
chromatography R (5 pm); 0.5 p er cent;
— temperature: 40 °C. — total: maximum 1.5 p er cent;
— reporting threshold: 0.1 p er cent.
Mobile phase:
— mobile phase A: a mixture prepared with carbon dioxide-free Sulfate
water R, containing 1.8 g/L o f sodium octanesulfonate R, 23.3 per cent to 25.8 per cent (dried substance).
20 g/L o f anhydrous sodium sulfate R l, 1.4 per cent V/V o f Dissolve 0.250 g in 100 m L o f water R and adjust the
tetrahydrofuran R, and 5 per cent V/V of 0.2 M potassium solution to p H 11 using concentrated ammonia R.
dihydrogen phosphate R previously adjusted to p H 3.0 with A dd 10.0 m L o f 0.1 M barium chloride and about 0.5 m g of
dilute phosphoric acid R; degas; phthalein purple R Titrate with 0.1 M sodium edetate adding
— mobile phase B: a mixture prepared with carbon dioxide-free 50 m l. of ethanol (96 per cent) R when the colour o f the
water R, containing 1.8 g/L o f sodium octanesulfonate R, solution begins to change and continue the titration until the
28 g/L o f anhydrous sodium sulfate R l , 1.4 per cent V/V of violet-blue colour disappears.
2016 Amikacin Sulfate 1-137
1 m l. o f 0.1 M barium chloride is equivalent to 9.606 mg of

sulfate (SO 4).
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
A. 4-0-(3-amino-3-deoxy-a-D-glucopyranosyl)-6-0-(6-amino-
containing 25 m g o f the substance to be examined in water
6 -deoxy-a-D-ghicopyranosyl)-1-N-[(2S) -4-amino-2 -
for injections R.
hydroxybutanoyl] - 2-deoxy-L-streptamine,
A SSAY
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. B. 4-0-(3-amino-3-deoxy-a-D-ghicopyranosyl)-6-0-(6-amino-
Mobile phase A mixture prepared with carbon dioxide-free 6-deoxy-a-D-glucopyranosyl)-1,3-N-bis [(25)-4-amino-2-
water R, containing 1.8 g/L o f sodium octanesulfonate R, hydroxybutanoyl] -2-deoxy-L-streptamine,
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
dilute phosphoric acid R; degas.
Flow rate 1.0 m l7min.
Injection 20 |iL.
Rim time 1.3 times the retention time of amikacin.
Retention time Amikacin = about 30 min.
— symmetry factor, maximum 1.5 for the peak due to C. 4-0-(6-amino-6-deoxy-a-D-glucopyranosyl)-6-0-[3-[[(25)-
amikacin; if necessary, adjust the am ount o f acetonitrile R l 4-amino-2-hydroxybutanoyl] amino]-3-deoxy-a-D-
in the mobile phase; peak splitting may be observed when glucopyranosyl] -2-deoxy-D-streptamine,
the retention tim e becomes too short;
— repeatability: m axim um relative standard deviation of
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 D . 6-0-(3-amino-3-deoxy-a-D-ghicopyranosyl)-4-0-
present at a sufficient level, be detected by one or other of (6-amino-6-deoxy-a-D-glucopyranosyl)-2-deoxy-D-streptamine
the tests in the m onograph. They are limited by the general (kanamycin),
by die general m onograph Substances for pharmaceutical use
impurities for dem onstration o f compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use): C, D,
E, G.
1-138 Amiloride Hydrochloride 2016
Amiloride Hydrochloride
(Ph. Eur. monograph 0651) *
O NH
CK .N. JLN J k NH,
H2N
XX H
Y
N NH2
, HCI, 2 H ,0 .
E. 4-0-(3-amino-3-deoxy-a-D-giucopyranosyl)-6-0-[6- [ [(25)- C6HgQ2N70j2H20 302.1 17440-83^1

4-amino-2-hydroxybutanoyI]amino]-6-deoxy-a-D-
glucopyranosyfl-2-deoxy-L-streptamine, Action and use
Sodium channel blocker; potassium-sparing diuretic.
Preparations
Amiloride Tablets
Co-amilofruse Tablets
Co-amilozide Oral Solution
Co-amilozide Tablets
PhEur__________________________________________________________
DEFINITION
3,5-Diarnino-Ar-carbamirnidoyl-6-chloropyrazine-2-
F. 6-0-(3-amino-3-deoxy-a-D-glucopyranosyl)-4-0- [6- [(25)- carboxamide hydrochloride dihydrate.
4-amino-2-hydroxybutanoyl] amino-6-deoxy-a-D-
Content
glucopyranosyQ-l-N- [(25)-4-amino-2-hydroxybutanoyi]-2-
deoxy-D -streptam ine}
CHARACTERS
Appearance
Pale yellow or greenish-yellow powder.
Solubility
Slightly soluble in water and in anhydrous ethanol.
IDENTIFICATION
First identification A, D.
Comparison amiloride hydrochloride CRS.
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-2-
hydroxybutanoyl]-2-deoxy-D-streptamine, 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
same solvent.
Mobile phase dilute ammonia R l, water R, dioxan R
(6:6:88 VIV/V); freshly prepared mixture.
Application 5 |iL.
Drying In air.
H . 6-0-(3-amino-3-deoxy-a-D-gJucopyranosyJ)-l-N-[(25)-4- Detection Examine in ultraviolet light at 365 nm.
amino-2-hydroxybutanoyI] -4-0- (2,6-diamino-2, 6-dideoxy-a- Results T h e principal spot in the chrom atogram obtained with
D-gJucopyranosyl)-2-deoxy-D-streptaminej the test solution is similar in position, fluorescence and size
to the principal spot in the chrom atogram obtained w ith the
H° 2 C ^ ^ , N H 2 reference solution.
H OH 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
I. (25)-4-amino-2-hydroxybutanoic add. hydroxide solution R and 1 m L o f bromine water R. A greenish-
yellow colour is produced. Add 2 m L o f dilute hydrochloric
___________________________________________________________ PhEur
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).
2016 Aminobenzoic Acid 1-139
TESTS ASSAY
Free acid Dissolve 0.200 g in a mixture o f 5.0 m L o f 0.01 M
Dissolve 1.0 g in a mixture o f 50 m L o f methanol R and hydrochloric add and 50 m L of ethanol (96 per cent) R. Carry
50 m L o f water R and titrate with 0.1 M sodium hydroxide, out a potentiom etric titration (2.2.20), using 0.1 M sodium
determ ining the end-point potentiometrically (2.2.20). hydroxide. Read the volume added between the 2 points o f
N o t m ore than 0.3 m L o f 0.1 M sodium hydroxide is required inflexion.
to reach the end-point. 1 m L of 0.1 M sodium hydroxide is equivalent to 26.61 m g
Related substances Of C6H 9CI2N 7O.
Liquid chrom atography (2.2.29). STORAGE
Test solution Dissolve 20.0 m g o f the substance to be Protected from light.
examined in a mixture of 1 volume o f acetonitrile R and
IM P U R IT IE S
3 volumes o f water R and dilute to 10.0 m L with the same
mixture o f solvents.
present at a sufficient level, be detected by one o r other o f
Reference solution (a) Dilute 1.0 m L o f the test solution to the tests in the monograph. T hey are limited by the general
100.0 m L with a mixture o f 1 volume o f acetomtrUe R and
3 volumes o f water R. by the general monograph Substances for pharmaceutical use
Reference solution (b) Dilute 1.0 m L o f reference solution (a) (2034). It is therefore not necessary to identify these
to 10.0 m l, with a mixture o f 1 volume o f acetonitrUe R and impurities for demonstration o f compliance. See also 5.10.
3 volumes of water R. Control of impurities in substances for pharmaceutical use): A .
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 ,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 HjN N NH2
3 volumes o f water R.
Column: A. methyl 3,5-diamino-6-chloropyrazine-2-carboxylate.
— sizer. I = 0.25 m , 0 = 4.6 mm;
PhEur
— 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 Aminobenzoic Acid *
★ ★
phosphoric add R and 9 volumes o f water R. Adjust the
(4-Aminobenzoic Add, Ph Eur monograph 1687) * * * * *
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
h 2n
XT
in the concentration results in a shorter retention time for
CzHyNOa 137.1 150-13-0
amiloride).
Flow rate 1 mL/min. A ctio n a n d u se
Detection Spectrophotom eter at 254 run. Skin protective.
Irqection 20 pL. PhEtr__________ __
Run time 5 times the retention time o f amiloride.
D E F IN IT IO N
4-Aminobenzoic acid.
— signal-to-noise ratio: m inim u m 5.0 for the peak due to
amiloride. C o n te n t
Limits: 99.0 p er cent to 101.0 per cent (anhydrous substance).
— unspecified impurities: for each impurity, n o t more than CHARACTERS
0.2 tim es the area of the peak due to impurity A in the A p p e a ra n c e
chrom atogram obtained with reference solution (c) W hite o r slightly yellow, crystalline powder.
(0.10 per cent);
S o lu b ility
— total: not m ore than the area o f the peak due to
Slightly soluble in water, freely soluble in alcohol. It dissolves
im purity A in the chromatogram obtained with reference
in dilute solutions o f alkali hydroxides.
— disregard limit. 0.1 times the area of the peak due to ID E N T IF IC A T IO N
im purity A in the chromatogram obtained with reference First identification B
solution (c) (0.05 per cent). Second identification A , C
Water (2.5.12) A. M elting point (2.2.14): 186 °C to 189 °C.
11.0 per cent to 13.0 per cent, determ ined on 0.200 g. B. Infrared absorption spectrophotometry (2.2.24).
Sulfated ash (2.4.14) Comparison 4-aminobenzoic add CRS.
M axim um 0.1 p er cent, determined on 1.0. g. C. Thin-layer chromatography (2.2.27).
1-140 Aminobenzoic Acid 2016
Test solution Dissolve 20 mg o f the substance to be examined — any other impurity: not more than 0.5 times the area o f the
in methanol R and dilute to 20 m L w ith the same solvent. peak due to im purity A in the chrom atogram obtained
Reference solution (a) Dissolve 20 m g o f 4-aminobenzoic with the reference solution (0.1 p er cent),
add CRS in methanol R and dilute to 20 m L with the same — total: n o t more than 2.5 times the area o f the peak due to
solvent. impurity A in the chrom atogram obtained with the
reference solution (0.5 p er cent),
Reference solution (b) Dissolve 10 m g o f 4-nitrobenzoic acid R
— disregard limit: 0.1 times the area o f the peak due to
in 10 m L o f reference solution (a).
impurity A in the chrom atogram obtained with the
Plate Suitable silica gel with a fluorescent indicator having an reference solution (0.02 per cent).
optimal intensity at 254 nm as the coatin g substance.
Impurity C and im purity D
Mobile phase glacial acetic add R, hexane R, methylene
Gas chromatography (2.2.28).
' chloride R (5:20:75 VIVIV).
Internal standard solution Dissolve 20.0 m g o f lauric add R in
Application 1 |iL.
methylene chloride R and dilute to 100.0 m L with the same
Development Over a path of 10 cm. solvent.
Drying In air. Test solution Dissolve 1.000 g o f the substance to be
Detection Examine in ultraviolet light at 254 nm. examined in 10.0 m L o f an 84 g/L solution o f sodium
System suitability T he chromatogram obtained with reference hydroxide R and extract with 2 quantities, each o f 10 mT, of
solution (b) shows 2 clearly separated spots. methylene chloride R. Combine and wash with 5 m L of
Results T he principal spot in the chrom atogram obtained with water R; filter through anhydrous sodium sulfate R. W ash the
the test solution is similar in position and size to the principal filter with methylene chloride R. Evaporate in a water-bath at
spot in the chromatogram obtained with reference 50-60 °C to obtain a volume o f about 1-5 mL. A dd 1.0 m L
solution (a). o f the internal standard solution and dilute to 10.0 m L with
methylene chloride R.
TESTS
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
The solution is clear (2.2.1) and no t m ore intensely coloured
solvent.
than reference solution B5 (2.2.2, Method II).
Reference solution (b) Dissolve 20.0 m g o f p-tohddine R in
Dissolve 1.0 g in alcohol R and dilute to 20 m L with the
methylene chloride R and dilute to 100.0 m L with the same
same solvent.
solvent.
Related substances Reference solution (c) Dilute 0.50 m L o f reference solution (a),
Liquid chromatography (2.2.29). 0.50 m L o f reference solution (b) and 10.0 m L o f the
Test solution Dissolve 25.0 mg o f the substance to be internal standard solution to 100.0 m L with methylene
examined in the mobile phase and dilute to 100.0 m L with chloride R.
the mobile phase. Column:
Reference solution Dissolve 25.0 m g o f 4-nitrobenzoic add R — material: fused silica,
and 25.0 m g o f benzocaine R in methanol R and dilute to — size. I = 30 m , 0 = 0.32 m m ,
100.0 m L with the same solvent. D ilute 1.0 m L to 50.0 m L — stationary phase. poly[methyl(95)phenyl(5)JsUoxane R (film
with the mobile phase. Dilute 1.0 m L o f this solution to thickness 0.5 nm).
10.0 m L with the mobile phase. Carrier gas helium for chromatography R.
Column: Flow rate 1.0 mL/min.
— size. I — 0.12 m , 0 = 4.0 m m ,
Split ratio 1:10.
— stationary phase: octylsUyl silica gel for chromatography R
(5 nm). Temperature:
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 H um Temperature
of a solution containing 1.5 g/L o f potassium dihydrogen (min) (°C)
Column 0 -4 130
phosphate R and 2.5 g/L of sodium octanesulfoncae R adjusted
to p H 2.2 with phosphoric add R. 4-6.5 130->180
Flow rate 1.0 mlVmin. 6.5 -11.5 180
Detection Spectrophotom eter at 270 nm . Injection port 280
Injection 20 |iL. 300
Detector
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; Detection Flame ionisation.
impurity B = about 9. Injection 2 nL; inject the test solution and reference
Limits: solution (c).
— impurity A: not m ore than the area o f the corresponding Retention time Internal standard = about 9.5 min.
peak in the chromatogram obtained with the reference Limits:
solution (0.2 per cent), — impurity C: calculate th e ratio (K) o f the area o f the peak
— impurity B: n o t more than the area o f the corresponding due to impurity C to the area of the peak due to the
peak in the chromatogram obtained with the reference internal standard from the chromatogram obtained with
solution (0.2 per cent), 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
2016 Aminocaproic Acid 1-141
the test solution: this ratio is not greater than R CHARACTERS

(10 ppm ), A white or almost white, crystalline pow der or colourless
— impurity D: calculate the ratio (R) of the area o f the peak crystals, fredy soluble in water, slightly soluble in alcohol.
due to impurity D to the area of the peak due to the It melts at about 205 °C with decomposition.
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 First identification A.
internal standard from the chromatogram obtained with Second identification B, C, D.
the test solution: this ratio is not greater than R A. Examine by infrared absorption spectrophotometry
(10 ppm ). (2.2.24), comparing with the spectrum obtained with
Ir o n (2.4.9) aminocaproic acid CRS. Examine the substances prepared as
M axim um 40 ppm . discs.
Dissolve 0.250 g in 3 m L of alcohol R and dilute to 10.0 m L B. Examine the chromatograms obtained in the test for
w ith water R. 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
M axim um 20 ppm .
chromatogram obtained with reference solution (a).
1.0 g complies with test C . Prepare the reference solution
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) heating on a water-bath. Dry the residue in a desiccator.
M axim um 0.2 p er cent, determined on 1.00 g. Dissolve the residue in about 2 m L o f boiling ethanol R.
S u lfa te d a s h (2.4.14) Allow to cool and maintain at 4 °C to 8 °C for 3 h. Filter
M axim um 0.1 per cent, determined on 1.0 g. 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
A SSA Y
(2.2.14) at 131 °C to 133 °C.
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 D . Dissolve about 5 mg in 0.5 m L o f distilled water R.
end-point potentiometrically (2.2.20). 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
STORA GE
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. R = C 0 2H , R ' = N 0 2: 4-nitrobenzoic ad d , A b so rb a n c e (2.2.25)
B. R = C 0 - 0 - C 2H 5, R ' = N H 2: ethyl 4-aminobenzoate A. T h e absorbance o f solution S at 287 nm is not more
(benzocaine), th an 0.10 and at 450 nm is not more th an 0.03.
C. R = H , R ' = N H 2: aniline, 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
D . R = C H 3, R ' = N H 2: 4-methylaniline (p-toluidine).
72 h. Dissolve in water R and dilute to 10.0 m L with the
__________________________________________________________ PhEur 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
Aminocaproic Acid i***% suitable silica gel as the coating substance.
*+ Test solution (a) Dissolve 0.10 g of the substance to be
(Ph Eur monograph 0874) * 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 Reference solution (a) Dissolve 10 mg o f aminocaproic
acid CRS in water R and dilute to 50 m L with the same
A c tio n a n d u s e solvent.
Antifibrinolytic.
Reference solution (b) Dilute 5 m L o f test solution (b) to
PhEur__________________________________________________________ 20 m L with water R.
D E F IN IT IO N Reference solution (c) Dissolve 10 mg o f aminocaproic acid CRS
Aminocaproic a d d contains not less than 98.5 p er cent and and 10 mg o f leucine CRS in water R an d dilute to 25 m L
not more th an the equivalent of 101.0 per cent of with the same solvent.
6-am inohexanoic ad d , calculated with reference to the dried Apply separately to the plate 5 (iL o f each solution. Allow the
substance. plate to dry in air. D evdop over a p ath of 15 cm using a
1-142 Aminoglutethimide 2016
mixture of 20 volumes o f glacial acetic acid R, 20 volumes of C. Thin-layer chromatography (2.2.27).

water R and 60 volumes o f butanol R. Dry the plate in a Test solution Dissolve 25 m g of the substance to be examined
current of w arm air. Spray with mnhydrin solution R and heat in acetone R and dilute to 5 m L with the same solvent.
at 100 °C to 105 °C for 15 min. Any spot in the Reference solution (a) Dissolve 25 m g of
chromatogram obtained with the test solution (a), apart from aminoglutethimide CRS in acetone R and dilute to 5 m L with
the principal spot, is n o t more intense than the spot in the the same solvent.
(0.5 per cent). T he test is not valid unless the chromatogram
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.
separated principal spots.
Plate TLC stHca gel F 254 plate R.
12 m L of solution S complies with test A for heavy metals Mobile phase glacial acetic add R, methanol R, ethyl acetate R
(10 ppm ). Prepare the reference solution using lead standard (0.5:15:85 VIVIV).
solution (2 ppm Pb) R. Application 5 |oL.
L oss o n d ry in g (2.2.32) Development Over 3/4 of the plate.
N ot more than 0.5 per cent, determined on 1.000 g by Drying In air.
drying in an oven at 105 °C. Detection Examine in ultraviolet light at 254 nm.
S u lfa te d a sh (2.4.14) System suitability: reference solution (b):
N ot more than 0.1 per cent, determined on 1.0 g. — the chromatogram shows 2 clearly separed spots.
A SSA Y Results T h e principal spot in the chromatogram obtained with
Dissolve 0.100 g in 20 m L o f anhydrous acetic acid R. Using the test solution is similar in position and size to the principal
0.1 m L of crystal violet solution R as indicator, titrate with spot in the chromatogram obtained with reference
0.1 M perchloric add until the colour changes from bluish- solution (a).
violet to bluish-green. TESTS
1 m L o f 0.1 M perchloric add is equivalent to 13.12 m g o f S o lu tio n S
C sH jaN O * Dissolve 1.0 g in methanol R and dilute to 20.0 m L with the
__________________________________________________________ PhEur same solvent.
Aminoglutethimide ***** O p tic a l ro ta tio n (2.2.7)
★. * —0.10° to + 0.10°, determined on solution S.
H Liquid chromatography (2.2.29).
O ^N Ô
Solvent mixture methanol R, acetate buffer solution pH 5.0 R
I L J \ NH2 and enantiomer (50:50 VIV).
H3< /
examined in the solvent mixture and dilute to 50.0 m L with
the solvent mixture.
C i 3H16N 20 2 232.3 125-84-8
Reference solution (a) Dissolve 5.0 m g o f aminoglutethimide
A c tio n a n d u se impurity A CRS in the solvent mixture and dilute to 25.0 m L
Inhibitor of adrenal corticosteroid synthesis; used in chemical with the solvent mixture.
adrenalectomy. Reference solution (b) Dilute 1.0 m L of reference solution (a)
PhEur__________________________________________________________
Reference solution (c) Dilute 1.0 m L o f the test solution to
D E F IN IT IO N 100.0 m L with the solvent mixture.
(3i?5)-3-(4-Aminophenyl)-3-ethylpiperidine-2j6-dione. Reference solution (d) Dilute 1.0 m L o f the test solution to
C o n te n t 10.0 m L with reference solution (a). -
98.0 per cent to 101.5 per cent (dried substance). Column:
— size. I = 0.15 m, 0 = 3.9 mm;
CHARACTERS
— stationary phase, octadecylsüyl silica gel for chromatography R
A p p e a ra n c e
(4 Jim);
W hite or slightly yellow, crystalline powder.
S olubility
Mobile phase Mix 27 volumes of methanol R and 73 volumes
o f acetate buffer solution pH 5.0 R
soluble in methanol.
First identification B
Injection 10 pL of the test solution and reference
Second identification A, C solutions (b), (c) and (d).
A. Melting point (2.2.14): 150 °C to 154 °C. Run time 4 times the retention time o f aminoglutethimide.
Comparison aminoglutethimide CRS.
2016 Aminoglutethimide 1-143
Identification of impurities Use the chromatogram obtained Prepare the reference solution vising lead standard solution
with reference solution (b) to identify die peak due to (1 ppm Pb) obtained by diluting lead standard solution
impurity A. (100 ppm Pb) R with a mixture o f 5 m L o f water R and
Relative retention W ith reference to aminoglutethimide 15 m L o f acetone R
(retention time = about 9 min): impurity A = about 1.3. L oss o n d ry in g (2.2.32)
System suitability Reference solution (d): M axim um 0.5 p er cent, determined on 1.000 g by drying in
— resolution: m inim u m 2.0 between the peaks due to an oven at 105 °C.
am in oglu teth im id e and impurity A. S u lfa te d a sh (2.4.14)
Limits: M axim um 0.1 per cent, determined on 1.0 g.
— impurity A: not m ore than twice die area o f the principal
A SSA Y
peak in the chromatogram obtained w ith reference
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
— 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) 1 m L of 0.1 M perchloric acid is equivalent to 23.23 mg
(0.10 per cent); Of C 13H 16N 2O 2.
— sum of impurities other than A: not more than the area o f IM P U R IT IE S
the principal peak in die chromatogram obtained with Specified impurities A, D .
— total: maximum 2.0 per cent for the sum of die contents
present at a sufficient level, be detected by one o r other o f
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 impurities for demonstration o f compliance. See also 5.10.
Liquid chromatography (2.2.29). Carry out die test protected Control of impurities in substances for pharmaceutical use): B, C.
from light Use shaking, not sordcadon or heat, to dissolve the
reference substance and the substance to be examined.
examined in dimethyl sulfoxide R and dilute to 100.0 m L with 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 A. R3 = N H 2) R4 = H: (3RS)-3-(3-aminophenyl)-3-
solution to 100.0 m l, with dimethyl sulfoxide R. ethylpiperidine-2,6-dione (3-aminoglutethimide),
Column: B. R3 = N O * R4 = H:
— sizer. I = 0.12 m , 0 = 4 mm; (3i?S)-3-ethyl-3-(3-nitrophenyl)piperidine-2,6-dione,
— stationary phase: octadecylsUyl silica gel for chromatography R C. R3 = H , R4 = N 0 2:
(5 pm). (3i?5)-3-ethyl-3-(4-nitrophenyl)piperidine-2,6-dione,
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.
Injection 10 |iL.
System suitability T est solution: D . 3,3'-[diazenediylbis(4,l-phenylene)]bis(3-ethylpiperidine-
— number of theoretical plates: minimum 3300, calculated for 2, 6-dione) (azoglutethimide).
the principal peak; PhEur
— 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 .
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.
1-144 Aminophylline 2016
E. W ater (see Tests).

Aminophylline ;***%
F . T h e pred p itate gives the reaction o f xanthines (2.3.1).
*★ ★*
(TheophyUine-ethyknediamine, anhydrous, *
TESTS
Ph Eur monograph 0300)
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
C 16H 24N 10O 4 420.4 317-34-0 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
A c tio n a n d u se phase.
N on-sdective phosphodiesterase inhibitor; treatm ent o f
reversible airways obstruction. 100.0 m L with the mobile phase. D ilute 1.0 m l . o f this
P re p a r a tio n s solution to 10.0 m l . with the mobile phase.
Aminophylline Injection Reference solution (b) Dissolve 10 m g o f theobromine R
Aminophylline Tablets (impurity G ) in the mobile phase, add 5 m L o f the test
Prolonged-release Aminophylline Tablets 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 — sizer. I = 0.25 m , 0 = 4 mm;
C o n te n t — stationary phase: octadecylsUyl silica gelfor chromatography R
— theophylline (C 7H&N4O 2; 180.2): 84.0 per cent to (7 nm).
87.4 per cent (anhydrous substance); Mobile phase M ix 7 volumes o f acetomtrUefor
— ethylenedianane (C 2HgN 2; 60.1): 13.5 p er cent to chromatography R and 93 volumes o f a 1.36 g/L solution of
15.0 per cent (anhydrous substance). sodium acetate R containing 0.50 per cent V/V o f glacial acetic
CHARACTERS add R.
A p p e a ra n c e Flow rate 2.0 mL/min.
W hite or slightly yellowish powder, sometimes granular, Detection Spectrophotom eter at 272 nm .
hygroscopic. Irqection 20 jjL .
S o lu b ility Run time 3.5 times the retention time o f theophylline.
Freely soluble in water (the solution becomes cloudy through Relative retention W ith reference to theophylline (retention
absorption o f carbon dioxide), practically insoluble in tim e = about 6 min): impurity G = about 0.6.
anhydrous ethanol.
ID E N T IF IC A T IO N — resolution: m in im u m 2.0 between the peaks due to
First identification B, C, E. impurity G and theophylline.
Second identification A , C, D, E, F. Limits:
Dissolve 1.0 g in 10 m L o f water R and add 2 m l . o f dilute — unspecified impurities: for each impurity, n o t more than the
hydrochloric acid R dropwise with shaking. Filter. U se the area o f the prindpal peak in the chrom atogram obtained
predpitate for identification tests A, B, D and F and the with reference solution (a) (0.10 per cent);
filtrate for identification test C. — total: n o t m ore than the area o f the prin d p al peak in the
A. M d tin g point (2.2.14): 270 °C to 274 °C, determ ined
(0.1 p er cent);
after w ash in g the predpitate with water R and drying at
— disregard Umit. 0.5 times the area o f the principal peak in
105 °C.
B. Infrared absorption spectrophotom etry (2.2.24). (0.05 per cent).
Preparation P redpitate, washed with water R and dried at
105 °C.
M aximum 20 ppm.
Comparison theophylline CRS. Solvent water R.
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
T h e substance predpitates after addition o f bitjfer solution
water R, dissolve in 5 m L o f h o t ethanol (96 per cent) R and
pH 3.5 R. D ilute to 100 m L with water R, the substance
add 5 m L o f water R. A predpitate is formed, which, when
re-dissolves completely.
washed and dried at 105 °C, m d ts (2.2.14) at 248 °C to
252 °C. Water (2.5.12)
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 Sulfated ash (2.4.14)
90 °C for 3 m in, then add 1.0 m L o f diazodsed sulfanUic acid M aximum 0.1 per cent, determ ined on 1.0 g.
solution R. A red colour slowly devdops. C an y out a blank
te s t
2016 Aminophylline Hydrate 1-145
A SSA Y
^ n^ v n
Etfaylenediamine
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 0
n>< 1 H
add until a green colour is obtained. ch3
1 m L of 0.1 M hydrochloric add is equivalent to 3.005 m g of

C2HgN2‘ E. l,3-dim ethyl-7,9-dihydro-lH-purine-2,6,8(3/i)-trione,
T h eo p h y llin e
H eat 0.200 g to constant mass in an oven at 135 °C. OH
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 o
X x> I
sodium hydroxide.. ch3
1 m L of 0.1 M sodium hydroxide is equivalent to 18.02 m g o f
C 7H 8N 4O 2. F. 7-(2-hydroxyethyl)-1,3-dimethyl-3,7 -dihydro-1H -purine-
2, 6-dione (etofylline),
STO RA G E
In an airtight container, protected from light
0 pi
IM P U R IT IE S 1 '
present at a sufficient level, be detected by one or other of X V> !
ch3
(2034). It is therefore not necessary to identify these G. 3,7-dimethyl-3,7-dihydro-lH-purine-2,6-dione
impurities for demonstration of compliance. See also 5.10. (theobromine).
Control of impurities in substances for pharmaceutical use): A, B, PhEur
C, D, E, F, G.
0 pt
1 ' ★ ★
Aminophylline Hydrate
lx > 1
Çlheophyïïme-eàiylenediamine Hydrate,
★ ★
*****
ch3 Ph Eur monograph 0301)
A. 1,3,7-trimethyl-3,7-dihydro-lH-purine-2,6-dione
(caffeine),
, xH 20
H2N'
1 1 >
I
ch3
HN
X L //>
C 16H 24N 10O 4PCH2O 420.4 72487-55-9
CH,
B. 3-methyl-3 37-dîhydro-1H-purine-236-dione3
Non-selective phosphodiesterase inhibitor; treatm ent of
A J. CHO
reversible airways obstruction.
O
JjC. N NH2
P re p a r a tio n
Aminophylline Injection
CH,
Aminophylline Tablets
Prolonged-release AminopyQine Tablets
C. AT-(6-am ino-l^-dim ethyl-2,4-dioxo-l,2,3,4-
tetrahydropyrimidin-5-yl)formamide, PhEur.
D E F IN IT IO N
C o n te n t
h3c x
— theophylline (C7H8N402; 180.2): 84.0 per cent to
»V>
hn ^ n
87.4 per cent (anhydrous substance);
— ethylenediamine (C 2H 8N 2; 60.1): 13.5 per cent to
CH, 15.0 per cent (anhydrous substance).
CHARACTERS
D. N-methyl-5-(methylamino)- lH-imidazole-4-carboxamide, 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 Injection 20 pL.

Freely soluble in water (the solution becomes cloudy through Run time 3.5 times the retention time o f theophylline.
absorption o f carbon dioxide), practically insoluble in
Relative retention W ith reference to theophylline (retention
anhydrous ethanol.
tim e = about 6 min): im purity G = about 0.6.
IDENTIFICATION System suitability: reference solution (b):
First identification B, C, E. — resolution: minimum 2.0 between the peaks due to
Second identification A , C, D, E, F. im purity G and theophylline.
Dissolve 1.0 g in 10 m L of water R and add 2 m L o f dilute Limits:
hydrochloric acid R dropwise with shaking. Filter. Use the — unspecified impurities: for each impurity, n o t m ore than the
precipitate for identification tests A, B, D and F and the area of the principal peak in the chrom atogram obtained
filtrate for identification test C. with reference solution (a) (0.10 per cent);
— total: n o t more than the area o f the principal peak in the
A. Melting point (2.2.14): 270 °C to 274 °C, determined
after washing the precipitate with water R and drying at
(0.1 per cent);
105 °C.
B. Tnfrared absorption spectrophotom etry (2.2.24).
Preparation Precipitate, washed with water R and dried at (0.05 per cent).
105 °C.
Comparison theophylline CRS. M aximum 20 ppm.
C. T o the filtrate add 0.2 m L o f benzoyl chloride R, make Solvent water R.
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
T h e substance precipitates after addition o f buffer solution
add 5 m L o f water R. A precipitate is formed, which, when
pH 3.5 R. D ilute to 100 m L with water R; the substance
washed and dried at 105 °C, melts (2.2.14) at 248 °C to
re-dissolves completely.
252 °C.
W a te r (2.5.12)
D . H eat about 10 m g o f the precipitate with 1.0 m L o f a
3.0 per cent to 8.0 per cent, determ ined on 0.50 g.
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 S u lfa te d a s h (2.4.14)
solution R. A red colour slowly develops. Carry out a blank M aximum 0.1 per cent, determ ined on 1.0 g.
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
TESTS
acid until a green colour is obtained.
1 mT. o f 0.1 M hydrochloric acid is equivalent to 3.005 m g of
T he solution is not m ore opalescent than reference
suspension II (2.2.T) and n o t m ore intensely coloured than C 2H8N2.
reference solution GY6 (2.2.2, Method II). Theophylline
Dissolve 0.5 g with gentle warming in 10 m l. of carbon H eat 0.200 g to constant mass in an oven at 135 °C.
dioxide-free water R 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.
Test solution Dissolve 50 mg o f the substance to be examined 1 mT. o f 0.1 M sodium hydroxide is equivalent to 18.02 m g of
C rH sN ^ .
phase.
Reference solution (a) D ilute 1.0 m l, o f the test solution to STORAGE
100.0 m L w ith the mobile phase. D ilute 1.0 m l. o f this In a well-filled, airtight container, protected from light.
solution to 10.0 m L w ith the mobile phase. IMPURITIES
Reference solution (b) Dissolve 10 m g o f theobromine R Other detectable impurities (the following substances would, if
(impurity G ) in the mobile phase, add 5 m L o f the test present at a sufficient level, be detected by one or other of
solution and dilute to 100 m L with the mobile phase. D ilute the tests in the m onograph. T hey are limited by the general
5 m L o f this solution to 50 m L with the mobile phase. acceptance criterion for other/unspecified impurities and/or
Column: by the general monograph Substances for pharm aceutical use
— size. I — 0.25 m, 0 = 4 mm; (2034). It is therefore no t necessary to identify these
— stationary phase: octadecylsifyl silica gel for chromatography R impurities for dem onstration o f compliance. See also 5.10.
(7 pm). Control of impurities in substances fin- pharmaceutical use): A , B,
Mobile phase M ix 7 volumes o f acetortitrile for C, D, E, F, G.
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.
2016 Amiodarone Hydrochloride 1-147
i ? Hs Amiodarone Hydrochloride *****
1Y>
★ ★
H,c' N' l V ' N
o^ n- ^ n
ch3
H,C
A. 1,3,7-trim ethyl-3,7-dihydro-lH-purine-2,6-dione , HQ
(caffeine), N. .CH,
C25H3oCn2N 0 3 682 19774-82-4

lV > I A ctio n a n d u se
ch3
Potassium channel blocker; class ID antiarrhythmic.
P re p a ra tio n s
B. 3-methyl-3,7-dihydro-lH-purine-2,6-dione,
Amiodarone Intravenous Infusion
Amiodarone Oral Suspension
h3c^ I B . Amiodarone Tablets
N | c
PhEir.
O ^N NH2
D E F IN IT IO N
ch3
(2-Butylben2ofuran-3-yl)[4-[2-(diethylamino)ethoxy]-3j5-
diiodophenyljmethanone hydrochloride.
C. N -(6-am ino-l,3-dim ethyl-2,4-dioxo-l,2,3,4-
tetrahydropyrimidin-5-yl)formamide, C o n te n t
CHARACTERS
H,C A p p e a ra n c e
rV s W hite or almost white, fine, crystalline powder.
H N N S olu b ility
I
CH3 Very slightly soluble in water, freely soluble in methylene
chloride, soluble in methanol, sparingly soluble in ethanol
D . N-methyl-5-(methylamino)-lH-imidazole-4-carboxamide, (96 per cent).
Comparison amiodarone hydrochloride CRS.
H*C'1n\ A[ ) ==0
o ^ n ^ n B. It gives reaction (b) o f chlorides (2.3.1).
I M TESTS
CH,
T h e solution is clear (2.2.1) and n o t more intensely coloured
E. 1,3-dim ethyl-7,9-dihydro-lii-purine-2,6,8(3H)-trione,
than reference solution GY5 or BY5 (2.2.2, Method II).
OH same solvent
pH (2.2.3)
1
oIA
JL >
HA > 3.2 to 3.8.
I
ch3 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
F . 7-(2-hydroxyethyl)-1,3-dimethyl-3,7-dihydro-1H -purine- Im p u rity H
2,6-dione (etofylline), Thin-layer chromatography (2.2.27). Prepare the solutions
immediately before use and keep protectedfrom bright light.
X " 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
ÍJC> I
the same solvent.
Reference solution (a) Dissolve 10.0 m g of
ch3 (2-chloroethyl)diethylamine hydrochloride R (impurity H) in
methylene chloride R and dilute to .50.0 m L with the same
G . 3,7-dim ethyl-3,7-dihydro-lii-purine-2,6-dione solvent. Dilute 2.0 m L o f the solution to 20.0 m L with
(theobromine). methylene chloride R.
PhEur
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 — disregard limit. 0.25 times the area o f the peak due to
chloride R (5:10:85 VIVIV). amiodarone in the chrom atogram obtained with the
Application 50 (iL o f the test solution and reference reference solution (0.05 per cent).
solution (a); 100 pL o f reference solution (b). Io d id e s
Development Over 2/3 o f the plate. M axim um 150 ppm.
Drying In a current o f cold air. Prepare the test and reference solutions simultaneously.
Detection Spray with potassium iodobismuthate solution R l and Solution A A dd 1.50 g o f the substance to be examined to
then with dilute hydrogen peroxide solution R', exam in e 40 m L o f water R at 80 °C and shake until completely
immediately in daylight. dissolved. Cool and dilute to 50.0 m L w ith water R.
System suitability: reference solution (b): Test solution T o 15.0 m L o f solution A add 1.0 m L o f
— the spot due to im purity H is clearly visible. 0.1 M hydrochloric acid and 1.0 mT. o f 0.05 Mpotassium
Limit: iodate. Dilute to 20.0 m L with water R. Allow to stand
— impurity H: any spot with the same Rp as the spot due to protected from light for 4 h.
im purity H in the chromatogram obtained with reference Reference solution T o 15.0 m L o f solution A add 1.0 m L of
solution (b) is n o t m ore intense th an the spot in the 0.1 M hydrochloric add, 1.0 m L of an 88.2 mg/L solution o f
chrom atogram obtained w ith reference solution (a) potassium iodide R and 1.0 m L o f 0.05 Mpotassium iodate.
(0.02 per cent). 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 ,
Buffer solution pH 49 T o 800 mT. o f water R add 3.0 m L o f using a mixture o f 15.0 m L o f solution A and 1.0 m L o f
glacial acetic add R, adjust to p H 4.9 with dilute ammonia R l 0.1 M hydrochloric acid diluted to 20.0 m L with water R as
and dilute to 1000 m L with water R. 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.
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 H e a v y m e ta ls (2.4.8)
solvents. M axim um 20 ppm .
Reference solution Dissolve 5 m g o f amiodarone 1.0 g complies with test C. Prepare the reference solution
impurity D CRS, 5 m g of amiodarone impurity E CRS and using 2 m L o f lead standard solution (10 ppm Pb) R.
5.0 m g o f amiodarone hydrochloride CRS in methanol R and L o ss o n d ry in g (2.2.32)
dilute to 25.0 m L w ith the sam e solvent. Dilute 1.0 m L o f Maximum 0.5 per cent, determ ined on 1.000 g by drying at
the solution to 20.0 m L with a mixture o f equal volumes o f 50 °C at a pressure not exceeding 0.3 kPa for 4 h.
acetonitrile R and water R. S u lfa te d a s h (2.4.14)
Column: M aximum 0.1 per cent, determ ined on 1.0 g.
— size: I = 0.15 m , 0 = 4.6 mm;
— stationary phase, end-capped octadecylsUyl silica gel for A SSA Y
chromatography R (5 pm); Dissolve 0.600 g in a mixture o f 5.0 m L o f
— temperature: 30 °C. 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
Mobile phase Buffer solution p H 4.9, methanol R, acetonitrile R
0.1 M sodium hydroxide. Read the volume added between the
(30:30:40 VIVIV).
2 points o f inflexion.
Flow rate 1 mL/min.
1 mT. o f 0.1 M sodium hydroxide is equivalent to 68.18 m g of
Detection Spectrophotom eter at 240 nm . C 25H 3oCU2N 0 3 .
Injection 10 (iL.
STORAGE
Run time Twice the retention time o f am iodarone. Protected from light, at a tem perature n o t exceeding 30 °C.
Relative retention W ith reference to amiodarone (retention
time = about 24 m in): im purity A = about 0.26; IM P U R IT IE S
im purity D = about 0.29; im purity E = about 0.37; Specified impurities A, B, C , D , E, F , G, H
impurity B = about 0.49; im purity C = about 0.55;
im purity G = about 0.62; im purity F = about 0.69.
impurities D and E.
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
A. (2-butyIbenzofuran-3-yl) [4-[2-(diethylamino)ethoxy]
chrom atogram obtained w ith the reference solution
phenyl] methanone,
(0.2 per cent);
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);
2016 Amisulpride 1-149
and enantiomer ★* *
Amisulpride ★ ★
★ ★
o\w/o
h 3c .
N
H u^
' ON and enantiomer.
B. (2-butylbenzofuran-3-yI) [4-[2-(ethyiamino)ethoxy]-3,5-
diiodophenyl] methanone, h2n och3 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]-3- Amisulpride Tablets
iodophenyl] methanone,
P h E tr ______________________________________________________________
DEFINITION
4-Amino-N- [ [(2i?5)-1-ethylpyrrolidin-2-yl] methyl] -5-
(ethylsulfonyl)-2-methoxybenzamide.
Content
CHARACTERS
D . (2-butyIbenzofuran-3-yl)(4-hydroxy-3,5- Appearance
diiodophenyl)methanone, 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,
Comparison amisulpride CRS.
TESTS
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
F. (2-butylbenzofuran-3-yl) (4-hydroxy-3-iodophenyl) water R.
methanone, Impurity A
Test solution Dissolve 0.20 g of th e substance to be examined
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)- Plate TLC silica gel G plate R.
1-methoxybutyl] benzofuran-3-yl] methanone,
Mobile phase 50 per cent VIV solution of concentrated
ammonia R, anhydrous ethanol R, di-isopropyl ether R
,c h 3
r
,N_XH3
(10:25:65 VIVIV); use the upper layer obtained after shaking
the mixture.
Application 10 (iL.
H . 2-chloro-N,N-diethylethanamine (2-chlorotriethylamine, Development Over 2/3 of the plate.
(2-chloroethyl)diethyIamine). Drying In air.
1-150 Amisulpride 2016
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)
Retardation factors Im purity A = about 0.2; M axim um 10 ppm.
amisulpride = about 0.5. Dissolve 4.0 g by gently heating in 5 m L o f dilute acetic
System suitability T he chrom atogram obtained with reference add R. Allow to cool and dilute to 20 m L w ith water R.
solution (b) shows 2 clearly separated spots. 12 m L o f the solution complies with test A. Prepare the
Limit. reference solution using lead standard solution (2 ppm Pb) R.
— impurity A: any spot due to impurity A is not more L o ss o n d ry in g (2.2.32)
intense than the corresponding spot in the chromatogram Maximum 0.5 per cent, determ ined on 1.000 g by drying in
obtained with reference solution (a) (0.1 per cent). an oven at 105 °C for 3 h.
R e la te d su b s ta n c e s S u lfa te d a s h (2.4.14)
liq u id chromatography (2.2.29). M axim um 0.1 per cent, determ ined on 1.0 g.
Solvent mixture acetomtrUe R l, methanol R2, mobile phase A A SSA Y
(12:16:72 V/V/V).
Dissolve 0.300 g with shaking in a mixture o f 5 m L o f acetic
Test solution Dissolve 0.10 g o f the substance to be examined anhydride R and 50 m L o f anhydrous acetic acid R. T itrate
in 16 m L o f methanol R2, add 12 m L of acetomtrUe R l and w ith 0.1 M perchloric add, determining the end-point
dilute to 100.0 m L with mobile phase A. potentiometrically (2.2.20).
Reference solution (a) Dilute 1.0 m L o f the test solution to 1 m L o f 0.1 M perchloric add is equivalent to 36.95 m g o f
100.0 m L with the solvent mixture. Dilute 1.0 m L o f this C 17H 2 7N 3O 4 S .
IM P U R IT IE S
Reference solution (b) Dissolve the contents o f a vial of
Spedfied impurities A
amisulpride for system suitabUity CRS (containing impurity B)
in 1.0 m L of the solvent mixture. Other detectable impurities (the following substances w ould, if
Column:
the tests in the monograph. They are lim ited by th e general
— size: I = 0.25 m , 0 = 4.6 mm ;
— stationary phase:, base-deactivated oaylsüyl sUica gel for
impurities for demonstration o f compliance. See also 5.10.
Mobile phase:
Control of impurities in substances for pharmaceutical use): B, C,
— mobile phase A: dissolve 0.7 g o f sodium octanesulfonate R
D, E, F, G3 H.
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 h2n
to 1000 m L with water R; H N and enantiomer
— mobile phase B: methanol R2;
CH3
— mobile phase C: acetomtrUe R l :;
A. [(1RS) -1 -ethylpyrrolidin-2-yI] m ethanam ine,

Time Mobile phase A Mobile phase B Mobile phase C
(min) (per cent V/V/V) (per cent V/V/V) (percent V/V/V)
o
0 -1 8 72 16 12
18 - 35 72 50 16 38 12
\
and enantiomer

Detection Spectrophotom eter a t 225 nm . B . 4 - am in o - A/-[[(2i?«S)-1 -ethylpyrrolidin-2-yl] methyl]-5-
Injection 10 ¿iL. (ethylsulfonyl)-2-hydroxybenzamide,
Identification of impurities U se the chromatogram obtained
im purity B.
and enantiomer
Relative retention W ith reference to amisulpride (retention
tim e = about 17 min): im purity B = about 1.1. H,N
above the baseline o f the peak due to impurity B and C . 4 - am ino - iV- [[(2ÆS)-1 -ethylpyirolidin-2-yl]methyl] -5-iodo-
Hv = height above the baseline o f the lowest point o f the 2-methoxybenzamide,
amisulpride.
Calculation of percentage contents Use the concentration of
amisulpride in reference solution (a).
Limits:
0.10 per cent;
— total: m aximum 0.3 per cent;
2016 Amitriptyline Hydrochloride 1-151
o o Content
*o
h 3c N -^ O 99.0 per cent to 101.0 per cent (dried substance).
and enantiomer
H H N
h2n och3
CHARACTERS
CH3 Appearance
W hite or almost white powder or colourless crystals.
D . 4-am ino-N - [ [ (2RS)-1 -ethylpyrrolidin-2-yl] methyl] -2- Solubility
methoxy-5-(methylsulfonyl)benzamide, Freely soluble in water, in ethanol (96 per cent) and in
methylene chloride.
o o
IDENTIFICATION
H2N.XX. ^ 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
T he solution is clear (2.2.1) and not more intensely coloured
na QN
H and enantiomer than reference solution B7 (2.2.2, Method II).
I '0
h2n 0CH3 I Dissolve 1.25 g in water R and dilute to 25 m L with the
ch3
same solvent
F. 4-am ino-N- [[(2i?S)-1-ethyl-1-oxidopyrrolidin-2-yl] Acidity or alkalinity

methyl]-5-(ethylsulfonyl)-2-methoxybenzamide, 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.
o o
❖'! T he solution is yellow. Add 0.4 m L of 0.01 M hydrochloric
h 3c s ^CH3
N acid. T he solution is red.
H H
and enantiomer Related substances
H,N 0CH3
G . 4-amincHN-[(3i?S)-l-ethylpiperidin-3-yl]-5-(ethylsulfonyl)- Test solution Dissolve 50.0 mg o f the substance to be
2-methoxybenzamide, 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 (impurity B) in 5.0 m L o f the test solution and dilute to
H!N ' ^ A o c g3H>H ^ 100.0 m L with the mobile phase.
CH,
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:
— sizer. I = 0.15 m, 0 = 4.6 mm;
PhEur
— stationary phase: end-capped polar-embedded octadecylsUyl
amorphous organosUica polymer R (5 pm);
Mobile phase Mix 35 volumes of acetonitrile R and 65 volumes
★ ★
Amitriptyline Hydrochloride ★ ★ 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.
Injection 10 |iL.
CH3
N HCI Run time 3 times the retention time of amitriptyline.
I
CH3 Relative retention W ith reference to amitriptyline (retention
im purity A = about 2.2.
C 20H 24Q N 549-18-8 — resolution: minimum 2.0 between the peaks due to
impurity B and amitriptyline.
M onoam ine reuptake inhibitor; tricyclic antidepressant. Limits:
— impurity B: no t more than the area of the corresponding
Preparation peak in the chromatogram obtained with reference
Amitriptyline Tablets solution (b) (0.1 p er cent);
PhEur. — impurity A: n o t more than 0.5 times the area of the
corresponding peak in the chromatogram obtained with
D E F IN IT IO N reference solution (b) (0.05 per cent);
3-(10jl 1-Dihydro-5//-dibenzo [a,d\ [7] annulen-5-ylidene) -
iVJV-dimethylpropan-1-amine hydrochloride.
1-152 Amlodipine Besilate 2016

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
— disregard limit. 0.5 times the area o f the peak due to
amitriptyline in the chromatogram obtained with D . R = C H 2-C H 2-C H 2-N (C H 3)2:
reference solution (b) (0.05 per cent). 5-[3-(dimethylamino)propyl]-10,l l-d ihydro-5//-
dibenzo [a,d\ [7] annulen-5-ol,
M axim um 20 ppm. G . R = H: 10,1 l-dihydro-5H-dibenzo[a,if] [7]annulen-5-ol
(dibenzosuberol),
1.0 g complies with test F. Prepare the reference solution
ch3
N.
M axim um 0.5 per cent, d eterm ined on 1.000 g by drying in CH3
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 E. NyN-àimexhÿ{-3-( 1,2,3,4,4a, 10,11,11 a-octahydro-5H -
with 0.1 M sodium hydroxide, determ ining the end-point 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
IM P U R IT IE S
Other detectable impurities (the following substances would, if F . (5£Z, 10£S>5-[3-(dim ethylam ino)propylidene]-10,11-
present at a sufficient level, be detected by one or other of dihydro-5/f-dibenzo [a^d] [7]annulen-10-ol.
the tests in the monograph. They are limited by the general PhEur
★ ★
Control of impurities in substances for pharmaceutical use): C, D, Amlodipine Besilate ★ ★
E, F, G. *****
A
h3c n^ ^ o ^ s^ nh2
SOjH
,0 . Jl 1 .0 , .c h 3
H
*c Yo t
:
CT
o
^
A. 10,11 -dihydro-5H-dibenzo [a,d\ [7] annulen-5-one
(dibenzosuberone), o ci and enantiomer
CH3 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)-isyV-
D E F IN IT IO N
dimethylpropan-1-amine (cydobenzaprine),
3-Ethyl 5-methyl (4R^)-2-[(2-aminoethoxy)methyl]-4-
(2-chlorophenyl)-6-methyl-l,4-dihydropyridine-3,5-
dicarboxylate benzenesulfonate.
.C H ,
C o n te n t
CHARACTERS
A p p e a ra n c e
C. 3-(10,l l-dihydro-5H-dibenzo[a,d] [7]annulen-5-ylidene)- W hite or almost white powder.
N -m ethylpropan-1-am ine (nortriptyline),
2016 Amlodipine Besilate 1-153
Solubility System suitability: reference solution (b):

Slightly soluble in water, freely soluble in methanol, sparingly — resolution: minimum 2.0 between the peaks due to
soluble in anhydrous ethanol, slightly soluble in 2-propanol. impurities G and B.
ID E N T IF IC A T IO N Limits:
Infrared absorption spectrophotometry (2.2.24). — correction factors: for the calculation o f content, multiply
Comparison amlodipine besilate CRS.
corresponding correction factor: impurity D = 1.7;
TESTS impurity F = 0.7;
O p tic a l ro ta tio n (2.2.7) — impurity D: not more than 3 times the area o f the
— 0.10° to + 0.10°. principal peak in the chromatogram obtained with
Dissolve 0.250 g in methanol R and dilute to 25.0 m L with reference solution (a) (0.3 per cent);
the same solvent. — impurity A: n o t more than 1.5 times the area of the
Related substances
reference solution (d) (0.15 p er cent);
Liquid chromatography (2.2.29). Carry out the test protected
— impurities E, F: for each impurity, not more than
front light.
Test solution (a) Dissolve 50.0 mg of the substance to be chromatogram obtained with reference solution (a)
examined in the mobile phase and dilute to 50.0 m L with (0.15 per cent);
the mobile phase. — unspecified impurities: for each impurity, not more than the
Test solution (b) Dilute 5.0 m L of test solution (a) to area o f the prindpal peak in th e chromatogram obtained
100.0 m L with the mobile phase. w ith reference solution (a) (0.10 per cent);
Reference solution (a) Dilute 1.0 m L o f test solution (a) to — total: maximum 0.8 per cent;
10.0 m L with the mobile phase. Dilute 1.0 m L o f this — disregard limit. 0.5 times the area of the principal peak in
solution to 100.0 m L with the mobile phase. the chromatogram obtained w ith reference solution (a)
Reference solution (b) Dissolve 5 mg of amlodipine (0.05 per cent); disregard any peak due to benzene
impurity B CRS and 5 mg o f amlodipine impurity G CRS in sulfonate (relative retention = about 0.14).
the mobile phase and dilute to 50.0 m L with the mobile W a te r (2.5.12)
phase. Dilute 1.0 m L o f the solution to 10.0 m L with the M aximum 0.5 p er cent, determined on 1.000 g.
mobile phase. S u lfa te d a s h (2.4.14)
Reference solution (c) Dissolve 5 mg o f amlodipine for peak M aximum 0.2 per cent, determined on 1.0 g.
identification CRS (containing impurities D , E and F) in
ASSAY
10 m L of the mobile phase.
Reference solution (d) Dissolve 5.0 mg of amlodipine related substances with the following modification.
impurity A CRS in acetomtrUe R and dilute to 5.0 m L with
Injection T est solution (b), reference solution (e).
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 Calculate the percentage content o f C26H31C1N208S from
10.0 m L with the mobile phase. the declared content o f amlodipine besilate CRS.
Reference solution (e) Dissolve 50.0 mg o f amlodipine STO RA G E
besilate CRS in the mobile phase and dilute to 50.0 m L with In an airtight container, protected from light.
the mobile phase. Dilute 5.0 m L of the solution to 100.0 m L
IM P U R IT IE S
with the mobile phase.
Specified impurities A, D , E, F
Column:
— size: I = 0.25 m , 0 = 4.0 mm;
— stationary phase: octadecylsüyl silica gel for chromatography R
(5 |im );
Mobile phase 2.3 g/L solution of ammonium acetate R, (2034). It is therefore n o t necessary to identify these
methanol R (30:70 VIV). impurities for demonstration of compliance. See also 5.10.
Flow rate 1.5 mlVmin. Control of impurities in substances for pharmaceutical use): B, G,
Detection Spectrophotom eter at 237 nm. H.
Injection 20 |iL o f test solution (a) and reference solutions (a),
(b), (c) and (d).
Run time Twice the retention time o f amlodipine.
with amlodipine for peak identification CRS and the
the peaks due to impurities D , E and F; use the
identify the peak due to impurity A.
Relative retention W ith reference to amlodipine (retention
time = about 20 min): impurity G = about 0.21; A. 3-ethyl 5-methyl (4&S)-4-(2-chlorophenyl)-2-[[2-(l,3-
impurity B = about 0.25; impurity D = about 0.5; dioxo-1,3-dihydro-2//-isoindol-2-yl) ethoxy] methyl]-6-
impurity F = about 0.8; impurity E = about 1.3. m ethyl-1,4-dihydropyridine-3,5-dicarboxylate,
1-154 Ammonia 2016
h3c x
NH
a
and enantiomer
H . 2- [ [2- [[(4&S)-4-(2-chlorophenyl)-3 -(ethoxycarbonyl)-S-

(m ethoxycaibonyl)-6-methyl-1,4-dihydropyridin-2-yI]
B. 3-ethyl 5-methyl (4&S)-4-(2-chlorophenyi)-6-methyl-2- methoxy] ethyl] carbamoyl]benzoic acid.
[ [2-[[2-(methylcarbamoyl)benzoyl] amino] ethoxy] methyl] -1,4- ___________________________________________________________ PhEur
dihydropyridine-3,5-dicarboxylate,
Strong Ammonia Solution *****

*★ **
(Ammonia Solution, Concentrated, *
NH3 17.03
Preparation
D. 3-ethyl 5-methyl 2-[(2-aminoethoxy)methyl]-4- D ilute Ammonia Solution
(2-chlorophenyl)-6-methylpyridine-3,5-dicarboxyiate, 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- IDENTIFICATION

(2-chlorophenyl)-6-methyl-l,4-dihydropyridine-3,5- A. Relative density (2.2.5): 0.892 to 0.910.
dicarboxylate, 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
Evaporate 220 m L almost to dryness on a water-bath. Cool,
F. dimethyl (4RS)-2-[(2-aminoethoxy)methyi]-4- add 1 m L o f dilute acetic add R and dilute to 20 m L with
(2-chlorophenyl) -6-m ethyl-134-dihydropyridme-3,5- distilled water R.
dkarboxylate,
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-1,4-

Pyridine and related substances
Maximum 2 ppm , calculated as pyridine.
dihydropyridine-3,5-dicarboxylate,
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.
2016 Ammonio Methacrylate Copolymer 1-155
Carbonates ★ ★
M aximum 60 ppm.
Ammonio Methacrylate Copolymer ★ ★
T o 10 m L in a test-tube with a ground-glass neck add (Type A) *****
10 m l, o f calcium hydroxide solution R. Stopper immediately (Ph Eur monograph 2081)
and mix. Any opalescence in the solution is not more intense
than that in a standard prepared at the same time and in the
r
° 1
0
same manner using 10 m L o f a 0.1 g/L solution of anhydrous
J k 0 / * \ ch3
O
X
H2C ^
«0
\
sodium carbonate R.
O
2
-
X
C h lo rid e s (2.4.4) ch3
M aximum 1 ppm.
S u lfates (2.4.13)
cr
Maximum 5 ppm.
Iro n (2.4.9)
A ctio n a n d u se
M aximum 0.25 ppm.
Excipient.
H eavy m e ta ls (2.4.8) PhEir.
M aximum 1 ppm. D E F IN IT IO N
Dilute 4 m L o f solution S to 20 m L with water R. 12 m L o f Poly(ethyl propenoate-co-methyl 2-methylpropenoate-co-2-
the solution complies with test A. Prepare the reference (trimethylammonio)ethyl 2-methylpropenoate) chloride
solution using lead standard solution (2 ppm Pb) R. having a m ean relative molecular mass of about 150 000.
R esid u e o n e v a p o ra tio n T h e ratio o f ethyl propenoate groups to methyl
Maximum 20 m g/L 2-methylpropenoate groups to 2-(trimethylammonio)ethyl
Evaporate 50 m L to dryness on a water-bath and dry at 2-methylpropenoate groups is about 1:2:0.2.
100-105 °C for 1 h. T he residue weighs a maximum of 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
1 mg. 8.9 per cent to 12.3 per cent (dried substance).
ASSAY CHARACTERS
Weigh accurately a flask with a ground-glass neck containing A p p e a ra n c e
50.0 m L o f 1 M hydrochloric add. A dd 2 m L of the substance Colourless to white or almost white granules or powder.
to be examined and re-weigh. Add 0.1 m L of methyl red S o lu b ility
solution R as indicator. T itrate with 1 M sodium hydroxide Practically insoluble in water, freely soluble in anhydrous
until the colour changes from red to yellow. ethanol and in methylene chloride giving clear to cloudy
1 m L of 1 M hydrochloric acid is equivalent to 17.03 m g of solutions. D ue to the polymeric nature o f the substance, a
N H 3. stirring time of u p to 5 h may be necessary.
STORAGE ID E N T IF IC A T IO N
Protected from air, at a temperature not exceeding 20 °C. 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).
Ammonio Methacrylate Copolymer * * * * *
★ ★
Solution A Dissolve 3.5 g o f sodium perchlorate R in water for (Type B) * * * * *
chromatograpky R and dilute to 100 m L with the same (Ph Eur monograph 2082)
solvent.
Test solution Dissolve 5.00 g of the substance to be examined o
in methanol R and dilute to 50.0 m L with the same solvent.
HjC ^ A ^ C H s
T o 10.0 m L o f this solution add 5.0 m L o f solution A, o ch3
dropwise, while continuously stirring. Remove the ch3
precipitated polymer by centrifugation. U se the clear
supernatant solution. 9 H,C CH3
Reference solution Dissolve 50.0 m g o f ethyl acrylate R and H*0 -"•'C H , cr
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.
Column: A c tio n a n d u se
— sizer. I = 0.12 m , 0 = 4.6 mm; Excipient
— stationary phase: octadecylsUyl silica gel for chromatography R PhEur_____________
(7 Jim).
D E F IN IT IO N
Mobile phase Dilute phosphoric add R with water for
chromatography R to obtain a solution at p H 2.0; Poly(ethyl propenoate-co-m ethyl 2-m ethylpropenoate-co-2-
(trimethylammpnio)ethyl 2-m ethylpropenoate) chloride
mix 800 m L o f this solution and 200 m L o f methanol R, filter
and degas. having a m ean relative m olecular mass o f about 150 000.
Flow rate 2.0 mL/min. T h e ratio o f ethyl propenoate groups to methyl
Detection Spectrophotom eter at 202 nm . 2-methylpropenoate groups to 2-(trimethylam monio)ethyl
2-methylpropenoate groups is about 1:2 :0 . 1.
Injection 50 pL.
System suitability: reference solution: 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
— resolution: minimum 1.5 between the peaks due to 4.5 per cent to 7.0 per cent (dried substance).
impurity A and im purity B. CHARACTERS
Limits: A p p e a ra n c e
— impurity A: not m ore than the area of the corresponding Colourless to white or almost white granules or powder.
peak in the chrom atogram obtained w ith the reference
S o lu b ility
solution (100 ppm );
— impurity B: not m ore than 2.5 times the area of the ethanol and in methylene chloride giving clear to cloudy
corresponding peak in the chrom atogram obtained with solutions. D ue to the polymeric nature o f die substance, a
the reference solution (50 ppm). 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 ID E N T IF IC A T IO N
M axim um 20 ppm. Comparison Ph. Eur. reference spectrum of ammonio methacrylate
1.0 g complies with test C. Prepare the reference solution copolymer (type B).
using 2.0 m L of lead standard solution (10 ppm Pb) R. B. Viscosity (see Tests).
L o ss o n d ry in g (2.2.32) C. It complies w ith the limits of the assay.
M axim um 3.0 per cent, determ ined on 1.000 g by drying TESTS
in vacuo at 80 °C for 5 h. S o lu tio n S
A SSA Y Dissolve a quantity o f die substance to be examined
Dissolve 1.000 g in a mixture o f 3 mL o f anhydrous formic corresponding to 12.5 g o f the dried substance in a m ixture
add R and 30 m L o f anhydrous acetic add R and heat to of 35.0 g of acetone R and 52.5 g o f 2-propanol R.
dissolve. A dd 20 m L o f acetic anhydride R. T itrate with 0.1 M V iscosity (2.2.10)
perchloric acid, determining the end-point potentiometrically M axim um 15 mPa-s, determ ined on solution S.
(2.2.20).
Apparatus Rotating viscometer.
o f C 9H 1802 N C 1 (ammonio methacrylate groups).
Dimensions:
— spindle: diam eter = 25.15 m m ; height = 90.74 m m ; shaft
IM P U R IT IE S diam eter = 4.0 mm;
Specified impurities A, B — cylinder, diam eter = 27.62 m m ; height = 0.135 m.
Stirring speed 30 r/min.
HjC Volume of sdution 16 m L of solution S.
Temperature 20 °C.
A. R = H , R ; = C 2H 5: ethyl propenoate (ethyl acrylate), 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
B. R = R ' = C H 3: methyl 2-m ethyipropenoate (methyl
drying a clear film is formed.
methacrylate).
PhEur
2016 Ammonium Bicarbonate 1-157
M o n o m e rs
L iquid chromatography (2.2.29).
Ammonium Bicarbonate ?**%
Solution A Dissolve 3.5 g of sodium perchlorate R in water for *★ ★*
(Ammonium Hydrogen Carbonate, *
chromatography R and dilute to 100 m L with the same Ph Eur monograph 1390)
solvent. NHtHCOa 79.1 1066-33-7
in methanol R and dilute to 50.0 m L with the same solvent. A ctio n a n d u se
T o 10.0 m L of this solution add 5.0 m L o f solution A, Expectorant
dropwise, while continuously stirring. Remove the P re p a ra tio n s
precipitated polymer by centrifugation. Use the clear Aromatic Ammonia Solution
supernatant solution.
Strong Ammonium Acetate 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 Aromatic Ammonia Spirit
50.0 m L with the same solvent. Dilute 1.0 m L of the PhEur__________________________________________________________
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. D E F IN IT IO N
Column: C o n ten t
— sizer. I = 0.12 m , 0 = 4.6 mm; 98.0 per cent to 101.0 per cent.
— stationary phase: octadecylsSyl silica gel for chromatography R CHA RACTERS
(7 pm). A p p e a ra n c e
Mobile phase Dilute phosphoric acid R with water for Fine, white or almost white, crystalline powder or white or
chromatography R to obtain a solution at p H 2.0; almost white crystals, slightly hygroscopic.
mix 800 m L of this solution and 200 m L o f methanol R, filter Solubility
and degas. Freely soluble in water, practically insoluble in ethanol
Flow rate 2.0 mL/min. (96 per cent).
Detection Spectrophotometer at 202 nm. It volatilises rapidly at 60 °C. T h e volatilisation takes place
Injection 50 (iL. slowly at ambient temperatures if the substance is slightly
System suitability: reference solution: moist. It is in a state of equilibrium with ammonium
— resolution: minimum 1.5 between the peaks due to carbamate.
impurity A and impurity B.
Limits: A. It gives the reaction o f carbonates and bicarbonates
— impurity A: n o t more than the area o f the corresponding
(2.3.1).
B. Dissolve 50 m g in 2 m L of water R. T he solution gives the
solution (100 ppm);
— impurity B: not more than 2.5 times the area o f the reaction o f amm onium salts (2.3.1).
corresponding peak in the chromatogram obtained with TESTS
the reference solution (50 ppm). S o lu tio n S .
M e th a n o l (2.4.24, System A) Dissolve 14.0 g in 100 m L of distilled water R. Boil, to remove
M aximum 1.5 per c e n t the ammonia, allow to cool and dilute to 100.0 m L with
H eav y m e ta ls (2.4.8) distilled water R.
M axim um 20 ppm. C h lo rid es (2.4.4)
1.0 g complies with test C. Prepare the reference solution Maximum 70 ppm .
using 2.0 m L of lead standard solution (10 ppm Pb) R. Dilute 5 m L of solution S to 15 m L with water R.
L oss o n d ry in g (2.2.32) S u lfates (2.4.IS)
M axim um 3.0 p er cent, determined on 1.000 g by drying Maximum 70 ppm , determined on solution S.
in vacuo at 80 °C for 5 h.
Iro n (2.4.9)
A SSAY Maximum 40 ppm .
Dissolve 2.000 g in a mixture of 3 m L of anhydrous formic
Dilute 1.8'm L o f solution S to 10 m L with water R.
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 H eav y m e ta ls (2.4.8)
perchloric add, determining the end-point potentiometrically Maximum 10 ppm .
( 2.2.20). Dissolve cautiously 2.5 g in 25 m L o f 1 M hydrochloric acid.
1 m L of 0.1 M perchloric add is equivalent to 20.77 mg 12 m L of the solution complies with test A. Prepare the
o f C 9H i80 2N C 1 (ammonio methacrylate groups). reference solution using lead standard solution (1 ppm Pb) R.
IM P U R IT IE S ASSAY
Specified impurities A, B Dissolve cautiously 1.0 g in 20.0 m L o f 0.5 M sulfuric acid
o 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
HiCY ^ 0' R methyl red solution R as indicator.
R 1 m L o f 0.5 M sulfuric acid is equivalent to 79.1 mg
A. R = H , R ; = C 2H 5: ethyl propenoate (ethyl acrylate), o fN H tH C O s.
B. R = R ' = C H 3: methyl 2-methylpropenoate (methyl ST O R A G E
methacrylate). In an airtight container.
________________________________ !___________ ;______________ PhEur _________________________________________________________ PhEur
1-158 Ammonium Bromide 2016
Flow rate 0.4 m U m in.

Ammonium Bromide *****
+*
Detection Conductivity detector equipped with a suitable ion
(Ph. Eur. monograph 1389) * suppressor.
N H iB r 97.9 12124-97-9 Irqection 50 pL o f test solution (b), reference solutions (a)
PhEur___________________________________________________________
and (b) and the blank solution.
Run time 2.5 times the retention time o f bromide.
D E F IN IT IO N
C o n te n t
Retention tone C hloride = about 5 min; brom ide = about
8 m in; sulfate = about 16 min.
CHARACTERS — resolution: m inim um 8.0 betw een the peaks due to
A p p e a ra n c e chloride and brom ide.
W hite or almost white, crystalline pow der or colourless
Calculation of percentage contents'.
crystals, hygroscopic.
— for chlorides, use the concentration of chloride in
S o lu b ility reference solution (a); correct the area o f the peak due to
Freely soluble in water, sparingly soluble in ethanol chloride in the chrom atogram obtained w ith reference
(96 p er cent). solution (a) by subtracting the area of the peak due to
It becomes yellow w hen exposed to light or air. chloride in the chrom atogram obtained with test
solution (b);
— for sulfates, use the concentration o f sulfate in reference
A. It gives reaction (a) o f bromides (2.3.1).
solution (a); correct the area o f the peak due to sulfate in
B. 10 m L of solution S (see Tests) gives the reaction o f the chrom atogram obtained with reference solution (a) by
amm onium salts (2.3.1). subtracting the area o f the peak due to sulfate in the
TESTS chrom atogram obtained w ith test solution (b).
S o lu tio n S Limits:
Dissolve 10.0 g in carbon dioxide-free water R and dilute to — chlorides: maximum 0.6 p er cent;
100 m L with the sam e solvent. — sulfates: m axim um 0.01 p er cent.
A p p e a ra n c e o f so lu tio n Iodides
Solution S is clear (2.2.1) and colourless (2.2.2, Method II). T o 5 m L o f solution S add 0.15 m L o f ferric chloride
A c id ity o r a lk a lin ity
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).
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 Iron (2.4.9)
sodium hydroxide is required to change the colour o f the Maximum 20 ppm .
indicator. D ilute 5 m L o f solution S to 10 m L with water R.
B ro m a te s M agnesium and alkaline-earth m etals (2.4.7)
T o 10 m L o f solution S add 1 m L o f starch solution R, Maximum 200 ppm , calculated as Ca.
0.1 m L of a 100 g/L solution o f potassium iodide R and 10.0 g complies w ith the test for magnesium and alkaline-
0.25 m L of 0.5 M sulfuric add and allow to stand protected earth metals. T h e volume o f 0.01 M sodium edetate used does
from light for 5 min. N o blue or violet colour develops. n o t exceed 5.0 m L
C h lo rid e s a n d su lfa te s Heavy m etals (2.4.8)
L iquid chrom atography (2.2.29). Maximum 10 ppm .
Test solution (a) Dissolve 0.400 g o f the substance to be 12 m L o f solution S complies with test A. Prepare the
examined in 50 m L o f water for chromatography R and dilute 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 Maximum 1.0 per cent, determ ined on 1.000 g by drying in
50.0 m L with water for chromatography R.
an oven at 105 °C.
Reference solution (a) T o 25.0 m L o f test solution (a) add Sulfated ash (2.4.14)
1.0 m L o f sulfate standard solution (10 ppm SO 4) R and
Maximum 0.1 p er cent, determ ined on 1.0 g.
12.0 m L of chloride standard solution (50 ppm Cl) R and dilute
to 50.0 m L with water for chromatography R. A SSA Y
Reference solution (b) D ilute 10.0 m L o f test solution (a) to Dissolve 80.0 m g in water R , add 5 m L o f dilute nitric add R
100.0 m L with water for chromatography R. T o 2.0 m l, of this and dilute to 50 m L with water R. T itrate with 0.1 M silver
solution add 8.0 m L o f chloride standard solution nitratey determ ining the end-point potentiometrically (2.2.20).
(50 ppm Cl) R and dilute to 20.0 m L w ith water for 1 m L o f 0.1 M silver nitrate is equivalent to 9.794 m g of
chromatography R. N H iB r.
Blank solution water for chromatography R. Calculate the percentage content of N H ^B r using the
Column: following expression:
— size. I — 0.25 m , 0 = 2 m m ;
— stationary phase, strongly basic anion-exchange resin for o - 2.763 b
chromatography R (13 pm).
Mobile phase Dissolve 0.600 g o f potassium hydroxide R in a = percentage content o f N H jB r and NH4CI obtained in
water for chromatography R and dilute to 1000.0 m L with the the assay and calculated as NH*Br;
same solvent. b = percentage content o f Cl obtained in the test for
chlorides.
2016 Ammonium Glycyrrhizinate 1-159
STO RA G E L oss o n d ry in g (2.2.32)

In an airtight container, protected from light. M aximum 1.0 per cent, determined on 1.00 g by drying in
__________________________________________________________ PhEur
S u lfa ted a s h (2.4.14)
ASSAY
Dissolve 1.000 g in 20 m L of water R and add a mixture of
Ammonium Chloride
5 m L of formaldehyde solution R, previously neutralised to
*+ +*
(Ph Eur monograph 0007) * phenolphthalein solution R, and 20 m l. o f water R. After
NH 4CI 53.49 12125-02-9 1-2 min, titrate slowly with 1 M sodium hydroxide, using a
further 0.2 m L of the same indicator.
A ction a n d use 1 m L of 1 M sodium hydroxide is equivalent to 53.49 mg
Used for the acidification of urine and to correct metabolic o f N H 4CI.
alkalosis. __________________________________________________________PhEur
P re p a r a tio n
Ammonium Chloride Mixture
PhEtr---------------------------------------------------------------------------------------- Ammonium Glycyrrhizinate *****

D E F IN IT IO N *★ ★*
(Ammonium Gfycyrrhizate, Ph Eur monograph 1772) *
C o n ten t
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.
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).
GKHssNOje 840 53956-04-0
TESTS
PhEtr__________________________________________________________
S o lu tio n S
Dissolve 10.0 g in carbon dioxide-free water R prepared from D E F IN IT IO N
distilled water R an d dilute to 100 m L with the same solvent. M ixture o f amm onium 18a- and 18^-glycyrrhizate
A p p e a ra n c e o f so lu tio n (ammonium salt of (20P)-3^-[[2-0-(^- d-
Solution S is clear (2.2.1) and colourless (2.2.2, Method II). glucopyranosyhironic acid)-a-D-glucopyranosyluronic
acid]oxy]-ll-oxoolean-12-en-29-oic ad d ), the 18|3-isomer
A cidity o r alk a lin ity
being the m ain com ponent
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 C o n te n t
sodium hydroxide is required to change the colour of the 98.0 per cent to 102.0 per cent (anhydrous substance).
indicator. CHARACTERS
Bromides and iodides A p p e a ra n c e
T o 10 m l. of solution S add 0.1 m L of dilute hydrochloric W hite or yellowish-white, hygroscopic powder.
acid R and 0.05 m l. of chloramme solution R. After 1 min, add S olu b ility
2 m l. o f chloroform R and shake vigorously. T h e chloroform Slightly soluble in water, very slightly soluble in anhydrous
layer remains colourless (2.2.2, Method I). ethanol, practically insoluble in acetone. It dissolves in dilute
S u lfates (2.4.13) solutions of adds and of alkali hydroxides.
M aximum 150 ppm . ID E N T IF IC A T IO N
Dilute 10 m L o f solution S to 15 m L with distilled water R. A Infrared absorption spectrophotometry (2.2.24).
C a lc iu m (2.4.3) Comparison ammonium gfycyrrkizate CRS.
M axim um 200 ppm . B. Dissolve 0.1 g in 20 m L of water R, add 2 m L o f dilute
Dilute 5 m L of solution S to 15 m L with distilled water R. sodium hydroxide solution R and heat cautiously. O n heating,
Iro n (2.4.9) the solution gives off vapours th at may be identified by the
M aximum 20 ppm . 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
H eavy m e ta ls ( 2.4.8) A p p e a ra n c e o f so lu tio n
M aximum 10 ppm . T he solution is clear (2.2.1) and n o t more intensely coloured
than reference solution BY7 (2.2.2, Method I).
reference solution using lead standard solution (1 ppm Pb) R. Dissolve 1.0 g in ethanol (20 per cent V/V) R and dilute to
1-160 Amobarbital 2016
Specific optical rotation (2.2.7) STO RA G E

+ 49.0 to + 54.0 (anhydrous substance). In an airtight container.
Dissolve 0.5 g in ethanol (50 per cent V/V) R and dilute to IM P U R IT IE S
Related substances
the mobile phase.
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
Column:
— size: I = 0.25 m , 0 = 4.0 mm, A. (4(3,20P)-3fH[2-0-({^-i>giucopyranosyluronic add)-a-D-
— stationary phase: octadecykifyl silica gelfor chromatography R glucopyranosyluronic add]oxy]-23-hydroxy-l l-oxoolean-12-
(5-10 pm). en-29-oic a d d (24-hydroxyglycyrrhizinic a d d ).
Mobile phase glacial acetic acid R, acetomtrUe R, water R ___________________________________________________________ PhEur
(6:380:614 V/V/V).
Injection 10 pL. Amobarbital
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
(retention tim e = about 8 min): impurity A = about 0.8;
18a-glycyrrhizic a d d = about 1.2 .
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
C n H 18N 20 3 226.3 57-43-2
reference solution (a) ( 10.0 per cent),
— impurity A: not m ore than the sum o f the areas o f the A c tio n a n d u se
peaks in the chrom atogram obtained with reference Barbiturate.
solution (a) (5.0 p e r cent),
— any other impurity: for each impurity, not more than PhEur__________________________________________________________
0.4 times the sum o f the areas o f the peaks in the D E F IN IT IO N
chromatogram obtained with reference solution (a)
Amobarbital contains n o t less than 99.0 p er cent and not
(2.0 per cent),
more than the equivalent of 101.0 p er cent o f 5-ethyl-5-
— sum of other impurities: not more than 1.4 times the sum o f
(3-methylbutyI)pyrimidin-2,4,6 (1 H,3H,5H) -trione, calculated
the areas o f the peaks in the chromatogram obtained with
with reference to die dried substance.
reference solution (a) (7.0 per cent),
— disregard limit: 0.04 times the sum o f the areas o f the CHARACTERS
peaks in the chrom atogram obtained with reference A white or alm ost white, crystalline powder, very slightly
solution (a) (0.2 p er cent). soluble in water, freely soluble in alcohol, soluble in
Heavy m etals (2.4.8) methylene chloride. It forms water-soluble compounds with
Maximum 20 ppm . alkali hydroxides and carbonates and with ammonia.
1.0 g complies with limit test C. Prepare the reference ID E N T IF IC A T IO N

solution vising 2 m L o f lead standard solution (10 ppm Pb) R. First identification A , B.
Water (2.5.12) Second identification A , C, D.
M aximum 6.0 per cent, determ ined on 0.250 g. A. D eterm ine the m d tin g point (2.2.14) o f the substance to
Sulfated ash (2.4.14) be ex a m in ed . Mix equal parts o f the substance to be
M axim vim 0.2 per cent, determ ined on 1.0 g. 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
ASSAY points (which are about 157 °C) is n o t greater than 2 °C.
Dissolve 0.600 g in 60 m L o f anhydrous acetic acid R heating
B. Examine by infrared absorption spectrophotom etry
at 80 °C if necessary. Cool. T itrate with 0.1 M perchloric acid,
determining the end-point potentiometrically (2.2.20).
amobarbital CRS.
1 m L o f 0.1 M perchloric add is equivalent to 84.0 m g
C. Examine by thin-layer chromatography (2.2.27), using
Of C 42H 65N O 16.
silica gel GF2 5 4 R as the coating substance.
2016 Amobarbital Sodium 1-161

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 (Ph Eur monograph 0166) *
alcohol R and dilute to 100 m L with the same solvent.
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. C 1iH 17N aN a03 248.3 6443-7
D . It gives the reaction of non-nitrogen substituted
barbiturates (2.3.1). A ctio n a n d u se
Barbiturate.
TESTS
A p p e a ra n c e o f so lu tio n PhEur__________________________________________________________
Dissolve 1.0 g in a mixture of 4 m L o f dilute sodium hydroxide D E F IN IT IO N
solution R and 6 m L o f water R. T he solution is clear (2.2.1) Amobarbital sodium contains not less than 98.5 per cent and
and not more intensely coloured than reference solution Y 6
not more than the equivalent of 102.0 per cent o f sodium
(2.2.2, Method II). derivative o f 5-ethyl-5-(3-methylbutyi)pyrimidin-
A cid ity o r alk alin ity 2,4,6(lH ,3H ,5ii)-trione, calculated with reference to the
T o 1.0 g add 50 m L o f water R and boil for 2 m in. Allow to dried substance.
cool and filter. T o 10 m L of the filtrate add 0.15 m L of
CHARACTERS
methyl red solution R and 0.1 m L of 0.01 M sodium hydroxide.
A white or almost white, granular powder, hygroscopic, very
T he solution is yellow. Add 0.2 m L of 0.01 M hydrochloric
soluble in carbon dioxide-free water (a small fraction may be
add. T h e solution is red.
insoluble), freely soluble in alcohol.
Examine by thin-layer chromatography (2.2.27), using silica ID E N T IF IC A T IO N
gel GF254 R as the coating substance. First identification A , B, E
Test solution Dissolve 1.0 g of the substance to be examined Second identification A , C, D, E.
in alcohol R and dilute to 100 m L with the same solvent. A. Acidify 10 m L of solution S (see Tests) with dilute
Reference solution D ilute 0.5 m L of the test solution to hydrochloric add R and shake with 20 m L o f ether R. Separate
100 m L with alcohol R. the ether layer, wash w ith 10 m L of water R, dry over
anhydrous sodium sulfate R and filter. Evaporate the filtrate to
Apply separately to the plate 20 |jL o f each solution. Develop
dryness and dry the residue at 100 °C to 105 °C (test
over a p ath of 15 cm using the lower layer from a mixture of
residue). Repeat the operations using 0.1 g of amobarbital
5 volumes of concentrated ammonia R, 15 volumes o f alcohol R
sodium CRS (reference residue). Determine the melting point
and 80 volumes o f chloroform R. Examine the plate
immediately in ultraviolet light at 254 nm . Any spot in the
(2.2.14) of the test residue. Mix equal parts of the test
residue and the reference residue and determine the melting
chromatogram obtained with the test solution, apart from the
point of the mixture. T h e difference between the melting
principal spot, is n o t more intense than the spot in the
points (which are about 157 °C) is not greater th an 2 °C.
chromatogram obtained with the reference solution. Spray
with diphenylcarbazone mercuric reagent R. Allow die plate to B. Examine by infrared absorption spectrophotometry
dry in air and spray with freshly prepared alcoholic potassium (2.2.24), comparing the spectrum obtained with the reference
hydroxide solution R diluted 1 in 5 with aldehyde-free alcohol R. residue prepared from amobarbital sodium CRS with that
Heat at 100 °C to 105 °C for 5 min and examine obtained with the test residue (see identification test A).
immediately. Any spot in the chromatogram obtained with C. Examine by thin-layer chromatography (2.2.27), using
the test solution, apart from the principal spot, is n o t more silica gel GF254 R as the coating substance.
intense than the spot in the chromatogram obtained with the Test solution Dissolve 0.1 g of the substance to be examined
reference solution (0.5 per cent). in alcohol R and dilute to 100 m l, with the same solvent.
L oss o n d ry in g (2.2.32) Reference solution Dissolve 0.1 g of amobarbital sodium CRS in
N ot m ore than 0.5 per cent, determined on 1.000 g by alcohol R and dilute to 100 m L with the same solvent.
drying in an oven at 105 °C. Apply separately to the plate 10 nL of each solution. Develop
S u lfa te d a sh (2.4.14) over a path of 18 cm using the lower layer of a mixture of
N ot m ore than 0.1 per cent, determined on 1.0 g. 5 volumes o f concentrated ammonia R, 15 volumes o f alcohol R
and 80 volumes of chloroform R. Examine immediately in
A SSAY
ultraviolet light at 254 nm . T he principal spot in the
Dissolve 0.100 g in 5 m L of pyridine R. A dd 0.5 m L of
chromatogram obtained with the test solution is similar in
thymolphthalein solution R and 10 m L o f silver nitrate solution
in pyridine R. Titrate with 0.1 M ethanolic sodium hydroxide
obtained with the reference solution.
until a pure blue colour is obtained. Carry out a blank
titration. D . It gives the reaction o f non-nitrogen substituted
barbiturates (2.3.1).
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. E. It gives reaction (a) o f sodium (2.3.1).
____________________________________________ _____________ Ph Eur

1-162 Amoxicillin Sodium 2016
TESTS ***
Amoxicillin Sodium ★ *
S o lu tio n S ★ ★
Dissolve 5.0 g in alcohol (50 per cent V/V) R and dilute to (Ph. Eur. monograph 0577) *****
A p p e a ra n c e o f so lu tio n ! COzHa
Solution S is clear (2.2.1) and not m ore intensely coloured
p H (2.2.3)
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.
C 16H18N 3N a 0 5S 387.4 34642-77-8
Examine by thin-layer chromatography (2.2.27), using silica A c tio n a n d u se
gel GF254 R as the coating substance. Penicillin antibacterial.
Test solution Dissolve 1.0 g o f the substance to be examined P re p a r a tio n s
in alcohol R and dilute to 100 m L w ith the same solvent. Amoxicillin Injection
Reference solution D ilute 0.5 m L of the test solution to Co-amoxiclav Injection
100 m L with alcohol R.
PhEur.
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 D E F IN IT IO N
5 volumes o f concentrated ammonia R, 15 volumes o f alcohol R Sodium (2S,5R,6R)-6-[[(2i?)-2-amino-2-(4-hydroxyphenyl)
and 80 volumes of chloroform R. Examine the plate acetyl] amino] -3,3-dimethyl-7 -oxo-4-thia-1 -
immediately in ultraviolet light at 254 nm . Spray with azabicydo[3.2.0]heptane-2-carboxylate.
diphenylcarbazone mercuric reagent R. Allow the plate to dry in Semi-synthetic product derived from a fermentation product.
air and spray with freshly prepared alcoholic potassium
C o n te n t
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 CHARACTERS
spraying, any spot in the chromatogram obtained with the A p p e a ra n c e
test solution, apart from the principal spot, is no t more W hite or almost white, very hygroscopic, powder.
intense than the spot in the chromatogram obtained with the S o lu b ility
reference solution (0.5 per cent). Disregard any spot at the Very soluble in water, sparingly soluble in anhydrous ethanol,
point o f application. very slightly soluble in acetone.
L oss o n d ry in g (2.2.32) ID E N T IF IC A T IO N
N o t m ore than 3.0 per cent, determined on 0.50 g by drying
First identification A , D.
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 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
in pyridine R. T itrate with 0.1 M ethanolic sodium hydroxide
iced water. Filter die crystals and wash with 2-3 m L o f a
until a pure blue colour is obtained. Carry out a blank
titration. 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.
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 ^ .
Comparison amoxicillin trihydrate CRS.
STORAGE
Test solution Dissolve 25 m g o f die substance to be examined
Store in an airtight container.
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-
2016 Amoxicillin Sodium 1-163
System suitability: reference solution (b): — mobile phase B: mix 20 volumes of acetonitrüe R and
— the chromatogram shows 2 clearly separated spots. 80 volumes o f a 25 per cent V/V solution o f 0.2 M
Results T h e principal spot in the chromatogram obtained with potassium dihydrogen phosphate R adjusted to p H 5.0 with
the test solution is similar in position, colour and size to the dilute sodium hydroxide solution R\
solution (a). Time Mobile phase A Mobile phase B
C. Place about 2 m g in a test-tube about 150 m m long and (min) (per cent V/V) (per cent V/V)
about 15 mm in diameter. M oisten with 0.05 m L o f water R 0 -t, 92 8
and add 2 m L o f sulfuric acid-formaldehyde reagent R. M ix the f ,- ( f , + 25) 92 -» 0 8 4 100
contents o f the tube by swirling; the solution is practically
colourless. Place the test-tube in a water-bath for 1 min; (Í, + 25) - (Í, + 40) 0 100
a dark yellow colour develops. (t^ + íO M ^ + ss) 92 8
D . It gives reaction (a) o f sodium (2.3.1). t, = retention time of amoxicillin determined with reference solution (c)
TESTS
A p p e a ra n c e o f so lu tio n I f the mobile phase has been adjusted to achieve the required
T h e solution is n o t more opalescent than reference resolution, the adjusted composition will apply at time zero
suspension II (2.2. 1), it may show an initial, b u t transient, in the gradient and in the assay.
pink colour, and after 5 min, its absorbance (2.2.25) at
430 nm is not greater than 0.20.
same solvent Examine immediately after dissolution.
Injection 50 |iL o f reference solutions (b) and (c) with
isocratic elution at the initial mobile phase composition and
p H (2.2.3) 50 |iL o f test solution (b) and reference solution (d)
8.0 to 10.0. according to the elution gradient described under Mobile
Dissolve 2.0 g in carbon dioxide-free water R and dilute to phase; inject mobile phase A as a blank according to the
20 m L with the same solvent. elution gradient described under Mobile phase.
S p ecific o p tic a l ro ta tio n (2.2.7) Identification of impurities Use the chromatogram obtained
+ 240 to + 290 (anhydrous substance). w ith reference solution (d) to identify the 3 principal peaks
Dissolve 62.5 m g in a 4 g/L solution of potassium hydrogen eluted after the m ain peak corresponding to impurity C,
phthalate R and dilute to 25.0 m L with the same solution. amoxicillin dim er (impurity J; n = 1) and amoxicillin trim er
(impurity J; n = 2).
Liquid chromatography (2.2.29). Relative retention W ith reference to amoxicillin:
im purity C = about 3.4; impurity J (n = 1) = about 4.1;
Test solution (a) Dissolve 30.0 mg o f the substance to be
impurity J (n = 2) = about 4.5.
examined in mobile phase A and dilute to 50.0 m L with
mobile phase A. System suitability, reference solution (b):
amoxicillin and cefadroxil; if necessary, adjust the ratio
A:B o f the mobile phase.
mobile phase A. Prepare immediately before use.
Limits:
Reference solution (a) Dissolve 30.0 m g o f amoxicillin — impurity J (n = 1): not more than 3 times the area o f the
trihydrate CRS in mobile phase A and dilute to 50.0 m L with principal peak in the chromatogram obtained with
mobile phase A. reference solution (c) (3 per cent);
Reference solution (b) Dissolve 4.0 m g of cefadroxü CRS in — any other impurity: for each impurity, n o t more than twice
mobile phase A and dilute to 50 m L with mobile phase A. the area o f th e principal peak in the chromatogram
T o 5.0 m L o f this solution add 5.0 m L of reference obtained w ith reference solution (c) (2 per cent);
solution (a) and dilute to 100 m L with mobile phase A. — total: not m ore than 9 times the area o f the principal peak
Reference solution (c) Dilute 2.0 m L o f reference solution (a) in the chromatogram obtained with reference solution (c)
to 20.0 m L with mobile phase A. Dilute 5.0 m L of this (9 per cent);
solution to 20.0 m L with mobile phase A. — disregard limit. 0.1 times the area of the principal peak in
Reference solution (d) T o 0.20 g of amoxicillin trihydrate R add the chromatogram obtained w ith reference solution (c)
1.0 m L o f water R. Shake and add dropwise dilute sodium (0.1 per cent).
hydroxide solution R to obtain a solution. T he p H of the iV ^V -D im ethylaniline (2.4.26, Method A or B)
solution is about 8.5. Store the solution at room tem perature M aximum 20 ppm.
for 4 h. Dilute 0.5 m L of this solution to 50.0 m L with 2 -E th y lh ex a n o ic a c id (2.4.28)
mobile phase A. Maximum 0.8 per cent m/m.
Column:
— sizer. I = 0.25 m , 0 = 4.6 mm;
M aximum 20 ppm.
— stationary phase: octadecylsüyl silica gel for chromatography R
(5 pm ). 1.0 g complies with test C. Prepare the reference solution
Mobile phase:
— mobile phase A: mix 1 volume o f acetonitrüe R and W a te r (2.5.12)
99 volumes o f a 25 per cent V/V solution o f 0.2 M M axim um 3.0 p er cent, determined on 0.400 g.
potassium dihydrogen phosphate R adjusted to p H 5.0 with
dilute sodium hydroxide solution R]
1-164 Amoxicillin Sodium 2016
B a c te ria l e n d o to x in s (2.6.14) H\jP O2H

Less than 0.25 IU/mg, if intended for use in the manufacture H NH* HN ^CH ,
o f parenteral preparations w ithout a further appropriate j î ^ ^ / ^ c H j 3nd epimer a tC *.
procedure for the removal of bacterial endotoxins. I I H

1 O
A SSAY
related substances with the following modifications. E. (2RS,4S)-2-[ [[(2Æ)-2-amino-2-(4-hydroxyphenyl) acetyl]
amino]methyI] -5,5-dimethylthiazolidine-4-carboxylic a d d
Mobile phase Initial composition o f the mixture of mobile
(penilloic adds of amoxidllin),
phases A and B, adjusted where applicable.
Injection T est solution (a) and reference solution (a).
Calculate the percentage content of amoxicillin sodium by
multiplying the percentage content o f amoxicillin by 1.060.
STORAGE F. 3-(4-hydroxyphenyl)pyrazin-2-ol,
In an airtight container. If the substance is sterile, store in a
IM P U R IT IE S
o V ^ H
V nA ^ h,
H2n - -|— k -S CHa
H H
G. (2S,5R,6R)-6 - [[(2R)-2-[[(2Æ)-2-amino-2-
A. (2S,5/?,6i?)-6-amino-3,3-dimethyl-7-oxo-4-thia-l- (4-hydroxyphenyl) acetyl] amino] 2-
azabicyclo[3.2.0]heptane-2-carboxylic a d d (4-hydroxyphenyl) acetyl] amino]-3,3-dim ethyl-7-oxo-4-thia-1-
(6-aminopenidllanic acid), azabicydo[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-1- H . (2i?)-2-[(2,2-dimethylpropanoyl)amino]-2-
azabicyclo[3.2.0]heptane-2-caiboxylic acid (L-amoxicillin), (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] -5,5-

dimethylthiazolidine-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
J. co-oligomers of amoxidllin and penidlloic ad d s of
a d d (penidlloic ad d s o f amoxicillin),
amoxicillin,
2016 Amoxicillin Trihydrate 1-165
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
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.
K. oligomers o f penicilloic ad d s of amoxicillin. Development Over a path of 15 cm.
____ ______________________________________________________ PhEur Drying la air.
examine in daylight.
— 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
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
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
TESTS
A ctio n a n d u se S o lu tio n S
Penicillin antibacterial. 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.
Amoxicillin Capsules
Amoxicillin Oral Suspension p H (2.2.3)
3.5 to 5.5 for solution S.
Co-amoxiclav Oral Suspension
Co-amoxiclav T ablets
+ 290 to + 315 (anhydrous substance), determined on
Dispersible Co-amoxiclav Tablets
solution S.
PhEur------------------------------------------------------------------------------------------ R e la te d su b sta n c e s
D E F IN IT IO N
(25j5i?j6i?)-6-[[(2R)-2-Amino-2-(4-hydroxyphenyl)acetyl] Buffer solution pH 50 T o 250 m L o f 0.2 M potassium
amino]-3,3-dimethyl-7 -oxo-4-thia-1 - dihydrogen phosphate R add dilute sodium hydroxide solution R
azabicyclo [3.2.0]h ep tane-2-carboxylic a d d trihydrate. to p H 5.0 and dilute to 1000.0 m L with water R
Semi-synthetic product derived from a fermentation product. Test solution (a) Dissolve 30.0 m g of the substance to be
C o n te n t
mobile phase A.
Test solution (b) Dissolve 30.0 m g of the substance to be
CHARACTERS examined in mobile phase A and dilute to 20.0 m l. with
A p p e a ra n c e mobile phase A Prepare immediately before use.
White or almost white, crystalline powder. Reference solution (a) Dissolve 30.0 m g o f amoxicillin
Solubility trihydrate CRS in mobile phase A and dilute to 50.0 m L with
Slightly soluble in w ater, very slightly soluble in ethanol mobile phase A
(96 per cent), practically insoluble in fatty oils. It dissolves in Reference solution (b) Dissolve 4.0 m g of cefadroxU CRS in
dilute a d d s and dilute solutions o f alkali hydroxides. mobile phase A and dilute to 50 m L with mobile phase A.
ID E N T IF IC A T IO N T o 5.0 m L o f this solution add 5.0 m L o f reference
First identification A solution (a) and dilute to 100 m L with mobile phase A
Second identification B, C Reference solution (c) Dilute 2.0 m L o f reference solution (a)
A. Infrared absorption spectrophotometry (2.2.24). to 20.0 m L with mobile phase A. D ilute 5.0 m L o f this
Comparison amoxicillin trihydrate CRS.
Column:
B. Thin-layer chrom atography (2.2.27). — size: I = 0.25 m , 0 = 4.6 mm;
Test solution Dissolve 25 m g o f the substance to be examined — stationary phase: octadecylsHyl silica gel for chromatography R
in 10 m l, o f sodium hydrogen carbonate solution R. (5 pm).
Reference solution (a) Dissolve 25 mg of amoxicillin Mobile phase:
trihydrate CRS in 10 m L o f sodium hydrogen carbonate — mobile phase A: acetonitrüe R, buffer solution p H 5.0
solution R. (1:99 V!V)\
1-166 Amoxicillin Trihydrate 2016
mobile phase B: acetonitrUe R, buffer solution p H 5.0

(20:80 V!V)\

0-h 92 8
B. (25, 5R,6R)-6-[[(25)-2-amino-2-(4-hydroxyphenyl)acetyl]
f, * (i* + 25) 92 -> 0 8 -> 100
amino] -3,3-dimethyl-7 -oxo-4-thia-1 -
(f* + 25) - (/a + 40) 0 100 azabicyclo[3.2.0]heptane-2-carboxylic a d d (L-amoxicillin),
(fÄ+ 4 0 )-(f, + 55) 92 8
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.
Injection 50 (iL of reference solutions (b) and (c) with C. (45)-2-[5-(4-hydroxyphenyl)-3,6-dioxopiperazin-2-yl]-5,5-
isocratic elution at the initial mobile phase composition and dimethylthiazolidine-4-carboxylic a d d (amoxicillin
50 jiL of test solution (b) according to the elution gradient diketopiperazines),
described under Mobile phase; inject mobile phase A as a
blank according to the elution gradient described under h c o 2h
M obile phase.
H N *h
CH*
amoxicillin and cefadroxil; if necessary, adjust the ratio C02H
A:B o f the mobile phase. ;
Limit. D . (45)-2-[[[ (2R)-2-amino-2-(4-hydroxyphenyl)acetyl]
— arty impurity: for each impurity, not more than the area of amino] carboxymethyl] -5,5-dimethylthiazolidine-4-carboxylic
the principal peak in the chromatogram obtained with a d d (penicilloic ad d s o f amoxicillin),
reference solution (c) (1 per cent).
N ,N -D im e th y la n ilin e (2.4.26, Method A orB) CO2H
M aximum 20 ppm.
h nh2 h n ^ c h 3
W a te r (2.5.12) k g ^ X H s and epimer at C*
11.5 per cent to 14.5 per cent, determined on 0.100 g.
ASSAY E. (2RS,45)-2-[[[ (2R)-2-amino-2-(4-hydroxyphenyl)acetyl]
Liquid chromatography (2.2.29) as described in the test for amino]methyl]-5,5-dimethylthiazolidine-4-carboxylic a d d
related substances w ith the following modifications. (penilloic ad d s o f amoxicillin),
Mobile phase Initial composition o f the mixture o f mobile
Injection T e st solution (a) and reference solution (a).
Calculate the percentage content of C i6H 19N 30 5S taking F . 3-(4-hydroxyphenyl)pyrazin-2-ol,
into account the assigned content o f amoxicillin
trihydrate CRS.
STORAGE
IM P U R IT IE S
O > C°2H
V i ><CH3 G . (2S,5R,6R)-6-[ [(2R)-2-[[(2J?)-2-amino-2-
CHa (4-hydroxyphenyI) acetyl] amino]-2-(4-hydroxy-
H H
phenyl)acetyl]amino]-3,3-dimethyi-7-oxo-4-thia-l-
azabicydo[3.2.0]heptane-2-carboxylic a d d
A. (25,5J?,6i?)-6-ammo-3,3-dimethyl-7-oxo-4-thia-l- (D-(4-hydroxyphenyl)glycylamoxidllin),
azabicydo [3.2.0]heptane-2-carboxylic a d d
(6-aminopenidllanic a d d ),
2016 Amphotericin 1-167
CH3 o
H3 Amphotericin * ★
★ ★
H3C H NH
(Amphotericin B, Ph Eur monograph 1292)
C 02H
H . (2i?)-2- [(2 j 2-dimethylpropanoyl) amino] -2-

(4-hydroxyphenyl) acetic add,
I. (2iQ-2-amino-2-(4-hydroxyphenyl) acetic add.
C 4 7H 73 N O 17 924 1397-89-3
Action and use

Antifungal.
Preparation
Amphotericin for Infusion
PhEur______________________
D E F IN IT IO N
Mixture o f antifungal polyenes produced by the growth o f
certain strains o f Streptomyces nodosus or obtained by any
other means. It consists mainly o f amphotericin B which is
J. co-oligomers of amoxicillin and o f penidlloic adds of (1R,3S,5R,6R,9R,UR,15S,16R,11R,18S,19E,21E,23E,~
amoxicillin, 25E,21E,29E,31E,33R,35S,36R,31S)-33-[(3-aiDmo-3,6-
dideoxy-P-D-mannopyranosyl)oxy]-13j5,6,9,ll,17,37-
octahydroxy-15,16,18-trimethyl-13-oxo-14,3 9-dioxa-
bicyclo [33.3.1] nonatriaconta-19,21,23,25,27,29,31-heptaene-
36-caiboxylic ad d .
Content
M inim um 750 IU /m g (dried substance).
CHARACTERS
Appearance
Yellow or orange, hygroscopic powder.
K . oligomers o f penidlloic adds o f amoxicillin, Solubility
Practically insoluble in water, soluble in dimethyl sulfoxide
and in propylene glycol, slightly soluble in
dim ethylfonnamide, very slightly soluble in methanol,
practically insoluble in ethanol (96 per cent).
It is sensitive to light in dilute solutions.
IDENTIFICATION
First identification: B, D.
Second identification A , C
(2.2.25).
L. (2S,5£,6i$-6-[[(2S,5Æ ,6i3-6-[[(2i?)-2-am ino-2-
(4-hydroxyphenyl) acetyl] amino] -3j3-dimethyl-7 -oxo-4-thia-1 - Test solution Dissolve 25 m g in 5 m L of dimethyl sulfoxide R
azabicydo [3.2.0]heptane-2-carbonyl] amino]-3,3-dimethyl-7- and dilute to 50 m L w ith methanol R. Dilute 2 m L o f the
oxo-4-thia-l-azabicydo[3.2.0]heptane-2-carboxylic a d d solution to 200 m L with methanol R.
(6-APA amoxicillin amide). Spectral range 300-450 nm .
PhEur Absorption maxima A t 362 nm , 381 nm and 405 nm .
Absorbance ratios:
— -^ 362^381 = 0.57 to 0.61;
— A^ qi/A^qs — 0.87 to 0.93.
Comparison amphotericin B CRS.
1-168 Amphotericin 2016
If the spectra obtained show differences, dry the substance to adjusted to p H 3.9 using concentrated ammonia R and
be examined and reference substance at 60 °C at a pressure 68 volumes o f acetonitrile R;
not exceeding 0.7 kPa for 1 h and record new spectra.
C. T o 1 m L o f a 0.5 g/L solution in dimethyl sulfoxide R, add Time Mobile phase A Mobile phase B
5 m L o f phosphoric acid R to form a lower layer, avoiding (min) (percent V/V) (per cent V/V)
mixing the 2 liquids. A blue ring is immediately produced at 0 -3 100 0
the junction o f the liquids. Mix, an intense blue colour is
3 -2 3 100-»70 0*30
produced. A dd 15 m L o f water R and mix; the solution
becomes pale yellow. 23 - 33 70-» 0 30-» 100
D. Examine the chromatograms obtained in the test for 3 3 -4 0 0 100
related substances.
with the test solution at 383 nm is similar in retention time Flow rate 0.8 mL/min.
to the principal peak in the chromatogram obtained with Detection Spectrophotom eter
reference solution (a). — at 303 nm: detection of tetraenes;
— at 383 nm: detection o f heptaenes.
TESTS
Related substances Injection 20 |iL of the test solution and reference
solutions (b), (c), (d) and (e).
Liquid chromatography (2.2.29). Protect the solutions from light
and use within 24 h of preparation, except for reference Identification of impurities Use the chromatograms supplied
solution (c) which should be injected immediately after its with amphotericin B for peak identification CRS and the
preparation. chromatograms obtained with reference solution (e) to
identify the peaks due to impurities A and B.
Solvent mixture 10 g/L solution of ammonium acetate R,
N-metkylpyrrolidone R, methanol R (1:1:2 VIVIV). Relative retention W ith reference to amphotericin B (retention
im purity A = about 0.8; nystatin = about 0.85.
examined in 15 m L o f N-methylpyrrolidone R and within 2 h
dilute to 50.0 m L w ith the solvent mixture. Dilute 5.0 m L of System suitability at 383 nm Reference solution (d):
this solution to 25.0 m L with the solvent mixture. — resolution: minimum 1.5 between the 2 peaks presenting a
relative retention o f about 0.7.
Reference solution (a) Dissolve 20.0 mg o f amphotericin B CRS
in 15 m L o f N-methylpyrrolidone R and within 2 h dilute to Limits:
50.0 m L with the solvent mixture. Dilute 5.0 m L of this — impurity A at 303 nm: not more than 2.5 times the area of
solution to 25.0 m L with the solvent mixture. the principal peak in the chromatogram obtained with
reference solution (c) (5.0 per cent); if intended for use in
the m anufacture of parenteral preparations: not more
than the area o f the principal peak in the chromatogram
Reference solution (c) Dissolve 20.0 m g o f nystatin CRS in obtained with reference solution (c) (2.0 per cent);
15 m L o f N-methylpyrrolidone R and within 2 h dilute to — any other impurity at 303 nm: for each impurity, not more
50.0 m L with the solvent mixture. D ilute 5.0 m L of the than 0.5 times the area o f the principal peak in the
solution to 25.0 m L with reference solution (a). Dilute chromatogram obtained with reference solution (c)
2.0 m L o f this solution to 100.0 m L with the solvent (1.0 per cent);
mixture. — impurity B at 383 nm: not more than 4 times the area of
Reference solution (d) In order to prepare impurities B and C, the principal peak in the chromatogram obtained with
dissolve 10 m g o f the substance to be examined in 5 m l . of reference solution (b) (4.0 per cent);
N-methylpyrrolidone R and within 2 h add 35 m L o f a mixture — any other impurity at 383 nm: for each impurity, n o t more
of 1 volume o f methanol R and 4 volumes of anhydrous than 2 times the area o f the principal peak in the
ethanol R A dd 0.10 m L o f dilute hydrochloric acid R, mix and chrom atogram obtained with reference solution (b)
incubate at 25 °C for 2.5 h. Add 10 m L of 10 g/L solution (2.0 per cent);
of ammonium acetate R and mhc. — total at 303 and 383 nm: maximum 15.0 per cent;
Reference solution (e) Dissolve 4 m g o f amphotericin B for peak — disregard limit at 303 nm: 0.05 times the area of the
identification CRS (containing impurities A and B) in 5 m l. o f principal peak in the chromatogram obtained with
N-methylpyrrolidone R and within 2 h dilute to 50 m L with reference solution (c) (0.1 per cent);
the solvent mixture. — disregard limit at 383 nm: 0.1 times the area o f the
Blank solution T he solvent mixture. principal peak in the chromatogram obtained with
Column: reference solution (b) (0.1 per cent).
— size. I = 0.15 m , 0 = 4.6 mm; Loss on drying (2.2.32)
— stationary phase: base-deactivated end-capped octadecylsQyl M axim u m 5.0 per cent, d eterm in ed on 1.000 g by drying in
silica gel for chromatography R (3 pm); an oven at 60 °C at a pressure not exceeding 0.7 kPa.
— temperature: 20 °C. Sulfated ash (2.4.14)
Mobile phase: M aximum 3.0 per cent, determ ined on 1.0 g; if intended for
— mobile phase A: mix 1 volume o f methanol R, 3 volumes o f use in the m anufacture o f parenteral preparations: maximum
acetonitrile R and 6 volumes o f a 4.2 g/L solution o f citric 0.5 p er cent.
acid R previously adjusted to p H 4.7 using concentrated Bacterial endotoxins (2.6.14)
ammonia R\ Less than 1.0 IU/mg, if intended for use in the manufacture
— mobile phase B: mix 12 volumes o f methanol R, o f parenteral preparations without a further appropriate
20 volumes of a 4.2 g/L solution o f citric acid R previously procedure for the removal o f bacterial endotoxins.
2016 Ampicillin 1-169
A SSA Y
Protect all solutions from light throughout the assay Dissolve
25.0 m g in dimethyl sulfoxide R and dilute, with shaking* to
25.0 m L with the same solvent. U nder constant stirring of
this stock solution, dilute with dimethyl sulfoxide R to obtain
solutions of appropriate concentrations (the following
concentrations have been found suitable: 44.4, 66.7 and
100 IU /m L). Prepare final solutions by diluting 1:20 with
0.2 M phosphate buffer solution p H 10.5 so that they all
contain 5 per cent V/V o f dimethyl sulfoxide. Prepare the
reference and the test solutions simultaneously. Carry out the
microbiological assay of antibiotics (2.7.2).
STORAGE
C. amphotericin X 2 (13-O-ethyl-amphotericin B).
Protected from light, at a tem perature of 2 °C to 8 °C in an
airtight container. I f the substance is sterile, store in a sterile, PhEur
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 Ampicillin ★ ★
★ ★
preparations.
(Anhydrous Ampicillin, Ph Eur monograph 0167)
IM P U R IT IE S
Specified impurities: A , B.
impurities for dem onstration o f compliance. See also 5.10. CieH iiNsO iS 349.4 69-53-4
Control of impurities in substances for pharmaceutical use): C.
Penicillin antibacterial.
Ampicillin Capsules
Ampicillin Oral Suspension
PhEur_____________________________
D E F IN IT IO N
(2 S, 5R, 6R)-6- [[(22Q-2-Amino-2-phenylacetyl] amino] -3,3-
dimethyl-7 -oxo-4-thia-1 -azabicyclo [3.2.0] heptane-2-
carboxylic acid.
C o n te n t
96.0 per cent to 102.0 p er cent (anhydrous substance).
A. amphotericin A (28,29-dihydro-amphotericin B),
CHARACTERS
A p p e a ra n c e
S o lu b ility
Sparingly soluble in water, practically insoluble in acetone, in
ethanol (96 per cent) and in fatty oils. It dissolves in dilute
solutions of acids and o f alkali hydroxides.
I t shows polymorphism (5.9).
Preparation Discs o f potassium bromide R.
B. amphotericin X I (13-O-methyi-amphotericin B), Comparison anhydrous ampicUHn CRS.
in 10 m L o f sodium hydrogen carbonate solution R.
1-170 Ampicillin 2016
Reference solution (a) Dissolve 25 mg of anhydrous Column:

ampicHlin CRS in 10 m l . o f sodium hydrogen carbonate — size. I = 0.25 m , 0 = 4.6 mm;
solution R. — stationary phase: octadecylsüyl silica gd for chromatography R
Reference solution (b) Dissolve 25 mg of amoxicillin (5 [im).
tnhydrate CRS and 25 mg o f anhydrous ampicillin CRS in Mobile phase:
10 m L o f sodium hydrogen carbonate solution R. — mobile phase A: mix 0.5 m L o f dilute acetic add R, 50 m L
Plate TLC süamsed silica gel plate R. o f 0.2 Mpotassium dihydrogen phosphate R and 50 m L o f
acetomtrüe R, then dilute to 1000 m L w ith water R;
Mobile phase M ix 10 volumes of acetone R and 90 volumes of
— mobile phase B: mix 0.5 m L o f dilute acetic add R, 50 m l.
o f 0.2 Mpotassium dihydrogen phosphate R and 400 m l. o f
to p H 5.0 w ith glacial acetic add R.
acetomtrüe R, then dilute to 1000 m L w ith water R;
Application 1 |iL.
Development Over a path of 15 cm.
Drying In air. (min) (percent V/V) (per cent V/V)
Detection Expose to iodine vapour until the spots appear and 0-* 85 15
f, - (f, + 30) 85 -> 0 15+ 100
(f„ + 30) - (f, + 45) 0 100
Results T he principal spot in the chromatogram obtained with (f* + 45) - (f, + 60) 85 15
the test solution is similar in position, colour and size to the f, = retention time of ampicillin determined with reference solution (c)
solution (a).
C . Place about 2 mg in a test-tube about 150 m m long and
about 15 m m in diameter. M oisten with 0 .0 5 m L o f water R
at time zero in the gradient and in the assay.
and add 2 m L of sulfuric acid-formaldehyde reagent R. Mix the
contents o f the tube by swirling; the solution is practically Flow rate 1.0 mL/min.
colourless. Place the test-tube in a water-bath for 1 min; Detection Spectrophotom eter at 254 nm .
a dark yellow colour develops. Irgecdon 50 pL o f reference solutions (b) and (c) with
D. W ater (see Tests). isocratic elution at the initial mobile phase composition and
50 pL of test solution (b) according to the elution gradient
TESTS
described under Mobile phase; inject mobile phase A as a
A p p e a ra n c e o f so lu tio n blank according to the elution gradient described under
T h e solutions are not more opalescent than reference
M obile phase.
suspension II (2.2./).
Dissolve 1.0 g in 10 m L o f 1 M hydrochloric acid. Separately — resolution: minimum 3.0 between the peaks due to
dissolve 1.0 g in 10 m L of dilute ammonia R2. Examine ampicillin and cefradin; if necessary, adjust the ratio A:B
immediately after dissolution. o f die mobile phase.
p H (2.2.3) Lima:
3.5 to 5.5. — any impurity, for each impurity, n o t m ore than the area of
Dissolve 0.1 g in carbon dioxide-free water R and dilute to the principal peak in the chrom atogram obtained with
40 m L with the same solvent. reference solution (c) (1.0 per cent).
S p ecific o p tic a l ro ta tio n (2.2.7) N jA f-D im ethylaniline (2.4.26, Method B)
+ 280 to + 305 (anhydrous substance). M axim um 20 ppm .
Dissolve 62.5 m g in water R and dilute to 25.0 m L with the W a te r (2.5.12)
sam e solvent. M axim um 2.0 per cent, determ ined on 0.300 g.
L iquid chromatography (2.2.29). 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 A SSA Y
examined in mobile phase A and dilute to 50.0 m L with liq u id chromatography (2.2.29) as described in the test for
m obile phase A. related substances with the following modifications.
Test solution (b) Dissolve 27.0 mg o f the substance to be Mobile phase Initial composition o f the mixture o f mobile
examined in mobile phase A and dilute to 10.0 m L with phases A and B, adjusted where applicable.
mobile phase A. Prepare immediately before use.
Irgecdon T est solution (a) and reference solution (a).
Reference solution (a) Dissolve 27.0 mg pf anhydrous
ampicillin CRS in mobile phase A and dilute to 50.0 m L with — repeatability: maximum relative standard deviation of
mobile phase A.
1.0 p er cent after 6 injections.
Reference solution (b) Dissolve 2.0 mg of cefradine CRS in Calculate die percentage content o f C ie H ig ^ C ^ S from die
mobile phase A and dilute to 50 m L with mobile phase A.
declared content o f anhydrous ampicillin CRS.
T o 5.0 m L o f this solution add 5.0 m l, o f reference
solution (a). STORAGE
Reference solution (c) Dilute 1.0 m L of reference solution (a) In an airtight container, at a tem perature n o t exceeding
to 20.0 m L w ith mobile phase A. 30 °C.
2016 Ampicillin 1-171
im p u r it ie s
0 > C°2H
V t >< CH3
^ " r r s CHj
H H H . 3-phenylpyrazin-2-ol,
A. (2S,5R, 6i?)-6-amino-3j3-dimethyl-7-oxo-4-thia-1- h nh 2
azabicydo [3.2.0] heptane-2-carboxyHc a d d
° 0 J > C°2H
(6-am inopenidllanic ad d ),
H NH H V n A ^ C H 3
H H
I. (2S,5R,6R)-6- [ [(2R)-2- [ [(2R)-2-amino-2-phenylacetyl]

amino]-2-phenyiacetyl] amino]-3,3-dimethyl-7 -oxo-4-thia-1-
azabicyclo[3.2.0]heptane-2-carboxylic a d d
B. (2S,5R,6K)-6-[ [(2.S)-2-amino-2-phenylacetyl] amino]-3,3- (D-phenylglycylampicillin),
dim ethyl-7-oxo-4-thia-l -azabicydo [3.2.0] heptane-2-
carboxylic a d d (L-ampicillin),
° v _ N*- V\ *^CH3
CH,
II H H
0
J. (2S,5i?j6i?)-6-[(2,2-dimethylpropanoyl) amino] -3,3-

dimethy 1-7-oxo-4-thia-1-azabicydo [3.2.0] heptane- 2-
carboxylic ad d ,
C. (4S)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5-
dimethylthiazolidine-4-caiboxylic a d d (diketopiperazines o f CH3 0
am pidtlin),
H3C H NH
^ . c o 2h CO2H
H NH2 h H N ''A <,CH3
K . (2R) -2-[(2,2-dimethylpropanoyi) amino] -2-phenylacetic

ad d ,
D . R = CO 2H : (4<S)-2-[[[(2i?)-2-amino-2-phenylacetyl]
amino] carboxymethyi]-5}5-dimethyithiazolidine-4-carboxylic
a d d (penicilloic a d d s of ampidllin),
F. R = H : (2itS,45)-2-[[[(2i?)-2-amino-2-phenylacetyl]
amino]methyl] -5,5-dimediyidiiazoüdine-4-carboxylic a d d
(penilloic a d d s o f ampidllin),
L . (2R)-2-amino-2-phenylacetic a d d (ü-phenyigiycine),
E. (2K)-2- [[ [(25,5/?,6R)-6- [ [(2i?)-2-amino-2-phenylacetyl]

amino]-3,3-dimethyi-7-oxo-4-thia-l -azabicydo
[3.2.0] hept-2-yI] carbonyl]amino]-2-phenylacetic add
(ampidllinyl-D-phenylglydne),
M . co-oligomers of am pidllin and of penicilloic adds o f

ampicillin.
PhEur
G. (3.R,6i?)-3,6-diphenylpiperazine-2j5-dione,
1-172 Ampicillin Sodium 2016

Ampicillin Sodium f * \ — the chromatogram shows 2 clearly separated spots.
(Ph Eur monograph 0578) * Results T h e principal spot in the chrom atogram obtained with
0 j^COjNa
solution (a).
C . Place about 2 m g in a test-tube about 150 m m long and
H H
and add 2 m L of sulfuric acid-formaldehyde reagent R. Mix the
contents of the tube by swirling; the solution is practically
C 16H 18N3Na04S 371.4 69-52-3
a dark yellow colour develops.
A ctio n a n d u s e D . It gives reaction (a) of sodium (2.3.1).
Penicillin antibacterial. TESTS
P re p a ra tio n A p p e a ra n c e o f so lu tio n
Ampicillin Injection Solutions A and B are not m ore opalescent than reference
suspension II (2.2.1) and the absorbance (2.2.25) of
PhEur__________________________________________________________
solution B at 430 nm is n o t greater than 0.15.
D E F IN IT IO N Place 1.0 g in a conical flask and add slowly and with
Sodium (2S,5R, 6R)-6- [ [(22^-2-amino-2-phenylacetyl] continuous swirling 10 m L of 1 M hydrochloric add
amino]-3j3-dimethyl-7 -oxo-4-thia-1 - (solution A). Separately dissolve 1.0 g in water R and dilute
azabicyclo [3.2.0] heptane-2-carboxylate. to 10.0 m l. with the same solvent (solution B). Examine
Semi-synthetic product derived from a fermentation p ro d u c t im m ediately after dissolution.
C o n te n t p H (2.2.3)
91.0 per cent to 102.0 per cent (anhydrous substance). 8.0 to 10.0.
CHARACTERS
20 m L with the same solvent. M easure 10 min after
A p p e a ra n c e
dissolution.
White or almost white powder, hygroscopic.
Solubility
Freely soluble in water, sparingly soluble in acetone,
practically insoluble in fatty oils and in liquid paraffin. Dissolve 62.5 mg in a 4 g/L solution of potassium hydrogen
phthalate R and dilute to 25.0 m L with the same solvent.
R e lated su b s ta n c e s
First ideritification A, D
Second identification B, C, D
Test sdution (a) Dissolve 31.0 m g o f the substance to be
A. Infrared absorption spectrophotometry (2.2.24). examined in mobile phase A and dilute to 50.0 m L with
Preparation Dissolve 0.250 g in 5 m L of water R, add 0.5 m L mobile phase A.
of dilute acetic add R, swirl and allow to stand for 10 min in Test solution (b) Dissolve 31.0 m g of the substance to be
iced water. Filter the crystals through a small sintered-glass examined in mobile phase A and dilute to 10.0 m L with
filter (40) (2.1.2), applying suction, wash with 2-3 m L o f a mobile phase A. Prepare immediately before use.
mixture of 1 volume o f water R and 9 volumes of acetone R,
then dry in an oven at 60 °C for 30 min.
Reference solution (a) Dissolve 27.0 m g of anhydrous
ampicillin CRS in mobile phase A and dilute to 50.0 m L with
Comparison ampidEin trihydrate CRS. mobile phase A.
B. Thin-layer chromatography (2.2.27). Reference solution (b) Dissolve 2.0 m g of cefradine CRS in
Test solution Dissolve 25 m g o f the substance to be examined mobile phase A and dilute to 50 m L with mobile phase A.
in 10 m L of sodium hydrogen carbonate solution R. T o 5.0 m L o f this solution add 5.0 m L o f reference
Reference solution (a) Dissolve 25 mg of ampidOin solution (a).
trihydrate CRS in 10 m L of sodium hydrogen carbonate Reference solution (c) Dilute 1.0 m L of reference solution (a)
solution R to 20.0 m L with mobile phase A.
Reference solution (b) Dissolve 25 mg of amoxicillin Reference solution (d) T o 0.20 g o f the substance to be
trihydrate CRS and 25 m g of ampidBn tnhydrate CRS in examined add 1.0 m l. of water R. H eat the solution at 60 °C
10 m L of sodium hydrogen carbonate solution R. for 1 h. D ilute 0.5 m L of this solution to 50.0 m L with
Plate TLC sUamsed silica gel plate R. mobile phase A.
Mobile phase M ix 10 volumes of acetone R and 90 volumes of Column:
a 154 g/L solution o f ammonium acetate R previously adjusted — size: I = 0.25 m , 0 = 4.6 mm;
to p H 5.0 with glacial acetic add R. — stationary phase, octadecylsifyl silica gel for chromatography R
Application 1 |iL. (5 pm).
Development Over a path of 15 cm. Mobile phase:
— mobile phase A: mix 0.5 m L o f dilute acetic add R, 50 m L
Drying In air. of 0.2 M potassium dihydrogen phosphate R and 50 m L of
Detection Expose to iodine vapour until the spots appear and acetonitrüe R, then dilute to 1000 m L with water R;
examine in daylight
2016 Ampicillin Sodium 1-173
— mobüe phase B : mix 0.5 m L o f dilute acetic acid R, 50 m L — stationary phase: diatomaceous earth for gas
of 0.2 M potassium ¡Mhydrogen phosphate R and 400 m L o f chromatography R impregnated with 10 per cent mtm of
acetomtrüe R, then dilute to 1000 m L with water R-} macrogol 1000 R.
Carrier gas nitrogen for chromatography R.
Time Mobile phase A Mobile phase B Flow rate 40 mlVmin.
(min) (percent V/V) (per cent V/V) Temperature:
o -f. 85 15 — column: 60 °C;
i, - (f, + 30) 85 -> 0 15-» 100 — irgecdon pore. 100 °C;
(fj, + 30) - (fg + 45) 0 100
Detection Flam e ionisation.
(fj, + 45) - (fg + 60) 85 15
Calculate the content o f methylene chloride taking its density
tR= retention time of ampidllin determined with reference solution (c) at 20 °C to be 1.325 g/mL.
Lima:
If the mobile phase composition has been adjusted to achieve — methylene chloride: maximum 0.2 per cent mlm.
the required resolution, the adjusted composition will apply H eav y m e ta ls (2.4.8)
at time zero in the gradient and in the assay. M aximum 20 ppm .
Flow rate 1.0 mL/min. 1.0 g complies with test C . Prepare the reference solution
Detection Spectrophotom eter at 254 nm. using 2 m L o f lead standard solution (10 ppm Pb) R.
Irgecdon 50 (iL o f reference solutions (b) and (c) with W a te r (2.5.12)
isocratic elution at the initial mobile phase composition and M aximum 2.0 per cent, determined on 0.300 g.
50 yL o f test solution (b) and reference solution (d) B a c te ria l en d o to x in s (2.6.14)
according to the elution gradient described under Mobile Less than 0.15 IU/mg, if intended for use in the manufacture
phase; inject mobile phase A as a blank according to the o f parenteral preparations without a further appropriate
elution gradient described under M obile phase. procedure for the removal of bacterial endotoxins.
Identification of peaks Use the chromatogram obtained with A SSAY
reference solution (d) to identify the peaks due to ampicillin
and am pid llin d im er.
Relative retention W ith reference to ampicillin: am pidllin
Mobile phase Initial composition of the mixture o f mobile
dim er = about 2.8.
— resolution: m in im u m 3.0 between the peaks due to
ampicillin and cefradin; if necessary adjust the ratio A:B System suitability: reference solution (a):
of the mobile phase. — repeatability: m axim um relative standard deviation of
Limits:
— ampiaDin dimer, n o t more than 4.5 times the area of the Calculate the percentage content of am pidllin sodium by
principal peak in the chromatogram obtained with multiplying the percentage content of ampicillin by 1.063.
reference solution (c) (4.5 per cent); STO RA G E
— any other impurity: for each impurity, not more than twice In an airtight container. I f the substance is sterile, store in a
the area o f the principal peak in die chromatogram sterile, airtight, tam per-proof container.
obtained with reference solution (c) (2 per cent).
IM P U R IT IE S
iV ,iV -D im ethylaniline (2.4.26, Method B)
M aximum 20 ppm.
O > C°2H
2 -E th y lh ex a n o ic a c id (2.4.28)
V nA xh3
M aximum 0.8 per cent m/m.
M eth y len e c h lo rid e
CHa
H H
Internal standard solution Dissolve 1.0 m L o f ethylene A. (2S,5R, 6/?)-6-amino-3,3-dimethyl-7 -oxo-4-thia-1 -
chloride R in water R and dilute to 500.0 m L with the same azabicydo[3.2.0]heptane-2-carboxylic a d d
solvent. (6-aminopenidllanic a d d ),
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. B. (25,5i?,6i?)-6-[[(25)-2-amino-2-phenylacetyI]amino]-3,3-
T o 1.0 m L o f this solution add 1.0 m L o f the internal dimethyl-7 -oxo-4-thia-1 -azabicyclo [3.2.0] heptane-2-
standard solution and dilute to 10.0 m L with water R. carboxylic a d d (L-ampicillin),
Column:
— material: glass;
— size. / = 1.5 m , 0 = 4 mm;
1-174 Ampicillin Sodium 2016
J. (2S,5i?,6.R)-6-[(2,2-dimethylpropanoyl)amino]-3,3-
dim ethyl-7-oxo-4-thia-l-azabicydo[3.2.0]heptane-2-
C. (45)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5- carboxylic ad d ,
dimethylthiazolidme-4-caiboxylic a d d (diketopiperazines of
ampidllin), CH3 o
H3C^ _ ^
COjH H3C H NH
H ^ H N - \ .C H 3 CO2H
X ^ Nn î A S CH3
I 0 R
K. (2R)-2-[(2,2-dimethjdpropanoyl)amino]-2-phenylacetic
add,
D . R = C 0 2H : (4<S)-2-[[[(2iQ-2-amino-2-phenylacetyl]
amino]carboxymeth5d]-5,5-dimethylthiazolidine-4-carboxylic
H <NH2
a d d (penicilloic ad d s o f ampidllin),
F. R = H: (2i?5',45)-2-[[[(2Ä)-2-amino-2-phenylacetyl] co2h
amino]methyI]-5,5-dimeth^thiazolidine-4-carboxylic a d d
(penilloic ad d s of ampidllin),
L. (2Æ)-2-amino-2-phenylacetic a d d (D-phenylglycine),
CO2H
E. (2i?)-2-[[[(2S’,5£,6i?)-6-[[(2.R)-2-amino-2-phenylacetyl]
amino]-3,3-dimethyl-7 -oxo-4-thia-1-azabicydo [3.2.0]hept-2-
yl] carbonyl] amino]-2-phenylacetic add (ampicillinyl-D-
phenylglycine),
M . co-oligomers of am pidllin and o f penicilloic a d d s of

ampidllin,
G . (3Æ,6i?)-3,6-diphenylpiperazme-2,5-dione,
N . oligomers o f penicilloic a d d s o f ampidllin.

PfiBr
H . 3-phenylpyrazin-2-ol,
h nh2
I. (2S,5i?,6Æ)-6-[[(2i?)-2-[[(2Æ)-2-amino-2-phenylacetyl]
amino]-2-phenylacetyl] amino]-3,3-dimethjd-7-oxo-4-thia 1-
azabicydo[3.2.0]heptane-2-carboxylic add
(D-phenylglycylampicillin),
2016 Ampicillin Trihydrate 1-175
Results T h e prindpal spot in the chrom atogram obtained with

Ampicillin Trihydrate ★
* * * * *
★ the test solution is sim ilar in position, colour and size to the
* * * * *
(Fh Eur monograph 0168) prindpal spot in the chromatogram obtained with reference
solution (a).
C. Place about 2 mg in a test-tube about 150 mm long and
3 H 20 and add 2 m L of sulfuric acid-formaldehyde reagent R. M ix the
contents of the tube by swirling; th e solution is practically
a dark yellow colour devdops.
D . W ater (see Tests).
C 16H 19N 30 4 S 3 H 20 403.5 7177-48-2
TESTS
A ctio n a n d u se A p p e a ra n c e o f so lu tio n
Penicillin antibacterial. T he solutions are not more opalescent than reference
P re p a ra tio n s suspension II (2.2.1).
Ampicillin Capsules Dissolve 1.0 g in 10 m L of 1 M hydrochloric acid. Separately
Ampicillin Oral Suspension dissolve 1.0 g in 10 m L of dilute ammonia R2. Examine
Co-fluampicil Capsules immediately after dissolution.
Co-fluampidl O ral Suspension p H (2.2.3)

3.5 to 5.5.
Ph Eur_____________________________ Dissolve 0.1 g in carbon dioxide-free water R and dilute to
D E F IN IT IO N 40 m L with the same solvent.
(2S,5R, 6R)-6- [ [(2i?) -2-Amino-2 -phenylacetyl] amino] -3,3- S p ecific o p tic a l ro ta tio n (2.2.7)
dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0] heptane-2- + 280 to + 305 (anhydrous substance).
carboxylic acid trihydrate. Dissolve 62.5 m g in water R and dilute to 25.0 m l, with the
Semi-synthetic product derived from a fermentation product. same solvent.
C o n ten t R e la te d su b sta n c e s
96.0 per cent to 102.0 per cent (anhydrous substance). Liquid chromatography (2.2.29).
CHARACTERS Test solution (a) Dissolve 31.0 m g o f the substance to be
A p p e a ra n c e examined in mobile phase A and dilute to 50.0 m L with
W hite or alm ost white, crystalline powder. mobile phase A.
S olubility Test solution (b) Dissolve 31.0 m g o f the substance to be
Slightly soluble in water, practically insoluble in ethanol examined in mobile phase A and dilute to 10.0 m L with
(96 per cent) and in fatty oils. It dissolves in dilute solutions mobile phase A. Prepare immediately before use.
of adds and o f alkali hydroxides. Reference solution (a) Dissolve 27.0 m g of anhydrous
ampicUUn CRS in mobile phase A and dilute to 50,0 m L with
mobile phase A.
Reference solution (b) Dissolve 2 m g of cefradine CRS in
mobile phase A and dilute to 50 m l. with mobile phase A.
A. Infrared absorption spectrophotometry (2.2.24). T o 5 m L of this solution, add 5 m L of reference solution (a).
Comparison ampicUUn trihydrate CRS. Reference solution (c) Dilute 1.0 m L o f reference solution (a)
B. Thin-layer chromatography (2.2.27). to 20.0 m L with mobile phase A.
Test solution Dissolve 25 mg of the substance to be examined Column:
in 10 m L of sodium hydrogen carbonate solution R. — size: I = 0.25 m , 0 = 4.6 mm;
Reference solution (a) Dissolve 25 mg of ampidUin — stationary phase. octadecylsHyl silica gel for chromatography R
trihydrate CRS in 10 m L of sodium hydrogen carbonate (5 jim).
solution R. Mobile phase.
Reference solution (b) Dissolve 25 mg of amoxicillin — mobile phase A: mix 0.5 m L o f dilute acetic acid R, 50 m L
trihydrate CRS and 25 mg o f ampicUUn trihydrate CRS in of 0.2 M potassium dUrtydrogen phosphate R and 50 m L of
10 m L o f sodium hydrogen carbonate solution R. acetonitrUe R, then dilute to 1000 m L with water R;
— mobile phase B: mix 0.5 m L o f dilute acetic acid R, 50 m L
Plate TLC sHanised silica gel plate R.
of 0.2 M potassium dihydrogen phosphate R and 400 mT. of
Mobile phase M ix 10 volumes of acetone R and 90 volumes of acetonitrUe R, then dilute to 1000 m L with water R,
Application 1 jiL.
Development Over a path o f 15 cm. o -f, 85 15
Drying In air.
f* - (t, + 30) 85 ->0 15-» 100
(Í, + 30) - (fR+ 45) 0 100
System suitability: reference solution (b): (i* + 45) - (ij, + 60) 85 15
— the chromatogram shows 2 clearly separated spots. tR=retention time*of ampicillin determined with reference solution (c)
1-176 Ampicillin Trihydrate 2016

at tim e zero in the gradient and in the assay.
Injection 50 pJL o f reference solutions (b) and (c) with
isocratic elution at the initial mobile phase composition and
50 |iL o f test solution (b) according to the ehition gradient C. (45)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5-
described under M obile phase; inject mobile phase A as a dimethylthiazolidine-4-carboxylic a d d (diketopiperazines o f
blank according to the elution gradient described under am pidllin),
M obile phase.
System suitability: reference solution (b): ^ .COjH
— resolution: m in im u m 3.0 between die peaks due to
h nh2 h n ''\x h 3
am pidllin and cefradin; if necessary, adjust the ratio A:B
o f the mobile phase.
Limit. k J O R
— any impurity, for each impurity, not more than the area of
the prindpal peak in the chromatogram obtained with
D . R = C O 2H: (46)-2-[[[(2i?)-2-amino-2-phenylacetyl]
amino] carboxymethyl] -5,5-dimethylthiazolidine-4-carboxylic
A îV -D im ethylaniline (2.4.26, Method E) a d d (penicilloic adds of am pidllin),
M aximum 20 ppm.
F. R = H : (2RS,4S)-2-[ [[ (2R)-2-amino-2-phenylacetyl]
W a te r (2.5.12) amino] methyl] -5,5-dimethylthiazolidine-4-carboxylic a d d
12.0 per cent to 15.0 per cent, determined on 0.100 g. (penilloic a d d s of am pidllin),
A SSA Y
Mobile phase Initial composition o f the mixture o f mobile
Injection T est solution (a) and reference solution (a).
System suitability: reference solution (a): E. (2i?)-2-[[[(2S,5i?,6i?)-6-[[(2i?)-2-amino-2-phenylacetyI]
— repeatability, m axim um rdative standard deviation of amino] -3,3-dimethyl-7 -oxo-4-thia-1 -azabicydo [3.2.0] hept-2-
1.0 per cent after 6 injections. yl] carbonyl] amino]-2-phenylacetic a d d (ampicillinyl-D-
phenylglycine),
Calculate the percentage content of ampidllin from the
declared content of anhydrous ampicQUn CRS.
STORAGE
IM P U R IT IE S
G . (3i?,6i?)-3,6-diphenylpiperazine-2,5-dione,
A. (2S,5R, 6i?)-6-amino-3,3-dimethyl-7-oxo-4-thia-1-
(6-aminopemdllanic a d d ),
H . 3-phenylpyrazin-2-ol,
H NH,
B. (25',5i?,6i?)-6-[[(2»S)-2-amino-2-phenylacetyl] amino] -3,3-

dimethyl-7-oxo-4-thia-1-azabicydo [3.2.0] heptane-2-
carboxylic a d d (t-a m p id llin ) ,
I. (25,5i?, 6K)-6- [[(2R) -2- [ [(2jR)-2-amino-2-phenylacetyl]
amino]-2-phenylacetyl] amino]-3,3-dimethyl-7 -oxo-4-thia-1 -
(D-phenylglycylampicillin),
2016 Amylmetacresol 1-177
o J ^ C°2H ★ ★
Amylmetacresol ★ ★
(Ph. Eur. monograph 2405) *****
3 II H H
O
h3c ^ ôh
J. (2S,5R,6R)-6 -[(2,2-dimethylpropanoyl)amino]-3,3-
ch3
dimethyl-7-oxo-4-thia-1-azabicydo [3.2.0] heptane-2-
carboxylic a d d ,
Ci2H180 178.3 1300-94-3
CH3 o
h3c ^_ Y Action and use
H3C h NH Antiseptic.
CO2H PhEur.
DEFINITION
5-Methyl-2-pentylphenol.
K. (21$-2-[(2,2-dimethylpropanoyl)amino]-2-phenylacetic Content
ad d , 98.0 per cent to 102.0 per cent.
CHARACTERS
Appearance
C lear or alm ost clear liquid, or solid crystalline mass,
colourless or slightly yellow when freshly prepared.
T h e substance changes colour during storage by darkening
and/or discolouration to dark yellow, brownish-yellow or
L. (2jR)-2-amino-2-phenylacetic a d d (D-phenylglycine),
pink.
Solubility
Practically insoluble in water, very soluble in acetone and in
It solidifies at about 22 °C.
IDENTIFICATION
Preparation Film between 2 plates of potassium bromide R.
Comparison amylmetacresol CRS.
TESTS
Related substances
Gas chrom atography (2.2.25): use the normalisation
procedure.
M . co-oligomers o f ampicillin and of penidlloic adds of
Internal standard solution Dissolve 0.100 g o f
ampicillin,
butylkydroxytoluene R in 2-propanol R and dilute to 10.0 m l.
with the same solvent.
CO2H examined in 2-propanol R and dilute to 10.0 m L with the
sam e solvent.
Test solution (b) T o 2.0 m L of test solution (a) add 2.0 m L of
the internal standard solution and dilute to 10.0 m L with
2-propanol R.
N . (3iS)-6-[[(2Æ)-2-amino-2-phenylacetyl] amino]-2,2-
dimethyl-7-oxo-2,3,4,7-tetrahydro-l,4-thiazepine-3-carboxylic
Reference solution (a) Dissolve 10 m g of m-cresol R
(impurity B) and 10 mg o f p-cresol R (impurity D ) in
ad d .
2-propanol R and dilute to 100.0 m L with the same solvent.
PhEur
amylmetacresol for peak identification CRS (containing
impurities A, G an d K) in 1.0 m l o f 2-propanol R.
Reference solution (c) Dissolve 0.1000 g of amylmetacresol CRS
in 2-propanol R and dilute to 10.0 m L with the same solvent.
T o 2.0 m L o f this solution add 2.0 m L o f the internal
standard solution and dilute to 10.0 m L with 2-propanol R.
Reference solution (d) Dilute 1.0 m L of test solution (a) to
100.0 m L w ith 2-propanol R. Dilute 1.0 m L of this solution
to 20.0 m L with 2-propanol R
Column:
— size. I = 30 m , 0 = 0.25 mm;
1-178 Amylmetacresol 2016
— stationary phase: macrogol 20 000 R (film thickness

0.5 pm).
Unear velocity 33 cm/s.
Split ratio 1:30.
Temperature:
Time Temperature
(min) rc)
Column 0-17.5 100-»240 B. 3-methylphenol (m-cresol),
17.5 - 32.5 240
H3C' ^ \ ^ 0H
Injection port 250 || H CH3 and enantiomer
Detector 250
C. 5-methyl-2-[(2i?S)-2-methyIbutyl]phenol,
Injection 1.0 jiL o f test solution (a) and reference
solutions (a), (b) and (d).
with amylmetacresolfor peak identification CRS and the
chrom atogram obtained with reference solution (b) to
identify the peaks due to impurities A, G and K.
Relative retention W ith reference to amylmetacresol (retention
time = about 16 min): impurity G
(diastereoisomer 1) = about 0.51; impurity G
(diastereoisomer 2) = about 0.53; impurity D = about 0.77;
O
im purity B = about 0.78; impurity K = about 0.95;
E. 1-(2-hydroxy-4-methylphenyl)pentan-1-one,
impurities D and B.
Limits:
— impurity A: maximum 0.6 per cent;
— impurities G (sum o f the 2 diastereoisomers), K. for each O
impurity, maximum 0.15 p er cent;
— unspecified impurities: for each impurity, maximum F. 1-(2-hydroxy-5-methylphenyl)pentan-1-one,
0.10 per cent;
— disregard limit, the area of the peak due to amylmetacresol
in the chrom atogram obtained with reference solution (d)
(0.05 per cent).
A SSA Y
Gas chrom atography (2.2.28) as described in the test for o
related substances with the following modification.
Injection 1.0 jiL o f test solution (b) and reference solution (c). H . ethyl pentanoate,
Calculate the percentage content o f C 12H i80 from the
declared content of amylmetacresol CRS.
STORAGE
In an airtight, non-metallic container, protected from lig h t
IM P U R IT IE S I. 3-methylphenyl pentanoate,
Specified impurities A, G , K
J. 4-methylphenyl pentanoate,
(2034). It is therefore no t necessary to identify these
impurities for dem onstration o f compliance. See also 5.10. K. unknow n structure.
Control of impurities in substances for pharmaceutical use): B, C, _____________________________________________________ _ PhEur
D, E, F, H, I, J.
2016 Anastrozole 1-179
Mobile phase'.
Anastrozole ***** — mobile phase A: phosphoric add R, water for
*+ +* chromatography R (0.1:100 V/V)',
— mobile phase B: phosphoric add R, acetonitrUe R l
CN (0.1:100 V/V)',

0 -2 95 5
2 - 54 95 * 3 5 5 ->65
CnHigNg 293.4 120511-73-1

A c tio n a n d u se Detection Spectrophotom eter at 215 nm.
Aromotase inhibitor; treatm ent of breast carcinoma. Irgecdon 20 |iL o f test solution (a) and reference solutions (a)
PhEir __________________________________________________________
and (b).
D E F IN IT IO N
2j2 '-[5-( 1H - 1,2 j 4-Triazol-1-ylme thyl) benzene-1,3-diyI] impurity E.
bis(2-methylpropanenitrile).
Relative retention W ith reference to anastrozole (retention
C o n te n t time = about 29 min): impurity E = about 1.05.
98.0 per cent to 102.0 per cent (anhydrous substance). System suitability: reference solution (b):
CHARACTERS — resolution: m in im u m 3.5 between the peaks due to
A p p e a ra n c e anastrozole and impurity E.
White or almost white powder. Calculation ofpercentage contents:
S o lu b ility — for each impurity, use the concentration of anastrozole in
Very slightly soluble in water, freely soluble in anhydrous reference solution (a).
ethanol, practically insoluble in cyclohexane. Limits'.
It shows polymorphism (5.9). — unspecified impurities: for each impurity, m axim um
0.10 per cent;
ID E N T IF IC A T IO N — total: maximum 0.2 per cent;
Infrared absorption spectrophotometry (2.2.24). — reporting threshold: 0.05 per cent.
Comparison anastrozole CRS. W a te r (2.5.32)
If the spectra obtained in the solid state show differences, M aximum 0.3 per cent, determined on 50.0 mg.
dissolve the substance to be exam ined and the reference S u lfa te d a sh (2.4.14)
substance separately in acetone R, evaporate to dryness and M aximum 0.1 per cent, determined on 1.0 g.
record new spectra using the residues.
ASSAY
TESTS
R e la te d su b s ta n c e s related substances with the following modification.
Irgecdon T est solution (b) and reference solution (c).
Solvent mixture acetonitrUe R l, waterfor chromatography R
Calculate the percentage content o f C 17H 19N 5 taking into
(50:50 V/V).
account the assigned content of anastrozole CRS.
Test solution (a) Dissolve 25 m g of the substance to be
exam in ed in th e solvent mixture and dilute to 100.0 m L with IM P U R IT IE S
the solvent mixture. Other detectable impurities (the following substances would, if
Test solution (b) Dissolve 25.0 mg of the substance to be present at a sufficient level, be detected by one or other of
examined in the solvent mixture and dilute to 200.0 m L with the tests in the monograph. They are lim ited by the general
the solvent mixture. acceptance criterion for other/unspecified impurities and/or
Reference solution (a) Dilute 1.0 mL o f test solution (a) to (2034). It is therefore not necessary to identify these
Control of impurities in substances for pharmaceutical use): A, B,
Reference solution (b) Dissolve 2.5 mg of anastrozole C, D, E, F, G, H, I.
impurity E CRS in 20.0 mL o f the solvent mixture. Dilute
1.0 m L o f the solution to 50.0 m L with test solution (a). CN
Reference solution (c) Dissolve 25.0 m g of anastrozole CRS in
the solvent mixture and dilute to 200.0 m l. with the solvent N i T | CH3
mixture. ''c=5 N J and enantiomer
Column-. H3C - L
— size. I = 0.15 m , 0 = 4.6 mm; h T CN
— stationary phase, end-capped, polar-embedded octadecylsUyl
amorphous organosUica polymer R (3.5 nm). A. 2-[3-[(litS )-l-cyanoethyl]-5-(l/f-l,2,4-triazol-l-
ylmethyl) phenyl] -2-methylpropanenitrile,
1-180 Antazoline Hydrochloride 2016
and enantiomer H . 2,2 '-(5-methylbenzene-1,3-diyl)

bis (2-methylpropanenitrile),
B. (2J?S)-2,3-bis [3-( 1-c y a n o -l-m e th y ie th y l)-5 -(l1,2,4-

triazol-1-ylmethyl)phenyl] -2-methylpropanenitrile,
I. 2 ,2 '-[5-(chloromethyl)benzene-1,3-diyI]
H3C -7 ^
bis(2-methylpropanenitrile).
PhEur
C. 2 , 2 [ 5 -(bromomethyl)benzene-1,3-diyi]
Antazoline Hydrochloride *****

★ ★
* * * * *
(Ph Ever monograph 0972)
HCI
D . 2 ,2 '-[5-(dibromomethyl)benzene-1,3-diyi]
Q 7H20CIN3 301.8 2508-72-7
Histamine H I receptor antagonist] antihistamine.
PhEur__________________________________________________________
D E F IN IT IO N
Antazoline hydrochloride contains n o t less than 99.0 per cent
E. 2 , 2 [5-(hydroxymethyl)benzene-1,3-diyI] and not more than the equivalent o f 101.0 per cent o f
bis(2-methylpropanenitrile), N-benzyl-N-[(4,5-dihydro-lH-irnidazol-2-yl)methyl]aniline
hydrochloride, calculated w ith reference to the dried
substance.
CHARACTERS
A white or almost white, crystalline powder, sparingly soluble
in water, soluble in alcohol, slightly soluble in methylene
F. 4-methylbenzenesulfonic ad d , chloride.
I t melts at about 240 °C, with decomposition.
A Examine by infrared absorption spectrophotometry
antazoline hydrochloride CRS. Examine the substances as discs
G. 2 ,2 '-[5-(4H-1,2,4-triazol-4-ylmethyl) benzene-1,3-diyI] prepared using potassium chloride R.
bis (2-methylpropanenitrile), B. Examine the chromatograms obtained in the test for
related substances in daylight after spraying. T he principal
spot in the chromatogram obtained with test solution (b) is
similar in position, colour and size to the principal spot in
the chromatogram obtained with reference solution (b).
C. T o 5 m L o f solution S (see Tests) add, drop by drop,
dilute sodium hydroxide solution R until an alkaline reaction is
2016 Apomorphine Hydrochloride Hemihydrate 1-181
produced. Filter. T h e precipitate, washed with two 1 m L of 0.1 M alcoholic potassium hydroxide is equivalent to
quantities, each o f 10 m L, o f water R and dried in a 30.18 m g o f C 17H 20CIN 3.
desiccator under reduced pressure, melts (2.2.14) at 119 °C IM P U R IT IE S
to 123 °C.
TESTS
S o lu tio n S
Dissolve 2.0 g in carbon dioxide-free water R prepared from
distilled water R, heating at 60 °C if necessary. Allow to cool
A p p e a ra n c e o f so lu tio n A. N - (2-aminoethyI) -2-(benzylphenylamino) acetamide.
................... PhEur
than reference solution (2.2.2, Method II).
A cidity o r a lk a lin ity
T o 10 m L o f solution S add 0.2 m L o f methyl red solution R.
N ot more than 0.1 mT. o f 0.01 M hydrochloric add or 0.01 M
sodium hydroxide is required to change the colour of the Apomorphine Hydrochloride *****
indicator.
Hemihydrate *****
R elated su b s ta n c e s
Examine by thin-layer chromatography (2.2.27), using silica
gel GF2 5 4 R as the coating substance. H eat the plate at
110 °C for 15 m in before using.
examined in methanol R and dilute to 5 m L with the same
solvent.
with methanol R.
C nH ^C IN C V /iH zO 312.8 41372-20-7
100 m L with methanol R. A c tio n a n d u se
Reference solution (b) Dissolve 20 mg o f antazoUne D opam ine receptor agonist; treatm ent o f Parkinson’s disease.
P r e p a r a tio n
same solvent.
Apomorphine Hydrochloride for Homoeopathic Preparations
Reference solution (c) Dissolve 20 mg o f xylometazoUne
hydrochloride CRS in 1 m L o f test solution (a) and dilute to PhFtr __________________________________________
5 m L with methanol R. D E F IN IT IO N
Apply to the plate 5 jiL o f each solution. Develop over a (6aR)-6-M ethyl-5,6,6a,7-tetrahydro-4ii-dibenzo[d«^]
path of 15 cm using a mixture of 5 volumes of quinoline- 10,11 -diol hydrochloride h em ih ydrate.
dietkylamme R, 10 volumes of methanol R and 85 volumes of
C o n te n t
ethyl acetate R. D ry the plate in a current o f warm air for
98.5 p er cent to 101.5 p e r cent (dried substance).
15 min. Examine in ultraviolet light at 254 nm . T he test is
not valid unless the chromatogram obtained with reference CHARACTERS
solution (c) shows two clearly separated prindpal spots. A p p e a ra n c e
Spray with a mixture of equal volumes of a 200 g/L solution W hite or slightly yellowish-brown or green-tinged greyish,
of ferric chloride R and a 5 g/L solution of potassium crystalline pow der or crystals; on exposure to air and light,
ferruyanide R. Examine immediately in daylight. Any spot in the green tinge becomes more pronounced.
the chrom atogram obtained with test solution (a), apart from S o lu b ility
the prindpal spot, is not more intense than the spot in the Sparingly soluble in water and in ethanol (96 per cent),
chromatogram obtained with reference solution (a) practically insoluble in toluene.
(0.5 per cent).
First identification B, D
1.0 g complies with test C for heavy metals (20 ppm).
Prepare the reference solution using 2 m L o f lead standard Second identification A, C, D
solution (10 ppm Pb) R. A. Ultraviolet and visible absorption spectrophotometry
(2.2.25).
N ot more than 0.5 per cent, determined on 1.000 g by Test solution Dissolve 10.0 mg in a 10.3 g/L solution of
drying in an oven at 105 °C for 3 h. hydrochloric add R and dilute to 100.0 m L with the same a d d
solution. Dilute 10.0 m L of the solution to 100.0 m L with a
10.3 g/L solution o f hydrochloric add R.
N ot more than 0.1 per cent, determined on the residue
obtained in the test for loss on drying. Spectral range 230-350 nm
Absorption maximum A t 273 nm.
ASSAY
Dissolve 0.250 g in 100 m L of alcohol R. A dd 0.1 m L of
Shoulder A t 300-310 nm.
phenolphthalein solution R l. Titrate with 0.1 M alcoholic Spedfic absorbance at the absorption maximum 530 to 570.
potassium hydroxide. B. Infrared absorption spectrophotom etry (2.2.24).
1-182 Apomorphine Hydrochloride Hemihydrate 2016
Comparison apomorphine hydrochloride hemihydrate CRS. Flow rate 1.5 mlVmin.

C. T o 5 m L of solution S (see Tests) add a few millilitres of Detection Spectrophotom eter at 280 nm .
sodium hydrogen carbonate solution R until a perm anent, white Injection 10 pL.
precipitate is formed. T h e precipitate slowly becomes Identification of impurities U se the chrom atogram obtained
greenish. A dd 0.25 m L o f 0.05 M iodine and shake. with reference solution (b) to identify the peak due to
T he precipitate becomes greyish-green. Collect the impurity B.
precipitate. T he precipitate dissolves in methylene chloride R
Relative retention W ith reference to apom orphine (retention
giving a violet-blue solution and in ethanol (96 per cent) R
giving a blue solution.
boldine = about 0.9.
D . T o 2 m L of solution S (see Tests) add 0.1 m L o f nitric
System suitability: Reference solution (d):
acid R. Mix and filter. T h e filtrate gives reaction (a) of
— resolution: minim um 2.5 between the peaks due to boldine
chlorides (2.3.1).
and apomorphine.
TESTS Limits:
S o lu tio n S — impurity B: n o t more than 0.75 times the area o f the
Dissolve 0.25 g without heating in carbon dioxide-free water R corresponding peak in th e chrom atogram obtained with
and dilute to 25 m L w ith the same solvent. reference solution (c) (0.15 per cent);
A p p e a ra n c e o f so lu tio n — unspecified impurities, for each impurity, not more than the
Solution S is clear (2.2.1) and not m ore intensely coloured area o f the principal peak in the chromatogram obtained
than reference solution BY5 or GY ¡-(2.2.2, Method IT). with reference solution (a) (0.10 per cent);
p H (2.2.3)
S pecific o p tic a l ro ta tio n (2.2.7) (0.05 per cent).
- 5 2 to - 4 8 (dried substance).
Dissolve 0.25 g in a 2.06 g/L solution o f hydrochloric add R 2.5 per cent to 4.2 per cent, determ ined on 1.000 g by
and dilute to 25.0 m L with the same acid solution. drying in an oven at 105 °C for 2 h.
R e la te d su b stan ces S u lfa ted a s h (2.4.14)
Liquid chromatography (2.2.29). Maximum 0.1 per cent, determ ined on 1.0 g.
A SSAY
exam in ed in a 1 per cent V/V solution of glacial acetic acid R
and dilute to 20.0 m L with the same solution. Dissolve 0.250 g in a mixture of 5.0 m L o f 0.01 M
hydrochloric add and 50 m L of ethanol (96 per cent) R. Carry
Reference solution (a) D ilute 1.0 m L of the test solution to out a potentiometric titration (2.2.20), using 0.1 M sodium
100.0 m L with a 1 per cent VfV solution of glacial acetic
hydroxide. Read the volume added betw een the first 2 points
acid R. Dilute 1.0 m L o f this solution to 10.0 m L w ith a o f inflexion.
1 per cent V/V solution o f glacial acetic acid R.
1 m L of 0.1 M sodium hydroxide is equivalent to 30.38 mg of
Reference solution (b) Dissolve 12.5 m g of apomorphine
c 17h 18c i n o 2.
impurity B CRS in a 1 per cent V/V solution of glacial acetic
add R and dilute to 10.0 m L with the same solution. STORA GE
Reference solution (c) D ilute 2.0 m L o f reference solution (b) In an airtight container, protected from lig h t
to 10.0 m L with a 1 per cent V/V solution o f glacial acetic IM P U R IT IE S
acid R. Dilute 2.0 m L o f this solution to 100.0 m L w ith a Spedfied impurities B
1 per cent V/V solution o f glacial acetic add R. Other detectable impurities (the following substances would, if
Reference solution (d) Dissolve 25 mg o f boldine R in a present at a sufficient level, be detected by one or other o f
1 per cent V/V solution o f glacial acetic add R and dilute to the tests in the monograph. They are limited by the general
10.0 m L with the same solution. T o 1 m L of this solution acceptance criterion for other/unspecified impurities and/or
add 1 m L o f the test solution and dilute to 10.0 m L with a by the general m onograph Substances for pharmaceutical use
1 per cent V/V solution o f glacial acetic add R (2034). It is therefore no t necessary to identify these
Column: impurities for dem onstration o f compliance. See also 5.10.
— size. I = 0.15 m, 0 = 4.6 mm; Control of impurities in substances for pharmaceutical use): A, C.
— stationary phase: end-capped octadecykUyl silica gelfor
H3CO
Mobile phase:
— mobile phase A: 1.1 g/L solution o f sodium
octanesulfonate R, adjusted to pH 2.2 with a H I
CH3
50 per cent m/m solution o f phosphoric add R,
— mobile phase B: acetonitrile R \;
A (6aJ?)-l 0-methoxy-6-methyl-5,6,6a,7-tetrahydro-4if-
Time Mobile phase A Mobile phase B dibenzo [deg\quinolin-1 l-o l (apocodeine),
(min)_____________ (per cent V/V)_________ (per cent V/V)
0 -2 85 15
2 - 32 85 -> 68 15 -* 32
32-37 68 32
2016 Aprotinin 1-183
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 IDENTIFICATION
(m orphine). 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.
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.
Drying In air.
C. (6aR)-9-[7,8-didehydro-4,5a-epoxy-3-hydroxy-17-
Detection Spray with a solution of 0.1 g of ninhydrin R in a
m ethylm orphinan- 6a-yl] -6-methyl-5,6 , 6 a, 7-tetrahydr o-4H-
mixture o f 6 m L o f a 10 g/L solution of cupric chloride R,
dibenzo [de,g\ quinoline- 10, 11-diol (m orphine-apomorphine
21 m L of glacial acetic acid R and 70 m L o f anhydrous
dimer).
ethanol R. Dry the plate at 60 °C.
__________________________________________________________ PhEi*
Results T he principal spot in the chromatogram obtained with
reference solution.
Aprotinin ***** 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
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 -
solution pH 7.2 R.
-------------------------------------- . t(J
Trypsin solution Dissolve 10 mg of trypsin BRP in 0.002 M
T h r-G ly -P ro -C y s -L y s -A )a -A rg — lie— lie—Arg-
I______________________20 hydrochloric add and dilute to 100 m L with the same acid.
Tyr - Phe - Tyr - Asn - Ala - Lys - Ala - Gly - Leu - Cys -
_____________________________ m Casein solution Dissolve 0.2 g of casein R in buffer solution
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 — pH 7.2 R and dilute to 100 m L w ith the same buffer
I_____________ i 2_____
Lys - Arg - Asn - Asn - Phe - Lys - Ser - Ala - Glu - Asp -
solution.
_I 50 Predpitating solution gladd acetic add R, water R, anhydrous
Cys - Met - Arg - Thr - Cys - Gly - Gly - Ala - OH
----------------------- - 58 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
C 284H 432N 84O79S7 6511 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
A ctio n a n d u se
water and add 0.5 m L o f the precipitating solution. Shake
Antifibrinolytic. and allow to stand at room temperature for 15 min.
PhEtr__________________________________________________________ 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
D E F IN IT IO N test solution. T he solution is not cloudy.
Aprotinin is a polypeptide consisting of a chain of 58 amino
acids. It inhibits stoichiometrically the activity of several
TESTS
proteolytic enzymes such as chymotrypsin, kallikrein, plasmin Solution S
and trypsin. It contains not less than 3.0 Ph. Eur. U . Prepare a solution o f the substance to be examined
of aprotinin activity per milligram, calculated with reference containing 15 Ph. Eur. UVmL, calculated from the activity
to the dried substance. stated on the label.
P R O D U C T IO N
Solution S is clear (2.2.1).
T he animals from which aprotinin is derived must fulfil the
requirem ents for the health of animals suitable for hum an Absorbance (2.2.25)
consumption. Maximum 0.80 by measuring at the absorption maximum at
277 nm.
T he m ethod o f manufacture is validated to demonstrate that
the product, if tested, would comply with the following tests. Prepare a solution o f the substance to be examined
containing 3.0 Ph. Eur. U ./ mL.
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 Des-Ala-aprotinin and des-Ala-des-Gly-aprotinin
examined containing 2 Ph. Eur. U. dissolved in a sufficient Capillary zone electrophoresis (2.2.47): Use the normalisation
quantity o f waterfor injections R to give a volume o f 0.5 mL. procedure.
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 .
1-184 Aprotinin 2016
Test solution Prepare a solution o f the substance to be dihydrate R and 66.07 g o f ammonium sulfate R in
ex am in ed inwater R containing n o t less than 1000 m L of water; filter and degas;
1 Ph. E ur. U ./m L.
Reference solution D ilute aprotinin solution BRP in water R to Time Mobile phase A Mobile phase B
obtain the same concentration as the test solution. (min) (percent V/V) (percent V/V)
Capillary: 0 -2 1 92 64 8*36
— material: uncoated fused silica; 21 -3 0 64 0 3 6 * 100
— sizer, effective length = 45-60 cm, 0 = 75 Jim.
Temperature 25 °C.
CZE buffer Dissolve 8.21 g o f potassium ¿¡hydrogen
Detection Spectrophotom eter a t 210 nm .
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 Irgection 40 |xL.
through a m em brane filter (nominal pore size 0.45 Jim). Relative retention W ith reference to aprotinin (retention
Detection Spectrophotom eter a t 214 nm . tim e = 17.0 min to 20.0 min): impurity C = about 0.9.
Between-run rinsing Rinse the capillary for at least 1 min with System suitability: reference solution:
— resolution: minimum 1.5 betw een the peaks due to
0.1 M sodium hydroxide filtered through a m em brane filter
impurity C and aprotinin;
(nominal pore size 0.45 Jim) and for 2 min with the C ZE
—■symmetry factor, maximum 1.3 for the peak due to
buffer.
aprotinin.
Irgection U nder pressure or vacuum (for example, 3 s a t a
differential pressure o f 3.5 kPa).
Limits:
— impurity C: m aximum 1.0 p er cent;
Migration Apply a field strength o f 0.2 kV/cm, using the C ZE — any other impurity: m axim um 0.5 p er cent;
buffer as the electrolyte in b oth buffer reservoirs. — sum of impurities other than C: m aximum 1.0 p er c e n t
Run time 30 min.
Aprotinin oligomers
Identification of impurities Use the electropherogram supplied Size-exclusion chromatography (2.2.30): U se the
with aprotinin solution BRP and the electropherogram norm alisation procedure.
obtained with the reference solution to identify the peaks due
Test solution Prepare a solution o f the substance to be
to impurities A and B.
examined in water R containing about 5 Ph. Eur. UVmL.
Relative migration W ith reference to aprotinin (migration Reference solution T reat the substance to be examined to
tim e = about 22 m in): impurity A = about 0.98;
obtain about 2 per cent aprotinin oligomers. F or example,
im purity B = about 0.99.
heat freeze-dried aprotinin at about 110 °C for about 4 h.
System suitability Reference solution after at least 6 injections: T h en dissolve in water R to obtain a concentration o f about
— migration time: aprotinin = 19.0 m in to 25.0 m in ; 5 Ph. Eur. UVmL.
Column 3 columns coupled in series:
impurities A and B; minimum 0.5 between the peaks due
— size. I = 0.30 m , 0 = 7.8 m m ;
to im purity B and aprotinin;
— stationary phase, hydrophilic silica gelfor chromatography R
— peak distribution: the electrophoregram obtained is
o f a grade suitable for fractionation o f globular proteins in
qualitatively and quantitatively sim ilar to the
the relative molecular mass range o f 20 000 to
electropherogram supplied with aprotinin solution BRP,
10 000 000 (8 jam).
— height of the prindpal peak: at least 1000 times the height
o f the baseline noise. If necessary, adjust the sample load Mobile phase acetomtrUe R, glacial acetic add R, water R
to give peaks o f sufficient height. (2:2:6 V/VIV)-, filter and degas.
Limits: Flow rate 1.0 mlVmin.
— impurity A: maximum 8.0 per cent; Detection Spectrophotom eter at 277 nm .
— impurity B: m axim um 7.5 per cent. Irgection 100 |iL.
Pyroghitamyl-aprotinin and related compounds Run lime 40 min.
L iquid chrom atography (2.2.29): use the normalisation Relative retention W ith reference to aprotinin m onom er
procedure. (retention time = 24.5 min to 25.5 min): aprotinin
Test solution Prepare a solution of the substance to be dim er = about 0.9.
e x a m in ed in mobile phase A, containing about System suitability: reference solution:
5 Ph. E ur. UVmL. — resolution: minimum 1.3 betw een the peaks due to
Reference sdudon Dissolve the contents of a vial o f aprotinin aprotinin dim er and monom er;
for system suitability CRS in 2.0 m L o f mobile phase A. — symmetry factor, maximum 2.5 for the peak due to
Column: aprotinin m onom er.
— size. I = 0.075 m , 0 = 7.5 mm; Limit:
— stationary phase, strong cation-exchange silica gelfor — total: maxim um 1.0 p er cent.
chromatography R (10 pm); Loss on drying (2.2.32)
— temperature. 40 °C. Maximum 6.0 per cent, determ ined on 0.100 g by drying
Mobile phase: in vacuo.
— mobile phase A: dissolve 3.52 g o f potassium dihydrogen
B a c te r ia l e n d o to x in s (2.6.14)
phosphate R and 7.26 g o f disodium hydrogen phosphate Less than 0.14 IU per European Pharm acopoeia U nit o f
dihydrate R in 1000 m L o f water; filter and degas; aprotinin, if intended for use in the m anufacture of
— mobile phase B: dissolve 3.52 g o f potassium dihydrogen
parenteral preparations w ithout a further appropriate
phosphate R, 7.26 g o f disodium hydrogen phosphate procedure for the removal o f bacterial endotoxins.
ASSAY L A B E L L IN G
T he activity of aprotinin is determined by measuring its The label states:
inhibitory action on a solution of trypsin o f known activity. — the num ber o f European Pharmacopoeia Units of
T he inhibiting activity of the aprotinin is calculated from the aprotinin activity per milligram;
difference between the initial activity and the residual activity — where applicable, that the substance is suitable for use in
of die trypsin. the m anufacture o f parenteral preparations.
T he inhibiting activity of aprotinin is expressed in European IM P U R IT IE S
Pharmacopoeia Units. 1 Ph. Eur. U . inhibits 50 p er cent of
the enzymatic activity of 2 micro katals of trypsin. Ra - Arg - Pro - A s p -P h e -C y s -L e u -G lu -P ro -P ro - Tyr -
___ !_________________ | to
Use a reaction vessel with a capacity of about 30 m L, T h r-G ly -P ro -C y s -L y s -A la -A rg — lie— lie—Arg -
provided with: I «
Tyr - Phe - Tyr - Asn - Ala - Lys - Ala - Gly - Leu - Cys -
— a device that will maintain a temperature of 25 ± 0.1 ° Q _____________________________ m
— a stirring device, such as a magnetic stirrer; Gin - Thr - Ptie - Val - Tyr - Gly - Gly - Cys - Arg - Ala-
I w
— a lid with 5 holes for accommodating the electrodes, the Lys - Arg - Asn - Asn - Phe - Lys - Ser - Ala - Glu - Asp -
tip of a burette, a tube for the admission of nitrogen and —i 30
Cys - M e t-A rg -T h r-C y s -G ly -R b
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 A. Ra = H , Rb = OH: aprotinin-(1-56)-peptide,
pH -m eter is provided with a wide reading scale and glass and
B. Ra = H , Rb = Gly-OH: aprotinin-(l-57)-peptide,
calomel or glass-silver-silver chloride electrodes.
C. Ra = Glp, Rb = Gly-Ala-OH: (5-oxoprolyl)aprotinin
Test solution Prepare a solution of the substance to be (pyroglutamylaprotinin).
examined in 0.0015 M borate buffer solution pH 8.0 R
expected to contain 1.67 Ph. Eur. UVmL (about 0.6 mg __________________________________________________ PhEur
(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 Aprotinin Concentrated Solution
prepared solution and keep in iced water.
Trypsin and aprotinin solution T o 4.0 m L o f the trypsin (Ph Eur monograph 0579) *
solution add 1.0 m L of the test solution. Dilute immediately
H - Arg - Pro - Asp - Phe - Cys - Leu - Glu - Pro - Pro - Tyr-
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 T h r-G ly -P ro -C y s -L y s -A la -A rg — lie— lie—Arg-
keep in iced water. Use within 6 h o f preparation. |_________________ 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 -
Dilute trypsin solution Dilute 0.5 m L of the trypsin solution to ________________________________________ 221
Gin - Thr - Phe - Val - Tyr - Gly - Gly - Cys - Arg - Ala -
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 Lys - Arg - Asn - Asn - Phe - Lys - Ser - Ala - Glu - Asp -
—i 30
iced water. Cys - Met - Arg- Thr - Cys - Gly - Gly - Ala - OH
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 C 284H 432N 84O79S7 6511
solution of benzqylarginine ethyl ester hydrochloride R. Adjust to
p H 8.0 with 0.1 M sodium hydroxide. W hen the tem perature A c tio n a n d u se
has reached equilibrium at 25 ± 0.1 °C, add 1.0 m L of die Antifibrinolytic.
trypsin and aprotinin solution and start a timer. M aintain at PhEur_________________________________________________________
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 D E F IN IT IO N
6 m in . D eterm ine the num ber of m illilitres of 0.1 M sodium Aprotinin concentrated solution is a solution o f aprotinin, a
hydroxide used p er second (nx mL). Carry out, under the polypeptide consisting of a chain of 58 amino adds, which
same conditions, a titration using 1 .0 m L of the dilute inhibits stoichiometrically the activity o f several proteolytic
trypsin solution. Determ ine the num ber o f millilitres of enzymes such as chymotrypsin, kallikrem, plasmin and
0.1 M sodium hydroxide used per second (n2 mL). trypsin. It contains not less than 15.0 Ph. Eur. U.
Calculate the aprotinin activity in European Pharmacopoeia o f aprotinin activity per millilitre.
Units per milligram using the following expression: P R O D U C T IO N
T h e animals from which aprotinin is derived m ust fulfil the
4000 (2na — ni) requirem ents for the health of anim als suitable for hu m an
m consumption.
T h e m ethod of manufacture is validated to demonstrate that
T he estimated activity is n o t less than 90 per cent and not the product, if tested, would comply with the following tests.
more than 110 per cent o f the activity stated on the label. A b n o rm a l to x icity (2.6.9)
Inject into each mouse a quantity of the preparation to be
ST O R A G E
examined containing 2 Ph. Eur. U . diluted with a suffident
In an airtight, tam per-proof container, protected from light.
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 2016
CHARACTERS — size, effective length = 45-60 cm , 0 = 75 pm.

A p p e a ra n c e Temperature 25 °C.
Clear, colourless liquid. CZE buffer Dissolve 8.21 g o f potassium dihydrogen
ID E N T IF IC A T IO N phosphate R in 400 m L o f water R, adjust to p H 3.0 with
A. Thin-layer chromatography (2.2.27). phosphoric add R, dilute to 500.0 m L w ith water R and filter
Test solution Solution S (see Tests). through a m em brane filter (nom inal pore size 0.45 pm).
Reference solution D ilute aprotinin solution BRP in water R to Detection Spectrophotom eter at 214 nm .
obtain a concentration o f 15 Ph. Eur. U ./m L. Between-run rinsing Rinse the capillary for at least 1 min with
Plate TLC silica gel G plate R. 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
Mobile phase water R, glacial acetic acid R (80:100 V/V)
buffer.
containing 100 g/L o f sodium acetate R.
Injection U nder pressure or vacuum (ibr example, 3 s at a
Application 10 jiL.
differential pressure o f 3.5 kPa).
Migration Apply a field strength o f 0.2 kV/cm, using the C ZE
Drying In air. buffer as the electrolyte in b oth buffer reservoirs.
Detection Spray with a solution of 0.1 g o f mnhydrin R in a Run time 30 min.
m ixture o f 6 m L o f a 10 g/L solution o f cupric chloride R,
Identification of impurities Use the electropherogram supplied
21 m L o f glacial acetic acid R and 70 m L o f anhydrous
with aprotinin solution BRP and die electropherogram
ethanol R. D ry the plate at 60 °C.
obtained with the reference solution to identify the peaks due
Results T he principal spot in the chromatogram obtained with to impurities A and B.
Relative migration W ith reference to aprotinin (migration
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 System suitability Reference solution after at least 6 injections:
inhibit trypsin activity using the m ethod described below. — migration time, aprotinin = 19.0 m in to 25.0 min;
Test solution D ilute 1 m L o f solution S to 50 m L with buffer — resolution: m in im u m 0.8 between the peaks due to
solution pH 7.2 R impurities A and B; minim um 0.5 between the peaks due
Trypsin solution Dissolve 10 m g of trypsin BRP in 0.002 M to im purity B and aprotinin;
hydrochloric add and dilute to 100 m L with the same acid. — peak distribution: the electrophoregram obtained is
Casein solution Dissolve 0.2 g o f casein R in buffer solution qualitatively and quantitatively similar to the
pH 7.2 R and dilute to 100 m L with the same buffer electropherogram supplied with aprotinin solution BRP,
solution. — height of the principal peak: at least 1000 times the height
o f the baseline noise. I f necessary, adjust the sample load
Predpitating solution glacial acetic add R, water R, anhydrous
to give peaks o f a sufficient height.
ethanol R (1:49:50 VfVIV).
Limits.
M ix 1 m L o f the test solution with 1 m L o f the trypsin
— impurity A: m axim um 8.0 per cent;
solution. Allow to stand for 10 min and add 1 m L of the
— impurity B: maximum 7.5 p er cent.
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 Pyroglutamyl-aprotinin and related compounds
and allow to stand at room tem perature for 15 min. Liquid chrom atography (2.2.29): use die normalisation
T h e solution is cloudy. Carry out a blank test under the procedure.
sam e conditions using buffer solution pH 7.2 R instead o f the Test solution D ilute th e preparation to be examined in mobile
test solution. T h e solution is n o t cloudy. phase A to a concentration o f about 5 Ph. Eur. UVmL.
TESTS Reference solution Dissolve the contents o f a vial o f aprotinin
S o lu tio n S for system suitability CRS in 2.0 m L o f mobile phase A.
Prepare a solution c o n tain in g 15 Ph. Eur. U./m L, if Column:
necessary by dilution, on the basis o f the activity stated on — size. I = 0.075 m , 0 = 7.5 mm ;
the label. — stationary phase, strong cation-exchange silica gel for
Solution S is clear (2.2.1). — temperature. 40 °C.
Mobile phase.
A b so rb a n c e (2.2.25)
— mobile phase A: dissolve 3.52 g o f potassium dihydrogen
M axim um 0.80 by measuring at the absorption m m m n m at
phosphate R and 7.26 g o f disodium hydrogen phosphate
277 nm .
dihydrate R in 1000 m L o f water; filter and degas;
Prepare a solution containing 3.0 Ph. Eur. IJ /mT.. — mobile phase B: dissolve 3.52 g o í potassium dihydrogen
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 phosphate R, 7.26 g o f disodium hydrogen phosphate
Capillary zone electrophoresis (2.2.47) U se the normalisation dihydrate R and 66.07 g of ammonium sulfate R in
procedure. 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. Time Mobile phase A Mobile phase B
Reference solution D ilute aprotinin solution BRP in water R to (min) (percent V/V) (percent V/V)
obtain the same concentration as the test solution. 0 -2 1 92 -»64 8*36
Capillary: 2 1 -3 0 64*0 3 6 * 100
— material: u nfoated fused silica;
Flow rate 1.0 mL/min. T he inhibiting activity of the aprotinin is calculated from the
Detection Spectrophotom eter at 210 nm. difference between the initial activity and the residual activity
o f the trypsin.
Irgection 40 (iL.
T h e inhibiting activity of aprotinin is expressed in European
Relative retention W ith reference to aprotinin (retention
Pharmacopoeia Units. 1 Ph. Eur. U . inhibits 50 per cent o f
time = 17.0 m in to 20.0 m in); impurity C = about 0.9.
the enzymatic activity o f 2 microkatals of trypsin.
— resolution: minim um 1.5 between the peaks due to U se a reaction vessel with a capacity o f about 30 mL,
impurity C and aprotinin; provided with:
— symmetry factor, maximum 1.3 for the peak due to — a device th at will maintain a tem perature of 25 + 0.1 °C;
— a stirring device, such as a magnetic stirrer;
aprotinin.
— a lid with 5 holes for accommodating the electrodes, the
Limits:
tip o f a burette, a tube for the admission of nitrogen and
— impurity C: maximum 1.0 per cent;
the introduction o f the reagents.
— any other impurity, maximum 0.5 per cent; -
— sum of impurities other than C: maximum 1.0 per cent. 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
Aprotinin oligomers pH -m eter is provided with a wide reading scale and glass and
Size-exclusion chromatography (2.2.30) Use the calomel or glass-silver-silver chloride electrodes.
normalisation procedure.
Test solution W ith 0.0015 M borate buffer solution pH 8.0 R
Test solution Dilute the preparation to be examined in water R prepare an appropriate dilution (D) of the aprotinin
to obtain a concentration of about 5 Ph. E ur. UVmL.
concentrated solution expected, on the basis of the stated
Reference solution T reat the substance to be examined to potency, to contain 1.67 Ph. Eur. IJ VmT -
obtain about 2 p er cent aprotinin oligomers. F or example, Trypsin solution Prepare a solution o f trypsin BRP containing
heat freeze-dried aprotinin at about 110 °C for about 4 h.
about 0.8 microkatals per millilitre (about 1 mg/mL), using
T hen dissolve in water R to obtain a concentration o f about
0.001 M hydrochloric acid as the solvent. Use a freshly
5 Ph. E ur. U./mL. prepared solution and keep in iced water.
Column 3 columns coupled in series: Trypsin and aprotinin solution T o 4.0 m L of the trypsin
— sizer. I = 0.30 m , 0 = 7.8 mm; solution add 1.0 m L o f the test solution. Dilute immediately
— stationary phase: hydrophilic silica gel for chromatography R
to 40.0 m L with 0.0015 M borate buffer solution pH 8.0 R.
o f a grade suitable for fractionation of globular proteins in
Allow to stand at room tem perature for 10 min and then
the relative molecular mass range of 20 000 to keep in iced water. U se within 6 h of preparation.
10 000 000 (8 nm).
Dilute trypsin solution Dilute 0.5 m L of the trypsin solution to
Mobile phase acetordtrUe R, glacial acetic add R, water R 10.0 m L with 0.0015 M borate buffer solution pH 8.0 R. Allow
(2:2:6 VIV/V); filter and degas. to stand at room temperature for 10 min and then keep in
Flow rate 1.0 mL/min. iced water.
Detection Spectrophotom eter at 277 nm. Maintain an atmosphere of nitrogen in the reaction flask and
Irgection 100 (iL. stir continuously; introduce 9.0 m L of 0.0015 M borate buffer
Run time 40 min. 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
Relative retention W ith reference to aprotinin m onom er
p H 8.0 with 0.1 M sodium hydroxide. W hen the tem perature
(retention time = 24.5 min to 25.5 min): aprotinin
has reached equilibrium at 25 ± 0.1 °C, add 1.0 m L of the
dimer = about 0.9.
trypsin and aprotinin solution and start a timer. M aintain at
System suitability, reference solution: 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
aprotinin dim er and monomer;
6 min. Determine the num ber o f millilitres of 0.1 M sodium
— symmetry factor, m axim u m 2.5 for the peak due to
hydroxide used per second ( ^ m L). Carry out, under the
aprotinin m onom er. same conditions, a titration using 1.0 mL of the dilute
Limit: trypsin solution. Determine the num ber of millilitres of
— total: maxim um 1.0 per cen t 0.1 M sodium hydroxide used per second (n2 mL).
Specific activity o f the dry residue Calculate the aprotinin activity in European Pharmacopoeia
Minimum 3.0 Ph. Eur. U. o f aprotinin activity per milligram U nits per millilitre vising the following expression:
of dry residue.
Evaporate 25.0 m l, to dryness in a water-bath, dry the 4000 (2ri2 — n i )
residue at 110 °C for 15 h and weigh- From the mass of the m
residue and the activity determined as described below,
calculate the nu m b er o f European Pharmacopoeia U nits per
D = dilution factor of the aprotinin concentrated
milligram of dry residue.
solution to be examined in order to obtain a
Bacterial endotoxins (2.6.14) solution containing 1.67 Ph. Eur. UVmL.
Less than 0.14 IU per European Pharmacopoeia U nit of
aprotinin, if intended for use in the manufacture of T h e estimated activity is not less than 90 per cent and not
parenteral preparations without a further appropriate m ore than 110 per cent of the activity stated on the label.
procedure for the removal o f bacterial endotoxins. STO RA G E
ASSAY In an airtight, tam per-proof container, protected from light.
T he activity of aprotinin is determined by measuring its
inhibitory action on a solution of trypsin of.known activity.
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
— the num ber of European Pharmacopoeia U nits o f 0.4 per cent;
aprotinin activity p er millilitre; — palmitic acid: 5.0 per cent to 14.0 per cent;
— where applicable, th at the substance is suitable for use in — stearic add. 1.3 p er cent to 6.5 per cent;
the m anufacture o f parenteral preparations. — 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;
IM P U R IT IE S
— linolenic add: maximum 0.6 per cent;
Ra - *Arg
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 - — arachidic add: 0.5 per cent to 3.0 p er cent;
_!_________________ I 10 — eicosenoic acid: 0.5 per cent to 3.0 per cent;
T h r-G ly -P ro -C y s -L y s -A la -A r g — Ile— Ile—Arg-
I
I_____________________________________________20
_________________________________________20 — behenic acid: 1.0 per cent to 5.0 p er cent;
Tyr - Phe - Tyr - Asn - Ala - Lys - Ala - Gly - Leu - Cys- — erucic add: maximum 0.5 p er cent;
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 — — Ugnoceric acid: 0.5 per cent to 3.0 p er c e n t
Lys - Arg - Asn - Asn - Phe - Lys - Ser - Ala - Glu - Asp - W a te r (2.5.32)
—I 50 M aximum 0.1 per cent, determ ined on 1.00 g.
Cys - Met - Arg - Thr - Cys - Gly - Rb
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 ★* * * ★ PhEtr------------------------------------------------------------------------------------------
★ ★
P eanut Oil ***** D E F IN IT IO N
(Refined Arachis OH, Ph. Eur. monograph 0263) Oil obtained by refining, bleaching, hydrogenating and
d eod orisin g oil obtained from the shelled seeds o f Arachis
P r e p a r a tio n hypogaea L. Each type o f hydrogenated arachis oil is
Arachis Oil Enem a characterised by its nom inal drop point.
Ph Fir _______________________________________
CHARACTERS
D E F IN IT IO N A p p e a ra n c e
T h e refined fatty oil obtained from the shelled seeds o f W hite or faintly yellowish, soft mass which m elts to a clear,
Arachis hypogaea L. A suitable antioxidant may be added. pale yellow liquid w hen heated.
CHARACTERS S o lu b ility
A p p e a ra n c e Practically insoluble in water, freely soluble in methylene
C lear, yellowish, viscous liquid. chloride and in light petroleum (bp: 65-70 °C ), very slightly
soluble in ethanol (96 per cent).
S o lu b ility
Very slightly soluble in ethanol (96 per cent), miscible with ID E N T IF IC A T IO N
light petroleum. First identification A , B
R e lativ e d e n sity Second identification A , C
A bout 0.915. 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 (2.3.2).
Identification o f fatty oils by thin-layer chromatography Results T h e chromatogram obtained is similar to the
(2.3.2). chrom atogram for arachis oil shown in Figure 2.3.2.-1.
Results T he chrom atogram obtained is similar to the C. Composition o f fatty ad d s (see Tests).
corresponding chrom atogram shown in Figure 2.3.2.-1. TESTS
TESTS D ro p p o in t (2.2.17)
A c id v alu e (2.5.1) 32 °C to 43 °C, and within 3 °C o f th e nom inal value.
M axim um 0.5, determ ined on 10.0 g. A c id v a lu e (2.5.1)
P e ro x id e v a lu e (2.5.5, Method A) M aximum 0.5.
M axim um 5.0. Dissolve 10.0 g in 50 m L o f the prescribed solvent by
U n sa p o n ifia b le m a t te r (2.5.7) heating on a water-bath.
M axim um 1.0 per cent, determ ined on 5.0 g. P e ro x id e v a lu e (2.5.5, Method A)
A lk alin e im p u ritie s (2.4.19) M axim um 5.0.
It complies with the test. Dissolve 5.0 g in 30 m L o f the prescribed solvent by heating
C o m p o sitio n o f fa tty a d d s on a water-bath.
(2.4.22, Method A). Use the mixture o f calibratin g substances U n sa p o n ifia b le m a t te r (2.5.7)
in T able 2.4.22.-3. M axim um 1.0 per c e n t
2016 Arginine 1-189
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) (Ph. Eur. monograph 0806) *
U se the mixture o f calibrating substances in Table 2A.22.-3.
Column: H H* -NHj
— material: fused silica; H2N . .N . X .
Y co 2h
— size: I = 25 m , 0 = 0.25 mm; NH
— stationary phase: poly(cyanopropyl)sUoxane R (film thickness
0.2 |im).
CfiH iiNÂ 174.2 74-79-3
Flow rate 0.7 mL/min. A c tio n a n d u se
Split ratio 1:100. Amino acid; nutrient.
Temperature: PhEur__________________________________________________________
— column: 180 °C for 20 min;
— injection port and detector. 250 °C. D E F IN IT IO N
Detection Flame ionisation. (2S)-2-Amino-5-guanidinopentanoic add.
Composition of the fatty-acid fraction of the oil: Ferm entation product, extract or hydrolysate of protein.
— saturated fatty adds of chain length less than Crf. maximum C o n te n t
0.5 per cent; 98.5 per cent to 101.0 p er cent (dried substance).
— myrisac add: maximum 0.5 per cent; CHARACTERS
— palmitic add: 7.0 per cent to 16.0 per cent; A p p e a ra n c e
— stearic acid. 3.0 per cent to 19.0 per cent; W hite o r almost white, crystalline powder or colourless
— oleic acid and isomers: 54.0 per cent to 78.0 per cent; crystals, hygroscopic.
— Imoleic add and isomers: maximum 10.0 per cent;
— arachidic acid: 1.0 per cent to 3.0 per cent; S o lu b ility
— eicosenoic acids: maximum 2.1 p er cent; Freely soluble in water, very slightly soluble in ethanol
— behenic add: 1.0 per cent to 5.0 per cent; (96 per cent).
— erucic acid and isomers: maximum 0.5 per cent; ID E N T IF IC A T IO N
— lignoceric add\ 0.5 per cent to 3.0 per cent. First identification A , C
Nickel 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
Comparison arginine CRS.
into the substance a wick formed from twisted ashless filter
paper. Ignite the wick. W hen the substance has ignited stop I f the spectra obtained show differences, dry the substance to
heating. After combustion, ignite in a muffle furnace at about be examined and the reference substance in an oven, at
600 ± 50 °C. Continue ignition until white ash is obtained. 105 °C and record new spectra.
After cooling, take up the residue with 2 quantities, each of D . Thin-layer chromatography (2.2.27).
2 mT, o f dilute hydrochloric acid R and transfer into a 25 m L Test solution Dissolve 10 m g of the substance to be examined
graduated flask. Add 0.3 m L of nitric add R and dilute to in a 10.3 g/L solution o f hydrochloric acid R and dilute to
25.0 m L with water R. 50 m L with the same solution.
Reference solutions Prepare 3 reference solutions by adding Reference solution. Dissolve 10 mg of arginine CRS in a
1.0 mT, 2.0 m L and 4.0 m L o f nickel standard solution 10.3 g/L solution o f hydrochloric acid R and dilute to 50 m l,
(0.2 ppm Ni) R to 2.0 m L of the test solution and diluting to with the same solution.
10.0 m L with water R. Hate TLC silica gel plate R.
Source Nickel hollow-cathode lamp. Mobile phase concentrated ammonia R, 2-propanol R
Wavelength 232 nm. (30:70 VIV).
Atomisation device Graphite furnace. Application 5 |iL.
Carrier gas argon R. Development Over 2/3 of the plate.
STO RA G E Drying At 105 °C until the ammonia disappears completely.
Protected from light. Detection Spray with mnhydrin solution R and heat at 105 °C
L A B E L L IN G for 15 min.
T he label states the nominal drop point. Results T he principal spot in the chromatogram obtained with
the T est solution is similar in position, colour and size to the
__________________________________________________________ PhEur
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 T o 5 m L o f solution S add 0.5 m L o f dilute nitric add R and

S o lu tio n S dilute to 15 m L w ith water R.
Dissolve 2.5 g in distilled water R and dilute to 50 m L with S u lfa tes (2.4.13)
the same solvent. Maximum 300 ppm .
A p p e a ra n c e o f so lu tio n T o 10 m L o f solution S, add 1.7 m L o f dilute hydrochloric
Solution S is clear (2.2.1) and not m ore intensely coloured add R and dilute to 15 m L with distilled water R.
than reference solution BY6 (2.2.2, Method II). Amm onium
Specific o p tic a l ro ta tio n (2.2.7) Amino a d d analysis (2.2.56) as described in the test for
+ 25.5 to + 28.5 (dried substance). ninhydrin-positive substances with the following
Dissolve 2.00 g in hydrochloric add R1 and dilute to 25.0 m L modifications.
with the same acid. Irgection T est solution, reference solution (c) and blank
N in h y d rin -p o sitiv e su b s ta n c e s solution.
Amino acid analysis (2.2.56). F or analysis, use M ethod 1. Limit:
T he concentrations o f the test solution and the reference — ammonium at 570 mn: not m ore than the area of the
solutions may be adapted according to the sensitivity o f the corresponding peak in the chrom atogram obtained with
equipm ent used. T he concentrations o f all solutions are reference solution (c) (0.02 per cent), taking into account
adjusted so that the system suitability requirements described the peak due to am m onium in the chromatogram
in general chapter 2.2.46 are fulfilled, keeping the ratios of obtained with the blank solution.
concentrations between all solutions as described. Ir o n (2.4.9)
Solution A water R ot a sample preparation buffer suitable for M axim um 10 ppm.
the apparatus used. In a separating funnel, dissolve 1.0 g in 10 m L o f dilute
Test solution Dissolve 30.0 m g of the substance to be hydrochloric add R. Shake with 3 quantities, each o f 10 mL,
examined in solution A and dilute to 50.0 m L with o f methyl isobutyl ketone R l, shaking for 3 m in each time.
solution A. 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 H eav y m e ta ls (2.4.8)
10.0 m L with solution A. 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
solution A and dilute to 100.0 mT. w ith solution A. Dilute solvent. 12 m L o f the solution complies with test A. Prepare
1.0 m L o f the solution to 250.0 m L with solution A. the reference solution using lead standard solution
Reference solution (c) D ilute 6.0 mT. o f ammonium standard (1 ppm Pb) R.
solution (100 ppm N H J R to 50.0 m L with solution A. Dilute L oss o n d ry in g (2.2.32)
1.0 m L of this solution to 100.0 m l. with solution A. M axim um 0.5 per cent, determined on 1.000 g by drying in
Reference solution (d) Dissolve 30 m g o f isoleucine R and an oven at 105 °C.
30 m g o f leudne R in solution A and dilute to 50.0 m l. with S u lfa te d a s h (2.4.14)
solution A. Dilute 1.0 m L o f the solution to 200.0 m L with M axim um 0.1 per cent, determined on 1.0 g.
solution A.
A SSA Y
Blank solution Solution A. Dissolve 0.150 g in 50 m L of water R. T itrate with 0.1 M
Inject suitable, equal am ounts of the test, blank and reference hydrochloric add, determining the end-point potentiometrically
solutions into the amino acid analyser. R un a program (2.2.20).
suitable for the determination o f physiological amino adds. 1 mT. o f 0.1 M hydrochloric add is equivalent to 17.42 mg of
System suitability Reference solution (d): C 6HX4N 4O 2.
isoleucine and leudne. STORAGE
In an airtight container, protected from light.
— for any ninhydrin-positive substance detected at 570 nm , IM P U R IT IE S
use the concentration of arginine in reference solution (a); Other detectable impurities (the following substances would, if
— for any ninhydrin-positive substance detected at 440 nm , present at a suffident levd, be detected by one or other of
use the concentration of proline in reference solution (b); the tests in the m onograph. They are limited by the general
if a peak is above the reporting threshold at both acceptance criterion for other/unspecified impurities. It is
w avdengths, use the result obtained at 570 n m for therefore not necessary to identify these impurities for
quantification. 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; h nh2
Hj N ^ ' s ^ x - ^ S‘C02H
— reporting threshold: 0.05 p er c e n t
T he thresholds indicated u n d er R d a te d substances
A. (25)-2,6-diaminohexanoic a d d Qysine),
(Table 2034.-1) in the general m onograph Substances for
pharmaceutical use (2034) do not apply.
M axim um 200 ppm.
2016 Arginine Aspartate 1-191

25.0 m L with the same ad d .
N in h y d rin -p o sitiv e su b stan ces
B. (2S)-2-amino-5-(caibamoylamino)pentanoic a d d Test solution (a) Dissolve 0.20 g of the substance to be
(dtrulline), 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
C. (2S)-2,5-diaminopentanoic a d d (ornithine). of aspartic acid R in water R and dilute to 25 m L with the
PhEur same solvent.
Reference solution (b) Dilute 2 m L o f reference solution (a) to
50 m L with water R.
Arginine Aspartate ★ ★ Mobile phase ammonia R, propanol R (36:64 VIV).
★ ★
*****
Application 5 pL.
(Ph. Eur. monograph 2096) Development Over 2/3 of the plate.
Drying At 100-105 °C for 10 min.
H N
n h2 Detection Spray with ninhydrm solution R and heat at
ho 2c
C02H 100-105 °C for 10 min.
— the chromatogram shows 2 clearly separated prindpal
C ioH21N50 6 307.3 7675-83-4 spots.
Limit, test solution (a):
A ctio n a n d u se
— any impurity: any spots, apart from the 2 principal spots,
Amino add; n u trien t
are no t more intense than each of the 2 prindpal spots in
PhEur_________________ the chromatogram obtained with reference solution (b)
(0.2 p er cent).
D E F IN IT IO N
(2S)-2-Amino-5-guanidinopentanoic a d d (2S)-2- C h lo rid e s (2.4.4)
aminobutanedioate. M aximum 200 ppm.
Dilute 2.5 m L of solution S to 15 m L with water R.
C o n te n t
99.0 per cent to 101.0 per cent (dried substance). S u lfates (2.4.13)
M aximum 300 ppm.
CHARACTERS
T o 0.5 g add 2.5 m L of dilute hydrochloric acid R and dilute
A p p e a ra n c e
to 15 m L with distilled water R. Examine after 30 min.
W hite or almost white granules or powder.
A m m o n iu m (2.4.1)
S olubility
Very soluble in water, practically insoluble in alcohol and in M aximum 100 ppm, determined on 100 mg.
methylene chloride. 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
Comparison arginine aspartate CRS.
C . Examine the chromatograms obtained in the test for an oven at 60 °C for 24 h.
ninhydrin-positive substances.
Resubs T he 2 principal spots in the chromatogram obtained M aximum 0.1 per cent, determined on 1.0 g.
w ith test solution (b) are sim ilar in position, colour and size
to the 2 prindpal spots in the chromatogram obtained with ASSAY
reference solution (a). 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
TESTS
perchloric acid, determining the end-point potentiometrically
S o lu tio n S (2.2 .20).
o f C 10H 21N 5O 6.
__________________________________________________________PhEur
than reference solution Y 7 (2.2.2, Method II).
p H (2.2.3)
+ 25 to + 27 (dried substance).
1-192 Arginine Hydrochloride 2016
TESTS
Arginine Hydrochloride }***%
S o lu tio n S
(Ph Eur. monograph 0805) * Dissolve 2.5 g in distilled water R and dilute to 50 m L with
the same solvent.
H H* -NH2 A p p e a ra n c e o f so lu tio n
• HCI Solution S is clear (2.2.1) and not m ore intensely coloured
than reference solution BY6 (2.2.2, Method II).
NH
C eH uC m A 210.7 1119-34-2
Dissolve 2.00 g in hydrochloric add R1 and dilute to 25.0 m L
A c tio n a n d u se with the same ad d .
Amino add; nutrient. N in h y d rin -p o sitiv e su b s ta n c e s
P r e p a r a tio n Amino a d d analysis (2.2.56). F or analysis, use M ethod 1.
Arginine Hydrochloride Infusion T h e concentrations o f the test solution and die reference
Arginine Hydrochloride Oral Suspension solutions may be adapted according to the sensitivity o f the
Sterile Arginine Hydrochloride C oncentrate equipm ent used. T h e concentrations o f all solutions are
adjusted so that the system suitability requirem ents described
PhEur___________________________________________________________ in general chapter 2.2.46 are fulfilled, keeping the ratios o f
D E F IN IT IO N concentrations betw een all solutions as described.
(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
examined in solution A and dilute to 50.0 m L with
solution A.
CHARACTERS Reference solution (a) D ilute 1.0 m L o f the test solution to
A p p e a ra n c e 100.0 m L with solution A. D ilute 2.0 m L of this solution to
W hite or alm ost white, crystalline pow der o r colourless 10.0 m L with solution A.
crystals.
Reference solution (b) Dissolve 30.0 m g o f proUne R in
S o lu b ility solution A and dilute to 100.0 m L with solution A. Dilute
Freely soluble in w ater, very slightly soluble in ethanol 1.0 m L o f the solution to 250.0 m L with solution A.
(96 per cent). Reference solution (c) Dilute 6.0 m L o f ammonium standard
ID E N T IF IC A T IO N solution (100 ppm N H J R to 50.0 m L with solution A. D ilute
First identification A , B, E 1.0 m L o f this solution to 100.0 m L with solution A.
Second identification A , C, D} E Reference solution (d) Dissolve 30 m g of isoleucine R and
A. Specific optical rotation (see Tests). 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.
Comparison arginine hydrochloride CRS.
C . Thin-layer chromatography (2.2.27).
Inject suitable, equal amounts o f the test, blank and reference
Test solution Dissolve 10 m g o f the substance to be examined solutions into die amino add analyser. Rim a program
in water R and dilute to 50 m L with the same solvent. suitable for the determ ination o f physiological amino adds.
Reference solution. Dissolve 10 m g o f arginine System suitability Reference solution (d):
hydrochloride CRS in water R and dilute to 50 m L with the — résolution: minimum 1.5 between die peaks due to
sam e solvent. isoleucine and leucine.
Plate TLC silica gel plate R. Calculation of percentage contents:
Mobile phase concentrated ammonia R, 2-propanol R — for any ninhydrin-positive substance detected at 570 nm ,
(30:70 VIV). use die concentration o f arginine in reference solution (a);
Application 5 fiL. — 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
Drying At 105 °C until the am m onia disappears completely. wavelengths, use the result obtained at 570 n m for
Detection Spray with mnhydrin solution R and heat at 105 °C quantification.
for 15 min. Limits:
Results T he p rindpal spot in the chrom atogram obtained with — any ninhydrin-positive substance: for each impurity,
the T est solution is similar in position, colour and size to the m axim um 0.2 p er cent;
p rindpal spot in the chrom atogram obtained with the — total: maximum 0.5 per cent;
reference solution. — 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 T h e thresholds indicated under Related substances
a-naphthol solution R and 2 m L o f a mixture o f equal volumes (Table 2034.-1) in the general m onograph Substances for
o f strong sodium hypochlorite solution R and water R. A red pharmaceutical use (2034) do n o t apply.
colour develops.
E. I t gives reaction (a) o f chlorides (2.3.1).
2016 Argon 1-193
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
B. (2S)-2-amino-5-(carbamoylamino)pentanoic a d d
A m ino acid analysis (2.2.56) as described in the test for
(dtrulline),
ninhydrin-positive substances with the following
modifications.
Irgection T est solution, reference solution (c) and blank H,N,
solution.
Limit.
C. (2S)-2,5-diaminopentanoic a d d (ornithine).
— ammonium at 570 nm: not more than the area of the
corresponding peak in the chromatogram obtained with PhEur
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.
I r o n (2.4.9) Argon *****
★ ★
M aximum 10 ppm.
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, Ar 39.95 7440-37-1
of methyl tsobutyl ketone R l, shaking for 3 min each time. PhEur_____________________________
T o the combined organic layers add 10 m L o f water R and
shake for 3 min. U se the aqueous layer. D E F IN IT IO N
Gas obtained by fractional distillation of ambient air.
M axim u m 10 ppm. C o n te n t
M inim um 99.995 per cent V/V o f Ar, calculated by
solvent. 12 m L o f the solution complies with test A. Prepare deduction of the sum of impurities found when performing
the reference solution using lead standard solution 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) CHARACTERS
M axim um 0.5 per cent, determined on 1.000 g by drying in A p p e a ra n c e
an oven at 105 °C. Colourless gas.
S u lfa te d a s h (2.4.14) S o lu b ility
M axim um 0.1 per cent, determined on 1.0 g. 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. ID E N T IF IC A T IO N
A dd 30 m L o f anhydrous acetic acid R. T itrate with 0.1 M A. Verify that the gas is not oxygen using a paramagnetic
perchloric acid, determining the end-point potentiometrically analyser (2.5.27).
(2. 2. 20). B. Gas chromatography (2.2.28).
1 m L of 0.1 M perchloric acid is equivalent to 21.07 mg of Gas to be examined T he substance to be examined.
C e H is C lN ^ .
Reference gas Use the following mixture of gases in argon R l:
STORA GE methane R l (5 ppm V/V), nitrogen R l (5 ppm V/V), oxygen R
Protected from light. (5 ppm V/V).
IM P U R IT IE S 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).
therefore not necessary to identify these impurities for Carrier gas helium for chromatography R.
dem onstration o f compliance. See also 5.10. Control of Flow rate 10 mL/min.
impurities in substances for pharmaceutical use): A , B, C. Temperature:
— column: 50 °C;
H,N Detection Thermal conductivity.
Irgection 25 JiL.
A. (2S)-2,6-diaminohexanoic a d d (lysine), System suitability. Reference gas:
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 if* it
if ★
Im p u ritie s
Aripiprazole ★ ★
Gas chromatography (2.2.28). (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•
— 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 448.4 129722-12-9
Carrier gas argon R l.
Flow rate 70 mL/min. A c tio n a n d u se
D opam ine D 2 receptor antagonist; neuroleptic
Temperature.
— column: 80 °C; PhEur___________________________________________________________
D E F IN IT IO N
Detection Discharge ionisation.
7-[4- [4- (2,3-Dichlorophenyl)piperazin-1-yl] butoxy]-3,4-
Injection 1 m l- dihydroquinolin-2(lii)-one.
Sample rate 100 mL/min. C o n te n t
Relative retention W ith reference to im purity C (retention 98.0 per cent to 102.0 per cent (dried substance).
tim e = about 4.7 min): im purity A = about 0.4;
CHARACTERS
A p p e a ra n c e
System suitability: Reference gas:
W hite or alm ost white crystals o r crystalline powder.
impurities A and B and m in im u m 2.0 between the peaks S o lu b ility
due to impurities B and C. Practically insoluble in water, soluble in methylene chloride,
Limits: very slighty soluble in ethanol (96 per cent).
— impurity A: not m ore than the area of the corresponding It shows polym orphism (5.9).
peak in the chrom atogram obtained with the reference gas ID E N T IF IC A T IO N
(5.0 p pm V!V)\ Infrared absorption spectrophotom etry (2.2.24).
— total: m axim um 0.0040 p er cent o f the sum of the areas
Comparison aripiprazole CRS.
o f all the peaks (40.0 p pm VIV).
If the spectra obtained in the solid state show differences,
W a te r (2.5.28)
dissolve the substance to be examined and the reference
M axim um 10.0 ppm VIV, d eterm ined u sin g an electrolytic
substance separately in methylene chloride R, evaporate to
hygrometer.
dryness and record new spectra using the residues.
STORAGE
TESTS
In gaseous or liquid state, in suitable con tainers, complying
w ith the legal regulations.
I f intended for use in the m anufacture o f parenteral
IM P U R IT IE S preparations, the solution is clear (2.2.1) and n o t more
Specified impurities A, D intensely coloured than reference solution GY 5 (2.2.2,
Other detectable impurities B, C. Method II).
A. oxygen, 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
B. nitrogen,
the same mixture of solvents. Sonicate for about 15 m in,
C. m ethane, shaking occasionally, until dissolution is complete.
D . water.
------ ----------------------------------------------------------------------------- PhEur 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).
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
100.0 m L with the solvent mixture. D ilute 1.0 m L o f this
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
2016 Aripiprazole 1-195
mixture. Dilute 1 m L of the solution to 50 m L with the STO RA G E

solvent mixture. Protected from light. If the substance is sterile, store in a
Reference solution (c) Dissolve 50.0 mg of aripiprazole CRS in sterile, airtight, tam per-proof container.
the solvent mixture and dilute to 50.0 m L with the solvent L A B E L L IN G
mixture. Dilute 5.0 m L of the solution to 50.0 m L with the T h e label states, where applicable, that the substance is
solvent mixture. suitable for use in the manufacture o f parenteral
Column: preparations.
— sizer. I = 0.10 m , 0 = 4.6 mm;
IM P U R IT IE S
— stationary phase: end-capped. octadecylsUyl silica gel for
chromatography R (3 pm). Other detectable impurities (the following substances would, if
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)',
Control of impurities in substances for pharmaceutical use): A, B,
Time Mobile phase A Mobile phase B C, D} E, F, G.
( o i l) (per cent V/V) (per cent V/V)
0 -2 80 20
2 - 10 80 ->65 20-»35
10-20 65-» 10 35-»90
2 0 -2 5 10 90
A. 7-hydroxy-3,4-dihydroquinolirt-2(lfi)-one,
Flow rate 1.2 m l/m in .

Injection 20 |xL o f the test solution and reference solutions (a)
and (b).
Relative retention W ith reference to aripiprazole (retention a
time — about 11 min): impurity F = about 1.1.
System suitability: reference solution (b): B. l-(2,3-dichlorophenyl)piperazine,
aripiprazole and impurity F.
Calculation of percentage contents:
— for each impurity, use the concentration of aripiprazole in
Limits:
— unspecified impurities', for each impurity, maximum
0.10 per cent; C . 7- [4- [4-(2-chlorophenyl)piperazin-1-yl] butoxy] -3,4-
— total: maximum 0.2 per cent; dihydroquinolin-2( 1H)-ons,
— reporting threshold: 0.05 per cent.
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) ci
Less than 5 IU/mg, if intended for use in the m anufacture o f
parenteral preparations without a further appropriate D . 7-[4-[4-(3-chlorophenyl)piperazin-l-yl]butoxy]-3,4-
procedure for the removal o f bacterial endotoxins. dihydroquinolin-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
Injection T est solution and reference solution (c).
ci
— symmetry factor, maximum 2 .0 .
E. 7- [4- [4-(2,3-dichlorophenyl)piperazin-1-yl]butoxy]
Calculate the percentage content o f C 23H 27CI2N 3O 2 taking quinolin -2 (lii)-o n e ,
into account the assigned content o f aripiprazole CRS.
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.
F. 7-[4- [4-(2,3-dichlorophenyi)-1-oxidopiperazin-1-yl] C. Thin-layer chrom atography (2.2.27).
butoxy] -3,4-dihydroquinolin-2 (l/f)-o n e , 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.
Drying In air.
G. 7 , 7 [ethane-1,1 -diylbis [(2,3-dichlorobenzene-4,1 -diyl) Detection Examine in ultraviolet light at 254 nm.
piperazine-4, l-diyibutane-4, l-diyloxy]]bis[3,4- Results T h e principal spot in the chrom atogram obtained with
dihydroquinolin-2 (1 if)-o n e]. the test solution is similar in position and size to the principal
PhEur
solution.
D . It gives reaction (a) o f chlorides (2.3.1).
TESTS
Articaine Hydrochloride * * * * *
★ ★ S o lu tio n S
* * * * * Dissolve 0.50 g in water R and dilute to 10 m L with the
(Ph. Eur. monograph 1688) same solvent.
H CHS Solution S is clear (2.2.1) and n o t more intensely coloured
- HCI than reference solution BY 6 (2.2.2, Method I).
p H (2.2.5)
and enanb'omer 4.2 to 5.2.
C13H21CIN2O3S 320.8 23964-57-0 20.0 m L with the same solvent.
A ctio n a n d u se
Local anaesthetic.
PhEur. exam in ed in the mobile phase and dilute to 10.0 m L with
D E F IN IT IO N the mobile phase.
M ethyl 4-m ethyl-3- [[(2i?S)-2-(propyiamino)propanoyi] Reference solution (a) D ilute 1.0 m L o f the test solution to
amino]thiophene- 2-carboxylate hydrochloride. 100.0 m L with the mobile phase. D ilute 1.0 m L o f this
C o n te n t
98.5 per cent to 101.0 per cent (dried substance). 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
CHARACTERS the mobile phase and dilute to 50.0 m L with the mobile
A p p e a ra n c e phase. D ilute 1.0 m L o f the solution to 50.0 m L w ith the
W hite or alm ost white, crystalline powder. mobile phase.
S o lu b ility Column:
Freely soluble in w ater and in ethanol (96 per cent). — size: I = 0.25 m , 0 = 4.6 mm ;
ID E N T IF IC A T IO N — stationary phase: spherical end-capped octadecylsUyl silica gel
First identification B, D for chromatography R (5 pm);
Mobile phase M ix 25 volumes o f acetomtrUe R and 75 volumes
A. Dissolve 50.0 m g in a 1 g/L solution of hydrochloric add R
o f a solution prepared as follows: dissolve 2.02 g o f sodium
and dilute to 100.0 m L with the same acid. Dilute 5.0 m L o f
heptanesulfonate R and 4.08 g o f potassium dihydrogen
the solution to 100.0 m L with a 1 g/L solution o f hydrochloric
phosphate R in water R and dilute to 1000 m L with the same
add R. Exam ined between 200 n m and 350 nm (2.2.25), the
solvent. A djust to p H 2.0 with phosphoric acid R.
solution shows an absorption m axim um at 272 nm .
T he specific absorbance at the m axim u m is 290 to 320. Flow raze 1 mL/min.
B. Infrared absorption spectrophotom etry (2.2.24). Detection Spectrophotom eter at 276 nm.
Preparation Place dropwise 20 |iL o f the test solution on Irgecdon 10 pL.
300 mg discs. Run time 5 times the retention tim e o f articaine.
2016 Articaine Hydrochloride 1-197
HOzC L1 H CH
CH3
Relative retention W ith reference to articaine (retention
tim e = about 9 min): impurity A = about 0.8; and enantiomer
impurity E = about 0.86. O

CH3
System suitability Reference solution (b):
— resolution: m inim u m 1.2 between the peaks due to
impurities A and £ . B. 4-methyl-3-[[(22?S)-2-(propylamino)propanoyl]amino]
Limits: thiophene-2-carboxylic acid (articaine add),
— impurity A: not more than the area of the corresponding
h ch3
/ \ ^ ch3 and enantiomer
— sum of impurites other than A: not more than 5 times the
area of the principal peak in the chromatogram obtained C. 1-methylethyl 4-methyl-3-[ [(2RS)-2-(propjdamino)
with reference solution (a) (0.5 per cent); propanoyl]amino]thiophene-2-carboxylate (articaine
— disregard limit. 0.5 times the area o f the principal peak in isopropyl ester),
(0.05 p er cent).
H CH3
M aximum 5 ppm.
Dissolve 4.0 g in 20.0 m L o f water R 12 m L of the solution
and enantiomer
lead standard solution (1 ppm Pb) R.
D . methyl 3-[[(2i?S)-2-(ethylamino)propanoyl]amino]-4-
L oss on d ry in g (2.2.32)
M aximum 0.5 per cent, determined on 1.000 g by drying in methylthiophene-2-caiboxylate (ethylarticaine),
A SSA Y
Dissolve 0.250 g in a mixture o f 5.0 m L o f 0.01 M and enantiomer
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 E. methyl 4-methyl-3- [[(2ftS)-2-[( 1-methylethyl) amino]
hydroxide. Read the volume added between the 2 points of propanoyl] amino] thiophene-2-carboxylate
inflexion. (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 H H- #CH3
IM P U R IT IE S o
CHj and enantiomer
F. 4-methyl-N-propyl-3- [ [(2RS)-2-(propylamino)
propanoyl] amino] thiophene-2-carboxamide (articaine a d d
propionamide),
D ,E ,F ,G ,H ,I,J .
G. methyl 3- [ [(2RS)-2-(butylamino)propanoyl] amino] -4-

x\ ^ ch3
methylthiophene-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] -4-

methylthiophene-2-Carboxylate (dipropylarticaine),
1-198 Ascorbic Acid 2016
nh 2 A. Ultraviolet and visible absorption spectrophotom etry
(2.2.25).
ch3 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
I. methyl 3-am ino-4-methylthiophene-2-caiboxylate
dilute to 100.0 m L with water R
(3-aminoarticaine),
Absorption maximum At 243 nm , determ ined immediately
after dissolution.
i Spedfic absorbance at the absorption maximum 545 to 585.
Ch3 and enantiomer B. Infrared absorption spectrophotom etry (2.2.24).
Comparison ascorbic add CRS.
CH3
C. p H (2.2.3): 2.1 to 2.6 for solution S (see Tests).
J. methyl 3-[[(2i?S)-2-bromopropanoyl]amino]-4- D. T o 1 m L o f solution S add 0.2 m L o f dilute nitric acid R

methylthiophene- 2 -carboxylate (bromo compound). and 0.2 m L o f silver nitrate solution R2. A grey predpitate is
formed.
TESTS
S o lu tio n S
*****
Ascorbic Acid ★ ★ A p p e a ra n c e o f so lu tio n
(Ph. Eur. monograph 0253) * * * * * Solution S is clear (2.2.1) and n o t m ore intensdy coloured
S p ecific o p tic a l r o ta tio n (2.2.7)
+ 20.5 to + 21.5.
same solvent.
HO OH
I m p u rity E
M axim um 0.2 per cent.
CsHgOg 176.1 50-81-7
Test solution Dissolve 0.25 g in 5 m L o f water R. Neutralise
A c tio n a n d use using dilute sodium hydroxide solution R and add 1 m L o f dilute
Vitam in C. acetic add R and 0.5 m L o f caldum chloride solution R.
P re p a r a tio n s Reference solution Dissolve 70 m g o f oxalic add R in water R
Ascorbic A d d Injection and dilute to 500 m L with the same solvent; to 5 m L o f this
Ascorbic A d d Tablets solution add 1 m L o f dilute acetic add R and 0.5 m L o f
caldum chloride solution R
Chewable Ascorbic A d d Tablets
Allow the solutions to stand for 1 h. Any opalescence in the
Paediatric Vitam ins A, C and D Oral Drops
test solution is no t m ore intense than that in the reference
Potassium Ascorbate Eye D rops solution.
Vitamins B and C Injection R e la te d su b s ta n c e s
W hen Vitamin C is prescribed or dem anded. Ascorbic A d d Liquid chrom atography (2.2.29). Prepare the solutions
shall be dispensed or supplied. immediately before use.
PhEur___________________________________________________________
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
D E F IN IT IO N same solvent. Filter through a m em brane filter (nominal pore
(5i?)-5- [(1S) -1 ,2-Dihydroxyethyl] -3,4-dihydroxyfuran-2 (5H) - size 0.45 pm) and dilute to 1000 m L with water R.
one. Test solution Dissolve 0.500 g o f the substance to be
C o n te n t examined in the mobile phase and dilute to 10.0 m L with
99.0 per cent to 100.5 per cent. the mobile phase.
CHARACTERS Reference solution (a) Dissolve 10.0 m g o f ascorbic add
A p p e a ra n c e impurity C CRS in the mobile phase and dilute to 5.0 m L
W hite or alm ost white, crystalline pow der or colourless with the mobile phase.
crystals, becoming discoloured on exposure to air and Reference solution (b) Dissolve 5.0 m g o f ascorbic add
moisture. impurity D CRS and 5.0 m g o f ascorbic add CRS in the
S o lu b ility mobile phase, add 2.5 m L o f reference solution (a) and
Freely soluble in water, sparingly soluble in ethanol dilute to 100.0 m L with the mobile phase.
(96 per cent). Reference solution (c) D ilute 1.0 m L of the test solution to
200.0 m L with the mobile phase. M ix 1.0 m L of this
mp
A bout 190 °C, with decomposition. solution with 1.0 m L of reference solution (a).
Column:
— sizer. I — 0.25 m , 0 = 4.6 mm;
First identification: B , C.
2016 Ascorbic Acid 1-199
— stationary phase: aminopropylsifyl silica gel for Adjust the zero of the apparatus using 0.1 M nitric add.
chromatography R (5 nm)i H eav y m e ta ls (2.4.8)
— temperature: 45 °C. M aximum 10 ppm.
Mobile phase Phosphate buffer solutionj acetonitrUe R1 Dissolve 2.0 g in water R and dilute to 20 m L with the same
(25:75 VIV). solvent. 12 m L of the solution complies with test A. Prepare
Flow rate 1.0 mlVmin. the reference solution using lead standard solution
Detection Spectrophotom eter at 210 nm. (1 ppm Pb) R.
Injection 20 |iL o f the test solution and reference solutions (b) S u lfa te d a s h (2.4.14)
and (c). M aximum 0.1 per cent, determined on 1.0 g.
Run time 2.5 times the retention time of ascorbic acid. A SSAY
Identification of impttrities Use the chromatogram obtained Dissolve 0.150 g in a mixture of 10 m L of dilute sulfuric
with reference solution (b) to identify the peaks due to add R and 80 m L of carbon dioxide-free water R. Add 1 m L of
impurities C and D. starch solution R. Titrate with 0.05 M iodine until a persistent
Relative retention W ith reference to ascorbic acid (retention violet-blue colour is obtained.
time = about 11 min): impurity D = about 0.4; 1 m L of 0.05 M iodine is equivalent to 8.81 mg of CgHgOg.
impurity C = about 1.7.
STORA GE
System suitability. In a non-metallic container, protected from light.
ascorbic a d d and impurity C in the chromatogram IM P U R IT IE S
obtained with reference solution (c); Specified impurities C, D , E
— signal-to-noise ratio: m in im u m 20 for the peak due to Other detectable impurities (the following substances would, if
impurity C in the chromatogram obtained with reference present at a suffident level, be detected by one or other o f
solution (b). the tests in the monograph. They are limited by the general
Limits: acceptance criterion for other/unspecified impurities and/or
— impurities C, D: for each impurity, n o t more than by the general monograph Substances for pharmaceutical vise
1.5 times the area of the corresponding peak in the (2034). It is therefore not necessary to identify these
chrom atogram obtained with reference solution (b) impurities for demonstration of compliance. See also 5.10.
(0.15 per cent); Control of impurities in substances for pharmaceutical use): A , F,
— unspecified impurities: for each impurity, n o t more than the G, H.
area o f the peak due to ascorbic a d d in the
chromatogram obtained with reference solution (b) /° V -C H O
(0.10 per cent); i 1
— 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
A. 2-furaldehyde,
(0.2 per cent);
OH oh o
— 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).
rV/™
OH OH O
C opper
M aximum 5 ppm . C. D-xy/o-hex-2-ulosonic a d d (D-sorbosonic ad d ),
OH OH o
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 , OH OH O
0.4 ppm and 0.6 ppm ) by diluting copper standard solution
(10 ppm Cu) R w ith 0.1 M nitric add. D . methyl D-xyfo-hex-2-ulosonate (methyl D-sorbosonate),
Source C opper hollow-cathode lamp.
Adjust the zero o f the apparatus using 0.1 M nitric add.
V" o
Ir o n
M aximum 2 ppm . 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 HO OH
(20 ppm Fe) R w ith 0.1 M nitric acid.
Source Iron hollow-cathode lamp. F. (5R)-5-[(lR)-l,2-dihydroxyethyl]-3,4-dihydroxyfuran-
Wavelength 248.3 nm. 2 (5/i)-one,
1-200 Ascorbyl Palmitate 2016

.OH
HO + 21 to + 24 (dried substance), determ ined on solution S.
T h e thresholds indicated under Related substances
HO OH (Table 2034.-1) in the general m onograph Substances for
pharmaceutical use (2034) do n o t apply.
G. (2R)-2-[(2Æ)-3j4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl]-
2-hydroxyacetic acid. H e a v y m e ta ls (2.4.8)
M axim um 10 ppm.
M axim um 1.0 p er cent, determ ined on 1.000 g by drying
HO OH m vacuo at 60 °C for 5 h.
H . methyl (2i?)-2-[(22?)-3j4-dihydroxy-5-oxo-2j5- M aximum 0.1 per cent, determ ined on 1.0 g.
dihydrofuran-2-yl]-2-hydroxyacetate.
A SSA Y
PhEur
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
*
Ascorbyl Palmitate ★ ★ C 22H 38O 7.
(Ph. Eur. monograph 0807) ***** STORA GE

In an airtight container, protected from lig h t
___________________________________________________________PhEur
Asparagine Monohydrate *****

★ ★
* * * * *
C22H 3807 414.5 137-66-6 E** monograph 2086)
Excipient.
X x™ 1 , H20
h2n ' ^ ^ v co2h
PhEur.
C4H8N20 3iH 20 150.1 5794-13-8
D E F IN IT IO N
(25)-2-[(2i?)-3,4-Dihydroxy-5-oxo-2,5-dihydrofuraii-2-yl]-2- A c tio n a n d u se
hydroxyethyl hexadecanoate. Amino add.
C o n te n t
PhEur_____________
D E F IN IT IO N
CHARACTERS
(2S)-2,4-Diam ino-4-oxobutanoic a d d m onohydrate.
A p p e a ra n c e
W hite or yellowish-white powder. C o n te n t
S o lu b ility
Practically insoluble in water, freely soluble in ethanol CHARACTERS
(96 p er cent) and in m ethanol, practically insoluble in A p p e a ra n c e
methylene chloride and in fatty oils. W hite or almost white, crystalline pow der or colourless
crystals.
A. Specific optical rotation (see Tests). S o lu b ility
Slightly soluble in water, practically insoluble in ethanol
(96 per cent) and in methylene chloride.
Comparison ascorbyl palmitate CRS.
C. Dissolve about 10 mg in 5 m L o f methanol R.
First identification A , B
T he solution decolourises dichlorophenolindophenol standard
solution R. Second identification A , C
TESTS
S o lu tio n S B. Infrared absorption spectrophotom etry (2.2.24).
Dissolve 2.50 g in methanol R and dilute to 25.0 m L with the Comparison asparagine monohydrate CRS.
same solvent C. Examine the chrom atograms obtained in the test for
A p p e a ra n c e o f so lu tio n ninhydrin-positive substances.
Solution S is clear (2.2.1) a n d n o t m ore intensely coloured Results T h e principal spot in th e chrom atogram obtained with
than reference solution BY4 (2.2.2, Method I). test solution (b) is similar in position, colour and size to the
2016 Aspartame 1-201
principal spot in the chromatogram obtained with reference organic phases w ith 10 m L of water R for 3 min.
solution (c). T h e aqueous phase complies with th e limit test for iron.
TESTS H eav y m e ta ls (2.4.8)
S o lu tio n S M aximum 10 ppm .
Dissolve with heating 2.0 g in carbon dioxide-free water R and Dissolve 2.0 g in a mixture of 3 m L of dilute hydrochloric
dilute to 100 m L with the same solvent. acid R and 15 m L of water R with gentle warming if
A p p e a ra n c e o f so lu tio n necessary. Dilute to 20 m L with water R. 12 m L o f the
Solution S is d ear (2.2.1) and colourless (2.2.2, Method II). solution complies with test A. Prepare the reference solution
p H (2.2.3)
4.0 to 6.0 for solution S. 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)
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 A SSA Y
Thin-layer chromatography (2.2.27). 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
examined in water R, heating to not more than 40 °Cj and
( 2 .2.20).
dilute to 10 m L w ith the same solvent.
o fC 4 H 8N 20 3.
w ith water R.
Reference solution (a) Dilute 1.0 m L o f test solution (a) to IM P U R IT IE S
200 m L with water R. Specified impurities: A, B.
Reference solution (b) Dissolve 25 mg o f glutamic acid R in
H nh2
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 A. (25)-2-aminobutanedioic a d d (aspartic ad d ),
sam e solvent.
Hate TLC silica gel G plate R. h nh 2
V
Mobile phase glacial acetic acid R, water R, butanol R ho2c / S s ^ n‘co2h
(25:25:50 V/VIV).
Application 5 pL. B. (2S)-2-aminopentanedioic a d d (glutamic ad d ).
Development Over h alf of the plate. PhEur
Drying A t 110 °C for 15 min.
Detection Spray with ninhydrin solution R and heat at 110 °C
for 10 min.
System suitability: reference solution (b): ★★* * ★
— the chromatogram shows 2 clearly separated principal
Aspartame ★ ★
spots. (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
(0.5 per cent).
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 5 294.3 22839-47-0
T o 0.75 g add 2.5 m L o f dilute hydrochloric acid R and dilute A c tio n a n d u se

to 15 m L with distilled water R. Examine after 30 min. Sweetening agent.
PhEur_____________
M axim um 0.1 per cent, determined on 10 mg.
I r o n (2.4.9) D E F IN IT IO N
M axim um 10 ppm. (3S)-3-Amino-4- [ [(25) -1 -methoxy-1-oxo-3-phenylpropan-2-
yl] amino]-4-oxobutanoic a d d (methyl a-L-aspartyl-L-
phenylalaninate).
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 C o n te n t
1-202 Aspartame 2016
CHARACTERS Calculate the conductivity o f die solution o f the substance to

A p p e a ra n c e be examined using the following expression:
W hite or alm ost white, slightly hygroscopic, crystalline
powder. C i - 0.992 C2
S o lu b ility
Sparingly soluble or slightly soluble in water and in ethanol Specific optical rotation (2.2.7)
(96 per cent), practically insoluble in hexane and in + 14.5 to + 16.5 (dried substance).
methylene chloride. Dissolve 2.00 g in a 690 g/L solution of anhydrous formic
ID E N T IF IC A T IO N add R and dilute to 50.0 m L with the same solution.
First identification B. M easure within 30 min o f preparation.
Second identification A , C, D. Related substances
A. Ultraviolet and visible absorption spectrophotometry Liquid chromatography (2.2.29).
(2.2.25). Test solution Dissolve 0.60 g o f the substance to be examined
Test solution Dissolve 0.1 g in ethanol (96 per cent) R and in a mixture o f 1.5 volumes o f glacial acetic add R and
dilute to 100 rnT. with the same solvent. 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
Absorption maxima A t 247 nm , 252 nm , 258 nm and
impurity A CRS in a mixture o f 1.5 volumes o f glacial acetic
264 nm .
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. Reference solution (b) Dissolve 30.0 m g o f phenylalanine R
Comparison aspartame CRS. (impurity C) in a mixture o f 15 volumes o f glacial acetic
C. Thin-layer chrom atography (2.2.27). 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 with the same mixture o f solvents. Dilute 1.0 m L o f this
in 2.5 m L o f water R and dilute to 10 m L with acetic acid R 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 Reference solution (c) D ilute 5.0 m L o f the test solution to
of water R and dilute to 10 m L with acetic add R. 10.0 m L with water R D ilute 3.0 m L o f this solution to
Reference solution (d) Dissolve 30.0 m g o f L-aspartyl-L-
Mobile phase water R, anhydrous formic add R> methanol R,
phenylalanine R (impurity B) in a mixture o f 15 volumes of
methylene chloride R (2:4:30:64 V/V/V/V).
glacial acetic add R and 85 volumes o f water R and dilute to
Application 20 |±L. 100.0 m L with die same mixture o f solvents. D ilute 1.0 m L
Development O ver a p ath o f 15 cm. o f the solution to 10.0 m L with water R. M ix 1.0 m L o f this
Drying In air. solution with 1.0 m L o f reference solution (b).
Detection Spray with nmhydrm solution R and heat at Column
100-105 °C for 15 min. — sizer. I = 0.25 m , 0 = 4.0 mm ;
Results T he spot in the chrom atogram obtained with the test — stationary phase: octadecylsQyl silica gel for chromatography R
solution is similar in position, colour and size to the spot in (5-10 nm).
the chrom atogram obtained w ith the reference solution. Mobile phase M ix 10 volumes o f acetordtri1e R and 90 volumes
D . Dissolve about 20 m g in 5 m L o f methanol R and add o f a 6.8 g/L solution o f potassium dihydrogen phosphate R
1 m L o f alkaline hydroxylamme solution R l. H eat on a water- previously adjusted to p H 3.7 with phosphoric add R.
bath for 15 m in. Allow to cool and adjust to about p H 2 Flow rate 1 mL/min.
with dilute hydrochloric acid R. A dd 0.1 m L o f ferric chloride Detection Spectrophotom eter at 220 nm .
solution R l. A brownish-red colour is pro d u ced Injection 20 |±L.
TESTS Run time Twice the retention tim e o f aspartame.
S o lu tio n S System suitability Reference solution (d):
Dissolve 0.8 g in carbon dioxide-free water R and dilute to — resolution: minimum 3.5 betw een the peaks due to
100 m L with the sam e solvent. impurities B and C.
A p p e a ra n c e o f so lu tio n Limits:
Solution S is clear (2.2.1) and not m ore intensely coloured — impurity A: n o t m ore than die area o f the principal peak
than reference solution GY6 (2.2.2, Method II). in the chrom atogram obtained with reference solution (a)
C o n d u c tiv ity (2.2.38) (1.5 p er cent);
M axim um 30 nS-cm-1 . — impurity C: n o t m ore than die area o f th e principal peak
(0.5 p er cent);
distilled water R and dilute to 100.0 m L with the same
— sum of impurities other than A and C: n o t more th an the
solvent. M easure the conductivity o f the solution (C x) and
area o f die principal peak in die chrom atogram obtained
that o f the w ater used for preparing the solution (C 2).
w ith reference solution (c) (1.5 per cent);
T he readings m ust be stable within 1 p er cent over a period
— disregard limit: disregard any peak due to the solvent.
o f 30 s.
Maximum 10 ppm.
1.0 g complies with test C. Prepare die reference solution
2016 Aspartic Acid 1-203
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
Aspartic Acid ★ ★
★ ★
an oven at 105 °C. (Ph. Eur. monograph 0797) *★ *
M aximum 0.2 per cent, determined on 1.0 g. H NH 2
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 C4H7N 0 4 133.1 56-84-8
0.1 M perchloric add, determining the end-point
potentiometrically (2.2.20). A c tio n a n d u se
1 m L of 0.1 M perchloric acid is equivalent to 29.43 mg Amino a d d .
Of C 14H 18N 2O 5.
P hB r_____________
STORAGE
D E F IN IT IO N
Aspartic a d d contains not less th an 98.5 per cent and not
IM P U R IT IE S m ore than the equivalent of 101.5 p e r cent of
Specified impurities A, C (25)-2-am inobutanedioic add, calculated with reference to
Other detectable impurities (the following substances would, if the dried substance.
present at a sufficient level, be detected by one or other of CHARACTERS
the tests in the m onograph. They are limited by the general A white o r almost white, crystalline powder or colourless
acceptance criterion for other/unspecified impurities and/or crystals, slightly soluble in water, practically insoluble in
by the general m onograph Substances for pharmaceutical use alcohol. I t dissolves in dilute mineral adds and in dilute
(2034). I t is therefore not necessary to identify these solutions o f alkali hydroxides.
impurities for dem onstration of compliance. See also 5.10.
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).
COzH B. A suspension of 1 g in 10 m L o f water R is strongly a d d
(2.2.4).
C. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with aspartic
A. 2-[(25',53)-5-benzyl-3,6-dioxopiperazm-2-yl]acetic ad d . 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
TESTS
HOjC 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
B. (35)-3-amino-4- [[(13)-1 -carboxy-2-phenylethyI] amino] -4-
more intensely coloured than reference solution BY 6 (2.2.2,
oxobutanoic a d d (a-L-aspartyl-L-phenylalanine),
Method II).
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.
C . (2S)-2-amino-3-phenylpropanoic a d d (L-phenylalanine).
N in h y d rin -p o sitiv e su b stan ces
PhEir
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

Aspirin *. * * * ★
10 m g of glutamic add CRS in 2 m L of dilute ammonia R1 ★ ★
and dilute to 25 m L with water R. *****
(Acetylsalicylic Add, Ph Eur monograph 0309)
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 C02H
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 ch 3
solution (a), apart from the principal spot, is n o t more

intense than the spot in die chromatogram obtained with C9H 804 180.2 50-78-2
reference solution (b) (0.5 per cent). T h e test is n o t valid
unless the chromatogram obtained with reference solution (c) A c tio n a n d u se
shows 2 clearly separated principal spots. Salicylate; non-selective cyclo-oxygenase inhibitor;
C h lo rid e s (2.4.4) antipyretic; analgesic; anti-inflammatory.
Dissolve 0.25 g in 3 m L o f dilute nitric add R and dilute to P re p a ra tio n s
15 m L with water R. T h e solution, to which 1 m L o f water R Aspirin Tablets
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
Dispersible Aspirin Tablets
a
Effervescent Soluble Aspirin Tablets
Gastro-resistant Aspirin Tablets
Aspirin and Caffeine Tablets
limit test for sulfates (300 ppm ). Carry out the evaluation o f Co-codaprin Tablets
the test after 30 min. Dispersible Co-codaprin Tablets
A m m o n iu m PhEur.
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 D E F IN IT IO N
(100 ppm N H J R. 2-(Acetyloxy)benzoic acid.
Ir o n (2.4.9) C o n te n t
In a separating funnel, dissolve 1.0 g in 10 m L o f dilute 99.5 per cent to 101.0 per cent (dried substance).
hydrochloric add R. Shake with 3 quantities, each of 10 mT, CHARACTERS
of methyl isobutyl ketone R l, shaking for 3 min each time. A p p e a ra n c e
T o d ie combined organic layers add 10 m L o f water R and W hite or almost white, crystalline pow der or colourless
shake for 3 min. T he aqueous layer complies with the limit crystals.
test for iron (10 ppm ).
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
drying in an oven at 105 °C. ID E N T IF IC A T IO N
First identification A, B
Sulfa ted ash (2.4.14)
N ot m ore than 0.1 per cent, d eterm ined on 1.0 g. Second identification B, C, D
A Infrared absorption spectrophotometry (2.2.24).
A SSA Y
Dissolve 0.100 g in 50 m L o f carbon dioxide-free water R, with
Comparison acetylsalicylic add CRS.
slight heating if necessary. Cool and add 0.1 m L o f B. T o 0.2 g add 4 m L o f dilute sodium hydroxide solution R
bromothymol blue solution R l. T itrate with 0.1 M sodium and boil for 3 min. Cool and add 5 mT. o f dilute sulfuric
hydroxide until the colour changes from yellow to blue. add R. A crystalline precipitate is formed. Filter, wash the
precipitate and dry at 100-105 °C. T h e melting point
1 m L o f 0.1 M sodium hydroxide is equivalent to 13.31 m g of
C4H7N 0 4. (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.
STO RA G E
H eat the mixture and expose to the fumes produced a piece
Protected from light. o f filter paper impregnated with 0.05 m L o f nitrobenzaldehyde
PhEur 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
Method 11).
2016 Aspirin 1-205
Dissolve 1.0 g in 9 m L o f ethanol (96 per cent) R. Prepare the reference solution using lead standard solution
Related substances (1 ppm Pb) obtained by diluting lead standard solution
L iquid chromatography (2.2.29). Prepare the solutions (100 ppm Pb) R with a mixture o f 6 volumes of water R and
immediately before use. 9 volumes of acetone R.
Test solution Dissolve 0.100 g of the substance to be L oss o n d ry in g (2.2.32)

exam ined in acetonitrile for chromatography R and dilute to M axim um 0.5 p er cent, determined on 1.000 g by drying
10.0 m L w ith the same solvent. in vacuo.
Reference solution (a) Dissolve 50.0 m g o f saUcyUc add R S u lfa te d a s h (2.4.14)
(im purity C) in th e mobile phase and dilute to 50.0 m L with M axim um 0.1 p e r cent, determined on 1.0 g.
the mobile phase. Dilute 1.0 m L of the solution to 100.0 m L A SSAY
w ith the mobile phase. In a flask with a ground-glass stopper, dissolve 1.000 g in
Reference solution (b) Dissolve 10 mg o f salicylic add R 10 m L o f ethanol (96 per cent) R. Add 50.0 m L of 0.5 M
(impurity C) in th e mobile phase and dilute to 10.0 m L with sodium hydroxide. Close die flask and allow to stand for 1 h.
the mobile phase. T o 1.0 m L of the solution add 0.2 m L of U sing 0.2 m L o f phenolphthalan solution R as indicator, titrate
the test solution and dilute to 100.0 m L with the mobile with 0.5 M hydrochloric add. Carry out a blank titration.
phase. 1 m L of 0.5 M sodium hydroxide is equivalent to 45.04 m g o f
Reference solution (c) Dissolve with the aid o f ultrasound the C 9H 8O 4.
contents o f a vial of aceiylsalicylic add for peak
STO R A G E
identification CRS (containing impurities A, B, D , E and F)
in 1.0 m L of acetonitrile R.
Column: IM P U R IT IE S
— size. I = 0.25 m , 0 = 4.6 mm; Specified impurities A, B, C , D , E , F
— stationary phase: octadecylsUyl silica gelfor chromatography R
(5 pm). CO2H
Mobile phase phosphoric add R, acetonitrile for
chromatography R, water R (2:400:600 VIVfV). HO
Flow rate 1 mL/min.

Detection Spectrophotom eter at 237 nm . A. 4-hydroxybenzoic ad d ,
Injection 10 |iL.
h o 2c CO2H
Run time 7 times the retention time o f acetylsalicylic ad d .
Identification of impurities U se the chrom atogram obtained OH
w ith reference solution (a) to identify the peak due to
im purity C; use the chromatogram supplied with B. 4-hydroxybenzene-1,3-dicarboxylic a d d
acetylsalicylic acid for peak identification CRS and the (4-hydroxyisophthalic ad d ),
the peaks due to impurities A, B, D , E and F.
COjH
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;
a OH
im purity D = about 2.3; impurity E = about 3.2;

C . 2-hydroxybenzenecarboxylic a d d (salicylic ad d ),
im purity F = about 6.0.
acetylsalicylic a d d and impurity C .
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
(0.15 p e r cent);
— unspecified impurities', for each impurity, n o t more than D . 2-[[2-(acetyloxy)benzoyl]oxy]benzoic a d d
0.5 times the area of the principal peak in the (acetylsalicylsalicylic ad d ),
COjH
(0.05 p e r cent);
— total: n o t m ore than 2.5 times the area o f the prindpal
— 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) E. 2-[(2-hydroxybenzoyl)oxy]benzoic a d d (salsalate,
M axim um 20 ppm . salicylsalicylic a d d ),
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.
1-206 Atenolol 2016
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.
er ch3 Drying In air.
F. 2 -(acetyloxy)benzoic anhydride (acetylsalicylic anhydride). Results The principal spot in the chrom atogram obtained with
___________________________________________________ PhEir the test solution is similar in position and size to the principal
solution.
TESTS
Atenolol ★★* * ★ S o lu tio n S

★ ★ Dissolve 0.10 g in water R and dilute to 10 m L with the
(Ph Ettr. monograph 0703) * * * * * same solvent.
H OH
H Solution S is clear (2.2.1) and n o t more intensely coloured
N CH3 than degree 6 of the range of reference solutions o f the m ost
T and enant'omer
appropriate colour (2.2. 2, Method II).
CH3
+ 0.10° to —0.10°, determined on solution S.
C 14H 22N 2O 3 266.3 29122-68-7 R elated su b sta n c e s
A ctio n a n d u se
Beta-adrenoceptor antagonist. 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
P re p a r a tio n s the mobile phase.
Atenolol Injection
Reference solution (a) Dissolve 2 m g of atenololfor system
Atenolol Oral Solution
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__________________________
D E F IN IT IO N Column:
2- [4- [(2ftS)-2-Hydroxy-3- [(1 -methylethyl) amino] propoxy] — size. I = 0.125 m , 0 = 4.0 m m ;
phenyl] acetamide. — stationary phase, end-capped octadecylsilyl silica gel for
C o n te n t chromatography R (5 Jim).
99.0 per cent to 101.0 per cent (dried substance). Mobile phase Dissolve 1.0 g of sodium octanesulfonate R and
CHARACTERS 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
A p p e a ra n c e
W hite or alm ost white powder. methanol R2, and 800 volumes o f a 3.4 g/L solution of
potassium dihydrogen phosphate R; adjust the apparent p H to
S o lu b ility 3.0 with phosphoric add R
Sparingly soluble in water, soluble in anhydrous ethanol,
slightly soluble in methylene chloride.
Injection 10 jiL.
First identification C.
Run time 5 times the retention time o f atenolol.
A. Melting point (2.2.14)? 152 °C to 155 °C.
with atenololfor system suitability CRS and the chromatogram
B. Ultraviolet and visible absorption spectrophotometry obtained with reference solution (a) to identify the peaks due
(2.2.25). to impurities B, F , G , I and J.
Test solution Dissolve 0.100 g in methanol R and dilute to Relative retention W ith reference to atenolol (retention
100 m L with the same solvent. Dilute 10.0 m L o f this time = about 8 min): impurity B = about 0.3;
solution to 100 m L with methanol R. impurity J = about 0.7; impurity I = about 0.8;
Speared range 230-350 nm. impurity F = about 2.0 (pair o f peaks);
Absorption maxima A t 275 nm and 282 nm. impurity G = about 3.5.
Absorbance ratio A?7s/A?8? = 1.15 to 1.20. System suitability: reference solution (a):
impurities J (unidentified impurity) and I.
Comparison atenolol CRS.
Limits:
D. Thin-layer chromatography (2.2.27). — correction factor, for the calculation o f content, multiply the
Test solution Dissolve 10 mg o f the substance to be examined peak area of impurity I by 1.5;
in 1 m L o f methanol R.
2016 Atomoxetine Hydrochloride 1-207
OH
— impurity B: not m ore than twice the area of the principal
— impurities F, G, I : for each impurity, not more than E. 2,2'- [(2-hydroxypropane-1,3-diyl)bis(oxy-4,1-phenylene)]
1.5 times the area of the principal peak in the diacetamide,
h 3c ch3
(0.15 p er cent); oh j
— unspecified impurities, for each impurity, not more th an the
— totd: n o t more th an 5 limes the area of the principal peak F. 2,2'-[[(l-methylethyl)imino]bis[(2-hydroxypropane-3,l-
in the chrom atogram obtained with reference solution (b) diyI)oxy-4,1-phenylene]] diacetamide,
(0.5 per cent);
— disregard limit. 0.5 times the area o f the principal peak in V H H
^ ^ ch3
the chrom atogram obtained with reference solution (b) and enantiomer
(0.05 per cent). HO2C CH3
M axim um 0.1 per c e n t G . 2- [4- [(2i?5)-2-hydroxy-3- [(1 -methylethyl)amino]
Dissolve 50 mg in a mixture o f 1 m L o f dilute nitric acid R propoxy]phenyl] acetic a d d ,
and 15 m L o f water R. T he solution, without further addition
H OH
of dilute nitric acid R, complies with the test. H
N. .C H 3
L oss o n d ry in g (2.2.32) and enantiomer
M axim um 0.5 per cent, determined on 1.000 g by drying in CH3

an oven at 105 °C.
S u lfa te d a s h (2.4.14) H . 2-[4- [(2i?i)-2-hydroxy-3- [(1-methylethyl)amino]
M axim um 0.1 per cent, determined on 1.0 g. propoxy]phenyl] acetonitrile,
ASSAY H OH
Dissolve 0.200 g in 80 m L o f anhydrous acetic acid R. T itrate N CH3 and enantiomer
with 0.1 M perchloric add, d eterm ining the end-point
I. 2-[4- [(2i?5)-3-(ethylamino)-2-hydroxypropoxy]
1 m L o f 0.1 M perchloric acid is equivalent to 26.63 m g o f phenyl] acetamide.
C 14H 22N 2O 3.
PhEur
IMPURITIES
Specified impurities B, F , G, I
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 Atomoxetine Hydrochloride ★ ★
acceptance criterion for other/unspecified impurities and/or (Ph. Ettr. monograph 2640) *****
Control of impurities in substances for pharmaceutical use): A , D, , HCI
E, H.
C 17H 22CINO 291.8 82248-59-7

H,N
A ctio n a n d u se
A. R-H : 2-(4-hydroxyphenyl)acetamide, Noradrenaline reuptake inhibitor, treatm ent of attention
defidt hyperactivity disorder (A DH D).
H OH
and enantiomer PhEur.
D E F IN IT IO N
(3i?)-N-Methyl-3-(2-methylphenoxy)-3-phenylpropan-l-
B. 2-[4- [(2R5)-23-dihydroxypropoxy]phenyl] acetamide,
amine hydrochloride.
H OH C o n te n t
and enantiomer 98.0 per cent to 102.0 p er cent (dried substance).
CHARACTERS
D . 2-[4- [(2jR5)-3-chloro-2-hydroxypropoxy]phenyl] A p p e a ra n c e
acetamide, 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 2016
It shows polymorphism (5.9). acid R previously adjusted to p H 2.5 w ith a 280 g/L solution
o f potassium hydroxide R
IDENTIFICATION
A. Infrared absorption spectrophotom etry (2.2.24). Test solution (a) Dissolve 25 m g o f the substance to be
Comparison atomoxetine hydrochloride CRS.
the mobile phase.
substance separately in anhydrous ethanol R, evaporate to
the mobile phase.
B. Isomeric purity (see Tests).
100.0 m L with the mobile phase. D ilute 1.0 m L o f this
C. It gives reaction (a) o f chlorides (2.3.1). solution to 10.0 m L with the mobile phase.
TESTS Reference solution (b) Dissolve 7.5 m g o f 3-(methylammo)-l-
Isomeric purity phenylpropan-l-ol R (impurity H ) and 5 mg o f mandeHc add R
Liquid chromatography (2.2.29): use the normalisation (impurity E) in test solution (b) and dilute to 50 m L with
procedure. test solution (b).
Test solution Dissolve 35.0 m g of the substance to be Reference solution (c) Dissolve 5 m g o f atomoxetine for
examined in 2.5 m L o f anhydrous ethanol R, sonicate until impurity A identification CRS in the mobile phase and dilute
dissolution is complete and dilute to 10.0 m L with heptane R. to 20 m L with the mobile phase.
Reference solution (a) Dissolve 3.5 mg o f atomoxetine Reference solution (d) Dissolve 25.0 mg of atomoxetine
impurity B CRS and 1 mg o f atomoxetine impurity D CRS in hydrochloride CRS in the mobile phase and dilute to
5 m L of anhydrous ethanol R, sonicate until dissolution is 100.0 m L with the mobile phase.
complete and dilute to 20.0 m L with heptane R. Column:
Reference solution (b) Dissolve 35.0 m g of the substance to be — size. I = 0.15 m , 0 = 4.6 mm ;
examined in 2.5 m L o f anhydrous ethanol R. Add 1.0 m L of — stationary phase: end-capped octylsUyl silica gel for
reference solution (a) and dilute to 10.0 m L with heptane R. chromatography R (3.5 pm);
Reference solution (c) Dilute 1.0 m L of reference solution (a) — temperature. 40 °C.
to 100.0 m L with heptane R. Mobile phase propanol R, solution A (27:73 VIV).
Column: Flow rate 1.0 mL/min.
— size. I — 0.25 m , 0 = 4.6 mm; Detection Spectrophotom eter at 215 nm .
— stationary phase. cellulose derivative of silica gelfor chiral
Injection 10 pL of test solution (a) and reference solutions (a),
separation R (5 pm).
(b) and (c).
Mobile phase M ix 1.5 m L of diethylamine R, 2.0 m L of
Run time 2.5 times the retention time o f atomoxetine.
trifluoroacetic acid R and 150.0 m L o f 2-propanol R and dilute
to 1000 m l. with heptane R. 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
Detection Spectrophotom eter at 273 nm . atomoxetine for impurity A identification CRS and the
Irqecdon 10 pL of the test solution and reference solutions (b) chrom atogram obtained with reference solution (c) to identify
and (c). the peak due to impurity A.
Run time 1.3 times the retention time o f atomoxetine. Relative retention W ith reference to atomoxetine (retention
Identification of impurities U se the chromatogram obtained tim e = about 10 min): impurity E = about 0.2;
with reference solution (b) to identify the peaks due to impurity H = about 0.3; impurity A = about 0.7.
impurities B and D. System suitability: reference solution (b):
Relative retention W ith reference to atomoxetine (retention — resolution: m in im u m 5.0 between the peaks due to
time = about 12 min): impurity B = about 0.5; impurities E and H.
im purity D = about 0.6. Calculation ofpercentage contents:
System suitability: reference solution (b): — for each impurity, use the concentration o f atomoxetine
— resolution: minimum 1.8 between the peaks due to hydrochloride in reference solution (a).
impurities B and D . Limits:
Limits: — impurity A: maximum 0.3 per cent;
— impurity B: maximum 0.5 per cent; — unspecified impurities: for each impurity, maximum
— impurity D: maximum 0.15 per cent; 0.10 per cent;
— unspecified impurities', for each impurity, maximum — total: maximum 0.5 per cent;
0.10 per cent; — reporting threshold: 0.05 per cent.
— disregard limit: the area o f the peak due to impurity B in H e a v y m e ta ls (2.4.8)
the chromatogram obtained with reference solution (c) M axim um 10 ppm.
(0.05 per cent); disregard any peak with a relative Solvent mixture water R, methanol R (20:80 VIV).
retention with reference to atomoxetine o f about 0.7
0.250 g complies with test H. Prepare the reference solution
(impurity A).
using 0.25 m l . of lead standard solution (10 ppm Pb) R.
Related substances
M axim um 0.5 per cent, determined on 1.000 g by drying
Solution A Dissolve 5.9 g of sodium octanesulfonate in vacuo at 105 °C for 2 h.
monohydrate R in 1000 m L o f a 2.9 g/L solution of phosphoric
2016 Atorvastatin Calcium Trihydrate 1-209

ASSAY
Injection T est solution (b) and reference solution (d). F. (35)-3-(3-fluoro-2-methylphenoxy)-N-methyl-3-
Calculate the percentage content o f C i 7H 22C lN O taking into pheny lpropan-1-amine.
account the assigned content of atomoxetine
hydrochloride CRS.
IMPURITIES
Specified impurities A, B, D h3Cv xCH3
acceptance criterion for other/unspecified impurities and/or G. 3 j3 /-[(2-methylbenzene-lj3-diyl)bis(oxy)]bis(N-methyl-3-
phenylpropan-1-amine),
Control of impurities in substancesfor pharmaceutical use): C, E,
F, G, H.
H . 3-(methylamino)-1 -phenylpropan-1-ol.
PhEur
A. N-methyl-3-phenoxy-3-phenylpropan-1-amine,
Atorvastatin Calcium Trihydrate ★ ★
★ ★
o (Ph Ettr. monograph 2191) *****
B . (3S)-N-methyl-3-(2-methylphenoxy)-3-phenylpropan-1-
amine,
HaC
C66H 68CaF2N40 1(b3H20 1209 344423-98-9
Action and use

H M G Co-A reductase inhibitor; lipid-regulating drug.
C . (3i?)-iV-methyl-3-(4-methylphenoxy)-3-phenylpropan-1-
amine, PhEur.
DEFINITION
Calcium (3i?,5jR)-7-[2-(4-fluorophenyl)-5-(l-methyiethyl)-3-
phenyl-4-(phenylcarbamoyi)-1//-pyrrol- l-yQ-3,5-
dihydroxyheptanoate trihydrate.
H ,C
Content
97.0 p er cent to 102.0 p er cent (anhydrous substance).
D . (3R)-N-methyl-3-(3-methylphenoxy)-3-phenylpropan-1-
amine,
CHARACTERS
Appearance
W hite o r almost white powder.
HO H
Solubility
COjH
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
Comparison atorvastatin calcium trihydrate CRS.
1-210 Atorvastatin Calcium Trihydrate 2016
If the spectra obtained in the solid state show differences, impurity D CRS and 2.5 m g o f the substance to be examined
dissolve the substance to be examined and the reference in dimethylformamide R and dilute to 50.0 m L with the same
substance separately in methanol R, evaporate to dryness and solvent.
record new spectra using the residues. Column:
B. Enantiom eric purity (see Tests). — size: I = 0.25 m , 0 = 4.6 mm;
C. W ater (see Tests). — stationary phase: octylsifyl silica gel for chromatography R
(5 pm);
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. Mobile phase:
— mobile phase A: tetrahydrofuran R, acetonitrüe R, 3.9 g/L
TESTS solution o f ammonium acetate R adjusted to p H 5.0 with
Enantiomeric purity glacial acetic acid R (12:21:67 VIVIV)',
Liquid chrom atography (2.2.29). — mobile phase B: tetrahydrofuran R, 3.9 g/L solution o f
Solvent mixture anhydrous ethanol R, methanol R (50:50 V!V). ammonium acetate R adjusted to p H 5.0 with glacial acetic
Test solution Dissolve 10 mg o f the substance to be examined 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. Time Mobile phase A Mobile phase B
Reference solution (a) Dissolve 2 mg o f atorvastatin (min) (per cent V7V) (per cent V/V)
impurity E CRS in methanol R and dilute to 20.0 m L with the 0 • 40 100 0
same solvent (solution A). Dissolve 10 mg of the substance
40 - 70 100->20 0 + 80
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 7 0 -8 5 20 0 80 -» 100
10.0 m L w ith hexane R.
Reference solution (b) T o 2.0 m L o f the test solution add Flow rate 1.5 mlVmin.
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 Detection Spectrophotom eter at 244 nm .
solvent mixture and dilute to 20.0 m L with hexane R. Injection 20 )iL o f test solution (b) and reference solutions (b)
Column: and (c).
— size: I = 0.25 m, 0 = 4.6 mm; Identification of impurities U se the chrom atogram obtained
— stationary phase: amylose derivative of silica gel for with reference solution (c) to identify the peaks due to
chromatography R (10 jun). impurities A, B, C and D .
Mobile phase trifluoroacetic acid R, anhydrous ethanol R, Relative retention W ith reference to atorvastatin (retention
hexane R (0.1:6:94 VIVIV). time = about 33 min): impurity A = about 0.8;
Flow rate 1.0 mlVmin. im purity B = about 0.9; impurity C = about 1.2;
If necessary5 adjust the mobile phase by increasing or
Injection 20 )lL.
decreasing the percentage o f acetonitrile o r the p H o f the
Run time 1.2 times the retention time o f atorvastatin. am m onium acetate solution to achieve a retention time o f
Relative retention W ith reference to atorvastatin (retention about 33 rnin for atorvastatin. For example, raising the p H
time = about 44 min): impurity E = about 0.8. would decrease die retention time o f atorvastatin.
System suitability: reference solution (a): System suitability: reference solution (c):
— resolution: m inim um 2.0 between the peaks due to — resolution: m inim um 1.5 between the peaks due to
im purity E and atorvastatin. im purity B and atorvastatin.
Limit. Limits:
— impurity E: not m ore than the area o f the principal peak — impurities A , B: for each impurity, n o t m ore than 3 times
in the chrom atogram obtained with reference solution (b) the area o f the principal peak in the chrom atogram
(0.3 per cent). obtained with reference solution (b) (0.3 per cent);
Related substances — impurities C, D: for each impurity, n o t m ore than
Liquid chrom atography (2.2.29). 1.5 times die area o f the principal peak in the
Test solution (a) Dissolve 40.0 m g o f the substance to be
(0.15 p er cent);
examined in dimethytformamide R and dilute to 100.0 mT.
— unspecified impurities: for each im purity, not m ore than the
Test solution (b) Dissolve 50 m g o f the substance to be with reference solution (b) (0.10 p er cent);
examined in dimethylformamide R and dilute to 50.0 m L with — total: n o t m ore than 15 times the area o f the principal
the same solvent. peak in die chrom atogram obtained with reference
Reference solution (a) Dissolve 40.0 m g o f atorvastatin calcium solution (b) (1.5 p er cent);
trihydrate CRS in dimethylformamide R and dilute to — disregard limit. 0.5 tim es the area o f the principal peak in
100.0 m L with the same solvent. the chrom atogram obtained w ith reference solution (b)
Reference solution (b) D ilute 1.0 m L o f test solution (b) to (0.05 p er cent); disregard the peak due to
100.0 m L with dimethylformamide R. Dilute 1.0 m L o f this dimethylformamide.
solution to 10.0 m L w ith dimethylformarnide R. Sodium
Reference solution (c) Dissolve 2.5 mg o f atorvastatin M axim u m 0.4 per cent (anhydrous substance).
impurity A CRS, 2.5 m g o f atorvastatin impurity B CRS, Atomic absorption spectrometry (2.2.23, Method I).
2.5 m g o f atorvastatin impurity C CRS, 2.5 mg o f atorvastatin
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 . C. (3Æj5Æ)-7-[2j3-bis(4-fluorophenyl)-5-(l-methylethyl)-4-
Atomisation device Air-acetylene flame. (phenylcarbam oyl)-l//-pyrrol-l-yl]-3j5-dihydroxyheptanoic
a d d (fluoroatorvastatin),
M axim um 20 ppm .
h 3c n__ ch 3
Solvent mixture water R, methanol R (10:90 VIV).
It complies with test H with the following modifications.
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. D . 3-[(4-fluorophenyl)carbonyl]-2-(2-methylpropanoyl)-2Vj3-
W a te r (.2.5.12) diphenyloxirane-2-carboxamidej
A SSA Y
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.
E. (3S,5S)-7-[2-(4-fluorophenyI)-5-(l-methylethyl)-3-phenyl-
4-(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.
F . (3i?,5/$-7-[[(3i?,5£)-7-[2-(4-fluorophenyl)-5-
( 1-methylethyl)-3-phenyl-4-(phenylcarbamoyl)-1//-pyrrol-1-
yl] -3,5-dihydroxyheptanoyI] amino]-3,5-dihydroxyheptanoic
A. (3i?j5iR)-3j5-dihydroxy-7-[5-(l-methylethyl)-2j3-diphenyl-
add,
4-(phenylcarbamoyI)-1//-pyrrol-1-yl]heptanoic a d d
(desfluoroatorvastatin),
G . (3R,5R)-7- [2-(4-fluorophenyl)-i>-( 1-methylethyl)-3-

B . (3RS,5SK)-7- [2-(4-fluorophenyl) -5-( 1-methylethyl)-3- phenyl-4- (phenylcarbamoyl)-1//-pyrrol-1-yl] -5-hydroxy-3-
phenyl-4-(phenylcarbamoyl)-1//-pyrrol-1-yl] -3,5- m ethoxyheptanoic a d d (3-0-methylatorvastatin)j
dihydroxyheptanoic ad d .
1-212 Atovaquone 2016
o
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.
H. (4R,6R)-6-[2-[2-(4-fluorophenyI)-5-( 1-methylethyI)-3- Reference solution (c) Dilute 1.0 m L o f the test solution to
phenyl-4-(phenylcarbam oyl)-lif-pyrrol-l-yI]ethyl]-4- 100.0 m L with die solvent mixture. Dilute 1.0 m L o f this
hydroxytetrahydro-2Jï-pyran-2-one. 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
Mobile phase phosphoric add R, methanol R2, water for
Atovaquone ****** chromatography R, acetomtrUe R1 (0.5:17.5:30:52.5 VIVIVIV).
(Ph E ut monograph 2192) * Flow rate 2.5 mL/min.
Injection 20 jiL of the test solution and reference solutions (b)
and (c).
Run time Twice the retention time of atovaquone.
O with atovaquone for system suitability CRS and the
C22H19C103 366.8 95233-18-4 identify the peaks due to impurities B and C.
Relative retention W ith reference to atovaquone (retention
Action and use time = about 15 min): impurity B = about 0.85;
Antiprotozoal (malaria). impurity C = about 0.90.
PhEur__________________________________________________________ System suitability: reference solution (b):
DEFINITION impurity C and atovaquone;
2- [trans-4-(4-ChlorophenyI) cyclohexyl]-3- — peak-to-vaUey ratio: minim um 1.5, where Hp = height
hydroxynaphthalene-1,4-dione. above the baseline of the peak due to impurity C and
Content Hv ~ height above the baseline o f the lowest point of the
97.5 per cent to 102.0 per cent (anhydrous substance). curve separating this peak from the peak due to
impurity B.
CHARACTERS
Appearance Calculation of percentage contents:
Yellow, crystalline powder. — for each impurity, use the concentration o f atovaquone in
reference solution (c).
Solubility
Limits:
— impurity B: maximum 0.5 per cent;
chloride, very slightly soluble in methanol.
— impurity C: maximum 0.2 per cent;
It shows polymorphism (5.9). — unspecified impurities: for each impurity, maximum
IDENTIFICATION 0.10 per cent;
Infrared absorption spectrophotometry (2.2.24). — total: maximum 0.6 per cent;
Comparison atovaquone CRS. — reporting threshold: 0.05 per cent.
If the spectra obtained show differences, dissolve 0.1 g o f the W a te r (2.5.32)

substance to be examined and 0.1 g o f the reference M axim um 0.3 per cent, determ ined on 0.100 g using the
substance separately in 2.5 m L of a 50 g/L solution of evaporation technique:
potassium hydroxide R in methanol R. Filter the solutions and — temperature. 160 °C;
add each filtrate dropwise to a mixture o f 0.8 m L o f acetic — heating time. 3 min;
add R and 1.5 m L o f methanol R, stirring continuously. — flow rater. 50 mL/min.
Filter, wash the residues with methanol R and then with S u lfa te d a s h (2.4.14)
water R, and dry under vacuum at 55 °C. Record new Maximum 0.1 per cent, determ ined on 1.0 g.
spectra using the residues.
A SSA Y
TESTS Liquid chromatography (2.2. 29) as described in the test for
Related substances related substances with the following modification.
Liquid chromatography (2.2.29). Cany out the test protected Injection T est solution and reference solution (a).
from light. Calculate the percentage content o f C 22H 19CIO 3 taking into
Solvent mixture water R, acetonitrüe R1 (20:80 ViV). account the assigned content o f atovaquone CRS.
2016 Atracurram Besilate 1-213
IM P U R IT IE S *****
Specified impurities B, C
Atracurium Besilate ★ ★
Other detectable impurities (the following substances would, if (Ph Ettr monograph 1970) *****
th e tests in the monograph. They are limited by die general
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
B. 2 -[cw-4-(4-chlorophenyl)cyclohexyl]-3- Mixture of the cis-cis, cis-trans and trans-trans isomers of
hydroxynaphthalene-l,4-dione, 2 ,2 '- [pentane-1,5-diylbis [oxy(3-oxopropane-l ,3-diyl)]] bis [ 1-
(3,4-dimethoxybenzyl)-6,7-dimethoxy-2-methyl-l ,2,3,4-
tetrahydroisoquinolinium] dibenzenesulfonate.
C o n te n t
OH and enantiomer CHARACTERS
A p p e a ra n c e
White or yellowish-white, slightly hygroscopic powder.
C . 2- [( l.RS)-4-(4-chlorophenyl)cyclohex-3-en- 1-yl] -3- S olubility
hydroxynaphthalene-l,4-dione, Soluble in water, very soluble in acetonitrile, in ethanol
(96 per cent) and in methylene chloride.
A Infrared absorption spectrophotometry (2.2.24).
Comparison atracurium besilate CRS.
Results T he 3 principal isomeric peaks in the chromatogram
D . 2-[mms-4-(4-chlorophenyl)cyclohexyl]-3- obtained with test solution (a) are similar in retention tim e to
methoxynaphthalene-1,4-dione. those in the chromatogram obtained with reference
solution (a).
PhEur
TESTS
S o lu tio n S
Dissolve 1.00 g in water R and dilute to 100 m L with the
same solvent.
Test solution (a) Dissolve 50.0 m g of the substance to be
mobile phase A.
Test solution (b) Dissolve 0.100 g of the substance to be
mobile phase A.
1-214 Atracurium Besilate 2016
Reference solution (a) Dissolve 50.0 m g o f atracurium im purity B = about 1.15; im purity C l = ab o u t 1.2;
besilate CRS in mobile phase A and dilute to 50.0 m L with 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
100.0 m L with mobile phase A. atracurium trans-trans isomer an d the atracurium cis-trans
Reference solution (c) Dissolve 20.0 m g o f methyl isomer, and m inim um 1.5 betw een the peaks due to the
benzenesulfonate R in aceîonitrüe R and dilute to 100.0 m l. atracurium cis-trans isom er and th e atracurium cis-ds
with the same solvent. Dilute 50 nL o f the solution to isomer in the chrom atogram obtained with reference
100.0 m L with mobile phase A. solution (a);
— pedk-to-valley ratio: m inim um 1.2, w here HP — height
Reference solution (d) Dissolve 2.0 m g o f atracurium for peak
above the baseline o f the peak due to im purity A l and
identification CRS (containing impurities A l, A2, B, C l , C2,
Hv = heigjit above the baseline o f the lowest point o f the
D l, D 2, E, G and K) in 2.0 m L o f mobile phase A.
curve separating this peak from th e peak due to the
Reference solution (e) Dissolve 2.0 m g o f atracurium for atracurium cis-cis isomer in the chrom atogram obtained
impurity F identification CRS in 2.0 m L o f mobile phase A. with reference solution (d).
Column: Limits:
— size: I = 0.25 m , 0 = 4.6 mm; — correction factor, for the calculation o f content, multiply the
— stationary phase: base-deactivated end-capped octadecylsifyl peak area of impurity G by 0.5;
silica gelfor chromatography R (5 |im ). — impurity E: not more than 1.5 tim es the sum o f the areas
Mobile phase: of the peaks due to the atracurium cis-cis, trans-trans and
— mobile phase A: mix 5 volumes of methanol R, 20 volumes cis-trans isomers in the chrom atogram obtained with
o f acetonitrüe R and 75 volumes o f a 10.2 g/L solution of reference solution (b) (1.5 per cent);
potassium dihydrogen phosphate R previously adjusted to — impurities A, D: for each im purity, for the sum o f the
p H 3.1 with phosphoric add R; areas o f the 2 isomer peaks, n o t m ore than 1.5 times the
— mobile phase B: mix 20 volumes o f acetomtrüe R, sum o f the areas o f the peaks due to the atracurium
30 volumes of methanol R and 50 volumes o f a 10.2 g/L cis-cis, trans-trans and cis-trans isomers in the
solution o f potassium dihydrogen phosphate R previously chromatogram obtained with reference solution (b)
adjusted to p H 3.1 with phosphoric add R; (1.5 per cent);
— impurity C: for the sum o f the areas o f the 2 isom er peaks,
Urne Mobile phase A Mobile phase B
not m ore than the sum o f the areas o f the peaks due to
(min) (per cent V/V) (percent V/V) the atracurium cis-cis, trans-trans and cis-trans isomers in
0 -5 80 20 the chrom atogram obtained w ith reference solution (b)
(1.0 p er cent);
5 -15 80 ->40 20 ->60
— impurities F, G: for each im purity, n o t m ore than the sum
15-25 40 60 o f the areas of the peaks due to th e atracurium cis-ds,
2 5 -3 0 40 ->0 60-> 100
trans-trans and cis-trans isomers in the chrom atogram
obtained with reference solution (b) (1.0 p er cent);
30 -4 5 0 100 — 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
Flow rate 1 mlVmin.
and cis-trans isomers in the chrom atogram obtained with
Detection Spectrophotom eter at 280 run. reference solution (b) (1.0 per cent);
Injection 20 pL o f test solution (a) and reference solutions (a), — unspecified impurities: for each im purity, n o t m ore th an
(b), (d) and (e). 0.1 times the sum o f the areas o f the peaks due to the
Identification of impurities Use the chromatogram obtained atracurium cis-cis, trans-trans and cis-trans isomers in the
with reference solution (d) and the chromatogram supplied chromatogram obtained with reference solution (b)
with atracurium for peak identification CRS to identify the (0.10 p er cent);
peaks due to impurities A l, A2, B, C l, C2, D l, D 2, E, G — total: n o t more than 3.5 times th e sum o f the areas o f the
and K; use the chromatogram obtained with reference peaks due to the atracurium cis-cis, trans-trans and cis-trans
solution (e) and the chromatogram supplied with atracurium isomers in the chrom atogram obtained with reference
for impurity F identification CRS to identify the peak due to solution (b) (3.5 per cent);
im purity F. — disregard Umit. 0.05 times the sum o f the areas of the
Relative retention W ith reference to the atracurium tis-ds peaks due to the atracurium cis-cis, trans-trans an d cis-trans
isomer (retention time = about 30 min): isomers in the chrom atogram obtained with reference
impurity E = about 0.2; impurity F = about 0.25; solution (b) (0.05 per cent).
im purity G = about 0.3; impurity D l = about 0.45; Impurity J
impurity D 2 = about 0.5; atracurium Liquid chromatography (2.2.29) as described in the test for
trans-trans isom er = about 0.8; atracurium related substances with the following modifications.
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;
2016 Atracurium Besilate 1-215
Mobile phase:

0 -5 80 20
5 - 15 8 0 -»75 20 ->25
15 - 25 75 25 o o
2 5 -3 0 75 55 25 ->45
3 0 -3 8 55 -» 0 45-» 100
38 - 45 0 100 A . l-(3,4-dimethoxybenzyl)-2-[13-[l-(3,4-dimethoxybenzyl)-
6,7-dim ethoxy-3,4-dihydroisoquinolin-2(l/i)-yI]-3,ll-dioxo-
4 ,1 0-dioxatridecyI]-6,7-dimethoxy-2-inethyl-l,2,3,4-
Detection Spectrophotom eter at 217 nm. tetrahydroisoquinolinium (A1 = cis-trans isomer,
A2 — cis-cis isomer),
Irtjection 100 jiL o f test solution (b) and reference
solution (c). o o
Retention time Im purity J = about 25 min; atracurium
trans-trans isomer = about 38 min. r a A q/ W ^ o^ r
Limit.
B. pentane-l,5-diyl bis[3-[l-(3,4-dimethoxybenzyl)-6,7-
— impurity J: n o t more than the area of the principal peak in
dimethoxy-3,4-dihydroisoquinolin-2( li/)-yl] prop anoate],
(10 ppm). o o
Isomer composition
related substances with the following modifications. U se the
C . 1-(3,4-dimethoxybenzyl)-2-(3,11 -dioxo-4,10-dioxatridec-
12-enyl)-6,7-dimethoxy-2-methyl-1,2,3,4-
Irtjection T est solution (a). tetrahydroisoquinolinium (C l = trans isomer,
Limits: C 2 = cis isomer),
— 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, o
— 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. D . l-(3,4-dimethoxybenzyl)-2-[3-[(5-hydroxypentyl)oxy]-3-
Sulfated ash (2.4.14) oxopropyl] -6,7-dimethoxy-2-methyl-1,2,3,4-
M aximum 0.1 p er cent, determined on 1.0 g. tetrahydroisoquinolinium (D1 = trans isomer,
D 2 = cis isomer),
ASSAY
Liquid chromatography (2.2.29) as described in the test for . 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 E . 2-(2-carboxyethyl)- l-(3,4-dimethoxybenzyl)-6,7-
the sum of the areas o f the peaks due to the 3 isomers. dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinolinium,
STORAGE F . R+-C H 3: l-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2,2-
In an airtight containerj protected from light, at a dim ethyl-1,2,3,4-tetrahydroisoquinolinium,
tem perature o f 2 °C to 8 °C. G . R -C H 3: l-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2-
IM P U R IT IE S m ethyl-1,2,3,4-tetrahydroisoquinoline,
Specified impurities A, C, D , E, F, G , H , I, J, K o
the tests in the monograph. T hey are limited by die general O
by the general monograph Substances for pharmaceutical use H . 2,2 [hexane- 1,6-diylbis [oxy(3-oxopropane-1,3-
(2034). It is therefore n o t necessary to identify these dfyl)]]bis[l-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2-methyl-
impurities for dem onstration of compliance. See also 5.10. I.2,3,4-tetrahydroisoquinolinium] (H I = cis-trans isomer,
Control of impurities in substances for pharmaceutical usé): B. H 2 = cis-cis isomer),
I. 2,2 [(3-methylpentane-l ,5-diyl)bis [oxy(3-oxopropane-1,3-

diyl)] ]bis [ 1-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2-methyl-
1,2,3,4-tetrahydroisoquinolinitim] (II = cis-trans isomer,
12 = cis-cis isomer),
1-216 Atropine 2016
o o
Detecdon After cooling, spray with dilute potassium
’sV CHs iodobismuthate solution R.
Results T h e principal spot in the chrom atogram obtained with
J. methyl benzenesulfonate, principal spot in the chrom atogram obtained with the
reference solution.
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-methyl- E. Optical rotation (see Tests).
1,2,3,4-tetrahydroisoquinolinium]. TESTS
PhEur O p tic a l ro ta tio n (2.2.7)
—0.70° to + 0.05° (measured in a 2 dm tube).
★ ★ R e la te d su b s ta n c e s
Atropine ★ ★
*****
(Ph Eter monograph 2056) 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 with the test solution. D ilute 5.0 m L o f this solution to
A ctio n a n d use
Reference solution (c) Dissolve the contents o f a vial of atropine
Anticholinergic.
for peak identification CRS (containing impurities A, D , E, F,
PhEur. G and H ) in 1.0 m L o f mobile phase A
D E F IN IT IO N 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
(1 i?,3i?,53)-8-Methyl-8-azabicyclo[3.2. l]oct-3-yl (2RS)-3-
hydroxy-2-phenylpropanoate. 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
C o n te n t 10.0 m L with mobile phase A.
Column:
CHARACTERS — size. I = 0.10 m , 0 = 4.6 mm;
A p p e a ra n c e — stationary phase: octadecylsUyl silica gel for chromatography R
W hite or almost white, crystalline pow der or colourless (3 nm).
crystals. Mobile phase:
S o lu b ility — mobile phase A: dissolve 3.5 g o f sodium dodecyl sulfate R in
Very slightly soluble in water, freely soluble in ethanol 606 m L o f a 7.0 g/L solution o f potassium dihydrogen
(96 per cent) and in methylene chloride. 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;
First identification: A, B, E. — mobile phase B: acetomtrüe Rl;
Second identification: A , C, D, E
A. M elting point (2.2.14): 115 °C to 119 °C. Time Mobile phase A Mobile phase B
B. Infrared absorption spectrophotom etry (2.2.24). (min) (per cent V7V) (per cent V/V)
Comparison atropine CRS. 0 -2 95 5
C. Thin-layer chrom atography (2.2.27). 2-20 95->70 5 -> 30

in methanol R and dilute to 10 m L w ith die same solvent
Flow rate 1 mL/min.
Reference solution Dissolve 10 m g o f atropine CRS in
methanol R and dilute to 10 m L with the same solvent Detection Spectrophotom eter at 210 nm.
Plate TLC silica gel plate R. Injection 10 (iL.
Mobile phase concentrated ammonia R, water R, acetone R Identification of impurities U se the chrom atogram supplied
with atropine for peak identification CRS and the
(3:7:90 VIVIV).
Application 10 jiL. the peaks due to impurities A, D , E , F , G and H ; use the
Development Over half of the plate. chrom atogram obtained w ith reference solution (b) to
Drying A t 100-105 °C for 15 min. identify the peak due to impurity B; use the chromatogram
2016 Atropine 1-217

to im purity C. CO2H
and enantiomer
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; C. (2i?S)-3-hydroxy-2-phenylpropanoic a d d (tropic add),
impurity H = about 0.93; impurity G = about 1.1;
— resolution: m in im u m 2.5 between the peaks due to and epimer at C*
im purity B and atropine.
Limits:
corresponding correction facto r impurity A = 0.6; D . (1 i?,3S,5i?,6RS)-6-hydroxy-8-methyl-8-
im purity C = 0.6; azabicyclo [3.2.1] oct-3-yl (23)-3-hydroxy-2-phenylpropanoate
— impurities E, H: for each impurity, not more than 3 times (6-hydroxyhyoscyamine),
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 and epimer at C*
(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 E. (1 S,3i?,5Sj6i?5)-6-hydroxy-8-methjd-8-
w ith reference solution (a) (0.10 per cent); azabicydo[3.2.l]oct-3-yl (25)-3-hydroxy-2-phenylpropanoate
— total: no t more than 5 times th e area o f the principal peak (7-hydroxyhyoscyamine),
in the chrom atogram obtained w ith reference solution (a)
(0.5 per cent);
the chrom atogram obtained w ith reference solution (a)
(0.05 per cent). X °* w
Loss on drying (2.2.32) v H *
OH H
F. (li?,2i?,4S,55,7j)-9-methyl-3-oxa-9-
A SSA Y azatricyclo[3.3.1 .O ^ n o n ^ - y i (26)-3-hydroxy-2-
Dissolve 0.250 g in 40 mT. of anhydrous acetic acid R, heating phenylpropanoate (hyoscine),
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).
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
IM P U R IT IE S G. (lÄ,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl (2RS)-2-
Specified impurities A, B, C , D , E, F , G, H . hydroxy-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-2-

phenylpropanoate (noratropine),
1-218 Atropine Sulfate 2016
★* * Test solution Dissolve 24 m g of the substance to be examined

★ ★
Atropine Sulfate ★ ★ in mobile phase A and dilute to 100.0 m L with mobile
Atropine Sulphate ***** phase A.
(Ph E ut. monograph 0068) Reference solution (a) Dilute 1.0 m L o f the test solution to
100.0 m L with mobile phase A. Dilute 1.0 m L o f this
Reference solution (b) Dissolve 5 m g o f atropine
, h2s o 4 , h2o 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 and enantiomer
Reference solution (c) Dissolve the contents o f a vial o f atropine
for peak identification CRS (containing impurities A, D , E, F,
C34H 48N 201oS,H20 695 5908-99-6 G and H ) in 1 m L of mobile phase A.
Reference solution (d) Dissolve 5 m g o f tropic add R
Action and use
(impurity C) in mobile phase A and dilute to 10 m L with
Anticholinergic.
mobile phase A. Dilute 1 m L o f the solution to 100 mT. with
Preparations mobile phase A. Dilute 1 m L o f this solution to 10 m L with
Atropine Eye Drops mobile phase A.
Atropine Eye O intm ent Column:
Atropine Injection — size. I = 0.10 m , 0 = 4.6 mm;
Atropine Tablets — stationary phase: octadecylsifyl silica gel for chromatography R
(3 nm).
PhEtr____________________ Mobile phase:
D E F IN IT IO N — mobile phase A: dissolve 3.5 g o f sodium dodecyl sulfate R in
Bis [(li£j3r 35S)-8-methyl-8-azabicydo [3.2. 1] oct-3-yl (2RS)-3- 606 m L o f a 7.0 g/L solution of potassium dihydrogen
hydroxy-2-phenylpropanoate] sulfate monohydrate. phosphate R previously adjusted to p H 3.3 with
0.05 M phosphoric add, and mix with 320 m L of
Content
acetomtrüe Rl;
— mobile phase B: acetomtrüe R l ;
CHARACTERS
Appearance Mobile phase B
Tune Mobile phase A
W hite or alm ost white, crystalline pow der or colourless (min) (per cent V7V) (per cent V/V)
crystals. 0 -2 95 5
Solubility 2-20 95->70 5-> 30
Very soluble in water, freely soluble in ethanol (96 per cent).
First identification A , B, E. Flow rate 1 mL/m in.
Second identification C, D, E, F. Detection Spectrophotom eter a t 210 nm.
A. Optical rotation (see Tests). Injection 10 |xL.
B. Infrared absorption spectrophotom etry (2.2.24). Identification of impurities U se the chromatogram supplied
with atropine for peak identification CRS and the
Comparison atropine sulfate CRS.
C. Dissolve about 50 m g in 5 m L o f water R and add 5 m L the peaks due to impurities A, D , E, F, G and H. U se the
of picric, acid solution R. T he p redpitate, washed with water R chrom atogram obtained with reference solution (b) to
and dried at 100-105 °C for 2 h, m d ts (2.2.14) at 174 °C to identify the peak due to impurity B, and use the
179 °C. chrom atogram obtained with reference solution (d) to
D. T o about 1 m g add 0.2 m L o f fuming nitric add R and identify the peak due to impurity C.
evaporate to dryness in a water-bath. Dissolve the residue in Relative retention W ith reference to atropine (retention
2 m L o f acetone R and add 0.1 m L o f a 30 g/L solution of tim e = about 11 min): impurity C = about 0.2;
potassium hydroxide R in methanol R!TA violet colour devdops. im purity E = about 0.67; impurity D = about 0.73;
E. It gives the reactions of sulfates (2.3.1). im purity F = about 0.8; impurity B = about 0.89;
F. It gives the reaction o f alkaloids (2.3.1). impurity H = about 0.93; impurity G = about 1.1;
TESTS
pH (2.2.3) System suitability: reference solution (b):
4.5 to 6.2. — resolution: minimnm 2.5 between the peaks due to
im purity B and atropine.
Limits:
— correction factors: for the calculation o f content, multiply
Optical rotation (2.2.7) the peak areas o f the following impurities by the
-0 .5 0 ° to + 0.05° (measured in a 2 d m tube). corresponding correction facto r impurity A = 0.6;
Dissolve 2.50 g in water R and dilute to 25.0 m L with the impurity C = 0.6;
same solvent. — impurities E, H: for each impurity, not m ore than 3 times
Related substances the area o f the principal peak in the chromatogram
Liquid chrom atography (2.2.29). obtained with reference solution (a) (0.3 per cent);
2016 Attapulgite 1-219
— 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
and epimer at C*
(0.2 per cent);
area of the principal peak in the chrom atogram obtained
with reference solution (a) (0.10 per cent); E. (1 S,3R,5S,6i?3)-6-hydroxy-8-methyl-8-
— total: n o t m ore than 5 times the area o f the principal peak azabicydo[3.2.1]oct-3-yl (25)-3-hydroxy-2-phenylpropanoate
in the chromatogram obtained with reference solution (a) (7-hydroxyhyoscy amine),
(0.5 per cent);
(0.05 per cent).
W a te r (2.5.12)
S u lfa te d a s h (2. 4.14)
M aximum 0.1 per cent, determined on 1.0 g. F . (li?,2jR,4S,5S,7i)-9-methyl-3-oxa-9-
azatricyclo [3.3.1 .O ^non-T -yl (25)-3-hydroxy-2-
A SSAY phenylpropanoate (hyoscine)j
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
(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
H . unknow n structure.
Spedfied impurities: A, B, C , D , E, F , G , H .
PhEur
Attapulgite
A. (IR ,3r,55)-8-methyl-8-azabicydo[3.2.l]oct-3-yl Excipient.
2-phenylpropenoate (apoatropine)j
D E F IN IT IO N
Attapulgite is a purified native hydrated magnesium
alum inium silicate essentially consisting of the clay mineral
and enantiomer palygorskite.
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-2-
phenylpropanoate (noratropine),
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
CO2H washings to the filtrate. Reserve the residue for test B.
and enantiomer
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.
C . (2RS)-3-hydroxy-2-phenylpropanoic a d d (tropic ad d )j A white, gelatinous predpitate is produced.
B. W ash the residue reserved in test A with water and
H OH 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.
« ^ /•4 — r H
and epimer at C* A n intense red colour is produced.
, v -o -\
C. T o 2 m L of the solution obtained in test B add 1 m L of
\ h I strong sodium hydroxide solution and filter. T o the filtrate add
3 m L of ammonium chloride solution. A gelatinous white
D . (IR,3S,5R,6RS)- 6-hydroxy-8-methyl-8 - predpitate is produced.
a2abicydo[ 3 .2 . l]oct-3-yl (25)-3-hydroxy-2-phenylpropanoate D . T o 2 m L o f the solution obtained in test B add
( 6-hydiroxyhyoscyamine), ammonium chloride and an excess o f 13.5m ammonia and
1-220 Attapulgite 2016
filter. T o the filtrate add 0.15 m L o f magneson reagent and an Loss on ignition
excess o f 5 m sodium hydroxide. A blue precipitate is produced. W hen ignited at 600°, loses 15.0 to 27.0% o f its weight.
Use 1 g.
TESTS
Acidity or alkalinity
p H o f a 5% wN suspension in carbon dioxide-free water, after
shaking for 5 minutes, 7.0 to 9.5, Appendix V L.
Adsorptive capacity Activated Attapulgite
M oisture adsorption, 5 to 14% when determined by the
following method. D ry in air and pow der a sufficient quantity Action and use
o f th e substance being examined and pass through a sieve Antidiarrhoeal. '
with a nominal m esh aperture of 150 jam. Spread 0.5 g as a
DEFINITION
thin layer on a previously weighed piece o f aluminium foil
Activated Attapulgite is a purified native hydrated magnesium
(60 m m x 50 mm ) o f nominal gauge 17.5 nm and transfer
alum inium silicate essentially consisting o f the clay mineral
to a desiccator containing a dish o f sodium chloride crystals
palygorskite that has been carefully heated to increase its
partially immersed in saturated brine at 25°. After 4 hours,
adsorptive capacity.
remove from the desiccator and weigh immediately. D ry in
an oven at 110° for 4 hours, allow to cool in a desiccator and CHARACTERISTICS
weigh. T he moisture adsorption is the gain in weight o f the A light, cream or buff, very fine powder, free or almost free
substance being examined expressed as a percentage o f its from gritty particles.
oven-dried w eight
IDENTIFICATION
Arsenic A. Ignite 0 .5 g with 2 g o f anhydrous sodium carbonate for
T o 0.13 g add 5 m L o f water, 2 m L o f sulfuric add and 2 0 minutes, cool and extract with 2 5 m L o f boiling water.
10 m L of sulfur dioxide solution and evaporate on a water bath Cool, filter, wash the residue with water and add the
until the sulfur dioxide solution is removed and the volume washings to the filtrate. Reserve the residue for test B.
reduced to about 2 mL. Transfer the solution to the Cautiously acidify the combined filtrate and washings with
generator flask with the aid o f 5 m L o f water. T he resulting hydrochloric add, evaporate to dryness, m oisten the residue
solution complies with the limit test for arsenic, Appendix VII with 0 .2 m L o f hydrochloric add, add 10 m L of water and stir.
(8 ppm ). A white, gelatinous precipitate is produced.
Heavy metals B. W ash the residue reserved in test A with water and
A. N o t m ore than 2 0 ppm when determined by the following dissolve in 10 m L o f 2 m hydrochloric add. T o 2 m L of the
m ethod. Shake 6 .0 g with 4 0 m L o f 0 .5 m hydrochloric add at solution add a 10% w/v solution of ammonium thiocyanate.
3 7 ° for 3 0 minutes, cool and filter. W ash the residue with A n intense red colour is produced.
water and dilute the combined filtrate and washings to 5 0 m L C. T o 2 m l, o f the solution obtained in test B add 1 m L of
with water. T o 2 0 m L add 2 g each o f ammonium chloride and strong sodium hydroxide solution and filter. T o the filtrate add
ammonium tkiocyanate and dissolve. Shake the solution with 3 m L of ammonium chloride sdution. A gelatinous white
8 0 m L of a mixture o f equal volumes o f ether and isoamyl precipitate is produced.
alcohol and separate, retaining the aqueous layer. Extract with
D . T o 2 m L o f the solution obtained in test B add
a further 8 0 m L of the mixture. T o the aqueous layer add
ammonium chloride and an excess o f 1 3 .5 m ammonia and
2 g o f citric add, neutralise with 1 3 .5 m ammonia and dilute to
filter. T o the filtrate add 0 .1 5 m L o f magneson reagent and an
25 m L with water. 12 m L o f the resulting solution complies
excess o f 5 m sodium hydroxide. A blue precipitate is produced.
with limit test A for heavy metals, Appendix VII. Use lead
standard solution (2 ppm Pb) to prepare the standard. TESTS
B. N o t more than 10 ppm when determined by the following Acidity or alkalinity
m ethod. Shake 6.0 g with 40 m L o f 0.5m sodium hydroxide at p H o f a 5% w/v suspension in carbon dioxide-free water, after
37° for 30 minutes, cool and filter. W ash the residue with shaking for 5 minutes, 7 .0 to 9 .5 , Appendix V L.
water and dilute the combined filtrate and washings to 50 mT. Arsenic
with water. Neutralise 20 m L o f the solution with hydrochloric T o 0 .1 3 g add 5 mT, o f water, 2 m L o f sulfuric add and
add and dilute to 25 m L with water. 12 m L o f the resulting 10 mT, o f sulfur dioxide solution and evaporate on a w ater bath
solution complies with limit test A for heavy metals, until the sulfur dioxide solution is removed and the volume
A ppendix VH. Use lead standard solution (1 ppm Pb) to reduced to about 2 mT. Transfer the solution to the
prepare the standard. generator flask with the aid o f 5 m L o f water. T h e resulting
Acid-soluble m atter solution complies with the limit test for arsenic, Appendix VII
Boil 2 g with 1 0 0 m L o f 0 .2 m hydrochloric add under a reflux (8 ppm).
condenser for 5 minutes, cool and filter. Evaporate 5 0 mT, o f Heavy m etals
the filtrate to dryness. T he residue, after ignition at about A. N o t m ore than 2 0 ppm when determ ined by the following
6 0 0 ° for 3 0 minutes, weighs not m ore than 0 .2 5 g. m ethod. Shake 6 .0 g with 4 0 m L of 0.5m hydrochloric add at
W ater-soluble matter 3 7 ° for 3 0 minutes, cool and filter. W ash the residue with
Boil 10 g with 100 m L o f water under a reflux condenser for water and dilute die combined filtrate and washings to 5 0 m L
5 m inutes, cool and filter. Evaporate 50 m L o f the filtrate to with water. T o 2 0 mT, add 2 g each of ammonium chloride and
diyness. T h e residue, after ignition a t 600° for 30 minutes, ammonium thiocyanate and dissolve. Shake the solution with
weighs n o t m ore than 50 mg. 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
Loss on drying
a further 8 0 m L o f the mixture. T o the aqueous layer add
W hen dried to constant weight at 105°, loses n o t more than
2 g o f citric add, neutralise with 13.5m ammonia and dilute to
17.0% o f its weight. Use 1 g.
2 5 m L w ith water. 12 m L o f the resulting solution complies
2016 Azapropazone 1-221
with limit test A for heavy metals, Appendix VII. Use lead IDENTIFICATION
standard solution (2 ppm Pb) to prepare die standard. A. T he infrared absorption spectrum, Appendix II A, is
B. N o t m ore than 10 ppm w hen determined by the following concordant with die reference spectrum of azapropazone
method. Shake 6.0 g with 40 m L o f 0.5m sodium hydroxide at (RS 016).
37° for 30 m in u tes, cool and filter. W ash die residue with B. T he light absorption, Appendix I I B , in the range 230 to
water and dilute the combined filtrate and washings to 50 m L 350 nm of a 0.0008% w/v solution in 0.1 m sodium hydroxide
with water. Neutralise 20 m L o f the solution with hydrochloric exhibits two maxima, at 255 nm and 325 nm . T he
acid and dilute to 25 m L with water. 12 m L of the resulting absorbances at 255 nm and 325 nm are about 0.86 and 0.18
solution complies with limit test A for heavy metals, respectively.
Appendix VII. U se lead standard solution (1 ppm Pb) to
TESTS
prepare the standard.
Acetic add
A d d -s o lu b le m a t te r N o t more than 0.2%, determined by the following m ethod.
Boil 2 g with 100 m L o f 0.2m hydrochloric acid under a reflux Dissolve 10 g in 25 m L o f methanol, add 75 m L of water and
condenser for 5 m inutes, cool and filter. Evaporate 50 m L of carry out a potendometric titration, Appendix V H IB , using
the filtrate to dryness. T h e residue, after ignition at about 0.1 m sodium hydroxide KS as titrant to a p H o f 5.9. Each m L
600° for 30 minutes, weighs n o t more than 0.25 g. o f 0.1 m sodium hydroxide FS is equivalent to 6.005 mg of
W ate r-so lu b le m a t te r acetic ad d , C 2H 4O 2.
Boil 10 g with 100 m L of water under a reflux condenser for Related substances
5 minutes, cool and filter. Evaporate 50 m L of the filtrate to Carry out the following operations in subdued light using
dryness. T h e residue, after ignition at 600° for 30 minutes, low-actinic glassware without delay. Carry out the method
weighs not more than 50 mg. for liquid chromatography, Appendix HI D , using the following
A d so rp tiv e c a p a c ity solutions in a mixture of 1 volume of phosphate buffer pH 4.0
In a stoppered bottle shake 1.0 g, in very fine powder, with and 3 volumes of methanol.
50 m L o f a 0.12% w/v solution of methylene blue for ( 1) 0 . 10% w/v of die substance being examined.
5 minutes, allow to setde and centrifuge. T h e colour o f the (2) 0.00010% w/v o f azapropazone impurity A BPCRS.
clear supernatant solution is no t more intense than that of a
(3) 0.00025% w/v of azapropazone impurity B BPCRS.
0.0012% w/v solution of methylene blue.
(4) 0.00025% w/v of azapropazone impurity C BPCRS.
L oss o n d ry in g
W hen dried to constant weight at 105°, loses not more than (5) 0.0001% w/v o f the substance being examined.
4.0% of its weight. U se 1 g. ( 6) 0.00005% w/v of the substance being examined.
L oss o n ig n itio n (7) 0.1% w/v o f azapropazone impurity standard BPCRS.
W hen ignited at 600°, loses n o t more than 9.0% of its CHROMATOGRAPHIC CONDITIONS
weight. U se 1 g. (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
Azapropazone below.
(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
1 volume o f glacial acetic acid, 36 volumes of methanol and
63 volumes o f a 0.068% w/v solution of sodium
Ci6H 2oN40 2,2H20 336.4 13539-59-8 (anhydrous) butanesulfonate in water.
SYSTEM SUITABILITY
A ction a n d u se
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
Azapropazone Capsules
solution (7) closely resembles the reference chromatogram
Azapropazone Tablets supplied with the azapropazone impurity standard.
I f necessary adjust the proportion o f methanol in the mobile
D E F IN IT IO N
phase to give the required retention times.
Azapropazone is 5-dimethylamino-9-methyl-2-
LIMITS
propylpyrazolo [ 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 In the chromatogram obtained w ith solution (1):
101.0% of C 16H 2oN402 , calculated with’reference to the the area of any peak corresponding to azapropazone
anhydrous substance. im purity A is n o t greater than the area o f the corresponding
C H A R A C T E R IS T IC S peak in the chromatogram obtained with solution (2) (0 . 1%);
A white to pale yellow, crystalline powder. the area of any peak corresponding to azapropazone
Very slightly soluble in water, soluble in ethanol (96%)-, im purity B is n o t greater than the area of the corresponding
it dissolves in solutions of alkali hydroxides..
1-222 Azathioprine 2016
peak in the chromatogram obtained with solution (3)

(0.25% );
Me > t~ C
the area of any peak corresponding to azapropazone V rV N'N o
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 3. 5-hydroxy-9-methyl-2-propylpyrazolo [1,2-
area of the peak in the chromatogram obtained with solution a] [1,2,4]benzotriazine-1,3 (2//)-dione (impurity B),
(5) (0.1%).
Calculate the content of impurities A, B and C using the HOOC Pr"
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%. M e Y Y N ' N 0
Disregard any peak with an area less than the area o f the
principal peak in the chromatogram obtained with solution
(6) (0.05%). 4. a-(3-dimethylamino-7-methyl-1,2-dihydro-1,2,4-
H eav y m e ta ls benzotriazin-2-ylcaibonyl)valeric a d d (impurity C).
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). Azathioprine ★ ★
*****
C h lo rid e (Ph. Eur. monograph 0369)
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 ,n o 2
n -—y
(80 ppm ).
S u lfate
<N x S
A mixture o f 10 m L o f solution A and 5 m L o f water
complies with the limit test for sulfates, Appendix VII
(240 ppm ).
cx>
W a te r
10.0 to 11.5% w/w, Appendix IX C. Use 0.25 g. C9H7N70 2S 277.3 446-86-6
S u lfa te d a s h
A ctio n a n d u se
N o t m ore than 0.1%, Appendix IX A
Immunosuppressant.
A SSA Y P re p a ra tio n s
Carry out M ethod I for non-aqueous titration, Azathioprine Tablets
A ppendix V III A, using 0.25 g and determining the end
Azathioprine Oral Suspension
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. PhEur__________________________
IM P U R IT IE S 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
'N
X
N NM62 CHARACTERS
A p p e a ra n c e
1. 3-dimethylamino-7-methyi-l ,2,4-benzotriazine Pale-yellow powder.
(impurity A ), S o lubility
Practically insoluble in water and in ethanol (96 p er cent).
Bun It is soluble in dilute solutions of alkali hydroxides and
sparingly soluble in dilute mineral adds.
Me H L
N
A NMe2 Infrared absorption spectrophotom etry (2.2.24).
Comparison azathioprine CRS.
TESTS
2. 3-dimethylamino- l,2-dihydro-7-methyl-2-valeryl-1,2,4-
benzotriazine,
Solution A 2.76 g/L solution o f sodium dihydrogen phosphate
monohydrate R adjusted to p H 2.5 with phosphoric acid R.
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.
2016 Azathioprine 1-223
Reference solution (a) Dissolve 5 mg o f azathioprine S u lfa te d a s h (2.4.14)

impurity A CRS and 5 m g o f mercaptopurine R (impurity B) in M aximum 0.1 p er cent, determined on 1.0 g.
8.75 m L o f a 0.8 g/L solution of sodium hydroxide R and ASSAY
dilute to 25.0 m L with solution A. T o 1.0 m L o f this
Dissolve 0.250 g in 25 m L of dimethylformamide R. Titrate
solution, add 35 m L of a 0.8 g/L solution of sodium
with 0.1 M tetrabutylammonium hydroxide, d eterm in in g the
hydroxide R and dilute to 100.0 m L with solution A.
end-point potentiometrically (2.2.20).
Reference solution (b) Dissolve 2.5 mg o f azathioprine
1 m L of 0.1 M tetrabutylammonium hydroxide is equivalent to
impurity G CRS and 2.5 mg o f the substance to be examined
27.73 mg of C 9H 7N 7O 2S.
in 8.8 m L of a 0.8 g/L solution o f sodium hydroxide R and
dilute to 25.0 m L with solution A. T o 1.0 m L o f this STORAGE
solution, add 17.5 m L of a 0.8 g/L solution of sodium Protected from light.
hydroxide R and dilute to 50.0 m L with solution A. IM P U R IT IE S
Reference solution (c) Dilute 1.0 m L of die test solution to Specified impurities A, B
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: the tests in the monograph. They are limited by the general
— sizer. I = 0.15 m , 0 = 4.6 mm; acceptance criterion for other/unspecified impurities and/or
— stationary phase: phenylsüyl silica gel for chromatography R by the general m onograph Substances for pharmaceutical use
(5 nm); (2034). It is therefore n o t necessary to identify these
— temperature: 30 °C. impurities for demonstration of compliance. See also 5.10.
Mobile phase'. Control of impurities in substances for pharmaceutical use): C, D,
— mobile phase A: methanol R, solution A (5:95 VIV)', E, F, G.
— mobile phase B: solution A, methanol R (40:60 VIV)',
ino 2
N- V
Time
(min)
Mobile phase A
(per cent V7V)
Mobile phase B
(per cent V/V) <.x nh 2
0 -5 100 0 h 3c
5 - 15 100->0 0 -> 100

A. l-methyl-4-nitro-lH-imidazol-5-amine,
1 5 -20 0 100
SH

Detection Spectrophotometer at 240 nm . k ï !>
Injection 20 |iL.
Identification of impurities Use the chromatogram obtained B. 7H-purine-6-thiol (mercaptopurine),
with reference solution (a) to identify the peaks due to
impurities A and B. Use the chromatogram obtained with
<x
no 2
reference solution (b) to identify the peak due to impurity G.
Relative retention W ith reference to azathioprine (retention N ^C I
tim e = about 15 min): impurity A = about 0.3; H3C
im purity B = about 0.4; impurity G = about 0.97.
System suitability: C. 5-chloro-l-methyl-4-nitro- l//-im idazole,
impurities A and B in the chromatogram obtained with NOj
reference solution (a); minimum 2.0 between the peaks n —/
due to impurity G and azathioprine in the chromatogram <N x
-^ S H
obtained with reference solution (b).
H3C
Limits:
— impurities A, B: for each impurity, not more than
1.5 times the area o f the principal peak in the D . 1-methyl-4-nitro- li/-imidazole-5-thiol,
(0.15 per cent); ,NOj
N—/
<N 1 OH
with reference solution (c) (0.10 per cent); H,C
— total: n o t more than 5 times the area of the principal peak
in the chromatogram obtained with reference solution (c) E. l-methyl-4-nitro-lH-imidazol-5-ol,
(0.5 per cent);
— disregard Hmir. 0.5 times the area o f the principal peak in
(0.05 per cent).
an oven at 105 °C. F. 1,7-dihydro-6//-purin-6-one (hypoxanthine),
1-224 Azelastine Hydrochloride 2016

<X
h3c
n^ s
Hj N
xX> 100.0 m L w ith the solvent mixture. D ilute 1.0 m L o f this
Reference solution (b) Dissolve 1 m g o f azdastine
G . 6- [(1 -m ethyl-4-nitro-1//-imidazol-5-yl) sulfanyl] -7//-p u rin- impurity B CRS, 1 m g o f azdastine impurity D CRS and 1 mg
2-amine (thiamiprine). o f azdastine impurity E CRS in the test solution and dilute to
PhEur 20 m L w ith the test solution.
Column:
— sizer. I = 0.25 m , 0 = 4.6 m m ,
— stationary phase: nitrUe silica gd for chromatography R
★ ★ (10 pm ),
Azelastine Hydrochloride ★ ★ — temperature: 30°C.
(Ph Eur monograph 1633) ***** Mobile phase Dissolve 2.16 g o f sodium octanesulfonate R and
0.68 g o f potassium dihydrogen phosphate R in 740 m L o f water
for chromatography R, adjust to p H 3.0-3.1 w ith dilute
phosphoric add R, add 260 m L o f acetonitrile for
chromatography R and mix.
, Ha Flow rate 2.0 m lA nin.
Irgecdon 10 |iL.
Run time Twice the retention tim e of azelastine.
Relative retention W ith reference to azelastine (retention
C 22H 25Q 2N 3O 418.4 79307-93-0 tim e = about 8-9 min): im purity A = about 0.2;
impurity B = about 0.3; im purity C = about 0.4;
A ctio n a n d u se
impurity D = about 0 .6; im purity E = about 1.4.
Histamine H x receptor antagonist; antihistamine.
PhEir. — resolution: m inim um 4.0 between the peaks due to
impurities B and D ,
D E F IN IT IO N
— the peaks due to impurities D and E are baseline
4-(4-Chlorobenzyi)-2- [(4i25)-l-methylhexahydro- lH -azepin-
separated from the principal peak.
4-yI]phthalazin-1 (2H)-onc hydrochloride.
Limits:
C o n te n t — correction factors: for the calculation o f content, multiply
99.0 per cent to 101.0 p er cent (dried substance). the peak areas o f the following impurities by the
CHARACTERS corresponding correction factor, im purity B = 3.6;
A p p e a ra n c e impurity D = 0.7; im purity E = 2 . 1;
W hite or alm ost white, crystalline powder. — impurities A , B, C, D, E: for each impurity, n o t m ore than
the area of the principal peak in the chrom atogram
S o lu b ility
obtained with reference solution (a) (0.1 p er cent);
Sparingly soluble in water, soluble in ethanol and in
— any other impurity: for each impurity, n o t m ore than the
methylene chloride.
area o f th e principal peak in the chrom atogram obtained
ID E N T IF IC A T IO N with reference solution (a) (0.1 per cent);
A Infrared absorption spectrophotometry (2.2.24). — totah n o t m ore than twice the area o f the principal peak in
Comparison azdastine hydrochloride CRS. the chrom atogram obtained with reference solution (a)
B. Solution S (see Tests) gives reaction (a) o f chlorides (0.2 per cent);
(2.3.1). — disregard limit. 0.5 times the area of the principal peak in
TESTS (0.05 p er cent).
S o lu tio n S
M axim um 0.5 p e r cent, determ ined on 1.000 g by drying in
100 m L w ith the same solvent.
an oven at 105 °C.
ASSAY
In order to avoid overheating in the reaction medium, mix
A cid ity o r a lk a lin ity thoroughly throughout and stop the titration immediately after the
T o 10 m L o f solution S add 0.2 m L o f bromothymol blue end-point has been reached.
solution R l. N o t m ore than 0.1 m L o f 0.01 M hydrochloric
Dissolve 0.300 g in 5 m L o f anhydrous formic add R.
acid or 0.01 M sodium hydroxide is required to c h a n g e the
A dd 30 m L o f acetic anhydride R. T itrate quickly w ith 0.1 M
colour of the solution.
perchloric acid, d eterm in in g the end-point potentiom etrically
R e la te d su b s ta n c e s (2. 2. 20).
1.0 m L o f 0.1 M perchloric add is equivalent to 41.84 m g o f
Solvent mixture acetomtrUe for chromatography R, water R C 22H 25CI2N 3O.
(45:55 V/V).
2016 Azithromycin 1-225
IM P U R IT IE S ★>*★★
Azithromycin * ★
Specified impurities: A , B, C, D, E.
ch 3
NH
i
nh2
A. benzoyldiazane (benzohydrazide),
and enantiomer
sCH3
0
B. 1-benzoyl-2-[(4ftS)-l -methylhexahydro- l//-azepin-4-

yl]diazane, C3gH72N20i2>xH20 749
with x = 1 o r 2 (anhydrous substance)
Azithromycin monohydrate 121470-24-4
Azithromycin dihydrate 117772-70-0
A ctio n aind u se
Macrolide antibacterial.
PhEur_____________________________________
C. 2-[(4-chlorophenyl)acetyl]benzoic add, D E F IN IT IO N
(2R,3S,4R,5R,8R, 10R, 11R, 125,135,14R)-13- [(2,6-Dideoxy-
o 3-C-methyl-3-0-methyl-oc-L-nfco-hexopyranosyl) oxy] -2-ethyl-
3,4,1O-trihydroxy-3,5,6,8, 10,12,14-heptamethyl-l 1-[[3,4,6-
trideoxy-3- (dimethylamino)-P-D-ry/o-hexopyranosyl] oxy] -1 -
oxa-6-azacydopentadecan-15-one. T he degree of hydration is
1 or 2.
Semi-synthetic product derived from a fermentation p roduct
C o n te n t
D. 4-(4-chlorobenzyl)phthalazm-l (2/i)-one, CHARACTERS
A p p e a ra n c e
0 W hite or almost white powder.
S olu b ility
o
ethanol and in methylene chloride.
Comparison azithromycin CRS.
prepare further spectra using 90 g/L solutions in methylene
E. 3-(4-chlorobenzylidene)isobenzofuran-l(3if)-one. chloride R.
PhEur TESTS
S o lu tio n S
Dissolve 0.500 g in anhydrous ethanol R and dilute to
p H (2.2.3)
9.0 to 11.0.
Dissolve 0.100 g in 25.0 m L of methanol R and dilute to
50.0 m L with carbon dioxide-free water R.
—45 to —49 (anhydrous substance), determined on
solution S.
R e la te d su b stan c es
1-226 Azithromycin 2016
Solvent mixture Prepare a 1.73 g/L solution o f ammonium Limits:

dihydrogen phosphate R adjusted to p H 10.0 with ammonia R. — correction factors: for the calculation o f content, multiply
T ransfer 350 m L o f this solution to a suitable container. the peak areas o f die following impurities by the
A dd 300 m L of acetonitrile R1 and 350 m L o f methanol R l. corresponding correction fa c to r im purity F = 0.3;
M ix well. im purity G = 0.2; im purity H = 0.1; im purity L = 2.3;
Test solution Dissolve 0.200 g o f the substance to be im purity M = 0.6; impurity N = 0.7;
examined in the solvent mixture and dilute to 25.0 m L with — impurity B: n o t m ore than twice the area o f the principal
the solvent mixture. peak in the chrom atogram obtained with reference
solution (a) (2.0 p er cent);
— impurities A , C, E, F, H, I, L, M , N, O, P. for each
im purity, not m ore than 0.5 times the area o f the
Reference solution (b) Dissolve the contents o f a vial o f principal peak in the chrom atogram obtained with
azithromycin for system suitability CRS (containing impurities reference solution (a) (0.5 per cent);
F , H and J) in 1.0 m L o f the solvent mixture and sonicate
— sum of impurities D andj: n o t m ore than 0.5 times the
for 5 min.
Reference solution (c) Dissolve 8.0 m g o f azithromycin for peak w ith reference solution (a) (0.5 p er cent);
identification CRS (containing impurities A, B, C , E, F , G , I, — impurity G: n o t m ore than 0.2 times the area o f the
J, L , M , N , O and P) in 1.0 m L o f the solvent mixture. principal peak in the chrom atogram obtained with
Column: reference solution (a) (0.2 p er cent);
— sizer. I = 0.25 m , 0 = 4.6 mm; — any other impurity: for each impurity, not m ore than
— stationary phase, end-capped octadecylsUyl amorphous 0.2 times the area o f the principal peak in the
orgahosUica polymerfor mass spectrometry R (5 pm); chrom atogram obtained with reference solution (a)
— temperature: 60 °C. (0.2 p er cent);
Mobile phase". — total: n o t more than 3 times the area of the principal peak
— mobile phase A: 1.80 g/L solution o f anhydrous disodium in the chrom atogram obtained with reference solution (a)
hydrogen phosphate R adjusted to p H 8.9 with dilute (3.0 p er cent);
phosphoric acid R o r with dilute sodium hydroxide solution R; — disregard limit: 0.1 times the area o f the principal peak in
— mobile phase B: methanol R l, acetonitrile R l (250:750 V/V); the chrom atogram obtained with reference solution (a)
(0.1 p er cent); disregard the peaks eluting before
im purity L and after impurity B.
(min) (per cent V/V) (per cent V/V) H eav y m e ta ls (2.4.8)
0 -2 5 50 -> 45 50 ->55 M axim um 25 ppm .
25 - 30 45->40 55 -> 60 Dissolve 2.0 g in a mixture o f 15 volumes o f water R and
85 volumes o f anhydrous ethanol R and dilute to 20 m L with
30 - 80 40-> 25 60-> 75 the sam e mixture of solvents. 12 m L o f the solution complies
80 - 81 25 -> 50 75 •» 50 with test B. Prepare the reference solution using lead
81 - 93 50
standard solution (2.5 ppm Pb) obtained by diluting lead
50
standard solution (100 ppm Pb) R with a mixture of
15 volumes o f water R and 85 volumes o f anhydrous
Flow rate 1.0 mL/min. ethanol R.
Detection Spectrophotom eter at 210 nm . W a te r (2.5.12)
Injection 50 (iL. 1.8 p er cent to 6.5 per cent, determ ined on 0.200 g.
Identification of impurities Use the chrom atogram supplied S u lla te d a s h (2. 4.14)
with azithromycin for peak identification CRS and the M axim um 0.2 p er cent, determ ined on 1.0 g.
chrom atogram obtained with reference solution (c) to identify A SSA Y
the peaks due to impurities A, B, C , E , F , G, I, J, L , M , N , L iquid chrom atography (2.2.29).
O and P; use the chrom atogram supplied with azithromycin
Solution A Mix 60 volumes o f acetonitrile R l and 40 volumes
for system suitability CRS and the chromatogram obtained
o f a 6.7 g/L solution o f dipotassium hydrogen phosphate R
w ith reference solution (b) to identify the peak due to
adjusted to p H 8.0 w ith phosphoric acid R.
im purity H .
Relative retention W ith reference to azithromycin (retention
examined in 2 m L o f acetonitrile R l and dilute to 100.0 m L
tim e = 45-50 min): im purity L = about 0.29;
with solution A.
im purity M = about 0.37; im purity E = about 0.43;
im purity F = about 0.51; im purity D = about 0.54; Reference solution (a) Dissolve 53.0 m g o f azithromycin CRS
im purity J = about 0.54; im purity I = about 0.61; in 2 m L o f acetonitrile R l and dilute to 100.0 m L with
im purity C = about 0.73; im purity N = about 0.76; solution A.
im purity H = about 0.79; im purity A = about 0.83; Reference solution (b) Dissolve 5 m g o f the substance to be
im purity P = about 0.92; im purity O = about 1.23; examined and 5 m g o f azithromycin impurity A CRS in
im purity G = about 1.26; im purity B = about 1.31. 0.5 mT. o f acetonitrile R l and dilute to 10 mT. with
System suitability: reference solution (b): solution A.
— peak-to-vaUey ratio: minim um 1.4, where Hp = height Column:
above the baseline o f the peak due to impurity J and — size. I = 0.25 m , 0 = 4.6 m m ;
H v = height above the baseline o f the lowest point o f the — stationary phase. octadecylsUyl vinyl polymer for
curve separating this peak from the peak due to chromatography R (5 Jim);
im purity F. — temperature: 40 °C.
2016 Azithromycin 1-227
ch3
Mobile phase Mix 60 volumes of acetomtrüe R1 and h 3c /— u ch3
40 volumes o f a 6.7 g/L solution o f dipotassium hydrogen
phosphate R adjusted to p H 11.0 with a 560 g/L solution of
potassium hydroxide R
ch3
Detection Spectrophotometer at 210 nm .
cladinosyl
Injection 10 |xL.
h ch3
Run time 1.5 times the retention time of azithromycin.
Retention time Azithromycin = about 10 min.
im purity A and azithromycin. R1 = dadinosyl R2 = ( NH2
Calculate th e percentage content of C 38H 72N 2O i 2 from the

declared content o f azithromycin CRS.
STO RA G E E. 3 '-(N ^N -didem ethâzithrom ycin (am inoazhhrom ydn),
IMPURITIES
Specified impurities A, B, C, D , E, F , G , H , I, J, L, M , N , O, R1 = cladinosyl R2 =
P
the tests in the monograph. They are limited by the general G. 3 '-N-demethyl-3 '-N-
acceptance criterion for other/unspecified impurities and/or [(4-methylphenyl)sulfonyl] azithromycin,
Control of impurities in substancesfor pharmaceutical use): K. o
Í
R1 = dadinosyl R2 = h3C N
R2 H
H . 3 '-N- [[4-(acetylamino)phenyl] sulfonyl]-3 '-N-

demethylazithromydn,
R1 = H R2 =
J. 13-O-decladinosylazithromycin,
A. R1 = O H , R2 = R6 = H , R3 = R4 = R5 = C H 3: ch3
6-dem ethylazithromyán,
H c / o
B. R1 = R 6 = H , R2 = R3 = R4 = R5 = C H 3: R1 = dadinosyl R2 = 3 ^ n —c h
3-deoxyazithromydn (azithromycin B),
C. R1 = O H , R2 = R3 = R5 = C H 3, R4 = R6 = H : 3 "-0 - OH
dem ethylazithrom ydn (azithromycin C),
D. L. azithromycin 3 '-iV-oxide,
R1 = O H , R2 = R3 = R4 = CH3, R5 = C H 2O H , R6 = H:
14-dem ethyl-14- (hydroxymethyl) azithromycin (azithromycin
0 f»
F ),
F. R1 = O H , R2 = R4 = R5 = C H 3, R3 = C H O , R6 = H: R1 = dadinosyl R2 = HM r
C NH
3 '-N -dem ethyl-3 '-AT-foimylazithromycin,
I. R1 = O H , R2 = R4 = R5 = C H 3, R3 = R6 = H: 3 '-N- OH
demethylazithromycin,
O. R1 = O H , R2 = R3 = R4 = R5 = R6 = C H 3: M . 3 '-(N,N-didem ethyl)-3 '-N’-foimylazithromycin,
2-desethyl-2-propylazithromycin,
ch3
R1 * cladinosyl R2 =
O ÓH
N . 3 '-de(dime&ylamino)-3 '-oxoazithromycin,
1-228 Bacampicillin Hydrochloride 2016
ch3
Mobile phase M ix 10 volumes o f a 272 g/L solution o f sodium
acetate R adjusted to p H 5.0 with glacial acetic add R}
40 volumes o f water R and 50 volumes o f ethanol
(96 per cent) R.
Application 1 pL.
Development Over a p ath o f 15 cm.
Drying In a current o f warm air.
Detection Spray with nmhydrin solution R1 and heat a t 60 °C
for 10 min.
System suitability, reference solution (b):
— the chrom atogram shows 3 clearly separated spots.
K. C 14j 1 "-epoxyazithromydn (azithromycin E), Results T h e principal spot in the chrom atogram obtained with
P. unknow n structure.
PhEur solution (a).
C. Place about 2 m g in a test-tube about 150 mm long and
15 m m in diameter. M oisten with 0.05 m L o f water R and
add 2 m L o f sulfuric add-formaldehyde reagent R. M ix the
Bacampicillin Hydrochloride ★ ★ contents of the tube by swirling; the solution is practically
★ ★
colourless. Place the test-tube on a water-bath for 1 min;
a dark yellow colour develops.
D . Dissolve about 25 m g in 2 m L o f water R. A dd 2 m L of
dilute sodium hydroxide solution R and shake. W ait a few
h | V H3i
m inutes and add 3 m L o f dilute nitric add R and 0.5 m l. o f
H NH Y n^ A ^ ch ,
H'. / NH2h O V c h 3 silver nitrate sohaion R l. A white precipitate is formed.
A dd 0.5 m l. o f concentrated ammonia R T h e precipitate
H H dissolves.
® and epimer at C*
TESTS
C21H28CIN3O7S 502.0 37661-08-8
Dissolve 0.200 g in 20 m L o f water R; the solution is not
A c tio n a n d u se m ore opalescent than reference suspension II (2.2.1).
Penicillin antibacterial. Dissolve 0.500 g in 10 m L o f water R; the absorbance
(2.2.25) o f the solution at 430 nm is n o t greater th an 0.10.
PhEur. p H (2.2.3)
D E F IN IT IO N 3.0 to 4.5.
( IRS ) - 1-[(Ethoxycarbonyl)oxy]ethyl (2S,5R,6R)-6-[[(2R)-2- Dissolve 1.0 g in carbon dioxide-free water R and dilute to
amino-2-phenylacetyl] amino] -3 ,3-dime thyl-7 -oxo-4-thia-1 - 50 m L with the same solvent.
azabicyclo [3.2.0]heptane-2-carboxylate hydrochloride. S p ecific o p tic a l ro ta tio n (2.2.7)
Semi-synthetic product derived from a fermentation product. + 175 to + 195 (anhydrous substance).
C o n te n t Dissolve 0.250 g in w ater R and dilute to 25.0 m L with the
95.0 p er cent to 102.0 per cent (anhydrous substance). same solvent.
CHARACTERS R e la te d su b s ta n c e s
A p p e a ra n c e L iquid chrom atography (2.2.29). Prepare the test solution and
W hite or alm ost white pow der or granules, hygroscopic. reference solutions (a), (b) and (d) immediately before use.
S o lu b ility Phosphate buffer A Dissolve 1.4 g of sodium dihydrogen
Soluble in water, freely soluble in ethanol (96 per cent), phosphate monohydrate R in water R and dilute to about
soluble in methylene chloride. 800 m l. with the same solvent. Adjust to p H 3.0 w ith dilute
phosphoric add R and dilute to 1000.0 m L with water R.
Phosphate buffer B Dissolve 2.75 g o f sodium dihydrogen
First identification A , D phosphate monohydrate R and 2.3 g o f disodium hydrogen
Second identification B, C, D phosphate dihydrate R in water R and dilute to about 1800 m L
A Infrared absorption spectrophotom etry (2.2.24). with the sam e solvent. Adjust to p H 6.8, if necessary, using
Comparison bacampidUin hydrochloride CRS. dilute phosphoric add R or dilute sodium hydroxide solution R
and dilute to 2000.0 m L w ith water R.
Test solution Dissolve 10 mg o f the substance to be examined Test solution Dissolve 30.0 m g o f the substance to be
in 2 m L o f methanol R. examined in phosphate buffer A and dilute to 100.0 m L with
phosphate buffer A.
Reference solution (a) Dissolve 10 m g o f bacampidUin
hydrochloride CRS in 2 m L o f methanol R. Reference solution (a) Dissolve 30.0 m g o f bacampidllin
hydrochloride CRS in phosphate buffer A and dilute to
Reference solution (b) Dissolve 10 m g o f bacampidUin 100.0 m L w ith phosphate buffer A
hydrochloride CRS, 10 m g o f talampidUin hydrochloride CRS
and 10 mg o f pwampidUm CRS in 2 m l. o f methanol R.
Reference solution (b) D ilute 1.0 m L o f reference solution (a)
to 100.0 m L with phosphate buffer A.
Plate TLC silamsed silica gel plate R.
2016 Bacampicillin Hydrochloride 1-229
Reference solution (c) Dissolve 30 mg of the substance to be System suitability: reference solution (a):
examined in phosphate buffer B and dilute to 100 m L with — repeatability: maximum rdative standard deviation of
phosphate buffer B. H eat at 80 °C for about 30 min. 1.0 p er cent after 6 injections.
Reference solution (d) Dissolve 20 mg of ampidUin Calculate the percentage content of C 21H 28CIN 3O 7S from
trihydrate CRS (impurity I) in phosphate buffer A and dilute the d edared content of bacampicUHn hydrochloride CRS.
to 250 m L with phosphate buffer A. Dilute 5 m L o f this STORAGE
solution to 100 m L with phosphate buffer A.
Column:
— size. I = 0.05 m , 0 = 3.9 mm; IM P U R IT IE S
— stationary phase. octadecylsHyl silica gel for chromatography R
(5 |im ). H
Mobile phase Mix 30 volumes o f acetonitrile R l and ,CH3
70 volumes of a 0.06 per cent m/m solution o f
tetrahexylammonium hydrogen sulfate R in phosphate buffer B. H H
Flow rate 1.0 m lA nin.
Detection Spectrophotometer at 220 ran. A. (25,5i?,6JR)-6-amino-3,3-dimethyl-7-oxo-4-thia-l-
azabicydo[3.2.0]heptane-2-caiboxylic a d d
Injection 20 |iL o f the test solution and reference
( 6-am inopenidllanic ad d ),
Run time 3.5 times the retention time of bacampidllin. H NHa
System suitability".
— the peak due to impurity I is separated from the peaks
due to the solvent in the chromatogram obtained with
reference solution (d);
— relative retention with reference to bacampidllin: B. (2i?)-2-amino-2-phenylacetic a d d (D-phenylglycine),
degradation product eluting just after
bacam pidllin = 1.12 to 1.38 in the chromatogram
obtained with reference solution (c); if necessary, adjust
the concentration of tetrahexyiammonium hydrogen
sulfate in the mobile phase.
Limits:
— any impurity", for each impurity, not more than 1.5 times
obtained with reference solution (b) (1.5 p er cent); C . (2RS,45)-2-[[[(2i?)-2-annno-2-
— total: not more than 3 times the area o f the principal peak phenylacetyl] amino] methyl] -5,5-dimethylthiazolidine-4-
in the chromatogram obtained with reference solution (b) carboxylic a d d (penilloic adds of ampicillin),
(3 per cent);
— disregard limit. 0.1 times the area of the principal peak in *1 .c o 2h
the chromatogram obtained with reference solution (b) H NH2 H H N^C^CH a
(0.1 per cent).
B u ty l a c e ta te a n d eth y l a c e ta te (2.4.24> System A)
M axim um 2.0 per cent of butyl acetate, maximum
4.0 per cent of ethyl acetate and maximum 5.0 per cent for
the sum o f the contents. D . (45)-2-[[[(2JR)-2-amino-2-
Sample solution Dissolve 50.0 m g o f the substance to be phenylacetyl] amino] carboxymethyl]-535-dimethylthiazolidine-
examined in water R and dilute to 10.0 m L with the same 4-carboxylic a d d (penidUoic adds o f ampicillin),
solvent.
Use the method o f standard additions.
Static head-space conditions that may be used:
— equilibration temperature. 60 °C;
— equilibration time. 20 min.
iVyV-Dim etfaylaniline (2.4.26j, Method A)
M axim um 20 ppm .
W a te r (2.5.12)
M axim um 0.8 per cent, determined on 0.300 g. E. (4S)-2-(3,6-dioxo-5-phenylpiperazin-2-yl)-5,5-
S u lfa te d ash (2.4.14) dimethylthiazolidine-4-carboxylic a d d (diketopiperazines of
M axim um 1.5 p er cent, determined on 1.0 g. ampicillin),
A SSA Y
HS CH3
related substances with the following modifications. H3C ^ X C° 2H anc*enantiomer
H NH2
Injection Test solution and reference solution (a).
F. (2ÄS)-2-amino-3-methyl-3-sulfanylbutanoic a d d
(DL-penicillamine),
1-230 Bacitracin 2016
H NH2
CHARACTERS
A p p e a ra n c e
W hite or almost white powder, hygroscopic.
S o lu b ility
G . methyl (2R)-2-amino-2-phenylacetate (methyl Freely soluble in w ater and in ethanol (96 p e r cent).
D -phenylgiycinate), ID E N T IF IC A T IO N
First identification B, C
H ? H- / H3i Second identification A , C
0 ^ 0 O' 'CH, A. Thin-layer chromatography (2.2.27).
Test solution Dissolve 10 m g o f the substance to be exam in ed
in a 3.4 g/L solution of hydrochloric add R and dilute to
and epimer at C* 1.0 m L with the sam e solution.
Reference solution Dissolve 10 m g o f bacitradn zinc CRS in a
H . ( IRS ) - 1- [(ethoxycarbonyl)oxy]ethyl (2.S,5/?,6i?)-6-[[(2.R)- 3.4 g/L solution o f hydrochloric acid R and dilute to 1.0 m L
2-(acetylamino)-2-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4- w ith the same solution.
thia-l-azabicyclo[3.2.0]heptane-2-caiboxylate Plate TLC silica gd plate R.
(N-acetyibacampicillin), Mobile phase glacial acetic add R, water R, butanol R
(1:2:4 VIVIV).
o Application 10 |iL.
Development Over h alf o f the plate.
Drying A t 100-105 °C.
H H
Detection Spray with nmhydrm solution R1 and heat at 110 °C
for 5 min.
I. (2S,5i?,6i?)-6-[[(2i?)-2-amino-2-phenylacetyl]amino]-3,3-
Results T h e spots in the chrom atogram obtained with the test
solution are similar in position, size and colour to the spots
dim ethyl-7-oxo-4-thia-l-azabicydo [3.2.0]heptane-2-
in the chrom atogram obtained w ith the reference solution.
carboxylic a d d (am pidllin).
B. Composition (see Tests).
PhEur
C . Ignite 0.2 g. A n insignificant residue rem ains which is not
yellow at high tem perature. Allow to cool. Dissolve the
residue in 0.1 m L o f dilute hydrochloric add R. A dd 5 m L of
**★ water R and 0.2 m L of strong sodium hydroxide solution R.
Bacitracin * ★ N o white p redpitate is formed.
★ ★
(Ph. Eur. monograph 0465) * * * * * TESTS
S o lu tio n S
h,n V H’ 25 m L with the sam e solvent.
:' XN Solution S is clear (2.2.1).
p H (2.2.3)
L-Leu d-GIu Y—L- Lyso-OmX D-Phe-] 6.0 to 7.0 for solution S.
O t*- L-Asn— D-Asp -*• l-H is J C o m p o sitio n
L iquid chromatography (2.2.29): use the norm alisation
Name Mol. Formula X Y R procedure. Prepare the solutions immediately before use.
Bacitradn A CeeHi03Ni7OieS L-lle L-lle ch3 Test solution Dissolve 0.100 g of the substance to be
Bacitradn B1 CbsH io iN i 7 0 16S L-lle L-lle H e xam in ed in 50.0 m l . of the mobile phase.
Bacitradn B2 C «H io iN i70ieS L-Val L-lle ch3
Reference solution (a) Suspend 20.0 m g o f bacitradn zinc CRS
Bacitradn B3 C85H10iNi7O i6S L-lle L-Val ch3
in water R, add 0.2 m L o f dilute hydrochloric add R and dilute
to 10.0 m L with water R.
1405-87-4 Reference solution (b) Dilute 5.0 m L o f the test solution to

A c tio n a n d u se Reference solution (c) Dilute 1.0 m L o f reference solution (b)
Polypeptide antibacterial. to 10.0 m L with the mobile phase.
Reference solution (d) Dissolve 50.0 m g o f the substance to be
PhEtr.
exam ined in 25.0 m l. of a 40 g/L solution o f sodium edetate R
D E F IN IT IO N adjusted to p H 7.0 with dilute sodium hydroxide solution R.
M ixture of antimicrobial polypeptides produced by certain H eat in a boiling w ater-bath for 30 min. Cool to room
strains of Bacillus lichemformis or Bacillus subtSts, the main tem perature.
com ponents being b a d tra d n s A, B l, B2 and B3. Blank sdution A 40 g/L solution o f sodium edetate R adjusted
C o n te n t to p H 7.0 with dilute sodium hydroxide sdution R.
M inim um 60 IU /m g (dried substance).
2016 Bacitracin 1-231
1. impurity A 3. impurity C 5. bacitracin B2 7. bacitracin A

2. impurity B 4. bacitracin B1 6. bacitracin B3
Figure 0465.-1. - Chromatogram of the test for composition tn bacitracin obtained with the test solution at 254 nm
Column: R e la te d p e p tid e s
— size: I = 0.25 m , 0 = 4.6 mm; Liquid chromatography (2.2.29) as described in the test for
— stationary phase: end-capped octadecylsUyl silica gel for composition.
chromatography R (5 (im). See Figure 0465.-1.
Mobile phase A dd 40 volumes o f acetordtrile R, 300 volumes of Limit.
water R and 520 volumes of methanol R1 to 100 volumes o f a — sum of the areas of all peaks eluting before the peak due to
34.8 g/L solution o f dipotassium hydrogen phosphate R adjusted bacitracin Bl: maximum 20.0 per cent.
to p H 6.0 with a 27.2 g/L solution of potassium dihydrogen
Im p u rity E
phosphate R. Liquid chromatography (2.2.29) as described in the test for
Flow rate 1.0 m I7m in. composition.
Detection Spectrophotom eter at 254 nm. See Figure 0465.-2.
Injection 100 pL; inject the blank, the test solution and Detection Spectrophotometer at 254 nm; spectrophotometer
reference solutions (a) and (c). at 300 nm for reference solution (d).
Run time 3 times the retention time o f bacitracin A. Injection T est solution and reference solutions (b) and (d).
Relative retention W ith reference to bacitracin A (retention Limit:
time = 1 5 min to 25 min): bacitracin B1 = about 0.6; — impurity E: n o t more than 1.2 times the area o f the
bacitracin B3 = about 0.8; impurity E = about 2.5. principal peak in the chromatogram obtained with
If necessary, adjust the composition o f the mobile phase by reference solution (b) (6.0 per cent).
changing the am ount o f organic modifier whilst keeping the L oss o n d ry in g (2.2.32)
ratio constant betw een methanol and acetonitrile. M axim um 5.0 per cent, determined on 1.000 g by drying at
System suitability: reference solution (a): 60 °C over diphosphorus pentoodde R at a pressure not
— peak-to-valley ratio: minimum o f 1.2, where Hp = height exceeding 0.1 kPa for 3 h.
above the baseline o f the peak due to bacitracin B1 and S u lfa te d a s h (2.4.14)
Hv = height above the baseline o f the lowest point o f the M aximum 1.0 per cent, determined on 1.0 g.
bacitracin B2. S te rility
(2.6.1). If intended for the preparation of ophthalmic dosage
Limits:
forms without a further appropriate sterilisation procedure, it
— bacitracin A: minimnm 40.0 per cent;
complies with the test for sterility.
— sum of bacitracins A , B l, B2 and B3: minimum
70.0 per cent; B a c te ria l en d o to x in s (2.6.14)
— disregard limit: the area o f the peak due to bacitracin A in Less than 0.8 IU/mg, if intended for use in the manufacture
the chrom atogram obtained with reference solution (c) o f ophthalmic dosage forms without a further appropriate
(0.5 per cent); disregard any peak observed in the blank procedure for the removal of bacterial endotoxins.
run.
1-232 Bacitracin Zinc 2016
1. bacitracin B1 2. bacitracin B3 3. bacitracin A 4. impurity E

Figure 0465.-2. - Chromatogram of the test for impurity E m bacitracin obtained with reference solution (d) at 300 nm
ASSAY ★ ★
Bacitracin Zinc ★ ★
Carry out the microbiological assay o f antibiotics (2.7.2).
*****
Use bacitracin zinc CRS as the reference substance. (Ph. Eur. monograph 0466)
STORAGE
Action and use
In an airtight container at 2 °C to 8 °C. If the substance is
Polypeptide antibacterial.
sterile, store in a sterile, airtight, tam per-proof container.
Preparations
IMPURITIES Polymyxin and Bacitracin O intm ent
Polymyxin and Bacitracin Eye O intm ent
PhEur.
DEFINITION
Zinc complex o f bacitracin, which consists o f a mixture o f
antimicrobial polypeptides produced by certain strains of
Bacillus lichemformis or Bacillus subtiHs, the m ain com ponents
A. X = L-Val, Y = L-Ile, R = H : bacitracin C l, being bacitracins A, B l, B2 and B3.
B. X = L -Ü e, Y = L-Val, R = H: bacitracin C2, Content
Minimum 60 IU /m g (dried substance).
C. X = Y = L-Val, R = C H 3: bacitracin C3,
D . X = Y = L-Val, R = H : bacitracin E, CHARACTERS
Appearance
W hite or light yellowish-grey powder, hygroscopic.
Solubility
Slightly soluble in water and in ethanol (96 p er cent).
IDENTIFICATION
E. X = Y = L-Üe, R = C H 3: bacitracin F , A. Thin-layer chrom atography (2.2.27).
F. X = Y = L-Üe, R = H : bacitracin H I, Test solution Dissolve 10 m g o f the substance to be examined
in 0.5 m l. o f dilute hydrochloric add R and dilute to 1.0 m L
G . X = L-Val, Y = L-Üe, R = C H 3: bacitracin H 2,
with water R.
H . X = L-Ile, Y = L-Val, R = C H 3: bacitracin H 3,
Reference solution Dissolve 10 m g o f badtradn zinc CRS in
I. X = L-Val, Y = L-Üe, R = H : badtradn II, 0.5 m L o f dilute hydrochloric add R and dilute to 1.0 m L with
J. X = L-Ile, Y = L-Val, R = H : bacitracin 12, water R
K. X = Y = L-Val, R = C H 3: bacitracin 13. Plate TLC silica gel plate R.
PhEw
2016 Bacitracin Zinc 1-233
1. im purity A 3. im purity C 5. bacitracin B2 7. bacitracin A

2. im purity B 4. bacitracin B1 6. bacitracin B3
Figure 0466.-1. - Chromatogram of the test for composition in bacitracin zinc obtained with the test solution at 254 nm
Mobile phase glacial acetic acid R, water R, butanol R Reference solution (d) Dissolve 50.0 mg of the substance to be
(1:2:4 VIVIV). examined in 25.0 m L of a 40 g/L solution of sodium edetate R
Application 10 |iL. adjusted to p H 7.0 with dilute sodium hydroxide solution R.
H eat in a boiling water-bath for 30 min. Cool to room
Development Over half of the plate.
temperature.
Drying h i 100-105 °C.
Blank solution A 40 g/L solution o f sodium edetate R adjusted
Detection Spray with ninhydrin solution R l and heat at 110 °C to p H 7.0 with dilute sodium hydroxide R.
for 5 min.
Column:
Residts T h e spots in the chromatogram obtained with the test — size: I = 0.25 m , 0 = 4.6 mm ;
solution are similar in position, size and colour to the spots — stationary phase: end-capped octadecylsHyl silica gel for
in the chrom atogram obtained with the reference solution. chromatography R (5 Jim).
B. Com position (see Tests). Mobile phase Add 520 volumes of methanol R l, 40 volumes of
C. Ignite about 0.15 g, allow to cool and dissolve the residue acetonitrile R and 300 volumes o f water R to 100 volumes of a
in 1 m L o f dilute hydrochloric acid R. Add 4 m L of water R. 34.8 g/L solution o f dipotassium hydrogen phosphate R,
T he solution gives the reaction of zinc (2.3.1). adjusted to p H 6.0 with a 27.2 g/L solution of potassium
TESTS dihydrogen phosphate R.
p H (2.2.3) Flow rate 1.0 mL/min.
6.0 to 7.5. Detection Spectrophotom eter at 254 nm.
Shake 1.0 g for about 1 min with 10 m L o f carbon dioxide-free Injection 100 nL; inject the blank, the test solution and
water R and filter. reference solutions (a) and (c).
C o m p o sitio n Run time 3 times the retention time of bacitracin A.
Liquid chromatography (2.2.29): use the normalisation Relative retention W ith reference to bacitracin A (retention
procedure. Prepare the solutions immediately before use. time = 15 min to 25 min): bacitracin B1 = about 0.6;
Test solution Dissolve 0.100 g of the substance to be bacitracin B3 = about 0.8; impurity E = about 2.5.
examined in 50.0 m L o f a 40 g/L solution of sodium edetate R If necessary, adjust the composition of the mobile phase by
adjusted to p H 7.0 with dilute sodium hydroxide solution R. changing the am ount o f organic modifier whilst keeping the
Reference solution (a) Dissolve 20.0 m g of bacitracin zinc CRS ratio constant between methanol and acetonitrile.
in 10.0 m L of a 40 g/L solution of sodium edetate R adjusted System suitability: reference solution (a):
to p H 7.0 with dilute sodium hydroxide solution R. — peak-to-valley ratio: minimum o f 1.2, where Hp = height
Reference solution (b) Dilute 5.0 m L of the test solution to above the baseline o f the peak due to bacitracin B1 and
100.0 m L with water R. Hv = height above the baseline o f the lowest point o f the
Reference solution (c) Dilute 1.0 m L o f reference solution (b) curve separating this peak from the peak due to
to 10.0 m L with water R. bacitracin B2.
1-234 Bacitracin Zinc 2016
1. bacitracin B1 2. bacitracin B3 3. bacitracin A 4. im purity E

Figure 0466.-2. - Chromatogram of the test for impurity E in bacitracin zinc obtained with reference solution (d) at 300 nm
Limits: I m L o f 0.01 M sodium edetate is equivalent to 0.654 m g o f

— bacitracin A: m inim um 40.0 per cent; Zn.
— sum of bacitracins A , B l, B2 and B3: minimum L o ss o n d ry in g (2.2.32)
70.0 per cent; M axim um 5.0 p er cent, determ ined on 1.000 g by drying at
— disregard limir. the area o f the peak due to bacitracin A in 60 °C over diphosphorus pentoxide R a t a pressure not
the chrom atogram obtained with reference solution (c) exceeding 0.1 kPa for 3 h.
(0.5 per cent); disregard any peak observed in the blank
S te rility (2. 6.1)
run.
If intended for ad m in istration by spraying into internal body
R e la te d p e p tid e s cavities w ithout a further appropriate sterilisation procedure,
Liquid chrom atography (2.2.29) as described in the test for it complies with the test for sterility.
composition.
P y ro g e n s (2.6.8)
See Figure 0466.-1. If intended for administration by spraying into internal body
Limit: cavities w ithout a further appropriate procedure for the
— sum of the areas of all peaks eluting before the peak due to removal o f pyrogens, it complies with the test for pyrogens.
bacitradn B l: maximum 20.0 per cent. Inject per kilogram o f the rabbit's mass 1 m l. o f the
I m p u rity E supernatant obtained by centrifuging a suspension containing
Liquid chrom atography (2.2.29) as described in the test for I I m g per millilitre in a 9 g/L solution o f sodium chloride R.
composition. A SSA Y
See Figure 0466.-2. Suspend 50.0 m g in 5 m l. o f water Rt add 0.5 m l. o f dilute
Detection Spectrophotom eter at 254 nm; spectrophotom eter hydrochloric acid R and dilute to 100.0 m L with water R.
at 300 nm for reference solution (d). Allow the solution to stand for 30 min. C an y o u t the
Injection T est solution and reference solutions (b) and (d). microbiological assay o f antibiotics (2.7.2).
Limit. STO RA G E
— impurity E: not m ore than 1.2 times the area o f the In an airtight container. I f die substance is sterile, store in a
principal peak in the chrom atogram obtained with sterile, airtight, tam per-proof container.
reference solution (b) (6.0 p er cent).
Z inc
4.0 p er cent to 6.0 per cent (dried substance).
Dissolve 0.200 g in a mixture o f 2.5 m L o f dilute acetic add R
and 2.5 m L o f water. A dd 50 m L o f water R, 50 m g of
xylend orange triturate R and sufficient
hexamethylenetetramine R to produce a red colour. Add 2 g o f
hexamethylenetetramme R in excess. T itrate with 0.01 M
sodium edetate until a yellow colour is obtained.
2016 Baclofen 1-235
IM P U R IT IE S dilute mineral ad d s and in dilute solutions of alkali

hydroxides.
Second identification A, C.
(2.2.25).
Test solution Dissolve 70 mg in water R and dilute to
A. X = L-Val, Y = L-Ue, R = H: bacitracin C l, 100.0 m L with the same solvent.
B. X = L-H e, Y = L-Val, R = H: bacitradn C2, Spectral range 220-320 nm.
C. X = Y = L-Val, R = C H 3: bacitradn C3, Absorption maxima At 259 nm, 266 nm and 275 nm.
D . X = Y = L-Val, R = H: badtracin E, Resolution (2.2.25): minimum 1.5 for the absorbance ratio.
Specific absorbance at the absorption maxima:
— at 259 nm : 9.8 to 10.8;
— at 266 nm : 11.5 to 12.7;
— at 275 nm : 8.4 to 9.3.
Preparation Discs prepared using 3 mg of substance and
300 mg o f potassium bromide R.
Comparison baclofen CRS.
E . X = Y = L-He, R = C H 3: badtracin F,
dissolve 0.1 g o f each of the substances separately in 1 m L of
F . X = Y = L -H e, R = H: badtracin H I, dilute sodium hydroxide solution R and add 10 m L o f ethanol
G . X = L-Val, Y = L-Ue, R = C H 3: badtracin H 2, (96 per cent) R and 1 m L of dilute acetic acid R. Allow to
H . X = L-U e, Y = L-Val, R = C H 3: badtracin H 3, stand for 1 h. Filter, wash die predpitate with ethanol
I. X = L-Val, Y = L-U e, R = H: badtracin II , (96 per cent) R and dry in vacuo. Prepare new discs and
record the spectra.
J. X = L-U e, Y = L-Val, R = H: badtracin 12,
K . X = Y = L-Val, R = C H 3: badtracin 13.
__________________________________________________________ PhEur
in the mobile phase and dilute to 10 m L with the mobile
phase.
Reference solution Dissolve 10 m g o f baclofen CRS in the
mobile phase and dilute to 10 m L with the mobile phase.
Baclofen f %
★ .* Plate TLC silica gel G plate R.
(Ph. Eur. monograph 0653) * Mobile phase anhydrous formic acid R, water R, methanol R,
chloroform R, ethyl acetate R (5:5:20:30:40 V/V/V/V/V).
NH2
( H
Application 5 jiL.
Drying Allow the solvents to evaporate.
Cl Detection Spray with ninhydrin solution R3 until the plate is
slightly w e t Place in an oven maintained at 100 °C for
C 10H 12C1N02 213.7 1134-47-0 10 min Examine in daylight.
Action and use Results T h e prindpal spot in the chromatogram obtained with
Skeletal m u sd e relaxant
prindpal spot in the chromatogram obtained with the
Preparations reference solution.
Baclofen Oral Solution
TESTS
Baclofen Tablets
PhEtr__________________________________________________________ T h e solution is n o t m ore opalescent than reference
suspension II (2.2.1) and not m ore intensely coloured than
D E F IN IT IO N
reference solution BY5 (2.2.2, Method II).
(3&S)-4-Amino-3-(4-chlorophenyl)butanoic ad d .
Dissolve 0.50 g in 1 M sodium hydroxide and dilute to 25 m L
Content with the same solvent.
98.0 p er cent to 101.0 per cent (anhydrous substance).
CHARACTERS Liquid chromatography (2.2.29).
Appearance Test solution Dissolve 25.0 mg o f the substance to be
W hite or almost white powder. examined in the mobile phase and dilute to 10.0 m L with
S o lu b ility the mobile phase.
(96 per cent), practically insoluble in acetone. It dissolves in
1-236 Bambuterol Hydrochloride 2016
Reference sohaion (a) Dissolve 25.0 m g of baclofen

V -n h 2
impurity A CRS in the mobile phase and dilute to 10.0 m L
< H
with the mobile phase. CO 2 H and enantiomer
to 100.0 m L with the mobile phase. Cl Xj
Reference sdution (c) D ilute 2.0 m L o f the test solution to
100.0 m L with the mobile phase. B. (3ÆS)-5-amino-3-(4-chlorophenyi)-5-oxopentanoic ad d .
Reference solution (d) D ilute 2.0 m L o f the test solution and ___________________________________________________________ PhEur
2.0 m L of reference solution (a) to 100.0 m L with the
mobile phase.
Column:
— size. I = 0.25 m , 0 = 4.0 mm ;
★ ★
— stationary phase: octadecylsUyl silica gd for chromatography R Bambuterol Hydrochloride ★ ★
(10 nm). *****
(Ph. Eux. monograph 1293)
Mobile phase Dissolve 1.822 g o f sodium hexanesulfonaie R in
1 L o f a mixture o f 560 volumes o f water R, 440 volumes of |3 H OH
methanol R and 5 volumes o f glacial acetic acid R.
O ly l h3° ch3 HCI
Iiyection 20 (iL o f the test solution and reference
H,C y
O and enantiomer
Run time 5 times the retention time o f baclofen.
C18H30CIN3O5 403.9 81732-46-9
baclofen and im purity A A c tio n a n d u se
Limits: Beta2-adrenoceptor agonist; bronchodilator.
— impurity A: not m ore than th e area o f the principal peak
in the chrom atogram obtained with reference solution (b) PhEur________________________________________
(1.0 per cent); D E F IN IT IO N
— total: not m ore than the area of the principal peak in the 5 - [(1RS) -2- [(1 j 1-Dimethylethyl) amino] -1 -hydroxyethyl] -1,3-
chrom atogram obtained w ith reference solution (c) phenylene bis(dimethylcarbamate) hydrochloride.
(2.0 per cent).
C o n te n t
W a te r (2.5.12) 98.5 p er cent to 101.5 per cent (anhydrous substance).
CHARACTERS
A p p e a ra n c e
W hite o r almost white, crystalline powder.
A SSA Y
S o lu b ility
Dissolve 0.1500 g in 50 m L o f anhydrous acetic add R. F red y soluble in water, soluble in ethanol (96 p er cent).
T itrate with 0.1 M perchloric add, determining the end-point
1 m L o f 0.1 M perchloric acid is equivalent to 21.37 mg ID E N T IF IC A T IO N
o f C 10H 12CIN O 2. A. Infrared absorption spectrophotom etry (2.2.24).
IM P U R IT IE S
Preparation Discs.
Specified impurities A Comparison bambuterol hydrochloride CRS.
Other detectable impurities (the following substances would, if If the spectra obtained show differences, dissolve the
present at a sufficient level, be detected by one or other of substance to be examined and the reference substance
the tests in the m onograph. T hey are limited by the general separatdy in a m ixture o f 1 volume o f water R and 6 volumes
acceptance criterion for other/unspecified impurities and/or o f acetone R, cool in ice to p redpitate and dry both
by th e general m onograph Substances for pharmaceutical use predpitates in vacuo at 50 °C to constant w tig h t Record new
(2034). It is therefore n o t necessary to identify these spectra using the residues.
impurities for dem onstration o f compliance. See also 5.10. B. It gives reaction (a) o f chlorides (2.3.1).
Control of impurities in substances for pharmaceutical use): B. TESTS
S o lu tio n S
and enantiomer
H
T o 10 m L o f solution S add 0.2 m L o f methyl red sdution R
a and 0.2 m L o f 0.01 M hydrochloric acid. T h e solution is red.
A dd 0.4 m L o f 0.01 M sodium hydroxide. T h e solution is
A. (4RS)-4-(4-chlorophenyl)pyrrolidin-2-one, yellow.
O p tic a l r o ta tio n (2.2.7)
-0 .1 0 ° t o + 0.10°.
2016 Barbital 1-237
H OH
Dilute 1 m L o f solution S to 10 m L with carbon dioxide-free
water R.
and enantiomer
Test solution Dissolve 5.0 mg of the substance to be examined
phase. A. R l = N H -C (C H 3)3, R2 = R3 = H: (li?S )-l-(3,5-
dihydroxyphenyl)-2-[(l,l-dimethylethyl)amino]ethanol
Reference solution (a) Dissolve 1.0 mg of formoterol jumarate
(terbutaline),
dihydrate CRS in die mobile phase and dilute to 10.0 m L
with the mobile phase. Mix 0.8 m L o f this solution with B. R l = O H , R2 = R3 = C O -N (C H 3)2: 5 -[(li?S )-l,2-
0.4 m L of the test solution and dilute to 100.0 m L with the dihydroxyethyl] -1,3-phenylene bis (dimethylcarb amate),
mobile phase. C . R l = N H -C (C H 3)3, R2 = H , R3 = C O -N (C H 3)2:
Reference solution (b) Dilute 1.0 m L of the test solution to 3- [(lftS )-2 -[(1,1 -dimethylethyl) amino] -1 -hydroxyethyl] -5-
50.0 m L with the mobile phase. Dilute 2.0 m L of this hydroxyphenyl dimethylcarbamate,
solution to 20.0 m L with the mobile phase. D . R l = H , R2 = R3 = C O -N (C H 3)2: 5-[(lRS)-l-
Column: hydroxyethyl]-l,3-phenylene bis(dimethylcarbamate),
— size: I = 0.15 m , 0 = 4.6 mm;
— stationary phase, base-deactivated octadecylsHyl silica gelfor ch3 o
Mobile phase Dissolve 1.3 g of sodium octanesulfonate R in
430 m L of a mixture of 25 volumes of acetonitrile R l and
75 volumes o f methanol R; then mix this solution with O
H3C" Y
570 m L of 0.050 M phosphate buffer p H 3.0 prepared as
follows: dissolve 6.90 g of sodium dütydrogen phosphate
monohydrate R in water R and dilute to 1000 m L with
E. R = H : 5-acetyl-l,3-phenylene bis(dimethylcarbamate),
water R, adjust to p H 3.0 with a 50 g/L solution of dilute
phosphoric acid R. F . R = N H -C (C H 3)3: 5- [ [( 1,1 -dimethylethyl)amino] acetyl] -
1,3-phenylene bis (dimethylcarbamate).
PhEur
Injection 20 |iL; inject the mobile phase as a blank.
Run time 1.5 times the retention time o f bambuterol.
Retention time Form oterol = about 7 min; ★ ★
bambuterol = about 9 min. If necessary, adjust the Barbital ★ ★
composition o f the mobile phase; increase the content o f *****
(Ph. Ever, monograph 0170)
phosphate buffer to increase the retention time.
bambuterol and formoterol.
Limits:
— impurities A , B, C, D, E, F: for each impurity, n o t more
than the area o f the principal peak in the chromatogram
obtained w ith reference solution (b) (0.2 per cent); C8Hi2N 20 3 184.2 57-44-3
in the chromatogram obtained with reference solution (b) A c tio n a n d u se
(0.6 per cent); Barbiturate.
PhEur____________
(0.05 per cent); disregard any peak due to the mobile D E F IN IT IO N
phase. Barbital contains not less than 99.0 per cent and not m ore
W a te r (2.5.12) than the equivalent of 101.0 per cent of
M aximum 0.5 p er cent, determined on 0.500 g. 5,5-diethyipyrimidine-2,4,6(lH,3H,5.H)-trione, calculated
with reference to die dried substance.
M aximum 0.1 p er cent, determined on 1.0 g. CHARACTERS
A white or alm ost white, crystalline powder or colourless
A SSAY
crystals, slightly soluble in water, soluble in boiling water and
Dissolve 0.320 g in 50 m L o f ethand (96 per cent) R and add
in alcohol. It forms water-soluble compounds with alkali
5 m L of 0.01 M hydrochloric acid. C an y out a potentiometric
hydroxides and carbonates and w ith ammonia.
titration (2.2.20), using 0.1 M sodium hydroxide. Read
the volume added between the 2 points o f inflexion. ID E N T IF IC A T IO N
1 m L of 0.1 M sodium hydroxide is equivalent to 40.39 mg of First identification A , B
C 13H 30CIN 3O 5. Second identification A, C, D.
IM P U R IT IE S A. Determ ine the melting point (2.2.14) of the substance to
be examined. M ix equal parts o f the substance to be
Specified impurities A, B, C, D , E, F.
examined and barbital CRS and determine the melting point
1-238 Barium Sulfate 2016
o f the mixture. T he difference between the melting points in pyridine R. T itrate with 0.1 M ethanolic sodium hydroxide
(which are about 190 °C) is n o t greater than 2 °C. until a pure blue colour is obtained. Carry o u t a blank
B. Examine by infrared absorption spectrophotometry titration.
(2.2.24), comparing with the spectrum obtained with 1 m L o f 0.1 M ethanolic sodium hydroxide is equivalent to
barbital CRS. 9.21 m g o f C8H 12N 20 3.
C. Examine by thin-layer chrom atography (2.2.27), using PhEtr
silica gel GF254 R as the coating substance.
in alcohol R and dilute to 25 m L with the same solvent.
Reference solution Dissolve 75 m g o f barbital CRS in alcohol R Barium Sulfate ★ ★
★ ★
(Ph Eiar monograph 0010) *****
Apply separately to the plate 10 |iL of each solution. Develop
over a path o f 18 cm using the lower layer o f a mixture o f BaS0 4 233.4 7727-43-7
5 volumes of concentrated ammonia R, 15 volumes o f alcohol R A c tio n a n d u se
and 80 volumes o f chloroform R. Examine immediately in Radio-opaque substance used in the investigation o f the
ultraviolet light at 254 nm. T h e principal spot in the gastro-intestinal tra c t
chrom atogram obtained with the test solution is similar in
position and size to the principal spot in the chromatogram P re p a r a tio n
obtained with the reference solution. Barium Sulfate for Suspension
D. It gives the reaction o f non-nitrogen substituted PhEur__________________________________________________________

barbiturates (2.3.1). CHARACTERS
TESTS A p p e a ra n c e
A p p e a ra n c e o f so lu tio n Fine, white or alm ost white powder, free from gritty particles.
Dissolve 1.0 g in a mixture o f 4 m L o f dilute sodium hydroxide S o lu b ility
solution R and 6 m L o f water R. T he solution is clear (2.2.1) Practically insoluble in w ater and in organic solvents. It is
and not m ore intensely coloured than reference solution Y 6 very slightly soluble in ad d s and in solutions o f alkali
(2.2.2, Method II). hydroxides.
A cid ity ID E N T IF IC A T IO N
Boil 1.0 g with 50 m L o f water R for 2 min, allow to cool A. Boil a suspension o f 0.2 g with 5 m L o f a 500 g/L
and filter. T o 10 m L o f the filtrate add 0.15 m L o f methyl red solution of sodium carbonate R for 5 min, add 10 m L of
solution R. T h e solution is orange-yellow. N o t more than water R, filter and addify a part o f the filtrate with dilute
0.1 m L o f 0.1 M sodium hydroxide is required to produce a hydrochloric acid R. T h e solution gives the reactions o f sulfates
pure yellow colour. (2.3.1).
R e la te d s u b s ta n c e s B. W ash the residue collected in the preceding test with
Examine by thin-layer chromatography (2.2.27), using silica 3 successive small quantities o f water R. T o the residue add
gel GF2 $ 4 R as the coating substance. 5 m L of dilute hydrochloric acid R, filter and add to the filtrate
Test solution Dissolve 1.0 g o f the substance to be examined 0.3 m L of dilute sulfuric add R. A white predpitate is formed
in alcohol R and dilute to 100 m L with the same solvent. th at is insoluble in dilute sodium hydroxide solution R.
Reference solution Dilute 0.5 m l. o f the test solution to TESTS
100 m L with alcohol R. S o lu tio n S
Apply separately to the plate 20 |xL o f each solution. Develop T o 20.0 g add 40 m L o f distilled water R and 60 m L o f dilute
over a path o f 15 cm using the lower layer o f a mixture of acetic add R. Boil for 5 m in, filter and dilute the cooled
5 volumes of concentrated ammonia R, 15 volumes o f alcohol R filtrate to 100 m L with distilled water R.
and 80 volumes o f chloroform R. Examine immediately in A c id ity o r alk a lin ity
ultraviolet light at 254 nm. Spray with diphenylcarbazone H eat 5.0 g w ith 20 m L o f carbon dioxide-free water R on a
mercuric reagent R. Allow the plate to dry in air and spray w ater-bath for 5 min and filter. T o 10 m L o f the filtrate add
with freshly prepared alcoholic potassium hydroxide solution R 0.05 m L o f bromothymol blue solution R l. N o t m ore than
diluted 1 in 5 w ith aldehyde-free alcohol R. H eat at 100 °C to 0.5 m L o f 0.01 M hydrochloric acid or 0.01 M sodium
105 °C for 5 m in and examine immediately. W hen examined hydroxide is required to change the colour o f the indicator.
in ultraviolet light and after spraying, any spot in die
A c id -so lu b le su b sta n c e s
chrom atogram obtained with the test solution, apart from the
Maximum 0.3 per c e n t
p rindpal spot, is not m ore intense than the spot in the
chrom atogram obtained with the reference solution Evaporate 25 m L of solution S to dryness o n a water-bath
(0.5 per cent). and dry to constant mass at 100-105 °C. T h e residue w dghs
a maximum o f 15 mg.
N o t m ore than 0.5 per cent, determined on 1.00 g by drying O x id isa b le s u lfu r c o m p o u n d s
in an oven at 105 °C. Shake 1.0 g with 5 m L of water R for 30 s and filter. T o the
filtrate add 0.1 m L o f starch solution R, dissolve 0.1 g o f
S u lfa te d a s h (2.414)
potassium iodide R in the m ixture, add 1.0 m L o f a freshly
N ot m ore than 0.1 per cent, determined on 1.0 g.
prepared 3.6 m g/L solution o f potassium iodate R and 1 m L
A SSA Y o f 1 M hydrochloric add and shake well. T h e colour o f the
Dissolve 85.0 m g in 5 m L o f pyridine R. A dd 0.5 m L o f solution is m ore intense than th at o f a standard prepared at
thymolphthalein solution R and 10 m L o f silver nitrate solution the same tim e and in the same m anner, b u t omitting the
potassium iodate.
2016 Beclometasone Dipropionate 1-239
S o lu b le b a riu m salts ASSAY

M axim um 10 ppm. T o a quantity containing 0.6 g o f Barium Sulfate in a
T o 2.5 m L of a 0.2 mg/L solution o f barium nitrate R in a platinum dish add 5 g of sodium carbonate and 5 g o f
mixture o f 30 volumes of ethanol (96 per cent) R and potassium carbonate sesquihydrate and mix. H eat to 1000° and
70 volumes of water R, add 10 m L o f dilute sulfuric arid R. maintain at this temperature for 15 minutes. Allow to cool
Shake and allow to stand for 5 min. T o 1 m L of this solution and suspend the residue in 150 m L of water. W ash the dish
add 10 m L of solution S. Prepare a standard in die same with 2 m L of 6 m acetic acid and add the washings to the
manner using 10 m l. of barium standard solution suspension. Cool in ice and decant the supernatant liquid,
(2 ppm Ba) R instead of solution S. transferring as litde of the solid m atter as possible to the
After 10 min, any opalescence in the test solution is not filter. W ash the residue with successive quantities o f a
2% w/v solution of sodium carbonate until the washings are
more intense than that in the standard.
free from sulfate and discard the washings. Add 5 m L of 2m
H eav y m e ta ls (2.4.8) hydrochloric arid to the filter, wash through into the vessel
M axim um 10 ppm. containing the bulk of the solid m atter with water, add 5 m L
D ilute 10 m L of solution S to 20 m L with water R. 12 m L of of hydrochloric acid and dilute to 100 m L with water.
the solution complies with test A Prepare the reference Add 10 m l. of a 40% w/v solution of ammonium acetate,
solution using lead standard solution (1 ppm Pb) R 25 m L o f a 10% w/v solution o f potassium dichromate and
L oss o n ig n itio n 10 g o f urea. Cover and digest in a hot-air oven at 80° to 85°
M axim um 2.0 p er cent, determined on 1.0 g at for 16 hours. Filter whilst still hot through a sintered-glass
600 ± 50 °C. filter (ISO 4793, porosity grade 4, is suitable), washing the
precipitate initially with a 0.5% w/v solution o f potassium
______________________________________________________________ PhEur
dichromate and finally with 2 m L of water. D ry to constant
weight at 105°. Each g o f the residue is equivalent to
0.9213 g of barium sulfate, B aS04.
Barium Sulfate for Suspension

Barium Sulphate for Suspension
A c tio n a n d use
Anhydrous Beclometasone *****
Radio-opaque preparation used in the investigation of the Dipropionate *****
gastro-intestinal tract. (Ph. Eur. monograph 0654)
P re p a r a tio n
Barium Sulfate Oral Suspension O
D E F IN IT IO N
Barium Sulfate for Suspension is a dry mixture of Barium
Sulfate with a suitable dispersing agent and may contain
suitable flavours and suitable antimicrobial preservatives.
C o n te n t o f b a riu m sulfate, B a S 0 4
90.0 to 110.0% o f the stated amount.
A fine, white or creamy white powder.
ID E N T IF IC A T IO N A ctio n a n d u se
A Ignite 1 g to constant weight. T o 0.2 g of the residue add Glucocorticoid.
5 m L o f a 50% w/v solution o f sodium carbonate and boil for P re p a ra tio n s
5 m in u tes. Add 10 m l. of water and filter. Reserve the
Beclometasone Cream
residue for test B. Acidify a portion of the filtrate with
Beclometasone Aqueous Nasal Spray
2m hydrochloric acid. T he solution yields the reactions
characteristic of sulfates, Appendix VI. Beclometasone Inhalation Powder
B. W ash the residue reserved in test A with water, add 5 m L Beclometasone Inhalation Powder, pre-dispensed
of 2m hydrochloric acid, m ix well and filter. Add 0.3 m L o f Beclometasone O intm ent
1m sulfuric acid to the filtrate. A white precipitate is produced Beclometasone Pressurised Inhalation
which is insoluble in 2m hydrochloric acid.
PhEur______________________________________________________
TESTS
A c id ity o r alkalinity D E F IN IT IO N
p H o f an aqueous suspension containing the equivalent o f 9-Chloro-l 1ß-hydroxy-16ß-methyi-3,20-dioxopregna-l,4-
60% w/w o f Barium Sulfate or, for lower strengths, the diene-17,21-diyl dipropanoate.
aqueous suspension at the strength o f intended use, 3.5 to C o n te n t
8.5, Appendix V L. 96.0 per cent to 102.0 per cent (dried substance).
L oss o n d ry in g CHA RACTERS
W hen dried at 105° for 4 hours, loses not more than 1.0% of A p p e a ra n c e
its weight. Use 1 g. White or almost white, crystalline powder.
1-240 Beclometasone Dipropionate 2016
S o lu b ility Identification of impurities Use the chrom atogram supplied

Practically insoluble in water, freely soluble in acetone, w ith beclometasone dipropionate for peak identification CRS and
sparingly soluble in ethanol (96 per cent). the chromatogram obtained w ith reference solution (c) to
ID E N T IF IC A T IO N identify the peaks due to impurities A, B, C , F , L, M and N;
use the chrom atogram supplied with beclometasone
dipropionate for system suitability CRS and the chromatogram
Comparison anhydrous beclometasone dipropionate CRS. obtained with reference solution (b) to identify the peak due
B. T reat 25 m g by the oxygen-flask m ethod (2.5.10). U se a to impurity D.
mixture of 1 m L of 1 M sodium hydroxide and 20 m L o f Relative retention W ith reference to beclometasone
water R to absorb the combustion products. T h e solution dipropionate (retention time = about 25 min):
gives reaction (a) o f chlorides (2.3.1). im purity A = about 0.3; im purity B = about 0.6;
C. Loss on drying (see Tests). im purity D = about 1.1; im purity M = about 1.2;
TESTS impurity L = about 1.3; im purity C = about 1.8;
S pecific o p tic a l ro ta tio n (2.2.7) impurity N = about 2.0; im purity F = about 2.2.
+ 108 to + 115 (dried substance). System suitability: reference solution (b):
— peak-to-vaUey ratio: m in im u m 1.5, where Hp = height
above th e baseline o f the peak due to impurity D and
R e la te d su b s ta n c e s curve separating this peak from the peak due to
Liquid chromatography (2.2.29). beclometasone dipropionate.
Solvent mixture M obile phase A, mobile phase B (45:55 V!V). Limits:
Test solution (a) Dissolve 50.0 m g o f the substance to be — correction factors: for the calculation o f content, multiply
examined in 28 m L of mobile phase B and dilute to 50.0 m L the peak areas o f the following impurities by the
with mobile phase A. corresponding correction fa c to r im purity F = 1.3;
Test solution (b) Dilute 1.0 m L of test solution (a) to im purity M = 2.0;
50.0 m L with the solvent mixture. — impurity L: n o t m ore than 6 times the area o f the
Reference solution (a) Dilute 5.0 m L o f test solution (b) to
100.0 m L with the solvent mixture. reference solution (a) (0.6 p e r cent);
— impurities B, F, M: for each impurity, n o t m ore than
Reference solution (b) Dissolve 5 mg o f beclometasone 5 times the area o f the principal peak in the
dipropionate for system suitability CRS (containing im purity D) chrom atogram obtained w ith reference solution (a)
in 3 m L o f mobile phase B and dilute to 5 m L with mobile
(0.5 per cent);
phase A.
— impurities A, D, N: for each impurity, n o t m ore than twice
Reference solution (c) Dissolve 5 mg o f beclometasone the area of the principal peak in the chrom atogram
dipropionate for peak identification CRS (containing impurities obtained with reference solution (a) (0.2 p er cent);
A, B, C , L and M ) in 3 m L o f mobile phase B and dilute to — impurity C: n o t more than 1.5 times the area o f the
5 m L with mobile phase A. U se 1 m L o f this solution to principal peak in the chrom atogram obtained with
dissolve the contents o f a vial o f beclometasone dipropionate reference solution (a) (0.15 p er cent);
impurities F and N CRS. — unspecified impurities: for each im purity, n o t m ore than die
Reference solution (d) Dissolve 50.0 m g o f anhydrous area o f the principal peak in the chrom atogram obtained
beclometasone dipropionate CRS in 28 m L o f mobile phase B with reference solution (a) (0.10 per cent);
and dilute to 50.0 m L with mobile phase A. Dilute 1.0 m L — total: n o t more than 15 times the area o f the principal
of this solution to 50.0 m L with the solvent mixture. peak in the chrom atogram obtained with reference
Column: solution (a) (1.5 per cent);
— size: I = 0.25 m , 0 = 4.6 mm; — disregard limit. 0.5 times die area o f the principal peak in
— stationary phase: spherical difunctional bonded end-capped the chromatogram obtained w ith reference solution (a)
octadecylsHyl silica gelfor chromatography R (5 pm); (0.05 per cent).
— temperature: 50 °C. L o ss o n d ry in g (2.2.32)
Mobile phase: M axim um 0.5 per cent, determ ined on 1.000 g by drying in
— mobile phase A: 2.72 g/L solution o f potassium dihydrogen an oven at 105 °C for 3 h.
phosphate R adjusted to p H 2.35 w ith phosphoric add R;
A SSA Y
— mobile phase B: tetrahydrofuran R, acetonitrile R, methanol R
(5:23:25 V/V/V);
Irgection T est solution (b) and reference solution (d).
Time Mobile phue A Mobile phase B
(min)
Calculate die percentage content o f C 28H 37CIO 7 from the
(per cent V/V) (per cent V/V)
declared content o f anhydrous beclometasone dipropionate CRS.
0 -4 40 60
4 -1 2 4 0 -»45
IM P U R IT IE S
60-» 55
Spedfied impurities A, B, C , D , F , L , M , N
12-59 45 55
Flow rate 1.4 mlVmin. the tests in the m onograph. T h ey are limited by the general
by die general m onograph Substances for pharmaceutical use
Injection 20 jil o f test solution (a) and reference solutions (a), (2034). It is therefore n o t necessary to identify these
(b) and (c).
2016 Beclometasone Dipropionate 1-241

Control of impurities in substances for pharmaceutical use): E, H}
I, J> O, Q, R, S} U, V.
J. R1 = R2 = CO -C 2H 5: 9,lip-epoxy-16p-m ethyl-3,20-
dioxo-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-methyl-
9P-pregna-l,4-diene-3,20-dione,
U . R1 = C O -C 2H 5, R2 = H: 9,HP-epoxy-21-hydroxy-16P-
methyl-3,20-dioxo-9p-pregna-l,4-dien-17-yl propanoate,
A. R1 = R3 = H , R2 = Cl, R4 = C O -Q H g: 9-chloro-
V. R 1 = H , R2 = CO-C 2H 5: 9,llp-epoxy-17-hydroxy-16P-
lip,17-dihydroxy-16p-m ethyl-3,20-dioxopregna-l,4-dien-21-
methyl-3,20-dioxo-9P-pregna-l,4-dien-2 1-yl propanoate,
yl propanoate (beclometasone 21-propionate),
B. R1 = H , R2 = Cl, R3 = CO-C2H 5, R4 = C O -C H 3: o
2 l-(acetyloxy)-9-chloro-l 1P-hydroxy-16P-methyi-3,20-
dioxopregna-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 2-
C 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 - L. 9-chloro-l 1P-hydroxy-16P-methyl-3,20-dioxopregn-4-ene-
hydroxy-16 P-methyl-3^0-dioxopregna-1,4-diene-17,21 -diyl 17,21-diyl dipropanoate,
dipropanoate,
o
F . R1 = Br, R2 = Cl, R3 = R4 = C O -Q H g: 6a-bromo-9-
chloro-11P-hydroxy-16P-methyl-3,20-dioxopregna-l,4-diene-
17,21-diyl dipropanoate,
M . 9-chloro-l 1P-hydroxy-16P-methyl-3,20-dioxopregna-4,6-
diene-17,21-diyl dipropanoate,
o
E. R1 = Cl, R2 = CO -C 2H 5: 6a,9-dichloro-lip-hydroxy-
16p-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-methyl-
3,20-dioxopregna-l,4-dien-17-yl propanoate (beclometasone
17-propionate),
N . 2-bromo-9-chloro-l 1P-hydroxy-16P-methyl-3,20-
dioxopregna-1,4-diene-17,21-diyl dipropanoate,
O
O
0 ^ y ° 0 'w-CH,
ChT H T \-C H 3
I. 16P-methyi-3,20-diQxopregna-l,4,9(l l)-triene-17,21-diyl
O. R1 = R2 = Cl: 9,liP-dichloro-16P-methyl-3,20-
dipropanoate,
dioxopregna-l,4-diene-17,21-diyl dipropanoate,
Q. R1 = R2 = H : 16P-methyl-3,20-dioxopregna-l,4-diene-
S. R1 = O -C O -C 2H 5, R2 = Cl: 9-chloro-l6p-m ethyl-3,20-
dioxopregna-l,4-diene-l ip,17,21-triyl tripropanoate
(beclometasone tripropionate).
__________________________________________________________ PhEur
1-242 Beclometasone Dipropionate Monohydrate 2016
Reference solution (c) Dissolve 5 m g o f beclometasone

Beclometasone Dipropionate ***** dipropionate for peak identification CRS (containing
Monohydrate ***** impurities B, C and L) in 3 m L o f mobile phase B and dilute
(Ph. Eur. monograph 1709) 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
O impurities F and N CRS.
o fV lJ H i Reference solution (d) Dissolve 50.0 m g o f anhydrous
beclometasone dipropionate CRS in 28 m L of mobile phase B
and dilute to 50.0 m L with mobile phase A. D ilute 1.0 m L
h° X ' s 5 v ° ' ^ CHi ■ o f this solution to 50.0 m L w ith the solvent mixture.
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 — temperature: 50 °C.
Mobile phase:
Action and use
— mobile phase A: 2.72 g/L solution o f potassium dihydrogen
Glucocorticoid.
phosphate R adjusted to p H 2.35 w ith phosphoric add R',
Preparations — mobile phase B: tetrahydrofuran R, acetomtrile R, methanol R
Beclometasone Aqueous Nasal Spray (5:23:25 VIVIV)\
Beclometasone Inhalation Powder
Beclometasone Inhalation Powder, pre-dispensed
PhEur___________________________________________________________ 0 -4 40 60
D E F IN IT IO N 4 -1 2 40-» 45 60 55
9-C hloro-l 1{5-hydroxy-16 P-methyi-3,20-dioxopregna-1,4- 12 -5 9 45 55
diene-17,21-diyl dipropanoate monohydrate.
Content Flow rate 1.4 mL/min.
97.0 per cent to 102.0 p er cent (dried substance). Detection Spectrophotom eter at 254 nm .
CHARACTERS Injection 20 pi o f test solution (a) and reference solutions (a),
Appearance (b) and (c).
W hite or alm ost w hite powder. Identification of impurities U se the chrom atogram supplied
Solubility with beclometasone dipropionate for peak identification CRS and
Practically insoluble in water, freely soluble in acetone, the chrom atogram obtained with reference solution (c) to
sparingly soluble in ethanol (96 per cent). identify the peaks due to impurities B, C , F and L; use the
chrom atogram supplied with beclometasone dipropionate for
ID E N T IF IC A T IO N system suitability CRS and the chrom atogram obtained with
A. Infrared absorption spectrophotom etry (2.2.24). reference solution (b) to identify the peak due to impurity D .
Comparison beclometasone dipropionate monohydrate CRS. Relative retention W ith reference to beclometasone
B. T reat 25 m g by th e oxygen-flask m ethod (2.5.10). U se a dipropionate (retention time = about 25 min):
mixture of 1 m L o f 1 M sodium hydroxide and 20 m L o f im purity B = about 0.6; im purity D = about 1.1;
water R to absorb the com bustion products. T h e solution impurity L = about 1.3; im purity C = about 1.8;
gives reaction (a) o f chlorides (2.3.1). im purity F = about 2.2.
C. Loss on drying (see Tests). System suitability: reference solution (b):
— peak-to-vaHey ratio: m inim um 1.5, where Hp = height
TESTS
above the baseline o f the peak due to im purity D and
Specific optical rotation (2.2.7) Hv — height above the baseline o f the lowest point o f the
Dissolve 0.100 g in ethanol (96 per cent) R and dilute to beclom etasone dipropionate.
10.0 m L with the sam e solvent. Limits:
Related substances — correction factor, for the calculation o f content, multiply the
Liquid chrom atography (2.2.29). peak area o f im purity F by 1.3;
Solvent mixture M obile phase A, mobile phase B (45:55 VIV). — impurity B: n o t m ore than 5 times the area o f the
Test solution (a) Dissolve 50.0 m g o f the substance to be principal peak in the chrom atogram obtained with
examined in 28 m L o f mobile phase B and dilute to 50.0 m L reference solution (a) (0.5 per cent);
with mobile phase A. — impurities C, F, L: for each impurity, not m ore than
1 .5 times the area o f the principal peak in the
Test solution (b) D ilute 1.0 m L o f test solution (a) to
chrom atogram obtained w ith reference solution (a)
(0.15 p er cent);
Reference solution (a) D ilute 5.0 m L o f test solution (b) to — unspecified impurities: for each impurity, n o t more than the
100.0 m L with the solvent mixture. area of the principal peak in the chrom atogram obtained
Reference solution (b) Dissolve 5 m g o f beclometasone w ith reference solution (a) (0.10 per cent);
dipropionate for system suitability CRS (containing im purity D ) — total: n o t m ore than 10 times the area of the principal
in 3 m L o f mobile phase B and dilute to 5 m L with mobile peak in die chrom atogram obtained with reference
phase A. solution (a) (1.0 per cent);
2016 Beclometasone Dipropionate Monohydrate 1-243

(0.05 per cent).
A SSAY
related substances with the following modification. I. 16P-methyl-3,20-dioxopregna-l,4,9(l l)-trien e-17,21-diyl
dipropanoate,
Injection T est solution (b) and reference solution (d).
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
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,
(2034). I t is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10. R. R I = R2 = H : 9,lip-epoxy-17,21-dihydroxy-16P-m ethyl-
Control of impurities in substances for pharmaceutical use): A, D, 9 P-pregna-l,4-diene-3,20-dione,
E, H, I, J, M, N, O, Q, R, S, U, V. U . R I = CO -C 2H 5, R 2 = H: 9,llp-epoxy-21-hydroxy-16P-
methyl-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-16P-
methyl-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-chloro-

1 1P, 17 -dihydroxy-16 P-methyl-3 ,2 0-dioxopregna-1,4-dien-2 1-
D. R I = H , R2 = Br, R3 = R4 = CO -C 2H 5: 9 -b ro m o -llp -
L. 9-chloro-liP-hydroxy-16P-methyl-3,20-dioxopregn-4-ene-
hydroxy-16 P-methyl-3,20-dioxopregna-1,4-diene-17,21 -diyl
dipropanoate,
E. R I = R2 = Cl, R3 = R4 = C O -C 2H 5: 6a,9-dichloro-
11 P-hydroxy-16P-methyI-3,20-dioxopregna-l ,4-diene-l 7,2 1-
diyl dipropanoate,
H . R I = R4 = H , R2 = Cl, R3 = C O -C 2H 5: 9-chloro-
11 P,21-dihydroxy-16P-methyl-3,20-dioxopregna- 1,4-dien-l 7-
-R 2
M . 9-chloro-l 1P-hydroxy-16P-methyl-3,20-dioxopregna-4,6-
diene-17 ,2 1-diyl dipropanoate,
H R1
B. R I = H , R2 = C O C H 3: 21-(acetyloxy)-9-chloro-lip-
hydroxy-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)-pregna-
l,4-dien-21-yl butanoate (beclometasone 21-butyrate N . R I = Br, R2 = O H , R3 = Cl: 2-brom o-9-chloro-l 1P-
17-propionate), hydroxy-16P-methyl-3,20-dioxopregna-l,4-diene-l 7,2 1-diyl
F. R I = Br, R2 = CO-C 2H 5: 6a-brom o-9-chloro-lip- dipropanoate,
hydroxy-16 P-methyl-3,20-dioxopregna- 1,4-diene-17,21 -diyl O. R I = H , R2 = R3 = Cl: 9,11 P-dichloro-16P-methyl-
dipropanoate, 3,20-dioxopregna-l,4-diene-17,21-diyl dipropanoate,
1-244 Beeswax 2016
Q. R l = R2 = R3 = H : 16P-methyl-3,20-dioxopregna-l,4- of ethanol (96 per cent) R and xylene R and a few glass beads.
diene-17,21-diyl dipropanoate, H eat until the substance is dissolved. Add 25.0 m L o f 0.5 M
S. R l = H , R2 = 0 - C 0 - C 2H 5, R3 = Cl: 9-chloro-16p- alcoholic potassium hydroxide and heat under a reflux
methyl-3,20-dioxopregna-l ,4 -d ien e-lip ,1 7 321-triyl condenser for 3 h. Titrate the hot solution immediately with
tripropanoate (bedom etasone tripropionate). 0.5 M hydrochloric acid, using 1 m L o f phenolphthalein
solution R l as indicator (nx mL). Reheat the solution to
__________________________________________________________ PhEur
boiling several times during the course o f the titration. Carry
out a blank test (n2 mL).
.* * * ★ 28.05 (na — m )
★ Saponification value =
White Beeswax ★ ★
★+ ★
Ceresin, paraffins and certain other waxes
Action and use T o 3.0 g, in a 100 m L round-bottom ed flask, add 30 m L of
Excipient 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.
PhEur_______________________
Remove the condenser and immediately insert a
DEFINITION thermom eter. Place the flask in a water-bath at 80 °C and
Wax obtained by bleaching yellow beeswax. allow to cool, swirling the solution continuously.
N o precipitate is formed until 65 °C, although the solution
CHARACTERS
may be slightly opalescent. Beginning at 65 °C, the solution
Appearance may become cloudy and precipitates may be formed.
White or yellowish-white pieces or platesj translucent when
At 59 °C, the solution is cloudy.
thin, with a fine-grained, m att and non-crystalline fracture;
when w anned in the hand they become soft and malleable. Glycerol and other polyols
M axim um 0.5 per cent m/m, calculated as glycerol.
It has an odour similar to that o f yellow beeswax, though
fainter and never rancid. It is tasteless and does not stick to T o 0.20 g add 10 m L o f alcoholic potassium hydroxide
the teeth. 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
Solubility
filter. Rinse the flask and the filter with dilute sulfuric add R.
Practically insoluble in water, partially soluble in hot ethanol
Combine the filtrate and washings and dilute to 100.0 m L
(90 p er cent V/V) and completely soluble in fatty and
w ith dilute sulfuric add R. Place 1.0 m L of the solution in a
essential oils.
test-tube, add 0.5 m L o f a 10.7 g/L solution o f sodium
Relative density periodate R, mix and allow to stand for 5 min. Add 1.0 m L of
About 0.960. decolorisedfuchsin solution R and mix. Any precipitate
TESTS disappears. Place the tube in a beaker containing water at
Drop point (2.2.17) 40 °C. D uring cooling observe for 10-15 min. Any violet-
61 °C to 66 °C. blue colour in the solution is not more intense than that in a
standard prepared at the same time and in the same m anner
M elt the beeswax by heating on a water-bath, pour onto a
using 1.0 m L of a 10 m g/L solution o f glycerol R in dilute
glass plate and allow to cool to a semi-solid mass. Fill the
sulfuric add R.
metal cup by inserting the wider end into the beeswax and
__________________________________________________________ PhEur
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. *****
Yellow Beeswax ★ ★
Acid value *****
17.0 to 24.0. (Ph. Eur. monograph 0070)
To 2.00 g (m g), in a 250 m L conical flask fitted with a
Action and use
reflux condenser, add 40 m L of xylene R and a few glass
Excipient.
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 PhEur.
solution R l and titrate the hot solution with 0.5 M alcoholic
DEFINITION
potassium hydroxide until a red colour persists for at least 10 s
Wax obtained by melting the walls o f the honeycomb made
(«i mL). Carry out a blank test («2 mL).
by the honey-bee, Apis meUifera L., with hot water and
removing foreign matter.
Add value = 28-°^ (ni ~ ni)
tn CHARACTERS
Appearance
Ester value (2.5.2) Yellow or light brown pieces or plates with a fine-grained,
70 to 80. m att and non-crystalline fracture; when w anned in the hand
they become soft and malleable.
Saponification value
87 to 104. It has a faint odour, characteristic of honey. It is tasteless and
does n o t stick to the teeth.
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
2016 Benazepril Hydrochloride 1-245
S olubility filter. Rinse the flask and the filter with dilute sulfuric acid R.
Practically insoluble in water, partially soluble in h o t ethanol Combine the filtrate and washings and dilute to 100.0 m L
(90 per cent V/V) and completely soluble in fatty and with dilute sulfuric add R. Place 1.0 m l. o f the solution in a
essential oils. test-tube, add 0.5 m L of a 10.7 g/L solution of sodium
R elativ e d e n sity periodate R, mix and allow to stand for 5 min. Add 1.0 m L of
A bout 0.960. decolorisedfuchsm solution R and mix. Any precipitate
disappears. Place the tube in a beaker containing water at
TESTS 40 °C. D uring cooling observe for 10-15 min. Any violet-
D ro p p o in t (2.2.17) blue colour in the solution is not more intense than that in a
61 °C to 66 °C. standard prepared at the same time and in the same m anner
M elt the beeswax by heating on a water-bath, p our onto a using 1.0 m L of a 10 mg/L solution o f glycerol R in dilute
glass plate and allow to cool to a semi-solid mass. Fill the sulfuric add R.
metal cup by inserting the wider end into the beeswax and __________________________________________________________ PhEur
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. Benazepril Hydrochloride ****%
A cid value *. *
17.0 to 22.0.
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).
C24H 29CIN 2O 5 461.0 86541-74-4
A #J , 28.05 ( n i - n 2)
A cid value = ---------------------
m A c tio n a n d u se
Angiotensin converting enzyme inhibitor.
E s te r v alu e (2.5.2)
PhEir__________________________________________________________
70 to 80.
S ap o n ific a tio n v alu e D E F IN IT IO N
87 to 102. [(3S)-3-[ [( 1S )-1-(Ethoxycarbonyl)-3-phenylpropyl] amino] - 2 -
T o 2.00 g (m g), in a 250 m L conical flask fitted with a oxo-2}3,4,5-tetrahydro- 1/f-l-benzazepin- 1-yl] acetic a d d
reflux condenser, add 30 m L of a mixture o f equal volumes hydrochloride.
o f ethanol (96 per cent) R and xylene R and a few glass beads. C o n te n t
H eat until the substance is dissolved. Add 25.0 m L o f 0.5 M 97.5 per cent to 102.0 per cent (dried substance).
alcoholic potassium hydroxide and heat under a reflux CHARACTERS
condenser for 3 h. T itrate the hot solution immediately with
A p p e a ra n c e
0.5 M hydrochloric acid, using 1 m L o f phenolphthalein W hite or almost white, crystalline powder, hygroscopic.
solution R1 as indicator («j mL). Reheat the solution to
boiling several times during the course o f the titration. Carry S o lu b ility
out a blank test («2 mL)- Slightly soluble in water, freely soluble in anhydrous ethanol,
very slightly soluble in ethyl acetate, practically insoluble in
cyclohexane.
S ap o n ificatio n value = It shows polymorphism (5.9).
m
C e re sin , p a ra ffin s a n d c e rta in o th e r w axes Carry out either tests A, B, D or tests B, C, D.
T o 3.0 g, in a 100 m L round-bottom ed flask, add 30 m L of A. Specific optical rotation (2.2.7): —141 to -1 3 6 (dried
a 40 g/L solution of potassium hydroxide R in aldehyde-free substance).
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. B. Infrared absorption spectrophotometry (2.2.24).
N o precipitate is formed until 65 °C, although the solution Comparison benazepril hydrochloride CRS.
may be slightly opalescent. Beginning at 65 °C, the solution If the spectra obtained in the solid state show differences,
may become cloudy and precipitates may be formed. dissolve the substance to be examined and the reference
At 59 °C, the solution is cloudy. substance separately in methanol R, evaporate to dryness and
G lycerol a n d o th e r polyols record new spectra using the residues.
M aximum 0.5 per cent m/m, calculated as glycerol. C. Enantiomeric purity (see Tests).
T o 0.20 g add 10 m L o f alcoholic potassium hydroxide D . It gives reaction (a) o f chlorides (2.3.1).
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
1-246 Benazepril Hydrochloride 2016
TESTS — disregard limit. 0.25 times the area of the principal peak in
Related substances the chromatogram obtained with reference solution (c)
Liquid chromatography (2.2.29). (0.05 per cent).
Test solution (a) Dissolve 50.0 mg o f the substance to be E n a n tio m e ric p u rity
examined in the mobile phase and dilute to 50.0 m L with Liquid chromatography (2.2.29).
the mobile phase. Buffer solution pH 60 Dissolve 3.58 g o f disodium hydrogen
Test solution (b) Dilute 10.0 m L o f test solution (a) to phosphate R and 9.66 g o f potassium dihydrogen phosphate R in
100.0 m L with the mobile phase. water R and dilute to 1000.0 m L with the same solvent
Reference solution (a) Dissolve 50.0 mg o f benazepril Test solution Dissolve 50.0 mg o f the substance to be
hydrochloride CRS in the mobile phase and dilute to 50.0 m L examined in the mobile phase and dilute to 50.0 m L with
with the mobile phase. Dilute 10.0 m L of this solution to the mobile phase.
100.0 m L with the mobile phase. Reference solution (a) Dissolve 5.0 m g o f benazepril
Reference solution (b) Dissolve the contents of a vial of impurity A CRS in the mobile phase and dilute to 50.0 m L
benazeprilfor system suitability CRS (containing impurities B, with the mobile phase.
C, D , E, F and G) in 1.0 m L of test solution (a). 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
— size: I = 0.30 m, 0 = 3.9 mm ; solution to 10.0 m L with the test solution.
— stationary phase: end-capped octadecylsüyl silica gelfor Column:
chromatography R (10 pm). — size: I = 0.10 m , 0 = 4.0 mm;
Mobile phase Add 0.2 m L of glacial acetic acid R to 1000 m L — stationary phase: spherical silica gel AGP for chiral
o f a mixture o f 360 volumes o f water R and 640 volumes of chromatography R (5 pm);
methanol R2; add 0.81 g o f tetrabutylammonium bromide R and — temperature: 30 °C.
stir to dissolve. Mobile phase methanol R2, buffer solution p H 6.0
Flow rate 1.0 mlVmin. (20:80 V/V).
Detection Spectrophotometer at 240 nm . Flow rate 0.9 mL/min.
Injection 25 pL of test solution (a) and reference solutions (b) Detection Spectrophotom eter at 240 nm .
and (c). Injection 50 pL of the test solution and reference solutions (b)
Run time 3 times the retention time o f benazepril. and (c).
Relative retention W ith reference to benazepril (retention Run time 3.5 times the retention tim e of benazepril.
time = about 6 min): impurity E = about 0.3; Relative retention W ith reference to benazepril (retention
impurity F = about 0.4; impurity C = about 0.5; time — about 6 min): impurity A = about 1.9.
impurity B = about 1.8; impurity D = about 2.0; System suitability: reference solution (c):
impurity G = about 2.5. — peak-to-valley ratio: minimum 2.5, where Hp = height
Identification of impurities Use the chromatogram supplied above the baseline o f the peak due to impurity A and
with benazepril for system suitability CRS and the Hv = height above the baseline o f the lowest point o f the
chrom atogram obtained with reference solution (b) to curve separating this peak from the peak due to
identify the peaks due to impurities B, C, D , E, F and G. benazepril.
System suitability.: reference solution (b): Limit.
— resolution: m in im u m 2.5 between the peaks due to — impurity A: n o t more than the area of the corresponding
benazepril and impurity B and minimum 1.5 between the peak in the chromatogram obtained with reference
peaks due to impurities E and F. solution (b) (0.1 per cent).
Limits: H eav y m e ta ls (2.4.8)
— correction factors: for the calculation o f content, multiply M aximum 20 ppm.
the peak areas of the following impurities by the
corresponding correction factor, impurity E = 0.5;
using 2 mT. o f lead standard solution (10 ppm Pb) R.
im purity F = 0.7;
— impurity B: n o tm o re than 2.5 times the area o f the L oss o n d ry in g (2.2.32)
principal peak in the chromatogram obtained with M aximum 1.5 per cent, determined on 1.000 g by drying
reference solution (c) (0.5 per cent); in vacuo at 105 °C for 3 h.
— impurity C: not m ore than 1.5 times die area of the S u lfa te d a s h (2.4.14)
principal peak in the chromatogram obtained with M aximum 0.1 per cent, determined on 1.0 g.
A SSA Y
— impurities D, E, F, G: for each impurity, not more than
obtained with reference solution (c) (0.2 per cent);
— unspecified impurities: for each impurity, not more than Irqection T est solution (b) and reference solution (a).
0.5 times the area o f the principal peak in the Calculate the percentage content o f C 24H 29CIN 2O 5 from the
chrom atogram obtained with reference solution (c) declared content o f benazepril hydrochloride CRS.
(0.10 per cent);
STORAGE
— total: not m ore than 10 times the area of the principal
Protected from light, in an airtight container.
2016 Bendroflumethiazide 1-247
IM P U R IT IE S
Bendroflumethiazide ★
★
★
★
Specified impurities A, B, C, D , E 3 F 3 G
(Ph. Eitr. monograph 0370) *****
O O O 0
* '/ * //
C02h
H2n
,s s.
‘NH
and enantiomer
f3c A -
H H
A. [(3i?)-3- [ [(If ?)-1-(ethoxycarbonyl)-3-phenylpropyI] amino] - 421.4 73-48-3

2 -o x o -2 ,3 34 35-tetrahydro- 1H- 1-benzazepin - 1-yl] acetic acid,
Thiazide diuretic.
c o 2h Bendroflumethiazide Tablets
and enantiomer
Bendroflumethiazide Oral Suspension
PhEir.
D E F IN IT IO N
B. [(3i?S)-3-[[(1 SR)- 1-(ethoxycarbonyl)-3- (3^5)-3-Benzyl-6-(trifluorom ethyl)-3,4-dihydro-2H-l,2,4-
phenylpropyl]am ino]-2-oxo-2,3j4j5-tetrahydro-lii-l- benzothiadiazine-7-sulfonamide 1, 1-dioxide.
benzazepin-l-yl] acetic acid.
C o n te n t
98.0 p er cent to 102.0 per cent (dried substance).
o
H H || CHARACTERS
A p p e a ra n c e
W hite o r almost white, crystalline powder.
S o lu b ility
C. R = H: (2S)-2-[[(3<S)-l-(carboxymethyl)-2-oxo-2,3,4,5-
tetrahydro- IH -l -benzazepin-3-yI] amino] -4-phenyIbutanoic ID E N T IF IC A T IO N
acid, Infrared absorption spectrophotometry (2.2.24).
G. R = C 2H 5: ethyl (25)-2-[[(35)-l-(2-ethoxy-2-oxoethyl)-2- Comparison bendroflumethiazide CRS.
oxo-2,3,4 j5-tetrahydro- IH- l-benzazepin-3-yl] amino] -4- TESTS
phenylbutanoate, R e la te d su b sta n c e s
Liquid chrom atography (2.2.29). Prepare the solutions
Solvent mixture M ix 40 volumes of methanol R and
CO,H
60 volumes of a 2.0 gfL solution o f citric acid R.
examined in the solvent mixture and dilute to 50.0 m L w ith
D . [(35)-3-[[(15)-3-cyclohexyl-1- Reference solution (a) Dissolve 2 m g of bendroflumethiazide
(ethoxycarbonyl)propyl]amino]-2-oxo-2,3,4,5-tetrahydro-lfi- impurity A CRS and 2.5 mg of altizide CRS in the solvent
1-benzazepin-1-yl] acetic ad d , m ixture and dilute to 10 m L with the solvent mixture.
M ix 1 m L of this solution with 1 m L of the test solution and
dilute to 100 m L with the solvent mixture.
H,N. Reference solution (b) Dilute 1.0 m l. of the test solution to
Column:
— size: I = 0.15 m , 0 = 3.0 mm;
E. R = H: [(35)-3-am ino-2-oxo-2^,4,5-tetrahydro-lH -l-
chromatography R (5 jam);
benzazepin-l-yl] acetic add,
F. R = C (C H 3) 3: 1,1-dimethylethyl [(3S)-3-amino-2-oxo-
Mobile phase M ix 15 volumes of tetrahydrqfuran R,
2,3,4,5-tetrahydro- IH -l -benzazepin- 1-yl] acetate.
25 volumes of methanol R and 60 volumes of a 2.0 g/L
PhEur solution o f citric add R.
Injection 20 jiL.
Run time Twice the retention time of bendroflumethiazide.
1-248 Benorilate 2016
Relative retention W ith reference to bendroflumethiazide C H A R A C T E R IS T IC S

(retention time = about 8 m in): im purity A = about 0.2; A white or almost white, crystalline powder.
altizide = about 0.5. Practically insoluble in water, soluble in acetone, sparingly
System suitability: reference solution (a): soluble in ethanol (96%) and in methanol.
— resolution: m inim u m 10 between the peaks due to altizide
and bendroflumethiazide.
A. T he infrared absorption spectrum, Appendix II A, is
Limits:
concordant with the reference spectrum o f benorilate (RS 023).
— impurity A: not more than the area o f the principal peak
in the chromatogram obtained with reference solution (b) B. T o 10 mg add 10 m L o f 6m hydrochloric acid and boil
( 0.1 per cent); until completely dissolved- T o 5 m L of the resulting solution
— unspecified impurities: for each impurity, n o t more th an the add 0.1 m L o f strong 1-naphthol solution, mix and add
area o f the principal peak in the chromatogram obtained sufficient 1m sodium hydroxide to make the solution just
w ith reference solution (b) (0.10 per cent); alkaline. A blue colour is produced which is extracted into
— total: not more than twice the area o f the principal peak in butan-l-ol.
the chromatogram obtained with reference C. T he light absorption, Appendix II B, in the range 230 to
solution (b) (0.2 per cent); 350 nm o f a 0.001% w/v solution in absolute ethanol exhibits
— disregard limit: 0.5 times the area o f the principal peak in a maximum only at 240 nm . T h e absorbance at 240 nm is
the chromatogram obtained with reference solution (b) about 0.74.
(0.05 per cent). TESTS
L oss on d ry in g (2.2.32) M e ltin g p o in t
M aximum 0.5 per cent, determ ined on 1.000 g by drying in 178° to 181°, Appendix V A.
an oven at 105 °C.
H eav y m e ta ls
S u lfa te d a s h (2.4.14) 1.0 g complies with limit test C for heavy metals,
M aximum 0.1 per cent, determ ined on 1.0 g. Appendix VII. Use 2 m L o f lead standard solution
A SSAY (10 ppm Pb) to prepare the standard (20 ppm).
Dissolve 0.150 g in 50 m L of dimethyl sulfoxide R T itrate to 4 -A m in o p h en o l
the 2nd point of inflexion w ith 0.1 M tetrabutylammonium Shake 2.5 g with 100 m L of water for 15 minutes and filter.
hydroxide in 2-propartol, d eterm in in g the end-point T o 20 m L o f the filtrate add 0.2 m L o f sodium nitroprusside-
potentiometrically (2.2.20). C an y out a blank titration. carbonate solution, mix and allow to stand for 30 minutes.
1 m L of 0.1 M tetrabutylammonium hydroxide in 2-propanol is T he solution is n o t more intensely coloured than a solution
equivalent to 21.07 mg o f C 15H 14F 3N 304S2. prepared at the same time and in the same m anner b u t using
IM P U R IT IE S 2 m L o f a solution of 4-aminophenol con tain in g 5 \ig per m L
and 18 m L of water in place o f the filtrate (20 ppm).
Salicylic a d d
O O o o Shake 0.50 g with 20 m L o f water for 15 m in u tes and filter.
* // \ ''/
,s s. Transfer 10 m L o f the filtrate to a Nessler cylinder, dilute to
h2n' NH,
50 m L with water, add 0.2 m L o f a 0.5% w/v solution of
f3c NH2 iron(m) chloride and allow to stand for 1 m in u te. T h e colour
obtained is not m ore intense than that o f a solution prepared
A. 4-ammo-6-(trifluoromethyl)benzene-1,3-disulfonamide.
at the same time by adding 0.2 m L o f a 0.5% w/v solution of
PfiEur inm(m) chloride to a m ixture o f 1 m L o f a 0.025% w/v
solution o f salicylic acid in ethanol (96%) and sufficient water
to produce 50 m L (0.1%).
Benorilate Carry out the m ethod for thin-layer chromatography,
Appendix m A, using the following solutions in a mixture of
1 volume of methanol and 9 volumes o f dichloromethane.
(2) 0.040% w/v o f the substance being examined.
(3) 0.0080% w/v o f the substance being exam in ed.
(4) 0.0080% w/v o f paracetamol
c 17h 15n o 5 313.3 5003-48-5 CHROMATOGRAPHIC CONDITIONS
Develop the plate in the first mobile phase (mobile phase A).
After development dry in air and develop in the second
Salicylate-paracetamol derivative; antipyretic; analgesic; anti
mobile phase (mobile phase B).
inflammatory.
(a) Use a silica gel H F 254 precoated plate (Analtech plates
are suitable).
Benorilate Oral Suspension
(b) Use mobile phase A as described below.
Benorilate Tablets
(c) Apply 10 pL o f each solution.
D E F IN IT IO N (d) Develop the plate to 15 cm.
Benorilate is 4-acetamidophenyl O-acetylsalicylate. It c o n tains (e) After removal o f the plate, dry in air and examine under
not less than 99.0% and no t more than 100.5% of ultraviolet light (254 nm).
C 17H 15N O 5, calculated w ith reference to the dried substance.
2016 Benperidol 1-249
M OBILE PHASE Comparison benperidol CRS.

Mobile phase A 5 volumes o f glacial acetic acid, 15 volumes I f the spectra obtained in the solid state show differences,
of ether, and 80 volumes o f dichloromethane. dissolve the substance to be examined and the reference
Mobile phase B 10 volumes of formic add, 45 volumes of substance separately in the minimum volume o f methyl
ether, and 45 volumes of 2,2,4-trimethylpentane. isobutyl ketone R, evaporate to dryness and record new spectra
using the residues.
LIMITS
In the chrom atogram obtained with solution (1):
any spot corresponding to paracetamol is not more intense
in the mobile phase and dilute to 10 mL with the mobile
than the spot in the chromatogram obtained with solution (4)
phase.
(0.2%);
Reference solution (a) Dissolve 30 m g of benperidol CRS in the
any secondary spot with an R f value slightly higher than th a t of
mobile phase and dilute to 10 m L with the mobile phase.
the principal spot is not more intense than the principal spot
in the chrom atogram obtained with solution (2) ( 1%); Reference solution (b) Dissolve 30 mg of benperidol CRS and
30 mg of droperidol CRS in the mobile phase and dilute to
any other secondary spot is not more intense than the spot in
10 m L with the mobile phase.
the chrom atogram obtained with solution (3) (0.2%).
Loss on drying
W hen dried to constant weight at 105°, loses not more than
Mobile phase acetone R, methanol R (10:90 VW).
0.5% o f its weight. Use 1 g. Application 10 |iL.
Sulfated ash Development Over 3/4 of the plate.
N ot m ore than 0.1%, Appendix IX A. Drying In. air.
ASSAY Detection Examine in ultraviolet light at 254 nm .
Carry out M ethod I for the determination of nitrogen, System suitability: reference solution (b):
Appendix V IIIH , using 0.6 g and 10 m L of nitrogen-free — the chromatogram shows 2 clearly separated spots.
sulfuric acid. Each m L of 0.05 m sidfuric add FS is equivalent Results T h e principal spot in the chromatogram obtained with
to 31.33 m g of C i 7H 15N 0 5. the test solution is similar in position and size to the principal
solution (a).
C. Dissolve about 10 mg in 5 m L of anhydrous ethanol R.
Benperidol i***% Add 0.5 m L of dinitrobenzene solution R and 0.5 m L of 2 M
★. * alcoholic potassium hydroxide R. A violet colour is produced
(Ph Eur monograph 1172) * which becomes brownish-red after 20 min.
D . Mix about 5 m g with 45 m g o f 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 add R to render the solution
colourless. Filter. T o a freshly prepared mixture of 0.1 m L of
alizarin S solution R and 0.1 m L o f zirconyl nitrate solution R,
add 1.0 m L of the filtrate. Mix, allow to stand for 5 min and
C22H 24FN 3O2 381.4 2062-84-2 compare the colour of the solution with that o f a blank
prepared in the same manner. T h e test solution is yellow and
Action and use the blank is red.
D opam ine receptor antagonist; neuroleptic.
TESTS
PhEur__________________________________________________________ Related substances
DEFINITION Liquid chromatography (2.2.29). Prepare the solutions
1- [ 1- [4-(4-FluorophenyI) -4-oxobutyl] piperidin-4-yl] -1,3- immediately before use.
djhydro- 2f/-benzimidazol- 2-one. Test solution Dissolve 0.10 g of the substance to be examined
in dimethylformamide R and dilute to 10.0 mL with the same
Content
solvent.
Reference solution (a) Dissolve 2.5 m g of benperidol CRS and
CHARACTERS 2.5 mg o f droperidol CRS in dimethylformamide R and dilute to
Appearance 100.0 m L with the same solvent.
Reference solution (b) Dilute 1.0 m L of die test solution to
Solubility 100.0 m L with dimethylformamide R. Dilute 5.0 m L of this
Practically insoluble in water, freely soluble in solution to 20.0 m L with dimethylformamide R.
dimethylformamide, soluble in methylene chloride, slightly Column:
soluble in ethanol (96 per cent). — size: I — 0.1 m , 0 = 4.6 mm;
It shows polymorphism (5.9). — stationary phase: base-deactivated octadecylsilyl silica gelfor
IDENTIFICATION chromatography R (3 nm).
First identification A Mobile phase:
— mobile phase A: 10 g/L solution o f tetrabutylammonium
hydrogen sulfate R ;
1-250 Benserazide Hydrochloride 2016
ov
— mobile phase B: acetonitrUe R; •Y-NH
Time
(min)
Mobile phase A
(per cent V/V)
Mobile p h u t B
(per cent V/V)
C f 't )
0 -15
15-20
100 ->60
60
0*40
40
o ^ ~ 'r
20-25 100 0 B . 1- [ 1- [4-(2-fluorophenyl)-4-oxobutyl]piperidin-4-yI] -1,3-
d£hydro-2//-benzim idazol- 2 -one,

Irgection 10 |iL.
Relative retention W ith reference to benperidol (retention
time = about 6.5 min): impurity A = about 0.2;
impurity B = about 0.9; droperidol = about 1.1;
impurity D = about 1.2; impurity E = about 1.3; -
benperidol and droperidol.
C .1- [ 1- [4-oxo-4-[4- [4-(2-oxo-2,3-dihydro-1//-benzim idazol-
Limits:
l-yl)piperidin-l-yl]phenyl]butyl]piperidin-4-yl]-l,3-dihydro-
— impurities A , B, C, D, E: for each impurity, not more than
2//-benzim idazol- 2-one,
obtained with reference solution (b) (0.25 per cent); ov
\- N H
(0.10 per cent);
— total: not m ore than twice the area o f the principal peak in
(0.5 per cent); D. cis-1- [1 -[4-(4-fluorophenyl)-4-oxobutyl]piperidin-4-yl
— disregard limit. 0.2 times the area o f the principal peak in 1 -oxide] -1,3-dihydro-2H-benzimidazol-2-one,
the chromatogram obtained w ith reference solution (b)
(0.05 per cent).
Maximum 0.5 per cent, determ ined on 1.000 g by drying in
an oven at 105 °C.
M aximum 0.1 per cent, determ ined on 1.0 g in a platinum
crucible.
ASSAY E. trans- 1- [ 1- [4- (4-fluorophenyl)-4-oxobutyl] piperidin-4-yl
1 -oxide]-1,3-dihydro-2//-benzimidazol-2-one.
Dissolve 0.300 g in 50 m L o f a mixture o f 1 volume of
anhydrous acetic add R and 7 volumes o f methyl ethyl ketone R. PhEur
Titrate with 0.1 M perchloric add, using 0.2 m L of

naphtholbenzein solution R as indicator.
1 m L of 0.1 M perchloric acid is equivalent to 38.14 mg ★
*** ★
of C 22H 24F N 3O 2. Benserazide Hydrochloride ★ ★
STORA GE (Ph. Eur. monograph 1173) *****
IM P U R IT IE S H V Nh2
Spedfied impurities A, B, C, D , E. n r if? 0 “ and enantiomer , HQ
ov
V -N H OH
C10H16CIN3O5 293.7 14919-77-8

jC T O A c tio n a n d u se
D opa decarboxylase inhibitor.
A. 1-(piperidin-4-yl)- 1,3-dihydro-2#-benzim idazol-2-one,
Co-beneldopa Capsules
Dispersible Co-beneldopa Tablets
Prolonged-release Co-beneldopa Capsules
2016 Benserazide Hydrochloride 1-251
PhEur--------------------------------------------------------------------------- — — - Time Mobile phase A Mobile phase B

D E F IN IT IO N
0 -1 5 100-»0 0 -» 100
(2RS)-2-Amino-34iydroxy-2 '-(2,3,4-
trihydroxybenzyI)propanohydrazide hydrochloride. 15-25 0 100
Content
98.5 per cent to 101.0 per cent (anhydrous substance). Flow rate 1.3 m U m in.
C H A R A C T ER S Detection Spectrophotometer at 210 nm.
Appearance Injection 5 pL.
White or yellowish-white or orange-white, crystalline powder.
Identification of impurities Use die chromatogram supplied
S olubility with benserazide for peak identification CRS and die
Freely soluble in water, very slightly soluble in anhydrous chromatogram obtained with reference solution (c) to identify
ethanol, practically insoluble in acetone. the peaks due to impurities A, B and C; doubling of die peak
It shows polymorphism (5.9). due to impurity C , related to separation o f the (£Z)-isomers,
may be observed.
A. Infrared absorption spectrophotometry (2.2.24). Relative retention W ith reference to benserazide (retention
Comparison benserazide hydrochloride CRS.
impurity C = about 1.2; impurity B = about 1.5.
If die spectra obtained show differences, dissolve the System suitability: reference solution (a):
substance to be examined and die reference substance — resolution: minimum 5.0 between die peaks due to
separately in hot methanol R, evaporate to dryness and record benserazide and impurity C; use the 1st peak of
new spectra using the residues. impurity C if 2 peaks occur.
B. Solution S (see Tests) gives reaction (b) of chlorides Limits:
(2.3.1). — correction factor, for die calculation o f content, multiply the
TESTS peak area o f impurity B by 0.7;
Solution S — impurity A: not more than die area o f the corresponding
Dissolve 1.0 g in carbon dioxide-free water R and dilute to peak in the chromatogram obtained with reference
100 m L with die same solvent solution (a) (0.5 per cent);
— impurity B: not more than die area of the peak due to
benserazide in the chromatogram obtained with reference
— impurity C: not more than die area o f the corresponding
p H (2.2.5) peak or pair of peaks in the chromatogram obtained with
4.0 to 5.0 for solution S. reference solution (a) (0.5 per cent);
R elated su b stan ces — unspecified impurities: for each impurity, not more than the
Liquid chromatography (2.2.29). area o f the peak due to benserazide in die chromatogram
AM sohaions must be irgected immediatdy or stored at 4 °C. obtained with reference solution (b) (0.10 per cent);
— sum of impurities other than A: n o t more than twice the
Test solution Dissolve 0.100 g of die substance to be
area of die peak due to benserazide in die chromatogram
examined in methanol R2 and dilute to 50.0 m L with the
ob tain ed with reference solution (a) (1.0 per cent);
same solvent
— disregard Umar. 0.5 times die area of the peak due to
Reference solution (a) Dissolve 5.0 m g o f benserazide benserazide in the chromatogram obtained with reference
impurity A CRS, 5.0 m g of benserazide impurity C CRS angl solution (b) (0.05 per cent).
5.0 mg of benserazide hydrochloride CRS in methanol R2 and
dilute to 50.0 m L with the same solvent Dilute 5.0 m L of H eavy m e ta ls (2.4.8)
this solution to 50.0 m L with methanol R2. Maximum 20 ppm.
Reference solution (b) Dilute 2.0 m L of reference solution (a) 1.0 g complies with test C. Prepare the reference solution
to 10.0 m L with methanol R2. using 2 m L o f lead standard solution (10 ppm Pb) R
Reference solution (c) Dissolve 5 m g of benserazide for peak W a te r (2.5.12)
identification CRS (containing impurities A, B and C) in Maximum 1.0 per cent, determined on 0.500 g.
methanol R2 and dilute to 5.0 mL with the same solvent S u lfated a sh (2.4.14)
Column: Maximum 0.1 per cen^ determined on 1.0 g.
— size: I = 0.25 m, 0 = 4 mm; ASSAY
— stationary phase: octylsQyl silica gelfor chromatography R In order to avoid overheating during the titration, mix thoroughly
(5 Jim); throughout and stop the titration immediately after the end-point
— temperature. 30 °C.
has been reached
Mobile phase:
Dissolve 0.250 g in 5 m L o f anhydrous formic add R
— mobile phase A: dissolve 2.2 g of sodium heptanestdfbnate
Add 70 m L of anhydrous acetic add R Titrate immediately
monohydrate R and 6.8 g of potassium dihydrogen
with 0.1 M perchloric add, determining the end-point
phosphate R in 900 m L o f water R, add 50 m L of
methanol R2 and adjust to pH 3.5 with phosphoric add R;
— mobile phase B: dissolve 2.2 g of sodium heptanesulfonate 1 m L of 0.1 M perchloric add is equivalent to 29.37 mg of
monohydrate R and 6.8 g of potassium dihydrogen C 10H 16CIN 3O 5.
phosphate R in 500 m L o f water R, adjust to p H 3.5 with ST O R A G E
phosphoric add R and add 500 m L of methanol R2; Protected from light.
1-252 Bentonite 2016
IM P U R IT IE S for each portion to settle. Allow to stand for 2 h.

Specified impurities A , B, c. T he apparent volume of the sediment is n o t less than 22 mL.
c . 0.25 g gives the reaction o f silicates (2.3.1).
TESTS
A r- = Alkalinity
T o 2 g add 100 m L of carbon dioxide-free water R and shake
for 5 mm. T o 5 m L of this suspension add 0.1 m L of
xhymòiphừuủem solution R T he liquid becomes bluish.
Add 0.1 m L o f 0.1 M hydrochloric add. The liquid is
HJ V v ”o2 h
■n and enantiomer decolourised within 5 min.
H2N
o C o a rse p a rtic le s
M aximum 0.5 per c e n t
A. (2J?S)-2-amino-3-hydroxypropanohydrazide, T o 20 g add 1000 m L of water R and mix for 15 mm using a
high-speed mixer capable o f operating at not less than
H H* 'NHỉ
5000 iv'min. Transfer the suspension to a wet sieve (75),
X . .OH tared after drying at 100-105 °c. Wash with 3 quantities,
and enantiomer
I 1l each of 500 m L, o f water R, ensuring that any agglomerates
At ổ have been dispersed. Dry the sieve at 100-105 °c and weigh.
The p a ra d e s on the sieve weigh a maximum o f 0.1 g.
B. (2ÄS)-2-amino-3-hydroxy-2 ',2 /-bis(2,3}4- H eav y m e ta ls (2.4.8)
trihydroxybenzyI)propanohydrazide, M aximum 50 ppm.
T o 5.0 g add 7.5 m L of dilute hydrochloric add R and
H HV NHi 27.5 m L o f water R. Boil for 5 min. Centrifuge and filter tile
Ar N s y c OH Hb (Z)-isomer and
their enantiomers supernatant W ash the centrifugation residue with water R
0 and filter. Dilute the combined filtrates to 50.0 m L with
water R T o 5 m L o f this solution add 5 m L of water R,
C. (2& S)-2-amino-3-hydroxy-2'-[(l£Z)-(2,3,4- 10 m L o f hydrochloric add R and 25 m L of methyl isobutyl
trihydroxybenzylidene)] propanohydraãde. ketone R and shake for 2 min. Separate the layers. Evaporate
PhEur the aqueous layer to dryness on a water-bath. Dissolve the
residue in 1 m L o f acetic add R, dilute to 25 m L with
water R and filter. 12 m L o f the filtrate complies with test A.
Prepare the reference solution using lead standard solution
**★ (1 ppm Pb) R
Bentonite ★ ★
★ ★ L oss o n d ry in g (2.2.32)
(Ph. Eur. monograph 0467) *** Maximum 15 per cent, determined on 1.000 g by drying in
an oven a t 105 °c.
1302-78-9
PhEur.
TAM C: acceptance criterion 103 C FU/g (2.6.12).
D E F IN IT IO N
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
N atural clay containing a high proportion of montmorillonite,
a native hydrated alu m in iu m silicate in which some
recognised as being relevant control parameters far one or more
aluminium and silicon atoms may be replaced by other atoms
functions of die substance when used as an excipient (see chapter
such as magnesium and iron.
5.15J. This section ừ a non-mandatory part of the monograph
CHARACTERS and ừ Knot necessary to verify the characteristics to demonstrate
A p p e a ra n c e compliance. Control af these characteristics can however contribute
Very fine, homogeneous, greyish-white powder with a more to the quality of a medicinal product by improving the consistency
or less yellowish or pinkish tint. of ứte manufacturing process and ứie performance of the medicinal
S olubility product during use. Where control methods are cited, they art
Practically insoluble in water and in aqueous solutions. recognised as being suitable for the purpose, but other methods can
also be used. Wherever results for a particular characteristic are
It swells with a little w ater forming a malleable mass.
reported, die control method must be indicated.
ID E N T IF IC A T IO N The following characteristics may be relevant for bentonite used as
A. T o 0.5 g in a m etal crudble add 1 g of potassium nitrate R viscosity-increasing agent or suspending agent
and 3 g o f sodium carbonate R and heat until the mixture
S e d im e n ta tio n v o lu m e
melts. Allow to cool. T o this residue add 20 m L of boiling
T o 6.0 g add 200 m L o f water R and mix for 20 min using a
water R, mix and filter. W ash the insoluble residue with
high-speed Tnnrer capable o f operating at 10 000 r/min.
50 m L o f water R T o this residue add 1 m L of hydrochloric
Transfer 100 m L of this suspension to a graduated cylinder.
add R and 5 m L o f water R. Filter. T o the filtrate add 1 m l.
Allow to stand for 24 h. The volume o f tile d ear supernatant
o f strong sodium hydroxide solution R and filter. T o this filtrate
is n o t greater than 2 mL.
add 3 m L o f ammonium chloride solution R A gelatinous white
precipitate is formed. Swelling power with water
See Identification B.
B. A dd 2.0 g in 20 portions to 100 m L of a 10 g/L solution
o f sodium laurilsulfate R in a 100 m L graduated cylinder PhEur
about 30 m m in diam eter. Allow 2 min between additions
2016 Benzalkonium Chloride 1-253
Benzalkonium Chloride *****

Benzaldehyde ★ ★
★> *
(Ph. Eur. monograph 0372) *★*
CHO
H3C CH3
cr
8001-54-5
CrHfiO 106.1 100-52-7
Action and use
Action and use
Antiseptic.
Flavour.
PhEir____________
DEFINITION
Benzaldehyde c o n tains not less than 98.0% w/w and not DEFINITION
more than 100.5% w/w of G/HÔ. M ixture of alkylbenzyldrmethylaxnmonium chlorides, the
alkyl groups mainly having chain lengths o f C 12, C J4 and
CHARACTERISTICS
A clear, colourless liquid.
c16.
Content
Slightly soluble in water, miscible with ethanol (96%) and
95.0 per cent to 104.0 p er cent of
with ether.
alkylbenzyldimethylammonium chlorides (anhydrous
TESTS substance) calculated using the average relative molecular
Refractive index m ass (see Tests).
1.544 to 1.546, Appendix V E. CHARACTERS
Weight per mL Appearance
1.043 to 1.049 g, Appendix V G. W hite or yellowish-white powder or gelatinous, yellowish-
Free add white fragments, hygroscopic. On heating it forms a d ear
N ot more than 1.0% w/v, calculated as benzoic ad d , m olten mass.
CyHfiO^ w hen determ ined by the following method. Solubility
T o 10 m L add 2 0 m L of ethanol (96%) previously Very soluble in water and in ethanol (96 p er cent).
neutralised to phenolphthalein solution R1 and titrate with A n aqueous solution froths copiously when shaken.
0 .1 m sodium hydroxide F 5 using phenolphthalein solution R1 as
IDENTIFICATION
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.
First identification B, E
Second identification A, C, D, E
Chlorinated compounds
N o t more than 0.05% w/v, calculated as Cl, when A . Ultraviolet and visible absorption spectrophotometry
determined by the following method. T o 5 m L add 50 m L of (2.2.25).
isoamyl alcohol and 3 g o f sodium and boil under a reflux Test solution Dissolve 80 m g in water R and dilute to
condenser for 1 hour. Cool, add 50 m L o f water and 15 m l, 100.0 m L with the same solvent.
of nitric acid, cool, add 5 m L o f 0.1m silver nitrate FS, shake Spectral range 220-350 nm .
and titrate the excess silver nitrate with 0 . 1m ammonium Absorption maxima At 257 nm, 263 nm and 269 nm.
thiocyanate VS using ammonium iron(III) sulfate solution R2 as
indicator. Repeat the procedure without the substance being
Shoulder A t about 250 nm .
examined. T h e difference between die titrations represents B. Examine the chromatograms obtained in the test for
the am ount o f silver nitrate required. Each m L o f 0 . 1m silver average rdative molecular mass and ratio o f alkyl
nitrate F51is equivalent to 3 .5 4 5 mg o f CL components.
Results T he prindpal peaks in the chromatogram obtained
ASSAY
w ith the test solution are similar in retention time to the
Carry out the m ethod for determination of aldehydes,
p rin d p al peaks in the chromatogram obtained with the
Appendix X K, using 0.5 g. Each m L o f 0.5m potassium
reference solution.
hydroxide in ethanol (60%) FiS is equivalent to 53.06 mg o f
CrHfiO. 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
STORAGE solution R. A white predpitate is formed. Filter. Dissolve the
Benzaldehyde should be kept in a well-filled container, p redpitate in a mixture o f 1 m L o f acetone R and 5 m L o f
protected from light and stored at a tem perature not ethanol (96 per cent) R, heating to not m ore than 70 °C.
exceeding 15°. 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 2016
colourless. Add 0.1 m L of solution S and shake. Calculate the percentage of each homologue, using the
T he methylene chloride layer becomes blue. 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
100(I)
TESTS C = product of the relative molecular mass o f the given
S o lu tio n S homologue and the area of the corresponding peak
Dissolve 1.0 g in carbon dioxide-free water R and dilute to in the chromatogram obtained with the test
100 m L with the same solvent. solution;
A p p e a ra n c e o f so lu tio n D = sum o f the C values for all homologues quantified.
Solution S is clear (2.2.1) and not more intensely coloured Limits:
than reference solution Y 6 (2.2.2, MethodII). — C1 2 homologue: minimum 40 per cent;
A cid ity o r alk alin ity — C14 homologue: minimum 20 p er cent;
T o 50 m L of solution S add 0.1 m L o f bromocresolpurple — sum of C 1 2 and C14 homologues: minim um 70 per cent.
solution R. N ot more than 0.1 m L of 0.1 M hydrochloric add C 1 2 and C14 homologues
or 0.1 M sodium hydroxide is required to change the colour of Im p u ritie s A , B a n d C
the indicator. Liquid chromatography (2.2.29). Prepare the solutions
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 immediately before use.
co m p o n e n ts Test solution Dissolve 0.50 g o f the substance to be examined
L iquid chromatography (2.2.29). in methanol R1 and dilute to 10.0 m L with the same solvent.
Test solution Dissolve 0.400 g o f the substance to be Reference solution (a) Dissolve 25.0 mg of benzyl alcohol CRS
examined in water R and dilute to 100.0 m L with the same (impurity A) in methanol R1 and dilute to 100.0 m L with the
solvent. same solvent.
Reference solution Dissolve the contents o f a vial of Reference solution (b) Dissolve 75.0 m g of benzaldehyde CRS
benzalkonium chloridefor system suitability CRS in 5.0 m L o f (impurity B) in methanol R1 and dilute to 100.0 m L with the
water R. same solvent. Dilute 1.0 m L o f this solution to 10.0 m L with
Column: methanol R l.
— sizer. I = 0.25 m , 0 = 4.6 mm; Reference solution (c) Dilute 1.0 m L o f reference solution (a)
— stationary phase: end-capped nitrUe silica gelfor to 10.0 m L with methanol R l.
chromatography R (5 pm). Column:
Mobile phase Mix 45 volumes o f acetonitrUe R and 55 volumes — size. I = 0.15 m, 0 = 4.6 mm;
of a 13.6 g/L solution o f sodium acetate R previously adjusted — stationary phase, end-capped octadecylsUyl silica gel for
to p H 5.0 with glacial acetic add R. chromatography R (5 jam);
Flow rate 2.0 mL/min. — temperature: 30 °C.
Detection Spectrophotom eter at 254 nm. Mobile phase.
— mobile phase A: dissolve 1.09 g o f sodium hexanesulfimate R
Injection 10 (iL.
and 6.9 g of sodium dihydrogen phosphate monohydrate R in
Identification of homologues Use the chromatogram supplied water R; adjust to p H 3.5 with phosphoric add R and
with benzalkonium chloride for system suitability CRS and the dilute to 1000.0 m L with the same solvent;
chromatogram obtained with die reference solution to — mobile phase B: m ethanol R l ;
identify the peaks due to Q jj, C 14 and C i6.
Relative retention W ith reference to C 12 homologue (retention
Time Mobile phase A Nobile phase B
time = about 6 min): C 14 homologue = about 1.3;
C 16 homologue = about 1.7.
0-10 80 20
— resolution: minim um 1.5 between the peaks due to the C i 2 10-14 80 —> 50 2 0 -* 5 0
and C 14 homologues. 14-35 50 50
Calculate the average relative molecular mass o f the sample 35 -3 6 50 —> 20 5 0 -» 8 0
by summing the products for each homologue, using the
following expression: 3 6-55 20 80

-(i) Detection Spectrophotom eter at 210 nm for impurities A and
C, and at 257 n m for impurity B.
A = area o f the peak due to the given homologue in the Injection 20 |iL.
chrom atogram obtained with the test solution; Relative retention W ith reference to impurity A (retention
B = sum of the areas o f the peaks due to all time = about 10 min): impurity B = about 1.3;
homologues in the chromatogram obtained with impurity C = about 2.4.
the test solution;
System suitability A t 210 nm:
W = relative molecular mass for the given homologue: — signal-to-noise ratio: minimum 10 for the principal peak in
340, 368 and 396 for the C J2, C 14 and C 16 the chromatogram obtained w ith reference solution (c);
homologues, respectively.
2016 Benzalkonium Chloride 1-255
— symmetry factor, minimum 0.6 for the peak due to

im purity A in the chromatogram obtained with reference OH
solution (a).
Limits:. 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 CHO
B. benzaldehyde,
— impurity B: n o t more than the area of the corresponding
— impurity C: n o t more than 0.1 times the area o f the
reference solution (a) (0.05 per cent). 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 Benzalkonium Chloride Solution *****
12.0 m L of 0.1 M tetrabutylammomum hydroodde and record *. *
the potentiometric titration curve (2.2.20). If the curve shows (Ph. Eur. monograph 0371) *
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 A ctio n a n d u se
point o f inflexion, the substance to be examined does not Antiseptic.
comply with the te s t If the curve shows 1 point o f inflexion,
PhEur__________________________________________________________
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. D E F IN IT IO N
If the titration curve after addition o f 12.0 m L o f the titrant Aqueous solution of a mixture of
shows only 1 point of inflexion, the substance to be alkylbenzyldimethylammonium chlorides, the alkyl groups
examined does n o t comply with the test. mainly having chain lengths of C 121 C 14 and C 16.
W a te r (2.5.12) C o n te n t
M axim um 10 per cent, determined on 0.300 g. 475 g/L to 525 g/L o f alkylbenzyldimethylammonium
chlorides, calculated using the average relative molecular
S u lfa te d a sh ( 2.4.14)
M axim um 0.1 per cent, determined on 1.0 g. mass (see Tests). T h e solution may contain ethanol
(96 per cent).
A SSA Y
CHARACTERS
A p p e a ra n c e
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 Clear, colourless or slightly yellowish liquid.
of 0.1 M sodium hydroxide and 10.0 m L of a freshly prepared S olu b ility
50 g/L solution o f potassium iodide R. Shake well, allow to Miscible with water and with ethanol (96 per cent).
separate and discard the methylene chloride layer. Shake the It froths copiously when shaken.
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 Second identification A , C, D, E
colour is almost discharged. Add 5 m L o f methylene chloride R A. Ultraviolet and visible absorption spectrophotometry
and continue the titration, shaking vigorously, until the (2.2.25).
m ethylene chloride layer no longer changes colour. Carry out Test solution D ilute 0.3 m L to 100.0 mL with water R.
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. Absorption maxima A t 257 nm, 263 nm and 269 nm.
1 m L of 0.05 M potassium iodate is equivalent to
Shoulder A t about 250 nm .
average relative molecular mass and ratio o f alkyl
10 components.
Results T h e principal peaks in the chromatogram obtained
m g of benzalkonium chloride where x is the average relative with the test solution are similar in retention time to the
molecular mass o f the sample. principal peaks in the chromatogram obtained with the
reference solution.
STORA GE
In an airtight container. 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
IM P U R IT IE S solution R. A white precipitate is formed. Filter. Dissolve the
Specified impurities A, B, C. 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 2016
Add water R dropwise to the warm solution until a slight

opalescence forms. H eat gently until the solution is clear and "(s )
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
A = area o f the peak due to the given homologue in the
diphosphorus pentoxide R or anhydrous silica gel R a t a chromatogram obtained with the test solution;
tem perature not exceeding 50 °C. T h e crystals melt (2.2.14) B — sum o f the areas of th e peaks due to all
at 127 °C to 133 °C. homologues in the chrom atogram obtained with
D . T o 5 m L o f dilute sodium hydroxide solution R add 0.1 m L the test solution;
of bromophenol blue solution R1 and 5 m l. of methylene W = relative molecular mass for the given homologue:
chloride R and shake. T he methylene chloride layer is 340, 368 and 396 for the C 12, C j4 and C i6
colourless. A dd 0.05 m L o f the solution to be examined and homologues, respectively.
shake. T he methylene chloride layer becomes blue.
Calculate the percentage o f each homologue, using the
E. T o 0.05 m L add 1 m L o f dilute nitric add R. A white following expression:
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
100(§)
S o lu tio n S
Dilute 2.0 g to 100 m L with carbon dioxide-free water R.
C = product o f the relative molecular mass o f the given
homologue and the area o f the corresponding peak
A p p e a ra n c e o f so lu tio n in the chromatogram obtained with the test
Solution S is clear (2.2.1) and not m ore intensely coloured solution;
than reference solution Y6 (2.2.2, Method IT). D = sum o f the C values for all homologues quantified.
A cid ity o r alk a lin ity Limits:
T o 50 m L o f solution S add 0.1 m L of bromocresol purple — C;2 homologue: minimum 40 per cent;
solution R. N o t m ore than 0.1 m L o f 0.1 M hydrochloric acid — C14 homologue: m inim u m 20 per cent;
or 0.1 M sodium hydroxide is required to change the colour of — sum of C ] 2 and C14 homologues: minimum 70 per cent.
the indicator.
Im p u ritie s A , B a n d C
A verage re la tiv e m o le c u la r m a ss a n d ra tio o f alkyl Liquid chromatography (2.2.29). Prepare the solutions
c o m p o n e n ts immediately before use.
Test solution Determ ine the density (2.2.5) o f the solution to
Test solution D eterm ine the density (2.2.5) of the solution to be examined. Dilute a quantity o f the solution to be
be examined. D ilute a quantity of the solution to be examined equivalent to 2.5 g o f benzalkonium chloride to
examined equivalent to about 0.400 g o f benzalkonium 50.0 m L with methanol R l.
chloride to 100.0 m L with water R
Reference solution (a) Dissolve 25.0 mg o f benzyl alcohol CRS
Reference solution Dissolve the contents of a vial of (impurity A) in methanol R l and dilute to 100.0 m L with the
benzalkonium chloride for system suitability CRS in 5.0 m l. of same solvent.
water R.
Reference solution (b) Dissolve 75.0 mg of benzaldehyde CRS
Column: (impurity B) in methanol R l and dilute to 100.0 m l. with the
— size: I = 0.25 m , 0 = 4.6 mm; same solvent. D ilute 1.0 m L of this solution to 10.0 m L with
— stationary phase: end-capped nitrUe silica gel for methanol R l.
Reference solution (c) Dilute 1.0 m L of reference solution (a)
Mobile phase M ix 45 volumes o f acetomtrUe R and 55 volumes to 10.0 m L with methanol R l.
of a 13.6 g/L solution of sodium acetate R previously adjusted
Column:
— sizer. I = 0.15 m , 0 = 4.6 m m ;
Flow rate 2.0 mlVmin. — stationary phase: end-capped octadecylsHyl silica gelfor
Detection Spectrophotom eter at 254 nm . chromatography R (5 pm);
Injection 10 pL. — temperature: 30 °C.
Identification of homologues U se the chromatogram supplied Mobile phaser.
with benzalkonium chloride for system suitability CRS and the — mobile phase A: dissolve 1.09 g o f sodium hexanesulfonate R
chrom atogram obtained with the reference solution to and 6.9 g o f sodium dihydrogen phosphate monohydrate R in
identify the peaks due to homologues C 12s Q 4 and C 16. water R; adjust to p H 3.5 w ith phosphoric add R and
dilute to 1000.0 m L with the same solvent;
Relative retention W ith reference to C 12 homologue (retention
— mobile phase B: methanol Rl;
tim e = about 6 min): C 14 homologue = about 1.3;
C i6 homologue = about 1.7.
System suitability: reference solution: Time Mobile phase A Mobile phase B
— resolution: minim um 1.5 between the peaks due to the C i2 (min)____________ (percent V/V)________ (percent V/V)
and C 14 homologues. 0 -10 80 20
Calculate the average relative molecular mass o f the sample 10- 14 80-» 50 20-» 50
by summing the products for each homologue, using the 50
14-35 50
following expression:
3 5 -3 6 50-» 20 50-»80
3 6 -5 5 20 80
2016 Benzathine Benzylpenicillin 1-257
Flow rate 1.0 mL/min. L A B E L L IN G

Detection Spectrophotom eter at 210 nm for impurities A and 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. Specified impurities: A, B, C.
Relative retention W ith reference to impurity A (retention
— signal-to-noise ratio: m inim u m 10 for the principal peak in
A. benzyl alcohol,
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). CHO
Limits:
— correction factor, for the calculation of contentj multiply the B. benzaldehyde,
— impurity A: n o t m ore than the area of the corresponding
— impurity B: n o t more than the area of the corresponding
peak in the chromatogram obtained with reference C. (chloromethyl)benzene.
solution (b) (0.15 per cent); PhEur
— impurity C: n o t more than 0.1 times the area of the
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
Benzathine Benzylpenicillin ★ ★
★ ★
3 volumes of 1 M hydrochloric acid and 97 volumes o f (Ph. Eur. monograph 0373) *★ *
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 O > C°2H
the potentiometric titration curve (2.2.20). If the curve shows
h iT x ™ 3
2 points o f inflexio n , the volume o f titrant added between the N- - r r s^ ch3
2 points is not greater than 5.0 mL. If the curve shows no H H
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 C48H56N6O8S2 909 1538-09-6
dimethyldecylamme R in 2-propanol R before the titration.
If the titration curve after the addition of 12.0 m L o f the A c tio n a n d use
titrant shows only 1 point of inflexion, the solution to be Penicillin antibacterial.
examined does n o t comply with the test.
PhEtr___________________
M aximum 0.1 p er cent, determined on 1.0 g. D E F IN IT IO N
ATjN'-Dibenzylethane-1,2-diamine compound (1:2) with
ASSAY
(2S,5i?,6i?)-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-
D eterm in e the density (2.2.5) of the solution to be examined.
thia- 1-azabicyclo [3.2.0]heptane-2-carboxylic add.
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 Substance produced by the growth of certain strains of
methylene chloride R, 10 m L of 0.1 M sodium hydroxide and PenicUUum notatum or related organisms, or obtained by any
10.0 m L o f a freshly prepared 50 g/L solution of potassium other means.
iodide R. Shake well, allow to separate and discard the C o n te n t
methylene chloride layer. Shake the aqueous layer with — benzathine benzylpenicillin: 96.0 per cent to 102.0 per cent
3 quantities, each of 10 mT, of methylene chloride R and (anhydrous substance);
discard the methylene chloride layers. T o the aqueous layer — NyN'-dibenzylethylenedicunine (benzathine C 16H 20N 2;
add 40 m L o f hydrochloric acid R, allow to cool and titrate 240.3): 24.0 per cent to 27.0 p er cent (anhydrous
with 0.05 M potassium iodate until the deep-brown colour is substance).
almost discharged. Add 5 m L of methylene chloride R and It contains a variable quantity o f water. Dispersing or
continue the titration, shaking vigorously, until the methylene suspending agents may be added.
chloride layer no longer changes colour. Carry out a blank
CHARACTERS
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 A p p e a ra n c e
40 m L o f hydrochloric acid R. W hite or almost white powder.
1 m L of 0.05 M potassium iodate is equivalent to x 10 m g o f S o lu b ility

benzalkonium chloride where x is the average relative Very slightly soluble in water, freely soluble in
molecular mass o f the sample. dimethylformamide and in formamide, slightly soluble in
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. to 100.0 m L with mobile phase A.
Second identification B, C, D. Column:
A. Infrared absorption spectrophotometry (2.2.24). — size: I = 0.25 m , 0 = 4.0 mm;
— stationary phase: end-capped octadecylsüyl silica gelfor
Comparison benzathine benzylpenicillin CRS.
chromatography R (5 ^m);
B. Thin-layer chromatography (2.2.27). — temperature: 40 °C.
Test solution Dissolve 25 mg o f the substance to be examined Mobile phase:
in 5 m L o f methanol R. — mobile phase A: mix 10 volumes o f a 34 g/L solution of
Reference solution Dissolve 25 m g o f benzathine potassium dihydrogen phosphate R adjusted to p H 3.5 with
benzylpenicillin CRS in 5 m L o f methanol R. phosphoric add R, 30 volumes o f methanol R and
Hate TLC sOamsed silica gel plate R. 60 volumes o f water R;
— mobile phase B: mix 10 volumes of a 34 g/L solution of
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.
phosphoric acid R, 30 volumes o f water R and 60 volumes
of methanol R;
Application 1 [lL.
Drying In air. (min) (per cent V/V) (per cent V/V)
Detection Expose to iodine vapour until the spots appear and 0 - 10 75 25
examine in daylight
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 5 5 -7 0 75 25

with the test solution are similar in position, colour and size
to the 2 principal spots in the chromatogram obtained with
the reference solution. Flow rate 1 mlVmin.
C. Place about 2 mg in a test-tube about 150 mm long and Detection Spectrophotom eter at 220 nm.
15 m m in diameter. M oisten with 0.05 m L of water R and Injection 20 |iL.
add 2 m L o f sulfuric acid-formaldehyde reagent R. M ix the System suitability: reference solution (a):
contents of the tube by swirling; the solution is practically — relative retention with reference to benzylpenicillin:
colourless. Place the test-tube on a water-bath for 1 min; benzathine = 0.3 to 0.4; impurity C = about 2.4;
a reddish-brown colour develops. if necessary, adjust the concentration o f methanol in the
D . T o 0.1 g add 2 m l- of 1 M sodium hydroxide and shake for mobile phase.
2 m in. Shake the mixture with 2 quantities, each of 3 mT, of Limits:
ether R. Evaporate the combined ether layers to dryness and — impurity C: n o t more than twice the sum o f the areas of
dissolve the residue in 1 m L o f ethanol (50 per cent V/V) R. the 2 principal peaks in the chrom atogram obtained with
Add 5 m L o f picric acid solution R, heat at 90 °C for 5 min reference solution (b) (2 per cent);
and allow to cool slowly. Separate the crystals and — any other impurity, for each impurity, n o t more than the
recrystallise from ethanol (25 per cent V/V) R containing sum o f the areas of the 2 principal peaks in the
10 g/L of picric acid R. T he crystals melt (2.2.14) at about chromatogram obtained with reference solution (b)
214 °C. (1 per cent);
— disregard limit. 0.05 times the sum of the areas of the
TESTS
2 principal peaks in the chromatogram obtained with
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). W a te r (2.5.12)
T o 20 m L o f the filtrate add 0.1 m L o f bromothymol blue 5.0 per cent to 8.0 per cent, determined on 0.300 g.
solution R l. T he solution is green or yellow. N ot m ore than B a c te ria l en d o to x in s (2.6.14, Method E)
0.2 m L of 0.02 M sodium hydroxide is required to change the Less than 0.13 IU /m L, if intended for use in the
colour of the indicator to blue. manufacture of parenteral preparations w ithout a further
R e la te d su b stan ces appropriate procedure for the removal o f bacterial
Liquid chromatography (2.2.29). Prepare the solutions endotoxins.
immediately before use, using somcation (for about 2 min) to Suspend 20 mg in 20 m L o f a solution o f 0.1 M sodium
dissolve the samples. Avoid any overheating during the sample hydroxide diluted 1 to 100, shake thoroughly and centrifuge.
preparation. Examine the supernatant.
Test solution Dissolve 70.0 m g o f the substance to be A SSAY
examined in 25 m L o f methanol R and dilute to 50.0 m L Liquid chromatography (2.2.29) as described in the test for
with a solution containing 6.8 g/L o f potassium dihydrogen related substances with the following modifications.
phosphate R and 1.02 g/L of disodium hydrogen phosphate R.
Mobile phase phosphate buffer solution pH 3.5 R, methanol R,
Reference solution (a) Dissolve 70.0 m g o f benzathine water R (10:35:55 VIV/V).
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 Calculate the percentage contents o f benzathine and
phosphate R. b enzathine benzylpenicillin. Calculate the percentage content
2016 Benzatropine Mesilate 1-259
of benzathine benzylpenicillin by multiplying the percentage I.C O 2H

content o f benzylpenicillin by 1.36. hn - ^
C , ch3
. . . { ^ / S h3
STORAGE
H
sterile, airtight, tam per-proof container. and epimer at C*
IM P U R IT IE S F . (2RS,4S)-2 -[[(phenylacetyl)amino] methyl]-5,5-

Specified impurities C dimethylthiazolidine-4-carboxylic acid (penilloic a d d s of
Other detectable impurities (the following substances would, if benzylpenicillin).
present at a sufficient level, be detected by one or other of PhEur
(2034) . It is therefore not necessary to identify these
impurities for demonstration o f compliance. See also 5. 10.
Benzatropine Mesilate
Control of impurities in substances for pharmaceutical use): A , B,
D, E} F. Me
^ ^ 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.
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, 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
,c o 2h reference spectrum of benzatropine mesilate (RS 026).
ch 3 B. T h e light absorption, Appendix II B, in the range 230 to
ch 3 350 nm of a 0.1% w/v solution in 2m hydrochloric acid
H02C I H exhibits two maxima, at 253 and 258 nm . T he absorbance at
n
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
D . (35,7i?,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7a-
picric arid solution R1 and allow to c o o l T he melting point of
tetrahydroimidazo [5,1 -b]thiazole-3,7~dicarboxylic a d d
the predpitate, after drying at 105°, is about 185°,
(penillic a d d o f benzylpenicillin),
Appendix V A.
TESTS
I.COzH
T ro p in e
h n - A .c h 3
C arry out the m ethod for thin-layer chromatography,
Appendix HI A, using silica gel G as the coating substance
o co 2h
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
E. (45)-2-[caiboxy[(phenylacetyl)amino]methyl]-5,5- acetone containing (1) 4.0% w/v of the substance being
dimethylthiazoüdine-4-carboxylic a d d (penicilloic ad d s of examined and (2) 0.020% w/v o f tropine. After removal o f the
benzylpenicillin), 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 ASSAY

th an the spot in the chrom atogram obtained with Dissolve 0 .6 g in 2 5 m L of water, add 5 m L o f dilute sodium
solution (2). carbonate solution and extract with four 10 m L quantities of
Related substances chloroform. Wash the combined extracts with 10 m L o f water,
Carry out the m ethod for liquid chromatography, extract the washings with 5 m L of chloroform and add the
Appendix DDE D , using the following solutions. For solution chloroform to the com bined extracts. Filter and wash the
(1) mix with the aid o f ultrasound 50 m g of the substance filter with 5 m L o f chloroform. T o the combined filtrate and
being examined with 15 m L o f mobile phase A, dilute to washings add 2 5 m L o f 1,4-dioxan and titrate with
50 m L with the same solvent and filter. For solution (2) 0 .1 m perchloric add F51using 0 .1 5 m L of a 0.1% w fv solution
dilute 1 volume of solution (1) to 100 volumes with mobile o f methyl red in methanol as indicator. Each m L of
phase A and further dilute 1 volume o f the resulting solution 0 .1 m perchloric add P'S is equivalent to 4 0 .3 5 m g of
to 5 volumes with the same solvent. F o r solution (3) mix C2iH25N0,CH403S.
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 ★ ★
Benzbromarone ★ ★
0.01% wfv each of benzatropine mesUate BPCRS and desmethyl
*****
benzatropine hydrochloride BPCRS in mobile phase A. (Ph. Eur. monograph 1393)
T he chromatographic procedure may be carried out using
h3c
(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.
Mobile phase A A mixture o f 5 volumes o f a 1m potassium
phosphate buffer prepared as described for mobile phase B, Ci7Hi2Br20 3 424.1 3562-84-3
20 volumes o f acetonitrUe and 75 volumes o f water.
Mobile phase B A mixture o f 35 volumes of water, Action and use
6 0 volumes o f acetonitrUe and 5 volumes of a 1m potassium Uricosuric; treatm ent of hyperuricaemia.
phosphate buffer prepared in the following m anner: dissolve
Ph Eur_____________________________________
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 D E F IN IT IO N
1 0 0 0 mL. (3j5-Dibromo-4-hydroxyphenyl)(2-ethylbenzofuran-3-
yl)methanone.
Tima Mobile phase A M obile phase B Comment

Content
(M inutes) (% v/v) (% W v)
0-20 70->30 30-»70 linear gradient CHARACTERS

20-30 30->0 70-+100 linear gradient
Appearance
30-55 0 100 isocratic
Solubility
55-65 70 30 isocratic
Practically insoluble in water, freely soluble in acetone and in
methylene chloride, sparingly soluble in ethanol
Inject 20 |iL of solution (4). T he test is not valid unless the (96 per cent),
resolution factor between the two principal peaks is at least 1. mp: about 152 °C.
If necessary adjust the concentration o f acetonitrile or adjust IDENTIFICATION
the time program of the linear gradient elution.
Inject separately 20 |iL o f mobile phase A as a blank and
Comparison benzbromarone CRS.
20 jiL each o f solutions (1), (2) and (3). In the
chromatogram obtained with solution (1) the area o f any B. By means of a copper wire, previously ignited, introduce a
peak corresponding to desmethyl benzatropine is n o t greater small am ount o f the substance to be examined into the non-
than the area o f the principal peak in the chromatogram luminous part o f a flame. T h e colour o f the flame becomes
obtained with solution (3) (0.5%), the area o f any other green.
secondary peak is not greater that the area o f the principal TESTS
peak in the chromatogram obtained with solution (2) (0.2%) Appearance o f solution
and the sum o f the areas of any such peaks is not greater T h e solution is clear (2.2. i) and n o t more intensely coloured
than 2.5 times the area o f the principal peak in the than reference solution Y5 (2.2.2, Method U).
chrom atogram obtained with solution (2) (0.5%). In solution Dissolve 1.25 g in dimethylformamide R and dilute to 25 m L
(1) disregard any peaks corresponding to the peaks in the with the same solvent.
chrom atogram obtained with the blank solution.
Loss on drying Shake 0.5 g with 10 m L o f carbon dioxide-free water R for
W hen dried to constant weight at 105°, loses not more than 1 m in and filter. T o 2.0 m L o f the filtrate add 0.1 m L of
5.0% o f its weight. U se 1 g. methyl red solution R and 0.1 m L o f 0.01 M hydrochloric add.
Sulfated ash T h e solution is red. Add 0.3 m L o f 0.01 M sodium hydroxide.
N ot more than 0.1%, Appendix IX A. T h e solution is yellow.
2016 Benzethonium Chloride 1-261
Related substances L oss o n d ry in g (2.2.32)

Liquid chromatography (2.2.29). M aximum 0.5 per cent, determined on 1.000 g by drying
Test solution Dissolve 0.125 g o f the substance to be in vacuo at 50 °C for 4 h.
examined in 30 m L of methanol R and dilute to 50.0 m L S u lfa te d a s h (2.4.14)
with the mobile phase. M axim um 0.1 per cent, determined on 1.0 g.
Reference solution (a) Dilute 1.0 m L of the test solution to ASSAY
100.0 m L with the mobile phase. Dilute 1.0 m L o f this Dissolve 0.300 g in 60 m L of methanol R. Stir until
solution to 10.0 m L with the mobile phase. completely dissolved and add 10 m L of water R. T itrate with
Reference solution (b) Dissolve 10 m g o f benzarone CRS 0.1 M sodium hydroxide, determining the end-point
(impurity C) in the mobile phase and dilute to 20 m L with potentiometrically (2.2.20).
the mobile phase. T o 5 m L of this solution add 1 m L of the 1 m L o f 0.1 M sodium hydroxide is equivalent to 42.41 mg
test solution and dilute to 100 m L with the mobile phase. o f C i 7H i 2Br 203 .
Column:
STORA GE
— sizer. I = 0.25 m , 0 = 4.6 mm;
— stationary phase: octadecylsQyl silica gel far chromatography R Protected from light.
(5 |im). IM P U R IT IE S
Mobile phase glacial acetic acid R, acetonitrUe R, water R, Specified impurities A, B.
methanol R (5:25:300:990 VIVIVIV). Other detectable impurities (the following substances would, if
Flow rate 1.5 mL/min. present at a sufficient level, be detected by one o r other o f
Detection Spectrophotom eter at 231 nm . the tests in the monograph. They are limited by the general
Injection 20 |iL.
Run time 2.5 times the retention time of benzbromarone. (2034). It is therefore n o t necessary to identify these
Relative retention W ith reference to benzbromarone: impurities for demonstration of compliance. See also 5.10.
impurity A = about 0.6; impurity B = about 2. Control of impurities in substances far pharmaceutical use): C.
impurity C (1st peak) and benzbromarone (2nd peak).
Limits:
— impurity A: n o t more than 4 times the area of the
— impurity B: n o t more than 10 times the area of the
principal peak in the chromatogram obtained with A. R l = R2 = H , R3 = Bn (3-bromo-4-
reference solution (a) ( 1.0 per cent); hydroxyphenyl) (2-ethylbenzofuran-3-yl)methanone,
— unspecified impurities: for each impurity, n o t more than the B. R l = R2 = R3 = Br. (6-bromo-2-ethylbenzofuran-3-
area of the principal peak in the chromatogram obtained yl) (3,5-dibromo-4-hydroxyphenyl)methanone,
with reference solution (a) ( 0.10 per cent); C. R l = R2 = R3 = H: (2-ethylbenzofuran-3-
— sum of impurities other than A and B: no t more than twice yl)(4-hydroxyphenyl)methanone (benzarone).
____________________________ :_____________________________ PhEur
obtained with reference solution (a) ( 0.2 per cent);
(0.02 per cent).
H a lid e s e x p ressed a s ch lo rid e s (2.4.4) Benzethonium Chloride
M axim um 400 ppm. (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
C 27H 42O N O 2 448.1 121-54-0
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. A ctio n a n d u se
Allow to cool. Rinse the crucible with water R , collect the Antiseptic.
rinsings and dilute to 25 m L with water R. Dilute 2 m L of
this solution to 10 m L with water R. PhEur__________________________________________________________
H eav y m e ta ls (2.4.8) D E F IN IT IO N
M axim um 20 ppm . N -B enzyl-N ^-dim ethyl-2- [2- [4-( 1,1,3,3-
0.5 g complies with test C. Prepare the reference solution tetramethylbutyl)phenoxy] ethoxy] ethanaminium chloride.
using 1 m L o f lead standard solution (10 ppm Pb) R. C o n te n t
1-262 Benzocaine 2016
CHARACTERS ASSAY
A p p e a ra n c e Dissolve 2.000 g in water R and dilute to 100.0 m L with the
W hite or yellowish-white powder. same solvent. Transfer 25.0 m L o f the solution to a
S o lubility separating funnel, add 10 m L of a 4 g/L solution o f sodium
Very soluble in water and in ethanol (96 per cent), freely hydroxide R, 10.0 m L of a freshly prepared 50 g/L solution of
soluble in methylene chloride. potassium iodide R and 25 m L o f methylene chloride R. Shake
vigorously, allow to separate and discard the lower layer.
An aqueous solution froths copiously when shaken.
Shake the upper layer with 3 quantities, each of 10 mT, of
ID E N T IF IC A T IO N methylene chloride R and discard the lower layers. T o the
A. Melting point (2.2.14): 158 °C to 164 °C, after drying at upper layer add 40 m L o f hydrochloric add R, allow to cool
105 °C for 4 h. and titrate with 0.05 M potassium iodate until the deep brown
B. Thin-layer chromatography (2.2.27). colour is almost discharged. Add 4 m L o f methylene chloride R
Test solution Dissolve 25 mg o f the substance to be examined and continue the titration, shaking vigorously, until the lower
layer is no longer brown. C any ou t a blank titration using a
in water R and dilute to 5 m L with the same solvent.
mixture of 10.0 m L o f a freshly prepared 50 g/L solution of
Reference solution Dissolve 25 m g of benzethonium chloride CRS potassium iodide R, 20 m L o f water R and 40 m L of
in water R and dilute to 5 m L with the same solvent.
hydrochloric acid R.
Plate TLC silica gel F2 5 4 plate R. 1 m L of 0.05 Mpotassium iodate is equivalent to 44.81 m g of
Mobile phase glacial acetic acid R, water R, methanol R C 27H 42CINO 2.
(5:5:100 V/V/V).
STORAGE
Application 20 pL.
PhEur
Drying In a current of warm air.
the test solution is similar in position and size to the principal ★ ★
spot in the chromatogram obtained with the reference Benzocaine ★ ★
solution. (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 'CH,
colour develops in the lower layer.
h2n
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 CçHuNOa 165.2 94-09-7
reaction (a) o f chlorides (2.3.1).
TESTS Local anaesthetic.
S o lu tio n S
Dissolve 5.0 g in carbon dioxide-free water R and dilute to PhEur.
50 m L with the same solvent. DEFINITION
Appearance o f solution Ethyl 4-aminobenzoate.
Solution S is clear (2.2.1) and not more intensely coloured Content
than reference solution Y6 (2.2.2, Method II). 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
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 Appearance
sodium hydroxide. T h e solution is pink. Add 0.1 m L of methyl
crystals.
red solution R and 0.5 m L o f 0.01 M hydrochloric acid
T he solution is orange-red. Solubility
Very slightly soluble in water, freely soluble in ethanol
V olatile b a se s a n d sa lts o f v o latile b ase s (2.4.1,
Method B) (96 per cent).
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 IDENTIFICATION
solution (100 ppm N H J R. Replace heavy magnesium oxide Infrared absorption spectrophotometry (2.2.24).
by 2.0 m L o f strong sodium hydroxide solution R. Comparison benzocaine CRS.
L oss o n d ry in g (2.2.32) If the spectra obtained show differences, dissolve the
M aximum 5.0 per cent, determined on 1.000 g by drying in substance to be examined and the reference substance
an oven at 105 °C for 4 h. separately in anhydrous ethanol R, evaporate to dryness and
S u lfa te d a s h (2.4.14) record new spectra using the residues.
Maximum 0.1 per cent, determined on 1.0 g. TESTS
Related substances
2016 Benzocaine 1-263
Solvent mixture acetonitrile R l, waterfor chromatography R - IM P U R IT IE S

(50:50 V/V). Other detectable impurities (the following substances would, if
Test solution Dissolve 25.0 mg of the substance to be present at a sufficient level, be detected by one or other of
examined in 5 m L of acetonitrile R l and dilute to 50.0 m L the tests in the monograph. They are limited by the general
with the solvent mixture. acceptance criterion for other/unspecified impurities and/or
Reference solution (a) Dilute 1.0 m L of the test solution to by the general monograph Substances for pharmaceutical use
100.0 m L with the solvent mixture. Dilute 1.0 m L of this (2034). It is therefore n o t necessary to identify these
solution to 10.0 m L with the solvent mixture. impurities for demonstration of compliance. See also 5.10.
Reference solution (b) Dissolve 5 mg of the substance to be C, D, E, F, G, H.
examined and 5 mg of 4-rdtrobenzoic acid R (impurity E) in
10.0 m L of the solvent mixture. Dilute 1.0 m L o f the
Column:
— sizer. I = 0.10 m, 0 = 4.6 mm;
— stationary phase: end-capped octadecylsilyl silica gel for
A. (4-aminophenyl)methanol,
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;
B. (2-aminophenyl)methanol,
mix 9 volumes of this solution and 1 volume of
acetonitrile R l ; o
— mobile phase B: acetonitrile Rl;
Tune Mobile phase A Mobile phase B

NHj
0 -2 100 0
2 - 15 100 -> 38.5 0 61.5
C . ethyl 3-aminobenzoate,
Flow rate 1.5 mL/min. o

Injection 10 (iL.
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):
impurity E and benzocaine.
E . 4-nitrobenzoic ad d ,
— for each impurity, use the concentration of benzocaine in
Limits:
nh2
0.10 per cent;
— reporting threshold: 0.05 per cent. F . (3-aminophenyl)methanol,
in vacuo.
G. 4-aminobenzoic acid,
A SSA Y
Carry out the determination of primary aromatic amino- o
nitrogen (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.
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 H . methyl 4-aminobenzoate.
Protected from light. __________________________________________________________PhEur
1-264 Benzoic Acid 2016
*** , washings to 25.0 m L with water R. T his solution is used to

★ ★
Benzoic Acid ★ ★ preparer solution A.
*****
(Ph. Eur. monograph 0066) 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
Ç T * 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)
C 7H 0O 2 122.1 65-85-0 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
A c tio n a n d u se swirling 2 m L o f nitric add R and 5 m L o f mercuric
Antimicrobial preservative. thiocyanate solution R. Shake. Dilute the contents o f each flask
P re p a r a tio n s to 25.0 m L with water R and allow the solutions to stand in
C om pound Benzoic A d d O intm ent a water-bath at 20 °C for 15 min. M easure a t 460 n m the
Benzoic Acid Solution absorbance (2.2.25) o f solution A using solution B as the
compensation liquid, and the absorbance o f solution C using
PhEur. 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
D E F IN IT IO N
greater than that o f solution C.
Benzenecarboxyiic ad d .
C o n te n t
M axim um 10 ppm .
12 m L o f solution S complies with test B. Prepare the
CHARACTERS reference solution using a mixture o f 5 m L o f lead standard
A p p e a ra n c e solution (1 ppm Pb) R and 5 m L of ethanol (96 per cent) R.
W hite or alm ost white, crystalline pow der or colourless
crystals.
M aximum 0.1 p er cent, determined on 1.0 g.
S o lu b ility
Slightly soluble in water, soluble in boiling water, freely A SSA Y
soluble in ethanol (96 per cent) and in fatty oils. 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
ID E N T IF IC A T IO N red solution R as indicator, until the colour changes from
A. M dting point (2.2.14): 121 °C to 124 °C. yellow to violet-red.
B. Solution S (see Tests) gives reaction (a) of benzoates 1 m L of 0.1 M sodium hydroxide is equivalent to 12.21 m g of
(23.1). C 7H 6O 2.
TESTS ___________________________________________________________PhEir
S o lu tio n S
A p p e a ra n c e o f so lu tio n ★ ★
Hydrous Benzoyl Peroxide ★ ★
* * * * *
C a rb o n is a b le su b sta n c e s (Ph. Eur. monograph 0704)
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).
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 C 14H 10O4 242.2 94-36-0
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
U sed topically in the treatm ent of acne.
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 P re p a r a tio n s
glassware be reservedfor this test Benzoyl Peroxide Cream
Solution (a) Dissolve 6.7 g in a mixture o f 40 m L o f 1 M Benzoyl Peroxide G d
sodium hydroxide and 50 m L o f ethanol (96 per cent) R and Benzoyl Peroxide Lotion
dilute to 100.0 m L with water R. T o 10.0 mT. o f this solution Potassium Hydroxyquinoline Sulfate and Benzoyl Peroxide
add 7.5 m L o f dilute sodium hydroxide solution R and 0.125 g Cream
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 PhEur.
25 m L volumetric flask and wash w ith 3 quantities, each of D E F IN IT IO N
2 m L , o f ethanol (96 per cent) R. Dilute the filtrate and C o n te n t
<— dibenzoyl peroxide: 70.0 per cent to 77.0 p er cent;
2016 Benzoyl Peroxide 1-265
— water, m inim um 20.0 per cent. phase. Dilute 1.0 m L of the solution to 10.0 mT. with the
mobile phase.
CHARACTERS
Appearance Reference solution (c) Dissolve 50.0 mg of ethyl benzoate R in
White o r alm ost white, amorphous or granular powder. the mobile phase and dilute to 100.0 m L with the mobile
phase. Dilute 1.0 m L o f the solution to 100.0 m L with the
S o lu b ility mobile phase.
Practically insoluble in water, soluble in acetone, soluble in
methylene chloride with the separation of water, slightly
Reference solution (d) Dissolve 50.0 mg of benzaldehyde R in
It loses w ater rapidly on exposure to air with a risk of
mobile phase.
explosion.
Reference solution (e) Dissolve 30.0 mg of benzoic add R and
Mix the entire sample thoroughly before carrying out the following 30.0 m g of benzaldehyde R in the mobile phase and dilute to
tests. 100.0 m L with the mobile phase. Dilute 1.0 m L of the
ID E N T IF IC A T IO N solution to 10.0 m L with the mobile phase.
First identification B Column:
Second identification A y C, D. — size: I = 0.25 m , 0 = 4.6 mm;
A. U ltraviolet and visible absorption spectrophotometry — stationary phase: octadecylsüyl silica gel for chromatography R
(10 |im).
(2.2.25).
Solution A Dissolve 80.0 mg in ethanol (96 per cent) R and Mobile phase glacial acetic acid R, acetonitrüe R, water R
dilute to 100.0 m L with the same solvent. Dilute 10.0 m L o f (1:500:500 V/V/V).
the solution to 100.0 m L with ethanol (96 per cent) R. Flow rate 1 mL/min.
Solution B D ilute 10.0 m L of solution A to 100.0 m L with Detection Spectrophotometer at 235 nm.
ethanol (96 per cent) R. Injection 20 |iL loop injector.
Spectral ranges 250-300 n m for solution A; 220-250 nm for Run time 2 times the retention time of dibenzoyl peroxide.
solution 6 . Relative retention W ith reference to dibenzoyl peroxide
Absorption maxima A t 274 nm for solution A; at 235 nm for (retention time = about 28.4 min): impurity B = about 0.15;
solution B. impurity A = about 0.2; impurity C = about 0.4.
Shoulder A t about 282 nm for solution A. System suitabüity: reference solution (e):
Absorbance ratio A 2 3 5 /A 2 7 4 = 1.17 to 1.21. — resolution: minimum 6 between the peaks due to benzoic
acid and benzaldehyde.
Limits:
Comparison Ph. Eur. reference spectrum of hydrous benzoyl
— impurity A: n o t m ore than the area of the principal peak
peroxide.
in the chromatogram obtained with reference solution (d)
C. Dissolve about 25 m g in 2 m L o f acetone R. Add 1 m L o f (0.25 per cent);
a 10 g/L solution o f diethylphenylenediamme sulfate R and mix. — impurity B: not more than the area of the principal peak
A red colour develops which quickly darkens and becomes in the chromatogram obtained with reference solution (b)
dark violet within 5 mm. (1.5 per cent);
D . T o 1 g add 5 mT. of ethanol (96 per cent) R, 5 m L of — impurûy C: not more than the area of the principal peak
dilute sodium hydroxide solution R and 10 m L of water R Boil in the chromatogram obtained with reference solution (c)
the m ix ture under reflux for 20 min. Cool. T he solution (0.25 per cent);
gives reaction (c) o f benzoates (2.3.1). — unspecified impurities: for each impurity, not m ore than the
TESTS area of the principal peak in the chromatogram obtained
A cid ity with reference solution (a) (0.10 per cent);
Dissolve a quantity o f the substance to be examined — disregard limit:. 0.2 times the area of the principal peak in
containing the equivalent o f 1.0 g o f dibenzoyl peroxide in the chromatogram obtained with reference solution (a)
25 m L o f acetone R, add 75 m L of water R and filter. Wash (0.02 per cent).
the residue with two quantities, each of 10 mL, o f water R. Chlorides (2A. 4)
Com bine the filtrate and the washings and add 0.25 m L of M aximum 0.4 per cent.
phenolphthalein solution R l. N o t more than 1.25 m L of 0.1 M Dissolve a quantity of the substance to be ex a m in ed
sodium hydroxide is required to change the colour of the containing the equivalent of 0.5 g of dibenzoyl peroxide in
indicator. Carry out a blank test. 15 m L of acetone R. Add, while stirring, 50 m L o f 0.05 M
Related substances nitric acid. Allow to stand for 10 min and filter. W ash the
Liquid chrom atography (2.2.29). Prepare the solutions residue with 2 quantities, each of 10 mL, of 0.05 M nitric
immediately before use. acid. Combine the filtrate and the washings and dilute to
Test solution Dissolve a quantity of the substance to be 100 m L with 0.05 M nitric acid. Dilute 2.5 m L of the
examined containing the equivalent of 0.10 g of dibenzoyl solution to 15.0 m L w ith water R.
peroxide in acetonitrüe R and dilute to 50 m L with the same A SSAY
solvent. Solution (a) Dissolve 2.500 g immediately before use in
Reference solution (a) Dilute 1.0 m L of the test solution to 75 m L of dimethylformamide R and dilute to 100.0 m L with
100.0 m L with acetonitrüe R. D ilute 1.0 m L of this solution the same solvent.
to 10.0 m L with acetonitrüe R. Dibenzoyl peroxide
Reference solution (b) Dissolve 30.0 m g of benzoic acid R in T o 5.0 m L of solution (a) add 20 m L of acetone R and 3 m L
the m obile phase and dilute to 100.0 m L with the m obile, o f a 500 g/L solution o f potassium iodide R and mix. Allow to
1-266 Benzydamine Hydrochloride 2016
stand for 1 m in. T itrate with 0.1 M sodium tkiosulfate using D E F IN IT IO N

1 m L of starch solution R, added towards die end o f die Benzydamine Hydrochloride is 3-( 1-benzylindazol-3-
titration, as indicator. Carry o u t a blank titration. yloxy)propyldimethylamine hydrochloride. It contains n o t less
1 m L o f 0.1 M sodium tkiosulfate is equivalent to 12.11 mg of than 99.0% and not m ore than 101.0% o f C i9H 23N30 ,H C l,
Q 4H 10O 4. calculated with reference to the dried substance.
W a te r (2.5.12) P R O D U C T IO N
Carry out the semi-micro determination o f water, using T h e m ethod o f manufacture is such that the level o f
5.0 m L o f solution (a). Use as die solvent a mixture o f 3-chloropropyl(dimethyl)amine hydrochloride is n o t more
20.0 m L of anhydrous methanol R and 3.0 m L o f a 100 g/L than 5 ppm when d eterm in ed by a suitable method.
solution o f potassium iodide R in dimethylformamide R. After
CHARACTERISTICS
adding solution (a), stir for 5 m in before starting the
A white crystalline powder.
titration. C a n y out a blank determination.
Very soluble in water, freely soluble in ethanol (96%);
Calculate the percentage content o f w ater using the following
expression:
(n i — n a ) x w x 2 A. T h e infrared absorption spectrum, Appendix II A, is
+ ( p x 0.0744) concordant with the reference spectrum of b en zyd am in e
m
hydrochloride (RS 027).
«1 n um b er o f millilitres o f iodosidfurous reagent R used in B. Yields reaction A characteristic o f chlorides, Appendix VI.
the sample determination,
TESTS
«2 num ber of millilitres o f iodosutfurous reagent R used in
Clarity and colour o f solution
the blank determination,
A 10.0% w/v solution is dear, Appendix IV A, and not more
water equivalent of iodosutfurous reagent R in
intensely coloured than reference solution Y6, Appendix IV B,
milligrams o f w ater per millilitre o f reagent,
M ethod II.
mass o f the substance to be examined used for the
preparation o f solution (a) in grams, A c id ity
P = percentage content o f dibenzoyl peroxide. p H o f a 10% w/v solution, 4.0 to 5.5, A ppendix V L.
Heavy metals
ST O R A G E
12 m l . o f a 10% w/v solution complies with limit test A for
In a container that has been treated to reduce static discharge
heavy metals, Appendix VII (10 ppm ). Use 1 m L o f lead
and that has a device for release o f excess pressure, at a
standard solution (10 ppm Pb) to prepare the standard.
tem perature o f 2 °C to 8 °C, protected from light.
Related substances
IM P U R IT IE S
Carry out the m ethod for liquid chromatography,
Appendix IQ D , using the following solutions in methanol
(50%). Solution (1) contains 0.25% w/v o f the substance
b ein g examined. Solution (2) contains 0.0005% w/v o f
o 1 - 3-dimethylaminopropyl 2-benzylaminobenzoate
hydrochloride BPCRS (impurity A) and 0.00125% w/v of
A. R = H: benzaldehyde, 3-(l,5dibenzyl-lH-mdazole-3-yl)oxypropyldimethylamirie
hydrochloride BPCRS (impurity B). Solution (3) contains
B. R = OH: benzoic acid,
0.00025% w/v o f 1-benzyl-lH-mdazol-3-ol BPCRS
C. R = 0 -C H 2-C H 3: ethyl benzoate. (impurity C). Solution (4) contains 0.00025% w/v o f the
PhEur substance being examined. Solution (5) contains equal
volumes o f solutions (1), (2) and (3).
T h e chromatographic procedure may be carried out using a
stainless steel colum n (25 cm x 4.6 mm ) packed with end-
capped octadecylsUyl silica gelfor chromatography (5 fun)
Benzydamine Hydrochloride (Suplex pK B-100 is suitable), fitted with a stainless steel
guard colum n (2 cm x 4.6 mm ) packed with the same
m aterial U se as the initial mobile phase with a flow rate of
1.5 m L per m inute, a m ixture o f 50 volumes o f solution A
and 50 volumes o f methanol. Solution A contains
0.01m potassium dihydrogen orthophosphate and 0.005m sodium
octyl sulfate in water, adjusted to p H 3.0 ± 0 . 1 with
orthophosphoric add Carry out a linear gradient elution
increasing the percentage o f methanol to 70% over
20 m inutes from die m om ent of injection and then
C19H23N3OJHCI 132-69-4 decreasing the percentage o f methanol to 50% over 2 minutes
and maintain the final mobile phase until the completion of
Action and use th at run. U se a detection wavelength o f 320 nm .
Cyclo-oxygenase inhibitor; analgesic; anti-inflam m atory
Inject 20 jiL of solution (5) and modify the rate o f change o f
Preparations the mobile phase, if necessary, to obtain a retention time of
Benzydamine Cream about 10 m inutes for the substance being examined.
Benzydamine M outhwash
Benzydamine Oromucosal Spray
2016 Benzyl Alcohol 1-267
T he test is not valid unless, in the chromatogram obtained

Benzyl Alcohol ★* * * ★
with solution (5), the resolutionfactor between any two ★ ★
adjacent peaks is at least 2.5. (Ph. Eur. monograph 0256) *****
Inject 20 |iL of solution (1) and allow the chromatography to
proceed for 30 m in u tes. In the chromatogram obtained the
areas o f any peak corresponding to impurity A o r impurity B
is not greater than the area of the corresponding peak in the
CT”
chromatogram obtained with solution (2) (0.2% of
impurity A and 0.5% o f impurity B), the area o f any peak CjU80 108.1 100-51-6
corresponding to impurity C is not greater than the area of
the peak in the chromatogram obtained with solution (3) A c tio n a n d u se
(0.1%) and the area of any other secondary peak is not greater Local anaesthetic; disinfectant.
than the area of the peak in the chromatogram obtained with
PhEur.
solution (4) (0.1%). T he sum of the areas of any sudh peaks
is not greater than 1%. D E F IN IT IO N
P rim a ry a m in e s Phenylmethanol.
Dissolve 50 mg o f the substance being examined in 10 m L of C o n te n t
ethanol (96%), add 0.1 m L o f hydrochloric acid and 2 m L o f a 98.0 per cent to 100.5 per cent.
5% w/v solution o f 4-dimethylaminobenzaldehyde in ethanol
CHARACTERS
(96%). T h e yellow colour obtained is not m ore intense than
A p p e a ra n c e
that obtained by treating 10 m L o f a 0.00005% w/v solution
Clear, colourless, oily liquid.
of 2-aminobenzoic add in ethanol (96%) in the same manner.
S o lu b ility
Loss o n d ry in g
Soluble in water, m isdble with ethanol (96 per cent) and
W hen dried for 3 hours at 100° to 105° at a pressure not
with fatty and essential oils.
exceeding 0.7 kPa, loses not more than 0.5% o f its weight.
Use 1 g. R e lativ e d e n sity
1.043 to 1.049.
S u lfated a sh
N ot more than 0.1% , Appendix IX A. Use 1 g. ID E N T IF IC A T IO N
ASSAY
Dissolve 0.3 g in 100 m L o f anhydrous acetic add and carry Comparison benzyl alcohol CRS.
out M ethod I for rum-aqueous titration, Appendix VIII A, TESTS
determining the end point potentiometrically. Each m L o f A p p e a ra n c e o f so lu tio n
0.1 m perchloric add FS is equivalent to 34.59 m g o f Shake 2.0 m L with 60 m L of water R. It dissolves
Ci9H23N 30 ,H C l. completely. T he solution is clear (2.2.1) and colourless
IM P U R IT IE S (2.2.2, Method II).
A cid ity
T o 10 m L add 10 m L of ethanol (96per cent) R and 1 m l. of
phenolphthalein solution R. N ot m ore than 1 m L of 0.1 M
sodium hydroxide is required to ch an ge the colour of the
indicator to pink.
R e fra c tiv e in d e x (2.2.6)
1.538 to 1.541.
P e ro x id e v alu e (2.5.5)
M axim um 5.
A. 3-dimethylaminopropyl 2-benzylaminobenzoate, Related substances
Test solution T h e substance to be examined.
Standard solution (a) Dissolve 0.100 g o f ethylbenzene R in the
test solution and dilute to 10.0 m L with the same solution.
Dilute 2.0 m L of this solution to 20.0 m L with the test
solution.
O v NMe2 Standard solution (b) Dissolve 2.000 g o f dicydohexyl R in the
test solution and dilute to 10.0 m L with the same solution.
B. 3-( 1j5-dibenzyl-1H-indazol-3-yl)oxypropyldimethylamine, Dilute 2.0 m L o f this solution to 20.0 m L with the test
solution.
Reference solution (a) Dissolve 0.750 g o f benzaldehyde R and
0.500 g of cydohexyhnethanol R in the test solution and dilute
to 25.0 m L with the test solution. Add 1.0 m L of this
solution to a mixture o f 2.0 m L o f standard solution (a) and
3.0 m L o f standard solution (b) and dilute to 20.0 m L with
the test solution.
Reference solution (b) Dissolve 0.250 g o f benzaldehyde R and
C. 1-benzyl- liî-indazol-3-ol. 0.500 g o f cycbhexyhnethanol R in the test solution and dilute
1-268 Benzyl Alcohol 2016
to 25.0 m L with the test solution. A dd 1.0 m L o f this — disregard litrtir. 0.01 times the area o f the peak due to
solution to a mixture o f 2.0 m L o f standard solution (a) and ethylbenzene in the chrom atogram obtained with
2.0 m L of standard solution (b) and dilute to 20.0 m L with reference solution (a) corrected if necessary as
the test solution. described above (0.0001 p er cent).
Column: Benzyl alcohol intendedfor parenteral administration
— material: fused silica; Injection W ithout air-plug, 0.1 nL o f the test solution and
— size: / = 30 m, 0 = 0.32 m m ; reference solution (b).
Relative retention W ith reference to benzyl alcohol (retention
0.5 nm).
time = about 26 min): ethylbenzene = about 0.28;
Carrier gas helium for chromatography R. dicyclohexyl = about 0.59; impurity A = about 0.68;
Linear velocity 25 cm/s. impurity B = about 0.71.
Temperature: System suitability: reference solution (b):
Time Temperature
impurities A and B.
(min) CC) If any peaks in the chrom atogram obtained with the test
Column 0 - 34 50*220 solution have die same retention times as the peaks due to
ethyl benzene o r dicyclohexyl, substract the areas o f any such
3 4 -6 9 220
peaks from die peak areas at these retention times in the
Injection port 200 chromatograms obtained with reference solutions (a) o r (b)
Detector 310 (corrected peak areas of ethyl benzene and dicyclohexyl).
Any such peaks in the chrom atogram obtained with the test
solution are to be included in the assessments for the sum of
Detection Flam e ionisation. other peaks.
Benzyl alcohol not intended for parenteral administration Limits:
Injection W ithout air-plug, 0.1 jtL o f the test solution and — impurity A: n o t m ore than the difference between the
reference solution (a). area o f die peak due to impurity A in the
Relative retention W ith reference to benzyl alcohol (retention chrom atogram obtained w ith reference solution (b)
time = about 26 min): ethylbenzene = about 0.28; and the area o f the peak due to impurity A in the
dicyclohexyl = about 0.59; im purity A = about 0.68; chrom atogram obtained w ith the test solution
impurity B = about 0.71. (0.05 per cent);
— impurity B: n o t m ore than the difference between the
area o f die peak due to impurity B in the
impurities A and B.
and the area o f the peak due to impurity B in the
If any peaks in the chrom atogram obtained with the test chrom atogram obtained with the test solution
solution have the sam e retention tim e as the peaks due to (0.10 p er cent);
ethyl benzene or dicyclohexyl, substract the areas o f any such — sum of other peaks with a relative retention less than that
peaks from the peak areas at these retention times in the of benzyl alcohol: n o t m ore than twice the area of the
chromatograms obtained with reference solutions (a) o r (b) peak due to ethylbenzene in the chromatogram
(corrected peak areas o f ethyl benzene and dicyclohexyl). obtained with reference solution (b) corrected if
Any such peaks in the chrom atogram obtained w ith the test necessary as described above (0.02 p er cent);
solution are to be included in die assessments for the sum of — sum of peaks with a relative retention greater than that of
other peaks. benzyl alcohol: n o t m ore than the area o f the peak due
Limits: to dicyclohexyl in the chrom atogram obtained with
— impurity A: n o t m ore than the difference betw een the reference solution (b) corrected if necessary as
area o f the peak due to impurity A in the described above (0.2 per cent);
chrom atogram obtained with reference solution (a) — disregard limit: 0.01 times the area o f the peak due to
and the area o f the peak due to impurity A in the ethylbenzene in the chrom atogram obtained with
chrom atogram obtained w ith the test solution reference solution (b) corrected if necessary as
(0.15 per cent); described above (0.0001 per cent).
— impurity B: n o t m ore than the difference Jbetween the
Residue on evaporation
area o f the peak due to impurity B in the
M aximum 0.05 p er cent.
and the area o f the peak due to impurity B in the After ensuring th at th e substance to be examined complies
chrom atogram obtained with the test solution with the test for peroxide value, evaporate 10.0 g to dryness
(0.10 p er cent); in a tared quartz o r porcelain crucible or platinum dish on a
— sum of other peaks with a relative retention less than that hot plate a t a tem perature n o t exceeding 200 °C. Ensure that
of benzyl alcohol: no t m ore than 4 times die area of the the substance to be examined does no t boil during
peak due to ethylbenzene in the chromatogram evaporation. D ry the residue on the ho t plate for 1 h and
obtained with reference solution (a) corrected if allow to cool in a desiccator. T h e residue weighs a maximum
necessary as described above (0.04 per cent); of 5 mg.
— sum of peaks with a relative retention greater than that of ASSAY
benzyl alcohol: n o t m ore than the area o f the peak due T o 0.900 g ( m g ) add 15.0 m L o f a freshly prepared mixture
to dicyclohexyl in the chrom atogram obtained with of 1 volume o f acetic anhydride R and 7 volumes o f anhydrous
reference solution (a) corrected if necessary as pyridine R and heat u n d er a reflux condenser on a boiling
described above (0.3 p er cent);
2016 Benzyl Hydroxybenzoate 1-269
water-bath for 30 min. Cool and add 25 m L o f water R. ID E N T IF IC A T IO N

Using 0.25 m L o f phenolphthalein solution R as indicator, First identification A.
titrate with 1 M sodium hydroxide (nj mL). C any out a blank Second identification B, C.
titration (n 2 mL).
Calculate the percentage content of CyHgO using the
Comparison Ph. Eur. reference spectrum of benzyl benzoate.
B. T o 2 g add 25 m L o f alcoholic potassium hydroxide
10.81 ( n a — n j
solution R and boil under a reflux condenser for 2 h. Remove
the ethanol on a water-bath, ad d 50 m L of water R and
m distill. Collect about 25 mT. o f distillate and use it for
identification test C. Acidify th e liquid remaining in the
STORAGE distillation flask with dilute hydrochloric acid R. A white
In an airtight container, under nitrogen, protected from light precipitate is formed that, when washed with water R and
and at a tem perature between 2 °C and 8 °C. dried in vacuo melts ( 2.2.14) a t 121 °C to 124 °C.
L A B E L L IN G C. T o the distillate obtained in identification test B add 2.5 g
T he label states, where applicable, that the substance is o f potassium permanganate R and 5 m L o f dilute sodium
suitable for use in the manufacture o f parenteral hydroxide solution R. Boil under a reflux condenser for
preparations. 15 min, cool and filter. Acidify the filtrate with dilute
hydrochloric acid R. A white precipitate is formed that, when
IM P U R IT IE S
washed with water R and dried in vacuo, melts (2.2.14) at
Specified impurities: A, B. 121 °C to 124 °C.
CHO TESTS
A cid ity
Dissolve 2.0 g in ethanol (96 per cent) R and dilute to 10 m l,
with the same solvent. Titrate with 0.1 M sodium hydroxide
A. benzaldehyde, using phenolphthalein solution R as indicator. N ot more than
0.2 m L is required to change th e colour o f the indicator to
OH
pink.
R elativ e d en sity (2.2.5)
1.118 to 1. 122.
B. cyclohexylmethanol.
R efractiv e in d e x (2.2.6)
PhEur
1.568 to 1.570.
F re e z in g p o in t (2.2.18)
M inim um 17.0 °C.
* * *★
★ S u lfa ted a sh (2. 4.14)
Benzyl Benzoate ★ ★
ASSAY
T o 2.000 g add 50.0 m L of 0.5 M alcoholic potassium
hydroxide and boil gently under a reflux condenser for 1 h.
T itrate the hot solution with 0.5 M hydrochloric acid using
1 m L of phenolphthalein solution R as indicator. Carry out a
blank determination.
C 14H 12O 2 212.2 120-51-4 1 m L o f 0.5 M alcoholic potassium hydroxide is equivalent to
106.1 mg of C 14H 120 2.
A ction a n d u se
Used topically in the treatm ent of scabies.
STORAGE
In an airtight, well-filled container, protected from light.
P r e p a r a tio n
PhEur
Benzyl Benzoate Application
PhEti.
D E F IN IT IO N
Phenylmethyl benzoate. Benzyl Hydroxybenzoate
C o n te n t Benzylparaben
CHARACTERS
A p p e a ra n c e
Colourless or almost colourless crystals or colourless or
almost colourless, oily liquid.
S o lubility
Practically insoluble in water, miscible with ethanol
C 14H 12O3 228.3 94-18-8
(96 per cent), with methylene chloride and with fatty and
essential oils. A ctio n a n d u se
Eb: about 320 °C. Antimicrobial preservative.
1-270 Benzylpenicillin Potassium 2016
D E F IN IT IO N
Benzylpenicillin Potassium *****
Benzyl Hydroxybenzoate is benzyl 4-hydroxybenzoate.
It contains not less than 99.0% and n o t more than 101.0% (Ph. Eur. monograph 0113) *
of C 14H 12O 3.
0 H1 C O jK
Y - N '''\.C H 3
A white to creamy white, crystalline powder.
Practically insoluble in water, freely soluble in ethanol (96%) n - - |—4 ^s ch3
H H
and in ether. It dissolves in solutions o f alkali hydroxides.
A. T h e infrared absorption spectrum, Appendix II A, is Ci6H 17KN 20 4S 372.5 113-98-4
concordant with the reference spectrum of benzyl
hydroxybenzoate (RS 028). A c tio n a n d u se
B. T h e light absorption, Appendix II B, in the range 230 to Penicillin antibacterial.
350 nm of a 0.001% w/v solution in ethanol (96%) exhibits a P r e p a r a tio n
m axim um only at 260 nm . T h e absorbance at the maximum Benzylpenicillin Injection
at 260 nm is about 0.76.
C. Dissolve 0.1 g in 2 m L of ethanol (96%), boil and add
PhEur___________________________________________________
0.5 m L of nitric add solution of mercury. A precipitate is D E F IN IT IO N
produced slowly and the supernatant liquid becomes red. Potassium (2S,5Ä,6.R)-3,3-dimethyl-7-oxo-6-
D. Melting point, about 112°, Appendix V A. [(phenylacetyl) amino] -4-thia-1-azabicydo [3.2.0] heptane-2-
TESTS carboxylate.
A cid ity Substance produced by the growth of certain strains of
Dissolve 0.2 g in 10 m L of ethanol (50%) previously PenicUHum notation or related organisms, or obtained by any
neutralised to methyl red solution and titrate with 0 .1 m sodium other means.
hydroxide KS using methyl red solution as indicator. N o t more C o n te n t
than 0.1 m L of 0. 1m sodium hydroxide VS is required to 96.0 per cent to 102.0 per cent (dried substance).
change the colour o f the solution.
CHA RACTERS
A p p e a ra n c e
Carry out the m ethod for thin-layer chromatography,
Appendix IH A, using a plate precoated with silica gel F 254j
the surface o f which has been modified with chemically- S o lu b ility
bonded octadecylsilyl groups (W hatm an K C18F plates are Very soluble in water, practically insoluble in fatty oils and in
suitable) and a mixture o f 70 volumes o f methanol, liquid paraffin.
30 volumes o f water and 1 volum e o f glacial acetic add as the ID E N T IF IC A T IO N
mobile phase. Apply separately to the plate 2 |iL o f each of First identification A , D
two solutions o f the substance being examined in acetone
containing (1) 1.0% w/v and (2) 0.010% w/v. After removal
of the plate, allow it to dry in air and examine under A. Infrared absorption spectrophotometry (2.2.24).
ultraviolet light (254 nm). Any secondary spot in the Comparison benzylpemdOin potassium CRS.
chrom atogram obtained with solution ( 1) is not more intense B. Thin-layer chromatography (2.2.27).
than the spot in the chrom atogram obtained with Test solution Dissolve 25 mg of the substance to be examined
solution. (2). in 5 m l, of water R.
Sulfated ash
Reference solution (a) Dissolve 25 m g of benzylpenidllin
N ot m ore than 0.1%, A ppendix IX A. potassium CRS in 5 m L of water R
A SSA Y Reference solution (b) Dissolve 25 m g of benzylpenicHlin
Gently boil 0.12 g under a reflux condenser with 20 m L of potassium CRS and 25 m g o f phenoxymethylpemdUin
2m sodium hydroxide for 30 minutes. Cool and extract with potassium CRS in 5 mT. o f water R
three 20-m L quantities o f 1,2-dichhroethane. Wash the
com bined extracts with 20 m L o f 0.1m sodium hydroxide and
. add the washings to the m ain aqueous phase, discarding the Mobile phase Mix 30 volumes of acetone R and 70 volumes of
organic layer. T o the aqueous solution add 25 m L of a 154 g/L solution of ammonium acetate R previously adjusted
0.0333m potassium bromate VS, 5 m L o f a 12.5% w/v solution to p H 5.0 with glacial acetic acid R.
of potassium bromide and 10 m L o f hydrochloric add and Application 1 |iL.
immediately stopper the flask. Shake for 15 minutes and Development Over a path o f 15 cm.
allow to stand for 15 minutes. A dd 25 m L o f dilute potassium Drying In air.
iodide solution and shake vigorously. T itrate the liberated
iodine with 0 .1 m sodium thiosulfate VS using starch mucilage,
added towards the end of the titration, as indicator. Repeat
the operation w ithout the substance being examined. System suitability: reference solution (b):
T he difference between the titrations represents the am ount — the chromatogram shows 2 clearly separated spots.
of potassium brom ate required. T he volume of Results T he principal spot in the chromatogram obtained with
0 .0 3 3 3 m potassium bromate VS used is equivalent to h alf of the test solution is similar in position, colour and size to the
the volum e o f 0 . 1m sodium thiosulfate VS required for the principal spot in obtained with reference solution (a).
titration. Each m L o f 0 .0 3 3 3 m potassium bromate VS is C. Place about 2 m g in a test-tube about 150 m m long and
equivalent to 7.608 m g o f Q 4H 12O 3. 15 m m in diameter. Moisten with 0.05 m L o f water R and
2016 Benzylpenicillin Potassium 1-271
add 2 m L o f sulfuric acid-formaldehyde reagent R. Mix the If the mobile phase composition has been adjusted to achieve
contents of the tube by swirling; the solution is practically the required resolution, the adjusted composition will apply
colourless. Place the test-tube on a water-bath for 1 min; at time zero in the gradient and in the assay.
a reddish-brown colour develops. Flow rate 1.0 mL/min.
D . It gives reaction (a) of potassium (2.3.1). Detection Spectrophotometer at 225 nm.
TESTS Injection 20 jiL o f reference solutions (b) and (c) with
pH (2.2.3) isocratic elution at the initial mobile phase composition and
5.5 to 7.5. 20 nL o f test solution (b) according to the elution gradient
Dissolve 2.0 g in carbon dioxide-free water R and dilute to described under Mobile phase; inject water R as a blank
20 m L with the same solvent. according to the elution gradient described under Mobile
phase.
+ 270 to + 300 (dried substance). System suitability: reference solution (b):
impurity B and benzylpenicillin; if necessary, adjust the
ratio A:B o f the mobile phase.
Absorbance (2.2.25)
Limit:.
Dissolve 94.0 mg in water R and dilute to 50.0 m L with the
— any impurity: for each impurity, not more than the area of
same solvent. Measure the absorbance of the solution at
325 nm , 280 nm and at the absorption maximum at 264 nm,
diluting the solution, if necessary, for the measurement at
264 nm . T h e absorbances at 325 nm and 280 nm do not Loss on drying (2.2.32)
exceed 0.10 and that at the absorption maximum at 264 nm M axim um 1.0 per cent, determined on 1.000 g by drying in
is 0.80 to 0 .88, calculated on the basis o f the undiluted an oven at 105 °C.
(1.88 g/L) solution. Verify the resolution o f the apparatus Bacterial endotoxins (2.6.14, Method E)
(2.2.25); the ratio o f the absorbances is at least 1.7. Less than 0.16 IU/mg, if intended for use in the manufacture
Related substances o f parenteral preparations without a further appropriate
Liquid chromatography (2.2.29). Prepare the solutions procedure for the removal of bacterial endotoxins.
immediately before use. ASSAY
Test solution (a) Dissolve 50.0 mg o f the substance to be Liquid chromatography (2.2.29) as described in the test for
examined in water R and dilute to 50.0 m L with the same related substances with the following modifications.
solvent. Mobile phase Initial composition of the mixture of mobile
Test solution (b) Dissolve 80.0 mg of the substance to be phases A and B, adjusted where applicable.
examined in water R and dilute to 20.0 m L with the same Injection T est solution (a) and reference solution (a).
solvent.
Calculate the percentage content o f C 16H 17K N 20 4S by
Reference solution (a) Dissolve 50.0 mg o f benzylpenicillin multiplying the percentage content of benzylpenicillin sodium
sodium CRS in water R and dilute to 50.0 m L with the same by 1.045.
solvent.
STORAGE
Reference solution (b) Dissolve 10 mg of benzylpenicillin
sodium CRS and 10 mg of phenylacetic acid R (impurity B) in In an airtight container. If the substance is sterile, store in a
water R, then dilute to 50 m L with the same solvent. sterile, airtight, tamper-proof container.
Reference solution (c) Dilute 4.0 m L o f reference solution (a) IMPURITIES

to 100.0 m L w ith water R.
Column:
— sizer. I = 0.25 m , 0 = 4.6 mm;
(5 nm). H H
Mobile phase:
A. (2S,5i?,6i?)-6-amino-3,3-dimethyl-7-oxo-4-thia-1-
— mobile phase A: mix 10 volumes of a 68 g/L solution of
azabicyclo[3.2.0]heptane-2-carboxylic acid
(6-aminopenidllanic acid),
a 500 g/L solution of dilute phosphoric acid R, 30 volumes
o f methanol R and 60 volumes of water R;
— mobile phase B: mix 10 volumes of a 68 g/L solution of
a 500 g/L solution of dilute phosphoric acid R, 40 volumes
of water R and 50 volumes of methanol R;

0 -f, 70 30
U - (f* + 20) 7 0 ->0 30-» 100
(f, + 20) - (f„ + 35) 0 100
(f* + 35) - (f„ + 50) 70 30

C . (2S,5R, 6R) - 6- [ [(4-hydroxyphenyl) acetyl] amino] -3,3-
f„ = retention time of benzylpenicillin determined with reference dimethyl-7-oxo-4-thia-1 -azabicyclo [3.2.0] heptane-2-
solution (c) carboxylic acid,
1-272 Benzylpenicillin Sodium 2016
S o lu b ility
W I c o 2h Very soluble in water, practically insoluble in fatty oils and in
k V cc h 3 liquid paraffin.
ch3 ID E N T IF IC A T IO N
V P *
HOî C ^ H First identification A, D
D. (3«Sj7i?,7ai?)-5-benzyl-2j2-dimethyl-2j3j7j7a- A. Infrared absorption spectrophotometry (2.2.24).
tetrahydroimidazo [5,1 -b]thiazole-3,7-dicarboxyiic a d d Comparison benzylpemdUin sodium CRS.
(penillic a d d o f benzylpenicillin).
H .COî H Test solution Dissolve 25 m g o f the substance to be examined

in 5 m L o f water R.
h n - ^ x .c h 3
Reference solution (a) Dissolve 25 m g o f benzylpenidllin
sodium CRS in 5 m L of water R.
o c o 2h Reference solution (b) Dissolve 25 m g o f benzylpenicillin
sodium CRS and 25 mg o f phenoxymethylpemdMin
E. (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5- potassium CRS in 5 m L o f water R.
dimethylthiazoIidine-4-carboxyiic a d d (penidlloic a d d s of Plate TLC süamsed silica gel plate R.
benzylpenicillin). Mobile phase Mix 30 volumes of acetone R and 70 volumes of
H COjH to p H 5.0 with glacial acetic add R.
h n ^ \ x h 3 Application 1 |iL
» ancl eP'mer at c *
H
Drying In air.
F. (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5-
dimethylthiazoIidine-4-carboxylic a d d (penilloic a d d s of System suitability, reference solution (b):
benzylpenicillin). — the chromatogram shows 2 clearly separated spots.
PhEur
Results T h e prindpal spot in the chromatogram obtained with
p rindpal 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
Benzylpenicillin Sodium ★ ★
★ ★ 15 m m in diameter. Moisten with 0.05 m L o f water R and
***** add 2 m L of sulfuric add-formaldehyde reagent R. Mix the
colourless. Place the test-tube on a water-bath for 1 min;
0 H COjNa
a reddish-brown colour devdops.
N ^ A _ch3
D . It gives reaction (a) of sodium (2.3.1).
TESTS
p H (2.2.3)
5.5 to 7.5.
C 16H 17N 2N a 0 4S 356.4 69-57-8 Dissolve 2.0 g in carbon dioxide-free water R and dilute to
A c tio n a n d u se S p ecific o p tic a l ro ta tio n (2.2.7)
Penicillin antibacterial. + 285 to + 310 (dried substance).
P r e p a r a tio n Dissolve 0.500 g in carbon dioxide-free water R and dilute to
Benzylpenicillin Injection 25.0 m L with the same solvent.
P h E tr __________________________________ A b so rb a n c e (2.2.25)
Dissolve 90.0 m g in water R and dilute to 50.0 m L with the
D E F IN IT IO N
same solvent. M easure the absorbance o f the solution at
Sodium (2S,5R,6R)-3,3-dimethyl-7-oxo-6 -
325 nm , at 280 nm and at the absorption maximum at
[(phenylacetyl)amino]-4-thia-l-azabicyclo[3.2.0]heptane-2- 264 nm , diluting the solution, if necessary, for the
carboxylate.
m easurem ent at 264 nm. T h e absorbances at 325 nm and
Substance produced by the growth o f certain strains of 280 nm are not greater than 0.10 and the absorbance at the
PenicHtium notation or related organisms, or obtained by any absorption maximum at 264 nm is 0.80 to 0.88, calculated
other means. on the basis o f the undiluted (1.80 g/L) solution. Verify the
C o n te n t resolution o f the apparatus (2.2.25); the ratio of the
96.0 per cen t to 102.0 per cent (dried substance). absorbances is at least 1.7.
CHARACTERS R e la te d su b sta n c e s
A p p e a ra n c e Liquid chromatography (2.2.29). Prepare the solutions
W hite or alm ost white, crystalline powder. immediately before use.
2016 Benzylpenicillin Sodium 1-273
Test solution (a) Dissolve 50.0 mg o f the substance to be B a c te ria l en d o to x in s (2.6.14, Method E)
examined in water R and dilute to 50.0 m L with the same Less than 0.16 IU/m g, if intended for use in the manufacture
solvent. o f parenteral preparations without a further appropriate
Test solution (b) Dissolve 80.0 mg of die substance to be procedure for the removal of bacterial endotoxins.
examined in water R and dilute to 20.0 m L with the same ASSAY
solvent. Liquid chromatography (2.2.29) as described in the test for
Reference solution (a) Dissolve 50.0 m g of benzylpema&n related substances with the following modifications.
sodium CRS in water R and dilute to 50.0 m L with the same Mobile phase Initial composition o f the mixture of mobile
solvent. phases A and B, adjusted where applicable.
Reference solution (b) Dissolve 10 mg o f benzylpemdOin Injection T e st solution (a) and reference solution (a).
sodium CRS and 10 mg o f phenylacetic add R (impurity B) in
Calculate die percentage content o f C i 6H 17N 2N a 0 4S from
water R, then dilute to 50 m L with the same solvent.
die declared content o f benzylpemdSBn sodium CRS.
Reference solution (c) Dilute 4.0 m L o f reference solution (a)
to 100.0 m L with water R STO R A G E
Column: In an airtight container. If the substance is sterile, store in a
— size: I = 0.25 m , 0 = 4.6 mm; sterile, airtight, tamper-proof container.
— stationary phase: octadecylsifyl silica gelfor chromatography R IM P U R IT IE S
(5 Jim).
Mobile phase:
— mobile phase A: mix 10 volumes o f a 68 g/L solution of
potassium dihydrogen phosphate R adjusted to p H 3.5 with h 2n--
a 500 g/L solution of dilute phosphoric add R, 30 volumes
of methanol R and 60 volumes o f water R;
— mobile phase B: mix 10 volumes o f a 68 g/L solution of
A. (2S,5Æ,6i?)-6-amino-3,3-dimethyl-7-oxo-4-thia-l-
potassium dihydrogen phosphate R adjusted to p H 3.5 with azabicydo [3.2.0]heptane- 2-carboxylic ad d
a 500 g/L solution of dilute phosphoric add R, 40 volumes (6-aminopenidllanic add),
of water R and 50 volumes o f methanol R;
Time Mobik phase A Mobik phase B C P 00*

0 - 1, 70 30
B. phenylacetic ad d ,
f, - (t, + 20) 70 -» 0 30-» 100
(i„ + 20) - (f, + 35) 0 100
(t„ + 35) - (f„ + 50) 70 30
tj, = retention time of benzyipeniallm determined with reference

solation (c)

the required resolution, die adjusted composition will apply C. (2S,5i?,62?)-6-[[(4-hydroxyphenyI)acetyl]amino]-3j3-
at time zero in the gradient and in the assay. dimethyl-7-oxo-4-thia- l-azabicyclo[3.2.0]heptane-2-
Flow rate 1.0 mT7min. carboxylic add,
Injection 20 pL of reference solutions (b) and (c) with
isocratic elution at the initial mobile phase composition and H. .COjH
20 jiL o f test solution (b) according to the elution gradient / " " n - A . ch3
described under M obile phase; inject water R as a blank
according to the elution gradient described under Mobile V ^ s
HOzC' ' h h
Hs
phase.
— resolution: minimum 6.0 between die peaks due to D . (3S,7R,7 ai?)-5-benzyl-2,2-dimethyl-2>3j7,7a-
impurity B and benzylpenidllin; if necessary, adjust die tetrahydroimidazo [5,1 -è] thiazole-3,7-dicarboxylic a d d
ratio A:B o f the mobile phase. (penillic a d d of benzylpenidllin),
Untie.
— any impurity: for each impurity, n o t more than die area of H COjH
the principal peak in die chromatogram obtained with h n -A .c h 3

2-Ethyihexanoic add (2.4.28) O COaH
Maximum 0.5 per cent m/m.
E. (45)-2- [carboxy[(phenylacetyl) amino]methyl]-5,5-
dimethyhhiazolidine-4-carboxylic a d d (penicOloic adds o f
an oven at 105 °C.
benzylpenidllin),
1-274 Betacarotene 2016
H C02H TESTS
HN'^X.CHa Related substances
H U / \ ru and epimerat C*
Determine the absorbance (2.2.25) o f test solutions (b)
ç r r h and (a) used in Identification, at 455 nm an d at 340 nm
respectively.
Absorbance ratio A 4 S5 / A ^ : minimum 1.5.
F. (2i?5,45)-2-[[(phenyIacetyl)amino]methyi]-5,5-
dimetiiylthiazoHdine-4-caiboxylic a d d (penifloic ad d s o f T h e thresholds indicated under Related substances
(Table 2034.-1) in the general monograph Substances for
benzylpenidllin).
pharmaceutical use (2034) do n o t apply.
PhEur
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
★ ★
Betacarotene ★ ★ Loss on drying (2.2.32)
*
(Ph. Eur. monograph 1069) •k* 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.
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 <2.2.25) o f test solution (b) used in
Identification at the absorption maximum at 455 nm , using
cydohexane R as the compensation liquid.
Calculate the content o f C 40H 56 taking the specific
absorbance to be 2500.
STORAGE
In an airtight container, protected from light, at a
tem perature n o t exceeding 25 °C.
PhEur
C40H 56 536.9 7235-40-7
Action and use

Precursor of vitamin A.
★ ★
PhEur____________________ Betadex ★ ★
DEFINITION B etacydodextnn *****

(aU-£)-3,7,l 2,16 -T etram eth y i-l,l 8-bis(2,6,6- (Ph Eur. monograph 1070)
rrimerhylcyclohex - 1-enyl) octadeca-1 ,3,5,7,9,11, 13,15,17-
nonaene. OH HO
Content
96.0 per cent to 101.0 p e r cent (dried substance).
CHARACTERS
Appearance
Brown-red or brownish-red, crystalline powder.
Solubility
Practically insoluble in w ater, slightly soluble in cydohexane,
practically insoluble in anhydrous ethanol.
It is sensitive to air, heat a n d light) especially in solution.
Cany out all operations as rapidly as possible avoiding exposure to
actinic light; use freshly prepared solutions.
IDENTIFICATION
1135 7585-39-9
Ultraviolet and visible absoiption spectrophotometry (2.2.25).
Test solution (a) Dissolve 50.0 m g in 10 m L o f chloroform R Action and use
and dilute immediately to 100.0 m L with cydohexane R. Carrier molecule for drug delivery systems.
D ilute 5.0 m L o f this solution to 100.0 m L with
cydohexane R P h B r.
Test solution (b) Dilute 5.0 m L of test solution (a) to DEFINITION

50.0 m L with cydohexane R C ydoheptakis-(l ->4)-(a-D-giucopyranosyl)
Absorption maximum A t 455 n m for test solution (b). (cydomaltoheptaose or 0-cydodextrin).
Absorbance ratio ^455 / A 4 S3 = 1.14 to 1.18 for test Content
solution (b). 98.0 per cent to 101.0 per cent (dried substance).
2016 Betadex 1-275
CHARACTERS Reference solution (c) Dissolve 25.0 mg of betadex CRS in

Appearance water R and dilute to 25.0 m L with the same solvent.
White o r almost white, amorphous or crystalline powder. Column:
S o lu b ility — sizer. I — 0.25 m, 0 = 4.6 mm ;
Sparingly soluble in water and in propylene glycol, practically — stationary phase: octadecylsifyl silica gel for chromatography R
insoluble in anhydrous ethanol and in methylene chloride. (10 nm).
A. Specific optical rotation (see Tests). Flow rate 1.5 rnL/m in.
B. Examine the chromatograms obtained in the assay. Detection Differential refractometer.
Results T h e principal peak in the chromatogram obtained Equilibration W ith the mobile phase for about 3 h .
with test solution (b) is similar in retention time and size to Irtjection 50 |jL o f test solution (a) and reference solutions (a)
the principal peak in the chromatogram obtained with and (b).
reference solution (c). Run time 1.5 times the retention time of betadex.
C. Dissolve 0.2 g in 2 m L of iodine solution R4 by wanning Relative retention W ith reference to betadex (retention
on a w ater-bath, and allow to stand at room temperature. time = about 10 min): impurity B = about 0.3;
A yellowish-brown precipitate is formed. impurity A = about 0.45.
TESTS System suitability: reference solution (a):
S o lu tio n S — resolution: minim um 1.5 between the peaks d u e to
Dissolve 1.000 g in carbon dioxide-free water R with heating, impurities B and A; if necessary, adjust the concentration
allow to cool and dilute to 100.0 m L with the same solvent o f methanol in the mobile phase.
Limits:
Solution S is clear (2.2.1). — impurities A, B: for each impurity, not more than
0.5 times the area of the corresponding peak in the
p H (2.2.3) chromatogram obtained with reference solution (b)
5.0 to 8.0. (0.25 per cent);
T o 10 m L of solution S add 0.1 m L of a saturated solution — sum of impurities other than A and B: not m ore than
of potassium chloride R. 0.5 times the area of the peak due to betadex in die
S pecific o p tic a l ro ta tio n (2.2.7) chromatogram obtained with reference solution (b)
+ 160 to 4- 164 (dried substance), determined on solution S. (0.5 per cent).
R e d u c in g su g a rs Residual solvents
M aximum 0.2 per cent. Head-space gas chromatography (2.2.28) Use the standard
Test solution T o 1 m L o f solution S add 1 m L of cupri-tartaric additions method.
solution R4. H eat on a water-bath for 10 min, cool to room Internal standard ethylene chloride R.
tem perature. Add 10 m L of ammonium molybdate reagent R1 Test solutions In each o f 4 identical 20 m L flasks, dissolve
and allow to stand for 15 min. 0.5 g o f the substance to be examined in water R and add
Reference solution Prepare a reference solution at the same 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 Add 1 m L of reference solutions (a), (b), (c) and (d), adding
1 m L o f a 0.02 g/L solution of glucose R. 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 water R.
reference solution at the absorption maximum at 740 nm Reference solutions Prepare a 10 \sUL solution o f ethylene
using water R as the compensation liquid. T he absorbance of chloride R (reference solution (a)). Prepare reference
the test solution is not greater than that of the reference solutions (b), (c) and (d) from reference solution (a) to
solution. contain respectively, per litre, 5 nL, 10 |iL and 15 |iL of both
trichloroethylene R and toluene R.
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 Column:
230 nm and 350 nm , the absorbance (2.2.25) is not greater — material: fused silica;
than 0.10. Between 350 nm and 750 nm , the absorbance — sizer. I = 25 m , 0 = 0.32 mm ;
(2.2.2S) is not greater than 0.05. — stationary phase: macrogol 20 000 R (film thickness 1 nm).
Liquid chrom atography (2.2.29). Static head-space conditions which may be used:
— equilibration temperature: 45 °C;
— equilibration timer. 2 h.
examined in water R with heating, cool and dilute to
25.0 m L with the same solvent. Temperature:
— column: 50 °C;
Test solution (b) Dilute 5.0 m L of test solution (a) to
— infection port. 140 °C;
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 Irtjection 200 |jL of the head space, at least 3 times.
dilute to 50.0 m L with the same solvent. Retention time Toluene = about 10 min.
1-276 Betahistine Dihydrochloride 2016
System suitability:
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 .
M axim um 10 ppm. B. cyclooctakis-(l -+4)-(a-D-glucopyranosyl)
1.0 g complies with test C. Prepare the reference solution (cyclomaltooctaose or y-cyclodextrin).
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) Betahistine Dihydrochloride ★ ★
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 R
related substances w ith the following modifications. f CH3 ,2HCI
Injection T est solution (b) and reference solutions (a) and (c).
— repeatability: maximum relative standard deviation of the C8Hi4C12N 2 209.1 5579-84-0
area of the peak due to betadex of 2.0 per cent.
Calculate the percentage content of [C 6H 10O 5]7 from the
Histamine H ] receptor antagonist; antih istam in e.
assigned content of betadex CRS.
P r e p a r a tio n
STORAGE B etahistin e Dihydrochloride Tablets
PhEur___________________________________________________________
IM P U R IT IE S
Specified impurities A, B D E F IN IT IO N
N-M ethyl-2-(pyridin-2-yi)ethanamine dihydrochloride.
C o n te n t
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.
A. M eltin g point (2.2.14): 150 °C to 154 °C.
A. cyclohexakis-(l-*4)-(a-D-glucopyranosyl) (alfadex or
cyclomaltohexaose or a-cyclodextrin),
Comparison betahistine dihydrochloride CRS.
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.
2016 Betahistine Mesilate 1-277
Development Over 2/3 of the plate. — total: not more than 0.5 times the area of the principal
Drying At 110 °C for 10 min. peak in the chromatogram obtained with reference
Results T he principal spot in the chromatogram obtained with the chromatogram obtained with reference solution (c)
the test solution is similar in position and size to the prindpal (0.05 p er cent).
solution. L oss o n d ry in g (2.2.32)
an oven at 105 °C.
TESTS S u lfa te d a sh (2.4.14)
Solution S Maximum 0.1 p er cent, determined on 1.0 g.
Dissolve 5.0 g in carbon dioxide-free water R, and dilute to
50 m L with the same solvent. 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
than reference solution B 8 (2.2.2, Method II).
the second point of inflexion.
pH (2.2.3) 1 m L of 0.1 M sodium hydroxide is equivalent to 10.46 m g of
2.0 to 3.0 for solution S. C 8H 14C12N 2.
Related substances
STORA GE
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 IM P U R IT IE S
phase. 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. C T "
Dilute 2.0 m L o f the solution to 50.0 m L with the mobile
phase. A. 2-ethenylpyridine (2-vinylpyridine),
Reference solution (c) Dilute 2.0 m L of reference solution (b)
to 10.0 m L with the mobile phase. C P '* '
Column:
— sizer. I = 0.15 m , 0 = 3.0 mm; B. 2-(pyridin-2-yl)ethanol,
— stationary phase: base-deactivated end-capped octadecylsilyl
ch3
silica gel for chromatography R (5 Jim).
n.
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 O T '- '^ O
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 C. N-methyl-2-(pyridin-2-yl)-N-[2-(pyridin-2-
using dilute sodium hydroxide solution R and mix w ith 300 m L yl)ethyl] ethanamine.
of acetonitrile R.
PhEur
Flow rate 1 mlVmin.
Irtjection 20 (iL.
Run time 4 times the retention time of betahistine. ★ ★
Betahistine Mesiiate ★ ★
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.
— resolution: minim um 3.5 between the peaks due to CH3 , 2 H3C -S O 3H
2-vinylpyridine and betahistine.
Limits:
— correction factor, for the calculation of content, multiply the C 10H20N2O6S2 328.4 54856-23-4
peak area o f impurity B by 0.4;
— impurities A , B, C: for each impurity, n o t more th an the A ctio n a n d u se
area o f the principal peak in the chromatogram obtained Histamine H I receptor antagonist; antihistamine.
with reference solution (c) (0.2 per cent);
PhEur_________________________________________________________
— unspecified impurities: for each impurity, not m ore than
0.5 times o f the area o f the principal peak in the D E F IN IT IO N
chromatogram obtained with reference solution (c) N-Methyl-2-(pyridin-2-yl)ethanamine bis(methanesulfonate).
(0.10 p er cent); C o n te n t
1-278 Betahistine Mesilate 2016
P R O D U C T IO N Test solution Dissolve 50 mg o f the substance to be examined

It is considered that alkylsulfonate esters are genotoxic and in the mobile phase and dilute to 10.0 m L with the mobile
are potential impurities in betahistine mesilate. phase.
T h e manufacturing process should be developed taking into Reference solution (a) Dissolve 10 m g of betahistine
consideration the principles o f quality risk management, mesilate CRS and 10 m g o f 2-vinylpyridme R (impurity A) in
together with considerations o f the quality of starting the mobile phase and dilute to 50.0 m L with the mobile
materials, process capability and validation. T he general phase. Dilute 2.0 m L o f this solution to 50.0 m L with the
m ethods 2.5.37. Methyl, ethyl and isopropyl methanesulfonate in mobile phase.
methanesutfonic add, 2.5.38. Methyl, ethyl and isopropyl Reference solution (b) Dilute 1.0 m L o f the test solution to
methanesulfonate in active substances and 2.5.39. 100.0 m L with the mobile phase.
Methanesulfonyl chloride in methanesulfonic acid are available to
assist manufacturers.
CHARACTERS Column:
A p p e a ra n c e — size: I = 0.25 m , 0 = 4.6 mm;
W hite or alm ost white, crystalline powder, very hygroscopic. — stationary phase: octadecylsUyl silica gel for chromatography R
S o lu b ility (5 jun).
Very soluble in water, freely soluble in ethanol (96 per cent), Mobile phase Dissolve 2.0 g o f sodium dodecyl sulfate R in a
very slightly soluble in 2 -propanol. mixture o f 15 volumes o f a 10 per cent V/V solution of
ID E N T IF IC A T IO N sulfuric acid R, 35 volumes o f a 17 g/L solution of
First identification B. tetrabutylammonium hydrogen sulfate R and 650 volumes of
water R; adjust to p H 3.3 using dilute sodium hydroxide
Second identification A, C, D. solution R and mix with 300 volumes of acetonitrUe R.
A. M elting point (2.2.14): 108 °C to 112 °C.
Flow rate 1 mlVmin.
Comparison betahistine mesilate CRS. Injection 20 jjL.
Run time 3 times the retention tim e o f betahistine mesilate.
Test solution Dissolve 10 m g o f the substance to be examined Retention time Betahistine mesilate = about 8 min.
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 im purity A and betahistine mesilate.
ethanol (96 per cent) R and dilute to 2 m L with the same
Limits:
solvent.
— impurity A: n o t more than the area o f the principal peak
Plate TLC silica gel F2 5 4 plate R. in the chromatogram obtained with reference solution (c)
Mobile phase concentrated ammonia R, ethyl acetate R, (0.2 per cent);
methanol R ( 0.75:15:30 VIV/V). — unspecified impurities: for each impurity, n o t more than
Application 2 |iL. 0.1 times the area of the principal peak in the
Development O ver 3/4 o f the plate. chromatogram obtained with reference solution (b)
( 0.10 per cent);
Drying At 110 °C for 10 min. — total: not more than 0.5 times the area o f the principal
Detection Examine in ultraviolet light at 254 nm. peak in the chromatogram obtained with reference
Results T he principal spot in the chromatogram obtained with solution (b) (0.5 per cent);
the test solution is similar in position and size to the principal — disregard limit. 0.05 times the area of the principal peak in
spot in the chrom atogram obtained with the reference the chromatogram obtained with reference solution (b)
solution. (0.05 per cent).
D . T o 0.1 g add 5 m l. o f dilute hydrochloric acid R and shake 2 -P ro p a n o l (2.424)
for about 5 min. A dd 1 m L o f barium chloride solution R l. M axim um 0.5 p er c e n t
T he solution rem ains clear. T o a further 0.1 g add 0.5 g o f
C h lo rid e s (2.44)
anhydrous sodium carbonate R, mix and ignite until a white Maximum 35 ppm .
residue is obtained. Allow to cool and dissolve the residue in
T o 14 m L of solution S add 1 m L of water R.
7 m L of water R. T he solution gives reaction (a) o f sulfates
(2.3.1). S u lfa tes (2.413)
M axim um 250 ppm.
TESTS
S o lu tio n S D ilute 6 m L o f solution S to 15 m L with distilled water R.
Dissolve 5.0 g in carbon dioxide-free water R prepared from H ea v y m e ta ls (2.48)
distilled water R, and dilute to 50 m L with the same solvent. M axim um 20 ppm .
A p p e a ra n c e o f so lu tio n 12 m L o f solution S complies with test A. Prepare the
Solution S is clear (2.2.1) and colourless (2.2.2, Method II). reference solution using lead standard solution (2 ppm Pb) R.
p H (2.2.3) W a te r (2.5.12)
2.0 to 3.0 for solution S. Maximum 2.0 per cent, determ ined on 0.50 g.
R e la te d su b s ta n c e s A SSA Y
Liquid chromatography (2.2.29). 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.
2016 Betamethasone 1-279
T itrate with 0.1 M perchloric acid, determining the end-point chloride R, evaporate to dryness on a water-bath and record
potentiometrically (2.2.20). new spectra using the residues.
1 m L of 0.1 M perchloric add is equivalent to 16.42 mg C. Thin-layer chromatography (2.2.27).
of C 10H 20N 2O 6S 2. Solvent mixture methanol R, methylene chloride R (1:9 V/V).
STORA GE Solvent mixture
In an airtight container. Test solution Dissolve 10 mg of the substance to be examined
IM P U R IT IE S in the solvent mixture and dilute to 10 m L with the solvent
Specified impurities A mixture.
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
A. 2-ethenylpyridine (2-vinylpyridine).
solution (a).
PhEur
Mobile phase butanol R saturated with water R, toluene R,
ether R (5:10:85 VIVIV).
★ ★ Application 5 |iL.
Betamethasone ★ ★
*****
(Ph. Eur. monograph 0312) Drying In air.
Detection A Examine in ultraviolet light at 254 nm.
Results A T h e principal spot in the chromatogram obtained
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
C22H 29FO 5 392.5 378-44-9 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 the test solution is similar in position, colour in daylight,
Glucocorticoid. fluorescence in ultraviolet light at 365 nm and size to the
P re p a ra tio n principal spot in the chromatogram obtained with reference
Betamethasone T ablets solution (a).
PhEir ___________________ — the chromatogram shows 2 spots which may, however,
D E F IN IT IO N not be completely separated.
9-Fluoro-l 1P, 17 j2 1-trihydroxy-16 ^-methylpr egna-1,4- D . Mix about 5 m g with 45 mg of heavy magnesium oxide R
diene-3,20-dione. and ignite in a crucible until an almost white residue is
C o n te n t obtained (usually less than 5 min). Allow to cool, add 1 m L
97.0 per cent to 103.0 per cent (dried substance). 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
CHARACTERS colourless. Filter. Add 1.0 m L of the filtrate to a freshly
A p p earan ce prepared mixture o f 0.1 m L of alizarin S solution R and
White or almost white, crystalline powder. 0.1 m L o f zirconyl nitrate solution R. Mix, allow to stand for
Solubility 5 min and compare the colour of the solution with that o f a
Practically insoluble in water, sparingly soluble in anhydrous blank prepared in the same manner. T he test solution is
ethanol, very slightly soluble in methylene chloride. yellow and the blank is red.
ID E N T IF IC A T IO N E. Add about 2 m g to 2 m L of sidfuric acid R and shake to
First identification B, C. dissolve. W ithin 5 min, a deep reddish-brown colour
develops. Add this solution to 10 m L of water R and mix.
Second identification A , C, D, E.
T h e colour is discharged and a clear solution remains.
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 TESTS
solution in a stoppered tube, add 10.0 m L o f S p ecific o p tic a l ro ta tio n (2.2.7)
phertylhydrazine-sidfuric acid solution R, mix and heat in a + 118 to + 126 (dried substance).
water-bath at 60 °C for 20 min. Cool immediately. Dissolve 0.125 g in methanol R and dilute to 25.0 m L with
The absorbance (2.2.25) measured at 419 n m is n o t greater the same solvent.
than 0 . 10. R e la te d su b sta n c e s
B. Infrared absorption spectrophotometry (2.2.24). Liquid chromatography (2.2.29).
Comparison betamethasone CRS. Test solution Dissolve 25.0 mg o f the substance to be
If the spectra obtained in the solid state show differences, examined in a mixture of equal volumes o f acetonitrHe R and
dissolve the substance to be examined and the reference methanol R and dilute to 10.0 m L with the same mixture o f
substance separately in the minimum volume of methylene solvents.
1-280 Betamethasone 2016
Reference solution (a) Dissolve 2 m g o f betamethasone CRS and Calculate the content of C 22H 29F O 5 taking the specific
2 m g of methylprednisolone CRS in mobile phase A, then absorbance to be 395.
dilute to 100.0 m L with mobile phase A. STO RA G E
Reference solution (b) Dilute 1.0 m L of the test solution to Protected from light.
IM P U R IT IE S
Column'.
Specified impurities A, B, C, D , E, F, G , H , I, J
— size. I = 0.25 m , 0 = 4.6 mm;
— stationary phase, octadecylsilyl silica gel for chromatography R
(5 Jim);
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; A. 9 -flu o ro -lip , 17,21 -trihydroxy-16a-m ethylpregna-1,4-
— mobile phase B: acetomtrUe R; diene-3,20-dione (dexamethasone),

0 - 15 100 0
15-40 100*0 0 * 100
4 0-41 0 * 100 10 0 * 0
4 1 -4 6 100 0
B. 21-chloro-9-fluoro-l ip,17-dihydroxy-16P-methylpregna-
l,4-diene-3,20-dione,
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. C. 17,21-dihydroxy-16P-methylpregna-l,4,9(l l)-triene-3,20-
Retention time M ethylprednisolone = about 11.5 min; dione,
betam ethasone = about 12.5 min.
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) D . 9 -flu o ro -lip , 17-dihydroxy-16P-methyl-3,20-dioxopregna-
( 1.0 per cent), and not m ore than 1 such peak has an l,4-dien-21-yl ethoxycarboxylate,
area greater than 0.5 times the area of the principal peak
(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);
(0.05 per cent). E. 9,11 P-epoxy-17,21 -dihydroxy-16P-methyl-9 P-pregna-1,4-
L oss o n d ry in g (2.2.32) diene-3,20-dione,
an oven at 105 °C.
A SSA Y
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. F . 17,21-dihydroxy-16P-methylpregna-l,4,l l-triene-3,20-
dione,
2016 Betamethasone Acetate 1-281
CHARACTERS
A p p e a ra n c e
S olu b ility
soluble in ethanol (96 per cent) and in methylene chloride.
G . 1 la,17,21-trihydroxy-16ß-methylpregna-l,4-diene-3,20-
dione, ID E N T IF IC A T IO N
Second identification A , C, D, E, F
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
H . 14-fluoro-l lß,17,21-trihydroxy-16ß-methyl-8a,9ß,14ß- than 0 . 10.
pregna-1 ,4-diene-3,20-dione, B. Infrared absorption spectrophotometry (2.2.24).
Comparison betamethasone acetate CRS.
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.
Solvent mixture methanol R, methylene chloride R (1:9 VIV).
I. 8-fluoro-llß,17,21-trihydroxy-16ß-methyl-8a,9ß-pregna-
l 34-diene-3,20-dione, Test solution Dissolve 10 mg of the substance to be exam ined
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
Reference solution (b) Dissolve 10 mg of prednisolone
acetate CRS in reference solution (a) and dilute to 10 m L
J. 17,21 -dihydroxy-16 (J-methylpregna-134-diene-3 ,2 0-dione. Plate TLC silica gel F 254 plate R.
__________________________________________________________ PhEtr 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.
Betamethasone Acetate ******
Drying In. air.
(Ph Eur monograph 0975) * Detection A Examine in ultraviolet light at 254 nm.
Results A T h e principal spot in the chromatogram obtained
solution (a).
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 with the test solution is similar in position, colour in daylight,
fluorescence in ultraviolet light at 365 nm and size to the
A ctio n a n d u se
Glucocorticoid.
solution (a).
PhEur__________________________________________________________ System suitability: reference solution (b):
D E F IN IT IO N
D . Add about 2 m g to 2 m L o f sulfuric acid R and shake to
9-Fhioro-11(3,17-dihydroxy-16|}-inethyl-3,20-dioxopregna-
l,4-diene-21-yl acetate. dissolve. W ithin 5 min, a deep reddish-brown colour
develops. Add this solution to 10 m L of water R and mix.
C o n te n t T h e colour is discharged and a clear solution rem ain s.
1-282 Betamethasone Acetate 2016
E. M ix about 5 mg with 45 m g of heavy magnesium oxide R A SSAY

and ignite in a crucible until an almost white residue is Dissolve 0.100 g in ethanol (96 per cent) R and dilute to
obtained (usually less than 5 min). Allow to cool, add 1 m L 100.0 m L with the same solvent. D ilute 2.0 m L of this
of water R, 0.05 m L of phenolphthalein solution R1 and about solution to 100.0 m L with ethanol (96 per cent) R M easure
1 m L o f dilute hydrochloric acid R to render the solution the absorbance (2.2.25) at the absorption maximum at
colourless. Filter. T o a freshly prepared mixture o f 0.1 m L o f 240 nm .
alizarin S solution R and 0.1 m L of zirconyl nitrate solution R, Calculate the content of C 24H 3iF 0 6 taking the specific
add 1.0 m L o f the filtrate. Mix, allow to stand for 5 min and absorbance to be 350.
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 STO R A G E
the blank is red. Protected from light.
F. A bout 10 m g gives the reaction o f acetyl (2.3.1). IM P U R IT IE S
Specified impurities A, B, C , D
TESTS
Dissolve 0.250 g in dioxan R and dilute to 25.0 m L with the
sam e solvent.
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 A. 9-fluoro-lip,17,21-trihydroxy-16|3-niethylpregna-l,4-
diene-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
Reference solution (b) D ilute 1.0 m L o f the test solution to
Column:
— size: I = 0.25 m , 0 = 4.6 mm;
— stationary phase, octadecylsUyl silica gel for chromatography R B. 9-fluoro-l ip,l7-dihydroxy-16a-m ethyl-3,20-dioxopregna-
(5 \un).
l,4-dien-21-yl acetate (dexamethasone acetate),
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. C . 9-fluoro-17-hydroxy-16 |3-methyi-3,20-dioxopregna-1,4-
d ie n e -lip , 21 -diyl diacetate (betamethasone 11, 21 -diacetate),
Retention time Betam ethasone acetate = about 19 min;
im purity B = about 22 m in.
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 D . 9,11 (5-epoxy-17-hydroxy-l 6P-methyl-3,20-dioxo-9|3-
chrom atogram obtained with reference solution (b) pregna-1,4-diene-21-yl acetate.
(0.5 per cent); __________________ ________________________________ __ PhEur
— total: not m ore than 1.25 times the area of the principal
(0.05 per cent).
W a te r (2.5.12)
2016 Betamethasone Dipropionate 1-283
immediately pass a current of nitrogen R briskly through the

Betamethasone Dipropionate ****** 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.
O Mobile phase Add a mixture of 1.2 volumes o f water R and
Application 5 |iL.
Drying In. air.
Results A T he principal spot in each o f the chromatograms
obtained with the test solutions is similar in position and size
to the principal spot in the chromatogram obtained with the
Action and use corresponding reference solution.
Glucocorticoid. Detection B Spray with alcoholic solution of sulfuric acid R, heat
at 120 °C for 10 m in or until the spots appear, and allow to
PhEtr -----------------------------------------------------------------------------------------
cool; examine in daylight and in ultraviolet light at 365 nm.
D E F IN IT IO N Results B T he principal spot in each of the chromatograms
9-Fluoro-l 1 P-hydroxy-16P-methyl-3}20-dioxopregna-l ,4- obtained with the test solutions is similar in position, colour
diene-17j21-diyl dipropanoate. in daylight, fluorescence in ultraviolet light at 365 nm and
Content size to the principal spot in the chromatogram obtained with
97.0 per cent to 102.0 per cent (dried substance). the corresponding reference solution; the principal spot in
each of the chromatograms obtained with test solution (b)
CHARACTERS
and reference solution (b) has an Rp value distinctly lower
Appearance
than that o f the principal spot in each o f the chromatograms
obtained with test solution (a) and reference solution (a).
Solubility 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
methylene chloride, sparingly soluble in ethanol
develops. Add this solution to 10 m L o f water R and mix.
(96 per cent). T he colour is discharged and a clear solution remains.
IDENTIFICATION E. Mix about 5 mg with 45 mg o f heavy magnesium oxide R
First identification B. and ignite in a crucible until an alm ost white residue is
Second identification A , C, D, E. obtained (usually less than 5 min). Allow to cool, add 1 m L
A. Dissolve 10.0 m g in anhydrous ethanol R and dilute to o f water R, 0.05 m L of phenolphthalein solution R1 and about
100.0 m L with the same solvent Place 2.0 m L of the 1 m L of dilute hydrochloric add R to render the solution
solution in a ground-glass-stoppered tube, add 10.0 m L of colourless. Filter. Add 1.0 m L o f the filtrate to a freshly
phenylhydrazine-sulfuric add solution R, mix and heat in a prepared mixture o f 0.1 m L of alizarin S solution R and
water-bath at 60 °C for 20 min. Cool immediately. 0.1 m l , of zirconyl nitrate solution R. Mix, allow to stand for
T he absorbance (2.2.25) measured at 419 nm is not more 5 min and compare the colour o f th e solution with that of a
than 0 . 10. blank prepared in the same manner. T he test solution is
yellow and the blank is red.
Comparison betamethasone dipropionate CRS. TESTS
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. Related substances
Test solution (b) Transfer 2 m L of solution A to a 15 m L Liquid chromatography (2.2.29).
glass tube with a ground-glass stopper or Test solution (a) Dissolve 60.0 m g o f the substance to be
a polytetrafluoroethylene cap. Add 10 m L of saturated exam in e d in the mobile phase and dilute to 25.0 m l . with
methanoUc potassium hydrogen carbonate solution R and the mobile phase.
immediately pass a current o f nitrogen R briskly through the Test solution (b) Dilute 1.0 m L o f test solution (a) to
solution for 5 min. Stopper the tube. H eat in a water-bath at 10.0 m L with the mobile phase.
45 °C, protected from light, for 2 h. Allow to cool Reference solution (a) Dissolve 5 mg o f betamethasone
Reference solution (a) Dissolve 25 mg of betamethasone dipropionate for system suitability CRS (containing impurities B,
dipropionate CRS in methanol R with gende heating and dilute C, D , E and G) in the mobile phase and dilute to 2.0 m L
to 5 m l, w ith the sam e solvent (solution B). Dilute 2 m L o f with the mobile phase.
solution B to 10 m l. with methylene chloride R. Reference solution (b) Dilute 1.0 m L o f test solution (a) to
Reference solution (b) T ransfer 2 m L of solution B to a 15 m L 100.0 m L with the mobile phase. Dilute 1.0 m L o f this
glass tube with a ground-glass stopper or solution to 10.0 m l. with the mobile phase.
a polytetrafluoroethylene cap. Add 10 m L o f saturated
methanoUc potassium hydrogen carbonate solution R and
1-284 Betamethasone Dipropionate 2016
Reference solution (c) Dissolve 60.0 mg o f betamethasone L oss o n d ry in g (2.2.32)

dipropionate CRS in the mobile phase and dilute to 25.0 m L M aximum 1.0 per cent, determ ined on 0.500 g by drying in
w ith the mobile phase. Dilute 1.0 m L o f the solution to an oven at 105 °C.
10.0 m L with the mobile phase. ASSAY
Reference solution (d) Dissolve 5 mg o f betamethasone Liquid chromatography (2.2.29) as described in the test for
dipropionate for peak identification CRS (containing related substances with the following modification.
impurity H) in the mobile phase and dilute to 2.0 m L with
the mobile phase.
Calculate the percentage content o f C 28H 37F O 7 from the
Column:
declared content o f betamethasone dipropionate CRS.
— size. I = 0.10 m , 0 = 2.0 mm;
— stationary phase: octadecylsiJyl silica gel for chromatography R STO R A G E
(2.5 pm); Protected from light.
— temperature: 20 + 2 °C.
IM P U R IT IE S
Mobile phase M ix 35 m L o f water R and 56 m L of Specified impurities B, C, D , E, G , H
acetomtrUe R and allow to equilibrate; dilute to 100 m L with
water R and mix.
Flow rate 0.2 mL/min. the tests in the monograph. They are limited by the general
Detection Spectrophotom eter at 254 nm. acceptance criterion for other/unspecified impurities and/or
Injection 5 jiL o f test solution (a) and reference solutions (a), by the general monograph Substances for pharmaceutical use
(b) and (d). (2034). It is therefore not necessary to identify these
Run time 3 times the retention time o f betamethasone impurities for demonstration o f compliance. See also 5.10.
dipropionate. Control of impurities in substances for pharmaceutical use): A , F.
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
to impurity H.
Relative retention W ith reference to betamethasone A. 9-fluoro-lip,17,21-trihydroxy-16p-m ethylpregna-l,4-
dipropionate (retention time = about 10 min): diene-3,20-dione (betamethasone),
impurity D = about 0.7; impurity E = about 1.2; o 0H
im purity H = about 1.7; impurity G = about 2 . 1.
— peak-to-valley ratio: minimum 4.0, where Hp = height C hT h T V -C H 3
above the baseline of the peak due to impurity E and
Hv = height above the baseline o f the lowest point of the
betamethasone dipropionate.
B. 9-fluoro-l 1P,21-dihydroxy-16P-methyl-3,20-dioxopregna-
Limits:
l,4-dien-17-yl propanoate (betamethasone 17-propionate),
corresponding correction factor, im purity G = 1.3; o
impurity H = 1.4;
— impurity C: not m ore than 5 times the area of the
— impurities B, H: for each impurity, not more than 3 times
— impurities D, E, <7: for each impurity, not more than twice
the area o f the principal peak in the chromatogram C . 9-fluoro-11P, 17-dihydroxy-16P-methyl-3,20-dioxopregna-
obtained with reference solution (b) (0.2 per cent); l,4-dien-21-yl propanoate (betamethasone 21-propionate),
— unspecified impurities: for each impurity, n o t more than the
total: not m ore than 10 times the area o f the principal
solution (b) ( 1.0 per cent);
disregard limit. 0.5 times the area o f the principal peak in
(0.05 per cent).
2016 Betamethasone Sodium Phosphate 1-285
o
*****
Betamethasone Sodium ★ ★
o< \ / o CH3
Phosphate *****
r H V -C H 3
° ,ONa
0 " p\
"OH oNa
D. 2 l-(acetyloxy)-9-fluoro-l 1ß-hydroxy-16ß-methyl-3520-
dioxopregna-1,4-dien-17-yi propanoate (betamethasone
21-acetate 17-propionate),
QiTjH^sFNaÔuP 516.4 151-73-5
Action and use

Glucocorticoid.
Preparations
Betamethasone Eye Drops
Betamethasone Injection
Betamethasone Sodium Phosphate Tablets
E. 9-chloro-l 1ß-hydroxy-16ß-methyi-3,20-dioxopregna-1,4- PhEur_______________________________________

diene-17,21-diyl dipropanoate (beclometasone dipropionate),
D E F IN IT IO N
9-Fluoro-l 1P, 17 -dihydroxy-16f}-methyl-3320-dioxopregna-
l,4-dien-21-yl disodium phosphate.
o J Content
CHARACTERS
chv ° r a * ch3
Appearance
W hite or almost white powder, very hygroscopic.
Solubility
Freely soluble in water, slightly soluble in ethanol
F. 9j 11 ß-epoxy-16ß-m ethyW Ô -dioxo^ß-pregna-1,4-diene- (96 per cent), practically insoluble in methylene chloride.
17,21-diyi dipropanoate (9ß,llß-epoxybetam ethasone
dipropionate), ID E N T IF IC A T IO N
O Second identification A , C, D, E, F
CH3 J!
A. Dissolve 10.0 m g in 5 mL of water R and dilute to
s ^ ° 100.0 m L with anhydrous ethanol R. Place 2.0 m L o f this
solution in a ground-glass-stoppered tube, add 10.0 m L o f
chT h V - ch 3 phenylhydrazine-sulfuric add solution R, mix and heat in a
w ater-bath at 60 °C for 20 min. Cool immediately.
T h e absorbance (2.2.25) measured at the absorption
maximum at 450 nm is not more th an 0.10.
G. 9-fluoro-l 6 ß-methyi- 3^ 0-dioxopregna-lj4-diene- Comparison betamethasone sodium phosphate CRS.
llß,17,21-triyl tripropanoate (betamethasone tripropionate),
substance separately in the m inim u m volume o f ethanol
(96 per cent) R, evaporate to dryness on a water-bath and
in methanol R and dilute to 10 m L w ith the same solvent.
Reference solution (a) Dissolve 10 m g of betamethasone sodium
H Br phosphate CRS in methanol R and dilute to 10 m L with the
same solvent
H . 6a-brom o-9-fluoro-l 1ß-hydroxy-16ß-methyl-3,20- Reference solution (b) Dissolve 10 m g o f prednisolone sodium
dioxopregna-l,4-diene-17,21-diyl dipropanoate phosphate CRS in methanol R and dilute to 10 m L with the
(6a-brom obetam ethasone dipropionate). same solvent Dilute 5 m L of this solution to 10 m L with
PhEur reference solution (a).
1-286 Betamethasone Sodium Phosphate 2016
Mobile phase glacial acetic add R, water R, butanol R Test solution Dissolve 62.5 m g o f the substance to be
(20:20:60 VIVIV). examined in the mobile phase and dilute to 25.0 m L with
Application 5 pL. the mobile phase.
Development Over a path of 15 cm. Reference solution (a) Dissolve 25 m g of betamethasone sodium
Drying In air. phosphate CRS and 25 m g o f dexamethasone sodium
phosphate CRS in the mobile phase and dilute to 25.0 m L
Detection A Examine in ultraviolet light at 254 nm. with the mobile phase. D ilute 1.0 m L o f this solution to
Results A T he principal spot in the chromatogram obtained 25.0 m L with the mobile phase.
with the test solution is similar in position and size to the Reference solution (b) Dilute 1.0 m L of the test solution to
principal spot in the chromatogram obtained with reference 50.0 m L with the mobile phase.
solution (a).
Column:
Detection B Spray with alcoholic solution of sulfuric add R. H eat — size: I = 0.25 m , 0 = 4.6 mm;
at 120 °C for 10 min or until the spots appear. Allow to — stationary phase: octadecylsifyl silica gel for chromatography R
cool. Examine in daylight and in ultraviolet light at 365 nm. (5 pm).
Results B T h e principal spot in the chromatogram obtained Mobile phase In a 250 m L conical flask, weigh 1.360 g of
with the test solution is similar in position, colour in daylight, potassium dihydrogen phosphate R and 0.600 g o f hexylamine R,
fluorescence in ultraviolet light at 365 nm and size to the mix and allow to stand for 10 min and then dissolve in
principal spot in the chromatogram obtained with reference 185 m L o f water R\ add 65 m L of acetomtrUe R, mix and
solution (a). filter (0.45 pm).
System suitability: reference solution (b): Flow rate 1 mL/min.
— the chromatogram shows 2 spots which may, however,
not be completely separated.
Equilibration W ith the mobile phase for about 45 min.
D. Add about 2 mg to 2 m L o f sulfuric acid R and shake to
dissolve. W ithin 5 m in, an intense reddish-brown colour Injection 20 pL.
develops. Add the solution to 10 m L of water R and mix. Run time Twice the retention time o f betamethasone sodium
T he colour is discharged and a clear solution remains. phosphate.
E. Mix about 5 mg with 45 mg of heavy magnesium oxide R Retention time Betam ethasone sodium phosphate = about
and ignite in a crucible until an almost white residue is 14 min; dexamethasone sodium phosphate = about
obtained (usually less than 5 min). Allow to cool, add 1 m L 15.5 min.
o f water R, 0.05 m L o f phenolphthakin solution R1 and about System suitability, reference solution (a):
1 m L of dilute hydrochloric add R to render the solution — resolution: minim um 2.0 between the peaks due to
colourless. Filter. Add 1.0 m L of die filtrate to a freshly betam ethasone sodium phosphate and dexamethasone
prepared mixture o f 0.1 m L of alizarin S solution R and sodium phosphate; if necessary, increase the
0.1 m L o f zvrconyl nitrate solution R. Mix, allow to stand for concentration o f acetonitrile or increase the concentration
5 min and compare the colour of the solution with th at o f a of water in die mobile phase.
blank prepared in the same manner. T h e test solution is Limits:
yellow and the blank is red. — any impurity, for each impurity, no t more than the area of
F. T o about 40 m g add 2 m L of sulfuric acid R and heat the principal peak in the chromatogram obtained with
gently until white fumes are evolved. Add nitric add R reference solution (b) (2 per cent), and not more than
dropwise, continue the heating until the solution is almost 1 such peak has an area greater than 0.5 times the area of
colourless and cool. A dd 2 m L o f water R, heat until white the principal peak in the chromatogram obtained with
fumes are again evolved, cool, add 10 m L of water R and reference solution (b) (1 per cent);
neutralise to red litmus paper R with dilute ammonia R l. — total: n o t more than 1.5 times the area of the principal
T he solution gives reaction (a) of sodium (2.3.1) and peak in the chrom atogram obtained with reference
reaction (b) o f phosphates (2.3.1). solution (b) (3 per cent);
TESTS — disregard limit: 0.025 times the area o f the principal peak
S o lu tio n S
(0.05 per cent).
20 m L with the same solvent. In o rg a n ic p h o sp h a te
M aximum 1 per cent.
Solution S is clear (2 . 2 . 1 ) and n o t m ore intensely coloured Dissolve 50 m g in water R and dilute to 100 m L with the
than reference solution B 7 (2.2.2, Method II). same solvent. T o 10 m L o f this solution add 5 m L of
molybdovanadic reagent R, mix and allow to stand for 5 min.
p H (2.2.3)
Any yellow colour in the solution is not m ore intense than
7.5 to 9.0.
that in a standard prepared at the same time and in the same
Dilute 1 m L o f solution S to 5 m L with carbon dioxide-free maimer using 10 m L of phosphate standard solution (5 ppm
water R. PO4 ) R.
Specific o p tic a l r o ta tio n (2.2.7) W a te r (2.5.12)
+ 98 to + 104 (anhydrous substance). M aximum 8.0 per cent, determined on 0.200 g.
ASSAY
same solvent.
R elated su b s ta n c e s same solvent. D ilute 5.0 m L o f this solution to 250.0 m L
Liquid chrom atography (2.2.29). with water R. M easure the absorbance (2.2.25) at the
absorption maximum at 241 nm.
2016 Betamethasone Valerate 1-287
Calculate the content of C 22H 28F N a 20 8P taking the specific TESTS

absorbance to be 297. Specific o p tic a l ro ta tio n (2.2.7)
STORAGE
In an airtight container protected from light. Dissolve 0.250 g in anhydrous ethanol R and dilute to
__________________________________________________________ PhEur
Liquid chrom atography (2.2.29). Carry out the test protected
from light. Prepare the solutions immediately before use.
Solvent mixture glacial acetic acid R, mobile phase
Betamethasone Valerate (1:1000 V/V).
(Ph. Eur. monograph 0811) * Test solution Dissolve 50 m g of the substance to be examined
in the solvent mixture and dilute to 20.0 m L with the solvent
mixture.
Reference solution (b) Dissolve 12.5 mg of betamethasone
valerate for system suitability CRS (containing impurities D
and G) in 5.0 m L o f the solvent mixture. Use 1.0 m L of this
solution to dissolve the contents of a vial of betamethasone
valerate impurity mixture CRS (containing impurities C, H
and I).
Action and use Reference solution (c) Dissolve 6 mg of betamethasone CRS
Glucocorticoid.
(impurity A) and 3 mg o f betamethasone 21-valerate CRS
Preparations (impurity E) in 30.0 m L o f the solvent mixture. Dilute
Betam ethasone Valerate Scalp Application 1.0 m L o f this solution to 10.0 m L with the solvent mixture.
Betamethasone Valerate Cream Column:
Betamethasone and Clioquinol Cream — size: I — 0.25 m , 0 = 4.6 mm;
Betamethasone Valerate Lotion
chromatography R (5 |im);
Betamethasone Valerate Ointm ent
Betamethasone and Clioquinol O intm ent
Mobile phase acetonitrUe R, water R (50:50 V/V).
PhEur__________________________________________________________ Flow rate 1 mL/min.
DEFINITION Detection Spectrophotom eter at 239 nm.
9-Fluoro-l 1 P,21-dihydroxy-16 P-methyl-3320-dioxopregna- Injection 20 |iL.
lj4-dien-17-yl pentanoate. Run time 2.5 times the retention time of betamethas<?ne
Content valerate.
97.0 per cent to 103.0 per cent (dried substance). Identification of impurities Use the chromatogram supplied
with betamethasone valerate for system suitability CRS and the
CHARACTERS
Appearance
identify the peaks due to impurities C, D , G, H and I;
use the chrom atogram obtained with reference solution (c) to
Solubility identify the peaks due to impurities A and E.
Relative retendon W ith reference to betamethasone valerate
methylene chloride, soluble in ethanol (96 per cent).
(retention time = about 20 min): impurity A = about 0.3;
mp impurity I = about 0.6; impurity C = about 0.8;
A bout 192 °C, with decomposition. impurity H = about 1.3; impurity D = about 1.4;
IDENTIFICATION impurity E = about 1.6; impurity G = about 2.0.
A. Infrared absorption spectrophotometry (2.2.24). System suitability: reference solution (b):
— resolution: m in im um 1.7 between the peaks due to
Comparison betamethasone 17-valerate CRS.
impurities H and D.
Limits:
— impurity A: n o t more than 7 times the area of the
substance separately in the m inim um volume of methylene
chloride R, evaporate to dryness on a water-bath and record
new spectra using the residues.
— impurities E, G: for each impurity, not more th an 3 times
B. Examine the chromatograms obtained in the test for the area of the principal peak in the chromatogram
related substances. obtained with reference solution (a) (0.3 per cent);
Results T h e principal peak in the chrom atogram obtained — impurities C, H, 1: for each impurity, not more than
with the test solution is similar in retention time and size to 1.5 times the area of the principal peak in the
the principal peak in the chromatogram obtained with chrom atogram obtained with reference solution (a)
reference solution (b). (0.15 p er cent);
1-288 Betaxolol Hydrochloride 2016

w ith reference solution (a) ( 0.10 per cent);
— total: n o t m ore than 15 times the area of the principal
— disregard limit. 0.5 times die area o f the principal peak in
the chromatogram obtained with reference solution (a) B. R l = F, R 2 = R3 = H : 9-fluoro-l lß , 17-dihydroxy-16ß-
(0.05 per cent). methylpregna-1,4-diene-3,20-dione (21-deoxy-
L oss o n d ry in g (2.2.32) betam ethasone),
M aximum 0.5 per cent, determined on 1.000 g by drying in D . R l = Br, R2 = C O -[C H 2]3-C H 3, R3 = O H : 9-bromo-
an oven at 105 °C. 11 ß,21dihydroxy-16 ß-methyl-3,20-dioxopregna-l ,4-dien-17-
A SSA Y yl pentanoate (9-bromo-betamethasone valerate),
Dissolve 50.0 m 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 50.0 m L with ethanol (96 per cent) R. M easure the
absorbance (2.2.25) at the absorption maximum at 240 nm.
Calculate the content of C 27H 37F 0 6 taking the specific
absoibance to be 325.
STORA GE
F. 21-hydroxy-16ß-m ethyi-3,20-dioxopregna-l,4,9(l l)-trien-
IM P U R IT IE S 17-yl pentanoate (betamethasone valerate 5-9(11)).
Specified impurities A, C , E, G , H , I PhEtr
by the general monograph Substances for pharmaceutical use Betaxolol Hydrochloride ***\
★ ★
(2034). It is therefore n o t necessary to identify these (Ph Eur monograph 1072) *
Control of impurities in substances for pharmaceutical usé): B, D, H OH H
F. X V ° ^ A / Nv^ch3
I T I . HCI
CH3
V and enantiomer
C18H30C1NO3 343.9 63659-19-8
Preparations
Betaxolol Eye Drops, Solution
A. R l = R3 = R5 = R 6 = H , R2 = F , R4 = C H 3: 9-fluoro-
Betaxolol Eye Drops, Suspension
1 l ß ,17,21 -trihydroxy-16 ß-methylpregna-1,4-diene-3,20-dione
(betamethasone), PhEur-----------------------------------------------------------------------------------------
C. R l = R 4 = R 6 = H , R2 = F, R3 = C H 3, R5 = CO- D E F IN IT IO N
[C H 2] 3-C H 3: 9-fluoro-l 1ß,21-dihydroxy-l 6a-methyl-3,20-
(2RS) -1 -[4- [2-(Cyclopropylmethoxy) ethyl] phenoxy] -3-
dioxopregnal,4-dien-17-yl pentanoate (dexamethasone
[(l-m ethylediyl)amino]propan- 2-ol hydrochloride.
17-valerate),
C o n te n t'
E. R l = R3 = R5 = H , R2 = F , R4 = C H 3, R 6 = C O -
[ C H J 3-C H 3: 9-fluoro-l 1ß, 17-dihydroxy-l6ß-methyl-3,20-
dioxopregna 1,4-dien-21 -yl pentanoate (betamethasone CHARACTERS
21-valerate), A p p e a ra n c e
G. R l = Br, R2 = F, R3 = R 6 = H , R4 = C H 3, R5 = W hite or almost white, crystalline powder.
C O- [CH 2] 3-C H 3: 6a-brom o-9-fluoro-l 1ß,21-dihydroxy- S o lu b ility
16ßmethyl-3,20-dioxopregna-l ,4-dien-17-yl pentanoate Very soluble in water, freely soluble in ethanol (96 p er cent),
( 6a-brom o-betam ethasone valerate), soluble in methylene chloride.
H . R l = R3 = R 6 = H , R2 = Cl, R4 = C H 3, R5 = C O - ID E N T IF IC A T IO N
[C H J 3-C H 3: 9-chloro-11 ß,2 1-dihydroxy-16 ß-methyl-3,20-
First identification B, D.
dioxopregna 1,4-dien-17-yl pentanoate (beclomethasone
17-valerate), Second identification A , C, D.
I. R l = R3 = R4 = R 6 = H , R2 = F , R5 = C O - f C H ^ -
C H 3: 9-fluoro-l lß ,21-dihydroxy-3,20-dioxopregna-1,4-dien- B. Infrared absorption spectrophotom etry (2.2.24).
17-yi pentanoate (9-fluoro-prednisolone 17-valerate), Comparison betaxold hydrochloride CRS.
2016 Betaxolol Hydrochloride 1-289
C . Thin-layer chromatography (2.2.27). — stationary phase: octylsUyl silica gel for chromatography R
Test solution Dissolve 10 mg of the substance to be examined (5 nm).
in 1 m L o f methanol R. Mobile phase M ix 175 m L of acetonitrile R and 175 m L of
Reference solution (a) Dissolve 20 mg of betaxolol methanol R and dilute to 1 L with a 3.4 g/L solution of
hydrochloride CRS in 2 m L of methanol R. potassium dihydrogen phosphate R, previously adjusted to
p H 3.0 with phosphoric add R.
Reference solution (b) Dissolve 10 m g of oxprenolol
hydrochloride CRS in 1 m L of reference solution (a). Flow rate 1.5 mL/min.
Plate TLC octadecylsHyl silica gel F 254 pl&te R- Detection Spectrophotometer at 273 nm .
Mobile phase perchloric add R, methanol R, water R Injection 20 nL o f the test solution and reference
(0.5:50:50 VIVIV). solutions (a), (b) and (d).
Application 2 (xL. Run time 4.5 the retention time of betaxolol.
Development Over a path of 10 cm. Identification of impurities Use the chromatogram obtained
Drying In air.
impurity A; use the chromatogram supplied with betaxolol for
System suitability-, reference solution (b): peak identification CRS and the chromatogram obtained with
reference solution (d) to identify the peaks due to
Detection A Examine in ultraviolet light at 254 nm . impurities B, C, D and E.
Results A T h e principal spot in the chromatogram obtained Relative retention W ith reference to betaxolol (retention
with the test solution is similar in position and size to the time = about 8 min): impurity B = about 0.3;
principal spot in the chromatogram obtained with reference impurity A = about 0.8; impurity D = about 1.5;
solution (a). impurity E = about 2.2; impurity C = about 4.1.
Detection B Spray with a 50 g/L solution o f vanillin R in a System suitability: reference solution (a) :
mixture o f 5 volumes of sulfuric add R, 10 volumes of glacial — resolution: m in im u m 2.0 between the peaks due to
acetic acid R and 85 volumes of methanol R, heat at impurity A and betaxolol.
100-105 °C until the colour o f the spots reaches maximum Limits:
intensity (10-15 min), and examine in daylight. — impurities A , B, C, D, E: for each impurity, not more than
Results B T he principal spot in the chromatogram obtained 0.3 times the area o f the prindpal peak in the
w ith the test solution is similar in position, colour and size to chromatogram obtained with reference solution (b)
the prindpal spot in the chromatogram obtained with (0.3 per cent);
reference solution (a). — unspecified impurities: for each impurity, not more than
D . It gives reaction (a) of chlorides (2.3.1). 0.1 times the area o f the principal peak in the
TESTS
(0.10 per cent);
Appearance o f solution — total: not more than the area of the prindpal peak in the
Method II). (1.0 per cent);
Dissolve 0.5 g in water R and dilute to 25 m L w ith the same — disregard limit. 0.05 times the area o f the prindpal peak in
solvent. the chromatogram obtained with reference solution (b)
Acidity or alkalinity (0.05 per cent).
Dissolve 0.20 g in carbon dioxide-free water R and dilute to H eav y m e ta ls (2.4.8)
20 m L with the same solvent. Add 0.2 m L of methyl red M aximum 10 ppm .
solution R and 0.2 m L of 0.01 M hydrochloric add. Dissolve 2.0 g in 20 m L of water R. 12 m L of the solution
T h e solution is red. Add 0.4 m L of 0.01 M sodium hydroxide.
T h e solution is yellow.
10 m L o f lead standard solution (1 ppm Pb) R.
Related substances L oss o n d ry in g (2.2.32)
L iquid chromatography (2.2.29). Prepare reference solutions (c)
and (d) immediately before use. an oven at 105 °C.
phase.
Reference solution (a) Dissolve 8 mg of the substance to be ASSAY
examined and 4 m g o f betaxold impurity A CRS in 20.0 m L Dissolve 0.300 g in a mixture of 10.0 m L of 0.01 M
of the mobile phase. hydrochloric add and 50 m L of ethanol (96 per cent) R. Carry
out a potentiometric titration (2.2.20), using 0.1 M sodium
hydroxide. Read the volume added between the 2 points of
inflexion.
Reference solution (c) Dissolve 2 m g o f betaxolol
1 m L of 0.1 M sodium hydroxide is equivalent to 34.39 m g
impurity C CRS in 50 m L of the mobile phase. D ilute 5 m L
of C 18H 30CINO 3.
o f the solution to 20 m L with the mobile phase.
Reference solution (d) Dissolve 10 m g o f betaxolol for peak STORA GE
identification CRS (containing impurities B, D and E) in Protected from light.
5 m L of reference solution (c). IM P U R IT IE S
Column'. Specified impurities A, B, C, D, E
— sizer. I = 0.25 m , 0 = 4 mm; •
1-290 Bezafibrate 2016
H OH
H CHARACTERS
N ^ CH3
T
^ „
and enantiomer Appearance
H3C CH3 W hite or almost white, crystalline powder.
Solubility
A. (2ftS)-l-(4-ethylphenoxy)-3- Practically insoluble in water, freely soluble in
[(1 -methylethyl)amino] propan-2-ol, dimethylformamide, sparingly soluble in acetone and in
ethanol (96 per cent). It dissolves in dilute solutions o f alkali
H OH hydroxides.
N ^/C H 3
I and enantiomer It shows polymorphism (5.9).
CH3 IDENTIFICATION
First identification A , B.
B. (2RS)-1- [4-(2-hydroxyethyI)phenoxy]-3- Second identification A, C.
[(1 -methylethyl) amino] propan-2-ol, A. Melting point (2.2.14): 181 °C to 185 °C.
Comparison bezafibrate CRS.
and enantiomer
If the spectra obtained show differences, dissolve the
substance to be examined and the reference substance
separately in methanol R and evaporate to dryness. D ry the
C. (2.RS)-2-[[4-[2- residues in vacuo at 80 °C for 1 h and record new spectra
(cyclopropylmethoxy)ethyl] phenoxy] methyl] oxirane, using the residues.
OH Test solution Dissolve 10 mg o f the substance to be examined
in methanol R and dilute to 5 m L with the sam e solvent.
Reference solution Dissolve 10 mg o f bezafibrate CRS in
D . 4 -[2-(cyclopropylmethoxy)ethyl]phenol, Plate TLC silica gel F2sa R
Mobile phase glacial acetic acid R, methyl ethyl ketone R,
H OH xylene R (2.7:30:60 V/V/V).
Application 5 pL.
Development Over h alf of the plate.
and enantiomer Drying A t 120 °C for at least 15 min.
Detection Examine in ultraviolet light at 254 nm .
E. (2i?5)-l-[4-(2-butoxyethyl)phenoxy]-3- Results T he principal spot in the chrom atogram obtained with
[(1 -methylethyl) amino] propan-2-ol. the test solution is similar in position and size to the principal
PhEur spot in the chromatogram obtained with the reference
solution.
TESTS
Solution S
Bezafibrate ***** Dissolve 1.0 g in dimethylformamide R and dilute to 20 mL
★ ★
* ★ with the same solvent.
(Ph Eur monograph 1394) Appearance o f solution
Solution S is clear (2.2.1) and n o t m ore intensely coloured
O ^ C O jH
H3C CH3 Related substances
a Test solution Dissolve 50.0 m g o f the substance to be
C 19H 20CINO4 361.8 41859-67-0 the mobile phase.
Action and use Reference solution (a) Dilute 10.0 m L o f the test solution to
Fibrate; lipid-regulating drug. 100.0 m L with the mobile phase. D ilute 5.0 m L o f this
Preparations
Bezafibrate Tablets
Prolonged-release Bezafibrate Tablets
Reference solution (c) T o 1 m L o f the test solution, add 1 m L
PhEur. o f 0.1 M hydrochloric add and evaporate to diyness on a hot
plate. Dissolve the residue in 20 m L o f the mobile phase.
DEFINITION
Column:
2-[4-[2-[(4-Chlorobenzoyl)amino]ethyl]phenoxy]-2-
— sizer. I = 0.125 m , 0 = 4 mm ;
methylpropanoic acid.
— stationary phase: octadecylsHyl silica gd for chromatography R
Content (5 pm)'.
2016 Bicalutamide 1-291
Mobile phase M ix 40 volumes o f a 2.72 g/L solution of

potassium cHhydrogen phosphate R adjusted to p H 2.3 with
phosphoric acid R, and 60 volumes o f methanol R
Flow rate 1 mL/min.
A. 4-chloro-N-[2-(4-hydroxyphenyl)ethyl]benzamide
Injection 20 |iL.
(chlorobenzoyltyramine),
Run time T h e time necessary to detect the ester, which,
depending on the route o f synthesis, may be impurity C, D
or E.
Relative retention W ith reference to bezafibrate (retention
time = about 6.0 min): impurity A = about 0.5;
impurity B = about 0 .6; impurity C = about 1.5; B. 4-chlorobenzoic acid,
o
System suitability:
— resolution: m in im u m 5.0 between die 2 principal peaks in
the chrom atogram obtained with reference solution (c);
— signal-to-noise ratio: minimum 5 for the principal peak in
the chrom atogram obtained with reference solution (b).
Limits:
— impurities A , B, C, D, E: for each impurity, not more than C . methyl 2-[4-[2-[(4-chlorobenzoyl)amino]ethyl]phenoxy]-2-
the area o f the principal peak in the chromatogram methylpropanoate,
obtained w ith reference solution (a) (0.5 per cent);
— unspecified impurities: for each impurity, not more than o
0.2 times the area of the principal peak in the
(0.10 per cent);
— total: no t m ore than 1.5 times the area o f the principal
D . ethyl 2-[4-[2-[(4-chlorobenzoyl)amino]ethyl]phenoxy]-2-
methylpropanoate,
(0.05 per cent). o
C h lo rid es (2.4.4)
M aximum 300 ppm .
Dilute 10 m L o f solution S to 50 m L w ith water R Filter the
resultant suspension through a wet filter previously washed
with water R until free from chlorides. Prepare the standard
using 9 m L o f chloride standard solution (5 ppm CO R and
E . butyl 2-[4-[2-[(4-chlorobenzoyl)amino]ethyl]phenoxy]-2-
6 m L of water R.
methylpropanoate.
__________________________________________________________ PhEur
M aximum 10 ppm .
2.0 g complies w ith test C. Prepare the reference solution
Bicalutamide *****
an oven at 105 °C. *****
Ov O HO CH3
Maximum 0.1 p er cent, determined on 1.0 g. V ^ V ”C 3 ^ enantiomer
A SSAY ° v A cn
Dissolve 0.300 g in 50 m L o f a mixture o f 25 volumes of
water R and 75 volumes o f ethanol (96 per cent) R. Using C 18H 14F4N204S 430.4 90357-06-5
0.1 m L of phenolphthakin solution R as indicator, titrate with
0.1 M sodium hydroxide until a pink colour is obtained. C an y A ctio n a n d u se
out a blank titration. Antiandrogen; treatm ent o f prostate cancer.
1 m L o iO .lM sodium hydroxide is equivalent to 36.18 mg P r e p a r a tio n
o f C 19H 20C IN O 4. Bicalutamide Tablets
IM P U R IT IE S PhEur__________________________________________________________
Specified impurities A, B, C , D , E.
D E F IN IT IO N
(2 RS) -N- [4-Cyano-3-(trifluoromethyl)phenyl] -3-
[(4-fluorophenyl) sulfonyl] -2-hydroxy-2-methylpropanamide.
C o n te n t
1-292 Bicalutamide 2016
CHARACTERS Relative retention W ith reference to bicalutamide (retention

A p p e a ra n c e tim e = about 38 min): impurity B = about 0.98;
W hite or almost white powder. impurity C = about 1.1.
S o lu b ility System suitability: reference solution (b):
Practically insoluble in water, freely soluble in acetone, — peak-to-valley ratio: minim um 2.5, where Hp = height
slightly soluble in anhydrous ethanol and in methylene above the baseline o f the peak due to im purity B and
chloride. Hv = height above the baseline of the lowest point o f the
bicalutamide.
ID E N T IF IC A T IO N Limits:
Infrared absorption spectrophotometry (2.2.24). — impurity C: no t more than 1.5 times the area o f the
Comparison bicalutamide CRS. principal peak in the chromatogram obtained with
If the spectra obtained in die solid state show differences, reference solution (a) (0.15 per cent);
dissolve the substance to be examined and the reference — unspecified impurities: for each impurity, n o t more than the
substance separately in acetone R, evaporate to dryness and area of the principal peak in the chromatogram obtained
record new spectra using the residues. with reference solution (a) (0.10 per cent);
TESTS
R e la te d su b stan ce s
(0.5 per cent);
Liquid chromatography (2.2.29). — disregard limit: 0.5 times the area o f the principal peak in
Solvent mixture phosphoric acid R, acetonitrUe R l, water R the chrom atogram obtained with reference solution (a)
(0.05:50:50 V/V/V). (0.05 per cent).
Test solution (a) Dissolve 25.0 m g o f the substance to be H eav y m e ta ls (¿2.4.8)
examined in the solvent mixture and dilute to 25.0 m L with M aximum 20 ppm.
Solvent mixture water R, acetone R (10:90 V/V).
Test solution (b) Dilute 5.0 m L o f test solution (a) to
Reference solution (a) D ilute 1.0 m L o f test solution (a) to
100.0 m L w ith the solvent mixture. D ilute 1.0 m L o f this
Reference solution (b) Dissolve 5 mg o f bicalutamide for system
m ixture and dilute to 5.0 m L with the solvent mixture. M aximum 0.1 p er cent, determined on 1.0 g in a platinum
crucible.
Reference solution (c) Dissolve 25.0 m g o f bicalutamide CRS in
the solvent mixture and dilute to 25.0 m L with the solvent ASSAY
mixture. D ilute 5.0 m L o f the solution to 25.0 m L with the Liquid chromatography (2.2.29) as described in the test for
solvent mixture. related substances with the following modification.
Column: Injection T est solution (b) and reference solution (c) .
— size: I = 0.25 m, 0 = 4.0 mm; Calculate the percentage content o f C i8Hi4F4N20 4S taking
— stationary phase:, spherical end-capped octadecylsUyl silica gel into account the assigned content o f bicalutamide CRS.
for chromatography R (5 |im);
IM P U R IT IE S
Specified impurities C.
Mobile phase:
— mobile phase A: phosphoric acid R, acetonitrUe R l, water R Other detectable impurities (the following substances would, if
(1.9:100:1900 V/V/V); present at a sufficient level, be detected by one or other o f
— mobile phase B: phosphoric acid R, water R, acetonitrUe R l the tests in the monograph. T hey are limited by the general
(1.9:100:1900 V/V/V); acceptance criterion for other/unspecified impurities and/or
0 -3 92 8
D, E, F, H, J, K, L, M.
3 -2 3 92 —¥ 67 8 -> 3 3
23-43 6 7 —>50 33 —* 50
43-50 50 50

Detection Spectrophotom eter at 210 nm . A. (2RS)-N- [4r-cyano-3-(trifluoromethyl)phenyl]-2-hydroxy-2-
Injection 10 (iL of test solution (a) and reference solutions (a) methyl-3-(phenylsulfonyl)propanamide,
and (b).
with bicalutamide for system suitability CRS and the
identify the peaks due to impurities B and C.
2016 Bifonazole 1-293
h H3C OHO OHOCH3 h

f 3c Y y ny V s\ A ^ ny v cf3
and enantiomer
CCYXX 0 0
and enantiomer
B. (2RS)-N- [4-cyano-3-(trifluoromethyI)phenyl]-3-
[(2-fluorophenyI)sxilfonyI]-2-hydroxy-2-methyipropanaxnidej L. (2RS,2 'RS)-3,3 '-sulfonylbis [N- [4-cyano-3-
(trifluoromethyl)phenyl] -2-hydroxy-2-methylpropanamide],
o o h ch3
O OHO CH,
^ 3andenan**0mer *'/ \f
sn A co 2h and enantiomer
. 8
C. (2iiS)-N-t4-cyano-3-(trifluoromethyl)phenyl]-3-
[(4-fluorophenyl)sulfonyI]-2-methylpropanainidej
M. (2RS) -3-[(4-fluorophenyl) sulfonyl] -2-hydroxy-2-
methylpropanoic add.
H2NV ^ s^ CF3
XX CN
PhEur
D. 4-amino-2-(txifluoromethyl)benzonitxile3 ***
★ ★
Bifonazole ★ ★
° “ v /^ H *****
(Ph. Ettr. monograph 1395)
and enantiomer
x r^ x c
and enantiomer
E. (2RS)-N- [4-cyano3-(trifluoromethyI)phenyl]-3-[(&S)-
(4-fluorophenyl)sulfinyl]-2-hydroxy-2-methylpropanamide,
°,
C22H 18N 2 310.4 60628-96-8
III I
I I a
nde
nan
ti
o
mer
fA ^ o v A cn Action and use
Antifungal.
F. (2SR)-N -[4-cyano-3-(trifluoromethyI)phenyl]-3 - [(RS)-
PhEir ____________
(4-fluorophenyl)sulfmyI]-2-hydroxy-2-methylpropanamide,
DEFINITION
1- [(&S)-(Biphenyl-4-yl) phenylmethyl] -1 //-imidazole.
and enantiomer Content

CHARACTERS
Appearance
H . (2RS)-N- [4-cyano-3-(trifluoromethyl)phenyl]-2-
[(4-fluorophenyI)sulfonyI]-3-hydroxy-2-methylpropanamide, Solubility
Practically insoluble in water, sparingly soluble in anhydrous
ethanol.
and enantiomer
IDENTIFICATION
Comparison bifonazole CRS.
J. (2RS)-N- [4-cyano-3-(trifluoroniethyl)phenyl]-3-
[(4-fluorophenyl)sulfanyl]-2-hydroxy-2-methylpropanamide,
substance separately in the minimum volume o f 2-propanol R,
• , H3C OH 0 0 HO CH3 u
evaporate to dryness and record new spectra using the
f 3c cf 3
residues.
nc
x> y w y -toc CN TESTS
Related substances
K. (22?,2 'S)-3,3 '-sulfonylbis [N-[4-cyano-3- Liquid chrom atography (2.2.29).
(trifluoromethyl)phenyI]-2-hydroxy-2-methylpropanamide], Buffer solution pH 32 M ix 2.0 m L o f phosphoric acid R with
980 m L o f water R, adjust to p H 3.2 (2.2.3) with
tnethylamine R and dilute to 1000.0 m L with water R.
1-294 Bifonazole 2016
Test solution Dissolve 50.0 mg of the substance to be L oss o n d ry in g (2.2.32)

examined in 25 m L o f acetomtrik R and dilute to 50.0 m L M aximum 0.5 p er cent, determined on 1.000 g by drying in
with buffer solution p H 3.2. an oven at 105 °C.
Reference solution (a) Dilute 1.0 m L o f the test solution to S u lfa ted a s h (2.4.14)
100.0 m L with buffer solution p H 3.2. Dilute 1.0 m L of this M aximum 0.1 p er cent, determined on 1.0 g.
solution to 10.0 m L with buffer solution p H 3.2.
ASSAY
Reference solution (b) Dissolve 2 mg o f bifonazole for system Dissolve 0.250 g in 80 m l. o f anhydrous acetic acid R. Titrate
suitability CRS (containing impurities A, B, C , D and E) in with 0.1 M perchloric acid, determining the end-point
2 m L of acetonitrUe R and dilute to 10.0 m L with buffer potentiometrically (2.2.20).
solution p H 3.2.
1 m L o f 0.1 M perchloric acid is equivalent to 31.04 mg
Column:
of C 22H 18N 2.
— sizer. I = 0.125 m, 0 = 4.0 mm;
— stationary phase: octadecylsUjyl silica gel for chromatography R IM P U R IT IE S
(5 nm); Specified impurities A, B, C , D , E
Mobile phase:
— mobile phase A: acetomtrik R l, buffer solution p H 3.2
(20:80 VIV)\ and enantiomer
— mobile phase B: buffer solution p H 3.2, acetomtrik R l
(20:80 VfV);
A. (i?5)-(biphenyl-4-yl)phenylmethanol,
(min) (per cent VfV) (per cent V7V)
0 -8 60 40
and enantiomer
8 - 12 60-» 10 40-» 90
12 - 30 10 90
NH
Flow rate 1 mL/min. B. 4-[(.&S)-(biphenyl-4-yl)phenylmethyl]-1 H-imidazole,

Injection 50 |±L.
with bifonazole for system suitability CRS and the
identify the peaks due to impurities A, B, C, D and E. C. lH-imidazole,
Relative retention W ith reference to bifonazole (retention
time = about 4 min): im purity C = about 0.2;
impurity B = about 0.7; impurity A = about 3.2;
impurity B and bifonazole.
Limits:
— correction factor, for the calculation o f content, multiply the
peak area of impurity C by 2;
— impurities B, D: for each impurity, not more than 5 times
the area o f the principal peak in the chromatogram D. l,3-bis[(biphenyl-4-yl)phenylmethyl]-lH-imidazolium ion,
— impurities A , C: for each impurity, n o t more than twice
— impurity E: not m ore than 1.5 times the area o f the
— total: not more than 10 times the area o f the principal
peak in the chrom atogram obtained with reference E. 1,4-bis [(biphenyl-4-yl)phenylmethyl]-1H-imidazole.
PhEur
disregard limir. 0.5 times the area o f the principal peak in
(0.05 per cent).
2016 Biotin 1-295
-L.*** Reference solution (b) Dilute 1 m L o f test solution (b) to

Biotin 20 m L with glacial acetic add R.
(Ph. Ever, monograph 1073) ***** Reference solution (c) Dilute 1 m L o f test solution (b) to
40 m L with glacial acetic acid R.
Apply to the plate 10 ^L of each solution. Develop over a
path of 15 cm using a mixture of 5 volumes of methanol R,
25 volumes o f glacial acetic add R and 75 volumes of
toluene R. D ry the plate in a current o f warm air. Allow to
cool and spray with 4-dimeihylaminocmnamaldehyde solution R.
Examine immediately in daylight. Any spot in the
Cl0Hl6N2O3S 244.3 58-85-5
chromatogram obtained with test solution (a), apart from the
principal spot, is n o t more intense th an the spot in the
A ctio n a n d u se
Vitamin.
(0.5 per cent) and at most one such spot is more intense
PhEur_____________ than the spot in the chromatogram obtained with reference
solution (c) (0.25 per cent).
D E F IN IT IO N
Biotin contains n o t less than 98.5 per cent and n o t more H eav y m e ta ls (2.4.8)
than the equivalent o f 101.0 per cent o f 5-[(3a5,4536ai?)-2- 1.0 g complies with test C for heavy metals (10 ppm ).
oxohexahydrothieno[3,4-d]imidazol-4-yl]pentanoic ad d , Prepare the reference solution using 10 m L of lead standard
calculated with reference to the dried substance. solution (1 ppm Pb) R.
CHARACTERS
N o t more than 1.0 per cent, determined on 1.000 g by
A white or almost white, crystalline powder or colourless
crystals, very slighdy soluble in water and in alcohol,
practically insoluble in acetone. It dissolves in dilute solutions S u lfa te d a s h (2.4.14)
o f alkali hydroxides. N o t more than 0.1 per cent, determined on 1.0 g.
ID E N T IF IC A T IO N ASSAY
First identification A Suspend 0.200 g in 5 m L of dimethytformannde R. H eat until
the substance has dissolved completely. A dd 50 m L of
ethanol R and titrate with 0.1 M tetrabutylammonium
hydroxide, determining the end-point potentiometrically
( 2.2.24), comparing with the spectrum obtained with ( 2. 2. 20).
biomt CRS.
1 m L of 0.1 M tetrabutylammonium hydroxide is equivalent to
B. Examine the chromatograms obtained in the test for 24.43 mg o f CioH16N20 3S.
related substances (see Tests). The principal spot in the
chromatogram obtained with test solution (b) is similar in STORA GE
position and size to the principal spot in the chrom atogram Store protected from lig h t
obtained with reference solution (a). IM P U R IT IE S
C. Dissolve about 10 mg in 20 mL of water R with heating.
Allow to cool. Add 0.1 m L o f bromine water R. T he brom ine
water is decolourised.
*- = o = <
TESTS
S o lu tio n S
Dissolve 0.250 g in a 4 g/L solution o f sodium hydroxide R
and dilute to 25.0 m L with the same alkaline solution.
Solution S is clear (2.2.1) and colourless (2.2.2, Method IT). c o 2h
Specific o p tic a l r o ta tio n (2.2.7)

T he specific optical rotation is + 89 to + 93, determ ined on A. di[3- [(3aS,4S,6ai?)-2-oxohexahydrothieno [3,4-d\ imidazol-
solution S and calculated with reference to the dried 4-yl] propyl] acetic ad d ,
substance.
R elated su b s ta n c e s
Examine by thin-layer chromatography (2.2.27), using as the
coating substance a suitable silica gel (5 pm). Prepare the
solutions immediately before use and keep protected from bright
light B. 4-[(3aS,4.S,6aÆ)-2-oxohexahydrothieno [3,4-d]imidazol-4-
yl] butane-1,1-dicarboxylic add,
Test solution (a) Dissolve 50 mg of the substance to be
examined in glacial acetic acid R and dilute to 10 m L with the
h2n
same solvent.
with glacial acetic acid R. H*N X .
Reference solution (a) Dissolve 5 mg o f biotin CRS in glacial
acetic acid R and dilute to 10 m L with the same solvent. C. 5-(3,4-diamino-2-thienyl)pentanoic ad d ,
1-296 Biperiden Hydrochloride 2016
, cc>2h
and enantiomer Reference solution (a) Dissolve 25 m g of biperiden
H CH3 hydrochloride CRS in methanol R and dilute to 5 m L with the
same solvent.
D. 2-methyl-5-[(3a£,4S,6aR)-2-oxohexahydrothieno[3,4- Reference solution (b) Dissolve 5 m g o f biperiden
d\ imidazol-4-yl] pentanoic ad d , impurity A CRS in reference solution (a) and dilute to 2 m L
Plate TLC silica gel F2 5 a plate R.
Mobile phase diethylamine R, methanol R, toluene R
(1:1:20 VfVfV).
Application 5 |iL
Drying In air.
c o 2h
Results A T he prindpal spot in the chromatogram obtained
prindpal spot in the chromatogram obtained with reference
solution (a).
E. 5- [(385,45,6aR)-3-benzyl-2-oxohexahydrothieno [3,4-
d\imidazol-4-yl]pentanoic a d d and 5-[(3a£,4S,6ai?)-l-benzyl- Detection B Spray with dilute potassium iodobismuthate
2-oxohexahydrothieno [334-d] imidazol-4-yl] pentanoic add.
solution R and then with sodium nitrite solution R and exam in e
in daylight.
___________________________________________________ ________ PhEur
Results B T he prindpal spot in the chromatogram obtained
with the test solution is similar in position, colour and size to
the prindpal spot in the chromatogram obtained with
***
Biperiden Hydrochloride ★ ★ System suitability, reference solution (b):
★ ★
C. T o about 20 m g add 5 m L o f phosphoric add R. A green
colour develops.
TESTS
and enantiomer , HCI S o lu tio n S
Dissolve 0.10 g in carbon dioxide-free water R, heating gently if
necessary, and dilute to 50 m L with the same solvent.
Solution S is n o t more opalescent than reference
C^HaoClNO 347.9 1235-82-1 suspension II (2.2.1) and is colourless (2.2.2, Method II).
A c tio n a n d use p H (2.2.3)

Anticholinergic. 5.0 to 6.5 for solution S.
D E F IN IT IO N Test solution Dissolve 0.10 g o f the substance to be examined
(l/?5)-l-[(li?5,2S2?,4i?S)-Bicyclo[2.2.1]hept-5-en-2-yl]-l- in methanol R and dilute to 10 m L with the same solvent.
phenyl-3-(piperidin- l-yl)propan- l-ol hydrochloride. Reference solution (a) Dilute 0.5 m L of the test solution to
C o n te n t 100 m L with methanol R. Dilute 10 m L of this solution to
99.0 per cent to 101.0 per cent (dried substance). 50 m L with methanol R.
CHARACTERS Reference solution (b) Dissolve 5 m g o f the substance to be
A p p e a ra n c e examined and 5 m g o f biperiden impurity A CRSjn.
W hite or almost white, crystalline powder. methanol R and dilute to 5 m L with the same solvent. Dilute
1 m L o f the solution to 10 m L with methanol R.
S o lu b ility
Column:
Slighdy soluble in w ater and in alcohol, very slightly soluble
— material: fused silica,
in methylene chloride,
— sizer. I = 50 m , 0 = 0.25 m m ,
mp — stationary phase: poly(dimethyl) (diphenyl) (divinyl)süoxcme R
A bout 280 °C, with decomposition. (film thickness 0.25 pm).
ID E N T IF IC A T IO N Carrier gas nitrogen for chromatography R.
First identification A , D. Flow rate 0.4 mL/min.
A. Infrared absorption spectrophotometry (2.2.24). Split ratio 1:250.
Comparison biperiden hydrochloride CRS.
B. Thin-layer chrom atography (2.2.27).
2016 Biperiden Hydrochloride 1-297
Temperature:
Time Temperature and enantiomer

(min) (°C)
Column 0 -5 200
5 -40 200 270
Injection port 250

A. (li?5)-l-[(l5i?j25i?j45J?)-bicyclo[2.2.1]hept-5-en-2-yl]-l-
Detector 300
phenyl-3-(piperidin-l-yl)propan-l-ol (endo form),

Irgecdon 2 |jL.
Run time Twice the retention time o f biperiden. and enantiomer
Relative retention W ith reference to biperiden: impurities A, B
and C = between 0.95 and 1.05.
System suitability:
— resolution: minimum 2.5 between the peak due to
biperiden (1st peak) and the peak due to impurity A B. (li?5)-l-[(15i?,2i?5,45^)-bicyclo[2.2.1]hept-5-en-2-yl]-l-
(2nd peak) in the chromatogram obtained with reference phenyl-3-(piperidin- l-yl)propan-1-ol,
solution (b),
the chrom atogram obtained with reference solution (a).
Limits:
— impurities A , B, C: for each impurity, maximum and enantiomer
0.50 per cent o f the area of the principal peak,
— any other impurity: for each impurity, maximum
0.10 per cent o f the area o f the principal peak,
— total of impurities A, B and C: maximum 1.0 per cent of
the area of the principal peak, C . (li? S )-l-[(l RS,2RS,4RS)-bicyclo[2.2.1]hept-5-en-2-yl]-1 -
— total of impurities other than A, B and C: maximum phenyl-3-(piperidin-l-yl)propan-1-ol,
0.50 per cent o f the area of the principal peak,
— disregard limit: 0.05 per cent of the area o f the principal
peak. h h
I m p u rity F (2.4.24) and enantiomer

M axim um 2 ppm .
Maximum 20 ppm .
1.0 g complies with test D . Prepare the reference solution D . 1- [(1 RS,2SR, 4RS) -bicyclo [2.2.1 ]hept-5-en-2-yl] -3-
using 2 m l, o f lead standard solution (10 ppm Pb) R. (piperidin-1-yl)propan-1-one,
and enantiomer
S u lia te d a s h (2.4.14)
H
A SSAY
Dissolve 0.200 g in 60 m L o f alcohol R. In a closed vessel,
E. 1- [(1 RS,2RS,4RS)-bicyclo [2.2.1] hept-5-en-2-yl] -3-
titrate with 0.1 M alcoholic potassium hydroxide, determining
(piperidin-1-yl)propan-1-one,
the end-point potentiometrically [2.2.20).
F. benzene.
1 mT. of 0.1 M alcoholic potassium hydroxide is equivalent to
34.79 m g o f C 21H 30C1NO. PhEur
STORAGE
IM P U R IT IE S
Specified impurities A , B, C, F.
Control of impurities in substances fa pharmaceutical use): D, E.
1-298 Bisacodyl 2016
★ ★ Drying In air, if necessary heating at 100-105 °C.

Bisacodyl ★ ★
Detection Spray with a mixture o f equal volumes o f 0.05 M
(Ph Eur. monograph 0595) ***** iodine and dilute sulfuric add R.
h 3c the test solution is similar in position and size to the principal
Y 0"’ spot in the chromatogram obtained with the reference
0
solution.
TESTS
T o 1.0 g add 20 m L o f carbon dioxide-free water R, shake,
heat to boiling, cool and filter. Add 0.2 m L o f 0.01 M sodium
C 22H 19N 04 603-50-9 hydroxide and 0.1 m L o f methyl red solution R. T h e solution is
yellow. N o t more than 0.4 m L of 0.01 M hydrochloric add is
Action and use required to change the colour of the indicator to red.
Stim ulant laxative.
Related substances
Preparations Liquid chromatography (2.2.29). Prepare the solutions
Bisacodyl Suppositories immediately before use.
Gastro-resistant Bisacodyl Tablets Solvent mixture glacial acetic add R, acetonitrUe R, water R
PhEur______________________________
(4:30:66 V/V/V).
DEFINITION
in 25 m L o f acetonitrUe R and dilute to 50.0 m L with the
4,4(Pyridin-2-ylm ethylene)diphenyl diacetate.
solvent mixture.
Content Reference solution (a) Dilute 1.0 m L of the test solution to
98.0 per cent to 101.0 per cent (dried substance). 100.0 m L with the solvent mixture. Dilute 1.0 m L of this
CHARACTERS solution to 10.0 m L with the solvent mixture.
Appearance Reference solution (b) Dissolve 2.0 m g of bisacodylfor system
W hite or alm ost white, crystalline powder. suitability CRS (containing impurities A, B, C , D and E) in
Solubility 1.0 m L o f acetomtrile R and dilute to 2.0 m L with the solvent
Practically insoluble in water, soluble in acetone, sparingly mixture.
soluble in ethanol (96 per cent). It dissolves in dilute mineral Reference solution (c) Dissolve 5.0 m g of bisacodyl for peak
acids. identification CRS (containing impurity F) in 2.5 m L of
IDENTIFICATION acetomtrile R and dilute to 5.0 m L with the solvent mixture.
First identification C. Column:
— size: I = 0.25 m , 0 = 4.6 mm;
— stationary phase: end-capped octadecylsHyl silica gel for
A. M elting point (2.2.14): 131 °C to 135 °C. chromatography R (5 pm).
B. Ultraviolet and visible absorption spectrophotometry Mobile phase Mix 45 volumes o f acetomtrile R and 55 volumes
(2.2.25). of a 1.58 g/L solution o f ammonium formate R previously
Test solution Dissolve 10.0 m g in a 6 g/L solution of potassium adjusted to p H 5.0 with anhydrous formic add R.
hydroxide R in methanol R and dilute to 100.0 m L with the Flow rate 1.5 mL/min.
sam e solution. Dilute 10.0 m L o f this solution to 100.0 m L
with a 6 g/L solution o f potassium hydroxide R in methanol R.
Irtjection 20 (tL.
Run time 3.5 times the retention time of bisacodyl.
Absorption maximum A t 248 nm .
Shoulder At 290 nm.
with bisacodyl for system suitability CRS and the chromatogram
Specific absorbance at the absorption maximum 632 to 672. obtained with reference solution (b) to identify the peaks due
C. Infrared absorption spectrophotom etry (2.2.24). to impurities A, B, C, D and E.
Comparison bisacodyl CRS. Relative retention W ith reference to bisacodyl (retention
If the spectra obtained in the solid state show differences, time = about 13 min): impurity A = about 0.2;
dissolve the substance to be examined and the reference impurity B = about 0.4; impurity C = about 0.45;
substance separately in chloroform R, evaporate to dryness and impurity D = about 0.8; impurity E = about 0.9;
record new spectra using the residues. impurity F = about 2.6.
D. Thin-layer chromatography (2.2.27). System suitability: reference solution (b):
— peak-to-vaHey ratio: minim um 1.5, where Hp = height
above the baseline of the peak due to impurity E and
in acetone R and dilute to 10 m L w ith the same solvent.
Hv = height above the baseline of the lowest point of the
Reference solution Dissolve 20 mg o f bisacodyl CRS in curve separating this peak from the peak due to bisacodyl.
Limits:
Plate TLC silica gel GF254 plate R. — correction factor, for the calculation of content, multiply the
Mobile phase methyl ethyl ketone R, xylene R (50:50 V/V). peak area o f impurity A by 0.7;
Application 10 fiL.
2016 Bismuth Subcarbonate 1-299
— impurities A , B: for each impurity, not more than the area ★ ★

Bismuth Subcarbonate ★ ★
reference solution (a) (0.1 per cent); Bism uth Carbonate
— impurities C, E: for each impurity, not more than 5 times (Ph Eur. monograph 0012)
PhEur______________________________
— impurity D: n o t more than twice the area of the principal DEFINITION
peak in the chromatogram obtained with reference Content
solution (a) (0.2 per cent); 80.0 p er cent to 82.5 per cent of Bi (Ar 209.0) (dried
— impurity F: n o t more than 3 times the area of the substance).
CHARACTERS
Appearance
W hite or almost white powder.
w ith reference solution (a) ( 0.10 per cent); Solubility
— total: not m ore than 10 times the area of the principal Practically insoluble in water and in ethanol (96 per cent).
peak in the chromatogram obtained with reference I t dissolves with effervescence in mineral adds.
solution (a) ( 1.0 per cent); IDENTIFICATION
— disregard limit: 0.5 times the area of the principal peak in A. It gives the reaction of carbonates (2.3.1).
B. It gives the reactions o f bismuth (2.3.1).
(0.05 per cent).
TESTS
M axim um 0.5 p er cent, determined on 0.500 g by drying in Solution S
an oven at 105 °C. Shake 5.0 g with 10 m L of water R and add 20 m L of nitric
acid R. H eat to dissolve, cool and dilute to 100 m L with
water R.
A SSA Y Solution S is n o t m ore opalescent than reference
Dissolve 0.300 g in 60 m L o f anhydrous acetic arid R. T itrate suspension II (2.2.1) and is colourless (2.2.2, Method II).
with 0.1 M perchloric acid determining the end-point
Chlorides (2.4.4)
M aximum 500 ppm.
T o 6.6 m L o f solution S add 4 m L of nitric acid R and dilute
o f C 22H 19N O 4.
STO RA G E
Nitrates
Protected from light. M aximum 0.4 per c e n t
IM P U R IT IE S T o 0.25 g in a 125 m L conical flask, add 20 m L of water R,
Specified impurities A, B, C , D , E, F 0.05 m L of indigo carmine solution R1 and then, as a single
addition b u t with caution, 30 m L of sulfuric acid R. T itrate
immediately with indigo carmine solution R1 until a stable blue
colour is obtained. N o t more than n m L o f the titrant is
required, n being the volume corresponding to 1 mg of N 0 3.
and enantiomer
Alkali and alkaline-earth metals
T o 1.0 g add 10 m L o f water R and 10 m l. of acetic acid R.
Boil for 2 min, cool and filter. W ash the residue with 20 m L
A. R1 = R3 = O H , R2 = H : 4J4'-(pyridin-2- o f water R. T o the combined filtrate and washings add 2 m L
ylmethylene)diphenol, o f dilute hydrochloric acid R and 20 m L o f water R. Boil and
B. R1 = H, R2 = R3 = OH: 2-[(RS)~ pass hydrogen sulfide R through the boiling solution until no
(4-hydroxyphenyl) (pyridin-2-yl)methyl] phenol, further predpitate is formed. Filter, wash the residue with
C. R1 = OH, R2 = H , R3 = 0 -C 0 -C H 3: 4~[(RS)~ water R, evaporate the combined filtrate and washings to
(4-hydroxyphenyl) (pyridin-2-yl)methyI]phenyl acetate, dryness on a water-bath and add 0.5 m L of sulfuric acid R.
Ignite gently and allow to cool. T h e residue weighs a
E. R1 = H , R2 = R3 = 0 - C 0 -C H 3: 2-[(RS)-[4r
maximum o f 10 mg.
(acetyloxy)phenyl] (pyridin-2-yl)methyI]phenyl acetate,
D . unknow n structure,
Arsenic (2.4.2, Method A)
M aximum 5 ppm.
F. unknow n structure.
T o 0.5 g in a distillation flask add 5 m L of water R and
______________________________________________________ _____ PhEur
7 m L o f sulfuric acid R, allow to cool and add 5 g of reducing
mixture R and 10 m L of hydrochloric acid R. H eat the
contents of the flask to boiling gradually over 15-30 min and
continue heating at such a rate that the distillation proceeds
steadily until the volume in the flask is reduced by half or
until 5 m in after the air-condenser has become full of steam.
It is im portant that distillation be discontinued before fumes
o f sulfur trioxide appear. Collect the distillate in a tube
1-300 Bismuth SubgaUate 2016
containing 15 m L o f water R cooled in ice-water. W ash down

the condenser with water R and dilute the distillate to 25 m L
Bismuth Subgallate *****
**
with the same solvent. Prepare the standard using a mixture (Ph Em monograph 1493) *
of 2.5 mL o f arsenic standard solution (1 ppm As) R and
22.5 m L of water R.
C opper
Maximum 50 ppm.
OH
T o 5 m L of solution S, add 2 m L of ammonia R and dilute
to 50 m L with water R. Filter. T o 10 m L of the filtrate add
1 m L of a 1 g/L solution of sodium diethyldithiocarbamate R C7H 5B i06 394.1 99-26-3
T he solution is not m ore intensely coloured than a standard P h E ir __________________________________________________________________________________________
prepared at the same time in the same m anner using a D E F IN IT IO N
mixture of 0.25 m L o f copper standard solution (10 ppm Cu) R
Complex o f bismuth and gallic acid.
and 9.75 m L o f water R instead of 10 m L of the filtrate.
C o n te n t
L ead
48.0 per cent to 51.0 per cent of Bi (AT 209.0) (dried
Maximum 20 ppm.
substance).
Atomic absorption spectrometry (2.2.23, Method II).
CHARACTERS
Test solution Dissolve 12.5 g in 75 m l. o f a mixture of
A p p e a ra n c e
equal volumes of lead-free nitric add R and water R. Boil for
Yellow powder.
1 m in, cool and dilute to 100.0 m L with water R.
S o lubility
Reference solutions Prepare the reference solutions using
Practically insoluble in water and in ethanol (96 per cent).
appropriate quantities of lead standard solution and a
37 p er cent V/V solution o f lead-free nitric add R. It dissolves in mineral acids with decomposition and in
solutions o f alkali hydroxides, producing a reddish-brown
Source Lead hollow-cathode lam p.
liquid.
Wavelength 283.3 nm (depending on the apparatus, the line
at 217.0 nm may be used). ID E N T IF IC A T IO N
A. M ix 0.1 g with 5 m L of water R and 0.1 m L of phosphoric
add R. H eat to boiling and maintain boiling for 2 min. Cool
S ilv er and filter. T o the filtrate, add 1.5 m L of ferric chloride
M aximum 25 ppm. solution Rl; a blackish-blue colour develops.
T o 2.0 g add 1 m L of water R and 4 m L of nitric add R. B. It gives reaction (b) of bismuth (2.3.1).
H eat gently until dissolved and dilute to 11 m L with water R.
TESTS
Cool and add 2 m L o f 1 M hydrochloric add. Allow to stand
protected from light for 5 m in. Any opalescence in the S o lu tio n S
solution is n o t more intense than that in a standard prepared In a porcelain or quartz dish, ignite 1.0 g, increasing the
at the same tim e in the same m anner using a mixture o f tem perature very gradually. H eat in a muffle furnace at
10 m L of silver standard solution (5 ppm Ag) R, 1 m L o f nitric 600 ± 50 °C for 2 h. Cool and dissolve the residue with
add R and 2 m L of 1 M hydrochloric add. warming in 4 m L of a mixture o f equal volumes of lead-free
nitric add R and water R and dilute to 20 m L with water R.
L oss o h d ry in g (2.2.32)
Maximum 1.0 per cent, determined on 1.000 g by drying in A cid ity
an oven at 105 °C. Shake 1.0 g with 20 m L of water R for 1 min and filter.
T o the filtrate add 0.1 m L o f methyl red solution R. N o t more
A SSAY than 0.15 m L of 0.1 M sodium hydroxide is required to
Dissolve 0.500 g in 3 m L of nitric add R and dilute to change the colour o f the indicator to yellow.
250 m L with water R. Carry o u t the complexometric titration
of bism uth (2.5.11).
M aximum 200 ppm.
1 m L o f 0.1 M sodium edetate is equivalent to 20.90 m g of BL
T o 0.5 g add 10 m L of dilute nitric add R. H eat on a water-
ST O R A G E bath for 5 min and filter. Dilute 5 m L o f the filtrate to
Protected from light. 15 m L with water R.
___________________________________________________________________________________________P h E ir N itra te s
T o 1.0 g add 25 m L of water R then 25 m L o f a mixture of
2 volumes of sulfuric add R and 9 volumes of water R. H eat
at about 50 °C for 1 min with stirring and filter. T o 10 m L
of the filtrate, carefully add 30 m L o f sulfuric add R.
T he solution is not more intensely brownish-yellow than a
reference solution prepared at the same time as follows: to
0.4 g o f gallic add R, add 20 m L o f nitrate standard solution
(100 ppm NOj) R and 30 m L of a mixture o f 2 volumes of
sulfuric add R and 9 volumes of water R, then filter; to 10 m L
of the filtrate, carefully add 30 m L o f sulfuric add R.
C opper
M aximum 50 ppm.
2016 Bismuth Subnitrate 1-301
Test solution Solution S. ***

Heavy Bismuth Subnitrate ★ ★
★ ★
Reference solutions Prepare the reference solutions using copper
*****
standard solution (10 ppm Cu) R and diluting with a (Ph Eur monograph 1494)
6.5 per cent V/V solution o f lead-free nitric acid R. 4[BiN03(0H) 2],B i0(0H ) 1462 12
1304-85-4
Source Copper hollow-cathode lamp. P h E tr ______________________________________________________________________________
D E F IN IT IO N
Atomisation device Air-acetylene flame. C o n te n t
Lead 71.0 p er cent to 74.0 per cent o f Bi (At 209.0) (dried
M axim um 20 ppm . substance).
Atomic absorption spectrometry (2.2.23, Method II). CHARACTERS
Test solution Solution S. A p p e a ra n c e
Reference solutions Prepare the reference solutions using lead W hite or almost white powder.
standard solution (10 ppm Pb) R and diluting with a S o lu b ility
6.5 per cent V/V solution o f lead-free nitric add R. Practically insoluble in water and in ethanol (96 per cent).
Source Lead hollow-cathode lamp. It dissolves in mineral acids with decomposition.
Wavelength 283.3 nm (depending on the apparatus, the line ID E N T IF IC A T IO N
at 217.0 nm m ay be used). A. D ilute 1 m L o f solution S i (see Tests) to 5 m L with
Atomisation device Air-acetylene flame. water R and add 0.3 m l. of potassium iodide solution R.
SU ver A black precipitate is formed which dissolves into an orange
M aximum 25 ppm . solution with the addition of 2 m L o f potassium iodide
Atomic absorption spectrometry (2.2.23, Method I). solution R.
Test solution Solution S. B. It gives reaction (b) o f bism uth (2.3.1).
Reference solutions Prepare the reference solutions using silver C. It gives the reaction o f nitrates (2.3.1).
standard solution (5 ppm Ag) R and diluting with a D. p H (2.2.1): maximum 2.0 for solution S2 (see Tests).
6.5 per cent V/V solution of lead-free nitric acid R. TESTS
Source Silver hollow-cathode lamp. S o lu tio n S I
Wavelength 328.1 nm. Shake 5.0 g by gently heating in 10 m L of water R and add
Atomisation device Air-acetylene flame. 20 m L o f nitric add R. H eat until dissolution, cool and dilute
S u b sta n c e s n o t p re c ip ita te d b y am m onia
M aximum 1.0 p er c e n t S o lu tio n S2
Place 1.00 g in a 20 m L volumetric flask and add 2.0 m L of
In a porcelain o r quartz dish, ignite 2.0 g, increasing the
lead-free nitric add R. Allow a d d attack to take place w ithout
tem perature very gradually to 600 ± 50 °Cj allow to cool.
h eatin g and if necessary warm slightly at the end to
M oisten the residue with 2 m L o f nitric acid R, evaporate to
completely dissolve the test sample. Add 10 m L of water R,
dryness on a w ater-bath and carefully heat and ignite once
shake and add, in small fractions, 4.5 m L of lead-free
more at 600 ± 50 °C. After cooling, dissolve the residue in
ammonia R; shake and allow to cool. Dilute to 20.0 m L with
5 m L of nitric acid R and dilute to 20 m L with water R
water R, shake again and allow the solids to settle. The clear
T o 10 m L of this solution, add concentrated ammonia R until
supernatant solution is solution S2.
alkaline and filter. W ash the residue with water R and
evaporate the com bined filtrate and washings to dryness on a A cid ity
water-bath. A dd 0.3 m L of dilute sulfuric acid R and ignite. Suspend 1.0 g in 15 m L of water R and shake several times.
T h e residue weighs a maximum o f 10 mg. Allow to stand for 5 min and filter. T o 10 m L o f the filtrate,
add 0.5 m L o f phenolphthalein solution R l. N o t more than
0.5 m l. o f 0.1 M sodium hydroxide is required to change the
colour of the indicator to pink.
A SSA Y M aximum 200 ppm.
T o 0.300 g add 10 m L of a mixture o f equal volumes of
T o 5.0 m L of solution S 1, add 3 m L of nitric add R and
nitric add R and water R, heat to boiling and maintain boiling
dilute to 15 m L with water R.
for 2 m in. Add 0.1 g o f potassium chlorate R, heat to boiling
and maintain boiling for 1 min. Add 10 m L o f water R and C opper
heat until the solution becomes colourless. T o the hot M aximum 50 ppm .
solution, add 200 m L o f water R and 50 m g of xylenol orange Atomic absorption spectrometry (2.2.23, Method I).
triturate R. T itrate with 0.1 M sodium edetate until a yellow Test solution Solution S2.
colour is obtained.
1 m L of 0.1 M sodium edetate is equivalent to 20.90 m g o f Bi. standard solution (10 ppm Cu) R and diluting with a
STO R A G E 37 p er cent V/V solution o f lead-free nitric acid R.
Protected from light. Source Copper hollow-cathode lamp.
___________________________________________________________________________________________ PhEur Wavelength 324.7 nm.
L ead
M aximum 20 ppm.
1-302 Bismuth Subsalicylate 2016
Atomic absorption spectrometry (2.2.23, MethodII). filtrate for identification test B. Wash the residue with dilute
Test solution Solution S2. hydrochloric acid R and then with water R. Dissolve the
residue in 0.5-1 m L of diluxe sodium hydroxide solution R.
Add 15 m L of water R. Neutralise with dilute hydrochloric
standard solution (10 ppm Pb) R and diluting with a
acid R. T he solution gives reaction (a) of salicylates (2.3.1).
37 per cent V/V solution o f lead-free nitric acid R.
B. T he filtrate obtained in identification test A gives
reaction (b) of bismuth (2.3.1).
Wavelength 283.3 nm (depending on the apparatus, the line
at 217.0 nm may be used). TESTS
S o lu tio n S
In a porcelain or quartz dish, ignite 1.0 g, increasing the
Silver temperature very gradually. H eat in a muffle furnace at
M aximum 25 ppm. 600 ± 25 °C for 2 h. Cool and dissolve the residue with
Atomic absorption spectrometry (2.2.23, Method I). warming in 4 m L of a mixture o f equal volumes of lead-free
Test solution Solution S2. nitric add R and water R and dilute to 20 m L with water R.
Reference solutions Prepare the reference solutions using silver A cidity
standard solution (5 ppm Ag) R and diluting with a Shake 2.0 g with 30 m L of ether R for 1 min and filter.
37 per cent V/V solution of lead-free nitric acid R. T o the filtrate add 30 m L o f alcohol R and 0.1 m L of thymol
Source Silver hollow-cathode lamp. blue solution R. N ot more than 0.35 m L of 0.1 M sodium
hydroxide is required to change the colour o f the indicator to
blue.
S u b sta n c e s n o t p re c ip ita te d b y a m m o n i a Maximum 200 ppm.
Dissolve 0.250 g in a mixture of 2 m L o f nitric acid R, 5 m L
To 20 m L o f solution S i, add concentrated ammonia R until of water R and 8 m L o f methanol R.
an alkaline reaction is produced and filter. Wash the residue
N itra te s
with water R, and evaporate the combined filtrate and
washings to dryness on a water-bath. T o the residue, add
0.3 m L of dilute sulfuric acid R and ignite. T he residue weighs T o 0.1 g add 10 m L o f water R and, with caution, 20 m L of
a maximum of 10 mg. sulfuric acid R and stir. The solution is not more intensely
yellow coloured than a reference solution prepared at the
same time using 0.1 g o f salicylic add R, 6 m L o f water R,
4 m L of nitrate standard solution (100 ppm NO 3 ) R and
an oven at 105 °C.
20 m L o f sulfuric acid R.
A SSAY C opper
Dissolve with heating 0.250 g in 10 m L of a mixture of Maximum 50 ppm.
2 volumes o f perchloric add R and 5 volumes of water R.
T o the hot solution, add 200 m L o f water R and 50 m g o f
xylenol orange triturate R. Titrate with 0.1 M sodium edetate Test solution Solution S.
until a yellow colour is obtained. Reference solutions Prepare the reference solutions using copper
1 m L o f 0.1 M sodium edetate is equivalent to 20.90 m g of Bi. standard solution (10 ppm Cu) R and diluting with a
6.5 per cent V/V solution of lead-free nitric acid R.
PhEur
Source Copper hollow-cathode lamp.
Bismuth Subsalicylate ***** L ead
★ ★ M aximum 20 ppm.
C7H 5B i04 362.1 14882-18-9 Test solution Solution S.
PhEur__________________________________________________________
D E F IN IT IO N standard solution (10 ppm Pb) R and diluting with a
Complex of bismuth and salicylic add. 6.5 per cent V/V solution of lead-free nitric add R.
C o n te n t Source Lead hollow-cathode lamp.
56.0 per cent to 59.4 per cent of Bi (At 209.0) (dried Wavelength 283.3 nm (depending on the apparatus, the line
substance). at 217.0 nm may be used).
CHA RACTERS Atomisation device Air-acetylene flame.
A p p e a ra n c e S ilver
White or almost white powder. M aximum 25 ppm.
S olubility Atomic absorption spectrometry (2.2.23, Method I).
Practically insoluble in water and in alcohol. It dissolves in Test solution Solution S.
mineral acids with decomposition. Reference solutions Prepare the reference solutions using silver
ID E N T IF IC A T IO N standard solution (5 ppm Ag) R and diluting with a
A. T o 0.5 g add 10 m L of hydrochloric add R l. H eat on a 6.5 per cent V/V solution o f lead-free nitric add R.
boiling water-bath for 5 min. Cool and filter. Retain the Source Silver hollow-cathode lamp.
2016 Bisoprolol Fumarate 1-303
Wavelength 328.1 nm. ID E N T IF IC A T IO N

Atomisation device Air-acetylene flame. Infrared absorption spectrophotometry (2.2.24).
S oluble b is m u th Comparison bisoprololfumarate CRS.
M aximum 40 ppm. If the spectra obtained in the solid state show differences,
Atomic absorption spectrometry (2.2.23, Method I). dissolve the substance to be examined and the reference
substance separately in methanol R, evaporate and dry the
Test solution Suspend 5.0 g in 100 m L of water R. Stir
residues at 60 °C at a pressure not exceeding 0.7 kPa and
constantly for 2 h at 20-23 °C. Filter through filter paper
(slow filtration) then through a cellulose micropore
mem brane filter (0.1 (im). T o 10.0 m L of clear filtrate, add TESTS
0.1 m L of nitric add R. R e late d su b stan c es
Reference solutions Prepare the reference solutions using Liquid chromatography (2.2.29).
bismuth standard solution (100 ppm Bi) R and diluting with a Solvent mixture acetonitrile R l, water for chromatography R
mixture o f equal volumes of dilute nitric add R and water R. (20:80 VIV).
Source Bismuth hollow-cathode lamp. Test solution Dissolve 25 mg of the substance to be examined
Wavelength 223.06 nm. in the solvent mixture and dilute to 25.0 m L with the solvent
Atomisation device Air-acetylene flame. mixture.
solution to 10.0 m L with the solvent m ixture.
an oven at 105 °C.
Reference solution (b) Dissolve the contents of a vial of
A SSAY bisoprolol for peak identification CRS (containing impurities A
Dissolve with heating 0.300 g in 10 m L of a mixture of and E) in 1.0 m L of the solvent mixture.
2 volumes of perchloric add R and 5 volumes of water R.
Reference solution (c) Dissolve the contents of a vial of
T o the h o t solution, add 200 m L of water R and 50 mg of
bisoprolol for system suitability CRS (containing im purity G) in
xylenol orange triturate R. Titrate with 0.1 M sodium edetate
1.0 m L of the solvent mixture.
until a yellow colour is obtained.
Column:
1 m L o f 0.1 M sodium edetate is equivalent to 20.90 mg of Bi.
— sizer. I = 0.25 m , 0 = 4.6 mm;
STORAGE — stationary phase: octadecylsQyl silica gel for chromatography R
Protected from light. (5 pm);
PhEur
— temperature: 20 + 2 °C.
Mobile phase:
— mobile phase A: 10 g/L solution of phosphoric acid R;
— mobile phase B: 10 g/L solution o f phosphoric add R in
★ ★
Bisoprolol Fumarate ★ ★
acetonitrile Rl;
(FL Eier, monograph 1710) *****

ho 2c
\ P HH 0 -4 95
o ^ X ^ n ^ c h 3 ' 5
4 -8 95 -»80 5 -»20
ch3
c o 2h
8 - 15 80 20
15-34 80*20 20 -»80
and enantiomer
3 4-36 20 80
C40H66N2O12 767 104344-23-2
A ctio n a n d use Flow rate 1.0 mL/min.

Beta-adrenoceptor antagonist Detection Spectrophotometer at 225 nm.
P re p a r a tio n Injection 10 (iL.
Bisoprolol Tablets Identification of impurities Use the chromatogram supplied
with bisoprolol for peak identification CRS and the
PhEtr.
D E F IN IT IO N identify the peaks due to fumaric acid and impurities A and
(2RS)-l - [4- [[2- (1 -Methylethoxy) ethoxy] methyl] phenoxy]-3- E; use the chromatogram supplied with bisoprolol for system
[(l-methylethyl)amino]propan-2-ol fumarate. suitability CRS and the chromatogram obtained with
reference solution (c) to identify the peak due to impurity G.
C o n te n t
99.0 p er cent to 101.0 per cent (anhydrous substance). Relative retention W ith reference to bisoprolol (retention
CHARACTERS impurity G = about 1.1; impurity E = about 1.2.
A p p e a ra n c e
White o r almost white, slightly hygroscopic powder.
S o lu b ility above the baseline of the peak due to impurity G and
Very soluble in water, freely soluble in methanol. Hv = height above the baseline of the lowest point of the
1-304 Bisoprolol Fumarate 2016

N ^C H 3
bisoprolol.
Limits: ch3
— impurity G: not m ore than 2.5 times the area o f the
— impurity A: not m ore than 1.5 times the area of the
reference solution (a) (0.3 per cent); CH,
— impurity E: not m ore than the area o f the principal peak
C. l-[4-[4-(2-hydroxy-3-isopropylamino-
(0.2 per cent);
propoxy)benzyl]phenoxy]-3-isopropylaminopropan-2-ol,
— unspecified impurities: for each impurity, not more than
(0.10 per cent);
r
— total: not more than 2.5 times the area of the principal CH,
(0.05 per cent); disregard the peak due to fumaric acid.
W a te r (2.5.12) CH,
S u lfa te d a s h (2.4.14) D. l-[4-[4-(2-hydroxy-3-
M aximum 0.1 per cent, determined on 1.0 g. isopropylaminopropoxy)benzyloxylmethyl]phenoxy]-3-
A SSA Y isopropylaminopropan-2-ol,
Dissolve 0.300 g in 50 m L of anhydrous acetic add R. T itrate
with 0.1 M perchloric add, determining the end-point
of C4oH6$N20i2.
STO R A G E
In an airtight container, protected from light. E. (£Z)-[3-[4-(2-isopropoxy-
ethoxymethyl)phenoxy] allyl] isopropylamine,
IM P U R IT IE S
Specified impurities A, E , G OH
the tests in the m onograph. T hey are limited by the general ch3 n ch3
acceptance criterion for other/unspecified impurities and/or .
'0
A H
CH3 and enantiomer
F. (2RS)-2- [4-(2-isopropoxy-ethoxymethyl)phenoxy] -3-
isopropylaminopropan-2-ol,
D, F, K, L, N , a R} S} T, U.
H OH h
H OH
o .N ^ C H s ch3
ch3
CH,
and enantiomer O CH3 and enantiomer
G. (2/?S)-l-[4-
A. (2i?5)-1-(4-hydroxymethyl-phenoxy)-3- [ [(2-isopropoxyethoxy)methoxy] methyl] phenoxy] -3-
isopropylaminopropan-2-ol, isopropylaminopropan-2-ol,
H OH H OH
n ^ch 3
ch3
ch3
/\^ C H 3 and enantiomer
O CH3 and enantiomer
B. (2RS)-1 -isopropylamino-3-[4-(2-propoxy- K. 2-isopropoxyethyl 4- [ [(2/?5)-2-hydroxy-3-

ethoxymethyl)phenoxy] propan-2-ol, (isopropylamino)propyl] oxy] benzoate,
2016 Bleomycin Sulfate 1-305
H OH ***
Bleomycin Sulfate ★ ★
★ ★
Bleomycin Sulphate *****
OHC and enantiomer
L. 4-[[(2i?S)-2-hydroxy-3-
(isopropylamino)propyl] oxy] benzaldehyde,
O^ ,NH
V H« n3>-—s. ji y—s
H,N
\ __ I > H -y ^ N H N_ /
/ ~ \ . nh k .x y
^ O CH3 and enantiomer
*n3
, x H2S 04
N . (2JRS)-1 - [4- [(2-ethoxyethoxy)methyl] phenoxy] -3-
isopropylaminopropan-2-olj
hv 0H «
ch 3
bleomycin B2:
R = NH-[CH2]4-NH-C(=NH)-NH2
'OCH3 and enantiomer
Q. (2i?S)-l-(isopropylamino)-3-[4- 9041-93-4
(2-methoxyethoxy)methyl]phenoxypropan-2-ol,
A ctio n a n d u se
Cytotoxic antibacterial.
H OH
H
n^ ch3 P re p a r a tio n
Bleomycin Injection
ch 3
h3c and enantiomer PhEur.
D E F IN IT IO N
R. (2i?S)-l-(isopropylamino)-3-(4-methylphenoxy)propan-2-
Sulfate of a mixture of glycopeptides produced by
ol,
Streptomyces verdciUiis or by any other means; the 2 principal
components of the mixture are N-[3-
OH
(dimethylsulfonio)propyl]bleomycinamide (bleomycin A 2)
and N- [4-(carbamimidoylamino)butyI] bleomycinamide
OHC (bleomycin B2).
P o te n c y
S. 4-hydroxybenzaldehyde, Minimum 1500 IU/m g (dried substance).
CHARACTERS
A p p e a ra n c e
0"\ 1h 3
,c
W hite or yellowish-white, very hygroscopic powder.
ch 3 S o lu b ility
OHC
Very soluble in water, slightly soluble in anhydrous ethanol,
practically insoluble in acetone.
T . 4- [(3-isopropyl-2-oxo-1,3-oxazolidin-5- ID E N T IF IC A T IO N
yl) methoxy] benzaldehyde. A. Examine the chromatograms obtained in the test for
composition.
Results T he 2 principal peaks in the chromatogram obtained
o-"\ <3
ch with the test solution are similar in retention time and size to
> ^ L y N~< the 2 principal peaks in the chromatogram obtained with
ch3 reference solution (a).
HO
B. It gives the reactions o f sulfates (2.3.1).
TESTS
U . 5- [ [4-(hydroxymethyl)phenoxy] methyl] -3-isopropyl-1,3-
oxazolidin-2-one.
T h e solution is clear (2.2.1) and its absorbance (2.2.25) at
PhEur 430 nm is not greater than 0.10.
same solvent.
p H (2.2.3)
4.5 to 6.0.
1-306 Bleomycin Sulfate 2016
Dissolve 50 m g in carbon dioxide-free water R and dilute to Atomisation (iedce Air-acetylene flame.
10 m l. with the same solvent. L oss o n d ry in g (2.2.32)
C o m p o sitio n M aximum 3.0 per cent, determ ined on 50 mg by drying at
Liquid chrom atography (2.2.29): use the normalisation 60 °C at a pressure not exceeding 0.67 kPa for 3 h.
procedure. B a c te ria l e n d o to x in s (2.6.14)
Test solution Dissolve 25 .0 mg o f the substance to be Less than 5 IU/m g, if intended for use in the manufacture of
examined in water R a n d dilute to 50.0 m L with the same parenteral preparations without a further appropriate
solvent. procedure for the removal o f bacterial endotoxins.
Reference solution (a) Dissolve the contents of a vial of A SSAY
bleomycin sulfate CRS in water R and dilute to 10.0 m L with Carry out the microbiological assay o f antibiotics (2.7.2),
the same solvent. using the diffusion m ethod. Use bleomycin sulfate CRS as the
Reference solution (b) D ilute 1.5 m L of reference solution (a) chemical reference substance.
STO RA G E
Column:
In an airtight container, at a tem perature o f 2 °C to 8 °C.
— sizer. I = 0.25 m , 0 = 4.6 mm;
If the substance is sterile, store in a sterile, airtight, tam per
— stationary phase: octadecylsifyl silica gel for chromatograph R
p roof container.
(7 pm).
Mobile phase: IM P U R IT IE S
— mobile phase A: methanol R; Specified impurities D
— mobile phase B: dissolve 0.960 g o f sodium Other detectable impurities (the following substances would, if
pentanesulfonate R in 900 m L o f acetic a d d (4.8 g/L present at a suffident levd, be detected by one or other o f
C 2H 4O 2), add 1.86 g o f sodium edetate R, dilute to the tests in the monograph. They are limited by the general
1000 m L with the same solvent and adjust to p H 4.3 acceptance criterion for other/unspecified impurities and/or
with ammonia R] by the general monograph Substances for pharmaceutical use
(min) (per cent V/V) (per cent V/V) Control of impurities in substances for pharmaceutical use): A , B,
0 - 60 10 ->40 90 —» 60 C.
60 - end 40 60

In jection 2 0 j i L
Run time U ntil im purity D is eluted (about 80 min).

Relative retention W ith reference to bleomycin A2:
im purity D = 1.5 to 2.5.
System suitability:
— resolution: minimum 5 between the peaks due to
bleomycin A 2 (1st prindpal peak) and bleomycin B 2
( 2nd prindpal peak) in the chrom atogram obtained with
reference solution (a);
— signal-to-noise ratio: minimum 20 for the prindpal peak in
the chrom atogram obtained with reference solution (b);
— repeatability: maxim um relative standard deviation of
2 per cent for the prindpal peak after 6 injections o f
Limits: A. R = O H: bleomycinic a d d ,
— bleomycin A 2: 55 per cent to 70 per cent; B. R = N H -[ C H J 3- N H -[C H J 4-N H 2: bleomycin A 5 ,
— bleomycin B2: 25 per cent to 32 per cent; C. R = N H -[ C H J 4-N H -C (= N H )-N H -[C H J 4-N H -
— sum of bleomycin A 2 and B2: minim um 85 per cent; C (= N H )-N H 2: bleomycin B4,
— impurity D: maxim um 5.5 per cent; D . R = N H -[C H 2] 3-S-C H 3: demethylbleomycin A2.
— sum of impurities other than D: maximum 9.5 per cent;
PhEur
— disregard limit: 0.1 per cent o f the total.
C opper
M aximum 200 ppm.
Test solution Dissolve 50 mg in water R and dilute to 10.0 m L
Reference solution D ilute 1.0 m L of copper standard solution
(10 ppm Cu) R to 10.0 m L with water R.
Source Copper hollow-cathode lamp.
Wavelength 324.7 nm .
2016 Borax 1-307
★ ★ LA B E LL IN G
Refined Borage Oil ★ ★
T h e label states, where applicable, that the oil is suitable for
*****
Refined Starflower Oil use in the manufacture of parenteral preparations.
(Refined Borage (Starflower) OH, Ph Eur monograph 2105) ------------------------------------------------------------------------------------- -- PhEur
PhEur_________ _________________________________________________
D E F IN IT IO N
Fatty oil obtained firom seeds of Borago officinalis L. ***
by extraction and/or expression. It is then refined. A suitable Borax *
★
*
★
antioxidant may be added. Sodium Borate; Sodium Tetraborate *****
CHARACTERS (Ph. Eur. monograph 0013)
A p p e a ra n c e N a2B40 7,10H20 . 381.4 1303-96-4
CleaTj light yellow or yellow liquid.
PhEur_________________________________
S olubility
Practically insoluble in water and in ethanol (96 per cent), D E F IN IT IO N
misrible with light petroleum. Disodium tetraborate decahydrate.
R elativ e d en sity C o n te n t
About 0.921. 99.0 per cent to 103.0 per cent o f N a 2B407, 10H 20 .
Refractive index A bout 1.476. CHARACTERS
ID E N T IF IC A T IO N A p p e a ra n c e
First identification B W hite or almost white, crystalline powder, colourless crystals
or crystalline masses, efflorescent.
Second identification A
S olu b ility
A. Identification of fatty oils by thin-layer chromatography
Soluble in water, very soluble in boiling water, freely soluble
(2.5.2).
in glycerol.
Results T he chromatogram obtained is similar to the
corresponding chromatogram shown in Figure 2.3.2.-1. ID E N T IF IC A T IO N
A. T o 1 m L o f solution S (see Tests) add 0.1 m L of svifunc
B. Composition o f fatty adds (see Tests).
add R and 5 m L o f methanol R and ignite. The flame has a
TESTS green border.
A cid valu e (2.5. 1) B. T o 5 m L o f solution S add 0.1 m L of phenolphthalein
Maximum 0.5, or maximum 0.3 if intended for use in the solution R. T he solution is red. O n the addition of 5 m L o f
manufacture of parenteral preparations. glycerol (85 per cent) R the colour disappears.
P e ro x id e v alu e (2.5.5, Method A) C. Solution S gives the reactions of sodium (2.3.1).
M aximum 10.0, or maximum 5.0 if intended for use in the
TESTS
manufacture o f parenteral preparations.
S o lu tio n S
U n sap o n ifiab le m a tte r (2.5.7)
distilled water R and dilute to 100 m L with the same solvent.
A lkaline 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)
p H (2.2.3)
Use the mixture of calibrating substances in Table 2.4.22.-3. 9.0 to 9.6 for solution S.
Composition of the fatty-acid fraction of the oil:
S u lfates (2.4.13)
— saturated fatty acids of chain length less than C;<j: maximum
M aximum 50 ppm , determined on solution S.
0.3 per cent,
— palmitic acid: 9.0 per cent to 12.0 per cent, Use in this test 1.0 m L o f acetic acid R. Prepare the standard
— palmitoleic acid: maximum 0.6 per cent, using a mixture o f 3 m L of sulfate standard solution
— stearic acid: 2.0 per cent to 6.0 per cent, (10 ppm SO4) R and 12 m L of distilled water R.
— oleic acid: 12.0 per cent to 22.0 per cent, A m m o n iu m (2.4.1)
— linoleic acid: 30.0 per cent to 41.0 per cent, M aximum 10 ppm.
— gamma-Unolenic acid: 17.0 per cent to 27.0 per cent, Dilute 6 m L o f solution S to 14 m L with water R. Prepare
— alpha-linolenic acid: maximum 0.5 per cent, the standard using a mixture o f 2.5 m L of ammonium
— arachidic acid: maximum 0.5 per cent, standard solution (1 ppm N H J R and 7.5 m L of water R.
— eicosenoic acid: 2.8 per cent to 4.4 per cent,
— erucic acid: maximum 3.0 per cent,
Maximum 5 ppm , determined on 5 m L o f solution S.
— nervonic acid: maximum 4.5 per cent.
C a lc iu m (2.4.3)
B ra ssic a ste ro l (2.4.23)
M aximum 100 ppm , determined on solution S.
Maximum 0.3 per cent in the sterol fraction of the oil.
Prepare the standard using a mixture of 6 m L o f calcium
W a te r (2.5.32)
standard solution (10 ppm Ca) R and 9 m L o f distilled water R.
STO RA G E M aximum 25 ppm.
U nder an inert gas, in a well-filled, airtight container,
12 m L of solution S complies w ith test A. Prepare the
protected from light.
1-308 Boric Acid 2016
A SSAY hydroxide, using 0.5 m L of phenolphthalein solution R as

Dissolve 20 g of mcmnitol R in 100 m L of water R, heating if indicator, until a pink colour is obtained.
necessary, cool and add 0.5 m L o f phenolphthalein solution R 1 m L of 1 M sodium hydroxide is equivalent to 61.8 mg
and neutralise with 0.1 M sodium hydroxide until a pink of H 3BO 3.
colour is obtained. Add 3.00 g o f the substance to be
PhEur
examined, heat until dissolution is complete, cool, and titrate
with 1 M sodium hydroxide until the pink colour reappears.
1 m L o f 1 M sodium hydroxide is equivalent to 0.1907 g
o f N a 2B40 7, 10 H 2O. .★ **
Botulinum Toxin Type A ★ ★
__________________________________________________________ PhEur ★ ★
for Injection * * * * *

★*★
Boric Acid ★ ★
★ ★ D E F IN IT IO N
(Ph Eur monograph 0001) ***** Botulinum toxin type A for injection is a dried preparation
containing purified botulinum neurotoxin type A, which may
H3B03 61.8 10043-35-3
be present in the form o f a complex with haemaggjutinins
PhEur_____________________________ and non-toxic proteins. Botulinum neurotoxin type A or its
D E F IN IT IO N haemaggjutinin complex is prepared by a suitable purification
C o n te n t process o f the liquid supernatant from a broth-culture of a
99.0 per cent to 100.5 per cent. suitable strain of Clostridium botulinum type A.
T he purified complexes consist o f several proteins and can be
CHARACTERS
of various sizes. T he largest complex (relative molecular mass
A p p e a ra n c e
o f about 900 000) consists o f a 150 000 relative molecular
White or almost white, crystalline powder, colourless, shiny
mass neurotoxin, a 130 000 relative molecular mass non
plates greasy to the touch, or white or almost white crystals.
toxic protein and various haemagghitinins ranging between
S o lu b ility relative molecular mass 14 000 and 43 000. T he purified
Soluble in water and in ethanol (96 per cent), freely soluble toxin moiety is composed of only the same 150 000 relative
in boiling water and in glycerol (85 per cent). molecular mass neurotoxin as is found in the 900 000
ID E N T IF IC A T IO N relative molecular mass neurotoxin complex, which is initially
produced as a single chain and further cleaved (nicked) by
A. Dissolve 0.1 g by gendy heating in 5 m L of methanol R,
add 0.1 m L o f sulfuric acid R and ignite the solution. endogenous proteases into a fully active, disulfide-linked,
T he flame has a green border. 54 000 relative molecular mass light chain and a 97 000
relative molecular mass heavy chain.
B. Solution S (see Tests) is acid (2.2.4).
T he preparation is reconstituted before use, as stated on the
TESTS label.
S o lu tio n S
P R O D U C T IO N
Dissolve 3.3 g in 80 m L o f boiling distilled water R, cool and
GENERAL PROVISIONS
dilute to 100 m L with carbon dioxide-free water R prepared
from distilled water R. Production o f the toxin is based on seed cultures, managed
in a defined seed-lot system in which the ability to produce
A p p e a ra n c e o f so lu tio n toxin is conserved. T he production m ethod m ust be shown
Solution S is clear (2.2.1) and colourless (2.2.2, Method II). to yield consistently product o f activity and profile
p H (2.2.3) comparable to that o f lots shown in clinical studies to be of
3.8 to 4.8 for solution S. adequate safety and efficacy.
S o lu b ility in e th a n o l (96 p e r cen t) T h e production method is validated to dem onstrate that the
T he solution is not more opalescent than reference product, if tested, would comply with the general test of
suspension II (2.2.1) and is colourless (2.2.2, Method II). abnormal toxicity (2.6.9) using n o t less than the maximum
Dissolve 1.0 g in 10 m L o f boiling ethanol (96 per cent) R. human clinical dose, in the presence o f a suitable am ount of
specific botulinum type A antitoxin used for neutralisation.
O rg a n ic m a tte r
It does not darken on progressive heating to dull redness. T he production m ethod and stability o f the finished product
and relevant intermediates are evaluated using the tests
S u lfates (2.413)
below. Such tests include the specific toxin activity per
Maximum 450 ppm.
milligram o f protein of purified toxin in an appropriate
D ilute 10 m L o f solution S to 15 m L with distilled water R. functional model o f toxin activity and may be supported by
H eav y m e ta ls (2.48) tests confirming the presence of botulinum toxin type A, and,
M aximum 15 ppm. if appropriate, associated non-toxic proteins.
12 m L of solution S complies with test A. Prepare the B A C T E R IA L S E E D L O T S
reference solution using a mixture o f 2.5 m L o f lead standard A highly toxigenic strain of C. botulinum o f known toxin
solution (2 ppm Pb) R and 7.5 m L o f water R. type A and confirmed absence o f genes encoding other
ASSAY botulinum toxins (particularly botulinum toxin types B and
Dissolve 1.000 g with heating in 100 m L o f water R F), with known origin and history, is grown using suitable
containing 15 g of mannitol R. Titrate with 1 M sodium media. T he bacterial strain, used for the m aster seed lot,
shall be identified by historical records that include
2016 Botulinum Toxin Type A For Injection 1-309
information on its origin and the tests used to characterise Im m u n o lo g ic a l id e n tity

the strain. These will include morphological, cultural, T h e presence of specific type A toxin is confirmed by a
biochemical, genetic and serological properties o f the strain. suitable im munochemical method (2.7.2).
T he m aster seed lot and the working seed lot, where S pecific ac tiv ity
applicable, must be demonstrated to have identical profiles. T h e specific activity is confirmed in a mouse m o d d of
Only a seed lot that complies with the following requirements toxidty or by in mvolex vivo methods validated with respect
may be used. to the LD50 assay and expressed in mouse LD50 units per
Id e n tific a tio n milligram of protein. Specific activity must not be less than
E ach seed lot is identified as containing pure cultures of 1 x 108 mouse LD50 units per milligram o f protein for the
C. botulinum type A bacteria with no extraneous bacterial or 150 000 relative molecular mass neurotoxin and must not be
fungal contamination. less than 1 x 107 mouse LD50 units per milligram of protein
M ic ro b ia l p u rity for the 900 000 rdative molecular mass neurotoxin complex.
E ach seed lot complies with die requirements for absence of P ro te in
contaminating micro-organisms. T h e purity of bacterial T h e total protein concentration is determined by a suitable
cultures is verified by methods of suitable sensitivity. These m ethod. An acceptable value is established for the product
may include inoculation into suitable media and examination and each batch m ust be shown to comply with the limits.
of colony morphology. P ro te in p ro file
P h e n o ty p ic p a ra m e te rs Identity and protein composition are determined by
Each seed lot m ust have a known fatty a d d profile, sugar polyacrylamide gel electrophoresis (2.2.32) under reducing or
fermentation profile (glucose, lactose, mannose, etc.) and non-reducing conditions or by other suitable physicochemical
proteolytic activity and must demonstrate relevant lipase, methods such as size-exclusion chromatography (2.2.30),
ledthinase and gelatinase activity. comparing with suitable reference standards.
G en etic p u rity T o ta l viab le c o u n t
Each seed lot m ust have information on the toxin gene It complies with the limits approved for the particular
sequence and comply with requirements for the absence of p ro d u ct
other genes encoding other toxin serotypes.
FIN A L B U L K
P ro d u c tio n o f a ctiv e to x in T h e final bulk is prepared by adding approved exdpients to
A bacterial strain producing a high yield o f active toxin, as the bulk purified toxin. T he solution is filtered through a
determ ined by an acute toxidty assay, is suitable. Seed lots bacteria-retentive filter. If human albumin is added, it
dem onstrate a capability o f producing at least a minimum complies with the monograph Human albumin
toxidty level appropriate for the manufacturing process and solution (0255).
scale.
FINAL LOT
M A N U F A C T U R E R 'S R E F E R E N C E P R E P A R A T IO N S T he final bulk is distributed aseptically into sterile, tamper
D uring development, reference preparations are established proof containers. Uniformity of fill is verified during filling
for subsequent verification o f batch consistency during and the test for uniformity of content (2.9.6) is n o t required.
production and for control o f the bulk purified toxin and T h e containers are closed so as to prevent contamination.
finished p ro d u c t T hey are derived from representative Only a final lot that is within the limits approved for the
batches o f botulinum toxin type A that are characterised as particular product and is satisfactory with respect to each of
described under Bulk Purified Toxin. the requirements given below under Identification, Tests and
T he reference preparations are suitably characterised for their Assay may be released for use.
intended purpose and are stored in suitably sized aliquots p H (2.2.3)
under conditions ensuring their suitability. T h e p H of the reconstituted product is within ± 0.5
B U L K P U R IF IE D T O X IN p H units of the limit approved for the particular product.
C. botulinum type A strain is grown anaerobically, in suitable W a te r
media, from which cultures are selected for step-up N o t more than the limit approved for the particular product.
incubations under a suitably controlled anaerobic atmosphere
through th e seed culture and bulk fermentation stages to
T h e presence of botulinum toxin type A is confirmed by a
allow maximum production of toxin. T he toxin is purified by
suitable immunochemical method (2.7.2).
suitable methods to remove nucleic adds and components
likely to cause adverse reactions. TESTS
Only a purified toxin that complies with the following S te rility (2. 6.2)
requirem ents may be used in the preparation of the final It complies with the test for sterility.
bulk. F or each test and for each product, limits of acceptance B a c te ria l e n d o to x in s (2.6.14)
are established and each new purified toxin m ust comply Less than 10 IU per vial.
with these limits.
ASSAY
R e s id u a l re a g e n ts In accordance with the provisions of the European
Removal o f residual reagents used in purification steps is Convention for the Protection of Vertebrate Animals U sed
confirmed by suitable limit tests or by validation o f the for Experimental and O ther Sdentific Purposes, tests m ust
process. be carried out in such a way as to use the minimum num ber
N u cleic a c id s o f animals and to cause the least pain, suffering, distress or
Removal o f nucleic a d d s is confirmed by suitable limit tests lasting harm. T h e LD S0 assay is associated with severe
or by validation o f the process. suffering o f animals and manufacturers are strongly
encouraged to develop and validate assays that will reduce
1-310 Botulinum Toxin Type B for Injection 2016
the num ber o f animals used, or refine or replace the test proteases. T h e nicked protein is a fully active double-chain
procedure with the goal of prom oting animal welfare. polypeptide consisting o f a heavy chain (100 000 relative
T he potency of the reconstituted product is determined by molecular mass) and a light chain (50 000 relative molecular
an L D 50 assay in mice or by a m ethod validated with respect mass), connected by a disulfide bond.
to the L D 50 assay. T h e potency is expressed in terms of the P R O D U C T IO N
L D 50 for mice or relative to the reference preparation. GENERAL PROVISIONS
For determ ination of the LDsoj graded doses of the product Production of the toxin is based on seed cultures, managed
are injected intraperitoneally into groups o f mice and the in a defined seed-lot system in which the ability to produce
L D 50 is calculated by the usual statistical methods (5.3) from toxin is conserved. T h e production m ethod m ust be shown
the mouse lethality in each group. A suitable reference to yield consistently product o f activity and profile
preparation is assayed in parallel; the potency of the toxin is comparable to that of lots shown in clinical studies to be of
expressed relative to the reference or the value found for the adequate safety and efficacy.
reference is within suitable limits defined in terms of the T h e production m ethod is validated to dem onstrate that the
assigned potency. product, if tested, would comply with the general test of
After validation with respect to the L D 50 assay (reference abnorm al toxicity (2.6.9) using not less than the m axim um
m ethod), the product may also be assayed by other methods hum an clinical dose, in the presence o f a suitable am ount of
that are preferable in terms o f animal welfare, for example specific botulinum type B antitoxin used for neutralisation.
mouse bioassays using paralysis as the end-point, ex vivo T he production m ethod and stability o f th e finished product
assays using mouse phrenic nerve diaphragm, endopeptidase and relevant intermediates are evaluated using the tests
assays in vitro and cell-based assays. below. Such tests include the specific toxin activity per
For alternative replacement methods the potency is milligram o f protein o f purified toxin in an appropriate
calculated with respect to a suitable reference preparation functional model o f toxin activity and may be supported by
calibrated in mouse L D 50 units. tests confirming the presence o f botulinum toxin type B, and,
T he estim ated potency is n o t less than 80 per cent and not if appropriate, associated non-toxic proteins.
more than 125 per cent o f the stated potency. BACTERIAL SEED LOTS
T he confidence limits (P = 0.95) are n o t less than A highly toxigenic strain o f C. botulinum o f known toxin
80 per cent and not more than 125 p er cent of the estimated type B and confirmed absence o f genes encoding other
potency. botulinum toxins (particularly botulinum toxin types A
T he test may be repeated b u t when m ore than 1 test is and F ), with known origin and history, is grown using
performed, the results o f all valid tests m ust be combined in suitable media. T he bacterial strain, used for the m aster seed
the estimate o f potency. lot, shall be identified by historical records that include
information on its origin and the tests used to characterise
L A B E L L IN G
the strain. These will include morphological, cultural,
T he label states:
biochemical, genetic and serological properties o f the strain.
— the num ber of units of toxin per vial with a statem ent
T h e m aster seed lot and the working seed lot, where
that units are product specific and n o t applicable to other
applicable, m ust be dem onstrated to have identical profiles.
preparations containing botulinum toxin type A;
Only a seed lot that complies with the following requirem ents
— the nam e and the volume of the diluent to be added for
may be used.
reconstitution of the dried product.
Id e n tific a tio n
___________________________________________________________ PhEur
Each seed lot is identified as containing pure cultures of
C. botulinum type B bacteria with no extraneous bacterial or
fungal contamination.
M ic ro b ia l p u rity
Botulinum Toxin Type B for ***** Each seed lot complies with the requirem ents for absence o f
contaminating micro-organisms. T he purity of bacterial
Injection ***** cultures is verified by m ethods o f suitable sensitivity. These
(Ph Eur monograph 2581) may include inoculation into suitable media and examination
PhEur___________________________________________________________ of colony morphology.
D E F IN IT IO N P h e n o ty p ic p a ra m e te rs
Botulinum toxin type B for injection is a liquid preparation Each seed lot m ust have a known fatty a d d profile, sugar
containing purified botulinum neurotoxin type B, which may fermentation profile (glucose, lactose, m annose, etc.) and
be present in the form of a complex w ith haemagglutinins proteolytic activity and m ust demonstrate relevant lipase,
and non-toxic proteins. Botulinum neurotoxin type B or its ledthinase and gelatinase activity.
haemagglutinin complex is prepared by a suitable purification G e n etic p u rity
process o f the liquid supernatant from a broth-culture o f a Each seed lot must have information on the toxin gene
suitable strain o f Clostridium botulinum type B. Suitable genomic location and on the toxin gene sequence, and
stabilisers may be added. comply with requirements for the absence of other genes
T he toxin is present in its native form as a complex o f encoding other toxin serotypes.
neurotoxin and non-toxin proteins and haemagglutinins with P ro d u c tio n o f activ e to x in
a total relative molecular mass o f approximately 700 000. A bacterial strain producing a high yield o f active toxin, as
T he neurotoxin is synthesised by the bacterium as a single determined by an acute toxidty assay, is suitable. Seed lots
chain polypeptide o f approximately 150 000 relative demonstrate a capability o f producing at least a m inim um
molecular mass that is activated during the fermentation toxidty level appropriate for the manufacturing process and
process via a proteolytic cleavage (nicking) by endogenous scale.
2016 Botulinum Toxin Type B for Injection 1-311
MANUFACTURER S REFERENCE PREPARATIONS Only a final lot that is within the limits approved for the
D uring development, reference preparations are established particular product and is satisfactory with respect to each of
for subsequent verification of batch consistency during the requirements given bdow un d er Identification, Tests and
production and for control of the bulk purified toxin and Assay may be released for use.
finished p ro d u ct T h ey are derived from representative p H (2.2.3)
batches of botulinum toxin type 8 that are characterised as T he p H of the product is within ± 0.5 p H units of the limit
described under Bulk Purified Toxin. approved for the particular product.
T he reference preparations are suitably characterised for their
intended purpose and are stored in suitably sized aliquots
T he presence o f botulinum toxin type B is confirmed by a
under conditions ensuring their suitability.
suitable immunochemical m ethod (2.7.1).
BULK PURIFIED TOXIN
C. botulinum type B strain is grown anaerobically, in suitable TESTS
media, from which cultures are selected for step-up S te rility (2.6.1)
incubations under a suitably controlled anaerobic atmosphere It complies with the test for sterility.
through the seed culture and bulk fermentation stages to B a c te ria l e n d o to x in s (2.6.14)
allow maximum production of toxin. T he toxin is purified by Less than 10 IU per vial.
suitable methods to remove nucleic acids and components
ASSAY
likely to cause adverse reactions.
In accordance with the provisions o f the European
Only a purified toxin that complies with the following Convention for the Protection of Vertebrate Animals U sed
requirements may be used in the preparation o f the final for Experimental and O ther Scientific Purposes, tests must
bulk. F or each test and for each product, limits of acceptance be carried out in such a way as to use the minimum num ber
are established and each new purified toxin m ust comply of animals and to cause the least pain, suffering, distress or
with these limits. lasting harm. T h e L D 50 assay is associated with severe
R e sid u a l re a g e n ts suffering o f animals and manufacturers are strongly
Removal of residual reagents used in purification steps is encouraged to develop and validate assays that will reduce
confirmed by suitable limit tests or by validation of the the num ber of animals used, or refine or replace the test
process. procedure with the goal of prom oting animal welfare.
N ucleic a d d s T h e potency of the product is determ ined by an L D 50 assay
Removal of nucleic ad d s is confirmed by suitable limit tests in mice or by a method validated with respect to the L D 50
or by validation o f the process. assay. T he potency is expressed in terms o f the L D 50 for
Im m u n o lo g ic a l id e n tity mice or relative to the reference preparation.
T he presence of specific type B toxin is confirmed by a For determination of the L D 50, graded doses of the product
suitable immunochemical method (2.7.1). are injected intraperitoneally into groups o f mice and the
L D 50 is calculated by the usual statistical methods (5.5) from
Specific a ctiv ity
the mouse lethality in each group. A suitable reference
T he specific activity is confirmed in a mouse m o d d o f
preparation is assayed in parallel; the potency of the toxin is
toxidty or by in vivo/ex vivo methods validated with respect
expressed relative to the reference or the value found for the
to the L D 50 assay and expressed in mouse L D 50 units per
reference is within suitable limits defined in terms of the
milligram o f protein. Specific activity m ust not be less than
assigned potency.
1 x 108 mouse L D 50 units per milligram of protein.
After validation with respect to the L D 50 assay (reference
P ro te in
method), the product may also be assayed by other methods
T he total protein concentration is determined by a suitable
that are preferable in terms of animal welfare, for example
method. An acceptable value is established for the product
mouse bioassays using paralysis as the end-point, ex vivo
arid each batch m ust be shown to comply with the limits.
assays using mouse phrenic nerve diaphragm, endopeptidase
P ro te in pro file assays tn vitro and cell-based assays.
Identity and protein composition are determined by For alternative replacement methods the potency is
polyacrylamide g d electrophoresis (2.2.31) under reducing or calculated with respect to a suitable reference preparation
non-reducing conditions or by other suitable physicochemical calibrated in mouse L D 50 units.
m ethods such as size-exdusion chromatography (2.2.30),
T he estimated potency is not less than 80 per cent and not
comparing with suitable reference standards.
more than 125 p er cent of the stated potency.
T o ta l viab le c o u n t T he confidence limits (P = 0.95) are not less than
It complies with the limits approved for the particular 80 per cent and not more than 125 per cent of the estimated
product. potency.
FINAL BULK T he test may be repeated b u t w hen more than 1 test is
T he final bulk is prepared by adding approved exdpients to performed, the results o f all valid tests m ust be combined in
the bulk purified toxin. T h e solution is filtered through a the estimate of potency.
bacteria-retentive filter. If hum an albumin is added, it
L A B E LL IN G
complies with the monograph Human albumin
solution (0255). T he label states the num ber of units of toxin per vial with a
statement that units are product specific and not applicable
FINAL LOT to other preparations containing botulinum toxin type B.
T he final bulk is distributed aseptically into sterile, tam per
__________________________________________________________ PhEur
proof containers. Uniformity of fill is verified during filling
and the test for uniformity of content (2.9.6) is not required.
T he containers are d o sed so as to prevent contamination.
1-312 Bovine Serum 2016
A step or steps for virus inactivation/removal are applied to

Bovine Serum ******
serum intended for production o f immunological veterinary
_ _ ____ V .*
(Ph. Eur. monograph 2262) * medicinal products. Unless otherwise justified and authorised
for a particular medicinal product, a step or steps for virus
PhEur__________________________________________________________
inactivation/removal are applied to serum intended for
DEFINITION production o f hum an and non-immunological veterinary
Liquid fraction o f blood obtained from the ox (.Bos taurus L.) medicinal products.
and from which cells, fibrin and clotting factors have been
INACTIVATION
removed.
T h e inactivation procedure applied is validated with respect
Different types o f bovine serum are used: to a suitable representative range o f viruses covering different
— adult bovine serum obtained at slaughter from cattle that types (enveloped, non-enveloped, D N A , RNA viruses).
are declared fit for hum an consumption; T h e optimal choice of relevant and model viruses depends
— calf serum obtained at slaughter from animals, fit for strongly on the specific inactivation/removal procedure;
hum an consum ption, before the age o f 12 months; representative viruses with different degrees of resistance to
— new-born calf serum obtained at slaughter from animals the type o f treatm ent m ust be included. Bovine viral
before the age of 20 days; diarrhoea virus m ust be included in the viruses used for
— foetal bovine serum obtained from normal foetuses from validation. Serum free from antibodies against bovine viral
dams fit for hum an consumption; diarrhoea virus is used in p art or all o f the validation studies.
— donor bovine serum obtained by repeated bleeding o f donor
F o r bovine serum intended for m e in immunological
animals from controlled donor herds.
veterinary medicinal products, for inactivation by gamma
Thừ monograph provides a general quality specification for bovine irradiation a minim um dose o f 30 kGy is applied, unless
serum. Various measures are applied during the production of otherwise justified and authorised.
bovine serum aimed at obtaining a product that ừ acceptable as
Critical param eters for the m ethod o f virus
regards viral safely. No single measure, nor the combination of
inacdvation/removal are established and the param eters used
measures outlined below can guarantee complete viral safety but
in the validation study are strictly adhered to during
they rather reduce the risk involved in the use of serum in the
subsequent application o f the procedures to each batch o f
manufacture of medicinal products. It ừ therefore necessary for the
serum.
manufacturer of a medicinal product to take account of this when
choosing the serum for a particular use by making a risk F o r inactivation by gamma irradiation, critical param eters
assessment. include:
— the tem perature;
PRODUCTION — packaging configuration;
All stages o f serum production are subm ined to a suitable — distribution of dosimeters to assess the effective dose
quality managem ent system. received by the product whatever its position;
Traceability o f serum is m aintained from the final container — the m inim um and maximum dose received.
to die abattoừ of origin (for blood collected from slaughtered
QUALITY CONTROL TESTS APPLIED TO EACH
animals) o r to die herd o f origin (for blood collected from
BATCH
donor animals).
A suitable sample size for each batch is established. Specific
Further guarantee o f the safety and quality of serum may be tests for viral contaminants are validated with respect to
ensured by the use o f a controlled donor herd. Where serum sensitivity and specificity. T h e cell cultures used for general
is obtained from such a herd, the animals are subjected to tests for viral contaminants are shown to be sensitive to a
regular veterinary examination to ascertain theừ health status. suitable range o f potential contam inants. C ontrol cells used
Animals introduced into the herd are traceable as regards in the tests are cultivated, where relevant, with a bovine
source, breeding and rearing history. T h e introduction of serum controlled and inactivated as described in this
animals into the herd follows specified procedures, including m onograph. Serum free from antibodies to bovine viral
defined quarantine measures. D uring the quarantine period diarrhoea virus is required for validation o f the effect of
the animals are observed and tested to establish that they are antibodies on the detection limits for bovine viral diarrhoea
free from all agents and antibodies from which the donor virus.
herd is claimed to be free. It may be necessary to test the
animals in quarantine for freedom from additional agents, Tests carried out on the batch prior to treatment
depending on factors such as information available on their T h e following tests are carried out on the serum (before any
breeding and rearing history. It is recom m ended that animals virus inactivation/removal steps, where applicable).
in the herd should n o t be vaccinated against bovine viral Tests for viral contaminants General tests supplem ented by
diarrhoea virus. T ests are carried out for any agent and/or specific tests are carried out.
antibody from which the herd is claimed to be free. General tests Validated tests are carried out by inoculation of
Serum is obtained by separation o f the serum from blood the serum on at least 2 distinct cell lines, one o f which is of
cells and clot under conditions designed to minimise bovine origin. T h e cell lines used are suitable for detecting
microbial contam ination. Serum from a num ber o f animals is haem adsorbing viruses such as bovine parainfluenza virus 3
pooled and a batch num ber is allocated to the pool. and cytopathic agents such as bovine herpesvirus 1.
Appropriate steps are taken to ensure homogeneity of the Specific tests for viral contaminants (if not detected by general
harvested material, interm ediate pools and the final batch. tests), where relevant in view of the country of origin of the serum
Suitable measures (for example filtration) are taken to ensure Bluetongue virus, bovine adenovirus, bovine parvovirus,
sterility or a low bioburden. Before further processing, the bovine respiratory syncytial virus, bovine viral diarrhoea virus,
serum is tested for sterility or bioburden. General and rabies virus and reovirus. D epending on the country o f
specific tests for viral contam inants are carried out as origin, specific tests for other viruses may be needed.
described below.
2016 Bretylium Tosilate 1-313
T he animal health status of countries is defined by the where applicable, th at the serum has been inactivated and
‘Office International des Epizooties’ (OIE). the inactivation method;
F or serum to be subjected to a virus inactivation/removal where the serum has been inactivated by gamma
procedure, if evidence of viral contamination is found in any irradiation, the target minimum dose o f the irradiation
of the tests described above, the serum is acceptable only if procedure.
the virus is identified and shown to be present in an am ount PhEur
that has been shown in a validation study to be effectively
inactivated.
For serum that is not to be subjected to a virus
inactivation/removal procedure, if evidence of viral
contamination is found in any of the tests described above, Bretylium Tosiiate
the serum is not acceptable.
A test for bovine viral diarrhoea virus antibodies is carried •Br
out; an acceptance criterion for the titre is established taking s o 3-
account of the risk assessment.
Composition T h e content of a suitable selection o f the Me
following components is determined and shown to be within
the expected range for the type of serum: cholesterol, a-, p-
and y-globulin, albumin, creatinine, bilirubin, glucose, serum C18H 24BrN03S 414.4 61-75-6
aspartate transaminase (SAST, formerly SG O T - serum
glutamic-oxaloacetic transaminase), serum alanine A ctio n a n d u se
transaminase (SALT, formerly S G P T - glutamic-pyruvic Antiarrhythmic.
transaminase), phosphorus, potassium, calcium, sodium and P re p a ra tio n
pH . Bretylium Injection
T ests c a rr ie d o u t o n th e b a tc h p o s t-tr e a tm e n t
D E F IN IT IO N
If bovine viral diarrhoea virus was detected before virus
Bretylium Tosiiate is 2-bromobenzyl-N'-
inactivation/removal, the following test for bovine viral
ethyldimethylammonium-p-toluenesulfonate. It contains not
diarrhoea virus is carried out after virus inactivation/removal.
less than 99.0% and n o t more than 101.0% of
Test for bovine viral diarrhoea virus A validated test for bovine C i 8H 24BrN 0 3S, calculated with reference to the dried
viral diarrhoea virus is carried out, for example by inoculation substance.
into susceptible cell cultures, followed by n o t fewer than
3 subcultures and detection by immunostaining. N o evidence C H A R A C T E R IS T IC S
of the presence o f bovine viral diarrhoea virus is found. A white, crystalline powder. It melts at about 98°. It exhibits
polymorphism.
Freely soluble in water, in ethanol (96%) and in methanol.
A. T he electrophoretic pattern corresponds to that for serum
and is consistent with the type (foetal or other) o f bovine ID E N T IF IC A T IO N
serum. A. T he infrared absorption spectrum, Appendix II A, is
B. Bovine origin is confirmed by a suitable immunochemical concordant with the reference spectrum o f bretylium tosiiate
m ethod (2.7.1). (RS 030). If the spectra are not concordant, dissolve a
quantity of the substance being examined in the minimum
TESTS
volume o f acetone by heating on a water bath at 50°,
O sm o lality (2.2.35) evaporate to dryness at room tem perature under a current of
280 mosmol/kg to 365 mosmol/kg for foetal bovine serum nitrogen and prepare a new spectrum of the residue.
and 240 mosmol/kg to 340 mosmol/kg for other types.
B. Carry out the method for thin-layer chromatography,
T o ta l p ro te in (2.5.33) Appendix HI A, vising the following solutions in water.
30 m g/m L to 45 mg/mL for foetal bovine serum and
(1) 0.5% wtv o f the substance being examined.
minim um 35 mg/mL for other types.
(2) 0.5% w/v of bretylium tosiiate BPCRS.
H a em o g lo b in
Maximum 4 mg/mL, determined by a validated method, CHROMATOGRAPHIC CONDITIONS
such as spectrophotometry. (a) Use a silica gel F2m precoated plate (M erck plates are
B a c te ria l e n d o to x in s (2.6.14) suitable).
Less than 10 IU /m L for donor bovine serum, less than (b) Use the mobile phase as described below.
25 IU /m L for foetal bovine serum, less than 100 IU /m L for (c) Apply 10 (iL of each solution.
other types. (d) Develop the plate to 15 cm.
S te rility (2. 6.1) (e) After removal of the plate, dry it in a current o f air and
It complies with the test. Use 10 m L for each medium. examine under ultraviolet light (254 nm).
M y co p lasm a s (2.6.7) MOBILE PHASE
It complies with the test. 15 volumes o f glacial acetic acid, 30 volumes of water and
STO RA G E 75 volumes o f butan-l-oL
Frozen at —10 °C or below. CONFIRMATION
L A B E L L IN G T he two principal spots in the chromatogram obtained with
T he label states: solution (1) correspond to those in the chromatogram
— the type o f serum; obtained with solution (2).
1-314 Brimonidine Tartrate 2016
TESTS IM P U R IT IE S
A cid ity
pH o f a 5.0% w hr solutionj 5.0 to 6.5} Appendix V L.
Clarity and colour o f solution
A 5.0% w/v solution is dear, Appendix IV A, and colourless, C O " *
Appendix IV B, M ethod II.
Related substances A. 2-bromobenzyldimethylamine,
Appendix III D , using the following solutions in mobile
j* Ç j^ ^ N M e 2
phase.
(2) 0.002% w/v of the substance being examined. Br
(3) 0.05% w/v of bretytium tosilate BPCRS and 0.05% w/v o f
2-bromobenzyldimethylamine hydrochloride BPCRS. B. 3-bromobenzyldimethylamine,
(a) U se a stainless steel colum n (25 cm x 4.6 mm) packed
with particles o f silica the surface o f which has been modified
by chemically bonded phenyl groups (5 (im) (Spherisorb » X T “ -
Phenyl is suitable).
(b) U se isocratic elution and the mobile phase described C. 4-bromobenzyldimethylamine.
below.
(c) U se a flow rate o f 2 m L p er minute.
(d) U se an am bient column tem perature.
(e) U se a detection wavelength of 265 nm. ★* * +★
Brimonidine Tartrate ★ ★
(f) Inject 20 (iL o f each solution. ★ ★
MOBILE PHASE
0.5 volume o f triethylamine, 2 volumes o f glacial acetic add,
19 volumes o f acetonitrile and 81 volumes of 0.01m sodium,
o H OH
octanesulfonate. C 02H
SYSTEM SUITABILITY Y jc Y > • h<v >;Hx oOH
T he test is n o t valid unless, in the chromatogram obtained H
with solution (3), the resolution factor between the two
principal peaks is at least 6.0. C 15H 16BrN50 6 442.2 70359-46-5
LIMITS
Alpha2-adrenoceptor agonist; treatm ent o f hypertension
the area of any secondary peak is not greater than half the area
of the peak in the chrom atogram obtained with solution (2) PhEur.
(0.5% ); DEFINITION
the sum of the areas o f all such peaks is not greater than the 5-Bromo-N-(imidazolidin-2-ylidene)qiiinoxalin-6-amine
area o f the principal peak in the chromatogram obtained with (2i?,3.R)-2j3-dihydroxybutanedioate.
solution (2) (1%).
C o n te n t
Disregard the peak due to tosilate (retention time, about 99.0 per cent to 101.0 per cent (dried substance).
2 minutes) and any peak with an area less than 0.05 times
the area of the principal peak in the chromatogram obtained CHARACTERS
with solution (2) (0.05%). A p p e a ra n c e
W hite or slightly yellowish or slightly brownish powder.
Loss on drying
W hen dried to constant weight over phosphorus pentoxide at S o lu b ility
60° a t a pressure not exceeding 0.7 kPa, loses not m ore than Soluble in water, practically insoluble in anhydrous ethanol
3.0% of its weight. U se 1 g. and in toluene.
Sulfated ash ID E N T IF IC A T IO N
N ot m ore than 0.1%, Appendix IX A. Use 1 g. A. Specific optical rotation (see Tests).
A SSA Y B. Infrared absorption spectrophotometry (2.2.24).
Dissolve 0 .2 g in 5 0 m L of 1,4-dioxan and carry out Comparison brimonidine tartrate CRS.
M ethod I for rton-aqueous titration, Appendix VHI A, using TESTS
0 .0 2 5 m perchloric add F S as titrant and determining the
end-point potentiometrically. Each m L of 0.025m perchloric + 9.0 to + 10.5 (dried substance).
add VS is equivalent to 1 0 .3 6 mg o f C ] 8H 24BrN 03 S.
STO RA G E same solvent.
Bretylium Tosilate should be kept in an airtight container R e la te d su b s ta n c e s
and protected from lig h t Liquid chromatography (2.2.29).
2016 Brimonidine Tartrate 1-315

HN— v
solvent.
Reference solution (a) Dilute 1.0 m L of the test solution to H
100.0 m L with water R. Dilute 1.0 m L of this solution to
A. N-(imidazoUdin-2-yHdene)quinoxalin-6-amine,
Reference solution (b) Dissolve the contents o f a vial o f
brimonidine for system suitability CRS (containing impurity E)
in 1.0 m l. of water R.
Column: v V "
— size. I = 0.25 m , 0 = 4.6 mm;
U _
— stationary phase, end-capped octadecylstlyl silica gel for
chromatography R (5 nm); -
— temperature. 30 °C. B. 5-bromoquinoxalin-6-amine.
Mobile phase Dissolve 2.6 g o f sodium heptanesidfonate R in
310 m l. o f methanol R, add 2.5 m L of triethylamine R and
7.5 m L of glacial acetic acid R, and dilute to 1000 m L with
water R. Use a freshly prepared mixture.
Flow rate 1.0 m l7m in. V NH,
Injection 20 |iL. C. quinoxalin-6-amine,
Run time 3 times the retention time o f brimonidine.
Identification of impurities U se the chromatogram supplied
w ith brimonidine for system suitability CRS and the
chrom atogram obtained with reference solution (b) to V S r Brs
identify the peak due to impurity E.
N NH,
Relative retendon W ith reference to brimonidine (retention H 2
tim e = about 19 min): impurity E = about 0.9.
System suitability', reference solution (b): D . l-(5-bromoquinoxalin-6-yl)thiourea,
impurity E and brimonidine.
— for each impurity, use the concentration o f brimonidine in
reference solution (a). " t C N
r . NH2
Limits:
— unspecified impurities: for each impurity, maximum E. 2-(5-bromoquinoxalin-6-yl)guanidine,
0.10 per cent;
— reporting threshold. 0.05 p er cent.
N. Br
L o ss o n d ry in g ( 2.2.32)
an oven at 105 °C for 3 h. W > H h
M axim um 0.2 per cent, determined on 1.0 g. F . 5-bromo-N-(lii-imidazol-2-yl)qiiinoxalin-6-amine5
A SSA Y
Dissolve 0.350 g in 70 m L o f anhydrous acetic acid R using
sonication until complete dissolution. Titrate with 0.1 M
( 2. 2. 20). N N
H H
1 m L of 0.1 M perchloric acid is equivalent to 44.22 m g o f
C 15H 16BrN 50 6.
G . 1-(2-aminoethyl)-3-(5-bromoquinoxalin-6-yi)urea.
IM P U R IT IE S
PhEur
C, D, E, F, G.
1-316 Bromazepam 2016
★* ★ Injection 20 nL.
★ ★
Bromazepam ★ ★
Run tone 4 times the retention tim e o f brom azepam .
Identification of impurities U se the chrom atogram supplied
with bromazepam for system suitability CRS and the
chromatogram obtained w ith reference solution (b) to
identify the peaks due to impurities A, B, C , D and E.
Relative retention W ith reference to brom azepam (retention
time = about 5 min): im purity D = about 1.4;
impurity A = about 1.5; im purity C = about 1.6;
impurity E = about 2.1; im purity B = about 2.2.
Ci4H10BrN3O 316.2 1812-30-2 brom azepam and im purity D and m inim um 1.2 between
the peaks due to impurities A and C.
Action and use
Benzodiazepine.
Limits:
PhEir. the peak areas of the following impurities by the
corresponding correction fa c to r impurity A = 1.3;
D E F IN IT IO N
impurity B = 1.8; im purity E = 2.1;
7-Brom o-5-(pyridin-2-yl)-1,3-dihydro-2/i-l ,4-benzodiazepin-
— impurities A , B, E: for each impurity, no t more than the
2-one.
area of the principal peak in the chrom atogram obtained
C o n te n t with reference solution (a) (0.1 per cent);
99.0 per cent to 101.0 per cent (dried substance). — unspecified impurities', for each impurity, n o t m ore than the
CHARACTERS area of the principal peak in the chrom atogram obtained
A p p e a ra n c e with reference solution (a) (0.10 per cent);
W hite or yellowish, crystalline powder. — total: n o t more than twice the area of the principal peak in
S o lu b ility
(0.2 per cent);
Practically insoluble in water, slighdy soluble or sparingly
soluble in ethanol (96 per cent) and in methylene chloride.
ID E N T IF IC A T IO N (0.05 per cent).
Infrared absorption spectrophotom etry (2.2.24). L o ss o n d ry in g (2.2.32)
Comparison bromazepam CRS. M axim um 0.2 per cent, determ ined on 1.000 g by drying at
80 °C at a pressure not exceeding 2.7 kPa for 4 h.
TESTS
L iquid chrom atography (2.2.29). Prepare the solutions M axim u m 0.1 per cent, determ ined on 1.0 g.
immediately before use. A SSA Y
Test solution Dissolve 10.0 m g o f the substance to be Dissolve 0.250 g in 20 m l . o f anhydrous acetic acid R
examined in 9 m L o f a mixture of 1 volume of acetonitrüe R Add 50 m l . o f acetic anhydride R. T itrate w ith 0.1 M
and 8 volumes o f methanol R Dilute to 20.0 m L with an perchloric acid, determining the end-point potentiometrically
11.33 g/L solution o f potassium àihydrogen phosphate R (2. 2. 20).
previously adjusted to p H 7.0 with a 100 g/L solution of 1 m L of 0.1 M perchloric add is equivalent to 31.62 m g
potassium hydroxide R. o f C i 4H 10B rN 3O.
STORAGE
100.0 m L w ith the mobile phase. Dilute 1.0 m L o f this
solution to 10.0 m L with the mobile phase. Protected from light.
Reference solution (b) Dissolve 5 mg of bromazepam for system IM P U R IT IE S

suitability CRS (containing impurities A, B, C, D and E) in Specified impurities A, B, E
5 m L of a m ixture o f 1 volume of acetomtrüe R and Other detectable impurities (the following substances would, if
8 volumes o f methanol R. D ilute to 10.0 m L with an present at a sufficient level, be detected by one or other of
11.33 g/L solution o f potassium dihydrogen phosphate R the tests in the monograph. T hey are limited by the general
previously adjusted to p H 7.0 with a 100 g/L solution of acceptance criterion for other/unspecified impurities and/or
potassium hydroxide R. by the general monograph Substances for pharm aceutical use
Column: (2034). It is therefore not necessary to identify these
— size. I = 0.15 m, 0 = 4.6 mm; impurities for demonstration o f compliance. See also 5.10.
— stationary phase: end-capped octadecylsüyl silica gelfor Control of impurities in substances for pharmaceutical use): C, D.
chromatography R (3.5 pn);
Mobile phase M ix 5 volumes o f acetomtrüe R, 45 volumes of
methanol R and 50 volumes o f an 1 1.33 g/L solution of
potassium dihydrogen phosphate R previously adjusted to
p H 7.0 with a 100 g/L solution of potassium hydroxide R.
2016 Bromhexine Hydrochloride 1-317
First identification A , E.
Second identification B, C, D, E.
Comparison bromhexine hydrochloride CRS.
A. R = H : (2-amino-5-bromophenyl) (pyridin-2- dissolve the substance to be examined and the reference
yl)methanone} substance separately in methanol R, evaporate to dryness and
B. R = C O -C H 2-C l: N - [4-bromo-2-(pyridin-2-
ylcarbonyl) phenyl] -2-chloroacetamide, B. Thin-layer chromatography (2.2.27).
E. R = C O -C H 2-Br: 2-bromo-N- [4-bromo-2-(pyridm-2- Test solution Dissolve 20 mg of the substance to be examined
ylcarbonyl)phenyl]acetamide, in methanol R and dilute to 10 m L with the same solvent.
Reference solution Dissolve 20 mg of bromhexine
sam e solvent.
Plate TLC silica gel F254 plate R.
Mobile phase glacial acetic acid R, water R, butanol R
(17:17:66 VIVIV).
Application 20 |iL.
C. 7-brom o-5-(6-m ethylpyridin-2-yl)-l,3-dihydro-2H -l,4-
benzodiazepin-2-one, Drying In air.
Results T h e principal spot in the chromatogram obtained w ith
solution.
C. Dissolve about 25 m g in a mixture of 1 m L o f dilute
sulfuric acid R and 50 m L of water R. Add 2 m L o f methylene
chloride R and 5 m L of chloramine solution R and shake.
D . 3-am ino-6-bromo-4-(pyridin-2-yl)quinolin-2( \H)-one.
A brownish-yellow colour develops in the lower layer.
PhEur
D. Dissolve about 1 m g in 3 m L of 0.1 M hydrochloric acid.
T h e solution gives the reaction of primary aromatic amines
(2.3.1).
E. Dissolve about 20 mg in 1 m L o f methanol R and add
★ ★
Bromhexine Hydrochloride ★ ★ 1 m L of water R. T he solution gives reaction (a) o f chlorides
(2.3.1).
(Ph Eitr monograph 0706) *★ *
TESTS
Br A p p e a ra n c e o f so lu tio n
fS -H3 . HCI
nh2 same solvent.
Ci4H2iBr2ClN2 412.6 611-75-6 Liquid chromatography (2.2.29).
A ctio n a n d u se in methanol R and dilute to 10.0 m L with the same solvent.
Mucolytic.
Reference solution (a) Dissolve 5 m g o f bromhexine
P h E tr ____________________ impurity C CRS in methanol R, add 1.0 m L of the test
solution and dilute to 10.0 m L with the same solvent.
D E F IN IT IO N
AT-(2-Amino-3,5-dibromobenzyi)-2V-methylcyclohexanamine
100.0 m L with methanol R. Dilute 1.0 m L of this solution to
hydrochloride.
Content Column:
98.5 per cent to 101.5 per cent (dried substance). — size: I = 0.12 m, 0 = 4.6 mm,
CHARACTERS — stationary phase: end-capped octadecylsüyl silica gel for
Appearance chromatography R (3 nm).
W hite o r almost white, crystalline powder. Mobile phase Mix 0.50 m L of phosphoric acid R in 950 m L of
Solubility water R, adjust to p H 7.0 with triethylamine R (about
Very slightly soluble in water, slightly soluble in alcohol and 1.5 mL) and dilute to 1000 m L with water R,
in methylene chloride. mix 20 volumes o f this solution with 80 volumes of
acetonitrüe R.
It shows polym orphism (5.9).
1-318 Bromocriptine Mesilate 2016

Detection Spectrophotom eter a t 248 nm .
Injection 10 pL.
Run time 2.5 times the retention time o f bromhexine.
Relative retention W ith reference to brom hexine (retention
time = about 11 m in): impurity A = about 0.1; C. R = H: N-(2-am inobenzyl)-N-m ethylcydohexanamine,
D . R = B n JV-(2-amino-5-bromobenzyl)-N-
methylcydohexanamine,
System suitability, reference solution (a):
impurity C and bromhexine.
Limits:
— arty impurity, not m ore than twice the area o f the principal and enantiomer

solution (b) (0.2 p er cent), and n o t more than 1 such
peak has an area greater than the area of the principal
E. (3ÆS)-6,8-dibromo-3-cyclohexyl-3-methyl-1,2,3,4-
solution (b) (0.1 p er cent),
tetrahydroquinazolin-3-ium.
— total: not more th an 3 times the area of the principal peak
in the chrom atogram obtained w ith reference solution (b) PhEur
(0.3 per cent),

(0.05 per cent). ★* *
★ ★
Bromocriptine Mesiiate •* ★
L oss on d ry in g (2.2.32) *
(Ph. Eur. monograph 0596) ***
an oven at 105 °C.
S 0 3H
ASSAY
ch3
Dissolve 0.300 g in 70 m L o f alcohol R and add 1 m L o f
0.1 M hydrochloric acid. Carry out a potentiom etric titration
(2.2.20), \ising 0.1 M sodium hydroxide. Read the volume
between the 2 points o f inflexion.
1 m l. of 0.1 M sodium hydroxide is equivalent to 41.26 m g o f C33H44BrN50 8S 751 22260-51-1
C i4H 2iBr2C lN 2.
STORAGE A c tio n a n d u se
Protected from light. D opamine receptor agonist.
IM P U R IT IE S
Bromocriptine Capsules
Specified impurities: A, B, C , D .
Bromocriptine Tablets
present at a sufficient level, be detected by one or other o f PhEur________________________
D E F IN IT IO N
by the general monograph Substances for pharmaceutical use (6&R,9R)-5-Bvomo-N- [(2R,5S, 1OaS, 10bS )-l Ob-hydroxy-2-
(2034). It is therefore not necessary to identify these (l-methylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro-8//-
impurities for dem onstration o f compliance. See also 5.10. oxazolo [3,2-a]pyrrolo[2,1-c]pyrazin-2-yl] -7-methyl-
Control of impurities in substances for pharmaceutical use): E. 4,6,6a,7,8,9-hexahydroindolo [4,3 ~fg]quinoline-9-carboxamide
monomethanesulfonate.
C o n te n t
P R O D U C T IO N
It is considered that alkylsulfonate esters are genotoxic and
are potential impurities in brom ocriptine mesilate.
T h e manufacturing process should be devdoped taking into
A. R = C H 2OH: (2-am ino-3,5-dibromophenyl)methanol, consideration the prindples o f quality risk m anagement,
together with considerations of the quality o f starting
B. R = C H O : 2-amino-3,5-dibromobenzaldehyde,
materials, process capability and validation. T h e general
m ethods 2.5.37. Methyl, ethyl and isopropyl methanesulfonaxe in
methanesulfonic acid, 2.5.38. Methyl, ethyl and isopropyl
methanesulfonate in active substances and 2.5.39.
Methanesulfonyl chloride in methanesulfonic add are available to
assist manufacturers.
2016 Bromocriptine Mesilate 1-319
CHARACTERS Dissolve 0.25 g in methanol R and dilute to 25 m L with the

A p p e a ra n c e same solvent.
W hite or slightly coloured, fine crystalline powder. p H (2.2.3)
S olubility 3.1 to 3.8.
Practically insoluble in water, freely soluble in methanol, Dissolve 0.2 g in a mixture o f 2 volumes o f methanol R and
soluble in ethanol (96 per cent), sparingly soluble in 8 volumes of carbon dioxide-free water R and dilute to 20 m L
methylene chloride. with the same mixture of solvents.
It is very sensitive to light. S pecific o p tic a l ro ta tio n (2.2.7)
The identification, tests and assay are to be carried out as rapidly + 95 to + 105 (dried substance).
as possible, protected from light. Dissolve 0.100 g in a mixture o f equal volumes o f methanol R
ID E N T IF IC A T IO N and methylene chloride R and dilute to 10.0 m L with the same
First identification B mixture of solvents.
Second identification A, C, D, E R e late d su b sta n c e s
(2.2.25). Solvent mixture bitffer solution pH 2.0 R, methanol R
Test solution Dissolve 10.0 mg in 10 m L of methanol R and (50:50 V/V).
dilute to 200.0 m L with 0.01 M hydrochloric acid. Test solution Dissolve 0.500 g of the substance to be
Spectral range 250-380 nm. examined in 5.0 m L of methanol R and dilute to 10.0 m L
with buffer solution pH 2.0 R.
Absorption maximum At 305 nm.
Absorption minimum At 270 nm. 100.0 m L with the solvent mixture.
Specific absorbance at the absorption maximum 120 to 135
(dried substance).
Comparison bromocriptine mesilate CRS. bromocriptine mesilate for system suitability CRS (containing
C. Thin-layer chromatography (2.2.27). Prepare the solutions impurities A and B) in 1.0 m L of the solvent mixture.
immediately before use. Column:
Solvent mixture ethanol (96 per cent) R, methanol R, methylene — sizer. I = 0.12 m , 0 = 4 mm;
chloride R (30:30:40 VIVIV). — stationary phase: octadecylsHyl silica gel for chromatography R
Test solution Dissolve 10 mg o f the substance to be examined (5 Jim).
in the solvent mixture and dilute to 10 m L with the solvent Mobile phase:
mixture. — mobile phase A: 0.791 g/L solution of ammonium
Reference solution Dissolve 10 mg o f bromocriptine mesilate CRS carbonate R;
in the solvent mixture and dilute to 10 m L with the solvent — mobile phase B: acetonitrile R;
mixture.
Plate TLC silica gel G plate R. Time Mobile phase A Mobile phase B
Mobile phase concentrated ammonia R, water R, 2-propanol R,
0 -3 0 90->40 10 -» 60
methylene chloride R, ether R (0.1:1.5:3:88:100 V/V/V/V/V).
30-45 40 60
Application 10 (iL.
Development Immediately in an unsaturated tank, over a path
o f 15 cm. Flow rate 2 mlVmin.
Drying In a current of cold air for 2 min. Detection Spectrophotometer at 300 nm.
Detection Spray with ammonium molybdate solution R3 and dry Injection 20 pL.
at 100 °C until the spots appear (about 10 min). Identification of impurities Use the chromatogram supplied
Residts T h e principal spot in the chromatogram obtained with with bromocriptine mesilate for system suitability CRS and the
the test solution is similar in position, colour and size to the chromatogram obtained with reference solution (c) to identify
principal spot in the chromatogram obtained with the the peaks due to impurities A and B.
reference solution. Relative retention W ith reference to bromocriptine:
D . T o 0.1 g add 5 m L of dilute hydrochloric acid R and shake im purity C = about 1.2.
for about 5 min. Filter and add 1 m L o f barium chloride System suitability, reference solution (c):
solution R l. T h e filtrate remains clear. T o a further 0.1 g add — resolution: minimum 1.1 between the peaks due to
0.5 g o f anhydrous sodium carbonate R, mix and ignite until a impurities A and B.
white residue is obtained. Allow to cool and dissolve the
Limits:
residue in 7 m L of water R (solution A). Solution A gives
— impurity A: not more than 0.2 times the area o f the
reaction (a) o f sulfates (2.3.1).
E. Solution A obtained in identification test D gives reference solution (b) (0.02 per cent);
reaction (a) of bromides (2.3.1). — impurity C: not more than 4 times the area of the
TESTS principal peak in the chromatogram obtained with
A p p e a ra n c e o f so lu tio n reference solution (b) (0.4 per cent);
T he solution is clear (2.2.1) and not more intensely coloured — impurities B, D, E, F} G: for each impurity, n o t more than
than reference solution B5, BY5 or Y5 (2.2.2, Method II). twice the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.2 per cent) and
1-320 Bromocriptine Mesilate 2016
n o t m ore than 1 such peak has an area greater than the 4,6,6a,7,8,9-hexahydroindolo[4,3-j&]quinoline-9-carboxamide
area of the principal peak in the chromatogram obtained ((95)-2-bromo-a-ergocriptine),
w ith reference solution (b) (0.1 p er cent);
— total: not more than 1.5 times the area of the principal
— disregard limir. 0.5 times the area o f the principal peak in 'CO jH
(0.05 per cent), apart from the peak due to impurity A.
D . (6aR,9i?)-5-bromo-7-methyi-4,6,6a,7,8,9-
M axim um 3.0 per cent, determ ined on 0.500 g by drying
hexahydroindolo [4,3-^] quinoline-9-carboxylic acid,
ASSAY
Dissolve 0.500 g in 80 m L o f a mixture of 10 volumes of
anhydrous acetic add R and 70 volumes of acetic anhydride R
Titrate with 0.1 M perchloric add, determining the end-point
of C 33H 44B rN 508 S.
E. (6aR,9/?)-5-bromo-7-methyl-4,6,6a,7,8,9-
STO RA G E hexahydroindolo [4,3-/^] quinoline-9-carboxamide,
tem perature not exceeding —15 °C.
IM P U R IT IE S
Spedfied impurities A, B, C , D , E, F , G
F. (6aR,9i?)-5-bromo-N-[(25,55,10aS,10b5)-10b-hydroxy-2-
(l-m ethylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro-8/f-
oxazolo [3,2-a] pyrrolo [2,1 -c] pyrazin-2-yl] -7-methyl-
4,6,6a,7,8,9-hexahydroindolo[4,3-j^]quinoline-9-carboxamide
A. (6ai?,9i?)-5-bromo-N- [(2R,5S)-2-( 1-methylethyl)-5- ((2 '^ ^ -b ro m o -a-erg o crip tin e),
(2-methylpropyl)-3,6-dioxo-2,3,5,6,9,10-hexahydro-8/f-
oxazolo [3,2-a] pyirolo [2,1 -c] pyrazin-2-yl]-7-methyl-
4,6,6a,7,8,9-hexahydroindolo[4,3-/g]quinoline-9-carboxamide
(2-bromodehydro-a-ergocriptine),
G . (6aR,9i?)-5-bromo-N- [(2/?,55’, 10 a 5 ,10 b 5 )-l Ob-methoxy-

2-( 1-methylethyl) -5-(2-methylpropyl) -3 ,6-dioxooctahydro-
8/f-oxazolo [3,2-a] pyrrolo [2,1 -c]pyrazin-2-yl]-7-methjd-
4,6,6a,7,8,9-hexahydroindolo [4,3-^] quinoline-9-carboxamide
B. (6aR,9R)-N-[(2R,5S,10aS, 10 bS )-l Ob-hydroxy-2-
(2-brom o-10 'b-O-methyl-a-ergocriptine).
(l-m ethylethyl)-5-(2-methylpropyl)-3,6-dioxooctahydro-8/f-
oxazolo [3,2-a] pyrrolo [2,1-c] pyrazin-2-yl]-7-methyl- ___________________________________________________________ PhEur
4,6,6a,7,8,9-hexahydroindolo [4,3-fg] quinoline-9-carboxamide
(a-ergocriptine).
C. (6ai?,95)-5-brom o-N-[(2i?,55,10a5,10b5)-lOb-hydroxy-2-
(1 -methylethyl) -5-(2-methylpropyl)-3 ,6-dioxooctahydro-8//-
oxazolo [3,2-a] pyrrolo [2, l-c]pyrazin-2-yl]-7-methyl-
2016 Bromperidol 1-321
E. T o 0.1 g in a porcelain crucible add 0.5 g o f anhydrous

Bromperidol f**%
sodium carbonate R. H eat over an open flame for 10 min.
★. .★
(Ph Eur monograph 1178) * Allow to cool. Take up the residue with 5 m L o f dilute nitric
acid R and filter. T o 1 m L of the filtrate add 1 m L of
water R. T he solution gives reaction (a) o f bromides (2.3.1).
TESTS
T h e solution is clear (2.2.1) and not m ore intensely coloured
OH than reference solution Y7 (2.2.2, Method II).
Dissolve 0.2 g in 20 m L o f a 1 per cent VIV solution of lactic
C2iH23BrFN02 420.3 10457-90-6 acid R.
A ctio n a n d u se Liquid chromatography (2.2.29).
Dopam ine receptor antagonist; neuroleptic.
PhEtr __________________________________________________________ examined in methanol R and dilute to 10.0 m L with the same
solvent.
D E F IN IT IO N
4-[4-(4-Bromophenyl)-4-hydroxypiperidin- 1-yl]-1- Reference solution (a) Dissolve 2.5 mg of bromperidol CRS and
(4-fluorop heny 1)b utan-1-one. 5.0 mg o f haloperidol CRS in methanol R and dilute to
C o n te n t
100.0 m L with methanol R Dilute 1.0 mT. o f this solution to
CHARACTERS 10.0 m L with methanol R.
A p p e a ra n c e Column:
W hite or almost white powder. — size: I = 0.1 m , 0 = 4.0 mm;
S o lu b ility — stationary phase: base-deactivated octadecylsUyl silica gelfor
Practically insoluble in water, sparingly soluble in m ethanol chromatography R (3 pm).
and in methylene chloride, slightly soluble in ethanol Mobile phase:
(96 per cent). — mobile phase A: 17 g/L solution o f tetrabutylammonium
ID E N T IF IC A T IO N hydrogen sulfate i?;
— mobile phase B: acetonitrSe R;
B. Infrared absorption spectrophotometry (2.2.24). 0 -15 90-» 50 10->50
Comparison bromperidol CRS.
15-20 50 50
2 0-25 90 10 ,
Reference solution (a) Dissolve 10 mg o f bromperidol CRS in Flow rate 1.5 mlVmin.
methanol R and dilute to 10 m L with the same solvent. Detection Spectrophotometer at 230 nm .
Reference solution (b) Dissolve 10 mg of bromperidol CRS and Irqection 10 pL.
10-m g of haloperidol CRS in methanol R and dilute to 10 m L
Relative retention W ith reference to bromperidol (retention
Plate TLC octadecylsifyl silica gel plate R. impurity B = about 0.8; haloperidol = about 0.9;
Mobile phase tetrahydrqfuran R, methanol R, 58 g/L solution of impurity C — about 1.4; impurity D = about 1.5;
sodium chloride R (10:45:45 VIVIV). impurity E = about 1.8; impurity F = about 1.85.
Application 1 pL. System suitability: reference solution (a):
Development In an unsaturated tank, over 3/4 of the plate. — resolution: minimum 3.0 between the peaks due to
haloperidol and bromperidol.
Drying In air.
Limits'.
— impurities A, B, C, D, E, F: for each impurity, not more
System suitability,: reference solution (b): than the area of the principal peak in the chromatogram
— the chromatogram shows 2 spots which may, however, obtained with reference solution (b) (0.5 per cent);
not be completely separated. — unspecified impurities: for each impurity, n o t more than
Results T he principal spot in the chromatogram obtained with 0.2 times the area of the principal peak in the
the test solution is similar in position and size to the principal chromatogram obtained with reference solution (b)
spot in the chromatogram obtained with reference (0.10 per cent);
solution (a). — total: not more than twice the area o f the principal peak in
D . Dissolve about 10 m g in 5 m L o f anhydrous ethanol R. the chromatogram obtained with reference solution (b)
A dd 0.5 m L of dinitrobenzene solution R and 0.5 mT. of 2 M (1 p er cent);
alcoholic potassium hydroxide R. A violet colour is produced — disregard limit: 0.1 times the area o f the principal peak in
that becomes brownish-red after 20 min. the chromatogram obtained with reference solution (b)
(0.05 per cent).
1-322 Bromperidol Decanoate 2016
Br
an oven at 105 °C.
Maximum 0.1 per cent, determ ined on 1.0 g in a platinum
crudble.
Br
ASSA Y
Dissolve 0.300 g in 50 m L o f a mixture o f 1 volume o f
anhydrous acetic acid R and 7 volumes o f methyl ethyl ketone R. OH
T itrate with 0.1 M perchloric acidy using 0.2 m L of
naphthoJbenzein solution R as indicator.
E. 4-[4-(4-brom ophenyl)-4-hydroxypiperidin-l-yl]-l-[4-[4-
1 m L o f 0.1 M perchloric acid is equivalent to 42.03 mg (4-bromophenyl)-4-hydroxypiperidin-l-yl]phenyl]butan-l-
o f C 2iH 23B rFN 02. one,
STO RA G E
IM P U R IT IE S
Specified impurities A , By C, D, E, F.
F . 4- [4- (4 '-bromobiphenyl-4-yl)-4-hydroxypiperidin-1-yl] -1 -
(4-fluorophenyl)butan-1-one.
OH PhEur
A. 1-(4-fluorophenyl)-4-(4-hydroxy-4-phenylpiperidin-1-
yI)butan-l-one,
★ ★
Bromperidol Decanoate ★ ★
Br
(Ph Eitr monograph 1397) *****
OH Br
B. 4- [4-(4-bromophenyl)-4-hydroxypiperidin- 1-yl]-1-
(2-fluorophenyl)butan-1-one, H,C
C 3jH ^B rFN O j 574.6 75067-66-2
PhEur________________________________________
C . 4- [4-(biphenyl-4-yl)-4-hydroxypipeiidin- 1-yl] -1-
(4-fluorophenyl)butan-l-one, D E F IN IT IO N
4-(4-Brom ophenyl)-l-[4-(4-fluorophenyl)-4-
oxobutyl]piperidin-4-yl decanoate.
h 3c
C o n te n t
'XV ~ Q J C r * OH
CHARACTERS
A p p e a ra n c e
D . 4- [4-(4-bromophenyl)-4-hydroxypiperidin-1-yl] -1 -
S o lu b ility
(3-ethyl-4-fluorophenyl)butan- 1-one,
Practically insoluble in water, very soluble in methylene
chloride, soluble in ethanol (96 p er cent),
m p A bout 60 °C.
Comparison bromperidol decanoate CRS.
B. T o 0.1 g in a porcelain crucible add 0.5 g o f anhydrous
sodium carbonate R. H eat over an open flame for 10 min.
Allow to cool. T ake up the residue with 5 m L of dilute nitric
2016 Bromperidol Decanoate 1-323
acid, R and filter. T o 1 m L o f the filtrate add 1 m L of — disregard limit: 0.1 times the area of the principal peak in
water R. T h e solution gives reaction (a) of bromides (2.3.1). the chromatogram obtained with reference solution (b)
(0.05 per cent).
TESTS
A p p e a ra n c e o f so lu tio n L oss o n d ry in g (2.2.32)
T he solution is clear (2.2.1) and not m ore intensely coloured M aximum 0.5 p er cent, determined on 1.000 g by drying
than reference solution B 5 (2.2.2, Method II). m vacuo at 30 °C.
Dissolve 2.0 g in methylene chloride R and dilute to 20 mL S u lfa ted a s h (2.4.14)
with the same solvent. M aximum 0.1 p er cent, determined on 1.0 g in a platinum
crucible.
Liquid chromatography (2.2.29). Prepare the solutions ASSAY
immediately before use and protea from light. Dissolve 0.450 g in 50 m L of a mixture o f 1 volume of
Test solution Dissolve 0.100 g o f the substance to be anhydrous acetic acid R and 7 volumes of methyl ethyl ketone R.
examined in methanol R and dilute to 10.0 m L with the sam e T itrate with 0.1 M perchloric add, using 0.2 m L of
solvent. naphtholbenzein solution R as indicator.
Reference solution (a) Dissolve 2.5 mg o f bromperidol 1 m L o f 0.1 Mperchloric acid is equivalent to 57.46 mg
decanoate CRS and 2.5 mg o f haloperidol decanoate CRS in o f C 3iH 41B rFN 0 3.
methanol R and dilute to 50.0 m L w ith the same solvent. STORA GE
Reference solution (b) Dilute 5.0 m L of the test solution to Protected from light, at a temperature below 25 °C.
100.0 m L with methanol R. Dilute 1.0 m L o f this solution to
IM P U R IT IE S
Specified impurities A, B, C , D, E, F , G, H , I, J, K
Column:
— size. / = 0,1 m , 0 = 4.0 mm; Other detectable impurities (the following substances would, if
— stationary phase: base-deactivated octadecylsUyl silica gel for present at a sufficient level, be detected by one o r other o f
chromatography R (3 |im). the tests in the monograph. They are limited by the general
Mobile phase:
— mobile phase A: 27 g/L solution o f tetrabutylammonium
hydrogen sulfate R;
— mobile phase B: acetonitrüe R;
Control of impurities in substances for pharmaceutical use): L.

0 - 30 80 ->40 20-> 60
3 0 -3 5 40 60
3 5 -4 0 40 ->80 60-> 20
O
A. 1-[4-(4-fluorophenyl)-4-oxobutyl]-4-phenylpiperidin-4-yl
decanoate,
Injection 10 |iL.
Relative retention W ith reference to brom peridol decanoate
(retention time = about 24 min): impurity G = about 0.10;
impurity L = about 0.15; impurity H = about 0.8;
im purity A = about 0.89; impurity I = about 0.91;
impurity B = about 0.96s haloperidol
decanoate = about 0.98; impurity F = about 1.10;
im purity C = about 1.15; impurity K = about 1.2 ; o
im purity E = about 1.23; impurity D = about 1.25.
System suitability: reference solution (a): B. 4-(4-brom ophenyl)-l-[4-(2-fluorophenyl)-4-oxobutyl]-
— resolution: minimum 1.5 between the peaks due to piperidin-4-yl decanoate,
haloperidol decanoate and bromperidol decanoate.
Limits:
— impurities A , B, C, D, E, F, G, H, I, J, K, for each
im purity, n o t more than the area o f the principal peak in
(0.5 p er cent);
0.2 times the area o f the principal peak in the O
( 0.10 per cent); C . 4-(4-brom ophenyl)-l-[4-(3-ethyl-4-fluorophenyl)-4-
— total: not more than 3 times the area of the principal peak oxobutyl]-piperidin-4-yl decanoate,
(1.5 per cent);
1-324 Brompheniramine Maleate 2016
Br
Br
I. 4-(4-bromophenyl)-1- [4-(4-fluorophenyl)-4-
oxobutyl]piperidin-4-yl nonanoate,
D . 4-(4-brom ophenyl)-l -[4- [4-[4-(4-bromophenyl)-4-

hydroxypiperidin-1-yl] phenyl] -4-oxobutyI]piperidin-4-yl
decanoate,
J. 4-(4-bromophenyl)-l-[4-(4-fluorophenyl)-4-
oxobutyl]piperidin-4-yl undecanoate,
E. 4-(4'-bromobiphenyl-4-yl)-l-[4-(4-fluorophenyl)-4-
oxobutyl]piperidin-4-yl decanoate,
K. 4-(4-brom ophenyl)-l -[4-(4-fluorophenyl)-4-

oxobutyl]piperidin-4-yl dodecanoate,
ch3
L. l-(4-fluorophenyl)ethanone.
F. 4-(biphenyl-4-yl)-1- [4-(4-fluorophenyl)-4- PhEur
oxobutyl]piperidin-4-yl decanoate,
★ ★
Brompheniramine Maleate ★ ★
c o 2h
G. 4- [4-(4-bromophenyl)-4-hydroxypiperidin-1-yl] -1 -
(4-fluorophenyl)butan-1-one (bromperidol), and enantiomer
■( C02H
C20H23BIN2O4 435.3 980-71-2
Action and use

Histamine H i receptor antagonist; antihistamine.
Preparation
Brompheniramine Tablets
H. 4-(4-brom ophenyl)-1- [4-(4-fluorophenyl)-4-
oxobutyl]piperidin-4-yl octanoate, PhEur.
DEFINITION
(3/?5)-3-(4-Bromophenyi)-J\yV-dimethyl-3-(pyridin-2-
yl)propan-l-am ine (Z)-butenedioate.
Content
2016 1-325
CHARACTERS Test solution Dissolve 0.100 g of the substance to be

Appearance examined in 10.0 m L of methylene chloride R.
W hite or almost white, crystalline powder. Reference solution (a) Dilute 1.0 m L o f the test solution to
S o lu b ility 100.0 m L with methylene chloride R. D ilute 1.0 m L of this
Soluble in water, freely soluble in ethanol (96 per cent), in solution to 10.0 m L with methylene chloride R.
m ethanol and in methylene chloride. Reference solution (b) Dissolve 10 mg o f chlorphenamine
maleate CRS (impurity A) and 10 m g o f phemramme
maleate CRS (impurity C) in methylene chloride R and dilute
First identification C, F.
to 5 m L w ith the same solvent To 2.5 m L of the solution
Second identification A , B, D, E, F. add 2.5 m L o f the test solution.
A. Melting point (2.2.14): 130 °C to 135 °C. Column:
B. Ultraviolet and visible absorption spectrophotometry — material: fused silica;
(2.2.25). — sizer. I = 30 m , 0 = 0.32 mm;
Test solution Dissolve 65 mg in a 10.3 g/L solution of — stationary phase: pdymethylphenylsiloxane R (film thickness
hydrochloric add R and dilute to 100.0 m L with the same 0.5 ^m ).
solution. Dilute 5.0 m L of this solution to 100.0 m L with a Carrier gas nitrogen for chromatography R.
10.3 g/L solution o f hydrochloric add R. Flow rate 1.0 mL/min.
- Spectral range 220-320 nm. Split ratio 1:5.
Absorption maximum A t 265 nm. Temperature:
Spedfic absorbance at the absorption maximum 190 to 210. — column: 205 °C;
C. Infrared absorption spectrophotometry (2.2.24). — injection port and detector. 250 °C.
Comparison brompheniramine maleate CRS. Detection Flam e ionisation.
D . Thin-layer chromatography (2.2.27). Injection 1 (iL.
Test solution Dissolve 0.10 g o f the substance to be examined Run time 1.2 times the retention time o f brompheniramine.
in methanol R and dilute to 5.0 m L with the same solvent. Identification of impurities Use the chromatogram obtained
Reference solution Dissolve 56 mg of maleic add R in with reference solution (b) to identify the peaks due to
methanol R and dilute to 10 m L with the same solvent impurities A and C.
Plate TLC silica gel F2 5 4 plate R. Relative retention W ith reference to brompheniramine
(retention time = about 34 min): impurity C = about 0.4;
Mobile phase water R, anhydrous formic add R, methanol R,
di-isopropyl ether R (3:7:20:70 VIV/V/V).
Application 5 pL.
Development Over 2/3 o f the plate. impurity A an d brompheniramine.
Drying In a current of air for 5 min. Limits:
Detection Examine in ultraviolet light at 254 nm. — impurities A , C: for each impurity, n o t m ore than 4 times
Results T h e chrom atogram obtained with the test solution the area o f the principal peak in the chromatogram
shows 2 clearly separated spots; the upper spot is similar in obtained with reference solution (a) (0.4 per cent);
position and size to the spot in the chromatogram obtained — unspecified impurities: for each impurity, n o t more than the
with the reference solution. area o f the principal peak in the chromatogram obtained
E. T o 0 . 1 5 g i n a porcelain crucible add 0.5 g o f anhydrous with reference solution (a) (0.10 per cent);
sodium carbonate R. H eat over an open flame for 10 min. — total: n o t m ore than 10 times the area o f the principal
Allow to cool. T ake up the residue in 10 m L of dilute nitric peak in the chromatogram obtained with reference
add R and filter. T o 1 m L o f the filtrate add 1 m L o f solution (a) (1.0 per cent);
water R. T h e solution gives reaction (a) of bromides (2.3.1). — disregard limit. 0.5 times the area of the principal peak in
F. Optical rotation (see Tests).
(0.05 p er cent).
TESTS H eav y m e ta ls (2.48)
A p p e a ra n c e o f so lu tio n M axim um 20 ppm .
T he solution is clear (2.2.1) and no t more intensely coloured
1.0 g complies w ith test C. Prepare the reference solution
using 2 m l, o f lead standard solution (10 ppm Pb) R.
same solvent.
Maximum 0.5 p er cent, determined on 1.000 g by drying in
p H (2.2.3) an oven at 105 °C for 3 h.
4.0 to 5.0.
Dissolve 0.20 g in 20 m L o f carbon dioxide-free water R. Maximum 0.1 p er cent, determined on 1.0 g.
A SSAY
- 0 .2 0 ° to + 0.20° (m easured in a 2 dm tube).
Dissolve 0.260 g in 50 m L of anhydrous acetic add R. T itrate
Dissolve 2.5 g in water R and dilute to 25.0 m L w ith the with 0.1 M perchloric add, determining the end-point
same solvent. potentiometrically (2.2.20).
R e la te d s u b s ta n c e s 1 m L of 0.1 M perchloric acid is equivalent to 21.77 mg
Gas chromatography (2.2.28). of C2oH 23B rN 20 4 .
1-326 Bronopol 2016
STORAGE (1) 0.2% w/v of the substance being examined.

Protected from light. (2) Dilute a volume of solution (1) to produce a solution
IM P U R IT IE S containing 0.0002% w/v o f the substance being examined.
Specified impurities A , C (3) 0.001% w/v each of 2-methyl-2-nitropropan-l,3-diol and
tris(hydroxymethyl) nitromethane.
(4) 0.0002% w/v each o f 2-m ethyl-2-nitropropane-l,3-diol5
2-nitroethanol, sodium bromide and
tris(hydroxymethyl)nitromethane and 0.2% w/v o f the
substance being examined.
(a) Use a stainless steel column (15 cm x 4.6 mm) packed
A. (3i?¿)-3-(4-chlorophenyl)-AyV-dimethyl-3-(pyridin-2- with octadecylsüyl silica gel for chromatography (5 pm)
yl)propan-1-amine (chlorphenamine), (Phenomenex Luna C l 8 (2) is suitable).
(b) Use isocratic elution and the mobile phase described
below.
Q „ (c) Use a flow rate of 1 m L per minute.
and enantiomer
N (d) Use a column tem perature o f 35°.
i
CH3 (e) Use a detection wavelength of 214 nm.
(f) Inject 20 pL o f each solution.
C . (3i?¿)-AVV-dimethyl-3-phenyl-3-(pyridin-2-yl)propan-1- (g) For solution (1) allow the chromatography to proceed for
a m in e (pheniram ine). at least 3 times the retention time o f the principal peak.
___________________________________________________________________________________________ PhEur MOBILE PHASE
1 volume of a 10% v/v solution of orthophosphoric acid,
10 volumes of acetonitrile and 189 volumes o f water, adjust
the p H to 3.0 using 2m sodium hydroxide.
Bronopol SYSTEM SUITABILITY
T h e test is not valid unless, in the chromatogram obtained
Br N 02 with solution (4):
ho^ J K ^ oh the resolution factor between the peaks due to sodium brom ide
and tris(hydroxymethyl)nitromethane is at least 1.0;
C3H 6B rN 04 200.0 52-51-7 the resolution factor between the peaks due to
tris (hydroxymethyl) nitromethane and 2-nitroethanol is at
A c tio n a n d use least 1.5.
Antibacterial preservative.
LIMITS
D E F IN IT IO N In the chromatogram obtained with solution (1):

Bronopol is 2-brom o-2-nitropropane-l,3-diol. It contains not the area of any peak corresponding to 2-methyl-2-
less than 99.0% and no t more than 101.0% o f C jH ^B rN O ^ nitropropane-l,3-diol and tris(hydroxymethyl)nitromethane
calculated with reference to the anhydrous substance. are not greater than the area o f the corresponding peaks in
the chromatogram obtained with solution (3) (0.5% o f each);
W hite or almost white crystals or crystalline powder. the area o f any other secondary peak is not greater than the
area o f the principal peak in the chromatogram obtained with
Freely soluble in water and in ethanol (96%); slightly soluble
solution (2) (0.1%).
in glycerol and in liquid paraffin.
Sulfated ash
N o t more than 0.1%, Appendix IX A.
A. T he infrared absorption spectrum, Appendix II A, is
concordant with the reference spectrum o f bronopol (RS 031). Water
N o t more than 0.5% w/w, Appendix IX C , M ethod I B.
B. Dissolve 0.1 g in 10 m L o f water, add 10 m L 'o f
U se 5 g.
7.5 m sodium hydroxide and, carefully with constant stirring
and cooling, 0.5 g of nickel-alwninium alloy. Allow the A SSA Y
reaction to subside, filter and carefully neutralise with nitric In a flask fitted with a reflux condenser dissolve 0.4 g in
acid. T he resulting solution yields reaction A characteristic of 15 m L of water and add 15 m L o f 7.5m sodium hydroxide.
bromides, Appendix VI. Slowly, with caution, add 2 g o f nickel-aluminium alloy
C. Melting point, after drying over phosphorus pentoxide at a through the reflux condenser, agitating the flask whilst
pressure not exceeding 0.7 kPa, about 130°, A ppendix V A. cooling under running water. Allow the mixture to stand for
10 minutes and boil for 1 hour. Cool and filter under
TESTS reduced pressure, washing the condenser, flask and residue
A cid ity o r a lk alin ity with 150 m L o f water. Combine the filtrate and washings,
p H o f a 1% w/v solution, 5.0 to 7.0, Appendix V L. add 25 m L o f nitric acid and 40 m L of 0. 1m silver nitrate VS,
R e la te d su b sta n c e s shake vigorously and titrate with 0. Im ammonium thiocyanate
Carry out the m ethod for liquid chromatography, F 5 using ammonium iron(m) sulfate solution R2 as indicator.
Appendix HI D , using the following solutions in the mobile Repeat the operation without the substance being examined.
phase. T h e difference between the titrations represents the am ount
2016 Brotizolam 1-327
of silver nitrate required. Each m L o f 0.1m silver nitrate VS is mobile phase B: mix 25 volumes of a 2 g/L solution of
equivalent to 20.00 mg of C 3H 6B rN 0 4. sodium heptanesulfonate R and 75 volumes of acetonitrile R',
STORAGE
Bronopol should be protected from light. Time Mobile phase A Mobile phase B
(min) (per cent V/V) (percent V/V)
0 -4 63 37
4-15 6 3-» 12 3 7-> 8 8

★.* * *★
Brotizolam ★ ★
Injection 5 pL.
Relative retention W ith reference to brotizolam (retention
tim e = about 7.4 min): impurity A = about 0.5;
— resolution: minimum 5.0 between die peaks due to
impurity B and brotizolam.
C 15H 10BrClN4S 393.7 57801-81-7

Limits'.
— impurity B: n o t more than the area of the principal peak
Action and use in the chromatogram obtained with reference solution (a)
Benzodiazepine. (0.1 p er cent);
PhEur. area o f the principal peak in the chromatogram obtained
DEFINITION with reference solution (a) (0.10 per cent);
2-Brom o-4-(2-chlorophenyl)-9-methyl-6/i-thieno-[3,2- — total: n o t more than twice the area of the principal peak in
f\ [1 ,2,4]-triazolo [4,3-a] [l,4]diazepine. the chromatogram obtained with reference solution (a)
(0.2 p er cent);
Content — disregard limit: 0.5 times the area of the principal peak in
99.0 per cent to 101.0 per cent (dried substance). the chromatogram obtained w ith reference solution (a)
CHARACTERS (0.05 p er cent).
Appearance C h lo rid e s (2.4.4)
W hite or yellowish powder. M aximum 100 ppm .
Solubility Dissolve 0.67 g in 20.0 m L of methanol R, mix and filter.
Practically insoluble in water, sparingly soluble or slightly L o ss o n d ry in g (2.2.32)
soluble in m ethanol, slightly soluble in ethanol (96 per cent). M axim um 0.5 p er cent, determined on 1.000 g by drying in
IDENTIFICATION an oven at 105 °C.
Infrared absorption spectrophotometry (2.2.24). S u lfa te d a s h (2.4.14)
Comparison brotizolam CRS. M axim um 0.1 per cent, determined on 1.0 g.
TESTS A SSA Y
Related substances Dissolve 0.150 g in a mixture of 25 m L o f glacial acetic
Liquid chrom atography (2.2.29). Carry out the test protected acid R and 50 m L of acetic anhydride R. T itrate to the second
from light a n d prepare the solutions immediately before use. point o f inflexion with 0.1 M perchloric acid, determining the
Test solution Dissolve 50.0 mg of the substance to be end-point potentiometrically (2.2.20).
examined in acetonitrile R and dilute to 50.0 m L with the 1 m L of 0.1 M perchloric acid is equivalent to 19.68 mg
same solvent. of C 15H 10BrCIN 4S.
Reference solution (a) Dilute 1.0 m L of the test solution to IM P U R IT IE S
100.0 m L o f acetomtrüe R. Dilute 1.0 m L of this solution to Specified impurities B.
10.0 m L w ith acetonitrile R.
Reference solution (b) Dissolve 5 mg o f the substance to be present at a sufficient level, be detected by one or other o f
examined an d 5 m g o f brotizolam impurity B CRS in 50 m L the tests in the monograph. T hey are limited by the general
o f acetonitrile R. Dilute 2 m L o f this solution to 20 m L with acceptance criterion for other/unspecified impurities and/or
acetonitrile R. by the general monograph Substances for pharmaceutical use
Column: (2034). It is therefore n o t necessary to identify these
— sizer. I = 0.15 m , 0 = 4.6 mm; impurities for demonstration o f compliance. See also 5.10.
— stationary phase: oaylsüyl silica gel for chromatography R Control of impurities in substances for pharmaceutical use): A.
(5 nm);
Mobile phase:
— mobile phase A: 2 g/L solution o f sodium heptanesulfonate
monohydrate R',
1-328 Buclizine Hydrochloride 2016
45 volumes o f acetonitrUe and as the final mobile phase 0. 01m

sodium heptanesulfonate in a mixture of 20 volumes o f water
and 80 volumes o f acetonitrUe. Before use, adjust the p H of
both the initial and final mobile phases to 4.0 with 1m
orthophosphoric acid. C any out a linear gradient elution with a
flow rate of 2 m L per minute for 30 m inutes and maintain
the final mobile phase for 10 m inutes with the same flow
rate. Use a detection wavelength o f 230 nm.
A. R1 = C H 3j R2 = H: 4-(2-chlorophenyl)-9-methyl-6H- T h e test is not valid unless the chrom atogram obtained with
thieno[3j2-/| [ 1,2,4] triazolo [4,3-a] [l,4]diazepine solution (4) closely resembles the chromatogram supplied
(desbromobrotizolam), with buclizine hydrochloride impurity standard BPCRS.
B. R l = H , R2 = B n 2-bromo-4-(2-chlorophenyl)-6H- In the chromatogram obtained with solution (2) the area of

thieno[3,2-y) [ 1,2,4] triazolo [4,3-a] [l,4]diazepine any peak corresponding to 1,4-bis (4-chlorobenzhydryl)-
(desmethylbrotizolam). piperazine is not greater than the area o f the peak obtained in
the chromatogram with solution (3) and the area o f any
___________________________________________________________ PhEur
other secondary peak is not greater than the area of the peak
in the chromatogram obtained with solution (1).
L oss o n d ry in g
W hen dried to constant weight at 100° to 105°, loses not
Buclizine Hydrochloride more than 1.0% o f its weight. Use 1 g.
S u lfa te d a sh
N o t more than 0.1 %, Appendix IX A.
A SSAY
Carry out M ethod I for non-aqueous titration,
Appendix V IE A, using 0.4 g and determ in in g the end point
potentiometrically. Each m L o f 0. 1m perchloric add VS is
equivalent to 25.30 m g of C28H33C1N2j2HC1.
IM P U R IT IE S
C a ^ a N ^ d 506.0 129-74-8 A. l,4-bis(4-chlorobenzhydryl)piperazine,
B. 4-chlorobenzhydrol, l-(4-chlorobenzhydryl)piperazine,
A c tio n a n d u se 4-chlorobenzophenone.
H istamine H i receptor antagonist; antiemetic.
D E F IN IT IO N
Buclizine Hydrochloride is (i?5)-l-(4-terr-butylbenzyl)-4-
(4-chlorobenzhydryl)piperazine dihydrochloride. It contains
Budesonide *****
n o t less than 99.0% and not more than 100.5% o f
*+ +*
C 28H 33C 1N 2, 2H C 1, calculated with reference to the dried (Ph. Evr. monograph 1075) *
substance.
C H A R A C T E R IS T IC S o P H
A white or slightly yellowish, crystalline powder.
Practically insoluble in water, sparingly soluble in propane-1,2- Ch J h H and epimer at C*
diok very slightly soluble in ethanol (96%).
A. T h e infrared absorption spectrum, Appendix II A, is
concordant with the reference spectrum o f buclizine
hydrochloride (RS 032). C 25H340 6 430.5 51333-22-3
B. A 0.25% w/v solution in ethanol (50%) yields reaction A
characteristic o f chlorides, Appendix VI. A ctio n a n d use
Glucocorticoid.
TESTS
Budesonide Aqueous Nasal Spray
C arry out the m ethod for liquid chromatography,
Appendix IQ D , using four solutions in the initial mobile Budesonide Inhalation Powder
phase containing (1) 0.0010% w/v o f the substance being Budesonide Inhalation Powder, pre-dispensed
examined, (2) 0.50% w/v o f the substance being examined, Budesonide Nebuliser Suspension
(3) 0.0010% w/v of Budesonide Pressurised Inhalation
l,4-bis(4-chbrobenzhy<byl)piperazine BPCRS and (4)
0.50% w/v o f buclizine hydrochloride impurity standard BPCRS. PhEur__________________________________________________________
T h e chromatographic procedure may b e carried out using a D E F IN IT IO N

stainless steel column (20 cm x 4 m m ) packed with Mixture of the C-22S (epimer A) and the C-22R (epimer B)
octadecylsUyl silica gel for chromatography (10 jxm) (Nucleosil epimers of 16a, 17- [(1i?*S)-butylidenebis(oxy)]-11 ß,21-
C l 8 is suitable). Use as the initial mobile phase 0.01m sodium dihydroxypregna-1,4-diene-3,20-dione.
heptanesulfonate in a mixture o f 55 volumes o f water and
2016 Budesonide 1-329
Content TESTS
97.5 per cent to 102.0 per cent (dried substance). Related substances
CHARACTERS Liquid chromatography (2.2.29). Carry out the test protected
from light.
Appearance
W hite or almost white, crystalline powder. Solvent mixture acetomtrüe R, phosphate buffer solution pH 3.2 R
(32:68 VIV).
Solubility
Practically insoluble in water, freely soluble in methylene Test solution (a) Dissolve 50 mg of the substance to be
chloride, sparingly soluble in ethanol (96 p er cent). examined in 15 m L of acetonitrüe R and dilute to 50 m L with
phosphate buffer solution pH 3.2 R.
IDENTIFICATION
Test solution (b) Dissolve 25.0 mg o f the substance to be
First identification A. examined in 15 m L of acetomtrüe R and dilute to 50.0 m L
Second identification B, C, D. with phosphate buffer solution pH 3.2 R.
A. Infrared absorption spectrophotometry (2.2.24). Reference solution (a) Dilute 1.0 m L o f test solution (a) to
Comparison budesonide CRS. 10.0 m L with the solvent mixture. D ilute 1.0 m L of this
B. Thin-layer chromatography (2.2.27). solution to 100.0 m L with the solvent mixture.
Solvent mixture methanol R, methylene chloride R (10:90 VIV). Reference solution (b) Dissolve 5 m g o f budesonide for system
Test solution Dissolve 25 m g o f the substance to be examined suitabüity CRS (containing impurities A, D , G, K and L) in
in the solvent mixture and dilute to 10 m L with die solvent 1.5 m L of acetomtrüe R and dilute to 5 m L with phosphate
mixture.
buffer solution pH 3.2 R.
Reference solution (a) Dissolve 25 mg o f budesonide CRS in the Reference solution (c) Dissolve 25.0 mg o f budesonide CRS in
solvent mixture and dilute to 10 m L with the solvent 15 m L o f acetonitrüe R and dilute to 50.0 m L with phosphate
mixture.
buffer solution pH 3.2 R.
Column:
Reference solution (b) Dissolve 12.5 mg o f triamcinolone
— size: I — 0.15 m , 0 = 4.6 mm;
acetordde CRS in reference solution (a) and dilute to 5 m L
Plate TLC silica gel F2 5 4 plate R. — temperature: 50 °C.
Mobile phase Add a mixture o f 1.2 volumes o f water R and Mobile phase:
8 volumes of methanol R to a mixture o f 15 volumes of — mobile phase A: anhydrous ethanol R, acetonitrüe R,
ether R and 77 volumes of methylene chloride R. phosphate buffer solution pH 3.2 R (2:32:68 VIVIV)\
Application 5 jiL. — mobile phase B: acetomtrüe R, phosphate buffer solution
Development Over a path o f 15 cm. pH 3.2 R (50:50 VIV)-,
Drying \n air.
Detection A Examine in ultraviolet light at 254 nm. Time Mobile phase A Mobile phase B
Results A T h e principal spot in the chromatogram obtained 100 0
0 - 38
principal spot in die chromatogram obtained with reference 3 8 -5 0 100 -»0 0 * 100
solution (a). 5 0 -6 0 0 100
Detection B Spray with alcoholic solution of sulfuric acid R; heat
at 120 °C for 10 m in or until the spots appear and allow to
Flow rate 1 mL/min.
cool; examine the chromatograms in daylight and in
ultraviolet light at 365 nm. Detection Spectrophotom eter at 240 nm.
Results B T he principal spot in the chromatogram obtained Injection 20 jiL o f test solution (a) and reference solutions (a)
with the test solution is similar in position, colour in daylight, and (b).
fluorescence in ultraviolet light at 365 nm and size to the Identification of impurities Use the chrom atogram supplied
principal spot in the chromatogram obtained with reference with budesonide for system suitability CRS and the
solution (a). chromatogram obtained with reference solution (b) to
System suitability: reference solution (b): identify the peaks due to impurities A, D , G , K and L.
— the chromatogram shows 2 clearly separated spots. Relative retention W ith reference to budesonide epimer B
C. Dissolve about 2 m g in 2 m L o f sulfuric add R. W ithin (retention time = about 17 min): impurity A = about 0.1;
5 min a yellow colour develops. W ithin 30 min the colour epimers o f impurity D = about 0.63 and 0.67;
changes to brown or reddish-brown. Cautiously add the impurity L = about 0.95; epimers o f im purity G = about 1.2
solution to 10 m L of water R and mix. T h e colour fades and and 1.3; epimers of impurity K = about 2.9 and 3.0.
a clear solution remains. System suitability: reference solution (b):
D . Dissolve about 1 mg in 2 m L o f a solution containing 2 g — peak-to-vaEey ratio: minimum 2.5, where Hp = height
o f phosphomdybdic add R dissolved in a mixture o f 10 m L of above the baseline of the l n o f the 2 peaks due to
dilute sodium hydroxide solution R, 15 m L o f water R and impurity G and Hv = height above the baseline of the
25 m L o f glacial acetic acid R. H eat for 5 m in on a water- lowest point o f the curve separating this peak from the
bath. Cool in iced water for 10 min and add 3 m L o f dilute peak due to budesonide epimer A (the 2nd of the
sodium hydroxide solution R. T h e solution is blue. 2 principal peaks); and minimum 3, where Hp = height
above the baseline o f the peak due to impurity L and
budesonide epimer B (the 1st of the 2 principal peaks).
1-330 Budesonide 2016
Limits: IM P U R IT IE S
— correction factors: for the calculation of content, multiply Specified impurities A , D, K, L.
the peak areas o f the following impurities by the Other detectable impurities (the following substances would, if
corresponding correction facto r impurity D = 1.8; present at a sufficient level, be detected by one or other of
impurity K = 1.3; the tests in the monograph. They are limited by the general
— impurities A , L: for each impurity, n o t more than twice acceptance criterion for other/unspecified impurities and/or
the sum of the areas of the 2 peaks due to the budesonide by the general monograph Substances for pharmaceutical use
epimers in the chrom atogram obtained with reference (2034). It is therefore not necessary to identify these
solution (a) (0.2 p er cent); impurities for demonstration of compliance. See also 5.10.
— impurities D, K. for each impurity, for the sum of the Control of impurities in substances for pharmaceutical use): B, C,
areas of the 2 epim er peaks, not m ore than twice die sum E, F, G, H, I, J.
o f the areas of the 2 peaks due to the budesonide epimers
(0.2 per cent);
— unspecified impurities: for each individual peak, not more
than the sum o f the areas o f the 2 peaks due to the
budesonide epimers in the chromatogram obtained with
— total: not more than 5 times the sum o f the areas o f the
2 peaks due to the budesonide epimers in the
chromatogram obtained with reference solution (a) A. 1 ip,16a,17,21-tetrahydroxypregna-l,4-diene-3,20-dione,
(0.5 per cent);
— disregard limit: 0.5 times the sum of the areas o f the
2 peaks due to the budesonide epimers in the
(0.05 per cent).
E p im e r A
Mobile phase: B. R = H: 16a,17-[(LRS)-ethylidenebis(oxy)]-lip,21-
dihydroxypregna-1,4-diene-3,20-dione,

F. R = C H 3: 16a, 17-[ 1-methylethylidenebis(oxy )]-11 (3,21-
(min) (per cent V7V) (per cent V7V) dihydroxypregna-1,4-diene-3,20-dione,
0 -21 100 0
21-22 100 -> 0 0 ^ 100
22 - 31 0 100
Injection 20 |iL of test solution (b) and reference solutions (b)

and (c).
C . 16a,17-[(lJ?S)-butylidenebis(oxy)]-l ip-hydroxy-17-
Retention time Budesonide epim er B = about 17 min;
(hydroxymethyl)-Z>-homoandrosta-l ,4-diene-3,17a-dione,
budesonide epim er A = about 19 min.
System suitability:
— resolution: m inim um 1.5 between the 2 principal peaks
(budesonide epimers A and B) in the chromatogram
obtained w ith reference solution (c);
— peak-to-vaUey ratio: minim um 3, where Hp = height above
the baseline of the peak due to impurity L and
D . R = CHO: 16a,17-[(lJ?.S)-butylidenebis(oxy)]-lip-
budesonide epim er B (the 1st o f the 2 principal peaks) in
hydroxy-3,20-dioxopregna-l ,4-dien-21-al,
Limit: K . R = C H 2- 0 - C 0 - C H 3: 16ot,17-[(li?5)-
— epimer A: 40.0 per cent to 51.0 per cent of the sum of the butylidenebis(oxy)]-11 [3,21 -dihydroxypregna-1,4-diene-3,20-
dione- 21-acetate,
areas of the 2 peaks due to the budesonide epimers.
an oven at 105 °C.
ASSAY
Liquid chromatography (2.2.29). Examine the
chromatograms obtained in the test for epimer A.
Calculate the percentage content of C 25H 34O 6 from the sum
of the areas o f the 2 peaks due to the budesonide epimers E. 16a, 17-[(l/?5)-butylidenebis(oxy)]-l 1P,21-
and the declared content o f budesonide CRS. dihydroxypregna-1,4,14-triene-3,20-dione,
2016 Bufexamac 1-331
Bufexamac ★ ★
★ ★
„0 H and epimer at C*
H
G. 16a, 17- [(li? 5 ) -butylidenebis(oxy)] -11 p,21-

dihydroxyprega-4-ene-3j20-dione.
C 12H 17NO 3 223.3 2438-72-4
Action and use

. -0 -' ' X ^ CH3
„ 0 H and epimer at C* PhEtr.
DEFINITION
2-(4-Butoxyphenyl)-N-hydroxyacetamide.
Content
H . 16a,17-[(li?5)-butylidenebis(oxy)]-21- 98.5 per cent to 101.5 per cent (dried substance).
hydroxypregnal,4,9(l l)-triene-3j20-dionej CHARACTERS
Appearance
Solubility
Practically insoluble in water, soluble in dimethylformamide,
slightly soluble in ethyl acetate and in methanol.
IDENTIFICATION
I. 11P, 17,2 1-trihydroxy-3,20-dioxopregna-1,4-dien-16a-yl
butanoate,
(2.2.25).
Test solution Dissolve 20 mg in methanol R and dilute to
20 m L with the same solvent. D ilute 1 m L of the solution to
--0 ^ \ ^ c h 3 50 m L with methanol R.
..q H and epimer at C* Spectral range 210-360 nm.
H Absorption maxima At 228 nm, 277 n m and 284 nm.
Comparison bufexamac CRS.
J. 16a,17-[(liiS)-butylidenebis(oxy)]-9a-brom o-l ip ,2 1 - C. Thin-layer chromatography (2.2.27).
dihydroxypregna-1,4-diene-3,20-dione, Test solution Dissolve 10 mg of the substance to be examined
Reference solution (a) Dissolve 20 mg o f bufexamac CRS in
Reference solution (b) Dissolve 10 m g o f salicylic acid R in
H I X "0 *"* and epimer at C* reference solution (a) and dilute to 5 m l. with the same
H solution.
H
Plate TLC silica gel F2 5 4 plate R.
Mobile phase glacial acetic acid R, dioxan R, toluene R
L. 16a, 17- [(1RS) -butylidenebis (oxy) ] -21 -hydroxypregna-1, 4- (4:20:90 V/V/V).
diene-3,11,20-trione. Application 10 jiL.
PhEur Development Over 2/3 o f the plate.
Drying In a current o f warm air.
Results T he principal spot in the chromatogram obtained w ith
spot in the chromatogram obtained w ith reference
solution (a).
TESTS
Related substances
1-332 Buflomedil Hydrochloride 2016
Test solution. Dissolve 50.0 m g of the substance to be A SSA Y

examined in mobile phase A and dilute to 20.0 m L with Dissolve 0.200 g in 50 m L of dimethytformamide R. T itrate
mobile phase A. w ith 0.1 M lithium methoxide, determining the end-point
Reference solution (a) D ilute 5.0 m L o f the test solution to potentiometrically (2 . 2 . 2 0 ).
25.0 m L with mobile phase A. Dilute 1.0 m L o f this solution 1 m L o f 0.1 M lithium methoxide is equivalent to 22.33 m g of
to 100.0 m L w ith mobile phase A. C 12H 17N O 3.
Reference solution (b) Dissolve 5 m g o f bufexamac CRS, 5 mg STORAGE
o f bufexamac impurity A CRS, 5 mg o f bufexamac Protected from light.
impurity B CRS, 5 m g of bufexamac impurity C CRS and
5 m g o f bufexamac impurity D CRS in mobile phase A and IM P U R IT IE S
dilute to 10.0 m L w ith mobile phase A. Dilute 1.0 m L o f the Specified impurities A, B, C, D
Column:
— sizer. I = 0.25 m , 0 = 4.6 mm;
chromatography R (5 }im).
Mobile phase: A. 2-(4-butoxyphenyl) acetic add,
— mobile phase A: m ix 30 volumes of a 1.4 g/L solution of
dipotassium hydrogen phosphate R and 70 volumes of
methanol R, then adjust to p H 3.6 with dilute phosphoric
acid R’,
— mobile phase B: methanol R;
B. methyl 2-(4-butoxyphenyl)acetate,
0 - 10 100 0
10 - 13 100-> 70 0-> 30
1 3-40 70 30
C. butyl 2-(4-butoxyphenyl)acetate,
Flow rate 1 m IJm in .
Injection 20 |iL.
with reference solution (b) to identify the peaks due to
impurities A, B, C and D. D . 2-(4-butoxyphenyl)acetamide.
___________________________________________________________________________________ ' P h E ir
Relative retention W ith reference to bufexamac (retention
time = about 5.7 min): impurity D = about 1.3;
impurity A = about 1.8; impurity B = about 3.0;
System suitability. reference solution (b): Buflomedil Hydrochloride f**%
bufexamac and impurity D . (Ph. Eux. monograph 1398) *
Limits:
— impurities A , B, C, D: for each impurity, not more than
C 17H 26d N 0 4 343.9 35543-24-9
(0.10 per cent);
Vasodilator.
solution (a) (0.5 p e r cent);
— disregard limit. 0.25 times the area o f the principal peak in P h & x ________________________________________________________________________________________ —
D E F IN IT IO N
(0.05 per cent).
4-(Pyrrolidin-l-yl)-l-(2,4,6-trim ethoxyphenyl)butan-l-one
L oss o n d ry in g (2.2.32) hydrochloride.
C o n te n t
M axim um 0.1 per cent, determined on 1.0 g. CHARACTERS
A p p e a ra n c e
W hite or almost white, microcrystalline powder.
2016 Buflomedil Hydrochloride 1-333
S o lu b ility — stationary phase: end-capped octadecylsUyl sUica gel for

Freely soluble in water, soluble in ethanol (96 per cent), very chromatography R (5 (im);
slightly soluble in acetone. — temperature: 40 °C.
mp MobUe phase Mix 45 volumes of acetomtrUe R1 and
About 195 °Cj with decomposition. 55 volumes o f a 9.25 g/L solution of potassium dihydrogen
phosphate R adjusted to p H 2.5 with phosphoric acid R.
First identification: B, D. Flow rate 1 mL/min.
Second identification A , C, D. Detection Spectrophotometer at 210 nm.
A. Ultraviolet and visible absorption spectrophotometry Injection 10 pL of the test solution and reference solutions (a)
and (c).
(2.2.25).
Test solution Dissolve 25.0 mg in ethanol (96 per cent) R and Run time Twice the retention time of buflomedil.
dilute to 50.0 m L with the same solvent. Dilute 2.0 m L of Identification of impurities Use the chromatogram supplied
the solution to 20.0 m L with ethanol (96 per cent) R. with buflomedUfor peak identification CRS and the
the peaks due to impurities A, B and C.
Relative retention W ith reference to buflomedil (retention
Specific absorbance at the absorption maximum 143 to 149. time = about 5 min): impurity B = about 0.6;
B. Infrared absorption spectrophotometry (2.2.24). impurity C = about 0.7; impurity A = about 1.5.
Comparison buflomedU hydrochloride CRS. System suitabUay. reference solution (c):
C. Thin-layer chromatography (2.2.27). — resolution: minimum 1.5 between the peaks due to
Test solution Dissolve 40 mg o f the substance to be examined im purity B and impurity C.
in methanol R and dilute to 2 m L with the same solvent. Limitsr.
Reference solution Dissolve 40 mg o f buflomedU — impurities A , B, C: for each impurity, not m ore than the
hydrochloride CRS in methanol R and dilute to 2 m L with the area o f the principal peak in the chromatogram obtained
same solvent. with reference solution (a) (0.25 per cent);
— unspecified impurities: for each impurity, not m ore rhan
Plate TLC silica gel F2$4 plate R.
Mobile phase triethylamine R, 2-propanol R, toluene R chromatogram obtained with reference solution (a)
(5:50:50 VIVIV). (0.10 per cent);
Application 10 jiL — total: not more than twice the area o f the principal peak in
Development Over 3/4 of the plate. the chromatogram obtained with reference solution (a)
Drying In air. (0.5 per cent);
Results T h e principal spot in the chromatogram obtained w ith (0.05 per cent).
M axim um 10 ppm .
solution.
TESTS
S o lu tio n S M aximum 0.5 per cent, determined on 1.000 g by drying in
Dissolve 2.5 g in carbon dioxide-free water R and dilute to an oven at 105 °C for 2 h.
A p p e a ra n c e o f so lu tio n M axim um 0.1 per cent, determined on 1.0 g.
A SSA Y
p H (2.2.3)
Dissolve 0.300 g in 15 m L of anhydrous acetic acid R and add
35 m L o f acetic anhydride R. T itrate with 0.1 M perchloric
R e la te d su b sta n c e s acid, determining the end-point potentiometrically (2.2.20).
1 m L o f 0.1 M perchloric add is equivalent to 34.39 mg of
Test solution Dissolve 0.10 g o f the substance to be examined C 17H 26C1N04.
phase. IM P U R IT IE S
Specified impurities A, B, C.
Reference solution (b) Dissolve 2 m g of buflomedU
impurity B CRS in the mobile phase, add 0.5 m L o f the test
solution and dilute to 100.0 m L with the mobile phase.
Reference solution (c) Dissolve the contents o f a vial of
buflomedUfor peak identification CRS (containing impurities A A. l-(2-hydroxy-4j6-dimethoxyphenyi)-4-(pyrrolidin-l-
and C ) in 1.0 m L o f reference solution (b). yl)butan-l-one,
Column:
— sizer. I = 0.25 m , 0 = 4.6 mm;
1-334 Bumetanide 2016
OCH3 O r" \
TESTS
J Z K ^ O Appearance of solution
T he solution is clear (2.2.1) and colourless (2.2.2,
H O -^ ^ ^ O C H s Method II).
Dissolve 0.1 g in a 6 g/L solution o f potassium hydroxide R
B. 1-(4-hydroxy-2 j 6-dimethoxyphenyl)-4- (pyrrolidin - 1-
and dilute to 20 m L with the same solution.
yl)butan-l-one,
Related substances
och3 o r^ \ Liquid chromatography (2.2.29).
HO ^ OH phase.
C. 1-(2j4-dihydroxy-6-methoxyphenyl)-4-(pyrrolidin - 1- 100.0 m L with the mobile phase. Dilute 1.0 m L o f this
yl)butan-l-one. solution to 10.0 m L with the mobile phase.
PhEur Reference solution (b) Dissolve 2 m g o f bumetanide
impurity A CRS and 2 m g o f bumetanide impurity B CRS in
phase. Dilute 1.0 m L o f this solution to 100.0 m L with the
mobile phase.
Bumetanide ★ ★ Column:
***** — size: I = 0.15 m , 0 = 4.6 mm,
— stationary phase: end-capped octylsUyl silica gelfor
chromatography R (3.5 fim).
C02H Mobile phase Mix 70 volumes o f methanol R, 25 volumes of
H,N
water for chromatography R and 5 volumes of a 27.2 g/L
solution o f potassium dihydrogen phosphate R previously
adjusted to p H 7.0 with a 280 g/L solution o f potassium
hydroxide R ,’ add tetrahexylammonium bromide R to this
mixture to obtain a concentration of 2.17 g/L.
Flow raze 1.0 mL/min.
C 1 7 H 2 0 N 2 O 5S 364.4 28395-03-1 Detection Spectrophotometer at 254 nm .
A c tio n a n d u se Injection 10 (iL.
Loop diuretic. Run time 5 times the retention time of bum etanide.
P re p a r a tio n s Relative retention W ith reference to bum etanide (retention
Bum etanide Injection time = about 6 min): impurity B = about 0.4;
Bum etanide Oral Solution impurity A = about 0.6; Impurity C = about 2.5;
Bumetanide Tablets
Bum etanide and Slow Potassium Tablets — resolution: minimum 2.0 between the peaks due to
PhEur.
impurity A and im purity B.
L im its:
D E F IN IT IO N — impurities A, B, C, D: for each impurity, n o t m ore than
3-(Butylamino)-4-phenoxy-5-sulfamoylbenzoic acid. the area o f the principal peak in the chrom atogram
C o n te n t obtained with reference solution (a) (0.1 p er cent),
99.0 per cent to 101.0 per cent (dried substance). — any other impurity: for each impurity, n o t m ore th an the
CHARACTERS area o f the principal peak in the chrom atogram obtained
with reference solution (a) (0.1 per cent),
A p p e a ra n c e
— total: n o t more than twice the area of the principal peak in
S o lu b ility (0.2 per cent),
Practically insoluble in water, soluble in acetone and in — disregard limit: 0.5 times the area of the principal peak in
ethanol (96 p er cent), slightly soluble in methylene chloride. the chromatogram obtained with reference solution (a)
It dissolves in dilute solutions of alkali hydroxides. (0.05 per cent).
mp M aximum 0.5 per cent, determined on 1.000 g by drying in
A bout 233 °C. an oven at 105 °C for 4 h.
ID E N T IF IC A T IO N Sulfated ash (2.4.14)
Infrared absorption spectrophotometry (2.2.24). M aximum 0.1 per cent, determined on 1.0 g.
Comparison bumetanide CRS. ASSAY
If the spectra obtained in the solid state show differences, Dissolve 0.300 g in 50 m L o f alcohol R. Add 0.1 m L o f
dissolve the substance to be examined and the reference phenol red solution R. T itrate with 0.1 M sodium hydroxide
substance separately in acetone R, evaporate to dryness and until a violet-red colour is obtained. Carry out a blank
record new.spectra using the residues. titration.
2016 Bupivacaine Hydrochloride 1-335
1 m l, of 0.1 M sodium hydroxide is equivalent to 36.44 mg of ★* ★

★ ★
C j 7H20N2O5S.
Bupivacaine Hydrochloride ★ ★
*****
STORAGE (Ph. Eur. monograph 0541)
IMPURITIES
Specified impurities: A, B, C, D. and enantiomer , HCI , H20
CH3
0 O
*'/
.COoH
1 V ^ V
C 18H 29CIN 20 ,H 20 342.9 73360-54-0
Action and use

Local anaesthetic.
Preparations
Bupivacaine Injection
A. 3-nitro~4-phenoxy-5-sulfamoylbenzoic a d d . Bupivacaine Heavy Injection
Bupivacaine and Adrenaline Injection/Bupivacaine and
O o Epinephrine Injection
*'/
s - ^ sss^ - c 0 2H Bupivacaine and Diamorphine Injection
h 2n
>v nh2
Bupivacaine and Fentanyl Injection
PhEur.
DEFINITION
(2RS)-l -Butyl-N-(2 j6-dimethylphenyI)piperidine-2-
carboxamide hydrochloride monohydrate.
B. 3-amino-4-phenoxy-5-sulfamoylbenzoic acid. Content
98.5 per cent to 101.0 p e r cent (dried substance).
CHARACTERS
Appearance
White or almost white, crystalline powder or colourless
crystals.
Solubility
Soluble in water, freely soluble in ethanol (96 per cent).
IDENTIFICATION
C. butyl 3-(butylamino)-4-phenoxy-5-sulfamoylbenzoate, First identification A , D, E
Second identification B, C, D, E
Comparison bupivacaine hydrochloride CRS.
and enantiomer
Reference solution Dissolve 25 mg of bupivacaine
D . 3- [ [(2ftS)-2-ethylhexyl] amino] -4-phenoxy-5- same solvent.
sulfamoylbenzoic acid. Plate TLC silica gel G plate R.
PhEur Mobile phase concentrated ammonia R, methanol R
(0.1:100 VIV).
Application 5 pL.
Drying In. air.
Detection Spray with dilute potassium iodobismuthate solution R.
reference solution.
C. Dissolve 0.1 g in 10 m L of water R, add 2 m L of dilute
sodium hydroxide solution R and shake with 2 quantities, each
of 15 mL, o f 1,1-dimethylethyl methyl ether R. Dry the
combined upper layers over anhydrous sodium sulfate R and
filter. Evaporate the filtrate, recrystallise the residue from
1-336 Bupivacaine Hydrochloride 2016
ethanol (90 per cent VIV) R and dry under reduced pressure. Injection 1 jxL.
T he crystals m elt (2.2.14) at 105 °C to 108 °C. Identification of impurities Use the chrom atogram obtained
D. It gives reaction (a) o f chlorides (2.3.1). w ith reference solution (a) to identify the peaks due to
E. Optical rotation (see Tests). impurities B and E.
TESTS Relative retention W ith reference to bupivacaine (retention

tim e = about 10 min): impurity B = about 0.7;
S o lu tio n S
impurity E = about 1.1; internal standard = about 1.4.
50 m L with the same solvent. System suitability: reference solution (a):
bupivacaine and impurity E.
Limits:
A c id ity o r alk a lin ity — impurity B: calculate the ratio (Rx) of the area o f the
T o 10 m L of solution S add 0.2 m L o f 0.01 M sodium principal peak to the area o f the peak due to the internal
hydroxide; the p H (2.2.3) is n o t less than 4.7. A dd 0.4 m L of standard from the chromatogram obtained with reference
0.01 M hydrochloric add; the p H is not greater than 4.7. solution (c); from the chromatogram obtained with the
O p tic a l ro ta tio n (2.2.7) test solution, calculate the ratio of the area of the peak
- 0 . 10° t o + 0 . 10°. due to impurity B to the area o f the peak due to the
Dissolve 1.0 g in methanol R and dilute to 20.0 m L with the internal standard: this ratio is not greater than R\
same solvent. (0.5 per cent);
— unspecified impurities: calculate the ratio (R2) o f the area
o f the principal peak to the area o f the peak due to the
internal standard from the chromatogram obtained with
Internal standard solution Dissolve 25 mg of methyl behenate R reference solution (d); from the chromatogram obtained
in methylene chloride R and dilute to 500 m L with the same with the test solution, calculate for each im purity the ratio
solvent. o f the area o f any peak, apart from the principal peak, the
Test solution Dissolve 50.0 m g of the substance to be peak due to impurity B and the peak due to the internal
examined in 2.5 m L of water R, add 2.5 m L o f dilute sodium standard, to the area of the peak due to the internal
hydroxide solution R and extract with 2 quantities, each of standard: this ratio is n o t greater than R2 (0.10 p er cent);
5 m L, o f the internal standard solution. Filter the lower — total: calculate the ratio (R3) of the area o f the principal
layer. peak to the area o f the peak due to the internal standard
Reference solution (a) Dissolve 10 mg o f the substance to be from the chromatogram obtained with reference
examined, 10 m g of bupivacaine impurity B CRS and 10 mg solution (b); from the chromatogram obtained with the
of bupivacaine impurity E CRS in 2.5 m L of water R, add test solution, calculate the ratio o f the sum of the areas of
2.5 m L of dilute sodium hydroxide solution R and extract with any peaks, apart from the principal peak and the peak
2 quantities, each of 5 mL, o f the internal standard solution. due to the internal standard, to the area o f the peak due
Filter the lower layer and dilute to 20 m L with the internal to the internal standard: this ratio is not greater than R3
standard solution. ( 1.0 per cent);
Reference solution (b) Dilute 1.0 m L o f the test solution to — disregard limit: ratio less than 0.05 times R3
100.0 m L with the internal standard solution. (0.05 per cent).
Reference solution (c) D ilute 5.0 m L of reference solution (b) Im p u rity F
to 10.0 m L with the internal standard solution. Liquid chromatography (2.2.29). Prepare the solutions
Reference solution (d) Dilute 1.0 m L of reference solution (b)
to 10.0 m L with the internal standard solution. Test solution Dissolve 50 mg o f the substance to be examined
Column: in mobile phase A and dilute to 10.0 m L with mobile
— material: fused silica; phase A.
— size: I = 30 m , 0 = 0.32 m m ; Reference solution (a) Dissolve 5.0 m g o f bupivacaine
— stationary phase: poly (dimethyl) (diphenyl)sUoxane R (film impurity F CRS in mobile phase A and dilute to 100.0 m L
thickness 0.25 Jim). with mobile phase A. Dilute 1.0 m L o f the solution to
Carrier gas helium for chromatography R. 100.0 m L with mobile phase A. Dilute 1.0 m L o f this
solution t a 10.0 m L with mobile phase A.
Reference solution (b) Dissolve 20 mg o f methyl benzoate R and
Split ratio 1:12.
25 m g of bupivacaine impurity F CRS in mobile phase A and
Temperature: dilute to 50.0 m L with mobile phase A. Dilute 3.0 m L o f the
solution to 50.0 m L with mobile phase A. Dilute 1.0 m L of
T in e Temperature this solution to 10.0 m L with mobile phase A.
(min) CC) Column:
0 ISO — size. I = 0.25 m , 0 = 4.6 mm;
Column 0 - 10 180 -> 230 — stationary phase: end-capped octadecylsHyl silica gel for
chromatography R (5 Jim).
10 -15 230
Mobile phase:
Injection port 250 — mobile phase A: dissolve 0.23 g o f sodium dihydrogen
Detector 250 phosphate monohydrate R and 3.626 g of disodium hydrogen
phosphate dihydrate R in water R and dilute to 1000 m L
with the same solvent; mix equal volumes o f this solution
Detection Flame ionisation. (pH 8.0) and acetomtrüe R;
2016 Buprenorphine 1-337
mobûe phase B: acetomtrüe R ; Control of impurities in substances for pharmaceutical usé): A , C,

D, E.
Urne Mobile phase A Mobile phase B
(mln) (per cent V/V) (per cent V/V)
0 -10 100 0
10- 15 100 ->80 0 -» 20
15 - 25 80 20
A. N- (2,6-dimethylphenyl)pyridine-2-carboxamide,
Flow rate 1.0 m L/m in.
Injection 50 |iL. and enantiomer
impurity F.
B. (2R5)-iV-(2,6-dimethylphenyl)piperidine-2-carboxamide,
Relative retention w ith reference to bupivacaine (retention
time = about 20 min): impurity F = about 0.3; methyl
benzoate = about 0.4.
System suitability:
impurity F and methyl benzoate in the chromatogram
obtained w ith reference solution (b);
— signal-to-noise ratio: minimum 40 for the principal peak in C . 1-(2,6-dimethylphenyl)-l,5,6,7-tetrahydro-2/i'-azepin-2-
the chrom atogram obtained with reference solution (a). one,
Limit.
— impurity F: n o t more than die area of the principal peak
in the chrom atogram obtained with reference solution (a) Cl and enantiomer
(10 ppm ).
H eavy m e ta ls ( 2.4.8)
Maximum 10 ppm .
D . (2ÆS)-2,6-dichloro-N-(2,6-dimethylphenyl)hexanamide,
Dissolve 2.0 g in a m ixture of 15 volumes of water R and
85 volumes o f methanol R and dilute to 20 m L with the same CH3
mixture o f solvents. 12 m L of the solution complies with
test B. Prepare the reference solution using lead standard ¿ X " r—
solution (1 p p m Pb) obtained by diluting lead standard
CH3 and enantiomer
solution (100 ppm Pb) R with a mixture o f 15 volumes of
water R and 85 volumes o f methanol R.
E. 6-(butylamino)-N-(2,6-dimethylphenyl)hexanamide,
4.5 per cent to 6.0 per cent, determined on 1.000 g by ch 3
S ulfa te d a sh (2.4.14)
M aximum 0.1 p er cent, determined on 1.0 g. CH3
A SSA Y
Dissolve 0.250 g in a mixture of 20 m L o f water R and F . 2,6-dimethylaniline.
25 m L o f ethanol (96per cent) R. Add 5.0 m L o f 0.01 M PhEur
hydrochloric add. C arry out a potentiometric titration (2.2.20),
using 0.1 M ethanolic sodium hydroxide. Read the volume
added betw een the 2 points of inflexion.
1 m L o f 0.1 M ethanolic sodium hydroxide is equivalent to ★ *
32.49 m g o f C 18H 29CIN 2O.
Buprenorphine ★ ★
STO RA G E
IM P U R IT IE S
Specified impurities B, F
the tests in the m onograph. They are limited by the general
by the general m onograph Substances for pharmaceutical use C 29H 41NO 4 52485-79-7
impurities for dem onstration o f compliance. See also 5.10. A c tio n a n d u se
Opioid receptor partial agonist; analgesic.
1-338 Buprenorphine 2016
PhEur__________________________________________________________ Injection 5 pL.

D E F IN IT IO N Identification of impurities Use the chrom atogram supplied
(25)-2- [ 17-(Cydopropylmethyl)-4,5a-epoxy-3-hydroxy-6- w ith buprenorphine for system suitability CRS and the
methoxy- 6 a, 14-ethano-14a-m orphinan-7 a-yl] -3,3- chromatogram obtained with reference solution (b) to
dimethylbutan- 2-ol. identify the peaks due to impurities A, B, F, G, H and J.
C o n te n t Relative retention W ith reference to buprenorphine (retention

98.5 per cent to 101.5 per cent (dried substance). time = about 8.5 min): impurity B = about 0.4;
impurity J = about 1.1; impurity F = about 1.27;
CHARACTERS impurity H = about 1.33; impurity A = about 1.40;
A p p e a ra n c e impurity G = about 1.8 .
W hite or almost white, crystalline powder. System suitability: reference solution (b):
S o lubility — resolution: minim um 1.5 between the peaks due to
Very slightly soluble in water, fredy soluble in acetone, buprenorphine and impurity J.
soluble in methanol, slightly soluble in cyclohexane. Limits:.
It dissolves in dilute solutions of adds. — correction factor, for the calculation o f content, multiply the
mp peak area of impurity G by 0.3;
A bout 217 °C. — impurity H: n o t more than 2.5 times the area of the
prindpal peak in the chromatogram obtained with
Infrared absorption spectrophotom etry (2.2.24).
— impurities A, B, F, J: for each impurity, n o t more than
Comparison buprenorphine CRS. twice the area o f the prindpal peak in the chromatogram
TESTS obtained with reference solution (a) (0.2 per cent);
S o lu tio n S — impurity G: n o t more than 1.5 times the area o f the
Dissolve 0.250 g in anhydrous ethanol R and dilute to prindpal peak in the chromatogram obtained with
25.0 m L with the same solvent. reference solution (a) (0.15 per cent);
area of the prindpal peak in the chromatogram obtained
Specific o p tical ro ta tio n (2.2.7) — total: n o t more than 7 times the area o f the principal peak
- 1 0 3 to —107 (dried substance), determined on solution S. in the chromatogram obtained with reference solution (a)
R e la te d su b stan ces (0.7 per cent);
Liquid chromatography (2.2.29). — disregard limit. 0.5 times the area of the prindpal peak in
Test solution Dissolve 50.0 m g of the substance to be the chromatogram obtained with reference solution (a)
examined in methanol R and dilute to 10.0 m L with the same (0.05 per cent).
solvent. L oss o n d ry in g (2.2.32)
Reference solution (a) Dilute 1.0 m L o f the test solution to Maximum 1.0 per cent, determined on 1.000 g by drying in
100.0 m L with methanol R. D ilute 1.0 m L o f this solution to an oven at 105 °C.
10.0 m L with methanol R. A SSAY
Reference solution (b) Dissolve 5 m g o f buprenorphine for system Dissolve 0.400 g in 40 m L of anhydrous acetic acid R. T itrate
suitability CRS (containing impurities A, B, F, G, H and J) in with 0.1 M perchloric acid, determining the end-point
1.0 m L of methanol R. potentiometrically (2.2.20).
Column: 1 m L o f 0.1 M perchloric acid is equivalent to 46.76 mg o f
— size. I = 0.05 m, 0 = 4.6 mm; C 29H 41N O 4.
STO R A G E
chromatography R (3.5 pm);
Mobile phase: IM P U R IT IE S
— mobile phase A: mix 10 volumes o f acetonitrUe R and Specified impurities A , B, F, G, H, J.
90 volumes of the following solution: dissolve 5.44 g of Other detectable impurities (die following substances would, if
potassium dihydrogen phosphate R in 900 m l. of water R, present at a suffident levd, be detected by one or other of
adjust to pH 4.5 with a 5 per cent V/V solution of the tests in the monograph. T hey are limited by the general
phosphoric acid R and dilute to 1000 m L with water R; acceptance criterion for other/unspecified impurities and/or
— mobile phase B: acetonitrUe R; by the general m onograph Substances for pharmaceutical use
Time Mobile phase A Mobile phase B impurities for demonstration o f compliance. See also 5.10.
(min)____________ (per cent V/V)________ (per cent V/V) Control of impurities in substances for pharmaceutical use): C, D,
0 -2 89 11 E, I.
2 - 12 89->64 11-»36
12- 15 64 -» 41 36 -» 59
15 - 20 41 -> 39 59 -» 61

2016 Buprenorphine Hydrochloride 1-339
A. R = C H 2-C H 2-C H = C H 2: (2S)-2-[17-(but-3-enyl)-4,5a-

epoxy-3-hydroxy-6-methoxy-6a,14-ethano-14a-m orphinan- I. 17-(cyclopropylmethyl)-4//,4 //,5 ",5 "-tetramethyl-
7 a-yl] -3,3-dimethylbutan-2-ol, 4",5 "dihydro-(7 ß /f)- 6a , 14-ethano-(5 ß/f)-
B. R = H : (2.S)-2-(4,5a-epoxy-3-hydroxy-6-methoxy-6a,14- difurano[2/,3 /,4 /,5 /:4,12,13,5;2//,3":6,7]-14a-m orphinan-3-
ethano-14a-m orphinan-7a-yi)-3,3-dimethyibutan-2-ol ol,
(norbuprenorphine),
H . R = C H 2-C H 2-C H 2-C H 3: (2S)-2-[17-butyl-4,5a-epoxy-
3-hydroxy-6-methoxy-6ctj 14-ethano-14a-m orphinan-7 a-
yl] 3j3-dimethylbutan-2-olj
J. (25) -2-[ 17-(cydopropylmethyl)-4 ,5a-epoxy-3-hydroxy-6-

methoxy- 6a , 14-etheno-14a-morphinan-7 a-yl] -3,3-
dimethyIbutan-2-ol.
_______________________________________________ ____ PhEur
C . 4,5a-epoxy-7a - [( 1S)- 1-hydroxy-13232 -trimethylpropyl] -

336-dimethoxy-6a314-ethano-14a-m orphinan-17-carbonitrile 3
Buprenorphine Hydrochloride *****
** +*
(Ph. Eier, monograph 1181) *
D . R I = R2 = C H 3: ( 2.S)-2-[l 7-(cydopropylmethyl)-4J5a-
epoxy-3,6-dimethoxy-6a, 14-ethano-14a-m orphinan-7 a-yl] -
C 29H 42CINO 4 504.1 53152-21-9
3j3-dimethylbutan-2-ol (3-O-methylbuprenorphine),
E. R I = R2 = H : (25)-2-[17-(cyclopropylmethyl)-4,5a- A ctio n a n d u se
epoxy-3j6-dihydroxy-6a, 14-ethano-14oc-morphinan-7 a-yl] - Opioid receptor partial agonist; analgesic.
3,3-dimethylbutan-2-ol (6-O-desmethylbuprenorphine),
Buprenorphine Injection
Buprenorphine Transdermal Patches
Buprenorphine Sublingual Tablets
PhEur__________________________________________________________
D E F IN IT IO N
(2S)-2-[17-(Cyclopropylmethyl)-4,5a-epoxy-3-hydroxy-6-
methoxy- 6a , 14-ethano-14a-m orphinan-7 a-yl] -3,3-
F. 17-(cyclopropyimethyl)-4j5a-epoxy-6-methoxy-7a-[l-(l j 1
dimethylbutan- 2-ol hydrochloride.
dimethylethyi)ethenyl] - 6a, 14-ethano-14a-m orphinan-3-ol,
C o n te n t
CHARACTERS
A p p e a ra n c e
S o lu b ility
Sparingly soluble in water, freely soluble in methanol, soluble
in ethanol (96 per cent), practically insoluble in cyclohexane.
G. R-R; 17,17 '-diCcyclopropyimethyO^jSa^' jSa'-diepoxy-
7 a ,7 a /-di[(lS)-l-hydroxy-l,2,2-trim ethylpropyl]-6,6/-
dimethoxy-2,2 '-bi( 6a , 14-ethano-14a-m orphinan)-3,3 '-diol
(2,2 '-bibuprenorphine), Comparison buprenorpkine hydrochloride CRS.
B. 3 m L o f solution S (see Tests) gives reaction (a) of
chlorides (2.3.1).
1-340 Buprenorphine Hydrochloride 2016
TESTS Limits:
Solution S — correction factor, for the calculation of content, multiply the
Dissolve 0.250 g in 5.0 m L of methanol R and, while stirring, peak area of impurity G by 0.3;
dilute to 25.0 m L with carbon dioxide-free water R. — impurity H: not more than 2.5 times the area of the
Appearance o f solution principal peak in the chromatogram obtained with
Solution S is clear {2.2.1) and colourless (2.2.2, Method II). reference solution (a) (0.25 per cent);
— impurities A, B, F, J: for each impurity, not more than
A cid ity o r alk a lin ity twice the area of the principal peak in the chromatogram
T o 10.0 m L of solution S add 0.05 m L of methyl red obtained with reference solution (a) (0.2 per cent);
solution R. N o t m ore than 0.2 m L o f 0.02 M sodium hydroxide — impurity G: not m ore than 1.5 times the area o f the
or 0.02 M hydrochloric acid is required to change the colour of principal peak in the chromatogram obtained with
the indicator. reference solution (a) (0.15 per cent);
Specific optical rotation (2.2.7) — unspecified impurities: for each impurity, n o t m ore than the
—92 to - 9 8 (dried substance). area of the principal peak in the chromatogram obtained
Dissolve 0.200 g in methanol R and dilute to 20.0 m L with with reference solution (a) (0.10 per cent);
the same solvent. — total: not more than 7 times the area o f the principal peak
(0.7 per cent);
Test solution Dissolve 50.0 m g of the substance to be the chromatogram obtained with reference solution (a)
examined in methanol R and dilute to 10.0 m L with the same (0.05 per cent).
solvent.
L o ss on d ry in g (2.2.32)
M aximum 1.0 per cent, determ ined on 1.000 g by heating in
100.0 m L with methanol R. Dilute 1.0 m L of this solution to
an oven at 115-120 °C.
Reference solution (b) Dissolve 5 m g of buprenorphine for system ASSA Y
suitability CRS (containing impurities A, B, F, G, H and J) in Dissolve 0.400 g in a mixture o f 5 m L of 0.01 M hydrochloric
1.0 m l, of methanol R. acid and 50 m L o f ethanol (96 per cent) R. Carry out a
potentiometric titration (2.2.20), using 0.1 M sodium
Column:
— size. I = 0.05 m , 0 = 4.6 mm; hydroxide. Read the volume added between the 2 points of
inflexion. Carry out a blank titration.
chromatography R (3.5 pm); 1 m L o f 0.1 M sodium hydroxide is equivalent to 50.41 mg of
— temperature. 30 °C. C29H42CINO4.
Mobile phase: STO R A G E
— mobile phase A: mix 10 volumes of acetonitnle R and Protected from light.
90 volumes of the following solution: dissolve 5.44 g of
IM P U R IT IE S
potassium dihydrogen phosphate R in 900 m L o f water R,
adjust to p H 4.5 with a 5 per cent V/V solution of Specified impurities A , B, F, G, H, J.
phosphoric acid R and dilute to 1000 m L with water R', Other detectable impurities (the following substances would, if
— mobile phase B: acetonitnle R; present at a sufficient level, be detected by one or other of
the tests in the m onograph. They are limited by the general
0- 2 89 11
2 - 12 89 -»64 11 -» 36 Control of impurities in substances for pharmaceutical use): C, D,
12 - 15 64 -»41 36-» 59 E, I.
15 - 20 41 -» 39 59-» 61
,R
N

Injection 5 (iL.
w ith buprenorphine for system suitability CRS and the
chrom atogram obtained with reference solution (b) to A. R = C H 2-C H 2-C H = C H 2: (2S)-2-[17-(but-3-enyl)-4,5oc-
identify the peaks due to impurities A, B, F, G, H and J. epoxy-3-hydroxy-6-methoxy-6a, 14-ethano-14a-m orphinan-
7 a-yl]-3,3-dimethylbutan-2-ol,
Relative retention W ith reference to buprenorphine (retention
B. R = H : (2S)-2-(4,5a-epoxy-3-hydroxy-6-methoxy-6a,14-
tim e = about 8.5 min): impurity B = about 0.4;
impurity J = about 1.1; impurity F = about 1.27; ethano-14a-m orphinan-7a-yl)-3,3-dimethylbutan-2-ol
impurity H = about 1.33; impurity A = about 1.40; (norbuprenorphine),
impurity G = about 1.8. H . R = C H 2-C H 2-C H 2-C H 3: (2S)-2-[17-butyl-4,5oc-epoxy-
System suitability: reference solution (b): 3-hydroxy-6-methoxy-6a, 14-ethano-14a-m orphinan-7 a-
— resolution: m inim um 1.5 between the peaks due to yl] 3,3-dimethylbutan-2-ol,
buprenorphine and impurity J.
2016 Buserelin 1-341
Buserelin *****
★ ★
(Ph Ever monograph 1077) *
C. 4,5a-epoxy-7a-[(l.S)-l-hydroxy-l,2,2-triinethylpropyl]-
3, 6-dimethoxy-6a, 14-ethano-14a-m orphinan-17-carbonitrile, H j|-His-Trp-Ser-Tyr-D-Ser-Leu-Arg-Pro-N CH3
O
Q oH m N wO u 1239 57982-77-1
A ctio n a n d u se
Gonadotrophin releasing hormone (gonadorelin) analogue;
treatm ent of prostate cancer.
D . R I = R2 = C H 3: (25)-2-[ 17-(cyclopropylmethyi)-435a- PhEtr__________________________________________________________

epoxy-3,6-dimethoxy-6a,14-ethano-14a-m orphinan-7 a-yl] - D E F IN IT IO N
3j3-dimethylbutan-2-ol (3-0-methylbuprenorphine)j
5-Oxo-L-prolyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-0-(l, 1-
E: R l = R2 = H: (2S)-2-[17-(cydopropylmethyl)-4,5a- dimethylethyl)-i>seryl-L-leucyl-L-argmyl-N-ethyl-L-
epoxy-3, 6-dihydroxy- 6a , 14-ethano-14a-m orphinan-7 a-yl] - prolinamide.
3,3-dimethylbutan-2-ol (6-O-desmethylbuprenorphine),
Synthetic nonapeptide analogue of hum an gonadotrophin-
releasing horm one G nR H with agonistic activity to
gonadorelin. It is obtained by chemical synthesis and is
available as an acetate.
C o n te n t
95.0 per cent to 102.0 p er cent (anhydrous, acetic add-free
substance).
F. 17-(cyclopropylmethyl)-4,5a-epoxy-6-methoxy-7a- [1 -(1,1- CHARACTERS
dimethylethyl) ethenyl] - 6a, 14-ethano-14a-m orphinan-3-ol, A p p e a ra n c e
W hite or slightly yellowish powder, hygroscopic.
S o lu b ility
Sparingly soluble in water and in dilute adds.
Carry out either tests A and B o r tests A and C.
A. Examine the chromatograms obtained in the assay.
G . R-R: 17,17,-di(cyclopropylmethyl)-4,5a;4/,5 a /-diepoxy- Results T h e principal peak in the chromatogram obtained
7a,7 a/-d i[(l5 )-l -hydroxy-1,2,2-trimethylpropyl] - 6, 6 '- with the test solution is similar in retention time and size to
dimethoxy-2,2 '-bi (6a , 14-ethano-14a-m o rphinan) -3,3 '-diol the prindpal peak in the chromatogram obtained with
(2,2 '-bibuprenorphine), reference solution (b).
B. N uclear magnetic resonance spectrometry (2.2.64).
Preparation 4 m g/mL solution in a mixture of 20 volumes of
deuterated acetic acid R and 80 volumes of deuterium oxide R.
Comparison 4 mg'mL solution of buserelin CRS in a mixture
o f 20 volumes o f deuterated acetic acid R and 80 volumes of
deuterium oxide R (dissolve the contents o f a vial of
buserelin CRS in this solvent mixture to obtain the desired
concentration).
I. 17-(cyclopropylmethyl)-4 ",4 ",5 ",5 "-tetramethyl- Operating conditions:
4 ",5"dihydro-(7 ß /f)- 6a , 14-ethano-(5 ß /f)- — field strength: minimum 300 M Hz;
difurano[2/,3 /,4 /,5 /:4,12,13,5;2",3":6,7]-14a-m orphinan-3- — temperature: 27 °C.
ol, Results Examine the JH N M R spectrum from 0 to 9 ppm.
T h e *H N M R spectrum obtained is qualitatively similar to
the JH N M R spectrum obtained with buserelin CRS.
C. Amino a d d analysis (2.2.56). M ethod 1 for hydrolysis and
m ethod 1 for analysis are suitable.
Express the content of each amino a d d in moles. Calculate
the rdative proportions o f the amino ad d s, taking 1/6 of the
siim of the num ber o f moles of glutamic a d d , histidine,
J. (2S)-2- [ 17-(cydopropylmethyl)-4,5a-epoxy-3-hydroxy-6- tyrosine, leucine, arginine and proline as equal to 1.
methoxy- 6a , 14-etheno-14a-m orphinan-7 a-yl] -3,3- T h e values fall within the following limits: serine 1.4 to 2.0;
dim ethylbutan- 2-ol. proline 0.8 to 1.2; glutamic a d d 0.9 to 1.1; leucine
_____________________________________________ !____________ PhEtx 0.9 to 1.1; tyrosine 0.9 to 1.1; histidine 0.9 to 1.1; arginine
1-342 Buserelin 2016
0.9 to 1.1. N o t more than traces of other amino acids are Test solution Dissolve 20.0 mg of the substance to be
present. examined in a mixture o f 5 volumes of mobile phase B and
95 volumes of mobile phase A and dilute to 10.0 m L with
TESTS
the same mixture of solvents.
A 10 g/L solution is clear (2.2.1) and not more intensely W a te r (2.5.12)
coloured than reference solution Y 7 (2.2.2, Method IT). M aximum 4.0 per cent, determined on 80.0 mg.
S pecific o p tic a l ro ta tio n (2.2.7) B a c te ria l e n d o to x in s (2.6.14)
- 4 9 to - 5 8 (anhydrous, acetic acid-free substance), Less than 55.5 IU/mg, if intended for use in the manufacture
determined on a 10 g/L solution. of parenteral preparations without a further appropriate
49 to 56, m easured at the absorption maximum at 278 nm A SSAY
(anhydrous, acetic acid-free substance). Liquid chromatography (2.2.29) as described in the test for
Dissolve 10.0 mg in 100.0 m L of 0.01 M hydrochloric add. related substances with the following modification.
R e la te d su b s ta n c e s Injection T est solution and reference solution (b).
Liquid chromatography (2.2.29). Calculate the content o f buserelin ( C ^ H s ^ ^ O ^ ) using the
Test solution Dissolve 5.0 mg of the substance to be examined areas o f the peaks in the chromatograms obtained and the
in 5.0 m L of the mobile phase. declared content of C 60H 86N 16O i 3 in buserdin CRS.
Reference solution (a) Dissolve the contents o f a vial of D-His- STO R A G E
buserdin CRS in the mobile phase. Dilute an In an airtight container, protected from light, at a
appropriate volume o f this solution in the mobile phase to tem perature o f 2 °C to 8 °C. If the substance is sterile, store
obtain a final concentration o f 1 mg/mL. Add 1.0 m L o f the in an airtight, sterile, tam per-proof container.
test solution to 1.0 m L o f this solution.
L A B E L L IN G
Reference solution (b) Dissolve the contents of a vial of T h e label states:
buserdin CRS in the mobile phase. Dilute an — the mass of peptide in the container;
appropriate volume o f this solution in the mobile phase to — where applicable, that the substance is suitable for use in
obtain a final concentration o f 1.0 mg/mL. the manufacture o f parenteral preparations.
Reference solution (c) Dilute 1.0 m L of the test solution to
IM P U R IT IE S
Spedfied impurities: A, B, C, D , E.
Column:
— size: I = 0.25 m , 0 = 4 mm ;
— stationary phase: octadecylsilyl silica gd for chromatography R
(5 |un).
H
Mobile phase Mix 200 m L of acetomtrUe R and 700 m L o f an
11.2 g/L solution o f phosphoric add R and adjust to p H 2.5 H jp D-His - Trp - Ser - Tyr - D-Ser - Leu - Arg - Pro - N CH3
with triethylamine R. O
A. [2-D-histidine]buserelin,
Injection 10 pL of the test solution, reference solution (a) and
reference solution (c).
Relative retention W ith reference to buserelin (retention
time = about 36 m in): impurity B = about 0.76;
H |j—His - Trp - D-Ser - Tyr - D-Ser - Leu - Arg - Pro - N CH 3
impurity D = about 0.94; impurity E = about 0.94. O
— resolution: m inim um 1.5 between the peaks due to B. [4-D-serine]buserelin,
impurity A and buserelin.
Limits: H3C ^ H3
— sum of impurities D and E: not more than 3 times the area
o f the principal peak in the chromatogram obtained with H-Trp-Ser-Tyr-D-Ser-Leu-Arg-Pro-N CH3
H
reference solution (c) (3 per cent);
— arty other impurity: for each impurity, not more than
3 times the area o f the principal peak in the C. buserelin-(3-9)-peptide,
chrom atogram obtained with reference solution (c)
(3 per cent);
— total: not more than 5 times the area of the principal peak
in the chrom atogram obtained with reference solution (c)
(5 per cent); H His - Trp - Ser-D -Tyr-D-Ser-Leu-A rg-Pro-N CH3
— disregard limic. 0.1 times the area o f the principal peak in O
the chrom atogram obtained with reference solution (c)
(0.1 per cent).
D . [5-D-tyrosine]buserelin,
A cetic a c id (2.5.34)
2016 Buspirone Hydrochloride 1-343
Column:
ch 3 — size. I = 0.15 m , 0 = 4.6 mm,
Ç t H3C
p"CH3 — stationary phase: octadecylsHyl silica gelfor chromatography R
oI
H V -H is- Trp-Ser-Tyr-D-Ser-Leu-Arg-Pro-N CH3 (5 pm),
H
O — temperature: 40 °C.
Mobile phase:
E. [l-(5-oxo-D-proline)]buserelin. — mobile phase A: mix 950 volumes of a solution containing
PhEur 6.8 g/L of potassium dihydrogen phosphate R and 0.93 g/L
of sodium hexanesulfonate monohydrate R, previously
adjusted to p H 3.4 with phosphoric acid R and 50 volumes
of acetorntrik Rl;
>** ★ — mobile phase B: mix 250 volumes of a solution containing
★
Buspirone Hydrochloride ★ * 3.4 g/L of potassium dihydrogen phosphate R and 3.52 g/L
***** of sodium hexanesulfonate monohydrate R, previously
(Ph Eur monograph 1711) adjusted to p H 2.2 with phosphoric acid R and
750 volumes of acetorntrik R l,

HCI (min) (per cent V/V) (per cent V/V)
0 -6 90 10
6 -3 4 9 0* 42 10->58
34 - 45 42 58
45 - 55 42 -» 0 58 -» 100
C21H32CIN5O2 422.0 33386-08-2
55 - 56 0 -» 100 100 -> 0
Action and use
5 6 -6 0 100 0
Non-benzodiazepine hypnotic; treatment of anxiety.
60 - 61 100->90 0 -> 10
PhEtr__________________________________________________________
D E F IN IT IO N
Flow rate 1 mL/min.
8- [4- [4- (Pyrimidin-2-yl) piperazin-1-yl] butyl] -8 -
azaspiro[4.5]decane-7,9-dione hydrochloride. Detection Variable wavelength spectrophotometer capable of
operating at 240 nm and at 210 run.
Content
Injection 20 |oL.
CHARACTERS with buspirone for system suitability CRS and the
Appearance chromatogram obtained with reference solution (b) to
White or alm ost white, crystalline powder. identify the peaks due to impurities E, G, J, L and N .
Solubility Relative retention at 240 nm With reference to buspirone
Freely soluble in water and in methanol, practically insoluble (retention time = about 25 min): impurity A = about 0.2;
in acetone. impurity B = about 0.3; impurity C = about 0.6;
It shows polymorphism (5.9). impurity D = about 0.7; impurity E = about 0.8;
IDENTIFICATION impurity F = about 0.9; impurity G = about 1.05;
impurity H = about 1.1; impurity I = about 1.2;
im purity J = about 1.5.
Comparison buspirone hydrochloride CRS.
Relative retention at 210 nm With reference to buspirone
If the spectra obtained in the solid state show differences, (retention time = about 25 min): impurity K = about 0.6;
dissolve the substance to be examined and the reference impurity L = about 1.7; impurity M = about 1.8;
substance separately in methanol R, evaporate to dryness on a impurity N = about 1.9.
water-bath and record new spectra using the residues.
B. It gives reaction (a) of chlorides (2.3.1). — peak-to-valley ratio at 240 nm: m in im u m 5.0, where
TESTS Hp = height above the baseline of the peak due to
Related substances impurity G and Hv = height above the baseline of the
Liquid chromatography (2.2.29). lowest point o f the curve separating this peak from the
peak due to buspirone;
examined in mobile phase A and dilute to 25.0 m L with — resolution at 210 nm: minimum 4.0 between the peaks due
mobile phase A. to impurity L and impurity N ;
— the chromatograms obtained are similar to the
Reference solution (a) Dilute 1.0 mL o f the test solution to
chromatograms supplied with buspirone for system
100.0 m L with mobile phase A. Dilute 1.0 m L of this
suitability CRS.
Limits Spectrophotometer at 240 nm :
Reference solution (b) Dissolve the contents o f a vial of — correction factor, for the calculation of contentj multiply the
buspirone for system suitability CRS (containing impurities E, peak area of impurity J by 2,
G, J 3L and N ) in 2.0 ml of mobile phase A and sonicate for
10 min.
1-344 Buspirone Hydrochloride 2016
— impurity E: not more than 3 times the area o f the

— impurity J: not m ore than twice the area o f the principal N
solution (a) (0.2 p er cent),
— any other impurity, for each impurity, not more than the B. 8-(pyrimidin-2-yl)-8-aza-5-azoniaspiro [4.5] decane,
with reference solution (a) ( 0.1 p er cent),
— total: n o t more than 4 times the area o f the principal peak
(0.4 per cent),
the chrom atogram obtained with reference
solution (a) (0.05 per cent). C. X = [ C H J * 2,2'-[butane-l,4-diylbis(piperazine-l,4-
diyl)]dipyrimidine,
Limits Spectrophotom eter at 210 nm :
— impurity K: not more than the area o f the principal peak D . X = [ O y ^ O - f C H y ^ * 2 ,2 [oxybis[butane-1,4-
in the chrom atogram obtained with reference solution (a) diyl (piperazine-1,4-diyl)]] dipyrimidine,
( 0.1 per cent),
— arty other impurity ehmng with a relative retention greater
than 1.6: for each impurity, not m ore than the area o f the
ho 2c
— total: n o t m ore than twice the area o f the principal peak in
(0.2 per cent),
the chromatogram obtained with reference solution (a) E. [ l-[2-oxo-2-[[4-[4-(pyrimidin-2-yi)piperazin-l -
(0.05 per cent). yl] butyl] amino] ethyl] cyclopentyl] acetic acid,
M axim um 0.5 per cent, determined on 1.000 g by drying at
105 °C.
S u lfa te d a s h (’2.4.14)
A SSAY
Dissolve 0.150 g in 10 m L o f glacial acetic add R and add
50 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 21.10 mg F. X = N H : 4-[4-(pyrimidin-2-yl)piperazin-l-yl]butyl [l-[ 2 -
o f C 2JH 32CIN 5O 2. oxo-2- [ [4- [4-(pyrimidin-2-yl)piperazin-1-
STORAGE yl] butyl] amino] ethyl] cyclopentyl] acetate,
Protected from light. H . X = O: bis[4-[4-(pyrimidin-2-yl)piperazin-l-yl]butyl]
(cyclopentane- 1,1 -diyl) diacetate,
IM P U R IT IE S
a
Spedfied impurities E, J, K
present at a sufficient level, be detected by one or other o f N N
the tests in the monograph. T hey are limited by die general
G. 2, 2 '-(piperazine- 1,4-diyl) dipyrimidine,
C, D, F, G, H, I, L, M , N.
■v
Y l
n ' n^ x ci
A. 2-(piperazm-l-yl)pyrimidine,
I. 8-[4-[4-(5-chloropyrimidin-2-yl)piperazin-l-yl]butyl]-8-
azaspiro [4.5] decane-7,9-dione,
2016 Busulfan 1-345
First identification A.
Comparison busulfan CRS.
J. 4-(7,9-dioxo-8-azaspiro[4.5]dec-8-yl)butyl [l-[2-oxo-2-[[4- in 2 m L o f acetone R.
[4-(pyrimidin- 2-yl)piperazin - 1- Reference solution Dissolve 20 mg o f busulfan CRS in 2 m L of
yl] butyl] amino] ethyl] cyclopentyl] acetate. acetone R.
Mobile phase acetone R, toluene R (50:50 VIV).
Application 5 (¿L.
Y ' Development Over a p ath of 15 cm.
Drying In a current of warm air.
K. R = H: 8-a2aspiro [4.5] decane-7,9-dione, Detection Spray with anisaldehyde solution R and heat at
L. R = [CHJ4-CI: 8-(4-chlorobutyl)-8-azaspiro [4.5] decane- 120 °C.
7, 9-dione, Results T h e principal spot in the chromatogram obtained with
M . R — [C H ^ -B r: 8-(4-bromobutyl)-8-azaspiro[4.5]decane- the test solution is similar in position, colour and size to the
7,9-dione, principal spot in the chromatogram obtained with the
reference solution.
C. T o 0.1 g add 5 m L o f 1 M sodium hydroxide. H eat until a
clear solution is obtained. Allow to cool. T o 2 m L of the
solution add 0.1 m L o f potassium permanganate solution R.
T h e colour changes from purple through violet to blue and
finally to green. Filter and add 1 m L of ammoniacal silver
nitrate solution R. A precipitate is formed.
D . T o 0.1 g add 0.1 g o f potassium nitrate R and 0.25 g of
N . 8 ,8 '-(butane- 1,4-diyl)bis(8-azaspiro [4.5] decane-7,9-
sodium hydroxide R, mix and heat to fusion. Allow to cool and
dione).
dissolve the residue in 5 m L of water R. Adjust to pH 1-2
P h E ir using dilute hydrochloric acid R. T h e solution gives reaction (a)
of sulfates (2.3.1).
TESTS
Busulfan ★ ★
★ ★ T h e solution is clear (2.2.1) and n o t more intensely coloured
*★★★* than reference solution B 7 (2.2.2, Method II).
(Ph. Eitr. monograph 0542)
Dissolve 0.25 g in 20 m L o f acetonitrile R, dilute to 25 m L
0\\ //0 with water R and examine immediately.
, 0 ^ ,c h 3 A cid ity
h3c 0 s
//* Dissolve 0.20 g with heating in 50 m L of anhydrous
o o
ethanol R. Add 0.1 m L of methyl red solution R. N o t more
Q H Ô ^ 246.3 55-98-1 than 0.05 m L o f 0.1 M sodium hydroxide is required to
change the colour of the indicator.
A c tio n a n d u se L oss o n d ry in g (2.2.32)
Cytotoxic alkylating agent. M aximum 2.0 per cent, determined on 1.000 g by drying
P re p a r a tio n in vacuo at 60 °C.
Busulfan Tablets S u lfate d a sh (2.4.14)
P h E ir .
ASSAY
D E F IN IT IO N
T o 0.250 g add 50 m L o f water R. Shake. Boil under a reflux
B utane- 1,4-diyl di(methanesulfonate).
condenser for 30 min and, if necessary, make up to the
C o n te n t initial volume with water R. Allow to cool. Using 0.3 m L o f
99.0 per cent to 100.5 per cent (dried substance). phenolphthalein solution R as indicator, titrate with 0.1 M
CHARACTERS sodium hydroxide until a pink colour is obtained.
A p p e a ra n c e 1 m L of 0.1 M sodium hydroxide is equivalent to 12.32 mg of
W hite or almost white, crystalline powder. C sH uO ^.
S o lu b ility STORA GE
Very slightly soluble in water, freely soluble in acetone and in In an airtight container, protected from light.
acetonitrile, very slightly soluble in ethanol (96 per cent).
__________________________________________________________________________________________ P h E ir
mp
A bout 116 °C.
1-346 Butyl Hydroxybenzoate 2016
TESTS
Butyl Hydroxybenzoate ***** Solution S
Butylparaben * ** Dissolve 1.0 g in ethanol (96 per cent) R and dilute to
(Butyl Parahydroxybenzoate, Ph Eur monograph 0881) 10 m L with the same solvent.
Acidity
T o 2 m L o f solution S add 3 m L o f ethanol (96 per cent) R,
5 m l. of carbon dioxide-free water R and 0.1 m l. of bromocresol
C n H 140 3 194.2 94-26-8 green solution R. N o t more than 0.1 m L o f 0.1 M sodium
hydroxide is required to change the colour o f the indicator to
Action and use blue.
Excipient. Related substances
Ph Eur'___________________________________________________________
D E F IN IT IO N examined in 2.5 m L of methanol R and dilute to 50.0 m l.
Butyl 4-hydroxybenzoate. with the mobile phase. D ilute 10.0 m L o f the solution to
Content 100.0 m L with the mobile phase.
98.0 per cent to 102.0 per cent. Reference solution (a) Dissolve 5 m g o f 4-hydroxybenzoic acid R
CHARACTERS (impurity A), 5 m g of propyl parahydroxybenzoate R
Appearance (impurity D ) and 5 mg o f the substance to be examined in
W hite or alm ost white, crystalline powder or colourless the mobile phase and dilute to 100.0 m L with the mobile
crystals. phase. Dilute 1.0 m L o f the solution to 10.0 m L with the
mobile phase.
Solubility
Reference solution (b) Dissolve 50.0 mg o f butyl
Very slightly soluble in water, freely soluble in ethanol
parahydroxybenzoate CRS in 2.5 m L of methanol R and dilute
(96 per cent) and in m ethanol.
to 50.0 m L with the mobile phase. D ilute 10.0 m L o f the
IDENTIFICATION solution to 100.0 m L with the mobile phase.
First identification A , B Reference solution (c) Dilute 1.0 m L o f the test solution to
Second identification A , C 20.0 m L with the mobile phase. D ilute 1.0 m L of this
A. Melting point (2.2.14): 68 °C to 71 °C. solution to 10.0 m L with the mobile phase.
B. Infrared absorption spectrophotom etry (2.2.24). Reference solution (d) Dissolve 5 m g of butyl
Comparison butyl parahydroxybenzoate CRS. parahydroxybenzoate impurùy E CRS (iso-butyl
parahydroxybenzoate) in the mobile phase and dilute to
Reference solution (e) Dilute 0.5 m L o f reference solution (d)
examined in acetone R and dilute to 10 m L with the same
to 50.0 m L with reference solution (b).
solvent.
Column:
Test solution (b) D ilute 1 m L of test solution (a) to 10 m L — size. I = 0.15 m , 0 = 4.6 m m ;
with acetone R.
Reference solution (a) Dissolve 10 mg o f butyl chromatography R (5 pm);
parahydroxybenzoate CRS in acetone R and dilute to 10 m L — temperature: 35 °C.
with the sam e solvent.
Mobile phase 6.8 g/L solution o f potassium dihydrogen
Reference solution (b) Dissolve 10 mg o f propyl phosphate R, methanol R (50:50 V/V).
parahydroxybenzoate R in 1 m L of test solution (a) and dilute Flow rate 1.3 mL/min.
to 10 m L w ith acetone R.
Plate TLC octadecylsilyl silica gel F2 5 4 plate R.
Mobile phase glacial acetic add R, water R, methanol R
solutions (a), (c) and (e).
(1:30:70 VIVIV).
Run time 1.5 times the retention time o f butyl
Application 2 pL of test solution (b) and reference
parahydroxybenzoate.
solutions (a) and (b).
Drying In air. impurities A and D ; use the chrom atogram obtained with
Detection Examine in ultraviolet light at 254 nm. reference solution (e) to identify the peak due to im purity E.
System suitability: reference solution (b): Relative retention W ith reference to butyl parahydroxybenzoate
— the chrom atogram shows 2 clearly separated principal (retention time = about 22 min): impurity A = about 0.1;
spots. impurity D = about 0.5; im purity E = about 0.9.
Results T he principal spot in the chromatogram obtained with System suitability:
test solution (b) is similar in position and size to the principal — resolution:
spot in the chrom atogram obtained with reference
solution (a).
2016 Butylated Hydroxyanisole 1-347
minimum 5.0 betw een the peaks due to impurity D and

butyl parahydroxybenzoate in the chromatogram obtained
with reference solution (a);
HO
minimum 1 .5 betw een the peaks due to impurity E and butyl
parahydroxybenzoate in the chromatogram obtained with
D . propyl 4-hydroxybenzoate (propyl parahydroxybenzoate),
reference solution (e).
Limits: o
peak area o f im purity A by 1.4;
— impurity A: n o t m ore than the area of the principal peak
in the chromatogram obtained with reference solution (c)
(0.5 per cent); E. 2-methylpropyl 4-hydroxybenzoate (iso-butyl
— unspecified impurities: for each impurity, n o t more than the parahydroxybenzoate).
Ph Eur
( 1.0 per cent);
Butylated Hydroxyanisole ★ ★
— disregard limit: 0.2 times the area o f the principal peak in ★ ★
the chromatogram obtained with reference solution (c) *****
(Butylhydroxanisole, Ph Eur monograph 0880)
(0.1 per cent).
S u lfated a sh (2.4.14)
ASSAY H3CO
h3c ch 3
liq u id chromatography (2.2.29) as described in the test for
related substances w ith the following modification.
Q 1H 1602 180.3 25013-16-5
Injection T est solution and reference solution (b).
Calculate the percentage content o f C 11H 14O 3 from the A ctio n a n d u se
declared content o f butyl parahydroxybenzoate CRS. Antioxidant.
IM P U R IT IE S
PhEur.
D E F IN IT IO N
present at a sufficient level, be detected by one or other of Butylhydroxyanisole is 2-(l,l-dim ethylethyl)-4-
the tests in the m onograph. They are limited by the general methoxyphenol containing not m ore than 10 p er cent of
acceptance criterion for other/unspecified impurities and/or 3-( 1,1 -dimethylethyl)-4-methoxyphenol.
by the general m onograph Substances for pharmaceutical use CHARACTERS
(2034). It is therefore not necessary to identify these A white, yellowish or slighdy pinkish, crystalline powder,
impurities for dem onstration of compliance. See also 5.10. practically insoluble in water, very soluble in methylene
Control of impurities in substancesfor pharmaceutical use): B, C, chloride, freely soluble in alcohol and in fatty oils. It dissolves
D ,E. in dilute solutions of alkali hydroxides.
CO2H
A. Examine the chromatograms obtained in the test for
rd a te d substances. The prindpal spot in the chromatogram
HO
obtained with test solution (b) is similar in position, colour
and size to the prindpal spot in the chromatogram obtained
A. 4-hydroxybenzoic ad d , with reference solution (a).
B. T o 0.5 m L o f solution S (see Tests) add 10 m L of
aminopyrazolone solution R and 1 m L o f potassium ferricyanide
solution R. Mix and add 10 m L o f methylene chloride R. Shake
vigorously. After separation, the organic layer is red.
C. Dissolve about 10 m g in 2 m L o f alcohol R. A dd 1 m L o f
a 1 g/L solution o f testosterone propionate R in alcohol R and
B. methyl 4-hydroxybenzoate (methyl parahydroxybenzoate), 2 m L of dilute sodium hydroxide solution R. H eat in a water-
bath at 80 °C for 10 min and allow to cool. A red colour
develops.
o ch3 TESTS
S o lu tio n S
Dissolve 2.5 g in alcohol R and dilute to 25 m L with the
same solvent.
C. ethyl 4-hydroxybenzoate (ethyl parahydroxybenzoate),
than intensity 5 o f the range o f reference solutions of the
most appropriate colour (2.2.2, Method II).
1-348 Butylated Hydroxytoluene 2016
Butylated Hydroxytoluene ?***
gel G R as the coating substance. *★ ★*
(Butylhydroxytoluene, Ph Eur monograph 0581) *
examined in methylene chloride R and dilute to 10 m L with
the same solvent.
with methylene chloride R.
Reference solution (a) Dissolve 25 mg o f
butylhydroxycmisole CRS in methylene chloride R and dilute to
C i 5H 240 220.4 128-37-0
Reference solution (b) D ilute 1 m L o f reference solution (a) to
20 m L with methylene chloride R. A c tio n a n d u se
Reference solution (c) Dissolve 50 m g o f hydroquinone R in Antioxidant.
5 m L of alcohol R and dilute to 100 m L with methylene
chloride R. Dilute 1 m L of this solution to 10 m L with P h E i r ____________________________________________________________________________________________
methylene chloride R. D E F IN IT IO N
Apply separately to the plate 5 jiL of each solution. Develop Butylhydroxytoluene is 2,6-bis (1,1 -dimethylethyl)-4-
over a path o f 10 cm using methylene chloride R. Allow the m ethylphenol.
plate to dry in air and spray with a freshly prepared mixture
CHARACTERS
of 10 volumes of potassium ferricyanide solution R, 20 volumes
A white or yellowish-white, crystalline powder, practically
of ferric chloride solution R1 and 70 volumes of water R. In the
insoluble in water, very soluble in acetone, freely soluble in
chrom atogram obtained with test solution (a): any violet-blue
alcohol and in vegetable oils.
spot with an RF value o f about 0.35 (corresponding to
3-(l,l-dim ethylethyl)-4-m ethoxyphenol) is not m ore intense ID E N T IF IC A T IO N
than the principal spot in the chrom atogram obtained with First identification A , C.
reference solution (a) (10 per cent); any spot corresponding Second identification A , B, D.
to hydroquinone is n o t more intense than the principal spot
A. Freezing-point (see Tests).
B. Dissolve 0.500 g in ethanol R and dilute to 100.0 m L with
(0.2 per cent); any spot, apart from the principal spot and
the same solvent. Dilute 1.0 m L o f this solution to 100.0 m L
any spots corresponding to 3-(l,l-dim ethylethyl)-4-
w ith ethanol R. Examined between 230 n m and 300 nm
methoxyphenol and hydroquinone, is n o t more intense than
the principal spot in the chrom atogram obtained with
(2.2.25), the solution shows an absorption m aximum at
278 nm . T h e specific absorbance at the m aximum is 80 to
90.
C . Examine by infrared absorption spectrophotom etry
(2.2.24), com paring with the spectrum obtained with
Prepare the reference solution using 1 m L o f lead standard
butylhydroxytoluene CRS.
solution (10 ppm Pb) R.
D . Dissolve about 10 mg in 2 m L o f alcohol R. Add 1 m L o f
a 1 g/L solution o f testosterone propionate R in alcohol R and
N ot more than 0.1 per cent, determ ined on 1.0 g.
2 m L o f dilute sodium hydroxide solution R. H eat in a water-
STORAGE b ath at 80 °C for 10 min and allow to cool. A blue colour
Store protected from light. develops.
IM P U R IT IE S TESTS
same solvent. T h e solution is clear (2.2.1) and not m ore
intensely coloured than reference solution Y 5 or BY 5 (2.2.2,
Method II).
A. benzene- 1,4-diol (hydroquinone). F re e z in g -p o in t (2.2.18)
____________________________________________________________________________________________PhEur 69 °C to 70 °C.
Exam ine by thin-layer chromatography (2.2.27), using silica
gel G R as th e coating substance.
Reference solution Dilute 1 m L o f the test solution to 200 m L
w ith methanol R.
Apply separately to the plate 10 |iL o f each solution. Develop
over a p ath o f 15 cm using methylene chloride R. D ry the plate
in air and spray with a freshly prepared mixture of
10 volumes o f potassium ferricyanide solution R, 20 volumes o f
ferric chloride solution R1 and 70 volumes o f water R. Any spot
in the chrom atogram obtained with the test solution, apart
2016 Cabergoline 1-349
from the principal spot, is not more intense than the spot in Reference solution (a) Dissolve 30.0 mg of cabergoline CRS in
the chromatogram obtained with the reference solution the mobile phase and dilute to 25.0 m L with the mobile
(0.5 per cent). phase.
S u lfa te d a s h (2.4.14) Reference solution (b) Dilute 1.0 m L of the test solution to
N ot m ore than 0.1 per cent, determined on 1.0 g. 100.0 m L with the mobile phase. Dilute 10.0 m L of this
Ph Eur
Reference solution (c) Suspend 50 m g of the substance to be
examined in 10 m L of 0.1 M sodium hydroxide. Stir for about
15 min. T o 1 m L o f the suspension add 1 m L o f 0.1 M
.**★ ★ hydrochloric acid and dilute to 10 m L with the mobile phase.
★
Cabergoline ★ ★ Sonicate until dissolution is complete. T h e main degradation
***** product obtained is impurity A.
Column'.
— size. I = 0.25 m , 0 = 4.6 mm,
— stationary phase. octadecylsQyl silica gel for chromatography R
(10 nm).
Mobile phase Mix 16 volumes of acetonitrUe R and 84 volumes
o f a freshly prepared 6.8 g/L solution of potassium dihydrogen
phosphate R previously adjusted to p H 2.0 with phosphoric
H H « 11
0 0 add R. Add 0.2 volumes of triethylamine R.
C 26H 37N 5O 2 451.6 81409-90-7
Injection 20 |iL of the test solution and reference solutions (b)
A ctio n a n d u se and (c).
Dopam ine D 2 receptor agonist. Run time 4 times the retention time o f cabergoline.
PhEur___________________________
Relative retention W ith reference to cabergoline (retention
D E F IN IT IO N impurity B = about 0.6; impurity A = about 0.8;
1-Ethyl-3- [3-(dimethylamino)propyl] -3-[[(6ai?,9i?, 10ai?)-7- impurity C = about 2.9.
(prop-2-enyl)-4,6,6a,7,8,9,10,1 Oa-octahydroindolo [4,3- System suitability', reference solution (c):
j£]quinolin-9-yl] carbonyl]urea. — resolution: minimum 3.0 between the peaks due to
C o n te n t cabergoline and impurity A.
98.0 per cent to 102.0 per cent (anhydrous substance). Limits:
CHARACTERS — impurities A, C: for each impurity, not more than
A p p e a ra n c e 1.5 times the area of the principal peak in the
White or almost white, crystalline powder. chromatogram obtained with reference solution (b)
(0.3 per cent);
S o lu b ility
— impurities B, D: for each impurity, not more than
(96 per cent), very slightly soluble in hexane. It is slightly
soluble in 0.1 M hydrochloric add.
(0.1 per cent);
It shows polymorphism (5.9). — any other impurity, for each impurity, n o t more than
ID E N T IF IC A T IO N 0.5 times the area of the principal peak in the
A. Specific optical rotation (see Tests). chromatogram obtained with reference solution (b)
(0.1 per cent);
Comparison cabergoline CRS. in the chromatogram obtained with reference solution (b)
If the spectra obtained in the solid state show differences, (0.8 per cent);
dissolve 50 mg o f the substance to be examined and 50 mg — disregard limit. 0.25 times the area o f the principal peak in
o f the reference substance separately in 1 m L o f ethanol the chromatogram obtained with reference solution (b)
(96 per cent) R, evaporate to dryness and record new spectra (0.05 per cent).
using the residues.
W a te r (2.5.12)
TESTS M aximum 0.5 per cent, determined on 1.000 g.
S pecific o p tic a l ro ta tio n (2.2.7) A SSAY
—77 to —83 (anhydrous substance).
Dissolve 0.100 g in ethanol (96 per cent) R and dilute to related substances with the following, modification.
R e la te d su b sta n c e s Calculate the percentage content of C 26H 37N 5O 2 from the
Liquid chromatography (2.2.29). Prepare the solutions areas o f the peaks and the declared content of
immediately before use and protectedfrom light. cabergoline CRS.
STO RA G E
the mobile phase.
1-350 Caffeine 2016
IMPURITIES Content
Specified impurities A, B, C, D 98.5 per cent to 101.5 per cent (dried substance).
CHARACTERS
CH2
Appearance
W hite or almost white, crystalline powder or silky, white or
almost white, crystals.
Solubility
Sparingly soluble in water, freely soluble in boiling water,
slightly soluble in ethanol (96 per cent). It dissolves in
concentrated solutions of alkali benzoates o r salicylates.
A. ( 6 a/?,9/?, 10ai?)-7-(prop-2-enyl)-4,6,6a,7,8,9, 10,10a- It sublimes readily.
octahydroindolo [4,3 -fg] quinoline-9-carboxylic acid. IDENTIFICATION
First identification A , B, E
Second identification A , C, D, E, F.
r- n
Comparison caffeine CRS.
C. T o 2 m L of a saturated solution add 0.05 m L o f iodinated
potassium iodide solution R. T he solution remains clear.
Add 0.1 m L o f dilute hydrochloric add R; a brown precipitate
B. R = C O -N H -C 2H 5, R ' = H : (6ai? 39i?J10a/?)-iV9-[3-
is formed. Neutralise with dilute sodium hydroxide solution R\
(dimet±iylaxnino)propyI]-iV4-ethyl-7-(prop-2-enyl)-
the precipitate dissolves.
6a373839310jl Oa-hexahydroindolo [4 ,3 - ^ quinoline-4,9 (6H)-
dicarboxamide, D. In a ground-glass-stoppered tube, dissolve about 10 mg in
0.25 m L o f a mixture of 0.5 m L of acetylacetone R and 5 m L
C. R = R ' = C O -N H -C 2H 5: (6aÄ,9i? 310aÄ )-^ 9-[3-
o f dilute sodium hydroxide solution R. H eat in a water-bath at
(dimethylamino)propyl] -N 4-ethyl-iV 9-(et±ijdcaTbamoyl)-7 -
80 °C for 7 min. Cool and add 0.5 m L of
(prop-2-eriyl)-6a,7,83931031Oa-hexahydroindolo [4,3-
dimethylamiitobenzaldehyde solution R2. H eat again in a water-
fg] quinoline-4,9 (6/i)-dicarboxam ide,
bath at 80 °C for 7 min. Allow to cool and add 10 m L of
D. R = R ' = H: (6ai?, 9R, 1QaR)-N- [3- water R', an intense blue colour develops.
(dimethylamino)propyl] -7-(prop-2-enyl) -43636a,7,8,9,10,10a-
E. Loss on drying (see Tests).
octahydroindolo [4,3-fg] quinoline-9-carboxamide.
F. It gives the reaction o f xanthines (2.3.1).
PhEur
TESTS
Solution S
Dissolve 0.5 g with hearing in 50 m l. o f carbon dioxide-free
water R prepared from distilled water i?, cool and dilute to
★ ★
Caffeine ★ ★ 50 m L with the same solvent.
Anhydrous Caffeine ***** Appearance o f solution
Solution S is d ear (2.2.1) and colourless (2.2.2, Method II).
A cid ity
T o 10 m L o f solution S add 0.05 m L of bromothymol blue
X solution R l; the solution is green or yellow. N o t more than
XX> 0.2 m L o f 0.01 M sodium hydroxide is required to change the
colour o f the indicator to blue.
I
ch 3 Related substances
C 8H 10N 4O 2 194.2 58-08-2 Test solution Dissolve 0.100 g of the substance to be
Action and use the mobile phase. Dilute 1.0 m L o f this solution to 10.0 m L
Central nervous system stimulant. with the mobile phase.
Preparations Reference solution (a) Dilute 2.0 m L o f the test solution to
Aspirin and Caffeine Tablets 100.0 m L with the mobile phase. D ilute 1.0 m l. of this
Caffeine Citrate Injection solution to 10.0 mL with the mobile phase.
Caffeine C itrate Oral Solution Reference solution (b) Dissolve 5 m g o f caffdne for system
Paracetamol and Caffeine Capsules suitability CRS (containing impurities A, C, D and F ) in the
Paracetamol and Caffeine Tablets mobile phase and dilute to 5 m L with the mobile phase.
D ilute 2 m L o f this solution to 10 m L with the mobile
Paracetamol, Codeine Phosphate and Caffeine Capsules
phase.
Paracetamol, Codeine Phosphate and Caffeine Tablets Column:
PhEur______________________________________________________ — size: I = 0.15 m , 0 = 4.6 mm ;
— stationary phase: base-deactivated end-capped octadecylsilyl
DEFINITION silica gel for chromatography R (5 pm).
1,3,7 -Trimethyl-3,7 -dihydro-1H -purine-2, 6-dione.
2016 Caffeine 1-351
0
Mobile phase Mix 20 volumes of tetrahydrofuran R,
25 volumes of acetonitrile R and 955 volumes of a solution
containing 0.82 g/L of anhydrous sodium acetate R previously
adjusted to p H 4.5 with glacial acetic add R.
Flow rate 1.0 mL/min. ch 3

A. 1,3-dim ethyl-3,7-dihydro-li/-purine-2,6-dione
Injection 10 |xL.
(theophylline),
Run time 1.5 times the retention time of caffeine.
with caffeine for system suitability CRS and the chromatogram
obtained with reference solution (b) to identify the peaks due
to impurities A, C , D and F.
Retention time Caffeine = about 8 min. 1
System suitability: reference solution (b): ch 3
impurities C and D and minimum 2.5 between the peaks B. iV-(6-amino-l,3-dimethyl-2,4-dioxo-l,2,3,4-
due to impurities F and A. tetrahydropyrimidin-5-yl)formamide,
Limits:
— unspecified impurities: for each impurity, no t more than 0
0.5 times the area of the principal peak in the H3C N
N
(0.10 per cent); X N
>
— total: not more than 0.5 times the area o f the principal CH3 CH3
C. l,3j9-trimethyl-3,9-dihycko-lH-purine-2,6-dione
(isocaffeine),
(0.05 per cent).
S u lfates (2.4.13) 1 ,CHs
M aximum 500 ppm , determined on 15 m L of solution S. h n 'a V n
Prepare the standard using a mixture of 7.5 m L of sulfate o^ n^ - n
standard solution (10 ppm SO4 ) R and 7.5 m~L of distilled ch 3
water R.
H eav y m eta ls (2.4.8) D . 3,7-dimethyl-3,7-dihydro-l/f-purine-2,6-dione
M aximum 20 ppm . (theobromine),
1
S u lfa te d ash (2.4.14) ch 3
M aximum 0.1 p e r cent, determined on 1.0 g.
A SSA Y E. N, l-dimethyl-4-(methylamino)-lH-imidazole-5-
Dissolve 0.170 g with heating in 5 m L o f anhydrous acetic carboxamide (caffeidine),
acid R. Allow to cool, add 10 m L o f acetic anhydride R and
20 m L of toluene R. T itrate with 0.1 M perchloric acid,
I 'CH3
determining the end-point potentiometrically (2 .2 .2 0 ).
o ^ n ^ n
o f CjjHioNiiC^- H
IM P U R IT IE S
Other detectable impurities (the following substances would, if F . 1,7 -dimethyl-3,7 -dihydro- 1/i-purine- 2, 6-dione.
present at a sufficient level, be detected by one or other of _________________________________________________ .__ _____ PhEur
C, D, E, F.
1-352 Caffeine Hydrate 2016
A c id ity
Caffeine Hydrate ***** T o 10 m l . o f solution S add 0.05 m L o f bromothymol blue
*. *
(Caffeine Monohydrate, Ph Eur monograph 0268) * solution R l; the solution is green or yellow. N o t m ore than
0.2 m L o f 0.01 M sodium hydroxide is required to change the
colour o f the indicator to blue.
Related substances
I
ch 3 examined in the mobile phase and dilute to 50.0 m L with
the mobile phase. Dilute 1.0 m L of this solution to 10.0 m L
w ith the mobile phase.
CgHxoN.AjHiO 212.2 5743-12-4
A ctio n a n d u se 100.0 m L with the mobile phase. Dilute 1.0 m L of this
Central nervous system stimulant. solution to 10.0 m L with the mobile phase.
Reference solution (b) Dissolve 5 m g of caffeine for system
Ph Eur___________________________________________________________
suitability CRS (containing impurities A, C, D and F) in the
D E F IN IT IO N mobile phase and dilute to 5.0 m L with the mobile phase.
l,3,7-Trim ethyl-3,7-dihydro-lH -purine-2,6-dione D ilute 2.0 m L o f this solution to 10.0 m L with the mobile
monohydrate. phase.
C o n te n t Column:
98.5 per cent to 101.5 per cent (dried substance). — size. I = 0.15 m , 0 = 4.6 mm ;
— stationary phase, base-deactivated end-capped octadecylsHyl
CHARACTERS
silica gelfor chromatography R (5 Jim).
A p p e a ra n c e
Mobile phase Mix 20 volumes o f tetrahydrofuran R,
W hite or almost white, crystalline powder or silky, white or
25 volumes of acetonitrile R and 955 volumes o f a solution
alm ost white crystals.
c o n t a i n i n g 0.82 g/L of anhydrous sodium acetate R previously
S o lu b ility adjusted to p H 4.5 with glacial acetic add R.
Sparingly soluble in water, freely soluble in boiling water,
slightly soluble in ethanol (96 per cent). It dissolves in
concentrated solutions o f alkali benzoates or salicylates. Detection Spectrophotom eter at 275 nm.
It sublimes readily. Injection 10 |iL.
Run time 1.5 times the retention time o f caffeine.
First identification A, B, E. Identification of impurities U se the chrom atogram supplied
with caffeine for system suitability CRS and the chrom atogram
Second identification A, C, D, E, F. obtained with reference solution (b) to identify the peaks due
A. M elting point (2.2.14): 234 °C to 239 °C, determ ined to impurities A, C , D and F.
after drying at 100-105 °C.
Retention time Caffeine = about 8 min.
B. Infrared absorption spectrophotometry (2.2.24). System suitability: reference solution (b):
Preparation D ry the substance to be examined at 100-105 °C — resolution: minimum 2.5 between the peaks due to
before use. impurities C and D; m in im u m 2.5 between the peaks due
Comparison caffeine CRS. to impurities F and A.
C. T o 2 m L o f a saturated solution add 0.05 m L o f iodmazed Limits:
potassium iodide solution R; the solution remains clear. — unspecified impurities: for each impurity, no t m ore than
A dd 0.1 m L o f dilute hydrochloric add R; a brown precipitate 0.5 times the area of the principal peak in the
is formed. Neutralise with dilute sodium hydroxide solution R; chromatogram obtained with reference solution (a)
the precipitate dissolves. (0.10 per cent);
D . In a glass-stoppered tube, dissolve about 10 m g in — total: n o t more than 0.5 times the area of the principal
0.25 m L of a mixture of 0.5 m L of acetylacetone R and 5 m L peak in the chromatogram obtained with reference
o f dilute sodium hydroxide solution R. H eat in a w ater-bath at solution (a) ( 0.1 per cent);
80 °C for 7 min. Cool and add 0.5 m L of — disregard limit. 0.25 times the area o f the principal peak in
dxmethylaminobenzaldehyde solution R2. H eat again in a water- the chromatogram obtained with reference solution (a)
bath at 80 °C for 7 m in. Allow to cool and add 10 m L of (0.05 per cent).
water R; an intense blue colour develops. S u lfa te s (2.4.11)
E. Loss on drying (see Tests). M axim um 500 ppm , d e te rm in e d on 15 m L o f solution S.
F. It gives the reaction of xanthines (2.3.1). Prepare the standard using a mixture o f 7.5 m L o f sulfate
standard solution (10 ppm SO 4) R and 7.5 m l . of distilled
TESTS
water R.
S o lu tio n S
Dissolve 0.5 g with heating in 50 m L of carbon dioxide-free
M axim um 20 ppm .
water R prepared from distilled water R, cool, and dilute to
50 m L with the same solvent. 1.0 g complies with test C. Prepare the reference solution
2016 Calamine 1-353
h3c 1 -CHi
drying in an oven at 105 °C for 1 h. 'Hhn^X - >n
S u lfa te d a sh (2.4.14) I
ch3
A SSAY E. N ,l-dimethyl-4-(methylamino)-lff-imidazole-5-
Dissolve 0.170 g, previously dried at 100-105 °Cj with carboxamide (caffeidine).
heating in 5 m L o f anhydrous acetic acid R. Allow to cool, and
add 10 m L of acetic anhydride R and 20 m L of toluene R n Cl
T itrate with 0.1 M perchloric acid, determining the end-point
potentiometrically (2.2.20). Î Ï )
1 m L o f 0.1 M perchloric acid is equivalent to 19.42 mg H
o f C 8H 10N 4O 2.
IM P U R IT IE S F . l,7-dim ethyl-3,7-dihydro-lff-purine-2,6-dione.
Other detectable impurities (the following substances would, if PhEur
by the general monograph Substances for pharmaceutical use Calamine
Prepared Calamine
Control of impurities in substances for pharmaceutical use): A , B, A ctio n a n d u se
C, D, E, F. Antipruritic.
P re p a ra tio n s
Aqueous Calamine Cream
h’ c ' n Calamine Lotion
O ^N Calamine Ointm ent
ch3 Calamine and Coal T a r Ointment
D E F IN IT IO N
A. l,3-dim ethyl-3,7-dihydro-lff-purine-2,6-dione
Calamine is a basic zinc carbonate suitably coloured with
(theophylline),
iron(m) oxide.
o y -H An amorphous, impalpable, pink o r reddish brown powder,
HjC'N-A y - NH the colour depending on the variety and amount of iron(m)
oxide present and the process by which it is incorporated.
0^ N NH! Practically insoluble in water. It dissolves with effervescence
ch 3 in hydrochloric acid.
B. N -(6-am ino-l,3-dim ethyl-2,4-dioxo-l,2,3,4-
A. Yields the reactions characteristic of carbonates,
tetrahydropyrimidin-5-yl)formamide,
Appendix VI.
B. T o 2 g add 5 m L o f hydrochloric acid and heat to boiling;
if necessary, add hydrochloric acid drop wise until a bright
h 3c
yellow solution is obtained. Cool and add 13. 5m ammonia
or iV >
'n
1 I,
until the first sign of precipitate (solution A). T h e solution
yields reaction B characteristic o f iron salts, Appendix VI.
CHj Dilute 1 m L of solution A to 5 m L with water, the solution
yields the reaction characteristic o f zinc salts, Appendix VI.
C . 1,3,9-trimethyl-3,9-dihydro- lH -purine-2,6-dione TESTS
(isocaffeine),
C a lc iu m
Dissolve 0 .5 0 g in a mixture of 10 m L o f water and 2 .5 m l.
o f glacial acetic acid and filter. T o 0.5 m L of the filtrate add
1 'Cl 15 m L of 5 m ammonia and 2 m L of a 2 .5 % whr solution o f
x jC > ammonium oxalate and allow to stand for 2 minutes.
O' N T he solution remains clear.
1I
ch 3
S olu b le b a riu m salts
T o the remainder of the filtrate obtained in the test for
D . 3,7-dimethyl-3,7-dihydro-lff-purm e-2,6-dione Calcium add 2 m L o f 1m sulfuric acid and allow to stand for
(theobromine), 5 minutes. T he solution remains clear.
L ead
N o t more than 1 5 0 ppm when determined by atomic
absorption spectrophotometry, Appendix II D, M ethod II,
1-354 Calcifediol 2016
measuring at 283.3 n m or 217 nm and using an air-acetylene CHARACTERS

flame. Carefully add 5 g o f the substance being examined to A p p e a ra n c e
25 m L of hydrochloric add and allow to stand for 18 hours. W hite or almost white crystals.
A dd 5 m L of nitric add and sufficient water to produce S o lu b ility
200 mL. U se lead standard solution (100 ppm Pb) suitably Practically insoluble in water, freely soluble in ethanol
diluted with a 3.5% vfv solution o f nitric add to prepare the (96 per cent), soluble in fatty oils.
standard solution.
It is sensitive to air, heat and light.
Chloride
Dissolve 0.15 g in water with the addition of 1 m L o f nitric ID E N T IF IC A T IO N
add, filter and dilute to 30 m L with water. T he resulting A. Infrared absorption spectrophotom etry (2.2.24).
solution complies with die limit test for chlorides, A ppendix VII Preparation Mix 2 mg o f the substance to be examined and
(0.07%). 225 m g o f potassium bromide R.
Sulfate Comparison Ph. Eur. reference spectrum of caldfediol.
Dissolve 0.1 g in water with the addition of 3 m L o f 2m B. Examine the chromatograms obtained in the assay.
hydrochloric add, filter and dilute to 60 m L with water. Results T h e principal peak in the chrom atogram obtained
T he resulting solution complies with the limit test for sulfates, with the test solution is similar in retention time and size to
Appendix V II (0.6%). the principal peak in the chrom atogram obtained with
Ethanol-soluble dyes reference solution (a).
Shake 1.0 g with 10 m L o f ethanol (90%) and filter.
TESTS
T h e filtrate is colourless, Appendix IV B, M ethod n .
Matter insoluble in hydrochloric acid Liquid chromatography (2.2.29): use the normalisation
Dissolve 1 g in 20 m L o f w arm 2m hydrochloric add and filter. procedure. Carry out the test as rapidly as possible, avoiding
T he residue, w hen washed with water and dried to constant exposure to actinic light and air.
weight at 105°, weighs not more than 10 mg.
W ater-soluble dyes examined without hearing in 10.0 m L o f the mobile phase.
Shake 1.0 g w ith 10 m L o f water and filter. T he filtrate is Reference solution (a) Dissolve 1.00 m g of caldfediol CRS
colourless, Appendix IV B, M ethod II. w ithout heating in 10.0 m L o f the mobile phase.
Residue on ignition Reference solution (b) Dilute 1.0 m L o f reference solution (a)
68.0 to 74.0%, w hen ignited at a tem perature n o t lower than to 100.0 m L with the mobile phase. Dilute 1.0 m L o f this
900° until, after further ignition, two successive w e igh in gs do solution to 10.0 m L with the mobile phase.
n ot differ by more than 0 .2 % of the weight o f the residue. Reference solution (c) H eat 2 m L o f reference solution (a) in a
U se 1 g.
w ater-bath at 80 °C under a reflux condenser for 2 h and
cool.
Column:
— size. I = 0.15 m, 0 = 4.6 mm;
Calcifediol ***** — stationary phase: octylsUyl silica gel for chromatography R l
★ ★ (5 jun).
(Ph. Eur. monograph 1295) ** Mobile phase water R, methanol R (20:80 VIV).
Injection 50 jiL o f the test solution and reference solutions (b)
and (c).
Run time Twice the retention time of calcifediol.
Relative retention W ith reference to caldfediol (retention
impurity B = about 1.1; im purity C = about 1.2; pre-
caldfediol = about 1.3; impurity A = about 1.6 .
C 27H 4 402#20 418.7 63283-36-3 caldfediol and pre-caldfediol; if necessary, adjust the
proportions of the constituents in the mobile phase.
Action and use
Limits:
Vitamin D analogue.
— impurities A, B, C, D: for each impurity, maximum
PhEur______________________________ ____________________________ 0.5 p er cent;
DEFINITION
0.10 p er cent;
(5Z ,7£)-9, 10-Secocholesta-5,7, 10( 19)-triene-3 P,25-diol — total: maximum 1.0 per cent;
monohydrate. — disregard lirnir. 0.5 times the area o f the principal peak in
Content the chromatogram obtained with reference solution (b)
97.0 per cent to 102.0 p er cent (anhydrous substance). (0.05 p er cent); disregard the peak due to pre-calcifediol.
A reversible isomérisation to pre-caldfediol takes place in W a te r (2.5.32)
solution, depending on tem perature and time. T he activity is 3.8 p er cent to 5.0 per cent, determined on 10.0 mg.
due to both com pounds (see Assay). '
2016 Anhydrous Calcipotriol 1-355
ASSAY
Anhydrous Calcipotriol *****
★ ★
related substances with the following modifications. (Ph. Eur. monograph 2011) *
Injection T est solution and reference solutions (a) and (c).
1 per cent for the peak due to calcifediol after 6
injections.
Calculate the percentage content o f C 27H 44O 2 using the
chromatogram obtained with reference solution (a) and
taking into account the assigned content o f calcifedbl CRS
and, if necessary, the peak due to pre-caldfediol.
STO RA G E
U nder nitrogen, in an airtight container, protected from light,
at a tem perature o f 2 °C to 8 °C.
C jvK kA 412.6 112965-21-6
T he contents of an opened container are to be used
immediately. A ctio n a n d use
IM P U R IT IE S Vitamin D analogue.
Specified, impurities A, B, C, D. P re p a ra tio n s
Calcipotriol Cream
Calcipotriol Ointm ent
Calcipotriol Scalp Application
PhEur__________________________________________________________
D E F IN IT IO N
(5Z,7£,22£,245)-24-Cydopropyl-9,10-secochola-
5,7,10( 19),22-tetraene-l a,3P,24-triol.
A. 9 P, 10a-cholesta-5,7-diene-3 (3325-diol,
C o n te n t
A reversible isomérisation to pre-caldpotriol takes place in
solution, depending on tem perature and time. T h e activity is
due to both compounds.
CHARACTERS
A p p e a ra n c e
B. cholesta-5,7-diene-3P,25-diol, S o lubility
(96 per cent), slightly soluble in methylene chloride.
It is sensitive to heat and light.
Comparison Ph. Eur. reference spectrum of anhydrous calcipotriol.
B. Loss on drying (see Tests).
TESTS
Carry out the tests for related substances and the assay as rapidly
as possible and protected from actinic light and air.
C . (6£)-9,l0-secocholesta-5(l0),6,8-triene-3P,25-diol, R ela te d su b sta n ce s
A. Thin-layer chromatography (2.2.27).
Solution A T o 1 m L of triethylamine R add 9 m L o f
chloroform R.
in 100 jiL of solution A.
Reference solution (a) T o 10 (iL o f the test solution add
990 jiL o f solution A.
Reference solution (b) T o 250 (iL o f reference solution (a) add
750 (iL o f solution A.
Reference solution (c) T o 100 (iL o f reference solution (a) add
900 pL of solution A.
D . (5£,7£)-9,l0-secocholesta-5,7,10( 19)-triene-3P,25-diol.
!____________:____________ Ph Eur
1-356 Anhydrous Calcipotriol 2016
Reference solution (d) Place 2 mg o f the substance to be Run time Twice the retention time of caldpotriol.
examined in a vial and dissolve in 200 (iL of solution A. Relative retention W ith reference to caldpotriol (retention
Close the vial and keep it in a water b ath at 60 °C for 2 h. time = about 13.5 min): im purity B = about 0.86;
Plate TLC silica gel F2 $ 4 plate R. impurity C = about 0.92; impurity D = about 1.3.
Mobile phase 2-methylpropanol R, methylene chloride R System suitability: reference solution (c):
(20:80 VIV). — peak-UHvaHey ratio: m inim um 1.5, where Hp = height
Application 10 [iL of the test solution and reference above the baseline o f the peak due to im purity C and
solutions (b), (c) and (d). Hv = h d g h t above the baseline of the lowest point of
the curve separating this peak from the peak due to
caldpotriol;
Drying In air, then at 140 °C for 10 min. — the chrom atogram obtained is similar to the
Detection Spray the h o t plate with an alcoholic solution of chrom atogram supplied with caldpotriol
sulfuric add R, dry at 140 °C for not m ore than 1 m in and monohydrate CRS.
examine in ultraviolet light a t 366 nm . Limits:
Relative retention With reference to calcipotriol — impurity B: not m ore than 0.5 times the area of the
(Rp = about 0.4): impurity G = about 0.4; principal peak in the chrom atogram obtained with
im purity H = about 0.4; pre-caldpotriol = about 0.9; reference solution (a) (0.5 per cent);
impurity A = about 1.2 . — impurities C, D: for each impurity, not m ore than the
System suitability Reference solution (d): area of the prindpal peak in the chromatogram
— the chrom atogram shows a secondary spot due to pre- obtained with reference solution (a) ( 1.0 per cent);
caldpotriol. — unspecified impurities: for each impurity, n o t m ore than
Limits: the area o f the principal peak in the chrom atogram
— impurity A: any spot due to im purity A is not more obtained with reference solution (b) (0.10 per cent);
intense than the spot in the chrom atogram obtained — total: not m ore than 2.5 times the area o f the prindpal
with reference solution (b) (0.25 per cent); peak in the chrom atogram obtained with reference
— impurities G, H: any spot due to im purity G or H is solution (a) (2.5 p er cent);
not m ore intense than the spot in the chromatogram — disregard lim it 0.5 times the area o f the prindpal peak
obtained with reference solution (b) (0.25 per cent for in the chrom atogram obtained with reference
the sum ); solution (b) (0.05 per cent).
— unspecified impurities: any other spot is not m ore Loss on drying
intense than the spot in the chrom atogram obtained M aximum 1.0 p er cent, determ ined on 5 m g by
with reference solution (c) (0.1 per cent). thermogravimetry (2.2.34). H eat to 105 °C at a rate of
B. Liquid chrom atography (2.2.29). 10 °C/min and maintain at 105 °C for 60 min.
Solution A Dissolve 1.32 g o f ammonium phosphate R in ASSAY
water R and dilute to 10.0 m L with th e same solvent. Liquid chromatography (2.2.29) as described in the test for
Solvent mixture Solution A, water R, methanol R related substances with the following modification.
(0.3:29.7:70 V/V/V). Injection T est solution (b) and reference solution (d).
Test solution (a) Dissolve 2 m g of the substance to be Calculate the percentage content o f C 27H 40O 3 taking into
examined in the solvent mixture and dilute to 5.0 m L with account the assigned content o f caldpotriol monohydrate CRS.
STO RA G E
Test solution (b) Dissolve 2.00 mg o f the substance to be In an airtight container, protected from light, at —20 °C or
below.
the same solvent mixture.
IM P U R IT IE S
Reference solution (a) D ilute 1.0 m L o f test solution (a) to
100.0 m L with the solvent mixture. Spedfied impurities A, B, C , D , G , H
Reference solution (b) Dilute 1.0 m L o f reference solution (a) Other detectable impurities (the following substances would, if
to 10.0 m L w ith the solvent mixture. present at a suffident level, be detected by one or other o f
the tests in the m onograph. T hey are limited by the general
Reference solution (c) Dissolve 1 mg o f caldpotriol
monohydrate CRS (containing impurities B, C and D ) in the
solvent m ixture and dilute to 2.5 m L w ith the solvent
mixture.
Reference solution (d) Dissolve 2.00 m g o f caldpotriol Control of impurities in substances for pharmaceutical use): E, F,
monohydrate CRS in the solvent mixture and dilute to I.
By thin-layer chromatography: A , G, H, I.
Column:
— size: I = 0.10 m , 0 = 4.0 mm ;
By liquid chromatography: B, C, D, E, F.
— stationary phase: octadecylsUyl silica gel for
Mobile phase water R, methanol R (30:70 V!V).
Injection 20 |iL of test solution (a) and reference solutions (a),
(b) and (c).
2016 Anhydrous Calcipotriol 1-357
A. (5Z,7£,22£)-24-cydopropyl-l 0,3 ß-dihydroxy-9,10- E. rac-(5Zj7£j245)-24-cyclopropyl-9j 10-secochola-

secochola-5j7jl 0(19),22-tetraen-24-one, 5,7,10( 19)-triene-10Cj3 ß,24-triol,
B . (5Z,7Z,22£,24S)-24-cydopropyl-9,10-secochola- F . (5Z ,7£,22£,24S)-24-cydopropyl-la,3ß-bis[[(l, 1-

5,7,10(19),22-tetraene-laj3ßj24-ttiol ((72)-caldpotriol). dimediylethyl)dimethylsilyl] oxy]-9,10-secochola-
5,7,10(19) j22-tetraen-24-ol,
C. (5£j7£j22£j245)-24-cyclopropyl-9j 10-secochola-
5,7,10(19)j22-tetraene-laj3ßj24-ttiol ((5£)-caldpotriol),
G. 24,24 '-oxybis [(5Z,7£,22Ẹ,245)-24-cydopropyl-9,10-

secochola-5,7,10(19),22-tetraene-laj3ß-diol],
D . (5Z,7Ẹ,22.Ẹ,24j?!)-24-cyclopropyl-9,10-secochola-
5,7,10( 19),22-tetraene-1aj3ß,24-triol (24-ẹpt-calcipotriol),
H . (5Zj7£j22jEj24R)-24-cyclopropyl-24-[[(5Zj7£j22jEj245)-
24-cydopropyl-la,3ß-dihydroxy-9,10-secochola-
5 ,7 ,10(19),22-tetraen-24-yl]oxy]-9,10-secochola-
5,7,10(19) j22-tetraène-l aj3ß-diolj
1-358 Calcipotriol Monohydrate 2016
TESTS
Carry out the tests for related substances and the assay as rapidly
as possible and protected from actinic light and air.
Related substances
Solution A T o 1 m L of triethylamine R add 9 m l. o f
chloroform R.
Test solution Dissolve 1 mg of the substance to be exam in ed
in 100 pL o f solution A.
Reference solution (a) T o 10 |iL o f the test solution add
I. (65’j7iîj8i?j22£j245)-24-cyclopropyl-6j8:7j 19-dicyclo-9,10-
990 |jL o f solution A.
secochola-5(10),22-diene-la,3P,24-triol (suprasterol of
calcipotriol). Reference solution (b) T o 250 |iL of reference solution (a) add
750 pL o f solution A.
___________________________________________________________PhEur
Reference solution (c) T o 100 |iL o f reference solution (a) add
900 jjL o f solution A.
Reference solution (d) Place 2 mg o f the substance to be
examined in a vial and dissolve in 200 |iL o f solution A.
Calcipotriol Monohydrate Close the vial and keep it in a water bath at 60 °C for 2 h.
(Ph. Eur. monograph 2284) * Plate TLC silica gel F2 5 4 plate R
Mobile phase 2-methylpropanol R, methylene chloride R
(20:80 VIV).
Application 10 (iL o f the test solution and reference
Drying In air, then at 140 °C for 10 min.
Detection Spray the hot plate with an alcoholic solution of
sulfuric acid R, dry at 140 °C for n o t more than 1 min and
examine in ultraviolet light at 366 nm.
Relative retention W ith reference to calcipotriol
(Rp = about 0.4): impurity G = about 0.4;
im purity H = about 0.4; pre-calcipotriol = about 0.9;
C 27H 40O 332 O 430.6 147657-22-5 im purity A = about 1.2.
Action and use System suitability Reference solution (d):

— the chromatogram shows a secondary spot due to pre-
Vitamin D analogue.
calcipotriol.
Preparations
Limits:
Calcipotriol Cream
— impurity A: any spot due to im purity A is not m ore
Calcipotriol O intm ent intense than the spot in the chromatogram obtained
Calcipotriol Scalp Application with reference solution (b) (0.25 per cent);
— impurities G, H: any spot due to impurity G or H is
PhEur___________________________________________________________
not more intense than the spot in the chromatogram
DEFINITION obtained with reference solution (b) (0.25 per cent for
(5Z, 7E,22E, 243) -2 4-Cyclopropyl-9,1 0-secochola- the sum);
5,7,10(19),22-tetraene-la,3P,24-triol monohydrate. — unspecified impurities: any other spot is n o t more
Content intense than the spot in the chromatogram obtained
95.5 per cent to 102.0 per cent (anhydrous substance). with reference solution (c) (0.1 per cent).
A reversible isomérisation to pre-calcipotriol takes place in B. Liquid chromatography (2.2.29).

solution, depending on tem perature and time. T he activity is Solution A Dissolve 1.32 g of ammonium phosphate R in
due to both com pounds. water R and dilute to 10.0 m L with the same solvent.
CHARACTERS Solvent mixture Solution A, water R, methanol R
(0.3:29.7:70 VIVIV).
Appearance
W hite or almost white, crystalline powder. Test solution (a) Dissolve 2 m g o f the substance to be
Solubility
(96 p er cent), slightly soluble in methylene chloride. Test solution (b) Dissolve 2.00 mg o f the substance to be
It is sensitive to light.
the same solvent mixture.
IDENTIFICATION Reference solution (a) Dilute 1.0 m L o f test solution (a) to
A. Infrared absorption spectrophotom etry (2.2.24). 100.0 m L with the solvent mixture.
Comparison Ph. Eur. reference spectrum of calcipotriol Reference solution (b) Dilute 1.0 m L o f reference solution (a)
monohydrate. to 10.0 m L with the solvent mixture.
B. W ater (see Tests).
2016 Calcipotriol Monohydrate 1-359
Reference solution (c) Dissolve 1 mg of calcipotriol (2034). It is therefore not necessary to identify these
monohydrate CRS (containing impurities B, C and D ) in the impurities for demonstration of compliance. See also 5.10.
solvent mixture and dilute to 2.5 m L with the solvent Control of impurities in substances for pharmaceutical use): E, F,
mixture. I.
Reference solution (d) Dissolve 2.00 m g of calcipotriol By thin-layer chromatography: A, G, H, I.
monohydrate CRS in the solvent mixture and dilute to By liquid chromatography: B, C, D, E, F.
Column:
— size: I = 0.10 m, 0 = 4.0 mm;
— stationary phase: octadecylsOyl silica gel for
Mobile phase water R, methanol R (30:70 V!V).
Irgection 20 p L o f test solution (a) and reference solutions (a),
(b) and (c).
Run time Twice the retention time o f calcipotriol.
Relative retention W ith reference to calcipotriol (retention A. (5Z,7£,22JB)-24-cyclopropyl-1a,3P-dihydroxy-9,10-
time = about 13.5 min): impurity B = about 0.86; secochola-5,7,10(19),22-tetraen-24-one,
impurity C = about 0.92; impurity D = about 1.3.
— peak-to-valley ratio: minimum 1.5, where Hp = height
above the baseline o f 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
caldpotriol;
— the chromatogram obtained is similar to the
chromatogram supplied with calcipotriol
monohydrate CRS.
Limits:
— impurity B: not more than 0.5 times the area of the
reference solution (a) (0.5 per cent); B. (5Z,7Z,22£,245)-24-cydopropyl-9,10-secochola-
5,7,10(19),22-tetraene-1a,3p,24-triol ((7Z)-calcipotriol),
— impurities C, D: for each impurity, n o t more than the
area of the principal peak in the chromatogram
obtained with reference solution (a) ( 1.0 per cent);
— total: n o t more than 2.5 times the area o f the principal
— disregard limic. 0.5 times the area of the principal peak
in the chromatogram obtained with reference
solution (b) (0.05 per cent).
Water (2.5.12)
3.3 per cent to 5.0 per cent, determined on 0.100 g . C. (5£,7£,22£,245)-24-cydopropyl-9,10-secochola-
5,7,10(19),22-tetraene-la,3p,24-triol ((5£)-calcipotriol),
A SSAY
account the assigned content of calcipotriol monohydrate CRS.
STO RA G E
IM P U R IT IE S
Specified impurities A, B, C, D , G, H
present at a sufficient level, be detected by one or other of D. (5Z,7£,22£,24i?)-24-cyclopropyl-9,10-secochola-
the tests in the monograph. They are limited by the general 5,7,10(19),22-tetraene-1otJ3p,24-triol (24-ept-calcipotriol),
1-360 Calcitonin (Salmon) 2016
H H
I. (65’37i?,8i?,22£'j245)-24-cydopropyl-6,8:7,19-dicyclo-9,10-
E. rac-(5Z,7J5'j245)-24-cyclopropyl-9,10-secochola-
secochola-5 (10),22-diene-10,3(3,24-triol (suprasterol o f
5,7,10(19)-triene-1a,3 P,24-triol,
calapotriol).
___________________________________________________________PhEur
Calcitonin (Salmon) *****

** +*
H- Cys - Ser - Asn - Leu- Ser - Thr - Cys - Val - Leu - Gly-
l_______________________ i
h3c ch3 h3c ch3 Lys - Leu - Ser - Gin- Glu - Leu—His - Lys - Leu - Gin-
Thr - Tyr - Pro - Arg - Thr - Asn - Thr - Gly - Ser - Gly-
F. (5Z, 7£,222j,24S)-24-cyclopropyl-1a ,3 P-bis [[(1,1-
Thr-Pro-NH2
dimethylethyl)dimethylsilyl] oxy]-9,10-secochola-
5,7,10(19),22-tetraen-24-ol,
C 145H 240N 44O 48S2 3432 47931-85-1
Action and use

H orm one.
Preparation
C aldtonin (Salmon) Injection
PhEur_______________________________________________________________
DEFINITION
Polypeptide having the structure determ ined for salm on
caldtonin I. It lowers the caldum concentration in plasma of
mammals by dim in ishin g the rate o f bone resorption. It is
obtained by chemical synthesis or by a m ethod based on
recom binant D N A (rDNA) technology. It is available as an
acetate.
Content
G . 24,24 '-oxybis [(5Z,7£32 2 £ 3245)-24-cydopropyl-9j 10- 90.0 per cent to 105.0 per cent o f the peptide
secochola-5,7,10( 19) ,22-tetxaene-1a,3 P-diol], C 145H 240N 44O 48S 2 (anhydrous and acetic add-free
substance).
By convention, for the purpose o f labelling caldtonin
(salmon) preparations, 1 mg o f caldtonin (salmon)
(C j 45H 240N 44O 48S 2) is equivalent to 6000 IU o f biological
activity.
PRODUCTION
T h e following requirements apply only to caldtonin (salmon)
produced by a m ethod based on rD N A technology.
Prior to rdease the following tests are carried out on each
batch o f final bulk product unless exemption has been
granted by the competent authority.
Host-cell-derived proteins
T h e limit is approved by the competent authority.
H ost-cell or vector-derived DNA
T h e limit is approved by the com petent authority.
H . (5Z,72j,222j,24i?)-24-cyclopropyl-24- [ [(5Zj7£’j22£,245) -
24-cyclopropyl-1a,3 (J-dihydroxy-9,1O-secochola- CHARACTERS
5,7,10(19),22-tetraen-24-yl] oxy]-9,10-secochola- Appearance
5,7,10(19),22-tetraene-1a,3|3-diol, W hite or alm ost white powder.
2016 Calcitonin (Salmon) 1-361
S o lu b ility Equilibration A t initial conditions for at least 15 min. Carry

Freely soluble in water. out a blank run using the above-mentioned gradient.
ID E N T IF IC A T IO N Injection 20 pL.
A. Examine the chromatograms obtained in the assay. System suitability T h e chromatograms obtained with the test
Results T h e principal peak in the chromatogram obtained solution and the reference solution are qualitatively similar to
w ith the test solution is similar in retention time and size to the chrom atogram of caldtonin (salmon) digest supplied with
the principal peak in the chromatogram obtained with the calcitonin (salmon) CRS.
reference solution. Results T h e profile of the chromatogram obtained with the
The following requirement applies only to calcitonin (salmon) test solution corresponds to that o f the chromatogram
obtained by chemical synthesis obtained with the reference solution: the retention times of
the fragment peaks in the chromatogram obtained with the
B. A m in o acid analysis (2.2.56).
test solution are within 5 per cent of the retention times of
Express the content of each am ino a d d in moles. Calculate the fragments ôbtained with the reference solution; the peak
the relative proportions of the amino adds taking as area ratios of the fragment peaks in the chromatogram
equivalent to 1 the sum, divided by 20, of the num ber of obtained with the test solution, normalised to the area of
moles of aspartic ad d , glutamic add, proline, glycine, valine, peak T2 3 are within 5 per cent o f the corresponding peak
leucine, histidine, arginine and lysine. T he values fall within ratios in the chromatogram obtained with the reference
the following limits: aspartic add: 1.8 to 2 .2; glutamic ad d : solution.
2.7 to 3.3; proline: 1.7 to 2.3; glycine: 2.7 to 3.3; valine:
0.9 to 1.1; leucine: 4.5 to 5.3; histidine: 0.9 to 1.1; arginine: TESTS
0.9 to 1.1; lysine: 1.8 to 2.2; serine: 3.2 to 4.2; threonine: A cetic a c id (2.5.34)
4.2 to 5.2; tyrosine: 0.7 to 1.1; half-cystine: 1.4 to 2.1. 4.0 p er cent to 15.0 per c en t
The following requirement applies only to calcitonin (salmon) Test solution Dissolve 10.0 mg o f the substance to be
produced by a method based on rDNA technology. examined in a mixture o f 5 volumes of mobile phase B and
95 volumes o f mobile phase A and dilute to 10.0 m L with
C . Peptide mapping (2.2.55).
the same mixture of mobile phases.
SELECTIVE CLEAVAGE OP THE PEPTIDE BONDS
R e la te d su b stan c es
Test solution Prepare a 1 mg/mL solution of the substance to
Liquid chromatography (2.2.29): use the normalisation
be examined. Transfer 1.0 m L to a clean tube. Add 100 jxL
procedure.
o f 1 M tris-hydrochloride buffer solution pH 8.0 R and 20 pL of
a freshly prepared 1.0 mg/mL solution of trypsin for peptide The following requirement applies to calcitonin (salmon), whether
mapping R. Allow to stand at 2-8 °C for 16-20 h. Stop the obtained by chemical synthesis or by a method based on rDNA
reaction by adding 10 (iL o f a 50 per cent V/V solution of technology.
trifluoroacetic acid R. Cap the vial and mix. Centrifuge the A. Test solution. Prepare a 1.0 mg/mL solution o f the
vials to remove air bubbles. substance to be examined in mobile phase A.
Reference solution Prepare at the same time and in the same Reference solution Dissolve the contents o f a vial o f calcitonin
m an n er as for the test solution but using calcitonin (salmon) CRS in mobile phase A to obtain a concentration of
(salmon) CRS instead of the substance to be examined. 1.0 m g/m L
CHROMATOGRAPHIC SEPARATION Resolution solution Dissolve the contents of a vial of N-acetyl-
Liquid chromatography (2.2.29). Cys1-calcitonin CRS in 400 |iL o f mobile phase A and add
100 (iL o f the test solution.
Column:
— size: I = 0.25 m , 0 = 4.6 mm; Column:
— stationary phase: octadecylsifyl silica gel for — size: I = 0.25 m, 0 = 4.6 mm;
chromatography R (5 pm) with a pore size o f 30 nm . — stationary phase, octadecylsifyl silica gel for
Mobile phase:
— mobile phase A: mix 1 m L o f trifluoroacetic acid R and
1000 m L o f water R\ filter and degas; M obile phase:
— mobile phase B: mix 0.850 m L of trifluoroacetic acid R, — mobile phase A: dissolve 3.26 g of tetramethylammonium
200 m L of water R and 800 m L of acetonitrile for hydroxide R in 900 m L o f water R, adjust to pH 2.5
chromatography R; filter and degas; w ith phosphoric acid R and mix with 100 m L of
acetonitrile for chromatography R\ filter and degas;
— mobile phase B: dissolve 1.45 g of tetramethylammonium
Time Mobile phase A Mobile phase B hydroxide R in 400 m L of water R, adjust to pH 2.5
(min) (per cent V/V) (per cent V/V) with phosphoric add R and mix with 600 m L of
0 -5 0 10 0 * 6 5 0 * 35 acetonitrile for chromatography R; filter and degas;
5 0 -6 0 65 ->40 35*60
60 - 60.1 40*0 6 0 * 100 Time Mobile phase A Mobile phase B

60.1 - 65.1 0 100
0 -3 0 72*48 28*52
65.1 - 65.2 0 -» 100 10 0 *0
3 0 -3 2 4« * 7 2 52*28
65-2 - 802 100 0
3 2 -5 5 72 28
Flow rate 1.2 mlVmin. Flow rate 1.0 mL/min.

Detection Spectrophotometer at 214 nm. Detection Spectrophotometer at 220 nm.
1-362 Calcitonin (Salmon) 2016
Injection 20 pL. Limits:

Relative retention W ith reference to calcitonin (salmon) — impurity E: maximum 0.6 per cent;
(retention tim e = about 20 min): impurity B = about 0.8; — impurities F, G: for each impurity, maximum
im purity C = about 0.9; impurity D = about 1.05; 0.2 per cent.
im purity A = about 1.15. Water (2.5.32)
System suitability Resolution solution: M axim um 10.0 per cent.
— resolution: minimnm 5.0 between the peaks due to Acetic ad d and water
calcitonin (salmon) and im purity A, M axim um 20 p er cent, calculated by adding together the
— symmetry factor, maximum 2.5 for the peak due to percentage contents o f acetic a d d and w ater determ ined by
im purity A. the m ethods described above.
Limits:
Bacterial endotoxins (2.6.14)
— impurities A , B, C, D: for each impurity, maximum
Less than 25 IU /m g, if intended for use in the m anufacture
3.0 p er cent; other unidentified, specified impurities
of parenteral preparations w ithout a further appropriate
may occur that co-elute with impurities A, B, C
and D ; the acceptance criterion applies irrespective o f
w hether these impurities co-elute; ASSAY
— total-, maximum 5.0 per cent; Liquid chromatography (2.2.29) as described in the test for
— disregard limit: 0.1 per cent. related substances. Use m ethod A for caldtonin (salmon)
The following requirement applies only to calcitonin (salmon) obtained by chemical synthesis and m ethod B for caldtonin
produced by a method based on rDNA technology. (salmon) obtained by a m ethod based on rD N A technology.
B. Test solution. Prepare a 0.5 mg/mL solution o f the Calculate the content o f caldtonin (salmon)
substance to be examined. T o 1.0 m l. o f this solution add (C 145H 240N 44O 48S 2) from the area of the p rindpal peak in
100 pL of 0.25 M citrate buffer solution pH 3.0 R each o f the chromatograms obtained with the test solution
and the reference solution and the d ed ared content of
Resolution solution Prepare a 1 m g/m L solution o f the
C 145H 240N 44O 48S 2 in caldtonin (salmon) CRS. Proceed with
substance to be examined. Mix 1 volume of the solution and
tangential integration o f the peak areas.
1 volum e of caldtomn-Gly CRS. T o 1.0 mT. o f this mixture
add 100 pL o f 0.25 M citrate buffer solution pH 3.0 R. STORAGE
Column: Protected from light at a tem perature betw een 2 °C and
— size: I = 0.20 m , 0 = 4.6 mm ; 8 °C. If the substance is sterile, store in a sterile, airtight,
— stationary phase: a suitable polysulfoethylaspartamide tam per-proof container.
ion-exchange gel (5 pm). LABELLING
Mobile phase: The label states:
— mobile phase A: mix 15 volumes o f acetomtrUe for — the caldtonin peptide content ( Q 45H 240N 44O 48S2);
chromatography R and 85 volumes o f a 2.72 g/L — the origin: synthetic or rD N A technology.
solution of potassium dihydrogen phosphate R adjusted
to p H 5.0 with a 600 g/L solution o f potassium
IMPURITIES
hydroxide R; Spedfied impurities A, B, C, D , E, F , G
— mobile phase B: mix 15 volumes o f acetomtrUe for
R1 - Cys - Ser - Asn - Leu - Ser - Thr - Cys - Val —X —Gly-
chromatography R and 85 volumes o f a solution ------------------------------------ 1 10
containing 2.72 g/L o f potassium dihydrogen Lys - Leu - Ser - G in -G lu -L e u -H is -L y s -L e u -G in -
20
phosphate R and 29.22 g/L o f sodium chloride R Thr - Tyr - P r o -A rg -T h r -A s n -T h r -G ly -S e r -G ly -
30
adjusted to p H 4.6 with a 600 g/L solution o f
Thr-Pro-R2
potassium hydroxide R]
A. R1 = C O -C H 3, R2 = N H 2, X = L-Leu: acetylcaldtonin
Time Mobile phase A Mobile phase B (salmon),
B. R1 = H , R2 = N H 2, X = D-Leu: [9-D-Ieucine]caldtonin
0 - 10 100-»0 0 -» 100
(salmon),
1 0 - 15 0 100 E. R1 = H , R2 = N H -C H 2- C 0 2H , X = L-Leu: salmon
15 - 15.1 0 -» 100 100-»0 caldtoninylglydne,
15.1 - 22.1 100 0 H - Cys - Ser - Asn - Leu - Ser - Thr - Cys - Val - Leu - Gly-
1------------------------------------------------------- 1 10
Lys - Leu - Ser - Gin - Glu - Leu - His - Lys - Leu - Gin-
20
Flow rate 1.2 mL/min. Thr - Pro - Arg - T h r-A s n -T h r-G ly -S e r -G ly -T h r -
30
Detection Spectrophotom eter at 220 nm . Pro-NH2
Injection 50 pL; rinse the injector with a 40 p er cent V/V
solution o f acetomtrUe for chromatography R. C. des-22-tyrosine-caldtonin (salmon),
Relative retention W ith reference to calcitonin (salmon) D . O-acetyiated caldtonin (salmon),
(retention tim e = about 9 min): impurity G = about 0.4;
impurity F = about 0.6; impurity E = about 0.9.
System suitabUity Resolution solution:
im purity E and calcitonin (salmon).
2016 Calcitriol 1-363
s o 3h s o 3h
3I 3I TESTS
H- Ala - Ser - Asn - Leu - Ser - Thr - Ala - Val - Leu - Gly- Related substances
10
Lys - Leu - Ser-Gln-Glu - Leu - His - Lys - Leu - Gin - Liquid chromatography (2.2.29): use the normalisation
20
Thr - Tyr - Pro - Arg - Thr - Asn - Thr - Gly - Ser - Gly- procedure. Carry out the test as rapidly as possible, avoiding
30 exposure to actinic light and air.
Thr - Pro-R
Test solution Dissolve 1.00 mg o f the substance to be
exam in ed without heating in 10.0 m L of the mobile phase.
F. R = N H 2: [lj7-bis(3-sxalfo-L-alanme)]caldtonin (salmon),
Reference solution (a) Dissolve 1.00 m g o f caldtriol CRS
G . R = N H -C H 2- C 0 2H: [l,7-bis(3-sulfo-L-
w ithout heating in 10.0 m L of the mobile phase.
alanine)]caldtoninylglydne (salmon).
Reference solution (b) Dilute 1.0 m l. o f reference solution (a)
___________________________________________________________________________________________ Ph Eur
to 100.0 m L with the mobile phase. Dilute 1.0 m L of this
Reference solution (c) H eat 2 m L o f reference solution (a) at
80 °C for 30 min.
★ ★
Calcitriol ★ ★ Column:
**+★* — size: I = 0.25 m, 0 = 4.6 mm;
(Ph. Eur. monograph 0883) — stationary phase: octylsUyl silica gel for chromatography R1
(5 pm);
H3C H — temperature: 40 °C.
Mobile phase Mix 450 volumes o f a 1.0 g/L solution of
H V -C H 3
tris(kydroxymetkyl)aminomethane R adjusted to p H 7.0-7.5
IX y HO ch3 with phosphoric add R, and 550 volumes o f acetonitrile R.
I *■* Flow raxe 1.0 mL/min.
J L ^ ch2 Injection 50 |xL.
H O -y k ^ J^ O H Run time Twice the retention time o f caldtriol.
H H Relative retention W ith reference to caldtriol (retention
time = about 14 min): impurity C = about 0.4; pre-
416.6 32222-06-3 caldtriol = about 0.88; impurity A = about 0.95;
Action and use System suitability:
Vitamin D analogue. — resolution: m inim um 3.5 between the peaks due to pre-
Preparation caldtriol and caldtriol in the chromatogram obtained with
C aldtriol Capsules reference solution (c);
— number of theoretical plates: m inim u m 10 000, calculated
P h E ir ____________________________________________________________________ for the peak due to caldtriol in the chromatogram
D E F IN IT IO N obtained with reference solution (a).
(5Z,7£)-9,10-Secocholesta-5,7,10(19)-triene-l a,3P,25-triol. Limits:
Content — impurities A , B, C: for each impurity, maximum
0.5 per cent;
— unspecified impurities: for each impurity, maxim um
A reversible isomerisation to pre-caldtriol takes place in
0.10 per cent;
solutipn, depending on temperature and time. T he activity is
due to b oth com pounds (see Assay).
— disregard limit:. 0.5 times the area o f the prindpal peak in
CHARACTERS the chromatogram obtained w ith reference solution (b)
A p p e a ra n c e (0.05 per cent); disregard the peak due to pre-caldtriol.
White or almost w hite crystals. ASSAY
Solubility Liquid chromatography (2.2.29) as described in the test for
Practically insoluble in water, freely soluble in ethanol rd ated substances with the following modifications.
(96 per cent), soluble in fatty oils. Injection T est solution and reference solution (a).
It is sensitive to air, heat and light. System suitability: reference solution (a):
ID E N T IF IC A T IO N — repeatability: maximum relative standard deviation of
A. Infrared absorption spectrophotometry (2.2.24). 1 per cent for die peak due to caldtriol after 6 injections.
Comparison Ph. Eur. reference spectrum of caldtriol. Calculate the percentage content o f Q 7H 44O 3 taking into
account the assigned content o f calcitriol CRS and, if
necessary, the peak due to pre-caldtriol.
Results T h e prindpal peak in the chromatogram obtained
with the test solution is similar in retention time and size to STO RA G E
the prindpal peak in the chromatogram obtained with U nder nitrogen, in an airtight container, protected from light,
reference solution (a). at a tem perature of 2 °C to 8 °C.
T he contents o f an opened container are to be used
immediately.
1-364 Calcium Acetate 2016
IM P U R IT IE S
Specified impttrities A, B, C
Calcium Acetate *****
** +*
(Calcium Acetate, Anhydrous, *
Ph Ever monograph 2128)
Ca2+ [h 3C - C 0 2-]2
C ^C aO * 158.2 62-54-4
U sed in solutions for haemodialysis and peritoneal dialysis.
PhEur__________________________________________________________
D E F IN IT IO N
A. (5E,7E)-9,l0-secocholesta-5j7jl0( 19)-triene-laj3ß,25-triol
(rrans-calcitriol). C ald u m diacetate.
C o n te n t
CHARACTERS
A p p e a ra n c e
W hite or almost white, hygroscopic powder.
S o lu b ility
(96 per cent).
A. It gives reaction (b) of caldum (2.3.1).
B. It gives reaction (b) of acetates (2.3.1).
B. (5Zj7£)-9j 10-secocholesta-5j7j 10(19)-triene-l ß,3ß,25-triol TESTS
(lß-caldtriol), S o lu tio n S
p H (2.2.3)
7.2 to 8.2.
Dilute 5.0 m L o f solution S to 10.0 m L with carbon dioxide-
free water R.
R e ad ily o x id isab le su b stan ces
Dissolve 2.0 g in boiling water R and dilute to 100 m L with
boiling water R, add a few glass beads, 6 m L of 5 M sulfuric
C. (6aR,72?,9a/?)-l l-[(3S,5.R)-3,5-dihydroxy-2- acid and 0.3 m L of 0.02 Mpotassium permanganate, mix, boil
m ethylcyclohex-l-enyl]-7-[(lJ?)-5-hydroxy-l,5- gently for 5 min and allow the predpitate to settle. T h e pink
dim ethylhexyi]-6a-methyl-2-phenyl-5,6,6a,7,8,9,9a,l 1- colour in the supernatant is not completely discharged.
octahydro-l//j4aii-cy d o p en ta \f\ [1,2,4] triazolo [1,2- C h lo rid e s (2.4.4)
a]cinnoline-1,3 (2/i)-dione (triazoline adduct o f pre- M aximum 330 ppm.
caldtriol).
___________________________________________________________PhEur same solvent.
F lu o rid e s
M aximum 50 ppm.
Potentiom etry (2.2.36, Method I).
Test solution In a 50 m L volumetric flask, dissolve 0.200 g in
a 10.3 g/L solution of hydrochloric acid R, add 5.0 m L of
fluoride standard solution (1 ppm F) R and dilute to 50.0 m L
with a 10.3 g/L solution o f hydrochloric add R. T o 20.0 m L
of the solution add 20.0 m L of total-ionic-strength-adjiistment
buffer R and 3 m L o f an 82 g/L solution of anhydrous sodium
acetate R. Adjust to pH 5.2 with ammonia R and dilute to
50.0 m L with distilled water R.
Reference solutions T o 0.25 m l^ 0.5 mL, 0.75 m L and
1.0 m l. of fluoride standard solution (10 ppm F) R add
20.0 m L o f total-ionic-strength-adjustment buffer R and dilute to
50.0 m L w ith distilled water R.
2016 Calcium Acetate 1-365
Indicator electrode Fluoride selective. Reference solutions Prepare the reference solutions using
Reference electrode Silver-silver chloride. potassium standard solution (0.2 per cent K) R, diluted as
necessary with water R.
Take into account the addition of fluoride to the test solution
for the calculation. Wavelength 766.5 nm.
N itra te s S o d iu m
T o 10.0 m L o f solution S add 5 m g o f sodium chloride R, M aximum 500 ppm , if intended for use in the m anufacture
0.05 m L o f indigo carmine solution R and add with stirring, o f parenteral preparations or haemodialysis solutions.
10 m L o f nitrogen-free sulfuric acid R. T h e blue colour Atomic emission spectrometry (2.2.22, Method II).
remains for at least 10 min. Test solution Dissolve 1.00 g of the substance to be examined
S u lfates ( 2.4.13) in water R and dilute to 100.0 m L with the same solvent.
Maximum 600 ppm. Reference solutions Prepare the reference solutions using sodium
Dissolve 0.25 g in distilled water R and dilute to 15 m L with standard solution (200 ppm Na) R, diluted as necessary with
the same solvent. water R.
A lu m in iu m (2.4.17) Wavelength 589 nm.
Maximum 1 ppm , if intended for use in the manufacture of S tro n tiu m
peritoneal dialysis solutions, haemofiltration solutions or M aximum 500 ppm , if intended for use in the m anufacture
haemodialysis solutions. o f parenteral preparations or haemodialysis solutions.
Test solution Dissolve 4.0 g o f the substance to be examined Atomic emission spectrometry (2.2.22, Method II).
in 100 m l, o f water R and add 10 m L of acetate buffer solution Test solution Dissolve 2.00 g of the substance to be examined
pH 6.0 R. in water R and dilute to 100.0 m L with the same solvent.
Reference solution Mix 2 m L of aluminium standard solution Reference solutions Prepare the reference solutions using
(2 ppm AQ R, 10 m L of acetate buffer solution pH 6.0 R and strontium standard solution (1.0 per cent Sr) R, diluted as
98 m L o f water R. necessary with water R.
Blank solution M ix 10 m L o f acetate buffer solution pH 6.0 R Wavelength 460.7 nm.
and 100 m L o f water R.
A rsen ic (2.4.2) M aximum 10 ppm.
M axim um 3 ppm.
Dissolve 4.0 g in water R and dilute to 20 m L w ith the same
3.3 m L o f solution S complies with test A. solvent. 12 m L o f the solution complies with test A. Prepare
B a riu m the reference solution using lead standard solution
M aximum 50 ppm . (2 ppm Pb) R.
Inductively coupled plasma-atomic emission spectrometry W a te r (2.5.12)
(2.2.57). M axim um 7.0 per cent, determined on 0.100 g. A dd 2 m L
Test solution Dissolve 5.00 g of the substance to be examined of anhydrous acetic add R to the titration vessel in addition to
in water R and dilute to 100.0 m L w ith the same solvent. the methanol. Clean the titration vessel after each
Reference solutions Prepare the reference solutions using determination.
barium standard solution (0.1 per cent Ba) R, diluted as ASSAY
necessary with water R Dissolve 0.150 g in 100 m L of water R and carry out the
Wavelength 455.4 run. complexometric titration of calcium (2.5.11).
Ir o n (2.4.9) 1 m L o f 0.1 M sodium edetate is equivalent to 15.82 mg
Maximum 20 ppm , if intended for use in the manufacture o f o f C 4HôCa 0 4.
parenteral preparations or haemodialysis solutions. STORA GE
Dilute 5 m L o f solution S to 10 m L o f water R. In an airtight container.
M a g n e s iu m L A B E L L IN G
M axim um 500 ppm. T h e label states, where applicable, that the substance is
Atomic absorption spectrometry (2.2.23, Method II). suitable for u s e in the manufacture o f parenteral
Test solution Dissolve 50.0 m g of the substance to be preparations, peritoneal dialysis solutions, haemoflltration
exam in ed in water R and dilute to 100.0 m L with the same solutions or haemodialysis solutions.
solvent. __________________________________________________________ PhEur
magnesium standard solution (0.1 per cent Mg) R, diluted as
necessary with water R.
Source M agnesium hollow-cathode lamp.
P o ta s s iu m
Maximum 500 ppm , if intended for use in the manufacture
o f parenteral preparations or haemodialysis solutions.
Atomic emission spectrometry (2.2.22, Method II).
in water R and dilute to 25.0 mL w ith the same solvent.
1-366 Calcium Ascorbate 2016
ử ★ Potentiom etry (2.2.36, Method I).

Calcium Ascorbate ử ử
Test solution In a 50 m L volumetric flask, dissolve 1.000 g in
*****
(Ph. Eur. monograph 1182) a 10.3 g/L solution of hydrochloric acid R, add 5.0 m L of
fluoride standard solution (1 ppm F) R and dilute to 50.0 m L
with a 10.3 g/L solution o f hydrochloric acid R. T o 20.0 m L
o f the solution add 20.0 m L o f total-ionic-strength-adjustment
buffer R and 3 m L of an 82 g/L solution of anhydrous sodium
2H20 acetate R. Adjust to p H 5.2 with ammonia R and dilute to
Reference solutions T o 0.25 mL, 0.5 mL, 1.0 m i , 2.0 m L and
5.0 m L o f fluoride standard solution (10 ppm F) R add
C12H14C a0 12j2H20 426.3 5743-28-2 20.0 m L o f total-ionic-strength-adjustment buffer R and dilute to
A c tio n a n d u se Indicator electrode Fluoride selective.
Excipient. Reference electrode Silver-silver chloride.
PhEur_______________ Take into account the addition o f fluoride to the test solution
for the calculation.
D E F IN IT IO N
C opper
Calcium di[(1?)-2-[(5)-152-dihydroxyethyl]-4-hy droxy-5-oxo-
M axim um 5 ppm.
2//-furan-3-olate] dihydrate.
C o n te n t
99.0 per cent to 100.5 per cent of C 12H 14C a0i2j2H 20 . Test solution Dissolve 2.0 g in a 9.7 g/L solution o f nitric
acid R and dilute to 25.0 m L with the same a d d solution.
CHARACTERS
A p p e a ra n c e
standard solution (10 ppm Cu) R, diluting with a 9.7 g/L
W hite or slightly yellowish, crystalline powder.
solution of nitric acid R.
S o lu b ility Source Copper hollow-cathode lamp.
Freely soluble in water, practically insoluble in ethanol
(96 per cent).
Ir o n
First identification A , B, E
M aximum 2 ppm .
Test solution Dissolve 5.0 g in a 9.7 g/L solution o f nitric
B. Infrared absorption spectrophotometry (2.2.24). add R and dilute to 25.0 m L with the same a d d solution.
Comparison Ph. Eur. reference spectrum of calcium ascorbate. Reference solutions Prepare the reference solutions using iron
C. D ilute 1 m L of solution S (see Tests) to 10 m L with standard solution (10 ppm Fe) R, diluting with a 9.7 g/L
water R. T o 2 m L of the solution add 0.2 m L o f a 100 g/L solution o f nitric add R
solution of ferrous sulfate R. A deep violet colour develops. Source Iron hollow-cathode lamp.
D . T o 1 m L o f solution S add 0.2 m L of dilute nitric acid R Wavelength 248.3 nm.
and 0.2 m L o f silver nitrate solution R2. A grey precipitate is
formed.
E. T h e substance gives reaction (b) o f calcium (2.3.1). H eav y m e ta ls (2.4.8)
M aximum 10 ppm.
TESTS
S o lu tio n S
using 2.0 m L o f lead standard solution (10 ppm Pb) R.
50.0 m L with the same solvent. L oss o n d ry in g (2.2.32)
an oven at 105 °c for 2 h.
than reference solution Y 6 (2.2.2, MethodII). Examine the A SSA Y
colour of the solution immediately after preparation of the Dissolve 80.0 m g in a mixture o f 10 m L o f dilute sui/uric
solution. add R and 80 m L o f carbon dioxide-free water R. Add 1 m L of
p H (2.2.3)
starch solution R. T itrate with 0.05 M iodine until a persistent
violet-blue colour is obtained.
1 m L o f 0.05 M iodine is equivalent to 10.66 mg
o f C 12H14C a 0 12,2H20 .
+ 95 to + 97 (dried substance), determined using freshly
prepared solution S. STORA GE
R e la te d su b sta n c e s In a non-metaJlic container, protected from light.
T he thresholds indicated under Related substances __________________________________________________________ PhEur
pharmaceutical use (2034) do no t apply.
F lu o rid e s
M axim um 10 ppm.
2016 Calcium Chloride Dihydrate 1-367
***** changes and then add 2 m L in excess. H eat to boiling and

Calcium Carbonate ★ ★ add 50 m l. of h o t ammonium oxalate solution R. Allow to
* * * * *
(Ph. Ettr. monograph 0014) stand for 4 h, dilute to 100 m L with water R and filter
through a suitable filter. T o 50 m L of the filtrate add
C aC 03 100.1 471-34-1
0.25 m l. o f sulfuric acid R. Evaporate to dryness on a water-
A c tio n a n d u se b ath and ignite to constant mass at 600 ± 50 °C.
Antacid. T he residue weighs a maximum o f 7.5 mg.
P re p a r a tio n s H eav y m e ta ls (2.4.8)

C aldum Carbonate Chewable Tablets M aximum 20 ppm .
C aldum and Colecaldferol Tablets 12 m L o f solution S complies with test A. Prepare the
Chewable C ald u m and Colecaldferol Tablets
PhEtr__________________________________________ M aximum 2.0 per cent, determined on 1.000 g by drying in
D E F IN IT IO N an oven at 200 ± 10 °C.
C o n te n t A SSAY
98.5 per cent to 100.5 per cent (dried substance). Dissolve 0.150 g in a mixture of 3 m L o f dilute hydrochloric
CHARACTERS acid R and 20 m l. o f water R. Boil for 2 m in, allow to cool
and dilute to 50 m L with water R. C any out the
A p p e a ra n c e
complexometric titration of caldum (2.5.11).
1 m l. of 0.1 M sodium edetate is equivalent to 10.01 mg
S o lu b ility
o f C a C 0 3.
Practically insoluble in water.
A. It gives the reaction of carbonates (2.3.1).
B. 0.2 m L o f solution S (see Tests) gives the reactions of functions of the substance when used as an excipient (see
caldum (2.3.1). chapter 5.15). This section is a non-mandatory part of the
TESTS monograph and it is not necessary to verify the characteristics to
S o lu tio n S demonstrate compliance. Control of these characteristics can
Dissolve 5.0 g in 80 m L of dilute acetic acid R. W hen the however contribute to the quality of a medicinal product by
effervescence ceases, boil for 2 min. Allow to cool, dilute to improving the consistency of the manufacturing process and the
100 m L with dilute acetic acid R and filter, if necessary, performance of the medicinal product during use. Where control
through a sintered-glass filter (2.1.2). methods are cited, they are recognised as being suitable for the
purpose, but other methods can also be used. Wherever results for
S u b sta n c e s in so lu b le in acetic a c id
a particular characteristic are reported, the control method must be
M axim um 0.2 p er cen t
indicated
W ash any residue obtained during the preparation o f
The following characteristics may be relevantfor calcium carbonate
solution S with 4 quantities, each of 5 m L, of hot water R
used as filler in tablets and capsules.
and dry at 100-105 °C for 1 h. T he residue w dghs a
m aximum of 10 mg. P a rtic le -siz e d is trib u tio n (2.931 or 2.938).
C h lo rid e s (2.4.4) P o w d e r flow (2.936)
M axim um 330 ppm . __________________________________________________ • PhEur
D ilute 3 m L of solution S to 15 m L with water R.

S u lfates (2.4.13)
M aximum 0.25 p er cen t
Calcium Chloride Dihydrate ★* * * ★
D ilute 1.2 m L o f solution S to 15 m L with distilled water R. ★ ★
A rse n ic (2.4.2, Method A) *****
M axim u m 4 ppm , d eterm ined on 5 m L o f solution S.
CaCl2,2H20 147.0 10035-04-8
B a riu m
P re p a ra tio n s
T o 10 m L o f solution S add 10 m L of calcium sulfate
C ald u m Chloride Injection
solution R. After at least 15 min, any opalescence in the
solution is no t m ore intense than that in a mixture o f 10 m L Com pound Sodium Lactate Infusion
o f solution S and 10 m L of distilled water R. PhEur_____________________________________
Ir o n (2.4.9) D E F IN IT IO N
M axim u m 200 ppm . C o n te n t
Dissolve 50 m g in 5 m L of dilute hydrochloric acid R and 97.0 per cent to 103.0 per cent o f CaCl2,2H 20 .
dilute to 10 m L w ith water R.
CHARACTERS
M a g n e s iu m a n d alk ali m e ta ls A p p e a ra n c e
M axim um 1.5 p e r c e n t W hite or almost white, crystalline powder, hygroscopic.
Dissolve 1.0 g in 12 m L of dilute hydrochloric acid R. Boil the S o lu b ility
solution for about 2 min and add 20 m L o f water R, 1 g o f Freely soluble in water, soluble in ethanol (96 per cent).
ammonium chloride R and 0.1 m L o f methyl red solution R.
A dd dilute ammonia R1 until the colour of the indicator
1-368 Calcium Chloride 2016
ID E N T IF IC A T IO N 1 m L of 0.1 M sodium edetate is equivalent to 14.70 m g

A. Solution S (see Tests) gives reaction (a) o f chlorides o f CaCl2,2H20 .
(2.3.1). L A B E L L IN G
B. It gives the reactions of calcium (2.3.1). T h e label states, where applicable, th at die substance is
C. It complies with the limits o f the assay. suitable for use in the m anufacture o f dialysis solutions.
TESTS STO RA G E
S o lu tio n S In an airtight container.
Dissolve 10.0 g in carbon dioxide-free water R prepared from PhEur
than reference solution Y6 (2.2.2, Method IT).
★**★ ★
Calcium Chloride Hexahydrate ★ ★
T o 10 m L of freshly prepared solution S add 0.1 m L of (Ph Eur monograph 0707) *****
phenolphthalein solution R. If the solution is red, not more
CaCl2,6H20 219.1 7774-34-7
than 0.2 m L o f 0.01 M hydrochloric add is required to
discharge the colour and if the solution is colourless, not PhEur__________________________________________
m ore than 0.2 m L o f 0.01 M sodium hydroxide is required to D E F IN IT IO N
tu rn it red. C o n te n t
S u lfates (2.4.IT) 97.0 per cent to 103.0 per cent o f CaCl2,6 H 20 .
M aximum 300 ppm. CHARACTERS
D ilute 5 m L o f solution S to 15 m L with disdUed water R. A p p e a ra n c e
A lu m in iu m W hite or almost white, crystalline mass or colourless crystals.
T o 10 m L o f solution S add 2 m L of ammonium chloride S o lu b ility
solution R and 1 m L o f dilute ammonia R1 and boil the Very soluble in water, freely soluble in ethanol (96 per cent).
solution. N o turbidity or precipitate is formed. It solidifies at about 29 °C.
If intended for vise in the manufacture o f dialysis solutions,
the above test is replaced by the following test for aluminium
(2.417): maximum 1 ppm. A. Solution S (see Tests) gives reaction (a) of chlorides
(2.3.1).
Prescribed solution Dissolve 4 g in 100 m L o f water R and add
10 m L of acetate buffer solution pH 6.0 R. B. It gives the reactions of calcium (2.3.1).
Reference solution Mix 2 m L o f aluminium standard solution C. It complies with the limits of the assay.
(2 ppm Al) R, 10 m L o f acetate buffer solution pH 6.0 R and TESTS
98 m L of water R. S o lu tio n S
Blank solution M ix 10 m L of acetate buffer solution pH 6.0 R Dissolve 15.0 g in carbon dioxide-free water R prepared from
and 100 m L o f water R. distilled water R and dilute to 100 m L with the same solvent.
B a riu m A p p e a ra n c e o f solution
T o 10 m L o f solution S add 1 m L of calcium sulfate Solution S is clear (2.2.1) and n o t more intensely coloured
solution R. After at least 15 min, any opalescence in the th an reference solution Y6 (2.2.2, Method II).
solution is n o t more intense than that in a mixture of 1 m L Acidity or alkalinity
of distilled water R and 10 m l, o f solution S. T o 10 m L o f freshly prepared solution S add 0.1 m L of
Ir o n (2.4.9) phenolphthalein solution R. If the solution is red, not more
M aximum 10 ppm , determined on solution S. than 0.2 m L of 0.01 M hydrochloric add is required to
M a g n e s iu m a n d a lk ali m e ta ls discharge the colour and if the solution is colourless, n o t
M aximum 0.5 per cent. m ore than 0.2 m L o f 0.01 M sodium hydroxide is required to
tu rn it red.
T o a mixture o f 20 m L of solution S and 80 m L of water R
add 2 g of ammonium chloride R and 2 m l. of dilute S u lfa te s (2.4. IS)
ammonia R l, heat to boiling and pour into the boiling Maximum 200 ppm.
solution a h o t solution o f 5 g of ammonium oxalate R in D ilute 5 m L of solution S to 15 m L w ith distilled water R.
75 m L of water R. Allow to stand for 4 h, dilute to 200 m L A lu m in iu m
with water R and filter through a suitable filter. T o 100 m L T o 10 m L o f solution S add 2 m L of ammonium chloride
of the filtrate add 0.5 m L of sulfuric add R. Evaporate to solution R and 1 m l. of dilute ammonia R l. H eat to boiling.
dryness on a water-bath and ignite to constant mass at N o turbidity or precipitate is formed.
600 ± 50 °C. T he residue weighs a maximum of 5 mg
I f intended for use in the m anufacture o f dialysis solutions,
H eav y m e ta ls (2.4.8) the above test is replaced by the following test for aluminium
Maximum 20 ppm. (2.4.17): maximum 1 ppm.
12 m L of solution S complies with test A. Prepare the Prescribed solution Dissolve 6 g in 100 m L o f water R and add
reference solution using lead standard solution (2 ppm Pb) R. 10 m L o f acetate buffer solution pH 6.0 R.
A SSA Y Reference solution Mix 2 m L o f aluminium standard solution
Dissolve 0.280 g in 100 m L o f water R and carry out the (2 ppm Al) R, 10 m L of acetate biffer solution pH 6.0 R and
complexometric titration of calcium (2.5.11). 98 m L of water R.
2016 Calcium Dobesilate 1-369
Blank solution M ix 10 m L of acetate buffer solution pH 6.0 R Test solution Dissolve 0.100 g in water R and dilute to
and 100 m L of water R 200.0 m L with the same solvent. D ilute 5.0 m L o f this
B a riu m solution to 100.0 m L with water R.
T o 10 m L o f solution S add 1 m L o f calcium sulfate Spectral range 210-350 nm.
solution R. After at least 15 min, any opalescence in the Absorption maxima At 221 nm and 301 nm.
solution is not m ore intense than th at in a mixture of 1 m L Specific absorbance at the absorption maximum at 301 nm
of distilled water R and 10 m L of solution S. 174 to 181.
I r o n (2.4.9) B. M ix 1 m L o f ferric chloride solution R2, 1 m L o f a freshly
M axim um 7 ppm , determined on solution S. prepared 10 g/L solution of potassium ferricyanide R and
M a g n e s iu m a n d alk ali m etals 0.1 m L o f nitric add R. T o this mixture add 5 m L of freshly
M axim um 0.3 p e r cent. prepared solution S (see Tests): a blue colour and a
T o a mixture of 20 m L of solution S and 80 m L o f water R precipitate are immediately produced.
add 2 g of ammonium chloride R and 2 m L o f dilute ■C. 2 m L of freshly prepared solution S gives reaction (b) o f
ammonia R l, h eat to boiling and pour into the boiling calcium (2.3.1).
solution a h o t solution of 5 g of ammonium oxalate R in TESTS
75 m L of water R. Allow to stand for 4 h, dilute to 200 m L S o lu tio n S
with water R and filter through a suitable filter. T o 100 m L
o f the filtrate add 0.5 m L of sulfuric add R. Evaporate to 100 m L with the same solvent.
dryness on a w ater-bath and ignite to constant mass at
600 ± 50 °C. T h e residue weighs a maximum o f 5 mg. A p p e a ra n c e o f so lu tio n
Solution S, when freshly prepared, is clear (2.2.1) and
H eav y m e ta ls (2.4.8) colourless (2.2.2, Method II).
M axim um 15 ppm .
p H (2.2.3)
A SSAY Liquid chromatography (2.2.29). Keep all solutions at 2-8 °C.
Dissolve 0.200 g in 100 m L o f water R. Carry out the
Buffer solution Dissolve 1.2 g o f anhydrous sodium dihydarogen
complexometric titration of calcium (2.5.11).
phosphate R in 900 m L o f waterfor chromatography R, adjust
1 m L of 0.1 M sodium edetate is equivalent to 21.91 mg to p H 6.5 with disodium hydrogen phosphate solution R and
o f CaCl2,6H20 . dilute to 1000 m L with waterfor chromatography R.
L A B E L L IN G Test solution Dissolve 0.100 g o f die substance to be
T he label states, where applicable, that the substance is exam in e d in water R and dilute to 10.0 m L with the same
suitable for use in the manufacture o f dialysis solutions. solvent.
Ph Eur Reference solution (a) Dilute 1.0 m L of the test solution to
100.0 m L with water R Dilute 1.0 m L of this solution to
Reference solution (b) Dissolve 10 m g of the substance to b e
***** ex a m in ed and 10 mg of hydroquinone R (impurity A) in
Calcium Dobesilate Monohydrate ★ ★ water R and dilute to 10 m L with the same solvent. Dilute
(Ph Eur monograph 1183) *★ * 1 m l . o f this solution to 100 m l . with water R.
Column:
— size: 1 = 0.25 m , 0 = 4.6 mm ;
Caz
XX OH
, H20
— stationary phase: spherical end-capped octadecylsûyl silica gel
for chromatography R (5 (im).
M obile phase acetonitrile R l , buffer solution (10:90 V IV ).
C 12H 10CaO10S*H 2O 436.4 20123-80-2 Detection Spectrophotometer at 220 nm.
PhEir__________________
Irtjection 10 |jL.
D E F IN IT IO N Run time 2.5 times the retention time o f dobesilate.
Calcium di(2,5-dihydroxybenzenesulfonate) monohydrate. Relative retention W ith reference to dobesilate (retention
C o n te n t time = about 6 min): impurity A = about 1.7.
99.0 per cent to 101.0 per cent (anhydrous substance). System suitability: reference solution (b):
CHARACTERS — resolution: m inim um 8.0 between the peaks due to
dobesilate and impurity A.
A p p e a ra n c e
W hite or almost white, hygroscopic powder. Limits:
S o lu b ility
peak area o f im purity A by 0.6;
Very soluble in water, freely soluble in anhydrous ethanol,
— impurity A: n o t more than the area of the principal peak
very slightly soluble in 2-propanol, practically insoluble in
methylene chloride.
(0.1 per cent);
ID E N T IF IC A T IO N — unspecified impurities: for each impurity, no t m ore than the
A. Ultraviolet and visible absorption spectrophotometry area o f the principal peak in the chromatogram obtained
(2.2.25). w ith reference solution (a) (0.10 per cent);
1-370 Calcium Folinate 2016
— total: not more th a n twice the area o f the principal peak in — caldum (Ca; A r 40.08): 7.54 per cent to 8.14 per cent
the chromatogram obtained with reference solution (a) (anhydrous substance).
(0.2 per cent); It contains a variable quantity of water.
— disregard limir. 0.5 tim es the area o f the principal peak in
CHARACTERS
(0.05 per cent). A p p e a ra n c e
White or light yellow, amorphous or crystalline, hygroscopic
H e a v y m e ta ls (2.4.8) powder.
M axim um 15 ppm.
S o lu b ility
1.0 g complies with te s t C. Prepare the reference solution
Sparingly soluble in water, practically insoluble in acetone
using 1.5 m l. o f lead standard solution (10 ppm Pb) R.
and in ethanol (96 per cent).
Ir o n (2.4.9) T he amorphous form may produce supersaturated solutions
M axim um 10 ppm , determ ined on 10 m L of solution S. in water.
W a te r (2.5.12)
4.0 p e r cent to 6.0 p e r cent, determined on 0.500 g.
First identification A , B, D
A SSA Y Second identification A , C, D
Dissolve 0.200 g in a m ixture o f 10 m l, of water R and
40 m L of dilute sulfuric add R. T itrate with 0.1 M cerium
sulfate, d eterm ining th e end-point potentiometrically (2 .2 .2 0 ). B. Infrared absorption spectrophotometry (2.2.24).
1 m L o f 0.1 M cerium sulfate is equivalent to 10.45 m g of Preparation Discs.
Ci2HxoCaOioS2- Comparison caldum folinate CRS.
STO RA G E If the spectra obtained show differences, dissolve the
In an airtight container, protected from light substance to be examined and the reference substance
separately in the minim um volume of water R and add
IM P U R IT IE S dropwise suffident acetone R to produce a predpitate. Allow
Spedfied impurities A to stand for 15 min, collect the predpitate by centrifugation,
wash die predpitate with 2 small quantities o f acetone R and
HO
dry. Record new spectra using the residues.
OH
A. benzene-1,4-diol (hydroquinone). in a 3 per cent V/V solution of ammonia R and dilute to
PhEur
Reference solution Dissolve 15 m g o f caldum folinate CRS in a
3 per cent V/V solution o f ammonia R and dilute to 5 m l,
★ ★ Plate cellulose for chromatography F2 5 4 R as the coating
Calcium Folinate ★ ★
substance.
Mobile phase T he lower layer of a mixture of 1 volume of
isoamyl alcohol R and 10 volumes o f a 50 g/L solution of caric
add R previously adjusted to p H 8 with ammonia R.
Application 5 \iL.
Drying In air.
Results T h e prindpal spot in the chromatogram obtained with
C20H 21CaN7O7^ H 2O 511.5 the test solution is sim ilar in position and size to the prindpal
spot in die chromatogram obtained with the reference
(anhydrous substance) solution.
D . It gives reaction (b) o f caldum (2.3.1).
A c tio n a n d u se Carry out the tests and the assay as rapidly as possible, protected
A ntidote to folic a d d antagonists. from actinic light.
TESTS
C aldum Folinate Injection
S o lu tio n S
C ald u m Folinate Tablets Dissolve 1.25 g in carbon dioxide-free water R, heating at
PhEir------------------------------------ :-----------------------------------------
40 °C if necessary, and dilute to 50.0 m L with the same
solvent.
D E F IN IT IO N
C aldum (2S)-2-[[4- [ [[ (6 RS) -2-amino-5-formyl-4-
Solution S is clear (2.2.1) and its absorbance (2.2.25) at
oxo 1,4,5,6,7,8-hexahydropteridin-6-
420 nm is not greater than 0.60. Use water R as the
yl] methyl] amino]benzoyl] amino]pentanedioate.
compensation liquid.
C o n te n t
p H (2.2.3)
— caldum folinate ^ o ^ j C a N y O y ) : 97.0 per cent to
2016 Calcium Folinate 1-371
Specific optical rotation (2.2.7) phosphate R, previously adjusted to p H 7.8 with phosphoric
+ 14.4 to + 18.0 (anhydrous substance), determ ined on add R.
solution S. Flow rate 1 mL/min.
Acetone, ethanol and methanol Detection Spectrophotom eter at 280 nm.
Head-space gas chromatography (2.2.28) U se the standard Injection 10 pL o f the test solution and reference
additions method. solutions (b), (c), (d) and (e).
Test solution Dissolve 0.25 g o f the substance to be examined Run time 2.5 times the retention time o f folinate.
in water R and dilute to 10.0 m L with the same solvent.
System suitability: reference solution (e):
Reference solution Dilute 0.125 g of acetone R, 0.750 g of — resolution: minim um 2.2 between the peaks due to fo linate
anhydrous ethanol R and 0.125 g o f methanol R in water R and and impurity D .
dilute to 1000.0 m L with water R Limits:
Column: — impurity D: n o t m ore than the area o f the principal peak
— material: fused silica; in the chromatogram obtained with reference solution (c)
— sizer. I = 10 m , 0 = 0.32 mm; (1 per cent);
— stationary phase: styrene-divinylbenzene copolymer R. — impurities A , B, C, E, F, G: for each impurity, not more
Carrier gas nitrogen for chromatography R. than the area o f the principal peak in the chromatogram
Flow rate 4 mL/min. obtained with reference solution (b) (1 per cent);
Static head-space conditions that may be used: — sum of impurities other than D: not m ore than 2.5 times the
— equilibration temperature. 80 °C; area of the principal peak in the chromatogram obtained
— equilibration time: 20 min; with reference solution (b) (2.5 per cent);
— pressurisation time. 30 s. — disregard limit: the area of the principal peak in the
chromatogram obtained with reference solution (d)
Temperature:
(0.1 per cent).
Chlorides
Time Temperature
(min) CC)
Column 0 -6 125 -> 185 Dissolve 0.300 g in 50 m L of water R heating at 40 °C if
necessary. Add 10 m L o f 2 M nitric add and titrate with
6 - 15 185
0.005 M silver nitrate determining the end-point
Injection port 250 potentiometrically (2.2.20).
Detector 250 1 m L of 0.005 M silver nitrate is equivalent to 0.177 m g o f
Cl.
Detection Flam e ionisation. M aximum 50 ppm.
Irqecdon A t least 3 times. 1.0 g complies with test F. Prepare the reference solution
Limits: using 5 m L o f lead standard solution (10 ppm Pb) R.
— acetone: maximum 0.5 p er cent;
Platinum
— ethanol: maximum 3.0 per cent;
M aximum 20 ppm.
— methanol: m aximum 0.5 per c e n t
Related substances
Liquid chromatography (2.2.29). Test solution Dissolve 1.00 g in water R and dilute to
examined in water R and dilute to 10.0 m L with the same Reference solutions Prepare the reference solutions using
solvent. platinum standard solution (30 ppm Pt) R, diluted as necessary
w ith a mixture o f 1 volume of nitric add R and 99 volumes of
Reference solution (a) Dissolve 10.0 m g o f calcium folinate CRS
water R.
Source Platinum hollow-cathode lamp.
to 100.0 m L with water R. Wavelength 265.9 nm.
Reference solution (c) Dissolve 10.0 mg o f formylfotic acid CRS Water (2.5.12)
(impurity D) in the mobile phase and dilute to 100.0 m L M aximum 17.0 per cent.
with the mobile phase. Dilute 1.0 m L o f this solution to Dissolve 0.100 g in a mixture o f 50 m L o f the titration
10.0 m L with water R. solvent and 15 m L o f formamide R. Stir for about 6 m in
Reference solution (d) Dilute 1.0 m l, o f reference solution (b) before titrating and use a suitable titrant th at does not
to 10.0 m L with water R. contain pyridine.
Reference solution (e) Dilute 5.0 m L of reference solution (c) Bacterial endotoxins (2.6.14)
to 10.0 m L with reference solution (b). Less than 0.5 IU/mg, if intended for use in the m anufacture
Column: o f parenteral preparations without a further appropriate
— size. I — 0.25 m , 0 = 4 m m ; procedure for the removal of bacterial endotoxins.
— stationary phase, octadecylsifyl silica gel for chromatography R ASSAY
(5 pm); Calcium
— temperature. 40 °C. Dissolve 0.400 g in 150 m L o f water R and dilute to 300 m L
Mobile phase Mix 220 m L o f methanol R and 780 m L o f a with the same solvent. Carry out the complexometric
solution containing 2.0 m L o f tetrabutylammonium hydroxide titration o f calcium (2.5.11).
solution (400 g/L) R and 2.2 g o f disodium hydrogen
1-372 Calcium Glucoheptonate 2016
1 m L of 0.1 M sodium edetate is equivalent to 4.008 m g of O CHO

Ca. . O
n and enantiomer
Calcium folinate
Liquid chromatography (2.2.29) as described in the test for h 2n
A H H
Injection T est solution and reference solution (a). E. 4-[[[(6ftS)-2-amino-5-formyi-4-oxo-l,4,5,6,7,8-
System suitability: hexahydropteridin-6-yl] methyl] amino] benzoic a d d
— repeatability: maximum relative standard deviation of (5-formyltetrahydropteroic ad d ),
2.0 per cent after 6 injections o f reference solution (a).
Calculate the percentage content of C 2oH 2iC aN 707 from the
declared content of calcium folinate CRS.
STORAGE CC^H
In an airtight container, protected from light. If the substance
is sterile, store in a sterile, airtight, tam per-proof container. H,N
IMPURITIES
Specified impurities A, B, C, D , E, F , G F. R = C H O : (25)-2-[[4-[[(2-am ino-4-oxo-l,4,7,8-
tetrahydropteridin-6-
yl) methyl] formylamino] benzoyl] amino] pentanedioic a d d
9 H COjH
(10-formyldihydrofolic ad d ),
G. R = H : (25) -2- [ [4- [ [(2-amino-4-oxo-1,4,7,8-
H,N c o 2h
yl)methyl] amino] benzoyl] amino]pentanedioic a d d
(dihydrofolic add).
A. (2S)-2 [(4-aminobenzoyl) amino]pentanedioic acid,
PhEur
o H COjH
c o 2h Calcium Glucoheptonate *****

★ ★
H H and epimer at C*
B. (2«S)-2-[[4- [ [[(6i?S)-2-amino-5-formyl-4-oxo-l,4,5,6,7,8-
hexahydropteridin-6-
Ca2* and epimer at C*
yl]methyi]formylamino]benzoyl]amino]pentanedioic a d d
(5,10-diformyltetrahydrofolic ad d ),
O H CO2H
Ci4H26C a016 490.4
o
Action and use
COjH
U sed in treatm ent o f caldum defidency.
h2n ^
x Xn ^ Tn^ ' *
2 H PhEur_____________________________________
DEFINITION
C . (2S)-2-[[4-[[(2-amino-4-oxo-1,4-dihydropteridin-6-
Mixture in variable proportions, o f caldum di(u-glycero-D-
yl)methyl]amino]benzoyl]amino]pentanedioic a d d (folic
ûZo-heptonate) and caldum di(D-^?yccrio-D-tii>-heptonate).
ad d ),
Content
98.0 per cent to 102.0 per cent o f caldum 2,3,4,5,6,7-
o h c o 2h -
hexahydroxyheptanoate (dried substance).
o r i T N '
CHARACTERS
'CC^H Appearance
White or very slightly yellow, amorphous powder,
iH0 hygroscopic.
H
Solubility
D. (26)-2-[[4-[ [(2-amino-4-oxo-1,4-dihydropteridin-6- Very soluble in water, practically insoluble in acetone and in
yl)methyl]formylamino]ben 2oyl]amino]pentanedioic a d d ethanol (96 per cent).
(10-formylfolic ad d ), IDENTIFICATION
in 1 m L o f water R.
Reference solution (a) Dissolve 20 m g o f calcium
glucoheptonate CRS in 1 m l. of water R.
2016 Calcium Gluconate 1-373
Reference solution (b) Dissolve 10 mg of calcium gluconate CRS Ir o n (2.4.9)

in 0.5 m L o f die test solution and dilute to 1 m L with Maximum 40 ppm.
water R. D ilute 2.5 m L o f solution S to 10 m L w ith water R
Plate cellulose for chromatography R1 as the coating substance. H eav y m e ta ls (2.4.8)
Mobile phase anhydrous formic add R, water R, acetone R, M aximum 10 ppm .
butanol R (20:20:30:30 V/V/V/V); use a freshly prepared Dissolve 2.0 g in 10 m L of buffer solution pH 3.5 R and dilute
mixture. to 20 m L with water R. 12 m L o f the solution complies with
Application 10 jiL as bands o f 20 m m by 2 mm. test A. Prepare the reference solution using lead standard
Development In a tank previously allowed to saturate for solution (1 ppm Pb) R.
10 min, over a path o f 12 cm. L o ss o n d ry in g (2.2.32)
Drying In air. M aximum 5.0 per cent, determined on 1.000 g by drying in
Detection Spray with 0.02 M potassium permanganate. an oven at 105 °C for 3 h.
System suitability: reference solution (b): B a c te ria l en d o to x in s (2.6.14)
— the chromatogram shows 2 clearly separated spots. Less than 167 IU/g, if intended for use in the m a n u f a c t u r e of
Results T he principal spot in the chromatogram obtained with parenteral preparations without a further appropriate
the test solution is similar in position and size to the prindpal procedure for the removal of bacterial endotoxins.
spot in the chromatogram obtained with reference ASSAY
solution (a). Dissolve 0.800 g in a mixture o f 2 m L o f 3 M hydrochloric
Results add and 150 m L of water R. While stirring, add 12.5 m L of
B. 0.2 m L of solution S (see Tests) gives reaction (b) of 0.1 M sodium edetate, 15 m L of 1 M sodium hydroxide and
caldum (2.3.1). 0.3 g of hydroxynaphthol blue, sodium salt R. T itrate with
0.1 M sodium edetate until the colour changes from violet to
TESTS pure blue.
S o lu tio n S
of C 14H 26C aO i6.
STO R A G E
Solution S is clear (2.2.1) and not more intensdy coloured In an airtight container. If the substance is sterile, store in a
than reference solution Y6 (2.2.2, Method II). sterile, airtight, tam per-proof container.
p H (2.2.3) ----------------------------------------------------------------------------------- ----- PhEur

R e d u c in g su g a rs
M axim um 1 per cent, expressed as glucose.
Dissolve 1.0 g in 5 m L o f water R with the aid o f gentle heat.
Calcium Gluconate *****
*+ ■*.*
Cool and add 20 m L o f cupn-dtnc solution R and a few glass (Ph Eur. monograph 0172) *
beads. H eat so th at boiling begins after 4 min and maintain
boiling for 3 min. Cool rapidly and add 100 m L o f a
2.4 per cent V/V solution of glacial acetic add R and 20.0 m L
Ca*+
o f 0.025 M iodine. W ith continuous shaking, add 25 m L of a
mixture of 6 volumes o f hydrochloric add R and 94 volumes
of water R until the predpitate dissolves, titrate the excess of
iodine with 0.05 M sodium tkiostdfate using 1 m L o f starch
solution R added towards the end of the titration, as indicator. C ^H ^C aO Ô 448.4 18016-24-5
N o t less than 12.6 m L o f 0.05 M sodium tkiostdfate is
Action and use
required.
U sed in treatm ent of caldum defidency.
C y an id e
Dissolve 5.0 g in 50 m L o f water R and add 2.0 g o f tartaric
C ald u m G luconate Tablets
add R. Place this solution in a distillation apparatus (2.2.11).
T h e plain bend adapter attached to the end of the condenser Chewable C aldum Gluconate Tablets
has a vertical p art that is long enough to extend to 1 cm Effervescent C aldum Gluconate Tablets
from the bottom of a 50 m L test-tube used as a recdver.
PhEur__________________________________________________________
Place 10 m l. o f water R and 2 m L of 0.1 M sodium hydroxide
into the recdver. Distil, collect 25 m L o f distillate and dilute D E F IN IT IO N
to 50 m L with water R. T o 25 m L o f this solution add C ald u m bis[(2R,35,4/?,5i?)-2,3,4,5,6-
25 m g of ferrous sulfate R and boil for a short time. After pentahydroxyhexanoate] monohydrate (caldum d i(D -
cooling to about 70 °C add 10 m L of hydrochloric add R l. gluconate) monohydrate).
After 30 min, filter the solution and wash the filter. A yellow C o n te n t
spot appears on the filter; there is no blue or green spot. 98.5 per cent to 102.0 per cent o f C i2H 22C a0i4,H 20 .
CHARACTERS
M axim um 100 ppm.
A p p e a ra n c e
T o 5 m L of solution S, add 10 m L of water R. W hite or almost white, crystalline or granular powder.
S u lfa te s (2.4.13)
S o lu b ility
M axim um 100 ppm , determined on solution S. Sparingly soluble in water, fredy soluble in boiling water.
1-374 Calcium Gluconate 2016
ID E N T IF IC A T IO N Evaporate 100 m L o f the filtrate to dryness and ignite.

A. Thin-layer chromatography (2.2.27). T h e residue weighs a maximum o f 2 mg.
Test solution Dissolve 20 mg o f the substance to be examined H ea v y m e ta ls (2.4.8)
in 1 m L of water R, heating if necessary in a water-bath at M axim um 10 ppm.
60 °C. 2.0 g complies with test D . H eat the substance to be
Reference solution Dissolve 20 m g of calcium gluconate CRS in examined gradually and with care until it is almost
1 m L o f water R, heating if necessary in a water-bath at completely transformed into a white mass and then ignite.
60 °C. Prepare the reference solution using 2 m L of lead standard
Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel solution (10 ppm Pb) R.
plate R (2-10 pm)]. M ic ro b ia l c o n ta m in a tio n
Mobile phase concentrated ammonia R, ethyl acetate R, water R, T A M C : acceptance criterion 103 C FU /g (2.6.12).
ethanol (96 per cent) R (10:10:30:50 V/V/V/V). TY M C: acceptance criterion 102 C FU/g (2.6.12).
Application 1 pL. A SSA Y
Development Over 2/3 o f the plate. Dissolve 0.8000 g in 20 m L o f hot water R, allow to cool and
Drying At 100 °C for 20 min; allow to cool. dilute to 300 m L with water R. Carry out the
Detection Spray with a solution containing 10 g/L o f cerium complexometric titration o f calcium (2.5.11).
sulfate R and 25 g/L o f ammonium molybdate R in dilute 1 m L o f 0.1 M sodium edetate is equivalent to 44.84 mg
sulfuric acid R and heat at 105 °C for about 10 min. o f C i oH -^C aO ^H oO .
Results After 5 m in, the principal spot in the chromatogram __________________________________________________________ PhEur
obtained with the test solution is similar in position, colour
and size to-the principal spot in the chromatogram obtained
with the reference solution.
B. Solution S (see Tests) gives the reactions of calcium ★ ★
(2.3.1). Anhydrous Calcium Gluconate ★ ★
★ ★
TESTS
S o lu tio n S
Dissolve 1.0 g in water R heated to 60 °C and dilute to HO H CCV
Ca2+
At 60 °C, solution S is not more intensely coloured than
reference solution Y6. After cooling, it is not more opalescent
than reference suspension II (2.2.1). C ^H ^C aO n 430.4 299-28-5
O rg a n ic im p u ritie s a n d b o ric a c id
Introduce 0.5 g into a porcelain dish previously rinsed with A c tio n a n d u se
sulfuric acid R and placed in a bath o f iced water. Add 2 m L U sed in treatm ent o f calcium deficiency.
of cooled sulfuric add R and mix. N o yellow or brown colour PhEur____________________________________
develops. Add 1 m l, of chromotrope IIB solution R. A violet
colour develops and does n o t become dark blue. D E F IN IT IO N
T he solution is not m ore intensely coloured than that o f a Anhydrous calcium D-gluconate.
mixture of 1 m L of chromotrope IIB solution R and 2 m L of C o n te n t
cooled sulfuric acid R. 98.0 per cent to 102.0 per cent (dried substance).
S u cro se a n d re d u c in g su g a rs CHARACTERS
Dissolve 0.5 g in a mixture o f 2 m L o f hydrochloric acid R l A p p e a ra n c e
and 10 m L of water R. Boil for 5 m in, allow to cool, add W hite or almost white, crystalline or granular powder.
10 m L of sodium carbonate solution R and allow to stand.
Dilute to 25 m L with water R and filter. T o 5 m L of the S o lu b ility
filtrate add 2 m L of cupri-tartaric solution R and boil for Sparingly soluble in water, ireely soluble in boiling water.
1 min. Allow to stand for 2 min. N o red precipitate is ID E N T IF IC A T IO N
formed. A. Thin-layer chromatography (2.2.27).
C h lo rid es (2.4.4) Test solution Dissolve 20 m g o f the substance to be examined
M aximum 200 ppm . in 1 m L o f water R, heating if necessary in a water-bath at
Dilute 12.5 m L o f solution S to 15 m L with water R. 60 °C.
S u lfates (2.4.13) Reference solution Dissolve 20 m g o f caldum gluconate CRS in
M aximum 100 ppm. 1 m L of water R, heating if necessary in a water-bath at
60 °C.
Dissolve 10.0 g with heating in a mixture of 10 m L o f acetic
acid R and 90 m L o f distilled water R. Plate TLC silica gel plate R (5-40 pm) [or TLC silica gel
plate R (2-10 pm)].
M a g n e siu m a n d alk ali m e ta ls
M aximum 0.4 per c e n t Mobile phase concentrated ammonia R, ethyl acetate R, water R,
ethanol (96 per cent) R (10:10:30:50 V/V/V/V).
Dissolve 1.00 g in 100 m L o f boiling water R, add 10 m L of
ammonium chloride solution R, 1 m L o f ammonia R and, Application 1 pL.
dropwise, 50 m L of h o t ammonium oxalate solution R. Allow Development Over 2/3 of the plate.
to stand for 4 h, dilute to 200 m L with water R and filter. Drying A t 100 °C for 20 m in, then allow to cool.
2016 Calcium Gluconate 1-375
Detection Spray with a solution containing 25 g/L of A SSAY

ammonium molybdate R and 10 g/L of cerium sulfate R in cLUuxe Dissolve 0.350 g in 20 m L of hot water R, allow to cool and
sulfuric acid R, and heat at 100-105 °C for about 10 min. dilute to 300 m L with water R. C arry out the
Results T he principal spot in the chromatogram obtained with complexometric titration o f caldum (2.5.11).
the test solution is similar in position, colour and size to the 1 m L o f 0.1 M sodium edetate is equivalent to 43.04 mg
principal spot in the chromatogram obtained with the o f C i2H 22C aO i4.
reference solution.
------------------------------------------------------------------------------------- PhEur
B. Solution S (see Tests) gives the reactions o f calcium
(2.3.1).
C. Loss on drying (see Tests).
TESTS Calcium Gluconate for Injection *****
S o lu tio n S
Dissolve 1.0 g in water R heated to 60 °C and dilute to (Ph. Eur. monograph 0979) *
A t 60 °C, solution S is not more intensely coloured than
reference solution Y6 (2.2.2, Method II). After cooling, it is
not more opalescent than reference suspension II (2.2.1).
O rg a n ic im p u ritie s a n d b o ric a d d
Place 0.5 g in a porcelain dish previously rinsed with sulfuric
C 12H22Ca014# 20 448.4 18016-24-5
acid R and placed in a bath o f iced water. Add 2 m L of
cooled sulfuric acid R and mix. N o yellow or brown colour A c tio n a n d u se
devdops. Add 1 m L o f chromotrope IIB solution R. A violet U sed in treatm ent of caldum defidency.
colour develops and does not become dark blue. Compare
P r e p a r a tio n
the colour obtained with that o f a mixture of 1 m L o f
C ald u m Gluconate Injection
ckromotrope IIB solution R and 2 m L o f cooled sulfuric acid R.
S u cro se a n d re d u c in g su g a rs PhEur________________________________________________
Dissolve 0.5 g in a mixture o f 2 m L of hydrochloric acid R1 D E F IN IT IO N
and 10 m L o f water R. Boil for 5 min, allow to cool, add
C aldum bis[(2R,3S,4R,5/?)-2,3,4,5,6-
10 m L o f sodium carbonate solution R and allow to stand for
pentahydroxyhexanoate] monohydrate (calcium d i(D -
10 min. Dilute to 25 m L with water R and filter. T o 5 m L of
gluconate) monohydrate).
the filtrate add 2 m l, of cupri-tartaric solution R and boil for
1 min. Allow to stand for 2 min. N o red predpitate is C o n te n t
formed. 99.0 per cent to 101.0 per cent o f C 12H 22 C a0 14,H 20.
C h lo rid e s (2.4.4) CHARACTERS

M aximum 200 ppm . A p p e a ra n c e
Dilute 12.5 m L o f solution S to 15 m L w ith water R. W hite or almost white, crystalline o r granular powder.
Sulfate s (2.4.13) S o lu b ility

M aximum 100 ppm . Sparingly soluble in water, freely soluble in boiling water.
Dissolve 10.0 g with heating in a mixture o f 10 m L o f acetic ID E N T IF IC A T IO N
add R and 90 m L of distilled water R. A. Thin-layer chromatography (2.2.27).
M a g n e siu m a n d alk ali m e ta ls Test solution Dissolve 20 m g of the substance to be examined
Maximum 0.4 p er cent (expressed as M gO). in 1 m L of water R, heating if necessary in a water-bath at
Dissolve 1.00 g in 100 m L o f boiling water R, add 10 m L of 60 °C.
ammonium chloride solution R, 1 m L of ammonia R and, Reference sdution Dissolve 20 mg o f calcium gluconate CRS in
dropwise, 50 m L o f hot ammonium oxalate solution R. Allow 1 m L of water R, heating if necessary in a water-bath at
to stand for 4 h , dilute to 200 m L with water R and filter. 60 °C.
Evaporate 100 m L o f the filtrate to dryness and ignite. Plate TLC silica gel plate R (5-40 pm ) [or TLC silica gel
T he residue w dghs a maximum of 2 mg. plate R (2-10 |im)].
H eav y m e ta ls (2.4.8) Mobile phase concentrated ammonia R, ethyl acetate R, water R,
M aximum 10 ppm . ethanol (96per cent) R (10:10:30:50 V/V/V/V).
2.0 g complies with test D . H eat the substance to be Application 1 |iL.
examined gradually and with care until it is almost Development Over 2/3 o f the plate.
completely transform ed into a white mass, and then ignite.
Drying A t 100 °C for 20 min; allow to cool.
Prepare the reference solution using 2 m L of lead standard
solution (10 ppm Pb) R. Detection Spray with a solution containing 10 g/L o f cerium
sulfate R and 25 g/L of ammonium molybdate R in dilute
L oss o n d ry in g (2.2.32) sulfuric add R and heat at 105 °C for about 10 m in.
M axim um 2.0 p e r cent, determined on 1.000 g by drying in
Results After 5 m in, the principal spot in the chromatogram
obtained with the test solution is similar in position, colour
M ic ro b ia l c o n ta m in a tio n and size to the prindpal spot in the chromatogram obtained
TA M C: acceptance criterion 103 C FU/g (2.6.12). w ith the reference solution.
TYMC: acceptance criterion 102 CFU/g (2.6.12). B. About 20 mg gives reaction (b) o f caldum (2.3.1).
1-376 Calcium Gluconate 2016
TESTS S t x 50
S o lu tio n S S r —S t
T o 10.0 g add 90 m L of boiling distilled water Rand boil
with stirring, for not more than 10 s, until completely St = area of the peak due to oxalate in the
dissolved, then dilute to 100.0 m L with the same solvent. chromatogram obtained with die test solution;
A p p e a ra n c e o f so lu tio n Sr = area o f the peak due to oxalate in die
A t 60 °C, solution S is not more intensely coloured than chromatogram obtained with the reference solution.
reference solution B7 0- After cooling to 20 °C, it is not Limit.
more opalescent than reference suspension II (2.2.1). — oxalates: maximum 100 ppm.
p H (2.2.3) S u c ro se a n d re d u c in g su g a rs
6.4 to 8.3. Dissolve 0.5 g in a mixture of 2 m L o f hydrochloric add R1
Dissolve 1.0 g in 20 m L o f carbon dioxide-free water R, and 10 m L of water R. Boil for 5 m in, allow to cool, add
heating on a water-bath. 10 m L of sodium carbonate solution R and allow to stand for
O rg a n ic im p u ritie s a n d b o ric a c id 10 min. Dilute to 25 m L with water R and filter. T o 5 m L of
the filtrate add 2 m L o f cupri-tartaric solution R and boil for
Introduce 0.5 g into a porcelain dish previously rinsed with
1 min. Allow to stand for 2 m in. N o red precipitate is
sulfuric add R and placed in a bath of iced water. A dd 2 m L
formed.
of cooled sulfuric add R and mix. N o yellow or brown colour
develops. A dd 1 m L o f ckromotrope IIB solution R. A violet C h lo rid e s (2.4.4)
colour develops and does n o t become dark blue. M aximum 50 ppm.
T he solution is not more intensely coloured than that o f a T o 10 m L o f previously filtered solution S add 5 m L o f
mixture of 1 m L o f ckromotrope IIB solution R and 2 m L of water R.
cooled sulfuric add R.
P h o sp h a te s (2.4.11)
O x alates M aximum 100 ppm.
Liquid chromatography (2.2.29). D ilute 1 m L of solution S to 100 m L with water R.
Test solution Dissolve 1.00 g o f the substance to be examined S u lfates (2.4.13)
in water for chromatography R and dilute to 100.0 m L with
M aximum 50 ppm , determined on previously filtered
the same solvent.
solution S.
Reference solution Dissolve 1.00 g o f the substance to be Prepare the standard using a mixture o f 7.5 m L o f sulfate
examined in water for chromatography R, add 0.5 m L o f a standard solution (10 ppm SO J R and 7.5 m L of distilled
0.152 g/L solution of sodium oxalate R in water for water R.
chromatography R and dilute to 100.0 m L with the same
solvent. I ro n
M axim um 5 ppm.
Precolumn'.
— size. 1= 30 mm, 0 = 4 mm; Atomic absorption spectrometry (2.2.23, Method I).
— stationary phase, suitable strong anion-exchange resin Test solution Introduce 2.0 g into a 100 m L
(30-50 nm). polytetrafiuoroethylene beaker and add 5 m L o f nitric add R.
Columns 1 and 2: Boil, evaporating almost to dryness. Add 1 m L o f strong
— size. I = 0.25 m , 0 = 4 mm; hydrogen peroxide solution R and evaporate again almost to
— stationary phase, suitable strong anion-exchange resin dryness. Repeat the hydrogen peroxide treatm ent until a clear
(30-50 pm). solution is obtained. Using 2 m L o f nitric add R, transfer the
Anion-suppresser column Connected in series with the solution into a 25 m L volumetric flask. D ilute to 25.0 m L
precolumn and analytical columns and equipped with a with dilute hydrochloric add R. In the same m anner, prepare a
microm embrane that separates the mobile phase from the compensation solution using 0.65 g o f caldum chloride R1
instead of the substance to be examined.
suppressor regeneration solution, flowing countercurrent to
the mobile phase. Reference solutions Prepare the reference solutions from iron
Mobile phase Dissolve 0.212 g of anhydrous sodium carbonate R standard solution (20 ppm Fe) R, diluting with dilute
and 63 mg o f sodium hydrogen carbonate R in waterfor
hydrochloric add R.
chromatography R and dilute to 1000.0 m L with the same Source Iron hollow-cathode lamp.
solvent. Wavelength 248.3 nm.
Flow rate of the mobile phase 2 mL/min. Atomisation device Air-acetylene flame.
Suppressor regeneration solution 1.23 g/L solution o f sulfuric Carry out a basic correction using a deuterium lamp.
add R in water for chromatography R. M a g n e s iu m a n d a lk a li m e ta ls
Flow rate of the suppressor regeneration solution 4 m l Vmin. M aximum 0.4 per c e n t
Detection Conductance. T o 0.50 g add a mixture o f 1.0 m L o f dilute acetic add R and
Injection 50 |iL 10.0 m L o f water R and rapidly boil, whilst shaking, until
System suitability, reference solution: completely dissolved. T o the boiling solution add 5.0 m L of
— repeatability, maximum relative standard deviation of ammonium oxalate solution R and allow to stand for at least
2.0 per cent for die area o f the peak due to oxalate after 5 6 h. Filter through a sintered-glass filter ( 1. 6) (2.1.2) into a
injections. porcelain crucible. Carefully evaporate the filtrate to dryness
and ignite. T h e residue weighs no t more than 2 mg.
Inject 50 pL of each solution 3 times. Calculate the content
of oxalates in parts per million using die following H eav y m e ta ls (2.4.8)
expression: M axim um 10 ppm.
2016 Calcium Hydrogen Phosphate 1-377
12 m L of solution S complies with test A. Prepare the C itric a c id

reference solution using lead standard solution (1 ppm Pb) R. Shake 5.0 g w ith 20 m L o f carbon dioxide-free water R and
B a c te r ia l e n d o to x in s (2.6.14) filter. T o the filtrate add 0.15 m L o f sulfuric add R and filter
Less than 167 IU/g. again. T o the filtrate add 5 m L of mercuric sulfate solution R
and h eat to boiling. Add 0.5 m L o f a 3.2 g/L solution of
M ic ro b ia l c o n ta m in a tio n potassium permanganate R and again heat to boiling.
T A M C : acceptance criterion 102 CFU /g (2.6.12). N o precipitate is formed.
A SSA Y G ly c e ro l a n d e th a n o l (96 p e r c e n t)-so lu b le su b s ta n c e s
Dissolve 0.350 g in 20 m L of hot water R, allow to cool and M axim um 0.5 p er cent.
dilute to 300 m L with water R. Carry out the
Shake 1.000 g w ith 25 m L of ethanol (96 per cent) R for
complexometric titration of calcium (2.5.11). Use 50 mg of
1 m m . Filter. Evaporate the filtrate on a w ater-bath and dry
calconecarboxytic add triturate R. the residue at 70 °C for 1 h. The residue weighs a maximum
1 m L of 0.1 M sodium edetate. is equivalent to 44.84 mg o f 5 mg.
of C 12H 22Ca 0 i 4,H 20 .
__________________________________________________________ PhEur M axim um 500 ppm .
Dissolve 0.1 g in a mixture of 2 m L o f acetic add R and
8 m L o f water R and dilute to 15 m L with water R.
P h o s p h a te s (2.4.11)
Calcium Glycerophosphate M axim um 400 ppm .
*. *
(Ph. Eur. monograph 0980) * D ilute 2.5 m L o f solution S to 100 m L with water R.
CaHTCaOeP 210.1 27214-00-2 S u lfa te s (2.4.13)
M axim um 0.1 p er cent, determined on solution S.
A c tio n a n d u se A rse n ic (2.4.2, Method A)
Excipient. M axim um 3 ppm .
PhEur__________________________________________________________ Dissolve 0.33 g in water R and dilute to 25 m L w ith the
same solvent.
D E F IN IT IO N
Ir o n (2.4.9)
M ixture in variable proportions of the calcium salt of
M axim um 50 ppm , detemined on 0.20 g.
(ftS)-2,3-dihydroxypropyl phosphate and o f 2-hydroxy-1-
(hydroxymethyl)ethyl phosphate which may be hydrated. H e av y m e ta ls (2.4.8)
M aximum 20 ppm .
C o n te n t
18.6 per cent to 19.4 per cent o f C a (dried substance). Dissolve 2.0 g in 10 m L o f buffer solution pH 3.5 R and dilute
to 20 m L with water R. 12 m L of the solution complies w ith
CHARACTERS test A. Prepare the reference solution using lead standard
A p p e a ra n c e solution (2 ppm Pb) R.
W hite or almost white powder, hygroscopic.
S o lu b ility M axim um 12.0 p er cent, determined on 1.000 g by drying in
Sparingly soluble in water, practically insoluble in ethanol an oven at 150 °C for 4 h.
(96 per cent).
A SSA Y
Dissolve 0.200 g in water R. Carry out the complexometric
A. M ix 1 g with 1 g o f potassium hydrogen sulfate R in a test titration o f cald u m (2.5.11).
tube fitted with a glass tube. H eat strongly and direct the
1 m L o f 0.1 M sodium edetate is equivalent to 4.008 mg of
white vapour towards a piece o f filter paper im pregnated with
Ca.
a freshly prepared 10 g/L solution of sodium nitroprusside R.
T he filter paper develops a blue colour in contact with __________________________________________________________ PhEur
piperidine R.
B. Ignite 0.1 g in a crucible. Take up the residue with 5 m L
of nitric add R and heat on a water-bath for 1 min. Filter.
T h e filtrate gives reaction (b) of phosphates (2.3.1). Calcium Hydrogen Phosphate ?**%
C. It gives reaction (b) of calcium (2.3.1). *★ ★*
D ibasic C ald u m Phosphate *
TESTS
(Caldum Hydrogen Phosphate Dihydrate,
S o lu tio n S
Dissolve 1.5 g at room tem perature in carbon dioxide-free
water R prepared from distilled water R and dilute to 150 m L C a H P O ^ H aO 172.1 7789-77-7
with the same solvent. PhEtr__________________________________________________________
A p p e a ra n c e o f so lu tio n D E F IN IT IO N
Solution S is not more opalescent than reference C o n te n t
suspension ID (2.2.1). 98.0 per cent to 105.0 p er cent.
A c id ity o r a lk alin ity
CHARACTERS
T o 100 m L o f solution S add 0.1 m L o f phenolphthalein
A p p e a ra n c e
solution R. N o t m ore than 1.5 m L of 0.1 M hydrochloric add
or 0.5 m L o f 0.1 M sodium hydroxide is required to change
the colour o f the indicator.
1-378 Calcium Hydrogen Phosphate 2016
Solubility Carry out the m easurem ent on 20.0 m L o f the test solution.
Practically insoluble in water and in ethanol (96 per cent). A dd at least 3 times 0.10 m L of the reference solution and
It dissolves in dilute hydrochloric acid and in dilute nitric carry out the m easurem ent after each addition. Calculate the
add. concentration o f fluorides using the calibration curve.
ID E N T IF IC A T IO N S u lfates
A. Dissolve with heating 0.1 g in 10 m L of dilute hydrochloric Maximum 0.5 per cent.
add R. A dd 2.5 m L o f dilute ammonia R l, shake and add Test solution Dissolve 0.5 g in a mixture o f 5 m L o f water R
5 m L of a 35 g/L solution of ammonium oxalate R. A white and 5 m L o f dilute hydrochloric add R and dilute to 100 m L
precipitate is produced. w ith water R. Filter if necessary. T o 20 m L o f this solution,
B. Dissolve 0.1 g in 5 m L o f dilute nitric add R, add 2 m L of add 1 m L of dilute hydrochloric add R and dilute to 50 m l.
ammonium molybdate solution R and heat at 70 °C for 2 min. with water R.
A yellow precipitate is produced. Reference solution T o 1.0 m L o f 0.005 M sulfuric add, add
C. It complies with the limits of the assay. 1 m L of dilute hydrochloric acid R -and dilute to 50 m L with
water R. Filter if necessary.
TESTS
T o the test solution and to the reference solution, add 2 m L
S o lu tio n S
of a 120 g/L solution o f barium chloride R and allow to stand
Dissolve 2.5 g in 20 m L of dilute hydrochloric acid R, filter if
for 10 min. Any opalescence in the test solution is not more
necessary and add dilute ammonia R l until a precipitate is
intense than that in the reference solution.
formed. A dd just sufficient dilute hydrochloric add R to
dissolve the precipitate and dilute to 50 m L with distilled A rse n ic (2.4.2, Method A)
water R. M aximum 10 ppm , determ ined on 2 m L o f solution s.
A cid -in so lu b le su b sta n c e s B a riu m
M aximum 0.2 per cent. T o 0.5 g, add 10 m L of water R and heat to boiling. While
Dissolve 5.0 g in 40 m L of water R, add 10 m L o f stirring, add 1 m L of hydrochloric acid R dropwise. Allow to
hydrochloric add R and heat to boiling for 5 min. Cool, then cool and filter if necessary. Add 2 m L of a 10 g/L solution of
collect the insoluble substances using ashless filter paper. dipotassium sulfate R and allow to stand for 10 m in.
N o turbidity is produced.
W ash with water R until turbidity is no longer produced
when silver nitrate solution R2 is added to the filtrate. Ignite at Ir o n (2.4.9)
600 ± 50 °C. T he residue weighs n o t more than 10 mg. M aximum 400 ppm.
C a rb o n a te s Dilute 0.5 m L o f solution s to 10 m L with water R.
Shake 0.5 g with 5 m L of carbon dioxide-free water R and add H eav y m e ta ls (2.4.8)
1 m L of hydrochloric add R. N o effervescence is produced. M aximum 40 ppm.
C h lo rid e s D ilute 10 m L o f solution s to 20 m L with water R. 12 m L of
M aximum 0.25 per cent. the solution complies with test A. Prepare the reference
Test solution Dissolve 0.20 g in a mixture of 20 m L o f water R solution using lead standard solution (1 ppm Pb) R.
and 13 m L o f dilute nitric add R by warming if necessary, L o ss on ig n itio n
dilute to 100 m L with water R and filter if necessary. 24.5 per cent to 26.5 per cent, determined on 1.000 g by
Use 50 m L of this solution. ignition to constant mass at 800-825 °c.
Reference solution T o 0.70 m L of 0.01 M hydrochloric add, add A SSAY
6 m l, of dilute nitric add R and dilute to 50 m L with water R.
Dissolve 0.4 g in 12 m L o f dilute hydrochloric acid R by
Add 1 m L o f silver nitrate solution R2 to the test solution and heating on a water bath if necessary and dilute to 200 m l.
to die reference solution and mix. After standing for 5 min with water R. T o 20.0 m L o f this solution add 25.0 m L of
protected from light, any opalescence in the test solution is 0.02 M sodium edetate, 50 m L of water R, 5 m L o f ammonium
n ot more intense than that in the reference solution. chloride buffer solution pH 10.7 R and about 25 m g o f mordant
F lu o rid e s black 11 triturate R. T itrate the excess o f sodium edetate with
M aximum 100 ppm. 0.02 M zinc sulfate. Carry o u t a blank titration.
Potentiom etry (2.2.36, Method II). 1 m L of 0.02 M sodium edetate is equivalent to 3.44 mg
Chelating solution Dissolve 45 g of cydohexylenedinitrilotetra- o f C a H P 0 4,2H 20 .
acetic acid R in 75 m L of sodium hydroxide solution R and 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
dilute to 250 m L with water R. This section provides information on characteristics that are
Test solution Dissolve 1.000 g in 4 m L o f hydrochloric add R l, recognised as being relevant control parameters for one or more
add 20 m L o f chelating solution, 2.7 m L of glacial acetic functions of the substance when used as an excipient (see chapter
acid R and 2.8 g of sodium chloride R, adjust to p H 5-6 with 5.15). Some of the characteristics described in the Functionality-
sodium hydroxide solution R and dilute to 50.0 m L with related characteristics section may also be present in the mandatory
water R. part of the monograph since they also represent mandatory quality
Reference solution Dissolve 4.42 g o f sodium fluoride R, criteria. In such cases, a cross-reference to the tests described in the
previously dried at 300 °C for 12 h , in water R and dilute to mandatory part ừ included in the Functumality-related
1000.0 m L with the same solvent. Dilute 50.0 m L o f this characteristics section. Control of the characteristics can contribute
solution to 500.0 m L with total-tonic-strength-adjustment to the quality of a medicinal product by improving the consistency
buffer R (200 ppm F). of the manufacturing process and the performance of the medicinal
Indicator electrode Fluoride-selective. product during use. Where control methods are dted, they are
Reference electrode Silver-silver chloride. also be used Wherever results for a particular characteristic are
reported, the control 'method must be indicated
2016 Calcium Hydrogen Phosphate 1-379
The following characteristics may be relevant for calcium hydrogen A dd 1 m L o f silver nitrate solution R2 to the test solution and
phosphate dihydrate used as filler in tablets and capsules. to the reference solution and mix. A fter standing for 5 min
P a rtic le -siz e d is trib u tio n (2.9.31 or 2.9.38). protected from light, any opalescence in the test solution is
not m ore intense than that in the reference solution.
B u lk a n d ta p p e d d e n sity (2.9.34)
F lu o rid e s
P o w d e r flow (2.9.36) M axim um 100 ppm .
__________________________________________________________ PhEur
Potentiom etry (2.2.36> Method II).
Chdcamg solution Dissolve 45 g of cydohexylenedmitrilotetra-
acedc add R in 75 m L o f sodium hydroxide sdution R and
dilute to 250 m L with water R
Anhydrous Calcium Hydrogen ***** Test solution Dissolve 1.000 g in 4 m L o f hydrochloric add R l,
Phosphate ***** add 20 m L o f chelating solution, 2.7 m L o f glacial acetic
(Ph Eur monograph 0981) add R and 2.8 g o f sodium chloride R, adjust to p H 5-6 with
sodium hydroxide solution R and dilute to 50.0 m L with
C aH P04 136.1 7757-93-9 water R.
PhEur__________________________________________________________ Reference sdution Dissolve 4.42 g o i sodium fluoride R,
D E F IN IT IO N previously dried at 300 °C for 12 h , in water R and dilute to
C o n te n t 1000.0 m L with the same solvent. D ilute 50.0 m L of this
98.0 per cent to 103.0 per cent. solution to 500.0 m L with total-ionic-strength-adjustment
buffer R (200 ppm F).
CHA RACTERS
Indicator electrode Fluoride-selective.
A p p e a ra n c e
W hite or almost white, crystalline powder, or colourless Reference electrode Silver-silver chloride.
crystals. Carry o u t the m easurem ent on 20.0 m L of the test solution.
A dd at least 3 times 0.10 m L o f the reference solution and
S o lubility
carry out the m easurem ent after each addition. Calculate the
Practically insoluble in water and in ethanol (96 per cent).
It dissolves in dilute hydrochloric a d d and in dilute nitric concentration of fluorides using the calibration curve.
acid. S u lfa tes
A. Dissolve with heating 0.1 g in 10 m L o f dilute hydrochloric Test solution Dissolve 0.5 g in a mixture of 5 m L o f water R
acid R. A dd 2.5 m L o f dUute ammonia R l, shake, and add and 5 m L o f dilute hydrochloric add R and dilute to 100 m L
5 m L of a 35 g/L solution o f ammonium oxalate R. A white w ith water R Filter if necessary. T o 20 m L of this solution,
add 1 m L o f dilute hydrochloric acid R and dilute to 50 m L
precipitate is produced.
with water R
B. Dissolve 0.1 g in 5 m L o f dilute nitric acid R, add 2 m L of
ammonium molybdate solution R and heat at 70 °C for 2 min. Reference solution T o 1.0 m L of 0.005 M sulfuric add, add
1 m L o f dilute hydrochloric add R a n d dilute to 50 m L with
A yellow precipitate is produced.
water R Filter if necessary.
C . It complies with the limits o f the assay.
T o the test solution and to the reference solution, add 2 m L
TESTS o f a 120 g/L solution o f barium chloride R and allow to stand
S o lu tio n S for 10 m in. Any opalescence in th e test solution is not more
Dissolve 2.5 g in 20 m L o f dilute hydrochloric add R, filter if intense than that in the reference solution.
necessary and add dilute ammonia R l until a precipitate is
formed. Add just sufficient dilute hydrochloric acid R to
M axim um 10 ppm , determined on 2 m L o f solution S.
dissolve'the precipitate and dilute to 50 m L with distilled
water R. B a riu m
T o 0.5 g, add 10 m L o f water R an d heat to boiling. While
A cid -in so lu b le su b s ta n c e s
stirring, add 1 m L of hydrochloric acid R dropwise. Allow to
cool and filter if necessary. Add 2 m L o f a 10 g/L solution o f
Dissolve 5.0 g in 40 m L o f water R, add 10 m L of dipotassium sulfate R and allow to stand for 10 min.
hydrochloric add R and heat to boiling for 5 min. Cool, then N o turbidity is produced.
collect the insoluble substances using ashless filter paper.
Ir o n (2.4.9)
W ash with water R until turbidity is no longer produced
M axim um 400 ppm .
w hen silver nitrate solution R2 is added. Ignite at
600 ± 50 °C. T he residue weighs not more th an 10 mg. Dilute 0.5 m L of solution S to 10 m L with water R.
C a rb o n a te s H eav y m e ta ls (2.4.8)
Shake 0.5 g with 5 m L o f carbon dioxide-free water R and add M axim um 40 ppm .
1 m L o f hydrochloric add R. N o effervescence is produced. Dilute 10 m L of solution S to 20 m L with water R. 12 m L o f
C h lo rid e s the solution complies with test A. Prepare the reference
solution using lead standard solution (1 ppm Pb) R.
Test solution Dissolve 0.20 g in a mixture o f 20 m L o f water R L oss o n ig n itio n
and 13 m L o f dilute nitric add R by warming if necessary, 6.6 p er cent to 8.5 p er cent, determ ined on 1.000 g to
dilute to 100 m L w ith water R and filter if necessary. constant mass at 800-825 °C.
U se 50 m L o f this solution.
Reference solution T o 0.70 m L of 0.01 M hydrochloric acid, add
6 m L of dilute nitric add R and dilute to 50 m L with water R.
1-380 Calcium Hydroxide 2016
A SSAY TESTS
Dissolve 0.4 g in 12 m L o f dilute hydrochbric acid R by M a tte r in so lu b le in h y d ro c h lo ric a c id
heatm g on a water b ath if necessary and dilute to 200 m L M axim um 0.5 per c e n t
with water R. T o 20.0 m L o f this solution add 25.0 m L of Dissolve 2.0 g in 30 m L of hydrochloric acid R. Boil the
0.02 M sodium edetate, 50 m L o f water R, 5 m L of ammonium solution and filter. Wash the residue w ith hot water R.
chloride buffer solution pH 10.7 R and about 25 mg o f mordant T h e residue weighs a m axim um of 10 mg.
black 11 triturate R. T itrate the excess o f sodium edetate with
C a rb o n a te s
0.02 M zinc sulfate. C an y out a blank titration.
M axim um 5.0 per cent o f C a C 0 3.
Add 5.0 m L o f 1 M hydrochloric acid to the titrated solution
of C a H P 0 4.
obtained under Assay and titrate with 1 M sodium hydroxide
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 using 0.5 m L of methyl orange solution R as indicator.
This section provides information on characteristics that are 1 m L of 1 M hydrochloric acid is equivalent to 50.05 mg
recognised as bang relevant control parameters for one or more o f C a C 0 3.
C h lo rid e s (2.44)
5.15). Some of the characteristics described in the Functionality-
M aximum 330 ppm .
related characteristics section may also be present in the mandatory
part of the monograph since they also represent mandatory quality Dissolve 0.30 g in a mixture o f 2 m L o f nitric acid R and
criteria. In such cases, a cross-reference to the tests described in the 10 m L o f water R and dilute to 30 m L with water R.
mandatory part is included in the Functionality-related S u lfates (2.413)
characteristics section. Control of the characteristics can contribute M axim um 0.4 per cent.
to the quality of a medicinal product by improving the consistency Dissolve 0.15 g in a mixture o f 5 m L of dilute hydrochloric
of the manufacturing process and the performance of the medicinal acid R and 10 m L o f distilled water R and dilute to 60 m L
product during use. Where control methods are cited, they are with distilled water R.
A rse n ic (2.42, Method A)
M aximum 4 ppm.
Dissolve 0.50 g in 5 m L o f brominated hydrochloric arid R and
The following characteristics may be relevant for anhydrous
dilute to 50 m L with water R Use 25 m L o f this solution.
calcium hydrogen phosphate used as filler in tablets and capsules.
M a g n e s iu m a n d alkali m e ta ls
P a rd c le -s iz e d is trib u tio n (2.9.31)
Maximum 4.0 per cent calculated as sulfates.
or 2.9.38).
Dissolve 1.0 g in a mixture of 10 m L o f hydrochloric acid R
B u lk a n d ta p p e d d e n sity (2.9.34)
and 40 m L o f water R Boil and add 50 m L o f a 63 g/L
P o w d e r flow (2.9.36) solution o f oxalic acid R. Neutralise with ammonia R and
__________________________________________________________ PhEur dilute to 200 m L w ith water R Allow to stand for 1 h and
filter through a suitable filter. T o 100 m L of the filtrate, add
0.5 m L o f sul/uric acid R. Cautiously evaporate to dryness
and ignite. T he residue weighs a maximum of 20 mg.
★★*★★ H eav y m e ta ls (2.4.8)
Calcium Hydroxide ★ ★
M axim um 20 ppm.
Dissolve 1.0 g in 10 m L of hydrochloric acid R l and evaporate
Ca(OH)2 74.1 1305-62-0 to dryness on a water-bath. Dissolve the residue in 20 m L of
Preparation water R and filter. 12 m L of the filtrate complies with test A.
Calcium Hydroxide Solution Prepare the reference solution using lead standard solution
(1 ppm Pb) R.
PhEur_____________________________
ASSAY
D E F IN IT IO N
T o 1.500 g in a mortar, add 20-30 m L o f water R and
C o n te n t
0.5 m L o f phenolphthalein solution R. Titrate with 1 M
hydrochloric acid by triturating the substance until the red
CHARACTERS colour disappears. T he final solution is used in the tests for
A p p e a ra n c e carbonates.
W hite or almost white, fine powder. 1 m L of 1 M hydrochloric acid is equivalent to 37.05 mg
S o lubility o f C a(O H )2.
Practically insoluble in water. __________________________________________________________ PhEur
A. T o 0.80 g in a mortar, add 10 m L o f water R and 0.5 m L
of phenolphthalein solution R and m k . T h e suspension turns
red. On addition of 17.5 m L of 1 M hydrochloric acid, the
suspension becomes colourless w ithout effervescing. T he red
colour occurs again when the mixture is triturated for 1 min.
O n addition of a further 6 m L o f 1 M hydrochloric acid and
triturating, the solution becomes colourless.
B. Dissolve about 0.1 g in dilute hydrochloric arid R and dilute
to 10 m L with water R. 5 m l, of the solution give
reaction (b) of calcium (2.3.1). •
2016 Calcium Lactate 1-381
★ ★ T o 20 m L of solution S add 20 m L of water R, 2 g of

Anhydrous Calcium Lactate ★ ★ ammonium chloride R and 2 m L o f dilute ammonia R l. H eat to
★ . .*
(Ph. Ettr. monograph 2118) *★ * boiling and rapidly add 40 m L o f hot ammonium oxalate
sdution R. Allow to stand for 4 h , dilute to 100.0 m L with
water R and filter. T o 50.0 m L o f the filtrate add 0.5 m L of
HsC^/COî-
Ca2* and enantiomer sulfuric add R. Evaporate to dryness and ignite the residue to
H OH constant mass at 600 ± 50 °C. T h e residue w dghs a
m axim u m o f 5 m g.
C^HioCaOg 218.2 H eav y m e ta ls (2.4.8)

Maximum 10 ppm.
A c tio n a n d u se Dissolve 2.0 g in water R and dilute to 20 m L with the same
U sed in treatm ent of caldum defidency. solvent. 12 m L o f the solution complies with test A. Prepare
the reference solution using lead standard solution
PhEur.
(1 ppm Pb) R.
D E F IN IT IO N L oss o n d ry in g (2.2.32)
C aldum bis(2-hydroxypropanoate) or mixture o f caldum M aximum 3.0 p er cent, determined on 0.500 g by drying in
(2R)-, (25)- and (2R5)-2-iydroxypropanoates. an oven at 125 °C.
C o n te n t ASSAY
CHARACTERS same solvent. Carry out the complexometric titration of
A p p e a ra n c e caldum (2.5.11).
W hite or alm ost white, crystalline or granular powder. 1 m L of 0.1 M sodium edetate is equivalent to 21.82 mg
S o lu b ility o f CgHioCaOô.
Soluble in water, freely soluble in boiling water, very slightly __________________________________________________________ PhEur
IDENTIFICATION
A. Loss on drying (see Tests).
★*★
B. It gives the reaction of lactates (2.3.1). Calcium Lactate Monohydrate ★ ★
★ ★
C. It gives reaction (b) o f caldum (2.3.1).
TESTS
S o lu tio n S
H sC Ô Y
Dissolve 5.0 g w ith heating in carbon dioxide-free water R Ca2* and enantiomer , H20
prepared from distilled water R, allow to cool and dilute to H OH
A p p e a ra n c e o f so lu tio n CftHioCaOJFÔ 236.0
Solution S is n o t m ore opalescent than reference
suspension II (2.2.1) and not more intensdy coloured than A ctio n a n d u se
reference solution BY6 (2.2.2, Method II). U sed in treatm ent o f caldum defidency.
PhEir_____________________________________
solution R and 0.5 m L o f 0.01 M hydrochloric add. D E F IN IT IO N
T h e solution is colourless. N o t more than 2.0 m L o f 0.01 M C aldum bis(2-hydroxypropanoate) or mixture of caldum
sodium hydroxide is required to change the colour of the (2R)-, (2S)~ and (2RS)-2-hydroxypropanoates monohydrates.
indicator to pink. C o n te n t
C h lo rid e s (2.4.4) 98.0 per cent to 102.0 per cent (dried substance).
M axim um 200 ppm . CHARACTERS
D ilute 5 m L o f solution S to 15 m L with water R. A p p e a ra n c e
S u lfates (2.4.13) White or almost white, crystalline or granular powder.
M aximum 400 ppm . Solubility
D ilute 7.5 m L o f solution S to 15 m L with distilled water R. Soluble in water, freely soluble in boiling water, very slightly
B a r iu m soluble in ethanol (96 per cent).
T o 10 m L o f solution S add 1 m L o f caldum sulfate ID E N T IF IC A T IO N
solution R. Allow to stand for 15 min. Any opalescence in the A. Loss on drying (see Tests).
solution is n o t more intense than th at in a mixture o f 1 m L
B. It gives the reaction o f lactates (2.3.1).
o f distilled water R and 10 m L of solution S.
C. It gives reaction (b) o f caldum (2.3.1).
I r o n (2.4.9)
Maxim um 50 ppm . TESTS
S o lu tio n S
D ilute 4 m L o f solution S to 10 m L with water R.
Dissolve 5.4 g (equivalent to 5.0 g of the dried substance)
M a g n e s iu m a n d alk ali salts with heating in carbon dioxide-free water R prepared from
M axim um 1 p er cent. distilled water R, allow to cool and dilute to 100 m L with the
same solvent.
1-382 Calcium Lactate 2016
Calcium Lactate Pentahydrate ★* * * ★
Solution S is n o t more opalescent than reference ★ ★
suspension II (2.2.1) and n o t m ore intensely coloured than C ald u m Lactate *****
reference solution BY6 (2.2.2, Method II). (Ph. Eur. monograph 0468)
solution R and 0.5 m L of 0.01 M hydrochloric acid. Ca2* 'X and enantiomer , 5 H20
T he solution is colourless. N o t more than 2.0 m L of 0.01 M H OH
sodium hydroxide is required to change the colour o f the

indicator to pink.
C6Hi0CaO6j5H2O 308.3 5743-47-5
M aximum 200 ppm. A ctio n a n d u se
Dilute 5 m L o f solution S to 15 m L with water R. U sed in treatm ent o f caldum defiriency.
S u lfates (2.4.13) P re p a r a tio n s
M axim u m 400 ppm. C ald u m and Eigocaldferol Tablets
Dilute 7.5 m L o f solution S to 15 m L with distiUed water R. C aldum Lactate Tablets
B a riu m Chewable C aldum and Ergocalciferol Tablets
T o 10 m L of solution S add 1 m L of calcium sulfate PhEur__________________________________________________________
solution R. Allow to stand for 15 min. Any opalescence in the
solution is n o t more intense than that in a mixture o f 1 m L D E F IN IT IO N
of distilled water R and 10 m L o f solution S. C aldum bis(2-hydroxypropanoate) or mixture o f caldum
Iro n (2.4.9)
(2R)~, (2S)~ and (2&S)-2-hydroxypropanoates pentahydrates.
M aximum 50 ppm. C o n te n t
Dilute 4 m L of solution S to 10 m L with water R. 98.0 per cent to 102.0 per cent (dried substance).
M a g n e siu m a n d alk ali salts CHARACTERS

M aximum 1 per cen t A p p e a ra n c e
T o 20 m L of solution S add 20 m L of water R, 2 g of W hite or almost white, crystalline or granular powder,
slightly efflorescent.
ammonium chloride R and 2 m L o f dilute ammonia R l. H eat to
boiling and rapidly add 40 m L of hot ammonium oxalate S o lu b ility
solution R. Allow to stand for 4 h, dilute to 100.0 m L with Soluble in water, freely soluble in boiling water, very slightly
water R and filter. T o 50.0 m L of the filtrate add 0.5 m L o f soluble in ethanol (96 per cent).
sulfuric acid R. Evaporate to dryness and ignite the residue to ID E N T IF IC A T IO N
constant mass at 600 ± 50 °C. T h e residue weighs a
A. Loss on drying (see Tests).
maximum of 5 mg.
B. It gives the reaction of lactates (2.3.1).
C. It gives reaction (b) of caldum (2.3.1).
M axim um 10 ppm .
Dissolve a quantity equivalent to 2.0 g o f the dried substance TESTS
in water R and dilute to 20 m L with the same solvent. 12 m L S o lu tio n S
of the solution complies with test A. Prepare the reference Dissolve 7.1 g (equivalent to 5.0 g of the dried substance)
solution using lead standard solution (1 ppm Pb) R. with heating in carbon dioxide-free water R prepared from
distilled water R, allow to cool and dilute to 100 m T . with the
same solvent.
5.0 per cent to 8.0 per cent, determ ined on 0.500 g by
drying in an oven at 125 °C. A p p e a ra n c e o f so lu tio n
Solution S is not more opalescent than reference
A SSAY
Dissolve a quantity equivalent to 0.200 g of the dried reference solution BY6 (2.2.2, Method II).
substance in water R and dilute to 300 mT. with the same
solvent. Carry out the complexometric titration of calcium
(2.5.11). T o 10 m L o f solution S add 0.1 m L of phenolphthalein
sdution R and 0.5 m L of 0.01 M hydrochloric add.
1 m L of 0.1 M sodium edetate is equivalent to 21.82 m g
T h e solution is colourless. N ot more than 2.0 m L o f 0.01 M
of C6H 10C aO 6.
sodium hydroxide is required to change the colour o f the
_________________________________________________________________ PhEur indicator to pink.
M aximum 200 ppm .
Dilute 5 m L o f solution S to 15 m l. with water R.
S u lfates (2.4.13)
M aximum 400 ppm .
Dilute 7.5 m L of solution S to 15 mT. with distilled water R.
B a riu m
T o 10 m L o f solution S add 1 m L o f caldum sulfate
sdution R. Allow to stand for 15 min. Any opalescence in the
2016 Calcium Lactate 1-383
solution is not more intense than that in a mixture o f 1 m L ID E N T IF IC A T IO N

of distilled water R and 10 m L of solution S. A. Loss on drying (see Tests).
I r o n (.2.4.9) B. It gives the reaction o f lactates (2.3.1).
M axim um 50 ppm. C. It gives reaction (b) of calcium (2.3.1).
D ilute 4 m L of solution S to 10 m L with water R. TESTS
M a g n e s iu m a n d a lk a li salts S o lu tio n S
M axim um 1 per cent. Dissolve 6.2 g (equivalent to 5.0 g o f the dried substance)
T o 20 m L of solution S add 20 m L o f water R, 2 g of with heating in carbon dioxide-free water R prepared from
ammonium chloride R and 2 m L of dilute ammonia R l. H eat to distilled water R , allow to cool and dilute to 100 m L with the
boiling and rapidly add 40 m l . of hot ammonium oxalate same solvent
solution R. Allow to stand for 4 h, dilute to 100.0 m L with A p p e a ra n c e o f so lu tio n
water R and filter. T o 50.0 m L of the filtrate add 0.5 m L of Solution S is not more opalescent than reference
sulfuric add R Evaporate to dryness and ignite the residue to suspension II (2.2.1) and not more intensely coloured than
constant mass at 600 ± 50 °C. T he residue weighs a reference solution BY6 (2.2.2, Method IT).
m a x i m u m of 5 mg.
A cid ity o r alk alin ity
H e a v y m e ta ls (2.4.8) T o 10 m L of solution S add 0.1 m L of phenolphthalein
M axim um 10 ppm. solution R and 0.5 m L of 0.01 M hydrochloric add
Dissolve a quantity equivalent to 2.0 g of the dried substance T h e solution is colourless. N ot more than 2.0 m L of 0.01 M
in water R and dilute to 20 m L with the same solvent. 12 m L sodium hydroxide is required to change the colour o f the
of the solution complies with test A. Prepare the reference indicator to pink.
solution using lead standard solution (1 ppm Pb) R C h lo rid e s (2.4.4)
L o ss o n d ry in g (2.2.32) M aximum 200 ppm .
22.0 per cent to 27.0 per cent, determined on 0.500 g by D ilute 5 m L o f solution S to 15 m L with water R.
drying in an oven a t 125 °C.
S u lfate s (2.4.13)
A SSA Y M aximum 400 ppm .
Dissolve a quantity equivalent to 0.200 g of the dried D ilute 7.5 m L of solution S to 15 m L w ith distilled water R.
substance in water R and dilute to 300 m L with the same
B a riu m
solvent. C an y out the complexometric titration of calcium
T o 10 m L of solution S add 1 m L of caldum sulfate
(2.5.11).
solution R. Allow to stand for 15 min. Any opalescence in the
1 m L of 0.1 M sodium edetate is equivalent to 21.82 mg solution is not m ore intense than that in a mixture of 1 m L
of CeHioCaOg. o f distilled water R and 10 m L of solution S.
Ph Eur
Ir o n (2.4.9)
M aximum 50 ppm.
M a g n e siu m a n d alk ali salts
★ ★
Calcium Lactate Trihydrate ★ ★ M aximum 1 per c e n t
(Ph. Eur. monograph 0469) ***** T o 20 m L of solution S add 20 m L of water R, 2 g of

ammonium chloride R and 2 mL of dilute ammonia R l. H eat to
boiling and rapidly add 40 mL of ho t ammonium oxalate
h 3c co2- solution R. Allow to stand for 4 h, dilute to 100.0 m L with
Ca2* and enantiomer , 3 H20
A
H OH water R and filter. T o 50.0 mL o f the filtrate add 0.5 m L of
sulfuric add R. Evaporate to dryness and ignite the residue to
constant mass at 600 ± 50 °C. T h e residue weighs a
CeHxoCaOeÔ 272.3 4137-22-9 maximum of 5 mg.
A c tio n a n d use H eav y m e ta ls (2.4.8)

U sed in treatm ent o f calcium deficiency. M aximum 10 ppm.
P r e p a r a tio n Dissolve a quantity equivalent to 2.0 g of th e dried substance
C ald u m Lactate Tablets in water R and dilute to 20 mL with the sam e solvent 12 m L
o f the solution complies with test A. Prepare the reference
PhEur_____________________________________ solution using lead standard solution (1 ppm Pb) R.
D E F IN IT IO N L oss o n d ry in g (2.2.32)
Calcium bis(2-hydroxypropanoate) or mixture of calcium 15.0 per cent to 20.0 per cent, determ ined on 0.500 g by
(2 R)-, (25)- and (21&S)-2-hydroxypropanoates trihydrates. drying in an oven at 125 °C.
C o n te n t A SSAY
98.0 per cent to 102.0 per cent (dried substance). Dissolve a quantity equivalent to 0.200 g o f the dried
substance in water R and dilute to 300 m L with the same
CHARACTERS
solvent. C any out the complexometric titration of calcium
A p p e a ra n c e
(2.5.11).
W hite or almost white, crystalline or granular powder.
S o lu b ility
of CôHioCaOô.
Soluble in water, freely soluble in boiling water, very slightly
soluble in ethanol (96 per cent). __________________________________________________________PhEur
1-384 Calcium Levofolinate 20H

Calcium Levofolinate Pentahydrate ***\
Drying In air.
(Ph. Eur. monograph 1606) * Detection Examine in ultraviolet light at 254 nm .
Results T h e p rindpal spot in the chrom atogram obtained with
the test solution is similar in position and size to the prindpal
solution.
D . It gives reaction (b) o f cald u m (2.3.1).
Cany out the tests and the assay as rapidly as possible, protected
from bright tight
TESTS
C20H 2i CaN 707j5H20 511.5 80433-71-2
S o lu tio n S
(anhydrous substance) Dissolve 0.40 g in carbon dioxide-free water R, h e a t i n g at
40 °C if necessary, and dilute to 50.0 m L with the same
A c tio n a n d use solvent.
A ntidote to folic a d d antagonists. A p p e a ra n c e o f so lu tio n
PhEur___________________________________________________________ 420 nm has a maximum o f 0.25.
D E F IN IT IO N p H (2.2.3)
C ald u m (2<S)-2-[[4- [[[(65)-2-amino-5-formyl-4-oxo- 7.5 to 8.5 for solution S.
134j5j6j738-hexahydropteridin-6-
yl] methyl] amino] benzoyl] amino] pentanedioate pentahydrate.
—10 to —15 (anhydrous substance), measured at 25 °C.
C o n te n t Dissolve 0.200 g in tris(hydroxymethyl)aminometharie
— calcium levcfolinate (C20H 2iC aN 7O 7; 511.5): 97.0 p er cent solution R previously adjusted to p H 8.1 with sodium hydroxide
to 102.0 per cent (anhydrous substance); solution R or hydrochloric add R l and dilute to 20.0 m L with
— caldum (Ca; A T40.08): 7.54 per cent to 8.14 per cent the same solvent.
(anhydrous substance).
A c e to n e a n d e th a n o l
CHARACTERS Head-space gas chromatography (2.2.28Use the standard
A p p e a ra n c e additions method.
W hite or light yellow, amorphous or crystalline powder, Test solution Dissolve 0.25 g o f the substance to be examined
hygroscopic. in water R and dilute to 10.0 m L with the same solvent.
S o lu b ility Reference solution Dissolve 0.125 g o f acetone R and 0.750 g of
Slightly soluble in water, practically insoluble in acetone and anhydrous ethanol R in water R and dilute to 1000.0 m L with
in ethanol (96 per cent). water R.
First identification A , B, D — material: fused silica;
Second identification A , C, D — size. I — 10 m , 0 = 0.32 mm;
— stationary phase: styrene-divinylbenzene copolymer R.
Flow rate 4 m U m in.
Preparation Discs.
Static head-space conditions which may be used:
Comparison: caldum folinate CRS.
— equilibration temperature: 80 °C;
I f the spectra obtained show differences, dissolve the — equilibration time. 20 min;
substance to be examined and the reference substance — pressurisation time: 30 s.
separatdy in the minim um quantity o f water R and add
Temperature:
dropwise suffident acetone R to produce a predpitate. Allow
to stand for 15 m in, collect the predpitate by centrifugation,
w ash the predpitate twice with a m i n i m u m quantity o f Time Temperature
acetone R an d dry. Record new spectra u s i n g the residues. (min) CC)
Column 0 -1 4 80 ->220
C . Thin-layer chrom atography (2.2.27).
Test solution Dissolve 15 m g o f the substance to be e x a m in e d Injection port 110
in a 3 per cent V/V solution o f ammonia R and dilute to Detector 270

5 m L with th e sam e solvent.
Reference solution Dissolve 15 m g o f caldum folinate CRS in a
3 per cent V/V solution of ammonia R and dilute to 5 mT. Detection Flam e ionisation.
w ith the sam e solvent. Injection A t least 3 times.
Plate cellulose for chromatography F 254 R as the coating Limits:
substance. — acetone: maxim um 0.5 per cent,
— ethanol: maxim um 3.0 per cent.
Mobile phase T h e lower layer of a mixture of 1 volume of
isoamyl alcohol R and 10 volumes o f a 50 g/L solution o f citric R e la te d su b s ta n c e s
add R previously adjusted to p H 8 with ammonia R. L iquid chromatography (2.2.29).
Application 5 (iL.
2016 Calcium Levofolinate 1-385
Test solution Dissolve 10.0 mg of the substance to be Mobile phase Dissolve 9.72 g of sodium dihydrogen phosphate R
examined in water R and dilute to 10.0 m L w ith the same in 890 m l, of water R and adjust to p H 5.0 with sodium
solvent. hydroxide solution R; add 100 m L o f 2-propanol R and 10 m L
Reference solution (a) Dissolve 10.0 mg of calcium folinate CRS of acetonitrUe R.
in water R and dilute to 10.0 m L with the sam e solvent. Flow rate 1 mL/min.
Reference solution (b) Dilute 1.0 m L of reference solution (a) Detection Spectrophotometer at 286 nm.
to 100.0 m L with water R. Injection 10 pL.
Reference solution (c) Dissolve 10.0 m g o f forrnylfoHc acid CRS Retention times Levofolinate = about 9 min;
in the mobile phase and dilute to 100.0 m L with the mobile im purity H = about 19 min.
phase. Dilute 1.0 m L of this solution to 10.0 m L with System suitability.
water R. — resolution: m in im u m of 5.0 between the peaks due to
Reference solution (d) Dilute 1.0 m L o f reference solution (b) levofolinate and to impurity H in the chromatogram
to 20.0 m L with water R. obtained with reference solution (a). T he sum o f the
Reference solution (e) Dilute 5.0 m L o f reference solution (c) areas o f the 2 peaks is 100 per cent. T he peak area of
to 10.0 m L with reference solution (b). impurity H is 48 per cent to 52 per cent. In the
Column: chromatogram obtained with reference solution (b) 2
— size: I = 0.25 m, 0 = 4 mm; clearly visible peaks are obtained.
— stationary phase: octadecylsilyl silica gelfor chromatography R Limit:
(5 pm); — impurity H: maximum 0.5 per cent.
— temperature: 40 °C. C h lo rid e s
Mobile phase Mix 220 m L o f methanol R and 780 m L of a M axim u m 0.5 per cent.
solution containing 2.0 m L o f tetrabutylamrnonium hydroxide Dissolve 0.300 g in 50 m L of water R heating at 40 °C if
solution (400 g/L) R and 2.2 g o f disodium hydrogen necessary. Add 10 m L o f 2 M nitric acid and titrate with
phosphate R previously adjusted to p H 7.8 w ith phosphoric 0.005 M silver nitrate determining the end-point
acid R. If necessary adjust the concentration o f methanol R to potentiometrically (2.2.20).
achieve the prescribed resolution. 1 m L o f 0.005 M silver nitrate is equivalent to 0.177 mg o f
Flow rate 1 mL/min. Cl.
Detection Spectrophotometer at 280 nm. P la tin u m
Injection 10 pL. M axim um 10 ppm .
Run time 2.5 times the retention time of the principal peak in Atomic absorption spectrometry (2.2.23, Method II).
the chromatogram obtained with the test solution. Test solution Dissolve 1.0 g in water R and dilute to 100.0 m L
System suitability, reference solution (e): with the same solvent.
— resolution: minim um o f 2.2 between the peaks due to Reference solutions Prepare the reference solutions losing
folinate and to impurity D. platinum standard sdution (30 ppm Pt) R, diluted as necessary
Limits: w ith a m ixture o f 1 volume of nitric acid R and 99 volumes of
— impurity D: n o t more than 0.8 times the area o f the water R.
Source Platinum hollow-cathode lamp.
— any other impurity, not more than 0.8 times the area o f the
principal peak in the chromatogram obtained with H eav y m e ta ls (2.4.8)
reference solution (b) (0.8 per cent); M axim um 50 ppm .
— sum of other impurities: not more than twice the area o f the 1.0 g complies with test F. Prepare the reference solution
principal peak in the chromatogram obtained with using 5 m L of lead standard solution (10 ppm Pb) R.
reference solution (b) (2.0 per cent); W a te r (2.5.12)
— disregard Umif. area of the principal peak in the 10.0 per cent to 17.0 per cent, determined on 0.200 g
chromatogram obtained with reference solution (d) (ground to a very fine powder). Stir the substance to be
(0.05 per cent). examined in the titration solvent for about 15 m in before
Im p u rity H titrating and use iodosidfurous reagent R as titranL
Liquid chromatography (2.2.29): use the normalisation B a c te ria l en d o to x in s (2.6.14)
procedure. Less than 0.5 IU/mg, if intended for use in the m anufacture
Test solution Dissolve 50.0 mg of the substance to be o f parenteral preparations without a further appropriate
examined in water R and dilute to 100.0 m L with the same procedure for the removal of bacterial endotoxins.
solvent.
A SSA Y
Reference solution (a) Dissolve 10.0 mg of caldum folinate CRS C a lc iu m
Dissolve 0.400 g in 150 m L o f water R and dilute to 300 m L
Reference solution (b) Dilute 1.0 m L o f reference solution (a) w ith the same solvent. Carry out the complexometric
to 100.0 m L with water R. titration o f calcium (2.5.11).
Column: 1 m L of 0.1 M sodium edetate is equivalent to 4.008 mg o f
— size: I = 0.15 m , 0 = 4 mm; Ca.
— stationary phase: human albumin coated silica gel for
C a lc iu m fo lin ate
related substances.
1-386 Calcium Levulinate 2016
Calculate the percentage content o f C2oH2iCaN70 7 from the

areas o f the peaks in the chrom atograms obtained with the
test solution and reference solution (a) and the declared
content of calcium folinate CRS. co 2h
STORAGE H,N
In a n airtight container, protected from light. If the substance
is sterile, store in a sterile, airtight, tam per-proof container.
F. R = C H O : (2S)-2-[[4-[[(2-am ino-4-oxo-l,4,7,8-
IM P U R IT IE S tetrahydropteridin-6-
yl)methyl]formylamino]benzoyl]amino]pentanedioic a d d
(10-formyldihydrofolic ad d ),
G . R = H: (2S)-2-[[4-[[(2-am ino-4-oxo-l,4,7,8-
H,N C02H yl)methyl] amino]benzoyl] amino] pentanedioic a d d
(dihydrofolic ad d ),
A. (2S)-2-[(4-aminobenzoyl) amino] pentanedioic acid,
O h co 2h
o H C02H
co 2h
co 2h
h2n n n
H H
h2n n n
H H
H . (2S)-2-[[4-[[[(6i?)-2-am ino-5-fonnyl-4-oxo-l,4,5,6,7,8-
B. (25)-2-[[4-[[[(6i?)-2-am ino-5-form yl-4-oxo-l,4,5,6,7,8- hexahydropteridin-6-
hexahydropteridin-6- yl] methyl] amino]benzoyl]amino]pentanedioic ad d .
yl] methyl]fonnylamino]benzoyi] amino]pentanedioic acid PtiEur
(5,10-difonnyltetrahydrofolic ad d ),
9 H c o 2h
★ ★
Calcium Levulinate Dihydrate ★ ★
h2n n n
C02*
Ca2* h3CY ^ , 2 H20
C . (2S)-2- [[4- [[(2-amino-4-oxo-l,4-dihydropteridin-6-
yl)methyl] amino]benzoyl]amino]pentanedioic a d d (folic
a d d ),
C io H 14C a 0 6 ^ H 20 306.3 5743-49-7
A ctio n a n d u se
Source o f caldum .
0 f
C02H PhEur_____________________________
CHO D E F IN IT IO N
h2n n n
2 H C ald u m di(4-oxopentanoate) dihydrate.
C o n te n t
D . (2S)-2-[[4-[[(2-amm o-4-oxo-1,4-dihydropteridin-6- 98.0 per cent to 101.0 per cent (dried substance).
yl)methyi]fonnylamino]benzoyl] amino]pentanedioic a d d
(10-fonnylfolic ad d ), CHARACTERS
A p p e a ra n c e
»XT Solubility
F red y soluble in water, very slightly soluble in ethanol
(96 per cent), practically insoluble in methylene chloride.
h2n n n
H H ID E N T IF IC A T IO N
First identificatidn A , D, E.
E. 4-[[[(65)-2-am ino-5-form yl-4-oxo-l,4,5,6,7,8- Second identification B, C, D, E.
hexahydropteridin-6-yl]methyl]amino]benzoic a d d
(5-fonnyitetrahydropteroic a d d ),
Comparison calcium levulinate dihydrate CRS.
in water R and dilute to 1 m L with .the same solvent.
2016 Calcium Pantothenate 1-387
Reference solution Dissolve 60 mg of calcium leuulinate dryness on a water-bath and ignite to constant mass at
dihydrate CRS in water R and dilute to 1 m L with the same 600 ± 50 °C. T he residue wdghs a maximum of 5.0 mg.
solvent. H eav y m e ta ls (2.4.8)
Plate TLC silica gel plate R. M aximum 10 ppm .
Mobile phase concentrated ammonia R, ethyl acetate R, water R, 12 m L o f solution S complies with test A. Prepare the
ethanol (96 per cent) R (10:10:30:50 VIVIVIV). reference solution using lead standard solution (1 ppm Pb) R.
Application 10 |iL. L oss o n d ry in g (2.2.32)
Development Over a path of 10 cm. 11.0 per cent to 12.5 per cent, determined on 0.200 g by
Drying A t 100-105 °C for 20 min and allow to cool. drying at 105 °C.
Detection Spray with a 30 g/L solution of potassium P y ro g e n s (2.6.8)
permanganate R. D ry in a current of warm air for about If intended for use in the manufacture of parenteral
5 min or until the spots become yellow. Examine in daylight. preparations without a further appropriate procedure for the
Results T h e prindpal spot in the chromatogram obtained with removal o f pyrogens, it complies with the test for pyrogens.
the test solution is similar in position, colour and size to the Inject per kilogram of the rabbit’s mass 4 m L of a solution
principal spot in the chromatogram obtained with the containing per millilitre 50 mg of the substance to be
examined.
reference solution.
C. T o 1 m L of solution S (see Tests), add 20 m L of a ASSAY
2.5 g/L solution o f dinitrophenyütydrazine R in dilute Dissolve 0.240 g in 50 m L of water R. C any out the
hydrochloric acid R. Allow to stand for 15 min. Filter, wash complexometric titration o f caldum (2.5.11).
the precipitate with water R. Dry the precipitate in an oven at 1 m L of 0.1 M sodium edetate is equivalent to 27.03 mg
100-105 °C. T he melting point (2.2.14) is 203 °C to 210 °C. o f CioH14C a 0 6.
D. It gives reaction (b) o f caldum (2.3.1). STORA GE
E. Loss on drying (see Tests). Protected from light.
TESTS __________________________________________________________PhEur
S o lu tio n S
distilled water R and dilute to 100.0 m L with the same
solvent. ***
Calcium Pantothenate ★ ★
A p p e a ra n c e o f so lu tio n ★ ★
Solution S is d ear (2.2.1) and not more intensely coloured (Ph. Eur. monograph 0470) *****
p H (2.2.3) h3c c H30
6.8 to 7.8 for solution S. Cat1* H0- V n
O x id isab le su b sta n c e s / oh h
T o 1 m L o f solution S, add 10 m L o f water R, 1 m L o f dilute
sulfuric add R and 0.25 m L o f a 3.0 g/L solution o f potassium
permanganate R. Mix. After 5 min, the violet colour o f the C x8H 32CaN 2O10 476.5 137-08-6
mixture is still visible.
S u c ro se a n d re d u c in g su g a rs
C om ponent of vitamin B.
T o 5 m L o f solution S add 2 m L of hydrochloric add R l and
dilute to 10 m L with water R. H eat to boiling for 5 m in and PhE ir______________________
allow to .cool. Add 10 m L of sodium carbonate solution R.
D E F IN IT IO N
Allow to stand for 5 min, dilute to 25 m L with water R and
filter. T o 5 m L o f die filtrate add 2 m L o f cupri-tartaric C aldum pantothenate contains not less than 98.0 per cent
solution R and heat to boiling for 1 min. N o red predpitate is and not more than the equivalent of 101.0 per cent of
formed. caldum bis[3-[[(21?)-2,4-dihydroxy-3,3-
dimethyibutanoyl]amino]propanoate], calculated with
C h lo rid e s (2.4.4) reference to the dried substance.
M aximum 50 ppm.
CHARACTERS
A white or almost white powder, slightly hygroscopic, fredy
S u lfates (2.4.13) soluble in water, slightly soluble in alcohol.
M axim um 200 ppm.
Dilute 7.5 m L o f solution S to 15 m L with distilled water R.
M a g n e s iu m a n d alk ali m e ta ls
3-aminopropionic ad d . T he principal spot in the
T o 10 m L o f solution S, add 80 m L of water R, 10 m L of chromatogram obtained with test solution (b) is similar in
ammonium chloride solution R and 1 m L o f ammonia R. H eat position, colour and size to the prindpal spot in the
to boiling. T o the boiling solution, add dropwise 50 m L of chromatogram obtained with reference solution (a).
warm ammonium oxalate sdution R. Allow to stand for 4 h,
C . T o 1 m L o f solution S (see Tests) add 1 m L o f dilute
then dilute to 200 m L with water R and filter. T o 100 m L of
sodium hydroxide sdution R and 0.1 m L o f copper sulfate
the filtrate, add 0.5 m L of sulfuric add R. Evaporate to
solution R. A blue colour develops.
D . It gives reaction (a) of caldum (2.3.1).
1-388 Calcium Phosphate 2016
TESTS ★ ★
S o lu tio n S
Calcium Phosphate ★ ★
Dissolve 2.50 g in carbon dioxide-free water R and dilute to Tribasic C ald u m Phosphate *****
50.0 m L w ith the same solvent. (Ph. Eur. monograph 1052)
Solution S is clear (2.2.1) and colourless (2.2.2, Method II). A c tio n a n d u se
Exdpient.
p H (2.2.3)
T he p H o f solution S is 6.8 to 8.0. P re p a r a tio n
C aldum and Ergocalciferol Tablets
+ 25.5 to + 27.5, determined on solution S and calculated C aldum Phosphate for H om oeopathic Preparations
with reference to the dried substance. Chewable C ald u m and Ergocalciferol Tablets
3-A m in o p ro p io n ic a c id PhEui.
gel G R as the coating substance. D E F IN IT IO N
M ixture o f caldum phosphates.
examined in water R and dilute to 5 m L with the same C o n te n t
solvent. 35.0 per cent to 40.0 per cent o f C a (Ar 40.08).
Test solution (b) Dilute 1 m L o f test solution (a) to 10 m L CHARACTERS
w ith water R. A p p e a ra n c e
Reference solution (a) Dissolve 20 m g o f calcium W hite or almost white powder.
pantothenate CRS in water R and dilute to 5 m L with the S o lu b ility
same solvent Practically insoluble in water. It dissolves in dilute
Reference solution (b) Dissolve 10 mg o f 3-aminopropionic hydrochloric a d d and in dilute nitric ad d .
acid R in water R and dilute to 50 mT. with the same solvent.
Apply separately to th e plate 5 pL o f each solution. Develop A. Dissolve 0.1 g in 5 m L o f a 25 p er cent VIV solution of
over a path o f 12 cm using a mixture of 35 volumes of nitric acid R. T he solution gives reaction (b) o f phosphates
water R ahd 65 volumes of ethanol R. D ry the plate in a (2.3.1).
current of air and spray with nmhydrin solution R l. H eat at
B. It gives reaction (b) o f caldum (2.3.1). Filter before
110 °C for 10 min. Any spot corresponding to
adding potassium ferrocyanide solution R.
3-am inopropionic a d d in the chrom atogram obtained with
test solution (a) is n o t more intense than the spot in the C. It complies with the limits of the assay.
chrom atogram obtained with reference solution (b) TESTS
(0.5 per cent). S o lu tio n S
C h lo rid e s (2.4.4) Dissolve 2.50 g in 20 mT. o f dilute hydrochloric add R. If the
5 m L o f solution S diluted to 15 m L with water R complies solution is n o t clear, filter it. Add dilute ammonia R l dropwise
with the limit test for chlorides (200 ppm ). until a predpitate is formed. Dissolve the predpitate by
H eav y m e ta ls (2.48) adding dilute hydrochloric add R and dilute to 50 m L w ith
12 m L o f solution S complies with test A for heavy metals
distilled water R.
(20 ppm ). Prepare the reference solution using lead standard C h lo rid e s (2.44)
solution (1 ppm Fb) R. M aximum 0.15 per cent.
L oss o n d ry in g (2.2.32) Dissolve 0.22 g in a mixture o f 1 m L o f nitric add R and
N ot m ore than 3.0 per cent, determ ined on 1.000 g by 10 m L of water R and dilute to 100 m L with water R.
drying in an oven at 105 °C. F lu o rid e s
A SSA Y M axim um 75 ppm .
Dissolve 0.180 g in 50 m L o f anhydrous acetic acid R. T itrate Potentiom etry (2.2.36, Method II).
with 0.1 M perchloric acid determ in in g the end-point Test solution Dissolve 0.250 g in 0.1 M hydrochloric add, add
potentiometrically (2.2.20). 5.0 m L of fluoride standard solution (1 ppm F) R and dilute to
1 m L o f 0.1 M perchloric add is equivalent to 23.83 m g o f 50.0 mT. with 0.1 M hydrochloric acid. T o 20.0 m L o f this
Ci8H32C aN 2O 10. solution add 20.0 mT. o f total-ionic-strength-adjustment buffer R
and 3 m L o f an 82 g/L solution o f anhydrous sodium
STORAGE
acetate R. Adjust to p H 5.2 with ammonia R and dilute to
Store in an airtight container.
PhEtr Reference solution Fluoride standard solution (10 ppm F) R.
Indicator electrode Fluoride-selective.
Reference electrode Silver-silver chloride.
Carry out the measurem ents on the test solution, th en add at
least 3 quantities, each o f 0.5 m L, o f the reference solution,
carrying out a m easurem ent after each addition. Calculate
the concentration o f fluorides using the calibration curve,
taking into account the addition o f fluoride to the test
solution.
2016 Calcium Polystyrene Sulfonate 1-389
S u lfates (.2.4.13)
Calcium Polystyrene Sulfonate
Calcium Polystyrene Sulphonate
D ilute 1 m L o f solution S to 25 m L with distilled water R.
A rsen ic (2.4.2, Method A) A c tio n a n d u se
M axim um 4 ppm , determined on 5 m L of solution S. U sed in the treatm ent of hyperkalaemia.
Ir o n (2.4.9)
D E F IN IT IO N
M axim um 400 ppm.
Calcium Polystyrene Sulfonate is a cation-exchange resin
Dilute 0.5 m L of solution S to 10 m L with water R.
prepared in the calcium form containing not less than 6.5%
H eav y m e ta ls (2.4.8) w/w and n o t m ore than 9.5% w/w of calcium, calculated with
M axim um 30 ppm . reference to die dried substance. Each g exchanges not less
D ilute 13 m L o f solution S to 20 m L with water R. 12 m L of than 1.3 m E q and not more than 2.0 m E q of potassium,
the solution complies with test A. Prepare the reference calculated with reference to the dried substance.
solution using lead standard solution (1 ppm Pb) R. C H A R A C T E R IS T IC S
A cid -in so h ib le m a tte r A cream to light brown, fine powder.
M aximum 0.2 per cent. Practically insoluble in water and in ethanol (96%).
Dissolve 5.0 g in a mixture o f 10 m L o f hydrochloric acid R
and 30 m L of water R. Filter, wash the residue with water R
and dry to constant mass at 100-105 °C. T h e residue weighs
concordant with the reference spectrum of calcium polystyrene
a maximum o f 10 mg.
sulfonate (RS 037).
L oss o n ig n itio n
B. Yields reaction C characteristic o f calcium salts,
M aximum 8.0 per cent, determined on 1.000 g by ignition at
Appendix VI.
800 ± 50 °C for 30 min.
TESTS
A SSAY
P a rtic le size
Dissolve 0.200 g in a mixture of 1 m L of hydrochloric acid R1
N o t more than 1% w/w is retained on a 150-pm sieve,
and 5 m L o f water R. Add 25.0 m L of 0.1 M sodium edetate
Appendix X V IIB . Use 20 g and sieve for 5 minutes.
and dilute to 200 m L with water R. Adjust to about p H 10
with concentrated ammonia R. Add 10 m L o f ammonium P o ta s s iu m
chloride buffer solution pH 10.0 R and a few milligrams of N o t more than 0.1% of K when determined by atomic
mordant black 11 triturate R. Titrate the excess sodium edetate emission spectrophotometry, Appendix II D , measuring at
with 0.1 M zinc sulfate until the colour changes from blue to 766.5 nm and using a solution prepared in the following
violet. m anner. T o 1.1 g of the substance being examined add
5 m L of hydrochloric add, heat to boiling, cool and add
1 m L of 0.1 M sodium edetate is equivalent to 4.008 m g of
10 m L o f water. Filter, wash the filter and residue with water
Ca.
and dilute the filtrate and washings to 25 m L with water. Use
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 potassium standard solution (100 ppm K), suitably diluted with
This section provides mformaxion on characteristics that are water, to prepare the standard solutions.
recognised as being relevant control parameters for one or more S o d iu m
functions of the substance when used as an excipient (see chapter N o t more than 0.1% of N a when determined by atomic
5.15). This section is a non-mandatory pan of the monograph emission spectrophotometry, Appendix II D , measuring at
and it is not necessary to verify the characteristics to demonstrate 589.0 nm and using a solution prepared in the following
compliance. Control of these characteristics can however contribute m anner. T o 1.1 g of the substance being examined add
to the quality of a medicinal product by improving the consistency 5 m L of hydrochloric add, heat to boiling, cool and add
of the manufacturing process and the performance of the medicinal 10 m L of water. Filter, wash the filter and residue with water
product during use. Where control methods are cited, they are and dilute the filtrate and washings to 25 m L with water. Use
recognised as being suitable for the purpose, but other methods can sodium solution (200 ppm Na), suitably diluted with water, to
also be used Wherever results for a particular characteristic are prepare the standard solutions.
A rsen ic
The following characteristics may be relevant for calcium
1 g complies with the limit test for arsenic, Appendix VII
phosphate is used as a filler in tablets and capsules.
(1 ppm ).
P a r d d e - s iz e d is trib u tio n (2.931 or 2.938).
H eav y m e ta ls
B u lk a n d ta p p e d d e n sity (2.934) H eat 4 g until charred, cool, add 4 m L o f lead-free nitric acid
P o w d e r flow (2.9.36) and 0.5 m L o f sulfuric add drop wise and heat cautiously
PhEur until white fumes are no longer evolved. Ignite in a muffle
furnace at 500° to 600° until a white residue is obtained.
Cool, add 4 m L o f hydrochloric add and dilute to 20 mL.
T h e resulting solution complies with limit test A for heavy
metals, Appendix VII. U se 2 m L o f lead standard solution
(lOppmPb) to prepare the standard (10 ppm).
S ty re n e
Appendix ID D , using the following solutions.
1-390 Calcium Stearate 2016
(1) Shake 10 g o f the substance being examined with 10 m L

of acetone for 30 minutes, centrifuge and use the supernatant
Calcium Stearate *****
liquid. (Ph. Eur. monograph 0882) *
(2) 0.0001% w/v o f styrene in acetone. 1592-23-0
(a) Use a stainless steel colum n (30 cm x 4 mm) packed
Excipient.
w ith octadecylsUyl silica gel for chromatography (jiBondapak
C 18 is suitable). PhEur_______________________________________________________________
D E F IN IT IO N
below.
M ixture o f calcium salts o f different fatty ad d s consisting
(c) Use a flow rate o f 2 m L per minute. mainly of stearic (octadecanoic) a d d [(C i7H 35C O O )2Ca;
(d) Use an am bient column tem perature. 607] and palmitic (hexadecanoic) a d d [(C 15H 3iC O O )2Ca;
(e) Use a detection wavelength o f 254 nm. 550.9] with m inor proportions o f other fatty adds.
(f) Inject 20 |iL o f each solution. C o n te n t
MOBILE PHASE — caldum: 6.4 per cent to 7.4 per cent (AT40.08) (dried
substance);
Equal volumes o f acetonitrile and water.
— stearic add in the fatty add fraction: m inim u m
LIMITS 40.0 per cent;
In the chrom atogram obtained with solution (1): — sum of stearic add and palmitic add in the fatty add fraction:
the area of any peak corresponding to styrene is n o t greater minim um 90.0 per cent.
th an the area of the peak in the chromatogram obtained with CHARACTERS
solution (2) (1 ppm ). A p p e a ra n c e
P o ta s s iu m e x c h a n g e c ap a c ity Fine, white or almost white, crystalline powder.
T o 3 g of the substance being examined in a dry 250 m L S o lu b ility
glass-stoppered flask add 100 m L o f a solution co n tainin g Practically insoluble in water and in ethanol (96 per cent).
0.7455% w/v of potassium chloride and 0.4401% w/v of
potassium hydrogen carbonate in water (solution A), stopper
and shake for 15 minutes. Filter and dilute 2 m L o f the First identification: C, D.
filtrate to 1000 m L with water. D eterm ine the concentration Second identification A, B, D
o f unbound potassium in this solution by atomic emission A. Freezing point (2.2.18): m inim um 53 °C, for the residue
spectrophotometry, Appendix II D , measuring at 766.5 nm and obtained in the preparation of solution S (see Tests).
using solution A suitably diluted with water, to prepare the B. A d d value (2.5.1): 195 to 210.
standard solutions. Calculate the potassium exchange
Dissolve 0.200 g o f the residue obtained in the preparation of
capacity of the substance being examined in milliequivalents
solution S in 25 m L of the prescribed mixture of solvents.
taking the concentration o f potassium in solution A as
144 milliequivalents o f K per litre. C. Examine the chromatograms obtained in the test for fatty
a d d composition.
L oss o n d ry in g
W hen dried at 70° at a pressure n o t exceeding 0.7 kPa for
Results T h e retention times of the prindpal peaks in the
16 hours, loses n o t more than 8.0% o f its weight. Use 2 g. chromatogram obtained with the test solution are
approximately the same as those of the prindpal peaks in the
M ic ro b ia l c o n ta m in a tio n chromatogram obtained with the reference solution.
Carry out a quantitative evaluation for Enterobacteria and
D . Neutralise 5 m L o f solution S to red litmus paper R using
certain other Gram-negative bacteria, Appendix XVI B l.
strong sodium hydroxide solution R. T he solution gives
0.01 g of the substance being examined gives a negative
reaction (b) o f caldum (2.3.1).
result, Table I (m ost probable num ber of bacteria per gram
fewer than 102). TESTS
A SSA Y S o lu tio n S
T o 5.0 g add 50 m L o f peroxide-free ether R, 20 m L o f dilute
F o r c a lc iu m
nitric add R and 20 m l. of distilled water R. Boil under a
Carefully h eat 1 g in a platinum crucible until a white ash is
reflux condenser until dissolution is complete. Allow to cool.
obtained and dissolve in 10 m L o f 2 m hydrochloric add with
In a separating funnel, separate the aqueous layer and shake
the aid of h e a t Transfer the resulting solution to a conical
the ether layer with 2 quantities, each of 5 mL, o f distilled
flask using 20 m L o f water. A dd 50 m L of 0.05m disodium
water R. Combine the aqueous layers, wash with 15 m L of
edetate KS, 20 m l. o f ammonia buffer pH 10.9 and titrate the
peroxide-free ether R and dilute the aqueous layer to 50 m L
excess of disodium edetate w ith 0 .0 2 m zinc sulfate KS, using a
w ith distilled water R (solution S). Evaporate the ether layer to
0.5% w/v solution o f mordant black 11 in ethanol (96%) as
dryness and dry the residue at 100-105 °C. Keep the residue
indicator to a red purple end point. Each m L of 0.05m
for identification tests A and B.
disodium edetate VS is equivalent to 2.004 m g o f Ca.
STORAGE
T o 1.0 g add 20 m L o f carbon dioxide-free water R and boil
Calcium Polystyrene Sulfonate should be kept in an airtight for 1 m in with continuous shaking. Cool and filter.
container.
T o 10 m L of the filtrate add 0.05 m L of bromothymol blue
solution R l. N ot more than 0.5 m L of 0.01 M hydrochloric
add or 0.01 M sodium hydroxide is required to change the
colour o f the indicator.
2016 Calcium Stearate 1-391
C h lo rid es (2.4.4) and titrate with 0.1 M zinc sulfate until the colour changes
Maximum 0.1 p er c e n t from blue to violet Carry out a blank titration.
Dilute 0.5 m L o f solution S to 15 m L with water R. 1 m L o f 0.1 M sodium edetate is equivalent to 4.008 mg of
S u lfates (2.4.13) Ca.
Maximum 0.3 per c e n t C o m p o sitio n o f fatty acids
Dilute 0.5 m L of solution S to 15 m L with distilled water R. Gas chromatography (2.2.28): use the normalisation
procedure.
C a d m iu m
M aximum 3 ppm . Test solution In a conical flask fitted with a reflux condenser,
dissolve 0.10 g of the substance to be examined in 5 m L of
Atomic absorption spectrometry {2.2.23, Method II).
boron trifluoride-methanol solution R. Boil under a reflux
Test solution Place 50.0 mg in a polytetrafluoroethylene condenser for 10 min. Add 4 m L o f heptane R through the
digestion bom b and add 0.5 m L o f a mixture o f 1 volume of condenser. Boil under a reflux condenser for 10 min. Allow
hydrochloric add R and 5 volumes of cadmium- and lead-free to cool. Add 20 m L of saturated sodium chloride solution R.
nitric add R. Allow to digest at 170 °C for 5 h. Allow to cool. Shake and allow the layers to separate. Remove about 2 m L
Dissolve the residue in water R and dilute to 5.0 m L with the o f the organic layer and dry over 0.2 g of anhydrous sodium
same solvent. sulfate R. Dilute 1.0 m L of the solution to 10.0 m L with
Reference solutions Prepare the reference solutions using heptane R.
cadmium standard solution (10 ppm Cd) R, diluted if necessary Reference solution Prepare the reference solution in the same
with a 1 per cent VIV solution of hydrochloric add R. m anner as the test solution using 50.0 mg oipalmitic
Source Cadm ium Hollow-cathode lamp. add CRS and 50.0 mg o f stearic add CRS instead of calcium
Wavelength 228J3 nm . stearate.
Atomisation device Graphite furnace. Cdumn:
L ead
— size. I = 30 m , 0 = 0.32 mm;
M aximum 10 ppm .
Atomic absorption spectrometry (2.2. 23, Method II). 0.5 |im).
Test solution Use the solution described in the test for Carrier gas helium for chromatography R.
cadmium.
Reference solutions Prepare the reference solutions using lead Temperature.
standard solution (10 ppm Pb) R, diluted if necessary with
water R.
Source Lead hollow-cathode lamp. Time Temperature
(min) <°C)
Wavelength 283.3 nm ; 217.0 nm may be used depending on 0 -2 70
Column
the apparatus.
2 -3 6 70 -> 240
Atomisation device Graphite furnace.
36-41 240
N ickel
M aximum 5 ppm . Injection port 220 ..
Atomic absorption spectrometry (2.2.23, Method II). Detector 260
Test solution Use the solution described in the test for
cadmium.
Reference solutions Prepare the reference solutions using nickel Detection Flame ionisation.
standard solution (10 ppm Ni) R, diluted if necessary with Injection 1 ¿iL.
water R. Relative retention W ith reference to methyl stearate: methyl
Source Nickel hollow-cathode lamp. palmitate = about 0.9.
Wavelength 232.0 nm. System suitability: reference solution:
— resolution: minimum 5.0 between the peaks due to methyl
palmitate and methyl stearate.
Calculate the content of palmitic acid and stearic acid.
Disregard the peak due to the solvent.
an oven at 105 °C.
TA M C: acceptance criterion 103 C FU/g (2.6.12).
Junctions of the substance when used as an excipient (see chapter
Absence of Escherichia coli (2.6.13). 5.15). This section is a non-mandatory part of the monograph
Absence o f Salmonella (2.6.13). and it is not necessary to verify the characteristics to demonstrate
ASSA Y
compliance. Control of these characteristics can however contribute
C a lc iu m
of the manufacturijig process and the performance of the medicinal
T o 0.500 g in a 250 m L conical flask add 50 m L o f a
mixture of equal volumes of anhydrous ethanol R and
recognised as bring suitable for the purpose, but other methods can
butanol R, 5 m L o f concentrated ammonia R, 3 m L of
ammonium chloride buffer solution pH 10.0 R, 30.0 m L of
0.1 M sodium edetate and 15 mg o f mordant black 11
triturate R. H eat to 45-50 °C until the solution is clear. Cool
1-392 Calcium Sulfate 2016
The following characteristics may be relevant for calcium stearate ID E N T IF IC A T IO N

used as a lubricant in tablets and capsules. A. Loss on ignition (see Tests).
P a rtic le -s iz e d is trib u tio n (2.9.31) B. Solution S (see Tests) gives reaction (a) o f sulfates (2.3.1).
S p ecific s u rfa c e a r e a (2.9.26, Method I) C. Solution S gives reaction (a) o f caldum (2.3.1).
D eterm ine die specific surface area in the P/P0 range of TESTS
0.05 to 0.15. S o lu tio n S
Sample outgassing 2 h at 40 °C. Dissolve 1.0 g in 50 m L o f a 10 per cent VIV solution of
____________________________________________________________________________________________ P hEur hydrochloric acid R by heating at 50 °C for 5 min. Allow to
cool.
Shake 1.5 g with 15 m L o f carbon dioxide-free water R for
Dried Calcium Sulfate 5 min. Allow to stand for 5 min and filter. T o 10 m L o f the
Exsiccated Calcium Sulfate; Plaster o f Paris; Dried C ald u m filtrate add 0.1 m L of phenolphthalem solution R and 0.25 m l.
Sulphate of 0.01 M sodium hydroxide. T he solution is red.
C a S (V /iH 20 145.1 26499-65-0 A dd 0.30 m L of 0.01 M hydrochloric acid T h e solution is
colourless. A dd 0.2 m L o f methyl red solution R. T h e solution
D E F IN IT IO N is reddish-orange.
D ried Calcium Sulfate is prepared by heating powdered
gypsum, C aS 04 , 2H 20 , at about 150° in a controlled m anner
M axim um 300 ppm .
such that it is substantially converted into the hemihydrate,
C aS 04 ,ViiH20 , w ith minim um production of the anhydrous Shake 0.5 g with 15 m L o f water R for 5 m in. Allow to stand
phases of calcium sulfate. It may contain suitable setting for 15 min and filter. D ilute 5 m L of the filtrate to 15 m L
accelerators o r decelerators. with water R.
M aximum 10 ppm , determined on 5 m L o f solution S.
A white or alm ost white powder; hygroscopic.
Ir o n (2.4.9)
Slighdy soluble in water, m ore soluble in dilute mineral adds;
M axim um 100 ppm .
practically insoluble in ethanol (96%).
T o 0.25 g add a mixture o f 5 m L o f hydrochloric acid R and
ID E N T IF IC A T IO N 20 m L o f water R. H eat to boiling, cool and filter.
Yields the reactions characteristic of calcium salts and of
sulfates, A ppendix VI.
M aximum 20 ppm .
TESTS
T o 2.5 g add a mixture of 2 m L o f hydrochloric add R and
S e ttin g p ro p e rtie s 15 m L o f water R. H eat to boiling. Cool and then add
20 g mixed w ith 10 m L o f water at 15° to 20° in a cylindrical 0.5 m L of phenolphthaldn solution R. Cautiously add
m ould about 2.4 cm in diam eter sets in 4 to 11 minutes. concentrated ammonia R until the colour changes to pink.
T h e mass thus produced, after sta n d in g for 3 hours, Add 0.5 m L o f glacial acetic acid R and dilute to 25 m L with
possesses suffitient hardness to resist pressure o f the fingers water R. Filter. 12 m L o f the filtrate complies with test A.
at the edges, which retain their sharpness of outline and do Prepare the reference solution using lead standard solution
n o t crumble. (2 ppm Pb) R.
L oss o n ig n itio n L oss o n ig n itio n
W hen ignited to constant w right at red heat, loses 4.5% to 18.0 per cent to 22.0 per cent, determined on 1.000 g by
8.0% o f its w dghL U se 1 g. ignition to constant mass at 800 ± 50 °C.
A SSA Y
Dissolve 0.150 g in 120 m L o f water R. C an y out the
Calcium Sulfate Dihydrate ****** complexometric titration o f caldum (2.5.11).
1 m L o f 0.1 M sodium edetate is equivalent to 17.22 m g
C ald u m Sulphate * of CaS 04 , 2H 20 .
. 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
C aS 04^H 20 172.2 10101-41-4 This section provides information on characteristics that are
recognised as bang relevant control parameters for one or more
Excipient.
5.15). This section is a non-mandatory part of the monograph
PhEur_______________________________________________________________ and it is not necessary to verify the characteristics to demonstrate
D E F IN IT IO N
C o n te n t of the manufacturing process and the performance of the medicinal
98.0 per cent to 102.0 per cent of C aS 04 , 2H 20 . product during use. Where control methods are cited, they are
CHARACTERS recognised as being suitable for the purpose, but other methods can
A p p e a ra n c e also be used. Wherever results for a particular characteristic are
W hite or alm ost white fine powder. reported, the control method must be indicated.
S o lu b ility The following characteristics may be relevant for caldum sulfate
Very slighdy soluble in water, practically insoluble in ethanol dihydrate used as filler in tablets and capsules.
(96 per cent).
2016 Camphor 1-393
P a rtic le -s iz e d is trib u tio n (2.9.31 or 2.9.38). R e la te d su b sta n ce s

B u lk a n d ta p p e d d e n sity (2.9.34) Gas chromatography (2.2.28).
P o w d e r flow (2.9.36)
__________________________________________________________________________________________ P hEur
in heptane R and dilute to 25.0 m L with the same solvent.
Reference solution (a) Dilute 1.0 m l. of the test solution to
100.0 m L with heptane R.
to 20.0 m L with heptane R.
Natural Camphor ;* % Reference solution (c) Dissolve 0.50 g of bomeol R in heptane R
*★ and dilute to 25.0 m L with the same solvent. D ilute 5.0 m L
(o-Camphor, Ph Eur monograph 1400) *
of the solution to 50.0 m L with heptane R.
CH3 Reference solution (d) Dissolve 50 m g of linalol R and 50 mg
o f bomyl acetate R in heptane R and dilute to 100.0 m L with
the same solvent.
Column:
H — size. I = 30 m , 0 = 0.25 mm,
— stationary phase: macrogol 20 000 R (0.25 pm).
C 10H 16O 152.2 464-49-3
Split ratio 1:70.
Ph E tr _____________________ I ; -------------------------------------------------------------------------------------------------------
Flow rate 45 cm/s.
D E F IN IT IO N Temperature:
(li?,4/?)-l ,7,7-Trimethylbicyclo [2.2. l]heptan-2-one.
CHARACTERS Time Temperature
A p p e a ra n c e (min) (°C)
W hite or almost white, crystalline powder or friable, Column 0-10 50
crystalline masses.
10-35 50 -> 100
Highly volatile even at room temperature.
35-45 100 -> 200
S o lu b ility
Slightly soluble in water, very soluble in alcohol and in light 45-55 200
petroleum , freely soluble in fatty oils, very slightly soluble in Injection port 220
glycerol. Detector 250
First identification: A , C.
Second identification A, B, D
Injection 1 pL.
System suitability Reference solution (d).
B. M elting point (2.2.14): 175 °C to 179 °C.
— resolution: minimum 3.0 between the peaks due to bomyl
C. Infrared absorption spectrophotometry (2.2.24). acetate and to linalol.
Comparison racemic camphor CRS. Limits:
D. Dissolve 1.0 g in 30 m L of methanol R. Add 1.0 g of — bomeol: not m ore than the area of the principal peak in
hydroxylamine hydrochloride R and 1.0 g of anhydrous sodium the chromatogram obtained with reference solution (c)
acetate R. Boil under a reflux condenser for 2 h. Allow to (2.0 p er cent),
cool and add 100 m L of water R. Filter, wash die precipitate — any other impurity: n o t more than half o f the area of the
obtained with 10 inL of water R and recrystallise from 10 m L principal peak in the chromatogram obtained with
of a mixture of 4 volumes of alcohol R and 6 volumes of reference solution (a) (0.5 per cent),
water R. T h e crystals, dried in vacuo, melt (2.2.14) at 118 °C — total of other impurities: not m ore than 4 times the area of
to 121 °C. the principal peak in the chromatogram obtained with
TESTS
Cany out the weighings and dissolution rapidly.
S o lu tio n S (0.05 p er cent).
Dissolve 2.50 g in 10 m L o f alcohol R and dilute to 25.0 m L
H a lo g en s
Maximum 100 ppm.
A p p e a ra n c e o f so lu tio n Dissolve 1.0 g in 10 m L of 2-propanol R in a distillation flask.
A dd 1.5 m L o f dilute sodium hydroxide solution R and 50 mg
A c id ity o r alk alin ity of nickel-alwnmium alloy R. H eat on a water-bath until the 2-
T o 10 m L of solution S add 0.1 m L o f phenolphthalein propanol R has evaporated. Allow to cool and add 5 m L of
solution R l. T h e solution is colourless. N o t more than water R. Mix and filter through a wet filter previously washed
0.2 m L o f 0.1 M sodium hydroxide is required to change the with water R until free from chlorides. Dilute the filtrate to
colour o f the indicator. 10.0 m L with water R. T o 5.0 m L of the solution, add nitric
S p ecific o p tic a l ro ta tio n (2.2.7) acid R dropwise until the precipitate which forms is
+ 41.0 to + 44.0, determined on solution S. redissolved and dilute to 15 m L with water R. T h e solution
complies with the limit test for chlorides (2.4.4).
1-394 Camphor 2016
H
R e sid u e o n e v a p o ra tio n (2.8.9) OH
M axim um 0.05 per cent. fT ^ t-c h 3
and enantiomer
Evaporate 2.0 g on a w ater-bath and dry in an oven at ch 3
100-105 °C for 1 h. T h e residue weighs a maximum o f i Cl 3
ch
1 mg.
W a te r
G. exo-2J3J3-trim ethylbicyclo[2.2.l]heptan-2-ol
Dissolve 1 g in 10 m L o f light petroleum R. T h e solution is
(camphene hydrate),
clear (2.2.1).
IM P U R IT IE S
ch 3
ch 3 f T ^ i - OH
and enantiomer
ch 3
H
ch 3
and enantiomer
h3c
H3C
H H . endo-2j3j3-trimethylbicyclo[2.2. l]heptan-2-ol
(methylcamphenilol),
A. 2,6,6-trim ethylbicyclo[3.1.1]hept-2-ene (a-pinene),
wrlj
OH
H
! Cl 3
ch and enantiomer
ch 3 S i p 1“
Ct, and enantiomer
I. exo-1,7,7-trimethylbicyclo [2.2.1] heptan-2-ol (exo-bomeol),
B. 2,2-dim ethyl-3-methylenebicydo[2.2. ljheptane ch 3

(cam phene), H
HsC -I^ /N ^ O H
and enantiomer
CH,
H
and enantiomer
h3c
h3c J. fi?j<ib-l,7j7-trimethyIbicyclo[2.2. l]heptan-2-ol
(endo-bomeol).
P hE tr
C. 6,6-dimethyl-2-methylenebicyclo[3.1 .ljh ep tan e
(p-pinene),
CH3
★.* * * ★
Racemic Camphor ★ ★
*+ +*
ch 3
ch 3
CH,
D . l,3,3-trimethyl-2-oxabicyclo[2.2.2]octane (dneole), H3C-

and enantiomer
h3c '
CH,
and enantiomer C ioH I60 152.2 76-22-2

ch 3
ch 3
Counter-irritant.
E. 1,33-trim ethylbicyclo[2.2. l]heptan-2-one (fenchone), P r e p a ra tio n s
C am phorated O pium T incture
ch 3 C oncentrated Cam phorated O pium Tincture
oh
Concentrated Cam phor W ater
fV V -H
and enantiomer
CH3 P h E tr .
u Cl 3
ch
D E F IN IT IO N
(1 i?5,4i?5)-1,7,7-Trimethylbicyclo [2.2.1] heptan-2-one.
F. exo-1,3,3-trimediylbicyclo [2.2.1 ]heptan-2-ol (fenchol), CHARACTERS
A p p e a ra n c e
W hite or alm ost white, crystalline powder or friable,
crystalline masses, highly volatile even at room tem perature.
2016 Candesartan Cilexetdl 1-395
S olubility — signal-to-noise ratio: minimum 5 for the principal peak in

Slightly soluble in water, very soluble in ethanol the chromatogram obtained with reference solution (b).
(96 per cent) and in light petroleum, freely soluble in fatty Limits:
oils, very slightly soluble in glycerol. — arty impurity: for each impurity, not more than 2 per cent
ID E N T IF IC A T IO N of the area o f the principal peak;
— total: n o t more than 4 per cent o f the area o f the principal
First identification A , C
peak;
Second identification A , B, D — disregard Itmir. the area of the principal peak in the
A. Optical rotation (see Tests). chrom atogram obtained with reference solution (b).
B. Melting point (2.2.14): 172 °C to 180 °C. Halogens
C. Infrared absorption spectrophotometry (2.2.24). M axim um 100 ppm.
Preparation Mulls in liquid paraffin R. Dissolve 1.0 g in 10 m L of 2-propanol R in a distillation flask.
Comparison racemic camphor CRS. A dd 1.5 m L o f dilute sodium hydroxide solution R and 50 m g
D . Dissolve 1.0 g in 30 m L o f methanol R. Add 1.0 g o f o f nickel-aluminium alloy R. Heat on a water-bath until the
hydroxylamme hydrochloride R and 1.0 g of anhydrous sodium 2-propanol R has evaporated. Allow to cool and add 5 m L of
acetate R. Boil under a reflux condenser for 2 h. Allow to water R. M ix and filter through a wet filter previously washed
cool and add 100 m L o f water R. A precipitate is formed. with water R until free from chlorides. Dilute the filtrate to
Filter, wash with 10 m L o f water R and recrystallise from 10.0 m L w ith water R. T o 5.0 m L of this solution, add nitric
10 m L o f a mixture of 4 volumes o f ethanol (96 per cent) R acid R dropwise until the precipitate which forms is
and 6 volumes o f water R T h e crystals, dried in vacuo, melt redissolved and dilute to 15 mL with water R. T he solution
(2.2.14) at 118 °C to 121 °C. complies w ith the limit test for chlorides (2.4.4).
TESTS
Water
Dissolve 1 g in 10 m L of light petroleum R. The solution is
Carry out the weighings rapidly.
clear (2.2.1).
S o lu tio n S
Residue on evaporation
Dissolve 2.50 g in 10 m L of ethanol (96 per cent) R and
dilute to 25.0 m L with the same solvent.
Evaporate 2.0 g on a water-bath and diy at 100-105 °C for
1 h. T he residue weighs not more than 1 mg.
Solution S is d ear (2.2.1) and colourless (2.2.2, Method II).
__________________________________________________ PhEur
A cid ity o r a lk alin ity
Dissolve 1.0 g in 10 m l. o f ethanol (96 per cent) R and add
0.1 m l, of phenolphthalein solution R l. T he solution is
colourless. N o t more than 0.2 m L o f 0.1 M sodium hydroxide
is required to change the colour o f the indicator. Candesartan Cilexetil *****
O p tic a l ro ta tio n (2.2. 7)
(Ph Eur. monograph 2573) *
—0.15° to + 0.15°, determined on solution S.
R elated su b sta n c e s
in hexane R and dilute to 50.0 m L with the same solvent.
Reference solution (a) Dissolve 50 mg o f the substance to be
examined and 50 m g o f bomyl acetate R in hexane R and
200.0 m L with hexane R.
Column:
— sizer. I = 2 m , 0 = 2 mm;
— stationary phase: diatomaceous earth for gas C33H34N60 6 611 145040-37-5
chromatography R impregnated with 10 per cent mtm of
macrogol 20 000 R. A c tio n a n d u se
Carrier gas nitrogen for chromatography R. Angiotensis II (ATj) receptor antagonist
Flow rate 30 mlVmin. PhBr__________________________________________________________
Temperature.
— column: 130 °C; D E F IN IT IO N
— injection port and detector. 200 °C. (1 RS)-1- [ [(Cyclohexyloxy)carbonyI] oxy] ethyl 2-ethoxy-1-
[ [2'-(lH -tetrazol-5-yl) biphenyl-4-yI] methyl] -1H-
benzimidazole-7-carboxylate.
Injection 1 |iL.
C o n te n t
Run time 3 times the retention time o f camphor.
System suitability:
— resolution: minimum 1.5 between the peaks due to CHARACTERS
cam phor and bom yl acetate in the chromatogram A p p e a ra n c e
obtained with reference solution (a); W hite or almost white powder.
1-396 Candesartan Cilexetil 2016
Solubility System suitability: reference solution (b):

Practically insoluble in water, freely soluble in methylene — resolution: minimum 4.0 between the peaks due to
chloride and slightly soluble in anhydrous ethanol. impurities A and B.
It shows polym orphism (5.9). Limits:
IDENTIFICATION — correction factors: for the calculation o f content, multiply
corresponding correction facto r impurities A and
Comparison candesartan cUexetH CRS. G = 0.7; impurity H = 1.6;
If the spectra obtained show differences, dissolve the — impurity B: not m ore than 3 times the area o f the
substance to be exam ined and the reference substance principal peak in the chrom atogram obtained w ith
separately in anhydrous ethanol R, evaporate to dryness and reference solution (a) (0.3 per cent);
record new spectra using the residues. — impurities F, G: for each impurity, n o t m ore th an twice
TESTS the area o f the principal peak in the chrom atogram
Related substances obtained with reference solution (a) (0.2 p er cent);
Liquid chrom atography (2.2.29). Prepare the solutions — impurities A , H: for each impurity, n o t m ore than
immediately before use. 1.5 times the area o f the principal peak in the
Solvent mixture water R , acetonitrile R (40:60 V/V). (0.15 p er cent);
Test solution Dissolve 20 m g o f the substance to be examined — unspecified impurities: for each im purity, n o t m ore than die
in 50.0 m L o f the solvent mixture. area o f the principal peak in the chrom atogram obtained
Reference solution (a) D ilute 1.0 m L o f the test solution to with reference solution (a) (0.10 p er cent);
100.0 m L with the solvent mixture. Dilute 1.0 m L o f this — total: n o t more than 6 times the area o f the principal peak
solution to 10.0 m L w ith the solvent mixture. in the chromatogram obtained with reference solution (a)
Reference solution (b) Dissolve 5 mg o f candesartan cilexetil for (0.6 p er cent);
system suitability CRS (containing impurities A, B and F) in — disregard limit. 0.5 times the area o f the principal peak in
the solvent mixture a n d dilute to 10.0 m L with the solvent the chrom atogram obtained with reference solution (a)
mixture. (0.05 p er cent).
Reference solution (c) Dissolve 2.5 m g o f candesartan cilexetilfor W a te r (2.5.32)
peak identification C RS (containing impurities G and H ) in M axim um 0.3 per cent, determ ined on 60.0 mg.
the solvent mixture an d dilute to 5.0 m L with the solvent S u lfa te d a s h (2.4.14)
mixture. M aximum 0.1 per cent, determ ined on 1.0 g.
Column:
A SSA Y
— size: I — 0.15 m , 0 = 3.9 mm;
Dissolve 0.500 g in 60 mT. o f glacial acetic acid R. T itrate
— stationary phase: octadecylsHyl silica gelfor chromatography R
im mediately with 0.1 M perchloric add, determining the
(4 pm).
end-point potentiometrically (2.2.20) at the 1st inflexion
Mobile phase: point.
— mobile phase A: glacial acetic add R, water R, acetonitrile R
1 m L o f 0.1 M perchloric acid is equivalent to 61.1 m g of
(1:43:57 V/V/V);
C33H34N60 6.
— mobile phase B: glacial acetic add R, water R, acetonitrile R
(1:10:90 VIVIV)', IM P U R IT IE S
Specified impurities A, B, F , G, H
Time Mobile phase A Mobile phase B Other detectable impurities (the following substances would, if
(min) (per cent V/V) (per cent V/V) present a t a sufficient level, be detected by one o r other o f
0 -3 100 0 the tests in the monograph. They are limited by the general
3 - 33 100 * 0 0+100
by the general m onograph Substances for pharm aceutical use
3 3 -4 0 0 100 (2034). It is therefore not necessary to identify these
Control of impurities in substances for pharmaceutical use): C, D,
E, I.
Detection Spectrophotom eter at 254 n m .
Injection 10 |iL. O
Identification of impurities Use the c h r o m a t o g r a m supplied
with candesartan cilexetilfor system suitability CRS and the
identify the peaks due to impurities A, B and F; use the
chrom atogram supplied with candesartan cUexetUfor peak
iderttification CRS and the chrom atogram obtained with
reference solution (c) to identify the peaks due to
impurities G and H .
Relative retention W ith reference to
c a n d e s a r t a n cilexetil A. ethyl 2-ethoxy-1- [[2 '-( 1//-tetrazol-5-yl)biphenyl-4-
(retention time = about 11 min): im purity G = about 0.2; yl] methyl] -1 /J-benzimidazole-7 -carboxylate,
im purity A = about 0.4; impurity B = about 0.5;
im purity F = about 2.0; im purity H = about 3.5.
2016 Candesartan Cilexetil 1-397
and enantiomer
and enantiomer
B. (1 RS)-1- [ [(cydohexyloxy)carbonyl]oxy]ethyl 2-oxo-3-[[2'-

(1 H-tetrazol-5-yl)biphenyl-4-yl] methyl] -2,3-dihydro-1H- F. (1 RS)-1 - [ [(cydohexyloxy)carbonyl] oxy] ethyl 2-ethoxy-l-
benzimidazole-4-carboxylate, [ [2 /-(2-ethyl-2H-tetrazol-5-yI) biphenyl-4-yl] methyl] -IH -
benzimidazole-7 -carboxylate.
G . 2-ethoxy-1- [ [2'-(1 H-tetrazol-5-yl)biphenyl-4-yI]methyl]-

lH-benzimidazole-7-carboxylic a d d (candesartan),
C. ( 1RS)-1-[[(cydohexyloxy)carbonyl]oxy]ethyl 3-[[2'-
(1 -ethyl-1H-tetrazol-5-yI) biphenyl-4-yI] methyl] -2-oxo-2,3-
dihydro-lH-benzimidazole-4-carboxylate,
ch3
H . (1 RS)-1 - [ [(cydohexyloxy)carbonyl]oxy]ethyl 2-ethoxy-l-

[ [ 2 [ 1 -(triphenylmethyl)-l//-tetrazol-5-yl] biphenyl-4-
D . (1 RS)-1 - [ [(cydohexyloxy)carbonyl] oxy] ethyl 3 -[[2'- yl] methyl] - lii-benzimidazole-7-carboxylate
(2-ethyl-2H-tetra2ol-5-yI)biphenyl-4-yI]methyI]-2-oxo-2,3- (AT-tritylcandesartan),
dihydro-lH-benzimidazole-4-caiboxylate,
I. methyl 2-ethoxy-l-[[2/-(l//-tetrazol-5-yl)biphenyl-4-
yI]methyl]-lJi-benzimidazole-7-carboxylate.
PhEur
E. (1 RS ) - 1- [ [(cydohexyloxy)carbonyl]oxy]ethyl 2-ethoxy-l-
[ [2'-(1 -ethyl-1Ji-tetrazol-5-yI)biphenyl-4-yl] methyl] -IH -
benzimidazole-7-carboxylate,
1-398 Capecitabine 2016
Column:
Capecitabine ****** — size. I = 0.25 m , 0 = 4.6 mm;
(Ph. Eur. monograph 2762) * — stationary phase: end-capped octadecylsUyl silica gelfor
chromatography R (5 jxm);
o — temperature. 40 °C.
Mobile phase.
— mobile phase A: acetomtrUe R, methanol R, 0.1 per cent V/V
solution o f glacial acetic add R (5:35:60 VIVIV)’,
— mobile phase B: acetonitrUe R, 0.1 per cent V/V solution of
glacial acetic add R, methanol R (5:15:80 VIVIV);

OH OH (min) (per cent V/V) (percent V/V)
0 -5 100 0
C 15H22FN306 359.3 154361-50-9 5 -2 0 100 -»49 0 -> 51
2 0 -3 0 49 51
Pyrimidine analogue; cytotoxic; treatm ent of colorectal
cancer. Flow rate 1.0 mL/min.
P h E u r ____________________________________________________________________________________________
Injection 10 pL of the test solution and reference solutions (b)
D E F IN IT IO N
and (c).
Pentyl [l-(5-deoxy-|3-D-ribofuranosyl)-5-fluoro-2-oxo-l,2-
dihydropyrimidin-4-yl] carbamate.
with reference solution (c) to identify the peaks due to
C o n te n t impurities A, B and D.
Relative retention W ith reference to capecitabine (retention
CHARACTERS time = about 17 min): impurity A = about 0.18;
A p p e a ra n c e impurity B = about 0.19; impurities D and E = about 0.95.
W hite or alm ost white powder. System suitability: reference solution (c):
S o lu b ility — resolution: m inim um 1.5 between the peaks due to
Sparingly soluble in water, freely soluble in anhydrous impurities A and B; m inim um 2.0 between the peaks due
ethanol, practically insoluble in heptane. to impurity D and capecitabine.
— for each impurity, use the concentration o f capecitabine
in reference solution (b);
B. Infrared absorption spectrophotometry (2.2.24). — correction factor, multiply the peak area o f impurity B by
Comparison capecitabine CRS. 1.3.
TESTS Limits:
S p ecific o p tic a l ro ta tio n (2.2.7) — impurities A, B: for each impurity, maximum 0.3 per cent;
+ 96.0 to + 100.0 (anhydrous substance). — sum of impurities D and E: m aximum 0.2 p er cent;
Dissolve 0.250 g in methanol R and dilute to 25.0 mT. with — unspecified impurities: for each impurity, maximum
the same solvent. 0.05 per cent;
R e la te d su b s ta n c e s — reporting threshold: 0.03 per cent.
L iquid chrom atography (2.2.29). Prepare the solutions
immediately before use or store them at 2-8 °C. Water (2.5.32)
Solvent mixture acetomtrUe R, methanol R, water R
Inject 1.0 m L o f a 0.200 g/m L solution of the substance to
(5:35:60 V/V/V).
be examined in methanol R.
examined in 80 m L o f the solvent mixture, sonicate until Sulfated ash (2.4.14)
dissolution is complete and dilute to 100.0 m L with the Maximum 0.1 per cent, determ ined on 1.0 g in a platinum
solvent mixture. crucible.
Reference solution (a) Dissolve 60.0 m g o f capecitabine CRS in ASSAY
80 m L of the solvent mixture, sonicate until dissolution is Liquid chromatography (2.2.29) as described in the test for
com plete and dilute to 100.0 m L with the solvent mixture. related substances with the following modification.
Reference solution (b) Dilute 1.0 m L o f the test solution to Injection T est solution and reference solution (a).
100.0 m L w ith the solvent mixture. D ilute 1.0 m L o f this Calculate the percentage content o f C 15H 22F N 3O 6 taking
solution to 20.0 m L with the solvent mixture. into account the assigned content o f capecitabine CRS.
Reference solution (c) Dissolve 3 mg of capecitabine IMPURITIES
impurity A CRS, 3 m g o f capecitabine impurity B CRS and
Spedfied impurities A, B, D , E
5 m g of capecitabine impurity D CRS in 80 mT, of the solvent
m ixture, sonicate until dissolution is complete and dilute to Other detectable impurities (the following substances would, if
100.0 m L w ith the solvent mixture. D ilute 1 m L o f the present at a sufficient level, be detected by one or other of
solution to 50 m L w ith the test solution. the tests in the m onograph. T hey are limited by the general
2016 Caprylocaproyl Macrogolglycerides 1-399

im purities for demonstration of compliance. See also 5.10.
Control of impurities in substancesfor pharmaceutical use):
C, F, G.
NH2
OH OH
N
O ^N E. 3-methyIbutyl [l-(5-deoxy-P-D-ribofuranosyl)-5-fluoro-2-
CH3 n oxo-1j2-dihydropyrimidin-4-yl] carbamate,
OH OH
A. 4-am ino-1-(5-deoxy-P-D-ribofuranosyl)-5-fluoropyrimidin-
2 (1 //)-one (5'-deoxy-5-fluorocytidine)3
3 0 "
O ' N
F. pentyl [5-fluoro-1- [(3ai?34i?36R,6ai?)-6-methyl-2-

OH OH oxotetrahydrofuro [3,4-d] [ 1,3] dioxol-4-yI] -2-oxo-1,2-
dihydropyrimidm-4-yI] carbamate,
B. l-(5-deoxy-P-D-ribofiiranosyl)-5-fluoropyrimidine- 0
2 ,4(lH ,3if)-d io n e (5'-deoxy-5-fluorouridine)3 1
HN O
G. pentyl [l-(2,3-di-0-acetyi-5-deoxy-P-D-ribofiiranosyl)-5-
C . 1-(2j3-di-0-acetyi-5-deoxy-P-D-ribofuranosyI)-4-amino-5- fluoro-2-oxo-1,2-dihydropyrimidin-4-yl] carbamate.
fluoropyrim idin-2(l//)-one3 PhEur
Caprylocaproyl ★ ★
★ ★
Macrogolglycerides *****
Action and use

Excipient.
P h E tr ___________________
D . (2R5)-2-methylbutyl [l-(5-deoxy-P-D-ribofuranosyl)-5- DEFINITION

fluoro-2-oxo-1,2-dihydropyrimidin-4-yl] carbamate, Mixtures o f monoesters, diesters and triesters o f glycerol and
monoesters and diesters o f macro gols with a m ean relative
molecular mass between 200 and 400.
They are obtained by partial alcoholysis o f medium-chain
triglycerides using macrogol or by esterification o f glycerol
and macrogol with caprylic (octanoic) a d d and capric
(decanoic) a d d or a mixture o f glycerol esters and
condensates of ethylene oxide with caprylic a d d and capric
ad d . They may contain free macrogols.
1-400 Caprylocaproyl Macrogolglycerides 2016
CHARACTERS Ethylene oxide units Type o f macrogol Saponification value

per molecule
A p p e a ra n c e (nominal value)
Pale-yellow, oily liquid. 4 200 265 to 285
S o lu b ility 6 300 170 to 190
Dispersible in h o t water, freely soluble in methylene chloride.
8 400 85 to 105
D e n sity
A bout 1.0 at 20 °C.
R efractiv e in d e x A lk a lin e im p u ritie s
A bout 1.4 at 20 °C. Introduce 5.0 g into a test-tube and carefully add a mixture,
ID E N T IF IC A T IO N neutralised if necessary w ith 0.01 M hydrochloric acid o r with
0.01 M sodium hydroxide, o f 0.05 m L o f a 0.4 g/L solution of
bromophenol blue R in ethanol (96per cent) R, 0.3 m l, o f
Test solution Dissolve 1.0 g o f the substance to be examined water R and 10 m L o f ethanol (96 per cent) R. Shake and
in methylene chloride R and dilute to 20 m L with the same
allow to stand. N o t more than 1.0 m L o f 0.01 M hydrochloric
solvent.
add is required to change the colour o f die u p p er layer to
Plate TLC silica gel plate R yellow.
Mobile phase hexane R, ether R (30:70 V/V). F r e e glycero l
Application 50 fiL. M axim um 5.0 per c e n t
Development Over a p ath o f 15 cm. Dissolve 1.20 g in 25.0 m L of methylene chloride R. H eat if
Drying In air. necessary. After cooling, add 100 m L o f water R. Shake and
Detection Spray with a 0.1 g/L solution o f rhodamine B R in add 25.0 m L of periodic acetic acid solution R. Shake and allow
ethanol (96 per cent) R and examine in ultraviolet light at to stand for 30 min. Add 40 m L o f a 75 g/L solution o f
365 nm . potassium iodide R. Allow to stand for 1 min. A dd 1 m L o f
starch solution R. T itrate the iodine with 0.1 M sodium
Results T he chrom atogram shows a spot due to triglycerides
thiosulfate. Carry o u t a blank titration.
with an Rp value of about 0.9 (R„ 1) and spots due to
1,3-diglycerides (Ra 0.7), to 1,2-diglycerides (R^ 0.6), to 1 m L o f 0.1 M sodium thiosulfate is equivalent to 2.3 m g o f
monoglycerides (Ra 0.1) and to esters o f macrogol (R„ 0). glycerol.
B. Hydroxyl value (see Tests). C o m p o sitio n o f fa tty a c id s (2.4.22, Method A)
Composition of the fatty-acid fraction of the substance:
C. Saponification value (see Tests).
— caproic acid: maximum 2.0 p er cent;
D. Composition of fatty ad d s (see Tests). — caprytic acid: 50.0 per cent to 80.0 per cent;
TESTS — capric add. 20.0 per cent to 50.0 per cent;
V iscosity (2.2.9) — lauric add: m axim um 3.0 per cent;
Carry out the determ ination at 20 ± 0.5 °C. — myrisdc add: maximum 1.0 p er cent.
E th y le n e oxide a n d d io x a n (2.4.25)
Ethylene oxide onto Type of macrogol Viscosity Maximum 1 ppm o f ethylene oxide and m axim um 10 ppm
per molecule (mPa>s) o f dioxan.
(nominal value)
4 200 30 to 50
Maximum 10 ppm .
6 300 60 to 80
8 400 80 to 110 using 2 m L o f lead standard solution (10 ppm Pb) R.
W a te r (2.5.12)
Maximum 1.0 per cent, determ ined on 1.0 g. U se a mixture
A cid v alu e (2.5.1)
o f 30 volumes of anhydrous methanol R and 70 volumes of
M aximum 2.0, determined on 2.0 g.
methylene chloride R as solvent.
H y d ro x y l v a lu e (2.5.3, Method A)
T o ta l a s h (2.4.16)
Use 1.0 g.
L A B E L L IN G
Ethylene oxide units TYpe of macrogol Hydroxyl value
per molecule T h e label states th e type of macrogol used (m ean relative
(nominal value) m olecular mass) o r th e num ber o f ethylene oxide units p er
4 200 80 to 120 m olecule (nominal value).
6 300 140 to 180 __________________________________________________________________PhEtir
8 400 170 to 205
P e ro x id e v a lu e (2.5.5, Method A)
Maximum 6.0, determ ined on 2.0 g.
S a p o n ific a tio n v alu e (2.5.6)
Use 2.0 g.
2016 Captopril 1-401
Reference solution (b) Mix 0.25 m L o f reference solution (a)

Captopril ****** and 0.75 m L of butyl acetate R.
*. ■*
(Ph. Eur. monograph 1079) * Column:
0 u — size: I = 25 m , 0 = 0.32 mm;
II \ ..C02H — stationary phase: pofy (dimethyl) (diphenyl) sHoxane R (film
thickness 1 |im).
H CH3 1__ /
C,Hi5N03S 217.3 62571-86-2
SpUt ratio 1:20.
A ctio n a n d u se Temperature:
Angiotensin converting enzyme inhibitor.
P re p a ra tio n s Time Temperature
Captopril Oral Solution (min) CC)
Captopril Tablets Column 0 -1 0 200
10-14 200 -> 240

P hEur --------------------------------------------------------------------------------------------------------------------------------------------
14-34 240
D E F IN IT IO N
(2<S)-l-[(25)-2-Methyl-3-sulfanylpropanoyl]pyrrolidine-2- Injection port 270
carboxylic ad d . • Detector 300
C o n te n t 7
CHARACTERS Injection 1 |iL.
A p p e a ra n c e
Relative retention W ith reference to captopril (retention
time = about 6 min): impurity F = about 0.96.
S o lub ility System suitability.
Soluble in water, freely soluble in methanol and in methylene — resolution: minimum 1.5 between the peaks due to
chloride. It dissolves in dilute solutions of alkali hydroxides. impurity F and captopril in the chromatogram obtained
ID E N T IF IC A T IO N w ith reference solution (a);
A. Specific optical rotation (see Tests). — signal-to-noise ratio: minimum 10 for the peak due to
B. Infrared absorption spectrophotometry (2.2.24). im purity F in the chromatogram obtained with reference
solution (b).
Comparison captopril CRS.
Calculate the percentage content o f impurity F using the
TESTS following expression:
S o lu tio n S
A
25 m L w ith die same solvent. - — - x 100
A +B
A = area of the peak due to im purity F in the
p H (2.2.3) chromatogram obtained w ith the test solution;
2.0 to 2.6 for solution S. B = area o f the peak due to captopril in the
Specific o p tic a l ro ta tio n (2.2.7) chromatogram obtained w ith the test solution.
—132 to —127 (dried substance). Limit:
Dissolve 0.250 g in anhydrous ethanol R and dilute to — impurity F: maximum 0.2 per cent.
25.0 m L with the same solvent. R e la te d su b sta n c e s
Im p u rity F L iquid chromatography (2.2.29).
Gas chromatography (2.2.28). Solvent mixture phosphoric arid R, acetomtrUe R l, water R
Reagent solution A dd 2.8 m L o f acetyl chloride R dropwise to (0.08:10:90 VIVIV).
17.2 m L o f anhydrous methanol R at 0 °C and mix. Allow to Test solution Dissolve 0.125 g of th e substance to be
stand for 20 mm at room temperature before use. examined in the solvent mixture and dilute to 25.0 m L with
Test solution Introduce 20.0 mg o f the substance to be the solvent mixture.
examined into a vial and add 1.0 m L of the reagent solution. Reference solution (a) Dissolve 4.0 m g o f captopril
Mix and heat at 60 °C for 30 min. Evaporate to dryness impurity J CRS, 5.0 mg o f captopril impurity B CRS, 5.0 m g
under a stream o f nitrogen R. Dissolve the residue in 0.5 m L o f captopril impurity C CRS and 5.0 m g of captopril
of ethyl acetate R, add 0.5 m L of pentafluoropropionic impurity D CRS in the solvent mixture and dilute to 50.0 m L
anhydride R, mix and heat at 60 °C for 30 min. Evaporate to with the solvent mixture. Dilute 1.0 m L o f the solution to
dryness under a stream o f nitrogen R. Dissolve the residue in 20.0 m L with the solvent mixture. Prepare immediately
1.0 m L o f butyl acetate R. before use.
Reference solution (a) Dissolve the contents o f a vial of Reference solution (b) Dissolve 5 m g o f the substance to be
captoprilfor system suitability CRS (containing impurity F) in examined and 5 m g o f captopril impurity E CRS in
1.0 m L o f the reagent solution. Prepare as described for the acetomtrUe R and dilute to 25.0 m L with the same solvent.
test solution.
1-402 Captopril 2016
D ilute 4 m L o f the solution to 50.0 m L with the solvent — unspecified impurities: for each im purity, n o t m ore than the
mixture. area o f die principal peak in the chrom atogram obtained
Reference solution (c) In order to prepare impurity A in situ, w ith reference solution (d) (0.10 per cent);
introduce 1.0 m L of the test solution into a volumetric flask — total: maximum 1.2 p er cent;
and add 230 jiL of 0.05 M iodine. If the solution is not — disregard Omit: 0.5 times the area o f the principal peak in
colourless, add 0.1 M sodium tkiosulfate dropwise until it the chrom atogram obtained with reference solution (d)
becomes colourless, and dilute to 50.0 m L with the solvent (0.05 per cent).
mixture. D ilute 5.0 m L o f this solution to 20.0 m L with the H e a v y m e ta ls (.2.4.8)
solvent mixture. M axim um 20 ppm .
Reference solution (d) Dilute 1.0 m L o f the test solution to Solvent water R.
100.0 m L with the solvent mixture. Dilute 1.0 m L o f this 0.50 g complies with test H . Prepare the reference solution
solution to 10.0 m L w ith the solvent mixture. using 1 m L o f lead standard solution (10 ppm Pb) R.
Column:
— sizer. I = 0.3 m, 0 = 3.9 mm;
M axim um 1.0 per cent, determ ined on 1.000 g by drying
— stationary phase, end-capped octadecylsUyl silica gelfor
u n d er high vacuum at 60 °C for 3 h.
— temperature: 50 °C. S u lfa te d a s h (2.4.14)
Mobile phase: M axim um 0.2 per cent, determ ined on 1.0 g.
— mobile phase A: phosphoric add R, water R (0.08:100 VIV); A SSA Y
— mobile phase B: phosphoric add R, acetonitrile R l, water R Dissolve 0.150 g in 30 m L o f water R. T itrate with 0.05 M
(0.08:50:50 V/V/V); iodine, determining the end-point potentiom etrically (2.2.20).
U se a combined platinum electrode.
Time Mobile phase A Mobile phase B 1 m L o f 0.05 M iodine is equivalent to 21.73 m g of
(min) (per cent V7V) (percent V7V) c 9h 15n o 3s .
0 -5 90 10 IM P U R IT IE S
5 -2 0 90-» 50 10-» 50 Specified impurities A , B, C, D, E, F, J.
2 0 -4 5 50 50 Other detectable impurities (the following substances would, if
Flow rate 1.5 miVmin acceptance criterion for other/unspecified impurities and/or
Detection Spectrophotom eter at 210 run. by the general m onograph Substances for pharmaceutical use
Irgecdon 25 |iL. (2034). It is therefore n o t necessary to identify these
Identification of impurities Use the chromatogram obtained Control of impurities in substances for pharmaceutical use): G, H,
1, L, M, N , O.
impurities B, C , D and J; use the chromatogram obtained
im purity E; use the chrom atogram obtained with reference
solution (c) to identify the peak due to impurity A.
Relative retention W ith reference to captopril (retention
time = about 15 min): impurity C = about 0.6;
im purity D = about 0.8; impurity E = about 0.9; A. 1 ,1 '-[disulfanediylbis [(2S)-2-m ethyl-1-oxopropane-3,1-
im purity B = about 1.17; im purity J = about 1.22; diyl]]bis[(25)-pyrrolidine-2-carboxylic] acid (captopril
im purity A = about 1.7. disulfide),
System suitability:
— resolution: m inim um 1.5 betw een the peaks due to
, c o 2h
impurities B and J in the chromatogram obtained with
impurity E and captopril in the chromatogram obtained
w ith reference solution (b). B. (2S)-1 - [(2S)-3-bromo-2-methylpropanoyl]pyrrolidine-2-
Limits: carboxylic a d d ,
— impurity A: not m ore than 10 times the area of the
no and enantiomer
reference solution (d) (1.0 per cent); H CH3
— impurity J: not m ore than 2.5 times the area of the
corresponding peak in the chrom atogram obtained with
C. (2itS)-2-methyl-3-sulfanylpropanoic a d d ,
— impurities B, C, D: for each impurity, not more than
1.5 times the area o f the corresponding peak in the and enantiomer
chrom atogram obtained w ith reference solution (a) H CH3
(0.15 per cent);
— impurity E: not m ore than 1.5 times the area of the D . (2i?5)-3-bromo-2-methylpropanoic a d d ,
reference solution (d) (0.15 per cent);
2016 Carbachol 1-403
h o 2c . f h3c ch3
V ..CO2H
n' yç 's '^ s ' y: n'
~ & r H CH 3 H CH3
E. (25)-1 -(2-methylpropanoyl)pyrrolidine-2-carboxylic acid. O . 1,1 '-[propane-2,2-ctiylbis[sulfanediyl[(25)-2-methyl-l-

oxopropane-3,l-diyl]]]bis[(25)-pyrrolidine-2-carboxylic] acid.
..CO2H -------------------------------------------------------------------------------------- — ------------- -- -------------------------------- PhEur
F. (25)-l-[(2Æ)-2-methyl-3-sulfanylpropanoyl]pyrrolidine-2- ★
*** ★
carboxylic acid (ept-captopril),
Carbachol ★ ★
(Ph. Eitr. monograph 1971) *****
o
H *CI3O S /y ? h3C ch3

' \ and enantiomer cr
H CH3
h2n o ch3
G . (2i?5)-3-(acetylsulfanyl)-2-methylpropanoic acid,
C<>H15C 1N20 2 182.7 51-83-2
A ctio n a n d u se
Cholinoceptor agonist.
PhEtr.
H . (25)-1 -[(25)-3-[[(2i?)-3 -(acetylsulfanyl)-2- D E F IN IT IO N

methylpropanoyl] sulfanyl] -2-methylpropanoyl] pyrrolidine-2- 2-(Carbamoyloxy)-NJjVJiV-trimethy1ethanaminium chloride.
caiboxylic acid,
C o n te n t
, . c o 2h CHARACTERS
A p p e a ra n c e
W hite or almost white, crystalline, hygroscopic powder.
S o lu b ility
I. (25)-l-[(25)-3-[[[(25)-l-[(25)-2-m ethyl-3- Very soluble in water, sparingly soluble in alcohol, practically
sulfanylpropanoyl] pyrrolidin-2-yl] carbonyl] sulfanyl]-2- insoluble in acetone.
methylpropanoyl]pyrrolidine-2-carboxylic acid,
, . c o 2h
Comparison carbachol CRS.
J. (25)-l-[(25)-3-(acetylsulfanyl)-2- B. Examine the chromatograms obtained in the test for
methylpropanoyl]pyrrolidine-2-carboxylic acid related substances.
(acetylcaptopril), Results T he principal spot in the chromatogram obtained with
test solution (b) is similar in position, colour and size to the
H Q 2c !
..CC^H principal spot in the chromatogram obtained with reference
solution (a).
C. 0.5 m L o f solution S (see Tests) gives reaction (a) of
chlorides (2.3.1).
L. 1,1'- [methylenebis [sulfenediyl [(25)-2-m ethyl-1- TESTS
oxopropane-3, l-diyl]]]bis[(25)-pyrrolidine-2-carboxylic] acid, S o lu tio n S
h 3c h
..C O jH 25 m L with the same solvent.
H O 2C
X ^ S
. ^ ,
M . (25)-1 - [(25)-3- [ [(25)-2-carboxypropyl] disulfianyl] -2- T o 2.0 m L of solution S, add 0.05 m L o f methyl red mixed
methylpropanoyl]pyrrolidine-2-carboxylic acid, solution R. N o t m ore than 0.2 m L of 0.01 M hydrochloric acid
or 0.01 M sodium hydroxide is required to change the colour
h3c h
o f the indicator.
HOj C
H CH 3
Prepare the solutions immediately before use.
N . 3 ,3 /-disulfanediyIbis[(25)-2-methylpropanoic] acid,
1-404 Carbamazepine 2016

exam ined in methanol R and dilute to 5.0 m L with the same
Carbamazepine *****
★ ★
solvent. (Ph Eur. monograph 0543) *
Test solution (b) Dilute 2.0 m L o f test solution (a) to
Reference solution (a) Dissolve 20 mg o f carbachol CRS in
methanol R and dilute to 5.0 m L with the same solvent.
Reference solution (b) Dissolve 8 mg o f choline chloride R and
8 m g o f acetylcholine chloride CRS in methanol R and dilute to
10.0 m L with the same solvent. Dilute 5.0 m L to 10.0 m L
w ith methanol R.
Plate cellulose for chromatography R as the coating substance. Ci5H12N 20 236.3 298-46-4
Mobile phase water R, methanol R (10:90 VIV).
A ctio n a n d use
Application 10 jiL. Antiepileptic.
Development Over 2/3 o f the plate. P re p a r a tio n
Detection Spray with potassium iodobismuthate solution R3. Carbamazepine Tablets
System suitability T he chromatogram obtained with reference
PhEur__________________________________________________________
solution (b) shows 2 clearly separated spots.
Limits: in the chromatogram obtained with test solution (a): D E F IN IT IO N
— arty impurity, any spot, apart from the principal spot, is 5/f-D ibenzo [bj\ azepine-5-carboxamide.
n o t more intense than one or other of the 2 principal C o n te n t
spots in the chromatogram obtained with reference 98.0 per cent to 102.0 per cent (dried substance).
solution (b) (1 p er cent). Com pare the spots with the
CHA RACTERS
spot o f the most appropriate colour in the chromatogram
obtained with reference solution (b). A p p e a ra n c e
M axim um 20 ppm. S o lu b ility
Very slightly soluble in water, freely soluble in methylene
12 m L of solution S complies with test A. Prepare the
chloride, sparingly soluble in acetone and in ethanol
(96 per cent).
L o ss o n d ry in g (2.2.32) It shows polymorphism (5.9). T he acceptable crystalline form
M axim um 1.0 per cent, determined on 1.000 g by drying in corresponds to carbamazepine CRS.
A. Melting point (2.2.14): 189 °C to 193 °C.
M axim um 0.1 per cent, determined on 1.0 g of the residue
obtained in the test for loss on drying. B. Infrared absorption spectrophotometry (2.2.24).
A SSA Y
Comparison carbamazepine CRS.
Dissolve 0.150 g in a mixture of 10 m L of anhydrous acetic Preparation Examine the substances as discs without prior
acid R and 40 m L o f acetic anhydride R. Titrate with 0.1 M treatm ent.
perchloric acid. Determ ine the end-point potentiometrically TESTS
(2.2.20). Acidity or alkalinity
1 m L o f 0.1 M perchloric acid is equivalent to 18.27 m g of T o 1.0 g add 20 m L of carbon dioxide-free water R, shake for
C o H is C lN ^ . 15 min and filter. T o 10 m L o f the filtrate add 0.05 m L of
STORAGE
phenolphthalein solution R1 and 0.5 m L of 0.01 M sodium
hydroxide; the solution is red. Add 1.0 m L o f 0.01 M
In an airtight container, protected from light
hydrochloric acid; the solution is colourless. Add 0.15 m L of
IM P U R IT IE S methyl red solution R; the solution is red.
“ h 3c ch3
n: or Liquid chromatography (2.2.29).
HO CH3 Test solution (a) Dissolve 60.0 mg of the substance to be
examined in methanol R2 and dilute to 20.0 m L with the
A. 2-hydroxy-NjNjN-trimethylethanaminium chloride same solvent Sonicate. Dilute 10.0 m L o f this solution to
(choline chloride). 20.0 m L with water R.
--------------------------------------------------------------------- --------------------------PhEur Test solution (b) Dilute 10.0 m L o f test solution (a) to
50.0 m L with a mixture of equal volumes o f methanol R2 and
water R.
Reference solution (a) Dissolve 7.5 m g o f carbamazepine CRS,
7.5 m g o f carbamazepine impurity A CRS and 7.5 m g of
iminodibenzyl R (impurity E) in methanol R2 and dilute to
100.0 m L with the same solvent. Dilute 1.0 m L o f this
solution to 50.0 m L with a mixture o f equal volumes of
methanol R2 and water R.
2016 Carbamazepine 1-405
Reference solution (b) Dissolve 60.0 mg o f carbamazepine CRS IM P U R IT IE S

in methanol R2 and dilute to 20.0 m L with the same solvent. Specified impurities A, E
Sonicate. Dilute 5.0 m L of this solution to 50.0 m L with a Other detectable impurities (die following substances would, if
m ixture of equal volumes of methanol R2 and water R. present a t a sufficient level, be detected by one or other of
Column: the tests in the monograph. They are limited by the general
— size. I = 0.25 m , 0 = 4.6 mm; acceptance criterion for other/unspecified impurities and/or
— stationary phase. nitrUe silica gel for chromatography R1 by die general monograph Substances for pharmaceutical use
(10 pm). (2034). It is therefore no t necessary to identify these
Mobile phase tetrahydrofuran R, methanol R2, water R impurities for demonstration of compliance. See also 5.10.
(3:12:85 V/V/V); to 1000 m L o f this solution add 0.2 m L of Control of impurities in substances for pharmaceutical use): B, C,
anhydrous formic add R and 0.5 m L o f triethylamine R. D, F, G.
Injection 20 pL o f test solution (a) and reference solution (a).
Run time 8 times the retention time of carbamazepine.
Relative retention W ith reference to carbamazepine (retention
tim e = about 10 min): im purity A = about 0.9;
im purity E = about 3.5.
A. 10,11 -dihydro-5/i-dibenzo [bj\ azepine-5-carboxamide
System suitability:
(10,11-dihydrocarbamazepine),
im purity A and carbamazepine in the chromatogram
obtained with reference solution (a).
Limits:
— impurities A, E: for each impurity, not m ore than
1.5 times the area o f the corresponding peak in the
(0.15 p er cent);
— unspecified impurities: n o t more than the area of the peak B. 9-methylacridine,
due to carbamazepine in the chromatogram obtained with
— total: no t more than 5 times the area o f the peak due to
carbamazepine in the chromatogram obtained with n n nh2
h 2
carbamazepine in the chromatogram obtained with
reference solution (a) (0.05 p er cent).
C. (5H-dibenzo [bj\ azepin-5-ylcarbonyI)urea
Maximum 140 ppm. (N-carbamoylcarbamazepine),
Suspend 0.715 g in 20 m L o f water R and boil for 10 min.

Cool and dilute to 20 m L with water R. Filter through a
m em brane filter (nominal pore size 0.8 |im ). Dilute 10 mL of
the filtrate to 15 m L with water R.
M aximum 20 ppm .
using 2 m L o f lead standard solution (10 ppm Pb) R. D. 5H-dibenzo [bj\ azepine (iminostilbene),
M axim um 0.5 p er cent, determ ined on 1.000 g by drying in
ASSAY
L iquid chromatography (2.2.29) as described in the test for
E. 10,11 -dihydro-5/i-dibenzo [bj\azepine (iminodibenzyl),
related substances with die following modifications.
Injection T est solution (b) and reference solution (b).
System suitability:
— repeatability: reference solution (b). i a
Calculate the percentage content o f C 15H 12N 20 from the
declared content o f carbamazepine CRS.
STORAGE
In an airtight container. F. 5H-dibenzo [bj\ azepine-5-carbonyl chloride
(5-chlorocarbonyliminostiIbene),
1-406 Carbaryl 2016
Loss on drying
W hen dried to constant weight over phosphorus pentoxide at a
pressure not exceeding 0.7 kPa, loses not m ore than 0.5% of
its weight. Use 1 g.
ASSA Y
G. 10-brom o-5if-dibenzo [bj\ azepine- 5-carboxamide Appendix IE D , using the following solutions.
( 10-bromocarbam azepine). (1) 0.005% w/v o f the substance being examined in methanol.
PhEtr (2) 0.005% w/v of carbaryl BPCRS in methanol.
(3) 0.005% w/v of die substance being examined and
0.005% w/v of 1-naphthol in the mobile phase.
Carbaryl (a) U se a stainless steel column (10 cm x 4.6 mm) packed

with end-capped octadecylsOyl silica gel for chromatography
(5 pm) (Spherisoxb ODS 2 is suitable).
O NHMe below.
(c) U se a flow rate o f 2.5 m L per minute.
(d) Use ambient column tem perature.
(e) U se a detection wavelength of 280 nm.
C i2H u N 0 2 201.2 63-25-2 MOBILE PHASE
1 volume of glacial acetic acid, 25 volumes of acetonitrile and
Action and use
75 volumes of water.
Insecticide.
SYSTEM SUITABILITY
P r e p a r a tio n
Carbaryl Lotion T h e test is not valid unless in the chrom atogram obtained
with solution (3) the resolution factor between the two
D E F IN IT IO N principal peaks is at least 2.0.
Carbaryl is 1-naphthyl methylcarbamate. It contains not less DETERMINATION OF CONTENT
than 98.0% and not more than 102.0% of C ^ H n N O ^ Calculate the content of C 12H n N 0 2 using the declared
calculated with reference to the dried substance.
content of C i2H n N 0 2 in carbaryl BPCRS.
STO RA G E
A white to off-white or light grey powder, which darkens on
Carbaryl should be protected from light.
exposure to light. It melts at about 142°.
IM P U R IT IE S
Very slightly soluble in water, soluble in acetone and in
ethanol (96%).
with the reference spectrum o f carbaryl (R S 039).
TESTS
1 -N ap h th o l
Carry out the m ethod for liquid chromatography, A. 1-naphthol
Appendix III D , using the following solutions.
(1) 0.1% w/v o f the substance being examined in acetonitrile.
(2) 0.005% w/v of carbaryl BPCRS in methanol. **+ ★
★
(3) 0.005% w/v of the substance being examined and Carbasalate Calcium ★ ★
0.005% w/v o f 1-naphthol in the mobile phase. *****
T h e chromatographic conditions described under Assay may COj
be used. o
SYSTEM SUITABILITY Ca2* Ï
h2n nh2
T he test is not valid unless, in the chromatogram obtained
0' ch 3
with solution (3), the resolution factor between the two
principal peaks is at least 2.0.
LIMITS C i 9HigCaN 209 458.4 5749-67-7
Action and use
the area of any peak corresponding to 1-naphthol is not Salicylate; non-selecrive cyclo-oxygenase inhibitor;
greater than the area o f the principal peak in the antipyretic; analgesic; anti-inflammatory.
chrom atogram obtained with solution (2) (1%).
2016 Carbasalate Calcium 1-407
P hE ur ___________________________________________________________________________________________ Reference solution (d) Dilute 1.0 m L o f the test solution to

D E F IN IT IO N 10.0 m L w ith the solvent mixture. M ix 1.0 m L o f this
solution w ith 5.0 m L of reference solution (a), add 1.0 m L
Equim olecular com pound of calcium
o f reference solution (c) and dilute to 10.0 m L w ith the
di[2-(acetyloxy)benzoate] and urea.
solvent mixture.
Content Cdumn:
— size. I = 0.25 m , 0 = 4.0 mm;
CHARACTERS — stationary phase, spherical end-capped octadecylsilyl silica gel
Appearance for chromatography R (5 fim);
W hite or almost white, crystalline powder. — temperature. 40 °C.
Solubility Mobile phase phosphoric add R, acetomtrUe for
Freely soluble in water and in dimethylformamide, practically chromatography R, water R (0.5:40:60 VIVIV).
insoluble in acetone and in anhydrous methanol. Flow rate 1.8 mL/min.
Protect the substance from moisture during handling: Examination Detection Spectrophotom eter at 240 nm .
in aqueous solutions has to be performed immediately after Iiyecdon 10 jiL of the test solution and reference solutions (b)
preparation. and (d).
IDENTIFICATION Run time 8 times the retention time o f acetylsalicylic add.
First identification B, E Identification of impurities Use the chromatogram obtained
Second identification A , C, D, E with reference solution (d) to identify the peaks due to
A. Ultraviolet and visible absorption spectrophotometry impurities B and C.
(2.2.25). Relative retention W ith reference to acetylsalicylic a d d
Test solution Dissolve 0.250 g in water R and dilute to (retention time = about 2 min): impurity C = about 1.3;
100.0 m L with the same solvent. T o 1.0 m L o f the solution im purity B = about 2.5.
add 75 m L o f water R and 5 m L of dilute hydrochloric acid R, System suitability Reference solution (d):
mix and dilute to 100.0 m L with water R Examine — resolution: minimum 5.0 between the peaks due to
immediately. acetylsalicylic a d d and impurity C.
Spectral range 220-350 nm. Limits:
Absorption maxima A t 228 nm and 276 nm. — impurity C: not more than 5 times the area of the
Specific absorbance at the absorption maxima:
— at 228 nm: 363 to 379,
— impurity B: not more than 1.5 times the area of the
— at 276 nm: 49 to 53.
B. Infrared absorption spectrophotometry (2.2.24). reference solution (b) (0.15 per cent);
Comparison Ph. Eur. reference spectrum of carbasalate calcium. — unspecified impurities: for each impurity, not m ore than
C. Dissolve 0.1 g in 10 m L o f water R, boil for 2 min and 0.5 times the area o f the principal peak in the
cooL T he solution gives reaction (a) o f salicylates (2.3.1). chromatogram obtained with reference solution (b)
D . H eat 0.2 g with 0.2 g o f sodium hydroxide R; a yellow or (0.05 p er cent);
yellowish-brown colour is produced and the vapour turns red — total: n o t more than 7 times the area o f the prindpal peak
litmus paper R blue. in the chromatogram obtained w ith reference solution (b)
(0.7 per cent);
E. It gives reaction (a) o f calcium (2.3.1).
— disregard limit: 0.3 times the area o f the prindpal peak in
TESTS the chromatogram obtained with reference solution (b)
Appearance of solution (0.03 p er cent).
T he solution is n o t m ore opalescent than reference S o d iu m
suspension II (2.2.1) and is colourless (2.2.2, Method II). M aximum 0.1 per cent.
Dissolve 2.5 g in 50 m L of water R. Atomic emission spectrometry (2.2.22, Method I).
Related substances Test solution Dissolve 1.0 g in 500.0 m L of water R.
M aximum 10 ppm.
Solvent mixture phosphoric add R, methanol R, acetomtrUe for
Dissolve 2.0 g in 8 m L o f water R w ith heating, cool and add
chromatography R (0.5:8:92 VIVIV).
12 m L o f acetone R. 12 m L of the solution complies with
Test solution Dissolve 0.100 g of the substance to be test B. Prepare the reference solution using 10 m L o f lead
examined in 5 m L o f the solvent mixture, sonicate for standard solution (1 ppm Pb) R.
15 min and dilute to 10.0 m L with the solvent mixture.
W a te r (2.5.12)
Filter the solution through a membrane filter (nominal pore
M axim um 0.1 per cent, determined on 1.000 g. U se a
size 0.45 |im).
mixture o f 15 m L o f anhydrous methanol R and 15 m L of
Reference solution (a) Dissolve 10.0 m g of salicylic add CRS dimethylformamide R as the solvent.
(impurity C) in the solvent mixture and dilute to 100.0 m L
with the solvent mixture. A SSAY
In a flask with a ground-glass stopper, dissolve 0.400 g in
25 m L o f water R. Add 25.0 m L of 0.1 M sodium hydroxide.
Close the flask and allow to stand for 2 h. Titrate with 0.1 M
Reference solution (c) Dissolve 2 mg o f carbasalate hydrochloric add, using 0.2 m L of phenolphthalein solution R.
impurity B CRS in 20.0 m L o f the solvent mixture. Carry out a blank titration.
1-408 Carbidopa 2016
1 m L of 0.1 M sodium hydroxide is equivalent to 22.92 mg of ★* *

Carbidopa ★ ★
C i9 H 18C aN 20 9. ★ ★
★. ★
STORA GE
CO2H
IM P U R IT IE S
Specified impurities B, C HN 'CH3 , HzO

acceptance criterion for other/unspecified impurities and/or Q oH Ô Ô 244.2 38821-49-7
A ctio n a n d u se
D opa decarboxylase inhibitor.
Control of impurities in substances for pharmaceutical use): A, D. P r e p a r a tio n
Co-careldopa Tablets
P h E u r.
D E F IN IT IO N
(25)-3-(3,4-Dihydroxyphenyl)-2-hydrazino-2-
methylpropanoic a d d monohydrate.
C o n te n t
A. 2-(acetyloxy)benzoic anhydride,
CHARACTERS
A p p e a ra n c e
^ V C02H o
UL 0
„ a ,c h 3
o
W hite o r yellowish-white powder.
S o lu b ility
(96 per cent), practically insoluble in methylene chloride.
It dissolves in dilute solutions o f mineral adds.
B. 2-[[2-(acetyloxy)benzoyI]oxy]benzoic a d d First identification A , C.
(acetylsalicylsalicylic ad d ),
Second identification A, B, D, E.
C02H A. Specific optical rotation (see Tests).
B. Ultraviolet and visible absorption spectrophotom etry
a (2.2.25).
Test solution Dissolve 50 m g in an 8.5 g/L solution o f
C . 2-hydroxybenzenecarboxylic a d d (salicylic ad d ), hydrochloric acid R in methanol R and dilute to 100.0 m L with
the same solution. Dilute 10.0 m L o f this solution to
100.0 m l, with an 8.5 g/L solution o f hydrochloric add R in
methanol R.
a " O OH Spectral range 230-350 nm .
Absorption maximum At 283 nm .
Specific absorbance at the absorption maximum 135 to 150
(dried substance).
D . 2-[(2-hydroxybenzoyl)oxy]benzoic a d d (salicylsalicylic Comparison carbidopa CRS.
ad d ).
D . Shake vigorously about 5 mg with 10 m L o f water R for
PhEur 1 mfn and add 0.3 m L o f ferric chloride solution R2.
A n intense green colour is produced, which quickly changes
to reddish-brown.
E. Suspend about 20 mg in 5 m L o f water R and add 5 m L
o f cupri-tartaric solution R. O n heating, the colour o f the
solution changes to dark brown and a red predpitate is
formed.
TESTS
T he solution is d e a r (2.2.1) and n o t more intensdy coloured
than reference solution BY6 or B6 (2.2.2, Method II).
Dissolve 0.25 g in 25 m L o f 1 M hydrochloric acid.
-2 6 .5 to -2 2 .5 (dried substance).
2016 Carbidopa 1-409
Using an ultrasonic bath, dissolve completely 0.250 g in Related substances

aluminium chloride solution R and dilute to 25.0 m L with the Liquid chromatography (2.2.29). Prepare the solutions
same solution. trmnediatdy before use.
Hydrazine Bttffer solution Dissolve 6.9 g of sodium dihydrogen phosphate
Thin-layer chromatography (2.2.27). monohydrate R in 900 m L of water R, adjust to p H 2.2 with
Test solution (a) Dissolve 0.50 g in dilute hydrochloric acid R phosphoric add R and dilute to 1 L with water R.
and dilute to 2.0 m L with the same add. Test solution Dissolve 20.0 mg o f the substance to be
Test solution (b) Place 25 g of strongly basic anion-exchange examined in the mobile phase, add 20 jjL of hydrochloric
resin R into each of 2 conical flasks with ground-glass add R and dilute to 10.0 m L with the mobile phase.
stoppers. T o each, add 150 m L of carbon dioxide-free water R Reference solution (a) Dilute 1.0 m L of the test solution to
and shake from time to time during 30 min. D ecant the 100.0 m L with the mobile phase. Dilute 1.0 m L o f this
liquid from b oth flasks and repeat the process with further solution to 10.0 m L with the mobile phase.
quantities, each o f 150 mL, of carbon dioxide-free water R. Reference solution (b) Dissolve 4 m g of carbidopa for system
Take two 100 m L measuring cylinders 3.5-4.5 cm in internal suitability CRS (containing impurities A, D , E, I and J) in the
diameter and la b d these A and B. Into cylinder A, transfer as mobile phase, add 4 nL of hydrochloric add R and dilute to
completely as possible the resin from 1 conical flask using 2.0 m L with the mobile phase.
60 m L o f carbon dioxide-free water R; into cylinder B, transfer Reference solution (c) Dissolve the contents o f a vial of
the 2nd quantity o f resin, this time using 20 m L of carbon carbidopa impurity mixture CRS (impurities F and H ) in
dioxide-free water R. 1.0 m L o f reference solution (b).
Into each cylinder, insert a gas-inlet tube, the end o f which Column:
has an internal diameter o f 2-3 mm and which reaches — size: I = 0.15 m , 0 = 4.6 mm;
almost to the bottom of the cylinder. Pass a rapid stream o f — stationary phase: end-capped octadecylsilyl amorphous
nitrogen for chromatography R through each mixture so that organosUica polymer R (3.5 nm);
homogeneous suspensions are formed. After 30 min, without — temperature: 25 °C.
interrupting the gas flow, add 1.0 m L of test solution (a) to Mobile phase ethanol (96 per cent) R, buffer solution
cylinder A; after 1 min stop the gas flow into cylinder A and (7:93 V/V).
transfer the contents, through a moistened filter paper, into
cylinder B. After 1 min, stop the gas flow to cylinder B and
pour the solution immediately through a moistened filter Detection Spectrophotom eter at 280 nm.
paper into a freshly prepared mixture of 1 m L of a 200 g/L Injection 20 |iL.
solution o f salicylaldehyde R in methanol R and 20 m L of Run time 6 times the retention time of carbidopa.
phosphate buffer solution pH 5.5 R in a conical flask; shake Identification of impurities Use die chromatogram obtained
thoroughly for 1 min and heat in a water-bath at 60 °C for with reference solution (c) to identify the peaks due to
15 m in. T h e liquid becomes clear. Allow to cool, add impurities A, D + E, F , H , I and J.
2.0 m L o f toluene R and shake vigorously for 2 min. T ransfer
Relative retention W ith reference to carbidopa (retention
the mixture into a centrifuge tube and centrifuge.
time = about 3 min): impurity A = about 0.9; impurities D
Separate the toluene layer in a 100 m L separating funnel and and E = about 1.9; impurity H = about 2.5;
shake vigorously with 2 quantities, each of 20 mL, of a im purity I = about 3.7; impurity J = about 4.0;
200 g/L solution o f sodium metabisulfite R and finally with impurity F = about 4.4.
2 quantities, each o f 50 m í , o f water R. Separate the toluene
System suitability:
layer.
Reference solution (a) Dissolve 10 mg o f hydrazine sulfate R in impurity A and carbidopa in the chromatogram obtained
dilute hydrochloric acid R and dilute to 50 m L with the same with reference solution (b); minimum 1.5 betw een the
add. D ilute 1.0 m L o f this solution to 10.0 m L with dilute peaks due to impurities I and J in the chromatogram
hydrochloric add R. obtained with reference solution (b);
Reference solution (b) Prepare the solution at the same time — signal-to-noise ratio: minimum 30 for the prin d p al peak in
and in the same manner as described for test solution (b) the chromatogram obtained with reference solution (a).
using 1.0 m L o f reference solution (a) instead of 1.0 m L of Calculation ofpercentage contents:
test solution (a) . — correction factors: multiply the peak areas o f the following
Plate TLC süamsed silica gel plate R. impurities by the corresponding correction facto r
Mobile phase water R , methanol R (10:20 V/V). impurities D and E = 1.5; impurity I = 1.5;
impurity J = 1.5;
Application 10 jiL o f test solution (b) and reference
— for each impurity, use the concentration o f carbidopa in
solution (b).
Limits:
Drying In air. — impurity A: maximum 0.5 per cent;
Detection Examine in ultraviolet light at 365 nm. — impurity f . maximum 0.25 per cent;
Limit: — sum of impurities D and E: maximum 0.2 per cent;
— hydrazine: any spot showing a yellow fluorescence is not — impurities F} H, I: for each impurity, maximum
more intense than the corresponding spot in the 0.15 p er cent;
chrom atogram obtained with reference solution (b) — unspecified impurities: for each impurity, maximum
(20 ppm ). 0.10 per cent;
1-410 Carbidopa 2016
o
M axim um 20 ppm.
6.9 p er cent to 7.9 p er cent, determined on 1.000 g by
S u lfa te d a s h (2.4.14) D . methyl (2<S)-2-(2-cydohexyiidenehydrazino)-3-(3,4-
M axim um 0.1 per cent, determ ined on 1.0 g. dihydroxyphenyl)-2-m ethylpropanoate,
A SSA Y
Dissolve 0.150 g with gentle heating in 75 m L of anhydrous o
acetic add R. T itrate with 0.1 M perchloric add, determining
the end-point potentiometrically (2.2.20).
of C 10H 14N 2O 4.
STORAGE
Protected from light. E. methyl (25)-3-(3,4-dihydroxyphenyl)-2-hydrazino-2-
m ethylpropanoate,
IM P U R IT IE S
Spedfied impurities A, D , E, F, H , I, J
o
oh
impurities for demonstration of compliance. See also 5.10. F. ethyl (25)-3-(3,4-dihydroxyphenyl)-2-hydrazino-2-
Control of impurities in substances for pharmaceutical use): B, C, methylpropanoate,
G.
^ / \ X 0 2H
HO
JL J h2n ch3
OH
OH
G. l-(3,4-dihydroxyphenyl)propan-2-one,
A. (26)-2-amino-3-(3,4-ciihydroxyphenyl)-2-methylpropanoic
acid (L-methyldopa),
o
JL Jl HN ’CH3
h3c o nh2
OH
JL Jl h2n 'c h 3
. ho H . (25) - 2 -hydrazina-3-(3-hydroxy-4-methoxyphenyl)-2-
OH methylpropanoic add,
B. methyl (26)-2-amino-3-(3,4-dihydroxyphenyl)-2-
methylpropanoate,
Jl J HN CH3
nh2
OH
JL J HN CH3
HO Y nh2 I. (25)-3-(3-bromo-4,5-dihydroxyphenyl)-2-hydrazino-2-
och3 methylpropanoic ad d ,
C. (25)-2-hydrazino-3-(4-hydroxy-3-methoxyphenyl)-2- Br
methylpropanoic acid (3-O-methylcarbidopa),
I J HN CH3
ho' Y
OH
J. (25)-3-(2-bromo-4,5-dihydroxyphenyl)-2-hydrazino-2-
methylpropanoic add.
PhEur
2016 Carbimazole 1-411
Solvent mixture acetomtrUe Ry water R (20:80 V/V).

Carbimazole *****
*+ +* Test solution. Dissolve 25.0 mg of the substance to be
(Ph. Ettr. monograph 0884) * examined in the solvent mixture and dilute to 50.0 m L with
r= \ Reference solution (a) Dissolve 5 mg of Unamazole CRS
.0 , ,CH3
(impurity A) in the solvent mixture and dilute to 100.0 m L
H
aC YS TO with the solvent mixture. Mix 1 m L o f the solution with
2 m L of the test solution and dilute to 10.0 m L with the
solvent mixture.
C 7H 10N 2O2S 186.2 22232-54-8
Reference solution (b) Dissolve 5.0 m g o f thiamazole CRS
Action and use (impurity A) in the solvent mixture and dilute to 100.0 m L
Thionam ide antithyroid drug. with the solvent mixture. Dilute 1.0 m L o f the solution to
Preparation
Carbimazole Tablets Reference solution (c) Dilute 1.0 m L of the test solution to
P tiE tr ___________________________________________________________________________________________ solution to 10.0 m L with the solvent mixture.
DEFINITION Reference solution (d) Dissolve 25.0 mg o f carbimazole CRS in
Ethyl 3-methyl-2-thioxo-2,3-dihydro-1H-imidazole-1- the solvent mixture and dilute to 50.0 m L with the solvent
carboxylate. mixture.
Column:
Content
— sizer. I = 0.15 m , 0 = 3.9 mm;
CHARACTERS chromatography R (5 |xm).
Appearance Mobile phase acetomtrUe R, water R (10:90 V/V).
W hite or yellowish-white, crystalline powder.
Flow rate 1 mLAnin.
Solubility Detection Spectrophotom eter at 254 nm.
Slightly soluble in water, soluble in acetone and in ethanol
(96 per cent).
Injection 10 (jL o f the test solution and reference
solutions (a), (b) and (c).
IDENTIFICATION
Run time 1.5 times the retention time of carbimazole.
Second identification A , C, D. with reference solution (a) to identify the peak due to
A. M elting point (2.2.14): 122 °C to 125 °C. impurity A.
B. Infrared absorption spectrophotometry (2.2.24). Relative retention W ith reference to carbimazole (retention
Comparison carbimazole CRS. time = about 6 min): impurity A = about 0.2.
C. Thin-layer chromatography (2.2.27). System suitability. reference solution (a):
Test solution Dissolve 10 m g o f the substance to be examined — resolution: minimum 5.0 between the peaks due to
in methylene chloride R and dilute to 10.0 m L with the same impurity A and carbimazole.
solvent. Limits:
Reference solution Dissolve 10 m g of carbimazole CRS in — impurity A: n o t more than 2.5 times the area o f the
methylene chloride R and dilute to 10.0 m L with the same principal peak in the chromatogram obtained with
solvent.
Plate TLC silica gel F 254 plate R area o f the principal peak in the chromatogram obtained
Mobile phase acetone R, methylene chloride R (20:80 V/V). with reference solution (c) (0.10 per cent);
Application 10 joL. — total: m axim um 0.6 p er cent;
Development Over 3/4 o f the plate. — disregard limit. 0.5 times the area o f the principal peak in
Drying In air for 30 min. the chromatogram obtained with reference solution (c)
(0.05 p er cent).
a desiccator over diphosphorus pentoxide R at a pressure not
spot in the chrom atogram obtained with the reference
exceeding 0.7 kPa for 24 h.
solution.
D . Dissolve about 10 m g in a mixture of 0.05 m L o f dilute
M axim um 0.1 p e r cent, determined on 1.0 g.
hydrochloric acid R and 50 m L o f water R. A dd 1 m L of
potassium iodobismtahate solution R. A red precipitate is A SSAY
formed. Liquid chromatography (2.2.29) as described in the test for
TESTS related substances with the following modification.
Related substances Injection T est solution and reference solution (d).
Liquid chromatography (2.2.29). Prepare the solutions Calculate the percentage content of C 7H 10N 2O 2S taking into
immediately before use.Test solution. Dissolve 25.0 m g of the account the assigned content of carbimazole CRS.
substance to be examined in the solvent mixture and dilute IM P U R IT IE S
1-412 Carbocisteine 2016
r~ \ N in h y d rin -p o sitiv e su b s ta n c e s
h 3c ' Y Examine by thin-layer chromatography (2.2.27), using a
SH suitable silica gel as the coating substance.
A. 1-m ethyl-lH-imidazole-2-thiol (thiamazole). examined in dilute ammonia R2 and dilute to 10 mT. with the
PhEur same solvent.
Test solution (b) Dilute 1 m L o f test solution (a) to 50 mT.
with water R.
Reference solution (a) Dissolve 10 m g of carbocisteine CRS in
***** dilute ammonia R2 and dilute to 50 m L with the same
Carbocisteine * * solvent.
* * * * * Reference solution (b) D ilute 5 m L o f test solution (b) to
(Ph. Ever, monograph 0885)
20 m L with water R.
H NH, Reference solution (c) Dissolve 10 m g of carbocisteine CRS and
HOjC. 10 mg of arginine hydrochloride CRS in 5 m L o f dilute
COjH
ammonia R2 and dilute to 25 m L with water R.
Apply separately to the plate 5 (jL of each solution. Allow the
C5H 9N 04S 179.2 638-23-3
plate to dry in air. Develop over a path of 15 cm using a
A ctio n a n d u se mixture of 20 volumes o f glacial acetic add R, 20 volumes of
Mucolytic. water R and 60 volumes o f butanol R. Dry the plate in a
current o f warm air. Spray with ninhydrin solution R and heat
P h E u r ____________________ at 100 °C to 105 °C for 15 min. Any spot in the
D E F IN IT IO N chromatogram obtained with test solution (a), apart from the
principal spot, is not m ore intense than the spot in the
Carbocisteine contains not less than 98.5 per cent and not
m ore than the equivalent o f 101.0 per cent o f (2i?)-2-amino-
(0.5 per cent). T he test is not valid unless the chromatogram
3-[(carboxymethyl)sulfanyl]propanoic acid, calculated with
separated principal spots.
CHARACTERS
A white or alm ost white, crystalline powder, practically
Dissolve 33 mg in 5 m L o f dilute nitric acid R and dilute to
insoluble in w ater and in alcohol. It dissolves in dilute
15 m L with water R. T he solution, without further addition
mineral a d d s and in dilute solutions o f alkali hydroxides.
o f nitric acid, complies with the limit test for chlorides
ID E N T IF IC A T IO N (0.15 per cent).
First identification A, B. S u lfa tes (2.4.13)
Second identification A , C, D. Dissolve 0.5 g in 5 m L o f dilute hydrochloric add R and dilute
A. Specific optical rotation (see Tests). to 15 m L with distilled water R. T h e solution complies with
B. Examine by infrared absorption spectrophotometry the limit test for sulfates (300 ppm ).
(2.2.24), comparing with the spectrum obtained with H eav y m e ta ls (2. 4.8)
carbocisteine CRS. Examine the substances prepared as discs. 2.0 g complies with test D for heavy metals (10 ppm).
C. Examine the chromatograms obtained in the test for Prepare the reference solution using 2 m L o f lead standard
ninhydrin-positive substances. T he principal spot in the solution (10 ppm Pb) R
chrom atogram obtained with test solution (b) is similar in L oss o n d ry in g (2.2.32)
position, colour and size to the principal spot in the N o t more than 0.5 per cent, determined on 1.000 g by
chrom atogram obtained with reference solution (a). drying in an oven at 105 °C for 2 h.
D . Dissolve 0.1 g in 4.5 m L o f dilute sodium hydroxide S u lfa te d a s h (2.4.14)
solution R H eat on a water-bath for 10 min. Cool and add N o t more than 0.3 per cent, determined on 1.0 g.
1 m L of a 25 g/L solution o f sodium nitroprusside R A dark
ASSAY
red colour is produced, which changes to brown and then to
Dissolve 0.150 g in 10 m L o f anhydrous formic add R with
yellow within a few minutes.
slight heating and shake until dissolution is complete.
TESTS Add 50 m L o f anhydrous acetic add R. T itrate with 0.1 M
S o lu tio n S perchloric add, determining the end-point potentiometrically
Disperse 5.00 g in 20 m L o f water R and add dropwise with (2.2.20).
shaking 2.5 m L of strong sodium hydroxide solution R. Adjust 1 m L o f 0.1 M perchloric add is equivalent to 17.92 m g of
to p H 6.3 with 1 M sodium hydroxide and dilute to 50.0 m L C sH ^S .
w ith water R.
STO R A G E
Store protected from light.
_________________________________________________________________ PhEur
p H (2.2.3)
Shake 0.2 g with 20 m L o f carbon dioxide-free water R.
T h e pH of the suspension is 2.8 to 3.0.
—32.5 to -3 5 .5 , determ ined on solution S and calculated
w ith reference to the dried substance.
2016 Carbomers 1-413
— mobile phase B: mixture of equal volumes of a 1.361 g/L

Carbomers /* * * solution of potassium dihydrogen phosphate R and acetonitrüe
(Ph. Eur. monograph 1299) * for chromatography Ri
Action and use
Stabilizer in pharmaceutical products. Time MobQe phase A Mobile phase B
Preparation (min) (per cent V7V) (per cent V/V)
C arbom er Eye D rops 0 -8 100 0
PhEur__________________________________________________________ 8 -9 100->0 0-» 100
DEFINITION 9 -2 0 0 100
High-molecular-mass polymers of acrylic acid cross-linked
w ith alkenyl ethers o f sugars or polyalcohols.
Flow rate 1 mL/min.
Content Detection Spectrophotometer at 205 nm.
56.0 per cent to 68.0 per cent o f carboxylic acid (-C 0 2H)
Injection 20 jiL.
groups (dried substance).
Retention lime Acrylic acid = about 6.0 min.
CHARACTERS
Limit:
Appearance
— acrylic acid: not more than the area of the corresponding
W hite or almost white, fluffy, hygroscopic powder.
Solubility solution (0.25 per cent).
Swells in water and in other polar solvents after dispersion
B en z e n e
and neutralisation w ith sodium hydroxide solution.
G as chromatography (2.4.24, System A).
ID E N T IF IC A T IO N Solution A Dissolve 0.100 g of benzene R in dimethyl
First identification A sulfoxide R and dilute to 100.0 m L with the same solvent.
Second identification B, C} D D ilute 1.0 m L of the solution to 100.0 m L with water R.
A. Infrared absorption spectrophotometry (2.2.24). Dilute 1.0 m L o f this solution to 100.0 m L with water R.
Main bands A t 1710 ± 5 cm-1, 1454 ± 5 cm -1, Test solution Weigh 50.0 m g of the substance to be examined
1414 ± 5 cm -1 , 1245 ± 5 cm-1, 1172 ± 5 cm-1, into an injection vial and add 5.0 m L of water R and 1.0 m L
1115 ± 5 cm-1 and 801 ± 5 cm-1 , with die strongest band o f dimethyl sulfoxide R.
at 1710 ± 5 cm -1 . Reference solution Weigh 50.0 mg o f the substance to be
B. Adjust a 10 g/L dispersion to about p H 7.5 with examined into an injection vial and add 4.0 m L o f water R,
1 M sodium hydroxide. A highly viscous gel is formed, 1.0 m L o f dimethyl sulfoxide R and 1.0 m L o f solution A.
c . Add 2 m L o f a 100 g/L solution of cakiian chloride R, Close the vials with a tight rubber membrane stopper coated with
with continuous stirring, to 10 m L o f die gel from polytetrafiuoroethylene and secure with an afambuum crimped
identification test B. A white precipitate is immediately cap. Shake to obtain a homogeneous dispersion.
produced. Static head-space conditions that may be used:
D . Add 0.5 m L o f thymol blue solution R to 10 m L o f a — equilibration temperature: 80 ° Q
10 g/L dispersion. A n orange colour is produced. — equilibration timer. 60 min;
Add 0.5 m L o f cresol red solution R to 10 m L of a 10 g/L — transfer-line temperature: 90 °C.
dispersion. A yellow colour is produced. Injection 1 m L o f the gaseous phase o f the test solution and
1 m L of the gaseous phase of the reference solution; repeat
TESTS
these injections twice more.
Free acrylic acid
System suitability:
— repeatability: maximum relative standard deviation of the
Test solution M ix 0.125 g of die substance to be examined differences in area between the analyte peaks obtained
with a 25 g/L solution of aluminium potassium sulfate R and
from the 3 replicate pair injections of the reference
dilute to 25.0 m L w ith the same solution. H eat the
solution and the test solution is 15 per c e n t
suspension at 50 ° c for 20 min with shaking, then shake die
Limit:
suspension at room temperature for 60 min. Centrifuge and
— benzene: the m ean area o f the peak due to benzene in the
use the clear supernatant solution as the test solution.
chromatograms obtained with the test solution is not
Reference solution Dissolve 62.5 mg of acrylic acid R in a greater than 0.5 times the m ean area of the peak due to
25 g/L solution of aluminium potassium sulfate R and diỉutè to benzene in the chromatograms obtained with the
100.0 m L with the same solution. Dilute 1.0 m L of this reference solution (2 ppm).
solution to 50.0 m L with a 25 g/L solution o f aluminium
potassium sulfate R.
M aximum 20 ppm .
Column:
— size. I = 0.12 m , 0 = 4.6 mm; 1.0 g complies w ith test C. Prepare the reference solution
— stationary phase’, octadecyisHyl silica gelfor chromatography R using 2 m L of lead standard solution (10 ppm Pb) R.
(5 pm). L o ss o n d ry in g (2.2.32)
Mobile phase: M axim um 3.0 per cent, determined on 1.000 g by drying
— mobile phase A'. 1.361 g/L solution OĨpotassium dihydrogen in vacuo at 80 °C for 60 m in.
phosphate R, adjusted to p H 2.5 using dilute phosphoric S u lia te d ash (2.4.14)
acid R ; M aximum 4.0 per cent, determined on 1.0 g.
1-414 Carbon Dioxide 2016
A SSA Y
Carbon Dioxide *****
Slowly add 50 m L o f water R to 0.120 g whilst stirring and *. ★
heating at 60 °c for 15 min. Stop heating, add 150 m L o f (Ph. Eur. monograph 0375) *
water R and continue stirring for 30 min, Add 2 g o f
C 02 44.01 124-38-9
potassium chloride R and titrate with 0.2 M sodium hydroxide,
determining the end-point potentíometrically (2.2.20). Carbon Dioxide should be kept in approved metal cylinders which
are painted grey and cany a label stating ‘Carbon Dioxide3.
In addition, cCarbon Dioxide ’ or the symbol ‘C02 ’ should be
carboxylic acid (-C 0 2H) groups.
stencilled in paint on the shoulder of the cylinder.
STORAGE P h E u r ___________________________________________________________________________________________
D E F IN IT IO N
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 C o n te n t
This section provides information on characteristics that are M inim um 99.5 per cent V/V of C 0 2 in the gaseous phase.
This monograph applies to carbon dioxide for medicinal use.
junctions of the substance when used as an excipient (see chapter
5.15). Thừ section ừ a rum-mandatory part of die monograph CHARACTERS
and it is not necessary to verify the characteristics to demonstrate A p p e a ra n c e
compliance. Control of these characteristics can however contribute Colourless gas.
to the quality of a medừinơỉ product by improving the consistency S o lu b ility
of the manufacturing process and the performance of the medicinal At 20 °C and at a pressure o f 101 kPa, 1 volume dissolves in
product during use. Where control methods are coed, they are about 1 volume o f water.
recognised as being suừabỉe for the purpose, but ođier methods can
P R O D U C T IO N
reported, the control method, must be indicated. Examine the gaseous phase.
The following characteristics may be relevant for carbomers used as I f the testis performed on a cylinder of gas, keep the cylinder of
viscosity-increasing agents and gelling agents. the substance to be examined at room temperature for not less than
6 h before carrying out the tests. Keep the cylinder in the vertical
A p p a re n t visco sity (2.2. 10)
position with the outlet valve uppermost.
T he nominal apparent viscosity is typically between
300 m P a s and 115 000 mPa-s. F or a product with a C a rb o n m o n o x id e
nom inal apparent viscosity of 20 000 mPa-s or greater, die Gas chromatography (2.2.25).
apparent viscosity is typically 70.0 per cent to 130.0 per cent Gas to be examined T he substance to be examined.
o f die nominal value; for a product with a nominal apparent Reference gas A mixture containing 5 ppm V/V o f carbon
viscosity of less than 20 000 mPa-s, the apparent viscosity is monoxide R in nitrogen R l.
typically 50.0 per cent to 150.0 per cent of the nominal Column:
value. — material: stainless steel,
D ry the substance to be examined in vacuo at 80 °c for 1 h. — size. I — 2 m , 0 = 4 mm,
Carefully add 2.50 g of the previously dried substance to be — stationary phase, an appropriate molecular sieve for
examined to 500 m L of water R in a 1000 m L beaker while chromatography (0.5 nm).
stirring continuously at 1000 ± 50 r/min, with the stirrer Carrier gas helium for chromatography R.
shaft set at an angle o f 60° to one side o f the beaker. Add the
Flow rate 60 mL/min.
previously dried substance over a period of 45-90 s, at a
Temperature.
uniform rate, ensuring that loose agglomerates o f powder are
— column: 50 °C,
broken up, and continue stirring at 1000 ± 50 r/min for
— injection port and detector. 130 °C.
15 min. Remove die stiirer and place die beaker containing
die dispersion in a water-bath at 25 ± 1 °c for 30 m in. Detection Flame ionisation with methaniser.
Insert the stirrer to a depth necessary to ensure that air is n o t Injection Loop injector.
draw n into the dispersion and, while stirring at Adjust the injected volumes and the operating conditions so
300 ± 25 r/min, tìơate with a glass-calomel electrode system that the height o f the peak due to carbon monoxide in the
to p H 7.3-7.8 by adding a 180 g/L solution of sodium chromatogram obtained with the reference gas is at least
hydroxide R below die surface, determining die end-point 35 p er cent o f the full scale of the recorder.
potentiometrically (2.2.2Ơ). T h e total volume of the Ì80 g/L Limit.
solution o f sodium hydroxide R used is about 6.2 m ĩ- Allow — carbon monoxide, not more than the area o f the
2-3 min before the final p H determination. If the final p H corresponding peak in the chrom atogram obtained with
exceeds 7.8, discard the preparation and prepare another the reference gas (5 ppm V/V).
using a smaller am ount o f sodium hydroxide for titration.
N itro g e n m o n o x id e a n d n itro g e n d io x id e
R eturn die neutralised preparation to die water-bath at 25 °c
M axim um 2 ppm V/V in total, determ ined using a
for 1 h, then perform the viscosity determination without
delay to avoid slight viscosity changes that occur 75 min after chemiluminescence analyser (2.5.26).
neutralisation. Determ ine die viscosity using a rotating Gas to be examined T he substance to be examined.
viscometer with a spindle rotating at 20 r/min, using a Reference gas (a) Carbon dioxide R l.
spindle suitable for the expected apparent viscosity. Reference gas (b) A mixture containing 2 ppm V/V o f nitrogen
C arboxylic a c id g ro u p s monoxide R in carbon dioxide R l or in nitrogen R l.
See Assay. Calibrate the apparatus and set the sensitivity using reference
PhEur gases (a) and (b). M easure the content o f nitrogen monoxide
and nitrogen dioxide in the gas to be examined.
2016 Carbon Dioxide 1-415
t t
Photomultiplier Amplifier
Figure 0375.-1- UVFluorescence Analyser
If nitrogen is used instead of carbon dioxide in reference Assay

gas (b)3 multiply the result obtained by the quenching Infrared analyser (2.5.24).
correction factor in order to correct the quenching effect on Gas to be examined T he substance to be examined. It must be
the analyser response caused by the carbon dioxide matrix filtered to avoid stray light phenomena.
effect. Reference gas (a) Carbon dioxide R l.
T he quenching correction factor is determined by applying a
Reference gas (b) A mixture containing 95.0 per cent VIV of
known reference mixture of nitrogen monoxide in carbon carbon dioxide R l and 5.0 per cent VIV of nitrogen R l.
dioxide and comparing the actual content with the content
indicated by the analyser which has been calibrated with a
gases (a) and (b). Measure the content o f carbon dioxide in
N O /N 2 reference mixture.
the gas to be examined.
-v , . ,. e , _______ a c tu a l n it r o g en m o n o x id e co n te n t
Quenching correction factor—„ ¿ ¿ ic a te d n itr o g e n m o n o x id e co n te n t
IDENTIFICATION
First identification A
Total sulfur
M axim um 1 ppm V/V, determined using an ultraviolet A. Infrared absorption spectrophotometry (2.2.24y.
fluorescence analyser after oxidation o f the sulfur compounds Comparison Ph. Eur. reference spectrum of carbon dioxide.
by heating at 1000 °C (Figure 0375.-1). B. Place a glowing splinter of wood in an atmosphere of the
T he apparatus consists of the following: substance to be examined. It is extinguished.
— a system generating ultraviolet radiation with a C . Pass a stream o f the substance to be examined through
wavelength o f 210 nm, m ade up of an ultraviolet lamp, a barium hydroxide solution R. A white predpitate is formed
collimator, and a selective filter; the beam is blocked which dissolves with effervescence in dilute acetic acid R.
periodically by a chopper rotating at high speed,
TESTS
— a reaction chamber through which flows the previously
Examine the gaseous phase.
filtered gas to be examined,
— a system that detects radiation emitted at a wavelength of I f the testis performed on a cylinder of gas, keep the cylinder of
350 nm , made up of a selective filter, a photomultiplier the substance to be examined at room temperature for not less than
tube an d an amplifier. 6 h before carrying out the tests. Keep the cylinder in the vertical
position with the outlet valve uppermost.
Gas to be examined T h e substance to be examined.
Reference gas (a) Carbon dioxide R l. Carbon monoxide
M aximum 5 ppm VIV, determined using a carbon monoxide
Reference gas (b) A mixture containing between 0.5 ppm V/V detector tube (2.1.6).
and 2 p p m VIV of hydrogen sulfide R l in carbon dioxide R l.
Hydrogen sulfide
M axim um 1 ppm V/V, determined using a hydrogen sulfide
gases (a) and (b). Pass the gas to be examined through a
detector tube (2.1.6).
quartz oven heated to 1000 °C. Oxygen R is circulated in the
oven a t a tenth o f the flow rate of the gas to be examined. Nitrogen monoxide and nitrogen dioxide
M easure the sulfur dioxide content in the gaseous mixture M axim um 2 ppm V/V in total, determined using a nitrogen
leaving the oven. monoxide and nitrogen dioxide detector tube (2.1.6).
Water Sulfur dioxide
M axim um 67 ppm VIV, determined using an electrolytic M aximum 2 ppm V/V, determined using a sulfur dioxide
hygrom eter (2.5.28). detector tube (2.1.6).
1-416 Carbon Monoxide 2016
Water vapour Limit.

M axim um 67 ppm V/V, determined using a water vapour — carbon dioxide: not more than the area o f the
detector tube (2.1.6). corresponding peak in the chromatogram obtained with
STORAGE the reference gas (300 ppm V/V).
Store liquefied under pressure in suitable containers Methane
complying with the legal regulations. Gas chromatography (2.2.28).
IM P U R IT IE S Gas to be examined T he substance to be examined.
A. N O : nitrogen monoxide, Reference gas A mixture containing 100 ppm V/V of
B. N 0 2: nitrogen dioxide, methane R in carbon monoxide R.
C. CO: carbon monoxide, Column:
D . total sulfur, — material: stainless steel;
E. H 20 : water. — size. I = 2 m , 0 = 4 mm;
— stationary phase: ethylvirtyWenzene-divmylbenzene
___________________________________________________________________________________________ PhEur
copolymer R (177-250 nm).
Carbon Monoxide ***** Temperature:
*. * — column: 95 °C;
(Ph. Ever, monograph 2408) *
CO 28.00 630-08-0
P h E u r ___________________________________________________________________________________________
Irtjection 1 mL.
D E F IN IT IO N Run time 3 min.
Gas obtained by steam reforming (catalytic oxidation) of
Retention time M ethane = about 1.8 min.
hydrocarbons.
Limit.
Content — methane: h o t more than the area o f the corresponding
M inim um 99.5 per cent V/V o f CO. peak in the chrom atogram obtained with the reference gas
T his monograph applies to carbon monoxide for medicinal (100 ppm V/V).
use.
Hydrogen
CHARACTERS Gas chromatography.
Appearance Gas to be examined T he substance to be examined.
Colourless, flammable gas.
Reference gas A mixture containing 300 ppm V/V o f hydrogen
Solubility for chromatography R in carbon monoxide R.
A t 20 °C and at a pressure o f 101 kPa, 2.266 volumes of
Column:
carbon monoxide dissolve in 100 volumes o f water.
IDENTIFICATION — size. I = 2 m , 0 = 2 mm ;
Carry out either test A or B. — stationary phase, molecular sieve for chromatography
A. Infrared absorption spectrophotom etry (2.2.24). (149-177 nm) with a nominal pore size of 0.5 nm .
Comparison Ph. Eur. reference spectrum of carbon monoxide. Carrier gas argon for chromatography R.
B. I t complies with the limits o f the assay. Flow rate 30 mL/min.
TESTS Temperature:
Carbon dioxide — column: 100 °C;
Gas chromatography (2.2.28). — detector. 160 °C.
Gas to be examined T h e substance to be examined. Detection Therm al conductivity.
Reference gas A mixture containing 300 ppm V/V of carbon Irtjection 1 mT^
dioxide R l in carbon monoxide R. Rim time 4 min.
Column: Relative retention W ith reference to carbon monoxide
— material: stainless steel; (retention time = about 2.3 min): hydrogen = about 0.4.
— size. I = 2 m , 0 = 2 mm ; Limit.
— stationary phase: an appropriate divinylbenzene porous — hydrogen: n o t m ore than the area of the corresponding
polymer (149-177 nm). peak in the chrom atogram obtained with the reference gas
Carrier gas helium for chromatography R. (300 ppm V/V).
Flow rate 30 mL/min. Nickel tetracarbonyl and iron pentacarbonyl
Temperature: N o t detectable, using a detector tube having a limit of
— column: 50 °C; detection o f 0.1 ppm V/V (2.1.16).
— detector. 220 °C. Water
Detection Therm al conductivity. M aximum 10 ppm V/V, determined using an electrolytic
Injection 1 mL. hygrometer (2.5.25).
Run time 3 min. A SSA Y
Relative retention W ith reference to carbon monoxide Infrared analyser (2.5.25).
(retention tim e = about 0.4 min): carbon dioxide = about Gas to be examined T he substance to be examined, previously
3.5. filtered to avoid stray light phenomena.
2016 Carboplatin 1-417
Reference gas (a) Carbon monoxide R. Impurity B and acidity

Reference gas (b) A mixture containing 95.0 per cent V/V of M aximum 0.5 per cent, calculated as impurity B.
carbon monoxide R and 5.0 per cent V/V of nitrogen R l. T o 10 m L of solution S add 0.1 m L of phenolphthalein
Calibrate the apparatus and set the sensitivity using reference solution R l. T he solution is colourless. N o t more than
gases (a) and (b). M easure the content of carbon monoxide 0.7 m L o f 0.01 M sodium hydroxide is required to change the
in the gas to be examined. colour of the indicator to pink.
STORAGE Related substances

U nder pressure in suitable containers complying with the
legal regulations. Test solution Dissolve 20.0 mg of the substance to be
examined in a mixture of equal volumes o f acetonitrile R and
IMPURITIES water R and dilute to 20.0 mL with the same mixture of
Specified impurities A, B, C, D , E, F. solvents.
A. C 0 2: carbon dioxide, Reference solution Dilute 0.5 mL of the test solution to
B. C H 4: methane, 200.0 m L with the mobile phase.
C. H 2: hydrogen, Column:
D . N i(C O ) 4: nickel tetracarbonyl, — size: I = 0.25 m , 0 = 4.6 mm;
E. Fe(C O ) 5: iron pentacarbonyl, — stationary phase: ammopropylsUyl silica gel for
F. H 20 : water.
Mobile phase water R, acetomtrUe R (13:87 V/V).
_______________________________________________________________PhEur
Flow rate 2 mL/min.
Injection 10 pL.
★ ★ Run time 2.5 times the retention time of carboplatin.
Carboplatin ★ ★
Relative retention W ith reference to carboplatin (retention
(Ph. Eur. monograph 1081) ***** tim e = about 7 min): impurity A = about 0.3.
System suitability T est solution:
— number of theoretical plates', minimum 5000; if necessary,
adjust the concentration of acetonitrile in the mobile
phase;
— mass distribution ratio: minimum 4.0; if necessary, adjust
the concentration of acetonitrile in the mobile phase;
— symmetry factor, maximum 2.0; if necessary, adjust the
41575-944
concentration o f acetonitrile in the mobile phase.
Action and use Limits:
Platinum-containing cytotoxic. — impurity A: not more than the area of the principal peak
in the chromatogram obtained with the reference solution
Preparation
(0.25 per cent);
Carboplatin Injection
— total: n o t more than twice the area o f the principal peak in
PhEtr_____________________________ the chromatogram obtained with the reference solution
(0.5 per cent);
DEFINITION
— disregard limir. 0.2 times the area of the principal peak in
(5P-4-2)-Diammine [cyclobutan-1, 1-dicarboxylato (2-) -
the chromatogram obtained with the reference solution
OjO'Jplatin.
(0.05 per cent).
Content C h lo rid e s (2.44)
98.0 per cent to 102.0 per cent (dried substance). M axim um 100 ppm.
CHARACTERS Dissolve 0.5 g in water R, heating slightly if necessary, and
Appearance dilute to 20 m L with the same solvent. Filter if necessary.
Colourless, crystalline powder. Dilute 10 m L of this solution to 15 m L with water R.
Solubility Prepare the standard using 5 mL o f chloride standard solution
Sparingly soluble in water, very slightly soluble in acetone (5 ppm Cl) R.
and in ethanol (96 per cent). A m m o n iu m (2.41, Method B)
mp M axim um 100 ppm, determined on 0.20 g.
A bout 200 °C, with decomposition. Prepare the standard using 0.2 m L of ammonium standard
IDENTIFICATION solution (100 ppm N H J R.
Infrared absorption spectrophotometry (2.2.24). S ilv er
M aximum 10 ppm.
Comparison Ph. Eur. reference spectrum of carboplatin.
Inductively coupled plasma-atomic emission spectrometry
TESTS (2.2.57).
Solution S
Test solution Dissolve 0.50 g in a 1 per cent V/V solution of
nitric acid R and dilute to 50.0 m L with the same solution.
1-418 Carboprost Trometamol 2016
Reference solutions Prepare the reference solutions using silver CHARACTERS

standard solution (5 ppm Ag) R, diluting with a 1 p er cent V/V Appearance
solution of nitric acid R. W hite or almost white powder.
Wavelength 328.1 nm . Solubility
S o lu b le b a r iu m Soluble in water.
M axim um 10 ppm . IDENTIFICATION
Inductively coupled plasma-atomic emission spectrometry A. Specific optical rotation (see Tests).
(2.2.57). B. Infrared absorption spectrophotometry (2.2.24).
Test solution Use the solution described in the test for silver. Comparison Ph Eitr. reference spectrum of carboprost trometamol.
TESTS
barium standard solution (50 ppm Ba) R, diluting with a
1 p er cent V/V solution of nitric acid R.
L o ss o n d ry in g (2.2.32) 10.0 m L with the same solvent.
an oven at 105 °C. Related substances
A SSA Y
U se the residue obtained in the test for loss on drying. Ignite examined in a mixture of 23 volumes o f acetonitrUe R and
0.200 g of the residue to constant mass at 800 ± 50 °C. 77 volumes of water for chromatography R and dilute to
1 m g o f the residue is equivalent to 1.903 mg of 10.0 m L with the same mixture of solvents.
C 6H 12N 20 4Pt. Reference solution (a) Dissolve 15.0 mg of carboprost
STO RA G E trometamol CRS (containing impurity A) in a mixture of
Protected from light. 23 volumes of acetonitrUe R and 77 volumes o f water for
chromatography R and dilute to 10.0 m L with the same
IM P U R IT IE S
mixture o f solvents.
Civ x nh3 and 0.15 m L o f (15R)-15-methylprostaglandin F2a R
Pt (impurity B) to 100.0 m L with a mixture of 23 volumes of
/ x
Cl nh3 acetonitrUe R and 77 volumes of water for chromatography R.
A. ay-diamminedichloroplatinum(]I) (cisplatin), 20.0 m L with a mixture o f 23 volumes of acetonitrUe R and
77 volumes o f water for chromatography R. Dilute 2.0 m L of
CO2H
this solution to 20.0 m L with a mixture o f 23 volumes o f
< x C02H acetonitrUe R and 77 volumes of water for chromatography R.
Column:
B. cyclobutane-1,1-dicarboxyiic acid. — size. I = 0.15 m , 0 = 4.6 m m ,
PhEur
— stationary phase. octadecylsUyl sUica gel for
chromatography R1 (5 pm) with a pore size of 8-10 nm
and a carbon loading o f 12-19 per cent.
Mobile phase Mix 23 volumes o f acetonitrUe R1 and
77 volumes o f a 2.44 g/L solution of sodium dihydrogen
Carboprost Trometamol ★ ★
★ ★ phosphate R in water for chromatography R previously adjusted
***** to p H 2.5 with phosphoric acid R.
OH OH Detection Spectrophotom eter at 200 nm .
Injection 20 jiL.
Run time 1.3 times the retention time o f carboprost.
HO'
H H H,C OH Relative retention W ith reference to carboprost (retention
tim e = about 80 min): impurity B = about 0.85;
C 25H 47NO 8 489.7 58551-69-2 impurity A = about 0.9.
A c tio n a n d u se with reference solution (a) and the chromatogram supplied
Prostaglandin (PG F2a) analogue. with carboprost trometamol CRS to identify the peak due to
impurity A.
P hE ur.
System suitability:
D E F IN IT IO N — resolution: minimum 3.4 between the peaks due to
2-Amino-2-(hydroxymethyl)propane-1,3-diol (5Z)-7- im purity B and carboprost in the chromatogram obtained
[(1 R,2R,3R, 5.S)-3,5-dihydroxy-2- [(1 £,3.S)-3-hydroxy-3- with reference solution (b);
m ethyloct- 1-enyl] cydopentyl]hept-5-enoate ((155)-15- — peak-to-vaSey ratio: minim um 3.0, where Hp = height
m ethyl-PG F 2). above the baseline o f the peak due to impurity A and
C o n te n t Hv = h d g h t above the baseline o f the lowest point o f the
94.0 per cent to 102.0 per cent (anhydrous substance). curve separating this peak from the peak due to
2016 Carisoprodol 1-419
impurity B in the chromatogram obtained with reference ★ ★

solution (a).
Carisoprodol ★ ★
Limits: (Ph. Eur. monograph 1689) * * * * *
— impurity A: not more than 3 times the area o f the

principal peak in the chromatogram obtained with ch3 o 0
reference solution (c) (3.0 per cent), x
— impurity B: n o t more than the area o f the principal peak h3c ^ n ^ c O NH2 arKi enantiomer.

CH 3
( 1.0 per cent),
0.1 tim es the area of the principal peak in the c 12h 24n 2o 4 260.3 78-44-4
(0.10 p er cent), A c tio n a n d u se
— total: n o t m ore than 4 times the area o f the principal peak Skeletal muscle relaxant
PhEur______________________
(4.0 p er cent),
— disregard Hmir. 0.05 times the area o f the principal peak in D E F IN IT IO N
the chrom atogram obtained with reference solution (c) (2i?S)- 2- [(Carbamoyloxy) methyl] - 2-methylpentyl
(0.05 p e r cent). ( 1-methylethyl) carbamate.
W a te r (2.5.32) C o n te n t
M axim um 0.5 per cent, determined on 50 mg. 98.0 per cent to 102.0 per cent (dried substance).
A SSAY CHARACTERS
L iquid chromatography (2.2.29) as described in the test for A p p e a ra n c e
related substances with the following modifications. W hite or almost white, fine powder.
Mobile phase M ix 27 volumes of acetonitrile R l and S o lub ility
73 volumes o f a 2.44 g/L solution o f sodium dihydrogen Very slightly soluble in water, freely soluble in acetone, in
phosphate R in waterfor chromatography R previously adjusted alcohol and in methylene chloride.
to pH 2.5 with phosphoric add R.
Irtjection T est solution and reference solution (a). First identification A , B
Run time 1.2 times the retention time o f carboprost. Second identification A, C, D
Retention time Carboprost = about 29 min. A. M elting point (2.2.14): 92 °C to 95 °C.
Calculate the percentage content of C 25H 47N O 8 using the B. Infrared absorption spectrophotometry (2.2.24).
declared content o f carboprost trometamol CRS.
Comparison carisoprodol CRS.
STORAGE C. Examine the chromatograms obtained in the test for
A t a tem perature below - 1 5 0 C. related substances.
IM P U R IT IE S Results T he principal spot in the chromatogram obtained with
Specified impurities: A, B. test solution (b) is similar in position, colour and size to the
solution (d).
D . Dissolve 0.2 g in 15 m L of a 28 g/L solution o f potassium
hydroxide R in alcohol R and boil under a reflux condenser for
15 m in. Add 0.5 m L of glacial acetic add R and 1 m L of a
H H h 3c oh 50 g/L solution o f cobalt nitrate R in ethanol R An intense
blue colour develops.
A. (5£)-7-[(l£,2Æ ,3£,5S)-3,5-dihydroxy-2-[(lE,3S)-3- TESTS
hydroxy-3-methyloct-1 -enyi] cyclopentyl]hept-5-enoic acid, O p tic a l r o ta tio n (2.2.7)
- 0 . 10° to + 0 . 10°.
Dissolve 2.5 g in alcohol R and dilute to 25.0 m L w ith the
same solvent
ho' T
h ho ch3 Thin-layer chrom atography (2.2.27).
B. (52)-7-[(li?,2i?,3i?,55)-3,5-dihydroxy-2-[(l£,3i?)-3- examined in methylene chloride R and dilute to 10 m L with
hydroxy-3-methyloct-1 -enyi] cyclopentyl]hept-5-enoic acid. the same solvent.
PhEur
with methylene chloride R.
Reference solution (a) Dissolve 5.0 mg o f meprobamate CRS in
methylene chloride R and dilute to 50 m L with the same
solvent.
Reference solution (b) D ilute 1 m L of test solution (b) to
50 m L with methylene chloride R.
1-420 Carmellose 2016
o
Reference solution (c) Dilute 5 m L o f reference solution (b) to
10 m l. with methylene chloride R. o.A.o
Reference solution (d) Dissolve 20 mg of carisoprodol CRS in
h 3c
solvent.
ch3
Reference solution (e) Dissolve 10 mg o f carisoprodol
impurity A CRS in 5 m L of reference solution (d) and dilute B. 5-methyl-5-propyl-l,3-dioxan-2-one,
to 50 m L with methylene chloride R.
Mobile phase acetone R, methylene chloride R (20:80 V/V). h 3c X v
Application 5 |iL. ch3
Development Over a p ath of 15 cm.

C. 2-methyl-2-propylpropane-1,3-diol,
Drying In air for 15 min.
Detection Spray with a solution prepared as follows: dissolve o o
5 g o f phosphomolybdic acid R in a mixture of 50 m L o f glacial
h2n nh2
acetic add R and 10 m L of sulfuric add R, and dilute to
(c h 3
100 m L with glacial acetic add R. H eat the plate at
100-105 °C for 30 min.
h 3c
System suitability:
— the chrom atogram obtained with reference solution (c)
D . 2-m ethyl-2-propylpropane-l,3-diyl dicarbamate
shows 1 clearly visible spot,
(meprobamate).
— the chrom atogram obtained with reference solution (e)
PhEur
shows 2 clearly separated spots.
Limits: in die chromatogram obtained with test solution (a):
— impurity D: any spot due to impurity D is not more
intense than the spot in the chromatogram obtained with
**★
reference solution (a) (0.5 per cent), ★ ★
Carmellose ★ ★
— any other impurity: any spot, apart from the principal spot
*****
and any spot due to im purity D , is not more intense than (Ph. Eur. monograph 2360)
the spot in the chromatogram obtained with reference 9000-11-7
solution (b) (0.2 per cent).
H eav y m e ta ls (2.4.8) A c tio n a n d u se
Maximum 10 ppm. Excipient; bulk laxative.
2.0 g complies with test C. Prepare the reference solution P h E ir .
D E F IN IT IO N
Carboxymethylether of cellulose.
M axim um 0.5 per cent, determined on 1.000 g in vacuo at
60 °C for 3 h. Partly O-carboxymethylated cellulose.
S u lfa te d a s h (2.4.14) CHARACTERS

M axim um 0.1 per cent, determined on 1.0 g. A p p e a ra n c e
White or almost white powder, hygroscopic.
A SSA Y
Dissolve 0.100 g in 15 m L o f a 25 per cent V/V solution of S o lu b ility
sulfuric acid R and boil under a reflux condenser for 3 h. Practically insoluble in anhydrous ethanol. It swells with
Cool, dissolve by cautiously adding 30 m L of water R, cool w ater to form a suspension and becomes viscid in 1 M
again and place in a steam-distillation apparatus. Add 40 m l. sodium hydroxide.
of strong sodium hydroxide solution R and distil immediately by ID E N T IF IC A T IO N
passing steam through the mixture. Collect the distillate into A. p H (2.2.3): 3.5 to 5.0.
40 m L o f a 40 g/L solution o f boric add R until the Suspend 1.0 g in 100 m l. of carbon dioxide-free water R.
total volume in the receiver reaches about 200 mT-
Add 0.25 m L of methyl red mixed solution R. T itrate with
0.1 M hydrochloric add, until the colour changes from green Comparison carmellose CRS.
to violet. Carry out a blank titration. TESTS
1 m L oi 0.1 M hydrochloric add is equivalent to 13.02 m g of C h lo rid e s
C 12H 24N 2O 4. M axim um 0.36 per cent.
IM P U R IT IE S Shake 0.8 g with 50 m L o f water R , dissolve in 10 m L of
1 M sodium hydroxide and dilute to 100 m L with water R.
ch3 o
H eat on a water-bath a mixture o f 10 m L o f dilute nitric
Hî C ^ N ^ C add R and 20 m L of this solution until a flocculent
and enantiomer
precipitate is produced. Cool, centrifuge and take o u t the
CH 3 supernatant. Wash the precipitate with 3 quantities, each o f
10 m L , o f water R, centrifuging each time. C om bine the
A. ( 2RS)-2-(hydroxymethyl)-2-methylpentyl supernatant and the washings and dilute to 100 m L with
( 1-m ethylethyl)carhamate, water R. T o 25 m L of this solution add 6 m L of dilute nitric
2016 Carmellose calcium 1-421
add R and dilute to 50 m L w ith water R (test solution). ★ ★

Prepare the reference solution in the same m anner, using
Carmellose Calcium ★ ★
0.40 m L o f 0.01 M hydrochloric add. Add 1 m L o f silver (Ph. Eur. monograph 0886)
nitrate solution R2 to the test solution and the reference
solution. Allow to stand protected from light for 5 min.
9050-04-8
Any opalescence in the test solution is not m ore intense than A ctio n a n d u se
th at in the reference solution. Excipient in pharmaceutical products; bulk laxative.
Sulfates
P h E it.
Shake 0.40 g with 25 m L o f water R, dissolve in 5 m L of DEFINITION
1 M sodium hydroxide and add 20 m L o f water R. H eat this Calcium salt o f a partly O-carboxymethylated cellulose.
solution with 2.5 m L of hydrochloric add R in a water-bath
CHARACTERS
until a flocculent precipitate is produced. Cool, centrifuge,
A p p e a ra n c e
and take out the supernatant. W ash the precipitate w ith
W hite or yellowish-white powder, hygroscopic after drying.
3 quantities, each o f 10 m L, of water R, centrifuging each
time. Combine the supernatant and the washings, and dilute S o lu b ility
to 100 m L with water R. Filter, and discard the first 5 m L o f Practically insoluble in acetone, in alcohol and in toluene.
the filtrate. T o 25 m L o f th e filtrate add 1 m L of dilute It swells with water to form a suspension.
hydrochloric acid R and dilute to 50 m L with water R (test ID E N T IF IC A T IO N
solution). Prepare the reference solution in the same m anner, A. Shake 0.1 g thoroughly with 10 m L o f water R. Add 2 m L
using 1.5 m L o f 0.005 M sulfuric add. A dd 2 m L o f a o f dilute sodium hydroxide solution R and allow to stand for
120 g/L solution o f barium chloride R to the test solution and 10 min (solution A). Dilute 1 m L of solution A to 5 m L with
the reference solution. M ix and allow to stand for 10 min. water R. T o 0.05 m L add 0.5 m L of a 0.5 g/L solution of
T h e white turbidity produced in the test solution is not chromotropic add, sodium salt R in a 75 per cent m/m solution
thicker than that in the reference solution. o f sulfuric add R and heat on a water-bath for 10 min.
Heavy metals A reddish-violet colour develops.
M axim um 20 ppm . B. Shake 5 m L o f solution A obtained in identification test A
Place 1.0 g in a quartz or porcelain crudble. Cover loosely with 10 m L o f acetone R. A white, flocculent precipitate is
w ith a lid and carbonise by gentle ignition. Cool and add produced.
2 m L of nitric add R and 5 drops o f sulfuric add R. H eat C . Shake 5 m L o f solution A obtained in identification test A
cautiously until white fumes are no longer evolved and with 1 m L o f ferric chloride solution R l. A brown, flocculent
incinerate by ignition at 500-600 °C. Cool and add 2 m L o f precipitate is formed.
hydrochloric add R. Evaporate to dryness o n a water-bath. D . Ignite 1 g and dissolve the residue in a mixture of 5 m L
M oisten the residue with 3 drops o f hydrochloric add R , add o f acetic add R and 10 m L o f water R. Filter if necessary and
10 m L o f hot water R and heat for 2 min. Add 1 drop o f boil the filtrate for a few minutes. Cool and neutralise with
phenolphthalein solution R1, add dilute ammonia R1 dropwise dilute ammonia R l. T he solution gives reaction (a) of calcium
until the solution develops a pale red colour. Add 2 m L of (2.3.1).
dilute acetic acid R, filter if necessary, and wash with 10 m L
o f water R. Transfer the filtrate and washings to a test-tube, TESTS
and dilute to 50 m L with water R (test solution). Prepare the S o lu tio n S
reference solution as follows: evaporate a mixture o f 2 m L o f Shake 1.0 g with 50 m L o f distilled water R, add 5 m L of
nitric acid R, 5 drops of sulfuric acid R and 2 m L of dilute sodium hydroxide solution R and dilute to 100 m L with
hydrochloric add R on a water-bath, then evaporate to dryness distilled water R.
on a sand-bath. M oisten the residue with 3 drops o f A lk alin ity
hydrochloric acid R. Proceed as described for the test solution, Shake 1.0 g thoroughly with 50 m L of carbon dioxide-free
then add 2.0 m L o f lead standard solution (10 ppm Pb) R and water R and add 0.05 m L o f phenolphthalein solution R
dilute to 50 m L with water R. N o red colour develops.
A dd 0.1 m L o f sodium sulfide solution R1 to the test solution C h lo rid e s (2.4.4)
and the reference solution and allow to stand for 5 min. M aximum 0.36 p er cent.
T h e colour o f the test solution is n o t more intense than that H eat 28 m L o f solution S with 10 m L o f dilute nitric add R
o f the reference solution. on a water-bath until a flocculent precipitate is produced.
L o ss o n d ry in g (2.2.32) Cool, centrifuge and separate the supernatant. Wash the
M axim um 8.0 p er cent, determ ined on 1.000 g by drying ip precipitate with 3 quantities, each o f 10 m l , o f water R,
an oven at 105 °C for 4 h. centrifuging each time. Combine the supernatant and the
S u lfa te d a s h (2.4.14) washings and dilute to 100 m L with water R T o 25 m L add
M axim um 1.5 p er cent (dried substance), determined on 6 m L o f dilute nitric acid R and dilute to 50 m L with water R.
1.0 g. Dilute 10 m L o f the solution to 15 m L with water R.
STORA GE S u lfa tes (2.4.13)

M aximum 1 per c e n t
H eat 20 m L o f solution S with 1 m L o f hydrochloric add R
_________________________________________________________________ PhEur
o n a water-bath until a flocculent precipitate is produced.
Cool, centrifuge and separate the supernatant. W ash the
precipitate with 3 quantities, each of 10 m T, o f disdUed
water R, centrifuging each time. Combine the supernatant
1-422 Carmellose Sodium 2016
and the washings and dilute to 100 m L with distilled water R. A p p a re n t v iscosity
T o 25 m L add 1 m L of dilute hydrochloric add R and dilute While stirring, introduce a quantity o f the substance to be
to 50 m L with distilled water R. examined equivalent to 2.00 g o f the dried substance into
H eav y m e ta ls (2.4.8) 50 m L o f water R heated to 90 °C. F or a product o f low
M axim um 20 ppm. viscosity, use if necessary, the quantity required to give the
concentration indicated on the label. Allow to cool, dilute to
100.0 m L with water R and stir until dissolution is complete.
Determ ine the viscosity (2.2.10) using a rotating viscometer
L oss o n d ry in g (2.2.32) at 20 °C and a shear rate o f 10 s-1 . If it is impossible to
M aximum 10.0 per cent, determined on 1.000 g by drying in obtain a shear rate o f exactly 10 s-1 , use a shear rate slightly
an oven at 105 °C for 4 h. higher and a rate slightly lower and interpolate. T h e apparent
S u lfa te d a s h (2.4.14) viscosity is not less than 75 per cent and n o t m ore than
10.0 per cent to 20.0 per cent, determined on 1.0 g in a 140 per cent of the value stated on the label.
platinum crucible. S o d iu m glycollate
STORA GE Place a quantity o f the substance to be examined equivalent
In an airtight container. to 0.500 g o f dried substance in a beaker. A dd 5 m L of acetic
add R and 5 m L o f water R. Stir until dissolution is complete
_______________________________________________________________ PhEur
(about 30 min). Add 80 m L o f acetone R and 2 g o f sodium
chloride R. Filter through a fast filter paper impregnated with
acetone R into a volumetric flask, rinse the beaker and filter
with acetone R and dilute the filtrate to 100.0 m L with the
Carmellose Sodium ***** same solvent. Allow to stand for 24 h without shaking.
** +* Use the clear supernatant to prepare the test solution.
In a volumetric flask, dissolve 0.310 g of glycoBic add R,
9004-32-4 previously dried in vacuo over diphosphorus pentoxide R, in
water R and dilute to 1000.0 m L with the same solvent.
Place 5.0 m L o f this solution in a volumetric flask, add 5 m L
Excipient; bulk laxative.
o f acetic add R and allow to stand for about 30 min.
P r e p a r a tio n Add 80 m L of acetone R and 2 g o f sodium chloride R and
Carmellose Sodium Eye Drops dilute to 100.0 m L with acetone R. Use this solution to
prepare the reference solution.
P h E u r ___________________________________________________________________________________________
Place 2.0 m L o f each solution in a separate 25 m L
D E F IN IT IO N volumetric flask. H eat on a water-bath to eliminate acetone.
Carmellose sodium (carboxymethylcellulose sodium) is the Cool to room tem perature and add 5.0 m L o f 2,7-
sodium salt o f a partly O-carboxymethylated cellulose. dihydroxynaphthalene solution R to each flask. Shake and add
It contains not less than 6.5 per cent and not more than 15.0 m L of 2,7-dihydroxynaphthalene solution R. Close the
10.8 per cent o f sodium (N a), calculated with reference to flasks with aluminium foil and heat on a water-bath for
the dried substance. 20 min. Cool under running water and dilute to 25.0 m L
CHARACTERS with sulfuric add R. W ithin 10 min, transfer 10.0 m L o f each
A white or alm ost white, granular powder, hygroscopic after solution to a flat-bottomed tube. Examine the solutions
drying, practically insoluble in acetone, in ethanol and in viewing vertically. T he test solution is not more intensely
toluene. It is easily dispersed in water giving colloidal coloured than the reference solution (0.4 per cent).
solutions. C h lo rid e s (2.4.4)
ID E N T IF IC A T IO N Dilute 2 mT, o f solution S to 15 m L with water R.
T h e solution complies with the limit test for chlorides -
A. T o 10 m L o f solution S (see Tests) add 1 m L of copper
(0.25 per cent).
sulfate solution R. A blue, cotton-like precipitate is formed.
B. Boil 5 m L of solution S for a few minutes. N o precipitate H eav y m e ta ls (2.4.8)
is formed. T o the residue obtained in the determination o f the sulfated
ash, add 1 m L o f hydrochloric add R and evaporate on a
C. T h e solution prepared from the sulfated ash in the test for
water-bath. Take up the residue in 20 m L o f water R. 12 m L
heavy metals gives the reactions of sodium (2.3.1).
o f the solution complies with test A for heavy metals
TESTS (20 ppm). Prepare the reference solution using lead standard
S o lu tio n S solution (1 ppm Pb) R.
Sprinkle a quantity of the substance to be examined L oss o n d ry in g (2.2.32)
equivalent to 1.0 g of the dried substance onto 90 m L of N ot more than 10.0 per cent, determined on 1.000 g by
carbon dioxide-free water R at 40 °C to 50 °C stirring drying in an oven at 105 °C.
vigorously. Continue stirring until a colloidal solution is
obtained, cool and dilute to 100 m L with carbon dioxide-free
20.0 per cent to 33.3 per cent, determined on 1.0 g using a
water R.
mixture o f equal volumes o f sulfuric add R and water R and
A p p e a ra n c e o f so lu tio n calculated with reference to the dried substance. T hese limits
Solution S is not more opalescent than reference correspond to a content o f 6.5 per cent to 10.8 per cent of
suspension IH (2.2.1) and not more intensely coloured than sodium (Na).
reference solution Y6 (2.2.2, Method II).
p H (2.2.3)
T he p H o f solution S is 6.0 to 8.0.
2016 Carmellose Sodium 1-423
LA B ELLIN G 1 m L of 0.05 M silver nitrate is equivalent to 2.922 mg

T he label states the apparent viscosity in millipascal seconds of NaCl.
for a 20 g/L solution; for a product of low viscosity, the label Sodium glycollate Place a quantity o f the substance to be
states the concentration of the solution to be used and the examined equivalent to 0.500 g of the dried substance in a
apparent viscosity in millipascal seconds. beaker. Add 5 m L of glacial acetic add R and 5 mT, of
__________________________________________________________________________________________ PhEur water R and stir to ensure total hydration (about 30 min).
Add 80 m L of acetone R and 2 g o f sodium chloride R. Stir for
several minutes to ensure complete precipitation o f the
carboxymethylcellulose. Filter through a fast filter paper
im pregnated with acetone R into a volumetric flask, rinse the
Low-substituted Carmellose ?**% beaker and filter with acetone R and dilute the filtrate to
100.0 m L with the same solvent. Allow to stand for 24 h
Sodium ***** w ithout shaking. Use the clear supernatant as the test
(Ph. Eur* monograph 1186) solution.
9050-32-4 Prepare the reference solutions as follows: in a 100 mL
volumetric flask, dissolve 0.100 g of glycollic add R,
A ction a n d u se
previously dried in vacuo over diphosphorus pentoxide R, in
Excipient in pharmaceutical products; bulk laxative.
water R and dilute to 100.0 m L w ith the same solvent.
PhE ur ________________________________________________________________________________________
Transfer 0.5 mL, 1.0 mL, 1.5 m L and 2.0 mL of the
solution to separate volumetric flasks; dilute the contents o f
D E F IN IT IO N each flask to 5.0 m L with water R, add 5 m L of glacial acetic
Low-substituted sodium carboxymethylcellulose. Sodium salt acid R, dilute to 100.0 m L with acetone R and mix.
of a partly O-(carboxymethylated) cellulose.
Transfer 2.0 m L o f the test solution and 2.0 m L o f each o f
C o n te n t the reference solutions to separate 25 m L volumetric flasks.
2.0 per cent to 4.5 per cent of sodium (Na) (dried H eat the uncovered flasks in a water-bath to eliminate the
substance). acetone. Allow to cool and add 5.0 m L of 2,7-
CHARACTERS dihydroxynaphthalene solution R to each flask. Mix, add a
A p p e a ra n c e further 15.0 m L o f 2,7-dihydroxynaphthalene solution R and
White or almost white powder or short fibres. mix again. Close the flasks with aluminium foil and heat in a
water-bath for 20 min. Cool and dilute to 25.0 m L with
Solubility sulfuric add R.
Practically insoluble in acetone, in anhydrous ethanol and in
Measure the absorbance (2.2.25) o f each solution at 540 nm .
toluene. It swells in water to form a gel.
Prepare a blank using 2.0 mL of a solution containing
ID E N T IF IC A T IO N 5 per cent V/V each of glacial acetic add R and water R in
A. Shake 1 g with 100 m L of a 100 g/L solution of sodium acetone R. Prepare a standard curve using the absorbances
hydroxide R. A suspension is produced. obtained with the reference solutions. From the standard
B. Shake 1 g with 50 m L of water R. Transfer 1 m L of the curve and the absorbance of the test solution, determine the
mixture to a test tube, add 1 m L o f water R and 0.05 m L o f mass a, in milligrams, of glycollic a d d in the substance to be
a freshly prepared 40 g/L solution of ct-naphthd R in examined and calculate the content of sodium glycollate from
methanol R. Incline the test tube and add carefully 2 m L of the following expression:
sulfuric acid R down the side so that it forms a lower layer.
A reddish-purple colour develops at the interface. 10 x 1.29 x a
C. Sulfated ash (2.4.14) (see Tests). (100 — b)m
D. T he solution prepared for the test for heavy metals gives
reaction (a) of sodium (2.3.1). 1.29 = the factor converting glycollic a d d to sodium
TESTS glycollate,
p H (223)
b = the loss on drying as a percentage,
6.0 to 8.5.
m = the mass of the substance to be examined, in
grams.
Shake 1 g with 100 m L of carbon dioxide-free water R for
5 min. Centrifuge. W ate r-so lu b le su b stan ces
S o d iu m c h lo rid e a n d so d iu m glycollate
M aximum 0.5 per cent (dried substance) for the sum of the Disperse 5.00 g in 400.0 mL of water R and stir for 1 min
percentage contents. every 10 min during the first 30 min. Allow to stand for 1 h
and centrifuge, if necessary. D ecant 100.0 m L of the
Sodium chloride Place 5.00 g in a 250 m L conical flask, add
supernatant onto a fast filter paper in a vacuum filtration
50 m L o f water R and 5 m L of strong hydrogen peroxide
funnel, apply vacuum and collect 75.0 m L of the filtrate.
solution R and heat on a water bath for 20 min, stirring
Evaporate to dryness and dry the residue at 100-105 °C for
occasionally to ensure total hydration. Cool, add 100 m L of
4 h.
water R and 10 m L o f nitric acid R. Titrate with 0.05 M silver
nitrate determining the end-point potentiometrically (2.2.20) H eav y m e ta ls (2.4.8)
using a silver-based indicator electrode and a double-junction M aximum 20 ppm.
reference electrode containing a 100 g/L solution of potassium T o the residue obtained in the determination of the sulfated
nitrate R in the outer jacket and a standard filling solution in ash add 1 m L of hydrochloric acid R and evaporate on a
the inner jacket. water-bath. Take up the residue in 20 m L of water R (this
solution is used for identification test D). 12 m L o f the
1-424 Carmustine 2016
solution complies w ith test A. Prepare the reference solution ID E N T IF IC A T IO N

using lead standard solution (1 ppm Pb) R. Examine by infrared absorption spectrophotom etry (2.2.24),
L o ss o n d ry in g (2.2.32) comparing with the Ph. Eur. reference spectrum of carmustine.
M axim um 10.0 per cent, determ ined on 1.000 g by drying in Examine the m elted substances prepared as films.
an oven at 105 °C. TESTS
S u lfa te d a s h (2.4.14) 1 ,3-B is (2 -c h lo ro e th y l) u r e a (im p u rity A)
6.5 p er cent to 13.5 p e r cent (dried substance), Examine by thin-layer chromatography (2.2.27), using a
corresponding to a content o f 2.0 p er cent to 4.5 per cent of suitable silica gel as the coating substance.
Na. Test solution Dissolve 0.10 g of the substance to be examined
U se 1.0 g w ith a mixture of equal volumes of sulfuric acid R in methylene chloride R and dilute to 5 m l. with the same
and water R. solvent.
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 Reference solution (a) Dissolve 2 m g of carmustine
This section provides information on characteristics that are impurity A CRS in methylene chloride R and dilute to 10 m L
recognised as being relevant control parameters for one or more with the same solvent.
functions of the substance when used as an excipient (see chapter Reference solution (b) Dilute 1 m L o f the test solution to
5.15). This section is a non-mandatory part of the monograph 10 m L with methylene chloride R. T o 5 m L of this solution,
and it is not necessary to verify the characteristics to demonstrate add 5 m L of reference solution (a).
compliance. Control of these characteristics can however contribute Apply separately to the plate 2 jiL of each solution. Develop
to the quality of a medicinal product by improving the consistency over a path o f 10 cm using a mixture o f 10 volumes of
of the manufacturing process and the performance of the medicinal methanol R and 90 volumes o f methylene chloride R. Allow the
product during use. Where control methods are cited, they are plate to dry in air. Spray with diethylamine R and heat at
recognised as being suitable for the purpose, but other methods can 125 °C for 10 min. Allow to cool and spray with silver nitrate
also be used Wherever results for a particular characteristic are solution R2. Expose to ultraviolet light at 365 nm until brown
reported, the control method must be indicated to black spots appear. Any spot corresponding to carmustine
The following characteristic may be relevant for low-substituted impurity A in the chromatogram obtained with the test
carmellose sodium used as dismtegranL solution is not more intense than the spot in the
S e ttlin g v o lu m e chrom atogram obtained with reference solution (a)
(1 per cent). T he test is n o t valid unless the chromatogram
15.0 m L to 35.0 mL.
obtained with reference solution (b) shows two cleariy
In a 100 m L graduated cylinder, place 20 m L of
separated spots.
2-^ropanol R, add 5.0 g o f the substance to be examined and
shake vigorously. D ilute to 30 m L with 2-propanol R then to W a te r (2.5.12)
50 m L with water R an d shake vigorously. Within 15 min, N o t more than 1.0 per cent, determ ined on 0.50 g by the
repeat the shaking 3 times. Allow to stand for 4 h and semi-micro determination o f water.
determ ine the volume o f the settled mass. A SSA Y
____________________________________________________________________________________________ PhEur Dissolve 0.100 g in 30 m L of ethanol R and dilute to
100.0 m L with water R. Dilute 3.0 m L o f the solution to
100.0 m L with water R. M easure the absorbance (2.2.25) at
the maximum at 230 nm.
★ ★ Calculate the content o f C 5H 9CI2N 3O 2 taking the specific
Carmustine ★ ★ absorbance to be 270.
(Ph. Eur. monograph 1187) ***** STORAGE
Store in an airtight container, protected from light, at a
tem perature o f 2 °C to 8 °C.
IM P U R IT IE S
C 5H 9CI2N 3O 2 214.1 154-93-8 X
Cytotoxic alkylating agent. A. 1,3-bis (2 -chloroethyl)urea.
PhEur
P hE ur.
D E F IN IT IO N
Carm ustine contains n o t less than 98.0 per cent and n o t
m ore than the equivalent of 102.0 per cent of
1,3-bis (2-chloroethyl) -1 -nitrosourea, calculated with reference
to the anhydrous substance.
CHARACTERS
A yellowish, granular pow der, very slightly soluble in water,
very soluble in methylene chloride, freely soluble in ethanol.
It melts at about 31 °C with decomposition.
2016 Carrageenan 1-425
★ ★ A cid valu e
Camauba Wax ★ ★ 2 to 7.
★. ★
(Ph. Ever, monograph 0597) *★* T o 2.000 g (m g) in a 250 m L conical flask fitted with a
reflux condenser add 40 mT. o f xylene R and a few glass
A ctio n a n d u se beads. H eat with stirring until the substance is completely
Excipient. dissolved. Add 20 m L of alcohol R and 1 m L o f bromothymol
blue solution R3 and titrate the hot solution with
PhEur.
0.5 M alcoholic potassium hydroxide until a green colour
D E F IN IT IO N persisting for at least 10 s is obtained (n1 mL). C arry out a
Purified wax obtained from the leaves of Copemicia cerifera blank test (n2 mL). Calculate the acid value from the
M art. expression:
CHARACTERS
A p p e a ra n c e 2 8 .0 5 ( n i — n j )
Pale yellow or yellow powder, flakes or hard masses.
S o lu b ility
Practically insoluble in water, soluble on heating in ethyl S a p o n ifica tio n v alu e
acetate and in xylene, practically insoluble in alcohol. 78 to 95.
T o 2.000 g (m g) in a 250 m L conical flask fitted with a
R elativ e d e n sity
A bout 0.97. reflux condenser add 40 m L of xylene R and a few glass
beads. H eat with stirring until the substance is completely
ID E N T IF IC A T IO N dissolved. Add 20 m L o f alcohol R and 20.0 m L o f
Thin-layer chromatography (2.2.27). 0.5 M alcoholic potassium hydroxide. Boil under a reflux
Test solution Dissolve 0.10 g of the substance to be examined condenser for 3 h. Add 1 m L of phenolphthalein solution R l
with heating in 5 m L of chloroform R. Use the warm solution. and titrate the hot solution immediately with
Reference solution Dissolve 5 mg o f menthol R, 5 pL o f menthyl 0.5 M hydrochloric acid until the red colour disappears.
acetate R and 5 mg of thymol R in 10 m L of toluene R. Repeat the heating and titration until the colour no longer
reappears on heating (n3 mL). Carry out a blank test
Plate TLC silica gel plate R
(n4 m L). Calculate the saponification value from the
Mobile phase ethyl acetate R, chloroform R (2:98 VIV). expression:
Application 30 |iL of the test solution and 10 pL of the
reference solution as bands 20 m m by 3 mm. 28.05 (714 — TI3)
Development Over half of the plate. m
Drying In air. T o ta l a s h (2.4.16)
Detection Spray with a freshly prepared 200 g/L solution of M aximum 0.25 per cent, determined on 2.0 g.
phosphomolybdic acid, R in alcohol R (about 10 m L for a 20 cm
STORA GE
plate). H eat at 100-105 °C for 10-15 min.
Results T he chromatogram obtained with the reference
solution shows in the lower part a dark blue zone (menthol), PhEur
above this zone a reddish zone (thymol) and in the upper

part a dark blue zone (menthyl acetate). T he chromatogram
obtained with the test solution shows a large blue zone ★*★
(triacontanol = melissyl alcohol) at a level between the Carrageenan ★ ★
★ ★
thymol and menthol zones in the chromatogram obtained
(Ph. Eitr. monograph 2138) * * * * *
with the reference solution. Further blue zones are visible in
the upper p art o f the chromatogram obtained with the test PhEur ________________________
solution, at levels between those o f the menthyl acetate and
D E F IN IT IO N
thymol zones in the chromatogram obtained with the
Polysaccharides extracted from different Rhodophyceae with
reference solution; above these zones further zones are visible
boiling water or aqueous alkali solutions. Carrageenan is
in the chromatogram obtained with the test solution;
separated by alcohol precipitation, potassium chloride
the zone with the highest Rp value is very pronounced.
precipitation, gel pressing, drum drying or freezing.
A num ber of faint zones are visible below the triacontanol
T h e alcohol used during separation and purification is
zone and the point of application is coloured blue.
generally 2-propanol. T he main components are potassium,
TESTS sodium, calcium or magnesium salts of the sulfate esters of
A p p e a ra n c e o f so lu tio n D-galactose and 3,6-anhydro-D-galactose copolymers. They
T he solution is clear (2.2.1) and n o t more intensely coloured exist in different proportions depending on the biological
than a 50 m g/L solution o f potassium dickromate R (2.2.2, origin o f the polymer.
Method II). T he prevalent copolymers are designated as K-, 1- and
Dissolve 0.10 g with heating in chloroform R and dilute to X-carrageenan.
10 m L with the same solvent. CHARACTERS
M e ltin g p o in t (2.2.15) A p p e a ra n c e
80 °C to 88 °C. Yellowish, brownish, or white or almost white powder.
M elt the substance to be examined carefully on a water-bath S o lu b ility
before introduction into the capillary tubes. Allow the tubes Soluble in water giving a viscous or colloidal solution,
to stand in the refrigerator for 24 h or at 0 °C for 2 h. insoluble in organic solvents.
1-426 Carteolol Hydrochloride 2016
ID E N T IF IC A T IO N T o ta l a s h (2.416)
A. Prepare a 20 g/L dispersion and heat in a water-bath at M axim um 40.0 p er cent.
80 °C. M ix 1 volume o f this solution and about 4 volumes of A sh in so lu b le ỉn h y d ro c h lo ric a c id (2.8.1)
water R and add 2-3 drops o f a 0.5 g/L solution o f methylene M aximum 2.0 per c e n t
blue R in ethanol (96 per cent) R. A blue precipitate is formed.
This section provides information on characteristics that are ■
Preparation Prepare a 2 g/L solution o f the substance to be recognised as being relevant control parameters for one or more
examined; cast 4.5 m L o f the solution into a plastic flat- functions of the substance when used as an excipient (see chapter
bottom ed weighing b oat about 35 m m in diameter and allow 5.15). Some of the characteristics described in the Functionality-
to dry completely in an air-flow oven at 60 °C until a film, related characteristics section may also be present in the mandatory
about 10 jim thickj is obtained (about 4 h). part of the monograph since they also represent mandatory quality
C arrageenan has strong, broad absorption bands, typical o f criteria. In such cases, a cross-reference to the tests described in the
all polysaccharides, in the 1000-1100 cm -1 region. mandatory part ừ included in the Fttncttonatity-rdated
Absorption maxima are 1065 cm -1 and 1020 cm-1 for characteristics section. Control of the characteristics can contribute
gelling and non-gelling types, respectively. O ther to the quality of a medicinal product by improving the consistency
characteristic absorption bands and their intensities relative to of the manufacturing process and the performance of the medicinal
the absorbance at 1050 cm -1 are shown in Table 2138.-1. product during use. Where control methods are cited, they are
recognised as being suitable far the purpose, but other methods can
Table 2138.-1. - Characteristic absorption bands fo r also be used Wherever results for a particular characteristic are
carrageenan identification by infrared absorption reported, the control method must be indicated
spectrophotometry The following characteristics may be relevantfor carrageenan used
Absorbance relative to the as viscosity-increasing agent
Wanrennmber absorbance at 1050 cm '1 G e l fo rm a tio n
Molecular structure
(cm -1)
K 1 \ Prepare a 20 g/L dispersion and heat in a w ater-bath at
80 °c (solution A). Allow to cool; it becomes m ore viscous
0 .7 - 1.4 -
1220 - 1260 Ester sulfate
1.2
1.2 -2.0
2.0
u pon cooling and may form a gel.
T o 10 m L o f solution A, while still hot, add 4 drops o f a
3,6-Anhydro-D- 0.3 - 0.2 -
928 - 933
gaiactose 0.6 0.5
£ 0 .2 100 g/L solution of potassium chloride R, mix and allow to
cool. A ‘brittle’ gel indicates a carrageenan o f a
0.3 - 0.2 - predom inantly K-type; an ‘elastic’ gel indicates a
840 -8 5 0 Galactose-4-sulfate -
0.5 0.4
predom inantly 1-type; if the solution does n o t form a gel, the
825 - 830 Galactose-2-sulfate - -
0.2 - carrageenan is of a predom inantly X-type; a weak or pourable
0.4 gel obtained on cooling indicates a carrageenan comprising a
0.1 - m ixture o f K- and X-types, which can be confirmed by
810 - 820 GaIactose-6-sulfate - -
0.3 infrared absorption spectrophotometry.
3,6-Anhydro-D- 0.2 - V iscosity
800 - 805 <0.2 -
gaIactose-2-sulfate 0.4 (see Tests).
____________________________________________________________________________________________ PhEur
T able 2138.-1 shows absorbance ratios corresponding to

copolymer types isolated from carrageenans. Native extracts
o f carrageenan from m ost cold-water seaweed species
comprise k-, i- and X-structures from the natural mix o f
haploid and diploid plants. Such extracts exhibit, in practice, Carteolol Hydrochloride *****
the characteristic absorbance peaks o f K-, i- and X-structures
and yield absorbance ratios somewhere between the above-
m entioned individual ranges.
TESTS
V iscosity (2.2.10)
M inim um 5 mPa-s. H eat a 15 g/L dispersion (dried
substance) at 80 °C for at least 15 min to dissolve.
C om pensate for any loss of w ater by evaporation, allow to
cool to 75 °C and c an y out th e test at this temperature. C 16H 25N 2O 3CI 328.8 51781-21-6
A rse n ic (2.427)
M axim um 3.0 ppm.
C a d m iu m (2.427)
P r e p a r a tio n
M axim um 2.0 ppm.
Carteolol Eye Drops
L e a d (2.427)
M axim um 5.0 ppm . P h E u r ____________________________________________________________________________________________
M e rc u ry (2.427) D E F IN IT IO N
M axim um 1.0 ppm. 5 - [(2RS) -3- [(1,1 -Dimethylethyl) amino] -2-hydroxypr opoxy] -
L oss o n d ry in g (2.2.32) 3,4-dihydroquinolin-2(l/i)-one hydrochloride.
M axim um 12.0 per cent, determ ined on 1.000 g by drying in C o n te n t
an oven at 105 °C. 99.0 per cent to 101.0 per cent (dried substance).
2016 Carteolol Hydrochloride 1-427
CHARACTERS Limits:
Appearance — impurity H: not more than twice the area o f the principal
W hite or almost white crystals or crystalline powder. peak in the chromatogram obtained with reference
Solubility solution (b) (0.2 per cent);
Soluble in water, sparingly soluble in methanol, slightly — unspecified impurities: for each impurity, not more Than the
soluble in ethanol 96 per cent, practically insoluble in area o f the principal peak in the chromatogram obtained
methylene chloride. with reference solution (b) (0.10 per cent);
— total: not more than half the area of the principal peak in
ID E N T IF IC A T IO N the chromatogram obtained with reference solution (a)
A. Infrared absorption spectrophotometry (2.2.24). (0.5 p er cent);
Comparison Ph. Eur. reference spectrum of carteolol hydrochloride. — disregard limit: 0.2 times the area o f the principal peak in
B. It gives reaction (a) o f chlorides (2.3.1). the chromatogram obtained with reference solution (b)
(0.02 per cent).
TESTS
an oven a t 105 °C for 3 h.
Method II).
Dissolve 0.300 g in water R and dilute to 10 m L with the S u lfa te d a s h (2.414)
same solvent. M aximum 0.1 per cent, determined on 1.0 g.
pH (2.2.3) A SSAY
5.0 to 6.0. Dissolve 0.250 g in 60 m L of ethanol (96 per cent) R.
Dissolve 0.250 g in carbon dioxide-free water R and dilute to A dd 5.0 m L o f 0.01 M hydrochloric acid. C any out a
25 m L w ith the same solvent. potentiometric titration (2.2.20), using 0.1 M sodium
hydroxide. Read the volume added between the 2 points o f
Related substances inflexion.
Test solution Dissolve 20.0 mg o f the substance to be C 16H 25N 2O 3CI.
the mobile phase. STORA GE
Reference solution (a) Dilute 1.0 m L of the test solution to In an airtight container.
100.0 m L with the mobile phase. IM P U R IT IE S
Reference solution (b) Dilute 1.0 m L o f reference solution (a) Specified impurities H
to 10.0 m L with the mobile phase. Other detectable impurities (the following substances would, if
Reference solution (c) Dissolve 10 m g of carteololfor system present at a sufficient level, be detected by one or other o f
suitability CRS in the mobile phase and dilute to 5 m L with the tests in the monograph. They are limited by the general
the mobile phase. acceptance criterion for other/unspecified impurities and/or
Reference solution (d) Dilute 5.0 m L o f reference solution (b) by the general monograph Substances for pharmaceutical use
to 10.0 m L with the mobile phase. (2034). I t is therefore not necessary to identify these
Column:
— size: I = 0.25 m , 0 = 4.6 mm;
C, D, E, F, G, I.
(5 nm).
Mobile phase Mix 1 volume of methanol R2, 20 volumes of
acetonitrile R and 79 volumes of a 2.82 g/L solution of sodium
hexartesulfonate R.
Flow rate 1 mL/min.
A. 4,6,7,8-tetrahydroquinoline-2,5(l//,3/f)-dione,
Irgecdon 20 [iL.
with carteolol for system suitability CRS to identify the peak
due to impurity H .
System suitability:
— the chromatogram obtained with reference solution (c) is
similar to the chromatogram provided with carteololfor
system stdtabHay CRS; the peaks due to impurity H and B. 5-hydroxy-3,4-dihydroquinolin-2(l.H)-one,
carteolol show base-line separation;
the chromatogram obtained with reference solution (d); -s^ °
.-x vvJ and enantiomer
— number of theoretical plates: minimum 6000, calculated for
C . 5-[ [(2i?S)-oxiran-2-yl]methoxy]-3,4-dihydroquinolin-
2 (1 //)-one,
1-428 Carvedilol 2016
Solubility
R Practically insoluble in water, Sparingly soluble in methylene
and enantiomer chloride, slightly soluble in ethanol (96 per cent). It is
practically insoluble in dilute adds.
R1 It shows polymorphism (5.9).
IDENTIFICATION
D . R = Cl, R ' = H: 5-[(2ÄS)-3-chloro-2-hydroxypropoxy]- Infrared absorption spectrophotometry (2.2.24).
3 j4-dihydroquinolin-2 ( 1if)-o n e, Comparison carvedüol CRS.
F. R = O C H 3, R ' = H : 5-[(2ÄS)-2-hydroxy-3- I f the spectra obtained show differences, dissolve the
methoxypropoxy] -3,4-dihydroquinolin-2 ( lü )-o n e , substance to be examined and the reference substance
G. R = O H , R ' = H: 5-[(2RS)-2,3-dihydroxypropoxy]-3,4- separately in 2-propanol R, evaporate to dryness and record
dihydroquinolin-2 ( 1H)-one, new spectra using the residues.
I. R = N H -C (C H 3)3, R ' = B n 7-brom o-5-[(2i?5)-3-[(l,l- TESTS
(dimethylethyl) amino] -2-hydroxypropoxy]-3,4-
Related substances
dihydroquinolin-2 ( 1if)-o n e, L iquid chromatography (2.2.29).
in the mobile phase and dilute to 25.0 m L w ith the mobile
phase.
E. 5 ,5 '-[(2-hydroxypropan-l,3-diyl)bis(oxy)]bis(3,4- Reference solution (b) Dissolve 5 mg o f carvedüol
dihydroquinolin-2 (l//)-o n e )3 impurity C CRS in 5.0 m L o f the mobile phase and dilute to
100.0 m L with the mobile phase. Dilute 4.0 m L o f the
solution to 100.0 m L with the mobile phase. D ilute 1.0 m L
hv 0H h o f this solution to 10.0 m L with the mobile phase.
O N CH 3 and enantiomer
Reference solution (c) Dissolve 5 mg o f carvedüolfor system
H3C ch3
sttitabüày CRS (containing impurities A and D ) in the mobile
phase and dilute to 50.0 m L with the mobile phase.
H . 5 -[ (2RS)-3-[( 1,1 -dimethylethyl)amino] -2- Column:
hydroxypropoxy] quinolin-2 (1H) -one. — size. I = 0.150 m , 0 = 4.6 mm;
— stationary phase, end-capped octylsüyl süica gel for
____________________________________________________________________________________________ PhEur
chromatography R (5 |im );
Mobile phase Dissolve 1.77 g of potassium dihydrogen
phosphate R in water R and dilute to 650 m L w ith the same
★ ★
Carvedilol ★ ★ solvent; adjust to p H 2.0 with phosphoric add R and add
350 m L o f acetonitrUe R.
H OH Detection Spectrophotom eter at 240 nm .
Injection 20 |iL.
Run time 6 times the retention rime o f carvedilol.
with carvedüolfor system suitabüùy CRS and the
the peaks due to impurities A and D ; use the chrom atogram
C24H 26N 20 4 406.5 72956-09-3 obtained with reference solution (b) to identify the peak due
to im purity C.
A c tio n a n d u s e
Relative retention W ith reference to carvedilol (retention
Beta-adrenoceptor antagonist; arteriolar vasodilator.
P r e p a r a tio n im purity C = about 2.9; im purity D = about 3.8.
Carvedilol Tablets System suitability:
P h E u r ____________________________________________________________________________________________
im purity A and carvedilol in the chrom atogram obtained
D E F IN IT IO N w ith reference solution (c);
(2RS ) - 1-(9/Jr-Carbazol-4-yloxy)-3- [ [2- — signal-to-noise ratio: minimum 10 for the peak due to
(2-methoxyphenoxy)ethyl]amino]propan-2-ol. im purity C in the chromatogram obtained w ith reference
C o n te n t solution (b).
99.0 per cent to 101.0 per cent (dried substance). Limits:
CHARACTERS
peak area of impurity A by 2.0;
A p p e a ra n c e
2016 Castor Oil 1-429
— impurity A: n o t more than twice the area of the principal

— impurity D: n o t more than 1.5 times the area o f the
— impurity C: no t more than the area of the corresponding
area of the principal peak in the chromatogram obtained B. 1,1'-[ [2-(2-methoxyphenoxy) ethyl] nitrilo] bis [3-(9H-
with reference solution (a) (0.10 per cent); carbazol-4-yloxy)propan-2-ol],
— sum of impurities other than C: not more than 5 times the
the chromatogram obtained with reference solution (a) and enantiomer
(0.05 per cent).
M aximum 10 ppm .
Solvent dimethyl sulfoxide R
2.0 g complies with test H. Prepare the reference solution C . (2RS)-1- [benzyl [2-(2-methoxyphenoxy)ethyl] amino] -3-
using 2 m L of lead standard solution (10 ppm Pb) R. (9J/-carbazol-4-yloxy)propan-2-ol,
an oven at 105 °C.
OCH,
ASSAY
Dissolve 0.350 g in 60 m L of anhydrous acetic acid R. Titrate
with 0.1 M perchloric acid, determining the end-point
1 m L of 0.1 M perchloric acid is equivalent to 40.65 mg of D . 1-(9i?-carbazol-4-yloxy)-3- [4- [2-hydroxy-3- [ [2-
C 24H 26N 2O 4. (2-methoxyphenoxy)ethyl]amino]propoxy]-9Ji-carbazol-9-
IM P U R IT IE S yl]propan-2-ol.
Specified impurities A, C, D PhEur

acceptance criterion for other/unspecified impurities and/or Hydrogenated Castor Oil ★ ★
★ ★
by the general monograph Substances for pharmaceutical use **+★*
(2034). It is therefore not necessary to identify these (PL Eur. monograph 1497)
A ctio n a n d u se
Control of impurities in substances for pharmaceutical use): B.
Excipient.
P h E u r ____________________________________________________
D E F IN IT IO N
Fatty oil obtained by hydrogenation of Virgin Castor
oil (0051). It consists mainly of th e triglyceride of
12-hydroxystearic (12-hydroxyoctadecanoic) acid.
CHARACTERS
A p p e a ra n c e
Fine, almost white or pale yellow powder or almost white or
pale yellow masses or flakes.
A. 1- [ [9- [2-hydroxy-3- [[2-
(2-methoxyphenoxy)ethyI] aminojpropyl] -9if-carbazol-4- S o lu b ility
yl] oxy] -3- [[2-(2-methoxyphenoxy) ethyl] amino]propan-2-ol, Practically insoluble in water, slightly soluble in methylene
chloride, very slightly soluble in anhydrous ethanol,
practically insoluble in light petroleum.
B. Hydroxyl value (see Tests).
C. Composition o f fatty adds (see
1-430 Castor Oil 2016
TESTS Rc = 1 for peaks corresponding to each o f the other

A c id v alu e (2.5.1) specified fatty a d d s or any unspecified fatty ad d ;
M axim um 4.0, determ ined on 10.0 g dissolved in 75 m L of mass o f methyl 12-hydroxystearate in the
hot ethanol (96 per cent) R. reference solution;
H y d ro x y l v a lu e (2.5.3, Method A) m2s = mass o f methyl stearate in the reference solution;
145 to 165, determ ined on a warm solution. A \s = area o f any peak due to methyl
12-hydroxystearate in the chrom atogram obtained
Io d in e v alu e (2.5.4, Method A) with the reference solution;
M axim um 5.0. area o f any peak due to methyl stearate in the
A lk alin e im p u ritie s chromatogram obtained with the reference
Dissolve 1.0 g by gentle heating in a mixture of 1.5 m L o f solution;
ethanol (96 per cent) R and 3 m L of toluene R. Add 0.05 m L A XyS = area o f the peaks due to any specified or
o f a 0.4 g/L solution o f bromophenol blue R in ethanol unspecified fatty a d d methyl esters.
(96 per cent) R. N o t m ore th an 0.2 m L of 0.01 M hydrochloric Composition of the fatty add fraction of the oU:
acid is required to change the colour o f the indicator to — palmitic add: n o t more than 2.0 per cent;
yellow. — stearic add. 7.0 per cent to 14.0 per cent;
C o m p o sitio n o f Catty a d d s (2.4.22) — arachidic add: n o t more than 1.0 per cent;
U se the m ixture o f calibrating substances in Table 2.4.22.-3. — 12-oxostearic add: not m ore than 5.0 per cent;
Test solution Introduce 75 m g o f the substance to be — 12-hydroxystearic add. 78.0 per cent to 91.0 per cent;
exam ined into a 10 m L centrifuge tube with a screw cap. — any other fatty acid: n o t m ore than 3.0 per cent.
Dissolve in 2 m L o f 1,1-dimethylethyl methyl ether R1 by N ick el (2.4.31)
shaking and heat gently (50-60 °C). A dd, when still warm, M axim um 1 ppm.
1 m L o f a 12 g/L solution o f sodium R in anhydrous
STORAGE
methanol R, prepared with the necessary precautions, and mix
In a well-filled container.
vigorously for at least 5 min. A dd 5 m L o f distilled water R
and mix vigorously for about 30 s. Centrifuge for 15 min at IM P U R IT IE S
1500 g. Use the upper layer. -COaH
h 3c '
Reference solution Dissolve 50 m g of methyl
12-hydroxystearate CRS and 50 m g of methyl stearate CRS in
10.0 m L o f 1,1-dimethylethyl methyl ether R l. A. 12-oxostearic ad d .
Column: PhEur
— size. I = 30 m; 0 = 0.25 mm ;
★ ★
0.25 pm). Polyoxyl Castor Oil ★ ★
Carrier gas helium for chromatography R. * * * * *
(Macrogolgfycerol Ricmoleate,
Flow rate 0.9 mL/min. Ph Eur monograph 1082)
Split ratio 1:100.
Temperature: A c tio n a n d u se
Excipient.
Time Temperature
PhEur______________________________________________________ ;---------
(min) CC)
Column 0-55 215 D E F IN IT IO N
Injection port 250 Contains mainly ricinoleyi glycerol ethoxylated with
30-50 molecules o f ethylene oxide (nom inal value), with
Detector 250 small amounts of macrogol ricinoleate and o f the
corresponding free glycols. It results from the reaction of
Detection Flam e ionisation. castor oil with ethylene oxide.
Injection 1 pL. CHARACTERS
Calculate the fraction o f each fatty-acid using the following A p p e a ra n c e
expression: Clear, yellow viscous liquid o r semi-solid.
S o lu b ility
A x,t,e/ ^ 2 x 100 p e r cent m /m Freely soluble in water, very soluble in m ethylene chloride,
freely soluble in ethanol (96 per cent).
AXyStC — corrected peak area o f the fatty a d d in the test R e la tiv e d e n sity
solution: A bout 1.05.
Viscosity 500 mPa-s to 800 mPa-s at 25 °C.
A x ,t,e — X Rc
Rc = rdative correction factor for the peak due to A. Iodine value (see Tests).
methyl 12-hydroxystearate:
B. Saponification value (see Tests).
m i ,, x Aa,r C. Thin-layer chromatography (2.2.27).

Re = Test solution T o 1 g o f the substance to be examined add
A i , r X 7712,r
100 m i. o f a 100 g/L solution o f potassium hydroxide R and
boil under a reflux condenser for 30 min. Allow to cool. R e sid u a l eth y len e oxide a n d d io x an (2.4.25)
Acidify the solution with 20 m L of hydrochloric acid R. Shake M aximum 1 ppm of residual ethylene oxide and 10 ppm of
the mixture with 50 m L of ether R and allow to stand until residual dioxan.
separation of the layers is obtained. Transfer the clear u p p er H eav y m e ta ls (2.4.8)
layer to a suitable tube, add 5 g of anhydrous sodium sulfate R, M aximum 10 ppm .
close the tube and allow to stand for 30 min. Filter and
12 m L o f solution S, filtered if necessary, complies with
evaporate the filtrate to dryness on a water-bath. Dissolve
test A. Prepare die reference solution using lead standard
50 m g of the residue in 25 m L of ether R.
Reference solution Dissolve 50 m g o f ricindeic acid R in
W a te r (2.5.12)
solvent.
Plate TLC octadecylsUyl silica gel plate R. T o ta l a sh (2.4.16)
Mobile phase methylene chloride R, glacial acetic acid R,
acetone R (10:40:50 VIVIV). STORA GE
Application 2 pL. Protected from light.
Development Over a path of 8 cm. L A B E LL IN G
Drying In a current of cold air. The label states:
Detection Spray w ith an 80 g/L solution of phosphomoJybdic — the am ount o f ethylene oxide reacted with castor oil
add R in 2-propanol R and heat at 120 °C for 1-2 min. (nominal value),
— where applicable, that the substance is suitable for use in
Restdts T he principal spot in the chromatogram obtained with
the manufacture of parenteral preparations.
the test solution is similar in position and colour to the
principal spot in the chromatogram obtained with the ______________________________________________________________ PhEur
reference solution.
D . Place about 2 g o f the substance to be examined in a test-
tube and add 0.2 m L of sulfuric add R. Close the tube using
a stopper fitted with a glass tube bent twice at right angles.
H eat the tube until white fumes appear. Collect the fumes in
Hydrogenated Polyoxyl Castor Oil *****
*. *
1 m L of mercuric chloride solution R. A white precipitate is (Macrogolgfycerol Hydroxystearate, *
formed and the fumes turn a filter paper impregnated with Ph Eur monograph 1083)
alkaline potassium tetraiodomercurate solution R black. PhEir__________________________________________________________
TESTS D E F IN IT IO N
S o lu tio n S Contains mainly trihydroxystearyl glycerol ethoxylated with
Dissolve 5.0 g in carbon dioxide-free water R and dilute to 7 to 60 molecules of ethylene oxide (nominal value), with
50 m L with the same solvent. small amounts o f macrogol hydroxystearate and o f the
A p p e a ra n c e o f so lu tio n corresponding free glycols. It results from the reaction of
Solution S is not m ore opalescent than reference hydrogenated castor oil with ethylene oxide.
suspension HI ('2.2.1) and not more intensely coloured th an
CHA RACTERS
reference solution BY5 (2.2.2, Method II). If intended for use
A p p e a ra n c e
in the manufacture o f parenteral preparations, solution S is
— if less than 10 units o f ethylene oxide p er molecule:
not m ore intensely coloured than reference solution BY6
yellowish, turbid, viscous liquid;
(2.2.2, Method II).
— if m ore than 20 units of ethylene oxide p er molecule:
A lk alin ity white or yellowish semi-liquid o r pasty mass.
Dissolve 2.0 g in a hot mixture of 10 m L o f water R and
S o lu b ility
10 m L o f ethanol (96 per cent) R. Add 0.1 m L of bromothymol
— if less than 10 units o f ethylene oxide per molecule:
blue sohaton R l. N o t more than 0.5 m L of 0.1 M hydrochloric
practically insoluble in water, soluble in acetone,
add is required to change the colour of the indicator to
dispersible in ethanol (96 p er cent);
yellow.
— if m ore than 20 units of ethylene oxide per molecule:
A cid v alu e (2.5.1) freely soluble in water, in acetone and in ethanol
M axim um 2.0, determined on 5.0 g. (96 per cent), practically insoluble in light petroleum.
H y d ro x y l v alu e (2.5.3, Method A) ID E N T IF IC A T IO N
See T able 1082.-1.
A. Iodine value (see Tests).
Io d in e v alu e (2.5.4) B. Saponification value (see Tests).
25 to 35.
S a p o n ific a tio n v a lu e (2.5.6)
Test sducion T o 1 g o f the substance to be examined, add
See T able 1082.-1.
100 m L o f a 100 g/L solution of potassium hydroxide R and
boil under a reflux condenser for 30 min. Allow to cool.
Table 1082.-1 Acidify the solution with 20 m L o f hydrochloric add R. Shake
Ethylene oxide units per Hydroxyl value Saponification value the mixture with 50 m L of ether R and allow to stand until
nxdcculg (nominal valn»|_________________________________ separation o f the layers is obtained. Transfer the clear upper
30 - 35 65 - 82 60 - 75 layer to a suitable tube, add 5 g o f anhydrous sodium sulfate R,
50 48 - 68 38 - 52
• close the tube and allow to stand for 30 min. Filter and
1-432 Castor Oil 2016
evaporate the filtrate to dryness on a water-bath. Dissolve R e sid u a l e th y le n e o x id e a n d d io x a n (2.4.25)

50 m g o f the residue in 25 m L of ether R. Maximum 1 ppm o f residual ethylene oxide and 10 ppm of
Reference solution Dissolve 50 m g o f 12-kydroxystearic acid R in residual dioxan.
methylene chloride R and dilute to 25 m l. with the same H ea v y m e ta ls (2.4.8)
solvent. Substances soluble in acetone!'anhydrous ethanol M axim um
Plate TLC octadecylsHyl silica gel plate R. 10 ppm.
Mobile phase methylene chloride R, glacial acetic acid R, 12 m L o f solution S complies with test B. Prepare the
acetone R (10:40:50 V/V/V). reference solution using lead standard solution (1 ppm Pb)
Application 2 pL. obtained by diluting lead standard solution (100 ppm Pb) R
with a mixture o f equal volumes of acetone R and anhydrous
ethanol R
Drying In a current o f cold air.
Substances soluble in water M axim um 10 ppm .
Detection Spray with a 80 g/L solution o f phosphomolybdic
12 m L o f solution S complies with limit test A. Prepare the
acid R in 2-propanol R and heat at 120 °C for about 1-2 min.
Results T he principal spot in the chrom atogram obtained with
W a te r (2.5.12)
the test solution is similar in position and colour to the
principal spot in the chrom atogram obtained with the
reference solution. T o ta l a s h (2.4.16)
D . Place about 2 g in a test-tube and add 0.2 m L of sulfuric
add R. Close the tube using a stopper fitted w ith a glass tube L A B E L L IN G
b ent twice at right angles. H eat the tube until white fumes T h e label states the num ber o f ethylene oxide units per
appear. Collect the fumes in 1 m L o f mercuric chloride molecule (nominal value).
solution R. A white precipitate is formed and the fumes turn a ___________________________________________ !___________________PhEur
filter paper im pregnated with alkaline potassium
tetraiodomercurate solution R black.
TESTS
S o lu tio n S
Dissolve 5.0 g of macrogolglycerol hydroxystearate with less
Refined Castor Oil ***”;
* *
than 40 units o f ethylene oxide per molecule in a mixture of (Ph Eur. monograph 2367) **
50 volumes o f acetone R and 50 volumes o f anhydrous
PhEur_______________________________________________________________
ethanol R and dilute to 50 m L with the same mixture of
solvents. D E F IN IT IO N
Dissolve 5.0 g of macrogolglycerol hydroxystearate with Fatty oil obtained from the seeds of Ridnus communis L.
40 units or m ore o f ethylene oxide p er molecule in carbon by cold expression. It is then refined. A suitable antioxidant
dioxide-free water R and dilute to 50 m L with the same may be added.
solvent. P R O D U C T IO N
A p p e a ra n c e o f so lu tio n D uring the expression step, the temperature of the oil m ust
Solution S is not m ore opalescent than reference n o t exceed 50 °C.
suspension HI (2.2.1) and n o t more intensely coloured than CHARACTERS
reference solution BY6 (2.2.2, Method II). A p p e a ra n c e
A lk alin ity Clear, almost colourless or slightly yellow, viscous,
T o 2 m L of solution S add 0.5 m l. o f bromothymol blue hygroscopic liquid.
solution R l. T h e solution is n o t blue. S o lu b ility
A cid v a lu e (2.5.1) Slightly soluble in light petroleum , m isdble with ethanol
M axim um 2.0, determined on 5.0 g. (96 per cent) and with glacial acetic add.
H y d ro x y l v a lu e (2.5.3, Method A) R elativ e d e n sity
See T able 1083.-1. About 0.958.
Io d in e v a lu e (2.5.4) Refractive index A bout 1.479.
M axim um 5.0. Viscosity A bout 1000 mPa-s.
S a p o n ific a tio n v a lu e (2.5.6) ID E N T IF IC A T IO N
See T able 1083.-1.
Table 1083.-1
A. A mixture of 2 m L o f the substance to be examined and
Ethylene oxide units per Hydroxyl value Saponification value 8 m L of ethanol (96 per cent) R is clear (2.2.1).
molecule (nominal value)
7 115 - 135 125 - 140 B. Specific absorbance (see Tests).
7 0 -9 0 7 0 -9 0
C. Composition of fatty ad d s (see Tests).
25
40 6 0 -8 0 4 5 -6 9 TESTS
A p p e a ra n c e
60 4 5 -6 7 40 -5 1
T h e substance to be examined is clear (2.2.1) and n o t more
intensdy coloured (2.2.2, Method II) than 20 m L of a
mixture o f 0.25 m L of blue primary solution, 0.25 m L o f red
primary solution, 0.8 m l. o f yellow primary solution, and
18.7 m L of a solution prepared by diluting 4.0 m L of Calculate the percentage content o f each fatty a d d by the
hydrochloric add R1 to 100.0 m L with water R. normalisation procedure.
O p tic a l ro ta tio n (2.2.7) C o rre a the area of the peak due to methyl ridnoleate, by
+ 3.5° to + 6.0°. multiplying by a factor R calculated using the following
expression:
Greater than 0.7 and mayi'mum 1.5, determined at the
m\ x A2
absorption maximum at 270 nm.
A\ x m 2
T o 1.00 g add ethand (96 per cent) R and dilute to 100.0 m L
m\ = mass o f methyl ridnoleate in the reference solution;
A d d v alu e (2.5.1) m2 = mass o f methyl stearate in the reference solution;
M aximum 0.8. A 1 = area o f the peak due to methyl ridnoleate in the
Dissolve 5.00 g in 25 m L of the prescribed mixture o f chromatogram obtained with the reference solution;
solvents. A 2 = area o f the peak due to methyl stearate in the
H ydroxyl v alu e (2.5.3, Method A) chromatogram obtained with the reference solution.
M inim um 160. Composition of the fatty-acid fraction of the oU:
P e ro x id e v alu e (2.5.5, Method A) — palmitic add: maximum 2.0 p e r cent;
M aximum 5.0. — stearic add: maximum 2.5 per cent;
— oleic add: 2.5 per cent to 6.0 p er cent;
U n sap o n ifiab le . m a tte r (2.5.7)
— linoleic add: 2.5 per cent to 7.0 per cent;
M aximum 0.8 per cent, determined on 5.0 g. — linolenic add. maximum 1.0 p er cent;
O il o b ta in e d b y e x tra c tio n a n d a d u lte ra tio n — eicosenoic add. maximum 1.0 p er cent;
In a ground-glass-stoppered tube about 125 m m long and — ridnoleic acid. 85.0 per cent to 92.0 per cent;
18 mm in internal diameter, thoroughly mix 3 m L o f the — any other fatty add: maximum 1.0 per cent.
substance to be examined with 3 m L o f carbon disulfide R.
W a te r (2.5.32)
Shake for 3 min with 1 m L of sulfuric add R. T h e mixture is
M aximum 0.3 per cent, or maximum 0.2 per cent if intended
less intensely coloured than a freshly prepared mixture of
for use in the manufacture of parenteral preparations,
3.2 m L o f ferric chloride solution R l, 2.3 m L o f water R and
determined on 1.00 g.
0.5 m L o f dilute ammonia R l.
STORAGE
C o m p o sitio n o f fa tty a d d s
In an airtight, well-filled container, protected from light.
Gas chromatography (2.4.22) with the following
modifications. L A B E L L IN G
Use the mixture o f calibrating substances in T able 2.4.22.-3. T he la b d states, where applicable, that the substance is
Test solution Introduce 75 mg o f the substance to be suitable for use in the manufacture of parenteral
examined into a 10 m L centrifuge tube with a screw cap. preparations.
Dissolve in 2 m L o f 1,1-dimeihylethyl methyl ether R l with -----------------------------------------------------------------------------PhEur
shaking and heat gently (50-60 °C). T o the still-warm
solution, add 1 m L o f a 12 g/L solution of sodium R in
anhydrous methanol R, prepared with the necessary
precautions, and shake vigorously for at least 5 min. Virgin Castor Oil *****
Add 5 m L o f distilled water R and shake vigorously for about
30 s. Centrifuge for 15 min at 1500 g. Use the u p p er layer. C astor Oil ****
Reference solution Dissolve 50 mg of methyl ridnoleate CRS and (Ph. Eur. monograph 0051)
50 mg o f methyl stearate CRS in 10.0 m L o f 1,1-dimethylethyl
methyl ether R l. A c tio n a n d u se
Stim ulant laxative; emollient.
Column.:
— material', fused silica; P re p a r a tio n
— sizer. I = 30 m , 0 = 0.25 mm; Zinc and Castor Oil Ointment
PhEur__________________________________________________
0.25 jim).
Carrier gas helium for chromatography R. D E F IN IT IO N
Fatty oil obtained by cold expression from the seeds of
Flow rate 0.9 m l An in.
Ridnus communis L. A suitable antioxidant may be added.
Split ratio 1:100.
P R O D U C T IO N
Temperature:
D uring the expression step, the tem perature of the oil m ust
not exceed 50 °C.
Time Temperature
(min) CC) CHARACTERS
Column 0 -5 5 215 A p p e a ra n c e
Injection port 250
Clear at 40 °C, slightly yellow, viscous, hygroscopic liquid.
S o lu b ility
Detector 250
Slightly soluble in light petroleum, m isdble with ethanol
(96 per cent) and with gladal acetic ad d .
Detection Flam e ionisation. R elativ e d e n sity
Injection 1 jiL . About 0.958.
1-434 Cefaclor 2016
Refractive index About 1.479. Calculate the percentage content o f each fatty a d d by the
First identification B, C. C orrect the area o f the peak due to methyl ridnoleate, by
multiplying by a factor R calculated using the following
Second identification A , B.
expression:
A. A mixture o f 2 m l. o f the substance to be examined and
8 m l. o f ethanol (96 per cent) R is d e a r (2.2.1). m i x A?
B. Specific absorbance (see Tests). A \ x m?
C. Com position of fatty a d d s (see Tests).
mi = mass o f methyl ridnoleate in the reference solution;
TESTS
m2 = mass o f methyl stearate in the reference solution;
O p tic a l ro ta tio n (2.2 . 7)
A i = area o f the peak due to methyl ridnoleate in the
+ 3.5° to + 6.0°.
chrom atogram obtained with the reference solution;
S p ecific a b so rb a n c e (2.2.25) A 2 = area o f the peak due to methyl stearate in the
M aximum 0.7, determ ined at the absorption m aximum at chrom atogram obtained with th e reference solution.
270 nm .
Composition of the fatty-acid fraction of the oik
T o 1.00 g add ethanol (96 per cent) R and dihite to 100.0 m l- — palmitic add: maximum 2.0 per cent;
with the same solvent. — stearic add: maxim um 2.5 per cent;
A cid v alu e (2.5.1) — oleic acid : 2.5 p er cent to 6.0 per cent;
M axim um 1.5. — linoleic add : 2.5 per cent to 7.0 per cent;
Dissolve 5.00 g in 25 m L o f the prescribed mixture of — Itnolenic acid : maximum 1.0 per cent;
solvents. — eicosenoic a d d : maximum 1.0 per cent;
— ridnoleic a d d : 85.0 per cent to 92.0 p er cent;
H y d ro x y l v a lu e (2.5.3, Method A)
— any otherfatty add: maximum 1.0 per cent.
M inim um 160.
W a te r (2.5.32)
P e ro x id e v alu e (2.5.5, Method A)
M axim um 10.0.
U n sa p o n ifia b le m a t t e r (2.5.7) STORAGE
M aximum 0.8 per cent, determ ined on 5.0 g. In an airtight, well-filled container, protected from light.
C o m p o sitio n o f fa tty a c id s _______________________________________________________________ PhEur
Gas chrom atography (2.4.22) with the following

modifications.
Use the mixture of calibrating substances in T able 2.4.22.-3.
Test solution Introduce 75 m g of the substance to be Cefaclor
examined into a 10 m L centrifuge tube with a screw cap.
(Ph. Eitr. monograph 0986) *
Dissolve in 2 m L o f 1,1-dimethylethyl methyl ether R l with
shaking and heat gendy (50-60 °C). A dd, while still warm, C02h
1 m L o f a 12 g/L solution o f sodium R in anhydrous
methanol R, prepared w ith the necessary precautions, and mix
vigorously for at least 5 min. Add 5 m L o f distilled water R
and mix vigorously for about 30 s. Centrifuge for 15 min at
1500 g. Use the upper layer.
Reference solution Dissolve 50 mg o f methyl ridnoleate CRS and
50 m g o f methyl stearate CRS in 10.0 m L o f 1,1-dimethylethyl C 15H 14C1N304S320 385.8 70356-03-5
methyl ether R l.
Column: Cephalosporin antibacterial.
— size: I = 30 m, 0 = 0.25 mm; P re p a r a tio n s
— stationary phase, macrogol 20 000 R (film thickness Cefaclor Capsules
0.25 ¿im). Cefaclor Oral Suspension
Carrier gas helium for chromatography R. Prolonged-release Cefaclor Tablets
Flow rate 0.9 m U m in. PhEur__________________________________________________________
Split ratio 1:100.
D E F IN IT IO N
Temperature:
(6R,7R)-7- [ [(2i?)-2-Amino-2-phenylacetyl] amino] -3-chloro-
8-oxo-5-thia-1-azabicydo [4.2.0]oct-2-ene-2-carboxylic a d d
lim e Temperature
monohydrate.
(min) CC)
Column 0 -5 5 215 Semi-synthetic product derived from a fermentation product.
Injection port 250

C o n te n t
96.0 per cent to 102.0 per cent o f C 15H 14C 1N 304 S
Detector 250 (anhydrous substance).
CHARACTERS
Detection Flam e ionisation. A p p e a ra n c e
Injection 1 (iL. W hite or slightly yellow powder.
2016 Cefaclor 1-435
S olubility — disregard limit. 0.1 times the area of the principal peak in
Slightly soluble in water, practically insoluble in methanol the chromatogram obtained with reference solution (b)
and in methylene chloride. (0.1 per cent).
ID E N T IF IC A T IO N H eav y m e ta ls (2.4.8)
Infrared absorption spectrophotometry (2.2.24). M aximum 30 ppm.
Comparison cefaclor CRS. 1.0 g complies with test C. Prepare the reference solution
TESTS
p H (2.2.3) W a te r (2.5.12)
3.0 to 4.5. 3.0 per cent to 6.5 per cent, determined on 0.200 g.
Suspend 0.250 g in carbon dioxide-free water R and dilute to ASSAY
10 m L with the same solvent. Liquid chromatography (2.2.29).
Specific o p tic a l ro ta tio n (2.2.7) Test solution Dissolve 15.0 mg of the substance to be
+ 101 t o + 111 (anhydrous substance). examined in the mobile phase and dilute to 50.0 m L w ith
the mobile phase.
Dissolve 0.250 g in a 10 g/L solution of hydrochloric acid R
and dilute to 25.0 m L with the same solution. Reference solution (a) Dissolve 15.0 mg o f cefaclor CRS in the
mobile phase and dilute to 50.0 m l. with the mobile phase.
Liquid chromatography (2.2.29). Reference solution (b) Dissolve 3.0 mg of cefaclor CRS and
3.0 mg o f deka-3-cefaclor CRS (impurity D) in the mobile
phase and dilute to 10.0 m L with the mobile phase.
examined in 10.0 m L o f a 2.7 g/L solution of sodium
dihydrogen phosphate R adjusted to p H 2.5 with phosphoric Column:
add R. — sizer. I = 0.25 m, 0 = 4.6 mm;
— stationary phase: octadecylstlyl silica gel for chromatography R
Reference solution (a) Dissolve 2.5 m g of cefaclor CRS and
(5 ^m).
5.0 mg of delta-3-cefaclor CRS (impurity D ) in 100.0 m L o f a
2.7 g/L solution of sodium dihydrogen phosphate R adjusted to Mobile phase Add 220 m L of methanol R to a mixture of
pH 2.5 with phosphoric add R. 780 m L of water R, 10 m L of triethylamine R and 1 g of
sodium pentanesulfonate R, then adjust to p H 2.5 with
phosphoric add R.
100.0 m L with a 2.7 g/L solution o f sodium dihydrogen
phosphate R adjusted to p H 2.5 with phosphoric add R. Flow rate 1.5 mlVmin.
Column: Detection Spectrophotometer at 265 nm.
— size: I = 0.25 m, 0 = 4.6 mm; Injection 20 (iL.
— stationary phase: end-capped octadecylsüyl silica gel for System suitability".
chromatography R (5 (im). — resolution: minimum 2.5 between the peaks due to cefaclor
Mobile phase: and impurity D in the chromatogram obtained with
— mobile phase A: 7.8 g/L solution o f sodium dihydrogen reference solution (b); if necessary, adjust the
phosphate R adjusted to p H 4.0 with phosphoric add R] concentration of methanol in the mobile phase;
— mobile phase B: mix 450 m L of acetonitrile R with 550 m L — symmetry factor, maximum 1.5 for the peak due to cefaclor
of mobile phase A; in the chromatogram obtained with reference, solution (b);
1.0 per cent after 6 injections of reference solution (a).
(min) (per cent V/V) (per cent V/V) IM P U R IT IE S
0 -3 0 9 5 -»75 5 -»25
3 0 -4 5 75 -» 0 25 -» 100
4 5 -5 5 0 100
Flow rate 1.0 mL/min. A. (2i?)-2-amino-2-phenylacetic a d d (phenylglycine),

Injection 20 nL. CO,H
— resolution: minimum 2 between the peaks due to cefaclor
and im purity D ; if necessary, adjust the acetonitrile
content in the mobile phase; H H
cefaclor; if necessary, adjust the acetonitrile content in the B. (6i?,7i?)-7-amino-3-chloro-8-oxo-5-thia-l-
mobile phase. azabicyclo[4.2.0]oct-2-ene-2-carboxylic ad d ,
Limits:
— any impurity, for each impurity, n o t more than 0.5 times
obtained w ith reference solution (b) (0.5 per cent);
— total: n o t m ore than twice the area of the principal peak in
(2 p e r cent);
1-436 Cefadroxil Monohydrate 2016
Cefadroxil Monohydrate *****

★ ★
c o 2h
H NH2
C. (6i?,7i?)-7-[[(25)-2-ainino-2-phenylacetyl]amino]-3- H20
chloro-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic
H H
acid,
c I6h 17n 3o 5s ä o 381.4 66592-87-8

and epimer at C* A c tio n a n d u se
Cephalosporin antibacterial.
Cefadroxil Capsules
D. (2R}6R,7R)- and (2S,6R,7R)-7-[[(2R)-2-ammo-2- Cefadroxil Oral Suspension
phenylacetyl] amino] -3-chloro-8-oxo-5-thia-
l-azabicydo[4.2.0]oct-3-ene-2-carboxylic acid PhEur_____________________
(delta-3-cefaclor)j
D E F IN IT IO N
(6R, 7R)-7-[ [(2J?)-2-Amino-2-
(4-hydroxyphenyl)acetyl]amino]-3-methyI-8-oxo-5-thia-l-
azabicyclo [4.2.0] oct-2-ene-2-carboxylie a d d monohydrate.
C o n te n t
CHARACTERS
E. 2 - [ [(2i?)-2-amino-2-phenylacetyl] amino] -2-(5-chloro-4-
A p p e a ra n c e
oxo-3}4-dihydro-2if-1,3-thiazin-2-yl) acetic acid}
N OH S o lu b ility
Slightly soluble in water} very slightly soluble in ethanol
(96 per cent).
F. 3-phenylpyrazin-2-olj Comparison cefadroxU CRS.
TESTS
p H (2.2.3)
4.0 to 6.0.
and epimer at C* Suspend 1.0 g in carbon dioxide-free water R and dilute to
G. (2R,6R,7K)~ and (2S,6R}7R)-7-[[(2K)-2-axamo-2- Dissolve 0.500 g in water R and dilute to 50.0 m L with the
phenylacetyl] amino] -3-methylene-8-oxo-5-thia-1- same solvent.
azabicyclo[4.2.0] octane-2-carboxylic acid (isocefalexine).
L iquid chromatography (2.2.29).
mobile phase A.
Reference solution (a) Dissolve 10.0 m g o f e h x -
(4-hydroxyphenyl)glycine CRS (impurity A) in mobile phase A
and dilute to 10.0 m L with mobile phase A.
Reference solution (b) Dissolve 10.0 m g o f 7-
H . (6J?}7.R)-7-[[(2i?)-2-[[(2.R)-2-amino-2-
aminodesaceioxycephahsporanic add CRS (impurity B) in
phenylacetyl] amino] -2-phenylacetyl] amino]-3-chloro-8-oxo-5-
phosphate buffer solution pH 7.0 R5 and dilute to 10.0 m L
thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic a d d
with the same buffer solution.
(N-phenylglycyl cefaclor).
Reference solution (c) Dilute 1.0 m L o f reference solution (a)
PhEur
and 1.0 m L o f reference solution (b) to 100.0 m L with
mobile phase A.
Reference solution (d) Dissolve 10 m g o f dimethylformamide R
and 10 m g o f dimethylacetamide R in mobile phase A and
2016 Cefadroxil Monohydrate 1-437
dilute to 10.0 m L with mobile phase A. Dilute 1.0 m L of Column:

this solution to 100.0 m L with mobile phase A. — size. I = 0.25 m , 0 = 4.6 mm,
Reference solution (e) Dilute 1.0 m L o f reference solution (c) — stationary phase. octadecylsOyl silica gel for chromatography R
to 25.0 m L with mobile phase A (5 nm).
Column: Mobile phase acetomtrüe R, a 2.72 g/L solution o f potassium
— size: I = 0.10 m , 0 = 4.6 mm, dihydrogen phosphate R (4:96 VfV).
— stationary phase: spherical octadecylsüyl silica gel for Flow rate 1 mL/min.
chromatography R (5 nm). Detection Spectrophotometer at 254 nm.
Mobile phase: Injection 20 |iL.
— mobile phase A: phosphate buffer solution pH 5.0 R,
— mobile phase B: methanol R2,
cefadroxil and to amoxicillin.
Time Mobile phase A Mobile phase B Calculate the percentage content o f cefadroxil.
(min)____________ (per cent V/V)________ (percent V/V)
0 -1 98 2 STO RA G E
1 -20 98 -» 70 2 30
IM P U R IT IE S

solutions (c), (d) and (e).
Relative retention W ith reference to cefadroxil (retention
A. (2£)-2-ammo-2-(4-hydroxyphenyl)acetic add,
time = about 6 min): dimethylformamide = about 0.4;
dimethylacetamide = about 0.75.
COjH
System suitability:
— resolution: minimum 5.0 between the peaks due to ° W V
impurities A and B in the chromatogram obtained with
reference solution (c), H2N" HHH V
— signal-to-notse ratio: minimum 10 for the 2nd peak in the
chromatogram obtained with reference solution (e). B. (6R,1R)-1 -amino-3-methyl-8-oxo-5-thia-1-
Limits: azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid (7-ADCA),
— impurity A: not more than the area of the 1st peak in the
chromatogram obtained with reference solution (c) CO,H
(1.0 per cent),
— arty other impurity: for each impurity, not more than the
and epimer ?t C*
area o f the 2nd peak in the chromatogram obtained with
reference solution (c) (1.0 per cent),
— total: not more than 3 times the area o f the 2nd peak in HO
(3.0 per cent), C. (2R,5RS)-2-[(!?)-[[(2Æ)-2-amino-2-
— disregard limit. 0.05 times the area of the 2nd peak in the (4-hydroxyphenyl) acetyl] amino] carboxymethyl]-5-methyl-5,6-
chromatogram obtained with reference solution (c) dihydro-2/i-1,3-thiazine-4-carboxylic ad d ,
(0.05 per cent); disregard the peaks due to
dimethylformamide and dimethylacetamide. COjH
N ,iV -D im eth y lan ilin e (2.4.26> Method B)
M axim um 20 ppm . H NH2
W a te r (2.5.12)
Q ’t t y
D . (6Æ,7£)-7-[[(2S)-2-amino-2-
A SSAY (4-hy droxyphenyl) acetyl] amino] -3-methyl-8-oxo-5-thia-1-
Liquid chromatography (2.2.29). azabicyclo [4.2.0] oct-2-ene-2-carboxylic a d d (L-cefadroxil),
examined in the mobile phase and dilute to 100.0 m l. with H ,n h 2
the mobile phase.
Reference solution (a) Dissolve 50.0 mg of cefadroxil CRS in and enantiomer
r y i h o
phase. U
X J HH
Reference solution (b) Dissolve 5 mg of cefadroxil CRS and
50 m g o f amoxicillin trihydrate CRS in the mobile phase and .E. (6ÆS)-3-(aminomethylene)-6-
dilute to 100 m L with the mobile phase. (4-hydroxyphenyl)piperazine-2,5-dione,
1-438 Cefalexin Monohydrate 2016
Comparison cefalexin monohydrate CRS.
TESTS
p H (2.2.3)
4.0 to 5.5.
F. (6R,7R)-7- [ [(2R)-2- [ [(2.&S)-2-ammo-2- Dissolve 50 mg in carbon dioxide-free water R and dilute to
(4-hydroxyphenyl) acetyl] amino] -2- 10 m L with the same solvent.
(4-hydroxyphenyI) acetyi] amino]-3-methyl-8-oxo-5-thia-1- S p ecific o p tic a l ro ta tio n (2.2.7)
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, + 149 to + 158 (anhydrous substance).
HO
Dissolve 0.125 g in phthalate buffer solution pH 4.4 R and
G . 3-hydroxy-4-methylthiophen-2 (5/f)-one,
CO,H m obile phase A.
Reference solution (a) Dissolve 10.0 m g o f D-phenylglycine R in
mobile phase A and dilute to 10.0 m L with mobile phase A.
Reference solution (b) Dissolve 10.0 m g o f
0 7-armnodesacetoocycephalosporanic acid CRS in phosphate buffer
solution pH 7.0 R5 and dilute to 10.0 m L with mobile
H . ( 6Æ, 7i?) -7 - [(2,2-dimethylpropanoyl) amino] -3-methyl-8- phase A.
oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid Reference solution (c) Dilute 1.0 m L of reference solution (a)
(7-AD CA pival amide). and 1.0 m L o f reference solution (b) to 100.0 m L with
__________ ^________________________________________________ PhEur mobile phase A.
Reference solution (d) Dissolve 10 m g o f dimethylformamide R
and 10 m g of dimethylacetamide R in mobile phase A and
dilute to 10.0 m L with mobile phase A. D ilute 1.0 m L of
Cefalexin Monohydrate ***** this solution to 100.0 m L with mobile phase A.
★, + Reference solution (e) Dilute 1.0 m L o f reference solution (c)
(Ph. Eur. monograph 0708) * to 20.0 m L with mobile phase A.
CO,H Reference solution (f) Dissolve 10 m g o f cefotaxime sodium CRS
in mobile phase A and dilute to 10.0 m L w ith mobile
phase A. T o 1.0 m L of this solution add 1.0 m L o f the test
solution and dilute to 100 m L with mobile phase A.
Column:
— size: I = 0.10 m , 0 = 4.6 mm ;
— stationary phase, spherical octadecylsüyl süica gel for
ClöH 17N 30 4S,H20 365.4 23325-78-2 chromatography R (5 pm).
A c tio n a n d u se Mobile phase:
— mobile phase A: phosphate buffer solution pH 5.0 R;
C ephalosporin antibacterial.
— mobile phase B: methanol R2~,
P r e p a r a tio n s
Cefalexin Capsules
Time Mobile phase A Mobile phaae B
Cefalexin Oral Suspension
Cefalexin Tablets 0 -1 98 2
PhEur_______________________________________________ 1 -20 98->70 2->30
D E F IN IT IO N
(6i?,7i?)-7-[[(2iQ-2-Amino-2-phenylacetyl]amino]-3-methyl- Flow rate 1.5 mL/min.
8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic a d d
m onohydrate.
Iiyection 20 (iL o f the test solution and reference
solutions (c), (d), (e) and (f).
C o n te n t
System suitability:
CHA RACTERS impurities A and B in the chrom atogram obtained with
A p p e a ra n c e reference solution (c) and minimum 1.5 betw een the
W hite or alm ost white, crystalline powder. peaks due to cefalexin and cefotaxime in the
S o lu b ility chrom atogram obtained with reference solution (f).
Sparingly soluble in water, practically insoluble in ethanol
(96 p er cent).
2016 Cefalotin Sodium 1-439
Limits:
— impurity B: n o t more than the area o f the 2nd peak in the
(1.0 per cent);
— any other impurity, not more than the area o f the 1st peak
(1.0 per cent);
— total: not m ore than 3 times the area o f the 1st peak in the C. (6£,7i?)-7-[[(2J?)-2-[[(2£)-2-amino-2-
chrom atogram obtained with reference solution (c) phenylacetyl] amino] -2-phenjdacetyl] amino]-3-methyl-8-oxo-
(3.0 per cent); 5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic ad d ,
— disregard limit, the area of the 2nd peak in the
chrom atogram obtained with reference solution (e)
(0.05 per cent); disregard any peaks due to
dimethylformamide or dimethylacetamide. h3c oh
N ,N -D im e th y la n ilin e (2.4.26, MethodB) D . 3-hydroxy-4-methylthiophen-2 (5 /i) -one,

M axim um 20 ppm .
COjH
COjH
W a te r (2.5.12)
O
4.0 per cent to 8.0 per cent, determined on 0.300 g. H3C ch 3
h3c
M axim um 0.2 p e r cent, determined on 1.0 g. Il H H
O
A SSAY
Liquid chromatography (2.2.29). E . (6i?,7i?)-7-[(2,2-dimethylpropanoyl)amino]-3-methyl-8-
oxo-5-thia-l-azabicyclo[4.2.0] oct-2-ene-2-carboxylic a d d
(7-ADCA pivalamide),
solvent. H COjH
Reference solution (d). Dissolve 50.0 mg of cefalexin
monokydrate CRS in water R and dilute to 100.0 m L with the and epimer at C*
same solvent.
Reference solution (b) Dissolve 10 mg of cefradine CRS in
20 m L o f reference solution (a) and dilute to 100 m L with
water R. F . (2RS,6R,7R)-7- [ [(2i?)-2-amino-2-phenylacetyl] amino]-3-
Column: methyi-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-3-ene-2-carboxylic
— sizer. I = 0.25 m , 0 = 4.6 mm; a d d (delta-2-cefalexin).
— stationary phase: octadecylstlyl silica gd for chromatography R « y fu r
(5 pm).
Mobile phase methanol R, acewnitrile R, 13.6 g/L solution of
potassium dihydrogen phosphate R, water R
(2:5:10:83 VIV/V/V).
Cefalotin Sodium ★
★ ★
*
Flow rate 1.5 mL/xnin. ★, .*
Detection Spectrophotom eter at 254 nm. (Ph. Ever, monograph 0987) *★ *
Injection 20 pL.
C02Na O
cefalexin an d cefradine.
Calculate the percentage content o f cefalexin monohydrate.
STORA GE
Protected from light. Ci6H15N2Na06S2 418.4 58-71-9
IM P U R IT IE S
Action and use
H NHj Cephalosporin antibacterial.
COjH
PhEtr.
O * D E F IN IT IO N
A. (2i?)-2-amino-2-phenylacetic add (D-phenylglycine), Sodium (6R,lR)-3- [(acetyloxy)methyl]-8-oxo-7- [(thiophen-2-
ylacetyl) amino] -5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-
ayn
carboxylate.
V h' V ^ Semi-synthetic product derived from a fermentation p ro d u ct
C o n te n t
H H 96.0 per cent to 102.0 per cent (anhydrous substance).
B. (6i?,7J?)-7-amino-3-methyl-8-oxo-5-thia-1- CHARACTERS
azabicydo[4.2.0]oct-2-ene-2-carboxylic A p p e a ra n c e
add(7-aminodesacetoxycephalosporanic ad d , 7-ADCA), W hite or almost white powder.
1-440 Cefalotin Sodium 2016
S o lu b ility Detection Spectrophotom eter at 220 nm.

Freely soluble in water, slightly soluble in anhydrous ethanol. Injection 20 |iL o f test solution (a) and reference
ID E N T IF IC A T IO N solutions (b), (c) and (d).
A. Infrared absorption spectrophotom etry (2.2.24). Relative retention W ith reference to cefalotin (retention
Comparison cefalotin sodium CRS. time = about 26 m in): im purity C = about 0.2;
impurity B = about 0.7; im purity D = about 0.88;
B. I t gives reaction (a) o f sodium (2.3.1).
TESTS System suitability: reference solution (c):
S o lu tio n S — resolution: m inim um 7.0 between the peaks due to
Dissolve 2.50 g in carbon dioxide-free water R and dilute to impurity D an d cefalotin.
25.0 m L with the same solvent. Limits:
A p p e a ra n c e o f so lu tio n — impurity B: no t more than the area o f the principal peak
Solution S is clear (2.2.1) and its absorbance (2.2.25) at in the chromatogram obtained w ith reference solution (b)
450 nm is not greater than 0.20. (1.0 p er cent);
p H (2.2.3) — impurity D: n o t more than 0.5 times the area o f the
4.5 to 7.0 for solution S. principal peak in die chrom atogram obtained with
— any other impurity: for each impurity, n o t m ore than
Dissolve 1.25 g in water R and dilute to 25.0 m L w ith the chrom atogram obtained with reference solution (b)
sam e solvent. (0.25 per cent);
R e la te d su b s ta n c e s — total: not m ore than 3 times the area o f the principal peak
L iquid chrom atography (2.2.29). Prepare the solutions in the chromatogram obtained with reference solution (b)
immediately before use. (3.0 per cent);
Test solution (a) Dissolve 75.0 mg o f the substance to be — disregard limit: 0.05 times the area o f the principal peak in
exam ined in water R and dilute to 25.0 m L with the same the chrom atogram obtained with reference solution (b)
solvent. (0.05 per cent).
Test solution (b) D ilute 5.0 m L o f test solution (a) to NjiV-Dim ethylaniline (2.426, Method B)
50.0 m L w ith water R. M axim um 20 ppm .
Reference solution (a) Dissolve 75.0 mg of cefalotin 2 -E th y lh e x a n o ic a d d (2.428)
sodium CRS in water R and dilute to 25.0 m L with the same M axim um 0.5 p er c e n t
solvent. D ilute 5.0 m L o f the solution to 50.0 m L with W a te r (2.5.12)
water R. M axim um 1.5 p e r cent, determined on 0.500 g.
Reference solution (b) D ilute 1.0 m L of test solution (a) to B a c te ria l e n d o to x in s (2.6.14)
100.0 m L w ith water R. Less fhan 0.13 IU/m g, if intended for use in the m anufacture
Reference solution (c) M ix 1 m L of test solution (a), 1 m L of of parenteral preparations without a further appropriate
hydrochloric add R1 and 8 m L o f water R H eat at 60 °C for procedure for the removal of bacterial endotoxins.
12 m in and cool to room tem perature in iced water. Inject
A SSA Y
immediately.
Reference solution (d) Dissolve 5 m g o f cefalotin for impurity B related substances with the following modifications.
identification CRS in water R and dilute to 5 m l. w ith the
Mobile phase M ix 14 volumes of acetonitrile R and 86 volumes
sam e solvent.
o f a 6.967 g/L solution o f dipotassium hydrogen phosphate R
Column:
previously adjusted to p H 6.0 with phosphoric add R.
— size. I = 0.25 m , 0 — 4.6 mm;
— stationary phase: end-capped octadecylsUyl silica gel for Detection Spectrophotom eter at 260 nm .
chromatography R (5 |im); Injection 5 |iL o f test solution (b) and reference solution (a).
— temperature: 40 °C. Run time 1.5 times die retention tim e of cefalotin (retention
Mobile phase. time = about 10 min).
— mobile phase A: mix 3 volumes o f acetonitrile R1 and Calculate the percentage content o f C 16H 15N 2N a 0 6S2 using
97 volumes o f a 1.742 g/L solution o f dipotassium the chrom atogram obtained with reference solution (a) and
hydrogen phosphate R previously adjusted to p H 2.5 with taking into account the assigned content o f cefabtin
phosphoric add R; sodium CRS.
— mobile phase B: m ix 40 volumes o f acetomtrüe R1 and
STORAGE
60 volumes o f a 1.742 g/L solution of dipotassium
Protected from light. If the substance is sterile, store in a
hydrogen phosphate R previously adjusted to p H 2.5 with
phosphoric add R; sterile, airtight, tam per-proof container.
IM P U R IT IE S
Time Mobile phase A Mobik phase B
Specified impurities B, D
(min) (percent V/V) (percent V/V) Other detectable impurities (the following substances would, if
0 -3 0 100*0 0 * 100 present at a sufficient level, be detected by one or other o f
3 0 -3 5 0 100
by the general m onograph Substances for pharm aceutical use
Flow rate 1.0 mL/min. (2034). It is therefore n o t necessary to identify these
2016 Cefamandole Nafate 1-441
impurities for demonstration o f compliance. See also 5.10. yl) sulfanyl] methyl] -8-oxo-5-thia-1-azabicydo [4.2.0] oct-2-ene-
Control of impurities in substances for pharmaceutical use): A , C. 2-carboxylate (cefamandole sodium), with sodium carbonate.
co 2h Semi-synthetic product derived from a fermentation p ro d u ct
Content
— cefamandole nafate (C 19H i7N $N a06S2): 93.0 per cent to
102.0 per cent (anhydrous and sodium carbonate-free
H H
c n r substance), for the sum of the content of cefamandole
nafate and cefamandole sodium expressed as cefamandole
A. (6R,7i?)-3-methyl-8-oxo-7- [(thiophen-2-ylacetyl)amino]-5- nafate;
thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic a d d — cefamandole sodium (Q gH nN gN aO sSj): maximum
(deacetoxycefalotin), 10.0 per cent (anhydrous and sodium carbonate-free
CO2H substance);
— sodium carbonate (Na2C 0 3): 4.8 per cent to 6.4 per cent.
CHARACTERS
cn ^ s Appearance
B . (6R, 7i?)-3-(hydroxymethyI)-8-oxo-7- [(thiophen-2- Solubility
ylacetyl)amino]-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2- Freely soluble in water, sparingly soluble in methanol.
caiboxylic a d d (deacetylcefalotin) , IDENTIFICATION
CO2H 0 A. Infrared absorption spectrophotometry (2.2.24).
Comparison cefamandole nafate CRS.
B. It gives reaction (a) of sodium (2.3.1).
H H S TESTS
Solution S
C. ( 6R,7i?)-3-[(acetyloxy)methyI]-7-amino-8-oxo-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic a d d (7-ACA),
pH
6.0 to 8.0 for solution S, measured after 30 min.
D . (5aR,6Æ)-6- [(thiophen-2-ylacetyl)amino]-5a,6-dihydro-
—35.0 to —45.0 (anhydrous and sodium carbonate-free
3/fj7H -azeto [2,1-b] furo [3,4-d] [1,3] thiazine-1,7 (4ü)-dione
substance).
(cefalotin lactone).
Dissolve 1.00 g in acetate buffer solution pH 4 7 R1 and dilute
Ph Eur
to 10.0 m L with the same solvent.
Related substances
***** immediately before use.
Cefamandole Nafate ★ ★
Solvent mixture M ix 18 volumes of acetonitrile R and
(Ph. Eur. monograph 1402) ***** 75 volumes of a 10 per cent V/V solution of triethylantine R
COjNa previously adjusted to p H 2.5 with phosphoric add R.
V -N ^ V ^ 5 examined in the solvent mixture and dilute to 10.0 m L with
if H H x / Reference solution (a) Dilute 1 mL of the test solution to
1 O N=N
10 m L with the solvent mixture, then heat at 60 °C for
Compound R Molecular Formula
30 min.
Mr
Cefamandole nafate CHO Ci9H17NgNaOeS2 512.5
Cefamandole sodium H C18H17N6NaOsS2 484.5
Column:
— sizer. I = 0.25 m, 0 = 4.6 mm;
A ctio n a n d use — stationary phase: octadecylsHyl silica gelfor chromatography R
Cephalosporin antibacterial. (5 nm).
P h B r ______________________________________
Mobile phase:
— triethylamine phosphate solution: dissolve 2.0 g of sodium
D E F IN IT IO N pentanesulfonate R in 350 m L of water R, add 40 m L of
Mixture o f sodium (6i?,7i?)-7-[[(2J?)-2-(f6rmyloxy)-2- triethylamine R, adjust to p H 2.5 with phosphoric add R
phenylacetyl]amino]-3-[[(l-m ethyl-lii-tetrazol-5- and dilute to 700 m L with water R;
yl) sulfanyl] methyl]-8-oxo-5-thia-l -azabicyclo [4.2.0] oct-2-ene- — mobile phase A: mix 1 volume of the triethylamine
2-carboxylate and sodium (6i?,7i?)-7-[[(2i?)-2-hydroxy-2- phosphate solution and 2 volumes of water R;
phenylacetyl] amino] -3- [ [(1 -methyl- lH-tetrazol-5-
1-442 Cefamandole Nafate 2016
mobile phase B: mix equal volumes of the triethylamine System suitability

phosphate solution, methanol R and acetomtrile R; — resolution: minim um 7.0 betw een the 2 principal peaks in
Time Mobile phase A Mobile phase B — repeatability, maxim um relative standard deviation of
(min) (percent V/V) (per cent V/V) 0.8 p er cent after a series o f single injections o f n o t less
0- 1 100 0 than 3 freshly prepared reference solutions (a).
1 - 35 100-»0 0 ^ 100 Calculate the percentage content of cefamandole nafate
(C i 9Hx7N 6N a 0 6S2) from the sum of the contents of
cefamandole nafate and cefamandole sodium expressed as
Flow rate 1.5 mL/m in. cefamandole nafate, using the declared content o f cefamandole
Detection Spectrophotom eter at 254 nm . nafate CRS.
Injection 20 pL loop injeaor. 1 m g o f cefamandole sodium is equivalent to 1.0578 m g of
Relative retention W ith reference to cefamandole nafate cefamandole nafate.
(retention tim e = about 24 min): cefamandole = about 0.8. S o d iu m c a rb o n a te
System suitability, reference solution (a): Dissolve 0.500 g in 50 m L o f water R. T itrate with 0.1 M
— resolution: m inim um 5.0 between the peaks due to hydrochloric add, determining the end-point potentiometrically
cefamandole and cefamandole nafate. (2.2.20).
Limits: 1 m L o f 0.1 M hydrochloric add is equivalent to 5.3 m g of
— arty impurity, for each impurity, n o t more than the area of N a2C 0 3.
the principal peak in the chrom atogram obtained with
STO R A G E
— total: n o t m ore th an 5 times the area of the principal peak In an airtight container, p ro te a e d from light. If the substance
in the chrom atogram obtained with reference solution (b) is sterile, store in a sterile, airtight, tam per-proof container.
(5.0 per cent); L A B E L L IN G
— disregard limit. 0.1 times the area o f the principal peak in T he label states that the substance contains sodium
the chrom atogram obtained with reference solution (b) carbonate.
(0.1 per cent).
IM P U R IT IE S
2 -E th y lh e x a n o ic a c id (2.4.28)
M axim um 0.3 per cent m/m.
M axim um 20 ppm .
W a te r (2.5.12) A. (6R,7R)-7- [[(2£)-2-(formyloxy)-2-phenylacetyI] amino]-3-
M axim um 2.0 per cent, determined on 0.500 g. methyl-8-oxo-5-thia-l-azabicyclo [4.2.0] oct-2-ene-2-carboxylic
B a c te ria l e n d o to x in s (2.6.14) acid (formylmandeloyl-7-amino-desacetoxy-cephalosporanic
Less than 0.15 IU /m g, if intended for use in the m anufacture acid),
o f parenteral preparations without a further appropriate
o c o 2h
A SSA Y
C e fa m a n d o le n a la te ^ V r i
N" H - s J n ^ n ' ch3
L iquid chrom atography (2.2.29). Prepare the solutions H H \ /
N—N
Test solution Dissolve 50.0 m g o f the substance to be C. (6i?,7i?)-7-[[(2i?)-2-(acetyloxy)-2-phenylacetyl]amino]-3-
examined in the mobile phase and dilute to 100.0 m L with [ [( 1-m ethyl-1/i-tetrazol-5 -yl) sulfanyl] methyl] -8-oxo-5-thia-1-
the mobile phase. azabicydo[4.2.0]oa-2-ene-2-carboxylic a d d
Reference solution (a) Dissolve 50.0 m g o f cefamandole (O-acetylcefiamandole),
nafate CRS in the mobile phase and dilute to 100.0 m L with SH
the mobile phase.
Reference solution (b) Dilute 1 m L o f the test solution to N N - ch 3
10 m L with the mobile phase, then heat at 60 °C for 30 m in. M-N
Column:
D . 1-m ethyl-1//-tetrazole-5-thiol,
— size: I = 0.25 m , 0 — 4.6 mm;
— stationary phase: octadecylsilyl silica gelfor chromatography R O COjH o
(5 pm ).
Mobile phase M ix 25 volumes o f acetonitrile R and 75 volumes
o f a 10 p er cent V/V solution o f triethylamine R previously
adjusted to p H 2.5 w ith phosphoric add R.
Detection Spectrophotom eter at 254 nm . E. (6Æ,7Æ)-7-[[(2Æ)-2-(fonnyloxy)-2-phenylacetyl] amino]-3-
[(acetyloxy)methyl]-8-oxo-5-thia-l -azabicydo [4.2.0] oct-2-
Injection 20 pL loop injeaor.
ene-2-carboxylic a d d (formylmanddoyl-7-ACA).
__ PhEur
2016 Cefapirin Sodium 1-443
Column:
Cefapirin Sodium — sizer. I = 0.30 m , 0 = 4 mm,
(Ph. Eur. monograph 1650) * — stationary phase: octadecylsUyl silica gelfor chromatography R
(10 nm).
CO^Na O
Mobile phase Mix 80 m l. o f dimetkylformamide R, 4.0 mT. o f
glacial acetic acid R and 20 m L of a 4.5 per cent mJm solution
o f potassium hydroxide R. Dilute to 2 L with water R.
O
C 17H 16N 3N a06S2 445.5 24356-60-3 Injection 20 )xL o f the test solution and reference
A c tio n a n d u se Run time Twice the retention time o f cefapirin.
Cephalosporin antibacterial. Relative retention W ith reference to cefapirin (retention
PhEur___________________________ _______________________________ time = about 13 min): impurity B = about 0.3;
impurity C = about 0.5; impurity A = about 0.75.
D E F IN IT IO N System suitability Reference solution (d):
Sodium ( 6R,7R)-3-[(acetyloxy)methyl]-8-oxo-7-[[[(pyridin-4- — resolution: minimum 2.0 between the peaks due to
yl) sulfanyl] acetyl] amino] -5-thia-1-azabicydo [4.2.0] oct-2-ene- cefapirin and impurity A.
2-carboxylate.
Limits:
Semi-synthetic product derived from a fermentation product. — any impurity, for each impurity, not more than the area o f
C o n te n t the principal peak in the chromatogram obtained with
96.0 per cent to 102.0 per cent (anhydrous substance). reference solution (b) (1.0 per cent), and not more than
1 such peak has an area greater than 0.3 times the area of
CHARACTERS
A p p e a ra n c e
reference solution (b) (0.3 per cent),
W hite or pale yellow powder.
— total: no t more than twice the area of the prindpal peak in
S o lu b ility the chrom atogram obtained with reference solution (b)
Soluble in water, practically insoluble in methylene chloride. (2.0 per cent),
ID E N T IF IC A T IO N — disregard limit, area o f the principal peak in the
A. Infrared absorption spectrophotometry (2.2.24). chromatogram obtained with reference solution (c)
(0.05 per cent).
Comparison cefapirin sodium CRS.
B. It gives reaction (a) o f sodium (2.3.1).
N,iV-Dimethylaniline (2.426, Method E)
M aximum 20 ppm .
TESTS
2-Ethylhexanoic acid (2.4.28)
M aximum 0.5 per c e n t
same solvent. T he solution is clear (2.2.1). D ilute 5.0 m L to W a te r (2.5.12)
10.0 m L with water R. T he absorbance (2.2.25) o f this M aximum 2.0 per cent, determined on 0.300 g.
solution at 450 n m is no t greater than 0.25. Bacterial endotoxins (2.6.14)
p H (2.2.3) Less than 0.17 IU/mg, if intended for use in the m anufacture
6.5 to 8.5. o f parenteral preparations without a further appropriate
10.0 m L with the same solvent. A SSAY
S p ecific o p tica l ro ta tio n (2.2.7) Liquid chromatography (2.2.29) as described in the test for
+ 150 to + 165 (anhydrous substance). related substances with the following modification.
Dissolve 0.500 g in water R and dilute to 25.0 m L with the Injection T est solution and reference solution (a).
sam e solvent. Calculate the percentage content of C 17H 16N 3 N a 0 6S2.
R e la te d su b sta n c e s STORAGE
Liquid chromatography (2.2.29). Prepare the solutions Protected from light. If the substance is sterile, store in a
immediately before use. sterile, tam per-proof container.
Test solution Dissolve 42 mg o f the substance to be examined IM P U R IT IE S
in the mobile phase and dilute to 200.0 m L with the mobile *Specified impurities A , B, C.
phase.
Reference solution (a) Dissolve 42 mg of cefapirin sodium CRS
phase.
Reference solution (d) Mix 1 m L o f the test solution, 8 m L of A. (5ai?, 6K)-6-[ [[(pyridin-4-yl)sulfanyl] acetyl] amino]-5a,6-
the mobile phase and 1 m L o f hydrochloric acid R l. H eat at dihydro-3/i,7if-azeto [2,1 -b\ furo [3,4-d\ [1,3] thiazine-1,7 (4H )-
60 °C for 10 min. dione (deacetylcefapirin lactone),
1-444 Cefatrizine Propylene Glycol 2016
co 2h
Dissolve 0.400 g in 1 M hydrochloric acid and dilute to
20.0 m L with the same add.
H ° ll ] R Propylene glycol
o
Solvent mixture acetone R, water R (20:80 VIV).
B. R = OH: (6i?,7i?)-3-(hydroxymethyl)-8-oxo-7-[[[(pyridin- Internal standard solution Dissolve 1.0 g o f dimethylacetamide R
4-yl)sulfanyl]acetyI]amino]-5-thia-l-azabicyclo[4.2.0]oct-2- in the solvent mixture and dilute to 50.0 m L with the solvent
ene-2-carboxylic a d d (deacetylcefapirin), mixture.
C. R = H: (6i?,7i?)-3-methyl-8-oxo-7-[[[(pyridin-4- Test solution Introduce 0.40 g of the substance to be
yl) sulfanyl] acetyl] am ino]-5-thia-1-azabicydo [4.2.0] oct-2-ene- examined into a ground-glass-stoppered test-tube.
2-carboxylic a d d (deacetoxycefapirin). Add 3.0 m L o f the internal standard solution, 1.0 m L o f the
solvent mixture and 2.0 m L o f hydrochloric acid R. Seal the
___________________________________________________________________________________________ PhEur
test-tube and shake.
Reference solution (a) Dissolve 2.0 g o f propylene glycol R in the
solvent mixture and dilute to 100.0 m L with the solvent
★*★ mixture.
★ ★
Cefatrizine Propylene Glycol ★ ★ Reference solution (b) Introduce into a ground-glass-stoppered
***** test-tube 1.0 m L o f reference solution (a) and 1.0 m L o f the
(Ph. Eur. monograph 1403) internal standard solution.
Column'.
— size: I — 2 m , 0 = 2 mm;
— stationary phase: ethylvinylbenzene-dwinylbeTizene
copolymer R (150-180 pm).
H OH Flow rate 30 mL/min.
X Ô H and enantioiDer Temperature:
h3c
— column: 200 °C;
C 18Hi8N60 5S2j(CyigO^n 462.5 (base) Detection R am e ionisation.
Injection 1 pL o f the test solution and reference solution (b).
Limit:
— propylene glycol: 13.0 per cent to 18.0 per cent.
P h E u r ________________________________________________________ Related substances
D E F IN IT IO N Liquid chromatography (2.2.29).
M ixture of (6i?,7i?)-7-[[(2i?)-2-amino-2- Test solution Dissolve 60.0 mg of the substance to be
(4-hydroxyphenyl) acetyl] amino] -8-oxo-3- [[(1H -1,2,3-triazol- examined in the mobile phase and dilute to 100.0 m L with
4-yl) sulfanyl] methyl] -5-thia-1-azabicydo [4.2.0] oct-2-ene-2- the mobile phase.
carboxylic a d d and propane-1,2-diol in molecular Reference solution (a) Dissolve 60.0 m g o f cefatrizine propylene
proportions o f about 1:1. glycol CRS in the mobile phase and dilute to 100.0 m L with
C o n te n t the mobile phase.
95.0 per cent to 102.0 per cent of C i8H i8N 60 5S2 Reference solution (b) Dissolve 30.0 mg o f cefatrizine
(anhydrous substance). impurity A CRS in buffer solution pH 7.0 R and dilute to
100.0 m L with the same buffer solution.
CHARACTERS
A p p e a ra n c e Reference solution (c) Dilute 0.6 m L o f reference solution (a)
W hite or alm ost white powder. to 100.0 m L with the mobile phase.
S o lu b ility Reference solution (d) Dilute 1.0 m L o f reference solution (b)
Slightly soluble in water, practically insoluble in ethanol to~l 00.0 m L with buffer solution pH 7.0 R.
(96 per cent) and in methylene chloride. Reference solution (e) T o 1.0 m L of reference solution (a) add
1.0 m L o f reference solution (b) and dilute to 10.0 m L with
the mobile phase.
A. Infiared absorption spectrophotometry (2.2.24).
Column:
Comparison cefatrizine propylene glycol CRS. — sizer. I = 0.25 m , 0 = 4 mm;
B. Examine the chromatograms obtained in the test for — stationary phase: octadecylsifyl silica gel for chromatography R
propylene glycol. (5 pm).
Results T he prindpal peak in the chromatogram obtained Mobile phase Mix 5 volumes of acetonitrile R and 95 volumes
w ith the test solution is similar in retention time and size to of a 2.72 g/L solution of potassium dihydrogen phosphate R in
the principal peak in the chrom atogram obtained with water R.
reference solution (b). Flow rate 2 m lA nin.
TESTS Detection Spectrophotometer at 272 nm.
2016 Cefazolin Sodium 1-445
Injection 20 jjL of the test solution and reference

solutions (c), (d) and (e).
Cefazolin Sodium *****
★. ★
Run time At least twice the retention time o f cefatrizine. (Ph. Eur. monograph 0988) *
System suitability, reference solution (e):
— resolution: minim um 5.0 between the peaks due to CO,Na
cefatrizine and impurity A.
Limits".
— impurity A: not m ore than the area of the corresponding
solution (d) (0.5 per cent);
— any other impurity: for each impurity, not more than the
C 14H13N8N a 0 4S3 476.5 27164-46-1
with reference solution (c) (0.6 p er cent);
— sum of impurities other than A: not more than 3.5 times the A ctio n a n d u se
area o f the principal peak in the chromatogram obtained Cephalosporin antibacterial.
with reference solution (c) (2.1 p er cent);
P re p a r a tio n
Cefazolin Injection
(0.03 per cent). P ftE ir ________________________________________________________________________________
W a te r (2.5.12) D E F IN IT IO N
Sodium (6R,7R)-3- [ [(5-methyl-1,3,4-thiadiazol-2-
S u lfated a sh (2.4.14) yl) sulfanyi] methyl] -8-oxo-7 - [(lfi-tetrazol-1-ylacetyl) amino] -
M aximum 0.1 per cent, determined on 1.0 g. 5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylate.
A SSAY Semi-synthetic product derived from a fermentation product.
liq u id chromatography (2.2.29) as described in the test for C o n te n t
related substances w ith the following modifications. 95.0 per cent to 102.0 per cent (anhydrous substance).
Injection T est solution and reference solution (a). CHARACTERS
System suitability: reference solution (a): A p p e a ra n c e
— repeatability: maximum relative standard deviation of White or almost white powder, very hygroscopic.
S o lu b ility
Calculate the percentage content o f C 18H 18N 60 5S2 from the Freely soluble in water, very slightly soluble in ethanol
declared content o f C 18H 18N 60 5S 2 in cefatrizine propylene (96 per cent).
glycol CRS.
IM P U R IT IE S
Specified impurities A.
CO,H Preparation Dissolve 0.150 g in 5 m L of water R, add 0.5 m L
of dilute acetic acid R, swirl and allow to stand for 10 m in in
iced water. Filter the precipitate and rinse with 1-2 m L of
water R. Dissolve in a mixture of 1 volume of water R and
9 volumes of acetone R. Evaporate the solvent almost to
N-NH dryness, then dry in an oven at 60 °C for 30 m in.
Comparison cefazolin CRS.
A. (6R,7R)-7-am ino-8-oxo-3-[ [(1 1,2,3-triazol-4-
B. It gives reaction (a) o f sodium (2.3.1).
yl) sulfanyi] methyl] -5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-
carboxylic a d d (7-ACA triazole). TESTS
puEir S o lu tio n S
p H (2.2.3)
- 2 4 to - 1 5 (anhydrous substance).
same solvent.
A b so rb a n c e (2.2.25)
same solvent. Dilute 2.0 mL of the solution to 100.0 m L
with sodium hydrogen carbonate solution R. Examined between
220 nm and 350 nm, the solution shows an absorption
1-446 Cefazolin Sodium 2016
1. impurity J 2. impurity E 3. unknow n structure 4. cefazolin 5. impurity L

Figure 0988.-1. - Chromatogram for the test for relaxed, substances of cefazolin sodium: reference solution (b) (in situ degradation)
maximum at 272 nm . The specific absorbance at the Flow rate 1.2 mL/min.
maximum is 260 to 300 (anhydrous substance). Detection Spectrophotom eter at 254 nm .
R e la te d su b s ta n c e s Injection 5 )iL.
L iquid chrom atography (2.2.29). System suitability: reference solution (b):
Test solution Dissolve 50.0 m g of the substance to be — resolution: minimum 2.0 between the peaks due to
examined in mobile phase A and dilute to 20.0 m L with cefazolin and impurity L (see Figure 0988.-1).
mobile phase A. Limits:
Reference solution (a) Dilute 1.0 m L of the test solution to — any impurity: for each impurity, no t m ore than the area of
100.0 m L with mobile phase A the principal peak in the chrom atogram obtained with
Reference solution (b) Dissolve 20 m g o f the substance to be reference solution (a) (1.0 p er cent);
examined in 10 m L o f a 2 g/L solution of sodium hydroxide R. — total: not more than 3.5 times the area of the principal
Allow to stand for 15-30 min. Dilute 1.0 m L of the solution peak in the chromatogram obtained with reference
to 20 m l- with mobile phase A. solution (a) (3.5 per cent);
Column:
— size. I = 0.125 m , 0 = 4.0 mm; the chromatogram obtained with reference solution (a)
— stationary phase: octadecylsûyl silica gel for chromatography R (0.05 per cent).
(3 nm); A ^ N -D im eth y lan ilin e (2.4.26, Method B)
— temperature: 45 °C. M aximum 20 ppm.
Mobile phase: W a te r (2.5.12)
— mobile phase A: solution containing 14.54 g/L o f disodium M aximum 6.0 per cent, determ ined on 0.300 g.
hydrogen phosphate R and J.53 g/L o f potassium dihydrogen B a c te ria l en d o to x in s (2.6.14)
phosphate R; Less than 0.15 IU/mg, if intended for use in the manufacture
— mobile phase B: acetonitrüe for chromatography R',
of parenteral preparations without a further appropriate
ASSAY
0 -2 98 2
2 -4 98-» 85 2-» 15
4 - 10 85-» 60 15 -»40 the mobile phase.
10 - 11.5 60-» 35 40-» 65 Reference solution (a) Dissolve 50.0 m g o f cefazolin CRS in the
mobile phase and dilute to 50.0 m L with the mobile phase.
11J - 12 35 65
Reference solution (b) Dissolve 5.0 m g o f cefuroxime
12- 15 3 5 -»98 6 5 -»2
sodium CRS in 10.0 m L o f reference solution (a) and dilute
15-21 98 2 to 100.0 m L with the mobile phase.
2016 Cefazolin Sodium 1-447
C02H 0
Column.
— size. I = 0.25 m, 0 = 4.6 mm; V -N
— stationary phase: octadecylsQyl silica gel for chromatography R
(5 nm).
Mobile phase M ix 10 volumes of acetomtrUe R and 90 volumes n* n o
i s
of a solution containing 2.77 g/L o f disodium hydrogen
phosphate R and 1.86 g/L o f citric acid R. D . (6R,7R)-3 -[(acetyloxy)methyl] -8-oxo-7- [(lH-tetrazol-1 -
ylacetyl)amino]-5-thia-l-azabicydo[4.2.0]oct-2-ene-2-
carboxylic ad d ,
SH
Injection 20 pL.
A
} -N
cefazoOn and cefuroxime. H,C
Calculate the percentage content o f cefazolin sodium by
multiplying the percentage content of cefazolin by 1.048. E. 5-m ethyl-1,3,4-thiadiazol-2-thiol (M M TD),
ST O R A G E
is sterile, store in a sterile, airtight, tam per-proof container.
IM P U R IT IE S
Other detectable impurities (the following substances would, if " i T s'
V n 0
acceptance criterion for other/unspecified impurities and/or G . (5a_R,6i?)-6- [(1 H-tetrazol-1-ylacetyl) amino] -5a, 6-dihydro-
3H ,7//-azeto [2,1 -¿]furo [3,4-d] [1,3] thiazine-1,7 (4H) -dione,
(2034). It is therefore not necessary to identify these c o 2h 0
°V - N
C, D} E, G} H, I} J, K, L H2N' * r ~ k r
H H
CO2H
H . (6i?,7i?) -3- [(acetyloxy)methyl] -7-amino-8-oxo-5-thia-1-
V _ n^ y ^ s azabicydo[4.2.0]oct-2-ene-2-carboxylic a d d (7-ACA),
S^N CC^H
h h \ ;
H,C n^ Y ^ s
A. (6R,7R)-7-am ino-3- [[(5-methyl-1,3,4-thiadiazol-2- 'n * N O COsH ^ )= N .

yl) sulfanyi] methyl] -8-oxo- 5-thia-1-azabicyclo [4.2.0] oct-2-ene- H3C
2-carboxylic acid,
I. 2- [carboxy [(1 H-tetrazol- 1-ylacetyl) amino] methyl] -5-
co 2h
[ [(5-methyl-l ,3,4-thiadiazol-2-yl) sulfanyi] methyl]-5,6-
ch 3 V nV
dihydro-2H-1,3-thiazine-4-carboxylic a d d (cefazoloic ad d ),
A
s
co^H
II h h \ r
O y=N
H3C
B. (6R,7R)-7-[(2,2-dimethylpropanoyl)amino]-3 -[[(5-methyl- 'N= n 0 c o 2h

1,3,4-thiadiazol-2-yI) sulfanyi] methyl]-8-oxo-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic ad d , J. 2 -[carboxy [(1 iT-tetrazol-1-ylacetyl) amino] methyl] -5-
m ethylidene-5,6-dihydro-2ii-1,3-thiazine-4-carboxylic ad d ,
co 2h
O^NH2
" V f j
« ° tl 7 I
n- n O
t t s
n* n o
C . (6i?,7i?)-3-methyl-8-oxo-7- [(1/7-tetrazol-1- h3c
ylacetyl) amino]-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-
carboxylic a d d , K . (6R,7R)-3- [ [(5-methyl-l ,3,4-thiadiazol-2-
yl) sulfanyi] methyl] -8-oxo-7- [(1/i-tetrazol-1-ylacetyl) amino] -
5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxamide
(cefazolinamide),
1-448 Cefepime Hydrochloride Monohydrate 2016
Im p u rity G
immediatefy before use.
examined in 0.01 M nitric acid and dilute to 10.0 m L with
the same ad d .
Reference solution (a) Dilute 0.250 g o f N-methylpyrrolidine R
L. (6Æ,7S)-3-[[(5-methyl-l,3,4-thiadiazol-2- (impurity G) to 100.0 m L with water R. D ilute 2.0 m L of
yl) sulfanyl] methyl] -8-oxo-7 - [( l//-tetrazo l-1-ylacetyl) amino] - this solution to 100.0 m L with 0.01 M nitric acid
5-thia-1-azabicydo [4.2.0] oct-2-ene-2-carboxylic ad d .
Reference solution (b) Dilute 0.250 g o f pyrrolidine R to
___________________________________________________________________________________________ PhEur
100 m L w ith 0.01 M nitric add D ilute 2 m L o f the solution
to 100 m L with 0.01 M nitric add. M ix 5 m L of this solution
with 5 m L o f reference solution (a).
Column:
Cefepime Hydrochloride ***** — size: I — 0.05 m , 0 = 4.6 mm;
— stationary phase', strong cation-exchange resin R (5 |im ).
Monohydrate ***** Mobile phase Mix 1 volume of acetonitrile R and 100 volumes
(Cefepime Dihydrochloride Monohydrate, o f 0.01 M nitric add, filter through a 0.2 |xm filter.
Ph Eur monograph 2126) Flow rate 1 mL/min.
Detection Conductivity detector.
Irqection 100 jiL.
Run time 1.1 times the retention time of cefepime.
Retention time Cefepime = about 50 min, eluting as a
broadened peak.
System suitability:
C 19H 26C12N 60 5S*H 20 571.5 123171-59-5 impurity G in the chromatogram obtained with reference
solution (a);
A ctio n a n d use — repeatability: maximum relative standard deviation of
Cephalosporin antibacterial. 5.0 per cent after 6 injections o f reference solution (a);
— peak-to-vaRey ratio: minimum 3 between the peaks due to
P hE ur ___________________________________________________________________________________________
pyrrolidine and im purity G in the chromatogram obtained
D E F IN IT IO N with reference solution (b).
(6R, 1R)-1- [ [(22)-(2-Aminothiazol-4- Calculate the percentage content of impurity G in the test
yl) (methoxyimino)acetyl] amino] -3- solution using reference solution (a).
[(1 -methylpyirolidinio) methyl] -8-oxo-5-ihia-l - Limit.
azabicydo[4.2.0]oct-2-ene-2-carboxylate dihydrochloride — impurity G: maximum 0.5 per cent.
monohydrate. Semi-synthetic product derived from a
fermentation product.
C o n te n t immediately before use or keep refrigerated at 4-8 °C for not more
97.0 per cent to 102.0 per cent (anhydrous substance). than 12 h.
CHARACTERS Test solution Dissolve 70.0 mg o f the substance to be
A p p e a ra n c e examined in mobile phase A and dilute to 50.0 m L with
W hite or almost white, crystalline powder. mobile phase A Sonicate for 30 s and stir for about 5 min.
S o lu b ility Reference solution (a) Dissolve 70.0 mg of cefepime
Freely soluble in water and in m ethanol, practically insoluble dihydrochloride monohydrate CRS in mobile phase A and dilute
in methylene chloride. to 50.0 m L with mobile phase A. Sonicate for 30 s and stir
for about 5 min.
A. Infrared absorption spectrophotometry (2.2.24). Reference solution (b) Dilute 1.0 m L o f the test solution to
10.0 m L w ith mobile phase A D ilute 2.0 m L of this solution
Comparison cefepime dihydrochloride monohydrate CRS.
B. It gives reaction (a) of chlorides (2.3.1).
Reference solution (c) Dissolve 7 mg o f cefepime dihydrochloride
TESTS monohydrate for system suitability CRS (containing impurities
A p p e a ra n c e o f so lu tio n A, B and F ) in mobile phase A and dilute to 5 m L with
T he solution is clear (2.2.1) and not more intensely coloured mobile phase A
than reference solution Y3 (2.2.2, Method II). Reference solution (d) Dissolve 2 mg of cefepime
Dissolve 2.0 g in water R and dilute to 20 m L with the same impurity E CRS in mobile phase A and dilute to 25.0 m L
solvent. with mobile phase A. Dilute 1.0 m L of the solution to
S pecific o p tic a l ro ta tio n (2.2.7) 10.0 m L w ith mobile phase A.
+ 40 to + 45 (anhydrous substance). Column:
— size: I = 0.25 m , 0 = 4.6 mm;
same solvent.
2016 Cefepime Hydrochloride Monohydrate 1-449
Mobile phase: B a c te ria l e n d o to x in s (2.6.14)

— mobile phase A: mix 10 volumes of acetomtrUe R and Less than 0.04 IU/mg, if intended for use in the manufacture
90 volumes o f a 0.68 g/L solution of potassium dihydrogen o f parenteral preparations without a further appropriate
phosphau R previously adjusted to p H 5.0 with 0.5 M procedure for the removal of bacterial endotoxins.
potassium hydroxide; ASSAY
— mobile phase B: mix equal volumes of acetomtrüe R and a
0.68 g/L solution o f potassium dihydrogen phosphate R
previously adjusted to p H 5.0 with 0.5 Mpotassium
hydroxide; Mobile phase Mobile phase A.
Time Mobile phase A Mobile phase B Run time 1.4 times the retention time o f cefepime.
(min) (per cent V7V) (per cent V/V) Calculate the percentage content o f Ci9H26Cl2N 60 5S2 from
0 - 10 100 0 the declared content of cefepime dihydrochloride
1 0 -3 0 100-»50 0*50
monohydrate CRS.
3 0 -3 5 50
STO RA G E
50
Protected from lig h t If the substance is sterile, store in a
35 - 36 50-» 100 50 0
3 6 -4 5 100 0
IM P U R IT IE S
Specified impurities A, B, E, F, G.
Flow rate 1 mL/min. Other detectable impurities (the following substances would, if
Detection Spectrophotom eter at 254 nm. present at a sufficient level, be detected by one or other o f
Identification of impurities Use the chromatogram supplied (2034). It is therefore n o t necessary to identify these
w ith cefepime dihydrochloride monohydrate for system impurities for demonstration of compliance. See also 5.10.
suitability CRS and the chromatogram obtained with Control of impurities in substances for pharmaceutical use): C, D.
reference solution (c) to identify the peaks due to
impurities A, B and F; use the chromatogram obtained with
reference solution (d) to identify the peak due to impurity E.
Relative retention W ith reference to cefepime (retention
tim e = about 7 min): impurity E = about 0.4;
im purity F = about 0.8; impurity A = about 2.5;
— resolution: minim um 1.5 between die peaks due to A. (6Ä,7i?)-7-[[(2E)-(2-aminothiazol-4-
impurity F and cefepime. yl) (methoxyimino) acetyl] amino] -3-
Limits: [ ( 1-methylpyrrolidinio) methyl] -8-oxo-5-thia-1-
— correction factors: for the calculation of content, multiply azabicydo[4.2.0]oct-2-ene-2-carboxylate (arm-cefepime),
nh2
corresponding correction factor, impurity A = 1.4;
impurity B = 1.4; im purity E = 1.8;
— impurity A: n o t more than 1.5 times the area o f the
— impurities B, F: for each impurity, n o t m ore than the area
o f the principal peak in the chromatogram obtained with
— impurity E: n o t m ore than 0.5 times die area of the
B. (6i?,7i^)-7-[[(2Z)-[2-[[(22)-(2-aminothiazol-4-
yl) (methoxyimino) acetyl] amino] thiazol-4-
yl] (methoxyimino)acetyl] amino]-3-
— unspecified impurities: for each impurity, no t m ore than
[(1 -methylpyrrolidmio) methyl] -8-oxo-5-thia-1-
. azabicydo [4.2.0] oct-2-ene-2-carboxylate,
(0.10 p er cent);
ch3
— total: n o t m ore than 5 times the area o f the principal peak
in the chrom atogram obtained with reference solution (b) N
(1.0 per cent);
— disregard Hmir. 0.25 times the area o f the principal peak in
(0.05 p er cent).
W a te r (2.5.12) C . (22) -2-(2-aminothiazol-4-yl)-N-(foimylmethyl)-2-
3.0 per cent to 4.5 per cent, determined on 0.400 g. (methoxyimino)acetamide,
1-450 Cefixime 2016
ch 3
C o n te n t
o
nT 95.0 per cent to 102.0 per cent (anhydrous substance).
n^ A co 2h
CHARACTERS
h2n - ^ T
A p p e a ra n c e
W hite or alm ost white, slightly hygroscopic powder.
D. (2Z)-(2-aminothiazol-4-yl)(methoxyimino)acetic ad d , S o lu b ility
Slightly soluble in water, soluble in methanol, sparingly
C02 soluble in anhydrous ethanol, practically insoluble in ethyl
acetate.
1 uyö“ H H
Comparison cefixime CRS.
E. (6R,7R)-7-am ino-3-[( 1-methylpyrroUdinio)methyl]-8-oxo- If the spectra obtained show differences, dissolve the
5-thia-1-azabicydo [4.2.0] oct-2-ene-2-carboxylate, substance to be examined and the reference substance
separately in methanol R, evaporate to dryness and record
TESTS
p H (2.2.3)
2.6 to 4.1.
Suspend 0.5 g in carbon dioxide-free water R and dilute to
10 m l. w ith the same solvent.
F. (6/?,72?)-7-[[[(6.R,7.R)-7-[[(2Z)-(2-aminothiazol-4- the mobile phase.
yl) (methoxyimino) acetyl] amino] -3- Reference solution (a) Dissolve 25.0 mg of cefixime CRS in the
[(1 -methylpyrrolidinio) methyl] -8-oxo-5-thia-1- mobile phase and dilute to 25.0 m l. with the mobile phase.
azabicydo [4.2.0] oct-2-en-2-yl] carbonyl] amino]-3-
[(1 -methylpyrrolidinio)methyl] -8-oxo-5-thia-1-
azabicydo [4.2.0] oct-2-ene-2-carboxylate,
Reference solution (c) Dissolve 10 m g o f cefixime CRS in
ch 3 10 m L of water R. H eat on a water-bath for 45 min and cool
N (in situ preparation o f impurity D ). Inject immediately.
Column:
o — size. I = 0.125 m , 0 = 4 mm;
G. 1-methylpyrrolidine (N-methylpyirolidine).
(5 nm);
PhEur — temperature. 40 °C.
— Mobile phase: mix 250 volumes o f acetonitrUe R and
750 volumes o f a tetrabutylammonium hydroxide solution
prepared as follows: dissolve 8.2 g o f tetrabutylammonium
★ ★ hydroxide R in water R and dilute to 800 m L with the
Cefixime ★ ★ same solvent; adjust to p H 6.5 with dilute phosphoric
(Ph. Eur. monograph 1188) *★* acid R and dilute to 1000 m L with water R.
Flow rate 1.0 m l/m in .
,C 0 2h
C02H Detection Spectrophotom eter at 254 nm.
N' ° Injection 10 [iL o f the test solution and reference solutions (b)
, 3 H20 and (c).
Run time 3 times the retention time o f cefixime.
Cx^xsNjOTS^HaO 507.5 cefixime and im purity D ; if necessary, adjust the
concentration o f acetonitrile in the mobile phase.
Action and use Limits:
Cephalosporin antibacterial. — any impurity, for each impurity, not more than 0.5 times
the area of the prindpal peak in the chromatogram
PhEur__________________________
DEFINITION — total: n o t more than 3 times the area o f the principal peak
(6R,7R)-7 - [ [(2)-2-(2-Aminothiazol-4-yl)-2- in the chromatogram obtained with reference solution (b)
[(carboxymethoxy)imino] acetyl] amino] -3-ethenyl-8-oxo-5- (3 per cent);
thia-1-azabicydo[4.2.0]oct-2-ene-2-carboxylic a d d trihydrate.
2016 Cefoperazone Sodium 1-451

(0.1 per cent).
Ethanol (2.4.24)
Head-space gas chromatography (2.2.28): use the standard
H2N—^
additions method.
Sample solution Dissolve 0.250 g of the substance to be
examined in a mixture of 1 volume of dimethylacetarmde R E. R = H, R; = CH3: (6i?,7/ 2)-7 -[[(Z)-2 -(2-aminothia2 ol-4-
and 4 volumes of water R and dilute to 25.0 mL with the yl)-2-[(carboxymethoxy)imino]acetyl]amino]-3-methyl-8-oxo-
same mixture of solvents. 5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic add,
Limit. F. R = C 2H5, R ' = CH=CH2:
— ethanol: maximum 1.0 per cent m/m. (6i?,7i?)-7-[[(Z)-2-(2-aminothiazol-4-yl)-2-[(2-ethoxy-2-
W ater (2.5.12) oxoethoxy)imino] acetjd] amino]-3-ethenyl-8-oxo-5-thia-1-
9.0 per cent to 12.0 per cent, determined on 0.200 g. azabicydo[4.2.0]oct-2-ene-2-carboxylic add.
Sulfa ted ash (2.4.14) __________________________________________________________ PhEur
ASSAY
related substances with the following modifications. Cefoperazone Sodium ★ ★
★ ★
Iryecdon The test solution and reference solution (a).
*****
Calculate the percentage content of C 16H 15N 5O7S2 from the
declared content of cefixime CRS.
STORAGE
In an airtight container, protected from light. •ch 3
IMPURITIES
C sEb M N aC V Sa 668 62893-20-3
Action and use

PhEur.
A. R = C 0 2H: 2-[[(Z)-2-(2-aminothiazol-4-yl)-2- DEFINITION
[(carboxymethoxy) imino] acetyl] ammo] -2- [(2.R)-5-methyl-7- Sodium (6R,7K)-7-[[(2R)-2-[[(4-ethyl-2,3-dioxopiperazin-1-
oxo-1 ,2,5,7-tetrahydro-4if-furo [3,4-d] [1,3] thiazin-2-yl] acetic yl) carbonyl] amino] -2-(4-hydroxyphenyl) acetyl] amino] -3-
acid, [[( 1-methyl-1H-tetrazol-5-yl) sulfanyl] methyl] -8-oxo-5-thia-1-
B. R = H: 2-[[[(Z)-\-(2-aminothiazol-4-yl)-2-[[[(2-R,5ftS)-5- azabicydo[4.2.0]oct-2-ene-2-carboxylate.
methyl-7-oxo-1,2,5,7 -tetrahydro-4//-furo [3,4-d] [1,3]thiazin- Semi-synthetic product derived from a fermentation product
2 -yl] methyl] amino] - 2 -oxoethylidene] amino] oxy] acetic acid,
Content
.CO,H 95.0 per cent to 102.0 per cent (anhydrous substance).
f c o 2h
CHARACTERS
N '°
Appearance
H jN -(
s
J
« y L S -H j
T
^
H H
White or slightly yellow, hygroscopic powder.
Solubility
Freely soluble in water, soluble in methanol, slightly soluble
C. ( 6 R, 1 S) - 1 -[[(2)-2-(2-aminothiazol-4-yl)-2- in ethanol (96 per cent).
[(carboxymethoxy) imino] acetyl] amino]-3-ethenyl-8-oxo5-
If crystalline, it shows polymorphism (5.9).
thia-l-azabicyclo [4.2.0]oct-2-ene-2-carboxylic add
(cefixime 7-epimer), IDENTIFICATION
HOjC
CO2H Preparation Dissolve die substance to be examined in
'I
methanol R and evaporate to dryness; examine die residue.
Comparison Ph. Eur. reference spectrum of cefoperazone sodium.
HjN— H H B. Examine the chromatograms obtained in the assay.
Results The prindpal peak in the chromatogram obtained
D. (6R,7K)-7- [[(JE)-2-(2-aminothiazol-4-yl)-2- with test solution (a) is similar in retention time and size to
[(carboxymethoxy) imino] acetyl] amino] -3-ethenyl-8-oxo5- the principal peak in the chromatogram obtained with
thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic add reference solution (a).
(cefixime (JE)-isomer), C. It gives reaction (a) of sodium (2.3.1).
1-452 Cefoperazone Sodium 2016
TESTS Solvent solution Dissolve 0.350 g of acetone R in water R and

Appearance of solution dilute to 100.0 mL with the same solvent. Dilute 10.0 mL of
The solution is clear (2.2.1) and its absorbance (2.2.25) at this solution to 100.0 mL with water R.
430 nm is not greater than 0.15. Prepare each of 4 injection vials as shown in the table below:
Dissolve 2.5 g in water R and dilute to 25.0 mL with the
same solvent.
Vial No. Sample solution Solvent solution Water R
pH (2.2.3) (mL) (mL) (mL)
4.5 to 6.5. 1 1.0 0 4.0
Dissolve 2.5 g in carbon dioxide-free water R and dilute to 2 1.0 1.0 3.0
10 mL with the same solvent.
3 1.0 2.0 2.0
Related substances
4 1.0 3.0 1.0
Test solution (a) Dissolve 25.0 mg of the substance to be Static head-space conditions that may be used:
examined in the mobile phase and dilute to 250.0 mL with — equilibration time: 15 min;
the mobile phase. — transfer-line temperature: 110 °C.
Test solution (b) Dissolve 25.0 mg of the substance to be Temperature:
examined in the mobile phase and dilute to 50.0 mL with — Column: 40 °C for 10 min.
the mobile phase. Heavy metals (2.4.8)
Reference solution (a) Dissolve 25.0 mg of cefoperazone Maximum 5 ppm.
dihydrate CRS in the mobile phase and dilute to 250.0 mL
using 1 mL of lead standard solution (10 ppm Pb) R.
Reference solution (b) Dilute 5.0 mL of reference solution (a)
W ater (2.5.12)
to 100.0 mL with the mobile phase.
Column:
— size: I = 0.15 m, 0 = 4.6 mm; Bacterial endotoxins (2.6.14)
— stationary phase: end-capped octadecylsUyl silica gelfor Less than 0.20 IU/mg, if intended for use in the manufacture
chromatography R (5 |im). of parenteral preparations without a further appropriate
Mobile phase Mix 884 volumes of water R, 1 10 volumes of
acetonitrUe R, 3.5 volumes of a 60 g/L solution of acetic add R ASSAY
and 2.5 volumes of a triethylammonium acetate solution Liquid chromatography (2.2.29) as described in the test for
prepared as follows: dilute 14 mL of triethylamine R and related substances with the following modifications.
5.7 mL of glacial acetic add R to 100 mL with water R. Injection Test solution (a) and reference solution (a).
Flow rate 1 mL/min. System suitability: reference solution (a):
Detection Spectrophotometer at 254 nm. — repeatability: maximum relative standard deviation of
Injection 20 |iL of test solution (b) and reference solutions (a) 1.0 per cent after 6 injections.
and (b). Calculate the percentage content of cefoperazone sodium by
Run time 2.5 times the retention time of cefoperazone. multiplying the percentage content of cefoperazone by 1.034.
Retention time Cefoperazone = about 15 min. STORAGE
System suitability-, reference solution (a): In an airtight container, protected from light, at a
— number of theoretical plates: minimum 5000, calculated for temperature of 2 °C to 8 °C. If the substance is sterile, store
the principal peak; if necessary, adjust the content of in a sterile, airtight, tamper-proof container.
acetonitrUe R in the mobile phase; IMPURITIES
— symmetry factor, maximum 1.6 for the principal peak;
if necessary, adjust the content of acetomtrUe R in the
mobile phase.
Limits'.
— any impurity: for each impurity, not more than 1.5 times
the chromatogram obtained with reference solution (b) A. (5ai?,6R)-6-[[(2K)-2-[[(4 -ethyl-2,3 -dioxopiperazin-1-
(0.1 per cent). yl) carbonyl] amino]-2-(4-hydroxyphenyl)acetyl]amino]-5a,6-
Acetone (2.4.24, System E) dihydro-3/7,7/f-azeto [2,1-6] furo [3,4-d] [l,3]thiazine-l,7 (4H)-
Maximum 2.0 per cent. dione,
Sample solution Dissolve 0.500 g of the substance to be
examined in water R and dilute to 10.0 mL with the same
solvent.
2016 Cefotaxime Sodium 1-453
Cefotaxime Sodium *****

*★ +**
ch3 CC^Na o
N "° ch 3
H H S
B. (6 R ,7R)-7-[[(2i?)-2-[[(4-ethyi-2 ,3 -dioxopiperazin-1-
yl)carbonyl] amino] -2-(4-hydroxyphenyl) acetyl] amino] -3-
[(4-methyl-5-thioxo-4j5-dihydro-l//-tetrazol-l-yI)methyl]-8- CjeHigNsNaOTSa 477.4 64485-93-4
oxo-5-thia-l-azabicydo[4.2.0]oct-2-ene-2-carboxylic add, Action and use
SH
Preparation
N ^ N '0"» Cefotaxime Injection
N -N
PhEir__________________________________________________________
C. l-methyl-l/f-tetrazole-5-thiol, DEFINITION
Sodium (6i?,7,R)-3-[(acetyloxy)methyl]-7-[[(22)-2-
co 2h (2-aminothiazol-4-yl)-2- (methoxyimino) acetyl] amino] -8-oxo-
5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylate.
Content
CHARACTERS
D. (6i?,7R)-7-amino-8-oxo-3-[(1H-1,2,3-triazol-4- Appearance
ylsulfanyl) methyl]-5-thia-1-azabicydo [4.2.0] oct-2-ene-2- White or slightly yellow powder, hygroscopic.
caiboxylic add (7-TACA),
Solubility
CO,H o
Freely soluble in water, sparingly soluble in methanol.
IDENTIFICATION
Comparison cefotaxime sodium CRS.
H H
B. It gives reaction (a) of sodium (2.3.1).
E. (6i?,7i?)-3-[(acetyloxy)methyl]-7-amino-8-oxo-5-thia-l- TESTS
azabicydo[4.2.0]oct-2-ene-2-carboxylic add (7-ACA), Solution S
Dissolve 2.5 g in carbon dioxide-five water R and dilute to
25.0 mL with the same solvent.
Solution S is clear (2.2.1). Add 1 mL of glacial acetic add R
to 10 mL of solution S. The solution, examined immediately,
is clear.
pH (2.2.3)
+ 58.0 to + 64.0 (anhydrous substance).
F. (6.R,7S)-7-[[(2 R)-2 - [[(4-ethyl-2,3-dioxopiperazine- 1- Dissolve 0.100 g in water R and dilute to 10.0 mL with the
yl) carbonyl] amino] -2-(4-hydroxyphenyl) acetyi] amino]-3- same solvent.
[[(1-methyl-1H-tetrazol-5-yl) sulfanyl] methyl] -8-oxo-5-thia-1- Absorbance (2.2.25)
azabicydo[4.2.0]oct-2-ene-2-carboxylic add. Maximum 0.40 at 430 nm for solution S.
__________________________________________________________ PhEur
Specific absorbance (2.2.25)
360 to 390, determined at the absorption maximum at
235 nm (anhydrous substance).
Dissolve 20.0 mg in water R and dilute to 100.0 mL with the
same solvent. Dilute 10.0 mL of the solution to 100.0 mL
with water R
Related substances
Solution A Mobile phase B, mobile phase A (14:86 V/V).
1-454 Cefotaxime Sodium 2016
Test solution Dissolve 40.0 mg of the substance to be — total: not more than 3 times the area of the principal peak
examined in solution A and dilute to 50.0 mL with the same in the chromatogram obtained with reference solution (b)
solution. (3.0 per cent);
Reference solution (a) Dissolve 8.0 mg of cefotaxime acid CRS — disregard limit. 0.05 times the area of the principal peak in
in solution A and dilute to 10.0 mL with the same solution. the chromatogram obtained with reference solution (b)
Reference solution (b) Dilute 1.0 mL of the test solution to
(0.05 per cent).
100.0 m l. with solution A. Ethanol (2.4.24System A)
Reference solution (c) Add 1.0 mL of dilute hydrochloric acid R Maximum 1 .0 per cent.
to 4.0 mL of the test solution. Heat the solution at 40 °C for N ,N-Dimethylaniline (2.4.26, Method B)
2 h. Add 5.0 m l. of buffer solution p H 6 .6 R and 1.0 m l. of Maximum 20 ppm.
dilute sodium hydroxide solution R. 2 -Ethylhexanoic a d d (2.4.28)
Reference solution (d) Dissolve 4 mg of cefotaximefor peak Maximum 0.5 per cent m/m.
identification CRS (containing impurities A, B, C, E and F) in W ater (2.5.12)
5 mL of solution A. Maximum 3.0 per cent, determined on 0.300 g.
Column:
— sizer. I = 0.15 m, 0 = 3.9 mm,
Less than 0.05 IU/mg, if intended for use in the manufacture
— stationary phase: octadecylsilyl silica gelfor chromatography R
(5 nm),
Mobile phase: ASSAY
— mobile phase A: 7.1 g/L solution of disodium hydrogen Liquid chromatography (2.2.29) as described in the test for
phosphate R adjusted to pH 6.25 using phosphoric acid R; related substances with the following modification.
— mobile phase B: methanol R; Injection Test solution and reference solution (a).
Calculate the percentage content of C 16H i 6N 5Na 0 7 S2 by
Time Mobile phase A Mobile phase B multiplying the percentage content of cefotaxime by 1.048.
(min) (per cent V/V) (per cent V/V) STORAGE
0,- 7 86 14
7 -9 86 -»82 1 4 * 18 is sterile, store in a sterile, airtight, tamper-proof container.
9 - 16 82 18 IMPURITIES
1 6 -4 5 82*60 18 * 40 Specified impurities A,B, C, D, E, F
4 5 -5 0 60 40
Other detectable impurities(the following substances would, if
5 0 -5 5 60*86 4 0 * 14 the tests in the monograph. They are limited by the general
55 - 60 86 14 acceptance criterion for other/unspecified impurities and/or
Flow rate 1 .0 mL/min. impurities for demonstration of compliance. See also 5.10.
Detection Spectrophotometer at 2 3 5 nm. Control of impurities in substances for pharmaceutical use): G.
Injection 1 0 joL of the test solution and reference

ch3 c o 2h
with cefotaximefor peak identification CRS and the
chromatogram obtained with reference solution (d) to HN— r H H
identify the peaks due to impurities A, B, C, E and F. R1' s
Relative retention With reference to cefotaxime (retention
time = about 13 min): impurity B = about 0.3; A. R = R' = H: (6i?,7i?)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2-
impurity A = about 0.5; impurity E = about 0.6; (methoxyimino) acetyl] amino] -3-methyl-8-oxo-5-thia-1-
impurity C = about 1.9; impurity D = about 2.3; azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
impurity F = about 2 .4 ; impurity G = about 3 .1 . (deacetoxycefotaxime),
System suitability: reference solution (c): B. R = OH, R' = H: (6/?,7i?)-7-[[(2Z)-2-(2-aminothiazol-4-
— resolution: minimum 3 .5 between the peaks due to yl)-2-(methoxyimino)acetyl] amino] -3-(hydroxymethyI)-8-oxo-
impurity E and cefotaxime; 5-thia-l -azabicydo[4.2.0] oct-2-ene-2-carboxylic add
— symmetryfactor, maximum 2.0 for the peak due to (deacetylcefotaxime),
cefotaxime. C. R = O-CO-CH 3, R' = CHO: (6R,7R)-3-
Limits: [(acetyloxy)methyl] -7-[[(2Z)-2-[2- (formylamino)thiazol-4-yl] -
— impurities A , B, C, D, E, F: for each impurity, not more 2-(methoxyimino)acetyl] amino] -8-oxo-5-thia-1-
than the area of the principal peak in the chromatogram azabicydo[4.2.0]oct-2-ene-2-carboxylic add
obtained with reference solution (b) ( 1.0 per cent); (iV-formylcefotaxime),
— any other impurity: for each impurity, not more than
(0.2 per cent);
2016 Cefoxitin Sodium 1-455
Cefoxitin Sodium *****

+**
D . (6/2,72?)-3-[(acetyloxy)methyl]-7-[[(2E)-2-
(2-aminothiazol-4-yl)-2-(methoxyimino)acetyl] amino]-8-oxo-
5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxy!ic add
(jg-cefotaxime),
C 16H 16N 3Na0 7S2 449.4 33564-30-6
Action and use

Preparation
Cefoxitin Injection
PhEur_________________________________________________________ r
DEFINITION
E. (5ai?,6i?)-6-[[(2Z)-2-(2-aiiiinothiazol-4-yl)-2-
(methoxyimino)acetyl]amino]-5a,6-dihydro*3.i/,7i£-azeto[2,l- Sodium (6R,7S)-3-[(carbamoyloxy)methyl]-7-methoxy-8-oxo-
è]furo[3,4-d] [1,3] thiazine-1,7(AH)-dione (deacetylcefotaxime 7- [[(thiophen-2-yl) acetyl] amino]-5-thia-1-
azabicydo [4.2.0] oct-2-ene-2-carboxylate.
lactone),
Content
CHARACTERS
Appearance
White or almost white, very hygroscopic powder.
Solubility
Very soluble in water, sparingly soluble in ethanol
(96 per cent).
IDENTIFICATION
F. (6R,7R)-3-[(acetyloxy)methyl]-7-[[(2 Z)-2 -[2-[[[(6R,7R)-7- A. Infrared absorption spectrophotometry (2.2.24).
[[(2Z)-2-(2-aminothiazol-4-yl)-2- Comparison cefoxitin sodium CRS.
(metiioxyimino) acetyl] amino]-2-carboxy-8-oxo-5-thia-1- B. It gives reaction (a) of sodium (2.3.1).
azabicydo[4.2.0] oct-2-en-2-yl] methyl] amino] thiazol-4-yl] -2-
(methoxyimino) acetyl] amino] -8-oxo-5-thia-1- TESTS
azabicydo [4.2.0] oct-2 -ene-2 -carboxylic add Solution S
(cefotaxime dimer), Dissolve 2.50 g in carbon dioxide-free water R and dilute to
Solution S is clear (2.2.1) and pot more intensely coloured
than intensity 5 of the range of reference solutions of the
most appropriate colour (2.2.2, Method II).
pH (2.2.3)
4.2 to 7.0.
Dilute 2 mL of solution S to 20 mL with carbon dioxide-free
water R.
G. (6R,7R)-3-[(acetyloxy) metiiyl] -7- [[(2 Z)-2 -[2- [[(2 Z)-2- Dissolve 0.250 g in methanol R and dilute to 25.0 mL with
(2-aminothiazol-4-yl)-2- (methoxyimino) acetyl] amino] thiazol- the same solvent.
4-yI] -2-(methoxyimino) acetyl] amino] - 8-oxo-5-thia-1- Related substances
azabicydo [4.2.0] oct-2 -ene-2 -carboxylic add Liquid chromatography (2.2.29). Prepare the solutions
(ATA cefotaxime). immediately before use.
____________________!_________________ PhEur Solution A Dissolve 1.0 g of potassium dihydrogen phosphate R
and 1.8 g of anhydrous disodium hydrogen phosphate R in
1000 mL of water R. To 100 mL of the solution add
800 mL of water R, adjust to pH 7.0 with phosphoric add R
or a 40 g/L solution of sodium hydroxide R and dilute to
1000 mL with water R.
in solution A and dilute to 50.0 mL with solution A.
1-456 Cefoxitin Sodium 2016
Reference solution (a) Dilute 1.0 mL of the test solution to — the area of the principal peak in the
disregard limit,
100.0 mL with solution A. chromatogram obtained with reference solution (b)
Reference solution (b) Dilute 1.0 mL of reference solution (a) (0.05 per cent).
to 20.0 mL with solution A. W ater (2.5.12)
Reference solution (c) Dissolve 5 mg of cefoxitin for peak Maximum 1.0 per cent, determined on 0.500 g.
identification CRS (containing impurities A, B, E, H, I and J) Bacterial endotoxins (2.6.14)
in solution A and dilute to 5 mL with solution A. Less than 0.13 IU/mg, if intended for use in the manufacture
Column: of parenteral preparations without a further appropriate
— size. I = 0.25 m, 0 = 4.6 mm; procedure for the removal of bacterial endotoxins.
— stationary phase: phertylsüyl silica gelfor chromatography R
ASSAY
(3.0 pm);
Mobile phase:
— mobile phase A: 1.0 g/L solution of ammoniumformate R
solvent.
adjusted to pH 2.7 with anhydrous formic acid R;
— mobile phase B: acetonitrüe R; Reference solution (a) Dissolve 25.0 mg of cefoxitin
sodium CRS in water R and dilute to 25.0 mL with the same
solvent.
Reference solution (b) Dissolve 20.0 mg of 2-(2-thienyl)acetic
acid R in water R and dilute to 25.0 mL with the same
0-5 92 8 solvent.
5-50 92->74 8-» 26 Reference solution (c) Mix 1.0 mL of reference solution (a)
5 0-8 5 74 26
and 5.0 mL of reference solution (b).
Column:
— size. I = 0.25 m, 0 = 4.6 mm;
Flow rate 1.0 mL/min. — stationary phase. octadecylsUyl silica gelfor chromatography R
Detection Spectrophotometer at 254 nm. (5 pm).
Injection 20 |xL. Mobile phase acetic acid R, acetomtrUe R, water R
(1:19:81 V/V/V).
with cefoxitin for peak identification CRS and the Flow rate 1 mL/min.
chromatogram obtained with reference solution (c) to identify Detection Spectrophotometer at 254 nm.
the peaks due to impurities A, B, E, H, I and J. Injection 20 jiL of the test solution and reference solutions (a)
Relative retention With reference to cefoxitin (retention and (c).
time = about 30 min): impurity A = about 0.83; Run time 12 min.
impurity I = about 0.98; impurity H = about 1.06; System suitability: reference solution (c):
impurity E = about 1.11; impurity B = about 1.18; — resolution: m inim u m 3.5 between the 2 principal peaks.
impurity J = about 1.66.
Calculate the percentage content of Ci 6H 16N 3Na0 7 S2 taking
into account the assigned content of cefoxitin sodium CRS.
impurities H and E; STORAGE
— peak-to-valley ratio: minimum 2.0, where Hp = height In an airtight container. If the substance is sterile, store in a
above the baseline of the peak due to impurity I and sterile, airtight, tamper-proof container.
H v = height above the baseline of the lowest point of the IMPURITIES
curve separating this peak from the peak due to cefoxitin. Specified impurities A,B, E, H, I, J
Limits:
— impurity I: not more than the area of the principal peak in present at a sufficient level, be detected by one or other of
the chromatogram obtained with reference solution (a) the tests in the monograph. They are limited by the general
( 1.0 per cent); acceptance criterion for other/unspecified impurities and/or
— impurities E, H: not more than 0.5 times the area of the
principal peak in the chromatogram obtained with (2034). It is therefore not necessary to identify these
reference solution (a) (0.5 per cent); impurities for demonstration of compliance. See also 5.10.
— impurity J: not more than 0.3 times the area of the
Control of impurities in substancesfor pharmaceutical use): C, D,
principal peak in the chromatogram obtained with F ,G .
— impurities A, B: for each impurity, not more than
(0.2 per cent);
twice the area of the principal peak in the chromatogram
— total: not more than 3 times the area of the principal peak A (6i?,75)-3-(hydroxymethyl)-7-methoxy-8-oxo-7-
in the chromatogram obtained with reference solution (a) [[(thiophen-2-yl)acetyl]amino]-5-thia-l-azabicyclo[4.2.0]oct-
(3.0 per cent); 2-ene-2 -carboxylic add (decarbamoylcefoxitin),
2016 Cefpodoxime Proxetil 1-457
H COjH 0 CO,H
N/ T A '0 NHj
and epimer at C*
B. (2RS,6 R ,75)-3-[(carbamoyloxy)methyl]-7-methoxy-8 -oxo-

7- [[(thiophen-2-yl) acetyl] amino] -5-thia-1-
azabicydo [4.2.0] oct-3-ene-2-carboxylic add
(delta-3-cefoxitiii),
G. (6i?,7S)-3-[[[[[[(6i?,7S)-2-carboxy-7-methoxy-8 -oxo-7-
[[2-(thiophen-2-yl) acetyl] amino] -5-thia-l-
azabicydo [4.2.0] oct-2-en-3-
yl] methyl] oxy] carbamoyl] oxy]methyl] -7-methoxy-8-oxo-7-
[[2-(thiophen-2-yl) acetyl] amino] -5-thia-l-
azabicyclo [4.2.0] oct-2-ene-2-carboxylic add
(cefoxitin dimer),
C. (5aR,6R)-6-[[(thiophen-2-yl)acetyl]amino]-5a,6-dihydro- H. unknown structure,
3//,7//-azeto [2,1 -b]furo [3,4-d\ [l 33 ]thiazine-1,7 (4H) -dione I. unknown structure,
(cefalotin lactone), J. unknown structure.
PhEur
Ov
V -o
V n- V
-----^ J ★ ★
Cefpodoxime Proxetil ★ ★
i l T CHj (Ph. Eur. monograph 2341)
* * * * *
D. (5aR, 6»S)-6-methoxy-6- [[(thiophen-2-yl)acetyl] amino]-

5a, ô-dihydro-3/i, 7//-azeto [2, l-i]furo [3a4-d] [l,3]thiazine-
l, 7 (4 H)-dione (cefoxitin lactone), 0 ^ .0 0 ch 3
çh3 y
n-° ° \ _ nA ^ \ 0 -CH,
S ® and
anc epimer at C*
C21H27N5O9S2 557.6 87239-81-4

E. (6R,7S)-3-[(carbamoyloxy)methyl]-7-methoxy-7-[[(2i?)-2-
Action and use
methoxy-2-(thiophen-2-yl)acetyI] amino] -8-oxo-5-thia-1-
azabicydo [4.2.0] oct-2-ene-2 -carboxylic add
((R)-methoxy cefoxitin), PhEur________________________
DEFINITION
COj H O
( 1R S)-1 -[ [( 1-Methylethoxy)carbonyl] oxy] ethyl (6R ,7R )-7-
H jC - 0 H n V | nh2 [[(22)-2-(2-aminothiazol-4-yI)-2-
(methoxyimino)acetyl]amino]-3-(methoxymethyl)-8-oxo-5-
( j I O^H S thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylate.
' — 0 ch3 Semi-synthetic product derived from a fermentation product.
Content
F. (6R,7S)-3-[(carbamoyloxy)methyl]-7-methoxy-7-[[(25)-2- 94.0 per cent to 102.0 per cent (anhydrous substance).
methoxy-2- (thiophen-2-yl) acetyl] amino] -8-oxo-5-thia-1-
azabicydo[4.2.0]oct-2-ene-2-carboxylic add CHARACTERS
((5)-methoxy cefoxitin), Appearance
White or pale yellow or light brown, amorphous powder.
Solubility
Very slightly soluble or practically insoluble in water, very
soluble in acetonitrile and in methanol, fredy soluble in
anhydrous ethanol.
IDENTIFICATION
Comparison cefpodoxime proxetil CRS.
1-458 Cefpodoxkne Proxetil 2016
TESTS System suitability:.

Diastereoisom er ratio — the chromatogram obtained with reference solution (b) is
Liquid chromatography (2.2.29) as described under Assay. similar to the chromatogram supplied with cefpodoxime
Use the normalisation procedure. proxetilfor peak identification CRS ;
Limit Test solution: — resolution: minimum 6.0 between the peaks due to
— the ratio of the area of the peak due to cefpodoxkne cefpodoxime proxetil diastereoisomers I and II in the
proxetil diastereoisomer II to the sum of the areas of the chromatogram obtained with reference solution (a);
peaks due to cefpodoxime proxetil diastereoisomers I — peak-to-vaRey ratio: minimum 1.1, where H p — height
and II is between 0.5 and 0.6. above the baseline of the peak due to diastereoisomer II
of impurity B and H v = height above the baseline of the
Related substances lowest point of the curve separating this peak from the
Liquid chromatography (2.2.29). Prepare the solutions peak due to impurity C in the chromatogram obtained
immediately before use or store them at 2-8 °C.
with reference solution (b).
Solvent mixture glacial acetic add R, acetomtrUe R, water R
Limits:
(2:99:99 V/VIV). — impurity C: not more than twice the sum of the areas of
Test solution Dissolve 50 mg of the substance to be examined the 2 principal peaks in the chromatogram obtained with
in the solvent mixture and dilute to 50.0 mL with the solvent reference solution (a) (2.0 per cent);
mixture. — impurity D (sum of the 2 diastereoisomers): not more than
Reference solution (a) Dilute 1.0 mL of the test solution to the sum of the areas of the 2 principal peaks in the
100.0 mL with the solvent mixture. chromatogram obtained with reference solution (a)
Reference solution (b) Dissolve 5 mg of cefpodoxime proxetilfor ( 1.0 per cent);
peak identification CRS (containing impurities B, C and D) in — impurity H (sum of the diastereoisomers): not more than the
5.0 mL of the solvent mixture. sum of the areas of the 2 principal peaks in the
Reference solution (c) Dissolve 5 mg of cefpodoxime proxetilfor
( 1.0 per cent);
impurity H identification CRS in 5.0 mL of the solvent
— impurity B (sum of the 2 diastereoisomers): not more than
mixture.
0.5 times the sum of the areas of the 2 principal peaks in
Column:
— sizer. I i= 0.15 m, 0 = 4.6 mm;
(0.5 per cent);
— stationary phase: end-capped octadecylsUyl silica gelfor
— any other impurity: for each impurity, not more than
— temperature: maintain at a constant temperature of 20 °C. the chromatogram obtained with reference solution (a)
Mobile phase: (0.2 per cent);
— mobile phase A: anhydrous formic add R, methanol R, — total: not more than 4 times the sum of the areas of the
water R (1:400:600 VIVIV)-, 2 principal peaks in the chromatogram obtained with
— mobile phase B: anhydrous formic add R, water R, reference solution (a) (4.0 per cent);
methanol R (1:50:950 VIVIV)-, — disregard limit. 0.05 times the sum of the areas of the
Time Mobile phase A Mobile phase B reference solution (a) (0.05 per cent).
(min) (per cent V7V) (per cent V7V) W ater (2.5.12)
0-65 95 5 Maximum 2.5 per cent, determined on 0.500 g.
65- 145 95 -» 15 5-» 85 ASSAY
145 - 155 15 85 Liquid chromatography (2.2.29).
Solution A 20 mg/L solution of anhydrous citric acid R in
acetomtrUe R.
Detection Spectrophotometer at 254 nm. examined in solution A and dilute to 50.0 mL with
Injection 20 pL. solution A.
Identification of impurities Use the chromatogram supplied Reference solution Dissolve 30.0 mg of cefpodoxime proxetil CRS
with cefpodoxime proxetilfor peak identification CRS and the in solution A and dilute to 50.0 mL with solution A.
chromatogram obtained with reference solution (b) to Column:
identify the peaks due to impurities B, C and D; use the — sizer. I = 0.15 m, 0 = 4.6 mm;
chromatogram supplied with cefpodoxime proxetilfor — stationary phase: end-capped octadecylsUyl silica gelfor
impurity H identification CRS and the chromatogram obtained chromatography R (5 pm);
with reference solution (c) to identify the peaks due to — temperature: 40 °C.
impurity H.
Relative retention With reference to cefpodoxime proxetil
diastereoisomer II (retention time = about 58 min):
diastereoisomer I of impurity B = about 0.68; Detection Spectrophotometer at 240 nm.
diastereoisomer I of cefpodoxime proxetil = about 0.74; Injection 10 pL.
impurity C = about 0.82; diastereoisomer II of Run time 1.2 times the retention time of cefpodoxime proxetil
impurity B = about 0.85; impurity D (2 peaks) = about 0.88 diastereoisomer II.
and 1.13; peaks due to diastereoisomers of impurity H: Retention time Cefpodoxime proxetil
between about 1.9 and 2.3. diastereoisomer II = about 30 min.
2016 Cefpodoxime Proxetil 1-459

— resolution: minimum 4.0 between the peaks due to "*V°yo °ych3ch’
0 ^ 0
cefpodoxirne proxetil diastereoisomers I and II. ch3
Calculate the percentage content of C21H 27N 5O9S2 from the oN
N ^
sum of the areas of the 2 peaks due to the diastereoisomers
and using the declared content of cefpodoxirne proxetil CRS. h2n- ^
STORAGE and epimer at C*

IMPURITIES D. (1RS)-1 -[[(1 -methylethoxy)carbonyl] oxy] ethyl (6i?,7i?)-7-
[ [(2E)-2-(2-aminothiazol-4-yl)-2-
Specified impurities B, C, D, H
(methoxyimino)acetyl]amino]-3-(methoxymethyl)-8-oxo-5-
Other detectable impurities (the following substances would, if thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylate
present at a sufficient level, be detected by one or other of (anti-cefpodoxime proxetil),
the tests in'the monograph. They are limited by the general
by the general monograph Substancesfor pharmaceutical use
(2034). It is therefore not necessary to identify these HsCV°Y°Y
impurities for demonstration of compliance. See also 5.10. CH3 V 0 o CHa
Control of impurities in substances for pharmaceutical use): A , E,
F } G.
N '° V « A v ^ o - ^ CH3
ch3 co2h
S ® and
an< epimer at C*
A
Y n ' V ' ' ' ° ' ch’
n^ a ^ n--J—¡\ j E. (1RS)-1 -[[(1 -methylethoxy)carbonyl]oxy]ethyl (6R,7R)-3-
H2N- f J T H HS (acetoxymethyl)-7-[[(2Z)-2~(2-aminothiazol-4-yl)-2-
(methoxyimino)acetyl] amino]-8-oxo-5-thia-1-
azabicydo[4.2.0]oct-2-ene-2-carboxy!ate (ACA-analogue of
A. (6R,7R)-7-[ [(2Z)-2-(2-aminothiazol-4-yl)-2- cefpodoxime proxetil),
(methoxyimino) acetyl] amino]-3-(methoxymethyI)-8-oxo-5-
thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylic add
l‘,\ ,CH3
(cefpodoxime), k* V y °Y
C H, Y ° CH"
°y
Y °y
y CH3 N > -N 0 3
o. .0
CH, V ‘o ch3
N '° S^ ® and epimer at C*
H!N~ 0• y W O ’ h - 7 F. (1ÄS)-1- [[(1-methylethoxy)carbonyl] oxy] ethyl (6i?,7i?)-7-

aand
n epimer at C*
[[(22)-2-[(2-formylamino)thiazol-4-y0-2-
(methoxyimino) acetyl] amino]-3- (methoxymethyl) -8-oxo-5-
B. (1RS)-1-[[(1 -methylethoxy)carbonyl]oxy]ethyl (6R,7R)-7- thia- 1-azabicyclo [4.2.0] oct-2-ene-2-carboxylate
[[(2Z)-2-(2-aminothiazol-4-yl)-2- (iV-formyl cefpodoxime proxetil),
(methoxyimino) acetyl] amino] -3-methyl-8-oxo-5-thia-1-
azabicyclo [4.2.0] oct-2-ene-2-carboxyIate (ADCA-analogue of
cefpodoxime proxetil), h3cV ° Y ° Y CH3
ch3 V ° 0 CH’
*cV y °y ch> ch3 n '° V n A ^ o ^ 3
and epimer at C*
and epimer at C*
G. (1RS)-1-[ [(1-methylethoxy) carbonyl] oxy] ethyl (6R,7R)-7-
[[(22)- [2-(2-acetylamino)thiazol-4-yI] -2-
(methoxyimino) acetyl] amino] -3- (methoxymethyl)-8-oxo-5-
C. (lÄS)-l-[[(l-methylethoxy)carbonyl]oxy]ethyl (6R,7R)-7- thia-l-azabicyclo [4.2.0] oct-2-ene-2 -carboxylate
[[(2Z)-2-(2-aminothiazol-4-yl)-2-
(N-acetyl-cefpodoxime proxetil),
(methoxyimino)acetyrjamino]-3-(methoxymethyl)-8-oxo-5-
thia- 1-azabicydo [4.2.0]oct-3-ene-2-carboxylate
(delta-2 -cefpodoxime proxetil),
1-460 Cefprozil Monohydrate 2016
TESTS
Related substances
examined in 1 mL of a 103 g/L solution of hydrochloric acid R
and dilute to 25.0 mL with mobile phase A.
Test solution (b) Dissolve 30.0 mg of the substance to be
solvent.
Reference solution (a) Dilute 1.0 mL of test solution (a) to
100.0 mL with mobile phase A.
Reference solution (b) Dissolve 5 mg of cefprozüfor peak
identification CRS (containing impurities B, H and M) in
0.05 mL of a 103 g/L solution of hydrochloric acid R and add
and diastereoisomers at C1 and C1' 1 mL of mobile phase A.
Reference solution (c) Dissolve 3 mg of cefprozü CRS and 6 mg
H. mixture of the diastereoisomers of
of cefprozü impurity mixture CRS (containing impurities D
1 -[[(1 -methylethoxy)carbonyl]oxy]ethyl (6R,7R)-7-[[(2Z)-2-
and F) in 2 mL of a 103 g/L solution of hydrochloric acid R
[2-[[(2i?)-2-[[(2Z)-2-(2-aminothiazol-4-yl)-2-
and dilute to 50 mL with mobile phase A.
(methoxyimino)acetyl] amino] -2- [(2.R)-5-(methoxymethyl)-4-
[[1-[[(1 -methylethoxy) caibonyl] oxy] ethoxy] carbonyl] -3,6- Reference solution (d) Dissolve 30.0 mg of cefprozü CRS in
dihydro-2/f-1,3-thiazin-2-yl] acetyl] amino]thiazol-4-yl] -2- water R and dilute to 100.0 mL with the same solvent.
(methoxyimino)acetyI]amino]-3-(methoxymethyl)-8-oxo-5- Reference solution (e) Dissolve 10.0 mg of cefadroxü CRS
thia-1 -azabicyclo [4.2 .0] oct-2 -ene-2-carboxylate (impurity B) in water R and dilute to 20.0 mL with the same
(cefpodoxime proxetil dimer). solvent. Dilute 1.0 mL of the solution to 20.0 mL with
water R.
___________________________________________________________PhEur
Reference solution (f) Dissolve 10.0 mg of cefprozü
impurity A CRS in water R and dilute to 100.0 mL with the
same solvent. Dilute 1.0 mL of the solution to 10.0 mL with
water R.
Cefprozil Monohydrate ***** Column.
— size. I = 0.25 m, 0 = 4.6 mm;
— stationary phase, end-capped octadecylsüyl silica gelfor
chromatography R (5 (im);
Mobile phase:
— mobile phase A: dissolve 11.5 g of ammonium dihydrogen
phosphate R in water R, adjust to pH 4.4 with phosphoric
add R and dilute to 1000 mL with water R;
— mobile phase B: acetomtrüe R, mobile phase A (50:50 V!V)\
C is H ^ O jS , H20 407.4 121123-17-9
Action and use (min) (per cent V/V) (per cent V7V)
Cephalosporin antibacterial 0 -8 81 19
8 - 20 81 -» 36 19->64
PhEur___________________________________________________________
20 -2 5 36 64
DEFINITION
Mixture of the 2 diastereoisomers of (6R,7R)-7-[[(2R)-2-
amino-2-(4-hydroxyphenyl) acetyl] amino] -8-oxo-3- [(1EZ) - Flow rate 1.0 mL/min.
prop- 1-enyl] -5-thia- 1-azabicydo [4.2.0] oct-2 -ene-2-carboxylic Detection Spectrophotometer at 230 nm.
add monohydrate. Injection 10 nL of test solution (a) and reference solutions (a),
Semi-synthetic product derived from a fermentation product. (b), (c), (e) and (f).
Content Identification of impurities Use the chromatogram supplied
96.0 per cent to 102.0 per cent (anhydrous substance). with cefprozUfor peak identification CRS and the
CHARACTERS chromatogram obtained with reference solution (b) to
identify the peaks due to impurities B} H and M; use the
Appearance
White or yellow, crystalline powder, slightly hygroscopic. chromatogram supplied with cefprozU impurity mixture CRS
and the chromatogram obtained with reference solution (c)
Solubility to identify the peaks due to impurities D and F; impurities G
Slightly soluble in water and in methanol, practically and I are identified by their rdative retention.
insoluble in acetone.
Relative retention With reference to cefprozil (Z)-isomer
IDENTIFICATION (retention time = about 7 min): impurity A = about 0.4;
Infrared absorption spectrophotometry (2.2.24). impurity B = about 0.5; impurity D = about 0.7;
Comparison cefprozU CRS. impurity F = about 0.9; cefprozil (£)-isomer = about 1.4;
2016 Cefprozil Monohydrate 1-461
impurity G = about 1.7; impurity H = about 2.0; Other detectable impurities (the fo llo w in g substances would, if
impurity I = about 2.1; impurity M = about 2.9. present at a sufficient level, be detected by one or other of
System suitability: reference solution (c): the tests in the monograph. They are limited by the general
— resolution: minimum 1.4 between die peaks due to acceptance criterion for other/unspecified impurities and/or
impurity F and cefprozil (2)-isomer. by the general monograph Substancesfor pharmaceutical use
Limits: (2034). It is therefore not necessary to identify these
— correction factor for the calculation of content, multiply the impurities for demonstration of compliance. See also 5.10.
peak area of impurity D by 2.3; Control of impurities in substancesfor pharmaceutical use): C, E,
— impurity B: not more than the area of the principal peak F , J , K ,L ,N .
in the chromatogram obtained with reference solution (e)
(0.5 per cent); H NHa
— impurities D, G, H , 1, M: for each impurity, not more
than 0.3 times the sum of the areas of the 2 principal
peaks in the chromatogram obtained with reference
H O ^
— impurity A: not more than the area of the principal peak A. (2i?)-2-amino-2-(4-hydroxyphenyl)acetic add
in the chromatogram obtained with reference solution (f) (p-hydroxyphenylglycme),
(0.2 per cent);
— any other impurity, for each impurity, not more than
(0.2 per cent);
— disregard lirnic. 0.05 times the sum of the areas of the
B. (6£,72?)-7-[[(22?)-2-ammo-2-
reference solution (a) (0.05 per cent). (4-hydroxyphenyl) acetyl] amino] -3-methyl-8-oxo-5-thia-1-
(E)-isomer ratio azabicydo [4.2.0] oct-2-ene-2-carboxylic add (cefadroxil),
Liquid chromatography (2.2.29) as described under Assay.
Determine the area of die peak due to the (£)-isomer in the
chromatogram obtained with test solution (b) and reference
solution (d). Calculate the ratio of the (£)-isomer to the sum
of both cefprozil isomers, as determined under Assay.
Limit:
— (E)-isomer ratio: 0.06 to 0.11.
Heavy metals (2.4.8) C. (6 RS)-3-(aminomethylene)-6 -
Maximum 20 ppm. (4-hydroxyphenyl)piperazine-2,5-dione,
1.0 g complies with test C. Prepare the reference solution COjH
using 2 m l, of lead standard solution (10 ppm Pb) R.
W ater (2.5.12)
V r
H 2N ” r K ^ C H j
H H
D. (6i?,7R)-7-amino-8-oxo-3- [(1Z)-prop- 1-enyl]-5-thia-1-
ASSAY azabicydo[4.2.0]oct-2-ene-2-carboxylic add,
Mobile phase Mobile phase B, mobile phase A (18:82 V!V).
Injection 10 |iL of test solution (b) and reference solution (d).
Run lime Twice the retention time of cefprozil (Z)-isomer.
Elution order (Z)-isomer, (£)-isomer. H2N H
Retention time Cefprozil (Z)-isomer = about 8 min.

E. (6R,7R)-7- [[(2i?)-2-amino-2-[4- [[(2i?)-2-amino-2-
(4-hydroxyphenyl)acetyl] oxy]phenyI] acetyl] amino]-8-oxo-3-
[(lZ)-prop- 1-enyl] -5-thia-l -azabicydo [4.2.0] oct-2-ene-2-
cefprozil (Z)-isomer and the (£)-isomer.
carboxylic add,
Calculate the percentage content of the sum of both isomers
of cefprozil (Ci8H 19N 30 3S) taking into account the assigned c o 2h
contents of both (£)-isomer and (Z)-isomer of cefprozil CRS.
STORAGE
h H
IMPURITIES
Specified impurities A, B, D, G, H, I, M F. (6 R,7R)-7-amino-8-oxo-3-[(1£)-prop- 1-enyl]-5-thia- 1-
azabicydo[4.2.0]oct-2-ene-2-carboxylic add,
1-462 Cefradine 2016
co 2h
H nh2
h° Vï T ^
n ” H V C h3
H H
G. (2i?)-2-[(2i?)-2-amino-2-(4-hydroxyphenyl)acetyl]amino- M. (6i?,7i?)-7-[[(2i?)-2-amino-2-[4-
2-[(2i?)-4-carboxy-5- [( 12)-prop-1-enyl] -3j6-dihydro-2H - 1,3- [(ethoxycarbonyl)oxy] phenyl] acetyl] amino] -8 -oxo-3-[(12 )-
thiazin-2-yl]-acetic add. prop-1-enyl]-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic
add,
h nh2
co 2h
co 2h
h nh2
° ^ y " cH
H H
3
H H
h3c
H. (6i?j7i?)-7-[[(2i?)-2-[[(2Ä)-2-amino-2- N. (6Ä,7Ä)-7-[[(2JR)-2-amino-2-[4-
(4-hydroxyphenyl)acetyi] amino]-2- (ethoxycarbonyl)oxy] phenyl] acetyl] amino] -8-oxo-3- [(1E)-
(4-hydroxyphenyl) acetyl] amino]-8-oxo-3- [( 12)-prop-l -enyl]- prop-1-enyl]-5-thia-l -azabicydo [4.2.0] oct-2-ene-2-caiboxylic
5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic add. add.
PhEur
co 2h
.**★★
★
Cefradine ★ ★
co 2h
I. (2i?)-2-[(2i?)-2-amino-2-(4-hydroxyphenyl)acetyl]amino-2-
[(2i?)-4-carboxy-5-[( 1£)-prop-l -enyl] -3j6-dihydro-2H-1,3- CHj
thiazin-2-yl]-acetic add,
Il H H
h nh2 O
co 2h
Compound Mol. Formula Mr
C15H19N3O4S 349.4
H H S
C1QH17N3O4S 347.4
J. (6£,72Q-7-[[(22Q-2-[[(2J$-2-amino-2-
(4-hydroxyphenyl) acetyl] amino] -2- ^16^21 N3O4S 351.4
(4-hydroxyphenyI) acetyl] amino] -8-oxo-3- [(1£)-prop-1-enyl] -
5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic add,
ch 3 Action and use

T V
hhn' W ^ h Preparations
Cefradine Capsules
^NHH Cefradine Injection
Cefradine Oral Suspension
and epimers at C*
PhEur________________________
K. mixture of 4 diastereoisomers of (3RS}6RS)-3-[(2R35R)-5- DEFINITION
ethyl-7-oxo-1,2,5,7-tetrahydro-4H-furo [3,4-d\ [1,3]thiazin-2- Main component (6i?,7i?)-7-[[(2i?)-amino(cyclohexa-l,4-
yl]-6-(4-hydroxyphenyl)piperazine-2,5-dione, dienyl) acetyl] amino]-3-methyl-8-oxo-5-thia-1-
azabicydo [4.2.0]oct-2-ene-2-carboxylic add (cefradine).
h nh2
Content
— cefradine: minimum 90.0 per cent (anhydrous substance);
— cefalexm: maximum 5.0 per cent (anhydrous substance);
— 4',5'-dibydrocefradine: maximum 2.0 per cent (anhydrous
L. 2-hydroxyethyl (2i?!)-2-amino-2-(4-hydroxyphenyl) acetate.
substance);
2016 Cefradine 1-463
— sum of the percentage contents of cefradine, cefalexin and Mobile phase:

4',5'-dihydrocefradine: 96.0 per cent to 102.0 per cent — mobile phase A: 2.72 g/L solution of potassium dihydrogen
(anhydrous substance). phosphate R adjusted to pH 3.0 with dilute phosphoric
add R;
CHARACTERS
— mobile phase B: methanol R2',
Appearance
White or slightly yellow, hygroscopic powder.
Solubility
Sparingly soluble in water, practically insoluble in ethanol
0 -2 .5 99-5*97 0 .5 * 3
(96 per cent) and in hexane.
2.5- 11 97*75 3 * 25
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24). 11 - 13 75*60 25*40
Comparison cefradine CRS. 13- 16 60 40
If the spectra obtained in the solid state show differences, 16 - 19 60*20 40*80
dissolve 30 mg of the substance to be examined and 30 mg
19- 19.1 20 * 99.5 80 * 0.5
of the reference substance separately in 10 mL of methanol R,
evaporate to dryness at 40 °C at a pressure less than 2 kPa 19.1 - 25 99.5 0.5
and record new spectra using the residues.
TESTS Flow rate 1 .0 m l/min.
Solution S Detection Spectrophotometer at 220 nm.
Dissolve 2.50 g in sodium carbonate solution R and dilute to
25.0 mL with the same solvent. Injection 25 |iL.
Solution S is not more opalescent than reference with cefradine for peak identification CRS and the
suspension II (2.2.1). Allow solution S to stand for 5 min. chromatogram obtained with reference solution (d) to
The absorbance (2.2.25) of solution S measured at 450 nm is identify the peaks due to impurities C, D and E; use the
not greater than 0.60. chromatogram obtained with reference solution (a) to
identify the peak due to impurity B; use the chromatogram
pH (2.2.3) supplied with cefradine impurity mixture CRS and the
3.5 to 6.0. chromatogram obtained with reference solution (e) to identify
Dissolve 0.100 g in carbon dioxide-free water R and dilute to the peaks due to impurities A and G.
10 mL with the same solvent. Relative retention With reference to cefradine (retention
Specific optical rotation (2.2.7) time = about 15 min): impurity A —about 0.27;
+ 80.0 to + 90.0 (anhydrous substance). impurity B = about 0.32; impurity C = about 0.53;
Dissolve 0.250 g in acetate buffer solution p H 4.6 R and dilute impurity D = about 0.63; impurity E = about 0.80;
to 25.0 mL with the same solution. impurity F = about 0.92; cefalexin = about 0.95; 4',5'-
dihydrocefradine = about 1.06; impurity G = about 1.32.
Related substances
cefalexin and cefradine.
examined in mobile phase A and dilute to 50.0 mL with
Limits'.
mobile phase A.
Reference solution (a) Dissolve 3.0 mg of cydohexa-1,4-
peak area of impurity B by 3.4;
dienylgiydne CRS (impurity B) in mobile phase A and dilute
— impurities A , B, C, D, E, F, G: for each impurity, not
to 100.0 mL with mobile phase A. more than 0.25 times the area of the principal peak in the
Reference solution (b) Dissolve 3 mg of the substance to be chromatogram obtained with reference solution (c)
exam in e d and 3 mg of cefalexin monohydrate CRS in mobile (0.25 per cent);
phase A and dilute to 25 mL with mobile phase A. — any other impurity, for each impurity, not more than
Reference solution (c) Dilute 1.0 mL of the test solution to 0.25 times the area of the principal peak in the
100.0 mL with mobile phase A chromatogram obtained with reference solution (c)
Reference solution (d) Dissolve 6 mg of cefradinefor peak (0.25 per cent);
identification CR S (containing impurities C, D and E) in — total: not more than twice the area of the principal peak in
1.0 mL of mobile phase A. the chromatogram obtained with reference solution (c)
Reference solution (e) Dissolve the contents of a vial of
(2.0 per cent);
cefradine impurity mixture CRS (impurities A and G) in
1.0 mL of mobile phase A. the chromatogram obtained with reference solution (c)
(0.05 per cent); disregard the peaks due to cefalexin and
Column:
4 ',5 '-dihydrocefradine.
— sizer. I — 0.15 m, 0 = 4.6 mm;
— stationary phase: octadecylsUyl silica gelfor chromatography R N J i- Dimethylaniline (2.4.26, Method B)
(5 Mm); Maximum 20 ppm.
— temperature: 30 °C. W ater (2.5.12)
Maximum. 0.2 per cent, determined on 1.0 g.
1-464 Cefradine 2016
ASSAY
Test solution Dissolve 50.0 mg of die substance to be
examined in phosphate buffer solution p H 5.0 R and dilute to
100.0 mL with die same solution. B. (2i?)-amino(cyclohexa-l,4-dienyl)acetic add
Reference solution (a) Dissolve 50.0 mg of cefradine CRS (D-dihydrophenylglycme, cyclohexa-1,4-dienylglydne),
(containing 4 /,5'-dihydrocefradine) in phosphate buffer solution
p H 5.0 R and dilute to 100.0 mL with the same solution. COj H
Reference solution (b) Dissolve 5.0 mg of cefalexin
monohydrate CRS in phosphate buffer solution pH 5.0 R and HNH2H
dilute to 100.0 mL with the same solution.
Reference solution (c) Dilute 1 ml. of reference solution (a) to
10 m l. with phosphate buffer solution p H 5.0 R. Mix 5 mL of
this solution and 5 m l, of reference solution (b).
C. (6i?,7i?)-7-[[(2i?)-amino(cyclohexa-l,4-
Column:
dienyl) acetyl] amino] -3-methyl-8-oxo-5-thia-1-
— size: I = 0.10 m, 0 = 4.6 mm; azabicyclo[4.2.0]oct-2-ene-2-carboxylic add 5-oxide
— stationary phase: octadecylsHyl silica gelfor chromatography R (isomer 1),
(5 |im).
D. (6i?,7i?)-7-[[(2i?)-amino(cyclohexa-l,4-
Mobile phase methanol R, phosphate buffer solution p H 5.0 R
dienyl) acetyl] amino] -3-methyl-8-oxo-5-thia-1-
(25:75 V!V).
azabicyclo[4.2.0]oct-2-ene-2-carboxylic add 5-oxide
Flow rate 1.5 mL/min. (isomer 2 ),
Injection 5 |xL. COj H
Run time Twice the retention time of cefradine. ch3
Relative retention With reference to cefradine (retention

time = about 3 min): cefalexm = about 0.7; H H
4 /j5 /-dihydrocefradine = about 1.5. O
OH
— resolution: minimum 4.0 between the peaks due to E. (6i?,7i?)-7-[[(2i?)-amino(2-hydroxyphenyl)acetyl]aniino]-
cefalexin and cefradine. 3-methyl-8-oxo-5-thia-l-azabicydo[4.2.0]oct-2-ene-2-
Calculate the percentage content of cefradine using the carboxylic add,
chromatogram obtained with reference solution (a) and
taking into account the assigned content of cefradine CRS. ho ch3
Calculate the percentage content of cefalexin using the
chromatogram obtained with reference solution (b) and
taking into account the assigned content of cefalexin
monohydrate CRS. Calculate the percentage content of
4/,5 /-dihydrocefradine using the chromatogram obtained F. 3-hydroxy-4-methylthiophen-2(5ii)-one,
with reference solution (b), taking into account the assigned
content of cefalexin monohydrate CRS and multiplying the CO2H
area of the peak due to 4 /,5'-dihydrocefradine by a
h 3c ch3
correction factor of 1.6 .
STORAGE A "
In an airtight container, protected from light, at a o
temperature of 2 °C to 8 °C.
G. (6i?,7i?)-7-[(2,2-dimethylpropanoyl)anuno]-3-methyl-8-
IMPURITIES oxo-5-thia-1 -azabicydo [4.2.0] oct-2-ene-2-carboxylic add
Specified impurities: A, B, C, D, E, F, G. (7-ADCA pivalamide).
COjH
_________________________ I___________ PhEur
H,N 1 I ' S '

H H
A. (6R,7R)-7-amino-3-methyl-8-oxo-5-thia-1-
azabicyclo [4.2.0] oct-2-ene-2-carboxylic add
(7-aminodeacetoxycephalosporanic add, 7-ADCA),
2016 Ceftazidime 1-465
Column'.
Ceftazidime Pentahydrate ★ ★ — sizer. I = 0.25 m, 0 = 4,6 mm;
Ceftazidime ***** — stationary phase, octadecylsüyl silica gelfor chromatography R

(5 pm);
— temperature'. 40 °C.
Mobile phase:
— mobile phase A: solution containing 3.6 g/L of disodium
hydrogen phosphate R and 1.4 g/L of potassium dxhydrogen
5 H 20
phosphate R, adjusted to pH 3.4 with a 10 per cent V/V
solution of phosphoric acid R',
— mobile phase B: acetonitrilefor chromatography R',
C^HzjNeOTS^HaO 637 78439-06-2 Time Mobfle phase A Mobile phase B

Action and use 0 -4 96*89 4 * 11
4 -5 89 11
Preparations
5 -8 89*84 11 * 16
Ceftazidime Injection
Ceftazidime Eye Drops 8 -11 84*80 1 6*2 0
11 - 15 80*50 20*50
PhEur__________________________________________________________
15-18 50*20 50*80
DEFINITION
(6i?j7i?)-7-[[(2Z)-2-(2-Aminothiazol-4-yl)-2-[(l-carboxy-l- 1 8-22 20 80
methylethoxy)imino] acetyl] amino] -8-oxo-3- [(pyridin- 1-ium-
1-yl)methyl] -5-thia-l -azabicyclo [4.2.0] oct-2-ene-2- Flow rate 1.3 mL/min.
caiboxylate pentahydrate.
Injection 10 jiL.
Content Relative retention With reference to ceftazidime (retention
95.0 per cent to 102.0 per cent (anhydrous substance). time = about 8 min): impurity F = about 0.4;
CHARACTERS impurity G = about 0.8; impurity A = about 0.9;
Appearance impurity B = about 1.4.
White or almost white, crystalline powder. Identification of impurities Use the chromatogram supplied
Solubility with ceftazidime for peak identification CRS and the
Slighdy soluble in water and in methanol, practically chromatogram obtained with reference solution (c) to identify
insoluble in acetone and in ethanol (96 per cent). It dissolves the peaks due to impurities A and G; use the chromatogram
in acid and alkali solutions. obtained with reference solution (b) to identify the peak due
to impurity B.
IDENTIFICATION
System suitability, reference solution (c):
Comparison ceftazidime CRS. impurity A and ceftazidime.
TESTS Limits:
Solution S — correction factor, for the calculation of content, multiply the
Dissolve 0.25 g in carbon dioodde-free water R and dilute to peak area of impurity G by 3.0;
50 mL with the same solvent. — impurities A, B, G: for each impurity, not more than the
Appearance of solution area of the principal peak in the chromatogram obtained
Solution S is clear (2.2.1) and colourless (2.2.2, Method IT). with reference solution (a) (0.2 per cent);
pH (2.2.3) 0.5 times the area of the principal peak in the
3.0 to 4.0 for solution S. chromatogram obtained with reference solution (a)
Related substances (0.10 per cent);
Liquid chromatography (2.2.29). — total: not more than 5 times the area of the principal peak
Test solution Suspend 0.150 g of the substance to be in the chromatogram obtained with reference solution (a)
examined in 5 mL of acetonitrUe R, dissolve by adding ( 1.0 per cent);
water R and dilute to 100 mL with water R. — disregard limit. 0.25 times the area of the principal peak in
Reference solution (a) To 1.0 mL of the test solution add the chromatogram obtained with reference solution (a)
5.0 mL of acetonitrUe R and dilute to 100.0 mL with water R. (0.05 per cent); disregard the peak due to impurity F.
Dilute 1.0 mL of this solution to 5.0 mL with water R. Im purity F
Reference solution (b) In order to prepare impurity B in situ, Liquid chromatography (2.2.29). Prepare the solutions
expose 5 mL of the test solution to ultraviolet light at immediately before use.
254 nm for about 24 h. Phosphate buffer solution Prepare a 10 per cent V/V solution of
Reference solution (c) Suspend 3 mg of ceftazidimefor peak phosphate buffer solution p H 7.0 R4.
identification CRS (containing impurities A and G) in 0.5 mL Test solution Dissolve 0.500 g of the substance to be
of acetonitrile R, dissolve by adding water R and dilute to examined in phosphate buffer solution and dilute to
2 mL with water R. 100.0 mL with the same solution.
1-466 Ceftazidime 2016
Reference solution (a) Dissolve 1.00 g of pyridine R in water R System suitability, reference
solution (b):
and dilute to 100.0 mL with the same solvent. Dilute 5.0 mL — resolution: minimum 1.5 between the peaks due to
of the solution to 200.0 mL with water R. Dilute 1.0 mL of impurity A and ceftazidime.
this solution to 100.0 mL with phosphate buffer solution. Calculate the content of ceftazidime (C22H 22N 60 7S2) taking
Reference solution (b) Dilute 1 mL of the test solution to into account the assigned content of C22H 22N 60 7S2 in
200 mL with phosphate buffer solution. To 1 mL of this ceftazidime CRS.
solution add 20 mL of reference solution (a) and dilute to STORAGE
200 mL with phosphate buffer solution.
Column: sterile, airtight, tamper-proof container.
— size: I = 0.25 m, 0 = 4.6 mm;
— stationary phase. octadecylsUyl silica gelfor chromatography R IMPURITIES
(5 nm). Specified impurities A,B, F, G
Mobile phase Mix 8 volumes of a 28.8 g/L solution of Other detectable impurities(the following substances would, if
ammonium dihydrogen phosphate R previously adjusted to present at a sufficient level, be detected by one or other of
pH 7.0 with ammonia R, 24 volumes of acetonitrüe R and the tests in the monograph. They are limited by the general
68 volumes of water R. acceptance criterion for other/unspecified impurities and/or
Flow rate 1.0 mL/min. by the general monograph Substances for pharmaceutical use
Detection Spectrophotometer at 255 nm. impurities for demonstration of compliance. See also 5.10.
Injection 20 |iL. Control of impurities in substances for pharmaceutical use): C, E,
Run time 10 min. H.
HjC\ CO,H
— resolution: m in im u m 7.0 between the peaks due to H3C-Y" h cor
ceftazidime and impurity F.
N
Limit.
— impurity F: not more than the area of the principal peak
in the chromatogram obtained with reference solution (a) H2N - f NY l f H H -
S"-^ 0 and epimer at C*
(500 ppm).
Heavy m etals (2.4.8) A. (2i?S,6i?,7i?)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2-
Maximum 20 ppm. [(1 -carboxy-1 -methylethoxy)imino] acetyl] amino]-8-oxo-3-
1.0 g complies with test F. Prepare the reference solution [(pyridin-1-ium-1-yl) methyl] -5-thia-1-azabicyclo [4.2.0] oct-3-
using 2.0 ml, of lead standard solution (10 ppm Pb) R. ene-2 -carboxylate (A-2-ceftazidime),
W ater (2.5.12)
ASSAY
Liquid chromatography (2.2.29). B. (6i?,7i?)-7-[[(2£)-2-(2-aminothiazol-4-yl)-2-[(l-carboxy- 1-
methylethoxy)imino] acetyl] amino]-8-oxo-3- [(pyridin- 1-him-
1-yl) methyl]-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-
ex a m in ed in the mobile phase and dilute to 25.0 mL with
carboxylate,
the mobile phase.
Reference solution (a) Dissolve 25.0 mg of ceftazidime CRS in C02”
the mobile phase and dilute to 25.0 mL with the mobile
phase.
Reference solution (b) Dissolve 5.0 mg of ceftazidime for peak
H H
identification CR S (containing impurities A and G) in the
mobile phase and dilute to 5.0 mL with the mobile phase. C. (6i?,7i?)-7-amino-8-oxo-3-[(pyridin-l-him-l-yl)methyl]-5-
Column: thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylate,
— size. I = 0.15 m, 0 = 4.6 mm;
h3c ch3
— stationary phase: hexylsifyl silica gelfor chromatography R V
(5 ^m). o ch3
Mobile phase Dissolve 4.3 g of disodium hydrogen phosphate R
and 2.7 g of potassium dihydrogen phosphate R in 980 mL of
water R, then add 20 mL of acetonitrüe R.
Flow rate 2 mlVmin.
Injection 20 |iL.
Run time 6 m in . E. (6Æ,7iQ-7-[[(22)-2-(2-aminothiazol-4-yl)-2-[[2-(l,l-
Relative retention With
reference to ceftazidime (retention dimethylethoxy)-l, 1-dimethyl-2 -
time = about 4.5 min): impurity A = about 0.7. oxoethoxy] imino] acetyl] amino] -8-oxo-3- [(pyridin-1-ium-1-
yl) methyl]-5-thia-1-azabicyclo [4.2.0] oct-2 -ene-2-carboxylate,
2016 Ceftazidime Pentahydrate with Sodium Carbonate for Injection 1-467
TESTS
Solution S
F. pyridine. 20.0 mL with the same solvent.
H3C r n u Solution S is clear (2.2.1) and its absorbance (2.2.25) at
„0
pH (2.2.3)
,CHO 5.0 to 7.5 for solution S.
H2N—^
Related substances
Test solution Suspend 0.150 g of the substance to be
G. 2-[ [[( 1Z )-1-(2-aminothiazol-4-yl)-2-[(oxoethyl) amino]-2 -
oxoethylidene] amino] oxy]-2-methyipropanoic acid, examined in 5 mL of acetonitrüe R, dissolve by adding
water R and dilute to 100 mL with water R.
och3 Reference solution (a) To 1.0 mL of the test solution add
5.0 mL of acetonitrüe R and dilute to 100.0 mL with water R.
Dilute 1.0 mL of this solution to 5.0 mL with water R.
Reference solution (b) In order to prepare impurity B in situ,
expose 5 mL of the test solution to ultraviolet light at
254 nm for about 24 h.
Reference solution (c) Suspend 3 mg of ceftazidime for peak
identification CRS (containing impurities A and G) in 0.5 ml.
H. (6i?,7i?)-7-[[(22)-2-(2-axninothiazol-4-yl)-2-[(2-met±ioxy- of acetonitrüe R, dissolve by adding water R and dilute to
1 j 1-dime thyl-2-oxoethoxy) imino] acetyl] amino] -8-oxo-3- 2 mL with water R.
[(pyridin-1-ium-1-yl) methyl]-5-thia-1-azabicyclo [4.2.0] oct-2- Column:
ene-2 -carboxylate. — sizer. I = 0.25 m, 0 = 4.6 mm;
PhEur — stationary phase: octadecylsQyl silica gelfor chromatography R
(5 pm);
Mobile phase:
— mobile phase A: solution con tainin g 3.6 g/L of disodium
Ceftazidime Pentahydrate with ;* * * *
hydrogen phosphate R and 1.4 g/L of potassium dihydrogen
Sodium Carbonate for Injection ***** phosphate R, adjusted to pH 3.4 with a 10 per cent VIV
(Ph Ettr monograph 2344) solution of phosphoric add 2?;
— mobile phase B: acetonitrüefor chromatography R;
Action and use
Preparation
Ceftazidime Injection
0 -4 9 6 -»89 4 - » 11
Ceftazidime Eye Drops
4 -5 89 11
PhEur__________________________________________________________
5 -8 8 9 -»84 11-»16
DEFINITION
Sterile mixture of Ceftazidime pentahydrate (1405) and 8-11 8 4 -»80 16-»20
Anhydrous sodium carbonate (0773). 11-15 8 0 -»50 20 -»50
Semi-synthetic product derived from a fermentation product. 15-18 5 0 -»20 5 0 -»80
Content 18-22 20 80
— ceftazidime: 93.0 per cent to 105.0 per cent (dried and
carbonate-free substance);
— sodium carbonate: 8.0 per cent to 10.0 per cent. Flow rate 1.3 mlVmin.
CHARACTERS Detection Spectrophotometer at 254 nm.
Appearance Injection 10 jiL.
White or pale yellow powder. Relative retention With reference to ceftazidime (retention
Solubility. time = about 8 min): impurity F = about 0.4;
Freely soluble in water and in methanol, practically insoluble impurity G = about 0.8; impurity A = about 0.9;
in acetone. impurity B = about 1.4.
IDENTIFICATION Identification of impurities Use the chromatogram supplied
A. Examine the chromatograms obtained in the assay. with ceftazidime for peak identification CRS and the
Results The principal peak in the chromatogram obtained
the peaks due to impurities A and G; use the chromatogram
with the test solution is similar in retention time to the
obtained with reference solution (b) to identify the peak due
principal peak in the chromatogram obtained with reference
to impurity B.
solution (a).
B. It gives the reaction of carbonates (2.3.1).
1-468 Ceftazidime Pentahydrate with Sodium Carbonate for Injection 2016
System suitability: reference solution (c): ASSAY

— resolution: minimum 4.0 between the peaks due to Ceftazidime
impurity A and ceftazidime. Liquid chromatography (2.2.29).
Limits'. Test solution Dissolve 25.0 mg of the substance to be
— correction factor,for the calculation of content, multiply the examined in the mobile phase and dilute to 25.0 m l , with
peak area of impurity G by 3.0; the mobile phase.
— impurities A, B, G: for each impurity, not more than the Reference solution (a) Dissolve 25.0 mg of ceftazidime CRS in
area of the principal peak in the chromatogram obtained the mobile phase and dilute to 25.0 mL with the mobile
with reference solution (a) (0.2 per cent); phase.
Reference solution (b) Dissolve 5.0 mg of ceftazidime for peak
identification CRS (containing impurities A and G) in the
mobile phase and dilute to 5.0 mL with the mobile phase.
(0.10 per cent);
Column:
in the chromatogram obtained with reference solution (a) — size. I = 0.15 m, 0 = 4.6 mm;
( 1.0 per cent); — stationary phase. hexylsUyl silica gelfor chromatography R
— disregard limit. 0.25 times the area of the principal peak in (5 pm).
the chromatogram obtained with reference solution (a) Mobile phase Dissolve 4.3 g of disodium hydrogen phosphate R
(0.05 per cent); disregard the peak due to impurity F. and 2.7 g of potassium dihydrogen phosphate R in 980 mL of
water R, then add 20 mL of acetonitrile R.
Im purity F
Liquid chromatography (2.2.29). Prepare the solutions Flow rate 2 mL/min.
immediately before use. Detection Spectrophotometer at 245 nm.
Phosphate buffer solution Prepare a 10 per cent V/V solution of Injection 20 pL.
phosphate buffer solution p H 7.0 R4. Run time 6 min.
Test solution Dissolve 0.500 g of the substance to be Relative retention With reference to ceftazidime (retention
examined in phosphate buffer solution and dilute to time = about 4.5 min): impurity A = about 0.7.
100.0 mL with the same solution. System suitability: reference solution (b):
Reference solution (a) Dissolve 1.00 g of pyridine R in water R — resolution: m in im u m 1.5 between the peaks due to
and dilute to 100.0 mL with the same solvent. Dilute 5.0 mL impurity A and ceftazidime.
of the solution to 200.0 mL with water R. Dilute 1.0 mL of Calculate the content of ceftazidime (C22H 22N 60 7S2) taking
this solution to 100.0 mL with phosphate buffer solution. into account the assigned content of C 22H 22N 60 7S2 in
Reference solution (b) Dilute 1 .0 mL of the test solution to ceftazidime CRS.
200.0 mL with phosphate buffer solution. To 1.0 mL of this Sodium carbonate
solution add 20.0 mL of reference solution (a) and dilute to Atomic absorption spectrometry (2.2.23, Method I).
200.0 mL with phosphate buffer solution.
Caesium chloride buffer solution To 12.7 g of caesium chloride R
Column:
add 500 mL of water R and 86 mL of hydrochloric acid R and
— size. I = 0.25 m, 0 = 4.6 mm;
dilute to 1000.0 mL with water R.
— stationary phase: octadecylsilyl silica gel for chromatography R
Sodium standard solution (1000 mg/L) Dissolve 3.70 g of
(5 pm).
sodium nitrate R in water R and dilute to 500 mL with the
same solvent, add 48.5 g of nitric acid R and dilute to
ammonium dihydrogen phosphate R previously adjusted to
pH 7.0 with ammonia R, 24 volumes of acetonitrUe R and
68 volumes of water R.
exam in ed in water R and dilute to 100.0 mL with the same
Flow rate 1.0 mL/min. solvent. To 10.0 mL of this solution add 5.0 mL of caesium
Detection Spectrophotometer at 255 nm. chloride buffer solution and dilute to 50.0 mL with water R.
Injection 20 pL. Reference solution Into 4 identical flasks, each containing
Run time 10 min. 20.0 mT. of caesium chloride buffer solution, introduce
System suitability: reference solution (b): respectively 0 mL, 5.00 ml,, 10.00 mL and 15.00 mL of
— resolution: m in im u m 7.0 between the peaks due to sodium standard solution (1000 mg/L) and dilute to
ceftazidime and impurity F. 200.0 mL with water R.
Limit. Source Sodium hollow-cathode lamp.
— impurity F: not more than 6 rimes the area of the Wavelength 330.2 nm to 330.3 nm.
principal peak in the chromatogram obtained with Atomisation device Air-acetylene flame.
reference solution (a) (0.3 per cent).
Calculate the percentage content of sodium carbonate.
Maximum 13.5 per cent, determined on 0.300 g. Dry at STORAGE
25 °C at a pressure not ex cee d in g 0.67 kPa for 4 h then heat In a sterile, airtight, tamper-proof container, protected from
the residue at 100 °C at a pressure not exceeding 0.67 kPa light and humidity.
for 3 h. LABELLING
Bacterial endotoxins (2.6.14) The label states the percentage content m/m of ceftazidime.
Less than 0.10 IU/mg, if intended for use in the manufacture IMPURITIES
of parenteral preparations without a further appropriate Specified impurities A, B, F, G.
2016 Ceftriaxone Sodium 1-469
H3C
Other detectable impurities (the following substances would, if h,c^ ' COîH
the tests in the monograph. They are limited by die general
acceptance criterion for other/unspecified impurities and/or ,CHO
by the general monograph Substancesfor pharmaceutical use h2n—^
Control of impurities in substancesfor pharmaceutical use): G. 2-[[[(12)-l-(2-aminothiazol-4-yl)-2- [(oxoethyl)amino]- 2-
C, E, H. oxoethylidene] amino] oxy] -2-methylpropanoic add,
h2n—^
and epimer at C*
A. (2i?S,6i?,7i?)-7-[[(22)-2-(2-aminothiazol-4-yI)-2- H. (6R,7R)-7-[ [(22)-2-(2-aminothiazol-4-yl)-2- [(2-methoxy-

[(1-carboxy-1-methylethoxy)imino] acetyl] amino] -8-oxo-3- I,1 -dimethyi-2-oxoethoxy)imino] acetyl] amino] -8-oxo-3-
[(pyridin-1-ium-1-yl)methyl]-5-thia-1-azabicyclo [4.2.0] oct-3- [(pyridin-1-ium-1-yl)methyl]-5-thia-1-azabicyclo [4.2.0] oct-2-
ene-2-carboxylate (A-2-ceftazidime), ene-2 -carboxylate.
PhEur
ho2c
Ceftriaxone Sodium *****

★ ★
* *
B. (6R,7R)-7-[[(2£)-2-(2-aminothiazol-4-yl)-2-[(1-carboxy-1-
methylethoxy)imino] acetyl] amino] -8-oxo-3- [(pyridin-1-him- ONa
1 -yl)methyl] -5-thia-1-azabicyclo [4.2.0] oct-2-ene-2- CH3 COjNa N Y
Ni
carboxylate. vV - N SA Ni
h2n- ^ 11
31/j H20
H,N- QgHjeNaNaiOvSa^ViHiO 662 104376-79-6

H H
Action and use
C. (6 R ,7R)-7-amino-8-oxo-3-[(pyridin-1-ium-1-yl)methyl]-5- Cephalosporin antibacterial.
thia- 1-azabicyclo [4.2.0]oct-2-ene-2-carboxylate, Preparation
Ceftriaxone Injection
PhEur_____________________________
DEFINITION
Disodium (6i?,7/Q-7-[[(22)-(2-aminothiazol-4-
yl) (methoxyimino) acetyl] amino] -3- [[(2-methyl-6-oxido-5-
oxo-2,5-dihydro-1,2,4-triazin-3-yl)sulfanyI] methyl] -8-oxo-5-
thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylate 3.5 hydrate.
Semi-synthetic product derived from a fermentation product
Content
E. (6R,7K)-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2- [[2-( 1,1- 96.0 per cent to 102.0 per cent (anhydrous substance).
dimethylethoxy)-l, l-dimethyl-2 - CHARACTERS
oxoethoxy] imino] acetyl] amino] -8-oxo-3- [(pyridin-1-ium-1-
Appearance
yl)mediyl]-5-thia-l-azabicyclo[4.2.0]oct-2-ene-2-carboxylate,
Almost white or yellowish, slighdy hygroscopic, crystalline
powder.
Solubility
Freely soluble in water, sparingly soluble in methanol, very
slighdy soluble in anhydrous ethanol.
F. pyridine, IDENTIFICATION
Comparison ceftriaxone sodium CRS.
1-470 Ceftriaxone Sodium 2016
B. It gives reaction (a) of sodium (2.3.1). W ater (2.5.12)

TESTS
Solution S Bacterial endotoxins (2.6.14)
Dissolve 2.40 g in carbon dioxide-free water R and dilute to Less than 0.08 IU/mg, if intended for use in the manufacture
20.0 mL with the same solvent. of parenteral preparations without a further appropriate
The solution is clear (2.2.1) and not more intensely coloured ASSAY
than reference solution Y5 or BY5 (2.2.2). Liquid chromatography (2.2.29) as described in the test for
Dilute 2 mL of solution S to 20 mL with water R. related substances with the following modification.
pH (2.2.3)
6.0 to 8.0 for solution S. Calculate the percentage content of CisHiôNsNaiOySa from
the declared content of ceftriaxone sodium CRS.
—155 t o —170 (anhydrous substance). STORAGE
Dissolve 0.250 g in water R and dilute to 25.0 mL with the In an airtight con tain er protected from light. If the substance
same solvent. is sterile, store in a sterile, airtight, tamper-proof container.
Related substances IMPURITIES
examined in the mobile phase and dilute to 100.0 mL with ch3 c o 2h N'
the mobile phase. -A.N „NH
Reference solution (a) Dissolve 30.0 mg of ceftriaxone N V -N S
sodium CRS in the mobile phase and dilute to 100.0 mL with
the mobile phase. S"' o iH i
Reference solution (b) Dissolve 5.0 mg of ceftriaxone
sodium CRS and 5.0 mg of ceftriaxone impurity A CRS in the
A. (6i?,7i?)-7-[[(2£)-(2-aminothiazol-4-
yl) (methoxyimino) acetyl] amino] -3- [[(2-methyl-5,6-dioxo-
Reference solution (c) Dilute 1.0 mL of the test solution to
l,2,5,6-tetrahydro-l,2,4-triazin-3-yl)sulfanyl]methyl]-8-oxo-5-
100.0 mL with the mobile phase. thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylic add
Column'. ((^-isomer),
— size: I = 0.25 m, 0 = 4.6 mm;
— stationary phase: octadecylsUyl silica gel for chromatography R
(5 |im). ch3 Vo
Mobile phase Dissolve 2.0 g of tetradecylammonium bromide R N^> V n' V
and 2.0 g of tetraheptylammomum bromide R in a mixture of
440 mL of water R, 55 mL of 0.067 Mphosphate bitffer
solution p H 7.0 R, 5.0 mL of citrate buffer solution pH 5.0 o— o
prepared by dissolving 20.17 g of citric add R in 800 mL of
water R, adjusting to pH 5.0 with strong sodium hydroxide
B. (5a£,6Æ)-6-[[(2Z)-(2-aminothiazol-4-
solution R and diluting to 1000.0 mL with water R, and
500 mT. of acetonitrüe R. yl) (methoxyimino) acetyl] amino] -5 a, 6-dihydro-3//, 1H-
azeto [2,1 -b]furo [3,4-d\ [1,3]thiazine-1,7 (4H)-dione,
Injection 20 jiL of the test solution and reference solutions (b)
and (c).
Run time Twice the retention time of ceftriaxone. HS
sK N.nh
I
System suitability: reference solution (b): ch3
ceftriaxone and impurity A. C. 2-methyl-3-sulfanyl-1,2-dihydro-1,2,4-triazine-5,6-dione,
Limits:
— any impurity: not more than the area of the principal peak ch 3
in the chromatogram obtained with reference solution (c) i
( 1.0 per cent);
(4.0 per cent);
h2n- ^
yrr
(0.1 per cent). D. 5-benzothiazol-2-yl (2Z)-(2-aminothiazol-4-
yl) (methoxyimino) thioacetate,
N ,N - Dimethylaniline (2.4.26, Method B)
Maximum 20 ppm.
2016 Cefuroxime Axetdl 1-471
o Test solution Dissolve 10.0 mg of the substance to be

CO2H H ^ fC examined in the mobile phase and dilute to 50.0 mL with
\V-N ^ Y ^ S JL the mobile phase.
^ N ,nh Reference solution (a) Dilute 1.0 mL of the test solution to
’n--Hv
h "I
H H
CH3 100.0 mL with the mobile phase.
Reference solution (b) In order to prepare in situ impurity A,
heat 5 mL of the test solution at 60 °C for 1 h.
E. (6i?j7i?)-7-amino-3-[[(2-methyl-5j6-dioxo-lj2j5j6-
Reference solution (c) In order to prepare in situ impurity B,
tetrahydro-1,2,4-triazin-3-yl)sulfanyl] methyl] -8-oxo-5-thia-1-
azabicydo [4.2.0] oct-2 -ene-2 -carboxylic add. expose 5 mL of the test solution to ultraviolet light at
254 nm for 24 h.
__________________________________________________________ PhEur
Reference solution (d) Dissolve 10.0 mg of cefuroxime
axetil CRS in the mobile phase and dilute to 50.0 mL with
the mobile phase.
Column:
Cefuroxime Axetil ★★*★ ★ — sizer. I = 0.25 m, 0 = 4.6 mm;
★ ★
— stationary phase: trimethylsUyl silica gelfor chromatography R
(Ph. Eicr. monograph 1300) * * * * *
(5 pm).
Mobile phase methanol R, 23 g/L solution of ammonium
H jC ^ O CH3
dihydrogen phosphate R (38:62 VtV).
O. .0
T IO Flow rate 1.0 mL/min.
ch3 Y O
N '° V -N ^ ^ Y ^ O ^ N H z Injection 20 pL of the test solution and reference
O V fr J
^—J O and epimer at C*
with reference solution (b) to identify the pair of peaks due
to impurity A and use the chromatogram obtained with
C20H22N4 0 1qS 510.5 64544-07-6 reference solution (c) to identify the pair of peaks due to
impurity B.
Action and use
Cephalosporin antibacterial. Relative retention With reference to cefuroxime axetil
diastereoisomer A: cefuroxime axetil
Preparations
diastereoisomer B = about 0.9, impurity A = about 1.2;
Cefuroxime Axetil Oral Suspension
impurity B = 1.7 and 2.1.
Cefuroxime Axetil Tablets System suitability: reference solution (b):
PhEur. — resolution: minimum 1.5 between the peaks due to
cefuroxime axetil diastereoisomer A and impurity A.
DEFINITION Limits:
Mixture of the 2 diastereoisomers of (li?5)-1-(acetyloxy)ethyl — impurity A: maximum 1.5 per cent for the sum of the pair
(6J?j7i?)-3-[(carbamoyloxy)methyi]-7-[[(Z)-2-(furan-2-yl)- of peaks;
2 (methoxyimino) acetyl] amino] -8-oxo-5-thia-1- — impurity B: maximum 1.0 per cent for the sum of the pair
azabicydo [4.2.0] oct-2-ene-2-carboxylate. of peaks;
Semi-synthetic product derived from a fermentation product. — impurity E: maximum 0.5 per cent;
Content — any other impurity: for each impurity, m axim um
96.0 per cent to 102.0 per cent (anhydrous substance). 0.5 per cent;
CHARACTERS — disregard limit: 0.05 times the area of the 2 principal peaks
Appearance in the chromatogram obtained with reference solution (a)
White or almost white powder. (0.05 per cent).
Solubility Diastereoisomer ratio
Slightly soluble in water, soluble in acetone, in ethyl acetate Liquid chromatography (2.2.29) as described in the test for
and in methanol, slightly soluble in ethanol (96 per cent). rdated substances.
IDENTIFICATION Limit Test solution:
A. Infrared absorption spectrophotometry (2.2.24). — the ratio of the area of the peak due to cefuroxime axetil
Comparison cefuroxime axetil CRS. diastereoisomer A to the sum of the areas of the peaks
B. Examine the chromatograms obtained in the assay. due to cefuroxime axetil diastereoisomers A and B is
between 0.48 and 0.55.
Results The principal peaks in the chromatogram obtained
with the test solution are similar in retention time and size to Acetone (2A. 24)
the peaks due to cefuroxime axetil diastereoisomers A and B M axim um 1.1 per cent
in the chromatogram obtained with reference solution (d). W ater (2.5.12)
TESTS M axim um 1.5 per cent, determined on 0.400 g.
Related substances ASSAY
Liquid chromatography (2.2.29): use the normalisation Liquid chromatography (2.2.29) as described in the test for
procedure. Prepare the test solution qnd reference solution (d) rdated substances with the following modifications.
1-472 Cefuroxime Sodium 2016
ov
Injection Test solution and reference solution (d). ch3 y -o
cefuroxime axetil diastereoisomers A and B;
2.0 per cent for the sum of the peaks due to cefuroxime
axetil diastereoisomers A and B after 6 injections. E. (5ai?, 6 R)-6 - [[(2Z)-2-(furan-2-yl)-2-
Calculate the percentage content of C 20H 22N 4O 10S from the (methoxyimino)acetyl] amino] -5a, 6-dihydro-3H, 7H-azeto [2,1-
sum of the areas of the 2 diastereoisomer peaks and the ¿]furo[3,4-d] [1,3]thiazine-1,7(4/i)-dione
declared content of C20H 22N 4O 10S in cefuroxime axetil CRS. (descarbamoylcefuroxime laaone).
PhEur
STORAGE
IMPURITIES
Specified impurities A, B, E. Cefuroxime Sodium ★ ★
★ ★
Other detectable impurities (the following substances would, if *****
the tests in the monograph. They are limited by die general CH3 C02Na O
by the general monograph Substances for pharmaceutical use N "° nh2
Control of impurities in substancesfor pharmaceutical use): C, D.
Ci6Hi 5N4Na0 8S 446.4 56238-63-2

H jC ^ O CH3
T T Action and use
ch3 °Y° ° 0 Cephalosporin antibacterial.
N "° \ - N xX^ i / ^ O ^ N H 2 Preparations
Cefuroxime Eye Drops
C r Y iv Cefuroxime Injection
Cefuroxime Intracameral Injection
A l-(acetyloxy)ethyl (6R,7R)-3-[(carbamoyloxy)methyl]-7- PhEur.

[[(Z)-2 -(furan-2 -yl)-2 -(methoxyimino) acetyl] amino] -8-oxo-5- DEFINITION
thia-1-azabicyclo [4.2.0]oct-3-ene-2-carboxylate (A3-isomers), Sodium (6/?,7/?)-3-[(carbamoyloxy)methyl]-7-[[CZ)-(furan-2-
yl) (methoxyimino) acetyl] amino] -8-oxo-5-thia-1-
H3C^.
Y ° Y ° H3 azabicyclo [4.2.0] oct-2-ene-2-carboxylate.
0 Semi-synthetic product derived from a fermentation product.
ch3 Y° °° Content
o^.
N 96.0 per cent to 102.0 per cent (anhydrous substance).
CHARACTERS
cA H H
and epimer at C* Appearance
White or almost white, slighdy hygroscopic powder.
B. (lÄS)-l-(acetyloxy)ethyl (6R,7R)-3- Solubility
[(carbamoyloxy)methyl]-7-[[(J5)-2-(furan-2-yl)-2- Freely soluble in water, very slighdy soluble in ethanol
(methoxyimino)acetyl] amino] -8-oxo-5-thia-1- (96 per cent).
azabicyclo[4.2.0]oct-2-ene-2caiboxylate ((jE)-isomers), IDENTIFICATION
CH3 COjH o
Comparison cefuroxime sodium CRS.
N
-O °v
V -N I ^ V O N B. It gives reaction (a) of sodium (2.3.1).
TESTS
Solution S
C. R = CO-CCI3 : (6i?,7/?)-7-[[(Z)-2-(furan-2-yl)-2- 20.0 mL with the same solvent.
(methoxyimino) acetyl] amino]-8-oxo-3- Appearance of solution
[[[(trichloroacetyl) carbamoyl] oxy]methyl] -5-thia-1- Solution S is not more opalescent than reference
azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid, suspension II (2.2.1). The absorbance (2.2.25) of solution S
D. R = H: cefuroxime. measured at 450 nm is not greater than 0.25.
pH (2.2.3)
5.5 to 8.5.
Dilute 2 mT. of solution S to 20 mL with carbon dioxide-free
water R
2016 Cefuroxime Sodium 1-473
Specific optical rotation (2.2.7) Injection Testsolution (b) and reference solution (a).
+ 59 to + 66 (anhydrous substance). Calculate the percentage content of cefuroxime sodium.
Dissolve 0.500 g in acetate buffer solution p H 4.6 R and dilute STORAGE
to 25.0 mL with the same buffer solution.
Related substances sterile, airtight, tamper-proof container.
IMPURITIES
immediately before use or keep at 2-8 °C.
Test solution (a) Dissolve 25.0 mg of the substance to be
examined in water R and dilute to 25.0 ml, with the same
solvent.
Test solution (b) Dilute 5.0 mL of test solution (a) to
50.0 mL with water R.
Reference solution (a) Dissolve 25.0 mg of cefuroxime
sodium CRS in water R and dilute to 25.0 mL with the same
A. R = OH: (6J?,7/?)-7-[[(Z)-(furan-2-
solvent. Dilute 5.0 mL to 50.0 mL with water R. yl) (methoxyimino) acetyl] amino]-3-(hydroxymethyl)-8-oxo-5-
Reference solution (b) Place 20 mL of reference solution (a) in thia- 1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic add
a water-bath at 80 °C for 15 min. Cool and inject (descarbamoylcefuroxime),
immediately. B. R = 0-C 0-C H 3: (6.R,7i?)-3-[(acetyloxy)methyl]-7-[[(Z)-
Reference solution (c) Dilute 1.0 mL of test solution (a) to (furan-2-yl) (methoxyimino) acetyl] amino]-8-oxo-5-thia-1-
100.0 mL with water R. azabicydo[4.2.0]oct-2-ene-2-carboxylic add,
Column: C. R = H: (6£,7£)-7-[[(2Hfuran-2-
— size. I = 0.125 m, 0 = 4.6 mm; yl) (methoxyimino) acetyl] amino]-3-methyl-8-oxo-5-thia-1-
— stationary phase: hexylstlyl silica gelfor chromatography R azabicyclo [4.2.0] oct-2-ene-2-carboxylic add,
(5 nm). D. R = 0-C0-NH-C0-CCl3: (6£,7/?)-7-[[(2)-(furan-2-
Mobile phase Mix 1 volume of acetonitrile R and 99 volumes yl) (methoxyimino) acetyl] amino]-8-oxo-3-
of an acetate buffer solution pH 3.4, prepared by dissolving [[[(trichloroacetyl) carbamoyl] oxy] methyl] -5-thia-1-
6.01g of glacial acetic acid R and 0.68 g of sodium acetate R azabicyclo [4.2.0] oct-2-ene-2-carboxylic add,
in water R and diluting to 1000 mL with the same solvent.
Injection 20 jiL loop injector; inject test solution (a) and
reference solutions (b) and (c).
Run time 4 times the retention time of cefuroxime.
— resolution: minimum 2.0 between the peaks due to E. R = 0-C 0-N H 2: (6R,7K)-3- [(carbamoyloxy)methyl]-7-
cefuroxime and impurity A. [ [(E)-(furan-2-yl) (methoxyimino) acetyl] amino] -8-oxo-5-thia-
Limits:
1-azabicydo [4.2.0] oct-2-ene-2-carboxylic add (trans-
— impurity A: not more than the area of the principal peak cefuroxime),
in the chromatogram obtained with reference solution (c) F. R = OH: (6fl,7i?)-7-[[(£)-(furan-2-
( 1.0 per cent); yl) (methoxyimino) acetyl] amino] -3-(hydroxymethyl)-8-oxo-5-
— any other impurity, not more than the area of the principal thia- 1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic add,
peak in the chromatogram obtained with reference G. R = O-CO-CH3: (6i?,7i?)-3-[(acetyloxy)methyl]-7-[[(£)-
solution (c) ( 1.0 per cent); (furan-2-yI) (methoxyimino) acetyl] amino]-8-oxo-5-thia-1-
— total: not more than 3 times the area of the principal peak azabicyclo[4.2.0]oct-2-ene-2-carboxylic add,
(3.0 per cent);
(0.05 per cent).
Af-THmethylanfline (2.4.26, Method E)
Maximum 20 ppm.
Maximum 0.5 per cent mim. H. (5ai?,6i?)-6-[[CZ)-(furan-2-
yl) (methoxyimino)acetyl] amino] -5 a, 6-dihydro-3/f,7//-
W ater (2.5.12) azeto [2,1-6]furo [3,4-d\ [1,3] thiazine-1,7(4i/)-di°ne,
ASSAY
Liquid chromatography (2.2.29) as described in the test for I. (Z)-(furan-2 -yl) (methoxyimino) acetic add.
related substances with the following modification. __________________________________________________________ PhEur
1-474 Celecoxib 2016
Mobile phase Mix 10 volumes of acetonitrile R l, 30 volumes of

Celecoxib f* * %
methanol R2 and 60 volumes of a 2.7 g/L solution of
(Ph Eur monograph 2591) * potassium dihydrogen phosphate R previously adjusted to
pH 3.0 with phosphoric add R.
and (c).
Run time 1.5 times die retention time of celecoxib.
with reference solution (b) to identify the peaks due to
impurities A and B.
Relative retention With reference to celecoxib (retention
C 17H 14F3N302S 381.4 169590-42-5
Action and use impurity B = about 1.1.
Cyclo-oxygenase (COX-2) inhibitor; analgesic; anti System suitability:
inflammatory. — resolution: minimum 1.8 between the peaks due to
impurity A and celecoxib and minimum 1.8 between the
PhEur___________________________________________________________ peaks due to celecoxib and impurity B in the
DEFINITION chromatogram obtained with reference solution (b).
4- [5- (4-Methylphenyl) -3-(trifluoromethyl)-1//-pyrazol-1- Calculation ofpercentage contents:
yl] benzenesulfonamide. — for all impurities, use the concentration of celecoxib in
Content reference solution (c).
CHARACTERS — unspecified impurities: for each impurity, maximum
Appearance 0.10 per cent;
White or almost white, crystalline or amorphous powder. — total: maximum 0.5 per cent;
Solubility — reporting threshold: 0.05 per cent.
Practically insoluble in water, freely soluble to soluble in Heavy metals {2.48)
anhydrous ethanol, soluble in methylene chloride. Maximum 20 ppm.
It shows polymorphism (5.9). Solvent mixture water R, acetone R (15:85 VIV).
IDENTIFICATION 0.50 g complies with test H. Prepare the reference solution
Infrared absorption spectrophotometry {2.2.24). using 1 mL of lead standard solution (10 ppm Pb) R.
Comparison celecoxib CRS. W ater {2.5.12)
If the spectra obtained show differences, dissolve the Maximum 0.5 per cent, determined on 0.400 g.
substance to be examined and the reference substance Sulfa ted ash {2.414)
separately in 2-propanol R, evaporate to dryness and record Maximum 0.2 per cent, d eterm ine d on 1.0 g in a platinum
new spectra using the residues. crucible.
TESTS ASSAY
Related substances Liquid chromatography {2.2.29) as described in the test for
Liquid chromatography {2.2.29). related substances with the following modifications.
Solvent mixture water R , methanol R2 (25:75 VIV). Injection Test solution and reference solution (a).
Test solution Dissolve 50.0 mg of the substance to be Calculate the percentage content of Q 7H 14F 3N 3O2S taking
examined in the solvent mixture and dilute to 100.0 mL with into account the assigned content of celecoxib CRS.
IMPURITIES
Reference solution (a) Dissolve 50.0 mg of celecoxib CRS in the
Spedfied impurities A.
solvent mixture and dilute to 100.0 mL with the solvent
mixture.
Reference solution (b) Dissolve 3 mg of celecoxib
impurity A CRS and 3 mg of celecoxib impurity B CRS in the
solvent mixture and dilute to 50.0 mL with the solvent
mixture. Dilute 1.0 mL of the solution to 25.0 mL with
Reference solution (c) Dilute 1.0 mT. of the test solution to Contrd of impurities in substances for pharmaceutical use): B.
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
solution to 10.0 mL with the solvent mixture.
Column:
— size. I = 0.25 m, 0 = 4.6 mm;
— stationary phase: end-capped phenylsifyl silica gelfor
2016 Celiprolol Hydrochloride 1-475

substance separately in methanol R, evaporate to dryness and
record new spectra using die residues.
B. It gives reaction (a) of chlorides (2.3.1).
TESTS
Optical rotation (2.2.7)
A. 4-[5-(3-methylphenyl)-3-(trifluoromethyl)-li?-pyrazol-l- - 0 .10° to + 0 . 10°.
yl] benzenesulfonamide,
same solvent.
Related substances
examined in mobile phase A and dilute to 20.0 mL with
mobile phase A
Reference solution (a) Dissolve 2 mg of the substance to be
-NH, examined and 2 mg of acebuzolol hydrochloride R in mobile
phase A and dilute to 50.0 mL with mobile phase A
Reference solution (b) Dissolve 10 mg of the substance to be
B. 4-[3-(4-mediylphenyl)-5-(trifluoromethyl)-1f/-pyrazol- 1- examined in 2 mL of mobile phase A and allow to stand for
yljbenzenesulfonamide. 24 h (for identification of impurity A).
PhEur Reference solution (c) Dilute 1.0 mL of the test solution to
100.0 mL with mobile phase A. Dilute 1.0 mL of this
solution to 10.0 mL with mobile phase A.
Reference solution (d) Dissolve 10 mg of cdiprololfor peak
identification CRS in mobile phase A and dilute to 2 mL with
Celiprolol Hydrochloride ★ ★
★ ★ mobile phase A.
(Ph. Eur. monograph 1632) *★* Reference solution (e) Thừ solution is only prepared if required
(see below) and Ù used to determine the identity of impurity I
Ow CH3 which co-duxes with impurity H (the 2 impurities originate from
H OHu different routes of synthesis). Dissolve the contents of a vial of
' t! pH3 celiprolol impurity I C R S in mobile phase A and dilute to
CH,
HCI 2.0 mL with mobile phase A.
' ' Nt CH,
Column'.
— size. I = 0.15 m, 0 = 4.6 mm,
— stationary phase: odylsOyl silica gelfor chromatography R
and enantiomer (5 |im),
— temperature: 30 °c.
C20H34CIN3O4 416.0 57470-78-7 Mobile phase:
— mobile phase A: mix 91 mL of tetrahydrqfuran R, 63 mL of
Action and use acetomtrữe R l, 0.6 mL of pentafluoropropanoic add R and
Beta-adrenoceptor antagonist. 0.2 mL of trifluoroacetic add R; dilute to 1000 mL with
Preparation water R;
Celiprolol Tablets — mobile phase B: acetoràtrũe R lm
,
PhEur__________________________
Urne Mobik phase A Mobile phase B
DEFINITION (min) (per cent V/V) (per cent V/V)
3-[3-Acetyl-4- [(2i?5)-3- [( 1, l-dimethylethyl)amino]-2- 0 -5 0 100-»80 0 + 20
hydroxypropoxy]phenyI]-l, 1-diethylurea hydrochloride. 50-51 80-» 100 20->0
Content 51 -65 100 0
CHARACTERS Flow rate 1.4 mUmin.
Appearance
White or very slightly yellow, crystalline powder.
Injection 10 (iL.
Solubility
Freely soluble in water and in methanol, soluble in ethanol
(96 per cent), very slightly soluble in methylene chloride. with celiprololfor peak identification CRS and the
It shows polymorphism (5.9). identify the peaks due to impurities B, E and F.
IDENTIFICATION Relative retention With reference to celiprolol (retention
A. Infrared absorption spectrophotometry (2.2.24). time = about 10 min): impurity A = about 0.3;
Comparison celiprolol hydrochloride CRS. impurity D = about 0.7; impurity G = about 1.2 ;
1-476 Cellacefate 2016
impurity B = about 1.4; impurity F = about 1.6;

impurity C = about 2.2; impurity H or I = about 2.5;
impurity E = about 3.9.
HN^-CHa H3C NH
X X
H3C ch3 H3C CH3
celiprolol and acebutolol.
Limits: B. 1,3-bis [3-acetyl-4- [3-[( 1,1 -dimethylethyl)amino] -2-
— correction factors: for the calculation of content, multiply hydroxypropoxy]phenyl] urea,
corresponding correction factor impurity A = 4.0; CH,
impurity B = 1.5; impurity E = 2.3; impurity F = 0.5; H,c.
^ch3 h3c^
impurity I = 1.7; OH OH
— any impurity, for each impurity, not more than twice the O^ Jx. „0
with reference solution (c) (0.2 per cent), and not more
than 1 such peak has an area greater than the area of the
(0.5 per cent); E. 1,1 /-[[(l,l-dimethylethyl)imino]bis[(2-hydroxypropane-
— if any of the above limits are exceeded and if a peak l,3-diyl)oxy(3-acetyl-l,4-phenylene)]]bis(3,3-diethylurea),
occurs with a relative retention of about 2.5 (impurity H
or I), the identity of this peak has to be clarified by use of
a UV spectrum recorded with a diode array detector;
if this spectrum is different from the one obtained with
reference solution (e), no correction factor is applied;
(0.05 per cent).
F. R1 = R3 = H, R2 = CO-CH3:
3-(3-acetyl-4-hydroxyphenyl)-1,1 -diethylurea,
an oven at 105 °C for 3 h. I. R1 = CO-CH 3, R2 = H, R3 = C 2H5:
1-acetyl-1- (4-ethoxyphenyl) -3,3 -diethylurea,
ASSAY
Dissolve 0.350 g under an atmosphere of nitrogen in 50 mL
of ethanol (96 per cent) R and add 1.0 mL of 0.1 M hv ,0
hydrochloric acid. Carry out a potentiometric titration (2.2.20), °^X i
using 0.1 M sodium hydroxide. Read the volume added II || | and enantiomer
between the 2 points of inflexion. H3C'''~''N N
H3CJ H
1 mL of 0.1 M sodium hydroxide is equivalent to 41.60 mg of
C 20H 34CIN3O4.
STORAGE G. 3- [3-acetyI-4- [[(i?5) -oxiranyl] methoxy] phenyl] -1,1-
Protected from light. diethylurea.
IM PURITIES PftEur
Specified impuritier. A, B, C, D, E, F, G, H, I.
Cellacefate *****
and enantiomer ★ ★
R1. * * * * *
(Cellulose Acetate Phthalate, Ph Eitr monograph 0314)
9004-38-0
A. R1 = H, R2 = NH-C(CH3)3:
Action and use
l-[5-amino-2-[(2.&S)-3-[(l, l-dimethylethyl)amino]-2-
Enteric coating in pharmaceutical products.
hydroxypropoxy]phenyl] ethanone,
C. R 1 = CO-NH-C(CH3)3, R2 = NH-C(CH3)3: PtiEur.
1-[3-acetyl-4-[(2i?5)-3-[(l, 1-dimethylethyl)amino] -2- DEFINITION
hydroxypropoxy]phenyl]-3-(l,l-dimethylethyl)urea, Partly O-acetylated and Ophthalylated cellulose.
D. R1 = CO-N(C 2H 5)2, R2 = N(C 2H 5)2: 3-[3-acetyl-4-
Content
[(2J?5)-3-(diethylamino)-2-hydroxypropoxy]phenyl]-l, 1-
diethylurea,
— phthaloyl groups (CgHsOj; M r 149.1): 30.0 per cent to
36.0 per cent (anhydrous and acid-free substance);
H. R 1 = CO-N(C 2H 5)2, R2 = Bn 3-[3-acetyl-4-[(2.RS)-3- — acetyl groups (C2H 3 0 ; M T 43.04): 21.5 per cent to
bromo-2 -hydroxypropoxy]phenyl]-l, 1-diethylurea 26.0 per cent (anhydrous and add-free substance).
(bromhydrin compound),
2016 Cellacefate 1-477
♦CHARACTERS Acetyl groups

Appearance To 0.100 g add 25.0 mL of 0.1 M sodium hydroxide and heat
White or almost white, free-flowing powder or colourless on a water-bath under a reflux condenser for 30 min. Cool,
flakes, hygroscopic. add about 0.1 mL of phenolphthalein solution R1 and titrate
Solubility with 0.1 M hydrochloric acid Carry out a blank titration.
Practically insoluble in water, freely soluble in acetone, Calculate the percentage content of acetyl groups using the
soluble in diethylene glycol, practically insoluble in ethanol following expression:
(96 per cent) and in methylene chloride. It dissolves in dilute
solutions of alkali hydroxides. ♦ 4305 (na —n i) 51.825
- 0.5772P
IDENTIFICATION (100 - a) (100 - 5) m (100 - S)
Comparison cellubse acetate phthalate CRS. a percentage content of water (see Tests);
TESTS
m mass of the substance to be examined, in grams;
Viscosity (2.2.9) n\ volume of 0.1 M hydrochloric acid used in the
titration, in millilitres;
45 mPa-s to 90 mPa-s, determined at 25 ± 0.2 °C.
n.2 volume of 0.1 M hydrochloric acid used in the blank
Dissolve 15 g, calculated with reference to the anhydrous titration, in millilitres;
substance, in 85 g of a mixture of 1 part by mass of water R p percentage content of phthaloyl groups;
and 249 parts by mass of acetone R. s percentage content of free acid (see Tests).
Free a d d
STORAGE
Maximum 3.0 per cent, calculated as phthalic acid
Shake 3.0 g for 2 h with 100 mL of a 50 per cent V/V FUNCTIONALITY-RELATED CHARACTERISTICS
solution of methanol R and filter. Wash the flask and the filter Thừ section provides information on characteristics that are
with 2 quantities, each of 10 m l, of a 50 per cent VIV recognised as being relevant control parameters for one or more
solution of methanol R. Combine the filtrate and washings, functions of the substance when used as an excipient (see chapter
add 0.1 mL of phenolphthalein solution R1 and titrate with 5.15). Some of the characteristics described in the Functionality-
0.1 M sodium hydroxide until a faint pink colour is obtained. related characteristics section may also be present in the mandatory
Carry out a blank titration using 120 mL of a part of the monograph since they also represent mandatory quality
50 per cent VIV solution of methanol R. criteria In such cases, a cross-reference to the tests described in the
mandatory part ừ included in the Functionality-related
characteristics section. Control of the characteristics can contribute
free acid, calculated as phthalic acid.
Heavy metals (2.4.8) of the manufacturing process and the performance of the medicinal
Maximum 10 ppm. product during use. Where control methods are cited, they are
2.0 g complies with test C. Prepare the reference solution recognised as being suitable for the purpose, but other methods can
using 2 mL of lead standard solution (10 ppm Pb) R. also be used. Wherever resultsfor a particular characteristic are
W ater (2.5.12) reported, the control method must be indicated
Maximum 5.0 per cent, determined on 0.500 g. The following characteristics may be relevant for ceữubse acetate
Carry out the test using a mixture of 2 volumes of methylene phthalate used as fihn former in gastro-resistant tablets and
chloride R and 3 volumes of anhydrous ethanol R. capsules.
Sulfated ash (2.4.14) Viscosity

Maximum 0.1 per cent, determ ined on 1.0 g. See Tests.
ASSAY Solubility of a film
Phthaloyl groups Dissolve about 0.15 g in 1 mL of acetone R and pour onto a
Dissolve 1.000 g in 50 mL of a mixture of 2 volumes of clear glass plate. A film is formed. Take a piece of the film
acetone R and 3 volumes of ethanol (96 per cent) R.
and place it in a flask co n taining 0 .1 M hydrochloric acid.
Add about 0.1 mT. of phenolphthalein solution R1 and titrate It does not dissolve. Then place the piece of film in a flask
with 0.1 M sodium hydroxide. Cany out a blank titration. containing phosphate buffer solution p H 6 .8 R. It dissolves.
Calculate the percentage content of phthaloyl groups (P) Phthaloyl groups
using the following expression: See Assay.
Acetyl groups
14910 (m - n 2)____ *I 179.55 See Assay.
(100 - a ) (1 0 0 - S) m ~ (100 - S) ____________________________________ PhEur
a = percentage content of water (see Tests);

m = mass of the substance to be examined, in grams;
«i = volume of 0.1 M sodium hydroxide used in the
titration, in millilitres;
«2 = volume of 0.1 M sodium hydroxide used in the blank
titration, in m illilitres;
5 = percentage content of free acid (see Tests).
1-478 Cellulose 2016
Loss on drying
Dispersible Cellulose When dried to constant weight at 105°, loses not more than
Action and use 8.0% of its weight. Use 1 g.
Pharmaceutical excipient. Sulfated ash
Not more than 5.0%, Appendix IX A. Use 2 g.
DEFINITION
ASSAY
Dispersible Cellulose is a colloid-forming, attrited mixture of
Microcrystalline Cellulose and Carmellose Sodium. Heat 2 g with 75 mL of anhydrous acetic add under a reflux
condenser for 2 hours, cool and carry out Method I for rum-
Content o f carmellose sodium aqueous titration, Appendix V1H A, determining the end point
75.0 to 125.0% w/w of the stated amount. potentiometrically. Each mL of 0.1m perchloric add FS is
CHARACTERISTICS equivalent to 29.6 mg of carmellose sodium.
A white or off-white, coarse or fine powder. STORAGE
Disperses in water producing a white, opaque dispersion or Dispersible Cellulose should be stored at a temperature of 8 °
gel; practically insoluble in organic solvents and in dilute to 15°.
acids.
LABELLING
IDENTIFICATION The label states (1) the percentage w/w of Carmellose
A. Mix 6 g with 300 mL of water stirring at Sodium; (2) the viscosity of a dispersion in water of a stated
18.000 revolutions per minute for 5 m in u tes. A white, percentage w/w of Carmellose Sodium.
opaque dispersion is obtained which does not produce a
supernatant liquid.
B. Add several drops of the dispersion obtained in test A to a
10% w/v solution of aluminium chloride. Each drop forms a
white, opaque globule which does not disperse on standing.
Microcrystalline Cellulose *****
*+ +**
C. Add 2 mL of iodinated potassium iodide solution to the
dispersion obtained in test A. No blue or purplish colour is
produced.
D. The solution obtained in the test for Heavy metals yields
the reactions characteristic of sodium salts, Appendix VI,
except that in test A the white precipitate produced may not
be dense.
TESTS
pH of the dispersion obtained in the test for Apparent
viscosity, 6.0 to 8.0, Appendix V L.
C6«H10n+2O5n+l 9004-34-6
Solubility
Add 50 mg to 10 mL of ammoniacal solution of copper Action and use
tetrammine and shake. It dissolves completely leaving no Excipient.
residue.
PhEur___________________________________________________________
A pparent viscosity
60 to 140% of the declared value when determined by the DEFINITION
following method. Calculate the quantity (x g) needed to Purified, partly depolymerised cellulose prepared by treating
prepare exactly 600 g of a dispersion of the stated percentage alpha-cellulose, obtained as a pulp from fibrous plant
w/w, with reference to the dried substance. To (600-*) g of material, with mineral acids.
water at 23° to 25° contained in a 1000 mL high-speed
CHARACTERS
blender bowl add x g of the substance b ein g examined,
Appearance
stirring at reduced speed, taking care to avoid contacting the
White or almost white, fine or granular powder.
sides of the bowl with the powder. C on tin u e stirring at low
speed for 15 seconds after the addition and then stir at Solubility
18,000 revolutions per minute for exactly 2 minutes. Practically insoluble in water, in acetone, in anhydrous
Immerse the appropriate spindle of a rotational viscometer, ethanol, in toluene, in dilute acids and in a 50 g/L solution of
switch on after 30 seconds and after a further 30 seconds sodium hydroxide.
determine the viscosity, Appendix V H, Method IE, using a IDENTIFICATION
speed of 20 revolutions per m in u te (2.09 radians per A. Place about 10 mg on a watch-glass and disperse in 2 mL
second). of iodinated zinc chloride solution R. The substance becomes
Heavy metals violet-blue.
To the residue obtained in the test for Sulfated ash add B. The degree of polymerisation is not more than 350.
1 mL of hydrochloric add, evaporate to dryness on a water Transfer 1.300 g to a 125 mL conical flask. Add 25.0 mL of
bath and dissolve the residue in 20 mL of water. 12 mL of water R and 25.0 mT, of cupriethylenediamine hydroxide
the resulting solution complies with limit test A for heavy solution R. Immediately purge the solution with nitrogen R,
metals, Appendix VII. Use lead standard solution (1 ppm) Pb to insert the stopper and shake until completely dissolved.
prepare the standard (10 ppm). Transfer an appropriate volume of the solution to a suitable
capillary viscometer (2.2.9). Equilibrate the solution at
25 ± 0.1 °C for at least 5 min. 'Record the flow time fa) in
2016 Cellulose 1-479
Table 0316.-1. - Intrinsic viscosity table

Intrinsic viscosity [ij], at different values of relative viscosity ^
[>tL
n* 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
1.1 0.098 0.106 0.115 0.125 0.134 0.143 0.152 0.161 0.170 0.180
lO 0.189 0.198 0.207 0J16 0025 0.233 0042 0050 0059 0068
U 0076 0.285 0-293 0302 0310 0318 0326 0334 0342 0350
1.4 0358 0.367 0375 0383 0391 0399 0.407 0.414 0.422 0.430
13 0.437 0.445 0.453 0.460 0.465 0.476 0.484 0.491 0.499 0307
1.6 0.515 0322 0329 0336 0344 0351 0358 0366 0373 0380
1.7 0387 0395 0.602 0.608 0.615 0.622 0.629 0.636 0.642 0349
1.8 0.656 0.663 0.670 0.677 0.683 0.690 0.697 0.704 0.710 0.717
1.9 0.723 0.730 0.736 0.743 0.749 0.756 0.762 0.769 0.775 0.7X2
2.0 0.788 0.795 0.802 0.809 0.815 0.821 0.827 0.833 0.840 0346
2.1 0.852 0.858 0.864 0370 0376 0.882 0.888 0.894 0.900 0306
20 0.912 0.918 0.924 0.929 0.935 0.941 0.948 0.953 0359 0365
23 0.971 0.976 0.983 0.988 0394 1.000 1.006 1.011 1.017 1.022
2.4 1.028 1.033 1.039 1.044 1.050 1.056 1.061 1.067 1.072 1378
23 1.083 1.089 1.094 1.100 1.105 1.111 1.116 1.121 1.126 1.131
2.6 1.137 1.142 1.147 1.153 1.158 1.163 1.169 1.174 1.179 1.184
2.7 1.190 1.195 1-200 1305 1010 1.215 1020 1025 1030 1035
2.8 1040 1.245 1050 1-255 1060 1065 1070 1075 1080 1085
IS 1090 1.295 1300 1305 1310 1314 1319 1324 1329 1333
3.0 1-338 1-343 1348 1352 1357 1362 1367 1371 1376 1381
3.1 1386 1.390 1395 1.400 1.405 1.409 1.414 1.418 L423 1.427
30 1.432 1.436 1.441 1.446 1.450 1.455 1.459 1.464 1.468 1.473
33 1.477 1.482 1.486 1.491 1.496 1300 1304 1308 1313 1317
3.4 1.521 1325 1329 1333 1337 1342 1346 1350 1354 1358
33 1362 1366 1370 1375 1379 1383 1387 1391 1395 L600
3.6 1.604 1.608 1.612 1.617 1.621 1.625 1.629 1.633 1-637 1.642
3.7 1.646 1.650 1-654 1.658 1.662 1.666 1.671 1.675 1.679 1383
3.8 L687 1.691 1.695 1.700 1.704 1.708 1.712 1.715 1.719 1.723
33 1.727 1.731 1.735 1.739 1.742 1.746 L750 1.754 1.758 1.762
4.0 1.765 1.769 1.773 1.777 1.781 1.785 1.789 1.792 1.796 1300
4.1 1.804 1.808 1.811 1315 1.819 1.822 1.826 1.830 1.833 1337
40 1.841 1.845 1348 1352 1.856 1359 1.863 L867 1370 1374
4J 1378 1.882 1385 1389 1.893 1396 1300 L904 1307 1311
4.4 1.914 L918 1321 1325 1329 1332 1336 1339 1343 1346
43 1.950 1.954 1.957 1.961 1364 1.968 1.971 1.975 1379 1382
4.6 1.986 1.989 1393 1396 2.000 2.003 2.007 1010 2X13 2X17
4.7 2.020 2.023 2.027 2.030 2.033 2.037 2.040 2.043 2.047 2.050
4.8 2.053 2.057 2.060 2.063 2.067 2.070 2.073 2.077 2.080 2.083
4.9 2.087 2.090 2X93 2.097 2.100 2.103 2.107 1110 2.113 2.116
5.0 2.119 2.122 2.125 2.129 2.132 2.135 2.139 2.142 2.145 2.148
5.1 2.151 2.154 2.158 2.160 2.164 2.167 2.170 2.173 2.176 2.180
SO 2.183 2.186 2.190 2.192 2.195 2.197 2000 2003 2006 2009
53 2-212 2015 2-218 2021 2024 2.227 2030 2033 2036 2040
5.4 2-243 1246 2-249 2052 2055 2.258 2061 2064 2067 2070
53 2073 2-276 2079 2082 2085 2088 2091 2094 2097 2300
5.6 2.303 2306 2309 2312 2315 2318 2320 2324 2326 2329
5.7 2-332 2335 ¿338 2-341 2344 2347 2350 2353 2355 2358
53 2361 2364 2367 2370 2373 2376 2379 2.382 2384 2387
5.9 2.390 2393 2396 2.400 2.403 2.405 2.408 2.411 2.414 2.417
6.0 2.419 2.422 2.425 2.428 2.431 2.433 2.436 2.439 2.442 2.444
6.1 2.447 2.450 2.453 145« 2.458 2.461 1464 2.467 2.470 2.472
60 2.475 2.478 2.481 2.483 2.486 2.489 2.492 2.494 2.497 2300
63 2-503 2305 2308 2311 2313 2316 2318 2321 2324 2326
6.4 2329 2332 2334 2337 2340 2342 2345 2347 2.550 2353
6.5 2355 2.558 2361 2363 2366 2368 2371 2374 2376 2379
6.6 2381 2384 2387 2390 2392 ‘ 2.595 2397 2.600 2.603 2.605
6.7 2.608 2.610 2.613 2.615 2.618 2.620 2.623 2.625 2.627 2.630
6.8 2.633 2.635 2.637 2.640 2.643 2.645 2.648 2.650 2.653 2.655
6.9 2.658 2.660 ' 2.663 2.665 2.668 2.670 2.673 2.675' 2378 2380
intrinsic viscosity [/f]c at different values of relative viscosity
M.
n* 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
7.0 2.683 2.685 2.687 2.690 2.693 2.695 2.698 2.700 2.702 2.705
7.1 2.707 2.710 2.712 2.714 2.717 2.719 2.721 2.724 2.726 2.729
72 2.731 2.733 2.736 2.738 2.740 2.743 2.745 2.748 2.750 2.752
7.3 2.755 2.757 2.760 2.762 2.764 2.767 2.769 2.771 2.774 2.776
7.4 2.779 2.781 2.783 2.786 2.788 2.790 2.793 2.795 2.798 2.800
73 2.802 2.805 2.807 2.809 2.812 2.814 2.816 2.819 2.821 2.823
7.6 2.826 2.828 2.830 2.833 2.835 2.837 2.840 2.842 2.844 2.847
7.7 2.849 2.851 2.854 2.856 2.858 2.860 2.863 2.865 2.868 2.870
7.8 2.873 2.875 2.877 2.879 2.881 2.884 2.887 2.889 2.891 2.893
7.9 2.895 2.898 2.900 2.902 2.905 2.907 2.909 2.911 2.913 2.915
8.0 2.918 2.920 2.922 2.924 2.926 2.928 2.931 2.933 2.935 2.937
8.1 2.939 2.942 2.944 2.946 2.948 2.950 2.952 2.955 2.957 2.959
8.2 2.961 2.963 2.966 2.968 2.970 2.972 2.974 2.976 2.979 2.981
8.3 2.983 2.985 2.987 2.990 2.992 2.994 2.996 2.998 3.000 3.002
8.4 3.004 3.006 3.008 3.010 3.012 3.015 3.017 3.019 3.021 3.023
8.5 3.025 3.027 3.029 3.031 3.033 3.035 3.037 3.040 3.042 3.044
8.6 3.046 3.048 3.050 3.052 3.054 3.056 3.058 3.060 3.062 3.064
8.7 3.067 3.069 3.071 3.073 3.075 3.077 3.079 3.081 3.083 3.085
8.8 3.087 3.089 3.092 3.094 3.096 3.098 3.100 3.102 3.104 3.106
8.9 3.108 3.110 3.112 3.114 3.116 3.118 3.120 3.122 3.124 3.126
9.0 3.128 3.130 3.132 3.134 3.136 3.138 3.140 3.142 3.144 3.146
9.1 3.148 3.150 3.152 3.154 3.156 3.158 3.160 3.162 3.164 3.166
92 3.168 3.170 3.172 3.174 3.176 3.178 3.180 3.182 3.184 3.186
93 3.188 3.190 3.192 3.194 3.196 3.198 3.200 3.202 3-204 3.206
9.4 3.208 3.210 3-212 3.214 3-215 3.217 3-219 3-221 3.223 3.225
9.5 3.227 3229 3-231 3.233 3.235 3.237 3J239 3.241 3-242 3.244
9.6 3.246 3.248 3.250 3252 3.254 3.256 3258 3260 3.262 3.264
9.7 3.266 3.268 3-269 3211 3273 3275 3277 3279 3.281 3-283
9.8 3.285 3.287 3-289 3-291 3.293 3295 3297 3298 3-300 3-302
9.9 3.304 3.305 3307 3.309 3.311 3.313 3.316 3.318 3.320 3-321
seconds between the 2 marks on the viscometer. Calculate Determine the relative viscosity (rjrJ) of die substance to be
the kinematic viscosity (vx) of the solution using the following examined using the following expression:
expression:
vi/v?
t\ (kx)
¿i is the viscometer constant.
Determine the intrinsic viscosity (faJJ by interpolation, using
Dilute a suitable volume of cupriethylenediamine hydroxide the intrinsic viscosity table (Table 0316.-1).
solution R with an equal volume of water R and measure the
flow time fe) using a suitable capillary viscometer. Calculate Calculate the degree of polymerisation (P) using the
the kinematic viscosity (V2) of the solvent using the following following expression:
expression:
95 [t?]c
ta (fca) m [(100 - 6 ) /100]
where k2 is the viscometer constant.

Intrinsic viscosity [»/]e at different values of relative viscosity r\M
M,
*1* 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
10 332 3.34 3.36 3.37 3.39 3.41 3.43 3.45 3.46 3.48
11 330 3.52 3.53 3.55 3.56 3.58 3.60 3.61 3.63 3.64
12 3.66 3.68 3.69 3.71 3.72 3.74 3.76 3.77 3.79 3.80
13 3.80 3.83 3.85 3.86 3.88 3.89 3.90 3.92 3.93 3.95
14 3.96 3.97 3.99 400 4.02 403 404 406 4.07 4.09
15 4.10 4.11 4.13 414 4.15 417 418 4.19 4.20 4.22
16 423 4-24 425 427 4.28 429 4.30 4.31 4.33 4.34
17 4.35 4.36 4.37 4.38 439 441 442 443 444 4.45
18 4.46 4.47 448 449 4.50 4.52 4.53 4.54 4.55 456
19 4.57 458 459 460 4.61 4.62 4.63 4.64 4.65 466
where m is the mass in grams of the substance to be Microbial contamination

examined and b is the loss on drying as a percentage. TAMC: acceptance criterion 103 CFU/g (2.6.12).
TESTS TYMC: acceptance criterion 102 CFU/g (2.6.12).
Solubility Absence of Escherichia coli (2.6.13).
Dissolve 50 mg in 10 mL of ammoniacal solution of copper Absence of Pseudomonas aeruginosa (2.6.13).
tetrammtne R. It dissolves completely, leaving no residue.
Absence of Staphylococcus aureus (2.6.13).
pH (2.2.3) Absence of Salmonella (2.6.13).
5.0 to 7.5 for the supernatant.
FUNCTIONALITY-RELATED CHARACTERISTICS
Shake 5 g with 40 mL of carbon dioxide-free water R for
20 min and centrifuge.
Conductivity (2.2.38) functions of the substance when used as an excipient (see chapter
The conductivity of the test solution does not exceed the 5.15). This section is a non-mandatory part of the monograph
conductivity of the water by more than 75 (iS-cm-1. and it is not necessary to verify the characteristics to demonstrate
Use as test solution the supernatant obtained in the test for compliance. Control of these characteristics can however contribute
pH. Measure the conductivity of the supernatant after a to the quality of a medicinal product by improving the consistency
stable reading has been obtained and measure the of the manufacturing process and the performance of the medicinal
conductivity of the water used to prepare the test solution. product during use. Where control methods are cited, they are
Ether-soluble substances recognised as being suitable for the purpose, but other methods can
M a x im u m 0.05 per cent (5 mg) for the difference between also be used. Wherever resultsfor a particular characteristic are
the weight of the residue and the weight obtained from a reported, the control method must be indicated.
blank determination. The following characteristics may be relevant for microcrystaUine
Place 10.0 g in a chromatography column about 20 mm in cellulose used as binder, diluent or disintegrant
internal diameter and pass 50 mL of peroxide-free ether R Particle-size distribution (2.9.31 or 2.9.38).
through the colu m n . Evaporate the eluate to dryness. Dry the
Powder flow (2.9.36)
residue at 105 °C for 30 min, allow to cool in a desiccator __________________________________________________________PhEur
and weigh. Carry out a blank determination.
Water-soluble substances
Maximum 0.25 per cent (12.5 mg) for the difference
between the mass of the residue and the mass obtained from
a blank determination. Microcrystalline Cellulose and *****
Shake 5.0 g with 80 mL of water R for 10 min. Filter Carmellose Sodium *****
through a filter paper with the aid of vacuum into a tared (Ph Eur. monograph 2050)
flask. Evaporate to dryness on a water-bath avoiding
charring. Dry at 105 °C for 1 h, allow to stand in a Action and use
desiccator and weigh. Carry out a blank determination. Excipient.
PhEtr__________________________________________________________
Maximum 10 ppm.
2.0 g complies with test C. Prepare the reference solution DEFINITION
using 2 mL of lead standard solution (10 ppm Pb) R. Colloid-forming, powdered mixture of MicrocrystaUine
ceUubse (0316) with 5 per cent to 22 per cent of CarmeUose
sodium (0472).
an oven at 105 °C for 3 h. Content
75.0 per cent to 125.0 per cent of the n o m in al amount of
carmellose sodium (dried substance).
CHARACTERS Calculate the quantity (x g) needed to prepare exactly 600 g

Appearance of a dispersion of the stated percentage mint (dried
White or off-whitej coarse or fine powder. substance). To (600 - x) g of water R at 23-25 °C contained
Solubility in a 1000 mL high-speed blender bowl, add x g of the
Dispersible in water producing a white, opaque colloidal substance to be examined and stir at reduced speed, taking
dispersion; practically insoluble in organic solvents and in care to avoid contacting the sides of the bowl with the
dilute adds. powder. Continue stirring at low speed for 15 s after the
addition of the powder and then stir at 18 000 r/min for
IDENTIFICATION exactly 2 min.
A. Mix 6 g with 300 mL of water R and stir at 18 000 r/min Determine the viscosity with a suitable relative rotational
for 5 min. A white opaque dispersion is obtained which does viscometer under the following conditions:
not produce a supernatant. — spindle: as appropriate;
B. Add several drops of the dispersion obtained in — speed: 20 r/min.
identification A to a 10 per cent m / v solution of akmàràum Immerse the spindle into the suspension immediately after
chloride R. Each drop forms a white, opaque globule which
preparation, switch on the rotation spindle after 30 s; after a
does not disperse on standing. further 30 s, take scale readings and calculate the viscosity
c . Add 2 mL of iodinated potassium iodide solution R to the according to the viscometer manual.
dispersion obtained in test A. No blue or purplish colour is
______________________________________________________________ PhEur
produced.
D. It complies with the limits of the assay.
TESTS
Solubility Powdered Cellulose
Add 50 mg to 10 mL of ammoniacal solution of copper
mrammme R and shake. It dissolves completely, leaving no (Ph. Eur. monograph 0315)
residue.
pH (2.2.3)
6.0 to 8.0 for the dispersion obtained in identification A.
an oven at 105 °c.
ASSAY
Heat 2.00 g with 75 mL of anhydrous acetic add R under a C6nHl0n+2O5n+1
reflux condenser for 2 h, cool and titrate with 0.1 M
perchloric acid, determining the end-point potentiometricaUy Action and use
(2.2.2Ớ). Excipient.
1 mL of 0.1 M perchloric acid is equivalent to 29.6 mg of PhEur _____________
caimellose sodium.
DEFINITION
LABELLING
Purified, mechanically disintegrated cellulose prepared by
The label states die nominal percentage m/m of carmeflose
processing alpha-cellulose obtained as a pulp from fibrous
sodium. plant material.
FUNCTIONALITY-RELATED CHARACTERISTICS CHARACTERS
Appearance
White or almost white, fine or granular powder.
5.15). Some of the characteristics described in the FunctionaHty- Solubility
rdated characteristics section may also be present in the mandatory Practically insoluble in water, slightly soluble in a 50 g/L
part of the monograph since they also represent mandatory quality solution of sodium hydroxide, practically insoluble in
criteria. In such cases, a cross-reference to the tests described in the acetone, in anhydrous ethanol, in toluene, in dilute adds and
mandatory part is included in the FtmctionaMty-related in most organic solvents.
characteristics section. Control of die characteristics can contribute IDENTIFICATION
to the quality of a medicinal product by improving ứie consistency A. Place about 10 mg on a watch-glass and disperse in 2 mL
of die manufacturing process and the performance of ứte medicinal of todinated zinc chloride solution R. The substance becomes
product during use. Where control methods are àĩed, they are violet-blue.
recognised as bàng suừabỉe for the purpose, but oứier methods can
B. The degree of polymerisation is greater than 440.
Transfer 0.250 g to a 125 mL conical flask. Add 25.0 mL of
water R and 25.0 mL of atpriethylenediamine hydroxide
The following characteristics may be relevant for microcrystaOine
solution R. Immediately purge the solution with nitrogen R,
ceJhdose and. carmeũose sodium used as a suspending agent.
insert the stopper and shake until completely dissolved.
Apparent viscosity (2. 2 .IƠ) Transfer an appropriate volume of the solution to a suitable
60 per cent to 140 per cent of the nominal value. capillary viscometer (2.2.9). Equilibrate the solution at
25 ± 0.1 °C for at least 5 min. Record the flow time fa) in
Table 0315.-1. - Intrinsic viscosity table
Intrinsic viscosity [//]t at different values of relative viscosity rj„,
M.
1m 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
1.1 0.098 0.106 0.115 0.125 0.134 0.143 0.152 0.161 0.170 0.180
1.2 0.189 0.198 0.207 0.216 0.225 0.233 0.242 0.250 0-259 0.268
1.3 0.276 0.285 0.293 0302 0310 0.318 0326 0.334 0342 0350
1.4 0.358 0.367 0.375 0383 0391 0.399 0.407 0.414 0.422 0.430
1.5 0.437 0.445 0.453 0.460 0.468 0.476 0.484 0.491 0.499 0.507
1.6 0.515 0.522 0.529 0.536 0344 0.551 0.558 0.566 0373 0380
1.7 0.587 0.595 0.602 0.608 0.615 0.622 0.629 0.636 0.642 0.649
1.8 0.656 0.663 0.670 0.677 0.683 0.690 0.697 0.704 0.710 0.717
1.9 0.723 0.730 0.736 0.743 0.749 0.756 0.762 0.769 0.775 0.782
2.0 0.788 0.795 0.802 0.809 0.815 0.821 0.827 0.833 0.840 0.846
2.1 0.852 0.858 0.864 0.870 0.876 0.882 0.888 0.894 0.900 0.906
2.2 0.912 0.918 0.924 0.929 0.935 0.941 0.948 0.953 0.959 0.965
2.3 0.971 0.976 0.983 0.988 0.994 1.000 1.006 1.011 1.017 1.022
2.4 1.028 1.033 1.039 1.044 1.050 1.056 1.061 1.067 1.072 1.078
2.5 1.083 1.089 1.094 1.100 1.105 1.111 1.116 1.121 1.126 1.131
2.6 1.137 1.142 1.147 1.153 1.158 1.163 1.169 1.174 1.179 1.184
2.7 1.190 1.195 1.200 1.205 U 10 1.215 1.220 1-225 1-230 1.235
2.8 1.240 1.245 1.250 1.255 1.260 1.265 1.270 1.275 1.280 1-285
2.9 1.290 1.295 1300 1305 1310 1.314 1319 1324 1329 1.333
3.0 1.338 1.343 1.348 1352 1357 1.362 1.367 1.371 1376 1381
3.1 1386 1390 1395 1.400 1.405 1.409 1.414 1.418 1.423 1.427
3.2 1.432 1.436 1.441 1.446 1.450 1.455 1.459 1.464 1.468 1.473
3-3 1.477 1.482 1.486 1.491 1.496 1.500 1.504 1308 1313 1317
3.4 1321 1.525 1.529 1333 1337 1.542 1.546 1.550 1354 1358
3.5 1.562 1.566 1.570 1375 1.579 1.583 1.587 1.591 1395 1.600
3.6 1.604 1.608 1.612 1.617 1.621 1.625 1.629 1.633 1.637 1.642
3.7 1.646 1.650 1.654 1.658 1.662 1.666 1.671 1.675 1.679 1.683
3.8 1.687 1.691 1.695 1.700 1.704 1.708 1.712 1.715 1.719 1.723
3.9 1.727 1.731 1.735 1.739 1.742 1.746 1.750 1.754 1.758 1.762
seconds between the 2 marks on the viscometer. Calculate where k2 is the viscometer constant.
the kinematic viscosity (vj) of the solution using the following Determine the relative viscosity of the substance to be
expression: examined using the following expression:
t \ (k i ) V\/V2
where kx is the viscometer constant. Determine the intrinsic viscosity by interpolation, using
Dilute a suitable volume of citprietkylenediamine hydroxide the intrinsic viscosity table (Table 0315.-1).
solution R with an equal volume of water R and measure the Calculate the degree of polymerisation (P) vising the
flow time (¿2) using a suitable capillary viscometer. Calculate following expression:
the kinematic viscosity (va) of the solvent using the following
expression: 95 [ij]c
m [(100 - b) /100]
£2 (fea)
Intrinsic viscosity [tj]t at different values of relative viscosity rj^
M.
0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
n*
40 1.765 1.769 1.773 1.777 1.781 1.785 1.789 1.792 1.796 1.800
4.1 1.804 1.808 1.811 1.815 1.819 1.822 1.826 1.830 1.833 1.837
42 1.841 1.845 1.848 1.852 1.856 1.859 1.863 1.867 1.870 1.874
43 1.878 1.882 1.885 1.889 1.893 1.896 1.900 1.904 1.907 1.911
4.4 1.914 1.918 1.921 1.925 1.929 1.932 1.936 1.939 1.943 1.946
4.5 1.950 1.954 1.957 1.961 1.964 1.968 1.971 1.975 1.979 1.982
4.6 1.986 1.989 1.993 1.996 2.000 2.003 2.007 2.010 2.013 2.017
4.7 2.020 2.023 2.027 2.030 2.033 2.037 2.040 2.043 2.047 2.050
48 2.053 2.057 2.060 2.063 2.067 2.070 2.073 2.077 2.080 2.083
4.9 2.087 2.090 2.093 2.097 2.100 2.103 2.107 2.110 2.113 2.116
5.0 2.119 2.122 2.125 2.129 2.132 2.135 2.139 2.142 2.145 2.148
5.1 2.151 2.154 2.158 2.160 2.164 2.167 2.170 2.173 2.176 2.180
5.2 2.183 2.186 2.190 2.192 2.195 2.197 2.200 2.203 2.206 2.209
5.3 2.212 2J215 2.218 2.221 2^24 2.227 2.230 2.233 2236 2.240
5.4 2.243 2-246 2.249 2252 2.255 2.258 2.261 2.264 2.267 2.270
5.5 ’ 2.273 2.276 2279 2282 2285 2.288 2.291 2.294 2297 2.300
5.6 2.303 2.306 2.309 2.312 2.315 2.318 2.320 2.324 2.326 2.329
5.7 2.332 2.335 2.338 2.341 2.344 2.347 2.350 2.353 2.355 2.358
5.8 2.361 2.364 2.367 2.370 2.373 2.376 2.379 2.382 2.384 2.387
5.9 2.390 2.393 2.396 2.400 2.403 2.405 2.408 2.411 2.414 2.417
6.0 2.419 2.422 2.425 2.428 2.431 2.433 2.436 2.439 2.442 2.444
6.1 2.447 2.450 2.453 2.456 2.458 2.461 2.464 2.467 2.470 2.472
62 2.475 2.478 2.481 2.483 2.486 2.489 2.492 2.494 2.497 2.500
6.3 2.503 2.505 2308 2.511 2.513 2.516 2.518 2.521 2.524 2.526
6.4 2.529 2.532 2.534 2.537 2.540 2.542 2.545 2.547 2.550 2.553
6.5 2.555 2.558 2.561 2.563 2.566 2.568 2.571 2.574 2.576 2.579
6.6 2.581 2.584 2.587 2.590 2.592 2.595 2.597 2.600 2.603 2.605
6.7 2.608 2.610 2.613 2.615 2.618 2.620 2.623 2.625 2.627 2.630
6.8 2.633 2.635 2.637 2.640 2.643 2.645 2.648 2.650 2.653 2.655
6.9 2.658 2.660 2.663 2.665 2.668 2.670 2.673 2.675 2.678 2.680
7.0 2.683 2.685 2.687 2.690 2.693 2.695 2.698 2.700 2.702 2.705
7.1 2.707 2.710 2.712 2.714 2.717 2.719 2.721 2.724 2.726 2.729
72 2.731 2.733 2.736 2.738 2.740 2.743 2.745 2.748 2.750 2.752
7.3 2.755 2.757 2.760 2.762 2.764 2.767 1769 2.771 2.774 2.776
7.4 2.779 2.781 2.783 2.786 2.788 2.790 2.793 2.795 2.798 2.800
7.5 2.802 2.805 2.807 2.809 2.812 2.814 2.816 2.819 2.821 2.823
7.6 2.826 2.828 2.830 2.833 2.835 2.837 2.840 2.842 2.844 2.847
7.7 2.849 2.851 2.854 2.856 2.858 2.860 2.863 2.865 2.868 2.870
7.8 2.873 2.875 2.877 2.879 2.881 2.884 2.887 2.889 2.891 2.893
7.9 2.895 2.898 2.900 2.902 2.905 2.907 2.909 2.911 2.913 2.915
Intrinsic viscosity [q]c at different values of relative viscosity tj„,
w,
1* 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09
8.0 2.918 2.920 2.922 1924 1926 1928 1931 1933 2.935 1937
8.1 2.939 2.942 2.944 2.946 1948 2.950 1952 1955 2.957 2.959
8.2 2.961 2.963 2.966 1968 1970 2.972 1974 1976 2.979 2.981
8.3 2.983 2.985 2.987 1990 1992 2.994 1996 1998 3.000 3.002
8.4 3.004 3.006 3.008 3.010 3.012 3.015 3.017 3.019 3.021 3.023
8.5 3.025 3.027 3.029 3.031 3.033 3.035 3.037 3.040 3.042 3.044
8.6 3.046 3.048 3.050 3.052 3.054 3.056 3.058 3.060 3.062 3.064
8.7 3.067 3.069 3.071 3.073 3.075 3.077 3.079 3.081 3.083 3.085
8.8 3.087 3.089 3.092 3.094 3.096 3.098 3.100 3.102 3.104 3.106
8.9 3.108 3.110 3.112 3.114 3.116 3.118 3.120 3.122 3.124 3.126
9.0 3.128 3.130 3.132 3.134 3.136 3.138 3.140 3.142 3.144 3.146
9.1 3.148 3.150 3.152 3.154 3.156 3.158 3.160 3.162 3.164 3.166
9.2 3.168 3.170 3.172 3.174 3.176 3.178 3.180 3.182 3.184 3.186
9.3 3.188 3.190 3.192 3.194 3.196 3.198 3.200 3.202 3.204 3.206
9.4 3.208 3.210 3.212 3.214 3-215 3.217 3.219 3.221 3.223 3.225
9.5 3227 3.229 3.231 3.233 3-235 3237 3.239 3.241 3-242 3.244
9.6 3.246 3.248 3.250 3.252 3.254 3256 3.258 3.260 3.262 3-264
9.7 - 3.266 3.268 3.269 3-271 3273 3.275 3277 3279 3-281 3.283
9.8 3.285 3.287 3.289 3.291 3293 3.295 3.297 3298 3300 3.302
9.9 3.304 3.305 3.307 3.309 3.311 3.313 3.316 3.318 3320 3321
Intrinsic viscosity [//]c at different values of relative viscosity
Me
0.0 0.1 02 03 0.4 0.5 0.6 0.7 0.8 0.9
10 3.32 3.34 3.36 3.37 3.39 3.41 3.43 3.45 3.46 3.48
11 3.50 3.52 3.53 355 3.56 3.58 3.60 3.61 3.63 3.64
12 3.66 3.68 3.69 3.71 3.72 3.74 3.76 3.77 3.79 3.80
13 3.80 3.83 3.85 3.86 3.88 3.89 3.90 3.92 3.93 3.95
14 3.96 3.97 3.99 400 4.02 403 404 406 407 4.09
15 410 4.11 413 414 4.15 417 418 419 4.20 422
16 4.23 4.24 4.25 427 4.28 429 430 431 433 4.34
17 435 436 437 438 439 441 442 443 4.44 445
18 446 447 4.48 4.49 430 452 453 454 4.55 456
19 4.57 458 459 4.60 461 462 4.63 464 4.65 466
where m is the mass in grams of the substance to be Ether-soluble substances

examined and b is the loss on drying as a percentage. Maximum 0.15 per cent (15 mg) for the difference between
TESTS the mass of the residue and the mass obtained from a blank
determination.
Solubility
Dissolve 50 mg in 10 mL of antmoniacal solution of copper Place 10.0 g in a chromatography column about 20 mm in
tetrammine R. It dissolves completely, leaving no residue. internal diameter and pass 50 mL of peroxide-free ether R
through the column. Evaporate the eluate to dryness in a
pH (2.2.3)
previously dried and tared evaporating dish, with the aid of a
5.0 to 7.5 for the supernatant.
current of air in a fume cupboard. After all the ether has
Mix 10 g with 90 mL of carbon dioxide-free water R and allow evaporated, dry the residue at 105 °C for 30 min, allow to
to stand with occasional stirring for 1 h. cool in a desiccator and weigh. Cany out a blank
determination.
1-486 Cellulose Acetate 2016
Water-soluble substances Content

Maximum 1.5 per cent (15.0 mg) for the difference between — acetyl groups (C2H30 ; 43.04): 29.0 per cent to
the mass of the residue and the mass obtained from a blank 44.8 per cent (dried substance); 90.0 per cent to
determination. 110.0 per cent of the nominal content (dried substance).
Shake 6.0 g with 90 mL of carbon dioxide-free water R for ^CHARACTERS
10 min. Filter with the aid of vacuum into a tared flask. Appearance
Discard the first 10 mL of the filtrate and pass the filtrate White, yellowish-white or greyish-white, hygroscopic powder
through the same filter a second time, if necessary, to obtain or granules.
a clear filtrate. Evaporate a 15.0 mL portion of the filtrate to
Solubility
dryness in a tared evaporating dish without charring. Dry at
Practically insoluble in water, soluble in acetone, in formic
105 °C for 1 h, allow to cool in a desiccator and weigh.
acid and in a mixture of equal volumes of methanol and
Carry out a blank determination.
methylene chloride, practically insoluble in ethanol
Heavy metals (2.4.8) (96 per cent).0
Maximum 10 ppm.
IDENTIFICATION
Comparison cellulose acetate CRS.
Preparation Prepare a 20 g/L solution of cellulose acetate,
an oven at 105 °C for 3 h. previously dried, in acetone R, and spread 1 drop of the
solution between 2 sodium chloride plates; separate the
Sulfated ash (2.4.14) plates, heat them both at 105 °C for 1 h, and reassemble the
Maximum 0.3 per cent (dried substance), determined on dried plates.
1.0 g.
TESTS
Microbial contamination
Free acid
TAMC: acceptance criterion 103 CFU/g (2.6.12).
Maximum 0.1 per cent, calculated as acetic acid (dried
TYMC: acceptance criterion 102 CFU/g (2.6.12). substance).
Absence of Escherichia coli (2.6.13). To 5.00 g in a 250 mL conical flask, add 150 mL of carbon
Absence of Pseudomonas aeruginosa (2.6.13). dioxide-free water R, insert the stopper, swirl the suspension
Absence of Staphylococcus aureus (2.6.13). gently and allow to stand for 3 h. Filter, then wash the flask
Absence of Salmonella (2.6.13). and the filter with carbon dioxide-free water R, adding the
washings to the filtrate. Add 0.1 mL of phenolphthalein
FUNCTIONALITY-RELATED CHARACTERISTICS solution R1 and titrate the combined filtrate and washings
This section provides information on characteristics that are with 0.01 M sodium hydroxide until a pale pink colour is
recognised as being relevant control parameters for one or more obtained.
Junctions of the substance when used as an excipient (see chapter
1 mL of 0.01 M sodium hydroxide is equivalent to 0.6005 mg
5.15). This section is a rum-mandatory part of the monograph
of free add, calculated as acetic acid.
and it is not necessary to verify the characteristics to demonstrate
compliance. Control of these characteristics can however contribute ♦H eavy metals (2.4.8)
to the quality of a medicinal product by improving the consistency Maximum 10 ppm.
of the manufacturing process and the performance of the medicinal 2.0 g complies with test D. Prepare the reference solution
product during use. Where control methods are cited they are using 2 ml, of lead standard solution (10 ppm Pb) R .+
recognised as being suitablefor the purpose but other methods can Loss on drying (2.2.32)
also be used. Wherever resultsfor a particular characteristic are Maximum 5.0 per cent, determined on 1.000 g by drying in
reported, the control method must be indicated an oven at 105 °C for 3 h.
The following characteristics may be relevantfor powdered celhdose
used as diluent or disintegrant.
Particle-size distribution (2.9.31 or 2.9.38).
Powder flow (2.9.36) TAMC: acceptance criterion 103 CFU/g (2.6.12).
___________________________________________________________ PhEur TYMC: acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
Absence of Salmonella (2.6.13).
Cellulose Acetate(1) ***** ASSAY
A. Cellulose acetate containing not more than 42.0 per cent
(Ph. Eur. monograph 0887) * of acetyl groups.
Action and use To 2.000 g in a 500 mL conical flask, add 100 mL of
Excipient acetone R and 5-10 mL of water R. Close the flask and stir
with a magnetic stirrer until dissolution is complete.
PhEir__________________________________________________________ Add 30.0 mL of 1 M sodium hydroxide with constant stirring.
DEFINITION A finely divided precipitate of regenerated cellulose, free from
Pardy or completely O-acetylated cellulose. lumps, is obtained. Close the flask and stir with a magnetic
stirrer for 30 min. Add 100 mL of water R at 80 °C, washing
down the sides of the flask, stir for 2 min and cool to room
temperature. Titrate with 0.5 M sulfuric add, using 0.1 mL of
2016 Cellulose Acetate Butyrate 1-487
as indicator. Cany out a blank

phenolphthalein solution R1 Viscosity
titration. Dissolve 10 g in a mixture of 50 mL of methanol R and
Calculate the percentage content of acetyl groups using the 50 mL of methylene chloride R by shaking. Determine the
following expression: viscosity of this solution at 20 ± 0.1 °C using a rotating
viscometer (2 .2 . 1 0 ).
4.305 (nạ —m ) Acetyl groups
X 100
(100 - d) X m See Assay.
The following characteristics may be relevant for cellulose acetate
d = loss on drying as a percentage; used as matrix former in prolonged-release tablets.
m = mass of the substance to be examined, in grams;
Viscosity
= volume of 0.5 M sulfuric add used in the titration,
See test above.
in millilitres;
«2 = volume of 0.5 M sulfuric acid used in the blank Acetyl groups
titration, in millilitres. See Assay.
B. Cellulose acetate containing more than 42.0 per cent of Molecular m ass distribution (2.2.30)
acetyl groups. Particle-size distribution (2.9.31)
To 2.000 g in a 500 mL conical flask, add 30 mL of dimethyl Powder flow (2.9.36)
sulfoxide R and 100 mL of acetone R. Close the flask and stir PhEur
with a magnetic stirrer for 16 h. Add 30.0 mL of 1 M sodium 1 This monograph has undergone pharmacopoeial harmonisation.
hydroxide with constant stirring. Close the flask and stir with See chapter 5.8. pharmacopoeial harmonisation.
a magnetic stirrer for 6 min. Allow to stand without stirring
for 60 min. Resume stirring and add 100 mL of water R at
80 °C, washing down the sides of the flask, stir for 2 min
and cool to room temperature. Titrate with 0.5 M
hydrochloric acid, using 0.1 mL of phenolphthalein solution R l Cellulose Acetate Butyrate *****★
★
as indicator. Add 0.5 mL of 0.5 M hydrochloric add in excess, *****
stir for 5 min and allow to stand for 30 min. Titrate with
0.5 M sodium hydroxide until a persistent pink colour is Action and use
obtained, stirring with a magnetic stirrer. Calculate the net Excipient.
amount of 0.5 M sodium hydroxide consumed, in millimoles,
taking the mean of 2 blank titrations into consideration. PhEur.
Calculate the percentage content of acetyl groups using the DEFINITION
following expression: Partly or completely O-acetylated and O-butyrated cellulose.
4.30Ỗ X n Content
X 100 — acetyl groups (C2H 30): 2.0 per cent to 30.0 per cent
(100 - d) X m
(dried substance); 90.0 per cent to 110.0 per cent of that
stated on the label (dried substance);
= loss on drying as a percentage;
— butyryl groups (C4H 7O): 16.0 per cent to 53.0 per cent
= mass of the substance to be examined, in grams;
(dried substance); 90.0 per cent to 110.0 per cent of that
= net amount of 0.5 M sodium hydroxide consumed,
stated on the label (dried substance).
in millimoles.
CHARACTERS
STORAGE Appearance
In an airtight container. White, yellowish-white or greyish-white powder or granules,
LABELLING slightly hygroscopic.
The label states the nominal percentage content of acetyl Solubility
groups. Practically insoluble in water, soluble in acetone, in formic
FUNCTIONALITY-RELATED CHARACTERISTICS acid and in a mixture of equal volumes of methanol and
methylene chloride, practically insoluble in ethanol
recognised as bang relevant control parameters for one or more
(96 per cent).
functions of the substance when used as an excipient (see chapter IDENTIFICATION
5.15). Some of the characteristics described in the Functionality- A. Infrared absorption spectrophotometry (2.2.24).
related characteristics section may also be present in the mandatory Comparison cellulose acetate butyrate CRS.
part of the monograph since they also represent mandatory quality
The intensity of the bands may vary according to the degree
criteria. In such cases, a cross-reference to the tests described in the
of substitution.
mandatory part ủ included in the Functionality-related
characteristics section. Control of the characteristics cơn contribute B. It complies with the limits of the assay.
to the quality of a medicinal product by improving the consistency TESTS
of the manufacturing process and die performance of the medicinal Acidity
product during use. Where control methods are cited, they are To 5.00 g in a 250 mL conical flask, add 150 mL of carbon
recognised as bang suitablefor the purpose, but other methods can dioxide-free water R, insert the stopper, swirl the suspension
also be used Wherever resultsfor a particular characteristic are gently and allow to stand for 3 h. Filter, wash the flask and
reported, the control method must be indicated the filter with carbon dioxide-free water R. Combine the filtrate
The following characteristics may be relevantfor cellulose acetate and washings. Add 0.1 mL of phenolphthalein solution R l.
used as film former.
1-488 Cetirizine Hydrochloride 2016
Not more than 3.0 mL of 0 .0 1 M sodium hydroxide is 1*** ★

★
required to change the colour of the indicator.
Cetirizine Hydrochloride ★ ★
Heavy m etals (.2.4.8) (Cetirizine Dihydrochloride, Ph Eur monograph 1084) *****
Maximum 20 ppm.
COjH
Loss on drying (2.2.32) 2 HCI
an oven at 105 °C for 3 h. and enantiomer
Total ash (2.4.16)
Maximum 0.1 per cent C 2 1H 2 7 C I3 N 2 O 3 461.8 83881-52-1
ASSAY
Liquid chromatography (2.2.29). Action and use
Histamine H I receptor antagonist; antihistamine.
Test solution To 1.000 g of the substance to be exam in ed in a
500 mL conical flask, add 100 mL of acetone R and 10 mL Preparations
of water R. Close the flask and stir with a magnetic stirrer Cetirizine Capsules
until dissolution is complete. Add 30.0 mL of 1 M sodium Cetirizine Oral Solution
hydroxide with constant stirring. Close the flask and stir with Cetirizine Tablets
a magnetic stirrer for 30 min. Add 100 mL of hot water R at
80 °C, washing down the sides of the flask and stir for PhEur______________________________________________
2 min. Cool, centrifuge or filter the suspension and wash the DEFINITION
residue with water R. Combine the filtrate and washings, (RS)-2- [2-[4-[(4-Chlorophenyl)phenylmethyl]piperazin-1-
adjust to pH 3 with dilute phosphoric acid R and dilute to yl] ethoxy] acetic add dihydrochloride.
500.0 mL with water R.
Content
Reference solution Dissolve 0.200 g of glacial acetic add R and
0.400 g of butyric add R in water R, adjust to pH 3 with
dilute phosphoric add R and dilute to 500.0 mL with water R. CHARACTERS
Column: Appearance
— sizer. I = 0.25 m, 0 = 4.6 mm; White or almost white powder.
— stationary phase:. octadecylsUyl silica gel for chromatography R Solubility
(5 nm). Freely soluble in water, practically insoluble in acetone and
Mobile phase: in methylene chloride.
— mobile phase A: methanol R; IDENTIFICATION
— mobile phase B: phosphate buffer solution p H 3.0 R1;
Second identification A, C, D
Time Mobile phase A Mobile phase B A. Ultraviolet and visible absorption spectrophotometry
(min) (percent V/V) (per cent V/V) (2.2.25).
0 -3 0 5 95
Test solution Dissolve 20.0 mg in 50 mL of a 10.3 g/L
3 0 -3 5 5->20 95-» 80 solution of hydrochloric acid R and dilute to 100.0 mL with
3 5 -6 0 20 80 the same add. Dilute 10.0 mL of this solution to 100.0 mL
with a 10.3 g/L solution of hydrochloric add R.
6 0 -6 1 5 95
Flow rate 1.2 mL/min. Specific absorbance at the absorption maximum 359 to 381.
Detection Spectrophotometer at 210 nm. B. Infrared absorption spectrophotometry (2.2.24).
Injection 20 jjL. Comparison cetirizine dihydrochloride CRS.
Calculate the percentage content of acetic acid and butyric C. Thin-layer chromatography (2.2.27).
acid using the chromatograms obtained with the 2 solutions.
To calculate the percentage content of acetyl (C2H 30 ) and
in water R and dilute to 5 mL with the same solvent.
of butyryl (C4H 7O) groups, multiply the percentage content
of acetic add and butyric add by 0.717 and 0.807, Reference solution (a) Dissolve 10 mg of cetirizine
respectively. dihydrochloride CRS in water R and dilute to 5 mL with the
same solvent.
STORAGE
Reference solution (b) Dissolve 10 mg of chlorphenamine
maleate CRS in water R and dilute to 5 mL with the same
LABELLING solvent. Mix 1 mL of the solution and 1 mL of reference
The label states the nominal percentage content of acetyl and solution (a).
butyryl groups. Plate TLC silica gel G F 254 plate R.
PhEur Mobile phase ammonia R, methanol R, methylene chloride R
(1:10:90 V/V/V).
5 |iL.
Application
2016 Cetirizine Hydrochloride 1-489
Drying In a current of cold air. corresponding correction factor impurity A = 0.7;

Detection Examine in ultraviolet light at 254 nm. impurity C = 1.9; impurity D = 0.6; impurity E = 1.3;
impurity F = 1.9;
— the chromatogram shows 2 clearly separated spots. — impurities A, B, C, D, E, F: for each impurity, not more
than 1.5 times the area of the principal peak in the
Results The principal spot in the chromatogram obtained with
the test solution is similar in position and size to the principal (0.15 per cent);
solution (a). area of the principal peak in the chromatogram obtained
D. It gives reaction (a) of chlorides {2.3.1). with reference solution (b) (0.10 per cent);
TESTS — total: not more than 3 times the area of the principal peak
Solution S in the chromatogram obtained with reference solution (b)
Dissolve 1.0 g in carbon dioxide-free water R and dilute to (0.3 per cent);
20 mL with the same solvent. — disregard limit. 0.5 times the area of the principal peak in
Appearance of solution (0.05 per cent).
than reference solution BY7 (2.2.2, Method II). Loss on drying (2.2.32)
pH (2.2.3)
an oven at 105 °C.
Related substances
ASSAY
in the mobile phase and dilute to 100.0 mL with the mobile Dissolve 0.100 g in 70 mL of a mixture of 30 volumes of
phase. water R and 70 volumes of acetone R. Titrate with 0.1 M
sodium hydroxide to the 2nd point of inflexion. Determine the
Reference solution (a) Dissolve 2 mg of cetirizine
end-point potentiometrically (2.2.20). Carry out a blank
dihydrochloride CRS and 2 mg of cetirizine impurity A CRS in
titration.
phase. Dilute 1.0 mL of the solution to 100.0 mL with the 1 mL of 0.1 M sodium hydroxide is equivalent to 15.39 mg of
mobile phase. C21H 27CI3N 2O3.
Reference solution (b) Dilute 1.0 mL of the test solution to STORAGE
100.0 m l. with the mobile phase. Dilute 1.0 mL of this Protected from light.
solution to 10.0 mL with the mobile phase. IMPURITIES
Reference solution (c) Dissolve the contents of a vial of Specified impurities A,B, C, D, E, F
cetirizine for peak identification CRS (containing impurities B,
C, Dj E and F) in 5.0 mL of the mobile phase.
Column:
— size. I = 0.25 m, 0 = 4.6 mm;
— stationary phase, silica gelfor chromatography R (5 pm). by the general monograph Substancesfor pharmaceutical use
Mobile phase dUute sulfuric acid R, water R, acetordtrUe R (2034). It is therefore not necessary to identify these
(0.4:6.6:93 VIVIV). impurities for demonstration of compliance. See also 5.10.
Flow rate 1 mL/min. Control of impurities in substancesfor pharmaceutical use): G.
Injection 20 pL.
Run time 3 times the retention time of cetirizine.
with cetirizinefor peak identification CRS and the
the peaks due to impurities B, C, D, E and F; use the
chromatogram obtained with reference solution (a) to
identify the peak due to impurity A.
Relative retention With reference to cetirizine (retention
— peak-to-vaUey ratio: minimum 5, where Hp = height above
the baseline of the peak due to impurity C and B. (RS)-2-[4-[(4-chlorophenyl)phenylmethyl]piperazin-1-
H v = height above the baseline of the lowest point of the yl] acetic acid,
curve separating this peak from the peak due to cetirizine.
Limits:
— for the calculation of content, multiply
correction factors:
the peak areas of th© following impurities by the
1-490 Cetostearyl Alcohol 2016
O__ COsH
CHARACTERS
Appearance
White or pale yellow, wax-like mass, plates, flakes or
granules.
and enantiomer Solubility
Practically insoluble in water, soluble in ethanol (96 per cent)
C. (RS)-2- [2- [4- [(2-chlorophenyl)phenylmethyl]piperazin-1- and in light petroleum. When mdted, it is misdble with fatty
yl] ethoxy] acetic acid, oils, with liquid paraffin and with mdted wool fat.
IDENTIFICATION
Examine the chromatograms obtained in the assay.
Results The 2 principal peaks in the chromatogram obtained
with the test solution are similar in retention time to the
prindpal peaks in die chromatogram obtained with the
reference solution.
TESTS
The solution is clear (2.2.1) and not more intensely coloured
D. 1,4-bis [(4-chlorophenyl)phenylmethyI] piperazine, than reference solution B6 (2.2.2, Method II).
Civ. Dissolve 0.50 g in 20 mL of boiling ethanol (96 per cent) R.
' 0 ' 'COzH Allow to cool.
Melting point (2.2.14)
49 °C to 56 °C.
Acid value (2.5.1)
and enantiomer
Maximum 1.0.
E. (i?S)-2-[2-[2-[4-[(4-chlorophenyI)phenylmethyl]piperazin- Hydroxyl value (2.5.3, Method A)
1-yl] ethoxy] ethoxy]acetic add (ethoxycetirizine), 208 to 228.
ov , co 2h Iodine value (2.5.4, Method A)
Maximum 2.0.
Dissolve 2.00 g in methylene chloride R and dilute to 25 mL
with the same solvent
Saponification value (2.5.6)
Maximum 2.0.
ASSAY
F. 2- [2- [4-(diphenylmethyl)piperazin- 1-yl] ethoxy] acetic add,
Gas chromatography (2.2.28): use the normalisation
CU ^ /-v. /-v .OH procedure.
Test sdudon Dissolve 0.100 g of the substance to be
'N \ / examined in ethanol (96 per cent) R and dilute to 10.0 mL
HW , with the same solvent.
\ — v and enantiomer Reference sdution Dissolve 60 mg of cetyl alcohd CRS and
40 mg of stearyl alcohol CRS in ethanol (96 per cent) R and
G. (RS)-2- [4- [(4-chlorophenyl)phenylmethyl]piperazin-l - dilute to 10 mL with the same solvent. Dilute 1 mL of this
yl]ethan-l-ol. solution to 10 mL with ethanol (96 per cent) R.
PhEur Column:
— sizer. I = 30 m, 0 = 0.32 mm,
— stationary phase, poly(dimethyl)sUoxane R (1 |im).
Carrier gas heliumfor chromatography R.
Cetostearyl Alcohol ★ ★ Flow rale 1 mL/min.

★ ★
Split ratio 1:100.
Temperature:
Action and use
Exdpient.
Time Temperature
PhEtr__________________________
fmin) CO
DEFINITION Column 0 -2 0 150->250
Mixture of solid aliphatic alcohols, mainly octadecan-l-ol 2 0 -4 0 250
(stearyl alcohol; C 18H 380 ; M, 270.5) and hexadecan-l-ol
Injection port 250
(cetyl alcohol; Ci6H340 ; 242.4), of animal or vegetable
origin. Detector 250
Content
— stearyl alcohol: m in im u m 40.0 per cent, Detection Flame ionisation.
— sum of the contents of stearyl alcohol and cetyl alcohol:
Irqection 1 jiL.
minimum 90.0 per cent.
2016 Cetostearyl Alcohol 1-491
System suitability: reference solution: to the principal spots in the chromatogram obtained
— resolution: minimum 5.0 between the peaks due to cetyl with reference solution (a);
alcohol and stearyl alcohol. — 2 of the spots in the chromatogram obtained with test
Calculate the percentage contents of C 16H 340 and Ci8H380. solution (b) are similar in position and colour to the
principal spots in the chromatogram obtained with
__________________________________________________________ PhEur
reference solution (b).
6 . Examine the chromatograms obtained in the assay of
cetostearyl alcohol.
Emulsifying Cetostearyl Alcohol ***** with the test solution are similar in retention time to the
2 principal peaks in the Chromatograms obtained with
(Type A) ***** reference solutions (a) and (b).
(Ph. Ettr. monograph 0801)
C. It gives a yellow colour to a non-luminous flame.
Action and use D. To 0.3 g add 20 mL of anhydrous ethanol R and heat to
Excipient. boiling on a water-bath with shaking. Filter the mixture
immediately, evaporate to dryness and take up the residue in
PhEir__________________________________________________________ 7 mL of water R. To 1 mL of the solution add 0.1 mL of a
DEFINITION 1 g/L solution of methylene blue R, 2 mL of dilute sulfuric
Mixture of cetostearyl alcohol and sodium cetostearyl sulfate. add R and 2 mL of methylene chloride R and shake. A blue
A suitable buffer may be added. colour develops in the lower layer.
Content TESTS
— cetostearyl alcohol: minimum 80.0 per cent (anhydrous Acid value (2.5.1)
substance); Maximum 2.0.
— sodium cetostearyl sulfate: minimum 7.0 per cent Iodine value (2.5.4, Method A)
(anhydrous substance). Maximum 3.0.
CHARACTERS Dissolve 2.00 g in 25 mL of methylene chloride R.
Appearance Saponification value (2.5.6)
White or pale yellow, waxy mass, plates, flakes or granules. Maximum 2.0.
Solubility Water (2.5.12)
Soluble in hot water giving an opalescent solution, practically Maximum 3.0 per cent, determined on 2.50 g.
insoluble in cold water, slightly soluble in ethanol
ASSAY
(96 per cent).
Cetostearyl alcohol
IDENTIFICATION Gas chromatography (2.2.28).
First identification B, C, D \ Internal standard solution Dissolve 0.200 g of
Second identification A, C, D. 1-nonadecanol CRS in anhydrous ethanol R and dilute to
A. Thin-layer chromatography (2.2.27). 100.0 mL with the same solvent.
Test solution (a) Dissolve 0.1 g of the substance to be Test solution Dissolve 0.200 g of the substance to be
examined in 10 mL of trimethylpentane R, heating on a water- examined in 25.0 mL of the internal standard solution.
bath. Shake with 2 mL of ethanol (70 per cent VIV) R and Add 25 mL of water R and shake with 4 quantities, each of
allow to separate. Use the lower layer as test solution (b). 25 mL, of pentane R, adding sodium chloride R , if necessary,
Dilute 1 mL of the upper layer to 8 mL with to facilitate the separation of the layers. Combine the upper
trimethylpentane R. layers, wash with 2 quantities, each of 30 mL, of water R, dry
Test solution (b) Use the lower layer obtained in the over anhydrous sodium sulfate R and filter.
preparation of test solution (a). Reference solution (a) Dissolve 0.100 g of cetyl alcohol CRS in
Reference solution (a) Dissolve 24 mg of cetyl alcohol CRS and 25.0 mL of the internal standard solution. Add 25 mL of
16 mg of stearyl alcohol CRS in 10 mL of trimethylpentane R. water R and shake with 4 quantities, each of 25 m l, of
pentane R, adding sodium chloride R, if necessary, to facilitate
Reference solution (b) Dissolve 20 mg of sodium cetostearyl
the separation of the layers. Combine the upper layers, wash
sulfate R in 10 mL of ethanol (70 per cent VIV) R, heating on
with 2 quantities, each of 30 mL, of water R, dry over
a water-bath.
anhydrous sodium sulfate R and filter.
Plate TLC octadecylsHyl silica gel F 254 plate R.
Reference solution (b) Dissolve 0.100 g of stearyl alcohol CRS
Mobile phase water R, acetone R, methanol R in 25.0 mL of the internal standard solution. Add 25 mL of
(20:40:40 V/VIV). water R and shake with 4 quantities, each of 25 mL, of
Application 10 jxL. pentane R, adding sodium chloride R, if necessary, to facilitate
Development Over 2/3 of the plate. the separation of the layers. Combine the upper layers, wash
Drying In air. with 2 quantities, each of 30 mL, of water R, dry over
anhydrous sodium sulfate R and filter.
Detection Spray with a 50 g/L solution of phosphomolybdic
Column:
acid R in ethanol (96 per cent) R; heat at 120 °C until spots
appear (about 5 min). — material: fused silica;
— size: I = 25 m, 0 = 0.25 mm;
Results:
— stationary phaser. poly(dimethyl)süoxane R (film thickness
— the 2 principal spots in the chromatogram obtained
0.25 pm).
with test solution (a) are similar in position and colour
1-492 Cetostearyl Alcohol 2016
Carrier gas helium for chromatography R. Sodium cetostearyl sulfate

Flow rate 1mL/min. Disperse 0.300 g in 25 mL of methylene chloride R.
1:100.
Split ratio
Add 50 mL of water R and 10 mL of dimidium bromide-sidfan
blue mixed solution R. Titrate with 0.004 M benzethonium
Temperature:
chloride, using sonication, heating, and allowing the layers to
separate before each addition, until the colour of the lower
Time Temperature layer changes from pink to grey.
(min) (°C) 1 mL of 0.004 M benzethonium chloride is equivalent to
Column 0 -2 0 150 -» 250
1.434 mg of sodium cetostearyl sulfate.
Injection port 250
LABELLING
Detector 250 The label states, where applicable, the name and
concentration of any added buffer.
___________________________________________________________PhEur
Injection 1 jiL.
Elution order Cetyl alcohol5 stearyl alcohol, 1-nonadecanol.
Calculate the percentage content of cetyl alcohol in the ★ ★
substance to be examined using the following expression and Emulsifying Cetostearyl Alcohol ★ ★
taking into account the assigned content of cetyl alcohol CRS : *.*★* ★
(Type B)
a A.2 TTIj « 1 ^
Ax x — x - 7- ^ x — x 100
Ax Ax m Action and use
Excipient.
= area of the peak due to cetyl alcohol in the PhEur.
chromatogram obtained with the test solution;
= area of the peak due to cetyl alcohol CRS in the DEFINITION
-chromatogram obtained with reference solution Mixture of cetostearyl alcohol and sodium laurilsulfate.
(a); A suitable buffer may be added.
Ai = area of the peak due to the internal standard in Content
the chromatogram obtained with the test — cetostearyl alcohol: minimum 80.0 per cent (anhydrous
solution; substance);
A2 = area of the peak due to the internal standard in — sodium laurilsulfate: minimum 7.0 per cent (anhydrous
the chromatogram obtained with reference substance).
solution (a); CHARACTERS
m = mass of the substance to be examined in the test Appearance
solution, in milligrams; White or pale yellow, waxy mass, plates, flakes or granules.
mXiy = mass of cetyl alcohol CRS in reference solution
(a), in milligrams. Solubility
Soluble in hot water giving an opalescent solution, practically
Calculate the percentage content of stearyl alcohol in the insoluble in cold water, slightly soluble in ethanol
substance to be examined using the following expression and (96 per cent).
taking into account the assigned content of stearyl
alcohol CRS: IDENTIFICATION
First identification B, C, D
j A3 n ij y 1 Second identification A , C, D.
Ax x —— x x — x 100
A\ A z,y m A. Thin-layer chromatography (2.2.27).
Az = area of the peak due to stearyl alcohol in the examined in 10 ml. of trimethylpentane R, heating on a water-
chromatogram obtained with the test solution; bath. Shake with 2 mL of ethanol (70 per cent V/V) R and
A Ziy = area of the peak due to stearyl alcohol CRS in the allow to separate. Use the lower layer as test solution (b).
chromatogram obtained with reference solution Dilute 1 mL of the upper layer to 8 mL with
(b); trimethylpentane R.
A\ = area of the peak due to the internal standard in Test solution (b) Use the lower layer obtained in the
the chromatogram obtained with the test preparation of test solution (a).
solution; Reference solution (a) Dissolve 24 mg of cetyl alcohol CRS and
Aj = area of the peak due to the internal standard in 16 mg of stearyl alcohol CRS in 10 mL of trimethylpentane R.
the chromatogram obtained with reference
Reference solution (b) Dissolve 20 mg of sodium
solution (b);
laurilsulfate CRS in 10 mL of ethanol (70 per cent V/V) R,
m = mass of the substance to be examined in the test
solution, in milligrams; heating on a water-bath.
mZjy = mass of stearyl alcohol CRS in reference solution Plate TLC octadecylsUyl silica gel F 254 phue &
(b), in milligrams. Mobile phase water R, acetone R, methanol R
The percentage content of cetostearyl alcohol corresponds to (20:40:40 V/V/V).
the sum of the percentage contents of cetyl alcohol and 10 jiL.

Application
stearyl alcohol. Development Over 2/3 of the plate.
2016 Cetostearyl Alcohol 1-493
Drying In air. with 2 quantities, each of 30 mL, of water R, dry over

Detection Spray with a 50 g/L solution of phosphomofybdic anhydrous sodium sulfate R and filter.
acid R in ethanol (96 per cent) R; heat at 120 °C until spots Column:
appear (about 5 min). — material: fused silica;
Results: — size. I = 25 m, 0 = 0.25 mm;
— the 2 principal spots in the chromatogram obtained — stationary phase, poly(dimethyl)sUoxane R (film thickness
with test solution (a) are similar in position and colour 0.25 (im).
to the principal spots in the chromatogram obtained Carrier gas helium for chromatography R.
with reference solution (a); Flow rate 1 mL/min.
— 1 of the spots in the chromatogram obtained with test Split ratio 1:100.
solution (b) is similar in position and colour to the
Temperature:
principal spot in the chromatogram obtained with
B. Examine the chromatograms obtained in the assay of Time Temperature
(min) (°C)
cetostearyl alcohol.
Column 0 -2 0 150->250
with the test solution are similar in retention time to the Injection port 250
2 principal peaks in the chromatogramS obtained with Detector 250
reference solutions (a) and (b).
C. It gives a yellow colour to a non-luminous flame.
D. To 0.3 g add 20 mL of anhydrous ethanol R and heat to
boiling on a water-bath with shaking. Filter the mixture Injection1 |iL.
immediately, evaporate to dryness and take up the residue in Elution order Cetyl alcohol, stearyl alcohol, 1-nonadecanol.
7 mL of water R. To 1 mL of the solution add 0.1 mL of a Calculate the percentage content of cetyl alcohol in the
1 g/L solution of methylene blue R, 2 mL of dilute sulfuric substance to be examined using the following expression and
arid R and 2 mL of methylene chloride R and shake. A blue taking into account the assigned content of cetyl alcohol CRS:
colour develops in the lower layer.
TESTS A? TTlj « 1
X -r—X X — X 100
A d d value (2.5.1) Ax m
Maximum 2.0.
Iodine value (2.5.4, Method A) = area of the peak due to cetyl alcohol in the
Maximum 3.0. chromatogram obtained with the test solution;
Dissolve 2.00 g in 25 mL of methylene chloride R. = area of the peak due to cetyl alcohol CRS in the
Saponification value (2.5.6) chromatogram obtained with reference solution
Maximum 2.0. (a);
Ai = area of the peak due to the internal standard in
W ater (2.5.12) the chromatogram obtained with the test
Maximum 3.0 per cent, determined on 2.50 g. solution;
ASSAY A2 = area of the peak due to the internal standard in
Cetostearyl alcohol the chromatogram obtained with reference
Gas chromatography (2.2.28). solution (a);
Internal standard solution Dissolve 0.200 g of
1 -nonadecanol CRS in anhydrous ethanol R and dilute to
solution, in milligrams;
mXjy = mass of cetyl alcohol CRS in reference solution
(a), in milligrams.
examined in 25.0 mL of the internal standard solution. Calculate the percentage content of stearyl alcohol in the
Add 25 mL of water R and shake with 4 quantities, each of substance to be examined using the following expression and
25 mL, of pentane R, adding sodium chloride R, if necessary, taking into account the assigned content of stearyl
to facilitate the separation of the layers. Combine the upper alcohol CRS:
layers, wash with 2 quantities, each of 30 mL, of water R, dry
over anhydrous sodium sulfate R and filter. ■ A$ 171» y
Ax x — x — x 100
Reference solution (a) Dissolve 0.100 g of cetyl alcohol CRS in ■^1
25.0 mL of the internal standard solution. Add 25 mL of
water R and shake with 4 quantities, each of 25 mL, of Az area of the peak due to stearyl alcohol in the
pentane R, adding sodium chloride R, if necessary, to facilitate chromatogram obtained with the test solution;
the separation of the layers. Combine the upper layers, wash A z¿ area of the peak due to stearyl alcohol CRS in the
with 2 quantities, each of 30 mL, of water R, dry over chromatogram obtained with reference solution
anhydrous sodium sulfate R and filter. (b);
Reference solution (b) Dissolve 0.100 g of stearyl alcohol CRS area of the peak due to the internal standard in
in 25.0 mL of the internal standard solution. Add 25 mL of the chromatogram obtained with the test
water R and shake with 4 quantities, each of 25 mL, of solution;
pentane R, adding sodium chloride R, if necessary, to facilitate area of the peak due to the internal standard in
the separation of the layers. Combine the upper layers, wash the chromatogram obtained with reference
solution (b);
1-494 Cetostearyl Isononanoate 2016
m — mass of the substance to be examined in the test Iodine value (2.5.4, Method A)
solution, in milligrams; Maximum 1.0.
mZiy = mass of stearyl alcohol CRS in reference solution Saponification value (2.5.6)
(b), in milligrams. 135 to 148, determined on 1.0 g.
The percentage content of cetostearyl alcohol corresponds to Heavy metals (2.4.8)
the sum of the percentage contents of cetyl alcohol and Maximum 10 ppm.
stearyl alcohol.
2.0 g complies with test D. Prepare the reference solution
Sodium laurilsulfate using 2 mL of lead standard solution (10 ppm Pb) R
Disperse 0.300 g in 25 mL of methylene chloride R
W ater (2.5.12)
Add 50 mL of water R and 10 mL of dimidium bromide-sulfan
blue mixed solution R. Titrate with 0.004 M benzethonium
chloride, using sonication, heating, and allowing the layers to Total ash (2.4.16)
separate before each addition, until the colour of the lower Maximum 0.2 per cent, determined on 2.0 g.
layer changes from pink to grey. _____________ '____________________________________________ PhEur
1 mL of 0.004 M benzethonium chloride is equivalent to
1.154 mg of sodium laurilsulfate.
LABELLING
The label states, where applicable, the name and Cetrlmide *****
* ★
concentration of any added buffer. (Ph. Eur. monograph 0378) *
__________________________________________________________ PhEur
H3C + CH3
H3C j > N n Br
K , ch3
Cetostearyl Isononanoate ***** Action and use

*. * Antiseptic.
Preparations
Action and use Cetrimide Cream
Excipient Cetrimide Emulsifying Ointment
PhEur__________________________________________________________ Strong Cetrimide Solution
DEFINITION PhEur__________________________________________________________
Mixture of esters of cetostearyl alcohol with isononanoic add, DEFINITION
mainly 3,5,5-trimethylhexanoic add.
Cetrimide consists of trimethyltetradecylammonium bromide
CHARACTERS and may contain smaller amounts of dodecyl- and hexadecyl-
Appearance trimethylammonium bromides.
Clear, colourless or slightly yellowish liquid. Content
Solubility 96.0 per cent to 101.0 per cent of alkyltrimethylammonium
Practically insoluble in water, soluble in ethanol (96 per cent) bromides, calculated as Ci 7H38BrN (336.4) (dried
and in light petroleum, miscible with fatty oils and with substance).
liquid paraffins. CHARACTERS
Viscosity 15 mPa-s to 30 mPa-s.
Appearance
Relative density White or almost white, voluminous, free-flowing powder.
0.85 to 0.86. Solubility
Refractive index 1.44 to 1.45. Freely soluble in water and in alcohol.
IDENTIFICATION IDENTIFICATION
A. On cooling, turbidity occurs bdow 15 °C. A Dissolve 0.25 g in alcohol R and dilute to 25.0 mL with
B. Saponification value (see Tests). the same solvent. At wavelengths from 260 nm to 280 nm,
C. Infrared absorption spectrophotometry (2.2.24). the absorbance (2.2.25) of the solution has a maximum
of 0.05.
Comparison Ph. Eur. reference spectrum of cetostearyl
isononanoate. B. Dissolve about 5 mg in 5 mL of buffer solution p H 8.0 R
Add about 10 mg of potassiumferruyanide R A yellow
TESTS predpitate is formed. Prepare a blank in the same manner
Appearance but omitting the substance to be examined: a yellow solution
The substance to be examined is dear (2.2.1) and not more is observed but no predpitate is formed.
intensely coloured than reference solution Y6
C. Solution S (see Tests) froths copiously when shaken.
(2.2.2, Method I).
D. Thin-layer chromatography (2.2.27).
Acid value (2.5.1)
Maximum 1 .0 , determined on 5.0 g.
in water R and dilute to 5 mL with the same solvent.
Hydroxyl value (2.5.3, Method A)
Reference solution Dissolve 0.10 g of
Maximum 5.0, determined on 4.0 g. Add 5.0 mL of
trimethyhetradecylammordttm bromide CRS in water R and
acetylating reagent.
dilute to 5 mL with the same solvent.
2016 Cetrimide Solution 1-495
Plate T LC sUamsed silica gel F 254 plate R.

Strong Cetrimide Solution
Mobile phase acetone R, 270 g/L solution of sodium acetate R,
methanol R (20:35:45 V/V/V). Action and use
Application 1 jiL. Antiseptic.
Development Over a path of 12 cm. Preparation
Drying In a current of hot air. Cetrimide Solution
Detection Mow to cool; expose the plate to iodine vapour and
DEFINITION
Strong Cetrimide Solution is an aqueous solution of
Result The principal spot in the chromatogram obtained with cetrimide. It contains 2 0 to 40% w/v of cetrimide, calculated
the test solution is similar in position, colour and size to the as C 17H 38BrN. It contains Ethanol (9 6 per cent) or
p rincip al spot in the chromatogram obtained with the Isopropyl Alcohol or both. It may be perfumed and may
reference solution. contain colouring matter.
E. It gives reaction (a) of bromides (2.3.1).
PRODUCTION
TESTS In making Strong Cetrimide Solution, Ethanol (9 6 per cent)
Solution S may be replaced by Industrial Methylated Spirit, provided
Dissolve 2.0 g in carbon dioxide-free water R and dilute to that the law and the statutory regulations governing the use
100 mL with the same solvent. of Industrial Methylated Spirit are observed.
Appearance of solution Content of cetrimide Ci 7H38BrN
Solution S is dear (2.2.1) and colourless (2.2.2, Method II). 9 5 .0 to 105.0% of the stated amount.
Acidity or alkalinity IDENTIFICATION
To 5 0 m l . of solution S add 0.1 mT. of bromocresolpurple A. Dilute a volume of the solution containing 0 .1 g of
solution R. Not more than 0.1 mL of 0.1 M hydrochloric add cetrimide to 5 mL with water and add 2 mL of a 5% w/v
or 0.1 M sodium hydroxide is required to change the colour of solution of potassium hexacyanoferrate(m). A yellow precipitate
the indicator. is produced.
Amines and am ine salts 6 . Shake together 5 mL of water, 1 mL of 2m sulfuric add,
Dissolve 5.0 g in 30 mL of a mixture of 1 volume of 1 M 2 mL of chloroform and 0 .0 5 ml. of methyl orange solution;
hydrochloric add and 99 volumes of methanol R and add the chloroform layer is colourless. Add 0.1 mL of the
100 mL of 2 -propanol R. Pass a stream of nitrogen R slowly solution being examined and shake; a yellow colour is
through the solution. Gradually add 15.0 mL of 0.1 M produced slowly in the chloroform layer.
tetrabutylammonium hydroxide and record the potentiometric
C. Yields reaction A characteristic of bromides, Appendix VI.
titration curve (2.2.20). If the curve shows 2 points of
inflexion, the volume of titrant added between the 2 points is TESTS
not greater than 2.0 mL. Acidity or alkalinity
Dilute a volume of the solution containing 10 g of cetrimide
to 1 00 mL and add 0.1 mL of bromocresol purple solution.
Not more than 1.0 mL of either 0.1m hydrochloric add KS or
0 .1 m sodium hydroxide VS is required to change the colour of
Sulfated ash (2.4.14) the solution.
Miscibility with ethanol
ASSAY Mix a volume of the solution containing 1.6 g of cetrimide
Dissolve 2.000 g in water R and dilute to 100.0 mL with the with a mixture of 2 mL of water and 16 mL of ethanol
same solvent. Transfer 25.0 mL of the solution to a (96%). The solution remains dear, Appendix IV A.
separating funnel, add 25 mL of chloroform R, 10 mL of
N eutral substances
0.1 M sodium hydroxide and 10.0 mL of a freshly prepared
To a volume of the solution containing 10 g of cetrimide add
50 g/L solution of potassium iodide R. Shake, allow to separate 2 5 mT. of ethanol (50%), acidify to bromophenol blue solution
and discard the chloroform layer. Shake the aqueous layer by the drop wise addition of hydhrochioric add and add
with 3 quantities, each of 10 mL, of chloroform R and discard 0 .0 5 mL in excess. Transfer quantitatively to the extraction
the chloroform layers. Add 40 mL of hydrochloric add R, compartment of an apparatus designed for continuous liquid-
allow to cool and titrate with 0.05 M potassium iodate until liquid extraction by fluids of a lesser density than water,
the deep brown colour is almost discharged. Add 2 mL of washing out the beaker with 10 ml. ethanol (50%) and
chloroform R and continue the titration, shaking vigorously,
adding the washings to the bulk of the solution in the
until the colour of the chloroform layer no longer changes. extractor. Add sufficient ethanol (50%), if necessary, to half
Carry out a blank titration on a mixture of 10.0 mL of the fill the extraction chamber to the level of the overflow limb.
freshly prepared 50 g/L solution of potassium iodide R, 20 mL Add sufficient purified hexane to fill the extraction chamber,
of water R and 40 mL of hydrochloric add R. secure an overflow volume of about 3 0 mL in the ebullition
1 mL of 0.05 Mpotassium iodate is equivalent to 33.64 mg of flask and heat using an electrically heated mantle. Ensure
C 17H 38BrN. that a continuous flow of hexane through the aqueous
PhEur ethanol layer is observed and continue the extraction for
16 hours. Transfer the hexane extract to a separating funnel,
washing out the flask with 10 mL of purified hexane. Shake
the combined extract and washings with 25 mL of ethanol
(50%) and discard the aqueous ethanol layer. Filter the
hexane layer through a dry filter paper (Whatman No. 1 is
1-496 Cetyl Alcohol 2016
suitable) into a tared flask and remove the solvent using a ★.**★ ★
rotary evaporator at 40° and then at room temperature at a
Cetyl Alcohol ★ ★
pressure not exceeding 0.7 kPa for 2 hours. The residue (Ph Eur. monograph 0540) *****
weighs not more than 0.4 g.
Non-quatem ised am ines Action and use
To a volume of the solution containing 10 g of cetrimide add Exdpient.
a mixture of 100 mL of propan-2 -ol, 0.1 mL of hydrochloric PhEur.
acid and 20 mL of methanoL Titrate with
0 .1 m tetrabutylammonium hydroxide VS passing a slow current DEFINITION
of nitrogen through the solution and determining the end Mixture of solid alcohols, mainly hexadecan-l-ol (C 16H 34O;
point potentiometrically using a platinum-glass electrode Afr 242.4), of animal or vegetable origin.
system. Inflections in the titration curve indicate (A) Content
neutralisation of excess hydrochloric add and (5) Minimum 95.0 per cent of C 16H 340 .
neutralisation of non-quatemised amine salts. The difference
CHARACTERS
between the volumes corresponding to A and B is not more
than 10 mL (2.4%, calculated as Ci6H 35N). Appearance
White or almost white, unctuous mass, powder, flakes or
Ethanol; Isopropyl alcohol granules.
Carry out one or both of the following methods according to
the dedared alcohol content of die solution being examined. Solubility
Practically insoluble in water, fredy soluble or sparingly
Ethanol Not more than 10.0% v/v, by the method for the
soluble in ethanol (96 per cent). When mdted, it is misdble
determination of ethanol, Appendix VH3 F. Use on-column
with vegetable and animal oils, with liquid paraffin and with
injection and do not heat the injection port. mdted wool fat.
Isopropyl alcohol Not more than 10.0% v/v, by the method
for the determination of ethanol, Appendix VIH F, with the IDENTIFICATION
following modifications. For solution (1) use a solution Examine the chromatograms obtained in the assay.
containing 5.0% v/v of propan-2-ol and 5.0% v/v of propan-1- Results The principal peak in the chromatogram obtained
ol (internal standard). For solution (2 ) vise the solution being with the test solution is similar in retention time to the
examined, diluted with water, if necessary, to contain about prindpal peak in the chromatogram obtained with reference
5.0% v/v of isopropyl alcohol. Maintain the colu m n solution (a).
temperature at 170°, use o n -co lu m n injection and do not TESTS
heat the injection port. Appearance of solution
ASSAY The solution is clear (2.2.1) and not more intensely coloured
Dilute a volume containing 4 g of cetrimide with suffident than reference solution B6 (2.2.2, Method II).
water to produce 100 m L Transfer 25 mL of the solution to Dissolve 0.50 g in 20 mL of boiling ethanol (96 per cent) R.
a separating funnel and add 25 mL of chloroform, 10 mL of Allow to cool.
0.1m sodium hydroxide and 10 mL of a freshly prepared Melting point (2.2.14)
8.0% w/v solution of potassium iodide. Shake well, allow to 46 °C to 52 °C.
separate and discard the chloroform layer. Wash the aqueous
layer with three 10 mL quantities of chloroform and discard Acid value (2.5.1)
the washings. Add 40 mL of hydrochloric acid, cool and titrate Maximum 1.0.
with 0.05m potassium iodate VS until the deep brown colour is Hydroxyl value (2.5.3, Method A)
almost discharged. Add 2 ml, of chloroform and continue the 218 to 238.
titration, with shaking, until the chloroform layer becomes Iodine value (2.5.4, Method A)
colourless. Carry out a blank titration on a mixture of 10 mT. Maximum 2.0.
of the freshly prepared potassium iodide solution, 20 mL of Dissolve 2.00 g in methylene chloride R and dilute to 25 mL
water and 40 mL of hydrochloric acid. The difference between
the titrations represents the amount of potassium iodate
required. Each mL of 0.05m potassium iodate FS is equivalent Saponification value (2.5.6)
to 33.64 mg of Ci 7H 38BrN. M axim u m 2.0.
STORAGE ASSAY
Strong Cetrimide Solution should be stored at a temperature Gas chromatography (2.2.28): use the normalisation
above 15°. procedure.
LABELLING
exam in ed in ethanol (96 per cent) R and dilute to 10.0 mL
The labd states whether Ethanol, Isopropyl Alcohol or both
are present and the percentage of cetrimide, wdght in
volume. Reference solution (a) Dissolve 50 mg of cetyl alcohol CRS in
ethanol (96 per cent) R and dilute to 5 mL with the same
solvent.
Reference solution (b) Dissolve 50 mg of stearyl alcohol R in
solvent.
Reference solution (c) Mix 1 mL of reference solution (a) and
1 mL of reference solution (b) and dilute to 10 mL with
ethanol (96 per cent) R.
2016 Cetyl Palmitate 1-497
Column: Dissolve 4.0 g in methylene chloride R and dilute to 20 mT.

— size. I = 30 m, 0 = 0.32 mm, with the same solvent.
— stationary phase: poly(dimethyl)sUoxane R (1 pm). Acid value (2.5.1)
Carrier gas helium for chromatography R. Maximum 4.0.
Flow rate 1 mL/min. Dissolve 10.0 g in 50 mL of the solvent mixture described by
Split ratio 1:100. heating under reflux on a water-bath for 5 min.
Temperature: Hydroxyl value (2.5.3, Method A)
Maximum 20.0.
Time Temperature
Iodine value (2.5.4, Method A)
(min) CC)
Maximum 2.0.
Column 0-20 150 ->250
Saponification value (2.5.6)
20-40 250
105 to 120.
Injection port 250
Heat under reflux for 2 h.
Detector 250 Alkaline impurities
Dissolve 2.0 g ‘with gentle heating' in a mixture of 1.5 mL
Detection Flame ionisation. of ethanol (96 per cent) R and 3 mL of toluene R.
Iitjection 1 pL of the test solution and reference solutions (a) Add 0.05 mL of a 0.4 g/L solution of bromophenol blue R in
and (c). ethanol (96per cent) R. Not more than 0.4 mL of 0.01 M
System suitability: reference solution (c): hydrochloric acid is required to change the colour of the
— resolution: minimum 5.0 between the peaks due to cetyl solution to yellow.
alcohol and stearyl alcohol. Nickel (2.4.31)
Calculate the percentage content of C 16H 34O. Maximum 1 ppm.
__________________________________________________________ PhEur W ater (2.5.12)
Maximum 0.3 per cent, determined on 1.0 g using a mixture
of equal volumes of anhydrous methanol R and methylene
chloride R as solvent.
Cetyl Palmitate ? **% Total ash (2.4.16)
Maximum 0.2 per cent, determined on 1 .0 g.
ASSAY
Action and use Gas chromatography (2.2.28): me the normalisation
Excipient. procedure.
PhEtr__________________________________________________________
examined in hexane R and dilute to 20.0 mL with the same
DEFINITION solvent.
Mixture of C 14-C 18 esters of lauric (dodecanoic), myristic Reference solution (a) Dissolve 20.0 mg of
(tetradecanoic), palmitic (hexadecanoic) and stearic cetyl palmitate 95 CRS in hexane R and dilute to 20.0 ml.
(octadecanoic) acids (‘Cetyl esters wax'). with the same solvent.
Content Reference solution (b) Dissolve 20.0 mg of
(expressed as hexadecyl hexadecanoate): cetyl palmitate 15 CRS in hexane R and dilute to 20.0 mL
10.0 per cent to 20.0 per cent for Cetyl palmitate 15, with the same solvent.
60.0 per cent to 70.0 per cent for Cetyl palmitate 65 and Column:
minimum 90.0 per cent for Cetyl palmitate 95. — material: stainless steel;
CHARACTERS — size: I = 10 m, 0 = 0.53 mm;
Appearance — stationary phase, poly(dimethyl)sUoxane R (film thickness
White or almost white, waxy plates, flakes or powder. 2.65 pm).
Solubility
Practically insoluble in water, soluble in boiling anhydrous Flow rate 6.5 mL/min.
ethanol and in methylene chloride, slightly soluble in light Split ratio 1:10.
petroleum, practically insoluble in anhydrous ethanol. Temperature:
mp
About 45 °C for Cetyl palmitate 15 and Cetyl palmitate 65 Time Temperature
and about 52 °C for Cetyl palmitate 95. (min) CC)
Column 0 -1 0 100 ->300
IDENTIFICATION
1 0-15 300
A. It complies with the limits of the assay and the
chromatogram obtained with the test solution shows the Injection port 350
typical main peak(s). Detector 350
TESTS
Appearance of solution Injection 1 pL.
The solution is not more intensely coloured than reference Relative retention With
reference to cetyl palmitate (retention
solution Y6 (2.2.2, Method II). time = about 9 min): cetyl alcohol = about 0.3; palmitic
1-498 Cetylpyridinium Chloride 2016
acid = about 0.4; lauric ester = about 0.8; myristic Acidity

ester = about 0.9; stearic ester = about 1.1. To 50 mL of solution S add 0.1 mL of phenolphthalein
System suitability’, reference solution (b): solution R. Not more than 2.5 mL of 0.02 M sodium hydroxide
— resolution: minimum of 1.5 between the peaks due to cetyl is required to change the colour of the indicator.
palmitate and cetyl stearate. Amines and amine salts
STORAGE Dissolve 5.0 g with heating in 20 mL of a mixture of
At a temperature not exceeding 25 °C. 3 volumes of 1 M hydrochloric acid and 97 volumes of
methanol R and add 100 mL of 2-propanol R. Pass a stream
LABELLING of nitrogen R slowly through the solution. Gradually add
The label states the type of cetyl palmitate. 12.0 mL of 0.1 M tetrabutylammonium hydroxide and record
__________________________________________________________ PhEur the potentiometric titration curve (2.2.20). If the curve shows
2 points of inflexion, the volume of titrant added between the
two points is not greater than 5.0 mL. If the curve shows no
point of inflexion, the substance to be examined does not
comply with the test. If the curve shows one point of
Cetylpyridinium Chloride inflexion, repeat the test but add 3.0 mL of a 25.0 g/L
(Ph. Eur. monograph 0379) * solution of dimethyldecylamine R in 2-propanol R before the
titration. If the titration curve after the addition of 12.0 mL
of the titrant shows only one point of inflexion, the substance
to be examined does not comply with the test.
W ater (2.5.12)
4.5 per cent to 5.5 per cent, determined on 0.300 g by the
C21H38CIN320 358.0 6004-24-6 semi-micro determination of water.
Action and use Sulfated ash (2.414)
Antiseptic. Not more than 0.2 per cent, determined on 1.0 g.
PhEur__________________________________________________________
ASSAY
DEFINITION same solvent. Transfer 25.0 mL of the solution to a
Cetylpyridinium chloride contains not less than 96.0 per cent separating funnel, add 25 mL of chloroform R, 10 mL of
and not more than the equivalent of 101.0 per cent of 0.1 M sodium hydroxide and 10.0 mL of a freshly prepared
1-hexadecylpyridinium chloride, calculated with reference to 50 g/L solution of potassium iodide R. Shake well, allow to
the anhydrous substance. separate and discard the chloroform layer. Shake the aqueous
CHARACTERS layer with three quantities, each of 10 mL, of chloroform R
A white or almost white powder, slightly soapy to the touch, and discard the chloroform layers. To the aqueous layer add
soluble in water and in alcohol. An aqueous solution froths 40 mL of hydrochloric add R, allow to cool and titrate with
copiously when shaken. 0.05 M potassium iodaie until the deep-brown colour is almost
discharged. Add 2 mL of chloroform R and continue the
IDENTIFICATION titration, shaking vigorously, until the chloroform layer no
First identification B, D longer changes colour. Carry out a blank titration on a
Second identification A , C, D mixture of 10.0 mL of the freshly prepared 50 g/L solution
A. Dissolve 0.10 g in water R and dilute to 100.0 mL with of potassium iodide R, 20 mL of water R and 40 mL of
the same solvent. Dilute 5.0 mL of this solution to 100.0 mL hydrochloric add R.
with water R. Examined between 240 nm and 300 nm 1 mL of 0.05 M potassium iodate is equivalent to 34.0 mg of
(2.2.25), the solution shows an absorption maxim um at C21H 38C1N.
259 nm and 2 shoulders at about 254 nm and at about __________________________________________________________ PhEur
265 nm. The specific absorbance at the maxim um is 126 to
134, calculated with reference to the anhydrous substance.
B. Examine by infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with Chalk
cetylpyridinium chloride CRS. Examine the substances in the
Prepared Chalk
solid state.
C. To 5 mL of dilute sodium hydroxide solution R add 0.1 mL CaC03 100.1
of bromophenol blue solution R1 and 5 mL of chloroform R and
Action and use
shake. The chloroform layer is colourless. Add 0.1 mL of
Antacid.
solution S (see Tests) and shake. The chloroform layer
becomes blue. DEFINITION
D. Solution S gives reaction (a) of chlorides (2.3.1). Chalk is a native form of calcium carbonate freed from most
TESTS of its impurities by élutriation and dried. It contains not less
Solution S than 97.0% and not more than 100.5% of CaC03,
Dissolve 1.0 g in carbon dioxide-free water R and dilute to calculated with reference to the dried substance.
100 mL with the same solvent. CHARACTERISTICS
Appearance of solution Chalk absorbs water readily.
Solution S is not more opalescent than reference Practically insoluble in water, slightly soluble in water
suspension 11*(2.2.1) and is colourless (2.2.2, Method II). containing carbon dioxide.
2016 Charcoal 1-499
Macroscopicd White or greyish white, small friable masses, ASSAY

visually conical in form, or in powder; amorphous; earthy; To 2 g in 100 mL of water add 50 mL of 1m hydrochloric acid
soft to the touch. FS, boil to remove carbon dioxide, cool and titrate the excess
Microscopical Consists of the calcareous shells and detritus of acid with 1m sodium hydroxide KS using methyl orange
of various foraminifera; the calcareous shells vary from about solution as indicator. Each mL of 1m hydrochloric add PS is
3 5 to 1 0 0 jim in breadth and from about 5 0 to 180 jim in equivalent to 50.04 mg of CaC03.
length; among the detritus are numerous small rings and
discs about 5 to 10 pm in diameter.
IDENTIFICATION
A. A solution in 6m acetic acid yields reaction C characteristic Activated Charcoal *****
of calcium salts, Appendix VI. **
Decolourising Charcoal *
B. Yields reaction A characteristic of carbonates, Appendix VI.
TfeSTS
Acidity or alkalinity Action and use
1 g , boiled with 5 0 mL of water and filtered, yields a filtrate Adsorbent.
which is neutral to bromothymol blue solution R3 or requires
not more than 0 .0 5 mL of 0.1m hydrochloric acid KS to make PhEtr_________________________________________________________ _
it so. DEFINITION
Aluminium, iron, phosphate and m atter insoluble in Obtained from vegetable matter by suitable carbonisation
hydrochloric a d d processes intended to confer a high adsorption power.
Dissolve 2 g in a mixture of 5 mL of hydrochloric acid and CHARACTERS
75 mL of water, boil to remove carbon dioxide and make
Appearance
alkaline with 5m ammonia using methyl red solution as
Black, light powder free from grittiness.
indicator. Boil for 1 minute, filter and wash the precipitate
with a hot 2% w/v solution of ammonium chloride. Dissolve Solubility
the precipitate as completely as possible by passing 2 0 mL of Practically insoluble in all usual solvents.
hot 2m hydrochloric acid through the filter and wash the filter IDENTIFICATION
with sufficient hot water to adjust the volume of the solution A. When heated to redness it bums slowly without a flame.
to 5 0 mL. Boil the solution and make alkaline with B. Adsorption power (see Tests).
5m ammonia using methyl red solution as indicator. Boil for
1 minute, filter through the same filter, wash the precipitate TESTS
with a hot 2 % w/v solution of ammonium nitrate, dry and Solution S
ignite at a temperature not lower than 1 0 00°. The residue To 2 .0 g in a conical flask with a ground-glass neck add
weighs not more than 4 0 mg. 5 0 mL of dilute hydrochloric acid R. Boil gently under a reflux
Arsenic condenser for 1 h, filter and wash the filter with dilute
hydrochloric add R. Evaporate the combined filtrate and
Dissolve 0 .5 g in 5 mL of brominated hydrochloric acid and
dilute to 5 0 mL with water. 25 mL of the resulting solution washings to dryness on a water-bath, dissolve the residue in
0.1 M hydrochloric acid and dilute to 5 0 .0 ml, with the same
complies with the limit testfor arsenic, Appendix VH (4 ppm).
acid.
Heavy metals
Dissolve 1.0 g in 10 mL of 2m hydrochloric acid, add 0.1 mL A ddity or alkalinity
of nitric acid and boil to remove carbon dioxide. Cool, make To 2 .0 g add 4 0 mL of water R and boil for 5 min. Cool,
alkaline with 5m ammonia, filter and wash the precipitate with restore to the original mass with carbon dioxide-free water R
water. Pass 5 mL of hot 2m hydrochloric acid through the
and filter. Reject the first 2 0 mL of the filtrate. To 10 mL of
filter, cool the filtrate, add 0 .5 g of ammonium tkiocyanate and the filtrate add 0 .2 5 ml, of bromothymol blue solution R1 and
0 .2 5 mL of 0.02 M sodium hydroxide. The solution is blue.
extract with two 5 mL quantities of a mixture of equal
volumes of isoamyl alcohol and ether. To the aqueous layer Not more than 0 .7 5 mL of 0.02 M hydrochloric acid is
add 0 .5 g of citric add and dilute to 2 0 mL with water. required to change the colour of the indicator to yellow.
12 mL of the resulting solution complies with limit test A for Add-soluble substances
heavy metals, Appendix VH. Use lead standard solution Maximum 3 per cent.
(2 ppm Pb) to prepare the standard (40 ppm). To 1.0 g add 25 mL of dilute nitric acid R and boil for 5 min.
Chloride Filter whilst hot through a sintered-glass filter (10) (2.1.2)
Dissolve 0.3 g in 2 mL of nitric acid and 10 mL of water, and wash with 10 mL of hot water R. Evaporate the
filter and dilute the filtrate to 30 mL with water. 15 mL of combined filtrate and washings to dryness on a water-bath,
the resulting solution complies with the limit testfor chlorides, add to the residue 1 mL of hydrochloric acid R, evaporate to
Appendix VII (330 ppm). dryness again and dry the residue to constant mass at
1 0 0 -1 0 5 °C. The residue weighs a maximum of 3 0 mg.
Sulfate
Dissolve 0 .2 5 g in 5 .5 mL of 2m hydrochloric add, dilute to Alkali-soluble coloured substances
3 0 mL with water and filter. 15 mL of the resulting solution To 0 .2 5 g add 10 mL of dilute sodium hydroxide solution R
complies with the limit testfor sulfates, Appendix VH (0 .1 2 % ). and boil for 1 min. Cool, filter and dilute the filtrate to
10 mL with water R. The solution is not more intensely
Loss on drying
coloured than reference solution GY4 (2.2.2, Method II).
When dried to constant weight at 105°, loses not more than
1.0% of its weight. Use 1 g. Ethanol (96 per cent) soluble substances
Maximum 0 .5 per cent.
1-500 Chenodeoxycholic Acid 2016
To 2.0 g add 50 mL of ethanol (96 per cent) R and boil under To 10.0 mL of the filtrate add 1.0 g of potassium bromide R
a reflux condenser for 10 min. Filter immediately, cool, and and 20 mL of dilute hydrochloric acid R. Using 0.1 mT. of
dilute to 50 mL with ethanol (96 per cent) R. The filtrate is methyl red solution R as indicator, titrate with 0.0167 M
not more intensely coloured than reference solution Y6 or potassium bromate until the red colour is discharged. Titrate
BY6 (2.2.2, Method II). Evaporate 40 mL of the filtrate to slowly (1 drop every 15 s) towards the end of the titration.
dryness and dry to constant mass at 100-105 °C. The residue Carry out a blank titration using 10.0 mL of the phenazone
weighs a maximum of 8 mg. solution.
Fluorescent substances Calculate the quantity of phenazone adsorbed per 100 g of
In an intermittent-extraction apparatus, treat 10.0 g with activated charcoal from the following expression:
100 mL of cychhexane R1 for 2 h. Collect the liquid and
dilute to 100 mL with cychhexane R l. Examine in ultraviolet 2.353 (a - b)
light at 365 nm. The fluorescence of the solution is not more m
intense than that of a solution of 83 ng of quinine R in
1000 mL of 0.005 M sulfuric acid examined under the same a = number of millilitres of 0.0167 M potassium bromate
conditions. used for the blank;
b = number of millilitres of 0.0167 M potassium bromate
Sulfides
used for the test;
To 1.0 g in a conical flask add 5 mL of hydrochloric acid R l
m = mass in grams of the substance to be examined.
and 20 mL of water R. Heat to boiling. The fumes released
do not turn lead acetate paper R brown. Minimum 40 g of phenazone is adsorbed per 100 g of
activated charcoal, calculated with reference to the dried
Copper
substance.
Maximum 25 ppm.
Atomic absorption spectrometry (2.2.23, Method I). M icrobial contamination
Test solution Use solution S.
TYMC: acceptance criterion 102 CFU/g (2.6.12).
standard solution (0.1 per cent Cu) R and diluting with 0.1 M STORAGE
hydrochloric acid. In an airtight container.
Source Copper hollow-cathode lamp. ___________________________________________________________ PhEur
Lead
M ax im u m 10 ppm. Chenodeoxycholic Acid /* * %
*. *
Atomic absorption spectrometry (2.2.23, Method I). (Ph. Eur. monograph 1189) *
standard solution (100 ppm Pb) R and diluting with 0.1 M
hydrochloric acid.
Wavelength 283.3 nm; 217.0 nm may be used depending on
the apparatus.
Zinc C24H40O4 392.6 474-25-9
Maximum 25 ppm.
Action and use
Atomic absorption spectrometry (2.2.23, Method I). Bile add; treatment of gallstones.
PhEur___________________________________________________________-
Reference solutions Prepare the reference solutions using zinc
standard solution (100 ppm Zn) R and diluting with 0.1 M DEFINITION
hydrochloric acid. Chenodeoxycholic add contains not less than 99.0 per cent
Source Zinc hollow-cathode lamp. and not more than the equivalent of 101.0 per cent of 3a,7a-
Wavelength 214.0 nm. dihydroxy-5 ^-cholan-24-oic add, calculated with reference to
the dried substance.
Loss on drying (2.2.32) CHARACTERS
Maximum 15 per cent, determined on 1.00 g by drying in an A white or almost white powder, very slightly soluble in
oven at 120 °C for 4 h. water, freely soluble in alcohol, soluble in acetone, slightly
soluble in methylene chloride.
Maximum 5.0 per cent, determined on 1.0 g. IDENTIFICATION
First identification A
Adsorption power
To 0.300 g in a 100 mL ground-glass-stoppered conical flask Second identification B, C
add 25.0 mL of a freshly prepared solution of 0.5 g of A. Examine by infrared absorption spectrophotometry
phenazone R in 50 mL of water R. Shake thoroughly for (2.2.24), comparing with the spectrum obtained with
15 min . Filter and reject the first 5 m l. of filtrate. chenodeoxycholic acid CRS. Examine the substances prepared
as discs using potassium bromide R.
2016 Chitosan Hydrochloride 1-501
B. Examine the chromatograms obtained in the test for (0.5 per cent); any spot apart from the principal spot and any
related substances. The principal spot in the chromatogram spots corresponding to lithocholic aad 5 ursodeoxycholic acid
obtained with test solution (b) is similar in positionj colour and cholic add, is not more intense than the principal spot in
and size to the principal spot in the chromatogram obtained the chromatogram obtained with reference solution (e)
with reference solution (a). (0.25 per cent). The test is not valid unless the
C. Dissolve about 10 mg in 1 mL of sulfuric acid R. chromatogram obtained with reference solution (f) shows two
Add 0.1 mL of formaldehyde solution R and allow to stand for clearly separated prindpal spots.
5 min. Add 5 mL of water R. The suspension obtained is Heavy metals (2.4.8)
greenish-blue. 1.0 g complies with test C for heavy metals (20 ppm).
TESTS Prepare the reference solution using 2 mL of lead standard
Dissolve 0.500 g in methanol R and dilute to 25.0 mL with Loss on drying (2.2.32)
the same solvent. The specific optical rotation is + 11.0 to Not more than 1.5 per cent, determined on 1.000 g by
+ 13.0j calculated with reference to the dried substance. drying in an oven at 105 °C.
Related substances Sulfated ash (2.4.14)
Examine by thin-layer chromatography (2.2.27), using a Not more than 0.1 per cent, determined on 1.0 g.
suitable silica gel as the coating substance. ASSAY
Test solution (a) Dissolve 0.40 g of the substance to be Dissolve 0.350 g in 50 mL of alcohol R, previously
examined in a mixture of 1 volume of water R and 9 volumes neutralised to 0.2 mL of phenolphthalem solution R.
of acetone R and dilute to 10 mL with the same mixture of Add 50 mL of water R and titrate with 0.1 M sodium
solvents. hydroxide until a pink colour is obtained.
Test solution (b) Dilute 1 mL of test solution (a) to 10 mL 1 mL of 0.1 M sodium hydroxide is equivalent to 39.26 mg of
with a mixture of 1 volume of water R and 9 volumes of C24H 40O 4.
acetone R.
IMPURITIES
Reference solution (a) Dissolve 40 mg of chenodeoxycholic
add CRS in a mixture of 1 volume of water R and 9 volumes
of acetone R and dilute to 10 mL with the same mixture of
solvents.
Reference solution (b) Dissolve 20 mg of lithocholic add CRS in
a mixture of 1 volume of water R and 9 volumes of acetone R
and dilute to 10 mL with the same mixture of solvents.
Dilute 2 mL of the solution to 100 mL with a mixture of
1 volume of water R and 9 volumes of acetone R.
Reference solution (c) Dissolve 20 mg of ursodeoxycholic A. R = H, Rl = OH, R2 = H, R3 = H: 3a-7 ^-dihydroxy-
acid CRS in a mixture of 1 volume of water R and 9 volumes 5P-cholan-24-oic add (ursodeoxycholic add),
of acetone R and dilute to 50 mL with the same mixture of B. R = H, Rl = H, R2 = OH, R3 = OH: 3o,7a,12a-
solvents. trihydroxy-5P-cholan-24-oic add (cholic add),
Reference solution (d) Dissolve 20 mg of cholic add CRS in a
C. R = H, Rl = H, R2 = H, R3 = H: 3a-hydroxy-5p-
mixture of 1 volume of water R and 9 volumes of acetone R cholan-24-oic add (lithocholic add),
D. R = H, Rl = OH, R2 = H, R3 = OH: 3a,7frl2a-
Reference solution (e) Dilute 0.5 mL of test solution (a) to
trihydroxy-5 (J-cholan-24-oic add (ursocholic add),
20 mL with a mixture of 1 volume of water R and 9 volumes
of acetone R. Dilute 1 mL of the solution to 10 mL with a E. R = H, Rl = H, R2 = H, R3 = OH: 3a,12a-dihydroxy-
mixture of 1 volume of water R and 9 volumes of acetone R. 5P-cholan-24-oic add (deoxycholic add),
Reference solution (f) Dissolve 10 mg of chenodeoxycholic
F. R = H, R1+R2 = = O, R3 = H: 3a-hydroxy-7-oxo-5p-
add CRS in reference solution (c) and dilute to 25 mL with
cholan-24-oic add,
the same solution. G. R = CH3, Rl = OH, R2 = H, R3 = H: methyl 3a,7p-
Apply separately to the plate 5 |iL of each solution. Develop dihydroxy-5 P-cholan-24-oate.
in an unsaturated tank over a path of 15 cm using a mixture __________________________________________________________ PhEur
of 1 volume of glacial acetic add R, 30 volumes of acetone R
and 60 volumes of methylene chloride R. Dry the plate at
120 °C for 10 min. Spray the plate immediately with a
47.6 g/L solution of phosphomofybdic acid R in a mixture of
1 volume of sulfuric add R and 20 volumes of glacial acetic Chitosan Hydrochloride *****
*. *
add R and heat again at 120 °C until blue spots appear on a (Ph. Eur. monograph 1774) *
lighter background. In the chromatogram obtained with test
PhEur__________________________________________________________
solution (a): any spot corresponding to lithocholic acid is not
more intense than the principal spot in the chromatogram DEFINITION
obtained with reference solution (b) (0.1 per cent); any spot Chitosan hydrochloride is the chloride salt of an unbranched
corresponding to ursodeoxycholic acid is not more intense binary heteropolysaccharide consisting of the two units
than the principal spot in the chromatogram obtained with iV-acetyl-D-glucosamine and D-glucosamine, obtained by
reference solution (c) (1 per cent); any spot corresponding to partial deacetylation of chitin normally leading to a degree of
cholic acid is not more intense than the principal spot in the deacetylation of 70.0 per cent to 95.0 per cent Chitin is
chromatogram obtained with reference solution (d) extracted from the shells of shrimp and crab.
1-502 Chitosan Hydrochloride 2016
PRODUCTION least squares linear regression. Use the standard curve to

The animals from which chitosan hydrochloride is derived d eterm ine the equivalent amount of N-acetylglucosamine for
must fulfil the requirements for the health of animals suitable the substance to be examined.
for human consumption to the satisfaction of the competent Calculate the degree of deacetylation (molar) using the
authority. It must have been shown to what extent the following expression:
method of production allows inactivation or removal of any
contamination by viruses or other infectious agents. 100 x M i x (C i - C2)
CHARACTERS (M i x C i) - [(M i - M 3) x Ca]
Appearance
White or almost white, fine powder. Ci = concentration of chitosan hydrochloride in the test
Solubility solution in micrograms per m illilitre;
Sparingly soluble in water, practically insoluble in anhydrous C2 = concentration of N-acetylglucosaminein the test
ethanol. solution, as determined from the standard curve
IDENTIFICATION prepared using the reference solution in
micrograms per millilitre;
Mi = 203 (relative molecular mass of
Preparation Discs. N-acetylglucosamine unit (C 8H 13N 0 5 ) in
Comparison chitosan hydrochloride CRS. polymer);
B. It gives reaction (a) of chlorides (2.3.1). Ai3 = relative molecular mass of chitosan hydrochloride.
C. Dilute 50 mL of solution S (see Tests) to 250 mL with a M3 is calculated from the pH in solution, assuming a pKa
25 per cent V/V solution of ammonia R. A voluminous value of 6 .8, using the following equations:
gelatinous mass is formed.
D. To 10 mL of solution S add 90 mL of acetone R. M3 = f x M2 + (1 - / ) x (M 2 + 36.5)
A voluminous gelatinous mass is formed.
TESTS
Solution S J 1+ p
Dissolve 1:0 g in 100 mL of water R and stir vigorously for p _ -^Q(pH-pKa)
20 min with a mechanical stirrer.
Appearance of solution M2 = 161 (relative molecular mass of deacetylated unit
Solution S is not more opalescent than reference (glucosamine) (C^HnNO^ in polymer).
reference solution BY5 (2.2.2, Method II). Chlorides
M atter insoluble in water
Maximum 0.5 per cent. Introduce 0.200 g into a 250 mL borosilicate flask fitted with
a reflux condenser. Add 40 mL of a mixture of 1 volume of
Add 2.00 g to 400.0 mL of water R while stirring until no
nitric acid R and 2 volumes of water R. Boil gently under a
further dissolution takes place. Transfer the solution to a 2 L
reflux condenser for 5 m in . Cool and add 25 mL of water R
beaker, and add 200 mL of water R. Boil the solution gently
through the condenser. Add 16.0 mL of 0 .1 M silver nitrate,
for 2 h, covering the beaker during the operation. Filter
shake vigorously and titrate with 0.1 M ammonium
through a sintered-glass filter (40) (2.1.2), wash die residue
thiocyanate, u sin g 1 mT. of ferric ammonium sulfate solution R2
with water and dry to constant weight in an oven at
as indicator, and shaking vigorously towards the end-point.
100-105 °C. The residue weighs a maximum of 10 mg.
Carry out a blank titration.
pH (2.2.3)
1 mL of 0.1 M silver nitrate is equivalent to 3.55 mg of Cl.
Heavy metals (2.48)
Viscosity (2.2.10)
Maximum 40 ppm.
80 per cent to 120 per cent of the value stated on the label,
determined on solution S. 1.0 g complies with test F. Prepare the reference solution
Determine the viscosity using a rotating viscometer at 20 °C
with a spindle rotating at 20 r/min, using a suitable spindle Loss on drying (2.2.32)
for the range of the expected viscosity. Maximum 10 per cent, determined on 1.000 g by drying in
an oven at 105 °C.
Degree of deacetylation
Test solution Dissolve 0.250 g in water R and dilute to Sulfa ted ash (2.414)
50.0 mL with the same solvent, stirring vigorously. Dilute M axim u m 1.0 per cent, determined on 1.0 g.
1.0 mL of this solution to 100.0 mL with water R. Measure STORAGE
the absorbance (2.2.25) from 200 nm to 205 nm as the first At a temperature of 2 °C to 8 °C, protected from moisture
derivative of the absorbance curve. Determine the pH of the and light.
solution.
LABELLING
Reference solutions Prepare solutions of 1.0 pg/mL, 5.0 Hg/mL,
The label states the nominal viscosity in millipascal seconds
15.0 ng/mL and 35.0 |ig/mL of N-acetylglucosamme R in
for a 10 g/L solution in water R.
water R. Measure the absorbance (2.2.25) from 200 nm to
205 nm of each solution as the first derivative of the _ _ _ ____________________________________________ _ PhEur
absorption curve. Make a standard curve by plotting the first
derivative at 202 nm as a function of the concentration of
N-acetylglucosamine, and calculate the slope of the curve by
2016 Chlorambucil 1-503
★**★ ★ phenolphthalem solution R as indicator. Titrate the neutralised

Chloral Hydrate ★ ★ solution with 0.1 M silver nitrate, using 0.2 mL of potassium
(Ph. Eitr. monograph 0265) ***** chromate solution R as indicator. Calculate the number of
millilitres of 1 M sodium hydroxide used by deducting from
OH the volume of 1 M sodium hydroxide, added at the b eginning
CI3C
X OH
of the titration, the volume of 0.5 M sulfuric acid used in the
1st titration and two-fifteenths of the volume of 0.1 M silver
nitrate used in the 2 nd titration.
C2H 3CI3O2 165.4 302-17-0
1 mL of 1 M sodium hydroxide is equivalent to 0.1654 g
Action and use of C2H 3CI3O2.
Hypnotic. STORAGE
Preparation In an airtight container.
Chloral Hydrate Oral Solution ________________________________________________ ______ __PhEur
PhEur___________________________
DEFINITION
2 ,2 ,2 -Trichloroethane-1,1 -diol. ★*★
Chlorambucil ★ ★
Content ★ ★
98.5 per cent to 101.0 per cent. (Ph. Eur. monograph 0137) *****
CHARACTERS
Appearance co 2h
Colourless, transparent crystals.

Solubility
Very soluble in water, freely soluble in ethanol (96 per cent).
IDENTIFICATION
A. To 10 mL of solution S (see Tests) add 2 mL of dilute C 14Hi9C12N 0 2 304.2 305-03-3
sodium hydroxide solution R. The mixture becomes cloudy
and, when heated, gives off an odour of chloroform. Action and use
B. To 1 mL of solution S add 2 mL of sodium sulfide Cytotoxic alkylating agent
solution R. A yellow colour develops which quickly becomes Preparation
reddish-brown. On standing for a short time, a red Chlorambucil Tablets
precipitate may be formed.
PhEur.
TESTS
Solution S DEFINITION
Dissolve 3.0 g in carbon dioxide-free water R and dilute to 4- [4- [Bis(2-chloroethyl) amino] phenyl] butanoic acid.
30 mL with the same solvent. Content
Appearance of solution 98.5 per cent to 101.0 per cent (anhydrous substance).
Solution S is clear (2.2.1) and colourless (2.2.2, MethodII). CHARACTERS
pH (2.2.3) Appearance
3.5 to 5.5 for solution S. White or almost white, crystalline powder.
Chloral alcoholate Solubility
Warm 1.0 g with 10 ml, of dilute sodium hydroxide solution R, Practically insoluble in water, freely soluble in acetone and in
filter the supernatant solution and add 0.05 M iodine ethanol (96 per cent).
dropwise until a yellow colour is obtained. Allow to stand for
IDENTIFICATION
1 h. No precipitate is formed.
Chlorides (2.4.4)
Comparison chlorambucil CRS.
Maximum 100 ppm.
Dilute 5 mL of solution S to 15 mL with water R. TESTS
Impurity G
Heavy m etals (2.4.8) Liquid chromatography (2.2.29). The solutions are stablefor
Maximum 20 ppm. 8 hat room temperature or for 24 h at 4-8 °C; protect themfrom
10 mL of solution S diluted to 20 mL with water R complies light.
with test A. Prepare the reference solution using lead standard
Test solution Dissolve 10 mg of the substance to be exam ined
in methanol R and dilute to 20.0 m l , with the same solvent.
Non-volatile residue Reference solution (a) Dilute 1.0 mL of the test solution to
Maximum 0.1 per cent. 50.0 mL with the mobile phase. Dilute 2.0 mL of this
Evaporate 2.000 g on a water-bath. The residue weighs a solution to 10.0 mL with the mobile phase.
m a x im u m of 2 mg.
Reference solution (b) Dissolve 5 mg of chlorambucil with
ASSAY impurity G CRS in methanol R and dilute to 10.0 mL with
Dissolve 4.000 g in 10 mL of water R and add 40.0 mL of the same solvent.
1 M sodium hydroxide. Allow to stand for exactly 2 min and Column:
titrate with 0.5 M sulfuric acid, using 0.1 mL of — size: I — 0.15 m, 0 = 3.9 mm;
1-504 Chlorambucil 2016
— stationary phase: phenylsUyl silica gelfor chromatography R Limits:

(5 nm). — impurity E: not more than 6 times the area of the
Mobile phase methanol R, 1 per cent V/V solution of principal peak in the chromatogram obtained with
trifluoroacetic acid R (50:50 V/V). reference solution (a) (0.6 per cent);
Flow raie 1.8 mL/min. — impurity B: not more than 4 times the area of the
Detection Spectrophotometer at 260 nm. reference solution (a) (0.4 per cent);
Injection 20 jxL. — unspecified impurities: for each impurity, not more than the
Run time Twice the retention time of chlorambucil. area of the principal peak in the chromatogram obtained
Relative retention With reference to chlorambucil (retention with reference solution (a) (0.10 per cent);
time = about 11 min): impurity G = about 1.2 . — total: not more than 10 times the area of the principal
— resolution: minimum 1.5 between the peaks due to solution (a) ( 1.0 per cent);
chlorambucil and impurity G. — disregard limit. 0.5 times the area of the principal peak in
Limit.
(0.05 per cent).
— impurity G: not more than the area of the principal peak
in the chromatogram obtained with reference solution (a) W ater (2.5.12)
(0.4 per cent). Maximum 0.5 per cent, determined on 1.00 g.
Related substances Sulfated ash (2.4.14)
Liquid chromatography (2.2.29). Prepare the solutions M axim um 0.1 per cent, determined on 1.0 g.
immediately before use and protect from light. ASSAY
Solvent mixture 10.3 g/L solution of hydrochloric acid R, Dissolve 0.200 g in 10 mL of acetone R and add 10 mL of
acetonitrüefor chromatography R (10:90 V/V). water R. Titrate with 0.1 M sodium hydroxide, using 0.1 mT,
Test solution Dissolve 25 mg of the substance to be examined of phenolphthalem solution R as indicator.
in the solvent mixture and dilute to 100.0 mL with the 1 mL of 0.1 M sodium hydroxide is equivalent to 30.42 mg of
solvent mixture. C 14H 19CI2NO 2.
Reference solution (a) Dilute 1.0 mL of the test solution to STORAGE
100.0 mL with the solvent mixture. Dilute 1.0 mL of this Protected from light.
IMPURITIES
Reference solution (b) Dissolve 5 mg of chlorambucilfor system
Spedfied impurities B, E, G
suitability CRS (containing impurities B and E) in the solvent
mixture and dilute to 20.0 mL with the solvent mixture. Other detectable impurities(the following substances would, if
Column: present at a sufficient level, be detected by one or other of
— size: I = 0.25 m, 0 = 3.0 mm; the tests in the monograph. They are limited by the general
— stationary phase, end-capped octadecylsüyl silica gelfor acceptance criterion for other/unspecified impurities and/or
chromatography R (5 nm). by the general monograph Substancesfor pharmaceutical use
Mobile phase:
— mobile phase A: 1.9 g/L solution of ammonium acetate R
Control of impurities in substancesfor pharmaceutical use): A , C,
adjusted to pH 3.9 with acetic add R\
D ,F .
— mobile phase B: acetonitrile for chromatography R;

0 -5 60 40
5 - 15 6 0 * 10 4 0 * 90
ci
1 5 -2 5 10 90
A. 4— [4—
[(2 -chloroethyl) (2 -hydroxyethyl)amino]phenyI] butanoic add,
Injection 10 jjL.
Identification of impurities Use the chromatogram supplied H
with chlorambucilfor system suitability CRS and the
chromatogram obtained with reference solution (b) to B. 4-[4- [(2-chloroethyl)amino]phenyljbutanoic add,
identify the peaks due to impurities B and E.
Relative retention With reference to chlorambucil (retention
time = about 12 min): impurity B = about 0.5; o
.Ox 1 .CL
impurity E = about 1.4. Cl P N

— resolution: minimum 5.0 between the peaks due to Cl
impurity B and chlorambucil.
C. 4-[4-[[2-[[bis(2-chloroethoxy)phosphoryl]oxy]ethyl]
(2 -chloroethyl) amino]phenyl] butanoic add,
2016 Chloramphenicol 1-505
/**★ ★
★
Chloramphenicol ★ ★
OH
? ' H
D. 2-chloroethyl 4-[4-
[bis(2 -chloroethyl)amino] phenyl] butanoate, H H OH
CnH 12Cl2N 20 5 323.1 56-75-7
Action and use

Antibacterial.
Preparations
Chloramphenicol Capsules
Chloramphenicol Ear Drops
Chloramphenicol Eye Drops
Chloramphenicol Eye Ointment
E. 4-[4-[[2-[[4-[4-[bis(2-chloroethyl)amino]phenyl]butanoyl]
PhEur.
oxy]ethylj (2 -chloroethyl)amino]phenyl]butanoic acid.
DEFINITION
Chloramphenicol is 2 ,2 -dichloro-N-[(li?32i?)-2-hydroxy-l-
(hydroxymethyl)-2-(4-nitrophenyl) ethyl] acetamide, produced
by the growth of certain strains of Streptomyces venezuelae in a
„Cl suitable medium. It is normally prepared by synthesis.
It contains not less than 98.0 per cent and not more than the
equivalent of 102.0 per cent of C n H ^ C ^ ^ O s , calculated
with reference to the dried substance.
CHARACTERS
A white, greyish-white or yellowish-white, fine, crystalline
powder or fine crystals, needles or elongated plates, slightly
r1
Cl
soluble in water, freely soluble in alcohol and in propylene
glycol.
A solution in ethanol is dextrorotatory and a solution in ethyl
F. 4-[4-[[2-[[4-[4-[[2-[[4-[4-[bis(2-dúoroethyl)amino]phenyI] acetate is laevorotatory.
butanoyl]oxy]ethyl](2 -chloroethyI)amino]phenyl]butanoyl]
oxy]ethyI] (2 -chIoroethyl)amino]phenyI]butanoic add. IDENTIFICATION
co 2h
chloramphenicol CRS.
Cl' 'Cl C. Examine the chromatograms obtained in the test for
related substances. The principal spot in the chromatogram
G. 4 -[2 -[bis(2 -chloroethyl)amino]phenyI]butanoic add or obtained with 1 pL of the test solution is similar in position
4-[3-[bis(2-chloroethyl)amino]phenyI]butanoic add (meta or and size to the prindpal spot in the chromatogram obtained
ortho chlorambucil). with reference solution (a).
Ph Eur D. Dissolve about 10 mg in 1 mL of alcohol
(50 per cent V/V) R, add 3 mL of a 10 g/L solution of calcium
chloride R and 50 mg of zinc powder R and heat on a water-
bath for 10 m in. Filter the hot solution and allow to cool.
Add 0.1 mL of benzoyl chloride R and shake for 1 min.
Add 0.5 mL of ferric chloride solution R1 and 2 mL of
chloroform R and shake. The aqueous layer is coloured light
violet-red to purple.
E. To 50 mg in a porcelain crudble add 0.5 g of anhydrous
sodium carbonate R. Heat over an open flame for 10 m in .
Allow to cool. Take up the residue with 5 mL of dilute nitric
acid R and filter. To 1 mL of the filtrate add 1 mL of
water R. The solution gives reaction (a) of chlorides (2.3.1).
1-506 Chloramphenicol Palmitate 2016
TESTS ★★*★★
Chloramphenicol Palmitate ★ ★
To 0.1 g add 20 mL of carbon dioxide-free water R, shake and (Ph. Eur. monograph 0473) *****
add 0.1 mL of bromothymol blue solution R l. Not more rhan
0.1 mL of 0.02 M hydrochloric add or 0.02 M sodium
hydroxide is required to ch an ge the colour of the indicator.
Dissolve 1.50 g in ethanol R and dilute to 25.0 mL with the
same solvent. The specific optical rotation is + 18.5 to H OH
+ 20.5.
Related substances C27H 42Q 2N 2O6 561.6 530-43-8
gel G F 254 R as the coating substance. Action and use
Test solution Dissolve 0.10 g of the substance to be examined Antibacterial.
in acetone R and dilute to 10 mL with the same solvent. PhEur.
Reference solution (a) Dissolve 0.10 g of chloramphenicol CRS
in acetone R and dilute to 10 mL with the same solvent. DEFINITION
Chloramphenicol palmitate contains not less than
98.0 per cent and not more than the equivalent of
to 100 mL with acetone R.
102.0 per cent of (2R,3R)-2-[(dichloroacetyl)amino]-3-
Apply separately to the plate 1 (iL and 20 (iL of the test hydroxy-3-(4-nitrophenyl)propyl hexadecanoate, calculated
solution, 1 (iL of reference solution (a) and 20 fiL of with reference to the dried substance.
reference solution (b). Develop over a path of 15 cm using a
mixture of 1 volume of water R, 10 volumes of methanol R
and 90 volumes of chloroform R. Allow the plate to dry in air CHARACTERS
and examine in ultraviolet light at 254 nm. Any spot in the A white or almost white, fine, unctuous powder, practically
chromatogram obtained with 20 pL of the test solution, apart insoluble in water, freely soluble in acetone, sparingly soluble
from the principal spot, is not more intense rhan the spot in in ethanol (96 per cent), very slightly soluble in hexane.
the chromatogram obtained with reference solution (b) It melts at 87 °C to 95 °C.
(0.5 per cent). It shows polymorphism (5.9). The thermodynamically stable
Chlorides (2.4.4) form has low bioavailability following oral administration.
To 1.00 g add 20 m l. of water R and 10 m l. of nitric add R
IDENTIFICATION
and shake for 5 min. Filter through a filter paper previously
A. Examine by thin-layer chromatography (2.2.27), using
washed by filtering 5 mL portions of water R until 5 mL of
TLC sUanised silica gel plate R.
filtrate no longer becomes opalescent on addition of 0.1 mL
of nitric acid R and 0.1 mL of silver nitrate solution R l. 15 m l. Test solution Dissolve 50 mg of the substance to be examined
of the filtrate complies with the limit test for chlorides in a mixture of 1 ml. of 1 M sodium hydroxide and 5 ml. of
(100 ppm). acetone R and allow to stand for 30 min. Add 1.1 mL of 1 M
hydrochloric add and 3 mL of acetone R.
Reference solution (a) Dissolve 10 mg of chloramphenicol CRS
Not more than 0.5 per cent, determined on 1.000 g by
drying in an oven at 105 °C. in acetone R and dilute to 5 mL with the same solvent.
Reference solution (b) Dissolve 10 mg of palmitic add R in
acetone R and dilute to 5 mL with the same solvent.
Not more than 0.1 per cent, determined on 2.0 g.
Reference solution (c) Dissolve 10 mg of the substance to be
Pyrogens (2.6.8)
examined in acetone R and dilute to 5 mL with the same
If intended for use in the manufacture of parenteral
solvent.
preparations without a further appropriate procedure for the
removal of pyrogens, it complies with the test for pyrogens. Apply to the plate 4 pL of each solution. Develop over a
Inject per kilogram of the rabbit's mass 2.5 mL of a solution path of 15 cm using a mixture of 30 volumes of a 100 g/L
containing per millilitre 2 mg of the substance to be solution of ammonium acetate R and 70 volumes of ethanol
examined. (96 per cent) R. Allow the plate to dry in air and spray with a
solution containing 0.2 g/L of dichlorcfluorescein R and
ASSAY 0.1 g/L of rhodamine B R in ethanol (96 per cent) R. Allow the
Dissolve 0.100 g in water R and dilute to 500.0 mL with the plate to dry in air and examine in ultraviolet light at 254 nm.
same solvent. Dilute 10.0 mL of this solution to 100.0 mL The chromatogram obtained with the test solution shows
with water R. Measure the absorbance (2.2.25) at the 3 spots corresponding in position to the principal spots in the
maximum at 278 nm. chromatograms obtained with reference solutions (a), (b) and
Calculate the content of C n H ^ C y ^ O s taking the specific (c).
absorbance to be 297. B. Dissolve 0.2 g in 2 mL of pyridine R, add 2 mL of a
STORAGE 100 g/L solution of potassium hydroxide R and heat on a
Store protected from light. If the substance is sterile, store in water-bath. A red colour is produced.
a sterile, airtight, tamper-proof container. C. Dissolve about 10 mg in 5 mL of ethanol (96 per cent) R
_____________________________________ PhEur and add 4.5 mL of dihite sulfuric add R and 50 mg of zinc
powder R. Allow to stand for 10 min and if necessary decant
the supernatant or filter. Cool the solution in iced water and
add 0.5 mL of sodium nitrite solution R. Allow to stand for
2016 Chloramphenicol Sodium Succinate 1-507
2 min and add 1 g of urea R, 2 ml. of strong sodium hydroxide Sulfated ash (2.4.14)
solution Rand 1 ml, of fi-naphthol solution R. A red colour Maximum 0.1 per cent, determined on 1.0 g.
develops. ASSAY
TESTS Dissolve 90.0 mg in ethanol (96 per cent) R and dilute to
Acidity 100.0 mL with the same solvent Dilute 10.0 mL of this
Dissolve 1.0 g in 5 mL of a mixture of equal volumes of solution to 250.0 mL with ethanol (96 per cent) R. Measure
ethanol (96 per cent) R and ether R, warming to 35 °C. the absorbance (2.2.25) of the solution at the m axim u m at
Add 0.2 mL of phertalphthalein solution R. Not more than 271 nm.
0.4 tnl. oi 0.1 M sodmm hydroxide is required to produce a Calculate the content of CjTliæCkÔôtaking the specific
pink colour persisting for 30 s. absorbance to be 178.
Specific optical rotation (2.2.7) STORAGE
Dissolve 1.25 g in anhydrous ethanol R and dilute to 25.0 mL Protected from light
with the same solvent. The specific optical rotation is
+ 22.5 to + 25.5. IMPURITIES
Free chloramphenicol
Maximum 450 ppm. Dissolve 1.0 g, with gentle heating, in
80 mL of xylene R. Cool and shake with 3 quantities, each of
15 mL, of water R. Dilute the combined aqueous extracts to
50mL with water R and shake with 10 mL of toluene R.
Allow to separate and discard the toluene layer. Centrifuge a
portion of the aqueous layer and measure the absorbance (A)
(.2.2.25) at the maximum at 278 nm using as the A. ( li?,2i?)-2-[(dichloroacetyl)amino]-3-hydroxy-1-
compensation liquid a blank solution having an absorbance (4-nitrophenyl)propyl hexadecanoate (chloramphenicol
not greater than 0.05. palmitate isomer),
Calculate the content of free chloramphenicol in parts per
m illion from the expression:
A x 104
5.96
Related substances
gel G F254 R as the coating substance.
B. ( 1R,2R)-2-[(dichloroacetyl)amino]-1-
(4-nitrophenyl)propane-1,3-diyl bishexadecanoate
in acetone R and dilute to 10 mL with the same solvent. (chloramphenicol dipalmitate).
Reference solution (a) Dissolve 20 mg of chloramphenicol
Ri Eur
palmitate isomer CRS in acetone R and dilute to 10 mL with
the same solvent. Dilute 1 mL of this solution to 10 mL with
acetone R
Reference solution (b) Dissolve 20 mg of chloramphenicol * **
dipalmitate CRS in acetone R and dilute to 10 mL with the Chloramphenicol Sodium ★
★ ★
*
same solvent. Dilute 1 mL of this solution to 10 mL with *****

acetone R
Succinate
Reference solution (c)Dissolve 5 mg of chloramphenicol CRS in
acetone R and dilute to 10 mL with the same solvent. Dilute
NOj
1 mL of this solution to 10 mL with acetone R.
Apply to the plate 10 pL of each solution. Develop over a
path of 15 cm using a mixture of 10 volumes of methanol R, H H 0 -R 1
40 volumes of chloroform R and 50 volumes of cydohexane R.
Allow the plate to dry in air and examine in ultraviolet light 1 isomer : R1 = CO-CHrCHrCOjNa, R3 = H
at 254 nm. In the chromatogram obtained with the test 3 isomer : R1 = H, R3 = CO-CHrCHrCX^Na
solution, any spots due to chloramphenicol palmitate isomer
and chloramphenicol dipalmitate are not more intense than C 15H 15Cl2N2Na0 8 445.2 982-57-0
the corresponding spots in the chromatograms obtained with
reference solutions (a) and (b) respectively (2.0 per cent) and Action and use
any spot, apart from the principal spot and the spots due to Antibacterial.
chloramphenicol palmitate isomer and chloramphenicol Preparation
dipalmitate, is not more intense than the principal spot in the Chloramphenicol Sodium Succinate Injection
(0.5 per cent). PhEur_______________________________________________________
Loss on drying (2.2.32) DEFINITION

Maximum 0.5 per cent, determined on 1.000 g by heating at Mixture in variable proportions of sodium (2R,3R)-2-
80 °C over diphosphorus pentoxide R at a pressure not [(dichloroacetyl) amino] -3-hydroxy-3-(4-nitrophenyl)propyl
exceeding 0.1 kPa for 3 h. butanedioate (3 isomer) and of sodium (lR,2R)-2-
1-508 Chloramphenicol Sodium Succinate 2016
[(dichloroacetyl) amino]-3-hydroxy-1-(4-nitrophenyl)propyl Reference solution (a) Dissolve 10.0 mg of chloramphenicol CRS

butanedioate (1 isomer). in the mobile phase and dilute to 100.0 mL with the mobile
Semi-synthetic product derived from a fermentation product. phase (solution A). Dilute 5.0 mL of this solution to
100.0 mL with the mobile phase.
Content
98.0 per cent to 102.0 per cent (anhydrous substance). Reference solution (b) Dissolve 10.0 mg of chloramphenicol
disodium disuccinate CRS in the mobile phase and dilute to
CHARACTERS 100.0 mL with the mobile phase (solution B). Dilute 5.0 mL
Appearance of this solution to 100.0 mL with the mobile phase.
White or yellowish-white powder, hygroscopic.
Reference solution (c) Dissolve 25 mg of the substance to be
Solubility exam in ed in the mobile phase, add 5 mL of solution A and
Very soluble in water, freely soluble in ethanol (96 per cent). 5 mL of solution B and dilute to 100 mL with the mobile
IDENTIFICATION phase.
A. Thin-layer chromatography (2.2.27). Column:

— sizer. I = 0.25 m, 0 = 4.6 mm;
in 2 mL of acetone R. — stationary phase. octadecylsUyl silica gelfor chromatography R

(5 pm).
Reference solution (a) Dissolve 20 mg of chloramphenicol sodium
Mobile phase 20 g/L solution of phosphoric acid R, methanol R,
succinate CRS in 2 mL of acetone R.
water R (5:40:55 VIVIV).
Reference solution (b) Dissolve 20 mg of chloramphenicol CRS
in 2 mL of acetone R. Flow rate 1.0 mL/min.
Plate TLC silica gel G F254 plate R.
Injection 20 j£L.
Mobile phase dilute acetic acid R, methanol R, chloroform R
(1:14:85 V/VIV). System suitability, reference solution (c):
— the 2 peaks corresponding to those in the chromatograms
Application 2 |iL.
obtained with reference solutions (a) and (b) are clearly
Development Over a path of 15 cm. separated from the peaks corresponding to the 2 principal
Drying In air. peaks in the chromatogram obtained with the test
Detection.Examine in ultraviolet light at 254 nm. solution; if necessary, adjust the methanol content of the
Results The 2 principal spots in the chromatogram obtained mobile phase.
with the test solution are similar in position and size to the Limits:
2 principal spots in the chromatogram obtained with — chloramphenicol: not more than the area of the principal
reference solution (a); their positions are different from that peak in the chromatogram obtained with reference
of the principal spot in the chromatogram obtained with solution (a) (2.0 per cent);
reference solution (b). — chloramphenicol disodium disuccmater. not more than the
B. Dissolve about 10 mg in 1 mL of ethanol area of the principal peak in the chromatogram obtained
(50 per cent VIV) R, add 3 mL of a 10 g/L solution of calcium with reference solution (b) (2.0 per cent).
chloride R and 50 mg of zinc powder R and heat on a water- W ater (2.5.12)
bath for 10 min. Filter the hot solution and allow to cool. Maximum 2.0 per cent, determined on 0.500 g.
Add 0.1 mL of benzoyl chloride R and shake for 1 min. Pyrogens (2.6.8)
Add 0.5 mL of ferric chloride solution R1 and 2 mL of If intended for use in the manufacture of parenteral
chloroform R and shake. The upper layer is light violet-red or preparations without a further appropriate procedure for
purple. removal of pyrogens, it complies with the test for pyrogens.
C. Dissolve 50 mg in 1 mL of pyridine R. Add 0.5 mL of Inject per kilogram of the rabbit's mass 2.5 mL of a solution
dilute sodium hydroxide solution R and 1.5 mL of water R. Heat in waterfor injections R containing 2 mg of the substance to
in a water-bath for 3 min. A red colour develops. Add 2 mL be examined per millilitre.
of nitric acid R and cool under running water. Add 1 mL of
ASSAY
0.1 M silver nitrate. A white precipitate is formed slowly.
D. It gives reaction (a) of sodium (2.3.1). same solvent. Dilute 5.0 mL of this solution to 100.0 mL
TESTS with water R. Measure the absorbance (2.2.25) at the
p H (2.2.3) -absorption maximum at 276 nm.
6.4 to 7.0. Calculate the content of C isH isC^^N aO g, taking the
Dissolve 2.50 g in carbon dioxide-free water R and dilute to specific absorbance to be 220 .
10 mL with the same solvent. STORAGE
Specific optical rotation (2.2.7) In an airtight container, protected from light If die substance
+ 5.0 to + 8.0 (anhydrous substance). is sterile, store in a sterile, airtight, tamper-proof container,
Dissolve 0.50 g in water R and dilute to 10.0 mL with the protected from light.
same solvent. _____________________________________ PhEur
Chloram phenicol and chloramphenicol disodium
disuccinate
examined in the mobile phase and dilute to 100.0 mL with
the mobile phase.
2016 Chlorcyclizine Hydrochloride 1-509
Chlorcyclizine Hydrochloride ★★** ★ Reference solution (a) Dissolve 10 mg of chlorcyclizine

★ ★ hydrochloride CRS in methanolR and dilute to 10 mT. with the
(Ph. Eitr. monograph 1086) ***** same solvent.
Reference solution (b) Dissolve 5 mg of methylpiperazine R in
methanol R and dilute to 50 mL with the same solvent
Reference solution (c) Dilute 1 mL of test solution (b) to
and enantiomer , HCI 25 mL with methanol R.
Reference solution (d) Dissolve 10 mg of hydroxyzine
hydrochloride CRS and 10 mg of chlorcyclizine
hydrochloride CRS in methanolR and dilute to 10 mT. with the
same solvent.
C 1&H22CI2N2 337.3 14362-31-3
Apply separately to the plate 10 pL of each solution and
Action and use devdop over a path of 15 cm using a mixture of 2 volumes
Histamine HI receptor antagonist; antihistamine. of concentrated ammonia R, 13 volumes of methanol R and
85 volumes of methylene chloride R. Allow the plate to dry in
PhEur---------------------------------------------------------------------------------------- air and expose it to iodine vapour for 10 min. In the
DEFINITION chromatogram obtained with test solution (a): any spot
corresponding to methylpiperazine is not more intense than
Chlorcyclizine hydrochloride contains not less than
99.0 per cent and not more than the equivalent of the spot in the chromatogram obtained with reference
101.0 per cent of (i?<S)-l-[(4-chlorophenyl)phenylmethyl]-4- solution (b) (0.5 per cent); any spot, apart from the principal
methylpiperazine hydrochloride, calculated with reference to spot and any spot corresponding to methylpiperazine, is not
more intense than the spot in the chromatogram obtained
with reference solution (c) (0.2 per cent). The test is not
CHARACTERS valid unless the chromatogram obtained with reference
A white or almost white, crystalline powder, freely soluble in solution (d) shows two clearly separated spots.
water and in methylene chloride, soluble in alcohol. Loss on drying (2.2.32)
IDENTIFICATION Not more than 1.0 per cent, determined on 1.000 g by
First identification B, D drying in an oven at 130 °C.
Second identification A , C, D Sulfated ash (2.4.14)
A. Dissolve 10.0 mg in a 5 g/L solution of sulfuric acid R and Not more than 0.1 per cent, determined on 1.0 g.
dilute to 100.0 mL with the same add. Dilute 10.0 mL of ASSAY
the solution to 100.0 mL with a 5 g/L solution of sulfuric Dissolve 0.200 g in a mixture of 1 mL of 0.1 M hydrochloric
add R. Examined between 215 nm and 300 nm (2.2.25), the acid and 50 mL of methanol R. Cany out a potentiometric
solution shows an absorption maximum at 231 nm. titration (2.2.20), using 0.1 M sodium hydroxide. Read
The specific absorbance at the maximum is 475 to 525, the volume added between the two points of inflexion.
B. Examine by infrared absorption spectrophotometry C 18H 22CI2N 2.
chlorcyclizine hydrochloride CRS. Examine the substances STORAGE
prepared as discs. Store protected from light.
C. Examine the chromatograms obtained in the test for IMPURITIES
related substances (see Tests). The prindpal spot in the
chromatogram obtained with test solution (b) is similar in .CH3
obtained with reference solution (a).
D. It gives reaction (a) of chlorides (2.3.1).
A. ftf-methylpiperazine.
TESTS
PhEur
Dissolve 0.5 g in water R and dilute to 10 mL with the same
solvent. The solution is clear (2.2.1) and colourless (2.2.2,
Method IT).
pH (2.2.3)
10 mL with the same solvent. The pH of the solution is
5.0 to 6.0.
Related substances
Examine by thin-layer chromatography (2.2.27), using a plate
coated with a suitable silica gel.
examined in methanol R and dilute to 10 mL with the same
solvent.
Test solution (b) Dilute 5 mL of test solution (a) to 100 mL
with methanol R.
1-510 Chlordiazepoxide 2016

Chlordiazepoxide *****
Injection 10 pL.
*+ +*
(Ph. Eur. monograph 0656) * Run time 6 times the retention time of chlordiazepoxide.
Relative retention With reference to chlordiazepoxide
(retention time = about 3.6 min): impurity A = about 0.7;
impurity A and chlordiazepoxide.
Limits:
— impurities A , B:for each impurity, not more than the area
C jfiH u C IN a O 299.8 58-25-3 reference solution (a) (0.2 per cent),
— impurity C: not more than the area of the principal peak
Action and use in the chromatogram obtained with reference solution (c)
Benzodiazepine. (0.2 per cent),
PhEur ______________________________________________________________
DEFINITION chromatogram obtained with reference solution (a)
7-Chloro-N-methyl-5-phenyl-3/7-1,4-benzodiazepin-2-amine (0.10 per cent),
4-oxide. — total: not more than 2.5 times the area of the principal
Content peak in the chromatogram obtained with reference
99.0 per cent to 101.0 per cent (dried substance). solution (a) (0.5 per cent),
CHARACTERS the chromatogram obtained with reference solution (a)
Appearance (0.05 per cent).
Almost white or light yellow, crystalline powder.
Solubility Maximum 0.5 per cent, determined on 1.000 g by drying in
Practically insoluble in water, sparingly soluble in ethanol an oven at 105 °C.
(96 per cent). Sulfated ash (2.414)
It shows polymorphism (5.9). Maximum 0.1 per cent, determined on 1.0 g.
IDENTIFICATION ASSAY
Infrared absorption spectrophotometry (2.2.24). Dissolve 0.250 g, with heating if necessary, in 80 mL of
Comparison chlordiazepoxide CRS. anhydrous acetic acid R. Titrate with 0.1 M perchloric acid
If the spectra obtained in the solid state show differences, determining the end-point potentiometrically (2.2.20).
dissolve the substance to be examined and the reference 1 mL of 0.1 M perchloric acid is equivalent to 29.98 mg
substance separately in methylene chloride R, evaporate to of C 16H 14C1N30 .
STORAGE
TESTS Protected from light.
Related substances
IMPURITIES
liquid chromatography (2.2.29). Carry out the test protected
Specified impurities A, B, C
from bright light and prepare the solutions immediately before use.
the mobile phase.
Reference solution (a) Dilute 1.0 ml. of the test solution to
100.0 mL with the mobile phase. Dilute 2.0 mL of this
solution to 10.0 mL with the mobile phase.
Reference solution (b) Dissolve 5 mg of chlordiazepoxide
impurity A CRS in the mobile phase, add 25.0 mL of the test
solution and dilute to 100.0 mL with die mobile phase. A. 7-chloro-5-phenyi-1,3-dihydro-2.fi-1,4-benzodiazepin-2-
Dilute 2.0 mL of this solution to 50.0 mL with the mobile one 4-oxide,
phase.
Reference solution (c) Dissolve 4.0 mg of
aminochlorobenzophenone R in the mobile phase and dilute to
Column:
— size. I = 0.15 m, 0 = 4.6 mm,
— stationary phase: octadecylsifyl silica gelfor chromatography R
(5 pm). B. 6-chloro-2-(chloromethyl)-4-phenylquinazoline 3-oxide,
Mobile phase acetonitrile R, water R (50:50 VIV).
2016 Chlordiazepoxide Hydrochloride 1-511
Related substances
Liquid chromatography (2.2.29). Cany out thefollowing
operations protectedfrom bright light and prepare the solutions
the mobile phase.
C. (2 -amino-5-chlorophenyl)phenylmethanone
Reference solution (a) Dilute 1.0 mL of the test solution to
(aminochlorobenzophenone).
_____________________________________________________________ PhEur solution to 10.0 mL with the mobile phase.
Reference solution (b) Dissolve 5 mg of chlordiazepoxide
impurity A CRS in the mobile phase, add 25.0 mL of the test
solution and dilute to 100.0 mL with the mobile phase.
Chlordiazepoxide Hydrochloride ****** Dilute 2.0 mL of this solution to 50.0 mL with the mobile
phase.
*+
(Ph. Eur. monograph 0474) * Reference solution (c) Dissolve 4.0 mg of
aminochlorobenzophenone R in the mobile phase and dilute to
100.0 mL with die mobile phase. Dilute 1.0 mL of this
Column:
— size. I — 0.15 m, 0 = 4.6 mm,
(5 pm).
Mobile phase acetonitrUe R, water R (50:50 V/V).
C 16H 15C12N30 336.2 438-41-5 Detection Spectrophotometer at 254 nm.
Injection 10 |jL.
Action and use
Run tone 6 times the retention time of chlordiazepoxide.
Benzodiazepine.
Relative retention With reference to chlordiazepoxide
PhEur __ __________________________________________________________ (retention time = about 3.6 min): impurity A = about 0.7;
DEFINITION impurity B = about 2.3; impurity C = about 3.9.
7-Chloro-iV-methyl-5-phenyl-3.fi-1,4-benzodiazepin-2-amine System suitability: reference solution (b):
4-oxide hydrochloride. — resolution: minimum 5.0 between the peaks due to
impurity A and chlordiazepoxide.
Content
99.0 per cent to 101.0 per cent (dried substance). Limits:
— impurities A, B: for each impurity, not more than the area
CHARACTERS of the principal peak in the chromatogram obtained with
Appearance reference solution (a) (0.2 per cent),
White or slightly yellow, crystalline powder. — impurity C: not more than the area of the principal peak
Solubility in the chromatogram obtained with reference solution (c)
Soluble in water, sparingly soluble in ethanol (96 per cent). (0.2 per cent),
It shows polymorphism (5.9). — unspecified impurities: for each impurity, not more than
IDENTIFICATION chromatogram obtained with reference solution (a)
A. Infrared absorption spectrophotometry (2.2.24). (0.10 per cent),
Comparison chlordiazepoxide hydrochloride CRS. — total: not more than 2.5 times the area of the principal
If the spectra obtained in the solid state show differences, peak in the chromatogram obtained with reference
dissolve 100 mg in 9 mT. of water R and add 1 mL of dilute solution (a) (0.5 per cent),
sodium hydroxide solution R. Extract with 10 mL of methylene — disregard limit: 0.25 times the area of the principal peak in
chloride R in a separating funnel. Evaporate the organic layer the chromatogram obtained with reference solution (a)
and dry the residue obtained at 100-105 °C. Proceed in the (0.05 per cent).
same way with the reference substance. Record new spectra Loss on drying (2.2.32)
using the residues. Maximum 0.5 per cent, determined on 1.000 g by drying
B. Dissolve 50 mg in 5 mL of water R, add 1 mL of dilute in vacuo at 60 °C for 4 h.
ammonia R1, mix, allow to stand for 5 min and filter. Acidify Sulfated ash (2.4.14)
the filtrate with dilute nitric acid R. The solution gives Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
TESTS Dissolve 0.250 g in 50 mL of water R. Titrate with
Appearance of solution 0.1 M silver nitrate, determining the end-point
The solution is clear (2.2.1) and not more intensely coloured potentiometrically (2.2.20).
than reference solution GY6 (2.2.2, Method IT). 1 mL of 0.1 M silver nitrate is equivalent to 33.62 mg
Dissolve 2.5 g in water R and dilute to 25 mL with the same of C 16H 15C12N 30 .
solvent.
1-512 Chlorhexidine Acetate 2016
STORAGE CHARACTERS
Protected from light. Appearance
IMPURITIES W hite or alm ost white, microcrystalline powder.
Specified impurities: A, B, C. Solubility
Sparingly soluble in water, soluble in ethanol (96 p er cent),
slightly soluble in glycerol and in propylene glycol.
IDENTIFICATION
Comparison chlorhexidine diacetate CRS.
B. Dissolve about 5 mg in 5 m L o f a w arm 10 g/L solution
A. 7-chloro-5-phenyl-1,3-dihydro-2//-1,4-benzodiazepin-2- o f cetrimide R and add 1 m L o f strong sodium hydroxide
one 4-oxide, solution R and 1 m L o f bromine water R. A deep red colour is
produced.
C. Dissolve 0.3 g in 10 m L o f a m ixture o f equal volumes o f
hydrochloric acid R and water R. A dd 40 m L o f water R, filter
if necessary and cool in iced w ater. M ake alkaline to titan
yellow paper R by adding dropwise, and w ith stirring, strong
sodium hydroxide solution R and ad d 1 m l. in excess. Filter,
wash the precipitate with water R until the washings are free
from alkali and recrystallise from ethanol (70 per cent V/V) R.
B. 6-chloro-2-(chloromethyI)-4-phenylquinazoline 3-oxide, D iy at 100-105 °C. T h e residue m elts (2.2.14) a t 132 °C to
136 °C.
D . It gives reaction (a) o f acetates (2.3.1).
TESTS
Impurity P (chloroaniline)
M axim um 500 ppm.
Test solution Dissolve 0.20 g in 25 m L o f water R with
shaking if necessary. Add 1 m L o f hydrochloric add R and
dilute to 30 m L with water R. A dd rapidly and w ith thorough
C. (2-amino-5-chlorophenyl)phenylmethanone mixing after each addition: 5 m L o f a 103 g/L solution o f
(am inochlorobenzophenone). hydrochloric add R, 0.35 m L o f sodium nitrite solution R, 2 m l.
PhEur o f a 50 g/L solution of ammonium sulfamate R, 5 m L o f a
1.0 g/L solution o f naphxhylethylenediamine dihydrochloride R
and 1 m L o f ethanol (96 per cent) R; transfer quantitatively to
a volumetric flask, dilute to 50.0 m L with water R and allow
** + to stand for 30 min.
★ ★
Chlorhexidine Acetate ★ ★ Reference solutions Prepare reference solutions representing
(Chlorhexidine Diacetate, Ph Eicr monograph 0657) ***** respectively 50 ppm , 100 ppm , 200 ppm , 500 ppm and
600 ppm o f chloroaniline R (im purity P) in the test sample as
follows: dilute 1.0 ml-, 2.0 ml-, 4.0 m l-, 10.0 m L and
12.0 mT. o f a solution containing 0.010 g/L o f chloroamline R
(impurity P) in dilute hydrochloric add R to 20 m L with
water R. T h en , add 10 m L o f water R. A dd rapidly and with
- C O jH
thorough mixing after each addition: 5 m L o f a 103 g/L
solution o f hydrochloric add R, 0.35 m L o f sodium nitrite
solution R, 2 m L o f a 50 g/L solution o f ammonium
sulfamate R, 5 m L o f a 1 g/L solution o f
naphxhykthylenediarmne dihydrochloride R and 1 m L o f ethanol
C26H38CI2N10O4 625.6 56-95-1 (96 per cent) R; transfer each solution quantitatively to a
volumetric flask, dilute to 50.0 m L with water R and allow to
Action and use stand for 30 min.
Antiseptic.
M easure th e absorbance (2.2.25) o f each reference solution
Preparation at 556 rnn and plot a calibration curve.
Chlorhexidine Irrigation Solution M easure th e absorbance (2.2.25) o f the test solution at
PhEur.
556 nm . D eterm ine the concentration of chloroaniline from
the calibration curve.
DEFINITION
Related substances
1j 1 '-(H exane-1,6-diyl)bis [5-(4-chlorophenyl)biguanide] Liquid chrom atography (2.2.29). Store the solutions at a
diacetate.
temperature not exceeding 12 °C.
Content
2016 Chlorhexidine Acetate 1-513
Test solution Dissolve 0.140 g of the substance to be chromatogram obtained with reference solution (b)
examined in mobile phase A and dilute to 100.0 m L with (0.10 p er cent);
mobile phase A. — total: not more than 0.8 times the area of the principal
Reference solution (a) Dissolve the contents o f a vial of peak in the chromatogram obtained with reference
Chlorhexidine for system suitability CRS (containing solution (b) (0.8 per cent);
impurities A, B, H , I, K, N and O) in 1.0 m L of mobile — disregard limit: 0.05 times the area of the principal peak in
phase A. the chromatogram obtained with reference solution (b)
(0.05 p er cent).
100.0 m L with mobile phase A. L oss on d ry in g (2.2.52)
Column: M aximum 3.5 p er cent, determined on 1.000 g by drying in
— size: I — 0.25 m , 0 = 4.6 mm; an oven at 105 °C.
— stationary phase: end-capped octadecylsüyl silica gel for Sulfated ash (2.4.14)
chromatography R (5 pm); Maximum 0.15 per cent, determined on 1.0 g.
— temperature: 30 °C. ASSAY
Mobile phase: — Dissolve 0.140 g in 100 m L o f anhydrous acetic acid R and
mobile phase A: mix 20 volumes of a 0.1 per cent V/V titrate with 0.1 M perchloric acid. Determine the end-point
solution of trifluoroacetic acid R in acetonitrUe R and potentiometrically (2.2.20).
80 volumes of a 0.1 per cent V/V solution of trifluoroacetic
add R in water R,
Of C 26H 38CI2N 10O 4.
— mobile phase B: mix 10 volumes o f a 0.1 per cent V/V
solution of trifluoroacetic acid R in water R and 90 volumes IM P U R IT IE S
o f a 0.1 per cent V/V solution of trifluoroacetic acid R in Specified impurities A, H, I, K, N, O, P
acetomtrüe R', Other detectable impurities (the following substances would, if
Time Mobile phase A Mobile phase B present at a sufficient level, be detected by one or other of
(min) (per cent V/V) (per cent V/V) the tests in the monograph. They are limited by the general
0- 2 100 0 acceptance criterion for other/unspecified impurities and/or
2 -3 2 100*80 0 * 20
3 2 -3 7 80 20 impurities for demonstration o f compliance. See also 5.10.
37 - 47 80 -»70 2 0 * 30 Control of impurities in substances for pharmaceutical use): B, E,
F, G, M.
4 7 -5 4 70 30
H H
Detection Spectrophotom eter at 254 nm . TNH
Injection 10 |iL.
Identification of impurities Use the chromatogram supplied Cl
NH NH
with chlorhexidine for system suitability CRS and the
X X
identify the peaks due to impurities A, B, H , I, K, N and O.
Relative retention W ith reference to chlorhexidine (retention
A. l-(4-chlorophenyl)-5-[6-
time = about 35 min): impurity N = about 0.35;
[(cyanocarbamimidoyl) amino]hexyl] biguanide,
impurity H = 0.85; impurity O = about 0.90;
impurity I = about 0.91; impurity K = about 1.4. H2N ^ / N N,
System suitability: reference solution (a): TO TNH
— peak-to-valUy ratio: m inim um 2.0, where Hp — height
above the baseline o f the peak due to impurity B and Cl
NH NH
curve separating this peak from the peak due to l X »N x Nx
impurity N . H H
Limits:
— sum cf impurities I and O: not more than 0.4 times the B. 1- [ [6- [ [ [(4-chlorophenyi) carbamimidoyl] carbamimidoyl]
area o f the principal peak in the chromatogram obtained amino]hexyl] carbamimidoyl]urea,
— impurity K: n o t more than 0.3 times the area of the
— impurities A , H, N: for each impurity, n o t more than "X x
H
a
2
(0.15 per cent); E. l-( 4-chlorophenyl)guanidine,
0.1 tim es the area of the principal peak in the
1-514 Chlorhexidine Gluconate Solution 2016
ci H H H
N
H
xo
NH,
*
F. l-(4-chlorophenyi)urea,
H,N.
O. 5-(2-chlorophenyl)-5 '-(4-chlorophenyl)-1,1 '- (hexane-1,6-

diyl) dibiguanide,
c l' v ^ S NH NH
I X Na NX N
H H H “Xi NH,
G. 1-( 6-aminohexyl)-5-(4-chlorophenyl)biguanide 3 P. 4-chloroaniline.

PhEur
★
Chlorhexidine Gluconate Solution ★
(Chlorhexidine Digluconate Solution,

H H H
H. 1 ,1 '-[iminobis(carbonimidoyliininohexane- 6 , 1-diyl)]bis [5-
(4-chlorophenyl)biguanide], Y y "'
NH NH
I. unknow n structure.
NH NH
H H H
T T H H
O NH
Cl
C 3 4 H 5 4 C I2 N 10 O 14 898 18472-51-0
CI' ' Y ^ S NH NH
Action and use
N N N Antiseptic.
H H H
Preparations
K. 1-(4-chlorophenyl)-3- [[6 - [[ [(4-chlorophenyl) Chlorhexidine G luconate Eye D rops
carbamimidoyl] carbamimidoyi] amino]hexyl] Chlorhexidine Gluconate Gel
carbamimidoyl] urea. Chlorhexidine Irrigation Solution
Chlorhexidine Mouthwash
H H H
Lidocaine and Chlorhexidine Gel
ny ny
NH NH
n'
PhEur.
Cl DEFINITION
NH NH
Aqueous solution o f l,l'-(hexane-l,6-diyl)bis[5-
LX na na n. (4-chlorophenyl)biguanide] di-D-gluconate.
H H H
Content
190 g/L to 210 g/L.
M. 5-(4-chlorophenyl)-5 '-phenyl- 1,1 '-(hexane-1,6-
diyl) dibiguanide, CHARACTERS
Appearance
Almost colourless or pale-yellowish liquid.
Solubility
Miscible with water, with no t m ore than 3 parts o f acetone
and with n o t more than 5 parts o f ethanol (96 per cent).
IDENTIFICATION
N . l-[6-(carbamimidoylam ino)hexyl]-5- A. Infrared absorption spectrophotom etry (2.2.24).
(4-chlorophenyl)biguanide, Preparation T o 1 m L add 40 m L o f water R, cool in iced
water, make alkaline to titan yellow paper R by adding
dropwise, and with stirring, strong sodium hydroxide solution R
2016 Chlorhexidine Gluconate Solution 1-515
and add 1 m L in excess. Filter, wash the precipitate with naphthylethylenediamine dihydrochloride R and 1 m L of ethanol
water R until the washings are free from alkali and (96 per cent) R] transfer each solution quantitatively to a
recrystallise from ethanol (70 per cent V/V) R. D ry at volumetric flask, dilute to 50.0 m L with water R and allow to
100-105 °C. Examine the residue. stand for 30 min.
Comparison chlorhexidine CRS. M easure the absorbance (2.2.25) o f each reference solution
B. Thin-layer chromatography (2.2.27). at 556 nm and plot a calibration curve.
Test solution Dilute 10.0 m L o f the preparation to be M easure the absorbance (2.2.25) o f the test solution at
examined to 50 m L with water R. 556 nm. D eterm ine the concentration of chloroaniline from
Reference solution Dissolve 25 mg of calcium gluconate CRS in
1 m L of water R. R e la te d su b sta n c e s
Plate TLC silica gel plate R. Liquid chromatography (2.2.29). Store the solutions at a
temperature not exceeding 12 °C.
Mobile phase concentrated ammonia R, ethyl acetate R, water R,
ethanol (96 per cent) R (10:10:30:50 VIVIVIV). Test solution Dilute 1.0 m L of the preparation to be examined
Application 5 jiL.
Reference solution (a) Dissolve the contents of a vial of
Development Over 1/2 of the plate. chlorhexidine for system suitability CRS (co n taining
Drying At 100 °C for 20 m in and allow to cool. impurities A, B, F, G , H , I, J, K, L, N and O) in 1.0 m L of
Detection Spray with a solution containing 25 g/L of mobile phase A.
ammonium molybdate R and 10 g/L o f cerium sulfate R in dilute Reference solution (b) Dilute 1.0 m L of the test solution to
sulfuric acid R, and heat at 110 °C for about 10 min. 100.0 m L with mobile phase A.
Results T he principal spot in the chromatogram obtained with Column:
the test solution is similar in position, colour and size to the — size. I = 0.25 m , 0 = 4.6 mm;
principal spot in the chromatogram obtained with the — stationary phase: end-capped octadecylsUyl silica gel for
reference solution. chromatography R (5 |im);
C. T o 1 m L add 40 m L o f water R, cool in iced water, make — temperature: 30 °C.
alkaline to titan yellow paper R by adding dropwise, and with Mobile phase:
stirring, strong sodium hydroxide solution R and add 1 m L in — mobile phase A: mix 20 volumes of a 0.1 per cent VIV
excess. Filter, wash the precipitate with water R until the solution of trifluoroacetic acid R in acetomtrUe R and
washings are free from alkali and recrystallise from ethanol 80 volumes of a 0.1 per cent VIV solution of trifluoroacetic
(70 per cent VIV) R. Dry at 100-105 °C. T h e residue melts acid R in water R ;
(2.2.14) at 132 °C to 136 °C. — mobile phase B: mix 10 volumes of a 0.1 per cent VIV
D. T o 0.05 m L add 5 m L of a 10 g/L solution of cetrimide R, solution of trifluoroacetic acid R in water R and 90 volumes
1 m L o f strong sodium hydroxide solution R and 1 m L o f of a 0.1 per cent VIV solution of trifluoroacetic acid R in
bromine water R; a deep red colour is produced. acetomtrUe R-,
TESTS
R elativ e d e n sity (2.2.5) Tune Mobile phase A Mobile phase B
1.06 to 1.07. (min) (per cent V/V) (per cent V/V)
0 -2 100 0
p H (2.2.3)
5.5 to 7.0. 2 - 32 10 0 * 80 0*20
Dilute 5.0 m L to 100 m L with carbon dioxide-free water R. 32-37 80 20
Im p u rity P (ch lo ro an ilin e) 37-47 80*70 20*30

M aximum 500 ppm , calculated with reference to 47-54 70 30
chlorhexidine digluconate solution.
Test solution Dilute 0.20 g o f the preparation to be examined
to 30 m L with water R. Add rapidly and with thorough Flow rater. 1.0 m l /m in.
m ixin g after each addition: 5 m L of a 103 g/L solution of Detection: spectrophotometer at 254 nm.
hydrochloric acid R, 0.35 m L of sodium nitrite solution R, 2 m L Injection 10 |iL.
of a 50 g/L solution of ammonium sidfamate R, 5 m L o f a
1 g/L solution o f naphthylethylenediamine dxhydrochloride R and
with chlorhexidine for system suitability CRS and the
1 m L of ethanol (96 per cent) R; transfer quantitatively to a
identify the peaks due to impurities A, B, F, G, H , I, J, K , L,
stand for 30 min.
N and O.
Reference solutions Prepare reference solutions Representing
respectively 50 ppm , 100 ppm , 200 ppm , 500 ppm and
time = about 35 min): impurity L = about 0.23;
600 ppm of chloroaniline R (impurity P) in the test sample as
impurity Q = about 0.24; impurity G = about 0.25;
follows: dilute 1.0 m T , 2.0 m T , 4.0 m l , 10.0 m L and
impurity N = about 0.35; impurity B = about 0.36;
12.0 m L of a solution containing 0.010 g/L o f chloroaniline R
impurity F = about 0.5; impurity A = about 0.6;
(impurity P) in dilute hydrochloric acid R to 20 m L with
impurity H = about 0.85; impurity O = about 0.90;
water R. T hen, add 10 m L o f water R. Add rapidly and with
impurity I = about 0.91; impurity J = about 0.96;
thorough mixing after each addition: 5 m L o f a 103 g/L
impurity K = about 1.4.
solution o f hydrochloric acid R, 0.35 m L o f sodium nitrite
solution R, 2 m L o f a 50 g/L solution of ammonium System suitability-, reference solution (a):
sidfamate R, 5 m L of a 1 g/L solution of
impurities L and G;
1-516 Chlorhexidine Gluconate Solution 2016
H H
— peak-to-vaRey ratio: minim um 2.0, where Hp = height
above the baseline of the peak due to impurity B and n^ Y '
NH
H v = height above the baseline of the lowest point o f the
curve separating this peak from the peak due to Cl
im purity N . NH NH
Limits: N N N'
— impurity N: not m ore than the area o f the principal peak H H H
( 1.0 per cent); A. 1-(4-chlorophenyl)-5- [6-
— impurity H: not more than 0.5 times the area o f the [(cyanocarbamimidoyl)amino]hexyl] biguanide,
h 2n ^ n ^ n ,
— impurities A , J ,K for each impurity, not more than
T T
0.4 times the area of the principal peak in the O NH
(0.4 per cent); Cl
NH NH
— sum of impurities I and O. n o t more than 0.4 times the
area of the principal peak in the chromatogram obtained H H H
w ith reference solution (b) (0.4 per cent);
— impurity G: not m ore than 0.3 times the area of the
B . 1- [ [6- [ [ [(4-chlorophenyl) carbamimidoyi] carbamimidoyl]
amino]hexyl] carbamimidoyl]urea,
— impurities B, F, L, Q. for each impurity, n o t more than
0.2 times the area o f the principal peak in the NH
( 0.2 per cent); N
X NH2
0.1 times the area of the principal peak in the E. l-(4-chlorophenyi)guanidine,
( 0.10 per cent);
N NH,
(3.0 per cent); H
the chrom atogram obtained with reference solution (b) F . l-(4-chlorophenyi)urea,
(0.05 per cent).
A SSA Y H,N.
D eterm ine the density (2.2.5) o f the preparation to be

examined. T ransfer 1.00 g to a 250 m L beaker and add
50 m L of anhydrous acetic add R. T itrate with 0.1 M Cl
NH NH
(2.2.20). N N N
H H H
of C 34H 54CI2N 10O 14.
G. l-(6-am inohexyl)-5-(4-chlorophenyl)biguanide,
STORAGE
Protected from light. Cl.
nh nh
IM P U R IT IE S
Spedfied impurities A , B, F, G, H, I, J, K, L, N, O, P, Q
> U nA nX n.
H H H
HN^NH
present at a sufficient level, be detected by one or other of Cl HN^NH
the tests in the monograph. T hey are limited by the general nh nh
acceptance criterion for other/unspecified im purities and/or I A N

a Na N
, nh
by the general m onograph Substances for pharmaceutical use H H H
impurities for dem onstration o f compliance. See also 5.10. H . 1,1 [iminobis(carbonimidoyliminohexane-6 , 1-diyl)]bis [5-
Control of impurities in substances for pharmaceutical use): E, M. (4-chlorophenyl)biguanide],
I. unknow n structure,
2016 Chlorhexidine Hydrochloride 1-517
H H H
l' Y ^ S NH NH O. 5-(2-chlorophenyl)-5'- (4-chlorophenyl)-1,1 '-(hexane-1,6-

diyl)dibiguanide,
^ ^ N ^H N H^ NH'
J. l-(4-chlorophenyl)-5-[6-[[4-[(4-chlorophenyl)amino]-6-
nh2
[(lS,2Æ ,3Æ ,4/Q-l,2,3,4,5-pentahydroxypentyl]-l,3,5-triazin-
2-yl] amino] hexyl]biguanide,
P. 4-chloroaniline,
H H H Q. unknown structure.
r r NT NY N' PhEur
o NH
NH NH
Chlorhexidine Hydrochloride ★ ★
U ^H AH -H ★ ★
★ft + ★
(Chlorhexidine Dihydrochloride, Ph. Eur. monograph *
K 1-(4-chlorophenyl)-3- [[6- [[ [(4-chlorophenyl) 0659)
carbamimidoyl] carbamimidoyl] amino] hexyl]
H H
carbamimidoyl] urea,
ny t
NH NH
2HCI
NH NH
I A N
a „
N
a
H H
C 22H 32CI4N 10 578.4 3697-42-5
L. (5i?,63)-2- [(4-chlorophenyl) amino] -5-hydroxy-6- [( li?,2i?)- Action and use

Antiseptic.
1,2 , 3-trihydroxypropyI] -5,6-dihydio-4H-1,3-oxazin-4-one,
PhEtr____________
h H H
^N, DEFINITION
NH NH
1,1 '-(H exane-1, 6-diyl)bis [5-(4-chlorophenyl)biguanide]
dihydrochloride.
a V ^ l NH NH
Content
U ^ na na n
H H H CHARACTERS
Appearance
M. 5-(4-chlorophenyl)-5 '-phenyl-1 , 1 '-(hexane-1,6- W hite or almost white, crystalline powder.
diyl)dibiguanide, Solubility
very slightly soluble in water, slightly soluble in propylene
H glycol, very slightly soluble in ethanol (96 per cent).
h 2n n ,
YNH IDENTIFICATION
First identification: A, D.
NH NH
U ^ V l Comparison chlorhexidine dihydrochloride CRS.
B. Dissolve about 5 mg in 5 m L o f a warm 10 g/L solution
N . l-[6-(carbamimidoylamino)hexyl]-5- o f cetrimide R and add 1 m L o f strong sodium hydroxide
(4-chlorophenyl)biguanide, solution R and 1 m L o f bromine water R. A dark red colour is
produced.
C. Dissolve 0.3 g in 10 m L o f a mixture o f equal volumes of
hydrochloric acid R and water R. A dd 40 m L of water R, filter
if necessary and cool in iced water. M ake alkaline to titan
yellow paper R by adding dropwise,* and with stirring, strong
1-518 Chlorhexidine Hydrochloride 2016
sodium hydroxide solution R and add 1 m L in excess. Filter, o f a 0.1 per cent VIV solution o f trifluoroacetic add R in
wash the precipitate with water R until the washings are free acetonitrUe R',
from alkali and recrystallise from ethanol (70 per cent VIV) R.
D ry at 100-105 °C. T h e residue melts (2.2.14) at 132 °C to
136 °C.
D . It gives reaction (a) of chlorides (2.3.1). 0 -2 100 0
TESTS 2 - 32 100*80 0 *20
Impurity P (chloroanfline)
32 - 37 80 20
M axim um 300 ppm.
Test solution T o 0.20 g add 1 m L of hydrochloric add R, shake 3 7 -4 7 80*70 20*30
for about 30 s, dilute to 30 m L with water R and shake until 47 - 54 70 30
a clear solution is obtained. A dd rapidly and with thorough
mixing after each addition: 5 m L o f a 103 g/L solution o f
hydrochloric acid R, 0.35 m L o f sodium nitrite solution R, 2 m L Flow rate 1.0 mL/min.
of a 50 g/L solution o f ammonium sidfamate R, 5 m l. o f a Detection Spectrophotom eter at 254 nm .
1 g/L solution of naphthykthylenediamine dihydrochloride R and Injection 10 |iL.
1 m L of ethanol (96 per cent) R; transfer quantitatively to a
w ith chlorhexidine for system suitability CRS and the
stand for 30 min.
Reference solutions Prepare reference solutions representing identify the peaks due to impurities A, B, H , I, K , N and O.
respectively 50 ppm , 100 ppm , 200 ppm , 300 ppm. and
500 ppm o f chloroanUme R (impurity P) in the test sample as
tim e = about 35 min): im purity N = about 0.35;
follows: dilute 1.0 m L, 2.0 m L, 4.0 m L, 6.0 m L and
10.0 m L of a solution containing 0.010 g/L of chloroani!ine R
impurity H = about 0.85; impurity O = about 0.90;
(impurity P) in dilute hydrochloric acid R to 20 m L with
im purity I = about 0.91; impurity K = about 1.4.
water R. T h en , add 10 m L o f water R. Add rapidly and with
thorough mixing after each addition: 5 m L o f a 103 g/L System suitability: reference solution (a):
solution o f hydrochloric acid i?, 0.35 mT. of sodium nitrite — peak-to-vaUey ratio: minim um 2.0, where Hp — height
solution R, 2 m L o f a 50 g/L solution of ammonium above the baseline o f the peak due to im purity B and
sidfamate R, 5 m L o f a 1 g/L solution of Hv — height above the baseline o f the lowest point o f the
naphthykthylenediamine dihydrochloride R and 1 m L o f ethanol curve separating this peak from the peak due to
(96 per cent) R; transfer each solution quantitatively to a impurity N .
volumetric flask, dilute to 50.0 m L with water R and allow to Limits:
stand for 30 min. — impurity H: no t more than 0.5 times the area of the
M easure the absorbance (2.2.25) o f each reference solution
reference solution (b) (0.5 p er cent);
at 556 nm and plot a calibration curve.
— impurity K. not more than 0.4 times the area of the
M easure the absorbance (2.2.25) of the test solution at
556 nm . D eterm ine the concentration o f chloroaniline from
reference solution (b) (0.4 p er cent);
— sum of impurities I and O: not m ore than 0.4 times the
Related substances area o f the principal peak in the chrom atogram obtained
Liquid chrom atography (2.2.29). Store the solutions at a with reference solution (b) (0.4 per cent);
temperature not exceeding 12 °C. — impurities A , N: for each impurity, not m ore than
Test solution Dissolve 0.130 g o f the substance to be 0.15 times the area o f the principal peak in the
examined in mobile phase A and dilute to 100.0 mT. with chrom atogram obtained with reference solution (b)
m obile phase A. (0.15 per cent);
Reference solution (a) Dissolve the contents of a vial o f — unspecified impurities: for each impurity, n o t m ore than
chlorhexidine for system suitability CRS (containing 0.1 times the area o f the principal peak in the
impurities A, B, H , I, K, N and O) in 1.0 m L of mobile chrom atogram obtained with reference solution (b)
phase A. (0.10 per cent);
— total: n o t m ore than the area o f the principal peak in the
Reference solution (b) D ilute 1.0 m L o f the test solution to
100.0 m L w ith mobile phase A.
( 1.0 p er cent);
Column: — disregard limit. 0.05 times the area of the principal peak in
— sizer. I = 0.25 m , 0 = 4.6 mm;
(0.05 p er cent).
chromatography R (5 fim);
— temperature: 30 °C. L o ss o n d ry in g (2.2.32)
Maximum 1.0 p er cent, determ ined on 1.000 g by drying in
Mobile phase:
an oven at 105 °C.
— mobile phase A: mix 20 volumes of a 0.1 per cent VIV
solution o f trifluoroacetic acid R in acetonitrUe R and S u lfa te d a s h (2.414)
80 volumes o f a 0.1 per cent VIV solution of trifluoroacetic M aximum 0.1 per cent, determ ined on 1.0 g.
add R in water R; A SSA Y
— mobile phase B: mix 10 volumes o f a 0.1 per cent VIV Dissolve 100.0 m g in 5 m L o f anhydrous formic add R and
solution o f trifluoroacetic add R in water R and 90 volumes add 70 mT. of acetic anhydride R. T itrate with 0.1 Mperchloric
addj determining the end-point potentiometrically (2.2.20).
2016 Chlorhexidine Hydrochloride 1-519
1 m l. o f 0.1 Mperchloric add is equivalent to 14.46 mg ClvY ^ S NH NH

o f C 22H 32CI4N 1Q. U - na na n
IM P U R IT IE S H H H
HN^NH
Specified impurities A, H , I, K , N , O, P
Other detectable impurities (the following substances would, if HN^NH
C ,N ^ S NH NH
present at a sufficient level, be detected by one or other of .NH
U „ A „ A
the tests in the monograph. T hey are limited by the general N N N
H H H
H . 1 , 1 [iminobis(carbonimidoyliminohexane-6 , 1-diyl)] bis [5-
(2034). It is therefore not necessary to identify these (4-chlorophenyl)biguanide],
Control of impurities in substances for pharmaceutical use): B, E, I. unknow n structure,
F, G, M.
H H H
N^
H H T0 N^TNHN-
w TNH
N NH NH
NH
■h « hK Kh
NH
X A „A
N
„A
N N
H H H
K. 1-(4-chlorophenyl)-3- [ [6- [[[(4-chlorophenyl)

carbamimidoyl] carbamimidoyl] amino]hexyl]
A. 1-(4-chlorophenyl)-5- [6-
carbamimidoyl]urea,
[(cyanocarb amimidoyl) amino] hexyl] biguanide,
H H H
N N N
h 2n ^
TO TNH
n n.
YNH YNH •
Cl
NH NH
NH NH
x A uA 1
N N N
‘N N H H H
H H
M . 5-(4-chlorophenyl)-5 '-phenyl-1,1 '-(hexane-1,6-

B. 1- [ [6- [ [ [(4-chlorophenyl) carbamimidoyl] carbamimidoyl]
diyl)dibiguanide,
amino]hexyl] carbamimidoyl] urea,
H
H,N^ ^N.
YNH
NH
^^N^NHz
H 2 CIY '5S NH NH
I A N
a „Na N
E. l-(4-chlorophenyl)guanidine, H H H
N . l-[6-(carbamimidoylamino)hexyI]-5-
0 (4-chlorophenyl)biguanide,
1
N NH2
H 2 H H H
.N ^ ^N,
F. l-(4-chlorophenyl)urea, NH NH
Cl NH NH
I A „NA NA N
H H H
NH NH
N
A..A
N
0.5-(2-chlorophenyl)-5'-(4-chlorophenyl)-l,l '-(hexane-1, 6-
diyl)dibiguanide,
H H
G. 1-(6-aminohexyl)-5-(4-chlorophenyl)biguanide,
^^N H ,
P. 4-chloroaniline.
1-520 Chlorinated Lime 2016
Comparison chlormadinone acetate CRS.

Chlorinated Lime
TESTS
A c tio n a n d u se S p ecific o p tic a l r o ta tio n (2.2.7)
Disinfectant. —14.0 to —10.0 (dried substance).
Dissolve 0.200 g in acetonitrile R and dilute to 10.0 m l. with
D E F IN IT IO N
the same solvent.
Chlorinated lim e contains n o t less than 30.0% w/w of
available chlorine, Cl. R e la te d su b sta n c e s
Test solution (a) Dissolve 20 m g o f the substance to be
A dull white powder.
examined in mobile phase B and dilute to 10.0 m L w ith
Partly soluble in water and in ethanol (96%). mobile phase B.
ID E N T IF IC A T IO N Test solution (b) Dissolve 10.0 m g o f the substance to be
A. Evolves chlorine copiously on the addition of examined in mobile phase B and dilute to 20.0 m L with
2m hydrochloric acid. mobile phase B.
B. W hen shaken with water and filtered, the filtrate yields Reference solution (a) Dissolve 4 m g o f chlormadinone acetate
reaction C characteristic of calcium salts and reaction A for system sidtabüày CRS (containing impurities A, B, E
characteristic of chlorides, Appendix VI. and K) in mobile phase B and dilute to 2.0 m L w ith mobile
A SSA Y phase B.
T riturate 4 g with successive small quantities o f water, dilute Reference solution (b) D ilute 1.0 m L of test solution (a) to
to 1000 m L with water and shake thoroughly. Mix 100 m L 100.0 m L with mobile phase B. D ilute 1.0 m L o f this
of the resulting suspension with a solution containing 3 g o f solution to 10.0 m L with mobile phase B.
potassium iodide in 100 m L o f water, acidify with 5 m L of Reference solution (c) Dissolve 5.0 m g of chlormadinone acetate
6m acetic add and titrate the liberated iodine with impurity G CRS in mobile phase B and dilute to 50.0 m L
0.1m sodium thiosulfate VS. Each m L o f 0.1m sodium thiosulfate with mobile phase B. D ilute 1.0 m L of the solution to
VS is equivalent to 3.545 m g o f available chlorine, Cl. 50.0 m L with mobile phase B.
STO RA G E Reference solution (d) Dissolve 10.0 m g o f chlormadinone
O n exposure to air Chlorinated Lime becomes m oist and acetate CRS in mobile phase B and dilute to 20.0 m L with
gradually decomposes, carbon dioxide being absorbed and mobile phase B.
chlorine evolved. Column:
— size: I = 0.15 m, 0 = 3.0 mm;
— stationary phase: end-capped extra-dense bonded octadecylsilyl
silica gelfor chromatography R (3.5 pm).
Mobile phase:
Chlormadinone Acetate ***** — mobile phase A: water R;
★. ★ — mobile phase B: acetonitrile Rj

0 -8 60 40
8 - 30 60* 5 40 * 95
3 0 -3 3 5 95

C23H29C104 404.9 302-22-7 Detection Spectrophotom eter at 236 nm .
Injection 10 pL o f test solution (a) and reference solutions (a),
A c tio n a n d u se (b) and (c).
Progestogen
PhEur________________________________________________________ __ with chlormadinone acetate for system suitability CRS an d the
D E F IN IT IO N identify the peaks due to impurities A, B, E and K; use the
6-Chloro-3,20-dioxopregna-4,6-dien-17-yl acetate. chrom atogram obtained with reference solution (c) to identify
C o n te n t th e peak due to im purity G.
97.5 p er cent to 102.0 per cent (dried substance). Relative retention W ith reference to chlormadinone acetate
CHA RACTERS (retention time = about 15 min): impurity K = about 0.75;
A p p e a ra n c e im purity G = about 0.80; im purity A = about 0.95;
W hite o r alm ost white, crystalline powder. im purity E = about 1.04; im purity B = about 1.2.
S o lu b ility
System suitability:
— signal-to-noise ratio: minim um 35 for the principal peak in
Practically insoluble in water, soluble in acetonitrile, slightly
the chrom atogram obtained w ith reference solution (b);
soluble in ethanol (96 p er cent).
— peak-to-vaUey ratio: m inim um 1.6, where Hp = height
ID E N T IF IC A T IO N above the baseline of th e peak due to impurity E and
Infrared absorption spectrophotom etry (2.2.24). Hv = height above the baseline o f the lowest point o f the
2016 Chlormadinone Acetate 1-521

Chlormadinone acetate in the chromatogram obtained
— correction factor multiply the peak area of impurity K by
1.7-,
— for impurities E, B and K , use the concentration of
chlormadinone acetate in reference solution (b);
— for impurities other than E, B and K, use the C. 6^-chloro-2^-methyl-3,20-dioxopregn-4-en-17-yl acetate,
concentration o f impurity G in reference solution (c).
Limits:
— impurities A, E, G, K: for each impurity, maximum
0.15 per cent;
0.10 p e r cent;
L oss o n d ry in g (2.2.32) D . 6-chloro-3,20-dioxopregna-1,4,6-trien-17-yl acetate,
an oven at 105 °C.
M axim u m 0.1 per cent, determined on 1.0 g.
ASSAY
Liquid chrom atography (2.2.29) as described in the test for
Mobile phase Mobile phase B, mobile phase A (45:55 V/V).
E. 6-bromo-3,20-dioxopregna-4,6-dien-17-yl acetate,
Run time Twice the retention time of chlormadinone acetate.
Retention time Chlormadinone acetate = about 12 min.
Calculate the percentage content of C 23H 29C 104 taking into
account the assigned content of chlormadinone acetate CRS.
IM P U R IT IE S
Specified impurities A, B, E, G , K
present at a sufficient level, be detected by one o r other of
acceptance criterion for other/unspecified impurities and/or F. 6^-methyl-3,20-dioxopregn-4-en-17-yl acetate,
Control of impurities m substances for pharmaceutical use): C, D,
F ,H ,I ,J ,L
G . 3,20-dioxopregn-4-en-17-yl acetate,
A. 6a-chloro-3,20-dioxopregn-4-en-17-yl acetate,
H . 3-methoxy-20-oxopregna-3,5-dien-17-yl acetate,
B. 2-bromo-6-chloro-3,20-dioxopregna- 1,4,6-trien-17-yl
acetate,
1-522 Chlormethine Hydrochloride 2016
A. Dissolve 50 m g in 5 m L o f water and add 1 m L o f
5m sodium hydroxide. Oily globules are produced which
dissolve on warming.
B. Dissolve 50 m g in 5 m L o f water and add 0.02 m L o f
potassium tetraiodomercurate solution. A cream precipitate is
produced.
C. Melting point, about 108°, A ppendix V A.
I. 6-chloro-3-ethoxy-20-oxopregna-3,5-dien-l 7-yl acetate,
A SSA Y
T o 0.2 g add 15 m L o f I m ethanolic potassium hydroxide and
15 m L o f water and boil under a reflux condenser for
2 hours. Evaporate the solution to h alf its volum e on a w ater
bath, dilute to 150 m L with water, add 3 m l. o f nitric add
and 50 m L o f 0.1m silver nitrate KS, shake vigorously and
filter. W ash the residue with water and titrate the excess of
silver nitrate in the combined filtrate and washings with
0.1m ammonium thiocyanate VS using 1 m L o f ammonium
J. 6-chloro-17-hydroxypregna-4,6-diene-3,20-dione, iron(m) surate solution R2 as indicator. E ach m L o f 0.1m silver
nitrate VS is equivalent to 6.418 m g o f C 5H n C l 2N ,H C l.
o. STO RA G E
Chlorm ethine Hydrochloride should be stored at a
tem perature o f 8 ° to 15°.
L A B E L L IN G
T h e label states th at the contents o f the container are
strongly vesicant.
K. 3>20-dioxopregna-4,6-dien-17-yl acetate,
★*★
★ ★
Chlorobutanol ★ ★
(Chlorobutanol Hemihydrate, Ph. Eux. monograph *****
0383)
h 3c oh
X . ’/.HjO
H3C CCta
L. 6 ß-chloro-3,20-dioxopregn-4-en-17-yl acetate.
Ph Eur GjHyCÔjViÔ 186.5 6001-64-5
Disinfectant preservative.
PhEur.
Chlormethine Hydrochloride
D E F IN IT IO N
1, 1,1 -Trichloro- 2-m ethylpropan- 2-ol hem ihydrate.
N' ,HCI
C o n te n t
Me 98.0 per cent to 101.0 per cent (anhydrous substance).
CHARACTERS
C sH nC ljN îC l 192.5 55-86-7
A p p e a ra n c e
A c tio n a n d u se W hite or almost white, crystalline pow der or colourless
Cytotoxic alkylating agent. crystals, sublimes readily.
P r e p a r a tio n S o lu b ility
Chlorm ethine Injection Slightly soluble in water, very soluble in ethanol
(96 per cent), soluble in glycerol (85 p er cent).
D E F IN IT IO N mp
Chlorm ethine Hydrochloride is A bout 78 °C (without previous drying).
bis(2-chloroethyl)methylamine hydrochloride. It contains n o t
less than 98.0% and n o t m ore than 101.0% o f
A. Add about 20 m g to a mixture o f 1 m L o f pyridine R and
C s H n C y s m C l.
2 m L o f strong sodium hydroxide solution R. H eat in a water-
C H A R A C T E R IS T IC S bath and shake. Allow to stand. T h e pyridine layer becomes
A white or almost white crystalline powder or mass; red.
hygroscopic; vesicant. B. A dd about 20 m g to 5 m L o f ammoniacal silver nitrate
Very soluble in water. solution R and warm slightly. A black precipitate is formed.
2016 Chlorobutanol 1-523
C . T o about 20 mg add 3 m L of 1 M sodium hydroxide and CHARACTERS

shake to dissolve. Add 5 m L of water R and then, slowly, A p p e a ra n c e
2 mT. o f iodinatedpotassium iodide solution R. A yellowish W hite or almost white, crystalline powder or colourless
precipitate is formed. crystals, sublimes readily.
D . W ater (see Tests). S o lu b ility
TESTS Slightly soluble in water, very soluble in ethanol
(96 per cent), soluble in glycerol (85 per cent).
S o lu tio n S
Dissolve 5 g in ethanol (96 per cent) R and dilute to 10 m L mp
with the same solvent. A bout 95 °C (without previous drying).
A p p e a ra n c e o f so lu tio n ID E N T IF IC A T IO N
Solution S is not more opalescent than reference A. Add about 20 mg to a mixture of 1 m L of pyridine R and
suspension II (2.2.1) and n o t more intensely coloured than 2 m L o f strong sodium hydroxide solution R. H eat in a water-
reference solution BY3 (2.2.2, Method II). bath and shake. Allow to stand. T he pyridine layer becomes
A cid ity red.
T o 4 m L of solution S add 15 m L o f ethanol (96 per cent) R B. Add about 20 mg to 5 mL of ammoniacal silver nitrate
and 0.1 mT. of bromothymol blue solution R l. N o t more than solution R and warm slightly. A black precipitate is formed.
1.0 mT. o f 0.01 M sodium hydroxide is required to change the C. T o about 20 m g add 3 mL o f 1 M sodium hydroxide and
colour o f the indicator to blue. shake to dissolve. Add 5 m L of water R and then, slowly,
C h lo rid e s (2.4.4) 2 m L of iodinated potassium iodide solution R. A yellowish
M axim um 100 ppm. precipitate is formed.
T o 1 mT. o f solution S add 4 m L o f ethanol (96 per cent) R D . W ater (see Tests).
and dilute to 15 m L with water R. W hen preparing the TESTS
standard, replace the 5 mT. o f water R by 5 m L o f ethanol
S o lu tio n S
(96 per cent) R. Dissolve 5 g in ethanol (96 per cent) R and dilute to 10 m L
W a te r (2.5.12) with the same solvent.
S ulfa te d a sh (2.4.14) Solution S is n o t more opalescent than reference
M axim um 0.1 per cent, determined on 1.0 g. suspension II (2.2.1) and not more intensely coloured than
ASSA Y reference solution BY5 (2.2.2, Method II).
Dissolve 0.100 g in 20 m L of ethanol (96 per cent) R. A cid ity
Add 10 mT. o f dilute sodium hydroxide solution R , heat in a T o 4 m L of solution S add 15 m L of ethanol (96 per cent) R
w ater-bath for 5 min and cool. A dd 20 m L o f dilute nitric and 0.1 m L o f bromothymol blue solution R l. N ot more than
acid R, 25.0 m L of 0.1 M silver nitrate and 2 m L of dibutyl 1.0 m L o f 0.01 M sodium hydroxide is required to change the
phthalate R and shake vigorously. A dd 2 m L of ferric colour o f the indicator to blue.
ammonium sulfate solution R2 and titrate with 0.1 M C h lo rid e s (2.4.4)
ammonium thiocyanate until an orange colour is obtained. Maximum 300 ppm .
1 m L o f 0.1 M silver nitrate is equivalent to 5.92 mg Dissolve 0.17 g in 5 m L of ethanol (96 per cent) R and dilute
Of C 4H 7 CI3 O. to 15 m L with water R. When preparing the standard,
ST O R A G E replace the 5 mT. o f water R by 5 m L o f ethanol
In an airtight container. (96 per cent) R.
__________________________________________________________ PhEur W a te r (2.5.12)
★ ★ ASSAY
Anhydrous Chlorobutanol ★ ★
Dissolve 0.100 g in 20 m L of ethanol (96 per cent) R.
(Ph. Eut. monograph 0382) ***** Add 10 m L o f dilute sodium hydroxide solution R, heat in a
water-bath for 5 min and cool. Add 20 m L of dilute nitric
h3c oh acid R , 25.0 m L of 0.1 M silver nitrate and 2 m L o f dibutyl
V phthalate R and shake vigorously. Add 2 m L of ferric
H3C CC(3
ammonium sulfate solution R2 and titrate with 0.1 M
ammonium thiocyanate until an orange colour is o b tained
C 4H 7C 130 177.5 57-15-8
1 m L of 0.1 M silver nitrate is equivalent to 5.92 m g
A c tio n a n d u se ofG iH vC laO .
Disinfectant preservative. STORAGE
PhEur.
PhEur
D E F IN IT IO N
1, 1,1 -Trichloro- 2-methylpropan- 2-ol.
C o n te n t
98.0 p e r cent to 101.0 per cent (anhydrous substance).
1-524 Chlorocresol 2016
★ ★ — stationary phase: süanised diatomaceous earth for gas

Chlorocresol ★ ★ chromatography R im pregnated with 3-5 p er cent m/m of
(Ph. Eur. monograph 0384) ***** pdymethylphenylsüoxane R.
OH
Temperature:
ch3
— injection port. 210 °C;
C 7H 7C10 142.6 59-50-7
Action and use Run time 3 times the retention tim e o f chlorocresol.
Antiseptic; antimicrobial preservative.
Retention time Chlorocresol = about 8 min.
PhEur__________________________________ Limits:
DEFINITION
0.10 per cent;
4-Chloro-3-methylphenol. — total: maximum 1 per cent;
Content — disregard limit, the area o f the principal peak in the
98.0 per cent to 101.0 per c e n t chromatogram obtained with the reference solution
CHARACTERS (0.05 per cent).
Appearance Non-volatile matter
W hite or almost white, crystalline powder or compacted M áxim um 0.1 per c e n t
crystalline masses supplied as pellets or colourless or white Evaporate 2.0 g to dryness on a w ater-bath and dry the
aystals. residue at 100-105 °C. T he residue weighs n o t m ore than
Solubility 2 mg.
Slightly soluble in water, very soluble in ethanol ASSAY
(96 per cent), freely soluble in fatty oils. It dissolves in In a ground-glass-stoppered flask, dissolve 70.0 m g in 30 m L
solutions of alkali hydroxides. of glacial acetic acid R. Add 25.0 m L o f 0.0167 M potassium
IDENTIFICATION brómate, 20 m L o f a 150 g/L solution of potassium bromide R
A. M elting point (2.2.14): 64 °C to 67 °C. and 10 m L o f hydrochloric add R. Allow to stand protected
from light for 15 m in . Add 1 g o f potassium iodide R and
B. T o 0.1 g add 0.2 m L of benzoyl chloride R and 0.5 m L o f
100 mT. o f water R. Titrate with 0.1 M sodium thiosulfate,
dilute sodium hydroxide solution R. Shake vigorously until a
shakin g vigorously and using 1 m L o f starch solution R, added
white, crystalline precipitate is formed. Add 5 m L o f water R
towards the end o f the titration, as indicator. Carry out a
and filter. T h e precipitate, reciystallised from 5 m L o f
methanol R and dried at 70 °C, melts (2.2.14) at 85 °C to blank titration.
88 °C. 1 m L of 0.0167 M potassium brómate is equivalent to
C. T o 5 m L o f solution S (see Tests) add 0.1 m L o f ferric 3.565 m g o f C 7H 7C10.
chloride solution R l. A bluish colour is produced. STORAGE
TESTS Protected from light.
Solution S PhEur
T o 3.0 g, finely powdered, add 60 m L o f carbon dioxide-free
water R, shake for 2 min and filter.
Appearance o f solution *** ★
★
T he solution is clear (2.2.1) and n o t more intensely coloured Chloroquine Phosphate ★ ★
than reference solution BY6 (2.2.2, Method II). * * * * *
(Ph Eur. monograph 0544)
25 m L with the same solvent. cu ^ „N ,
and enantiomer
Acidity
T o 10 m L o f solution S add 0.1 m l- of methyl red solution R. 2 H3 PO 4
T h e solution is orange or red. N o t more than 0.2 mT. of N CH3
0.01 M sodium hydroxide is required to produce a pure yellow H CH3 L
colour. ch3
Related substances C J8H 32Q N 3O 8P2 515.9 50-63-5

Gas chromatography (2.2.28): use the n o rm alisation
procedure. Action and use
Test solution Dissolve 1.0 g of the substance to be pvamineH Antiprotozoal (malaria).
in acetone R and dilute to 100 mT. with the same solvent. Preparation
Reference solution Dilute 1.0 m L o f the test solution to Chloroquine Phosphate Tablets
100.0 m L with acetone R. Dilute 5.0 mT. of this solution to
100.0 m L with acetone R. PhEur.
Column: DEFINITION
— material: glass; Chloroquine phosphate contains n o t less than 98.5 per cent
— size. I = 1.80 m , 0 = 3-4 mm ; and not more than the equivalent o f 101.0 p er cent o f
2016 Chloroquine Sulfate 1-525
iV4-(7-chloroquinolin-4-yl)-N/>N’i-diethylpentane-1,4-diamine obtained w ith the test solution, apart from the principal spot,
bis(dihydrogen phosphate), calculated with reference to the is no t more intense than the spot in the chromatogram
dried substance. obtained with reference solution (a) ( 1.0 p er cent) and not
m ore than one such spot is more intense than the spot in the
CHARACTERS
A white or almost white, crystalline powder, hygroscopic,
(0.5 per cent).
freely soluble in water, very slightly soluble in alcohol and in
methanol. H eav y m e ta ls (2.48)
Dissolve 2.0 gin 10 m L o f water R. Add 5 m L of concentrated
I t exists in 2 forms, one o f which melts at about 195 °C and
ammonia R and shake with 40 m L of methylene chloride R.
the other at about 218 °C.
Filter the aqueous layer and neutralise the filtrate with glacial
ID E N T IF IC A T IO N acetic acid R. H eat on a water-bath to elim in ate methylene
First identification B, D. chloride, allow to cool and dilute to 20.0 m L with water R.
Second identification A , C, D. 12 m L o f this solution complies with test A for heavy metals
A. Dissolve 0.100 g in water R and dilute to 100.0 m L with (20 ppm). Prepare the reference solution using lead standard
the same solvent. Dilute 1.0 m l. o f this solution to 100.0 m L solution (2 ppm Pb) R.
with water R. Examined between 210 nm and 370 n m L o ss on d ry in g (2.2.32)
(2.2.25), the solution shows absorption maxima at 220 nm , Maximum 2.0 per cent, determined on 1.000 g by drying in
235 nm , 256 nm , 329 n m and 342 nm . T he specific an oven at 105 °C.
absorbances at the maxima are respectively 600 to 660, ASSAY
350 to 390, 300 to 330, 325 to 355 and 360 to 390.
Dissolve 0.200 g in 50 m L o f anhydrous acetic acid R. T itrate
B. Examine by infrared absorption spectrophotometry with 0.1 M perchloric add determining the end-point
(2.2.24), comparing with the spectrum obtained w ith the potentiometrically (2.2.20).
base isolated from chloroquine sulfate CRS. Record the spectra
1 m L o f 0.1 M perchloric acid is equivalent to 25.79 mg o f
using solutions prepared as follows: dissolve separately 0.1 g
C \ 8H 32CIN 3O 3P 2-
of the substance to be examined and 80 mg of the reference
substance in 10 m L o f water R, add 2 m L o f dilute sodium STORA GE
hydroxide solution R and shake with 2 quantities, each of In an airtight container, protected from light.
20 mT, o f methylene chloride R; combine the organic layers, __________________________________________________________ PhEur
wash with water R, dry over anhydrous sodium sulfate R,
evaporate to dryness and dissolve the residues separately,
each in 2 m L of methylene chloride R.
C. Dissolve 25 m g in 20 m L o f water R and add 8 m L o f ★ ★
picric acid solution R l. T h e precipitate, washed with water R, Chloroquine Sulfate ★ ★
with alcohol R and finally w ith methylene chloride R, melts * * * * *
Chloroquine Sulphate
(2.2.14) at 206-209 °C.
D . Dissolve 0.1 g in 10 m L o f water R, add 2 m L o f dilute
sodium hydroxide solution R and shake with 2 quantities, each
of 20 m l , of methylene chloride R. T h e aqueous layer,
acidified by the addition o f nitric acid R, gives reaction (b) of
phosphates (2.3.1). , H2SO4 , h2o
TESTS
S o lu tio n S
C w H aO N aO A H aO 436.0 132-73-0
Solution S is clear (2.2.1) and not more intensely coloured A ctio n a n d u se
than reference solution BY 5 or GY 5 (2.2.2, Method IT). Antiprotozoal (malaria).
p H (2.2.3) P re p a r a tio n s
T he p H o f solution S is 3.8 to 4.3. Chloroquine Sulfate Injection
R elated su b s ta n c e s Chloroquine Sulfate Tablets
gel GF254 R as the coating substance. PhEir.
Test solution Dissolve 0.50 g o f the substance to be examined D E F IN IT IO N
in water R and dilute to 10 m L with the same solvent. Chloroquine sulfate contains n o t less than 98.5 per cent and
Reference solution (a) D ilute 1 m L o f die test solution to n o t more than the equivalent of 101.0 p er cent of
100 m L with water R. iST,-(7-<diloroquinolin-4-yl)-Ari>ZV/-diethylpentane-l,4-diamine
Reference solution (b) D ilute 5 m L o f reference solution (a) to sulfate, calculated with reference to the anhydrous substance.
10 m L with water R. CHARACTERS
Apply to the plate 2 pL o f each solution. Develop over a A white or alm ost white, crystalline powder, freely soluble in
path of 12 cm using a m ixture of 10 volumes of water and in methanol, very slightly soluble in ethanol
diethylamine R, 40 volumes o f cydohexane R and 50 volumes (96 per cent).
of chloroform R. Allow the plate to dry in air. Examine in It melts at about 208 °C (instantaneous method).
ultraviolet light at 254 nm . Any spot in the chromatogram
1-526 Chloroxylenol 2016
ID E N T IF IC A T IO N W a te r (2.5.12)
First identification B, D. 3.0 p er cent to 5.0 per cent, determined on 0.500 g.
Second identification A, C, D. S u lfa te d a s h (2.414)
A. Dissolve 0.100 g in water R and dilute to 100.0 m L with N o t more than 0.1 per cent, determined on 1.0 g.
the same solvent. Dilute 1.0 m L of this solution to 100.0 m L A SSA Y
with water R. Exam ined between 210 nm and 370 nm Dissolve 0.400 g in 50 m L o f anhydrous acetic add R T itrate
(2.2.25), the solution shows absorption maxima at 220 nm , with 0.1 M perchloric acid determining the end-point
235 nm , 256 nm , 329 nm and 342 nm . T he specific potentiometrically (2.2.20).
absorbances a t the maxima are respectively 730 to 810,
1 m L of 0.1 M perchloric add is equivalent to 41.8 mg o f
430 to 470, 370 to 410, 400 to 440 and 430 to 470.
C 18H 28CIN 3O 4S.
B. Examine b y infrared absorption spectrophotometry
(2.2.24), comparing with the spectrum obtained with the STO R A G E
base isolated from chloroqume sulfate CRS. Record the spectra Store in an airtight container, protected from light.
using solutions prepared as follows: dissolve separately 0.1 g _________________________________________________________ _ PhEur
of the substance to be examined and of the reference
substance in 10 m L of water R, add 2 m L of dilute sodium
hydroxide solution R and shake with 2 quantities, each of
20 m L, of methylene chloride R", combine the organic layers,
wash with water R, dry over anhydrous sodium sulfate R, Chloroxylenol
evaporate to dryness and dissolve the residues separately each
in 2 m L of methylene chloride R. OH
C. Dissolve 25 m g in 20 m L of water R and add 8 m L of
picric add solution R l. T he precipitate, washed with water R,
with ethanol (96 per cent) R and finally with ether R, melts
(2.2.14) at 206 °C to 209 °C.
D. It gives reaction (a) of sulfates (2.3.1). Cl
TESTS
S o lu tio n S CgHoClO 156.6 88-04-0
A ctio n a n d u se
Antiseptic.
P re p a r a tio n
Solution S is clear (2.2.1) and no t more intensely coloured
Chloroxylenol Solution
than reference solution BY5 or GY 5 (2.2.2, Method II).
p H (2.2.3) D E F IN IT IO N
T he p H of solution S is 4.0 to 5.0. Chloroxylenol is 4-chloro-3,5-xylenoL It contains not less
R e la te d su b s ta n c e s than 98.0% and no t more than 103.0% of CsHgClO.
Examine by thin-layer chromatography (2.2.27), using silica C H A R A C T E R IS T IC S
gel GF254 R as the coating substance. W hite or cream crystals or crystalline powder. It is volatile in
Test solution Dissolve 0.50 g o f the substance to be examined steam.
in water R and dilute to 10 m L with the same solvent. Very slightly soluble in water, freely soluble in ethanol (96%)\
Reference solution (a) Dilute 1 m l. o f the test solution to soluble in ether, in terpenes and in fixed oils. It dissolves in
100 m L with water R. solutions o f the alkali hydroxides.
Reference solution (b) Dilute 5 m L of reference solution (a) to ID E N T IF IC A T IO N
10 m L with water R. T h e infrared absorption spectrum, Appendix II A, is concordant
Apply separately to the plate 2 nL o f each solution. Develop with the reference spectrum of chloroxylenol (RS 055).
over a path of 12 cm using a mixture of 10 volumes of
TESTS
diethylamine R, 40 volumes of cydohexane R and 50 volumes
of methylene chloride R. Allow the plate to dry in air. E xam in e M e ltin g p o in t
114° to 116°, Appendix V A.
in ultraviolet light at 254 nm. Any spot in the chromatogram
obtained with the test solution, apart from the principal spot, T e tra c h lo ro e th y ie n e
is n o t more intense than the spot in the chromatogram Carry out the m ethod for gas chromatography,
obtained with reference solution (a) ( 1.0 per cent) and not Appendix HI B, using the following solutions. Prepare a
more than one such spot is more intense than the spot in the 0 .2 % v/v solution of butanol (internal standard) in methanol
chrom atogram obtained with reference solution (b) (solution A).
(0.5 per cent). ( 1) T o 4 g of the substance being exam ined , add 5 m l . o f
H eav y m e ta ls (2.48) solution A and dilute to 25 m L with methanol.
Dissolve 2.0 g in 10 m L of water R. Add 5 m l, of (2) T o 5 m L of a 0.2% v/v solution of tetrachloroethylene in
concentrated ammonia R and shake with 40 m L of ether R. methanol, add 5 m L o f solution A and dilute to 25 m L with
Filter the aqueous layer and neutralise the filtrate with glacial methanol (equivalent to 0.06488% w/v of tetrachloroethylene in
acetic add R. H eat on a water-bath to elim inate ether, allow methanol).
to cool and dilute to 20.0 m l. with water R. 12 m L o f this
solution complies with test A (20 ppm ). Prepare the
2016 Chlorphenamine Maleate 1-527
CHROMATOGRAPHIC CONDITIONS the flask and allow to stand protected from light for
(a) U se a fused silica capillary column (30 m x 0.53 mm) 15 minutes. A dd 1 g of potassium iodide and 100 m L of water
bonded with a 1 |im film of polyethylene glycol 20,000 (RH- and titrate w ith 0.1m sodium thiosulfate PIS, shaking vigorously
W ax is suitable). and using 1 m L o f starch solution, added towards the end of
(b) U se hydrogen as the carrier gas at 2 m L per m inute. the titration, as indicator. Repeat the operation without the
substance being examined. T he difference between the
(c) Use the gradient conditions described below.
titrations represents the amount o f potassium bromate
(d) Use an inlet temperature o f 240°. required. Each m L o f 0.0167m potassium bromate KS is
(e) Use a flame ionisation detector at a tem perature of 280°. equivalent to 3.915 m g of CgHgClO.
(f) Inject 0.5 |iL o f each solution.
(g) Use a split ratio o f 1:20.
Tim e Temperature Com m ent

Chlorphenamine Maleate *****
★, *
(Minutes) (Ph. Eur. monograph 0386) *
oo
"j
0 -4 isothermal
CO2H
4 -5 70°-»210° linear increase and enantiomer , |
isothermal
n ^CH3 CO2H
5 -1 5 210
i
CH3
15-18 210°->70° linear gradient
oo
re-equilibration
-j
18-20
CaoH^ClNaOi 390.9 113-92-8
A ctio n a n d u se
SYSTEM SUITABILITY Histamine H I receptor antagonist; antihistamine
T he test is n o t valid unless, in the chromatogram obtained
P re p a ra tio n s
with solution ( 2 ), the resolution between the peaks due to
Chlorphenamine Injection
tetrachloroethylene and the internal standard is at least 1.5.
Chlorphenamine Oral Solution
LIMITS
Chlorphenamine Tablets
In the chrom atogram obtained with solution (1), the ratio of
any peak due to tetrachloroethylene to th a t of the internal PhEtr___________________________________________________
standard is n o t greater than the corresponding ratio obtained D E F IN IT IO N
in the chrom atogram obtained with solution (2) (0.4%).
(3/?5)-3-(4-Chlorophenyl)-Ny^-dimethyl-3-(pyridin-2-
R e la te d su b s ta n c e s yI)propan-1-am ine hydrogen (Z)-butenedioate.
Carry out the m ethod for gas chromatography,
C o n te n t
Appendix HI B, using the following solutions in chloroform.
( 1) 2 .0 % w/v o f th e substance being examined.
CHARACTERS
(2) 2 .0 % w/v o f the substance being examined and
A p p e a ra n c e
0.040% w/v o f 4-chloro-o-crescl (internal standard).
S olu b ility
(a) Use a glass c o lu m n (1.5 m x 4 mm ) packed with acid- Freely soluble in water, soluble in ethanol (96 per cent).
washed diatomaceous support (80 to 100 mesh) coated with
3% w/w o f polyethylene glycol (Carbowax 20M is suitable). ID E N T IF IC A T IO N
(b) Use nitrogen as the carrier gas at 40 m L per minute.
(c) Use isotherm al conditions maintained at 160°.
Comparison chlorphenamine maleate CRS.
(d) Use an inlet tem perature of 200°.
C. Optical rotation (see Tests).
(e) Use a flame ionisation detector at a tem perature of 300°.
(f) Inject 1 |iL o f each solution. TESTS
S o lu tio n S
SYSTEM SUITABILITY
T he test is no t valid unless, in the chromatogram obtained same solvent.
w ith solution (2 ), the resolution between th e peaks due to
chloroxylenol an d 4-chloro-o-cresol is at least 1.5
LIMITS than reference solution BY 6 (2.2.2, Method II).
In the chrom atogram obtained with solution (2) the sum o f O p tic a l r o ta tio n (2.2.7)
the areas o f any secondary peaks is not greater than the area of - 0 . 10° to + 0 . 10°, determined on solution S.
the peak due to th e internal standard.
A SSAY Liquid chromatography (2.2.29).
Dissolve 70 m g in 30 m L o f glacial acetic acid, add 25 m L of Test solution Dissolve 0.100 g o f the substance to be
0.0167m potassium bromate VS, 20 m L o f a 15% w/v solution examined in the mobile phase and dilute to 100.0 m L with
o f potassium bromide and 10 m L of hydrochloric add, stopper the mobile phase.
1-528 Chlorphenamine Maleate 2016
Reference solution (a) Dilute 0.5 m L o f the test solution to S u lfa te d a s h (2.4.14)
100.0 m L with die mobile phase. M aximum 0.1 per cent, determined on 1.0 g.
Reference solution (b) Dilute 1.0 m L o f reference solution (a) ASSAY
to 10.0 m L with the mobile phase. Dissolve 0.150 g in 25 m L o f anhydrous acetic arid R. T itrate
Reference solution (c) Dissolve 5 mg o f chlorphenamine with 0.1 M perchloric acid, determining the end-point
impurity C CRS in 5 m L of the test solution and dilute to potentiometrically (2.2.20).
50.0 m L with the mobile phase. Dilute 2 m L of this solution 1 m L of 0.1 M perchloric acid is equivalent to 19.54 m g
to 20 m L with the mobile phase. o f C 20H 23C IN 2O 4.
Reference solution (d) Dissolve 5 mg o f 2,2'-dipyridylantine R
STO RA G E
(impurity 8 ) in the mobile phase and dilute to 100 m L with
the mobile phase.
Reference solution (e) Dissolve the contents of a vial of IM P U R IT IE S
chlorphenamine impurity A CRS in 2 m L of the test solution. Specified impurities: A, B, C, D .
Sonicate for 5 min.
Column:
— size. I = 0.30 m , 0 = 3.9 mm;
— stationary phase. octadecylsUyl silica gel for chromatography R
(10 pm).
^CH3
Mobile phase M ix 20 volumes o f aeetonitrile R and 80 volumes
of a 8.57 g/L solution o f ammonium dihydrogen phosphate R
previously adjusted to p H 3.0 with phosphoric acid R.
Flow rate 1.2 mL/min. A. 2-(4-dilorophenyi)-4-(dimethyiamino)-2-[2-
Detection Spectrophotom eter at 225 nm . (dimediyiamino)ethyl]butanenitrile,
Injection 20 pL.
Run time 3.5 times die retention time o f chlorphenamine.
Relative retention W ith reference to chlorphenamine (retention
time = about 11 min): maleic a d d = about 0 .2 ;
im purity A = about 0.3; impurity B = about 0.4;
(Vo
im purity C = about 0.9; im purity D = about 3.0. B. N-(pyridin- 2-yl)pyridin-2-amine (2,2/-dipyridylamine),
— resolution: minim nm 1.5 between the peaks due to
impurity C and chlorphenamine.
''= ( H and enantiomer
Limits: „CH3
— correction factors: for the calculation o f contents, multiply
corresponding correction factor impurity A = 1.5;
impurity B = 1.4;
C. (3i?S)-3-(4-chJorophenyi)-AT-methyi-3-(pyridin-2-
— impurity A: not m ore than 0.4 times the area of the
yl)propan - 1-amine,
principal peak in die chromatogram obtained with
— impurities B, C, D: for each impurity, not more than
0.2 times the area o f the principal peak in the and enantiomer
(0.1 per cent);
D . (2i?S)-2-(4-chlorophenyl)-4-(dimethylamino)-2-(pyridin-
( 0.10 per cent);
2-yl)butanenitrile.
— total: not m ore than the area of the principal peak in the
chrom atogram obtained with reference solution (a) PhEur
(0.5 per cent);
— disregard limit, the area o f the principal peak in the
(0.05 per cent); disregard the peaks due to the blank and
m aldc ad d .
M aximum 20 ppm .
M áximum 0.5 per cent, determ ined on 1.000 g by drying in
2016 Chlorpromazine Hydrochloride 1-529
*** ★
★
Chlorpromazine Chlorpromazine Hydrochloride ★ ★
(Fh Eur. monograph 0475) *****
ch3
N
I HCI
CH3
C 17H 19Q N 2S 318.9 50-53-3
A c tio n a n d u se 69-09-0
A ctio n a n d u se
P re p a r a tio n
D opamine receptor antagonist; neuroleptic.
Chlorpromazine Suppositories
P re p a ra tio n s
D E F IN IT IO N Chlorpromazine Injection
Chlorpromazine is [3-(2-chlorophenothiazin-10-yl)propyl]- Chlorpromazine Oral Solution
dimethylamine. It contains not less than 99.0% and n o t m ore Chlorpromazine Tablets
than 101.0 % o f C 17HX9CIN 2S, calculated with reference to
the dried substance. PhEur_______________________________________
C H A R A C T E R IS T IC S D E F IN IT IO N
A white or creamy white powder or waxy solid. 3-(2-Chloro-l OH-phenothiazin-10-yO -N ^-dim ethylprop an-
Practically insoluble in water, freely soluble in ethanol (96%) 1-amine hydrochloride.
and in ether. C o n te n t
ID E N T IF IC A T IO N 99.0 per cent to 101.0 p er cent (dried substance).
A. T he infrared absorption spectrum, Appendix II A, is CHARACTERS
concordant with the reference spectrum of chlorpromazine A p p e a ra n c e
(RS 056). White or almost white, crystalline powder.
B. Complies with the test for identification ofphenothiazines, S olu b ility
Appendix lH A, using chlorpromazine hydrochloride BPCRS to Very soluble in water, freely soluble in ethanol (96 per cent).
prepare reference solution. It decomposes on exposure to air and light.
TESTS It shows polymorphism (5.9).
M eltin g p o in t
Second identification A, C, D.
Complies with the test for related substances in phenothiazines,
Appendix IE A, using mobile phase A. A. Ultraviolet and visible absorption spectrophotometry
(2.2.25). Prepare the solutions protected from bright light and
L oss o n d ry in g measure the absorbances immediately.
W hen dried to constant weight over phosphorus pentoxide at a
Test solution Dissolve 50.0 mg in a 10.3 g/L solution of
pressure n o t exceeding 0.7 kPa, loses n o t more than 0.5% o f
hydrochloric add R and dilute to 500.0 m L with the same
its weight. Use 1 g.
solution. Dilute 5.0 m L of the solution to 100.0 m L with a
S u lfa te d a sh 10.3 g/L solution of hydrochloric acid R.
N ot more than 0.1%, Appendix IX A.
A SSA Y Absorption maxima At 254 nm and 306 nm.
Dissolve 0.8 g in 300 m L of acetone and carry out M ethod I Specific absorbance at the absorption maximum at 254 nm
for rum-aqueous titration, Appendix VIII A, using 3 m L o f a
890 to 960.
saturated solution of methyl orange in acetone as indicator.
Each m L of 0.1m perchloric acid K5 is equivalent to 31.89 m g
of C 17H 19CIN 2S. Preparation 60 g/L solutions in methylene chloride R using a
0.1 m m cell.
STORAGE
Comparison chlorpromazine hydrochloride CRS.
Chlorpromazine should be protected from light.
C. Identification o f phenothiazines by thin-layer
chromatography (2.3.3): use chlorpromazine hydrochloride CRS
to prepare the reference solution.
D . It gives reaction (b) o f chlorides (2.3.1).
TESTS
p H (2.2.3)
3.5 to 4.5. Carry out the test protected from light and use freshly
prepared solutions.
1-530 Chlorpromazine Hydrochloride 2016
Impurity F 0.5 per cent VIV solution o f trifiuoroacetic add R previously

Thin-layer chrom atography (2.2.27). Prepare the solutions adjusted to p H 5.3 with tetrametkylethylenediamirte R.
immediately before use and protea from light. Flow rate 1.0 mL/min.
Solvent mixture diethylamine R, methanol R (5:95 ViV). Detection Spectrophotom eter at 254 nm.
Test solution Dissolve 0.100 g o f the substance to be Injection 10 |iL.
examined in the solvent mixture and dilute to 5.0 m L with Run time 4 times the retention time of chlorpromazine.
Reference solution (a) Dissolve the contents of a vial o f with reference solution (c) to identify the peak due to
chlorpromazine impurity F CRS in 2.0 m L of the solvent impurity A; use the chromatogram obtained with reference
mixture.
solution (d) to identify the peaks due to impurities C and E;
Reference solution (b) D ilute 300 |iL of reference solution (a) use the chromatogram obtained with reference solution (a) to
to 10.0 m L w ith the solvent mixture. identify the peak due to impurity D .
Reference solution (c) Dissolve 0.10 g o f the substance to be Relative retention W ith reference to chlorpromazine (retention
examined in the solvent mixture, add 1.0 m L o f reference time = about 8 min): impurity A = about 0.4;
solution (a) and dilute to 5.0 m L with the solvent mixture. impurity B = about 0.5; impurity C = about 0.7;
Plate TLC silica gel F2 5 4 plate R impurity D = about 0.9; impurity E = about 3.4.
Mobile phase acetone R, diethylamine R, cydohexane R System suitability: reference solution (a):
(10:10:80 VIV/V). — resolution: minim um 2.0 between the peaks due to
Application 10 pL of the test solution and reference impurity D and chlorpromazine.
solutions (b) and (c). Limits:
Development Over 3/4 o f the plate. — impurities B, C, D: for each impurity, not m ore than
Drying In air.
Detection Examine in ultraviolet light at 254 nm. (0.3 per cent);
Retardation factors Im purity F = about 0.5; — impurity A: not more than 1.5 times the area o f the
chlorpromazine = abo u t 0 . 6 . corresponding peak in the chromatogram obtained with
System suitability: reference solution (c): reference solution (c) (0.15 per cent);
— the chromatogram shows 2 clearly separated spots due to — impurity E: not m ore than 1.5 times the area o f the
impurity F and chlorpromazine. corresponding peak in the chromatogram obtained with
Limit: reference solution (d) (0.15 per cent);
— impurity F: any spot due to impurity F is not more intense — unspecified impurities: for each impurity, not m ore than
than the spot in the chromatogram obtained with 0.2 times the area of the principal peak in the
reference solution (b) (0.15 per cent). chromatogram obtained with reference solution (b)
( 0.10 per cent);
Related substances
immediately before use and protect from light.
Test solution Dissolve 40.0 m g of the substance to be (0.05 per cent).
the mobile phase.
M aximum 10 ppm.
Reference solution (a) Dissolve 4 mg o f chlorpromazine
impurity D CRS in the mobile phase and dilute to 10.0 m L Solvent water R.
with the mobile phase. T o 1 m L of the solution add 1 m L of 0.25 g complies with test H . Prepare the reference solution
the test solution and dilute to 100.0 m L with the mobile using 0.25 m L of lead standard solution (10 ppm Pb) R.
phase. L o ss o n d ry in g (2.2.32)
Reference solution (b) D ilute 1.0 m L o f the test solution to Maximum 0.5 p er cent, determ ined on 1.000 g by drying in
20.0 m L with the mobile phase. D ilute 1.0 m L of this an oven at 105 °C.
solution to 10.0 m L with the mobile phase. S u lfa te d a s h (2.4.14)
Reference solution (c) Dissolve 4.0 m g o f chlorpromazine Maximum 0.1 per cent, determined on 1.0 g.
impurity A CRS in th e mobile phase and dilute to 100.0 m L ' A SSA Y
with the mobile phase. Dilute 1.0 m l, o f the solution to
Dissolve 0.250 g in a mixture of 5.0 m L of 0.1 M hydrochloric
add and 50 m L o f ethanol (96 per cent) R. C any out a
Reference solution (d) Dissolve 4 mg o f promazine potentiom etric titration (2.2.20), using 0.1 M sodium
hydrochloride CRS (impurity C) and 4.0 mg of chlorpromazine hydroxide. Read the volume added between the 2 points of
impurity E CRS in the mobile phase and dilute to 100.0 m L inflexion.
w ith the mobile phase. Dilute 1.0 m l. of the solution to
C 17H 2oC12N 2S.
Column:
— size. I — 0.25 m, 0 = 4.0 mm; STORA GE
— stationary phase, base-deactivated octylsUyl silica gel for In an airtight container, protected from light.
chromatography R (5 jim). IM P U R IT IE S
Mobile phase M ix 0.2 volumes of thiodiethylene glycol R with Specified impurities A, B, C , D , E, F
50 volumes o f acetonitrile R and 50 volumes of a
2016 Chlorpropamide 1-531
* *★
Chlorpropamide ★ ★
★ ★
° ° ?
i^ V s ' n - ^ n ' ^ - ch’
Cl X J H H
A. 3-(2-chloro-1Oif-phenothiazin-10-yl)-N,N-
dim ethylpropan- 1-amine 5-oxide (chlorpromazine sulfoxide).
C10H13CIN2O3S 276.7 94-20-2
A ctio n a n d u se
Inhibition o f A TP-dependent potassium channels
(sulfonylurea); treatm ent of diabetes mellitus.
P re p a r a tio n
Chlorpropamide Tablets
B. N - [3-(2-chloro-lOH-phenothiazin- 10-yl)propyl]-AyV'^N 7 PhEur.
trim ethylpropane-1,3-diamine,
D E F IN IT IO N
Chlorpropamide contains not less than 99.0 per cent and not
m ore than the equivalent of 101.0 per cent of
^ch 3 1 - [(4-chlorophenyl) sulfonyl] -3-propylurea, calculated with
CHARACTERS
A white o r almost white, crystalline powder, practically
insoluble in water, freely soluble in acetone and in methylene
C . 3-( 1Oif-phenothiazin-10-yl)-N>N'-dimethylpropan-1-amine chloride, soluble in alcohol. It dissolves in dilute solutions of
(promazine), alkali hydroxides.
First identification C, D
Second identification A , B, D
B. Dissolve 0.10 g in methanolR and dilute to 50.0 m l. with
the same solvent. Dilute 5.0 mL o f the solution to 100.0 m L
D . 3-(2-chloro-1Oif-phenothiazin-10-yl)-N’- m ethylpropan-1- with 0.01 M hydrochloric acid. Dilute 10.0 m l. of the solution
amine (desmethylchlorpromazme). to 100.0 m L w ith 0.01 M hydrochloric acid. Examined
between 220 n m and 350 nm (2.2.25), the solution shows an
absorption maximum at 232 nm. T h e specific absorption at
the maximum is 570 to 630.
C. Examine by infrared absorption spectrophotometry
chlorpropamide CRS. Examine the substances prepared as
discs. If the spectra obtained show differences, dissolve the
substance to be examined and the reference substance in
E. 2-chloro - 1OH-phenothiazine,
methylene chloride R, evaporate to dryness and record the new
spectra using the residues.
D . H eat 0.1 g with 2 g of anhydrous sodium carbonate R until
a dull red colour appears for 10 min. Allow to cool, extract
the residue with about 5 m L of water R, dilute to 10 m L
w ith water R and filter. T he solution gives the reaction (a) of
chloride (2.3.1).
TESTS
F . 3-(4-chloro - 1OH-phenothiazin-10-yI)-N,iV- Examine by thin-layer chromatography (2.2.27), using a
dimethylpropan - 1-amine. suitable silica gel as the coating substance.
PhEur Test solution Dissolve 0.50 g of the substance to be examined
in acetone R and dilute to 10 m L with the same solvent.
Reference solution (a) Dissolve 15 mg o f
4-chIorobenzenesidfonamide R (chlorpropamide im purity A) in
1-532 Chlorprothixene Hydrochloride 2016
Reference solution (b) Dissolve 15 m g o f chlorpropamide 1 m L o f 0.1 M sodium hydroxide is equivalent to 27.67 m g of
impurity B CRS in acetone R and dilute to 100 m L with the C 10H 13C lN 2O 3S.
sam e solvent. STO R A G E
Reference solution (c) Dilute 0.3 m l, o f the test solution to Store protected from light.
100 m L with acetone R.
IM P U R IT IE S
Reference solution (d) Dilute 5 m l, o f reference solution (c) to
15 m L with acetone R. o o
* //
Reference solution (e) Dissolve 0.10 g o f the substance to be Ss ,R
N
examined, 5 mg of 4-chlorobenzenesulfonamide R and 5 m g of H
chlorpropamide impurity B CRS in acetone R and dilute to
Apply to the plate 5 jxL of each solution. Develop over a A. R = H : 4-chlorobenzenesulfonamide,
path o f 15 cm using a mixture o f 11.5 volumes of C. R = C O -N H 2: [(4-chlorophenyl)sulfonyl]urea.
concentrated ammonia R, 30 volumes o f cydohexane R,
50 volumes o f methanol R and 100 volumes o f methylene O
chloride R. Allow the plate to dry in a current o f cold air, heat HaC„ A
at 110 °C for 10 min. At the bottom o f a chromatographic
tank, place an evaporating dish containing a mixture of
1 volum e o f hydrochloric add R, 1 volume of water R and B. 1,3-dipropylurea,
2 volumes of a 50 g/L solution of potassium permanganate R, PhEur
close the tank and allow to stand for 15 min. Place the dried
hot plate in the tank and close the tank. Leave the plate in
contact with the chlorine vapour for 2 min. W ithdraw the
plate and place it in a current o f cold air until the excess of J r *★
★ ★
chlorine is removed and an area of coating below the points Chlorprothixene Hydrochloride ★ ★
of application does n o t give a blue colour with a drop of *****
potassium iodide and starch sdution R. Spray with potassium
iodide and starch solution R. In the chromatogram obtained
with the test solution: any spot corresponding to im purity A
is n o t more intense than the spot in the chromatogram
obtained with reference solution (a) (0.3 per cent); any spot HCI
corresponding to im purity B is n o t m ore intense than the
solution (b) (0.3 per cent); any spot, apart from the principal
spot and any spot corresponding to impurity A and B, is not
m ore intense than the spot in the chromatogram obtained C 18H 19C12NS 352.3 6469-93-8
with reference solution (c) (0.3 per cent); not more than two
such spots are more intense than the spot in the
Dopam ine receptor antagonist; neuroleptic.
chrom atogram obtained with reference solution (d)
(0.1 per cent). T he test is not valid unless the chromatogram PhEur________________________________________
obtained with reference solution (e) shows three clearly
D E F IN IT IO N
separated spots with approximate Rp values o f 0.4, 0.6 and
(Z)-3-(2-Chloro-9H-thioxanthen-9-ylidene)-NJiV-
0.9 corresponding to chlorpropamide, impurity A and
dim ethylpropan- 1-amine hydrochloride.
im purity B respectively.
H e a v y m e ta ls (2.4.8) C o n te n t
Dissolve 2.0 g in a mixture o f 15 volumes of water R and
85 volumes o f acetone R and dilute to 20 m L with the same CHARACTERS
m ixture of solvents. 12 m L o f the solution complies with A p p e a ra n c e
test B for heavy metals (20 ppm ). Prepare the reference W hite or almost white, crystalline powder.
solution using lead standard solution (2 ppm Pb) prepared S o lu b ility
by diluting lead standard solution (100 ppm Pb) R with a Soluble in water and in ethanol (96 per cent), slightly soluble
m ixture of 15 volumes of water R and 85 volumes of
in methylene chloride,
acetone R.
mp: about 220 °C.
N ot m ore than 0.5 per cent, d eterm ined on 1.000 g by ID E N T IF IC A T IO N
drying in an oven at 100 °C to 105 °C. First identification A , E
S u lfa te d a s h (2.4.14) Second identification B} C, D, E
N o t m ore than 0.1 per cent, determ ined on 1.0 g. A. Infrared absorption spectrophotom etry (2.2.24).
A SSA Y Preparation Dissolve 25 m g in 1 m L of water R. A dd 0.1 m L
Dissolve 0.250 g in 50 m L o f alcohol R previously neutralised o f dilute sodium hydroxide solution R. Shake with 2 m L of
using phenolphthalein solution R1 as indicator and add 25 m L
methylene chloride R. Separate the organic layer and wash with
0.5 m L o f water R. Evaporate the organic layer to dryness
o f water R. T itrate with 0.1 M sodium hydroxide until a pink
colour is obtained. and dry the residue at 40-50 °C. Examine the residues
prepared as discs.
Comparison chlorprothixene hydrochloride CRS.
2016 Chlorprothixene Hydrochloride 1-533
8 . Dissolve 0.2 g in a mixture of 5 m L o f cHoxan R and Limits:

5 m L o f a 1.5 g/L solution o f sodium nitrite R. Add 0.8 m L of — impurity F: not more than 6.67 times the area of the
nitric add R. After 10 min add the solution to 20 m L o f principal peak in the chromatogram ob tained with
water R. 1 h later filter the predpate formed. T h e filtrate is reference solution (b) (2.0 per cent);
used immediately for identification test C. Dissolve the — impurity E: no t more than 3 times the area of the
precipitate by warming in about 15 m L o f ethanol principal peak in the chromatogram obtained with
(96 per cent) R and add the solution to 10 m L o f water R. reference solution (b) (0.3 per cent taking into a cco u n t a
Filter and dry the precipitate at 100-105 °C for 2 h. response factor of 3);
T h e melting point (2.2.14) is 152 °C to 154 °C. — arty other impurity, n o t more than the area of the principal
C . T o 1 m L o f the filtrate obtained in identification test B, peak in the chromatogram obtained with reference
add 0.2 m L o f a suspension o f 50 mg o f fast red B sab R in solution (b) (0.3 per cent);
1 m L of ethanol (96 per cent) R. Add 1 m L o f 0.5 M alcoholic — total of any other impurity, not m ore than 2.33 times the
potassium hydroxide. A dark red colour is produced. C an y out area o f the principal peak in the chromatogram obtained
a blank test. with reference solution (b) (0.7 per cent);
D . Dissolve about 20 mg in 2 m L of nitric acid R. A red — disregard limit: 0.1 times the area of the principal peak in
colour is produced. Add 5 m L of water R and examine in the chromatogram obtained with reference solution (b)
(0.03 p er cent).
ultraviolet light at 365 nm . T h e solution shows green
fluorescence. H eav y m e ta ls (2 A. 8)
E. It gives reaction (a) of chlorides (2.3.1). M aximum 20 ppm .
TESTS
S o lu tio n S
Dissolve 0.25 g in carbon dioxide-free water R and dilute to L oss o n d ry in g (2.2.32)
25 m L with the same solvent. M aximum 0.5 p er cent, determined on 1.000 g by drying
Solution S is clear (2.2.1) and colourless (2.2.2, Method II). S u lfa te d a s h (2.4.14)
p H (2.2.3)
4.4 to 5.2 for solution S. A SSAY
Dissolve 0.300 g in a mixture o f 5.0 m L o f 0.01 M
R e la te d s u b s ta n c e s
L iquid chrom atography (2.2.29). Cany out the test protected
hydrochloric add and 50 m L of ethanol (96 per cent) R. Carry
out a potentiometric titration (2.2.20), using 0.1 M sodium
from bright light
Test solution Dissolve 20.0 mg of the substance to be inflexion.
the mobile phase.
C 18H 19C12NS.
Reference solution (a) Dissolve 20.0 mg o f chlorprothixene
hydrochloride CRS in the mobile phase and dilute to 20.0 m L STO RA G E
with the mobile phase. Protected from light.
Reference solution (b) Dilute 2.0 m L o f the test solution to IM P U R IT IE S
100.0 m L w ith the mobile phase. Dilute 3.0 m L o f this Specified impurities Ay B, C, D, E, F.
Column:
— size. I = 0.12 m , 0 = 4.0 mm;
^CH3
— stationary phase, base-deactivated octadecylsüyl silica gelfor n
i.. and enantiomer
chromatography R (3 |im or 5 |jm). CH3
Mobile phase Solution containing 6.0 g/L o f potassium
dihydrogen phosphate R, 2.9 g/L of sodium laurUsulfate R and
9 g/L o f tetrabutylammcmium bromide R in a mixture of
50 volumes o f methanol R, 400 volumes of acetonitrüe R and A. C&S)-2-chloro-9- [3-(dimethylamino) propyl] -9H-
550 volumes o f distilled water R. thioxanthen-9-ol,
Flow rate 1.5 m l -/min.
Equilibration F o r about 30 min with the mobile phase.
n' CH*
Injection 20 |iL. 1
ch3
Run time Tw ice the retention time o f chlorprothixene.
Relative retention W ith reference to chlorprothixene:
System suitability: reference solution (a): B . NJV-dimethyl-3-(9//-thioxanthen-9-ylidene)propan-1-
— retention time, chlorprothixene = about 10 min; am in e.
— relative retention with reference to chlorprothixene:
1-534 Chlortalidone 2016
CHARACTERS
.c h 3 Appearance
W hite or yellowish-white powder.
Solubility
Very slightly soluble in water, soluble in acetone and in
methanol, practically insoluble in methylene chloride.
It dissolves in dilute solutions o f alkali hydroxides.
C. (Z)-3-(2-chloro-9i/-thioxanthen-9-ylidene)-N-
methylpropan - 1-amine, It shows polymorphism (5.9).
IDENTIFICATION
Comparison chlortalidone CRS.
substance separately in methanol R, evaporate to dryness and
D. (2)-3-(4-chloro-9i/-thioxanthen-9-ylidene)-iy,N- TESTS
dimethylpropan - 1-amine, Acidity
Dissolve 1.0 g with heating in a mixture o f 25 m L of
acetone R and 25 m L of carbon dioxide-free water R. Cool.
Titrate with 0.1 M sodium hydroxide, determ ining the
end-point potentiometrically (2.2.20). N o t more than
0.75 m L o f 0.1 M sodium hydroxide is required.
Related substances
Solvent mixture Mix 2 volumes o f a 2 g/L solution o f sodium
E. 2-chloro-9i/-thioxanthen-9-one, hydroxide R, 48 volumes of mobile phase B and 50 volumes
o f mobile phase A.
Test solution (a) Dissolve 50.0 m g of the substance to be
Test solution (b) Dilute 10.0 m L of test solution (a) to
F. (£)-3-(2-chloro-9f/-thioxanthen-9-ylidene)-N>iS/’- 100.0 m L with the solvent mixture. D ilute 1.0 m L of this
dim ethylpropan- 1-amine ((£)-isomer). solution to 10.0 m L with the solvent mixture.
PhEur Reference solution (b) Dissolve the contents o f a vial of
chlortalidone for peak identification CRS (containing
impurities B, G and J) in 1 m L of the solvent mixture.
Reference solution (c) Dissolve 50.0 m g o f chlortalidone CRS in
Chlortalidone ★ * the solvent m ixture and dilute to 50.0 m L with the solvent
★ ★ mixture. Dilute 10.0 m L o f this solution to 100.0 m L with
(Ph. Eur. monograph 0546) ***** the solvent mixture.
Column:
— sizer. I = 0.25 m , 0 = 4.6 mm;
— stationary phase: octylsQyl silica gel for chromatography R
and enantiomer
(5 pm);
Mobile phase.
C 14H n a N 204 S — mobile phase A: dissolve 1.32 g of ammonium phosphate R
338.8 77-36-1
in about 900 m L of water R and adjust to p H 5.5 with
Action and use dilute phosphoric add R; dilute to 1000 m L with water R;
Thiazide-like diuretic. — mobile phase B: methanol R2;
Preparations
Chlortalidone Tablets Time Mobile phase A Mobile phase B
Co-tenidone Tablets (min) (per cent V7V) (per cent V/V)
0 - 16 65 35
PhEur.
16-21 65*50 35*50
DEFINITION 2 1 -3 5 50 50
2-Chloro-5- [(1RS)-1-hydroxy-3-oxo-2,3-dihydro-1H-isoindol-
3 5 -4 5 50*65 50*35
1-yl] benzenesulfonamide.
Content
97.0 per cent to 102.0 per cent (dried substance). Flow rate 1.4 mL/min.
2016 Chlortetracycline Hydrochloride 1-535

Injection 20 |iL o f test solution (a) and reference solutions (a)
and (b).
w ith reference solution (b) and the chromatogram supplied
w ith chlortalidone for peak identification CRS to identify the A. R = H , R ' = OH: 2-(4-chloro-3-sulfobenzoyI)benzoic
peaks due to impurities B, G and J. acid,
Relative retention W ith reference to chlortalidone B. R = H , R ' = N H 2: 2-(4-chloro-3-
(retention time = about 7 m in): impurity B = about 0.7; sulfamoylbenzoyl) benzoic add,
im purity J = about 0.9; im purity G = about 6 . C. R = C 2H 5, R ' = N H 2: ethyl 2-(4-chloro-3-
System suitability: reference solution (b): sulfamoylbenzoyl) benzoate,
— resolution: m in im u m 1.5 between the peaks due to I. R = C H (C H 3) 2, R ' = N H 2: 1-methylethyl 2-(4-chloro-3-
im purity J and chlortalidone. sulfamoylbenzoyl) benzoate.
Limits:
— impurity B: n o t m ore than 7 times the area o f the
— impurity f . n o t more than 3 times the area o f the principal
D . R = O C 2H 5, R ' = S 0 2-N H 2: 2-chloro-5-[(lJ?S)-l-
— impurity G: n o t m ore than 2 times the area o f the
ethoxy-3-oxo-2,3-dihydro-lH-isoindol - 1-
yl] benzenesulfonamide,
reference solution (a) ( 0.2 per cent);
— unspecified impurities: for each impurity, not more than the E. R = H , R ' = S 0 2-N H 2: 2-chloro-5-[(lftS)-3-oxo-2,3-
area of the principal peak in the chromatogram obtained dihydro- l//-isoindol - 1-yl] benzenesulfonamide,
with reference solution (a) (0.10 per cent); G . R = O H, R ' = Cl: (3ftS)-3-(3,4-dichlorophenyl)-3-
— total: no t more than 12 times the area of the principal hydroxy-2 ,3-dihydro - 1ii-isoindol- 1-one,
peak in the chrom atogram obtained with reference H . R = O C H (C H 3) 2, R ' = S 0 2-N H 2: 2 -chloro-5-[(lflS )-l-
solution (a) ( 1.2 per cent); (1 -methylethoxy)-3-oxo-2,3-dihydro- lH -isoindol- 1-
— disregard limit: 0.5 times the area o f the principal peak in yl] benzenesulfonamide,
(0.05 per cent).
n oih o o os o ho n
M axim um 350 ppm .
T riturate 0.3 g finely, add 30 m L o f water R, shake for 5 m in \ y k X a Ha X / O
and filter. 15 m L o f the filtrate complies with the test.
Prepare the standard using 10 m L o f chloride standard solution F . bis[2-chloro-5-(l-hydroxy-3-oxo-2,3-dihydro-lii-isoindol-
(5 ppm Cl) R. 1-yI)benzenesulfonyI] amine,
L o ss o n d ry in g (2.2.32) J. impurity of unknow n structure with a relative retention of
M axim um 0.5 per cent, determined on 1.000 g by drying in about 0.9.
an oven at 105 °C.
--------------------------------------------------------------------- PhEur
A SSA Y
L iquid chromatography (2.2.29) as described in the test for ★* ★
Chlortetracycline Hydrochloride ★ ★
related substances with the following modification. ★ ★
Injection 20 |iL o f test solution (b) and reference solution (c). (Ph. Eur. monograph 0173) *****
Calculate the percentage content o f C 14H J 1CIN 2O 4S from
th e declared content of chlortalidone CRS.
IM P U R IT IE S
, ho
Specified impurities B, G , J
Other detectable impurities (the following substances would, if r ho ch 3 h n —c h 3
present at a sufficient level, b e detected by one or other of HjC
the tests in the m onograph. T hey are limited by the general Compound R Molecular formula K
Chlortetracycline hydrochloride Cl CaH^CljNjOg 5153
(2034). It is therefore not necessary to identify these Tetracycline hydrochloride H CjjHaClNjO, . 480.9
Control of impurities in substances for pharmaceutical use): A , C,
Chlortetracycline hydrochloride: [64-72-2]
D, E, F, H, I.
Tetracycline hydrochloride: [64-75-5]
Action and use

Tetracycline antibacterial.
1-536 Chlortetracycline Hydrochloride 2016
PhEur__________________________________________________________ the principal peak in the chromatogram obtained with

D E F IN IT IO N
Mixture o f antibiotics, the m ain component being the TESTS
hydrochloride o f (4S,4aS,5aS,6S,12aS)-7-chloro-4- p H (2.2.3)
(dimethylamino)-3,6,10,12,12a-pentahydroxy-6-methyl-1,11- 2.3 to 3.3.
dioxo-1,4,4a,5,5 a, 6,11,12a-octahydrotetracene-2-carboxamide Dissolve 0.1 g in 10 m L o f carbon dioxide-free water R,
(chlortetracycline hydrochloride), a substance produced by heating slightly.
the growth o f certain strains o f Streptomyces aureofaciens or
obtained by any other means.
—250 to —235 (anhydrous substance).
C o n te n t Dissolve 0.125 g in water R and dilute to 50.0 m L with the
— chlortetracycline hydrochloride (C 22H 24CI2N 2O 8): m inim u m same solvent
— tetracycline hydrochloride (C 22H 25CIN 2O 8): maximum A b so rb a n c e (2.2.25)
6.0 per cent (anhydrous substance); Maximum 0.40 at 460 nm.
— sum of the contents of chlortetracycline hydrochloride and Dissolve 0.125 g in water R and dilute to 25.0 m L with the
tetracycline hydrochloride. 94.5 per cent to 102.0 per cent same solvent.
(anhydrous substance). R e la te d su b sta n c e s
CHARACTERS Liquid chromatography (2.2.29). Prepare the solutions
A p p e a ra n c e immediately before use.
Yellow powder. Test solution Dissolve 25.0 m g o f the substance to be
examined in mobile phase B and dilute to 25.0 m L with
S o lu b ility
mobile phase B.
Slightly soluble in w ater and in ethanol (96 per cent).
It dissolves in solutions of alkali hydroxides and carbonates. Reference solution (a) Dissolve 25.0 m g o f chlortetracycline
hydrochloride CRS in mobile phase B and dilute to 25.0 m L
ID E N T IF IC A T IO N with mobile phase B.
First identification C, D
Second identification A , B, C 100.0 m L with mobile phase B.
A. Thin-layer chromatography (2.2.27). Reference solution (c) Dilute 1.0 m L o f reference solution (b)
Test solution Dissolve 5 mg o f the substance to be examined to 10.0 m L with mobile phase B.
in methanol R and dilute to 10 m L with the same solvent Reference solution (d) Dissolve 5 m g of chlortetracycline for
Reference solution (a) Dissolve 5 mg o f chlortetracycline system suitability CRS (containing impurities A, B, D , E, G,
hydrochloride CRS in methanol R and dilute to 10 m L with the H , J, K and L) in mobile phase B and dilute to 5 m L with
same solvent mobile phase B.
Reference solution (b) Dissolve 5 mg o f chlortetracycline Reference solution (e) Dissolve 25.0 m g of tetracycline
hydrochloride CRS, 5 m g of demecbcydine hydrochloride R and hydrochloride CRS in mobile phase B and dilute to 25.0 m L
5 m g of doxycycüne R in methanol R and dilute to 10 m L with with mobile phase B. Dilute 5.0 m L o f this solution to
the same solvent. 100.0 m L with mobile phase B.
Plate TLC octadecylsüyl silica gel F2â plate R Column:
Mobile phase M ix 20 volumes o f acetonitrüe R, 20 volumes of — size. I = 0.075 m , 0 = 4.6 mm;
methanol R and 60 volumes o f a 63 g/L solution of oxalic — stationary phase, end-capped octylsüyl silica gelfor
add R previously adjusted to p H 2 w ith concentrated chromatography with polar incorporated groups R (3.5 |jm);
ammonia R. — temperature. 45 °C.
Application 1 jiL. Mobile phase.
— mobile phase A: to 725 m L o f water R add 50 m L of
Development Over 31A of the plate.
perchloric add solution R, shake and add 225 m L o f
Drying In air. dimethyl sulfoxide R;
Detection Examine in ultraviolet light at 254 run. — mobile phase B: to 250 m L of water R add 50 m L of
System suitability T he chromatogram obtained with reference perchloric add solution R, shake and add 700 m L o f
solution (b) shows 3 clearly separated spots. dimethyl sulfoxide R;
the test solution is similar in position and size to the principal Time Mobile phase A Mobile phase B
spot in the chromatogram obtained w ith reference (min) (per cent V7V) (percent V/V)
solution (a). 0 -4 6 100*0 0 * 100
B. T o about 2 m g add 5 m l . of sulfuric add R. A deep blue
colour develops and becomes bluish-green. Add the solution
to 2.5 m L o f water R. T he colour becomes brownish.
C. It gives reaction (a) o f chlorides (2.3.1).
D . Liquid chromatography (2.2.29) as described in the test
for related substances with the following modification.
Iiyecdon T est solution and reference solution (a).
with chlortetracycline for system suitability CRS and the
Results T he principal peak in the chromatogram obtained chromatogram obtained with reference solution (d) to
with the test solution is similar in retention time and size to identify the peaks due to impurities A, B, D , E, G, H , J, K
and L.
2016 Chlortetracycline Hydrochloride 1-537
Relative retention W ith reference to chlortetracycline (retention C 22H 25CIN 2O 8 using the chromatogram obtained with
time = about 26 min): impurity D = about 0.5; reference solution (e) and taking into account the assigned
tetracycline = about 0.6; impurity E = about 0.7; content of tetracycline hydrochloride CRS.
impurity B = about 0.8, impurity A = about 0.86; STORA GE
impurity G = about 0.9; impurity H = about 1.1;
Protected from light. If the substance is sterile, store in a
impurity J = about 1 .4, impurity K = about 1.67;
impurity L = about 1.71.
System suitability Reference solution (d): IM P U R IT IE S
— resolution, minimum 1.5 between the peaks due to Specified impurities A, B, D , E, G , H , J, K, L
tetracycline and impurity E; m inim u m 1.5 between the Other detectable impurities (the following substances would, if
peaks due to impurities A and G; m in im u m 1.5 between present at a sufficient level, be detected by one or other of
the peaks due to impurities K and L; if necessary, adjust the tests in the monograph. They are limited by the general
the concentration o f dimethyl sulfoxide in mobile acceptance criterion for other/unspecified impurities and/or
phase A. by die general monograph Substances for pharmaceutical use
Limits. (2034). It is therefore n o t necessary to identify these
— correction factors: for the calculation o f content, multiply impurities for demonstration of compliance. See also 5.10.
the peak areas o f the following impurities by the Control of impurities in substances for pharmaceutical use): C, F,
corresponding correction factor im purity G = 1.4; I.
— impurity J = 0.3; impurity K = 0.4; impurity L = 0.4;
— impurity A: no t m ore than 4 times the area of the OH O OH__ O O
NHj
— impurities B, E: for each impurity, not m ore than the area
reference solution (b) ( 1.0 per cent);
— impurity J: n o t more than 3 times the area o f the principal
peak in the chromatogram obtained with reference A. (4i?,4aS,5aS,6S,12a5)-7-chloro-4-(dimethylamino)-
solution (c) (0.3 per cent); 3,6,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-
— impurities D, G, H, L: for each impurity, n o t m ore than l,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide
twice the area o f the principal peak in the chromatogram (4-epichlortetracycline),
— impurity K. n o t m ore than 1.5 times the area of the oh o oh o o
reference solution (c) (0.15 per cent); nh2
— unspecified impurities: for each impurity, no t more than the
1
a HO H H N—c h 3
h3c
— sum of impurities other than A: n o t more than twice the
with reference solution (b) (2.0 per cent); B. (45,4a5,5a5,65,12a5)-7-chloro-4-(dimethylamino)-
— disregard limit. 0.5 times the area o f the principal peak in 3,6,10,12,12a-pentahydroxy-l, 1 1-dioxo-1,4,4a,5 ,5a, 6,11,12a-
the chromatogram obtained with reference solution (c) octahydrotetracene- 2-carboxamide (demeclocycline),
(0.05 per cent).
oh o oh o o
M aximum 50 ppm . nh2
using 2.5 m L o f lead standard solution (10 ppm Pb) R.
Water (2.5.12) h3c
Sulfated ash (2.4.14) C. (4R,4a5,5a5,65,12aS)-4-(dimethylamino)-3,6,10,12,12a-
M axim u m 0.5 p er cent, determined on 1.0 g. pentahydroxy- 1,1 l-dioxo-l,4,4a,5,5a,6,l 1, 12a-
Bacterial endotoxins (2.6.14) octahydrotetracene- 2-carboxamide
Less than 1 IU/m g, if intended for use in the manufacture of (4-epidemethyltetracycline),
procedure for the removal of bacterial endotoxins. OH o 0H...0 o
ASSAY NHj
1
HO CH3 H N—CH3
Injection 10 nL o f the test solution and reference solutions (a) h3c
and (e).
Calculate the percentage content of C 22H 24CI2N 2O 8 using D . (4i?,4aS,5aS,6S, 12aS)-4-(dimethylamino)-3,6, 10, 12, 12a-
the chrom atogram obtained with reference solution (a) and pentahydroxy- 6-m eth y l-l,l l-dioxo-l,4,4a,5,5a,6, 11, 12a-
taking into account the assigned content o f chlortetracycline octahydrotetracene-2-carboxamide (4-epitetracycline),
hydrochloride CRS. Calculate the percentage ,content of
1-538 Cholesterol 2016
OH O OH O
OH I
Cl HO H H N-CH 3
h3c
E. (4/?,4aS’,5aS’,6.S, 12a£)-7-chloro-4-(dimethylamino)- J. (45’j4a5’j 12 aS )^(dim ethyiam ino)-3, 10,12,12a-

3 j6 jl0 jl2jl2a-pentah y d ro x y -ljl l-dioxo-l,4,4a,5,5a,6,l 1, 12a- tetrahydroxy-6 -m ethyi-l 311 -dioxo - 1,4,4a,5 , 11, 12 a-
octahydrotetracene- 2 -carboxamide hexahydrotetracene-2-carboxamide (anhydrotetracydine),
(4-epidemethylchlortetracydine)j
a ch 3 h n - ch 3
h3c
K. (4R,4aS, 12aS)-7-chloro-4-(dimethylamino)-3,10,12,12a-
tetrahydroxy-6-m ethyi-l, 11 -dioxo-1,4,4a,5 ,1 1,12a-
F. (4/?,4aS,6S,8aS)-6-[(lJ?)-7-chloro-4-hydroxy-l-methyl-3- hexahydrotetracene-2-caiboxamide
oxo-1,3-dihydro-2-benzofuran-1-yl]-4-(dimethylamino)-3,8a-
(4-epianhydrochlortetracycline)j
dihydroxy-1, 8-dioxo-1,4,4a,5 ,6,7,8,8a-octahydronaphthalene-
2-carboxamide (4-epiisochlortetracycline)j
Cl CH3 H N-CHs
h, c
ch 3 h n —c h 3
h3c
L. (4-S,4aS, 12aS)-7-chloro-4-(dimethylamino)-3,10,12,1 2 a-
tetrahydroxy-6-methyl- 1,1 l-d io x o -l,4 ,4 a,5 ,l 1, 12a-
G. (45,4a«S36S,8aS)-6-[(lK)-7-chloro-4-hydroxy-1 -methyl-3- hexahydrotetrac ene-2-carboxamide
oxo-1,3-dihydro-2-benzofuran-1-yl] -4-(dimethylamino)-3,8a- (anhydrochlortetracycline).
dihydroxy- 1, 8-dioxo - 1,4,4a,5,6,7,8,8a-octahydronaphthalene- __________________________________________________________ PhEur
2-carboxamide (isochlortetracycline),
OH O OH O
OH I
★*★
★ ★
Cholesterol ★ ★
d HO CH3 H N—CH3
h3c
H . (45,4a5,5a5,65,12a5)-2-acetyl-7-chloro-4-
(dimethylamino)-3j6,10,12,12a-pentahydroxy-6-methyl-
4a,5a,6,l 2a-tetrahydrotetracene-l, 11 (4//,5/i)-dione (2-acetyl-
2-decarboxamidochlortetracycline),
OH O OH o
OH I
C27H 44O 386.7 57-88-5
Action and use

ch3 H n -c h 3 Excipient.
h3c
PhEtr_____________
I. (4i?,4a£,12aS)-4-(dimethylamino)-3,10,12,12a- DEFINITION
tetrahydroxy- 6 -m ethyl- 1,1 l-dioxo-l,4,4a,5,l 1,12a- Cholest-5-en-3p-ol.
hexahydrotetracene-2-carboxamide Content
(4-epianhydrotetracycline), — cholesterol: m inim um 95.0 per cent (dried substance);
— total sterols: 97.0 per cent to 103.0 per cent (dried
substance).
CHARACTERS
Appearance
2016 Cholesterol 1-539
S o lu b ility Column:
Practically insoluble in water, sparingly soluble in acetone — material: fused silica;
and in ethanol (96 per cent). — size. I = 30 m , 0 = 0.25 mm;
It is sensitive to light. — stationary phase, poly (dimethyl)siloxane R (film thickness
0.25 Jim).
Flow rate 2 mL/min.
B. Thin-layer chromatography (2.2.27). Prepare the solutions
immediately before use. Split ratio 1:25.
Test solution Dissolve 10 mg of the substance to be examined Temperature:
in ethylene chloride R and dilute to 5 m L with the same — column: 275 °C;
— injection pore. 285 °C;
solvent.
Reference solution Dissolve 10 mg o f cholesterol CRS in ethylene
chloride R and dilute to 5 m L with the same solvent. Detection Flam e ionisation.
Plate TLC silica gel G plate R. Irgecdon 1.0 (iL.
Mobile phase ethyl acetate R, toluene R (33:66 ViV). System suitability: reference solution:
Application 20 jiL. pregnenolone isobutyrate and cholesterol;
Development Immediately, protected from light, over a path of — symmetry factor, m inim u m 0.6 for the peak due to
15 cm. cholesterol.
Drying In air. Calculate the percentage content of cholesterol taking into
Detection Spray 3 times with antimony trichloride solution R; account the assigned content of cholesterol CRS. Calculate the
examine within 3-4 min. percentage content of total sterols by adding together the
Results T h e principal spot in the chromatogram obtained with contents o f cholesterol and other substances with a retention
the test solution is similar in position, colour and si2e to the time less than or equal to 1.5 times the retention time of
principal spot in the chromatogram obtained with the cholesterol. Disregard the peaks due to the internal standard
reference solution. and the solvent.
C . Dissolve about 5 mg in 2 mL of methylene chloride R. STORAGE
A dd 1 m L of acetic anhydride R, 0.01 m L o f sulfuric acid R Protected from light.
and shake. A pink colour is produced which rapidly changes
L A B E L L IN G
to red, then to blue and finally to brilliant green.
T h e label states the source material for the production of
TESTS cholesterol (for example bovine brain and spinal cord, wool
S o lu b ility in e th a n o l (96 p e r c en t) fat or chicken eggs).
In a stoppered flask, dissolve 0.5 g in 50 m L o f ethanol
IM P U R IT IE S
(96 per cent) R at 50 °C. Allow to stand for 2 h. N o deposit
or turbidity is formed.
A cid ity
Dissolve 1.0 g in 10 m L o f ether R, add 10.0 m L o f 0.1 M
sodium hydroxide and shake for about 1 min. H eat gently to
eliminate ether and then boil for 5 min. Cool, add 10 m L of
water R and 0.1 m L o f phenolphthalem solution R as indicator
and titrate with 0.1 M hydrochloric acid until the pink colour
just disappears, stirring the solution vigorously throughout
the titration. Carry out a blank titration. T he difference A. 5a-cholest-7-en-3 (i-ol (lathosterol),
between the volumes of 0.1 M hydrochloric acid required to
change the colour of the indicator in the blank and in the test
is not m ore than 0.3 mL.
Maximum 0.3 per cent, determined on 1.000 g by drying
A SSA Y B. cholesta-5,24-dien-3|3-ol (desmosterol),
Gas chrom atography (2.2.28).
Internal standard solution Dissolve 0.100 g of pregnenolone
isobutyrate CRS in heptane R and dilute to 100.0 m L with the
same solvent.
examined in the internal standard solution and dilute to
25.0 m L w ith the same solution.
Reference solution Dissolve 25.0 m g of cholesterol CRS in the
internal standard solution and dilute to 25.0 m L with the C. 5a-cholesta-7,24-dien-3f}-ol.
same solution.
___________________________________________________ BhEur
1-540 Cholesterol for Parenteral Use 2016
Internal standard solution Dissolve 0.100 g of pregnenolone

Cholesterol for Parenteral Use ****** isobutyrate CRS in heptane R and dilute to 100.0 m l. with the
(Ph. Eur. monograph 2397) * same solvent.
examined in the internal standard solution and dilute to
25.0 m L with the same solution.
Reference solution Dissolve 25.0 m g of cholesterol CRS in the
internal standard solution and dilute to 25.0 m L with the
same solution.
Column:
— size. I = 30 m, 0 = 0.25 mm;
C ^H Ô 386.7 57-88-5 — stationary phase, poly (dimethyl)sUoxane R (film thickness
0.25 pm).
A ctio n a n d u se
Excipient
Flow rate 2 m D m in.
PhEur__________________________________________________________ Split ratio 1:25.
D E F IN IT IO N Temperature:
Cholest-5-en-3P~ol obtained from Wool fax (0134). — column: 275 °C;
— irgecdon port: 285 °C;
C o n te n t
— cholesterol: 99.0 p e r cent to 101.0 per cent (dried
substance). Detection Flam e ionisation.
Irgecdon 1.0 jjL.
CHARACTERS
A p p e a ra n c e Relative retention W ith reference to cholesterol (retention
W hite or almost white, crystalline powder. time = about 8.5 min): pregnenolone isobutyrate = about
0.8.
S o lu b ility
Practically insoluble in water, sparingly soluble in acetone
and in ethanol (96 p er cent).
pregnenolone isobutyrate and cholesterol.
It is sensitive to light.
Limits:
ID E N T IF IC A T IO N — total of other substances with a retention time less than or
A. M elting point (2.2.14): 147 °C to 150 °C. equal to 1.5 times the retention time of cholesterol: m axim um
B. Examine the chromatograms obtained in the assay. 0.5 per cent;
— disregard limit. 0.05 per cent; disregard the peak due to
the internal standard.
the principal peak in the chromatogram obtained with the Benzoyl ureas
reference solution. Liquid chromatography (2.2.29).
C. Dissolve about 5 m g in 2 m L of methylene chloride R. Test solution Dissolve 1.0 g of the substance to be examined
Add 1 m L o f acetic anhydride R and 0.01 mL o f sulfuric in 200 m L o f heptane R using a magnetic stirrer and add
acid R and shake. A pink colour is produced which rapidly 10 m L o f acetonitrUe R. Shake and allow the layers to
changes to red, then to blue and finally to bright green. separate. Isolate the lower layer (acetonitrile) and add 10 m L
of acetonitrile R to the heptane layer and extract again.
TESTS
Combine the lower layers and evaporate to dryness using a
S o lu b ility in e th a n o l (96 p e r cen t)
rotary evaporator (for example, at 40 °C and 17 kPa).
In a stoppered flask, dissolve 0.5 g in 50 m L of ethanol Add 0.5 m L of acetonitrile R then 0.5 m L of water R to the
(96 per cent) R at 50 °C. Allow to stand for 2 h. T he solution residue. Suspend with the aid of ultrasound for about 5 min.
is clear.
Centrifuge the suspension for 5 m in and use the supernatant.
A cid ity Reference solution (a) Dissolve 10.0 m g of difiubenzuron R
Dissolve 1.0 g in 10 m L of ether R, add 10.0 m L of 0.1 M (impiftity A) and 10.0 mg of triflwnuron R (impurity B) in
sodium hydroxide and shake for about 1 min. H eat gently to acetonitrile R and dilute to 100.0 m L with the same solvent.
eliminate the ether and then boil for 5 min. Cool, add Dilute 0.1 m L o f the solution to 100.0 m l. with acetonitrile R.
10 m L of water R and 0.1 m L o f phenolphthalem solution R as
Reference solution (b) Mix 0.5 m L o f reference solution (a)
indicator and titrate w ith 0.1 M hydrochloric add until die
and 0.5 m l. of water R.
pink colour just disappears, stirring the solution vigorously
throughout the titration. Carry out a blank titration. Reference solution (c) Dissolve 1.0 g of the substance to be
T he difference between the volumes o f 0.1 M hydrochloric examined in 200 m L of heptane R using a magnetic stirrer.
add required to change the colour o f the indicator in the Add 0.5 m L of reference solution (a) and 9.5 m L of
blank titration and in the test is not more than 0.1 m l. acetonitrile R. Shake and allow the layers to separate. Isolate
the lower layer (acetonitrile) and add 10 m L o f acetonitrile R
P e ro x id e v a lu e (2.5.5, Method A)
to the heptane layer and extract again. Combine the lower
M aximum 10.
layers and evaporate to dryness using a rotary evaporator (for
O th e r ste ro ls example, at e.g. 40 °C and 17 kPa). Add 0.5 m L of
Gas chromatography (2.2.28): use the normalisation acetonitrile R then 0.5 m L of water R to the residue. Suspend
procedure.
2016 Choline Salicylate 1-541
w ith the aid o f ultrasound for about 5 min. Centrifuge the

suspension for 5 m in and use the supernatant.
Column:
— sizer. I = 0.25 m , 0 = 3 mm;
— stationary phase: octadecylsUyl silica gd for chromatography R
(5 pm); A. l-(4-chIorophenyl)-3-(2,6-difluorobenzoyl)urea
— temperature: 40 °C. (diflubenzuron),
Mobile phase.
— mobile phase A: acetomtrUe R, water R (50:50 V/V);
— mobile phase B: acetomtrUe R)
9 x c r
H H
Time Mobile phase A Mobile phase B Cl
B . 1-(2-chlorobenzoyI)-3-[ (4-trifluoromethoxy)phenyl] urea
0 - 20 100 0 (triflumuron).
20 - 20.5 100->0 0 * 100 PhEur
20J - 30 0 100
After elution o f the components, a gradient is applied to

Choline Salicylate Solution
prevent a strong drifting baseline due to cholesterol during
the following run. A ctio n a n d u se
Flow rate 1 mL/min. Salicylate; non-selective cyclo-oxygenase inhibitor; analgesic;
Detection Spectrophotom eter at 254 am. anti-inflam m atory
Injection 100 jiL o f the test solution and reference P re p a r a tio n s

solutions (b) and (c). Choline Salicylate Ear Drops
Identification of impurities Use the chromatogram obtained Choline Salicylate Oromucosal Gel
w ith reference solution (b) to identify the peaks due to
impurities A and B. D E F IN IT IO N
Choline Salicylate Solution is an aqueous solution of choline
Retention lime Im purity A = about 10 min;
salicylate. It contains not less than 47.5% w/v and not m ore
impurity B = about 18 min.
than 52.5% w/v o f choline salicylate, C 12H 19N 0 4. It may
Limits:
contain a suitable antimicrobial preservative.
— impurity A: n o t more than 0.5 times the area o f the
corresponding peak in the chromatogram obtained with C H A R A C T E R IS T IC S
reference solution (c) (0.05 ppm); A clear, colourless liquid.
— impurity B: n o t more than 0.5 times the area o f the ID E N T IF IC A T IO N
A. Mix 0.5 m L w ith 10 m L o f methanol, dry with anhydrous
reference solution (c) ( 0.05 ppm).
sodium sulfate, filter and evaporate the methanol to dryness.
H e a v y m e ta ls (2.4.8) T h e infrared absorption spectrum o f the residue, Appendix II A,
M aximum 10 ppm . is concordant with the reference spectrum o f choline salicylate
2.0 g complies with test C. Prepare the reference solution (RS 059).
using 2.0 m L o f lead standard sdution (10 ppm Pb) R. B. Dilute 5 m L to 25 m L with water. T h e resulting solution
L oss o n d ry in g (2.2.32) yields the reactions characteristic of salicylates, Appendix VI.
Maximum 0.1 p er cent, determined on 1.000 g by drying TESTS
m vacuo at 60 °C for 4 h. A cid ity
S u lfa te d a s h (2.4.14) Dilute 4 m L to 20 m L with water and add 0.1 m L of phenol
M axim um 0.1 p er cent, determined on 1.0 g. red solution. T he solution is yellow and n o t more than 0.4 m L
M ic ro b ia l c o n ta m in a tio n o f 0.1m sodium hydroxide VS is required to change the colour
T A M C : acceptance criterion 102 CFU/g (2.6.12). o f the solution to reddish violet.
B a c te ria l e n d o to x in s (2.6.14) C la rity a n d c o lo u r o f solution

Less than 0.1 IU/mg. Dilute 1 volume o f the solution to 5 volumes with water.
T h e resulting solution is dear, Appendix IV A, and colourless,
A SSA Y Appendix IV B, M ethod II.
Gas chromatography (2.2.28) as described in the test for
W e ig h t p e r m L
other sterols.
1.070 to 1.110 g, Appendix V G.
Calculate the percentage content of C 27H 460 from the
C h lo rid e
declared content o f cholesterol CRS.
M ix 0.2 m L with 10 m L of water and add carefully, with
STORAGE mixing, 0.1 m L o f a mixture of 10 volumes o f silver nitrate
Protected from light. solution and 1 volume o f nitric acid T h e resulting solution is
IM P U R IT IE S n o t more opalescent than a standard prepared by treating
Specified impurities A, B 10 m L o f a 0.00164% w/v solution o f sodium chloride in the
same m an n er b e ginning at the words ‘add carefully . . . *
(0 . 1%)»
1-542 Choline Theophyllinate 2016
A SSA Y (d) Develop the plate to 15 cm.

T o 1 g add 50 m L of 1,4-dioxan and 5 m L of acetic anhydride (e) After removal o f the plate, dry in air, and examine under
and cany out M ethod I for rum-aqueous titration, ultraviolet light (254 nm).
Appendix VIII A, using 0.25 m L o f methyl orange-xylene
MOBILE PHASE
cyanol FF solution as indicator. Each m L o f 0.1m perchloric
5 volumes of ethanol (96%) and 9 5 volumes o f chloroform.
acid VS is equivalent to 24.13 m g of C 12H 19N O 4. U se the
weight per mL to calculate the percentage of C u H ^ O * LIMITS
weight in volume. Any secondary spot in the chrom atogram obtained with
solution ( 1) is not more intense than the spot in the
chromatogram obtained w ith solution (2) ( 1%).
L oss o n d ry in g
Choline Theophyllinate W hen dried to constant weight at 10 5 °, loses no t m ore than
0.5% o f its weight. Use 1 g.
O
S u lfa ted a sh
N ot more than 0.1% , Appendix IX A.
Me ASSAY
For choline
Me Dissolve 0 .6 g in 5 0 m L o f water and titrate with
0.05m sulfuric add FS, using methyl red mixed solution as
C 12H 21N 5O3 283.3 4499-40-5 indicator, until a violet end point is obtained. Each m L of
0.05m sulfuric add VS is equivalent to 1 2 .1 2 m g o f choline,
A c tio n a n d u se C 5H i 5N 0 2.
Non-selective phosphodiesterase inhibitor (xanthine); For theophylline
treatm ent of reversible airways obstruction. T o the solution obtained in the Assay for choline, add
P r e p a r a tio n 2 5 m L of 0.1m silver nitrate VS and warm on a water b ath for
Choline Theophyllinate Tablets 15 minutes. Cool in ice for 3 0 minutes, filter and wash the
residue with three 10 m L quantities o f water. T itrate the
D E F IN IT IO N combined filtrate and washings with 0 . 1m sodium hydroxide
C holine Theophyllinate is choline lj2,3,6-tetrahydro-l,3- VS. Each m L o f 0.1m sodium hydroxide FS is equivalent to
dim ethyl-2,6-dioxo-7i/-purin-7-ide. It contains not less than 1 8 .0 2 mg o f theophylline, C 7H 8N 4O 2.
41.9% and not more than 43.6% o f choline, C 5H 15N O 2, and
STORAGE
n ot less than 61.7% and not m ore than 65.5% of
Choline Theophyllinate should be protected from light.
theophylline, C7H9N4O2, each calculated with reference to
A white, crystalline powder. It melts betw een 187° and 192°,
★ ★
Appendix V A. Chondroitin Sulfate Sodium ★ ★
Very soluble in water, soluble in ethanol (96%); very slightly Chondroitin Sulphate Sodium *****
soluble in ether.
A. T h e light absorption, Appendix II B, in the range 2 3 0 to
3 5 0 nm of a 0 .0 0 2 % w/v solution in 0 .0 1 m sodium hydroxide
exhibits a maximum only at 2 7 5 nm . T h e absorbance at
2 7 5 nm is about 0 .8 3 . R = S 03Na and R' = H
or
B. T h e infrared absorption spectrum, Appendix II A, is R = H and R' = SOaNa
concordant with the reference spectrum o f choline
theophyllinate (RS 060).
TESTS
50 m L of a 10% w/v solution is clear, Appendix IV A, and H 20(C 14H 19N N a2014S)I
not m ore intensely coloured than reference solution GY'4 ,
A ctio n a n d u se
A ppendix IV B, M ethod I.
A d d mucopolysaccharide; treatm ent of osteoarthritis.
Carry out the m ethod for thin-layer chromatography, PhEur__________________________________________________________
A ppendix HI A, using the following solutions of the D E F IN IT IO N
substance being examined in ethanol (96%).
Natural copolymer based mainly on the 2 disaccharides: [4)-
( 1) 1. 0 % w/v o f the substance being examined. (P-D-glucopyranosyluronic a a d )-( l-> 3)- [2-(acetylamino)-2-
(2) 0 . 010 % w/v of the substance being examined. deoxy-^-D-galactopyranosyl 4-sulfate]-(l -►] and [4)-(J$-i>
glucopyranosyhironic ad d )-( 1-> 3)- [2-(acetylamino)-2-deoxy-
P-D-galactopyranosyl 6-sulfate ]-(1 -►], sodium sa lt
(a) U se as die coating silica gel HF2 5 4 .
On complete hydrolysis it liberates D-galactosamine,
(b) U se the mobile phase as described below. D-glucuronic ad d , acetic a d d and sulfuric add. It is obtained
(c) Apply 5 nL of each solution. from cartilage of both terrestrial and marine origins.
2016 Chondroitin Sulfate Sodium 1-543
D epending on the animal species of origin, it shows different viscosity range = 1 - 5 m m 2/s, internal diameter of
proportions of 4-sulfate and 6-sulfate groups. tube R = 0.53 m m , volume of bulb C = 5.6 m l , internal
C o n te n t diam eter of tube N — 2.8-3.2 mm) with a funnel-shaped
95 per cent to 105 per cent (dried substance). lower capillary end. Use the same viscometer for all
measurements; measure all outflow times in triplicate.
P R O D U C T IO N T h e test is not valid unless the results do not differ by m ore
T he animals from which chondroitin sulfate sodium is than 0.35 per cent from the mean and if the flow time t\ is
derived m ust fulfil the requirements for the health of animals n o t less than 1.6 x to and not more than 1.8 x to. If this is
suitable for hum an consumption. n o t the case, adjust the concentration of test solution (a) and
CHARACTERS repeat the procedure.
A p p e a ra n c e Calculation of the relative viscosities
W hite or almost white, hygroscopic powder. Since the densities of the chondroitin sulfate solutions and o f
S o lu b ility the solvent are almost equal, the relative viscosities Tin (being
Freely soluble in water, practically insoluble in acetone and T|ri, Tlr2311,3 and 1^ 4) can be calculated from the ratio of the
in ethanol (96 per cent). flow times for the respective solutions (being t1} t^ t3 and
14) to the flow time of the solvent to, but taking into account
the kinetic energy correction factor for the capillary
A. Infrared absorption spectrophotometry (2.2.24). (B = 30 800 s3), as shown below:
Preparation Discs o f potassium bromide R.
Comparison For chondroitin sulfate sodium o f terrestrial
origin use chondroitin sutfate sodium CRS and for chondroitin
sulfate sodium o f marine origine use chondroitin sulfate sodium
id
(marine) CRS. *° .2
co
B. Solution S I (see Tests) gives reaction (b) o f sodium
(2.3.1). Calculation of the concentrations
C. Examine the electropherograms obtained in the test for Calculate the concentration cx (expressed in kg/m3) of
related substances. chondroitin sulfate sodium in test solution (a) using the
Results T h e principal band in the electropherogram obtained following expression:
with the test solution is similar in position to the principal
band in the electropherogram obtained with reference x 100 - h
solution (a). m o p x 10 0 * - ¡o r x 1 0
TESTS
x = percentage content of chondroitin sulfate sodium as
S o lu tio n S I
determined in the assay;
. Dissolve 2.500 g in 50.0 m L of carbon dioxide-free water R.
h = loss on drying as a percentage.
S o lu tio n S2
Calculate the concentration c2 (expressed in kg/m3) of
D ilute 1.0 m L o f solution SI to 10.0 m L with water R.
chondroitin sulfate sodium in test solution (b) using the
p H (2.2.3) following expression:
5.5 to 7.5 for solution S i.
S p ecific o p tic a l ro ta tio n (2.2.7) ci x 0.75
—20 to —30 (terrestrial origin) or - 1 2 to - 1 9 (marine
origin) (dried substance), determined on solution S I. Calculate the concentration c3 (expressed in kg/m3) of
In trin s ic v isco sity chondroitin sulfate sodium in test solution (c) using the
0.01 m 3/kg to 0.15 m 3/kg. following expression:
Test solution (a) Weigh 5.000 g (mop) of the substance to be
examined and add about 80 m l. o f an 11.7 g/L solution of ci x 0.50
sodium chloride R at room temperature. Dissolve by shaking at
room tem perature for 30 min. Dilute to 100.0 m L with an Calculate the concentration c4 (expressed in kg/m3) of
11.7 g/L solution o f sodium chloride R. Filter through a chondroitin'sulfate sodium in test solution (d) using the
m em brane filter (nominal pore size 0.45 pm) and discard the following expression:
first 10 m L. T h e concentration of test solution (a) is only
indicative and m ust be adjusted after an initial measurement ci x 0.25
o f the viscosity o f test solution (a).
Calculation of the intrinsic viscosity
Test solution (b) T o 15.0 m L o f test solution (a) add 5.0 m L
o f an 11.7 g/L solution of sodium chloride R. T h e specific viscosity ti* of the test solution (being r[8l, r\s2i
and t | s4) is calculated from the relative viscosities T|n-
Test solution (c) T o 10.0 m L of test solution (a) add 10.0 m L
(being T|r l , T)r3 and Tir4) according to the following
o f an 1 1.7 g/L solution of sodium chloride R.
expression:
Test solution (d) T o 5.0 m L of test solution (a) add 15.0 m L
o f an 11.7 g/L solution o f sodium chloride R.
Vti - 1
D eterm ine the flow-time (2.2.9) for an 11.7 g/L solution o f
sodium chloride R (to) and the flow times for the 4 test T h e intrinsic viscosity [rj], defined as
solutions (rx, t^ r3 and *4), at 25.00 ± 0.03 °C. Use an
appropriate suspended level viscometer (specifications:
viscometer constant = about 0.005 m m 2/s2, kinematic ( 7)
1-544 Chondroitin Sulfate Sodium 2016
is calculated by linear least-squares regression analysis using Results Any secondary band in the electropherogram obtained
the following equation: with the test solution is no t more intense than the band in
the electropherogram obtained with reference solution (b)
(2 per cent).
— = a x fcn + [17]
C% P ro te in (2.5.33, Method 2)
M aximum 3.0 p er cent (dried substance).
C{ = concentration o f the substance to be examined Test solution Dilute 1.0 m L o f solution S I to 50.0 m L w ith
expressed in kg/m3; 0.1 M sodium hydroxide.
&h = H uggins' constant. Reference sdudons Dissolve about 0.100 g o f bovine albumin R,
R e la te d s u b s ta n c e s accurately weighed, m 0.1 M sodium hydroxide and dilute to
Electrophoresis (2.2.31). 50.0 m L with the same solvent. C a n y out all additional
Buffer solution A (0.1 M barium acetate p H 5.0). Dissolve dilutions using 0.1 M sodium hydroxide.
25.54 g o f barium acetate R in 900 m L o f water R. Adjust to C h lo rid e s (2.4.4)
p H 5.0 with glacial acetic add R and dilute to 1000.0 m L M aximum 0.5 per c e n t
w ith water R. D ilute 1 m L o f solution S2 to 15 m L with water R. D o n o t
Buffer solution B (1 M barium acetate p H 5.0). Dissolve add diluted nitric ad d . Prepare the standard using 5 m L of
255.43 g o f barium acetate R in 900 m L o f water R. Adjust to chloride standard solution (5 ppm CO R and 10 m L of water R.
p H 5.0 with glacial acetic acid R and dilute to 1000.0 m L H eav y m e ta ls (2.4.8)
w ith water R. M aximum 20 ppm .
Staining solution Dissolve 1.0 g of tohddine blue R and 2.0 g o f 1.0 g complies w ith test C . Prepare th e reference solution
sodium chloride R in 1000 m L of 0.01 M hydrochloric add using 2 m L o f lead standard solution (10 ppm Pb) R.
Filter.
Test solution Prepare a 30 mg/mL solution of the substance to M aximum 12.0 p er cent, determ ined on 1.000 g by drying in
be examined in water R.
Reference solution (a) Prepare a 30 m g/m L solution o f
chondroitin sulfate sodium CRS in water R.
TAM C: acceptance criterion 103 C FU /g (2.6.12).
Absence o f Staphylococcus aureus (2.6.13).
Reference solution (c) M ix equal volumes o f reference
solution (b) and water R. Absence o f Pseudomonas aeruginosa (2.6.13).
Procedure Allow the electrophoresis support to cool the plate Absence o f Escherichia cdi (2.6.13).
to 10 °C. Pre-equilibrate the agarose gel for 1 min in buffer Absence o f Salmonella (2.6.13).
solution A. Remove excess liquid by careful decanting. Absence o f bile-tolerant gram-negative bacteria (2.6.13).
D ry the gel for approximately 5 min. Place 400 m L o f buffer
A SSAY
solution B into each o f the containers o f the electrophoresis
equipm ent. Transfer 1 pL o f each solution to the slots of the Test solution (a) Weigh 0.100 g (m{) o f the substance to be
agarose gel. Pipette a few millilitres o f a 50 per cent V/V examined, dissolve in water R and dilute to 100.0 m L with
solution o f glycerol R onto the cooled plate of the the same solvent.
electrophoresis equipm ent and place the gel in the middle of Test solution (b) Dilute 5.0 m L o f test solution (a) to
the ceramic plate. Place a wick, saturated with buffer 50.0 m L with water R
solution B, at the positive and negative sides of the agarose Reference solution (a) Weigh 0.100 g (mo) o f chondroitin sulfate
gel. Ensure that there is good contact between the sodium CRS, previously dried as described in the test for loss
electrophoresis buffer and th e agarose gel. Perform the on drying, dissolve in water R and dilute to 100.0 m L with
electrophoresis under the following conditions: 75 mA/gel, the same solvent
resulting in a voltage o f 100-150 V (maximum 300-400 V) Reference solution (b) D ilute 5.0 m L o f reference solution (a)
for a gel o f about 12 cm x 10 cm. Carry out the to 50.0 m L with water R.
electrophoresis for 12 min. Place the gel in a mixture
Titrcmt solution (a) W dgh 4.000 g o f cetylpyridimurn chloride
consisting o f 10 volumes o f anhydrous ethand R and
monohydrate R and dilute to 1000 m L with water R.
90 volumes o f buffer solution A for 2 min. C any o u t the
electrophoresis for 20 min. Place the gel in a mixture Titrcmt sdution (b) Weigh 1.000 g o f cetylpyridxrdian chloride
consisting o f 30 volumes o f anhydrous ethand R and monohydrate R and dilute to 1000 m L with water R.
70 volumes o f buffer solution A for 2 min. Carry o u t the Perform either visual o r photom etric titration as follows:
electrophoresis for 20 m in. Stain the gel in the staining Visual titration Titrate 40.0 m L o f reference solution (a) and
solution for 10 min. D estain the gel for 15 min under 40.0 m L o f test solution (a) with titrant solution (a).
running tap water followed by 10-15 m in with wetter R until T h e solution becomes turbid. At the end point, the liquid
the band in the electropherogram obtained with reference appears d e ar, with an ahnost-white pred p itate in suspension.
solution (c) is visible. Allow the gel to dry. T h e predpitate is more apparent if 0.1 m L o f a 1 p er cent
System suitability: solution o f methylene blue R is added before starting the
— the electropherogram obtained w ith reference solution (c) titration. T h e predpitated partides are more apparent against
shows a visible band; the blue background.
— the band in the electropherogram obtained w ith reference Photometric titration T itrate 50.0 m L o f reference solution (b)
solution (b) is clearly visible and similar in position to the and 50.0 m L o f test solution (b) with titrant solution (b).
band in the electropherogram obtained with reference T o determine the end point, use a suitable autotitrator
solution (a).
2016 Chorionic Gonadotrophin 1-545
equipped with a phototrode at a suitable wavelength (none is ASSAY

critical) in the visible range. T he potency of chorionic gonadotrophin is estimated by
Calculate the percentage content o f chondroitin sulfate comparing under given conditions its effect of increasing the
sodium using the following expression: mass of the seminal vesicles (or the prostate gland) of
immature rats with the same effect of the International
vi x mo 100 Standard of chorionic gonadotrophin or of a reference
x x Z preparation calibrated in International Units.
vo x m i 100 — h
T he International U nit is the activity contained in a stated
Vo = volume o f appropriate titrant solution when amount of the International Standard, which consists of a
titrating the appropriate reference solution, in mixture of a freeze-dried extract of chorionic gonadotrophin
millilitres; from the urine of pregnant women with lactose.
= volume of appropriate titrant solution when T he equivalence in International Units o f the International
titrating the appropriate test solution, in millilitres; Standard is stated by the W orld Health Organization.
h = loss on drying of the substance to be examined, as Use immature male rats o f the same strain, 19 to 28 days
a percentage; old, differing in age by n o t more than 3 days and having
Z = percentage content o f H 20 (C i 4H i 9N N a 20 i 4S)x in body masses such that the difference between the heaviest
chondroitin sulfate sodium CRS. and the lightest rat is not more than 10 g. Assign the rats at
random to 6 equal groups of at least 5 animals. I f sets of
STORAGE
6 litter mates are available, assign one litter m ate from each
set to each group and mark according to litter.
L A B E L L IN G Choose 3 doses o f the reference preparation and 3 doses of
T he label states the origin o f the substance (marine or the preparation to be examined such that the smallest dose is
terrestrial). sufficient to produce a positive response in some of the rats
PhEur and the largest dose does not produce a m axim al response in
all the rats. Use doses in geometric progression and as an
initial approximation total doses of 4 IU , 8 IU and 16 IU
may be tried although the dose will depend on th e sensitivity
J r* * ★ of the anim als used, which may vary widely.
★
Chorionic Gonadotrophin ★ ★ Dissolve separately the total quantities o f the preparation to
(Ph Eur monograph 0498) ***** be examined and of the reference preparation corresponding
to the daily doses to be used in sufficient phosphate-albumin
A ctio n a n d u se buffered saline pH 7.2 R such that the daily dose is
Gonadotrophic hormone. administered in a volume of about 0.5 mL. Add a suitable
antimicrobial preservative such as 4 g/L o f phenol or
P r e p a r a tio n
0.02 g/L of thiomersal. Store the solutions at 5 + 3 °C.
Chorionic G onadotrophin Injection
Inject subcutaneously into each rat the daily dose allocated to
PhEur__________________________________ its group, on 4 consecutive days at the same tim e each day.
D E F IN IT IO N O n the 5th day, about 24 h after the last injection, euthanise
the rats and remove the seminal vesicles. Remove any
Chorionic gonadotrophin is a dry preparation of placental
glycoproteins which have luteinising activity. It is extracted extraneous fluid and tissue and weigh the vesicles
from the urine of pregnant women. T he potency is not less immediately. Calculate the results by the usual statistical
than 2500 IU/mg. methods, using the mass of the vesicles as the response. (The
precision of the assay may be improved by a suitable
P R O D U C T IO N correction of the organ mass with reference to the body mass
Chorionic gonadotrophin is extracted using a suitable of the animal from which it was taken; an analysis of
fractionation procedure. It is either dried under reduced covariance may be used).
pressure or freeze-dried.
T he estimated potency is not less than 80 per cent and not
CHARACTERS more than 125 per cent o f the stated potency.
A p p e a ra n c e The confidence limits (P = 0.95) of the estimated potency
White or yellowish-white, amorphous powder. are not less than 64 per cent and not more than 156 per cent
of the stated potency.
S o lu b ility
Soluble in water. ST O R A G E
ID E N T IF IC A T IO N In an airtight, tam per-proof container, protected from light at
a temperature of 2 °C to 8 °C. I f the substance is sterile,
W hen administered to immature rats as prescribed in the
store in a sterile, airtight» tam per-proof container.
assay, it causes an increase in the mass o f the seminal vesicles
and o f the prostate gland. LA B ELLIN G
TESTS The label states:
— the num ber o f International Units per container,
W a te r (2.5.52)
— the potency in International U nits per milligram.
M axim um 5.0 per c en t
B a c te ria l en d o to x in s (2.6.14)
_____________________________________ PhEur
Less than 0.02 IU per IU o f chorionic gonadotrophin, if
intended for use in the manufacture of parenteral
removal of bacterial endotoxins.
1-546 Chymotrypsin 2016
S p ecific a b so rb a n c e (2.2.25)
Chymotrypsin ?***
18.5 to 22.5, determined at the absorption maximum at
(Ph. Eitr. monograph 0476) * 281 nm ; maximum 8, determ ined at the absorption
minim um at 250 nm.
9004-07-3
Dissolve 30.0 m g in 0.001 M hydrochloric add and dilute to
A c tio n a n d u se 100.0 m L with the same acid.
Proteolytic enzyme. T ry p sin
Substrate solution T o 98.5 m g o f tosylargmine methyl ester
PhEur—_________________________________________
hydrochloride R, suitable for assaying trypsin, add 5 m L o f
D E F IN IT IO N tris(hydroxymethyl) aminomethane buffer solution pH 8.1 R and
Chymotrypsin is a proteolytic enzyme obtained by the swirl to dissolve. Add 2.5 m L of methyl red mixed solution R
activation o f chrymotrypsinogen extracted from the pancreas and dilute to 25.0 m L w ith water R.
of b eef (Bos taurus L .). It has an activity o f not less than Test solution T ransfer to a depression in a white spot-plate
5.0 microkatals per milligram. In solution it has maximal 0.01 m l, o f tris(kydroxymethyl)aminomethane buffer solution
enzymic activity at about p H 8; the activity is reversibly pH 8.1 R and 0.1 m L o f solution S. Add 0.2 m L o f the
inhibited at p H 3, the p H at which it is most stable. substrate solution.
P R O D U C T IO N Reference solution A t the sam e time and in the same m anner .
T he animals from which chymotrypsin is derived m ust fulfil as for the test solution, prepare a solution using the
the requirem ents for the health o f animals suitable for hum an substance to be examined to which n o t more than
consum ption. Furtherm ore, the tissues used shall n o t include 1 per cent m/m o f trypsin BRP has been added.
any specified risk material as defined by any relevant Start a timer. N o colour appears in the test solution within
international or, where appropriate, national legislation. 3-5 min after the addition o f the substrate solution. A purple
T he m ethod o f m anufacture is validated to demonstrate that colour is produced in the control solution.
the product, if tested, would comply with the following test. L oss o n d ry in g (2.2.32)
H is ta m in e C2.6.10) N o t m ore than 5.0 per cent, determ ined on 0.100 g by
N ot m ore than 1 |ig (calculated as histamine base) per drying at 60 °C at a pressure no t exceeding 0.7 kPa for 2 h.
5 microkatals o f chymotrypsin activity. Before carrying out
A SSAY
the test, heat the solution o f the substance to be examined on
T he activity o f chymotrypsin is determ ined by comparing the
a w ater-bath for 30 min.
rate at which it hydrolyses acetykyrosine ethyl ester R with the
CHARACTERS rate at which chymotrypsin BRP hydrolyses the same substrate
A p p e a ra n c e under the same conditions.
White or alm ost white, crystalline or amorphous powder, Apparatus U se a reaction vessel o f about 30 m L capacity
hygroscopic if amorphous. provided with:
S o lu b ility — a device that will maintain a tem perature o f
Sparingly soluble in water. 25.0 ± 0.1 °C;
— a stirring device, for example a magnetic stirrer;
— a lid with holes for the insertion o f electrodes, the tip o f a
A. D ilute 1 m L of solution S (see Tests) to 10 m L with
burette, a tube for the admission o f nitrogen and the
water R .J n a depression in a white spot-plate, mix 0.05 m L introduction o f reagents.
of this solution with 0.2 m L of the substrate solution.
An autom atic or m anual titration apparatus may be used.
A purple colour develops.
F o r the latter, the burette is graduated in 0.005 m L and the
Substrate solution T o 24.0 m g of acetykyrosine ethyl ester R add p H m eter is provided with a wide scale and glass-calomel or
0.2 m L o f ethanol (96 per cent) R and swirl to dissolve.
glass-silver-silver chloride electrodes.
Add 2.0 m L o f 0.067 Mphosphate buffer solution pH 7.0 R
and 1 m L o f methyl red mixed solution R and dilute to Test solution Dissolve 25.0 m g o f the substance to be
10.0 m L w ith water R. examined in 0.001 M hydrochloric add and dilute to
250.0 m L with the same acid.
B. D ilute 0.5 m L of solution S to 5 m L with water R.
Add 0.10 m L o f a 20 g/L solution of Reference solution Dissolve 25.0 m g o f chymotrypsin BRP in
tosylphenylaUtnylchloromethane R in ethanol (96 per cent) R. 0.001 M hydrochloric acid and dilute to 250.0 m L with the
same ad d .
Adjust to p H 7.0 and shake for 2 h. In a depression in a
white spot-plate, mix 0.05 m L o f this solution with 0.2 m l. Store the solutions at 0-5 °C. W arm 1 m L o f each solution
o f the substrate solution (see Identification test A). N o colour to about 25 °C over 15 min and use 50 (iL of each solution
develops within 3 min o f mhring, (corresponding to about 25 nanokatals) for each titration.
Carry out the titration in an atmosphere o f nitrogen. Transfer
TESTS
10.0 m L o f 0.01 M caldum chloride solution R to the reaction
S o lu tio n S
vessel and, while stirring, add 0.35 m L o f 0.2 M acetykyrosine
Dissolve 0.10 g in carbon dioxide-free water R and dilute to ethyl ester R. W hen the tem perature is steady at
25.0 ± 0.1 °C (after about 5 m in), adjust to p H 8.0 exactly
A p p e a ra n c e o f so lu tio n with 0.02 M sodium hydroxide. A dd 50 (iL o f the test solution
Solution S is n o t m ore opalescent than reference (equivalent to about 5 pg o f the substance to be examined)
suspension II (2.2.1). and start a timer. M aintain at p H 8.0 by the addition o f
p H (2.2.3) 0.02 M sodium hydroxide, noting the volume added every
3.0 to 5.0 for solution S. 30 s. Calculate the volume o f 0.02 M sodium hydroxide used
per second between 30 s and 210 s. Carry out a titration in
2016 Ciclesonide 1-547
the sam e m anner u sin g the reference solution and calculate B. Examine the chromatograms obtained in the assay.
the volume of 0.02 M sodium hydroxide used per second. Results T h e principal peak in the chromatogram obtained
Calculate the activity in microkatals per milligram using the with the test solution is similar in retention time and size to
following expression: the principal peak in the chromatogram obtained with
m' x V TESTS
x A
m xV ’ Related substances
mass o f the substance to be examined, in
Test solution Dissolve 50.0 mg of die substance to be
m illigram s;
examined in anhydrous ethanol R and dilute to 50.0 m L with
m' mass o f chymotrypsin BRP, in milligrams;
the same solvent.
V volume of 0.02 M sodium hydroxide used per
second by the test solution; Reference solution (a) Dissolve 50.0 mg o f ciclesonide CRS in
V volume of 0.02 M sodium hydroxide used per anhydrous ethanol R and dilute to 50.0 m L with the same
second by the reference solution; solvent.
A activity o f chymotrypsin BRP, in microkatals per Reference solution (b) Dissolve 3 mg o f ciclesonide
milligram. impurity B CRS, 3 mg o f ciclesonide impurity C CRS and 5 mg
o f ciclesonide containing impurity A CRS in anhydrous ethanol R
STORAGE and dilute to 10.0 m L with the same solvent.
In an airtight container at 2 °C to 8 °C, protected from light.
Reference solution (c) Dissolve 50 m g o f the substance to be
L A B E L L IN G examined in anhydrous ethanol R, add 1.0 m L of reference
The label states: solution (b) and dilute to 50.0 m L with anhydrous ethanol R.
— the quantity o f chymotrypsin and the total activity in Reference solution (d) Dilute 1.0 m L of the test solution to
microkatals per container; 100.0 m L with anhydrous ethanol R. Dilute 1.0 m L of this
— for the amorphous substance, that it is hygroscopic. solution to 10.0 m L with anhydrous ethanol R.
PhEur Column:
— size. I = 0.25 m, 0 = 4.6 mm;
— stationary phase: phenylsQyl silica gel for chromatography R
(5 nm);
*****
Ciclesonide ★ ★ Mobile phase water R, anhydrous ethanol R (38:62 VIV).
(Ph. Eur. monograph 2703) **★** Flow rate 1.0 mL/min.
Injection 20 (xL of the test solution and reference solutions (c)
and (d).
Run time 2.2 times the retention time of ciclesonide.
with reference solution (c) to identify the peaks due to
impurities A, B and C.
Relative retention W ith reference to ciclesonide (retention
impurity C = about 0.9; impurity A = about 1.4.
C 32H 44O7 540.7 12654447-6 System suitability, reference solution (c):
A ctio n a n d u se impurity C and ciclesonide.
Glucocorticoid.
PhEur____________ — for each impurity, use the concentration of ciclesonide in
reference solution (d).
D E F IN IT IO N
Limits:
(2'R)-2 '-Cyclohexyl- 11 J}-hydroxy-3,20-dioxo-l 6$H- — impurity A: maximum 1.0 per cent;
[l,3]dioxolo[4',5/:16,17]pregna-l,4-dien-21-yl
— impurities B, C: for each impurity, maximum
2-methylpropanoate.
0.15 per cent;
C o n te n t — unspecified impurities:, for each impurity, maximum
98.0 per cent to 102.0 per cent (anhydrous substance). 0.10 per cent;
CHARACTERS — total of unspecified impurities:, maximum 0.2 per cent;
A p p e a ra n c e — total: maximum 1.2 per cent;
W hite or yellowish-white, crystalline powder. — reporting threshold: 0.05 per cent.
Solubility Heavy metals (2.48)

Practically insoluble in water, freely soluble to soluble in M aximum 20 ppm.
acetone and in anhydrous ethanol. Solvent mixture water R, ethanol (96 per cent) R (15:85 VIV).
ID E N T IF IC A T IO N 0.250 g complies with test H. Prepare the reference solution
using 0.5 m L of lead standard solution (10 ppm Pb) R.
Comparison ciclesonide CRS.
1-548 Ciclopirox 2016
W a te r (2.5.12)
Ciclopirox ★**★ ★
M axim um 0.5 per cent, determined on 0.500 g. ★ ★
(Ph. Em. monograph 1407) *****
A SSA Y
Run time 1.6 times the retention time o f dclesonide.
— symmetry factor, maximum 2.2 for the peak due to C 12H 17N O 2 207.3 29342-05-0
dclesonide.
Calculate the percentage content o f C 32H 44O 7 taking into
Antifungal.
account the assigned content of dclesonide CRS.
IM P U R IT IE S PhEtr.
Specified impurities A, B, C. D E F IN IT IO N
6-Cyclohexyl-1-hydroxy-4-methylpyridin-2 (l/f)-o n e .
C o n te n t
CHARACTERS
A p p e a ra n c e
W hite o r yellowish-white, crystalline powder.
S o lu b ility
Slightly soluble in water, fredy soluble in anhydrous ethanol
and in methylene chloride.
A. (2 'S)-2 '-cydohexyl -1 1^-hydroxy-3,20-dioxo-16 $H-
[1,3] dioxolo[4 ',5 ': 16,17]pregna-l,4-dien-21-yl First identification B.
2-m ethylpropanoate (S-epimer of dclesonide), Second identification A , C.
A. M d tin g point (2.2.14): 140 °C to 145 °C.
Comparison ciclopirox CRS.
Reference solution Dissolve 20 m g o f ciclopirox CRS in
B. (2 'R)-2 '-cyclohexyl-1 113,21-dihydroxy- 16$H- Plate TLC silica gel F254 plate R.
[ 1,3] dioxolo [4 ',5 ' : 16 ,17] pregna-1,4-diene-3,20-dione,
Pretreatment Before use, predevelop with the mobile phase
until the solvent front has migrated to the top o f the plate.
Allow to dry in air for 5 min.
Mobile phase concentrated ammonia R, water R, ethanol
(96 per cent) R (10:15:75 V/V/V).
Application 10 (iL.
Drying In air for 10 mini
Detection A Examine in ultraviolet light at 254 nm .
Results A T h e prindpal spot in the chrom atogram obtained
C. (2'i?)-2'-[(l.RS)-cyclohex-3-enyi]-l 1 fi-hydroxy-3,20- w ith the test solution is sim ilar in position and size to the
dioxo-16 $H- [1,3] dioxolo [4 ',5 ' : 16,17] pregna-1,4-dien-21-yl principal spot in the chromatogram obtained with the
2-methylpropanoate. reference solution.
PhEtr
Detection B Spray with a 20 g/L solution of ferric chloride R in
anhydrous ethanol R.
Results B T h e prindpal spot in the chrom atogram obtained
the principal spot in the chromatogram obtained with the
reference solution.
2016 Ciclopirox 1-549
TESTS — impurities B, C at 298 nm: for each impurity, not more

A p p e a ra n c e o f so lu tio n than the area of the peak due to impurity B in the
T he solution is clear (2.2.1) and not more intensely coloured chromatogram obtained with reference solution (b)
than reference solution Y 5 (2.2.2, Method IT). (0.5 per cent);
Dissolve 2.0 g in methanol R and dilute to 10 m L with the — unspecified impurities at 298 nm: for each impurity, not
same solvent more than the area of the peak due to im purity B in the
R elated su b stan c es
(0.10 p er cent);
Liquid chromatography (2.2.29). Cany out the test avoiding
— sum of impurities other than B at 298 nm: not more than
exposure to actinic light. AH materials in direct contact with the
the area of the peak due to impurity B in the
substance to be examined like column materials, reagents, solvents,
etc. should contain only very low amounts of extractable metal
(0.5 per cent);
cadons.
— disregard limit at 298 nm: 0.5 times the area o f the peak
Solvent mixture acetoniarile R, mobile phase (10:90 VIV). due to impurity B in the chromatogram obtained with
Test solution Dissolve 30.0 mg of the substance to be reference solution (c) (0.05 per cent).
examined in 15 m L of the solvent mixture, using an H eav y m e ta ls (2.4.8)
ultrasonic bath if necessary, and dilute to 20.0 m L with the M aximum 10 ppm .
solvent mixture.
Reference solution (a) Dissolve 15.0 mg o f ciclopirox using 2 m l, of lead standard solution (10 ppm Pb) R.
impurity A CRS and 15.0 mg of ciclopirox impurity B CRS in
the solvent mixture and dilute to 10.0 m L with the solvent L oss o n d ry in g (2.2.32)
mixture. M aximum 1.5 per cent, determined on 1.000 g by drying
in vacuo at 60 °C over diphosphorus pentoxide R.
to 200.0 m L with the solvent mixture. S u lfa te d a s h (2. 4.14)
to 10.0 m L with the solvent mixture. ASSAY
Reference solution (d) M ix 5.0 m L of reference solution (a) Dissolve 0.150 g in 20 m L of methanol R. Add 20 m L of
and 5.0 m L of the test solution. water R and titrate with 0.1 M sodium hydroxide, determining
Column: the end-point potentiometrically (2.2.20). Carry out a blank
— size. I = 0.08 m , 0 = 4 mm; titration.
— stationary phase. nitrUe silica gel for chromatography R2 1 m L o f 0.1 M sodium hydroxide is equivalent to 20.73 m g of
(5 pm). C 12H 17N O 2.
In order to ensure desorption o f interfering metal ions, every STO RA G E
new colum n is to be rinsed with the rinsing solution over a Protected from light.
period o f no t less than 15 h and then with the mobile phase
IM P U R IT IE S
for not less than 5 h at a flow rate of 0.2 m l 7min.
Specified impurities A, B, C.
Rinsing solution glacial acetic add R, acetylacetone R,
acetomtrUe R, water R (0.1:0.1:50:50 VfVIVIV).
Mobile phase glacial acetic acid R, acetomtrUe R, 0.96 g/L
solution of sodium edetate R (0.01:23:77 V/VfV).
I O and enanbomer
—
Detection Spectrophotometer at 220 nm and at 298 nm . H3C
solutions (b), (c) and (d); inject the solvent mixture as a A. [(52?S)-3-cyclohexyl-5-methyl-4,5-dihydro-l,2-oxazol-5-
blank. yl] acetic acid,
Run time 2.5 times the retention time of ciclopirox.
Retention time Ciclopirox = 8 min to 11 min; if necessary
adjust the ratio of the 0.96 g/L solution of sodium edetate to o
acetonitrile in the mobile phase.
Relative retention W ith reference to ciclopirox:
impurity A = about 0.5; impurity C = about 0.9; ch 3
B. 6-cyclohexyl-4-methyl-2//-pyran-2-one,
ciclopirox and impurity B in the chromatogram obtained
with reference solution (d);
— symmetry factor. 0.8 to 2.0 for the principal peak in the o
chrom atogram obtained with the test solution.
Limits:
— impurity A at 220 nm: not more than the area of the ch 3
reference solution (b) (0.5 per cent); C. 6-cyclohexyl-4-methylpyridin-2(lii)-one.
PhEur
1-550 Ciclopirox Olamine 2016
★* ★ Results B T he principal spot in the chrom atogram obtained

Ciclopirox Olamine ★ ★
★ ★ with the test solution is similar in position, colour and size to
(Ph. E u t. monograph 1302) * * * * * the principal spot in the chrom atogram obtained with the
reference solution.
Detection C Spray the 2nd second plate with ninhydrin
solution R. H eat at 110 °C until the spots appear.
H,N Results C T h e principal spot in the chrom atogram obtained
the principal spot in the chrom atogram obtained with the
reference solution.
c 14h 24n 2o 3 268.4 41621-49-2 TESTS

A c tio n a n d u se A p p e a ra n c e o f so lu tio n
Antifungal T h e solution is clear (2.2.1) and not m ore intensely coloured
P hEur.
D E F IN IT IO N same solvent.
6-Cyclohexyl-1-hydroxy-4-methylpyridin-2( lii)-o n e and
p H (2.2.3)
2-am inoethanol.
8.0 to 9.0.
C o n te n t Dissolve 1.0 g in carbon dioxide-free water R and dilute to
— ciclopirox (C 12H 17N O 2; M t 207.3): 76.0 per cent to 100 m L with the same solvent.
78.5 per cent (dried substance);
— 2 -aminoethanol (C 2H 7N O ; Mt 61.1): 22.2 per cent to
Liquid chromatography (2.2.29). Carry out the test avoiding
23.3 per cent (dried substance).
exposure to actinic light All materials in direct contact with the
CHARACTERS substance to be examined, such as column materials, reagents,
A p p e a ra n c e solvents, etc. should contain only small amounts of extractable
W hite or pale yellow, crystalline powder. metal cations.
S o lu b ility Solvent mixture acetonitrile R, mobile phase (10:90 V!V).
Sparingly soluble in water, very soluble in ethanol Test solution Dissolve 40.0 m g o f the substance to be
(96 per cent) and in methylene chloride, slightly soluble in examined (corresponding to about 30 m g o f ciclopirox) in a
ethyl acetate, practically insoluble in cyclohexane. mixture o f 20 |iL o f anhydrous acetic acid R, 2 m L of
It shows polymorphism (5.9). acetonitrile R, and 15 m L o f the mobile phase, vising an
ID E N T IF IC A T IO N ultrasonic bath if necessary. Dilute the solution to 20.0 m L
Second identification B Reference solution (a) Dissolve 15.0 mg o f ciclopirox
impurity A CRS and 15.0 m g o f ciclopirox impurity B CRS in a
mixture o f 1 m L o f acetonitrile R and 7 m L o f the mobile
Comparison ciclopirox olamine CRS. phase, and dilute to 10.0 m L with the mobile phase.
If the spectra obtained in the solid state show differences, Reference solution (b) Dilute 1.0 m L o f reference solution (a)
dissolve the substance to be exam in ed and the reference to 200.0 m L with the solvent mixture.
substance separately in the minim um volume o f ethyl Reference solution (c) Dilute 2.0 m L of reference solution (b)
acetate R , evap orate to dryn ess o n a w ater-bath and record
Reference solution (d) Mix 5.0 m L of réference solution (a)
and 5.0 m L o f the test solution.
Test solution Dissolve 25 m g o f the substance to be examined Column:
in methanol R and dilute to 10 m L with the same solvent. — size. 1= 80 m m , 0 = 4 mm;
Reference solution Dissolve 25 mg o f ciclopirox olamine CRS in — stationary phase. mtrUe silica gel for chromatography R
methanol R and dilute to 10 m L with the same solvent. (5 Jim).
Plate TLC silica gel F2 5 4 plate R. In order to ensure desorption o f interfering metal ions, every
Pretreatment Before use, predevelop 2 plates with the mobile new colum n is to be rinsed with the rinsing solution over a
phase until the solvent front has migrated to the top o f the period o f n o t less than 15 h and then with th e mobile phase
plates. Allow to dry in air for 5 min. for not less than 5 h at a flow rate of 0.2 mL/min.
Mobile phase concentrated ammonia R, water R, anhydrous Rinsing solution acetylacetone R, anhydrous acetic acid R,
ethanol R (10:15:75 VIV/V). acetonitrile R, water R (0.1:0.1:50:50 V/VIVIV).
Application 10 |iL. Mobile phase anhydrous acetic acid R, acetonitrile R, 0.96 g/L
Development Over 2/3 of the plate. solution o f sodium edetate R (0.01:23:77 VIVIV).
Drying In air for 10 min. Flow rate 0.7 mL/min.
Detection A Examine in ultraviolet light at 254 m Detection Spectrophotom eter at 220 nm and at 298 nm .
Results A T h e principal spot in the chromatogram obtained Irgecdon 10 pL o f the test solution and reference
w ith the test solution is similar in position and size to the solutions (b), (c) and (d).
principal spot in the chrom atogram obtained with the Run time 2.5 times die retention time of ciclopirox.
reference solution.
Detection B Spray 1 plate with ferric chloride solution R3.
2016 Ciclosporin 1-551
Retendon time Ciclopirox = 8 min to 11 min; if necessary

adjust the ratio of die 0.96 g/L solution o f sodium edetate to and enantiomer
acetonitrile in the mobile phase.
Relative retention W ith reference to ciclopirox:
im purity A = about 0.5; impurity C = about 0.9;
A. [(5i?5)-3-cyclohexyl-5-methyl-4,5-dihydro-l,2-oxazol-5-
im purity B = about 1.3. yl] acetic acid,
System suitability At 298 nm:
— resolution: m in im u m of 2.0 between the peaks due to
im purity B and ciclopirox in the chromatogram obtained
CL JQ
w ith reference solution (d);
— symmetry factor. 0.8 to 2.0 for the principal peak in the
chrom atogram obtained with the test solution.
Limits'.
— impurity A at 220 nm: n o t more than the area of the
corresponding peak in the chromatogram obtained with B. 6-cyclohexyl-4-methyl-2ii-pyran-2-one,
— impurities B, C at 298 nm: for each impurity, not more
rhan the area o f the peak due to impurity B in the
(0.5 per cent);
— unspecified impurities at 298 nm: for each impurity, not
m ore than the area of the peak due to impurity B in the
chromatogram obtained with reference solution (c) C. 6-cyclohexyl-4-methylpyridin-2 ( lii)-o n e.
( 0.10 per cent); PhEur
— sum of impurities other than B at 298 nm: n o t more than
the area of the peak due to impurity B in the
(0.5 per cent);
Ciclosporin ★★* * ★
— disregard limit at 298 nm: 0.5 times the area of the peak ★ ★
due to impurity B in the chromatogram obtained with
M axim um 20 ppm . (♦Ala —o-Ala-*■MeLeu —MeLeu —MeVal- N.,H CH3
*- MeLeu Val MeLeu MeGly Abu
using 2 mT. o f lead standard solution (10 ppm Pb) R.
under high vacuum.
C62HmNu 0 12 1203 59865-13-3
M axim um 0.1 per cent, determined on 1.0 g. Action and use
A SSA Y C aldneurin inhibitor; immunosuppressant.
2 -A m in o e th a n o l Preparation
Dissolve 0.250 g in 25 m L o f anhydrous acetic add R. Titrate Ciclosporin Eye Drops
w ith 0.1 M perchloric add, determining the end-point Sterile Ciclosporin Concentrate
Ciclosporin O ral Solution
1 m L o f 0.1 M perchloric add is equivalent to 6.108 m g of
C 2H 7N O . PhEur.
Ciclopirox DEFINITION
Dissolve 0.200 g in 2 m L o f methanol R. Add 38 m L of Cyclo[[(25,3i?,4i?,6£)-3-hydroxy-4-methyl-2-
water R, swirl and titrate immediately with 0.1 M sodium (methylamino) oct-6-enoyI] -L-2-aminobutanoyl-iV-
hydroxide, determining the end-point potentiometrically methylglycyl-iV'-methyl-L-leucyl-L-valyl-iV'-methyl-L-leucyl-L-
(2.2.20). Carry out a blank titration. alanyl-D-alanyl-iV'-methyl-L-leucjd-iV'-methyl-L-leucyl-iV'-
U se 0.1 M sodium hydroxide, the titre of which has been methyl-L-valyl] (ddosporin A).
d eterm in ed under the conditions prescribed above using Substance produced by Beauveria rwuea (Tofypodadium
0.100 g of benzoic add RV. inflatum Gams) or obtained by any other means.
1 m L o f 0.1 M sodium hydroxide is equivalent to 20.73 mg of Content
c I2h I7n o 2. 97.0 per cent to 102.0 per cent (dried substance).
STORAGE CHARACTERS
Protected from light. Appearance
IM P U R IT IE S White or alm ost white powder.
Spedfied impurities A , B, C Solubility
ethanol and in methylene chloride.
1-552 Ciclosporin 2016
ID E N T IF IC A T IO N — total: not m ore than 1.5 times the area o f the principal
A Infrared absorption spectrophotom etry (2.2.24). peak in the chrom atogram obtained with reference
Comparison ciclosporin CRS. solution (b) (1.5 p er cent);
8 . Examine the chromatograms obtained in the assay.
Results The principal peak in the chromatogram obtained (0.05 per cent).
with the test solution is similar in retention time to the
principal peak in the chrom atogram obtained with reference H e av y m e ta ls (2.48)
solution (a). M axim um 20 ppm .
T h e residue obtained in the test for loss on drying complies
TESTS
with test C. Prepare the reference solution using 2 m L of
A p p e a ra n c e o f so lu tio n lead standard solution (10 ppm Pb) R.
T he solution is clear (2.2.1) and n o t m ore intensely coloured
than reference solution Y5, BY 5 or R 7 (2.2.2, Method II). L oss o n d ry in g (2.2.32)
M axim um 2.0 per cent, determ ined on 1.000 g at 60 °C at a
Dissolve 1.5 g in anhydrous ethanol R and dilute to 15 m L
pressure not exceeding 15 P a for 3 h.
Less than 0.84 IU/m g, if intended for use in the m anufacture
- 1 9 3 to - 1 8 5 (dried substance).
of parenteral preparations w ithout a further appropriate
Dissolve 0.125 g in methanol R and dilute to 25.0 m L with procedure for the removal o f bacterial endotoxins. Dissolve
the same solvent. 50 m g of the substance to be examined in a mixture of
R e la te d su b s ta n c e s 280 m g of ethanol (96 per cent) R and 650 m g o f
Liquid chrom atography (2.2.29). polyoxyethylated castor où R and dilute to the required
Solvent mixture acetonitrUe R, water R (50:50 VIV). concentration using water for BET.
Test solution Dissolve 30.0 m g of the substance to be A SSA Y
examined in the solvent mixture and dilute to 25.0 m L with Liquid chromatography (2.2.29) as described in the test for
the solvent mixture. rd a te d substances with the following modifications.
Reference solution (a) Dissolve 30.0 m g of ciclosporin CRS in Injection T est solution and reference solution (a).
the solvent m ixture and dilute to 25.0 m L with the solvent System suitability: reference solution (a):
mixture. — repeatability, m axim u m rdative standard deviation o f
Reference solution (b) D ilute 2.0 m L o f reference solution (a) 1.0 p er cent after 6 injections.
to 200.0 m L with the solvent mixture. Calculate the percentage content o f C ^ H m N n O ^ taking
Reference solution (c) Dissolve the contents o f a vial of into account the assigned content o f ddosporin CRS.
ciclosporin for system suitability CRS in 5.0 m l. of the mobile STORAGE
phase.
Column: is sterile, store in a sterile, airtight, tam per-proof container.
— size: I = 0.25 m , 0 = 4 mm ;
— stationary phase: octadecylsilyl silica gel for chromatography R IM P U R IT IE S
(3-5 pm);
R
r*- Ala—D-Ala—MeLeu—MeLeu—MeVal - N H CH3
T he column is connected to the injection port by a steel l 4 5 y *1 V .
capillary tube about 1 m long, having an internal diam eter o f *- MeLeu * Val-«- MeLeu * M e G l y * Abu—/ /> |
0.25 m m and m aintained at 80 °C. n 7 OH k,
Mobile phase phosphoric acid R, 1,1-dimethyleihyl methyl ether R,
acetonitrile R, water R (0.1:5:43:52 VIVIV/V).
Flow rate 1.5 mlVmin. A. different d dosporins [difference from d d o sp o rin
Detection Spectrophotom eter a t 210 nm . (R = C H 3: d d o sp o rin A)]: d d o sp o rin B [7-L-Ala];
Injection 20 p L o f the test solution and reference solutions (b) d d o sp o rin C [7-L-Thr]; d d o sp o rin D [7-L-VaI];
and (c). d d o sp o rin E [5-L-VaIJ; d d o sp o rin G [7-(l-2-
Run time 1.7 times the retention time o f ddosporin. aminopentanoyl)]; d d o sp o rin H [5-o-MeVal]; d d o sp o rin L
System suitability: reference solution (c): [R = H ]; d d o sp o rin T [4-L-Leu]; d d o sp o rin U [11-L-Leu];
— retention time, d d o sp o rin = 25 min to 30 min; d d o sp o rin V [1-L-Abu],
if necessary, adjust the ratio o f acetonitrile to water in the
ch3
mobile phase;
(-"-Ala—o-AJa— MeLeu— MeLeu— MeVal- N H CH3
— peak-to-vaUey ratio: minim um 1.4, where Hp = h d g h t
above the baseline o f the peak due to d d o sp o rin U and L
*- MeLeu * Val MeLeu * MeGly Abu -
Hv = hdgjht above the baseline of the lowest point o f the
ddosporin; if necessary, adjust the ratio of
1, 1-dimethylethyl methyl ether to acetonitrile in the
mobile phase. B. [6-[(2S,3£,4£)-3-hydroxy-4-m ethyl-2-
Limits: (methylamino)octanoic ad d ]]d d o sp o rin A,
any impurity: for each impurity, n o t m ore than 0.7 times C . isoddosporin A.
PhEur
2016 Cilastatin Sodium 1-553
Reference solution (c) Dissolve 3 m g o f cilastatin for system

Cilastatin Sodium /* * * * suitability 2 CRS (containing impurities C and G (epimer 1))
*+ +* in water R and dilute to 2.0 mL with the same solvent.
(Ph. Eicr. monograph 1408) *
Reference solution (d) Dissolve 32 m g o f mesityl oxide R
HO!C> ^ s^ ^ ^ r 0O!NacHi (impurity D ) in 100.0 m L of water R. Dilute 1.0 m L of the
solution to 50.0 m L with water R.
H hn { V ' ch) Column:
o — sizer. I = 0.25 m, 0 = 4.6 mm;
Ci6H 25N2Na05S 380.4 81129-83-1
chromatography compatible with 100 per cent aqueous mobile
phases R (5 |im);
A ctio n a n d use — temperature: 50 °C.
Dehydropeptidase-I inhibitor; inhibition of the renal Mobile phase:
metabolism o f imipenem. — mobile phase A: phosphate buffer solution pH 3.25 R;
— mobile phase B: acetonitrüe R l, phosphate buffer solution
PhEtr__ _______________________________________________________ pH 3.25 R (50:50 VIV)',
D E F IN IT IO N
Sodium (2)-7-[f(22^-2-anmo-2-carboxyethyI]sulfanyl]-2- Time Mobile phase A Mobile phase B
[ [ [(l<S)-2,2-dimethylcyclopropyI] carbonyl] amino]hept-2- (min) (per cent V/V) (per cent V/V)
enoate. 0-3 100 0
C o n te n t 3 - 28 100->90 0 -> 10
28 -3 8 90 10
CHARACTERS
38-63 90->50 10->50
A p p e a ra n c e
White or light yellow, amorphous, hygroscopic powder. 63 -7 8 50-» 30 50->70
S o lu b ility 78-88 30 70
Very soluble in w ater and in methanol, slightly soluble in
anhydrous ethanol, very slightly soluble in dimethyl sulfoxide,
practically insoluble in acetone and in methylene chloride. Flow rate 2.0 mL/min.
ID E N T IF IC A T IO N Detection Spectrophotometer at 210 nm.
A. Specific optical rotation (see Tests). Injection 20 jiL.
B. Infrared absorption spectrophotometry (2.2.24). Identification of impurities Use the chromatogram supplied
Comparison cilastatin sodium CRS. with cilastatin for system suitability 1 CRS and the
C. It gives reaction (a) of sodium (2.3.1).
identify the peaks due to impurities A, B, E, F, G (epimer 2)
TESTS and H ; use the chromatogram supplied with cilastatin for
S o lu tio n S system suitability 2 CRS and the chromatogram obtained with
Dissolve 1.0 g in carbon dioxide-free water R and dilute to reference solution (c) to identify the peaks due to impurities
100 m L with the same solvent. C and G (epimer 1); use the chromatogram obtained with
A p p e a ra n c e o f so lu tio n reference solution (d) to identify the peak due to impurity D .
Solution S is clear (2.2.1) and not more intensely coloured Relative retention W ith reference to cilastatin (retention
than reference solution Y 6 (2.2.2, Method II). time = about 50 min): impurity E = about 0 .2; impurity A
(epimer 1) = about 0.60; impurity A (epimer
p H (2.2.3)
2) = about 0.62; impurity D = about 0.9;
impurity F = about 0.98; impurity G (epimer 1) = about
S pecific o p tical ro ta tio n (2.2.7) 1.02; impurity G (epimer 2) = about 1.05;
+ 41.5 to + 44.5 (anhydrous substance). impurity H = about 1.06; impurity B = about 1.17;
Dissolve 0.250 g in a mixture of 1 volume o f hydrochloric impurity C = about 1.23.
acid R and 120 volumes of methanol R, then dilute to System suitability:
25.0 m L with the same mixture of solvents. — peak-to-vaUey ratio: minimum 10, where Hp = height
R e la te d su b sta n c e s above the baseline o f the peak due to impurity F and
Liquid chromatography (2.2.29). Prepare the solutions Hv = height above the baseline of the lowest point of the
immediately before use. curve separating this peak from the peak due to cilastatin
Test solution Dissolve 32 mg of the substance to be examined in the chromatogram obtained w ith reference solution (b);
in water R and dilute to 20.0 m L with the same solvent — peak-to-vaUey ratio: minimum 2.5, where Hp = height
above the baseline of the peak due to impurity G
(epimer 1) and Hv = height above the baseline of the
100.0 m L with water R. Dilute 1.0 m L of this solution to
lowest point of the curve separating this peak from the
peak due to cilastatin in the chromatogram obtained with
Reference solution (b) Dissolve 3 mg of cilastatin for system reference solution (c).
suitability 1 CRS (co n taining impurities A, B, E, F , G
(epimer 2) and H ) in water R and dilute to 2.0 m L with the
— for all impurities, use the concentration of cilastatin in
same solvent.
1-554 Cilastatin Sodium 2016
— correction factors: multiply the peak areas o f the following Ru = ratio of the area o f the solvent peak to the area of
impurities by the corresponding correction facto r the propanol peak in the chrom atogram obtained
im purity C = 1.3; im purity E = 3.3; impurity G with the test solution;
(epimer 1) and impurity G (epimer 2) = 1.6. Rs = ratio o f the area o f the solvent peak to the area of
Limits: the propanol peak in the chrom atogram obtained
— impurities A (sum of the epimers): m aximum 0.5 per cent; with the reference solution.
— impurity C: maximum 0.4 per cent; Limits:
— impurities E: maximum 0.3 p er cent; — acetone: maximum 1.0 per cent m/m;
— impurities B, F, H: for each impurity, m axim um — methanoh maximum 0.5 per cent m/m;
0.1 per cent; — impurity D: maximum 0.4 per cent m/m.
— impurity G: for each epimer, m axim um 0.1 per cent; H e a v y m e ta ls (2.4.8)
M axim um 10 ppm .
0.05 per cent;
— total: m axim um 1.0 per cent;
Solvent methanol R.
— reporting threshold: 0.03 per cent; disregard any peak due 1.0 g complies with test H . Prepare the reference solution
to im purity D in the chrom atogram obtained with using 1 m l. o f lead standard solution (10 ppm Pb) R.
reference solution (d). W a te r (2.5.12)
Impurity D , acetone and methanol M aximum 2.0 per cent, determ ined on 0.500 g.
Gas chrom atography (2.2.25). B a c te ria l e n d o to x in s (2.6.14)
Internal standard solution Dissolve 0.5 m l. of propanol R in Less than 0.17 IU/mg, if intended for use in the m anufacture
water R and dilute to 1000 m l. with the same solvent. o f parenteral preparations without a further appropriate
Test solution Dissolve 0.200 g o f the substance to be procedure for the removal o f bacterial endotoxins.
examined in water R, add 2.0 m l. o f the internal standard A SSA Y
solution and dilute to 10.0 m L with water R. Dissolve 0.100 g in 30 m L o f methanol R and add 5 m L of
Reference solution Dissolve 2.0 m l. o f acetone R, 0.5 m L o f water R. A dd 0.1 M hydrochloric acid to a p H o f about 3.0.
methanol R and 0.5 m L of mesityl oxide R (impurity D ) in Carry out a potentiom etric titration (2.2.20), using 0.1 M
water R and dilute to 1000 m L with the same solvent sodium hydroxide. T hree jum ps of potential are observed.
T o 2.0 m L o f this solution add 2.0 m L o f the internal Read the volume added between the 1st and the 3rd point of
standard solution and dilute to 10.0 m L with water R. This inflexion.
solution contains 316 jig o f acetone, 79 jig of m ethanol and 1 m l. o f 0.1 M. sodium hydroxide is equivalent to 19.02 mg
86 ng o f im purity D p er millilitre. o f C i 6H 25N 2N a 0 5S.
Column:
STO RA G E
In an airtight container, at a tem perature o f 2 °C to 8 °C.
— size. I = 30 m , 0 = 0.53 mm;
I f the substance is sterile, store in a sterile, airtight, tam per
proof container.
1.0 jim).
Carrier gas helium for chromatography R. IM P U R IT IE S
Flow rate 9 mL/min. Specified impurities A, B, C, D , E, F , G, H
Temperature:
H02CV ^ s
*'« H
H NH2 o
Tbne Temperature
and epimer at S
(min) CC)
Column 0 - 23 50
A. (Z)-7- [(i&S)- [(2i?)-2-amino-2-carboxyethyl] sulfinyl] -2-
2.5 - 5 50 -> 70 [ [[(15)-2,2-dimethylcydopropyI] carbonyl] amino] hept-2-enoic
5-5.5 70 add,
Injection port 160
Detector 220

Irgection 1 jiL.
B. (Z)-l-[ [(2i?)-2-carboxy-2-[[(1 iiS )-1-methyl-3-
Calculate the percentage contents o f acetone, m ethanol and
oxobutyl] amino] ethyl] sulfanyl] -2- [[[(1 S)-2,2-
im purity D using the following expression:
dimethylcyclopropyl] carbonyl] amino] hept-2-enoic ad d ,
c = concentration o f the solvent in the reference

solution, in )ig/mL;
w = quantity o f cilastatin sodium in the test solution, in C. (2)-7-[[(2i?)-2-carboxy-2-[(l,l-dim ethyl-3-
milligrams; oxobutyl)amino] ethyl] sulfanyl] -2- [[[(1 S)-2,2-
dimethylcyclopropyl]carbonyl] amino]hept-2-enoic ad d ,
2016 Cilazapril 1-555
( h3
c
S o lu b ility
Slightly soluble in water, freely soluble in m ethanol and in
h 3c c' h 3
methylene chloride.
0
D . 4-methylpent-3-en-2-one (mesityl oxide), A. Infrared absorption spectrophotometry (2.2.24).
H02C>^ \ S^ S^ ^ ^ V^ C 0 2H Comparison cUazaprU CRS.
H NH2 o
B. Specific optical rotation (see Tests).
TESTS
E. 7-[[(2i?)-2-amino-2-carboxyethyl] sulfanyl] - 2-oxoheptanoic Sp ecific o p tica l ro ta tio n (2.2.7)
add, — 383 to — 399 (anhydrous substance).
HOjC COjH Dissolve 0.200 g in 0.067 M phosphate buffer solution
pH 7.0 R, with the aid o f ultrasound if necessary, and dilute
to 50.0 m L with the same buffer solution. Carry o u t the
d eterm ination at 365 nm .
I m p u rity A
F. (2)-7-[[(2i?)-2-ainino-2-carboxyethyi] sulfanyl] -2-[(2,3- in methanol R and dilute to 5.0 m L with the same solvent.
dimethyIbut-3-enoyl)amino]hept-2-enoic ad d ,
Reference solution (a) Dissolve 2 mg of cilazapril
impurity A CRS in methanol R and dilute to 50.0 mT. with the
HO2C
same solvent
Reference solution (b) Dissolve 5 m g of cilazapril
impurity A CRS and 5 m g of the substance to be examined in
and epimer at C*
methanol R and dilute to 10.0 m L with the same solvent.
G. (£)-(2&S)-7-[[(2i?)-2-araino-2-carboxyethyl]sulfanyI]-2-
[ [[(15)-2,2-dimethylcydopropyI] carbonyl] amino]hept-3-enoic Mobile phase glacial acetic add. R, water R, hexane R,
add, methanol R, ethyl acetate R (5:5:15:15:60 V/VfV/V/V).
Application 5 pL.
h 2n ^ c o 2h
Drying In a current of cold air for 10 m in .
Detection Spray with a freshly prepared mixture o f 1 volume
o f potassium iodobismuthate solution R and 10 volumes o f dilute
H . (Z )-l- [(2-aminoethyI) sulfanyl]-2- [[ [( 1S)-2,2- acetic acid R and then with dilute hydrogen peroxide solution R.
dimethylcyclopropyi] carbonyl] amino] hept- 2 -enoic ad d . System suitability: reference solution (b):
PhEur
Lanit.
— 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).
★ ★
Cilazapril ★ ★ R e la te d su b sta n c e s
***** Liquid chromatography (2.2.29).
examined in the mobile phase and dilute to 50.0 mT. with
, HjO
the mobile phase.
C22H3xN305>H20 435.5 92077-78-6
Reference solution (b) Dissolve 5.0 mg of cUazaprU
A ctio n a n d u se impurity D CRS in the test solution and dilute to 10.0 m L
Angiotensin converting enzyme inhibitor. with the test solution.
Column:
PhEtr_________________________________________________________ — sizer. I = 0.25 m, 0 = 4.6 mm;
D E F IN IT IO N — stationary phase: octadecylsOyl silica gel for chromatography R
(15j95)-9-[[(15)-l -(Ethoxycarbonyl) -3-phenylpropyI] amino] - (5 pm).
10-oxooctahydro- 6//-pyridazino [1,2-a] [ l, 2]diazepine-l- Mobile phase Mix 10 volumes o f triethylamine R and
carboxylic a d d monohydrate. 750 volumes o f water R, adjust to p H 2.30 with phosphoric
C o n te n t add R, and add 200 volumes o f tetrahydrofuran R.
98.5 p er cent to 101.5 per cent (anhydrous substance). Flow rate 1.0 mL/min.
CHARACTERS Detection Spectrophotometer at 214 nm.
A p p e a ra n c e Injection 20 pL.
White o r almost white, crystalline powder.
1-556 Cimetidine 2016
Run time Twice the retention tim e o f cilazapril; when

im purity A is present, it may be necessary to continue the
chrom atography until it is eluted.
Relative retendon W ith reference to cilazapril:
im purity B = about 0.6; im purity D = about 0.9;
im purity C = about 1.6; im purity A = 4 to 5.
D . (lS,9S)-9-[[(2?)-l-(ethoxycarbonyl)-3-
System suitability: reference solution (b): phenylpropyl] amino] - 10-oxooctahydro- 6//-pyridazino- [ 1, 2-
— resolution: m in im u m 2.5 betw een the peaks due to a] [1,2] diazepine- 1-carboxylic ad d .
im purity D and cilazapril;
PhEur
— symmetry factor, maxim um 3.0 for the peak due to
cilazapril.
Limits:
— impurity B: n o t m ore than the area of the principal peak
in the chrom atogram obtained with reference solution (a) Cimetidine *****
★ ★
(0.5 per cent);
— impurity D: not m ore than 0.4 times the area o f the (Ph Eur. monograph 0756) *****
— impurity C: not m ore than 0.2 times the area of the
principal peak in th e chrom atogram obtained with
chrom atogram obtained w ith reference solution (a) c 10h 16n 6s 252.3 51481-61-9
(0.10 per cent);
— total: n o t m ore th an twice the area o f the principal peak in Action and use
th e chrom atogram obtained with reference solution (a) Histamine H 2 receptor antagonist; treatm ent o f peptic
(1 per cent); ulceration.
— disregard limit. 0.1 times th e area of the principal peak in Preparations
th e chrom atogram obtained with reference solution (a) Cim etidine Injection
(0.05 per cent); disregard any peak due to impurity A. Cim etidine Oral Solution
W a te r (2.5.12) Cimetidine Oral Suspension
3.5 p er cent to 5.0 p er cent, determ ined on 0.300 g.
Cim etidine Tablets
M aximum 0.1 per cent, determ ined on 1.0 g. PhEur___________________________________________________________
A SSA Y DEFINITION
Dissolve 0.300 g in 10 m L o f anhydrous ethanol R and add 2-C yano-1-methyl-3- [-2 -[ [(5-methyl-1//-im idazol-4-
50 m L of water R. T itrate w ith 0.1 M sodium hydroxide, yl) methyl] sulfanyl] ethyl] guanidine.
determ ining the end-point potentiometrically (2.2.20). Carry Content
out a blank titration. 98.5 per cent to 101.5 p er cent (dried substance).
1 m L o f 0.1 M sodium hydroxide is equivalent to 41.75 mg of CHARACTERS
C 22H 31N 3O 5.
Appearance
STO R A G E W hite or almost white powder.
Protected from light. Solubility
IM P U R IT IE S Slightly soluble in water, soluble in ethanol (96 p er cent),
Specified impurities A, B, C, D practically insoluble in methylene chloride. It dissolves in
dilute mineral adds.
Second identification A, C
A. M d tin g point (2.2.14): 139 °C to 144 °C.
A. R = C (C H 3)3, R ' = C 2H 5: 1,1-dimethylethyl (15,95)-9- If necessary, dissolve the substance to be examined in
[[(5)-1 -(ethoxycarbonyi)-3-phenyipropyl] amino] -10- 2-propanol R, evaporate to dryness and determ ine the m dting
oxooctahydro- 6H -pyridazino[l, 2 -a] [l, 2 ]diazepine-l- point again.
carboxylate, B. Infrared absorption spectrophotom etry (2.2.24).
B. R = R ' = H : (l5,95)-9-[[(5)-l-carboxy-3- Comparison cimetidine CRS.
phenylpropyl] am ino]- 10-oxooctahydro- 6 if-pyridazino [ 1,2 - If th e spectra obtained in the solid state show differences,
a ][ l, 2 ]diazepine-l-carboxylic a d d , dissolve die substance to be examined and the reference
C. R = R ' = C 2H 5: ethyl (l5 ,9 5 )-9 -[[(5 )-l- substance separately in 2-propanol R, evaporate to dryness
(ethoxycarbonyl)-3-phenylpropyl] amino] -10-oxooctahydro- and record new spectra using the residues.
6/f-pyridazino [ 1,2 -<z] [ l , 2]diazepine-l-carboxylate, C. Thin-layer chrom atography (2.2.27).
2016 Cimetidine 1-557
Test solution Dissolve 10 mg o f the substance to be examined identification CRS and the chromatogram obtained with
in methanol R and dilute to 10 m L with the same solvent reference solution (c) to identify the peak due to impurity F.
Reference solution Dissolve 10 m g of cimetidine CRS in Relative retention W ith reference to cimetidine (retention
methanol R and dilute to 10 m L with the same solvent. time = about 18 min): impurity G = about 0.2;
Plate TLC silica gel GF 254 plate R. impurity E = about 0.4; impurity D = about 1.5;
impurity C = about 1.6; impurity B = about 2 .0 ;
Mobile phase concentrated ammonia R, methanol R, ethyl
impurity H = about 2.3; impurity F = about 4.6.
acetate R (15:20:65 V/V/V).
Application 5 |iL.
Development Over 3/4 o f the plate. impurities D and C.
Drying In a current of cold air. Limits:
Detection Expose to iodine vapour until maximum contrast — correction factors: for the calculation o f content, multiply
has been obtained and examine in ultraviolet light at the peak areas o f the following impurities by the
254 nm. corresponding correction factor: impurity C = 2.5;
Results T he principal spot in the chromatogram obtained with impurity D = 3.3; impurity E = 0.7; impurity G = 0.6.
the test solution is similar in position and size to the principal — impurities B, C, D, E, F, G, H: for each impurity, not
spot in the chromatogram obtained with the reference more than the area o f the principal peak in the
solution. chromatogram obtained with reference solution (a)
(0.2 per cent);
TESTS — unspecified impurities, for each impurity, not more than
Appearance o f solution 0.5 times the area o f the principal peak in the
(0.10 per cent);
Dissolve 3.0 g in 12 m L o f 1 M hydrochloric acid and dilute — total: n o t m ore than 5 times the area o f the principal peak
to 20 m L with water R. in the chromatogram obtained with reference solution (a)
Related substances ( 1.0 per cent);
Liquid chromatography (2.2.29). — disregard limit: 0.25 times the area o f the principal peak in
Test solution Dissolve 20 m g of the substance to be examined the chromatogram obtained with reference solution (a)
in mobile phase A and dilute to 50.0 m L with mobile (0.05 per cent).
phase A. H eav y m e ta ls (2.4.8)
Reference solution (a) Dilute 1.0 m L of the test solution to M aximum 20 ppm .
100.0 m L with mobile phase A. Dilute 2.0 m L of this 1.0 g complies w ith test C. Prepare the reference solution
solution to 10.0 m L with mobile phase A using 2 m L o f lead standard solution (10 ppm Pb) R.
Reference solution (b) Dissolve the contents o f a vial o f L oss o n d ry in g (2.2.32)
cimetidine for system suitability CRS (containing impurities 6 , M aximum 0.5 per cent, determined on 1.000 g by drying in
C , D , E, G and H ) in 1.0 m L o f mobile phase A. an oven at 105 °C.
Reference solution (c) Dissolve 4 mg of cimetidine for peak S u lfa te d a s h (2.4.14)
identification CRS (containing impurity F) in mobile phase A M aximum 0.2 per cent, determined on 1.0 g.
A SSAY
Column:
Dissolve 0.200 g in 60 m L of anhydrous acetic acid R T itrate
— size: I = 0.25 m , 0 = 4.6 mm;
with 0.1 M perchloric acid determ inin g the end point
1 m L o f 0.1 M perchloric acid is equivalent to 25.23 mg of
Mobile phase A M ix 0.4 volumes of dieihylamine R and
c 10H16N6s
780 volumes o f a 1.1 g/L solution of sodium
hexanesulfonaxe R j adjust to p H 2.8 with phosphoric add R; STORAGE
add 250 volumes of methanol R2; Protected from light.
Mobile phase B methanol R2; IM P U R IT IE S
Specified impurities B, C , D , E, F , G, H
Time Mobile phase A Mobile phase B Other detectable impurities (the following substances would, if
(min) (per cent V/V) (per cent V/V) present at a sufficient level, be detected by one or other o f
0 -6 0 100 0 the tests in the monograph. They are limited by the general
6 0 -6 5 100*90 0 * 10 acceptance criterion for other/unspecified impurities and/or
65-120 90 10
Flow rate 1.1 mL/min. Control of impurities in substances for pharmaceutical use): A , I,
Irgection 50 |il.
with cimetidine for system suitability CRS and the
identify the peaks due to impurities B, C, D , E, G and H ;
use the chromatogram supplied with cimetidine for peak
1-558 Cimetidine Hydrochloride 2016
★*★
★ ★
Cimetidine Hydrochloride ★ ★
(Ph. Ever, monograph 1500) *****
A. methyl 3-cyano-1- [2- [[(5-methyl-1if-im idazol-4-

yl)methyl] sulfanyl] ethyl] carbamimidothioate, Ha
R1
I
N
HN
C 10H 17C1N6S 288.8 70059-30-2
H
H3C Action and use
H istamine H 2 receptor antagonist; treatm ent o f peptic
B. R I = C N , R2 = 0 -C H 3, X = S: methyl 3-cyano- 1-[ 2- ulceration.
[ [(5-methyl-1H-imidazol-4- Preparations
yl) methyl] sulfanyl] ethyl] carbamimidate, Cimetidine Injection
C. R I = C O -N H * R 2 = N H -C H 3, X = S:
1- [(m ethylamino) [[2- [ [(5-methyl-l //-im idazol-4- PhEur.
y 1) methyl] sulfanyl] ethyl] amino] methylidene] urea, D E F IN IT IO N
D . R I = H , R 2 = N H -C H 3, X = S: l-m ethyl-3-[2- 2-C yano-1-methyl-3- [-2- [[(5-methyi- 1if-im idazol-4-
[ [(5-methyl-1H-imidazol-4- yQmethyl] sulfanyl] ethyl] guanidine hydrochloride.
yl)methyl] sulfanyl] ethyl] guanidine,
Content
E. R I = C N , R2 = N H -C H 3, X = SO: 2-cyano - 1-methyl- 98.5 p e r cent to 101.5 per cent (dried substance).
3 [2- [ [(5-methyl- 1/i-im idazol-4-
CHARACTERS
yl) methyl] sulfinyl] ethyl] guanidine,
Appearance
S o lu b ility
Freely soluble in water, sparingly soluble in anhydrous
ethanol.
F. 2-cyano-1,3-bis [2- [ [(5-methyl- lH-imidazol-4- Second identffication A , C, D
yl) methyl] sulfanyl] ethyl] guanidine, A. Ultraviolet and visible absorption spectrophotom etry
(2.2.25).
, cn
N Test solution Dissolve 70 m g in 0 . 2 M sulfuric acid and dilute
h3C. . JL .CHj
N N
to 100.0 m L with the same ad d . D ilute 2.0 m L o f this
H H solution to 100.0 m L with 0 . 2 M sulfuric add.
Specific absorbance at the absorption maximum at 218 nm
G. 2-cyano-1 , 3 -dimethylguanidine, 650 to 705.
Comparison dmeudme hydrochloride CRS.
C . Thin-layer chromatography (2.2.27).
Reference solution Dissolve 10 mg o f dmetidine
hydrochloride CRS in methanol R and dilute to 10 m L w ith the
H . 1,1 '-(disulfanediyldiethylene)bis(2-cyano-3- same solvent.
methylguanidine), Plate TLC silica gel GF2 5 4 plate R.
/^ n acetate R (15:20:65 VIVIV).
HN
R1 Application 5 pL.
R2 Development Over 3/4 o f the plate.
Drying In a current o f cold air
I. R I = O H , R 2 = C 2H 5: (5-ethyl-1//-im idazol-4- Detection Expose to iodine vapour until maximum contrast
yl)methanol, has been obtained and examine in ultraviolet light at
J. R I = S-C H z-C H z-N H * R2 = C H 3: 2-[[(5-m ethyl-lH - 254 nm .
imidazol-4-yl)methyl] sulfanyl] ethanamine. Results T he principal spot in the chromatogram obtained with
PhEur the test solution is similar in position and size to the principal
spot in the chrom atogram obtained with the reference
solution.
2016 Cimetidine Hydrochloride 1-559
D . It gives reaction (a) of chlorides (2.3. 1). Limits:

TESTS — correction factors: for the calculation of content, multiply
corresponding correction factor: impurity C = 2.5;
impurity D = 3.3; impurity E = 0.7; impurity G = 0.6;
— impurities B, C, D, E, F, G, H: for each impurity, not
Dissolve 3.0 g in 12 m L of 1 M hydrochloric acid and dilute
more than the area o f the principal peak in the
to 20 m L with water R. chromatogram obtained with reference solution (a)
p H (2.2.3) (0.2 per cent);
4.0 to 5.0. — unspecified impurities: for each impurity, not more than
Dissolve 100 mg in carbon dioxide-free water R and dilute to 0.5 times the area of the principal peak in the
10.0 m L with the same solvent. chromatogram obtained with reference solution (a)
(0.10 per cent);
— total: not m ore than 5 times the area of die principal peak
Test solution Dissolve 20 mg of the substance to be examined ( 1.0 per cent);
in mobile phase A and dilute to 50.0 m L with mobile
phase A.
Reference solution (a) Dilute 1.0 m L of the test solution to (0.05 per cent).
100.0 m L with mobile phase A. Dilute 2.0 m L of this
M aximum 20 ppm.
cimetidine for system suitability CRS (containing impurities B,
C 3 D , Ej G and H ) in 1.0 m L of mobile phase A.
Reference solution (c) Dissolve 4 mg of cimetidine for peak
identification CRS (containing impurity F) in mobile phase A
an oven at 105 °G.
Column: S u lfa te d a sh (2.4.14)
— size: I = 0.25 m , 0 = 4.6 mm; M aximum 0.2 per cent, determ ined on 1.0 g.
— stationary phase: end-capped octadecylsüyl silica gel for A SSAY
chromatography R (5 pm). Dissolve 0.200 g in a mixture o f 5 m L of 0.01 M hydrochloric
Mobile phase A M ix 0.4 volumes of diethylamine R and acid and 50 m L of ethanol (96 per cent) R. Carry out a
780 volumes o f a 1.1 g/L solution of sodium potentiometric titration (2.2.20), using 0.1 M sodium
hexanesulfonate R. Adjust to p H 2.8 with phosphoric acid R hydroxide. Read the volume added between the 2 points o f
and add 250 volumes of methanol R2-, inflexion.
Mobile phase B methanol R2', 1 m L o f 0.1 M sodium hydroxide is equivalent to 28.88 m g of
C ioH i 7C1N6S.
Time Mobile phase A Mobile phase B STORA GE
(min) (per cent V7V) (per cent V/V) Protected from light.
0 -6 0 100 0
IM P U R IT IE S
6 0 -6 5 100*90 0 *10 Specified impurities B, C, D , E, F , G, H
65 - 120 90 10 Other detectable impurities (the following substances would, if
Flow rate 1.1 mlVmin. acceptance criterion for other/unspecified impurities and/or
Detection Spectrophotom eter at 220 nm. by the general monograph Substances for pharmaceutical use
Injection 50 pL. (2034). It is therefore not necessary to identify these
Control of impurities in substances for pharmaceutical use): A , I,
with cimetidine for system suitability CRS and the
chromatogram obtained with reference solution (b) to J■
identify the peaks due to the impurities B, C , D , E, G and
CN
H; use the chromatogram supplied with cimetidine for peak
/^ N N
identification CRS and the chromatogram obtained with
reference solution (c) to identify die peak due to impurity F.
Relathe retention W ith reference to cimetidine (retention
time = about 18 min): impurity G = about 0.2;
impurity E = about 0.4; impurity D = about 1.5; A. methyl 3-cyano-1- [2- [[(5-methyl- lii-im idazol-4-
im purity C = about 1.6; impurity B = about 2.0; yl) methyl] sulfanyl] ethyl] carbamimidothioate,
im purity H = about 2.3; impurity F = about 4.6.
System suitability-, reference solution (b):
impurities D and C.
1-560 Cinchocaine Hydrochloride 2016
***
★ ★
Cinchocaine Hydrochloride ★ ★
(PL Eur. monograph 1088) * * * * *
B. R1 = C N , R 2 = 0 - C H 3, X = S: methyl 3-cyano-l-[2- h3c

[ [(5-methyl-1i7-imidazol-4- HCI
yl)methyl] sulfanyl] ethyl] carbamimidate,
C. R1 = C O -N H * R2 = N H -C H 3, X = S:
l-[(m ethylam ino)[[2-[[(5-m ethyl-lii-im idazol-4-
yl) methyl] sulfanyl] ethyl] amino] methylidene] urea,
D . R 1 = H , R2 = N H -C H 3, X = S: l-m ethyl-3-[2- C 20H 30ClN 3O 2 379.9 61-12-1
[ [(5-methyl-1H-imidazol-4-
yl) methyl] sulfanyl] ethyl] guanidine,
Local anaesthetic.
E. R1 = C N , R2 = N H -C H 3, X = SO: 2-cyano-l-m ethyl-
3 [2-[ [(5-methyl- l#-im idazol-4- PhEur______________
yl) methyl] sulfinyl] ethyl] guanidine,
D E F IN IT IO N
Cinchocaine hydrochloride contains n o t less than
98.5 per cent and n o t more than the equivalent of
101.0 per cent o f 2-butoxy-Ar-[2-
(diethylamino)ethyl]quinoline-4-carboxamide hydrochloride,
CHARACTERS
F. 2-cyano - 1,3-bis [2-[[(5-methyl-lH-imidazol-4-yl)methyl]-
A white or almost white, crystalline powder o r colourless
sulfenyl] ethyl] guanidine.
crystals, hygroscopic, very soluble in water, freely soluble in
acetone, in alcohol and in methylene chloride.
_,CN
N It agglomerates very easily.
H3Cv
N
JL NxCH3 ID E N T IF IC A T IO N
H H
First identification B, E.
G. 2-cyano-1,3-dimethylguanidine,
A. Dissolve 60.0 m g in 1 M hydrochloric add and dilute to
100 m L with the same ad d . D ilute 2 m L o f the solution to
100 m L with 1 M hydrochloric add. Exam ined between
220 nm and 350 n m (2.2.25), the solution shows two
absorption maxima, at 246 nm and 319 nm . T h e ratio of the
absorbance m easured at 246 nm to that m easured a t 319 nm
is 2.7 to 3.0.
B. Examine by infrared absorption spectrophotom etry
H . 1,1 '-(disulfanediyldiethylenejbis (2-cyano-3- (2.2.24), comparing with the spectrum obtained with
methylguanidine), cinchocaine hydrochloride CRS. Examine the substances
prepared as discs using potassium chloride R.
/^ N C. Examine the chromatograms obtained in the test for
HN related substances. T he principal spot in the chromatogram
R1
obtained with test solution (b) is similar in position and size
R2
to the principal spot in the chromatogram obtained with
I. R l = O H , R2 = C 2H 5: (5-ethyl-1H-imidazol-4-
D . Dissolve 0.5 g in 5 m L o f water R. A dd 1 m L o f dilute
yl)methanol,
ammonia R2. A white preapitate is form ed. Filter, wash the
J. R l = S -C H 2-C H 2-N H 2, R 2 = C H 3: 2-[[(5-m ethyl-lH - preapitate with five quantities, each o f 10 m l,, o f water R
imidazol-4-yl) methyl] sulfanyl] ethanamine. and dry in a desiccator. It m d ts at 64 °C to 66 °C (2.2.14).
_____________________________________ PhEur E. It gives reaction (a) o f chlorides (2.3.1).
TESTS
S o lu tio n S
Dissolve 5.0 g in carbon dioxide-five water R prepared from
distilled water R, and dilute to 50 m L with the same solvent.
Solution S is d e a r (2.2.1) and not more intensdy coloured
p H (2.2.3)
D ilute 10 m L o f solution S to 50 m L with carbon dioxide-free
water R. T he pH o f the solution is 5.0 to 6.0.
2016 Cineole 1-561
R e la te d su b stan c es B. R1 = R2 = O H: 2-hydroxyquinoline-4-carboxylic acid,

Examine by thin-layer chromatography (2.2.27), using as the C. R1 = O H , R 2 = N H -[C H 2] 2-N (C 2H 5)2: N-[2-
coating substance a suitable silica gel with a fluorescent (diethylamino)ethyl]-2-hydroxyquinoline-4-carboxamide,
indicator having an optimal intensity at 254 run.
D . R1 = 0 -[C H 2] 3-C H 3, R2 = O H: 2-butoxyquinoline-4-
Test solution (a) Dissolve 0.20 g of the substance to be carboxylic add.
examined in methanol R and dilute to 5 m L w ith the same
PhEur
solvent.
Test solution (b) Dilute 1 m L o f test solution (a) to 10 m L
with methanol R.
Reference solution (a) Dissolve 20 mg o f cinchocaine ★*★
Cineole ★ ★
hydrochloride CRS in methanol R and dilute to 5 m L with the ★ ★
same solvent. (Ph. Eur. monograph 1973) *****
Reference solution (b) Dilute 1 m L of test solution (b) to
20 m l, w ith methanol R.
Reference solution (c) Dilute 1 m L of test solution (b) to
50 m L w ith methanol R.
Reference solution (d) Dissolve 20 mg o f benzocaine CRS in
methanol R and dilute to 5 m L with the same solvent. Dilute
1 m L o f the solution and 1 m L o f reference solution (a) to CioH 180 154.3 470-82-6
20 m l, w ith methanol R. PhEur____
Apply separately to the plate 5 pL of each solution. Develop
D E F IN IT IO N
over a path of 15 cm using a mixture of 1 volume of
1,3,3-Trimethyl-2-oxabicyclo[2.2.2] octane.
ammonia R, 5 volumes o f methanol R, 30 volumes of
acetone R and 50 volumes of toluene R. Dry the plate in a CHARACTERS
current o f warm air for 15 min. Examine in ultraviolet light A p p e a ra n c e
at 254 run. Any spot in the chromatogram obtained with test Clear colourless liquid.
solution (a), apart from the principal spot, is n o t more S o lu b ility
intense th an the principal spot in the chromatogram obtained Practically insoluble in water, miscible with alcohol and with
with reference solution (b) (0.5 p er cent) and at most one methylene chloride.
such spot is more intense than the spot in the chromatogram
It solidifies at about 0.5 °C.
obtained with reference solution (c) (0.2 per cent). T he test
is not valid unless the chromatogram obtained with reference ID E N T IF IC A T IO N
solution (d) shows two clearly separated spots. A. Refractive index (see Tests).
H eavy m e ta ls (2.4.8) B. Thin-layer chromatography (2.2.27).
12 m L o f solution S complies with test A for heavy metals Test solution Dilute 1 m L of solution S (see Tests) to 25 m L
(20 ppm ). Prepare the reference solution using lead standard with alcohol R.
solution (2 ppm Pb) R. Reference solution Mix 80 mg of cineole CRS with alcohd R
L oss o n d ry in g (2.2.32) and dilute to 10 m L with the same solvent.
N ot m ore than 2.0 per cent, determined on 0.500 g by Plate TLC silica gel plate R.
drying in vacuo at 60 °C.
Mobile phase ethyl acetate R, toluene R (10:90 VIV) .
S u lfa te d a s h (2.4.14) Application 2 (iL.
N ot m ore than 0.1 per cent, determined on 1.0 g.
ASSA Y Drying In a current of cold air.
Dissolve 0.300 g in a mixture o f 15.0 m L of 0.01 M
Detection Spray with anisaldehyde solution R, heat at
hydrochloric add and 50 m L of alcohol R. C any out a 100-105 °C for 5 min.
potentiom etric titration (2.2.20), using 0.1 M sodium
hydroxide. Read the volume added between the two points of Results T h e prindpal spot in the chromatogram obtained with
inflexion. the test solution is similar in position, colour and size to the
prindpal spot in the chromatogram obtained w ith the
reference solution.
C 20H 30CIN 3O 2.
C. T o 0.1 m L add 4 m L of sulfuric add R. An orange-red
STORA GE colour develops. Add 0.2 mL o f formaldehyde solution R.
Store in an airtight container, protected from light. T h e colour changes to deep brown.
IM P U R IT IE S TESTS
S o lu tio n S
Dilute 2.00 g to 10.0 m L with alcohol R.
Solution S is clear (2.2.1) and colourless (2.2.2, Method I).
C h ira l im p u ritie s
T h e optical rotation (2.2.7) of solution S is -0 .1 0 ° to
+ 0 . 10°.
A. R 1 = Cl, R 2 = N H -[C H 2] 2-N (C 2H 5) 2: 2-chloro-N-[ 2 - R efra c tiv e in d e x (2.2.6)
(diethylamino)ethyl] quinoline-4-carboxamide,
1.456 to 1.460.
1-562 Cinnamic Acid 2016
R e la te d su b s ta n c e s IM P U R IT IE S
Gas chrom atography (2.2.28).
ch 3
Internal standard solution Dissolve 1.0 g o f camphor R in
heptane R and dilute to 200 m L with the same solvent.
Test solution (a) Dissolve 2.5 g o f die substance to be
examined in heptane R and dilute to 25.0 m L with the same
solvent.
Test solution (b) Dissolve 2.5 g o f the substance to be
examined in heptane R, add 5.0 m L o f the internal standard A. l-methyl-4-(l-methylethyl)-7-oxabicyclo[2.2. l]heptane
solution and dilute to 25.0 m L with heptane R. (1,4-cineole).
Reference solution (a) T o 2.0 m L o f test solution (a) add ___________________________________________________________PhEur
20.0 m L of the internal standard solution and dilute to
100.0 m L w ith heptane R.
Reference solution (b) Dissolve 50 m g o f 1,4-cineole R and
50 m g o f the substance to be examined in heptane R and
dilute to 50.0 m L w ith the same solvent. Cinnamic Acid
Column:
— sizer. I = 30 m , 0 = 0.25 m m ,
0.25 jam).
Linear velocity 45 cm/s. G>H80 2 148.2 621-82-9
SpUt-ratio 1:70.
Temperature: Antimicrobial preservative; excipient.
lim e Temperature D E F IN IT IO N
(min) CO Cinnamic A d d is (£)-3-phenylprop-2-enoic acid. It contains
Column 0 - 10 50 n o t less than 99.0% and not more than 100.5% of CgH aO ^
10-35 5 0 * 100 calculated with reference to the dried substance.
35 -4 5 100*200
45 -5 5 200
Injection port 220 Colourless crystals.
Very slighdy soluble in water, freely soluble in ethanol (96%)',
Detector 250
soluble in ether.
Detection Flam e ionisation. A. T he infrared absorption spectrum, Appendix II A, is
Iiyecdon 1 |iL. concordant with the reference spectrum of cinnamic acid
System suitability, reference solution (b): (RS 062).
— resolution: m inim um 10 between the peaks due to B. T h e light absorption, Appendix II B, in the range 230 to
im purity A and to cineole. 350 nm o f a 0.0010% w/v solution in 0.1m sodium hydroxide
Limits: exhibits a maximum only at 267 nm . T he absorbance at
— total: calculate the ratio (R) o f the area of the peak due to 267 nm is about 1.4.
cineole to the area of the peak due to the internal
TESTS
standard from the chrom atogram obtained with reference
M e ltin g p o in t
solution (a); from the chrom atogram obtained with test
solution (b), calculate the ratio o f the sum of the areas of
any peaks, apart from the principal peak and the peak E th a n o l-in s o h ib le m a tte r
due to the internal standard, to the area o f the peak due A 10% w/v solution in ethanol (96%) is clear, Appendix IV A.
to internal standard: this ratio is no t greater than R~ R e la te d su b s ta n c e s
(2 per cent), Carry out the m ethod for thin-layer chromatography,
— disregard limit. 0.025 times the area o f the principal peak A ppendix m A, using the following solutions in methanol.
in the chrom atogram obtained with reference solution (a) (1) 5.0% w/v o f the substance being examined.
(0.05 per cent).
R e sid u e o n e v a p o ra tio n
(a) Use as the coating silica gel GF2S4.
T o 2.0 g add 5 m L o f water R, evaporate to dryness on a
water-bath and dry at 100-105 °C for 1 h. T h e residue (b) Use the mobile phase as described below.
weighs a m axim um o f 2 mg. (c) Apply 5 *iL o f each solution.
STORAGE (d) Develop die plate to 15 cm.
In an airtight container, protected from lig h t (e) After removal o f the plate, dry in air and examine under
ultraviolet light (254 nm).
MOBILE PHASE
10 volumes o f glacial acetic acid and 90 volumes o f toluene.
2016 Cinnarizine 1-563
LIMITS Mobile phase 58.4 g/L solution of sodium chloride R,

Any secondary spot in the chromatogram obtained with methanol R, acetone R (20:30:50 VIVIV).
solution ( 1) is n o t more intense than the spot in the Application 5 |xL.
chromatogram obtained with solution (2). Development In an unsaturated tank, over 3/4 of the plate.
Loss on drying Drying In air.
W hen dried to constant weight at 60° at a pressure not
exceeding 0.7 kPa, loses not more than 1.0% o f its weight.
Use 1 g.
Sulfated ash
N o t more than 0.1% , Appendix IX A.
ASSAY spot in the chromatogram obtained with reference
Dissolve 0.5 g in 15 m L of ethanol (96%) previously solution (a).
neutralised to phenol red solution and titrate with 0 . 1m sodium D . Dissolve 0.2 g of anhydrous citric acid R in 10 m L of acetic
hydroxide KS using phenol red solution as indicator. Each m L anhydride R in a water-bath at 80 °C and m aintain the
o f 0.1m sodium hydroxide FS is equivalent to 14.82 m g of tem perature o f the water-bath at 80 °C for 10 min.
C 9H 8O 2. Add about 20 m g of the substance to be examined. A purple
colour develops.
TESTS
★ ★
Cinnarizine ★ ★ T he solution is clear (2 .2 . 1 ) and n o t m ore intensely coloured
Dissolve 0.5 g in methylene chloride R and dilute to 20 m L
Suspend 0.5 g in 15 m L of water R. Boil for 2 min. Cool and
filter. Dilute the filtrate to 20 m L with carbon dioxide-free
water R. T o 10 m L of this solution add 0.1 m L of
phenolphthalein solution R and 0.25 m L o f 0.01 M sodium
hydroxide. T he solution is pink. T o 10 m L of the solution
C26H28N2 368.5 298-57-7 add 0.1 m L of methyl red solution R and 0.25 m l. of 0.01 M
hydrochloric add. T he solution is red.
Action and use Related substances
Histamine H I receptor antagonist; antihistamine. Liquid chromatography (2.2.29).
PhEir__________________________________________________________ Test solution Dissolve 25.0 mg of the substance to be
examined in methanol R and dilute to 10.0 m L with the same
DEFINITION solvent.
(fs)-l-(Diphenylmethyl)-4-(3-phenylprop-2-enyl)piperazine.
Reference solution (a) Dissolve 12.5 mg o f cinnarizine CRS and
Content 15.0 m g oiflunarizme dihydrochloride CRS in methanol R and
99.0 per cent to 101.0 per cent (dried substance). dilute to 100.0 m L with the same solvent. Dilute 1.0 m L of
CHARACTERS this solution to 20.0 m L with methanol R.
Appearance Reference solution (b) Dilute 1.0 m l. o f the test solution to
W hite or almost white powder. 100.0 m L with methanol R. Dilute 5.0 m l. o f this solution to
Solubility
Practically insoluble in water, freely soluble in methylene Column:
chloride, soluble in acetone, slightly soluble in ethanol — sizer. I = 0.1 m , 0 = 4.0 mm;
(96 per cent) and in methanol. — stationary phase, base-deactivated octadecylsifyl silica gel for
IDENTIFICATION
Mobile phase:
First identification A , B — mobile phase A: 10 g/L solution of ammonium acetate R;
Second identification A , C, D — mobile phase B: 0.2 per cent V/V solution of glacial acetic
A. Melting point (2.2.14): 118 °C to 122 °C. add R in acetomtrile R l;
Comparison cinnarizine CRS. Time Mobile phase A Mobile phase B
C . Thin-layer chromatography (2.2.27). (min) (per cent V/V) (per cent V/V)
0-20 75 * 10 25*90
Test solution Dissolve 10 m g of the substance to be exam in ed
in methanol R and dilute to 20 m L with the same solvent. 20 -2 5 10 90
Reference solution (a) Dissolve 10 m g o f cinnarizine CRS in
methanol R and dilute to 20 m L with the same solvent
Reference solution (b) Dissolve 10 mg of cinnarizine CRS and
10 m g oiflunarizme dihydrochloride CRS in methanol R and
dilute to 20 m L with the same solvent. Injection 10 |iL.
Plate TLC octadecylsifyl silica gel F2 5 4 plate R.
1-564 Ciprofibrate 2016
Relative retention W ith reference to rinnarizine (retention CeHs

time = about 11 min): im purity A = about 0.4; CeHs^N Cl'
flunarizine = about 1.05; impurity B = about 1.1; CeHs
im purity C = about 1.2; impurity D = about 1.6; c 6h 5

System suitability: reference solution (a): C. 4-(diphenylm ethyl)-1,1 -bis [(£)-3-phenylprop-2-
— resolution: m inim um 5.0 between the peaks due to enyl] piperazinium chloridej
rinnarizine and flunarizine.
Limits:
— impurities A , B, C, D, E: for each impurityj not more than
the area o f the principal peak in the chromatogram I I h ^ 5 and enantiomer
obtained w ith reference solution (b) (0.25 per cent); C eH s^N ^
— unspecified impurities: for each impurity, n o t more than CeH5
chrom atogram obtained with reference solution (b) D . 1-(diphenylmethyl)-4- [( 1i?.Sj3Ii)-4-phenyl-1- [(£)- 2-
(0.10 per cent); phenylethenyl]but-3-enyl]piperazinej
— total: not m ore than twice the area o f the principal peak in
the chromatogram obtained with reference solution (b) CeHs
(0.5 per cent);
— disregard limit. 0.2 times the area of the principal peak in I^ ^ N ^ C sH s
(0.05 per cent).
CeHs
M axim um 20 ppm . E. Ij4-bis(diphenylmethyl)piperazine.
Dissolve 1.0 g in a mixture o f 15 volumes of water R and Pit Eur
85 volumes of acetone R. Add dilute hydrochloric acid R until
dissolution is complete. Dilute to 20 m L with a mixture of
15 volumes o f water R and 85 volumes o f acetone R. 12 m L
o f the solution complies with test B. Prepare the reference ★★★
★ ★
solution using 10 m L o f lead standard solution (1 ppm Pb) Ciprofibrate ★ ★
obtained by diluting lead standard solution (100 ppm Pb) R *****
with a mixture o f 15 volumes o f water R and 85 volumes of (Ph. Eur. monograph 2013)
acetone R. O^ ^ CO î H
an oven in vacuo at 60 °C for 4 h. clvACl H
XT- X
l3C CH3 ancj enantiomer
S u lfa te d a sh (2. 4.14)

M axim um 0.1 per centj determined on 1.0 g. C 13H 14CI2O 3 289.2 52214-84-3
A SSA Y
Action and use
Dissolve 0.150 g in 50 m L o f a mixture o f 1 volume of
Fibrate; lipid-regulating drug.
anhydrous acetic add R and 7 volumes o f methyl ethyl ketone R.
T itrate with 0.1 Mperchloric add, using 0.2 m l. of
naphtholbenzein solution R as indicator.
DEFINITION
2- [4- [( \RS)-2 j2-Dichlorocyclopropyl]phenoxy]-2-
o f C 2&H28N 2. methylpropanoic ad d .
STO RA G E Content
Protected from light. 99.0 per cent to 101.0 per cent (anhydrous substance).
IM P U R IT IE S CHARACTERS
Spedfied impurities A, B, C, D , E Appearance
W hite or slighdy yellow, crystalline powder.
NH
r , Solubility
C9H5 ethanol, soluble in toluene.
mp
A. l-(diphenylmethyl)piperazinej A bout 115 °C.
IDENTIFICATION
ÇeHs
Comparison ciprofibrate CRS.
o TESTS
C9H5 Appearance o f solution
B. (Z)-l-(diphenylmethyl)-4-(3-phenylprop-2-enyl)piperazine, than reference solution BY4 (2.2.2, Method II).
2016 Ciprofibrate 1-565
Dissolve 1.0 g in anhydrous ethanol R and dilute to 10.0 m L — disregard limit. 0.5 times the area of the principal peak in
w ith the same solvent. the chromatogram obtained w ith reference solution (a)
R e la te d su b s ta n c e s (0.05 p er cent).
L iquid chromatography (2.2.29). C h lo rid e s (2.4.4)
Test solution Dissolve 0.125 g o f the substance to be M axim um 350 ppm.
exam ined in a mixture o f equal volumes o f acetomtrUe R and T o 0.190 g add 20 m l. o f water R and treat in an ultrasonic
water R and dilute to 50 m L with the same mixture of bath for 8 min. Filter. 15 mL of the filtrate complies with the
solvents. test.
Reference solution (a) Dilute 1.0 m L of the test solution to W a te r (2.5.12)
100.0 m L with a mixture of equal volumes o f acetomtrUe R M aximum 0.5 per cent, determined on 1.000 g.
and water R. D ilute 1.0 m L o f this solution to 10.0 m L with S u lfa te d a s h (2.4.14)
a m ixture of equal volumes of acetomtrUe R and water R.
Reference solution (b) Dissolve the contents o f a vial o f
A SSAY
ciprofibrate for system suitability CRS in 2.0 m L o f a mixture of
equal volumes o f acetomtrUe R and water R. Dissolve 0.250 g in a mixture o f 20 m L of water R and
40 m L o f anhydrous ethanol R. T itrate with 0.1 M sodium
Column'.
hydroxide, d eterm ining the end-point potentiometrically
— size: I = 0.15 m , 0 = 4.6 mm,
(2.2.20).
— stationary phase. octylsUyl silica gel for chromatography R
(5 pm). 1 m L o f 0.1 M sodium hydroxide is equivalent to 28.92 m g
of C 13H 14CI2O 3.
Mobile phase:
— mobile phase A: 1.36 g/L solution of potassium dihydrogen STORAGE
phosphate R adjusted to p H 2.2 with phosphoric acid R, In an airtight container, protected from light
— mobile phase B: acetomtrUe R,
IM P U R IT IE S
Specified impurities A, B, C, D , E.
0 -3 0 7 5 -» 3 0 2 5 -» 7 0 f Y ° x COlH
3 0 -40 30
h 1c » J v ) h >c
70
40-42 3 0 -» 7 5 7 0 -» 2 5
A. 2-(4-ethenylphenoxy)-2-methylpropanoic ad d ,
Flow rate 1.5 m L/m in. OH
Detection Spectrophotom eter at 230 nm. Cl and enantiomer

Injection 10 jiL. 'A " '
Cl H
w ith ciprofibrate for system suitability CRS to identify the peaks
due to impurities A, B, C, D and E. B. 4-[(li?5)-2,2-dichlorocyclopropyl]phenol,
Relative retention W ith reference to ciprofibrate (retention

im purity B = about 0.8; impurity C = about 0.95;
° xCH3
H3C
R and enantiomer
im purity D = ab o u t 1.3; impurity E = about 1.5.
Cl H
— resolution: baseline separation between the peaks due to
im purity C and ciprofibrate. C. R = C H 2O H: 2-[4-[(li?5)-2,2-
dichlorocyclopropyljphenoxy]-2-methylpropan - 1-ol,
Limits:
— correction factor, for the calculation o f content, multiply the D . R = CO-OCH 3 : methyl 2-[4-[(lflS)-2,2-
peak area o f im purity A by 2.3, dichlorocyclopropyl]phenoxy]-2-m ethylpropanoate,
— impurities A , C, D: for each impurity, n o t more than the E. R = C O -O C 2H 5: ethyl 2-[4-[(li?5)-2,2-
area o f the principal peak in the chromatogram obtained dichlorocyclopropyi]phenoxy]-2-methylpropanoate.
w ith reference solution (a) (0.1 per cent), PhEur
— impurity B: n o t m ore than twice the area o f the principal
peak in the chrom atogram obtained w ith reference
solution (a) (0.2 p er cent),
— impurity E: n o t m ore than 8 times the area o f the
— any other impurity: for each impurity, no t more than the
w ith reference solution (a) (0.1 per cent),
— total of other impurities’, not more than 5 times the area o f
1-566 Ciprofloxacin 2016
★ ★ Limit.
Ciprofloxacin ★ ★ — impurity A: any spot due to impurity A is n o t more
★ intense than the principal spot in the chrom atogram
obtained with the reference solution (0.2 p er cent).
R elate d su b s ta n c e s
Test solution T o 25.0 mg of the substance to be examined add
0.2 m L o f dilute phosphoric acid R and dilute to 50.0 m L with
C02H the mobile phase. T reat in an ultrasonic bath until a clear
solution is obtained.
C 17H 18FN 3O 3 331.4 85721-33-1 100.0 m L w ith the mobile phase. Dilute 1.0 m L of this
Action and use
Reference solution (b) Dissolve 2.5 m g of dprofloxadn
Fluoroquinolone antibacterial.
hydrochloride for peak identification CRS (containing
Preparations impurities B, C , D and E) in the mobile phase and dilute to
Ciprofloxacin Infusion 5.0 m L with the mobile phase.
Ciprofloxacin Eye Drops Column:
— size: I = 0.25 m , 0 = 4.6 mm;
PhEtr__________________________
— stationary phase: base-deactivated end-capped octadecylsifyl
D E F IN IT IO N silica gel for chromatography R (5 ¡mi);
1-Cyclopropyl-6-fluoro-4-oxo-7 - (piperazin-1-yl)-1,4- — temperature:: 40 °C.
dihydroquinoline-3-carboxylic ad d . Mobile phase Mix 13 volumes o f acetonitrUe R and 87 volumes
Content o f a 2.45 g/L solution of phosphoric add R, previously
99.0 per cent to 101.0 per cent (dried substance). adjusted to p H 3.0 with triethylamine R.
CHARACTERS Flow rate 1.5 mL/min.
Appearance Detection Spectrophotom eter at 278 nm .
Almost white or pale yellow, crystalline powder, slightly Injection 50 |iL.
hygroscopic. Run time Twice the retention time of ciprofloxacin.
Solubility Identification of impurities Use the chrom atogram supplied
Practically insoluble in water, very slightly soluble in with ciprofloxacin hydrochloride for peak identification CRS and
anhydrous ethanol and in methylene chloride. the chromatogram obtained with reference solution (b) to
ID E N T IF IC A T IO N identify the peaks due to impurities B, C , D and E.
Infrared absorption spectrophotometry (2.2.24). Relative retention W ith reference to ciprofloxacin (retention
Comparison ciprofloxacin CRS. time = about 9 min): impurity E — about 0.4;
TESTS impurity D = about 1.2.
Appearance o f solution System suitability: reference solution (b):
T he solution is clear (2.2.1) and not more intensely coloured — resolution: minimum 1.3 between the peaks due to
than reference solution GY 5 (2.2.2, Method II). impurities B and C.
Dissolve 0.25 g in a 10.3 g/L solution o f hydrochloric add R Limits:
and dilute to 20 m L with the same solution. — correction factors: for the calculation o f content, multiply
Impurity A the peak areas o f the following impurities by the
Thin-layer chromatography (2.2.27). corresponding correction factor, impurity B = 0.7;
Test solution Dissolve 50 m g of the substance to be examined impurity C = 0.6; impurity D = 1.4; impurity E = 6.7;
in dilute ammonia R1 and dilute to 5 m L with the same — impurities B, C, D, E: for each impurity, n o t more than
solvent. the area o f the principal peak in the chromatogram
obtained with reference solution (a) (0.2 p er cent);
Reference solution Dissolve 10 m g of ciprqfloxadn
— unspecified impurities: for each impurity, no t more than
impurity A CRS in a mixture o f 0.1 m L of dilute ammonia R1
and 90 m L o f water R and dilute to 100 m L with water R.
Dilute 2 m L o f the solution to 10 m l. with water R.
(0.10 p er cent);
Plate TLC silica gel F2 $ 4 plate R. — total: n o t more than 2.5 times the area of the principal
Mobile phase acetomtrUe R, concentrated ammonia R, peak in the chromatogram obtained with reference
methanol R, methylene chloride R (10:20:40:40 V/V/V/V). solution (a) (0.5 per cent);
Application 5 |iL. — disregard limit: 0.25 times the area of the principal peak in
Development A t the bottom of a chromatographic tank, place the chromatogram obtained with reference solution (a)
an evaporating dish containing 50 m L o f concentrated (0.05 p er cent).
ammonia R. Expose the plate to the ammonia vapour for H eav y m e ta ls (2.4.8)
15 m in in the closed tank. W ithdraw the plate, transfer to a M aximum 20 ppm.
2nd chromatographic tank and develop over 3/4 of the plate. Dissolve 0.5 g in dilute acetic add R and dilute to 30 m L with
Drying In air. the same solvent. A dd 2 m L of water R instead of 2 m L of
Detection Examine in ultraviolet light at 254 nm. buffer solution pH 3.5 R T he filtrate complies with test E.
2016 Ciprofloxacin Hydrochloride 1-567
Prepare the reference solution using 10 m L o f lead standard HN

M axim u m 1.0 per cent, determined on 1.000 g by drying
under vacuum at 120 °C.
M a x im u m 0.1 per cent, determined on 1.0 g in a platinum
E. 1-cyclopropyl-6-fluoro-7-(piperazin - 1-yl) quino lin-4 ( 1H)-
crucible. one (decarboxyiated compound),
A SSA Y
Dissolve 0.300 g in 80 m L o f glacial acetic acid R. Titrate
of C 17H 13F N 3O 3.
STO RA G E
F. 1-cyclopropyl-6-hydroxy-4-oxo-7-(piperazin - 1-yl)- 1,4-
IM P U R IT IE S dihydroquinoline-3-carboxylic ad d .
Specified impurities A, B, C , D , E
Ph Eur
by the general m onograph Substances for pharmaceutical use Ciprofloxacin Hydrochloride ★ ★
★ ★
Control of impurities in substances for pharmaceutical use): F.
a HCl , x HzO
X jÇ u
o
C02H
A. 7-chloro -1-cyclop ropyl-6-fluoro-4-oxo-1,4- CnHwCEFNaOs^HaO 367.8

dihydroquinoline-3-carboxylic a d d (fluoroquinolonic
(anhydrous)
ad d ),
p Y A c tio n a n d u se
Fluoroquinolone antibacterial.
Xxjx o
P r e p a r a tio n
Ciprofloxacin Tablets
PhEur.
B. l-cyclopropyl-4-oxo-7-(piperazin-l-yl)-l,4- D E F IN IT IO N
dihydroquinoline-3-carboxylic a d d (desfluoro compound),
1-Cydopropyl-6-fluoro-4-oxo-7-(piperazin - 1-yl)-1,4-
dihydroquinoline-3-carboxylic a d d hydrochloride. It contains
H,N'
. Y a variable quantity o f water.
C o n te n t
CHARACTERS
o
A p p e a ra n c e
C. 7-[(2-aminoethyI)amino] -1 -cyclopropyl-6-fluoro-4-oxo- Pale yellow, crystalline, slightly hygroscopic powder.
134-dihydroquinoline-3-carboxylic a d d (ethyienediamine S o lu b ility
com pound), Soluble in water, slightly soluble in methanol, very slightly
soluble in anhydrous ethanol, practically insoluble in acetone,
in ethyl acetate and in methylene chloride.
CO2H A. Infrared absorption spectrophotometry (2.2.24).
Comparison ciprofloxacin hydrochloride CRS.
B. 0.1 g gives reaction (b) o f chlorides (2.3.1).
D . 7-chloro- l-cydopropyl-4-oxo-6-(piperazin-1-yl)-1,4-
dihydroquinoline-3-carboxylic ad d ,
1-568 Ciprofloxacin Hydrochloride 2016
TESTS Run time 2.3 times the retention time of ciprofloxacin.

S o lu tio n S Identification of impurities Use the chrom atogram supplied
Dissolve 0.5 g in carbon dioxide-free water R and dilute to with ciprofloxacin hydrochloride for peak identification CRS and
20 m L with the same solvent. the chromatogram obtained with reference solution (b) to
A p p e a ra n c e o f so lu tio n identify the peaks due to impurities B, C , D and E.
T he solution is clear (2.2.1) and n o t m ore intensely coloured Relative retention W ith reference to ciprofloxacin (retention
than reference solution GY 5 (2.2.2, Method II). time = about 9 min): impurity E = about 0.4;
Dilute 10 m L o f solution S to 20 m L with carbon dioxide-free impurity B = about 0.6; im purity C = about 0.7;
water R. impurity D = about 1.2.
p H C2.2.3)
impurities B and C.
Impurity A
Limits:
— correction factors', for the calculation o f content, multiply
Test solution Dissolve 50 m g of the substance to be examined the peak areas o f the following impurities by the
in water R and dilute to 5 m L with the same solvent. corresponding correction facto r impurity B = 0.7;
Reference solution Dissolve 10 m g of ciprofloxacin impurity C = 0.6; impurity D = 1.4; impurity E = 6.7;
impurity A CRS in a mixture of 0.1 m L o f dilute ammonia R l — impurity E: n o t more than 1.5 times the area o f the
and 90 m L o f water R and dilute to 100 m L with water R. principal peak in the chromatogram obtained with
D ilute 2 m L o f the solution to 10 m L with water R. reference solution (c) (0.3 p er cent);
Plate TLC silica gel F254 plate R. — impurities B, C, D: for each impurity, n o t more than the
Mobile phase acetomtrUe R, concentrated ammonia R, area o f the principal peak in the chromatogram obtained
methanol R, methylene chloride R (10:20:40:40 V/V/V/V). with reference solution (c) (0.2 per cent);
Application 5 pL.
Development A t the bottom of a chromatographic tank, place chromatogram obtained with reference solution (c)
an evaporating dish containing 50 m L of concentrated (0.10 per cent);
ammonia R. Expose the plate to the ammonia vapour for — total: not more than 2.5 times the area of the principal
15 m in in the closed tank. W ithdraw the plate, transfer to a peak in the chromatogram obtained with reference
2nd chromatographic tank and develop over 3/4 of the plate. solution (c) (0.5 per cent);
Drying In air. — disregard limit. 0.25 times the area o f the principal peak in
Detection Examine in ultraviolet light at 254 nm . the chromatogram obtained with reference solution (c)
Limit. (0.05 per cent).
— impurity A: any spot due to impurity A is no t more H eav y m e ta ls (2.4.8)
intense than the principal spot in the chromatogram M aximum 20 ppm.
obtained with the reference solution (0.2 p er cent). Dissolve 0.25 g in water R and dilute to 30 m L with the
R e lated su b s ta n c e s same solvent. Carry out the prefiltration. T h e filtrate
Liquid chromatography (2.2.29). complies with test E. Prepare the reference solution using
Test solution Dissolve 25.0 mg o f the substance to be 5 m L of lead standard solution (1 ppm Pb) R.
examined in the mobile phase and dilute to 50.0 m L with W a te r (2.5.12)
the mobile phase. M aximum 6.7 per cent, determ ined on 0.200 g.
Reference solution (a). Dissolve 25.0 m g o f ciprofloxacin Sulfated ash (2.4.14)
hydrochloride CRS in the mobile phase and dilute to 50.0 m L M axim um 0.1 per cent, determ ined on 1.0 g in a platinum
with the mobile phase. crucible.
Reference solution (b) Dissolve 2.5 m g o f ciprofloxacin A SSAY
hydrochloride for peak identification CRS (containing Liquid chromatography (2.2.29) as described in the test for
impurities B, C , D and E) in the mobile phase and dilute to
Injection 10 pL o f the test solution and reference solution (a).
Reference solution (c) D ilute 1.0 m L o f the test solution to
50.0 m L with the mobile phase. Dilute 1.0 m L o f this Calculate the percentage content o f C 17H 19CIFN 3O 3 taking
into account the assigned content o f ciprofloxacin
hydrochloride CRS.
Column:
— size. I = 0.25 m , 0 = 4.6 mm; STORAGE
— stationary phase', base-deactivated end-capped octadecylsUyl In an airtight container, protected from light.
silica gel for chromatography R (5 pm); IM P U R IT IE S
Specified impurities A, B, C , D , E
Mobile phase M ix 13 volumes o f acetomtrUe R and 87 volumes
of a 2.45 g/L solution o f phosphoric acid R previously adjusted
present a t a sufficient level, be detected by one o r other of
to p H 3.0 w ith triethylamine R.
Flow rate 1.5 m U m in. acceptance criterion for other/unspecified impurities and/or
Detection Spectrophotom eter at 278 nm . by the general m onograph Substances for pharmaceutical use
Injection 50 pL of the test solution and reference solutions (b) (2034). It is therefore not necessary to identify these
and (c). impurities for dem onstration o f compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use): F.
2016 Cisplatin 1-569
>*★
Cisplatin ★ ★
★ ★
COjH
c iN x nh3
Pt
ay x nh3
A. 7-chloro-1-cydopropyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic a d d (fluoroquinolonic
add), PtCl2(NH 3)2 300.0 15663-27-1
Action and use

Platinum -containing cytotoxic.
Preparation
Cisplatin Injection
PhEur.
DEFINITION
B. 1-cyclopropyl-4-oxo-7-(piperazin - 1-yl) - 1,4- dy-Diamminedichloroplatinum(II).
dihydroquinoline-3-carboxylic a d d (desfluoro com pound), Content
CHARACTERS
Appearance
Yellow powder, o r yellow or orange-yellow crystals.
c o 2h
Solubility
Slightly soluble in water, sparingly soluble in
dimethylformamide, practically insoluble in ethanol
(96 per cent).
C . 7-[(2 -aminoethyI)amino]-1 -cyclopropyl-6-fluoro-4-oxo-
l,4-dihydroquinoline-3-carboxyIic a d d (ethylenediamine Cany out identification test B, the tests (except that for silver)
com pound), and the assay protected from light.
IDENTIFICATION
COjH Comparison cisplatin CRS.
Test solution Dilute 1 m L of solution S2 (see Tests) to 10 m L
with dimethylformamide R.
D . 7-chloro-1-cyclopropyl-4-oxo-6-(piperazin-1-yl)-1,4-
dihydroquinoline-3-carboxyüc ad d , Reference solution Dissolve 10 mg o f cisplatin CRS in 5 m L of
dimethylformamide R
Plate cellulose for chromatography R1 as the coating substance.
Y Pretreatment Activate the plate by hearing at 150 °C for 1 h.
:x)Çi o
Mobile phase acetone R , dimethylformamide R (10:90 VIV).
Application 2 |iL.
Drying In air.
E. l-cyclopropyl-6-fhioro-7-(piperazin-l-yl)quinolin-4(lif)- Detection Spray with a 50 g/L solution o f stannous chloride R
one (decarboxylated compound), in a mixture o f equal volumes of dilute hydrochloric acid R and
water R. Examine after 1 h.
reference solution.
C. Add 50 mg to 2 m L o f dilute sodium hydroxide solution R in
a glass dish. Evaporate to dryness. Dissolve the residue in a
mixture o f 0.5 m L of nitric add R and 1.5 m L of hydrochloric
F. 1-cyclopropyl-6-hydroxy-4-oxo-7-(piperazin-1-yl)- 1,4- add R. Evaporate to dryness. T he residue is orange. Dissolve
dihydroquinoline-3-carboxylic add. the residue in 0.5 m L of water R and add 0.5 m L of
PhEir ammonium chloride solution R. A yellow, crystalline predpitate
is formed.
1-570 Cisplatin 2016
TESTS Relative retention W ith reference to cisplatin (retention

S o lu tio n S I time = about 3.8 min): displacement peak = about 0.5;
Dissolve 25 m g in a 9 g/L solution of sodium chloride R in im purity A = about 0 .6 ; impurity B = about 0.7; cisplatin
carbon dioxide-free water R and dilute to 25 m L with the same aquo complex = about 1.2 .
solvent. System suitability Reference solution (d):
S o lu tio n S2 — resolution: minim um 2.5 between the peaks due to
Dissolve 0.20 g in dimethylformamide R and dilute to 10 m L impurities A and B, the displacement peak and the peak
with the same solvent. due to impurity A are well separated.
A p p e a ra n c e o f so lu tio n S I
Limits:
— impurity A: not m ore than the area of the corresponding
Solution S I is clear (2.2. 1) and not m ore intensely coloured
than reference solution GY 5 (2.2.2, Method II).
solution (d) (2.0 per cent);
A p p e a ra n c e o f so lu tio n S2 — impurity B: not m ore than the area o f the corresponding
Solution S2 is clear (2.2.1). peak in the chromatogram obtained with reference
p H (2.2.3) solution (d) ( 1.0 per cent);
4.5 to 6.0 for solution S I, m easured immediately after — unspecified impurities: for each impurity, not more than
preparation. 0.5 times the area o f the peak due to cisplatin in the
R e la te d su b s ta n c e s chromatogram obtained with reference solution (d)
Liquid chromatography (2.2.29). Carry out the test protected ( 0.10 per cent);
from light. Do not heat or sonicate any platinum-containing — sum of impurities other than A and B: not more than
solution. All solutions are to be used within 4 h. 2.5 times the area o f the peak due to cisplatin in the
(0.5 per cent);
examined in a 9.0 g/L solution of sodium chloride R and dilute
— disregard Umit. the area o f the peak due to cisplatin in the
to 25.0 m L with the same solution.
chromatogram obtained with reference solution (e)
Reference solution (a) Dissolve 25.0 mg of cisplatin CRS in a (0.05 per cent). Disregard any peak due to the cisplatin
9.0 g/L solution o f sodium chloride R and dilute to 25.0 m L aquo complex.
S ilv e r
Reference solution (b) Dissolve 5.0 m g o f cisplatin M aximum 250 ppm.
impurity A CRS in a 9.0 g/L solution o f sodium chloride R and
dilute to 50.0 m L w ith the same solution.
Reference solution (c) Dissolve 5.6 mg o f cisplatin Test solution Dissolve 0.100 g in 15 m L of nitric acid R,
impurity B CRS in a 9.0 g/L solution o f sodium chloride R and heating to 80 °C. Cool and dilute to 25.0 m L with water R.
dilute to 100.0 m L with the same solution. Reference solutions T o suitable volumes (10 m L to 30 m L) of
Reference solution (d) M ix 0.05 m L of the test solution with silver standard solution (5 ppm Ag) R add 50 m L o f nitric
5.0 m L of reference solution (b) and 5.0 m L of reference acid R and dilute to 100.0 m L with water R.
solution (c) and dilute to 25.0 m L with a 9.0 g/L solution of Source Silver hollow-cathode lamp, preferably using a
sodium chloride R. transmission band of 0.5 nm .
Reference solution (e) Dilute 5.0 m L of reference solution (d) Wavelenth 328 nm.
to 20.0 m L with a 9.0 g/L solution o f sodium chloride R. Atomisation device Fuel-lean air-acetylene flame.
Blank solution 9.0 g/L solution o f sodium chloride R. Carry out a blank determination.
Column: A SSA Y
— sizer. I = 0.25 m , 0 = 4.0 mm; Liquid chromatography (2.2.29) as described in the test for
— stationary phase: base-deactivated octylsüyl silica gel for related substances with the following modification.
Injection 10 nL of the test solution and reference solution (a).
Calculate the percentage content o f PtC l 2(N H 3)2 from the
Mobile phase Dissolve 1.08 g o f sodium octanesulfonate R,
sum of the areas o f the peaks due to cisplatin and cisplatin
1.70 g of tetrabutylammonium hydrogen sulfate R and 2.72 g of
aquo complex and from the declared content of
potassium dihydrogen phosphate R in waterfor chromatography R
and dilute to 950 m L with the same solvent. Adjust to cisplatin CRS.
p H 5.9 with 1 M sodium hydroxide and dilute to 1000 m L STORAGE
with water for chromatography R. In an airtight container, protected from light.
Flow rate 1.0 mlVmin. IM P U R IT IE S
Detection Spectrophotom eter at 210 nm . Specified impurities A, B
Injection 20 pL o f the test solution, reference solutions (d) Other detectable impurities (the following substances would, if
and (e), and the blank solution. present at a sufficient level, be detected by one or other of
Run time 7 times the retention time o f cisplatin. the tests in the monograph. T hey are limited by the general
T h e displacement peak is the latest eluting peak of the group acceptance criterion for other/unspecified impurities and/or
of injection peaks in the chrom atogram obtained with the by the general monograph Substances for pharmaceutical use
blank solution. (2034). It is therefore n o t necessary to identify these
Identification of cisplatin aquo complex Use the chromatogram
supplied with cisplatin CRS and the chromatogram obtained
cisplatin aquo complex.
2016 Citalopram Hydrobromide 1-571
C IN x nh3
Pt
✓ \ in mobile phase A and dilute to 100.0 m L w ith mobile
H3N CI
phase A.
A. rran$-diamminedichloroplatinum(II) (transplatin), Reference solution (a) Dilute 1.0 m L of the test solution to
100.0 m L with mobile phase A (solution A). Dilute 1.0 mT.
o f solution A to 10.0 m L with mobile phase A.
C IN x NH3"
Pt
a y v ci citalopram for system suitability CRS (containing impurities B,
D and G) in 1.0 m L o f solution A.
Column:
B. am m inetrichloroplatm ate(-)j
— size: I = 0.25 m , 0 = 4.6 m m ;
— stationary phase, end-capped octadecylsüyl sUica gel for
a \ , aT chromatography R (4 |im);
Pt — temperature: 40 °C.
a y yci
Mobile phase:
— mobile phase A: dissolve 1.58 g of ammonium formate R in
C. tetrachloroplatinate( 2-). 500 m L of a mixture of 4 volumes o f acetomtrUe R,
: _______________________________ ______ _ PhEur 32 volumes o f methanol R and 64 volumes o f water R;
— mobile phase B: dissolve 1.58 g of ammonium formate R in
500 m L of a mixture of 32 volumes o f water R and
68 volumes o f acetonitrUe R;
Citalopram Hydrobromide ?**%

(Th. Eitr. monograph 2288) *** (min) (per cent V/V) (per cent V/V)
0 -2 100 0
F
2 -2 5 100*40 0*60
and enantiomer
2 5 -3 0 40 60
,CH3 ’ HBr
CH3 Flow rate 1.0 mL/min.

Detection Spectrophotometer at 230 nm and, for impurity G,
at 254 nm.
C joH ^B rFN jO 405.3 59729-32-7 Injection 40 |iL.
Action and use Identification of impurities Use the chrom atogram supplied
Selective serotonin reuptake inhibitor; antidepressant. with citalopram for system suitability CRS and the
Preparations identify the peaks due to impurities B, D and G.
C italopram Tablets
Relative retention W ith reference to citalopram (retention
PhEur___________________________________________________________ time = about 19 min): impurity G = about 0.5;
im purity B = about 0.7; impurity D = about 0.9.
DEFINITION
( 1ÄS )-1 -[3-(Dimethylamino)propyI] -1 -(4-fluorophenyI)-l j3- System suitability: reference solution (b):
dihydroisobenzofuran-5-carbonitrile hydrobromide. — resolution: minimum 1.5 between the peaks due to
im purity D and citalopram at 230 nm .
Content
Limits:
99.0 p er cent to 101.5 per cen t (dried substance).
CHARACTERS peak area of impurity G by 0.6;
Appearance — impurity D: n o t more than twice the area o f the principal
W hite or almost white, crystalline powder. peak in the chromatogram obtained with reference
Solubility solution (a) ( 0.2 per cent);
Sparingly soluble in water and in anhydrous ethanol. — impurity B: n o t more than 1.5 times the area o f the
IDENTIFICATION reference solution (a) (0.15 p e r cent);
A. Optical rotation (see Tests). — impurity G at 254 nm: not m ore than 1.5 times the area of
B. Infrared absorption spectrophotometry (2.2.24). the principal peak in the chrom atogram obtained with
Comparison citalopram hydrobromide CRS. reference solution (a) (0.15 p e r cent);
C. It gives reaction (a) of bromides (2.3.1).
TESTS with reference solution (a) (0.10 per cent);
Optical rotation (2.2.7) — sum of impurities other than G: n o t m ore than 5 times the
- 0 . 10° to + 0 . 10°. area of the principal peak in the chrom atogram obtained
Dissolve 1.0 g in methanol R and dilute to 20 m L with the w ith reference solution (a) (0.5 per cent);
same solvent — disregard limit. 0.5 times the area of the principal peak in
Related substances the chromatogram obtained with reference solution (a)
L iquid chrom atography (2.2.29). (0.05 per cent).
1-572 Citalopram Hydrochloride 2016
M axim um 20 ppm .
Dissolve 0.5 g in ethanol (96 per cent) R and dilute to 20 mT. and enantiomer
w ith the same solvent. 12 m L o f the solution complies with
test B. Prepare the reference solution using lead standard
solution (0.5 ppm Pb) obtained by diluting lead standard
solution (100 ppm Pb) R w ith ethanol (96 per cent) R. Filter
the solutions through a m em brane filter (nominal pore size D . R l = C N , R2 = H: (1 RS) -1 - (4-fluorophenyl)-1- [3-
0.45 pm). (methylamino)propyl] -1,3-dihydroisobenzofuran-5-
carbonitrile,
M axim um 0.5 per cent, determ ined on 1.000 g by drying in F. R l = Br, R2 = C H 3: 3-[(lÄ S )-5-brom o-l-
an oven at 105 °C for 4 h. (4-fluorophenyl)-1,3-dihydro isobenzofuran-1-yl] -N,N-
dim ethylpropan- 1-amine,
M axim um 0.1 per cent, determ ined on 1.0 g in a platinum G. R l = C O -[C H 2] 3-N (C H 3)2, R2 = C H 3:
crucible. 4-(dimethylamino) -1 - [(1RS) -1 - [3-(dimethylamino)propyl] -1 -
(4-fluorophenyl)-1,3-dihydroisobenzofuran-5 -yl] b utan-1-one.
A SSA Y
__________________________________________________________ PhEur
Dissolve 0.300 g in 50 m L o f ethanol (96 per cent) R and add
0.5 m L o f 0.1 M hydrochloric acid Carry out a potentiom etric
titration (2.2.20), using 0.1 M sodium hydroxide. Read the
volum e added between the 2 points o f inflexion.
1 m L o i 0.1 M sodium hydroxide is equivalent to 40.53 mg o f Citalopram Hydrochloride ★★*★★
★ ★
C 2oH 22B rFN 20 .
IM P U R IT IE S
Specified impurities B, D, G
the tests in the monograph. T hey are limited by the general Ha
Control of impurities in substances for pharmaceutical use): A , C, C 20H 22CIFN 2O 360.9 85118-27-0
E ,F .
Selective serotonin reuptake inhibitor; antidepressant.
Citalopram Oral Drops
.c h 3 and enantiomer PhEur.
N
CH3 D E F IN IT IO N
(1 RS ) - 1-[3-(Dimethylamino)propyl]- l-(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carbonitrile hydrochloride.
C o n te n t
A. R = C O -N H 2, X = H 2: (l.ftS )-l-[3-
(dimethylamino)propyl] -1 -(4-fluorophenyl)-1,3-
dihydroisobenzofuran-5-carboxamide, CHARACTERS
C. R = C N , X = O: (3&S)-6-cyano-3-[3- A p p e a ra n c e
(dimethylamino)propyl]-3-(4-fluorophenyI)isobenzofuran- W hite or almost white, crystalline powder.
1 (3H)-one, S o lu b ility
E. R = Cl, X = H 2: 3-[( 1RS) -5-chloro-1- (4-fluorophenyl) - Very soluble in water, freely soluble in anhydrous ethanol.
1,3-dihydroisobenzofuran-l -yl] -N ^-d im eth y lp ro p an -1- ID E N T IF IC A T IO N
am ine, A. Optical rotation (see Tests).
Comparison citalopram hydrochloride CRS.
C. It gives reaction (a) of chlorides (2.3.1).
its epimer at C*
ch3
and their enantiomers TESTS
N
1 S o lu tio n S
ch3
same solvent.
B. l-[3-(dim ethylam ino)propyl]-l-(4-fluorophenyl)-3- Solution S, examined immediately after preparation, is clear
hydroxy-133dihydroisobenzofuran-5-carbonitrile, (2.2.1) and not m ore intensely coloured than reference
solution Y 6 (2.2.2, Method IT).
2016 Citalopram Hydrochloride 1-573
O ptical rotation (2.2.7) Dissolve 1.0 g in 20 mL of water R. 12 mL of the solution

-0.10° to + 0.10°, determined on solution S. complies with test A Prepare the reference solution using
Related substances lead standard solution (1 ppm Pb) R.
Liquid chromatography (2.2.29). Loss on drying (2.2.32)
Test solution Dissolve 50 mg of the substance to be examined Maximum 0.5 per cent, determined on 1.000 g by drying in
in mobile phase A and dilute to 100.0 mL with mobile an oven at 105 °C for 4 h.
phase A. Suliated ash (2.4.14)
Reference solution (a) Dilute 1.0 mL of the test solution to Maximum 0.1 per cent, determined on 1.0 g in a platinum
100.0 mL with mobile phase A (solution A). Dilute 1.0 mL crucible.
of solution A to 10.0 mL with mobile phase A. ASSAY
Reference solution (b) Dissolve the contents of a vial of Dissolve 0.250 g in 50 mL of ethanol (96 per cent) R and add
citalopram for system suitability CRS (impurities B and D) in 0.5 mL of 0.1 M hydrochloric acid. Carry out a potentiometric
1.0 mL of solution A. titration (2.2.20), using 0.1“M sodium hydroxide. Read the
Column: volume added between the 2 points of inflexion.
— size. I = 0.25 m, 0 = 4.6 mm; 1 mL of 0.1 M sodium hydroxide is equivalent to 36.09 mg of
— stationary phase: end-capped octadecylsüyl silica gelfor C20H 22CIFN2O.
chromatography R (4 Jim);
IMPURITIES
Specified impurities’. B.
Mobile phase:
— mobile phase A: dissolve 1.58 g of ammonium formate R in Other detectable impurities(the following substances would, if
500 mL of a mixture of 4 volumes of acetonitrüe R, present at a sufficient level, be detected by one or other of
32 volumes of methanol R and 64 volumes of water R; the tests in the monograph. They are limited by the general
— mobile phase B: dissolve 1.58 g of ammonium formate R in acceptance criterion for other/unspecified impurities and/or
500 mL of a mixture of 32 volumes of water R and by the general monograph Substances for pharmaceutical use
68 volumes of acetonitrüe R-, (2034). It is therefore not necessary to identify these
Control of impurities in substancesfor pharmaceutical use): A, C,
Time Mobile phase A Mobile phase B D, E, F.
0 -2 100 0
2 -2 5 100->40 0*60
2 5 -3 0 40 60
CH3 and enantiomer

Irqection 40 |iL.
Identification of impurities Use the chromatogram supplied A Rl = CO-NH 2, R2 = CH3, X = H2: (lRS)-l-[3-
with citalopram for system siätabüity CRS and the (dimethylamino)propyl] -1 -(4-fluorophenyI)-1,3-
chromatogram obtained with reference solution (b) to dihydroisobenzofuran-5-carboxamide,
identify the peaks due to impurities B and D. C. Rl = CN, R2 = CH3, X = O: (3ÆS)-6-cyano-3-[3-
Relative retention With reference to citalopram (retention
(dimethylamino)propyI]-3-(4-fluorophenyI)isobenzofuran-
time = about 19 min): impurity B = about 0.7; 1(3ff)-one,
impurity D = about 0.9. D. R l = CN, R2 = H, X = H2: (lÄS)-l-(4-fluorophenyl)-l-
System suitability: reference solution (b): [3- (methylamino) propyl] -1,3-dihydroisobenzofuran-5-
— resolution: minimum 1.5 between die peaks due to carbonitrile,
impurity D and citalopram. E. Rl = Cl, R2 = CH3, X = H2: 3-[(lÄS)-5-chloro-l-
Limits: (4-fluorophenyl) -1,3-dihydroisobenzofuran-1-yl] -NJJ-
— impurity B: not more than 1.5 times the area of the dimethylpropan- 1-amine,
principal peak in the chromatogram obtained with F. Rl = Br, R2 = CH3, X = H2: 3-[(lÄS)-5-bromo-l-
reference solution (a) (0.15 per cent); (4-fluorophenyI) -1,3-dihydroisobenzofuran- 1-yl] -NJJ-
— unspecified impurities: for each impurity, not more than the dimethylpropan- 1-amine,
(0.2 per cent);
(0.05 per cent).
Maximum 20 ppm. B. l-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-3-
hydroxy-1,3-dihydroisobenzofuran-5-carbonitrile.
1-574 Citric Acid 2016
★*** ★ colour in the solution is not more intense than that in a

Anhydrous Citric Acid ★ ★ standard prepared at the same time in the same manner
Citric Acid * * * * * using 4 mL of a 0.1 g/L solution of oxalic add R.
(Ph. Eur. monograph 0455) Sulfates (2.4. IS)
Maximum 150 ppm.
HO C 0 2H
H 02C ^X ^C02H Dissolve 2.0 g in distilled water R and dilute to 30 mL with
the same solvent.
Aluminium (2.4.17)
CôHgO? 192.1 77-92-9
Maximum 0.2 ppm, if intended for use in the manufacture of
PhEur___ dialysis solutions.
DEFINITION Prescribed solution Dissolve 20 g in 100 mL of water R and
2-Hydroxypropane-l,2,3-tricarboxylic add. add 10 mL of acetate buffer solution p H 6.0 R.
Content Reference solution Mix 2 mL of aluminium standard solution
99.5 per cent to 100.5 per cent (anhydrous substance). (2 ppm Al) R, 10 mL of acetate buffer solution p H 6.0 R and
98 mL of water R.
CHARACTERS
Blank solution Mix 10 mL of acetate buffer solution p H 6.0 R
Appearance
White or almost white, crystalline powder, colourless crystals and 100 mL of water R.
or granules. Heavy metals (2.4.8)
Solubility Maximum 10 ppm.
Very soluble in water, freely soluble in ethanol (96 per cent), Dissolve 5.0 g in several portions in 39 mL of dilute sodium
hydroxide solution R and dilute to 50 mL with distilled water R.
mp
12 mL of the solution complies with test A. Prepare the
About 153 °C, with decomposition.
IDENTIFICATION W ater (2.5.12)
First identification B, E.
A. Dissolve 1 g in 10 mL of water R. The solution is strongly Maximum 0.1 per cent, determined on 1.0 g.
addic (2.2.4).
B. Infrared absorption spectrophotometry (2.2.24). Less than 0.5 IU/mg, if intended for use in die manufacture
Preparation Dry the substance to be examined and die of parenteral preparations without a further appropriate
reference substance at 105 ± 2 °C for 2 h. procedure for the removal of bacterial endotoxins.
Comparison anhydrous citric add CRS. ASSAY
C. Add about 5 mg to a mixture of 1 mL of acetic Dissolve 0.550 g in 50 mL of water R. Titrate with 1 M
anhydride R and 3 mL of pyridine R. A red colour devdops. sodium hydroxide, using 0.5 mL of phenolphthalein solution R as
D. Dissolve 0.5 g in 5 mL of water R , neutralise using 1 M indicator.
sodium hydroxide (about 7 mL), add 10 mL of calaum chloride 1 mL of 1 M sodium hydroxide is equivalent to 64.03 mg
solution R and heat to boiling. A white predpitate is formed. of C 6H 80 7.
E. Water (see Tests). LABELLING
TESTS The label states, where applicable, that the substance is
Appearance of solution intended for use in the manufacture of dialysis solutions.
The solution is clear (2.2.1) and colourless or not more PhEur
intensdy coloured than reference solution Y7, BY7 or GY7
(2.2.2, Method II).
solvent. ★ ★
Citric Acid Monohydrate ★ ★
Readily carbonisable substances
To 1.0 g in a cleaned test tube add 10 mL of sulfuric acid R (Ph. Eur. monograph 0456)
and immediately heat the mixture in a water-bath at
HO C 0 2H
90 ± 1 °C for 60 min. Cool rapidly immediatdy afterwards.
The solution is not more intensdy coloured than a mixture HOjC ^ ^ X ^ C O jH 1 H2°
of 1 mL of red primary solution and 9 mL of yellow primary
solution (2.2.2, Method I). C6H80 7,H20 210.1 5949-29-1
Oxalic acid PhEur _________
Maximum 360 ppm, calculated as anhydrous oxalic add.
DEFINITION
Dissolve 0.80 g in 4 mL of water R. Add 3 mL of hydrochloric 2-Hydroxypropane-1,2,3-tricafboxylic add monohydrate.
acid R and 1 g of zinc R in granules. Boil for 1 min. Allow to
stand for 2 min. Transfer the supernatant to a test-tube Content
containing 0.25 mL of a 10 g/L solution of phenyJhydrazine
hydrochloride R and heat to boiling. Cool rapidly, transfer to a CHARACTERS
graduated cylinder and add an equal volume of hydrochloric Appearance
add R and 0.25 mL of a 50 g/L solution of potassium White or almost white, crystalline powder, colourless crystals
ferricyanide R. Shake and allow to stand for 30 min. Any pink or granules, efflorescent.
2016 Cladribine 1-575
Solubility Dissolve 5.0 g in several portions in 39 mL of dilute sodium

Very soluble in water, freely soluble in ethanol (96 per cent). hydroxide solution R and dilute to 50 mL with distilled water R.
12 mL of the solution complies with test A. Prepare the
IDENTIFICATION
First identification B,E.
Water (2.5.12)
Second identifkation A , C, D, E.
A. Dissolve 1 g in 10 mL of water R. The solution is strongly
acidic (2.2.4). Sulfa ted ash (2.414)
Preparation Dry the substance to be examined and the
reference substance at 105 ± 2 °C for 2 h.
Comparison citric acid monohydrate CRS. procedure for the removal of bacterial endotoxins.
C. Add about 5 mg to a mixture of 1 mL of acetic
ASSAY
anhydride R and 3 m l, of pyridine R. A red colour develops.
Dissolve 0.550 g in 50 mL of water R. Titrate with 1 M
D. Dissolve 0.5 g in 5 mL of water R, neutralise using 1 M sodium hydroxide, using 0.5 mL of phenolphthalein solution R as
sodium hydroxide (about 7 mL), add 10 mL of calcium chloride
indicator.
solution R and heat to boiling. A white precipitate is formed.
1 mL o i l M sodium hydroxide is equivalent to 64.03 mg
E. Water (see Tests). of CgHgOy.
TESTS STORAGE
Appearance of solution In an airtight container.
The solution is clear (2.2.1) and colourless or not more
intensely coloured than reference solution Y7, BY7 or GY7 LABELLING
(2.2.2, Method II). The label states, where applicable, that the substance is
Dissolve 2.0 g in water R and dilute to 10 mL with the same intended for use in the manufacture of dialysis solutions.
solvent. __________________________________________________________ PhEur

To 1.0 g in a cleaned test tube add 10 mL of sulfuric acid R
and immediately heat the mixture in a water-bath at
90 ± 1 °C for 60 min. Cool rapidly immediately afterwards. Cladribine ★ ★
The solution is not more intensely coloured than a mixture ★ '★
of 1 mL of red primary solution and 9 mL of yellow primary (Ph. Eur. monograph 2174) * * * * *
solution (2.2.2, Method I).

Oxalic acid NHj
Maximum 360 ppm, calculated as anhydrous oxalic acid.
Dissolve 0.80 g in 4 mL of water R. Add 3 mL of hydrochloric 1 X >
acid R and 1 g of zinc R in granules. Boil for 1 min. Allow to
stand for 2 min. Transfer the supernatant to a test-tube HO
containing 0.25 mL of a 10 g/L solution of phenylhydrazine
hydrochloride R and heat to boiling. Cool rapidly, transfer to a
graduated cylinder and add an equal volume of hydrochloric pOH
acid R and 0.25 mL of a 50 g/L solution of potassium
ferricyanide R. Shake and allow to stand for 30 min. Any pink
colour in the solution is not more intense than that in a CjoH^ClNgi^ 285.7 4291-63-8
standard prepared at the same time in the same manner Action and use
using 4 mL of a 0.1 g/L solution of oxalic acid R. Purine analogue; cytostatic.
Sulfates (2.413)
Maximum 150 ppm. PhEir _______________________
Dissolve 2.0 g in distilled water R and dilute to 30 mL with DEFINITION

the same solvent. 2-Chloro-9-(2-deoxy-p-D-e73'£/in>-pentofuranosyl)-9H-purin-6-
Aluminium (2.417) am ine.
Maximum 0.2 ppm, if intended for use in the manufacture of Content
dialysis solutions. 97.0 per cent to 102.0 per cent (anhydrous substance).
Prescribed solution Dissolve 20 g in 100 mL of water R and CHARACTERS
add 10 mL of acetate buffer solution p H 6.0 R. Appearance
Reference solution Mix 2 mL of aluminium standard solution White or almost white, crystalline powder.
(2 ppm Al) R, 10 mT. of acetate buffer solution p H 6.0 R and
Solubility
98 mL of water R.
Slightly soluble in water, soluble in dimethyl sulfoxide,
Blank solution Mix 10 mL of acetate buffer solution p H 6.0 R slightly soluble in methanol, practically insoluble in
and 100 mL of water R. acetonitrile.
Heavy metals (2.48) It shows polymorphism (5.9).
Maximum 10 ppm.
IDENTIFICATION
1-576 Cladribine 2016
B. Infrared absorption spectrophotometry (2.2.24). Reference solution (c) Dilute 1.0 mL of reference solution (b)
Comparison cladribine CRS. to 10.0 mL with the solvent mixture.
If the spectra obtained in the solid state show differences, Reference solution (d) Dissolve 1.0 mg of cladribine
dissolve the substance to be examined in the minimum impurity C CRS in reference solution (b) and dilute to
volume of methanol R and evaporate to dryness. Dry the 25.0 mL with the same solution.
precipitate at 100 °C for 2 h and record a new spectrum Reference solution (e) Dilute 5.0 mL of reference solution (c)
using the residue. to 10.0 mL with the solvent mixture.
TESTS Reference solution (f) Dissolve 3 mg of cladribine for peak
Appearance of solution identification CRS (containing impurities A, B, C and D) in
The solution is clear (2.2.1) and colourless (2.2.2, 2 mL of the solvent mixture.
Method II). Column:
Disperse 0.15 g in water R, dilute to 50 mL with the same — size:. I = 0.25 m, 0 = 4.6 mm;
solvent and sonicate until dissolution is complete. — stationary phase, base-deactivated octylsüyl silica gelfor
Mobile phase:
—21.0 to —27.0 (anhydrous substance).
— mobile phase A: waterfor chromatography R;
Dissolve 0.25 g in dimethyl sulfoxide R and dilute to 25.0 mL — mobile phase B: acetonitrile for chromatography R;
with the same solvent. — mobile phase C: 50 g/L solution of phosphoric acid R in
Im purity E waterfor chromatography R;
Test solution Dissolve 40.0 mg of the substance to be Time Mobile phase A Mobile phase B Mobile phase C
examined in dimethyJformamide R and dilute to 2.0 mL with (min) (per cent V/V) (per cent V/V) (per cent V/V)
the same solvent. 0 -1 0 80*70 10 * 2 0 10
Reference solution (a) Dissolve 5.0 mg of 2-deoxy-n-ribose R 10-25 70*20 20*70 10
(impurity E) in dimethylformamide R and dilute to 25.0 mL
25-30 20 70 10
with the same solvent. Dilute 3.0 mL of this solution to
10.0 mL with dimethylformamide R.
Reference solution (b) Dissolve 10.0 mg of 2-deoxy-u-ribose R Flow rate 0.8 mlVmin.
(impurity E) in dimethylformamide R and dilute to 5.0 mL Detection Spectrophotometer at 252 nm.
with the same solvent. Mix 9 volumes of this solution with
Injection 20 |iL of test solution (a) and reference
1 volume of the test solution.
solutions (c), (d), (e) and (f).
Mobile phase concentrated ammonia R, ethanol (96 per cent) R, with cladribine for peak identification CRS and the
ethyl acetate R (20:40:40 VIVIV). chromatogram obtained with reference solution (f) to identify
Application 5 nL as bands of 10 mm; thoroughly dry the the peaks due to impurities A, B, C and D.
points of application in a current of warm air. Relative retention With reference to cladribine (retention
Development Over 2/3 of the plate. time = about 10 min): impurity A = about 0.33;
Drying In air, then heat at 45 °C for 10 min. impurity B = about 0.44; impurity C = about 0.73;
Detection Spray with a solution containing 0.5 g of thymol R impurity D = about 0.92.
in a mixture of 5 mL of sulfuric acid R and 95 mL of ethanol System suitability Reference solution (d):
(96 per cent) R; heat at 110 °C for 20 min or until the spots — resolution', minimvim 4.5 between the peaks due to
appear. impurity C and cladribine.
System suitability: reference solution (b): Limits'.
— the chromatogram shows 2 clearly separated spots. — correction factors:for the calculation of content, multiply
Limit:. the peak areas of the following impurities by the
— impurity E: any spot due to impurity E is not more corresponding correction factor impurity B = 1.7;
intense than the spot in the chromatogram obtained with impurity C = 0.8;
reference solution (a) (0.3 per cent). — impurities A, C: for each impurity, not more than 3 times
Related substances obtained with reference solution (c) (0.3 per cent);
Liquid chromatography (2.2.29). — impurities B, D: for each impurity, not more than twice
Solvent mixture acetonitrile R, water R (10:90 VIV). the area of the principal peak in the chromatogram
Test solution (a) Dissolve 25.0 mg of the substance to be obtained with reference solution (c) (0.2 per cent);
examined in the solvent mixture and dilute to 5.0 mT, with — unspecified impurities: for each impurity, not more than the
the solvent mixture. area of the principal peak in the chromatogram obtained
Test solution (b) Dissolve 20.0 mg of the substance to be with reference solution (c) (0.10 per cent);
examined in the solvent mixture and dilute to 100.0 mT. with — total: not more than 10 times the area of the principal
the solvent mixture. peak in the chromatogram obtained with reference
solution (c) ( 1.0 per cent);
Reference solution (a) Dissolve 20.0 mg of cladribine CRS in
— disregard limit, the area of the principal peak in the
the solvent mixture and dilute to 100.0 mT. with the solvent
chromatogram obtained with reference solution (e)
mixture.
(0.05 per cent).
Reference solution (b) Dilute 1.0 mL of test solution (a) to
100.0 mL with the solvent mixture. W ater (2.5.32)
2016 Clarithromycin 1-577

Less than 3 IU/mg, if intended for use in the manufacture of
h 3c
ASSAY F. R = NH2: 4-methylbenzamide,
G. R = OCH3: methyl 4-methyIbenzoate.
PhEur
Injection Test solution (b) and reference solution (a).
Calculate the percentage content of CioH 12Q N 5 0 3 from the
declared content of cladribine CRS.
STORAGE Clarithromycin ★ ★
★ ★
Protected from light, at a temperature of 2 °C to 8 °C. If the *.jr jf-k *
substance is sterile, store in a sterile, airtight, tamper-proof (Ph. Ever, monograph 1651)
container.
LABELLING
The label states, where applicable, that the substance is
suitable for use in the manufacture of parenteral ch3
preparations.
IMPURITIES
Specified impurities A,B, C, D, E.
the tests in the monograph. They are limited by the general ch3
by the general monograph Substances for pharmaceutical use C38H$9N013 748 81103-11-9
impurities for demonstration of compliance. See also 5.10. Action and use
Control of impurities in substancesfor pharmaceutical use): F, G. Macrolide antibacterial.
Preparations
Clarithromycin for Infusion
Clarithromycin Tablets
Prolonged-release Clarithromycin Tablets
P h E tr ___________________________________________________________
DEFINITION
(3R,45,55,6R,7R,9R,nR, 12R, 135,14J?)-4-[(2,6-Dideoxy-3-
C-methyl-3-O-methyi-a-L-nJo-hexopyranosyl) oxy] -14-ethyl-
A. R = NH2: 9-(2-deoxy-ß-D-ö7yrÄn>-pentofuranosyl)-9H- 12,13-dihydroxy-7-methoxy-3,5,7,9,11,13-hexamethyl-6-
purin-2,6-diamine, [[3,4,6-trideoxy-3-(dimethylamino)-P-D-xyfo-
hexopyranosyl]oxy]oxacyclotetradecane-2 , l 0-dione (6 -0 -
B. R = OCH3: 9-(2-deoxy-ß-D-eo'£Äro-pentofuranosyl)-2-
methylerythromycin A).
methoxy-9H-purin-6-amine,
nh2
Content
n itA 96.0 per cent to 102.0 per cent (anhydrous substance).
Cl
a nI > CHARACTERS
Appearance
C. 2-chloro-7H-purin-6-amine (2-chloroadenine), White or almost white, crystalline powder.
Solubility
Practically insoluble in water, soluble in acetone and in
methylene chloride, slightly soluble in methanol.
IDENTIFICATION
Comparison: clarithromycin CRS.
D. 2-chloro-9-(2-deoxy-a-i>go'£Äro-pentofiiranosyl)-9H- TESTS
purin-6 -amine, Solution S
HOv Dissolve 0.500 g in methylene chloride R and dilute to
OH
Solution S is clear or not more opalescent than reference
OH
E. 2 -deoxy-D-eryifcro-pentofuranose (2-deoxy-i>ribose), reference solution Y7 (2.2.2, Method II).
1-578 Clarithromycin 2016
Specific optical rotation (2.2.7) Limits:

-9 4 to -102 (anhydrous substance), determined on — for the calculation of contents, multiply
correction factors:
solution S. the peak areas of the following impurities by die
Related substances corresponding correction factor impurity G = 0.27;
Liquid chromatography (2.2.29). impurity H = 0.15; use the chromatogram supplied with
clarithromycin for peak identification CRS to identify the
peaks;
examined in 25 mL of acetomtrUe R l and dilute to 50.0 mL
— any impurity: not more than twice the area of die principal
with water R. peak in the chromatogram obtained with reference
Reference solution (a) Dissolve 75.0 mg of clarithromycin CRS solution (c) (1.0 per cent), and not more than 4 such
in 25 mL of acetonitrile R1 and dilute to 50.0 mL with peaks have an area greater than 0.8 times the area of the
waxerR. principal peak in the chromatogram obtained with
Reference solution (b)Dilute 5.0 mL of reference solution (a) reference solution (c) (0.4 per cent);
to 100.0 mL with a mixture of equal volumes of — total: not more than 7 times the area of the principal peak
acetonitrile R1 and water R. in the chromatogram obtained with reference solution (c)
Reference solution (c) Dilute 1.0 mL of reference solution (b) (3.5 per cent);
to 10.0 mL with a mixture of equal volumes of acetonitrile R1 — disregard limit. 0.2 times the area of the principal peak in
and water R. the chromatogram obtained with reference solution (c)
Reference solution (d) Dissolve 15.0 mg of clarithromycin for
(0.1 per cent); disregard the peaks eluting before
peak identification CR S in 5.0 mL of acetonitrile R1 and dilute
impurity I and after impurity H.
to 10.0 mL with water R. Heavy metals (2.4.8)
Blank solution Dilute 25.0 mL of acetomtrUe R1 to 50.0 mL Maximum 20 ppm.
with water R and mix. Dissolve 1.0 g in a mixture of 15 volumes of water R and
Column: 85 volumes of dioxan R and dilute to 20 mL with the same
— size. I = 0.10 m, 0 = 4.6 mm, mixture of solvents. 12 mL of the solution complies with
— stationary phase: octadecylsUyl silica gelfor chromatography R test B. Prepare the reference solution using lead standard
(3.5 urn), solution (1 ppm Pb) obtained by diluting lead standard
— temperature. 40 °C. solution (100 ppm Pb) R with a mixture of 15 volumes of
Mobile phase: water R and 85 volumes of dioxan R.
— mobile phase A: a 4.76 g/L solution of potassium dihydrogen W ater (2.5.12)
phosphate R adjusted to pH 4.4 with dilute phosphoric Maximum 2.0 per cent, determined on 0.500 g.
acid R or a 45 g/L solution of potassium hydroxide R, Sulfated ash (2.4.14)
filtered through a C18 filtration kit, Maximum 0.2 per cent, determined on 0.5 g.
— mobile phase B: acetomtrUe R l,
ASSAY
Time Mobile phase A Mobile phase B related substances with the following modifications.
0 -3 2 7 5 -»40 25 ->60
Calculate the percentage content of C38H 69N 0 13.
32 - 34 40 60
IMPURITIES
Specified impurities: A, B, C, D, E, F, G, H , I, J, K , L, M , N,
Flow rate 1.1 mL/min. O, P.
Injection 10 nL of the blank solution, the test solution and o
h3c II ch 3
reference solutions (b), (c) and (d).
Relative retention r (not tq) with reference to clarithromycin
(retention time = about 11 min): impurity I = about 0.38;
impurity A = about 0.42; impurity J = about 0.63;
impurity L = about 0.74; impurity B = about 0.79;
impurity M = about 0.81; impurity C = about 0.89;
impurity D = about 0.96; impurity N = about 1.15;
impurity P = about 1.35; impurity O = about 1.41; ch 3
impurity K = about 1.59; impurity G = about 1.72;
impurity H = about 1.82.
A. Rl = CH3, R2 = OH, R3 = H: 2-demethyl-2-
System suitability:
(hydroxymethyl)-6-0-methylerythromycin A (clarithromycin
— symmetryfactor, maximum 1.7 for tile peak due to
F),
clarithromycin in the chromatogram obtained with
B. Rl = R2 = R3 = H: 6-O-methyl-15-norerythromycin A,
reference solution (b),
— peak-to-valley ratio: minimum 3.0, where Hp = height P. Ri = R3 = CH3, R2 = H: 4 ', 6-di-O-methylerythromydn
above the baseline of the peak due to impurity D and A,
H v = height above the baseline of the lowest point of the
clarithromycin in the chromatogram obtained with
reference solution (d).
2016 Clebopride Malate 1-579
R 3 -0 , ,0 —R
C. Rl = R2 = CH3, R3 = H: 6-O-methylerythromycin A L. R = H: 6-O-methylerythromycin A (Z)-9-oxime,

(£)-9-oxime, O. R = CH3: 6 -O-methylerythromycin A (Z)-9-(0-
G. R l = R2 = R3 = CH3: 6-0-methylerythromydn A methyloxime),
(£)-9-( O-methyloxime),
J. R l = CH3, R2 = R3 = H: erythromycin A (E)-9-oxime,
M. R l = R3 = H, R2 = CH3: 3"-N-demethyl-6-0-
methylerythromycin A (£)-9-oxme3
N. (10£)-10jl l-didehydro-l l-deoxy-6-O-

methylerythromycin A.
PhEur
D. R l = R2 = R3 = H: 3"-N-demethyl-6-0-
methylerythromycin A,
ic + ir
E. Rl = R2 = CH3, R3 = H: 6,1 1-di-O-methylerythromycin Clebopride Malate ★ ' ★
★ ★
A,
F. Rl = R3 = CH3, R2 = H: 6 , 12-di-O-methylerythromycin
A,
HOJ X »
H. Rl = CHO, R2 = R3 = H: 3 "-N-demethyl-3 '-iV-formyl-
6 -O-methylerythromycin A, a
■ H )
h o 2c
h 2n och3 and enantiomer
C24H3oC1N307 508.0 57645-91-7
Action and use

Dopamine receptor antagonist; antiprotozoal (veterinary).
PhEur__________________________________________________________
OH h ch3
DEFINITION
I. 3-0-decladinosyl-6-0-methylerythromycin A, 4-Amino-N-( 1-benzylpiperidin-4-yl)-5-chloro-2-
methoxybenzamide acid CRS)-2-hydroxybutanedioate.
Content
CHARACTERS
Appearance
Solubility
Sparingly soluble in water and in methanol, slightly soluble in
anhydrous ethanol, practically insoluble in methylene
K. (1S,2R35R,6S,7S,8R,9R, 112)-2-ethyl-6-hydroxy-9- chloride.
methoxy-lj5,7,9jlljl3-hexamethyi-8-[[3,4j6-trideoxy-3-
mp
(dimethylamino)-ß-D-xyto-hexopyranosyl] oxy] -3,15-
dioxabicydo[10.2.1]pentadeca-l l 313-dien-4-one (3-0-
decladinosyl-8,9:10,11-dianhydro-6-O- IDENTIFICATION
methylerythromycin A-9jl2-hemiketal)j First identification B, C .'
1-580 Clebopride Malate 2016
Second identification A , C, D. Column:

A. Ultraviolet and visible absorption spectrophotometry — size: I = 0.12 m, 0 = 4.0 mm;
(2.2.25). — stationary phase: octadecylsUyl silica gd for chromatography R
Test solution Dissolve 20.0 mg in water R and dilute to

(5 nm).
100.0 mL with the same solvent. Dilute 10.0 mL of the Mobile phase Mix 20 volumes of acetonitrile R l and
solution to 100.0 mL with water R. 80 volumes of a 1 g/L solution of sodium heptanesulfonate R
adjusted to pH 2.5 with phosphoric add R.
Flow rate 1 mUmin.
Absorption maxima At 270 nm and 307 nm.
Specific absorbance at the absorption maxima: Detection Spectrophotometer at 215 nm.
— at 270 nm: 252 to 278; Injection 20 (iL.
— at 307 nm: 204 to 226. Run time Twice the retention time of clebopride.
B. Infrared absorption spectrophotometry (2.2.24). Relative retention With reference to clebopride (retention
Comparison clebopride malate CRS. time = about 15 min): metoclopramide = about 0.45.
C. Dissolve 20 mg in 1 mL of sulfuric acid R, add 1 mL of System suitability, reference solution (b):
fi-naphthol solution R land mix. The solution examined in — resolution: minimum 5.0 between the peaks due to
daylight is yellow with blue fluorescence. metoclopramide and clebopride.
D. Thin-layer chromatography (2.2.27). Limits:
— for each impurity, not more than the
unspecified impurities-,
in anhydrous ethanol R and dilute to 10 mL with the same
solvent.
Reference solution (a) Dissolve 5 mg of clebopride malate CRS in the chromatogram obtained with reference solution (a)
in anhydrous ethanol R and dilute to 10 mL with the same (0.3 per cent);
solvent. — disregard limit: 0.5 times the area of the principal peak in
Reference solution (b) Dissolve 5 mg of clebopride malaxe CRS the chromatogram obtained with reference solution (a)
and 5 mg of metoclopramide hydrochloride CRS in anhydrous (0.05 per cent); disregard the 2 peaks eluting within the
ethanol Rand dilute to 10 mL with the same solvent. first 2 min.
Plate TLC silica gel F 254 plate R. Chlorides
Mobile phase concentrated ammonia R, acetone R, methanol R, Maximum 100 ppm.
toluene R (2:14:14:70 VIV/VIV). Prepare the solutions at the same time.
Application 5 |iL as bands of 10 mm by 3 mm. Test solution Dissolve 0.530 g in 20.0 mL of anhydrous acetic
Development Over 3/4 of the plate. add R, add 6 ml, of dilute nitric acid R and dilute to 50.0 mL
Drying In air. with water R.
Detection Examine in ultraviolet light at 254 nm. Reference solution To 1.5 mL of 0.001 M hydrochloric add add
System suitability, reference solution (b): 20.0 mL of anhydrous acetic add R and 6 mL of dilute nitric
acid R and dilute to 50.0 mL with water R.
— the chromatogram shows 2 clearly separated zones.
Results The principal zone in the chromatogram obtained Transfer both recently prepared solutions to separate test-
with the test solution is similar in position and size to the tubes. Add to each tube 1 mT. of silver nitrate solution R2.
principal zone in the chromatogram obtained with reference Allow to stand for 5 min protected from light. Examine the
solution (a). tubes laterally against a black background. Any opalescence
in the test solution is not more intense than that in the
TESTS reference solution.
Solution S
Sulfates
Maximum 100 ppm.
Prepare the solutions at the same time.
Test solution Dissolve 3.00 g in 20.0 mL of glacial acetic
Solution Sj examined immediately after preparation, is clear
add R, heating gently if necessary. Allow to cool and dilute
(2.2.1) and colourless (2 .2.2, Method I).
to 50.0 mT, with water R.
pH (2.2.3)
Reference solution To 9 mL of sulfate standard solution (10 ppm
SO 4J R l add 6 mL of glacial acetic add R.
Related substances
Into 2 test-tubes introduce 1.5 mL of sulfate standard solution
(10 ppm SO 4) R l and add 1 mL of a 250 g/L solution of
Test solution Dissolve 0.100 g of the substance to be barium chloride R. Shake and allow to stand for 1 min.
examined in the mobile phase and dilute to 100.0 mL with To one of the tubes add 15 mL of the test solution and to
the mobile phase. the other add 15 mL of the reference solution. After 5 min,
Reference solution (a) Dilute 1.0 mL of the test solution to any opalescence in the tube containing the test solution is not
100.0 mL with the mobile phase. Dilute 1.0 mT. of this more intense than that in the tube containing the reference
solution to 10.0 mL with the mobile phase. solution.
Reference solution (b) Dissolve 10 mg of the substance to be Heavy metals (2.4.8)
examined and 10 mg of metoclopramide hydrochloride CRS in Maximum 20 ppm.
the mobile phase and dilute to 100.0 mL with the mobile 1.0 g complies with test D. Prepare the reference solution
phase. Dilute 1.0 mL of the solution to 10.0 mT. with the using 2 mL of lead standard solution (10 ppm Pb) R.
mobile phase.
2016 Clemastine Fumarate 1-581
Loss on drying (2.2.32) PhEur.

Maximum 0.5 per cent, determined on 1.000 g by drying in DEFINITION
an oven at 105 °C. (2R)-2-[2- [ ( 1 i?)- 1 -(4-Chlorophenyl) - 1 -phenylethoxy] ethyl]-1 -
Sulfated ash (2.4.14) methylpyrrolidine hydrogen (£)-butenedioate.
Maximum 0.1 per cent, determined on 1.0 g. Content
ASSAY 99.0 per cent to 101.0 per cent (dried substance).
Dissolve 0.400 g in 50 mL of anhydrous acetic acid R. Titrate CHARACTERS
with 0.1 M perchloric acid, determining the end-point Appearance
potentiometrically (2.2.20). White or almost white, crystalline powder.
1 mL of 0.1 M perchloric acid is equivalent to 50.80 mg
Solubility
o f C 2 4 H 3 0 C IN 3 O 7.
Very slightly soluble in water, sparingly soluble in ethanol
STORAGE (70 per cent VIV), practically insoluble in heptane.
Protected from light. IDENTIFICATION
IMPURITIES First identification A, B
Other detectable impurities (the following substances would, if Second identification A, C, D
present at a sufficient level, be detected by one or other of A. Specific optical rotation (see Tests).
acceptance criterion for other/unspecified impurities and/or B. Infrared absorption spectrophotometry (2.2.24).
by the general monograph Substances for pharmaceutical use Comparison clemastinefumarate CRS.
(2034). It is therefore not necessary to identify these C. Thin-layer chromatography (2.2.27).
impurities for demonstration of compliance. See also 5.10. Test solution Dissolve 20.0 mg of the substance to be
Control of impurities in substancesfor pharmaceutical use): A, B, examined in methanol R and dilute to 10.0 mL with the same
C. solvent.
Cl COjH Reference solution. Dissolve 20.0 mg of clemastine
fumarate CRS in methanol R and dilute to 10.0 mL with the
H2N ' ^ OCH3 same solvent.
Plate TLC silica gel plate R
A. 4-amino-5-chloro-2-methoxybenzoic add, Mobile phase concentrated ammonia R, methanol R,
tetrahydrofwran R (1:20:80 VIVIV).
'N'
Application 5 (jL.
HjN Development Over 2/3 of the plate.
Drying In a current of cold air for 5 min.
B. l-benzylpiperidin-4-amine,
Detection Spray with a freshly prepared mixture of 1 volume
of potassium iodobismuthate solution R and 10 volumes of dilute
acetic add R and then with dilute hydrogen peroxide solution R;
N cover the plate immediately with a glass plate of the same
H size and examine the chromatograms after 2 mm.
h2n ' ^ ' och3 Results The prindpal spot in the chromatogram obtained with
The test solution is similar in position, colour and size to the
C. 4-amino-iV-(l -benzylpiperidin-4-yl)-2-methoxybenzamide. prindpal spot in the chromatogram obtained with the
_________________________ ;________________________________ PhEur reference solution.
D. Thin-layer chromatography (2.2.27).
Test soltaion Dissolve 40 mg of the substance to be examined
in methanol R and dilute to 2.0 mL with the same solvent.
Clemastine Fumarate *****
★ ★ Reference solution Dissolve 50 mg offumaric add CRS in
* * * * * ethanol (96 per cent) R and dilute to 10.0 mL with the same
Clemastine Hydrogen Fumarate
solvent.
Plate TLC silica gd plate R.
Mobilephase water R, anhydrousformic add R, di-isopropyl
ether R (5:25:70 V/VIV).
ho 2c
Application 5 (jL.
Drying At 100-105 °C for 30 min and allow to cool.
Detection Spray with a 16 g/L solution oi potassium
C25H30CINO5 460.0 14976-57-9 permanganate R and examine in daylight.
Results The Prindpal spot with the highest Rp value in the
Action and use chromatogram obtained with the test solution is similar in
Histamine Hx receptor antagonist; antihistamine. position, colour and size to the principal spot in the
Preparations chromatogram obtained with the reference solution.
Clemastine Oral Solution
Clemastine Tablets
1-582 Clemastine Fumarate 2016
TESTS System suitability, reference solution (b):

Solution S — resolution: minimum 2.0 between the peaks due to
Dissolve 0.500 g in methanol R and dilute to 50.0 mL with impurity B and clemastine.
the same solvent. Calculation ofpercentage contents:
Appearance of solution — for each impurity, use the concentration of clemastine in
Solution S is clear (2.2.1) and not more intensely coloured reference solution (a).
than reference solution BY7 (2.2.2, Method II). Limits:
pH (2.2.3)
0.10 per cent;
3.2 to 4.2.
— maximum 0.2 per cent;
total:
Suspend 1.0 g in 10 mL of carbon dioxide-free water R. — 0.05 per cent; disregard the peak due
reporting threshold:
Specific optical rotation (2.2.7) to fumaric add.
+ 15.0 to + 18.0 (dried substance), determined on
solution S.
Related substances Maximum 0.5 per cent, determined on 1.000 g by drying in
Liquid chromatography (2.2.29). Prepare the solutions an oven at 105 °C for 6 h.
Phosphate buffer solution p H 71 Mix 1.9 volumes of a 138 g/L Maximum 0.1 per cent, determined on 1.0 g.
solution of sodium dihydrogen phosphate monohydrate R,
6.8 volumes of an 89 g/L solution of disodium hydrogen ASSAY
phosphate dihydrate R and 91.3 volumes of waterfor Dissolve 0.350 g in 60 mL of anhydrous acetic add R. Titrate
chromatography R. with 0.1 M perchloric acid, determining the end-point
Solvent mixture acetonitrUe R l, waterfor chromatography R potentiometrically (2.2.20).
(20:80 VIV). 1 mL of 0.1 Mperchloric acid is equivalent to 46.00 mg
Test solution Dissolve 10 mg of the substance to be examined of C25H 3oC1N 0 5.
in 30 mL of the solvent mixture with the aid of ultrasound IMPURITIES
and dilute to 50.0 mL with the solvent mixture.
Reference solution (a) Dilute 1.0 mL of the test solution to Other detectable impurities (the following substances would, if
100.0 mL with the solvent mixture. Dilute 1.0 mL of this present at a sufficient level, be detected by one or other of
solution to 10.0 mL with the solvent mixture. the tests in the monograph. They are limited by the general
Reference solution (b) Dissolve the contents of a vial of acceptance criterion for other/unspecified impurities and/or
clemastinefor system suitability CRS (containing impurity B) in by the general monograph Substances for pharmaceutical use
1.0 mL of the solvent mixture with the aid of ultrasound for (2034). It is therefore not necessary to identify these
about 5 min. impurities for demonstration of compliance. See also 5.10.
Column: Control of impurities in substances for pharmaceutical use): A , B,
— sizer. I = 0.15 m, 0 = 4.6 mm; C.
— stationary phase: end-capped polar-embedded octadecylsilyl
amorphous organosUica polymer R (3.5 pm);
Mobile phase: o ' Nr ° and epimer at N
— mobile phase A: phosphate buffer solution pH 7.1; ° ' CH3
— mobile phase B: phosphate buffer solution pH 7.1,
acetonitrile R l (40:60 V!V)\
A. (li?5,2i?)-2-[2-[(li?)-l -(4-chlorophenyl)-1-
Time Mobile phase A Mobile phase B phenylethoxy] ethyl] -1 -methylpyrrolidine 1-oxide,
0-3 45 55
3 -2 3 45 -»5 55 -» 95
PH3 j ^ ^ N - CH3
23 - 26 5 95

Detection Spectrophotometer at 225 nm. B. 4-[(1R )-1-(4-chlorophenyl)-1-phenylethoxy] -1-
Injection 90 |iL. methylazepane,
with clemastinefor system suitability CRS and the
identify the peak due to impurity B. and enantiomer
Relative retention With reference to clemastine (retention

time = about 17 min): fumaric acid = about 0.1;
C. (1RS)-1-(4-chlorophenyl)-1-phenylethanol.
2016 Clenbuterol Hydrochloride 1-583
pH (2.2.3)
Clenbuterol Hydrochloride }***%
(Ph. Eur. monograph 1409) * Optical rotation (2.2.7)
- 0 .10° to + 0 .10°.
V HH Dissolve 0.30 g in water R and dilute to 10.0 mL with the
ci\ / V v ny ch3 same solvent. Filter if necessary.
I J H3C CH3 and enantlomer , HCI
Related substances
Cl
Test solution Disperse 100.0 mg of the substance to be
C 12H 1 9 Cl3N20 313.7 21898-19-1
the mobile phase.
Action and use Reference solution (a) Dilute 0.1 mL of the test solution to
Beta2-adrenoceptor agonist; bronchodilator. 100.0 mL with water R.
Reference solution (b) Dissolve 5 mg of clenbuterol
PhEur----------------------------------------------------------------------------------------
impurity B CRS in 10 mL of the mobile phase, add 2.5 mL
DEFINITION of the test solution and dilute to 25.0 mL with the mobile
(li?5)-l -(4-Amino-3j5-dichlorophenyl)-2-[(1,1 - phase.
dimethylethyl)amino]ethanol hydrochloride. Column:
Content — size: I = 0.125 m, 0 = 4 mm,
99.0 per cent to 101.0 per cent (anhydrous substance). — stationary phase: end-capped octadecylstlyl silica gel for
chromatography R (5 |im),
CHARACTERS — temperature: 40 °C.
Appearance
Mobile phase Mix 200 volumes of acetonitri"le R, 200 volumes
of methanol R and 600 volumes of a solution prepared as
Solubility follows: dissolve 3.0 g of sodium decanesulfonate R and 5.0 g
Soluble in water and in ethanol (96 per cent), slightly soluble of potassium dihydrogen phosphate R in 900 mL of water R,
in acetone. adjust to pH 3.0 with dilute phosphoric acid R and dilute to
mp 1000 mL with water R.
About 173 °C, with decomposition. Flow rate 0.5 mL/min.
IDENTIFICATION Detection Spectrophotometer at 215 nm.
First identification A, C. Injection 5 pL.
Second identification B, C. Run time 1.5 times the retention time of clenbuterol.
A. Infrared absorption spectrophotometry (2.2.24). Retention time Clenbuterol = about 29 min.
Comparison clenbuterol hydrochloride CRS. System suitability: reference solution (b):
B. Thin-layer chromatography (2.2.27). — resolution: minimum 4.0 between the peaks due to
impurity B and clenbuterol.
in 10 mL of methanol R Limits:
— impurities A, B, C, D, E, F: for each impurity, not more
Reference solution Dissolve 10 mg of clenbuterol
than the area of the principal peak in the chromatogram
hydrochloride CRS in 10 mL of methanol R
obtained with reference solution (a) (0.1 per cent),
Plate T L C silica gel F 254 plate R — any other impurity, for each impurity, not more than the
Mobile phase ammonia R, anhydrous ethanol R, toluene R area of the principal peak in the chromatogram obtained
(0.15:10:15 VIVIV). with reference solution (a) (0 . 1 per cent),
Application 10 jiL. — total: not more than twice the area of the principal peak in
Development Over a path of 10 cm. the chromatogram obtained with reference solution (a)
(0.2 per cent),
Drying In air.
Detection Spray with a 10 g/L solution of sodium nitrite R in the chromatogram obtained with reference solution (a)
1 M hydrochloric add and dip after 10 min in a 4 g/L solution (0.05 per cent).
of naphthylethylenediamme (¡¡hydrochloride R in methanol R
W ater (2.5.12)
Allow to dry in air.
the test solution is similar in position, colour and size to the Sulfated ash (2.4.14)
principal spot in die chromatogram obtained with the Maximum 0.1 per cent, determined on 1.0 g.
reference solution. ASSAY
C. It gives reaction (a) of chlorides (2.3.1). Dissolve 0.250 g in 50 mL of ethanol (96 per cent) R and add
5.0 mL of 0.01 M hydrochloric add. Titrate with 0.1 M
TESTS
sodium hydroxide, determining the end-point
Solution S potentiometrically (2.2.20). Read the volume added between
Dissolve 0.5 g in 10 mL of carbon dioxide-free water R. the 2 points of inflexion.
Appearance of solution 1 mL of 0.1 M sodium hydroxide is equivalent to 31.37 mg of
Solution S is not more opalescent than reference Ci2H i 9Cl3N 20 .
reference solution Y6 (2.2.2, Method IT).
1-584 Clindamycin Hydrochloride 2016
IMPURITIES IDENTIFICATION
Specified impurities: A , B, C, D, E, F. First identification: A , D.
Second identification B , C, D
R2
R1 Comparison clindamycin hydrochloride CRS.
H2N
1 / B. Thin-layer chromatography (2.2.27).
R2 Test solution Dissolve 10 mg of the substance to be examined
in methanol R and dilute to 10 mL with the same solvent.
A. R l = H, R2 = Cl: 4-amino-3,5-dichlorobenzaldehyde, Reference solution (a) Dissolve 10 mg of cBndamycm
B. R l = CH 2-NH-C(CH 3)33 R2 = Cl: l-(4-amino-3,5- hydrochloride CRS in methanol R and dilute to 10 mL with the
dichlorophenyl)-2- [( 1,1 -dimethylethyl)amino] ethanone, same solvent.
C. R l = CH3, R2 = Cl: l-(4-amino-3,5- Reference solution (b) Dissolve 10 mg of clindamycin
dichlorophenyl) ethanone, hydrochloride CRS and 10 mg of lincomycin hydrochloride CRS
D. R l = CH3, R2 = H: 1-(4-aminophenyl)ethanone, in methanol R and dilute to 10 mL with the same solvent.
E. R l = CH 2Br, R2 = Cl: 1-(4-amino-3,5-dichlorophenyl)- Plate TLC sQica gel G plate R.
2 -bromoethanone, Mobile phase Mix 19 volumes of 2-propanol R, 38 volumes of
a 150 g/L solution of ammonium acetate R adjusted to pH 9.6
H OH with ammonia R, and 43 volumes of ethyl acetate R.
Application 5 jiL.
Development Over a path of 15 cm using the upper layer of
the mobile phase.
and enantiomer
Drying Id. air.
F. (1RS)-1 -(4-amino-3-bromo-5-chlorophenyI)-2-[(l, 1- Detection Spray with a 1 g/L solution of potassium

dimethylethyl)amino] ethanol. permanganate R.
System suitability The chromatogram obtained with reference
PhEur
Clindamycin Hydrochloride ★*** ★ solution (a).
★ ★
(Ph. Eur. monograph 0582) ***** C. Dissolve about 10 mg in 2 mL of dilute hydrochloric acid R
and heat on a water-bath for 3 min. Add 3 mL of sodium
carbonate solution R and 1 mL of a 20 g/L solution of sodium
nitroprusside R. A violet-red colour develops.
D. Dissolve 0.1 g in water R and dilute to 10 mL with the
HCI same solvent. The solution gives reaction (a) of chlorides
(2.3.1).
TESTS
pH (2.2.3)
CigH^C^ÎÔgS 461.5 3.0 to 5.0.
21462-39-5
Action and use 10 mL with the same solvent.
Lincosamide antibacterial. Specific optical rotation (2.2.7)
Preparation + 135 to + 150 (anhydrous substance).
Clindamycin Capsules Dissolve 1.000 g in water R and dilute to 25.0 mL with the
same solvent.
PhEur______________________
Related substances
DEFINITION Liquid chromatography (2.2.29).
Methyl 7-chloro-6, 7,8 -trideoxy-6 - [[[(2S,4R)-1 -methyl-4-
propylpyrrolidin-2-yl] carbonyl] amino] -1 -xhio-L-threo-a-D-
â/aczo-octopyranoside hydrochloride. It contains a variable
the mobile phase.
quantity of water.
Reference solution (a) Dissolve 50.0 mg of clindamycin
Semi-synthetic product derived from a fermentation product
hydrochloride CRS in the mobile phase and dilute to 50.0 mL
Content with the mobile phase.
91.0 per cent to 102.0 per cent (anhydrous substance). Reference solution (b) Dilute 2.0 mL of the test solution to
CHARACTERS 100.0 mL with the mobile phase.
Appearance Column:
White or almost white, crystalline powder. — sizer. I = 0.25 m, 0 = 4.6 mm,
Solubility — stationary phase', octadecylsilyl silica gel for chromatography R
Very soluble in water, slightly soluble in ethanol (5 pm).
(96 per cent).
2016 Clindamycin Phosphate 1-585
Mobile phase Mix 45 volumes of acetomtrUe R and 55 volumes

of a 6.8 g/L solution of potassium dihydrogen phosphate R Clindamycin Phosphate *****
adjusted to pH 7.5 with a 250 g/L solution of potassium (Ph. Eur. monograph 0996) *
hydroxide R.
Flew rate 1 mL/min.
Injection 20pL.
Run time Twice the retention time of clindamycin.
— relative retendon with reference to clindamycin (retention
Limits: QsH^CnÔsPS 505.0 24729-96-2
— impurity B: not more than the area of the principal peak
in the chromatogram obtained with reference solution (b) Action and use
(2.0 per cent), Lincosamide antibacterial.
— impurity C: not more than twice the area of the principal Preparation
peak in the chromatogram obtained with reference Clindamycin Injection
solution (b) (4.0 per cent),
PhEir __________________________________________________________
— any other impurity not more than 0.5 times the area of the
principal peak in the chromatogram obtained with DEFINITION
reference solution (b) ( 1.0 per cent), Methyl 7-chloro-6,7,8-trideoxy-6- [[[(25,4i?)-1-methyl-4-
— total: not more than 3 times the area of the principal peak propylpyrrolidin-2 -yl] carbonyl] amino]- 2 -O-phosphono-1-
in the chromatogram obtained with reference solution (b) thio-L-r^co-a-D-âZacio-octopyranoside.
(6.0 per cent), Semi-synthetic product derived from a fermentation product.
— disregard limit. 0.025 times the area of the principal peak
Content
(0.05 per cent).
W ater (2.5.12) CHARACTERS
3.0 per cent to 6.0 per cent, determined on 0.500 g. Appearance
White or almost white, slightly hygroscopic powder.
Maximum 0.5 per cent, determined on 1.0 g. Solubility
Freely soluble in water, very slightly soluble in ethanol
ASSAY (96 per cent), practically insoluble in methylene chloride.
Injection 20 pL of the test solution and reference solution (a).
IDENTIFICATION
System suitability:
— repeatability: maximum relative standard deviation of Second identification B, C} D.
0.85 per cent after 6 injections of reference solution (a). A. Infrared absorption spectrophotometry (2.2.24).
STORAGE Comparison clindamycin phosphate CRS.

In an airtight container. If the spectra obtained in the solid state show differences,
dissolve 50 mg of the substance to be e x a m i n e d and the
IMPURITIES
reference substance separately in 0.2 mL of water R and heat
P *3 R2 R3 until completely dissolved. Evaporate to dryness under
reduced pressure, dry the residues at 100-105 °C for 2 h and
Test solution Dissolve 20 mg of the substance to be e x a m i n e d
OH ' CH*
Reference solution (a) Dissolve 20 mg of clindamycin
A. Rl = CH 2-CH 2-CH 33 R2 = OH, R3 = H: methyl phosphate CRS in methanol R and dilute to 10 mL with the
6, 8-dideoxy-6- [[ [(25,4/2)-1 -methyl-4-propylpyrrolidin-2- same solvent.
yl] carbonyl] amino] -1 -tiri.o-D-erythro-ai-D-galacto-
Reference solution (b) Dissolve 10 mg of lincomycin
octopyranoside (lincomycin),
hydrochloride CRS in 5 mL of reference solution (a).
B. Rl = C2H5, R2 = H, R3 = Cl: methyl 7-chloro-6,7,8-
Hate TLC silica gel plate R
trideoxy-6 - [[ [(2S,4R)-4-ethyl-l -methylpyrrolidin-2-
yl]caibonyI]arnino]-l-thio-L-ifcraKx-i>£aizcw-octopyranoside
(clindamycin B), (20:20:60 V/V/V).
C. Rl = CH 2-CH 2-CH3, R2 = Cl, R3 = H: methyl Application 5 pL.
7-chloro-6,7,8-trideoxy-6-[[[(25,4i?)-1-methyl-4- Development Over 2/3 of the plate.
propylpyrrolidin-2-yl] carbonyl] amino]-1-xhio-D-erythro-a-D- Drying At 100-105 °C for 30 min.
¿oZacw-octopyranoside (7-epiclindamycin). Detection Spray with a 1 g/L solution of potassium
___________________________________ PhEur permanganate R.
1-586 Clindamycin Phosphate 2016
System suitability:reference solution (b): Flow rate 1.1 mL/min.

— the chromatogram shows 2 principal spots. Detection Spectrophotometer at 210 nm.
Results The principal spot in the chromatogram obtained with Injection 20 pL of the test solution and reference solutions (b)
the test solution is similar in position, colour and size to the and (c).
principal spot in the chromatogram obtained with reference Identification of impurities Use the chromatogram supplied
solution, (a). with clindamycin phosphatefor system suitability CRS and the
C. Dissolve about 10 mg in 2 mL of dilute hydrochloric acid R chromatogram obtained with reference solution (c) to identify
and heat in a water-bath for 3 min. Add 4 mL of sodium the peaks due to impurities B, E, F, G, I, J, K and L.
carboTiate solution R and 1 mL of a 20 g/L solution of sodium
Relative retention With reference to clindamycin phosphate
nitroprusside R. Prepare a standard in the same manner using
(retention time = about 12 min): impurity F = about 0.15;
clindamycin phosphate CRS. The colour of the test solution
impurity G = about 0.19; impurity I = about 0.34;
corresponds to that of the standard. impurity B = about 0.45; impurity L = about 0.64;
D. Boil 0.1 g under a reflux condenser with a mixture of impurity J = about 1.20; impurity E = about 1.73;
5 mL of strong sodium hydroxide solution R and 5 mL of impurity K = about 1.90.
water R for 90 min. Cool and add 5 mL of nitric acid R. System suitability: reference solution (c):
Extract with 3 quantities, each of 15 mL, of methylene — resolution: minimum 2.0 between the peaks due to
chloride R and discard die extracts. Filter the upper layer impurities F and G.
through a paper filter. The filtrate gives reaction (b) of Calculation ofpercentage contents:
phosphates (2.3.1).
— for each impurity, use the concentration of clindamycin
TESTS phosphate in reference solution (b).
Solution S Limits:
Dissolve 1.00 g in carbon dioxide-free water R. Heat gently if — impurities B, L: for each impurity, maximum 1.0 per cent;
necessary. Cool and dilute to 25.0 mL with carbon dioxide-free — impurities E, F: for each impurity, maximum 0.5 per cent;
water R. — impurities G, I, J , K: for each impurity, maximum
Appearance of the solution 0.2 per cent;
Solution S is clear (2.2.1) and colourless (2.2.2, Method II). — unspecified impurities: for each impurity, maximum
0.10 per cent;
Related substances
— reporting threshold: 0.05 per cent
examined in mobile phase A and dilute to 10.0 mL with W ater (2.5.12)
mobile phase A. Maximum 5.0 per cent, determined on 0.200 g.
Reference solution (a) Dissolve 30.0 mg of clindamycin Bacterial endotoxins (2.6.14)
phosphate CR S in mobile phase A and dilute to 10.0 mL with Less than 0.6 IU/mg, if intended for use in the manufacture
mobile phase A. of parenteral preparations without a further appropriate
Reference solution (b) Dilute 1.0 mL of die test solution to
20.0 mL with mobile phase A. Dilute 1.0 mL of this solution ASSAY
to 10.0 mL with mobile phase A. Liquid chromatography (2.2.29) as described in the test for
Reference solution (c) Dissolve 3.0 mg of clindamycin phosphate related substances with the following modification.
for system suitability CRS (containing impurities B, E, F, G, I, Injection Test solution and reference solution (a).
J, K and L) in mobile phase A and dilute to 1.0 mL with System suitability: reference solution (a):
mobile phase A. — symmetryfactor, maximum 3.0 for the peak due to
Column: clindamycin phosphate.
— sizer. I = 0.15 m, 0 = 4.6 mm; Calculate the percentage content of CisH^CEÔsPS taking
— stationary phase: end-capped octadecylsüyl silica gelfor into account the assigned content of clindamycin
chromatography R (5 |xm); phosphate CRS.
STORAGE
Mobile phase:
In an airtight container. If the substance is sterile, the
— mobile phase A: mix 21 volumes of acetomtrUe R1 and
container is also sterile and tamper-proof.
79 volumes of a 13.6 g/L solution of potassium dihydrogen
phosphate R previously adjusted to pH 6.0 with a 450 g/L IMPURITIES
solution of potassium hydroxide R; Specified impurities B, E , F, G, I, J, K , L
— mobile phase B: mix 40 volumes of a 13.6 g/L solution of Other detectable impurities (the following substances would, if
potassium dihydrogen phosphate R previously adjusted to present at a sufficient level, be detected by one or other of
pH 6.0 with a 450 g/L solution of potassium hydroxide R, the tests in the monograph. They are limited by the general
and 60 volumes of acetonitrile R l; acceptance criterion for other/unspecified impurities and/or
Time Mobile phase A Mobile phase B (2034). It is therefore not necessary to identify these
(min)___________ (per cent V/V)________ (per cent V/V) impurities for demonstration of compliance. See also 5.10.
0 -1 3 100 0 Control of impurities in substances for pharmaceutical use): A , C,
D ,H .
13 -1 8 100 -» 50 0 50
18 -3 9 50 50
2016 Clindamycin Phosphate 1-587
A. methyl 6 }8 -dideoxy-6- [[[(2S,4R)~1-methyl-4-

propylpyrrolidin-2 -yl] carbonyl] amino] - 1-ûùo-D-erythro-a-D- F. methyl 6,8-dideoxy-6-[[[(25,4R)-l-methyl-4-
âZacZö-octopyranoside (lincomycin), propylpyrrolidin-2-yl] carbonyl] amino] -2-O-phosphono-1-
thio-D-e?yi/wt>-a-D-â/acZö-octopyranoside (lincomycin
2-phosphate),
B. methyl 7-chloro-6,7,8-trideoxy-6-[[[(2.S,4R)-4-ethyl-l-
methylpyrrolidin-2-yl] carbonyl] amino] -2-O-phosphono-1- G. methyl 6,8-dideoxy-2,4-0-(hydroxyphosphoryI)-6-
thio-L-iÄraHX-D-ôZacio-octopyranoside (clindamycin B [[[(25,4R)-1-methyl-4-propylpyrrolidin-2-yl] carbonyl] amino] -
2-phosphate), l-thio-i>^J3«Än>-a-D-â/aca>-octopyranoside (2,4-phosphatidyl
lincomycin),
h a
H-
C. methyl 7-chloro-6,7,8-trideoxy-6-[[[(2.S,4R)-l-methyl-4-
propylpyrrolidin-2-yl] carbonyl] amino] -3-O-phosphono-1- H. methyl 7-chloro-6,7,8-trideoxy-6-[[ [(25,4R)- 1-methyl-4-
thio-L-iâHX-D-â/acio-octopyranoside (clindamycin propylpyrrolidin-2-yl]carbonyl]amino]-2,3-di-0-phosphono-l-
3-phosphate), thio-L-i^«HX-i>â/acio-octopyranoside (clindamycin
2,3-bisphosphate),
H Cl
H-
/ /^
h3c ho o
D. methyl 7-chloro-6,7,8-trideoxy-6-[[[(2S,4R)-l-methyl-4-
propylpyrrolidin-2-yl] carbonyl] amino] -4-O-phosphono-1-
I. methyl 7-chloro-6,7,8-trideoxy-6- [[[(25,4R)-1-methyl-4-
thio-L-£Är«HX-i>â/aczo-octopyranoside (clindamycin
propylpyrrolidin-2-yl] carbonyl] amino] -2,4-di-O-phosphono-1-
4-phosphate),
thio-L-rÄreo-a-D-ô/acro-octopyranoside (clindamycin
2,4-bisphosphate),
E. methyl 7-chloro-6,7,8-trideoxy-6-[[[(2.S,4R)-l-methyl-4-
propylpyrrolidin-2-yl] carbonyl] amino]-1-ûùo-L-threo-a-D-
gcdacto-octopyranoside (clindamycin), J. methyl 7-chloro-6j7,8-trideoxy-6-[[[(25)-l-methyl-4-
propylidenepyrrolidin-2-yl] carbonyl] amino] -2-O-phosphono-
1-xhio-L-threo-a-D-galacto-octopyxanosidc (propylidene analog
of clindamycin 2-phosphate),
1-588 Clioquinol 2016
H Cl Solubility
H* chloride, very slightly soluble or slightly soluble in ethanol
(96 per cent).
IDENTIFICATION
First identification: B.
A. Dissolve 40.0 mg in methanol R and dilute to 100.0 mL
K. 2,2 '-oxybis(hydroxyphosphoryl)bis [methyl 7-chloro-6,7,8- with the same solvent. Dilute 10.0 mL to 100.0 mL with
trideoxy-6- [ [[(2S,4R)~1-methyl-4-propyipyrroIidin-2- methanol R (solution A). Examined between 280 nm and
yl] carbonyl] amino] -1 -diio-L-iAreo-a-D-â/aczo-octopyranoside] 350 nm (2.2.25), solution A shows an absorption maximum
(diclindamycin pyrophosphate), at 321 nm. Dilute 10.0 mL of solution A to 100.0 mL with
methanol R (solution B). Examined between 230 nm and
280 nm, solution B shows an absorption m a x im u m at
255 n m . The specific absorbance at this absorption
maximum is 1530 to 1660.
Preparation Discs of potassium bromide R.
Comparison clioquinol CRS.
C. When heated, violet fumes are produced.
D. Dissolve about 1 mg in 5 mL of ethanol (96 per cent) R.
Add 0.05 mL offerric chloride solution R l. A dark green
L. methyl 7-chloro-6 ,7,8-trideoxy-6- [[[(25,4R)-1-methyl-4- colour develops.
propylpyrrolidin-2 -yl] carbonyl] amino] - 2 -O-phosphono- 1-
TESTS
tMo-D-erythro-a-D-galacto-octopyranoside (7-epiclindamycin
2-phosphate). Acidity o r alkalinity
PhEur Shake 0.5 g with 10 mL of carbon dioxide-free water R and
filter. To the filtrate add 0.2 mL of phenolphthalein solution R.
The solution is colourless. Not more than 0.5 mL of 0.01 M
sodium hydroxide is required to change the colour of the
indicator to pink.
Clioquinol ★* * * ★ Related substances
★ ★
***** Liquid chromatography (2.2.29).
examined in methanol R and dilute to 50.0 mL with the same
solvent, heating gently if necessary. Dilute 10.0 mL of the
Reference solution (a) Dissolve 20.0 mg of 5-chloroquinolin-8-
ol R, 10.0 mg of 5,7-dichbroquinolin-S-ol R, 5 mg of the
substance to be examined and 10.0 mg of 5,7-diiodoquinolin-
8 -0 IR in methanol R, hearing gently if necessary and dilute to
C9H5CIINO 305.5 130-26-7 20.0 mL with the same solvent. Dilute 4.0 mL of the
Action and use Reference solution (b) Dilute 1.0 mL of reference solution (a)
Antibacterial; antiprotozoal. to 10.0 mL with the mobile phase.
Preparations Reference solution (c) Dilute 1.0 mL of the test solution to
Betamethasone and Clioquinol Cream 100.0 mL with the mobile phase. Dilute 1.0 mL of this
Betamethasone and Clioquinol Ointment solution to 20.0 mL with the mobile phase.
Fhimetasone and Clioquinol Ear Drops Column:
Hydrocortisone and Clioquinol Cream — size. I = 0.15 m, 0 = 3.9 mm,
Hydrocortisone and Clioquinol Ointment — stationary phase: octylsOyl silica gelfor chromatography R
(5 pm).
PhEir. Mobile phase Dissolve 0.50 g of sodium edetate R in 350 mL of
DEFINITION water R, add 4.0 mL of hexylamine R and mix. Adjust to
5-Chloro-7-iodoquinolin-8-ol. pH 3.0 with phosphoric add R. Add 600 mL of methanol R
and dilute to 1000 mL with water R.
Content
CHARACTERS
Injection 20 pL.
Appearance
Almost white, light yellow, brownish-yellow or yellowish-grey Run time 4 times the retention time of clioquinol.
powder. Relative retention With reference to clioquinol (retention
iitipurity B = about 0.7; impurity C = about 1.3.
2016 Clobazam 1-589

Clobazam *****
clioquinol and impurity C. (Ph. Eur. monograph 1974) *
Limits:
— impurity A: not more than the area of the corresponding
solution (b) (2.0 per cent),
— impurity B: not more than the area of the corresponding
solution (b) ( 1.0 per cent),
— impurity C: not more than the area of the corresponding
solution (b) ( 1.0 per cent),
— unspecified impurities: for each impurity, not more than C16Hi3a N 202- 300.7 22316-47-8
obtained with reference solution (c) (0.10 per cent), Action and use
— total of the nominal contents of impurities A , B, C and Benzodiazepine.
unspecified impurities: maximum 3.0 per cent, Preparation
— disregard limit, the area of the principal peak in the Clobazam Capsules
chromatogram obtained with reference solution (c) Clobazam Oral Suspension
(0.05 per cent).
PhEur__________________________________________________________
Halides
Maximum 140 ppm, expressed as chlorides. DEFINITION
Shake 0.5 g with 25 mL of water R for 1 min and filter. 7-Chloro-1-methyl-5-phenyl-1,5-dihydro-3//-1,5-
To the filtrate add 0.5 mL of dilute nitric acid R and 0.5 mL benzodiazepine-2,4-dione.
of stiver nitrate solution R2. Allow to stand for 5 min. Content
Any opalescence is not more intense than that in a standard 97.0 per cent to 103.0 per cent (dried substance).
prepared at the same time by adding 0.5 mL of silver nitrate
CHARACTERS
solution R2 to 25 mL of water R containing 0.2 mL of 0.01 M
Appearance
hydrochloric add and 0.5 mL of dilute nitric acid R.
Solubility
Maximum 0.5 per cent, determined on 1.000 g by drying
Slightly soluble in water, freely soluble in methylene chloride,
over diphosphorus pentoxide R at a pressure not exceeding
sparingly soluble in alcohol.
0.7 kPa for 24 h.
Sulfated ash (2.4.14) IDENTIFICATION
Maximum 0.1 per cent, determined on 1.0 g. Infrared absorption spectrophotometry (2.2.24).
Comparison Ph. Eur. reference spectrum of clobazam.
ASSAY
Dissolve 0.200 g in 20 mL of acetic anhydride R and add TESTS
30 mL of glacial acetic add R. Titrate with 0.1 M perchloric Related substances
acid, determining the end-point potentiometrically (2.2.20). Liquid chromatography (2.2.29).
1 mL of 0.1 M perchloric add is equivalent to 30.55 mg of Test solution Dissolve 10.0 mg of the substance to be
total quinolines, calculated as clioquinol. examined in the mobile phase and dilute to 50.0 mL with
the mobile phase.
STORAGE
Reference solution (a) Dissolve 5.0 mg of clobazam
impurity A CRS in the mobile phase and dilute to 50.0 mL
IMPURITIES with the mobile phase. Dilute 1.0 mL of the solution to
Specified impurities: A, B, C. 100.0 mL with the mobile phase.
Reference solution (b) Dissolve 5 mg of chlordtazepoxide CRS
and 5 mg of clonazepam CRS in the mobile phase and dilute
to 50 mL with the mobile phase. Dilute 1 mL of the solution
to 100 mL with the mobile phase.
R1 200.0 mL with the mobile phase.
Column:
A. R1 = Cl, R2 = H: 5-chloroquinolin-8-ol, — size: I = 0.25 m, 0 = 4.6 mm,
B. R1 = R2 = Cl: 5,7-dichloroquinolin-8-ol, — stationary phase: octadecylsUyl silica gelfor chromatography R
C. R 1 = R2 = I: 5,7-diiodoquinolin-8-ol. (5 pm).
PhEur Mobile phase acetomtrUe R, water R(40:60 VIV).
Flow rate 1 mL/min.
Injection 20 pL.
Run time 5 times the retention time of clobazam.
Retention time Clobazam = about 15 min.
1-590 Clobetasol Propionate 2016
System suitability:reference solution (b):

— minimum 1.3 between the peaks due to
resolution:
chlordiazepoxide and clonazepam.
Limits:
— impurity A: not more than die area of the principal peak
(0.5 per cent),
— any other impurity: not more than 0.4 times the area of the
principal peak in the chromatogram obtained with F. methyl 3-[[4-chloro-2-
reference solution (c) (0.2 per cent), (phenylamino)phenyl] methylamino] -3-oxopropanoate.
— total of other impurities: not more than twice the area of the PhEur
reference solution (c) ( 1.0 per cent),
Clobetasol Propionate ★** +★
(0.05 per cent). ★ ★
Loss on drying (2.2.32) * * * * *
(PL Eur. monograph 2127)
an oven at 105 °C.
Sulfated ash (2.4.14) CH,
Maximum 0.1 per cent, determined on the residue obtained
in the test for loss on drying.
ASSAY
Dissolve 50.0 mg in alcohol R and dilute to 100.0 mL with
the same solvent. Dilute 2.0 mL of the solution to 250.0 mL
with alcohol R Measure the absorbance (2.2.25) at the 25122-46-7
m a x i m u m at 232 n m .
Calculate the content of C 16H 13C1N 20 2 taking the specific Action and use
absorbance to be 1380. Glucocorticoid.
Preparations
IMPURITIES
Clobetasol Cutaneous Foam
Clobetasol Scalp Application
Clobetasol Shampoo
PhEur_________________________
DEFINITION
2 1-Chloro-9-fluoro-11 P-hydroxy-16|3-methyl-3,20-
dioxopregna-l,4-dien-l7-yi propanoate.
Content
A. R l = R3 = R4 = H, R2 = Cl: 7-chloro-5-phenyl-l,5- 97.0 per cent to 102.0 per cent (dried substance).
dihydro-3/f-1,5-benzodiazepme-2,4-dione, CHARACTERS
B. R l —CH3, R2 = R3 = R4 = H: 1-methyl-5-phenyl-1,5- Appearance
dihydro-3/i-1,5-benzodiazepine-2,4-dione, White or almost white, crystalline powder.
C. R l = R3 = CH3, R2 = Cl, R4 = H: (3J?S)-7-chloro-l,3- Solubility
dimethyl-5-phenyl-l,5-dihydro-3.£M,5-benzodiazepine-2,4- Practically insoluble in water, freely soluble in acetone,
dione, sparingly soluble in ethanol (96 per cent).
D. Rl = R3 = R4 = CH3, R2 = Cl: 7-chloro-1,3,3- IDENTIFICATION
trimethyi-5-phenyl- l,5-dihydro-3//-1,5-benzodiazepine-2,4- Infrared absorption spectrophotometry (2.2.24).
dione,
Comparison clobetasol propionate CRS.
TESTS
Dissolve 0.500 g in acetone R and dilute to 50.0 mL with the
same solvent
Related substances
E. N- [4-chloro-2-(phenylamino)phenyl]-N-methylacetamide, examined in the mobile phase and dilute to 20.0 mL with
the mobile phase.
the mobile phase.
2016 Clobetasol Propionate 1-591
Reference solution (a) Dissolve 20.0 mg of dobetasol — unspecified impurities:, for each impurity, not more than
propionate CRS in the mobile phase and dilute to 100.0 mL 0.2 times the area of the principal peak in the
with the mobile phase. chromatogram obtained with reference solution (d)
Reference solution (b) Dissolve the contents of a vial of (0.10 per cent);
clobetasol impurity J CRS in 2.0 mL of the mobile phase. — total: not more than 4 times the area of the principal peak
To 0.5 mL of this solution add 0.5 mL of test solution (b) in the chromatogram obtained with reference solution (d)
and dilute to 20.0 mL with the mobile phase. (2.0 per cent);
— disregard Umir. 0.1 times the area of the principal peak in
cbbetasolfor peak identification CRS (containing impurities
the chromatogram obtained with reference solution (d)
A, B, C, D, E, L and M) in 2 mL of the mobile phase. (0.05 per cent).
Reference solution (d) Dilute 1.0 mL of test solution (a) to Loss on drying (2.2.32)
50.0 mL with the mobile phase. Dilute 5.0 mL of this Maximum 0.5 per cent, determined on 1.000 g by drying in
•solution to 20.0 mL with the mobile phase. an oven at 105 °C for 3 h.
Column'. Sulfated ash (2.4.14)
— sizer. I = 0.15 m, 0 = 4.6 mm; Maximum 0.1 per cent, determined on 1.0 g.
— stationary phase: spherical octadecylsUyl silica gelfor ASSAY
chromatography R (5 pm); Liquid chromatography (2.2.29) as described in the test for
— temperature: 30 °C. related substances with the following modification.
Mobile phase Mix 10 volumes of methanol R, 42.5 volumes of Injection Test solution (b) and reference solution (a).
a 7.85 g/L solution of sodium dihydrogen phosphate
Calculate the percentage content of C 25H 32CIFO5 using the
monohydrate R adjusted to pH 5.5 with a 100 g/L solution of
chromatogram obtained with reference solution (a) and the
sodium hydroxide R and 47.5 volumes of acetonitrile R.
declared content of clobetasol propionate CRS.
STORAGE
Injection 10 pL of test solution (a) and reference
solutions (b)3 (c) and (d). IMPURITIES
Run time 3 times the retention time of clobetasol propionate. Specified impurities A,B, C, D, E, L, M
Identification of impurities Use the chromatogram supplied Other detectable impurities (the following substances would, if
with clobetasolfor peak identification CRS and the present at a sufficient level, be detected by one or other of
chromatogram obtained with reference solution (c) to identify the tests in the monograph. They are limited by the general
the peaks due to impurities A, B, C, D, E, L and M. acceptance criterion for other/unspecified impurities and/or
Relative retention With reference to clobetasol propionate
Control of impurities in substancesfor pharmaceutical use): F, G,
impurity J = about 1.1; impurity D = about 1.2; H ,I ,J ,K .
impurity L = about 1.3; impurity M = about 1 .6 ;
impurity E = about 2.1.
System suitability:
clobetasol propionate and impurity J in the chromatogram
— the chromatogram obtained with reference solution (c) is
similar to the chromatogram supplied with cbbetasolfor
peak identification CRS .
Limits: A. RI = CO-C2HS, R2 = OH: 9-fiuoro-1113,21-dihydroxy-
— correction factors: for the calculation of content, multiply 16fi-methyl-3,20-dioxopregna-l ,4-dien- 17-yl propanoate
the peak areas of the following impurities by the (betamethasone 17-propionate),
corresponding correction factor impurity B = 0.6; G. RI = H, R2 = Cl: 21-chloro-9-fluoro-11P, 17-dihydroxy-
impurity C = 1.5; 16P-methylpregna-l ,4-diene-3,20-dione (clobetasol),
— impurity E: not more than 1.4 times die area of the
principal peak in the chromatogram obtained with H. R1 = CO-C2H5, R2 = H: 9-fluoro-1 1^-hydroxy-16{i-
reference solution (d) (0.7 per cent); methyl-3,20-dioxopregna-1,4-dien-17-yl propanoate,
— impurity D: not more than the area of the principal peak I. R1 = CO-C2H5, R2 = 0-S0 2-CH3: 9-fluoro-lip-
in the chromatogram obtained with reference solution (d) hydroxy-16P-methyl-2 1- [(methylsulfonyl) oxy]-3,20-
(0.5 per cent); dioxopregna-1,4-dien-17-yl propanoate,
— impurities B, C: for each impurity, not more than K. RI = H, R2 = 0-C0-C 2H5: 9-fluoro-11P, 17-dihydroxy-
0.6 times the area of the principal peak in the 16P-methyl-3,20-dioxopregna-1,4-dien-21-yl propanoate
chromatogram obtained with reference solution (d) (betamethasone 21 -propionate),
(0.3 per cent);
— impurities A , L, M : for each impurity, not more than
(0.2 per cent);
1-592 Clobetasone Butyrate 2016
Clobetasone Butyrate ★★*★ ★

★ ★
(Ph. Ettr. monograph 1090) *****
Wl
/0
B. 21 -chloro-9-fluoro-11 P-hydroxy-16-methylpregna-1,4,16- ;-O ^ ^ C H 3

triene-3,20-dione,
Cl
°s J o
C26H 32CIFO5 479.0 25122-57-0
Action and use

Glucocorticoid.
Preparations
C. 2 l-chloro-9-fluoro-l 1P-hydroxy-16a-methyl-3j20- Clobetasone Cream
dioxopregna-1,4-dien- 17-yl propanoate, Clobetasone Ointment
PhEur___________________
r-J 0 DEFINITION
..0 A ^ ch,
21-Chloro-9-fluoro-16 P-methyl-3,11,20-trioxopregna-1,4-
dien- 17-yl butanoate.
Content
CHARACTERS
D. 21 -chloro-9-fluoro-11 P-hydroxy-16P-methyl-3,20-
Appearance
dioxopregn-4-en-17 -yl propanoate (1,2-dihydroclobetasol
17-propionate),
Solubility
a Practically insoluble in water, freely soluble in acetone and in
o methylene chloride, slightly soluble in ethanol (96 per cent).
mp
About 178 °C.
IDENTIFICATION
Comparison clobetasone butyrate CRS.
E. 2 1-chloro-l 6P-methyl-3,20-dioxopregna-l ,4-dien- 17-yl
TESTS
propanoate,
c o 2h + 131 to + 138 (dried substance).
H CH
hcv: Dissolve 0.250 g in ethanol R l and dilute to 25.0 mL with
the same solvent.
Related substances
F. 9-fluoro-l 1P-hydroxy-16P-methyl-3-oxopregna-l ,4,17 (20)- Solvent mixture anhydrous formic acid R, acetonitrUe R, water R
trien-21-oic add, (0.1:43:57 V/V/V).
in 5.0 mL of acetomtrile R and dilute to 25.0 mL with die
solvent mixture.
Reference solution (a) Dissolve 13 mg of clobetasone butyrate for
system suitability CRS (containing impurity F) in 1 mL of
acetonitrile R and dilute to 5.0 mL with the solvent mixture.
100.0 mL with the solvent mixture. Dilute 1.0 mL of this
J. (ni^^'-chloro-S'-ethyW -fluoro-l 1P-hydroxy-160-
Column:
methylspiro [androsta-1,4-diene-17,2' (3 7f)-furan]-3,3 '-dione
— sizer. I = 0.15 m, 0 = 4.6 mm;
(17a-spiro compound),
— stationary phase, end-capped octadecylsifyl silica gelfor
L. unknown structure, chromatography R (3.5 pm);
M. unknown structure. — temperature: 40 °C.
_________ :__________________________ PhEur
2016 Clobetasone Butyrate 1-593
Mobile phase:
— mobile phase A: anhydrous formic acid R, water R
(0.1:99.9 VIV);
— mobile phase B: anhydrous formic add R, acetomtrUe R
(0.1:99.9 VIV)',
Time Mobik phase A Mobik phase B

(min) (per cent V/V) (per cent V/V) A. 21-chloro-9-fluoro-17-hydroxy-16P-methylpregna-1,4-
0 -3 57 43 diene-3,ll,20-trione (clobetasone),
3 - 26 57->43 43*57
Cl
°\ JO
Flow rate 1.5 mL/min. L A r 'O CH3

Injection 10 |xL.
with clobetasone butyratefor system suitability CRS and the
identify the peak due to impurity F. C. 21 -chloro-9-fluoro-16P-methyl-3,11,20-trioxopregn-1-en-
17-yl butanoate (4,5-dihydroclobetasone butyrate),
Relative retention With reference to clobetasone butyrate
(retention time = about 14 min): impurity F = about 0.9.
System suitability'.
impurity F and clobetasone butyrate in the chromatogram CHa
obtained with reference solution (a);
Limits:
— for each impurity, not more than the
unspecified impurities:, D. 2-bromo-21-chloro-9-fluoro-16P-methyl-3,l 1,20-
area of the principal peak in the chromatogram obtained trioxopregna-l,4-dien-17-yl butanoate
with reference solution (b) (0.10 per cent); (2 -bromoclobetasone butyrate),
(0.5 per cent);
— disregard limit. 0.5 times the area of the principal peak in CH,
(0.05 per cent).
an oven at 105 °C.
E. 2 1-chloro-9-fluoro-16|3-methyl-3,l l,20-trioxopregn-4-en-
ASSAY 17-yl butanoate ( 1,2 -dihydroclobetasone butyrate),
Dissolve 20.0 mg in ethanol (96 per cent) R and dilute to
100.0 mL with the same solvent Dilute 5.0 mL of the
solution to 50.0 mL with ethanol (96 per cent) R. Measure the
absorbance (2.2.25) at the absorption maximum at 235 nm.
Calculate the content of C26H 32CIFO5, taking the specific
STORAGE
IMPURITIES F. 21 -chloro-9-fluoro-16a-methyl-3,1l,20-trioxopregna-l,4-
Other detectable impurities(the following substances would, if dien-17-yl butanoate (16a-methyl clobetasone butyrate),
by the general monograph Substancesfor pharmaceutical use
Control of impurities in substancesfor pharmaceutical use): A , C,
D, E, F, G, H, I.
G. 9-fluoro-l 6f3-methyl-3, 1l, 20 -trioxo-2 1-

(propanoyloxy)pregna-l,4-dien-17-yl butanoate,
1-594 Clofazimine 2016
o Cl If the spectra obtained in the solid state show differences,

substance separately in methylene chloride R> evaporate to
chT h T \-c h 3 dryness and record new spectra using the residues.
in methylene chloride R and dilute to 10 mL with the same
H. 21-chloro-9-fluoro-16P-methyi-3jl l,20-trioxopregna-l,4- solvent.
dien-17-yl propanoate (17-O-propionyl clobetasone), Reference solution Dissolve 10 mg of clofazimine CRS in
methylene chloride R and dilute to 10 mL with the same
solvent.
Plate TLC silica gel GF254 plate R.
Mobile phase propanol R, methylene chloride R (6:85 VIV).
Application 5 pL.
First development Over 2/3 of the plate.
Drying Horizontally in air for 5 min.
Second development Over 2/3 of the plate.
I. 21 -chloro-9-fluoro-16 |}-methyl-3, 11,20-trioxopregna-1,4-
dien-17-yl 2-methylpropanoate (17-O-isobtityryl Drying In air for 5 min.
clobetasone). Detection Examine in ultraviolet light at 254 nm.
____ ______________________________________________________ PhEur Results The principal spot in the chromatogram obtained with
solution.
Clofazimine C. Dissolve 2 mg in 3 mL of acetone R and add 0.1 mL of
kydrochloric acid R. An intense violet colour is produced.
(Ph. Eur. monograph 2054) * Add 0.5 ml. of a 200 g/L solution of sodium hydroxide R;
the colour changes to orange-red.
TESTS
Related substances
in the mobile phase and dilute to 100 mL with the mobile
phase.
h3c ch3 Reference solution (a) Dilute 1.0 mL of the test solution to
C27H 22CI2N4 473.4 2030-63-9 solution to 10.0 mL with the mobile phase.
Reference solution (b) Dissolve 5.0 mg of clofazimine for system
Action and use
suitability CRS in the mobile phase and dilute to 10.0 mL
Antileprosy drug.
P reparation Column:
Clofazimine Capsules — sizer. I = 0.25 m, 0 = 4.6 mm,
PhEur___________________________________________________________
— stationary phase: oaylsUyl silica gelfor chromatography R
(5 pm).
DEFINITION
Mobile phase Dissolve 2.25 g of sodium laurUsulfate R, 0.85 g
iV,5-Bis(4-chlorophenyl)-3- [( 1-methylethyl)imino] -3,5-
of tetrabutylammonium hydrogen sulfate R and 0.885 g of
dihydrophenazin-2-amine.
disodium hydrogen phosphate R in water R. Adjust to pH 3.0
Content with dilute phosphoric acid R and dilute to 500 mL with
99.0 per cent to 101.0 per cent (dried substance). water R. Mix 35 volumes of this solution and 65 volumes of
CHARACTERS acetomtrUe R.
Appearance Flow rate 1 m lVmin.
Reddish-brown, fine powder. Detection Spectrophotometer at 280 nm.
Solubility Injection 20 jiL.
Practically insoluble in water, soluble in methylene chloride, Run time 3 times the retention time of clofazimine.
very slightly soluble in ethanol (96 per cent).
Identification of impurities Use die chromatogram supplied
It shows polymorphism (5.9). with clofazimine for system suitability CRS to identify die peak
IDENTIFICATION due to impurity B.
First identification: A. Relative retention With reference to clofazimine (retention
Second identification: B, C. time = about 15 min): impurity A = about 0.7;
Comparison clofazimine CRS.
2016 Clofibrate 1-595

— resolution: baseline separation between the peaks due to Clofibrate *****
impurity B and clofazimine. *+ +*
Limits:
— impurity A: not more than the area of the principal peak O
(0.1 per cent),
— impurity B: not more than 3 times the area of the
reference solution (a) (0.3 per cent), C12H 15C103 242.7 637-07-0
— any other impttrity: for each impurity, not more than the
area of the principal peak in the chromatogram obtained Action and use
with reference solution (a) (0.1 per cent), Fibrate; lipid-regulating drug.
— total: not more than 5 tidies the area of the principal peak
Preparation
Clofibrate Capsules
(0.5 per cent),
— disregard limit. 0.5 times the area of the principal peak in PhEur__________________________________________________________
DEFINITION
(0.05 per cent).
Ethyl 2-(4-chlorophenoxy)-2-methylpropionate.
Maximum 10 ppm. CHARACTERS
2.0 g complies with test C. Prepare the reference solution Appearance
using 2 mL of lead standard solution (10 ppm Pb) R. Clear, almost colourless liquid.
Loss on drying (2.2.32) Solubility
Maximum 0.5 per cent, determined on 1.000 g by drying in Very slightly soluble in water, miscible with ethanol
an oven at 105 °C. (96 per cent).
Sulfated ash (2.4.14) IDENTIFICATION
Maximum 0.1 per cent, determined on 1.0 g. A Infrared absorption spectrophotometry (2.2.24).
Comparison clofibrate CRS.
ASSAY
Dissolve 0.400 g in 5 mL of methylene chloride R and add B. Ultraviolet and visible absorption spectrophotometry
20 mL of acetone R and 5 mL of anhydrous acetic acid R. (2.2.25).
Titrate with 0.1 M perchloric add, determining the end-point Test solution (a) Dissolve 0.10 g in methanol R and dilute to
potentiometrically (2.2.20). 100.0 mL with the same solvent. Dilute 10.0 mL of this
1 mL of 0.1 M perchloric acid is equivalent to 47.34 mg solution to 100.0 mL with methanol R.
of C27H 22CI2N 4. Test solution (b) Dilute 10.0 mL of test solution (a) to
100.0 mL with methanol R
IMPURITIES
Spectral range 250-350 nm for test solution (a); 220-250 nm
for test solution (b).
Absorption maxima At 280 nm and 288 nm for test
solution (a); at 226 nm for test solution (b).
Spedfic absorbances at the absorption maxima:
— at 226 nm: about 460 for test solution (b);
— at 280 nm: about 44 for test solution (a);
— at 288 nm: about 31 for test solution (a) .
TESTS
1
R2 Relative density (2.2.5)
1.138 to 1.147.
A. RI = Cl, R2 = H: N,5-bis(4-chlorophenyl)-3-imino-3,5- Refractive index (2.2.6)
dihydrophenazin-2-amine, 1.500 to 1.505.
B. RI = H, R2 = CH(CH3)2: 5-(4-chlorophenyl)-3- Acidity
[( 1-methylethyI)imino] -AT-phenyl-3,5-dihydrophenazin-2- To 1.0 g add 10 mL of anhydrous ethanol R and 0.1 mL of
amine. phenol red solution R Not more than 1.0 mL of 0.01 M
___________________________________ PhEur sodium hydroxide is required to change the colour of the
indicator.
Volatile related substances
Test solution To 10.0 g of the substance to be examined add a
mixture of 10 mL of dilute sodium hydroxide solution R and
10 mL of water R. Shake, separate die lower (organic) layer,
wash with 5 mL of water R and add the washings to the
aqueous layer. Dry the organic layer with anhydrous sodium
sulfate R and use as the test solution. Reserve the aqueous
layer for the test for 4-chlorophenol.
1-596 Clomethiazole 2016
Reference solution (a) Dissolve

0.12 g of the substance to be
examined in chloroform R and dilute to 100.0 mL with the
Clomethiazole
same solvent. Dilute 1.0 mL of this solution to 10.0 mL with
chloroform R
Reference solution (b) Dissolve 0.12 g of methyl
2-(4-chlorophenoxy)-2-methylpropionate CRS in thesubstance
to be examined and dilute to 10.0 mL with the same solvent.
Dilute 1.0 mL of the solution to 10.0 mL with the substance
to be examined. Dilute 1.0 mL of this solution to 10.0 mL QHsCINS 161.6 533-45-9
with the substance to be examined.
Action and use
Column:
— sizer. / = 1.5 m, 0 = 4 mm; Hypnotic.
— stationary phase. sUanised diatomaceous earth for gas Preparation
chromatography R (250-420 pm) impregnated with Clomethiazole Capsules
30 per cent m/m of poly(dimethyl)sUoxane R; or sUanised
diatomaceous earth for gas chromatography R (150-180 pm) DEFINITION
impregnated with 10 per cent m/m of Clomethiazole is 5-(2-chloroethyl)-4-methyl-thiazole.
poly(dimethyl)sUoxane R; It contains not less than 98.0% and not more than 101.0%
— temperature: 185 °C. o fC 6H 8ClNS.
Carrier gas nitrogen for chromatography R. CHARACTERISTICS
Detection Flame ionisation. A colourless to slightly yellowish brown liquid.
Injection 2 |iL. Slightly soluble in water, miscible with ethanol (96%) and
System suitability: reference solution
(b): with ether.
— peak-to-vaRey ratio: minimum 4, where Hp = height above IDENTIFICATION
the baseline of the peak due to methyl A. The light absorption, Appendix IT B, in the range 230 to
2-(4-chlorophenoxy)-2-methylpropionate and H v = height 350 nm of a 0.004% w/v solution in 0.1m hydrochloric acid
above the baseline of the lowest point of the curve exhibits a maximum only at 257 nm. The absorbance at the
separating this peak from the peak due to clofibrate. maximum is about 1.1.
Limit: B. The infrared absorption spectrum, Appendix II A, is
— total: not more than 10 times the area of the peak due to concordant with the reference spectrum of clomethiazole
clofibrate in the chromatogram obtained with reference (RS 051).
solution (a) (0.1 per cent). C. Mix 0.1 g with 0.2 g of powdered sodium hydroxide, heat
4-Chlorophenol to fusion and continue heating for a further few seconds.
Gas chromatography (2.2.28) as described in the test for Cool, add 0.5 mL of water and a slight excess of
volatile related substances with the following modifications. 2m hydrochloric acid and warm. Any fumes evolved do not
Test solution Shake the aqueous layer reserved in the test for turn moistened starch iodate paper blue (distinction from
volatile related substances with 2 quantities, each of 5 m l, of clomethiazole edisilate).
chloroform R and discard the organic layers. Acidify the
TESTS
aqueous layer by the dropwise addition of hydrochloric acid R. Acidity or alkalinity
Shake with 3 quantities, each of 3 mT, of chloroform R. pH of a 0.5% w/v solution, 5.5 to 7.0, Appendix V L.
Combine the organic layers and dilute to 10.0 mL with
chloroform R. Heavy metals
Moisten the residue obtained in the test for Sulfated ash with
Reference solution Dissolve
0.25 g of chlorophenol R in
2 mL of hydrochloric add and evaporate to dryness. Dissolve
chloroform R and dilute to100.0 mL with the same solvent.
the residue in water and add sufficient water to produce
Dilute 1.0 mL of this solution to 100.0 mL with
20 rnL 12 mL of the resulting solution complies with limit
chloroform R.
test A for heavy metals, Appendix VII. Use lead standard
Limit:
solution (1 ppm Pb) to prepare the standard (20 ppm).
— 4-chlorophenol: not more than the area of the peak due to
4-chlorophenol in the chromatogram obtained with the Related substances
reference solution (25 ppm). Carry out the method for liquid chromatography,
Appendix HI D, using the following solutions. Solution (1)
------------------------------------------------------ PhEur contains 0.20% w/v of the substance being examined in the
mobile phase. For solution (2) dilute 1 volume of solution
(1) to 1000 volumes with the mobile phase. For solution (3)
dilute 1 volume of a 0.030% w/v solution of 4-methyl-5-
vinylthiazole edisilate BPCRS in methanol (solution A) to
50 volumes with the mobile phase. For solution (4) dilute
1 volume of a 0.020% w/v solution of 5-(2-chloroethyl)-4-
methyl-3-[2-(4-methyhhiazol-5-yl)ethyl]-thiazotium
chloride BPCRS (quaternary dimer) in methanol (solution B)
to 50 volumes with the mobile phase. For solution (5) dilute
1 volume of a 0.020% w/v solution of 4-methyl-5-
(2-hydtroxyethyl)thiazole BPCRS in methanol (solution C) to
50 volumes with the mobile phase. For solution (6) add
1 mL each of solutions A, B and C to 0.10 g of the.
2016 Clomethiazole Edisilate 1-597
substance being examined and dilute to 50 mL with the IDENTIFICATION

mobile phase. A. Melting point, about 128°, Appendix V A.
The chromatographic procedure may be carried out using 8. The light absorption, Appendix IIB , in the range 230 to
(a) a stainless steel column (20 cm x 4 mm) packed with 350 nm of a 0.005% w/v solution in 0.1m hydrochloric acid
octadecykdyl silica gel far chromatography (10 pm) (Iichrosorb exhibits a maximum only at 257 nm. The absorbance at the
RP18 is suitable), (b) as the mobile phase with a flow rate of maximum is about 0.92.
1 mL per minute, a mixture of 70 volumes of a solution C. The infrared absorption spectrum, Appendix II A, is
containing 0.13% w/v of sodium hexanesidfonate and 2.7% w/v concordant with the reference spectrum of clomethiazole
of tetmmethylammoniim hydrogen sulfate, adjusted to pH 2.0 edisilate (RS 052).
with 5m sodium hydroxide, and 30 volumes of methanol and
D. Mix 0.1 g with 0.2 g of powdered sodium hydroxide, heat
(c) a detection wavelength of 257 nm.
to fusion and continue heating for a further few seconds.
The test is not valid unless in the chromatogram obtained Cool, add 0.5 mL of water and a slight excess of
with solution (6) baseline separation is achieved between the 2m hydrochloric add and warm. Fumes are evolved which turn
peaks due to the three specified impurities and also between moistened starch iodate paper blue (distinction from
the principal peak and the two adjacent specified clomethiazole).
impurity peaks.
TESTS
Calculate the content of each of the three specified impurities
Calcium
in the substance being examined expressing the content of
10 mL of a 10.0% w/v solution diluted to 15 mL with water
4-methyl-5-vinylthiazole as the base (1 mg of 4-methyl-5-
complies with the limit testfar calcium, Appendix VII
vinylthiazole edisilate is equivalent to 0.568 mg of base).
(100 ppm).
The total content of the three specified impurities is not
greater than 0.5%. In the chromatogram obtained with Heavy metals
solution (1) the area of any other secondary peak is not greater Moisten the residue obtained in the test for Sulfated ash with
than the area of the peak in the chromatogram obtained with 2 mL of hydrochloric add and evaporate to dryness. Dissolve
solution (2). the residue in water and add sufficient water to produce
20 mL. 12 mL of the resulting solution complies with limit
Sulfa ted ash
test A for heavy metals, Appendix VII. Use lead standard
Not more than 0.1%, Appendix IX A. Use 1 g.
solution (1 ppm Pb) to prepare the standard (20 ppm).
ASSAY Chloride
Carry out Method I for non-aqueous titration, 10 mL of a 10% w/v solution diluted to 15 mL with water
Appendix VIII A, using 0.3 g and determining the end point complies with the limit testfar chlorides, Appendix VII
potentiometrically. Each mL of 0.1m perchloric add FS is (50 ppm).
equivalent to 16.16 mg of CfrHgClNS.
Sulfate
STORAGE 10 mL of a 1.0% w/v solution diluted to 15 mL with distilled
Clomethiazole should be stored at a temperature of 2° to 8°. water complies with the limit testfor sulfates, Appendix VII
(0.15%).
Related substances
Carry out the method for liquid chromatography,
Clomethiazole Edisilate Appendix m D, using the following solutions. Solution (1)
contains 0.30% w/v of die substance being examined in the
mobile phase. For solution (2) dilute 1 volume of solution
(1) to 1000 volumes with the mobile phase. For solution (3)
Cl dilute 1 volume of a 0.030% w/v solution of 4-methyl-5-
Me
c S 'OH
S
.OH
// \\
vmylthiazole edisilate BPCRS in methanol (solution A) to
50 volumes with the mobile phase. For solution (4) dilute
1 volume of a 0.020% w/v solution of 5-(2-chloroethyl)-4-
o o
methyl-3-[2-(4-methylthiazol-5-yl)ethyl]-tkiazolium
chloride BPCRS (quaternary dimer) in methanol (solution B)
(CeHsClNS^CiHftOeSa 513.5 1867-58-9 to 50 volumes with the mobile phase. For solution (5) dilute
1 volume of a 0.020% w/v solution of 4-methyl-5-
Action and use (2-hydroxyethyl)thiazole BPCRS in methanol (solution C) to
Hypnotic. 50 volumes with the mobile phase. For solution (6) add
Preparations 1 mL each of solutions A, B and C to 0.15 g of the
Clomethiazole Infusion substance being examined and dilute to 50 mL with the
Clomethiazole Oral Solution mobile phase.
The chromatographic procedure may be carried out using
DEFINITION (a) a stainless steel column (20 cm x 4 mm) packed with
Clomethiazole Edisilate is 5-(2-chloroethyl) -4-methylthiazole octadecylsUyl silica gelfar chromatography (10 pm) (Iichrosorb
ethanedisulfonate. It contains not less than 99.0% and not RP18 is suitable), (b) as the mobile phase with a flow rate of
more than 101.0% of (C6H8dN S) 2,C2H60 6S2, calculated 1 mL per minute, a mixture of 70 volumes of a solution
with reference to the dried substance. containing 0.13% w/v of sodium hexanesidfonate and 2.7% w/v
CHARACTERISTICS of tetramethylammonium hydrogen sulfate, adjusted to pH 2.0
A white, crystalline powder. with 5m sodium hydroxide, and 30 volumes of methanol and
(c) a detection wavelength of 257 nm.
Freely soluble in water, soluble in ethanol (96%); practically
insoluble in ether.
1-598 Clomifene Citrate 2016
The test is not valid unless in the chromatogram obtained Preparation Discs of potassium bromide R.
with solution (6) baseline separation is achieved between the Comparison domifene citrate CRS.
peaks due to the three specified impurities and also between B. Dissolve about 5 mg in 5 mL of a mixture of 1 volume of
the principal peak and the two adjacent specified acetic anhydride R and 5 volumes of pyridine R, then heat in a
impurity peaks. water-bath. A deep red colour is produced.
Calculate the content of each of the three specified impurities
TESTS
in the substance being examined with reference to
Prepare the solutions protectedfrom light in brown-glass vessels
clomethia2ole base (1 mg of clomethiazole edisilate is
Ensure minimum exposure of the solutions to daylight until they
equivalent to 0.629 mg of base) expressing the content of
are requiredfor chromatography.
4-methyl-5-vinylthiazole as the base (1 mg of 4-methyl-5-
vinylthiazole edisilate is equivalent to 0.568 mg of base). Related substances
The total content of the three specified impurities is not Liquid chromatography (2.2.29).
greater than 0.5%. In the chromatogram obtained with Test solution Dissolve 12.5 mg of the substance to be
solution (1) the area of any other secondary peak is not greater examined in the mobile phase and dilute to 10.0 mL with
than the area of the peak in the chromatogram obtained with the mobile phase.
solution (2). Reference solution (a) Dissolve 12.5 mg of domifene citrate for
Loss on drying performance test CRS in the mobile phase and dilute to
When dried at 50° at a pressure not exceeding 0.7 kPa for 10.0 mL with the mobile phase.
6 hours, loses not more than 0.5% of its weight. Use 1 g. Reference solution (b) Dilute 1.0 mL of the test solution to
Sulfated ash 50.0 mL with the mobile phase.
Not more than 0.3%, Appendix IX A. Use 1 g. Column:
ASSAY — size. I = 0.25 m, 0 = 4.6 mm;
Dissolve 0.4 g in 50 mL of water and titrate with 0.1m sodium — stationary phase, butylsüyl silica gd for chromatography R
hydroxide VS using phenolphthalem solution R1 as indicator.

(5 pm).
Each mL of 0.1m sodium hydroxide KS is equivalent to Mobile phase Mix 400 mL of acetomtrüe R with 600 mL of
25.67 mg of (C6H8CINS)2,C2H60 6S2. water R and add 8.0 mL of diethylamine R ; adjust to pH 6.2
with about 1-2 mL oí phosphoric add R, taking care to reduce
progressively the volume of each addition as the required pH
is approached.
Clomifene Citrate ★ ★ Detection Spectrophotometer at 233 nm.
(Ph. Eur. monograph 0997) ***** Equilibration With the mobile phase for about 1 h.
Injection 10 pL.
Run time 4 times the retention time of domifene.
c o 2h
V C°2H — peak-to-vaUey ratio: minimum 15, where Hp — height
/ S h above the baseline of the peak due to impurity A and
c o 2h H v = height above the baseline of the lowest point of the
domifene; if necessary, adjust the concentration of
acetonitrile in the mobile phase;
C32H36C1N08 598.1 50-41-9 the chromatogram obtained is similar to the chromatogram
supplied with domifene citrate for performance test CRS.
Action and use
Limits:
Estrogen receptor modulator.
— impurity A: not more than the area of the prindpal peak
Preparation in the chromatogram obtained with reference solution (b)
Clomifene Tablets (2.0 per cent);
Ph Eur__________________________
— impurities B, C, D, E, F, G, H: for each impurity, not
more than 0.5 times the area of the prindpal peak in the
DEFINITION chromatogram obtained with reference solution (b)
Mixture of the (JE)- and (2)-isomers of 2-[4-(2-chloro- (1.0 per cent);
l,2diphenylethenyl)phenoxy]-N,N-diethylethanamine — total: not more than 1.25 times the area of the prindpal
dihydrogen citrate. peak in the chromatogram obtained with reference
Content solution (b) (2.5 per cent);
98.0 per cent to 101.0 per cent (anhydrous substance). — disregard limir. 0.025 times the area of the prindpal peak
CHARACTERS
(0.05 per cent); disregard any peak with a retention time
Appearance
rdative to the domifene peak of 0.2 or less.
White or pale yellow, crystalline powder.
(Z)-isomer
Solubility
Slightly soluble in water, sparingly soluble in ethanol
(96 per cent).
in 25 mL of 0.1 M hydrochloric add, add 5 mL of 1 M sodium
IDENTIFICATION hydroxide and shake with 3 quantities, each of 25 mL, of
2016 Clomipramine Hydrochloride 1-599
ethanol-free chloroform R. Wash the combined extracts with

10 mL of water R, dry over anhydrous sodium sulfate R and Ar
dilute to 100 mL with ethanol-free chloroform R To 20 mL of
this solution add 0.1 mL of triethylamine R and dilute to B. [4-[2-(diethylamino)ethoxy]phenyl]phenylmethanone,
100 mL with hexane R
0
Reference solution Dissolve 25 mg of domifene citrate CRS in
Ar* and enantiomer
25 mL of 0.1 M hydrochloric add, add 5 mL of 1 M sodium
hydroxide and shake with 3 quantities, each of 25 mT, of H Ar
ethanol-free chloroform R Wash die combined extracts with
10 mL of mater R, dry over anhydrous sodium sulfate R and C. (2RS)-2-[4-[2-(diethylamino)ethoxy]phenyi]-1,2-
dilute to 100 mL with ethanol-free chloroform R To 20 mL of diphenylethanone,
this solution add 0.1 mL of triexhylamme R and dilute to
100 mL with hexane R
Column:
— size. 1= 0.3 m, 0 = 4 mm;
— stationary phase: silica gd for chromatography R (10 pm).
D. 2,2-bis [4-[2-(diethylamino)ethoxy]phenyI]-1,2-
Mobile phase triethylamme R, ethanol-free chloroform R,
diphenylethanone,
hexane R (1:200:800 VfVfV).
Flow rate 2 ml/min.
and (£)-isomer
Equilibration With the mobile phase for about 2 h.
Injection 50 pL.
Identification ofpeaks The chromatogram obtained with the
reference solution shows a peak due to the (£)-isomer just
E. 2- [4- [1,2-bis(4-chlorophenyI) ethenyl]phenoxy] -N,N-
before a peak due to the (Z)-isomer.
diethyiethanamine,
— resolution: minimum 1.0 between the peaks due to the
(E)- and (Z)-isomers; if necessary, adjust the relative Ar1
and (E)-tsomer
proportions of ethanol-free chloroform and hexane in the
mobile phase.
Measure the area of the peak due to the (2)-isomer in the
chromatograms obtained with the test solution and the F. 2-[4- [2-chloro-2-(4-chlorophenyI)-l -
reference solution. Calculate the content of the (Z)-isomer, as phenylethenyQphenoxy] -N^-diethylethanamine,
a percentage of the total domifene citrate present, from the
declared content of domifene citrate CRS. 'N ch3
Limit: L or (ZHsomer
CH3
— (Z)-isomer. 30.0 per cent to 50.0 per cent
Water (2.5.12)
Maximum 1.0 per cent, determined on 1.000 g. GH. 2-[2-chloro-4-(2-chloro-lj2-diphenylethenyl)phenoxy] -
ASSAY N^-diethylethanamine (G. higher-melting-point isomer;
Dissolve 0.500 g in 50 mL of anhydrous acetic add R Titrate H. lower-melting-point isomer).
with 0.1 M perchloric add> determining the end-point PhEur
potentdometrically (2.2.20).
1 mL of 0.1 M perchloric add is equivalent to 59.81 mg
of C32H36C1N08. J
Clomipramine Hydrochloride ★r**★
STORAGE ★ ★
Protected from light. (Ph. Eur. monograph 0889) *****
IMPURITIES a
Specified impurities A, 8, C, D, E, F, G, H
, Ar = = . Ha
ch3
17321-77-6
Ar*
Ar and (ZHsomer Action and use
Ar Monoamine reuptake inhibitor; tricyclic antidepressant.
Preparation
A. 2- [4-( 1,2-diphenylethenyl)phenoxy] -NJsf- Clomipramine Capsules
diethylethanamine, Prolonged-release Clomipramine Tablets
1-600 2016

DEFINITION
immediately before use and protectedfrom light.
3-(3-Chloro-10,11 -dihydro-5//-dibenzo \bj\azepin-5-yl)-AT,N-
dimethylpropan-1-amine hydrochloride. Test solution Dissolve 20.0 mg of die substance to be
examined in a mixture of 25 volumes of mobile phase B and
Content 75 volumes of mobile phase A and dilute to 10.0 mL with
99.0 per cent to 101.0 per cent (dried substance). the same mixture of mobile phases.
CHARACTERS Reference solution (a) Dissolve 22.6 mg of imipramine
Appearance hydrochloride CRS, 4.0 mg of clomipramine impurity C CRS,
White or slightly yellow, crystalline powder, slightly 4.0 mg of clomipramine impurity D CRS and 2.0 mg of
hygroscopic. clomipramine impurity F CRS in a mixture of 25 volumes of
Solubility mobile phase B and 75 volumes of mobile phase A and
Freely soluble in water and in methylene chloride, soluble in dilute to 100.0 mL with the same mixture of mobile phases.
alcohol. Dilute 1.0 mL of this solution to 10.0 mL with a mixture of
It shows polymorphism (5.9). 25 volumes of mobile phase B and 75 volumes of mobile
phase A.
IDENTIFICATION
First identification: B, E.
100.0 mL with a mixture of 25 volumes of mobile phase B
Second identification A , C, D, E and 75 volumes of mobile phase A.
A. Melting point (2.2.14): 191 °C to 195 °C. Reference solution (c) Dissolve 10.0 mg of clomipramine
B. Infrared absorption spectrophotometry (2.2.24). hydrochloride CRS and 3.0 mg of clomipramine impurity C CRS
Preparation Discs of potassium bromide R. The transmittance in a mixture of 25 volumes of mobile phase B and
at about 2000 cm-1 (5 pm) is at least 65 per cent without 75 volumes of mobile phase A and dilute to 20.0 mL with
compensation. the same mixture of mobile phases. Dilute 1.0 mL of this
solution to 10.0 mL with a mixture of 25 volumes of mobile
Comparison clomipramine hydrochloride CRS.
phase B and 75 volumes of mobile phase A.
C. Thin-layer chromatography (2.2.27). Prepare the solutions
Column:
immediately before use and protectedfrom light.
— size. I = 0.25 m, 0 = 4.6 mm,
Test solution Dissolve 20 mg of the substance to be examined — stationary phase. cyanopropylsUyl silica gelfor
in methanol R and dilute to 10 mL with the same solvent chromatography R (5 pm),
Reference solution Dissolve 20 mg of clomipramine — temperature. 30 °C.
hydrochloride CRS in methanol R and dilute to 10 mL with the Mobile phase:
same solvent — mobile phase A: dissolve 1.2 g of sodium dihydrogen
Plate TLC silica gel G plate R. phosphate R in water R, add 1.1 mL of nonylamine R,
Mobile phase concentrated ammonia R, acetone R, ethyl acetate R adjust to pH 3.0 with phosphoric acid R and dilute to
(5:25:75 V/V/V). 1000 m l. with water R,
Application 5 pL. — mobile phase B: acetomtrUe R.

Drying In air. Time Mobile phase A Mobile phase B
Detection Spray with a 5 g/L solution of potassium
0 - 10 75 25
dichromate R in a 20 per cent V/V solution of sulfuric acid R.
Examine immediately. 10 -2 0 75->65 25->35
Results The principal spot in the chromatogram obtained with 2 0 -3 2 65 35

the test solution is similar in position, colour and size to the 3 2 -3 4 65->75 35->25
3 4 -4 4 75 25
reference solution.
D. Dissolve about 5 mg in 2 mL of nitric acid R. An intense
blue colour develops. Flow rate 1.5 mL/min.
E. Dissolve about 50 mg in 5 mL of water R and add 1 mL Detection Spectrophotometer at 254 nm.
of dilute ammonia R l. Mix, allow to stand for 5 min and
Injection 20 pL.
filter. Acidify the filtrate with dilute nitric add R. The solution
Relative retentions With reference to clomipramine (retention
gives reaction (a) of chlorides (2.3.1).
TESTS impurity B = about 0.7; impurity C = about 0.9;
Solution S impurity D = about 1.7; impurity E = about 2.5;
Dissolve 2.0 g in carbon dioxide-free water R and dilute to impurity F = about 3.4; impurity G = about 4.3.
20 mL with the same solvent. System suitability: reference solution (c):
Appearance of solution — resolution: minimum 3.0 between die peaks due to
Solution S is clear (2.2.1) and not more intensely coloured clomipramine and to impurity C.
than reference solution Y5 (2.2.2, Method I). Limits:
pH (2.2.3) — impurity B: not more than the area of the corresponding
3.5 to 5.0 for solution S. peak in the chromatogram obtained with reference
solution (a) (1.0 per cent),
2016 Clonazepam 1-601
ci
— impurity C} D". for each impurity, not more than the area
of the corresponding peak in the chromatogram obtained
with reference solution (a) (0.2 per cent), . ch3
— impurity F: not more than the area of the corresponding N
I
peak in the chromatogram obtained with reference ch3
solution (a) (0.1 per cent),
— arty other impurity, not more than 0.1 times the area of the
reference solution (b) (0.1 per cent), C. 3-(3-chloro-5//-dibenzo [bj\azepin-5-yl)-iV^V-
— total of other impurities: not more than 0.2 times the area dimethylpropan- 1-amine,
reference solution (b) (0.2 per cent), R1
— total: not more than the area of the principal peak in the
• chromatogram obtained with reference solution (b)
( 1.0 per cent),
(0.01 per cent). R3
Maximum 20 ppm. D. RI = R3 = Cl, R2 = CH2-CH 2-CH 2-N(CH3)2: 3-(3,7-
2.0 g complies with test C. Prepare the reference solution dichloro-10,11 -dihydro-5//-dibenzo [bj\ azepin-5-yl)-N,N-
using 4 mL of lead standard solution (10 ppm Pb) R. dimethylpropan-1-amine,
Loss on drying (2.2.32) E. RI = R2 = R3 = H: 10,ll-dihydro-5tf-
Maximum 0.5 per cent, determined on 1.000 g by drying in dibenzo [bj\ azepine (iminodibenzyl),
an oven at 105 °C. F. RI = Cl, R2 = R3 = H: 3-chloro-10,l l-dihydro-5ii-
Sulfated ash (2.4.14) dibenzo [bj\azepine,
Maximum 0.1 per cent, determined on 1.0 g. G. RI = Cl, R2 = CH2-CH=CH 2, R3 = H: 3-chloro-5-
ASSAY (prop-2-enyl)-l 0,11 -dihydro-5ii-dibenzo [bj\azepine.
Dissolve 0.250 g in 50 mL of alcohol R and add 5.0 mL of ______________________________________________________ ___ PhEur
0.01 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 of inflexion.
1 mL of 0.1 M sodium hydroxide is equivalent to 35.13 mg of ★ ★
C19H 24CI2N 2.
Clonazepam ★ ★
STORAGE (Ph. Eur. monograph 0890) *****
IMPURITIES
ci
.CH3
N N
1 1
ch3 ch3
C 15H 10ClN3O3 315.7 1622-61-3

Action and use
A. N - [3-(3-chloro-10,11 -dihydro-5ii-dibenzo [bj\azepin-5- Benzodiazepine.
yOpropylJ-NJVîV'-trimethylpropane-l,3-diamine, Préparations
Clonazepam Injection
Clonazepam Oral Suspension
, ch3 Clonazepam Tablets
n
1
ch3 PhEur
DEFINITION
5-(2-Chlorophenyl)-7-nitro-l,3-dihydro-2/f-l,4-
benzodiazepin-2 -one.
B. 3-(10,l 1-dihydro-5/i-dibenzo [bf\20£pm-5-yl)-N,N-
dimethylpropan- 1-amine (imipramine), Content
CHARACTERS
Appearance
Slightly yellowish, crystalline powder.
1-602 Clonidine Hydrochloride 2016
Solubility Loss on drying (2.2.32)

Practically insoluble in water, slightly soluble in alcohol and Maximum 0.5 per cent, determined on 1.000 g by drying in
in methanol, an oven at 105 °C for 4 h.
mp: about 239 °C. Sulfated ash (2.414)
IDENTIFICATION Maximum 0.1 per cent, determined on 1.0 g.
Infrared absorption spectrophotometry (2.2.24). ASSAY
Comparison Ph. Ever, reference spectrum of clonazepam. Dissolve 0.275 g in 50 mL of acetic anhydride R. Titrate with
0.1 M perchloric acid, determining the end-point
TESTS
potentiometrically (2.2.2(f).
Related substances
Liquid chromatography (2.2.29). Carry out the test protected
1 m l, of 0.1 Mperchloric add is equivalent to 31.57 mg
from light and prepare the solutions immediately before use.
of C 15H 10CIN3O3.
Solvent mixture tetrahydrcfuran R, methanol R, water R STORAGE
(10:42:48 V/V/V). Protected from light.
Test solution Dissolve 0.100 g of the substance to be IMPURITIES
examined in methanol R and dilute to 20.0 mL with the same Spedfied impurities: A, B.
solvent. Dilute 1.0 mL to 10.0 mL with the solvent mixture.
100.0 mL with the solvent mixture. Dilute 1.0 mL of the
examined and 5 mg of flunitrazepam R in the solvent mixture
and dilute to 100.0 mL with the solvent mixture.
Reference solution (c) Dissolve 1.0 mg of clonazepam
impurity B CR S in the solvent mixture and dilute to 20.0 mL A. (2-amino-5-nitrophenyl) (2 -chlorophenyl)methanone,
with the solvent mixture. Dilute 1.0 mL of the solution to
100.0 mL with the solvent mixture.
Column:
— sizer. I = 0.15 m, 0 = 4.6 mm,
— stationary phase. end-capped octylsUyl silica gel for
chromatography R (5 |iin).
Mobile phase Mix 10 volumes of tetrakydrofwran R,
42 volumes of methanol R and 48 volumes of a 6.6 g/L
solution of ammonium phosphate R previously adjusted to B. 3-amino-4-(2-chlorophenyl)-6-nitroquinolin-2( lü)-one.
pH 8.0 with a 40 g/L solution of sodium hydroxide R or dilute PhEur
phosphoric acid R.
Injection 10 |iL.
Clonidine Hydrochloride
Run time 3 times the retention time of clonazepam.
Relative retention With reference to clonazepam (retention (Ph. Eur. monograph 0477) *
— resolution-, minimum 1.8 between the peaks due to
flunitrazepam and to clonazepam. Cl
Limits-.
— impurity A : not more than the area of the principal peak C9HJ0Q 3N 3 266.6 4205-91-8
Action and use
(0.1 per cent),
Alpha2-adrenoceptor agonist; treatment of hypertension.
— impurity B: not more than the area of the principal peak
in the chromatogram obtained with reference solution (c) Preparations
(0.1 per cent) Clonidine Injection
— any other impurity, for each impurity, not more than the Clonidine Tablets
with reference solution (a) (0.1 per cent), PhEur__ ____________________________________________________ __
— total: not more than twice the area of the principal peak in DEFINITION
the chromatogram obtained with reference solution (a) 2,6-Dichloro-N-(imidazolidin-2-ylidene)aniline hydrochloride.
(0.2 per cent),
Content
— disregard lima: 0.5 times the area of the principal peak in
(0.05 per cent). CHARACTERS
Appearance
2016 Clonidine Hydrochloride 1-603
Solubility — stationary phase-, propylsifyl silica gd for chromatography R

Soluble in water and in anhydrous ethanol, (5 pm);
IDENTIFICATION — temperature: 40 °C.
Mobile phase:
— mobile phase A: dissolve 4 g of potassium dihydrogen
Second identification A , C, D.
phosphate R in 1000 mL of water R, and adjust to pH 4.0
A. Ultraviolet and visible absorption spectrophotometry with phosphoric acid R;
(2.2.25). — mobile phase B: mobile phase A, acetomtrüe R1
Test solution Dissolve 30.0 mg in 0.01 M hydrochloric acid and (25:75 V!V)-,
dilute to 100.0 mL with the same add.
Spectral range 245-350 nm. Time Mobile phase A Mobile phase B
Absorption maxima At 272 nm and 279 nm. (ndn) (ner cent V/V) (net cent V/V)
Point of inflexion At 265 nm. 0 90 ja
Specific absorbance at the absorption maxima: 0 -1 5 9 0 -» 3 0 1 0 -»70
— at 272 nm: about 18; 15 -15.1 3 0 -» 9 0 70 -»10
— at 279 nm: about 16.
15.1 - 20 90 10
Comparison donidine hydrochloride CRS.
C. Thin-layer chromatography (2.2.27). Flow rate 1.5 mL/min.
Test solution Dissolve 5 mg of the substance to be examined Detection Spectrophotometer at 210 nm.
in methanol R and dilute to 5 mL with the same solvent. Injection 5 pL
Reference solution Dissolve 5 mg of donidine hydrochloride CRS System suitability: reference solution (b):
in methanol R and dilute to 5 mL with the same solvent. — resolution: minimum 5 between the peaks due to clonidine
Plate T LC silica gel G plate R. and impurity B.
Mobile phase glacial acetic acid R, butanol R, water R Limits:
(10:40:50 V/VfV); allow to separate, filter the upper layer — unspecified impurities:for each impurity, not more than the
and use the filtrate. area of the principal peak in the chromatogram obtained
Application 10 pL.
Drying In air. (0.2 per cent);
Detection Spray with potassium iodobismuthate solution R2. — disregard limit. 0.5 times the area of the principal peak in
Allow to dry in air for 1 h. Spray again with potassium the chromatogram obtained with reference solution (a)
iodobismuthate solution R2 and then immediately spray with a (0.05 per cent).
50 g/L solution of sodium nitrite R. Loss on drying (2.2.32)
Results The principal spot in the chromatogram obtained with Maximum 0.5 per cent, determined on 1.000 g by drying in
the test solution is similar in position, colour and size to the an oven at 105 °C.
principal spot in the chromatogram obtained with the Sulfated ash (2.414)
reference solution. Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
TESTS Dissolve 0.200 g in 70 mL of ethanol (96 per cent) R. Titrate
Solution S with 0.1 M ethanoUc sodium hydroxide determining the
Dissolve 1.25 g in carbon dioxide-free water R and dilute to end-point potentiometrically (2.2.20).
25 mL with the same solvent. 1 m l. of 0.1 M sodium hydroxide is equivalent to 26.66 mg of
Appearance of solution C9H 10CI3N 3.
IMPURITIES
pH (2.2.3) present at a sufficient level, be detected by one or other of
4J0 to 5.0 for solution S. the tests in the monograph. They are limited by the general
Related substances acceptance criterion for other/unspecified impurities and/or
Liquid chromatography (2.2.29). by the general monograph Substances for pharmaceutical use
Test solution Dissolve 50 mg of the substance to be examined (2034). It is therefore not necessary to identify these
in mobile phase A and dilute to 50 mL with mobile phase A. impurities for demonstration of compliance. See also 5.10.
Control of impurities in substancesfor pharmaceutical use): A , B,
C.
Reference solution (b) Dissolve 5 mg of clonidine
impurity B CRS in 2 mL of acetonitrUe R and dilute to 5 mL
with mobile phase A. To 1 mL of this solution, add 1 mL of
the test solution and dilute to 10 mL with mobile phase A.
Column:
— sizer. I = 0.15 m, 0 = 3.0 mm; A. l-acetylimidazolidin-2-one,
1-604 Clopamide 2016
Test solution Dissolve 100 mg of the substance to be
çOo I H o>« c h 3
examined in
solvent.
methanol R and dilute to 10.0 mL with the same
Reference solution (a) Dissolve 10 mg of clopamide for system

suitability CRS (containing impurities B, C and H) in 1.0 mL
B. 1-acetyl-2- [(2,6-dichlorophenyl)amino] -4,5-dihydro-1H - of methanol R.
imidazole, Dilute 2.0 mL of the test solution to
Reference solution (b)
100.0 mL with methanol R. Dilute 2.0 mL of this solution to
40.0 mL with methanol R.
Column'.
— size. I = 0.15 m, 0 = 4.6 mm;
— stationary phase', end-capped octylsüyl silica gelfor
C. 2,6-dichloroaniline.
Mobile phase.
PhEur —mobile phase A: dissolve 1.0 g of ammonium acetate R in
950 ml. of water R, adjust to pH 2.0 with phosphoric
add R and dilute to 1000 mL with water R;
— mobile phase B: acetonitrüe R’,
★ ★ — mobile phase C: water R, tetrahydrofuran for
Clopamide ★ ★ chromatography R (20:80 V!V)\ this mobile phase allows
(Ph. Eur. monograph 1747) ***** adequate rinsing of the system;
Time Mobile phase A Mobile phase B Mobile phase C

(min) (per cent V/Y) (percent V7V) (per cent V/V\
0-35 95 -* 75 5 -* 2 5 0
35-45 75 -* 35 25 -»65 0
45-50 35 -* 30 65 -* 0 0 -* 70
C 14H 20CIN3O3S 345.8 636-54-4 50-60 30 0 70
Action and use

Thiazide-like diuretic.
PhEur_____________________________ Flow rate 0.4 mL/min.
DEFINITION Detection Spectrophotometer at 235 nm.
4-Chloro-iV-[(2i?5j65i?)-2j6-dimethylpiperidin-l-yl]-3- Injection 10 |iL.
sulfamoylbenzamide. Identification of impurities Use the chromatogram supplied
Content with clopamide for system suitability CRS and the
99.0 per cent to 101.0 per cent (dried substance). chromatogram obtained with reference solution (a) to
identify the peaks due to impurities B, C and H.
PRODUCTION
Relative retention With reference to clopamide (retention
The production method is evaluated to determine the
potential for formation of an N -nitroso compound (cis-2,6-
impurity H = about 1.2; impurity B = about 1.4.
dimethyl-l-nitrosopiperidine). Where necessary, the
production method is validated to demonstrate that the
iV-nitroso compound is absent in the final product — resolution: minimum 3 between the peaks due to
impurity C and clopamide.
CHARACTERS Limits:
Appearance — correction factory, for the calculation of content, multiply
White or almost white, hygroscopic, crystalline powder. the peak areas of the following impurities by the
Solubility corresponding correction factor impurity B = 0.5;
Slightly soluble in water and in anhydrous ethanol, sparingly impurity H = 0.4;
soluble in methanol. — impurities B, C, H: for each impurity, not more than twice
It shows polymorphism (5.9). the area of the principal peak in the chromatogram
IDENTIFICATION — unspecified impurities: for each impurity, not more than the
Comparison clopamide CRS. with reference solution (b) (0.10 per cent);
If the spectra obtained in the solid state show differences, — total: not more than 10 times the area of the principal
dissolve the substance to be examined and the reference peak in the chromatogram obtained with reference
substance separately in the minimum volume of methanol R, solution (b) (1.0 per cent);
evaporate to dryness on a water-bath and record new spectra — disregard limir. 0.5 times the area of the principal peak in
using the residues. the chromatogram obtained with reference solution (b)
(0.05 per cent).
TESTS
Related substances Heavy m etals (2.4.8)
Liquid chromatography (2.2.29). Maximum 20 ppm.
2016 Clopidogrel Hydrogen Sulfate 1-605
Dissolve 0.25 g in a mixture of 20 volumes of acetone R and

85 volumes of methanol R and dilute to 20 mL with the same
Clopidogrel Hydrogen Sulfate *****
★ ★
★ ★
mixture of solvents. 20 mL of the solution complies with (Ph. Eur. monograph 2531) *★*
modified test B. Prepare the reference solution by diluting
0.5 mL of lead standard solution (10 ppm Pb) R to 20 mL
with a mixture of 20 volumes of acetone R and 85 volumes of
methanol R. Prepare the blank solution by using 20 mL of a . H2SO4
mixture of 20 volumes of acetone R and 85 volumes of
methanol R.
Filter the solutions through a membrane filter (nominal pore
size 0.45 pm) to evaluate the result. Cl<)Hi8ClN06S2 419.9 120202- 66-6
Maximum 2.5 per cent, determined on 1.000 g by drying in Action and use
an oven at 105 °C. Inhibitor of ADP-mediated platelet aggregation.
Sulfated ash (2.4.14) PhEir.
Maximum 0.1 per cent, determined on 1.0 g. DEFINITION
ASSAY Methyl (2S)-(2 -chlorophenyl) [6,7-dihydrothieno[3,2-
Dissolve 0.280 g in 70 mL of anhydrous acetic acid R. Titrate c]pyridin-5(4if)-yl]acetate sulfate.
with 0.1 M perchloric acid, determining the end-point Content
potentiometrically (2.2.20). 99.0 per cent to 101.0 per cent (anhydrous substance).
CHARACTERS
of C 14H 20CIN3O3S.
Appearance
STORAGE White or almost white powder.
In an airtight container, protected from light. Solubility
IMPURITIES Freely soluble in water and in methanol, practically insoluble
Specified impurities B, C, H in cyclohexane.
Other detectable impurities (the following substances would, if It shows polymorphism (5.9).
present at a sufficient level, be detected by one or other of IDENTIFICATION
the tests in the monograph. They are limited by the general Cany out either tests A, B, D or tests B, C, D.
by the general monograph Substancesfor pharmaceutical use A. Specific optical rotation (2.2.7): + 54.0 to + 58.0
impurities for demonstration of compliance. See also 5.10. Dissolve 0.250 g in methanol R and dilute to 25.0 mL with
Control of impurities m substancesfor pharmaceutical use): A, G. the same solvent.
Comparison clopidogrel hydrogen sulfate CRS.
and enantiomer substance to be examined and the reference substance
separately in anhydrous ethanol R, evaporate to dryness and
record new spectra using the residues (the substance may
stick to the surface of the recipient used).
A. R = CH3: 4-chloro-iV-[(2ÄS,6ÄS)-2,6-dimethylpiperidin- C. Enantiomeric purity (see Tests).
1-yl] -3-sulfamoylbenzamide (rrans-clopamide), D. It gives reaction (a) of sulfates (2.3.1).
G. R = H: 4-chloro-N-[(2ÄS)-2-methylpiperidin-l-yl]-3-
sulfemoyibenzamide. TESTS
CO2H The solution is clear (2.2.1) and not more intensely coloured
than reference solution Y6 (2.2.2, Method I).
Cl Dissolve 1.0 g in methanol R and dilute to 20.0 mL with the
same solvent.
B. R = H: 4-chIorobenzoic add, Enantiomeric purity
C. R = SO2-NH 2: 4-chloro-3-sulfamoyIbenzoic acid, Liquid chromatography (2.2.29): use the normalisation
procedure.
in 25.0 mL of anhydrous ethanol R and dilute to 50.0 mL
h3c . ^ .s with heptane R.
N N
i Reference solution Dissolve 10 mg of clopidogrelfor system
ch3
suitability CRS (containing impurities B and C) in 2.5 mL of
anhydrous ethanol R and dilute to 5.0 mL with heptane R.
H. 4-chloro-3-[(£)-[(dimethylamino)methyiene]sulfamoyl]-N- Column:
[(2 ÆS,6SÆ)-2 ,6-dimethyipiperidin-l-yl]benzamide. — size. I = 0.25 m, 0 = 4.6 mm;
— stationary phase, silica gel OJ for chiral separations R
PhEur
•(10 pm).
1-606 Clopidogrel Hydrogen Sulfate 2016
MobUe phase anhydrous ethanol R, heptane R (15:85 VIV). Relative retention With reference to clopidogrel (retention
Flow rate 0.8 mL/min. time = about 25 min): impurity A = about 0.4;
Injection 10 pL.
— peak-to-valley ratio: minimum 10, where Hp — height
Run time 1.25 times the retention time of clopidogrel. above the baseline of the peak due to impurity B and
Identification of impurities Use die chromatogram supplied H v = height above the baseline of the lowest point of the
with clopidogrelfor system suitability CRS and the curve separating this peak from the peak due to
chromatogram obtained with the reference solution to clopidogrel.
identify the peaks due to impurities B and C. Limits:
Relative retention With reference to clopidogrel (retention — impurity B: not more than 3 times the area of the
time = about 18 min): impurity C = about 0.6; principal peak in the chromatogram obtained with
impurity B = about 0.7. reference solution (c) (0.3 per cent);
System suitability: reference solution: — impurity A: not more than twice the area of the principal
— resolution: minimum 2.0 between the peaks due to peak in the chromatogram obtained with reference
impurities C and B; solution (c) (0.2 per cent);
— signal-to-noise ratio: minimum 20 for the peak due to — unspecified impurities: for each impurity, not more than the
impurity C. area of the principal peak in the chromatogram obtained
Limit: with reference solution (c) (0.10 per cent);
— impurity C: maximum 0.5 per cent. — total: not more than 5 times the area of the principal peak
Related substances
(0.5 per cent);
Solvent mixture Mobile phase A, acetonitrile R1 (40:60 VIV). the chromatogram obtained with reference solution (c)
Test solution Dissolve 65 mg of the substance to be examined (0.05 per cent).
in the solvent mixture and dilute to 10.0 mL with the solvent Heavy metals (2.4.8)
mixture. Maximum 20 ppm.
Reference solution (a) Dissolve 5 mg of clopidogrel
impurity A CR S in the solvent mixture and dilute to 25.0 mL
with the solvent mixture.
W ater (2.5.12)
Reference solution (b) Dissolve 32 mg of clopidogrelfor system
mixture, add 0.5 mL of reference solution (a) and dilute to Replace the solvent after each titration.
5.0 mL with the solvent mixture. Sulfated ash (2.4.14)
Reference solution (c) Dilute 1.0 mL of the test solution to Maximum 0.1 per cent, determined on 1.0 g.
100.0 mL with the solvent mixture. Dilute 1.0 mL of this ASSAY
solution to 10.0 mL with the solvent mixture. Dissolve 0.160 g in a mixture of 10 mL of acetone R, 10 mL
Column: of methanol R and 30 mL of water R. Titrate with 0.1 M
— size: I = 0.15 m, 0 = 3.9 mm; sodium hydroxide, determining the end-point
— stationary phase: end-capped octadecylsUyl silica gelfor potentiometrically (2.2.20). A precipitate may be formed
chromatography R (5 pm); during the titration.
— temperature: 30 °C. 1 mL of 0.1 M sodium hydroxide is equivalent to 20.99 mg of
Mobile phase: c 16h 18o n o 6s 2.
— mobile phase A: mix 5 volumes of methanol R2 and
95 volumes of a 0.96 g/L solution of sodium STORAGE
pentanesulfonate monohydrate R adjusted to pH 2.5 with
phosphoric acid R; IMPURITIES
— mobile phase B: methanol R2, acetonitrile R1 (5:95 VIV); Specified impurities A,B, C
Other detectable impurities(die following substances would, if
Time Mobile phaae A Mobile phase B present at a sufficient level, be detected by one or other of
(min) (per cent V/V) (per cent V/V) the tests in the monograpB. They are limited by the general
0 -3 89.5 10.5
3 -4 « 89.5 •» 315 10.5 •» 68.5 by the general monograph Substances for pharmaceutical use
4 8 -6 8 31.5 68.5
Control of impurities in substances for pharmaceutical use): D.
and (c).
with clopidogrelfor system suitability CRS and the
identify the peaks due to impurities A and B. A. (2¿)-(2-chlorophenyl) [6,7-dihydrothieno [3,2-c]pyridin-
5 (4/f)-ylj acetic acid,
2016 Clotrimazole 1-607
IDENTIFICATION
B. methyl (25)-(2-chlorophenyl)[4,7-dihydrothieno[2,3- Comparison clotrimazole CRS.
c]pyridin-6 (5H)-yl] acetate. C. Thin-layer chromatography (2.2.27).
in ethanol (96 per cent) R and dilute to 5 mL with the same
solvent.
och3 Reference solution Dissolve 50 mg of clotrimazole CRS in.
solvent.
Plate TLC silica gel F 254 plate R
C. methyl (2i?)-(2-chlorophenyl) [6,7-dihydrothieno[3,2-
Mobile phase concentrated ammonia R l, propanol R, toluene R
c]pyridin-5 (4H)~yI] acetate,
(0.5:10:90 VIV/V).
Application 10 (iL.
Drying In air.
Detection Examine in ultraviolet light at 254 run.
D. methyl (2i?)-(2-chlorophenyl) [(23}-(2-chlorophenyl) [6,7- solution.
dihydrothieno [3,2-c]pyridin-5 (4H)-yl] acetyloxy] acetate.
TESTS
_______________________________________________ PhEur
Related substances
★★*★ ★ examined in acetomtrUe R l and dilute to 50.0 mL with the
Clotrimazole ★ ★ same solvent.
(Ph. Eicr. monograph 0757) ***** Reference solution (a) Dilute 1.0 mL of the test solution to
100.0 mL with acetonitrUe R l. Dilute 1.0 mL of this solution
to 10.0 mL with acetonitrUe R l.
clotrimazole for peak identification CRS (containing impurities
A, B and F) in 1.0 mL of acetonitrUe R l.
Reference solution (c) Dissolve 5.0 mg of imidazole CRS
(impurity D) and 5.0 mg of clotrimazole impurity E CRS in
acetonitrUe R l and dilute to 100.0 mL with the same solvent.
C22Hi7CIN2 23593-75-1 Dilute 1.0 mL of this solution to 25.0 mL with
acetonitrUe R l.
Action and use
Column'.
Annfungal.
— sizer. I = 0.15 m, 0 = 4.6 mm;
Preparations — stationary phase', spherical end-capped octylsUyl sUica gel for
Clotrimazole and Hydrocortisone Acetate Cream chromatography R (5 |im);
Clotrimazole Cream — temperature: 40 °C.
Clotrimazole Eye Drops Mobile phase:
Clotrimazole Pessaries — mobile phase A: dissolve 1.0 g of potassium dihydrogen
phosphate R and 0.5 g of tetrabutylammomum hydrogen
Clotrimazole Vaginal Tablets
sulfate R l in water R and dilute to 1000 mL with the
PhEur. same solvent;
— mobile phase B: acetonitrUe Rl',
DEFINITION
1- [(2-Chlorophenyl) diphenylmethyl] - lH-imidazole.
Content (min) (per cent V/V) (per cent V/V)
98.5 per cent to 100.5 per cent (dried substance). 0 -3 75 25
CHARACTERS 3 -2 5 75-»20 2 5 -»80
A ppearance 2 5 -3 0 20 80
White or pale yellow, crystalline powder.
Solubility
Practically insoluble in water, soluble in ethanol (96 per cent)
and in methylene chloride. Detection Spectrophotometer at 210 nm.
1-608 Cloxacillin Sodium 2016
Injection 10 |iL.
Relative retention With reference to clotrimazole (retention
impurity E = about 1.5; impurity A = about 1.8.
— resolution', minimum 1.5 between the peaks due to
impurity F and clotrimazole; B. R = Cl: l-[(4-chlorophenyl)diphenylmethyl]-lif-
— the chromatogram obtained is similar to the imidazole,
chromatogram supplied with clotrimazolefor peak F. R = H: l-(triphenylmethyl)-lif-imidazole
identification CRS. (deschloroclotrimazole),
Limits:
— impurities A , B: for each impurity, not more than twice
— impurities D, E: for each impurity, not more than the area
of the corresponding peak in the chromatogram obtained D. imidazole.
with reference solution (c) (0.2 per cent); PhEur
— impurity F: not more than the area of the principal peak
(0.1 per cent);
area of the principal peak in the chromatogram obtained Cloxacillin Sodium ★★* + +
★ +
(0.5 per cent);
— disregard limit: 0.5 times die area of the principal peak in
the chromatogram obtained with reference solution (a) h 2o
(0.05 per cent).
an oven at 105 °C.
Sulfated ash (2.4.14) CujHnClNaNaOsSjHjO 475.9 7081-44-9
Action and use
ASSAY Penicillin antibacterial.
Dissolve 0.300 g in 80 mL of anhydrous acetic acid R. Using
0.3 mL of naphtholbenzem solution R as indicator, titrate with PhEur_____________________________
0.1 M perchloric acid until the colour changes from brownish-
DEFINITION
yellow to green.
Sodium (2S,5R,6i?)-6- [[[3-(2-chlorophenyl)-5-
1 mL of 0.1 M perchloric acid is equivalent to 34.48 mg methylisoxazol-4-yl] carbonyl] amino]-3,3-dimethyl-7-oxo-4-
of C 22H 17CIN2. thia-l-azabicyclo[3.2.0]heptane-2-carboxylate monohydrate.
STORAGE Semi-synthetic product derived from a fermentation product.
Content
IMPURITIES 95.0 per cent to 102.0 per cent (anhydrous substance).
Specified impurities A, B, D, E, F
CHARACTERS
Other detectable impurities (die following substances would, if Appearance
present at a sufficient level, be detected by one or other of White or almost white, hygroscopic, crystalline powder.
acceptance criterion for other/unspecified impurities and/or Solubility
by the general monograph Substancesfor pharmaceutical use Freely soluble in water and in methanol, soluble in ethanol
(96 per cent).
impurities for demonstration of compliance. See also 5.10. IDENTIFICATION
Control of impurities in substancesfor pharmaceutical use): C. First identification A , D
Preparation Discs.
Comparison cloxacillin sodium CRS.
A. R = OH, R' = C$H5: (2-dilorophenyi)diphenylmethanol, B. Thin-layer chromatography (2.2.27).
C. R = Cl, R ' = C$H5: l-chloro-2- Test solution Dissolve 25 mg of the substance to be examined
(chlorodiphenylmethyl) benzene, in 5 mL of water R.
E. R + R' = O: (2-chlorophenyl)phenylmethanone Reference solution (a) Dissolve 25 mg of cloxacillin sodium CRS
(2-chlorobenzophenone), in 5 mL of water R.
2016 Cloxacillin Sodium 1-609
Reference solution (b) Dissolve 25 mg of chxadRin Flow rate 1.0 mL/min.

sodium CRS, 25 mg of didoxadUin sodium CRS and 25 mg of Detection Spectrophotometer at 225 nm.
flucloxacHlin sodium CRS in 5 mL of water R.
Injection 20 (iL of test solution (a) and reference solutions (b)
Plate T L C sUanised silica gel plate R. and (c).
Mobile phase Mix 30 volumes of acetone R and 70 volumes of Run time 5 times the retention time of cloxacillin.
a 154 g/L solution of ammonium acetate R, then adjust to System suitability: reference solution (c):
pH 5.0 with glacial acetic add R. — resolution: minimum 2.5 between the peaks due to
Application 1 jiL. cloxacillin (1st peak) and flucloxadllin (2nd peak).
Development Over a path of 15 cm. Limits:
Drying In air. — any impurity: not more than the area of the principal peak
Detection Expose to iodine vapour until the spots appear;

examine in daylight. (1.0 per cent);
(5.0 per cent);
Results The principal spot in the chromatogram obtained with — disregard limit:. 0.05 times the area of the principal peak in
the test solution is similar in position, colour and size to the the chromatogram obtained with reference solution (b)
principal spot in the chromatogram obtained with reference (0.05 per cent).
solution (a).
iV^V-Dimethylaniline (2.4.26, Method B)
C. Place about 2 mg in a test-tube about 150 mm long and Maximum 20 ppm.
15 mm in diameter. Moisten with 0.05 m l. of water R and
add 2 mL of sulfuric acid-formaldehyde reagent R. Mix the 2-Ethylhexanoic acid (2.4.28)
contents of the tube by swirling; the solution is slightly Maximum 0.8 per cent mlm.
greenish-yellow. Place the test-tube in a water-bath for W ater (2.5.12)
1 min; the solution becomes yellow. 3.0 per cent to 4.5 per cent, determined on 0.300 g.
D. It gives reaction (a) of sodium (2.3.1). Bacterial endotoxins (2.6.14)
TESTS Less than 0.20 IU/mg, if intended for use in the manufacture
Solution S
Dissolve 2.50 g in carbon dioxide-free water R and dilute to procedure for the removal of bacterial endotoxins.
25.0 mL with the same solvent. ASSAY
Appearance o f solution Liquid chromatography (2.2.29) as described in the test for
Solution S is clear (2.2.1) and its absorbance (2.2.25) at related substances with the following modifications.
430 nm is not greater than 0.04. Injection Test solution (b) and reference solution (a).
pH (2.2.3) System suitability:
5.0 to 7.0 for solution S. — repeatability: maximum relative standard deviation of
1.0 per cent after 6 injections of reference solution (a).
+ 160 to + 169 (anhydrous substance). Calculate the percentage content of Ci9Hi7dN3Na05S from
the declared content of doxadSin sodium CRS.
Dissolve 0.250 g in water R and dilute to 25.0 ml. with the
same solvent. STORAGE
Related substances In an airtight container, at a temperature not exceeding
Liquid chromatography (2.2.29). 25 °C. If the substance is sterile, store in a sterile, airtight,
tamper-proof container.
examined in the mobile phase and dilute to 50.0 mL with IMPURITIES
the mobile phase.
Test solution (b) Dilute 5.0 mL of test solution (a) to
C|—
50.0 mL with the mobile phase. \ / h, .c o 2h
Reference solution (a) Dissolve 50.0 mg of cloxacillin N = / H N - K .CH3
sodium CRS in the mobile phase and dilute to 50.0 mL with
' \ H I. X
oJ y nn ^ sA cH3
the mobile phase. Dilute 5.0 mL of this solution to 50.0 mL
with the mobile phase. ch3 o r
Reference solution (b) Dilute 5.0 mL of test solution (b) to

50.0 mL with the mobile phase. A. R = C 02H: (45)-2-[carboxy[[[3-(2-chlorophenyI)-5-
Reference solution (c) Dissolve 5 mg offlucbxadUm
methylisoxazol-4-yl] carbonyl] amino] methyl]-5,5-
sodium CRS and 5 mg of doxadSin sodium CRS in the mobile
dimethylthiazolidine-4-carboxylic acid (penicilloic add of
phase and dilute to 50.0 mL with the mobile phase. cloxaciUin),
Column: B. R = H: (2RS,4S)-2-[[[ [3-(2-chlorophenyl)-5-
— size. I = 0.25 m, 0 = 4 mm; methylisoxazol-4-yl] carbonyl] amino] methyl]-5,5-
— stationary phase. octadecylsUyl silica gd for chromatography R
dimethylthiazolidine-4-carboxylic add (penilloic add of
(5 pm). cloxacillin),
Mobile phase Mix 25 volumes of acetonitrile R and 75 volumes
of a 2.7 g/L solution of potassium dihydrogen phosphate R
adjusted to pH 5.0 with dilute sodium hydroxide solution R.
1-610 Clozapine 2016
Solubility
Practically insoluble in water, freely soluble in methylene
chloride, soluble in ethanol (96 per cent). It dissolves in
H H dilute acetic add.
IDENTIFICATION
C. (2S,5Ä,6i^-6-amino-3,3-dimethyI-7-oxo-4-thia-1- A. Mdting point (2.2.14): 182 °C to 186 °C.
azabicydo[3.2.0]heptane-2-carboxylic add
(6-aminopenirillanic add), B. Infrared absorption spectrophotometry (2.2.24).
Comparison clozapine CRS.
TESTS
Related substances
Solvent mixture water R, methanol R2 (20:80 V/V).
Solution A Dissolve 2.04 g of potassium dihydrogen phosphate R
in 1000 mL of water R and adjust to pH 2.4 ± 0.05 with
dilute phosphoric acid R.
D. 3-(2-chIorophenyi)-5-methylisoxazole-4-carboxylic add, Test solution Dissolve 75 mg of the substance to be examined
in 80 mL of methanol R2 and dilute to 100 mL with water R
o • »co2h Reference solution (a) Dilute 1.0 mL of the test solution to
V T ' V 0”3 10.0 mL with the solvent mixture. Dilute 1.0 mL of this
Cl— \ \ ° ^ n - - —L / X H a solution to 100.0 mL with the solvent mixture.
/ — 0 J - 'H H H Reference solution (b) Dissolve the contents of a vial of
N =( ^^N ^\.C H 3 clozapine for peak identification CRS (containing
O y X , N -i—Lg/^CHs impurities A, B, C and D) in 1.0 mL of the solvent mixture.
T H H H Column:
CH3 o
— size. I = 0.125 m, 0 = 4.6 mm;
— stationary phase, end-capped octadecylsUyl silica gelfor
E. (2S,5R,6R)-6- [[[(25,5R,6.R)-6-[ [[3-(2-chlorophenyl)-5- chromatography R (5 jjtm).
methyUsoxazol-4-yl]carbonyl]amino]-3,3-dimethyl-7-oxo-4-
Mobile phase.
thia-1-azabicydo [3.2.0]hept-2-yl] carbonyl] amino] -3,3-
— mobile phase A: acetonitrile for chromatography R,
dimethyl-7-oxo-4-thia-1 -azabicyclo [3.2.0] heptane-2-
methanol R2, solution A (1:1:8 V/V/V);
carboxylic add (6-APA cloxadllin amide).
— mobile phase B: acetonitrilefor chromatography R,
__________________________________________________________ PhEur methanol R2, solution A (4:4:2 V/V/V)',

0 -4 100 0
Clozapine *****
*, * 4 -2 4 100-»0 0 * 100
2 4 -2 9 0 100

Injection 20 jiL.
with clozapine for peak identification CRS and the
C18H19a N 4 326.8 5786-21-0 identify the peaks due to impurities A, B, C and D.
Relative retention With reference to clozapine (retention
Action and use
Dopamine D4 receptor antagonist; neuroleptic. time = about 11 min): impurity C = about 0.9;
impurity D = about 1.1; impurity A = about 1.6;
Preparation impurity B = about 1.7.
Clozapine Oral Suspension System suitability: reference solution (b):
PhEur__________________________________________________________ — resolution: minimum 2.5 between the peaks due to
impurity C and clozapine;
DEFINITION — the chromatogram obtained with reference solution (b) is
8-Chloro-l 1-(4-methylpiperazin-1-yl)-5H- similar to the chromatogram supplied with clozapinefor
dibenzo[6,^] [l,4]diazepine. peak identification CRS.
Content Limits:
99.0 per cent to 101.0 per cent (dried substance). — correctionfactor, for the calculation of content, multiply the
CHARACTERS peak area of impurity D by 2.7;
Appearance — impurity A: not more than the area of the prindpal peak
Yellow, crystalline powder. in the chromatogram obtained with reference solution (a)
(0.1 per cent);
2016 Cocaine 1-611
nh2
— Z>. for each impurity, not more than twice
impurities B,
obtained with reference solution (a) (0.2 per cent); nh o
— impurity C: not more than 3 times the area of the
reference solution (a) (0.3 per cent); 6 ^ 0 . CH,
area of the principal peak in the chromatogram obtained D . 1- [2- [(2-amino-4-chlorophenyl) amino]benzoyI] -4-
with reference solution (a) (0.10 per cent); methylpiperazine.
— total: not more than 6 times the area of the principal peak PhEur
(0.6 per cent);
(0.05 per cent). Cocaine
Maximum 20 ppm.
an oven at 105 °C.
ASSAY
Dissolve 0.100 g in 50 mL of anhydrous acetic acid R. Titrate
with 0.1 M perchloric arid, determining the end-point
potentiometrically (2.2.20). C17H2iN04 303.4 50-36-2
of CigHjgCIN^ Action and use
Local anaesthetic.
IMPURITIES
Specified impurities A, B, C, D. DEFINITION
Cocaine is methyl (1R,2R,3S,5S)-3-(benzoyloxy)-8-methyl-8-
azabicyclo[3.2.1]octane-2-carboxylate and may be obtained
from the leaves of Erythroxyhm coca Lam. and other species
of Erythroxyhim or by synthesis. It contains not less than
98.0% and not more than 101.0% of C 17H 2iN 0 4 , calculated
with reference to the dried substance.
CHARACTERISTICS
A. 8-chloro-5,l0-dihydro-1lH-dibenzo[V] [l,4]diazepin-l 1- Colourless crystals or a white, crystalline powder. Slightly
one, volatile.
Practically insoluble in water, freely soluble in ethanol (96%)
and in ether, soluble in arachis oil; slightly soluble in liquid
paraffin.
IDENTIFICATION
The infrared absorption spectrum, Appendix II A, is concordant
with the reference spectrum of cocaine (RS 071).
TESTS
Melting point
Specific optical rotation
B. 11,11 '-(piperazine-1,4-diyl)bis(8-chloro-5//- In a 2.4 % w/v solution in 0.1m hydrochloric add, —79 to
dibenzo[t,e] [l,4]diazepine), -8 1 , calculated with reference to the dried substance,
Appendix V F.
a NH Related substances
Carry out the method for liquid chromatography,
Appendix HI D, using the following solutions.
(1) 0.05% w/v of the substance being examined in the mobile
phase
(2) Dilute 1 volume of solution (1) to 50 volumes with the
C. 8-chloro-l l-(piperazin-l-yl)-5H- mobile phase, dilute 5.0 mL of this solution to 100.0 mL
dibenzo [b,e] [1,4] diazepine, with the mobile phase.
1-612 Cocaine Hydrochloride 2016
(3) Dissolve 25 mg of the substance being examined in H H

0.01m sodium hydroxide and dilute to 100.0 mL with the same Ar*
solvent. Allow the solution to stand for 15 minutes.
C H R O M A T O G R A P H IC C O N D IT IO N S R V i ■ Ar
II H H
(a) Use a stainless steel column (15 cm x 4.6 mm) packed O
with base-deactivated octadecylsUyl silica gelfor chromatography
(5 pm) (Waters Symmetry is suitable). B. bis[(li?,2i?,35,55)-2-(methoxycarbonyl)-8-methyl-8-
azabicydo[3.2.l]oct-3-yI] (lr,2c,3r,4t)-2,4-
diphenylcyclobutane-1,3-dicarboxylate (a-truxilline),
below.
(c) Use a flow rate of 1 mL per minute.
(d) Use a column temperature of 35°. Ar- C A
(f) Inject 20 |jL of each solution.
at' T 'T V '
MOBILE PHASE H H II
1 volume of triethylamine, 200 volumes of tetrahydrofuran,
860 volumes of acetomtrUe and 959 volumes of water. C. bis[(l-R,2R,3S,5S)-2-(methoxycarbonyl)-8-methyl-8-
SY STEM S U IT A B IL IT Y azabicyclo[3.2.1]oct-3-yl] (lr,2c,3r,4t)-3,4-
The test is not valid unless, in the chromatogram obtained diphenylcyclobutane-1,2-dicarboxylate (ß-truxilline).
with solution (3), the resolutionfactor between the peaks due
to cocaine (retention time, about 7 minutes) and the
degradation product is at least 5.0.
★ ★
L IM IT S Cocaine Hydrochloride ★ ★
In the chromatogram obtained with solution (1): *****
the area of any peak eluting after the principal peak is not
greater than the area of the peak in the chromatogram
obtained with solution (2) (0.1%);
the sum of the areas of any secondary peaks is not greater than HCI
5 times the area of the principal peak in the chromatogram
obtained with solution (2) (0.5%).
Disregard any peak with an area less than 0. 5 times the area
of the principal peak in the chromatogram obtained with C 17H22C1N04 339.8 53-21-4
solution (2) (0.05 %).
Loss on drying Action and use
When dried to constant weight at 80°, loses not more than Local anaesthetic.
0.5% of its weight, Appendix IX D. Use 1 g. Preparations
Sulfated ash Adrenaline and Cocaine Intranasal Solution
Not more than 0.1%, Appendix IX A. Cocaine Eye Drops
ASSAY Cocaine Paste
Carry out Method I for non-aqueoits titration,
PhEur_________________________________________
Appendix VUI A, using 0.7 g dissolved in 50 mL of
1,4-dioxan and crystal violet solution as indicator. Each mL of DEFINITION
0.1m perchloric acid FiS is equivalent to 30.34 mg of Methyl (li?,2i?J35,J55)-3-(benzoyloxy)-8-methyl-8-
C 17H 21NO 4. azabicyclo[3.2. l]octane-2-carboxylate hydrochloride.
STORAGE Content
Cocaine should be stored protected from light. 98.5 per cent to 101.0 per cent (dried substance).
IMPURITIES CHARACTERS
Appearance
crystals.
Ar> Solubility
Very soluble in water, freely soluble in alcohol, slightly
soluble in methylene chloride.
mp
IDENTIFICATION
A. methyl (lJ?,2i?,35,55)-8-methyl-3-[[(£)-3-
A. Dissolve 20.0 mg in 0.01 M hydrochloric add and dilute to
phenylpropenoyl]oxy]-8-azabicyclo[3.2.1]octane-2-
100.0 mL with the same add. Dilute 5.0 mL of the solution
carboxylate (dnnamoylcocaine),
to 50.0 mL with 0.01 M hydrochloric add. Examined between
2016 Cocaine Hydrochloride 1-613
220 nm and 350 nm (2.2.25), the solution shows Limits:

2 absorption maxima, at 233 nm and 273 nm. The specific — any impurity eluting after the principal peak:not more than
absorbance at 233 nm is 378 to 402. the area of the principal peak in the chromatogram
B. Infrared absorption spectrophotometry (2.2.24). obtained with reference solution (a) (0.1 per cent),
Comparison Ph. Eur. reference spectrum of cocaine hydrochloride.
C. Dissolve 0.1 g in 5 mL of water R and add 1 mL of dilute (0.5 per cent),
ammonia R2. A white precipitate is formed. Initiate — disregard limit. 0.5 times the area of the principal peak in
crystallisation by scratching the wall of the tube with a glass the chromatogram obtained with reference solution (a)
rod. The crystals, washed with water R and dried th vacuo, (0.05 per cent).
melt (2.2.14) at 96 °C to 99 °C.
E. It gives the reaction of alkaloids (2.3.1). an oven at 105 °C.
TESTS Sulfated ash (2.4.14)
Solution S Maximum 0.1 per cent, determined on the residue from the
Dissolve 0.5 g in water R and dilute to 25 mL with the same test for loss on drying.
solvent.
ASSAY
Appearance of solution Dissolve 0.250 g in a mixture of 5.0 mL of 0.01 M
Solution S is clear (2.2.1) and colourless (2.2.2, Method II). hydrochloric acid and 50 mL of alcohol R Carry out a
Acidity potentiometric titration (2.2.20), using 0.1 M sodium
To 10 mL of solution S add 0.05 mL of methyl red solution R. hydroxide. Read the volume added between the 2 points of
Not more than 0.2 mL of 0.02 Msodium hydroxide is inflexion.
required to change the colour of the indicator. 1 mL of 0.1 M sodium hydroxide is equivalent to 33.98 mg of
Specific optical rotation (2.2.7) C x7H 22CINO4.
—70 to —73 (dried substance). STORAGE
Dissolve 0.50 g in water R and dilute to 20.0 mL with the Protected from light.
same solvent.
IMPURITIES
To 0.2 g add 2 mL of sulfuric acid R. After 15 min, the
solution is not more intensely coloured than reference
solution BY5 (2.2.2, Method I).
Ar>
Related substances
Examine by liquid chromatography (2.2.29).
the mobile phase. O
Reference solution (a) Dilute 1.0 mL of die test solution to
Reference solution (b) Dissolve 25 mg of the substance to be A. methyl (li?,2i?,35,55)-8-methyl-3-[[(£)-3-
examined in 0.01 M sodium hydroxide and dilute to 10.0 mL phenylpropenoyl] oxy]-8-azabicyclo [3.2.1] octane-2 -
with the same solvent. Dilute 1.0 mL of the solution to carboxylate (dnnamoylcocaine),
10.0 ml. with 0.01 M sodium hydroxide. Allow the solution to
stand for 15 min. H H 11
Column:
— size: I = 0.15 m, 0 = 4.6 mm,
— stationary phase, end-capped octadecylsUyl silica gelfor R V n : At
Il H H
chromatography R (5 (im) with a specific surface area
of o
335 m2/g, a pore size of 10 nm and a carbon loading of
19.1 per cent, B. bis [( 1R,2R,3S,55)-2-(methoxycarbonyl)-8-methyl-8-
— temperature: 35 °C. azabicydo[3.2.1]oct-3-yI] (lr,2c,3r,4r)-2,4-
Mobile phase triethylamine R, tetrahydrofuran R, acetonitrile R, diphenylcyclobutane-l,3-dicarboxylate (a-truxilline),
water R (0.5:100:430:479.5 V/V/V/V).
Flow rate 1 mlVmin. H H
At*.
o
Injection 20 pL.
Relative retention With reference to cocaine (retention H h 11
rime = about 7.4 min): degradation product = about 0.7.
C. bis [(li?,2i?,35,55)-2-(methoxycarbonyl)-8-methyl-8-
— resolution: minimum of 5 between the peaks due to
azabicydo[3.2. l]oct-3-yI] (lr,2c,3r,4î)-3,4-
cocaine and to the degradation product
diphenylcyclobutane-1,2 -dicaiboxylate (ß-truxüline).
PhEur
1-614 Cochineal 2016
IDENTIFICATION
Cochineal A. Melting point (see Tests).
DEFINITION B. Composition of fatty acids (see Tests).
Cochineal is the dried female insect, Dactylopim coccus Costa,
containing eggs and larvae. TESTS
M elting point (2.2.14)
CHARACTERISTICS 23 °C to 26 °C.
Odour, characteristic.
Acid value (2.5.2)
Macroscopical Purplish black or purplish grey; about 3.5 to
Maximum 0.5, determined on 20.0 g.
5.5 mm long and 3 to 4.5 mm wide, plano-convex and
somewhat oval in outline; the convex dorsal surface is Peroxide value (2.5.5, Method A)
transversely wrinkled and shows about 11 segments; the flat Maximum 5.0.
or slightly concave ventral surface carries upon the anterior Unsaponifiable m atter (2.5.7)
part two seven-jointed straight antennae, three pairs of short Maximum 1.0 per cent, determined on 5.0 g.
legs, each terminating in a single claw, and a mouth from Alkaline im purities (2.419)
which projects the remains of a long filiform proboscis; these It complies with the test.
appendages are frequendy more or less broken. Easily
Composition of fatty acids (2.422, Method B)
reduced to powder, which is dark red or puce.
Refined coconut oil is melted under gende heating to a
Microscopical Scattered irregularly over the whole dermis are homogeneous liquid prior to sampling.
numerous solitary and grouped, short, tubular wax glands;
Reference solution Dissolve 15.0 mg of tricaproin CRS, 80.0 mg
within each insect are found numerous larvae, which are
of tristearin CRS, 0.150 g of tricaprin CRS, 0.200 g of
characterised by their proboscides appearing as two circular
tricaprylin CRS, 0.450 g of trimyristin CRS and 1.25 g of
coils.
trUaurin CR S in a mixture of 2 volumes of methylene
TESTS chloride R and 8 volumes of heptane R, then dilute to 50 mL
Colour value with the same mixture of solvents heating at 45-50 °C.
To 0.5 g in moderatelyfine powder add 60 mL of phosphate Transfer 2 mL of this mixture to a 10 mL centrifuge tube
buffer p H 8.0 and heat on a water bath for 30 minutes. Cool, with a screw cap and evaporate the solvent in a current of
add sufficient phosphate buffer p H 8.0 to produce 100 mL and nitrogen R. Dissolve with 1 mL of heptane R and 1 mL of
filter. Dilute 5 mL of the filtrate to 100 mL with phosphate dimethyl carbonate R and mix vigorously under gende heating
buffer p H 8.0. The absorbance of the resulting solution at the (50-60 °C). Add, while still warm, 1 mL of a 12 g/L solution
maximum at 530 nm is not less than 0.25, Appendix II B. of sodium R in anhydrous methanol R, prepared with the
Foreign m atter necessary precautions, and mix vigorously for about 5 min.
Complies with the test for foreign matter, Appendix XI D. Add 3 mL of distilled water R and mix vigorously for about
30 s. Centrifuge for 15 min at 1500 g. Inject 1 [iL of the
Water-insoluble m atter
organic phase.
When the insects are placed in water, no insoluble powder
separates. Calculate the percentage content of each fatty add using the
Ash
Not more than 7.0%, Appendix XIJ.
Axsc
M icrobial contam ination ^ x’s,c x 100 per cent m/m
1 g is free from Escherichia coli, 10 g is free from Salmonella,
Appendix XVI Bl.
A r r r Is
the corrected peak area of each fatly add in the test
solution:
Coconut Oil ★★* ★★ A x ,s ,c — A x ,a X R c

★ ★
(Refined Coconut OH, Ph Eur. monograph 1410) *****
Rc Is the relative correction factor:
8001-31-8
m x,r X A l tr
Action and use Rc =
A x,r X
Excipient
PhEur.
for the peaks due to caproic, caprylic, capric, lauric and
DEFINITION myristic add methyl esters.
Fatty oil obtained from the dried, solid part of the mass of tricaproin, tricaprylin, tricaprin, trilaurin or
endosperm of Cocos nucifera L., then refined. trimyristm in the reference solution, in milligrams;
mass of tristearin in the reference solution, in •
CHARACTERS
milligrams:
Appearance A ir = area of the peaks due to caproic, caprylic, capric,
White or almost white, unctuous mass. lauric and myristic add methyl esters in the
Solubility reference solution;
Practically insoluble in water, freely soluble in methylene area of the peak due to stearic add methyl ester in
chloride and in light petroleum (bp: 65-70 °C), very slighdy the reference solution;
soluble in ethanol (96 per cent). area of the peaks due to any specified or
Refractive index About 1.449, determined at 40 °C. unspecified fatty add methyl esters;
2016 Cocoyl Caprylocaprate 1-615
Rc = 1 for the peaks due to each of the remaining Iodine value (2.5.4, Method A)
specified fatty add methyl esters or any unspecified Maximum 1.0.
fatty add methyl ester. Saponification value (2.5.6)
Composition of thefatty-acidfraction of the oit 160 to 173.
— caproic acid (R& 0.11): maximum 1.5 per cent, Composition of fatty ad ds and fatty alcohols (2.4.22,
— capiyUc acid (R& 0.23): 5.0 per cent to 11.0 per cent, Method C)
— capric acid (Rri 0.56): 4.0 per cent to 9.0 per cent, Use the chromatogram obtained with the following reference
— lauric acid (R& 0.75): 40.0 per cent to 50.0 per cent, solution for identification of the peaks due to the fatty
— myrisdc acid (R& 0.85): 15.0 per cent to 20.0 per cent, alcohols.
— palmitic acid (Rri 0.93): 7.0 per cent to 12.0 per cent,
Reference solution Dissolve the amounts of the substances
— stearic acid (R& 1.00): 1.5 per cent to 5.0 per cent,
listed in the following table in 10 mL of heptane R.
— oleic acid (Rri 1.01): 4.0 per cent to 10.0 per cent,
— linoleic acid (R& 1.03): 1.0 per cent to 3.0 per cent,
— linolenic acid (R& 1.06): maximum 0.2 per cent, Substance Amount (mg)
— arachidic acid (R^ 1.10): maximum 0.2 per cent, Methyl caproate R 10
— eicosenotc add (R& 1.11): maximum 0.2 per cent
Methyl caprylate R 90
Water (2.5.32)
Maximum 0.1 per cent, determined on 1.00 g. Methyl decanoate R 50
Methyl laurate R 20
STORAGE
In a well-filled container, protected from light. Methyl myristate R 10
__________________________________________________________ PhEur Methyl palmitate R 10
Methyl stearate R 10
Decanol R 10
Lauryl alcohol R 100

Cocoyl Caprylocaprate *****
Myristyl alcohol R 40
*+
(Ph. Eur. monograph 1411) * Cetyl alcohol CRS 30
Action and use Stearyl alcohol CRS 20

Exdpient.
PhEur---------------------------------------------------------------------------------------- Consider the sum of the areas of the peaks due to the fatty
adds listed bdow to be equal to 100 and the stun of the
DEFINITION
areas of the peaks due to the fatty alcohols listed below to be
Mixture of esters of saturated C12 - C18 alcohols with
equal to 100.
capiylic (octanoic) and capric (decanoic) adds obtained by
Composition of thefatty acid fraction of the substance:
the reaction of these adds with vegetable saturated fatty
alcohols. — caproic add: maximum 2.0 per cent,
— capiylic add: 50.0 per cent to 80.0 per cent,
CHARACTERS — capric acid: 20.0 per cent to 50.0 per cent,
Appearance — lauric add: maximum 3.0 per cent,
Slightly yellowish liquid — myristic add: maximum 2.0 per cent.
Solubility Composition of thefatty alcoholfraction of the substance:
Practically insoluble in water, misdble with ethanol — capric alcohol: maximum 3.0 per cent,
(96 per cent) and with liquid paraffin. — lauryl alcohol: 48.0 per cent to 63.0 per cent,
Relative density — myristyl alcohol: 18.0 per cent to 27.0 per cent,
About 0.86. — cetyl alcohol: 6.0 per cent to 13.0 per cent,
— stearyl alcohol: 9.0 per cent to 16.0 per cent.
Refractive index About 1.445.
Viscosity About 11 mPa-s.
W ater (2.5.12)
IDENTIFICATION
Total ash (2.4.16)
A. Freezing point (2.2.18): maximum 15 °C. Maximum 0.1 per cent, determined on 1.0 g.
___________________________________ PhEur
Comparison cocoyl caprylocaprate CRS.
C. Composition of fatty adds and fatty alcohols (see Tests).
TESTS
Appearance
The substance to be examined is not more intensely coloured
than reference solution Y5 (2.2.2 Method I).
Add value (2.5.1)
Maximum 0.5, determined on 5.00 g.
Hydroxyl value (2.5.3, Method A)
Maximum 5.0, determined on 4.0 g. Add 5.0 mL of
acetylating reagent
1-616 Codeine 2016
Related substances
Codeine f * \ Liquid chromatography (2.2.29).
* ★
(Ph. Eur. monograph 0076) * Test solution Dissolve 0.100 g of the substance to be
examined and 0.100 g of sodium octanesulfonate R in the
Reference solution (a) Dissolve 5.0 mg of codeine
impurity A CRS in the mobile phase and dilute to 5.0 mL
C18H21N 03jH20 317.4 6059-47-8 50.0 mL with the mobile phase. Dilute 5.0 mL of this
solution to 100.0 mL with die mobile phase.
Action and use
Reference solution (¿0 To 0.25 mL of the test solution, add
Opioid receptor agonist; analgesic. 2.5 mL of reference solution (a).
PhEur__________________________________________________________ Column:
— size. I = 0.25 m, 0 = 4.6 mm;
DEFINITION
— stationary phase: end-capped octylsQyl silica gel for
7,8-Didehydro-4,5a-epoxy-3-methoxy-17-methylmorphinan- chromatography R (5 Jim).
6a-ol monohydrate.
Mobile phase Dissolve 1.08 g of sodium octanesulfonate R in a
Content mixture of 20 mL of glacial acetic acid R and 250 mL of
99.0 per cent to 101.0 per cent (dried substance). acetonitrile R and dilute to 1000 mL with water R
CHARACTERS Flow rate 2 mL/min.
Appearance Detection Spectrophotometer at 245 nm.
Injection 10 (iL.
crystals.
Run time 10 times the retention time of codeine.
Solubility
Relative retention With reference to codeine (retention
Soluble in boiling water, freely soluble in ethanol
(96 per cent).
impurity E = about 0.7; impurity A = about 2.0;
IDENTIFICATION impurity C = about 2.3; impurity D = about 3.6.
First identification A , C. System suitability Reference solution (d):
Second identification A , B, D, E — resolution: minimum 3 between the peaks due to codeine
A. Melting point (2.2.14): 155 °C to 159 °C. and impurity A.
B. Ultraviolet and visible absorption spectrophotometry Limits:
(2.2.25). — correction factor,for the calculation of content, multiply die
Test solution To 2.0 mL of solution S (see Tests) add 50 mL
— impurity A: not more than twice the area of the principal
of water R then 10 mL of 1 M sodium hydroxide and dilute to
100.0 mL with water R. peak in the chromatogram obtained with reference
— impurities B, C, D, E: for each impurity, not more than
Absorption maximum At 284 nm. twice the area of the principal peak in the chromatogram
Specific absorbance at the absorption maximum About 50 (dried obtained with reference solution (c) (0.2 per cent);
substance). — unspecified impurities: for each impurity, not more than the
C. Infrared absorption spectrophotometry (2.2.24). area of the principal peak in the chromatogram obtained
Preparation Dried substance prepared as a disc of potassium
— sum of impurities other than A: not more than 10 times the
bromide R.
Comparison codeine CRS.
D. To about 10 mg add 1 mL of sulfuric acid R and 0.05 mL — disregard limit. 0.5 times the area of the principal peak in
of ferric chloride solution R2 and heat on a water-bath. A blue the chromatogram obtained with reference solution (c)
colour develops. Add 0.05 mL of nitric acid R. The colour (0.05 per cent).
changes to red.
E. It gives the reaction of alkaloids (2.3.1). 4.0 per cent to 6.0 per cent, determined on 1.000 g by
TESTS drying in an oven at 105 °C.
Solution S Sulfated ash (2.414)
Dissolve 50 mg in carbon dioxide-free water R and dilute to Maximum 0.1 per cent, determined on 1.0 g.
ASSAY
A ppearance of solution Dissolve 0.250 g in 10 mL of anhydrous acetic acid R
Solution S is clear (2.2.1) and colourless (2.2.2, Method II). Add 20 mL of dioxan R Titrate with 0.1 M perchloric add,
Specific optical rotation (2.2.7) nsing 0.05 mL of crystal violet solution R as indicator.
-142 to -1 4 6 (dried substance). 1 ml. of 0.1 Mperchloric acid is equivalent to 29.94 mg
Dissolve 0.50 g in ethanol (96 per cent) R and dilute to of C18H21N 0 3.
2016 Codeine Hydrochloride 1-617
STORAGE
IMPURITIES
Specified impurities A,B, C, D, E
Other detectable impurities(die following substances would, if
the tests in the monograph. They are limited by the general E. 7,8-didehydro-4,5a-epoxy-3-methoxy-17-
acceptance criterion for other/unspecified impurities and/or methylmorphinan-6a, 10-diol,
Control of impurities in substancesfor pharmaceutical use): F, G.
F. 7,8-didehydro-4,5a-epoxy-3-methoxy-17-
methylmorphinan-óotj 14-diol,
A. 7,8-didehydro-4,5a-epoxy-3,6a-dimethoxy-17-
methylmorphinan (methylcodeine),
G. 6,7,8,14-tetradehydro-4,5a-epoxy-3,6-dimedioxy-17-
methylmorphinan (thebaine).
PhEur
B. 7,8-didehydro-4,5a-epoxy-17-methylmorphinan-3,6a-diol
(morphine),
***
Codeine Hydrochloride * ★
★ ★
(Codeine Hydrochloride Dihydrate, *****
, HCI , 2H20
C. 7,7',8,8/-tetradehydro-4,5a;4,,5 'a-diepoxy-3,3
dimethoxy-17,17 '-dimethyl-2,2 '-bimorphinanyl-6a, 6 'a-diol
(codeine dimer),
C18H22C1N03,2H20 371.9
Action and use

Opioid receptor agonist; analgesic.
PhEir.
DEFINITION
7,8-Didehydro-4,5a-epoxy-3-methoxy-17-methylmorphinan-
6a-ol hydrochloride dihydrate.
Content
D. 7,8-didehydro-2-[(7,8-didehydro-4,5a-epoxy-6a-hydroxy- 99.0 per cent to 101.0 per cent (anhydrous substance).
17-methylmorphinan-3-yl)oxy]-4,5a-epoxy-3-methoxy-17-
methylmorphinan-6a-ol (3-0-(codein-2-yl)morphine), CHARACTERS
Appearance
White or almost white, crystalline powder or small, colourless
crystals.
Solubility
Soluble in water, slightly soluble in ethanol (96 per cent),
practically insoluble in cyclohexane.
1-618 Codeine Hydrochloride 2016
IDENTIFICATION impurity E = about 0.7; impurity A = about 2.0;

First identification A, D. impurity C = about 2.3; impurity D = about 3.6.
Second identification B, C, D, E. System suitability Reference solution (d):
— resolution: minimum 3 between the peaks due to codeine
and impurity A.
Comparison Ph. Eur. reference spectrum of codeine hydrochloride
Limits:
dihydrate.
— for the calculation of content, multiply the
correction factor,
B. To 5 mL of solution S (see Tests) add 1 mL of a mixture peak area of impurity C by 0.25;
of equal volumes of strong sodium hydroxide solution R and — impurity A: not more than twice the area of the principal
water R and initiate crystallisation, if necessary, by scratching peak in the chromatogram obtained with reference
the wall of the tube with a glass rod and cooling in iced solution (b) (1.0 per cent);
water. Wash the precipitate with water R and dry at — impurities B, C, D, E: for each impurity, not more than
100-105 °C. It melts (2.2.15) at 155 °C to 159 °C. twice the area of the principal peak in the chromatogram
C. To about 10 mg add 1 mL of sulfuric acid R and 0.05 mL obtained with reference solution (c) (0.2 per cent);
of ferric chloride solution R2 and heat on a water-bath. A blue — unspecified impurities: for each impurity, not more than the
colour develops. Add 0.05 mL of nitric add R. The colour area of the principal peak in the chromatogram obtained
changes to red. with reference solution (c) (0.10 per cent);
D. Solution S gives reaction (a) of chlorides (2.3.1). — sum of impurities other than A: not more than 10 times the
E. It gives the reaction of alkaloids (2.3. T). area of the principal peak in the chromatogram obtained
TESTS — disregard limit. 0.5 times the area of the principal peak in
Solution S the chromatogram obtained with reference solution (c)
Dissolve 2.00 g in carbon dioxide-free water R prepared from (0.05 per cent).
distilled water R and dilute to 50.0 mL with the same solvent.
Sulfates (2.413)
Appearance of solution Maximum 0.1 per cent.
Dilute 5 mL of solution S to 20 mL with distilled water R.
W ater (2.5.12)
To 5 mL of solution S add 5 mL of carbon dioxide-free
water R. Add 0.05 mL of methyl red solution R and 0.2 mL of ASSAY
0.02 M hydrochloric acid; the solution is red. Add 0.4 mL of Dissolve 0.300 g in a mixture of 5 mL of 0.01 M hydrochloric
0.02 M sodium hydroxide", the solution becomes yellow. acid and 30 mL of ethanol (96 per cent) R. Carry out a
Specific optical rotation (2.2.7) potentiometric titration (2.2.20), using 0.1 M sodium
-117 t o -121 (anhydrous substance).
inflexion.
Dilute 5.0 mL of solution S to 10.0 mL with water R.
1 mL of 0.1 M sodium hydroxide is equivalent to 33.59 mg
Related substances of C 18H 22CINO3.
STORAGE
examined and 0.100 g of sodium octanesulfonate R in the Protected from light.
mobile phase and dilute to 10.0 mL with the mobile phase. IMPURITIES
Reference solution (a) Dissolve 5.0 mg of codeine Spedfied impurities A, B, C, D, E.
impurity A CRS in the mobile phase and dilute to 5.0 mL Other detectable impurities(the following substances would, if
with the mobile phase. present at a sufficient level, be detected by one or other of
Reference sdution (b) Dilute 1.0 mL of reference solution (a) the tests in the monograph. They are limited by the general
to 20.0 mL with the mobile phase. acceptance criterion for other/unspecified impurities and/or
Reference solution (c) Dilute 1.0 mL of the test solution to by the general monograph Substances for pharmaceutical use
50.0 mL with the mobile phase. Dilute 5.0 mL of this (2034). It is therefore not necessary to identify these
solution to 100.0 mL with the mobile phase. impurities for demonstration of compliance. See also 5.10.
Contrd of impurities in substancesfor pharmaceutical use): F, G.
Reference solution (d) To 0.25 mL of the test solution add
2.5 mL of reference solution (a).
Column:
— sizer. I = 0.25 m, 0 = 4.6 mm;
— stationary phase: end-capped octylsüyl silica gelfor
mixture of 20 mL of glacial acetic add R and 250 mL of
acetomtrüe R and dilute to 1000 mL with water R.
Flow rate 2 mUmin. A. 7,8-didehydro-4,5a-epoxy-3,6a-dimethoxy-17-
Detection Spectrophotometer at 245 nm. methylmorphinan (methylcodeine),
Injection 10 }iL.
2016 Codeine Phosphate 1-619
Codeine Phosphate ★ ★
★ ★
(Codeine Phosphate Hemihydrate, *****
B. 7,8-didehydro-4,5a-epoxy-l 7-methylmorphinan-3,6a-diol
(morphine).
C 18H 2 4N07 P,1/2H20 406.4 41444-62-6
Action and use

Preparations
C. 7,7/,8,8/-tetradehydro-4,5a:4/,5/a-diepoxy-3,3/- Co-codamol Capsules
dimethoxy-17,17 '-dimethyl-2,2'-bimorphinanyl-6a,6 'a-diol Co-codamol Tablets
(codeine dimer), Effervescent Co-codamol Tablets
Co-codaprin Tablets
Dispersible Co-codaprin Tablets
Codeine Linctus
Paediatric Codeine Linctus
Codeine Phosphate Injection
Codeine Phosphate Oral Solution
Codeine Phosphate Tablets
Paracetamol, Codeine Phosphate and Caffeine Capsules
D. 7,8-didehydro-2-[(7,8-didehydro-4,5a-epoxy-6a-hydroxy- Paracetamol, Codeine Phosphate and Caffeine Tablets
17-methyimorphinan-3-yI)oxy]-4,5a-epoxy-3-methoxy-17-
PhEir __________________________________________________________
methyimorphinan-6a-ol (3-0-(codein-2-yl)morphine),
DEFINITION
7,8-Didehydro-4,5a-epoxy-3-methoxy-17-methyimorphinan-
6a-ol phosphate hemihydrate.
Content
CHARACTERS
Appearance
E. 7,8-didehydro-4,5a-epoxy-3-methoxy-17- crystals.
methylmorphinan-6a, 10-diol,
Solubility
Freely soluble in water, slightly soluble or very slightly
IDENTIFICATION
First identification B, E , F.
Second identification A , C, D, E, F , G
(2.2.25).
F. 7,8-didehydro-4,5a-epoxy-3-methoxy-17- Test solution Dilute 1.0 mL of solution S (see Tests) to
methyImorphinan-6a, 14-diol, 100.0 mL with water R. To 25.0 mL of this solution add
25 mL of water R then 10 mL of 1 M sodium hydroxide and
Specific absorbance at the absorption maximum About 38 (dried
substance).
G. 6,7,8,14-tetradehydro-4,5a-epoxy-3,6-dimethoxy-17- Preparation Dissolve 0.20 g in 4 mL of water R. Add 1 mL of
methylmorphinan (thebaine). a mixture of equal volumes of strong sodium hydroxide
Ph Eur solution R and water R and initiate crystallisation, if necessary,
1-620 Codeine Phosphate 2016
by scratching the wall of the tube with a glass rod and Limits:
cooling in iced water. Wash the precipitate with water R and — correction factor, for the calculation of content, multiply the
dry at 100-105 °C. Examine the dried precipitate prepared as peak area of impurity C by 0.25;
discs using potassium bromide R. — impurity A: not more than twice the area of the principal
Comparison Ph. Eur. reference spectrum of codeine. peak in the chromatogram obtained with reference
C. Dissolve 0.20 g in 4 mL of water R. Add 1 mL of a solution (b) (1.0 per cent);
mixture of equal volumes of strong sodium hydroxide solution R — sum of impurities B and E: not more than 4 times the area
and water R and initiate crystallisation, if necessary, by of the principal peak in the chromatogram obtained with
scratching the wall of the tube with a glass rod and cooling in reference solution (c) (0.4 per cent);
iced water. The precipitate, washed with water R and dried at — impurities C, D: for each impurity, not more than twice
100-105 °C, melts (2.2.14) at 155 °C to 159 °C. the area of the principal peak in the chromatogram
D. To about 10 mg add 1 mL of sulfuric acid R and — unspecified impurities: for each impurity, not more than the
0.05 mL of ferric chloride solution R2 and heat on a water- area of the principal peak in the chromatogram obtained
bath. A blue colour develops. Add 0.05 mL of nitric acid R. with reference solution (c) (0.10 per cent);
The colour changes to red. — sum of impurities other than A: not more than 10 times the
E. Loss on drying (see Tests). area of the principal peak in the chromatogram obtained
F. Solution S gives reaction (a) of phosphates (2.3.1). with reference solution (c) (1.0 per cent);
G. It gives the reaction of alkaloids (2.3.1). — disregard limit. 0.5 times the area of the principal peak in
TESTS (0.05 per cent).
Solution S
Dissolve 1.00 g in carbon dioxide-free water R prepared from Sulfates (2.413)
distilled water R and dilute to 25.0 ml. with the same solvent.
Maximum 0.1 per cent
Dilute 5 mL of solution S to 20 mL with distilled water R.
pH (2.2.3)
4.0 to 5.0 for solution S. Loss on drying (2.2.32)
—98 to -102 (dried substance).
Dilute 5.0 mL of solution S to 10.0 mL with water R. ASSAY
Dissolve 0.350 g in a mixture of 10 mL of anhydrous acetic
Related substances
add R and 20 mL of dioxan R. Titrate with 0.1 M perchloric
acid using 0.05 mL of crystal violet solution R as indicator.
of C 18H 24NO 7P.
Reference solution (a) Dissolve 5.0 mg of codeine STORAGE
impurity A CRS in the mobile phase and dilute to 5.0 mL Protected from light.
with the mobile phase. IMPURITIES
Reference solution (b) Dilute 1.0 mL of reference solution (a) Spedfied impurities A, B, C, D, E
to 20.0 mL with the mobile phase. Other detectable impurities (the following substances would, if
Reference solution (c) Dilute 1.0 mL of the test solution to present at a sufficient level, be detected by one or other of
50.0 mL with the mobile phase. Dilute 5.0 mL of this the tests in the monograph. They are limited by the general
solution to 100.0 mL with the mobile phase. acceptance criterion for other/unspecified impurities and/or
Reference solution (d) To 0.25 mL of the test solution add by the general monograph Substances for pharmaceutical use
2.5 mL of reference solution (a). (2034). It is therefore not necessary to identify these
Column: impurities for demonstration of compliance. See also 5.10.
— size. I = 0.25 m, 0 = 4.6 mm; Control of impurities in substancesfor pharmaceutical use): F, G.
— stationary phase: end-capped octylsUyl silica gelfor
chromatography R (5 jam).
acetomtrile R and dilute to 1000 mL with water R.
Flow rate 2 ml 7min,
Injection 10 |iL.
A. 7,8-didehydro-4,5a-epoxy-3,6a-dimethoxy-17-
time = about 6 min): impurities B and E = about 0.7;
impurity A = about 2.0; impurity C = about 2.3;
and impurity A.
2016 Codeine Phosphate Sesquihydrate 1-621
Codeine Phosphate Sesquihydrate ★* * * ★

★ ★
(Ph. Em. monograph 0075) *****
B. 7,8-didehydro-4,5a-epoxy-17-methylmorphinan-3,6a-diol
(morphine).
C18H24N 07P,11/2H20 424.4 5913-76-8
Action and use

Preparations
Codeine Linctus
Paediatric Codeine Linctus
C. 7,7 '38j 8'-tetxadehydro-4j5a:4'j5 'a-diepoxy-3,3
dimethoxy-17,17 '-dimethyl-2,2 '-bimorphinanyl-6a,6 'a-diol Codeine Phosphate Oral Solution
(codeine dimer), Codeine Phosphate Tablets
PhEtr.
DEFINITION
7,8-Didehydro-4,5cc-epoxy-3-methoxy-17-methylmorphinan-
6a-ol phosphate sesquihydrate.
Content
CHARACTERS
Appearance
D . 7j8-didehydro-2- [(7,8-didehydro-4,5a-epoxy-6a-hydroxy-
17-methyhnorphinan-3-yl)oxy]-4,5a-epoxy-3-methoxy-l 7- crystals.
methylmorphinan-6a-ol (3-0-(codein-2-yl)morphine), Solubility
(96 per cent).
IDENTIFICATION
First identification B, E, F.
Second identification A ,
C, D, E, F, G
(2.2.25).
Test solution Dilute 1.0 mL of solution S (see Tests) to
E. 7,8-didehydro-4,5a-epoxy-3-methoxy-17-
100.0 mL with water R. To 25.0 mL of this solution add
methylmorphinan-ôa, 10-diol,
25 mL of water R then 10 ml. of 1 M sodium hydroxide and
Specific absorbance at the absorption maximum About 38 (dried
substance).
Preparation Dissolve 0.20 g in 4 m l. of water R. Add 1 mL of
F. 7,8-didehydro-4,5a-epoxy-3-methoxy-17- a mixture of equal volumes of strong sodium hydroxide
methylmorphinan-6a, 14-diol, solution R and water R and initiate crystallisation, if necessary,
by scratching the wall of the tube with a glass rod and
cooling in iced water. Wash the precipitate with water R and
dry at 100-105 °C. Examine the dried precipitate prepared as
discs using potassium bromide R.
Comparison Ph. Em. reference spectrum of codeine.
C. Dissolve 0.20 g in 4 mL of water R. Add 1 mL of a
mixture of equal volumes of strong sodium hydroxide solution R
and water R and initiate crystallisation, if necessary, by
G. 6,7,8,14-tetradehydro-4,5a-epoxy-3,6-dimethoxy-l 7- scratching the wall of the tube with a glass rod and cooling in
methylmorphinan (thebaine). iced water. The precipitate, washed with water R and dried at
PhEur 100-105 °C, melts (2.2.14) at 155 °C to 159 °C.
1-622 Codeine Phosphate Sesquihydrate 2016
D. To about 10 mg add 1 mT. of sulfuric add R and 0.05 mL — sum of impurities other than A:not more than 10 times the
of ferric chloride solution R2 and heat on a water-bath. A blue area of the principal peak in the chromatogram obtained
colour develops. Add 0.05 mL of nitric add R. The colour with reference solution (c) (1.0 per cent);
changes to red. — disregard limit: 0.5 times the area of the principal peak in
E. Loss on drying (see Tests). the chromatogram obtained with reference solution (c)
(0.05 per cent).
F. Solution S gives reaction (a) of phosphates (2.3.1).
G. It gives the reaction of alkaloids (2.3.1). Sulfates (2.4.13)
TESTS Dilute 5 mL of solution S to 20 mL with distilled water R.
Solution S
Dissolve 1.00 g in carbon dioxide-free water R prepared from Loss on drying (2.2.32)
distilled water R and dilute to 25.0 mL with the same solvent.
5.0per cent to 7.5 per cent, determined on 0.500 g by
pH (2.2.3)
4.0 to 5.0 for solution S. ASSAY
Dissolve 0.350 g in a mixture of 10 mL of anhydrous acetic
acid R and 20 mL of dioxan R. Titrate with 0.1 M perchloric
-9 8 to —102 (dried substance).
acid using 0.05 mL of crystal violet solution R as indicator.
Dilute 5.0 mL of solution S to 10.0 mL with water R.
Related substances of c 18h 24n o 7p .
STORAGE
mobile phase and dilute to 10.0 mL with die mobile phase. IMPURITIES
Reference solution (a) Dissolve 5.0 mg of codeine Speafied impurities A, B, C, D, E
impurity A CRS in the mobile phase and dilute to 5.0 mL Other detectable impurities (the following substances would, if
Reference solution (b) Dilute 1.0 mL of reference solution (a) the tests in the monograph. They are limited by the general
to 20.0 mL with the mobile phase. acceptance criterion for other/unspecified impurities and/or
50.0 mL with the mobile phase. Dilute 5.0 mL of this (2034). It is therefore not necessary to identify these
solution to 100.0 mL with the mobile phase. impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use): F, G.
Reference solution (d) To 0.25 mL of the test solution add
2.5 mL of reference solution (a).
Column:
— size. I = 0.25 m, 0 = 4.6 mm;
— stationary phase', end-capped octylsHyl silica gd for
chromatography R (5 jam).
acetomtrUe R and dilute to 1000 mL with water R
Flow rate 2 mL/min. A. 7,8-didehydro-4,5a-epoxy-3,6a-dimethoxy-17-
Injection 10 jiL.
impurity E = about 0.7; impurity A = about 2.0;
impurity C = about 2.3; impurity D = about 3.6.
B. 7,8-didehydro-4,5a-epoxy-17-methylmorphinan-3,6ac-diol
and impurity A.
(morphine),
Limits:
— correction factor, for the calculation of content, multiply die
— impurity A: not more than twice die area of the principal
— impurities B, C, D, E: for each impurity, not more than
obtained with reference solution (c) (0.2 per cent); h 3c ch3
area of the principal peak in the chromatogram obtained C. 7,7 ',8,8 '-tetradehydro-^Sa: 4 ',5 'a-diepoxy-3,3 '-
with reference solution (c) (0.10 per cent); dimethoxy-17,17 '-dimethyl-2,2/-bimorphinanyl-6a,6/a-diol
(codeine dimer),
2016 Codergocrine Mesilate 1-623
Codergocrine Mesilate ★ ★
★ ★
(Ph. Etar. monograph 2060) *****
D . 7,8-<iidehydro-2- [(7,8-didehydro-4,5a-epoxy-6a-hydroxy-
17-methylmorphinan-3-yi) oxy]-4,5a-epoxy-3-methoxy-17-
methylmorphinan-6a-ol (3-0-(codein-2-yl)morphine)J
H3C ^ C H 3
dihydroergocomine
C32 H4 5 N5 O 3 S 660
mesilate
h 3c o
dihydroergocristine
mesilate
C38 H45 N5 O 8 S 708
p ch3
E. 7,8-didehydro-4,5a-epoxy-3-methoxy-17- a-dihydroergocryptine C3 3 H4 7 N5 O3 S 674 | ^ ch 3

mesilate
methyimoiphinan-6a310-diol,
ch3
h 3c I
P-dihydroergocryptine C3 3 H4 7 N5 O8 S 674
mesilate
8067-24-1
Action and use

Vasodilator.
F. 7,8-didehydro-4,5a-epoxy-3-methoxy-17- PhEur.
methylmorphinan-ôa, 14-diol, DEFINITION
A mixture of;
— (6ai?j9jR, 10aR)-N- [(2R,5S, 1OaS, 10b5)-l Ob-hydroxy-2,5-
bis (1-methylethyl)-3,6-dioxooctahydro-8/i-oxazolo [3,2-
a] pyrrolo [2,1-c]pyrazin-2-yl] -7-methyl-
4,6,6a,7,8,9,10,1 Oa-octahydroindolo [4,3-J^] quinoline-9-
carboxamide methanesulfonate (dihydroergocomine
mesilate);
— (6aÆ, 9R, 1QaR)-N- [(2i?,55,1OaS, 10bS)-5-benzyl-10b-
G. 6,7,8j 14-tetradehydro-4,5a-epoxy-3,6-dimethoxy-17- hydroxy-2-(l -methylethyl)-3, 6-dioxooctahydro-8/i-
methyimorphinan (thebaine). oxazolo [3,2-a] pyrrolo [2,1-c]pyrazin-2-yl]-7-methyl-
4,6,6a,7,8,9,10,1 Oa-octahydroindolo [4,3-^] quinoline-9-
PhEir
carboxamide methanesulfonate (dihydroergocristine
mesilate);
— (6aR,9R, 10aR)-N~[(2R,5S, 1OaS, 10b.S)-10b-hydroxy-2-
(l-methylethyl)-5-(2-methyipropyl)-3,6-dioxooctahydro-
8/i-oxazolo [3,2-a] pyrrolo [2,1-c]pyrazin-2-yI] -7-methyi-
4,6,6a,7,8,9,10,1 Oa-octahydroindolo [4,3-j£] quinoline-9-
carboxamide methanesulfonate (a-dihydroergocryptine
mesilate);
— (6ai?,9i?, 10aR)-N- [(2iî,55,1OaS, 10bS)-l 0b-hydroxy-2-
(l-methylethyl)-5-[(lJ2S)-l-methylpropyl]-3,6-
dioxooctahydro-8/i-oxazolo [3,2-a] pyrrolo [2,1-c]pyrazin-2-
yI]-7-methyl-4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-
fg]quinoline-9-carboxamide methanesulfonate (P-
dihydroergocryptine mesilate or epicriptine mesilate).
Content
PRODUCTION
It is considered that alkylsulfonate esters are genotoxic and
are potential impurities in codergocrine mesilate.
The manufacturing process should be developed taking into
consideration the principles of quality risk management,
1-624 Codergocrine Mesilate 2016
together with considerations of the quality of starting — stationary phase: octadecylsUyl silica gd for chromatography R
materials, process capability and validation. The general (5 pm).
methods 2.5.37. Methyl, ethyl and isopropyl methanesidfonate in Mobile phase triethylamine R, acetonitrile R, water R
methanesulfonic acid, 2.5.38. Methyl, ethyl and isopropyl (2.5:25:75 V/VfV).
methanesulfonate in active substances and
Flow rate 1.5 mTVmin.
2.5.39. Methanesulfonyl chloride in methanesulfonic add are
available to assist manufacturers.
Irgection 20 pL.
CHARACTERS
Run time 20 min.
Appearance
White or yellowish powder. Elution order Dihydroergocomine, a-dihydroergocryptine,
dihydroergocristine, J3-dihydroergocryptine.
Solubility
System suitability Test solution:
Sparingly soluble in water, sparingly soluble to soluble in
— resolution: minimum 3 between any 2 consecutive
ethanol (96 per cent), slightly soluble in methylene chloride.
principal peaks.
IDENTIFICATION Composition:
A. Thin-layer chromatography (2.2.27). — dihydroergocomine: 30.0 per cent to 35.0 per cent;
Test solution Dissolve 0.20 g of the substance to be examined — a-dihydroergocryptine: 20.0 per cent to 25.0 per cent;
in a mixture of 1 volume of methanol R and 9 volumes of — dihydroergocristine: 30.0 per cent to 35.0 per cent;
methylene chloride R and dilute to 5 mL with the same — ¡i-dihydroergocryptine. 10.0 per cent to 13.0 per cent;
mixture of solvents. — disregard limit: 1.0 per cent.
Reference solution Dissolve 0.20 g of methanesulfonic acid R in a Related substances
mixture of 1 volume of methanol R and 9 volumes of Thin-layer chromatography (2.2.27). Perform the test as rapidly
methylene chloride R and dilute to 5 mL with the same as possible and protectedfrom direct light Prepare the test solution
mixture of solvents. last and immediately before application on the plate.
Plate TLC silica gd plate R. Test solution Dissolve 0.40 g of the substance to be examined
Mobile phase water R, concentrated ammonia R, butanol R, in a mixture of 1 volume of methanol R and 9 volumes of
acetone R (5:10:20:65 VIVIVIV). methylene chloride R and dilute to 5.0 mL with the same
Application 10 |iL. mixture of solvents.
Development Over 2/3 of the plate. Reference solution (a) Dissolve 40 mg of dihydroergocristine
mesilate CRS in a mixture of 1 volume of methanol R and
Drying In a current of cold air for not more than 1 min.
9 volumes of methylene chloride R and dilute to 10.0 mL with
Detection Spray with a 1 g/L solution of bromocresol purple R the same mixture of solvents. Dilute 3.0 mL of the solution
in methanol R, adjusted to a violet-red colour with 0.05 mL to 50.0 mL with a mixture of 1 volume of methanol R and
of dilute ammonia R l. 9 volumes of methylene chloride R.
Drying In a current of hot air at 100 °C. Reference solution (b) To 2.0 mL of reference solution (a),
Results The principal spot in the chromatogram obtained with add 1.0 mL of a mixture of 1 volume of methanol R and
the test solution is similar in position and colour to the 9 volumes of methylene chloride R.
principal spot in the chromatogram obtained with the Reference solution (c) To 1.0 mL of reference solution (a), add
reference solution. 2.0 mL of a mixture of 1 volume of methanol R and
B. Examine the chromatograms obtained in the test for 9 volumes of methylene chloride R.
composition. Reference solution (d) To 1.0 mL of reference solution (a),
Results The 4 principal peaks in the chromatogram obtained add 5.0 mL of a mixture of 1 volume of methanol R and
with the test solution are similar in retention time to the 9 volumes of methylene chloride R.
4 principal peaks in the chromatogram obtained with the Plate T LC silica gel plate R.
reference solution.
TESTS acetate R, methylene chloride R (1:3:50:50 VIVIVIV).
pH (2.2.3) Application 10 |iL.
4.2 to 5.2.
Drying In the dark for 2 min after the application of the last
Dissolve 0.10 g in carbon dioxide-free water R and dilute to solution.
First development In an unsaturated tank, over 2/3 of the
Composition plate.
Liquid chromatography (2.2.29): use the normalisation Drying In a current of cold air for not more than 1 min.
procedure.
Second development In an unsaturated tank, over 2/3 of the
plate; use freshly prepared mobile phase.
in a mixture of 1 volume of anhydrous ethanol R and
Drying In a current of cold air for not more than 1 min.
2 volumes of a 10 g/L solution of tartaric add R and dilute to
10 mL with the same mixture of solvents. Detection Spray thoroughly with dimethylaminobenzaldehyde
solution R7 and dry in a current of hot air until die spot in
Reference solution Dissolve 20 mg of codergocrine mesilate CRS
the chromatogram obtained with reference solution (d) is
in a mixture of 1 volume of anhydrous ethanol R and
2 volumes of a 10 g/L solution of tartaric add R and dilute to clearly visible.
10 mL with the same mixture of solvents. System suitability Test solution:
Column:
— the chromatogram shows at least 3 separated secondary
— size. I ~ 0.15 m, 0 = 4.6 mm; spots.
2016 Cod-liver Oil 1-625
Limits: with shifts similar to those in the spectrum shown in

— any impurity: any spots, apart from the principal spot, are Figure 2398.-1.
not more intense than the spot in the chromatogram The positional distribution (P(2)-acyI) for cervonic
obtained with reference solution (a) (0.3 per cent); (docosahexaenoic) add (C22:6 n-3; DHA), timnodonic
not more than 4 such spots are more intense than the (eicosapentaenoic) acid (C20:5 n-3; EPA) and moroctic add
spot in the chromatogram obtained with reference (C l8:4 n-3) complies with the limits.
solution (c) (0.1 per cent) and 2 of these may be more B. Linoleic add (see Tests).
reference solution (b) (0.2 per cent). TESTS
Loss on drying (2.2.32) Acid value (2.5.1)
Maximum 5.0 per cent, determined on 0.500 g by drying at Maximum 2.0.
120 °C under high vacuum. Anisidine value (2.5.36)
Maximum 10.0.
ASSAY
Dissolve 0.500 g in 60 mL of pyridine R. Pass a stream of Peroxide value (2.5.5, Method B)
nitrogen R over die surface of the solution and titrate with Maximum 5.0.
0.1 M tetrabutylammonium hydroxide, determining the Unsaponifiable m atter (2.5.7)
end-point potentipmetrically (2.2.20). Maximum 1.5 per cent, determined on 2.0 g, and extracting
1 mL of 0.1 M tetrabutylammonium hydroxide is equivalent to with 3 quantities, each of 50 mL, of peroxide-free ether R.
68.04 mg of codergocrine mesilate (average = 680). Stearin
STORAGE Heat at least 10 mL to 60-90 °C then allow to cool for 3 h
Protected from light. in a bath of iced water or a thermostatically controlled bath
at 0 ± 0.5 °C. If necessary, to eliminate insoluble matter,
__________________________________________________________ PhEur
filter the sample after heating. The sample remains clear.
Positional distribution (P(2)-acyl) of fatty adds
Nuclear magnetic resonance spectrometry (2.2.3J).
***** Test solution Dissolve 190-210 mg of the substance to be
Farmed Cod-liver Oil ★ ★
★ ★ examined in 500 pL of deuterated chloroform R. Prepare at
***** least 3 samples and examine within 3 days.
Apparatus High-resolution FT-NMR spectrometer operating
Action and use at minimum 300 MHz.
Source of vitamins A and D. Acquisition o f13C N M R spectra The following parameters may
be used:
PhEur___________________________
— sweep width: 200 ppm (-5 ppm to 195 ppm);
DEFINITION — irradiation frequency offset. 95 ppm;
Purified fatty oil obtained from the fresh livers of farmed cod, — time domain: 64 K;
Gadus morkua L., solid substances being removed by cooling — pulse delay. 2 s;
and filtering. — pulse program: zgig 30 (inverse gated, 30° exdtation
Content pulse);
— sian of the contents ofEPA and DHA (expressed as — dummy scans: 4;
triglycerides): 10.0 per cent to 28.0 per cent;
— number of scans: 4096.
— vitamin A: 50 IU (15 jig) to 500 IU (150 ng) per gram; Processing and plotting The following parameters may be used:
— vitamin D 3: maximum 50 IU (1.3 |ig) per gram. — size: 64 K (zero-filling);
A suitable antioxidant may be added. — window multiplication: exponential;
— Lorentzian broadeningfactor. 0.2 Hz.
PRODUCTION Use the CDC13 signal for shift referencing. The shift of the
The fish shall only be given feed with a composition that is central peak of the 1:1:1 triplet is set to 77.16 ppm.
in accordance with the relevant European Union or other
Plot the spectral region 5 171.5-173.5 ppm. Compare the
applicable regulations.
spectrum with the spectrum shown in Figure 2398.-1.
The content of dioxins and dioxin-like PCBs The shift values lie within the ranges given in Table 2398.-1.
(polychlorinated biphenyls) is controlled using methods and
limits in accordance with the requirements set in the
European Union or other applicable regulations. Table 2398.-1. - Shift values
Signal Shift range (ppm)
CHARACTERS
P DHA 172.05 -172.09
Appearance
Clear, pale yellowish liquid. a DHA 172.43 -172.47
Solubility P EPA 17252 -172.56
Practically insoluble in water, misdble with light petroleum,
a EPA 172.90 • 172.94
slightly soluble in ethanol (96 per cent).
P C18:4 172.56 -172.60
IDENTIFICATION
A. Examine the 13C NMR spectra obtained in the test for a C18:4 172.95 -172.99
positional distribution (p(2)-acyl) of fatty acids (see Tests).
The spectra contain peaks between 172 ppm and 173 ppm
1-626 Cod-liver Oil 2016
173.4 173.2 173.0 172.8 172.6 172.4 172.2 172.0 171.8 ppm
1. a C18:4 2. a EPA 3. p C18:4 4. P EPA 5. a DHA 6. p DHA
Figure 2398.-1. - 13CN M R spectrum: carbonyl region of farmed cod-liver oil
System suitability: 20:4 n-3, 20:5 n-3, 22:1 n-11, 22:1 n-9, 21:5 n-3, 22:5 n-3,
— signal-to-noise ratio:minimum 5 for the smallest relevant 22:6 n-3).
peak corresponding to a C 18:4 signal (in the Linoleic acid (2.4.29)
range 8 172.95-172.99 ppm); 3.0 per cent to 11.0 per cent.
— peak width at half-height, maximum 0.02 ppm for the
central CDCI3 signal (at 5 77.16 ppm). ASSAY
EPA and DHA (2.4.29)
Calculation of positional distribution ((¡(2)-acyl) Use the
See the chromatogram shown in Figure 2398.-2.
Vitamin A
100 x /3 Carry out the test as rapidly as possible, avoiding exposure to
actinic light and air, oxidising agents, oxidation catalysts (for
a+ 0
example, copper and iron) and adds.
a = peak area of the corresponding a-carbonyl peak; Use method A. If method A is found not to be valid, use
/? = peak area of {$-carbonyl peak from C22:6 n-3, method B.
C20:5 n-3 or C l8:4 n-3, respectively. M ETHOD A
Limits: Ultraviolet absorption spectrophotometry (2.2.25).
— positional distribution (fi(2)-acyl): Test solution To 1.00 g in a round-bottomed flask, add 3 mL
— cervomc (docosahexaenoic) acid (C22:6 n-3; DHA): of a freshly prepared 50 per cent m/m solution of potassium
71 per cent to 81 per cent; hydroxide R and 30 mL of anhydrous ethanol R. Boil under
— txmnodomc (eicosapentaenoic) acid (C20:5 n-3 EPA): reflux in a current of nitrogen R for 30 min. Cool rapidly and
32 per cent to 40 per cent; add 30 mL of water R. Extract with 50 mL of ether R. Repeat
— morocdc add ( C l8:4 n-3): 28 per cent to 38 per cent. the extraction 3 times and discard the lower layer after
Composition of fatty acids (2.4.29) complete separation. Wash the combined upper layers with
For identification of the peaks, see the chromatogram shown 4 quantities, each of 50 mT, of water R, and evaporate to
in Figure 2398.-2. dryness under a gentle current of nitrogen R at a temperature
not exceeding 30 °C or in a rotary evaporator at a
The 24 largest peaks of the methyl esters account for more
temperature not exceeding 30 °C under reduced pressure
than 90 per cent of the total area (these correspond to, in
(water ejector). Dissolve the residue in sufficient 2-
common elution order 14:0, 15:0, 16:0, 16:1 n-7, 16:4 n-1,
propanol R1 to give an expected concentration of vitamin A
18:0, 18:1 n-9, 18:1 n-7, 18:2 n-6 , 18:3 n-3, 18:4 n-3,
equivalent to 10-15 IU/mL.
20:1 n-11, 20:1 n-9, 20:1 n-7, 20:2 n-6 , 20:4 n-6 , 20:3 n-3,
2016 Cod-liver Oil 1-627
24
19
? 9
13
1011 20 23
12
22
'iV- n W i nAv-n/ X A-
mi|Tiii|iiii|iiii|iiiiii|n]'i[iinjiiii|iiii|iiii|mi|ii!i|in
8 10 12 14 16 18 20 22 24 26 min
1. C14:0 5. C16:4 n-1 9. C18:2 n-6 13. C20;l n-9 17. C20:3 n-3 21. C22:l n-9
2. C15:0 6. C18:0 10 C18:3 n-3 14. C20:l n-7 18. C20:4 n-3 22. C21:5 n-3
3. C16:0 7. C18:l n-9 11. C18:4 n-3 15. C20:2 n-6 19. C20:5 n-3 23. C22:5 n-3
4. C16:l n-7 8. C18:l n-7 12. C20:l tt-11 16. C20:4 n-6 20. C22:l n-11 24. C22:6 n-3
Figure 2398.-2. - Chromatogram for the test for composition of fatty acids of farmed cod-liver oil
Measure the absorbances of the solution at 300 nm, 310 nm, A Designates the absorbance at the wavelength indicated by
325 nm and 334 nm and at the wavelength of maximum the subscript
absorption with a suitable spectrophotometer in specially If ^325 has a value greater than A325jCon: /0.970, calculate the
matched 1 cm cells, using 2-propanol R1 as the compensation content of vitamin A using the following expression:
liquid.
Calculate the content of vitam in A, as all-irans-retinol, in
4 1 8 2 1
International Units per gram, using the following expression: ^325, corr X _ XV
100m
1821
¿325 x — T/
— x V The assay is not valid unless:
100 m — the wavelength of maximum absorption lies between
323 nm and 327 nm;
■^325 = absorbance at 325 nm; — the absorbance at 300 nm relative to that at 325 nm is at
m = mass of the substance to be examined, in most 0.73.
grams;
= total volume of solution containing 10-15 IU of M ETH O D B
vitamin A per millilitre; Liquid chromatography (2.2.29).
1821 = conversion factor for the specific absorbance of Test solution Prepare duplicates. To 2.00 g in a round-
all-rranî-retinol, in International Units. bottomed flask, add 5 mL of a freshly prepared 100 g/L
solution of ascorbic acid R, 10 mL of a freshly prepared
The above expression can be used only if ^325 has a value
800 g/L solution of potassium hydroxide R and 100 mL of
not greater than -^325,con- /0.970, where A 32^,can is the
anhydrous ethanol R. Boil under a reflux condenser on a
corrected absorbance at 325 nm and is given by the following
water-bath for 15 min. Add 100 mL of a 10 g/L solution of
equation:
sodium chloride R and cool. Transfer the solution to a 500 mL
separating funnel, rinsing the round-bottomed flask with
¿■325, corr = 6.815^-325 —.2.555^310 -r’ 4.260^334 about 75 mL of a 10 g/L solution of sodium chloride R and
1-628 Cod-liver Oil 2016
then with 150 mL of a mixture of equal volumes of ether R Column:

and light petroleum R l. Shake for 1 min. When the layers — size. I = 0.25 m, 0 = 4.6 mm;
have separated completely, discard the lower layer and wash — stationary phase: octadecylsUyl silica gelfor chromatography R
the upper layer, first with 50 mL of a 30 g/L solution of (5-10 pm).
potassium hydroxide R in a 10 per cent V/V solution of Mobile phase water R, methanol R (3:97 V/V).
anhydrous ethanol R and then with 3 quantities, each of
Flow rate 1 mL/min.
50 ml,, of a 10 g/L solution of sodium chloride R Filter the
upper layer through 5 g of anhydrous sodium sulfate R on a
fast filter paper into a 250 mL flask suitable for a rotary Injection10 pL; inject in triplicate the test solution and
evaporator. Wash the funnel with 10 mL of fresh extraction reference solution (b).
mixture, filter and combine the upper layers. Distil them at a Retention time All-rram-retinol = 5 + 1 min.
temperature not exceeding 30 °C under reduced pressure System suitability:
(water ejector) and fill with nitrogen R when evaporation is — the chromatogram obtained with the test solution shows a
completed. Alternatively, evaporate the solvent under a gentle peak corresponding to the peak due to all-trans-retinol in
current of nitrogen R at a temperature not exceeding 30 °C. the chromatogram obtained with reference solution (b);
Dissolve the residue in 2-propanol R, transfer to a 25 mL — the results obtained with the duplicate test solutions do
volumetric flask and dilute to 25 mL with 2-propanol R. not differ by more than 5 per cent;
Gentle heating in an ultrasonic bath may be required. A large — the recovery of all-rrans-retinol in reference solution (b) as
fraction of the white residue is cholesterol, constituting assessed by direct absorption spectrophotometry is greater
approximately 50 per cent mlm of the unsaponifiable matter of than 95 per cent
cod-liver oU.
Calculate the content of vitamin A using the following
Reference solution (a) Prepare a solution of retinol acetate CRS expression:
in 2-propanol R l so that 1 mL contains about 1000 IU of all-
trans-Teunol.
C x V 1
The exact concentration of reference solution (a) is assessed A\ x — ^— x —
A -2 m
by ultraviolet absorption spectrophotometry (2.2.25). Dilute
reference solution (a) with 2-propanol R l to a presumed
concentration of 10-15 IU/mL and measure the absorbance = area of the peak due to all-rrans-retinol in the
at 326 nm in matched 1 cm cells using 2-propanol R l as the chromatogram obtained with the test solution;
compensation liquid. A2 = area of the peak due to all-mms-retinol in the
chromatogram obtained with reference solution
Calculate the content of vitamin A in International Units per
(b);
millilitre of reference solution (a) using the following C = concentration of retinol acetate CRS in reference
expression, taking into account the assigned content of retinol solution (a) as assessed prior to the saponification,
acetate CRS:
in International Units per millilitre (= 1000
‘IU/mL);
1900 x V2 V = volume of reference solution (a) treated (2.00
100 x Vi mL);
A-326 = absorbance at 326 nm; solution (2.00 g).
V1 = volume of reference solution (a) used; Vitamin D 3
V2 = volume of the diluted solution; Liquid chromatography (2.2.29). Carry out the assay as rapidly
1900 = conversion factor for the specific absorbance of as possible, avoiding exposure to actinic light and air.
retinol acetate CRS, in International Units.
Internal standard solution Dissolve 0.50 mg of
Reference solution (b) Proceed as described for the test ergocaldferd CRS in 100 mL of anhydrous ethanol R.
solution but using 2.00 mL of reference solution (a) in place Test solution (a) To 4.00 g in a round-bottomed flask, add
of the substance to be examined. 5 mL of a freshly prepared 100 g/L solution of ascorbic
The exact concentration of reference solution (b) is assessed add R, 10 mL of a freshly prepared 800 g/L solution of
by ultraviolet absorption spectrophotometry (2.2.25). Dilute potassium hydroxide R and 100 mL of anhydrous ethanol R.
reference solution (b) with 2-propanol R l to a presumed all- Boil under a reflux condenser on a water-bath for 30 min.
rraw-retinol concentration of 10-15 IU/mL and measure the Add 100 mL of a 10 g/L solution of sodium chloride R and
absorbance at 325 nm in matched 1 cm cells using cool the solution to room temperature. Transfer the solution
2-propanol R l as the compensation liquid. to a 500 mL separating funnel, rinsing the round-bottomed
Calculate the content of all-zram-retinol in International flask with about 75 mL of a 10 g/L solution of sodium
Units per millilitre of reference solution (b), using the chloride R and then with 150 mL of a mixture of equal
following expression: volumes of ether R and light petroleum R l. Shake for 1 min.
When the layers have separated completely, discard the lower
1821 x V3 layer and wash the upper layer, first with 50 mL of a 30 g/L
100 x V4 solution of potassium hydroxide R in a 10 per cent V/V
solution of anhydrous ethanol R, and then with 3 quantities,
each of 50 mL, of a 10 g/L solution of sodium chloride R.
■^325 absorbance at 325 nm;
Filter the upper layer through 5 g of anhydrous sodium
v3 volume of the diluted solution;
sulfate R on a fast filter paper into a 250 mL flask suitable for
V4 volume of reference solution (b) used;
a rotary evaporator. Wash the funnel with 10 mL of fresh
1821 conversion factor for the specific absorbance of
extraction mixture, filter and combine the upper layers. Distil
all-iram-retinol, in International Units.
them at a temperature not exceeding 30 °C under reduced
2016 Cod-liver Oil (Type A) 1-629
pressure (water ejector) and fill with nitrogen R when A1 = area (or height) of the peak due to cholecalciferol
evaporation is completed. Alternatively, evaporate the solvent in the chromatogram obtained with test solution
under a gentle current of nitrogen R at a temperature not (a);
exceeding 30 °C. Dissolve the residue in 1.5 mL of the A2 = area (or height) of the peak due to cholecaldferol
mobile phase described under Purification. Gentle heating in in the chromatogram obtained with test solution
an ultrasonic bath may be required. A largefraction of the (b);
white residue is cholesterol, constituting approximately 50 per cent A3 = area (or hdght) of the peak due to ergocalciferol
mJm of the unsaponifiable matter of cod4ruer oH in the chromatogram obtained with reference
Test solution (b) Prepare duplicates. To 4.00 g add 2.0 mL of solution (b);
the internal standard solution and proceed as described for A4 = area (or hdght) of the peak due to ergocalciferol
test solution (a). in the chromatogram obtained with test solution
Reference solution (a) Dissolve 0.50 mg of cholecalciferol CRS (b);
in 100.0 mL of anhydrous ethanol R A5 = area (or hdght) of a possible peak in the
chromatogram obtained with test "Solution (a) with
Reference solution (b) In a round-bottomed flask, add 2.0 mL
the same retention time as the peak co-eluting
of reference solution (a) and 2.0 mL of the internal standard
with ergocalciferol in test solution (b);
solution and proceed as described for test solution (a).
A6 = area (or hdght) of the peak due to cholecalciferol
PURIFICATION in the chromatogram obtained with reference
Column: solution (b);
— sizer. I = 0.25 m, 0 = 4.6 mm; Vx — total volume of reference solution (a) (100 mL);
— stationary phase: nitrile silica gd for chromatography R V2 = volume of reference solution (a) used for
(10 pm). preparing reference solution (b) (2.0 mL).
Mobile phase isoamyl alcohol R, hexane R (1.6:98.4 VIV). STORAGE
Flow rate 1.1 mlVmin. In an airtight and well-filled container, protected from light
Detection Spectrophotometer at 265 nm. If no antioxidant is added, store under an inert gas.
Injection 350 pL of reference solution (b) and test Once the container has been opened, its contents are used as
solutions (a) and (b). Collect each ehiate from 2 min before soon as possible and any part of the contents not used at
until 2 min after the retention time of cholecalciferol, in a once is protected by an atmosphere of inert gas.
ground-glass-stoppered tube containing 1 mL of a 1 g/L LABELLING
solution of butylhydroxytoluene R in hexane R. Evaporate
The labd states:
separately to dryness at a temperature not exceeding 30 °C
— the concentration of EPA and DHA as a sum;
under a gentle current of nitrogen R Dissolve each residue in — the number of International Units of vitamin A per gram;
1.5 mL of acetomtrUe R.
— the number of International Units of vitamin D3 per
DETERMINATION gram.
Column: PhEur
— sizer. I = 0.15 m, 0 = 4.6 mm;
— stationary phase: octadecylsUyl silica gd for chromatography R
(5 pm).
Mobile phase phosphoric add R, 96 per cent VIV solution of
acetonitrUe R (0.2:99.8 VIV). Cod-liver Oil (Type A) ★ ★
★ ★
Flow rate 1.0 mlVmin. (Ph. Eur. monograph 1192) *****
Injection 2 quantities not exceeding 200 pL of each of the Action and use
3 solutions obtained under Purification. Source of vitamins A and D.
System suitability: Each IU of vitamin D3 is equivalent to 0.025 pg of
— resolution: minimum 1.4 between the peaks due to colecalciferol.
ergocalciferol and cholecalciferol in the chromatogram
PhEtr.
— the results obtained with the test solution (b) duplicates DEFINITION
do not differ by more than 5 per cent. Purified fatty oil obtained from the fresh livers of wild cod,
Calculate the content of vitamin D3 in International Units Gadus morhua L. and other spedes of Gadidae, solid
per gram using the following expression, taking into account substances being removed by cooling and filtering. A suitable
the assigned content of cholecaldfervl CRS: antioxidant may be added.
Content
Ai Az 7712 Vi — vitamin A: 600 IU (180 pg) to 2500 IU (750 pg) per
X ----- X — x 40 gram;
Ae As mi Vi
A4 - x A% — vitamin D 3: 60 IU (1.5 pg) to 250 IU (6.25 pg) per gram.
Ai
PRODUCTION
mi = mass of the sample in test solution (b), in grams; The content of dioxins and dioxin-like PCBs
m2 = total mass of cholecaldferol CRS used for the (polychlorinated biphenyls) is controlled using methods and
preparation of reference solution (a), in limits in accordance with the requirements set in the
European Union or other applicable regulations.
micrograms (500 pg);
1-630 Cod-liver Oil (Type A) 2016
CHARACTERS Composition of fatty acids. Gas chromatography (2.2.28).

Appearance Trivial name of Nomenclature Lower limit Upper Emit
Clear, yellowish liquid. fatty add area ana
_____________ _________________________ (per cent) (per cent)
Solubility Saturatedfatty acids:
Myristic add 14-0 2.0 6.0
Palmitic add 16:0 7.0 140
IDENTIFICATION Stearic add 18:0 1.0 40
First identification A, B, C Mono-unsaturatedfatty acids:
Second identification C, D Palmitoleic add 16:1 n-7 45 113
aj-Vaccenic add 18:1 n-7 2.0 7.0
A. In the assay for vitamin A using method A, the test
Oleic add 18:1 n-9 110 21.0
solution shows an absorption maximum (,2.2.25) at
Gaddeic add 20:1 n-11 1.0 5.5
325 ± 2 nm. In the assay for vitamin A using method B, the 5.0 17.0
Gondoic add 20:1 n-9
chromatogram obtained with the test solution shows a peak Erode add 22:1 n-9 0 1.5
corresponding to the peak due to all-trans-retmol in the Cetoleic add 22:1 n-11+13 5.0 1X0
chromatogram obtained with the reference solution. (211 n-11)
B. In the assay for vitamin D 3, the chromatogram obtained Poly-unsaturatedfatty adds:
with test solution (a) shows a peak corresponding to the peak Linoleic add 18:2 n-6 0J 3.0
due to cholecalciferol in the chromatogram obtained with a-Linolenic add 18:3 n-3 0 2.0
reference solution (b). Moroctic add 18:4 n-3 0J 45
Timnodonic 20:5 n-3 7.0 16.0
C. Composition of fatty acids (see Tests). (eicosapentaenoic)
D. To 0.1 g add 0.5 mL of methylene chloride R and 1 m l. of add (EPA)
antimony trichloride solution R. Mix. A deep blue colour Cervonic 22:6 n-3 6.0 18.0
(docosahexaenoic)
develops in about 10 s. add (DHA)___________________________________________________
TESTS
Appearance Test solution Introduce about 0.45 g of the substance to be
The substance to be examined is not more intensely coloured examined into a 10 mL volumetric flask, dissolve in hexane R
than a reference solution prepared as follows: to 3.0 mL of containing 50 mg of biaylhydroxytoluerie R per litre and dilute
red primary solution add 25.0 mL of yellow primary solution to 10.0 mL with the same solvent. Transfer 2.0 mL of this
and dilute to 50.0 mL with a 10 g/L solution of hydrochloric solution into a quartz tube and evaporate the solvent with a
acid R (2.2.2, Method II). gentle current of nitrogen R. Add 1.5 mL of a 20 g/L solution
Relative density (2.2.5) of sodium hydroxide R in methanol R, cover with nitrogen R,
0.917 to 0.930. cap tightly with a polytetrafluoroethylene-lined cap, mix and
Refractive index (2.2.6) heat on a water-bath for 7 min. Cool, add 2 mL of boron
trichloride-methanol solution R, cover with nitrogen R, cap
1.477 to 1,484.
tightly, mix and heat on a water-bath for 30 min. Cool to
Acid value (2.5.1) 40-50 °C, add 1 mL of trimethylpentane R, cap and vortex or
Maximum 2.0. shake vigorously for at least 30 s. Immediately add 5 mL of
Anisidine value (2.5.36) saturated sodium chloride solution R, cover with nitrogen R, cap
Maximum 30.0. and vortex or shake vigorously for at least 15 s. Allow the
Iodine value (2.5.4, Method B) upper layer to become clear and transfer it to a separate
150 to 180. tube. Shake the methanol layer once more with 1 mL of
trimethylpentane R and combine the trimethylpentane extracts.
Use starch solution R2.
Wash the combined extracts with 2 quantities, each of 1 mL,
Peroxide value (2.5.5, Method B) of water R and dry over anhydrous sodium sulfate R. Prepare
Maximum 10.0. 2 solutions for each sample.
Unsaponifiable m atter (2.5.7) Column:
Maximum 1.5 per cent, determined on 2.0 g, and extracting — material: fused silica;
with 3 quantities, each of 50 mT, of peroxide-free ether R. — size: I — 30 m, 0 = 0.25 mm;
Stearin — stationary phase: macrogol 20 000 R (film thickness
Heat at least 10 mL to 60-90 °C then allow to cool for 3 h 0.25 pm).
in a bath of iced water or a thermostatically controlled bath Carrier gas hydrogen for chromatography R or helium for
at 0 ± 0.5 °C. If necessary, to eliminate insoluble matter, chromatography R, where oxygen scrubber is applied.
filter the sample after heating. The sample remains clear. Split ratio 1:200.
Composition of fatty a d d s Temperature:
Time Temperature
____________________________ (min)_________________(*C)
Column 0 - 55 170 225
55 - 75 225
Injection port 250
Detector 280
Figure 1192.-1. - Chromatogram for the testfor composition offatty acids of cod-liver oil (type A)
Detection Flame ionisation. Use method A. If method A is found not to be valid, use
Injection 1 |iL, twice. method B.
System suitability: METHOD A
— the 15 fatty adds to be tested are satisfactorily identified Ultraviolet absorption spectrophotometry (2.2.25).
from the chromatogram shown in Figure 1192.-1; Test solution To 1.00 g in a round-bottomed flask, add 3 mL
— injection of a mixture of equal amounts of methyl of a freshly prepared 50 per cent m/m solution of potassium
palmitate R, methyl stearate R, methyl arachidate R and
hydroxide R and 30 mL of anhydrous ethanol R. Boil under
methyl behenate R gives area percentages of 24.4, 24.8,
reflux in a current of nitrogen R for 30 min. Cool rapidly and
25.2and 25.6 (± 0.5 per cent), respectively; add 30 mL of water R. Extract with 50 mL of ether R. Repeat
— resolution: minimum 1.3 between the peaks due to methyl the extraction 3 times and discard the lower layer after
oleate and methyl os-vaccenate; the resolution between complete separation. Wash the combined upper layers with
the pair due to methyl gadoleate and methyl gondoate is 4 quantities, each of 50 mL, of water R, and evaporate to
sufficient for purposes of identification and area dryness under a gentle current of nitrogen R a t a temperature
measurement. not exceeding 30 °c or in a rotary evaporator at a
Calculate the area per cent for each fatty add methyl ester temperature not exceeding 30 °c under reduced pressure
using the following expression: (water ejector). Dissolve the residue in suffirient
2-propanol R1 to give an expected concentration of vitamin A
x 100
At Measure the absorbances of the solution at 300 nm, 310 nm,
325 nm and 334 nm and at the wavelength of maximum
Ax = peak area of fatty add x; absorption with a suitable spectrophotometer in spedally
At = sum of the peak areas (up to C22:6 n-3). matched 1 cm cells, using 2-propanol R1 as the compensation
liquid.
The calculation is not valid unless:
— the total area is based only on peaks due solely to fatty Calculate thê content of vitamin A, as all-iTCOTj-retinol, in
add methyl esters; International Units per gram, using the following expression:
— the number of fatty add methyl ester peaks exceeding
0.05 per cent of the total area is at least 24; ¿325 X 1821 X V
T/
— the 24 largest peaks of the methyl esters account for more 100m
than 90 per cent of the total area (these correspond to, in
common elution order 14:0, 15:0, 16:0,16:1 n-7, A-Ì25 = absorbance at 325 nm;
16:4 n-1, 18:0, 18:1 n-9, 18:1 n-7, 18:2 n-6, 18:3 n-3, m = mass of the substance to be examined, in grams;
18:4 n-3, 20:1 n-11, 20:1 n-9, 20:1 n-7, 20:2 n-6, 20:4 V = total volume of solution containing 10-15 IU of
n-6, 20:3 n-3, 20:4 n-3, 20:5 n-3, 22:1 n-11, 22:1 n-9, vitamin A per millilitre;
21:5 n-3, 22:5 n-3, 22:6 n-3). 1821 = conversion factor for the specific absorbance of all-
mwis-retinol, in International Units.
ASSAY
V ita m in A The above expression can be used only if Aws has a value
Carry out the test as rapidly as possible, avoiding exposure to not greater than A^2ĩfifxJ0.91ồ, where A 325 corr is the
actinic light and air, oxidising agents, oxidation catalysts (for corrected absorbance at 325 nm and is given by the following
example, copper and iron) and acids. equation:
1-632 Cod-liver Oil (Type A) 2016
■4.325,corr = 6.815.4325 ~ 2.555.4.310 —4.260.4334 Vx = volume of reference solution (a) used;

V2 = volume of the diluted solution;
A Designates the absorbance at the wavelength indicated by 1900 = conversion factor for the specific absorbance of
the subscript. retinol acetate CRS, in International Units.
If A 325 has a value greater than A2,2sjO3J0.91Q, calculate the Reference solution (b)Proceed as described for the test
content of vitamin A using the following expression: solution but »sing 2.00 mL of reference solution (a) in place
of the substance to be examined.
1821 x T/
X ——— V The exact concentration of reference solution (b) is assessed
■4-325,e
100m by ultraviolet absorption spectrophotometry (2.2.25). Dilute
reference solution (b) with 2-propanol R l to a presumed all-
The assay is not valid unless: iram-retinol concentration of 10-15 IU/mL and measure the
— the wavelength of the maximum absorption lies between absorbance at 325 nm in matched 1 cm cells using
323 nm and 327 nm; 2-propanol R l as the compensation liquid.
— the absorbance at 300 nm relative to that at 325 nm is at Calculate the content of all-mzns-retinol in International
most 0.73. Units per millilitre of reference solution (b), using the
M ETHOD B following expression:
Test solution Prepare duplicates. To 2.00 g in a round- 1821 x V3
■¿325 x 100 x V4
bottomed flask, add 5 mL of a freshly prepared 100 g/L
solution of ascorbic acid R, 10 mL of a freshly prepared
800 g/L solution of potassium hydroxide R and 100 mL of ■^325 absorbance at 325 nm;
anhydrous ethanol R. Boil under a reflux condenser on a V3 volume of the diluted solution;
water-bath for 15 min. Add 100 mL of a 10 g/L solution of V4 volume of reference solution (b) used;
sodium chloride R and cool. Transfer the solution to a 500 mL 1821 conversion factor for the specific absorbance of
separating funnel, rinsing the round-bottomed flask with all-mzns-retinol, in International Units.
about 75 mL of a 10 g/L solution of sodium chloride R and Column:
then with 150 mL of a mixture of equal volumes of ether R — size. I — 0.25 m, 0 = 4.6 mm;
and light petroleum R l. Shake for 1 min. When the layers — stationary phase. octadecylsHyl silica gelfor chromatography R
have separated completely, discard the lower layer and wash (5-10 pm).
the upper layer, first with 50 mL of a 30 g/L solution of
Mobile phase water R, methanol R (3:97 VIV).
potassium hydroxide R in a 10 per cent V/V solution of
anhydrous ethanol R and then with 3 quantities, each of Flow rate 1 mL/min.
50 mT, of a 10 g/L solution of sodium chloride R. Filter the Detection Spectrophotometer at 325 nm.
upper layer through 5 g of anhydrous sodium sulfate R on a Injection 10 pL; inject in triplicate the test solution and
fast filter paper into a 250 mL flask suitable for a rotary reference solution (b).
evaporator. Wash the funnel with 10 mL of fresh extraction Retendon time All-mzns-retinol = 5 + 1 min.
mixture, filter and combine the upper layers. Distil them at a
System suitability.
temperature not exceeding 30 °C under reduced pressure
— the chromatogram obtained with the test solution shows a
(water ejector) and fill with nitrogen R when evaporation is
peak corresponding to the peak due to all-mzns-retinol in
completed. Alternatively, evaporate the solvent under a gentle
the chromatogram obtained with reference solution (b);
current of nitrogen R at a temperature not exceeding 30 °C.
— the results obtained with the duplicate test solutions do
Dissolve the residue in 2-propcmol R, transfer to a 25 mL
not differ by more than 5 per cent;
volumetric flask and dilute to 25 mL with 2-propanol R.
— the recovery of all-mzns-retinol in reference solution (b) as
Gentle heating in an ultrasonic bath may be required. A large
assessed by direct absorption spectrophotometry is greater
fraction of the white residue is cholesterol, constituting
than 95 per cent
approximately SOper cent m/m of the umapordfiable matter of
cod-liver otL Calculate the content of vitamin A using the following
expression:
Reference solution (a) Prepare a solution of retinol acetate CRS
in 2-propanol R l so that 1 m l. contains about 1000 IU of all-
trans-ictmol.
. C x V 1
A i X ---- ;----- X —
A2 m
The exact concentration of reference solution (a) is assessed
by ultraviolet absorption spectrophotometry (2.2.25). Dilute
reference solution (a) with 2-propanol R l to a presumed Ax = area of the peak due to all-mzns-retinol in the
concentration of 10-15 IU/mL and measure the absorbance chromatogram obtained with the test solution;
at 326 nm in matched 1 cm cells using 2-propanol R l as the A2 = area of the peak due to all-mzns-retinol in the
compensation liquid. chromatogram obtained with reference solution (b);
Calculate the content of vitamin A in International Units per C = concentration of retinol acetate CRS in reference
millilitre of reference solution (a) using the following solution (a) as assessed prior to the saponification, in
expression, taking into account the assigned content of retinal International Units per millilitre (= 1000 IU/mL);
acetate CRS: V = volume of reference solution (a) treated (2.00 mL);
1900 x V2 solution (2.00 g).
■¿326 x Vitamin D3
100 x Vi
Liquid chromatography (2.2.29). Cany out the assay as rapidly
A j 26 = absorbance at 326 nm; • as possible, avoiding exposure to actinic light and air.
Internal standard solution Dissolve 0.50 mg of Flow rate 1.0 mT Vmin.

ergocaldferol CRS in 100 mL of anhydrous ethanol R. Detection Spectrophotometer at 265 nm.
Test solution (a) To 4.00 g in a round-bottomed flask, add Injection 2quantities not exceeding 200 pL of each of thé
5 mL of a freshly prepared 100 g/L solution of ascorbic 3 solutions obtained under Purification.
acid R, 10 mL of a freshly prepared 800 g/L solution of
System suitability:
potassium hydroxide R and 100 mL of anhydrous ethanol R.
— resolution: minimum1.4 between the peaks due to
Boil under a reflux condenser on a water-bath for 30 min. ergocaldferol and cholecalciferol in the chromatogram
Add 100 mL of a 10 g/L solution of sodium chloride R and obtained with reference solution (b);
cool the solution to room temperature. Transfer the solution — the results obtained with test solution (b) duplicates do
to a 500 mL separating funnel, rinsing the round-bottomed not differ by more than 5 per cent.
flask with about 75 mL of a 10 g/L solution of sodium
chloride R and then with 150 mL of a mixture of
Calculate the content of vitamin D3 in International Units
equal volumes of ether R and light petroleum R l. Shake for per gram using the following expression, taking into account
1 min. When the layers have separated completely, discard the assigned content of cholecalciferol CRS:
the lower layer and wash the upper layer, first with 50 mL of
a 30 g/L solution of potassium hydroxide R in a Az 1712
x — x ^ x 40
10 per cent VIV solution of anhydrous ethanol R, and then At As mi Vi
A4- XA2
with 3 quantities, each of 50 mL, of a 10 g/L solution of
sodium chloride R. Filter the upper layer through 5 g of
anhydrous sodium sulfate R on a fast filter paper into a
250 mL flask suitable for a rotary evaporator. Wash the
mx mass of the sample in test solution (b), in grams;
m2 total mass of cholecalciferol CRS used for the
funnel with 10 mL of fresh extraction mixture, filter and
combine the upper layers. Distil them at a temperature not preparation of reference solution (a), in micrograms
(500 pg);
exceeding 30 °C under reduced pressure (water ejector) and
fill with nitrogen R when evaporation is completed. A\ = area (or height) of the peak due to cholecalciferol in
Alternatively, evaporate the solvent under a gentle current of the chromatogram obtained with test solution (a);
nitrogen R a t a temperature not exceeding 30 °C. Dissolve the
A2 = area (or height) of the peak due to cholecalciferol in
residue in 1.5 mL of the mobile phase described under the chromatogram obtained with test solution (b);
A 3 = area (or height) of the peak due to ergocaldferol in
Purification. Gentle heating in an ultrasonic bath may be
required. A large fraction of the white residue is cholesterol, the chromatogram obtained with reference solution
constituting approximately 50 per cent m/m of the unsaponifiable
(b);
A 4 = area (or hdght) of the peak due to ergocaldferol in
matter of cod-liver oiL
the chromatogram obtained with test solution (b);
Test solution (b) Prepare duplicates. To 4.00 g add 2.0 mL of
A 5 = area (or height) of a possible peak in the
the internal standard solution and proceed as described for chromatogram obtained with test solution (a) with
test solution (a). the same retention time as the peak co-duting with
Reference solution (a) Dissolve 0.50 mg of cholecalciferol CRS ergocaldferol in test solution (b);
in 100.0 mL of anhydrous ethanol R. Aa = area (or hdght) of the peak due to cholecalciferol in
Reference solution (b) Into a round-bottomed flask, add the chromatogram obtained with reference solution
2.0 mL of reference solution (a) and 2.0 mL of the internal (b);
standard solution and proceed as described for test total volume of reference solution (a) (100 mL);
solution (a). V2 volume of reference solution (a) used for preparing
reference solution (b) (2.0 mL).
PURIFICATION
Column’. STORAGE
— size'. I = 0.25 m, 0 = 4.6 mm; In an airtight and well-filled container, protected from light.
— stationary phase: nitrUe silica gd for chromatography R If no antioxidant is added, store under an inert gas.
(10 pm). Once the container has been opened, its contents are used as
Mobile phase isoamyl alcohol R, hexane R (1.6:98.4 VIV). soon as possible and any part of the contents not used at
Flow rate 1.1 mLVmin. once is protected by an atmosphere of inert gas.
Detection Spectrophotometer at 265 nm. LABELLING
Injection 350 pL of reference solution (b) and test The label states:
solutions (a) and (b). Collect each eluate from 2 min before — the number of International Units of vitamin A per gram;
until 2 min after the retention time of cholecalciferol, in a — the number of International Units of vitamin D3 per
ground-glass-stoppered tube containing 1 mL of a 1 g/L gram.
solution of butyUvydroxytoluerte R in hexane R. Evaporate ----------------------------------------------------- PhEur
separately to dryness at a temperature not exceeding 30 °C
under a gentle current of nitrogen R. Dissolve each residue in
1.5 mL of acetomtrUe R.
DETERMINATION
Column:
— size: I — 0.15 m, 0 = 4.6 mm;
— stationary phase: octadecylsUyl silica gelfor chromatography R
(5 pm).
Mobile phase phosphoric add R, 96 per cent VIV solution of
acetomtrUe R (0.2:99.8 VIV). •
1-634 Cod-liver Oil (Type B) 2016
Iodine value (2.5.4, Method B)

Cod-liver Oil (Type B) ?**%
150 to 180.
*+ -*•*
(Ph. Eur. monograph 1193) * Use starch solution R2.
Peroxide value (2.5.5, Method B)
Action and use
Maximum 10.0.
Source of vitamins A and D.
Unsaponifiable m atter (2.5.7)
Each IU of vitamin D 3 is equivalent to 0.025 pg of
Maximum 1 .5 per cent, determined on 2.0 g and extracting
colecaldferol.
with 3 quantities, each of 50 mL, of peroxide-free ether R.
PhEur__________________________________________________________ Stearin
DEFINITION Heat at least 10 mL to 60-90 °C then allow to cool for 3 h
Purified fatty oil obtained from the fresh livers of wild cod, in a bath of iced water or a thermostatically controlled bath
Gadus morkua L. and other species of Gadidae, solid at 0 ± 0.5 °C. If necessary, to eliminate insoluble matter,
substances being removed by cooling and filtering. A suitable filter the sample after heating. The sample remains clear.
antioxidant may be added. Composition of fatty acids
Content Gas chromatography (2.2.28).
— vitamin A : 600 IU (180 fig) to 2500 IU (750 pg) per
gram; Composition of fatty acids. Gas chromatography (2.2.28).
— vitamin D 3: 60 IU (1.5 pg) to 250 IU (6.25 pg) per gram. Trivial name Nomenclature Lower limit Upper limit
o f fatty acid area area
PRODUCTION (percent) (percent)
The content of d io x in s and dioxin-like PCBs Saturatedfatty adds:
(polychlorinated biphenyls) is controlled using methods and
Myristicadd 14-0 2.0 6.0
limits in accordance with the requirements set in the
Palmitic add 16:0 7.0 14.0
European Union or other applicable regulations. 1.0 40
Stearic add 18:0
CHARACTERS Mono-unsaturatedfatty adds:
Appearance Palmitoleic add 16:1 n-7 4.5 11.5
Clear, yellowish liquid. rii-Vaccenic add 18:1 n-7 2.0 7.0
Solubility Oldc add 18:1 n-9 1Z0 21.0
Gadoleic add 20:1 n-11 1.0 5.5
Gondoic add 20:1 n-9 5.0 17.0
Erudcadd 22:1 n-9 0 1.5
IDENTIFICATION Cetaldcadd 22:1 n-11+13 5.0 12.0
First identification A , B, C (22:1 n-11)
Poly-unsaturatedfatty adds:
Second identification C, D
Liaoltic add 18:2 n-6 0.5 3.0
A. In the assay for vitamin A using method A, the test
a-Linolenic add 18:3 n-3 0 2.0
solution shows an absorption maximum (2.2.25) at 18:4 n-3 0.5 45
Moroctic add
325 ± 2 nm. In the assay for vitamin A using method B, the 20:5 n-3 7.0 16.0
Timnodonic
chromatogram obtained with the test solution shows a peak (dcosapentaenoic)
corresponding to the peak due to all-mzm-retinol in the add (EPA)
chromatogram obtained with the reference solution. Cervonic 22:6 n-3 6.0 18.0
(docosahexaenoic)
B. In the assay for vitamin D3s the chromatogram obtained add (DHA)
with test solution (a) shows a peak corresponding to the peak
due to cholecalciferol in the chromatogram obtained with
Test solution Introduce about 0.45 g of the substance to be
examined into a 10 mL volumetric flask, dissolve in hexane R
C. Composition of fatty adds (see Tests). containing 50 mg of butyXhydroxytoluene R per litre and dilute
D. To 0.1 g add 0.5 mL of methylene chloride R and 1 mL of to 10.0 mL with the same solvent. Transfer 2.0 mL of the
antimony trichloride solution R. Mix. A deep blue colour solution into a quartz tube and evaporate the solvent with a
develops in about 10 s. gentle current of nitrogen R. Add 1.5 mL of a 20 g/L solution
TESTS of sodium hydroxide R in methanol R, cover with nitrogen R,
Appearance cap tightly with a polytetrafluoroethylene-lined cap, mix and
The substance to be examined is not more intensdy coloured heat on a water-bath for 7 min. Cool, add 2 mL of boron
than a reference solution prepared as follows: to 3.0 mL of trichloride-methanol solution R, cover with nitrogen R, cap
red primary solution add 25.0 mL of yellow primary solution tightly, mix and heat on a water-bath for 30 min. Cool to
and dilute to 50.0 mL with a 10 g/L solution of hydrochloric 40-50 °C, add 1 mL of trimethylpentane R, cap and vortex or
acid R (2.2.2, Method II). shake vigorously for at least 30 s. Immediately add 5 mL of
saturated sodium chloride solution R, cover with nitrogen R, cap
Relative density (2.2.5) and vortex or shake thoroughly for at least 15 s. Allow the
0.917 to 0.930.
upper layer to become clear and transfer to a separate tube.
Refractive index (2.2.6) Shake the methanol layer once more with 1 mL of
1.477 to 1.484. trimethylpentane R and combine the trimethylpentane extracts.
Acid value (2.5.1) Wash the combined extracts with 2 quantities, each of 1 mL,
Maximum 2.0. of water R and dry over anhydrous sodium sulfate R. Prepare
2 solutions for each sample.
2016 Cod-liver Oü (Type B) 1-635
Figure 1193.-1. - Chromatogram for the testfor composition offatty adds of cod-liver oU (type B)
Column: The calculation is not valid unless:

— material: fused silica; — the total area is based only on peaks due to solely fatty
— size. I — 30 m, 0 = 0.25 mm; adds methyl esters;
— stationary phase, macrogol 20 000 R (film thickness — the number of fatty add methyl ester peaks exceeding
0.25 pm). 0.05 per cent of the total area is at least 24;
Carrier gas hydrogen for chromatography R or helium for — the 24 largest peaks of the methyl esters account for more
chromatography R, where oxygen scrubber is applied. than 90 per cent of the total area (these correspond to, in
Split ratio 1:200. common elution order: 14:0, 15:0, 16:0,16:1 n-7,
16:4 n-1, 18:0, 18:1 n -9 ,18:1 n-7, 18:2 n-6 , 18:3 n-3,
Temperature:
18:4 n-3, 20:1 n-11, 20:1 n-9, 20:1 n-7, 20:2 n-6 , 20:4
n-6 , 20:3 n-3, 20:4 n-3, 20:5 n-3, 22:1 n-11, 22:1 n-9,
Time Temperature 21:5 n-3, 22:5 n-3, 22:6 n-3).
(min) i#C)
Column 0 -5 5 170 225 ASSAY
Vitamin A
55-75 225
Carry out the test as rapidly as possible, avoiding exposure to
Injection port 250 actinic light and air, oxidising agents, oxidation catalysts (for
Detector 280 example, copper and iron) and adds.
Use method A. If method A is found not to be valid, use
method B.
Injection 1 pL, twice. METHOD A
System suitability:
Ultraviolet absorption spectrophotometry (2.2.25).
— the 15 fatty adds to be tested are satisfactorily identified Test solution To 1.00 g in a round-bottomed flask, add 3 mL
from the chromatogram shown in Figure 1193.-1; of a freshly prepared 50 per cent mlm solution of potassium
— injection of a mixture of equal amounts of methyl hydroxide R and 30 mL of anhydrous ethanol R. Boil under
palmitate R, methyl stearate R, methyl arachidate R, and reflux in a current of nitrogen R for 30 mm. Cool rapidly and
methyl behenate R give area percentages of 24.4, 24.8, add 30 mL of water R. Extract with 50 mL of ether R Repeat
25.2 and 25.6 (± 0.5 per cent), respectively; the extraction 3 times and discard the lower layer after
— resolution: minimum of 1.3 between the peaks due to complete separation. Wash the combined upper layers with
methyl oleate and methyl cu-vaccenate; the resolution 4 quantities, each of 50 mL, of water R and evaporate to
between the pair due to methyl gadoleate and methyl dryness under a gende current of nitrogen R at a temperature
gondoate is sufficient for purposes of identification and not exceeding 30 °C or in a rotary evaporator at a
area measurement. temperature not exceeding 30 °C under reduced pressure
Calculate the area per cent for each fatty acid methyl ester (water ejector). Dissolve the residue in suffident 2-
propanol R1 to give an expected concentration of vitamin A
using the following expression:
Measure the absorbances of the solution at 300 nm, 310 nm,
4^x100 325 nm and 334 nm and at the wavelength of maximum
At
absorption with a suitable spectrophotometer in spedally
Ax = peak area of fatty add xr, matched 1 cm cells, using 2-propanol R1 as the compensation
At = sum of the peak areas (up to C22:6 n-3). liquid.
1-636 Cod-liver Oil (Type B) 2016
Calculate the content of vitamin A, as all-mzm-retinol, in Reference solution (a) Prepare a solution of retinol acetate CRS
International Units per gram using the following expression: in 2-propanol R l so that 1 mL contains about 1000 IU of
all-mzm-retinol.
ü 1821 Tr The exact concentration of reference solution (a) is assessed
^ X M0SXV by ultraviolet absorption spectrophotometry (2.2.25). Dilute
reference solution (a) with 2-propanol R l to a presumed
■^325 = absorbance at 325 nm; concentration of 10-15 IU/mL and measure the absorbance
m = mass of the substance to be examined, in at 326 nm in matched 1 cm cells using 2-propanol R l as the
grams; compensation liquid.
V total volume of solution containing 10-15 IU of Calculate the content of vitamin A in International Units per
vitamin A per millilitre; millilitre of reference solution (a) using the following
1821 conversion factor for the specific absorbance of expression, taking into account the assigned content of retinol
all-mzm-retinol, in International Units. acetate CRS:
The above expression can be used only if A 325 has a value
not greater than A 32s,con/0.970 where A 325,COn is the . 1900 x V2
corrected absorbance at 325 nm and is given by the 336 X 100 x Vi
equation:
-^326 = absorbance at 326 nm;
■^325,corr = 6 . 8 I 5 .A325 — 2 .5 5 5 ^ 3 1 0 — 4 .2 6 0 ^ 3 3 4 Vx = volume of reference solution (a) used;
V2 = volume of the diluted solution;
A Designates the absorbance at the wavelength indicated by 1900 = conversion factor for the specific absorbance of
the subscript. retinol acetate CRS, in International Units.
If A 325 has a value greater than A 325jCOJ0.970, calculate the Reference solution (b) Proceed as described for the test
content of vitamin A using the following expression: solution but using 2.00 mL of reference solution (a) in place
of the substance to be examined.
The exact concentration of reference solution (b) is assessed
■^325,coit X 1821 X 1V/ by ultraviolet absorption spectrophotometry (2.2.25). Dilute
100m
reference solution (b) with 2-propanol R l to a presumed
The assay is not valid unless: concentration of 10-15 IU/mL of all-mzm-retinol and
— the wavelength of maximum absorption lies between measure the absorbance at 325 nm in matched 1 cm cells
323 nm and 327 nm; using 2-propanol R l as the compensation liquid.
— the absorbance at 300 nm relative to that at 325 nm is at Calculate the content of all-mzm-retinol in International
most 0.73. Units per millilitre of reference solution (b) from the
M ETHODE expression:
liquid chromatography (2.2.29).
. 1821 x V3
Test solution Prepare duplicates. To 2.00 g in a round-
325 X 100 x VA
bottomed flask, add 5 mL of a freshly prepared 100 g/L
solution of ascorbic acid. R and 10 mL of a freshly prepared A 325 = absorbance at 325 nm;
800 g/L solution of potassium hydroxide R and 100 mL of V3 = volume of the diluted solution;
anhydrous ethanol R. Boil under a reflux condenser on a
F4 = volume of reference solution (b) used;
water-bath for 15 min. Add 100 mL of a 10 g/L solution of 1821 = conversion factor for the specific absorbance of
sodium chloride R and cool. Transfer the solution to a 500 mL
aU-mzm-retinol, in International Units.
separating funnel, rinsing the round-bottomed flask with
about 75 mL of a 10 g/L solution of sodium chloride R and Column:
then with 150 mL of a mixture of equal volumes of ether R — size. I = 0.25 m, 0 = 4.6 mm;
and light petroleum R l. Shake for 1 min. When the layers — stationary phase, octadecylsifyl silica gelfor chromatography R
have separated completely, discard the lower layer and wash (5-10 pm).
the upper layer, first with 50 mL of a 30 g/L solution of Mobile phase water R, methanol R (3:97 VIV).
potassium hydroxide R in a 10 per cent VIV solution of Flow rate 1 mlVmin.
anhydrous ethanol R and then with 3 quantities, each of Detection Spectrophotometer at 325 nm.
50 mL, of a 10 g/L solution of sodium chloride R. Filter the
Injection 10 pL; inject in triplicate the test solution and
upper layer through 5 g of anhydrous sodium sulfate R on a
fast filter paper into a 250 mL flask suitable for a rotary
evaporator. Wash the funnel with 10 mL of fresh extraction Retention time All-mzm-retinol = 5 + 1 min.
mixture, filter and combine the upper layers. Distil them at a System suitability:
temperature not exceeding 30 °C under reduced pressure — the chromatogram obtained with the test solution shows a
(water ejector) and fill with nitrogen R when evaporation is peak corresponding to the peak due to all-mzm-retinol in
completed. Alternatively evaporate the solvent under a gende the chromatogram obtained with reference solution (b);
current of nitrogen R at a temperature not exceeding 30 °C. — the results obtained with the duplicate test solutions do
Dissolve the residue in 2-propanol R, transfer to a 25 mL not differ by more than 5 per cent;
volumetric flask and dilute to 25 mL with 2-propanol R. — the recovery of all-mzm-retinol in reference solution (b) as
Gentle heating in an ultrasonic bath may be required. A large assessed by direct absorption spectrophotometry is greater
fraction of the white residue is cholesterol, constituting than 95 per cent
approximately 50 per cent m/m of the unsaponifiable matter of Calculate the content of vitamin A using the following
cod-liver criL expression:
2016 Cod-liver Oil (Type B) 1-637
C xV 1 Flow rate 1.1 mL/min.

Ai x — -— x —
A-i m
Injection 350 pL of reference solution (b) and test
Ai = area of the peak due to aH-iraw-retinol in the
chromatogram obtained with the test solution; solutions (a) and (b). Collect each eluate from 2 min before
A2 = area of the peak due to all-mzm-retinol in the
until 2 min after the retention time of cholecalciferol, in a
chromatogram obtained with reference solution ground-glass-stoppered tube c o n t a i n i n g 1 mL of a 1 g/L
solution of butylhydroxytoluene R in hexane R. Evaporate
(b);
C = concentration of retinol acetate CRS in reference separately to dryness at a temperature not e x c e e d i n g 30 °C
solution (a) as assessed prior to the saponification, under a gentle current of nitrogen R. Dissolve each residue in
in International Units per millilitre (= 1000 1.5 mL of acetonitrile R.
IU/mL); DETERMINATION
V = volume of reference solution (a) treated (2.00 Column:
mL); — size. I = 0.15 m, 0 = 4.6 mm;
m = mass of the substance to be examined in the test — stationary phase. octadecyhSyl silica gd for chromatography R
solution (2.00 g). (5 pm).
Vitamin D 3 Mobile phase phosphoric acid R, 96 per cent V/V solution of
Liquid chromatography (2.2.29). Carry out the assay as rapidly acetonitrile R (0.2:99.8 V/V).
as possible, avoiding exposure to actinic light and air. Flow rate 1.0 mL/min.
Internal standard solution Dissolve 0.50 mg of Detection Spectrophotometer at 265 nm.
ergocalciferol CRS in 100 mL of anhydrous ethanol R Injection 2quantities not e x c e e d i n g 200 pL of each of the
Test solution (a) To 4.00 g in a round-bottomed flask, add 3 solutions obtained under Purification.
5 mL of a freshly prepared 100 g/L solution of ascorbic System suitability:
add R 10 mL of a freshly prepared 800 g/L solution of — resolution: minimum1.4 between die peaks due to
potassium hydroxide R and 100 mL of anhydrous ethanol R. ergocalciferol and cholecalciferol in die chromatogram
Boil under a reflux condenser on a water-bath for 30 min. obtained with reference solution (b);
Add 100 mL of a 10 g/L solution of sodium chloride R and — the results obtained with the test solution (b) duplicates
cool the solution to room temperature. Transfer the solution do not differ by more than 5 per cent.
to a 500 mL separating funnel, rinsing the round-bottomed Calculate the content of vitamin D 3 in International Units
flask with about 75 mL of a 10 g/L solution of sodium per gram using the following expression, taking into account
chloride R and then with 150 mL of a mixture of
the assigned content of cholecalciferol CRS:
equal volumes of ether R and light petroleum R l. Shake for
1 min. When the layers have separated completely, discard
the lower layer and wash the upper layer, first with 50 mL of Aa A3 ma Va
x — x — x 40
a 30 g/L solution of potassium hydroxide R in a As As mi Vi
A 4 - x Ai
10 per cent V/V solution of anhydrous ethanol R, and then Ai
with 3 quantities, each of 50 mL, of a 10 g/L solution of
sodium chloride R Filter the upper layer through 5 g of
mass of the sample in test solution (b), in grams;
anhydrous sodium sulfate R on a fast filter paper into a
m2 total mass of cholecalciferol CRS used for the
250 mL flask suitable for a rotary evaporator. Wash the preparation of reference solution (a), in
funnel with 10 mL of fresh extraction mixture, filter and micrograms (500 pg);
combine the upper layers. Distil them at a temperature not A1 = area (or height) of the peak due to cholecalciferol
exceeding 30 °C under reduced pressure (water ejector) and in the chromatogram obtained with test solution
fill with nitrogen R when evaporation is completed.
(a);
Alternatively evaporate the solvent under a gentle current of = area (or height) of the peak due to cholecalciferol
nitrogen R at a temperature not exceeding 30 °C. Dissolve the
in the chromatogram obtained with test solution
residue in 1.5 mL of the mobile phase described under
Purification. Gentle heating in an ultrasonic bath may be
(b);
13 — area (or height) of the peak due to ergocalciferol
required. A large fraction of the white residue is cholesterol, in the chromatogram obtained with reference
constituting approximately 50 per cent m/m of the unsaponifiable
solution (b);
matter of cod-liver oiL
= area (or height) of the peak due to ergocalciferol
Test solution (b) Prepare duplicates. To 4.00 g add 2.0 mL of in the chromatogram obtained with test solution
the internal standard solution and proceed as described for (b);
test solution (a). = area (or height) of a possible peak in the
Reference solution (a) Dissolve 0.50 mg of cholecaldferol CRS chromatogram obtained with test solution (a) with
in 100.0 mL of anhydrom ethand R. the same retention time as the peak co-eluting
Reference solution (b) In a round-bottomed flask, add 2.0 mL with ergocalciferol in test solution (b);
of reference solution (a) and 2.0 mL of the internal standard area (or height) of the peak due to cholecalciferol
solution and proceed as described for test solution (a). in the chromatogram obtained with reference
solution (b);
PURIFICATION total volume of reference solution (a) (100 mL);
Vi
Column:
y2 volume of reference solution (a) used for
— size. I = 0.25 m, 0 = 4.6 mm; preparing reference solution (b) (2.0 mL).
— stationary phase, nitrile silica gd for chromatography R
(10 pm).
Mobile phase isoamyl alcohol R, hexane R (1.6:98.4 V/V).
1-638 Colchicine 2016
STORAGE C. To 0.5 mL of solution S (see Tests) add 0.5 mL of dilute

In an airtight and well-filled container, protected from light hydrochloric add R and 0.15 mL of ferric chloride solution R l.
If no antioxidant is added, store under an inert gas. The solution is yellow and becomes dark green on boiling for
Once the container has been opened, its contents are used as 30 s. Cool, add 2 mL of methylene chloride R and shake.
soon as possible and any part of the contents not used at The organic layer is greenish-yellow.
once is protected by an atmosphere of inert gas. D. Dissolve about 30 mg in 1 mL of ethanol (96 per cent) R
and add 0.15 mL of ferric chloride solution R l. A brownish-red
LABELLING
colour develops.
The label states:
— the number of International Units of vitamin A per gram; TESTS
— the number of International Units of vitamin D 3 per Solution S
gram. Dissolve 0.10 g in water R and dilute to 20 mL with the
___________________________________________________________PhEur
same solvent
than reference solution GY3 (2.2.2, Method II).
Colchicine To 10 mL of solution S add 0.1 mL of bromothymol blue
★ ★
solution R l. Either the solution does not change colour or it
becomes green. Not more than 0.1 mL of 0.01 M sodium
hydroxide is required to change the colour of the indicator to
blue.
-235 to —250 (anhydrous substance).
Dissolve 50.0 mg in ethanol (96 per cent) R and dilute to
Related substances
C22H25N 0 6 399.4 64-86-8
Solvent mixture methanol R, water R (50:50 V/V).
Action and use Test solution Dissolve 20.0 mg of the substance to be
Used in treatment of gout examined in the solvent mixture and dilute to 20.0 mL with
P reparation the solvent mixture.
Colchicine Tablets Reference solution (a) Dissolve 5 mg of colchicinefor system
suitability CRS in the solvent mixture and dilute to 5.0 mL
PhEur___________________________________________________________ with the solvent mixture.
DEFINITION Reference solution (b) Dilute 1.0 mL of the test solution to
[(75,12 aÆg)-l ,2,3,10-T etramethoxy-9-oxo-5,6,7,9- 100.0 mL with the solvent mixture.
tetrahydrobenzo [a]heptalen-7-yl] acetamide. Reference solution (c) Dilute 1 mL of reference solution (b) to
Content 20.0 mL with the solvent mixture.
97.0 per cent to 102.0 per cent (anhydrous substance). Column:
— sizer. I = 0.25 m, 0 = 4.6 mm;
C H A R A C TER S
— stationary phase. octylsHyl silica gel for chromatography R l
Appearance
(5 pm).
Yellowish-white, amorphous or crystalline powder.
Solubility
potassium dihydrogen phosphate R and 530 volumes of
Very soluble in water, rapidly recrystallising from
methanol R. After cooling to room temperature, adjust the
concentrated solutions as the sesquihydrate, freely soluble in
volume to 1000 m l. with methanol R. Adjust the apparent pH
ethanol (96 per cent), practically insoluble in cyclohexane.
to 5.5 with dilute phosphoric add R.
IDENTIFICATION Flow rate 1 mUmin.
Injection 20 jiL.
A Ultraviolet and visible absorption spectrophotometry Run time 3 times the retention time of colchicine.
(2.2.25).
Relative retention With reference to colchicine (retention
Test solution Dissolve 5 mg in ethanol (96 per cent) R and
dilute to 100.0 mL with the same solvent Dilute 5.0 mL of impurity E = about 0.7; impurity B = about 0.8;
this solution to 25.0 mL with ethanol (96 per cent) R. impurity A = about 0.94; impurity C = about 1.2.
Peak-to-vaUey ratio Minimum 2, where Hp = height above the
Absorbance ratio A 2^ A ^ o = 1.7 to 1.9. baseline of the peak due to impurity A and H v = height
B. Infrared absorption spectrophotometry (2.2.24). above the baseline of the lowest point of the curve separating
Preparation Discs of potassium bromide R. this peak from the peak due to colchicine.
Comparison colchicine CRS.
2016 Colchicine 1-639
Limits:
— impurity A: not more than 3.5 times the area of the
— any other impurity: not more than the area of the principal
solution (b) (1 per cent);
in the chromatogram obtained with reference solution (b) C. N-[(75'37bi?,10a5)-l,2,3,9-tetramethoxy-8-oxo-
(5 per cent); 5,6 ,7,7b,8,10a-
— disregard limit, the area of the principal peak in the hexahydrobenzo [a] cyclopenta [3,4] cyclobuta [1,2-
chromatogram obtained with reference solution (c) c]cyclohepten-7-yl]acetamide (P-lumicolchicine),
(0.05 per cent).
Colchiceine
Dissolve 50 mg in water R and dilute to 5 mL with the same
solvent. Add 0.1 mL of ferric chloride solution R l.
The solution is not more intensely coloured than a mixture
of 1 m l. of red primary solution, 2 mL of yellow primaiy
solution and 2 mL of blue primary solution (2.2.2,
Method II).
Chloroform (2.4.24)
D. N - [(7S, 12ai?J-3-(P-D-glucopyranosyloxy)-l,2,10-
Maximum 500 ppm.
trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
Ethyl acetate (2.4.24) yl]acetamide (colchicoside),
W ater (2.5.12)
ASSAY
Dissolve 0.250 g with gentle heating in a mixture of 10 mL
of acetic anhydride R and 20 mL of toluene R. Titrate with
0.1 M perchloric add, determining the end-point E. N-[(7S, 12a2?a)-3-hydroxy-l,2,10-trimethoxy-9-oxo-
potentiometrically (2.2.20). 5,6,7,9-tetrahydrobenzo [a] heptalen-7-yl] acetamide (3-0-
1 mL of 0.1 M perchloric acid is equivalent to 39.94 mg of demethylcolchicine),
C 22H 25NO 6.
STORAGE
IMPURITIES
F. N-[(7S, 12a/?a)-l0-hydroxy-1, 2,3-trimethoxy-9-oxo-

5,6,7,9-tetrahydrobenzo [a]heptalen-7-yl] acetamide
(colchiceine).
OCH3
-------------------- :____________________________________________ PhEur
A. N - [(75,12ai?J-l ,2,3,10-tetramethoxy-9-oxo-5,6,7,9-
tetrahydrobenzo [a]heptalen-7-yl]formamide (iV-deacetyl-N-
formylcolchicine),
OCH3
B. (-)-N-[(7S, 12 a 5 J-l,2,3,10-tetramethoxy-9-oxo-5,6,7,9-
tetrahydrobenzo[a]heptalen-7-yl]acetamide (conformational
isomer),
1-640 Colecalciferol 2016

Colecalciferol ***** ex a m in e d in trimethylpentane R without heating and dilute to
(Cholecalciferol, Ph Eur monograph 0072) * 10.0 mL with the same solvent.
Reference solution (a) Dissolve 10.0 mg of cholecalciferol CRS
in trimethylpentane R without heating and dilute to 10.0 mL
Reference solution (b) Dilute 1.0 mL of cholecalciferol for system
suitability CRS (containing impurity A) to 5.0 mL with the
mobile phase. Heat in a water-bath at 90 °C under a reflux
condenser for 45 min and cool (formation of pre-
cholecalciferol).
Reference solution (c) Dilute 10.0 mL of reference solution (a)
to 100.0 mL with the mobile phase. Dilute 1.0 mL of this
Column:
C27H 44O 384.6 67-97-0 — size. I — 0.25 m, 0 = 4.6 mm;
— stationary phase, silica gel for chromatography R (5 pm).
Action and use
Vitamin D3 analogue. Mobile phase pentanol R, hexane R (0.3:99.7 V!V).
Preparations Flow rate 2 mL/min.

Calcium and Colecalciferol Tablets Detection Spectrophotometer at 265 nm.
Chewable Calcium and Colecalciferol Tablets Iryection 5 pL of the test solution and reference solutions (b)
Colecalciferol Injection and (c).
Colecalciferol Tablets Run time Twice the retention time of cholecalciferol.
Paediatric Vitamins A, C and D Oral Drops Relative retention With reference to cholecalciferol (retention
time = about 19 min): pre-cholecalciferol = about 0.5;
When cholecalciferol or vitamin D3 is prescribed or
demanded, Colecalciferol shall be dispensed or supplied.
When calciferol or vitamin D is prescribed or demanded,
Colecalciferol or Ergocalciferol shall be dispensed or — resolution: m in im u m 1.5 between the peaks due to pre-
supplied. cholecalciferol and impurity A.
Limits:
PhEur___________________________________________________________ — impurity A: not more than the area of the principal peak
DEFINITION in the chromatogram obtained with reference solution (c)
(5Z,7£)-9,10-Secocholesta-5,7,10(19)-trien-3|3-ol. (0.1 per cent);
Content area of the principal peak in the chromatogram obtained
97.0 per cent to 102.0 per cent. with reference solution (c) (0.10 per cent);
A reversible isomerisation to pre-cholecalciferol takes place in — total: not more than 10 times the area of the principal
solution, depending on temperature and time. The activity is peak in the chromatogram obtained with reference
due to both compounds (see Assay). solution (c) ( 1.0 per cent);
1 mg of cholecalciferol is equivalent to 40 000 IU of — disregard limit. 0.5 times the area of the principal peak in
antirachitic activity (vitamin D) in rats. the chromatogram obtained with reference solution (c)
(0.05 per cent); disregard the peak due to pre-
CHARACTERS
cholecalciferol.
Appearance
White or almost white crystals. ASSAY
Solubility Liquid chromatography (2.2.29) as described in the test for
Practically insoluble in water, freely soluble in ethanol related substances with the following modification.
(96 per cent), soluble in trimethylpentane and in fatty oils. Injection Test solution and reference solution (a).
It is sensitive to air, heat and light. Solutions in solvents Calculate the percentage content of C27H 44O taking into
without an antioxidant are unstable and are to be used account the assigned content of cholecalciferol CRS and, if
immediately. necessary, the peak due to pre-cholecalciferol.
IDENTIFICATION STORAGE
Infrared absorption spectrophotometry (2.2.24). Under nitrogen, in an airtight container, protected from light,
Comparison cholecalciferol CRS. at a temperature of 2 °C to 8 °C.
The contents of an opened container are to be used
TESTS
immediately.
+ 105 to 4- 112, determined within 30 min of preparing the IMPURITIES
solution. Specified impurities A
Dissolve 0.200 g rapidly in aldehyde-free alcohol R without Other detectable impurities (the following substances would, if
heating and dilute to 25.0 mL with the same solvent. present at a sufficient level, be detected by one or other of
the tests in the monograph. They are limited by die general
Related substances
immediately before use, avoiding exposure to actinic light and air.
2016 Colecalciferol 1-641

Colecalciferol Concentrate *****
Control of impurities in substancesfor pharmaceutical use): B, C, (Oily Form) *****
D ,E . (Cholecalciferol Concentrate (Oily Form),
Action and use

Vitamin D analogue (Vitamin D3)
PhEur________ _________________________________________________
DEFINITION
Solution of Cholecalciferol (0072) in a suitable vegetable fatty
oil, authorised by the competent authority.
Content
90.0 per cent to 110.0 per cent of the cholecalciferol content
A. (5E,7E)-9, 1O-secocholesta-5,7,10(19)-trien-3^-ol (tram- stated on the label, which is not less than 500 000 IU/g.
cholecalciferol, mmi-vitamin D3), It may contain suitable stabilisers such as antioxidants.
CHARACTERS
Appearance
Clear, yellow liquid.
Solubility
Practically insoluble in water, slightly soluble in anhydrous
ethanol, miscible with solvents of fats.
Partial solidification may occur, depending on the
temperature.
B. cholesta-5j7-dien-3{J-ol (7,8-didehydrocholesterol,
provitamin D3), IDENTIFICATION
First identificadon A, C
A. Thin-layer chromatography (2.2.27). Prepare the solutions
Test solution Dissolve an amount of the preparation to be
examined corresponding to 400 000 IU in ethylene chloride R
containing 10 g/L of squalane R and 0.1 g/L of
butylhydroxytoluene R and dilute to 4 mL with the same
C. 9 p, 10a-cholesta-5j7-dien-3^-ol (himisterol3), solution.
Reference solution (a) Dissolve 10 mg of cholecalciferol CRS in
ethylene chloride R containing 10 g/L of squalane R and
0.1 g/L of butylhydroxytoluene R and dilute to 4 mL with the
same solution.
Reference solution (b) Dissolve 10 mg of ergocalciferol CRS in
0.1 g/L of bvaylhydroxytohiene R and dilute to 4 mL with the
same solution.
Mobile phase A 0.1 g/L solution of butylhydroxytoluene R in a
mixture of equal volumes of cydohexane R and peroxide-free
ether R.
D. (6E)-9,10-secocholesta-5(1 0)36j8(14)-trien-3f}-ol
(iso-tachysterol3), Application 20 |iL.
Development Immediately, protected from light, over a path of
15 cm.
Drying in air.
Detection Spray with sulfuric acid R.
Results The chromatogram obtained with the test solution
shows immediately a bright yellow principal spot which
rapidly becomes orange-brown, then gradually greenish-grey,
rem ain in g so for 10 min. This spot is similar in position,
colour and size to the spot in the chromatogram obtained
with reference solution (a). The chromatogram obtained with
reference solution (b) shows immediately at the same level an
E. (6E)-9,10-secocholesta-5(10),6,8-trien-3P-ol orange principal spot which gradually becomes reddish-
(tachysterol3). brown and remains so for 10 min.
PhEur
B. Ultraviolet and visible absorption spectrophotometry System suitability', reference solution (b):
(2.2.25). — resolution', m in im u m 1.0 between the
peaks due to pre-
Test solution Prepare a solution in cycbhexane R containing cholecaltiferol and mzw-cholecalciferol; if necessary adjust
the equivalent of about 400 IU/mL. the proportions of the constituents and the flow rate of
the mobile phase to obtain this resolution;
Absorption maximum At 267 nm. 1.0 per cent for the peak due to cholecalciferol after
C. Examine the chromatograms obtained in the assay. 6 injections.
Results The principal peak in the chromatogram obtained Calculate the conversion factor if) using the following
with the test solution is similar in retention time to the expression:
principal peak in the chromatogram obtained with reference
solution (a). K -L
TESTS M
A d d value (2.5./)
Maximum 2.0. K area (or height) of the peak due to cholecalciferol
Dissolve 5.0 g in 25 mL of the prescribed mixture of in the chromatogram obtained with reference
solvents. solution (d);
Peroxide value (2.5.5, Method A) = area (or height) of the peak due to cholecalciferol
Maximum 20. in the chromatogram obtained with reference
solution (e);
Related substances
M = area (or height) of the peak due to pre-
The thresholds indicated under Related substances
cholecalciferol in the chromatogram obtained with
The value of/ d eterm ined in duplicate on different days may
ASSAY
be used during the entire procedure.
Carry out the assay as rapidly as possible, avoiding exposure to
Calculate the content of cholecalciferol in International Units
actinic light and air.
per gram using the following expression:
Test solution Dissolve a quantity of the preparation to be
m V SD + ( f x Sp)
examined, weighed with an accuracy of 0.1 per cent, X — X x 40 000 x 1000
equivalent to about 400 000 IU, in 10.0 mL of toluene R and V7 m
dilute to 100.0 mL with die mobile phase.
Reference solution (a) Dissolve 10.0 mg of cholecalciferol CRS = mass of the preparation to be examined in the
without heating in 10.0 mL of toluene R and dilute to test solution, in milligrams;
100.0 mL with the mobile phase. = mass of cholecalciferol CRS in reference solution
(a), in milligrams;
Reference solution (b) Dilute 1.0 mL of cholecalciferolfor system
V = volume of the test solution (100 mL);
suitability CR S to 5.0 mL with the mobile phase. Heat in a
V = volume of reference solution (a) (100 mL);
water-bath at 90 °C under a reflux condenser for 45 min and
Sd = area (or height) of the peak due to cholecalciferol
cool.
in the chromatogram obtained with the test
Reference solution (c) Dissolve 0.10 g of cholecalciferol CRS
solution;
without heating in toluene R and dilute to 100.0 mL with the = area (or height) of the peak due to cholecalciferol
same solvent. in the chromatogram obtained with reference
Reference solution (d) Dilute 5.0 mL of reference solution (c) solution (a);
to 50.0 mL with the mobile phase. Keep the solution in iced = area (or height) of the peak due to pre-
water. cholecalciferol in the chromatogram obtained
Reference solution (e) Place 5.0 mL of reference solution (c) in with the test solution;
a volumetric flask, add about 10 mg of butylhydroxytoluene R f = conversion factor.
and displace air from the flask with nitrogen R. Heat in a
STORAGE
water-bath at 90 °C under a reflux condenser protected from
light and under nitrogen R for 45 min. Cool and dilute to ' In an airtight, well-filled container, protected from light.
50.0 mL with the mobile phase. The contents of an opened container are to be used as soon
as possible; any unused part is to be protected by an
Column'.
atmosphere of nitrogen.
— size. I = 0.25 m, 0 = 4.6 mm;
— stationary phase, silica gel for chromatography R (5 |im). LABELLING
Mobile phase pentanol R, hexane R (3:997 VIV). The label states:
— the number of International Units per gram;
Flow rate 2 mlVmin.
— the method of restoring the solution if partial
Detection Spectrophotometer at 254 nm. solidification occurs.
Injection The chosen volume of each solution (the same PhEur
volume for reference solution (a) and for the test solution);
automatic injection device or sample loop recommended.
Relative retention With reference to cholecalciferol: pre-
cholecalciferol = about 0.4; mzra-cholecaldferol = about 0.5.
orange principal spot, which gradually becomes reddish-

Colecalciferol Concentrate ; \ brown and remains so for 10 min.
(Powder Form) ***** B. Ultraviolet and visible absorption spectrophotometry
(Cholecalciferol Concentrate (Powder Form), (2.2.25).
Ph Eur monograph 0574) Test solution Place 5.0 mL of the test solution prepared for
the assay in a suitable flask and evaporate to dryness under
Action and use reduced pressure by swirling in a water-bath at 40 °C. Cool
Vitamin D analogue (Vitamin D3). under running water and restore atmospheric pressure with
nitrogen R. Dissolve the residue immediately in 50.0 mL of
PhEur.__ _______________________________________________________
cydohexane R.
DEFINITION Spectral range 250-300 nm.
Powder concentrate obtained by dispersing an oily solution of
Cholecalciferol (0072) in an appropriate matrix, which is
usually based on a combination of gelatin and carbohydrates C. Examine the chromatograms obtained in the assay.
of suitable quality, authorised by the competent authority. Results The principal peak in the chromatogram obtained
Content with the test solution is similar in retention time to the

90.0 per cent to 110.0 per cent of the cholecalciferol content principal peak in the chromatogram obtained with reference
stated on the label, which is not less than 100 000 IU/g. solution (a).
It may contain suitable stabilisers such as antioxidants. TESTS
Related substances
CHARACTERS
Appearance
(Table 2034.-1) in the general monograph Substancesfor
White or yellowish-white, small particles.
Solubility
Practically insoluble, swells, or forms a dispersion in water, ASSAY
Carry out the assay as rapidly as possible, avoiding exposure to
depending on the formulation.
IDENTIFICATION
Test solution Introduce into a saponification flask a quantity of
Second identification A, B.
the preparation to be examined, weighed with an accuracy of
A Thin-layer chromatography (2.2.27). Prepare the solutions 0.1 per cent, equivalent to about 100 000 IU. Add 5 mL of
immediately before use. water R, 20 mL of anhydrous ethanol R, 1 mL of sodium
Test solution Place 10.0 mL of the test solution prepared for ascorbate solution R and 3 mL of a freshly prepared
the assay in a suitable flask and evaporate to dryness under 50 per cent m/m solution of potassium hydroxide R. Heat in a
reduced pressure by swirling in a water-bath at 40 °C. Cool water-bath under a reflux condenser for 30 min. Cool rapidly
under running water and restore atmospheric pressure with under running water. Transfer the liquid to a separating
nitrogen R. Dissolve the residue immediately in 0.4 mL of funnel with the aid of 2 quantities, each of 15 m l, of
ethylene chloride R containing 10 g/L of squalane R and water R, 1 quantity of 10 mL of ethanol (96 per cent) R and
0.1 g/L of butylhydroxytoluene R. 2 quantities, each of 50 m l, of pentane R. Shake vigorously
Reference solution (a) Dissolve 10 mg of cholecalciferol CRS in for 30 s. Allow to stand until the 2 layers are clear. Transfer
ethylene chloride R containing 10 g/L of squalane R and the lower aqueous-alcoholic layer to a 2nd separating funnel
0.1 g/L of butylhydroxytoluene R and dilute to 4 mL with the and shake with a mixture of 10 mL of ethanol (96 per cent) R
same solution. . and 50 mL of pentane R. After separation, transfer the
Reference solution (b) Dissolve 10 mg of ergocalciferol CRS in
aqueous-alcoholic layer to a 3rd separating funnel and the
ethylene chloride R containing 10 g/L of squcdane R and
pentane layer to the 1st separating funnel, washing the
0.1 g/L of butylhydroxytoluene R and dilute to 4 mL with the 2nd separating funnel with 2 quantities, each of 10 mL, of
pentane R and ad d ing the w ashings to the 1st separating
same solution.
funnel. Shake the aqueous-alcoholic layer with 50 mL of
Plate T L C silica gel G plate R.
pentane R and add the pentane layer to the 1st funnel. Wash
Mobile phase A 0.1 g/L solution of butylhydroxytoluene R in a the pentane layer with 2 quantities, each of 50 m l, of a
mixture of equal volumes of cydohexane R and peroxide-free freshly prepared 30 g/L solution of potassium hydroxide R in
ether R. ethanol (10 per cent VIV) R, shaking vigorously, then wash
Application 20 pL. with successive quantities, each of 50 mL, of water R until
Development Immediately, protected from light, over a path of the washings are neutral to phenolphthalein. Transfer the
15 cm. washed pentane extract to a ground-glass-stoppered flask.
Drying In air.
Evaporate the contents of the flask to dryness under reduced
pressure by swirling in a water-bath at 40 °C. Cool under
Detection Spray with sulfuric acid R.
running water and restore atmospheric pressure with
Results The chromatogram obtained with the test solution nitrogen R. Dissolve the residue im m ediately in 5.0 mL of
shows im m ed iately a bright yellow principal spot, which toluene R and add 20.0 mL of the mobile phase to obtain a
rapidly becomes orange-brown, then gradually greenish-grey, solution containing about 4000 IU/mL.
rem a in in g so for 10 min. This spot is similar in position,
Reference solution (a) Dissolve 10.0 mg of cholecalciferol CRS,
colour and size to the spot in the chromatogram obtained without heating, in 10.0 mL of toluene R and dilute to
with reference solution (a). The chromatogram obtained with 100.0 mL with the mobile phase.
reference solution (b) shows immediately at the same level an
Reference solution (b) Dilute 1.0 mL of cholecalciferolfor system Sd = area (or height) of the peak due to cholecalciferol in
suitability CRS to 5.0 mL with the mobile phase. Heat in a the chromatogram obtained with the test solution;
water-bath at 90 °C under a reflux condenser for 45 min and S'd = area (or height) of the peak due to cholecalciferol in
cool. the chromatogram obtained with reference solution
Reference solution (c) Dissolve 0.10 g of cholecalciferol CRS, (a);
without heating, in toluene R and dilute to 100.0 mL with the Sp = area (or height) of the peak due to pre-
same solvent. cholecalciferol in the chromatogram obtained with
the test solution;
Reference solution (d) Dilute 5.0 mL of reference solution (c)
/ = conversion factor.
to 50.0 mL with die mobile phase. Keep the solution in iced
water. STORAGE
Reference solution (e) Place 5.0 mL of reference solution (c) in In an airtight, well-filled container, protected from light
a volumetric flask, add about 10 mg of butyJhydroxytoluene R The contents of an opened container are to be used as soon
and displace the air from the flask with nitrogen R. Heat in a as possible; any unused part is to be protected by an
water-bath at 90 °C under a reflux condenser, protected from atmosphere of nitrogen.
light and under nitrogen R, for 45 min. Cool and dilute to LABELLING
50.0 mL with the mobile phase. The label states the number of International Units per gram.
Column:
___________________________________________________________________________________________ PhEur
— size. I = 0.25 m, 0 = 4.6 mm;
— stationary phase, silica gelfor chromatography R (5 |im).
Mobile phase pentanol R, hexane R (3:997 VIV).
Flow rate 2 mL/min.
Detection Spectrophotometer at 254 nm. Colecalciferol Concentrate (Water- *****
Injection The chosen volume of each solution (the same dispersible Form) *****
volume for reference solution (a) and for the test solution); (Cholecalciferol Concentrate (Water-Dispersible Form),
automatic injection device or sample loop recommended. Ph Eur monograph 0598)
cholecalciferol = about 0.4; iram-cholecalciferol = about 0.5. Action and use
System suitability: reference solution (b): Vitamin D analogue (Vitamin D3).
— resolution: minimum 1.0 between the peaks due to pre-
Ph E u r ___________________________________________________________________________________________
cholecalciferol and irons-cholecalciferol; if necessary,
adjust the proportions of the constituents and the flow DEFINITION
rate of the mobile phase to obtain this resolution; Solution of Cholecalciferol (0072) in a suitable vegetable fatty
— repeatability: maximum relative standard deviation of oil, authorised by the competent authority, to which suitable
1.0 per cent for the peak due to cholecalciferol after solubilisers have been added.
6 injections. Content
Calculate the conversion factor (f) using the following 90.0 per cent to 115.0 per cent of the cholecalciferol content
expression: stated on the label, which is not less than 100 000 IU/g.
It may contain suitable stabilisers such as antioxidants.
K -L
CHARACTERS
M
Appearance
K = area (or height) of the peak due to cholecalciferol in Slightly yellowish liquid of variable opalescence and viscosity.
the chromatogram obtained with reference solution Highly concentrated solutions may become cloudy at low
(d); temperatures or form a gel at room temperature.
L — area (or height) of the peak due to cholecalciferol in IDENTIFICATION
the chromatogram obtained with reference solution First identification A , C, D
(e); Second identification A , B, D
M = area (or height) of the peak due to pre-cholecalriferol
in the chromatogram obtained with reference A. Thin-layer chromatography (2.2.27). Prepare the solutions
solution (e). immediately before use.
Test solution Place 10.0 mL of the test solution prepared for
The value of /determined in duplicate on different days may
be used during the entire procedure.
reduced pressure by swirling in a water-bath at 40 °C. Cool
Calculate the content of cholecalciferol in International Units under running water and restore atmospheric pressure with
per gram using the following expression: nitrogen R. Dissolve the residue immediately in 0.4 mL of
2L x Y- x gg ,+ (/ x Sp) x 40000 x 1000 0.1 g/L of btctylhydroxytoluene R.
V' m S'D Reference solution (a) Dissolve 10 mg of cholecalciferol CRS in
m = mass of the preparation to be examined in the test 0.1 g/L of butylhydroxytoluene R and dilute to 4 mL with the
solution, in milligrams; same solution.
m' = mass of cholecalciferol CRS in reference solution (a),
Reference solution (b) Dissolve 10 mg of ergocalciferd CRS in
in milligrams;
V = volume of the test solution (25 mL);
V = volume of reference solution (a) (100 mL);
0.1 g/L of butylhydroxytoluene R and dilute to 4 mL with the 50 mL of pentane R. After separation, transfer the aqueous-
same solution. alcoholic layer to a 3rd separating funnel and the pentane
Plate T L C silica gel G plate R. layer to the 1st separating funnel, washing the 2nd separating
funnel with 2 quantities, each of 10 m T , of pentane R and
Mobile phase A 0.1 g/L solution of butylhydroxytoluene R in a
adding the washings to the 1st separating funnel. Shake the
mixture of equal volumes of cydohexane R and peroxide-free
aqueous-alcoholic layer with 50 mL of pentane R and add the
ether R.
pentane layer to the 1st funnel. Wash the pentane layer with
Application 20 |iL. 2 quantities, each of 50 mL, of a freshly prepared 30 g/L
Development Immediately, protected from light, over a path of solution of potassium hydroxide R in ethanol
15 cm. (10 per cent V/V) R, shaking vigorously, and then wash with
Drying h i air. successive quantities, each of 50 m T , of water R until the
Detection Spray with sulfuric acid R. washings are neutral to phenolphthalein. Transfer the washed
pentane extract to a ground-glass-stoppered flask. Evaporate
the contents of the flask to dryness under reduced pressure
shows immediately a bright yellow principal spot, which
by swirling in a water-bath at 40 °C. Cool under ru nning
rapidly becomes orange-brown, then gradually greenish-grey,
water and restore atmospheric pressure with nitrogen R.
rem a in in g so for 10 m in . This spot is sim ilar in position,
Dissolve the residue im m ediately in 5.0 mL of toluene R and
colour and size to the principal spot in the chromatogram
add 20.0 mL of the mobile phase to obtain a solution
obtained with reference solution (a). The chromatogram
containing about 4000 IU/mL.
obtained with reference solution (b) shows immediately at
the same level an orange principal spot, which gradually Reference solution (a) Dissolve 10.0 mg of cholecalciferol CRS ,
becomes reddish-brown and remains so for 10 min. without heating, in 10.0 mL of toluene R and dilute to
B. Ultraviolet and visible absorption spectrophotometry
(2.2.25). Reference solution (b) Dilute 1.0 mL of cholecalciferolfor system
suitability CRS to 5.0 mL with the mobile phase. Heat in a
Test solution Place 5.0 mL of the test solution prepared for
water-bath at 90 °C under a reflux condenser for 45 min and
cool.
reduced pressure by swirling in a water-bath at 40 °C. Cool
under ru n n in g water and restore atmospheric pressure with Reference solution (c) Dissolve 0.10 g of cholecalciferol CRS,
nitrogen R. Dissolve the residue im m ediately in 50.0 mL of without heating, in toluene R and dilute to 100.0 mL with the
cycbhexane R. same solvent.
Spectral range 250-300 nm. Reference solution (d) Dilute 5.0 mL of reference solution (c)
to 50.0 mL with the mobile phase. Keep the solution in iced
water.
C. Examine the chromatograms obtained in the assay.
Reference solution (e) Place 5.0 mL of reference solution (c) in
Results The principal peak in the chromatogram obtained
a volumetric flask, add about 10 mg of butylhydroxytoluene R
with the test solution is similar in retention time to the and displace the air from the flask with nitrogen R. Heat in a
principal peak in the chromatogram obtained with reference water-bath at 90 °C under a reflux condenser, protected from
solution (a). light and under nitrogen R, for 45 min. Cool and dilute to
D. Mix about 1 g with 10 mL of water R previously wanned 50.0 mL with the mobile phase.
to 50 °C, and cool to 20 °C. Immediately after cooling, a Column:
uniform, slightly opalescent and slightly yellow dispersion is — sizer. I = 0.25 m, 0 = 4.6 mm;
obtained. — stationary phase: silica gelfor chromatography R (5 fun).
TESTS Mobile phase pentanol R, hexane R (3:997 VIV).
Related substances Flow rate 2 mL/min.
(Table 2034.-1) in the general monograph Substancesfor
pharmaceutical use (2034) do not apply. Injection The chosen volume of each solution (the same
volume for reference solution (a) and for the test solution);
ASSAY automatic injection device or sample loop recommended.
Cany out the assay as rapidly as possible, avoiding exposure to
cholecalciferol = about 0.4; mzw-cholecalciferol = about 0.5.
Liquid chromatography (2.2.29). System suitability: reference solution (b):
Test solution Introduce into a saponification flask a quantity of — resolution: minimum 1.0 between the peaks due to pre-
the preparation to be examined, weighed with an accuracy of cholecalciferol and mim-cholecalciferol; if necessary,
0.1 per cent, equivalent to about 100 000 IU. Add 5 mL of adjust the proportions of the constituents and the flow
water R, 20 mL of anhydrous ethanol R, 1 mL of sodium rate of the mobile phase to obtain this resolution;
ascorbate solution R and 3 mL of a freshly prepared — repeatability: maximum relative standard deviation of
50 per cent m/m solution of potassium hydroxide R. Heat in a 1.0 per cent for the peak due to cholecalciferol after
water-bath under a reflux condenser for 30 min. Cool rapidly 6 injections.
under running water. Transfer the liquid to a separating Calculate the conversion factor (f) using the following
funnel with the aid of 2 quantities, each of 15 ml., of expression:
water R, 1 quantity of 10 mL of ethanol (96 per cent) R and
2 quantities, each of 50 mT, of pentane R. Shake vigorously
K -L
for 30 s. Allow to stand until the 2 layers are clear. Transfer
the aqueous-alcoholic layer to a 2nd separating funnel and M
shake with a mixture of 10 mL of ethanol (96 per cent) R and
1-646 Colestipol Hydrochloride 2016
K — area (or height) of the peak due to cholecalciferol Practically insoluble in ethanol (96%) and in dichloromethaner,
in the chromatogram obtained with reference swells but does not dissolve in water and dilute aqueous
solution (d); solutions of acids and alkalis.
L = area (or height) of the peak due to cholecalciferol IDENTIFICATION
in the chromatogram obtained with reference
Carry out the method for gas chromatography,
solution (e);
Appendix m 5, using a suitable gas chromatograph fitted
M = area (or height) of the peak due to pre-
with a pyrolysis unit Operate the unit in accordance with the
cholecalciferol in the chromatogram obtained with
manufacturer’s instructions to obtain a pyrogram for colestipol
hydrochloride BPCRS that is similar to that supplied with the
The value of /determined in duplicate on different days may reference material.
be used during the entire procedure. (1) To prepare the sample, mix 1 part of n -eicosane and 4
Calculate the content of cholecalciferol in International Units parts of the substance being examined and grind the mixture
per gram vising the following expression: in a mortar with chloroform until the substance being
examined is un iform ly coated with the n-eicosane.
V w SD + (/ x Sp) (2) Prepare the standard in the same manner but adding 4
177 x x 40 000 x 1000
V' x ~
m parts of colestipol hydrochloride BPCRS in place of the
mass of the preparation to be exam ined in the C H R O M A T O G R A P H IC C O N D IT IO N S
test solution, in milligrams;
(a) Use a a glass column (1.8 m x 3 mm) packed with acid-
mass of cholecalciferol CRS in reference solution
washed, sUattised diatomaceous support (80 to 100 mesh)
(a), in milligrams;
V volume of the test solution (25 mL); (Chromosorb W is suitable) coated with 0.25% w/w of
potassium hydroxide and 5% w/w of polyethylene glycol 20,000
V volume of reference solution (a) (100 mL);
(Carbowax 20M is suitable).
SD area (or height) of the peak due to cholecalciferol
in the chromatogram obtained with the test (b) Use helium as the carrier gas at 60 mL per minute.
solution; (c) Use isothermal conditions maintained at 85°.
S 'D = area (or height) of the peak due to cholecalciferol (d) Use a pyrolysis unit capable of attaining a temperature of
in the chromatogram obtained with reference about 1000° when fined with a platinum ribbon probe.
solution (a); (e) Use a detector at a temperature of 270°.
area (or height) of the peak due to pre-
cholecalciferol in the chromatogram obtained (f) Load the sample and the standard separately into the
with the test solution; pyrolysis unit.
f conversion factor. C O N F IR M A T IO N
STORAGE The pyrogram obtained with the substance being examined is

concordant with that obtained with colestipol
In an airtight, well-filled co n tainer, protected from light, at
hydrochloride BPCRS.
the temperature stated on the label.
The contents of an opened container are to be used as soon TESTS
as possible; any unused part is to be protected by an Acidity or alkalinity
atmosphere of inert gas. Shake a 10% w/w suspension in a stoppered vial at
approximately 10-minute intervals for 1 hour and centrifuge.
LABELLING Transfer a portion of the clear supernatant liquid to a
The label states:
suitable container and record the pH as soon as the reading
— the number of International Units per gram; has stabilised. The pH is 6.0 to 7.5, Appendix V L.
— the storage temperature.
Water-soluble substances
PhEur Place 5 g in a glass-stoppered, 125 mL conical flask, add
80 mL of water, close die flask and shake in a water bath at
36° to 38° for 72 hours. Filter the contents of the flask
through a fine-porosity, sintered glass funnel or woven glass-
Colestipol Hydrochloride fibre filter, collectin g the filtrate in a tared 125 mL conical
flask. Rinse any residual contents in the flask with two 5 mL
37296-80-3 quantities of water, filter the washings and combine the
filtrates from the washings with the filtrate obtained
Action and use
previously. Evaporate the filtrate to dryness, using filtered air
Lipid-regulating drug.
or nitrogen, if necessary, to aid in the evaporation. Dry the
Preparation residue at 75° at a pressure of not more than 2 kPa for
Colestipol Granules 1 hour, allow to cool in a desiccator and weigh. Repeat the
procedure at the same time without the substance being
DEFINITION exam in ed b e gin n in g at the words ‘add 80 mL of water ... ’.
Colestipol Hydrochloride is a co-polymer of The difference in the weights of the residues is not more
diethylenetriamine and l-chloro-2 ,3-epoxypropane. Each g than 25 mg (0.5%).
binds not less than 1.1 mEq and not more than 1.7 mEq of
sodium cholate, determined in the test for Cholate b in ding Heavy metals
capacity and calculated with reference to the dried material. Transfer 1.0 g to a suitable crucible, wet with sulfuric acid
and carefully ignite at a low temperature until thoroughly
CHARACTERISTICS charred. To the carbonised substance add 2 mL of nitric acid
Yellow to orange beads; hygroscopic.
2016 Colestyramine 1-647
and 0.25 mL of sulfuric acid and heat cautiously until white (m g) of the substance being examined to a ground-glass-
fames are no longer evolved. Ignite, preferably in a muffle stoppered flask, add 100 mL of freshly prepared solution A
furnace, at 500° to 600°, until the carbon is completely and shake vigorously for 90 minutes with the flask positioned
removed and cool. Add 4 mL of 6m hydrochloric add, cover, horizontally. Remove the flask from the shaker and allow the
heat on a water bath for 15 minutes, uncover and slowly contents to settle for 5 minutes. Adjust the pH of a 20 mL
evaporate to dryness. Dissolve the residue using two 5 mL aliquot of the supernatant liquid to 10.5 by the drop wise
quantities of 2m hydrochloric acid. Add 0.1 mL of addition of 1 m sodium hydroxide and titrate potentiometrically
phenolphthalein solution and 13.5m ammonia drop wise until a with 0. 1m hydrochloric acid VS to the second inflection point
pink colour is produced. Cool, add glacial acetic add until the of the pH curve. Determine the volume of titrant added
solution is decolourised and add a further 0.5 mT- Filter if between the inflection points. Carry out a blank titration on
necessary and dilute the solution to 20 mL with water. 20 mL of freshly prepared solution A. The difference
12 mL of die resulting solution complies with limit test A for between the titrations represents the amount of hydrochloric
heavy metals, Appendix VII (20 ppm). Prepare the standard acid required (F mL). Calculate the cholate binding capacity
using a mixture of 2 mL of the test solution obtained above of the substance being examined in milliequivalents from the
and 10 mL of lead standard solution (2 ppm Pb). expression 0.5V/m.
Loss on drying Colestipol exchange capacity lim it
When dried at 75° at a pressure of not more than 2 kPa for Not less than 9.0 mEq per g and not more than 11.0 mEq
16 hours, loses not more than 1.0 % of its weight per g determined in the following manner. Transfer not less
Sulfated ash than 2 g and 100 mL of 1m sodium hydroxide to a stoppered
Not more than 0.3%, Appendix IX A. flask and shake for 4 hours. Filter the suspension through a
coarse-porosity sintered-glass funnel and wash the resin with
Chloride content 500 mL of water. Transfer the resin to a 1000 mL beaker,
Not less than 6.5% and not more than 9.0% calculated with add 200 mL of water and allow to stand for 10 minutes.
reference to the dried substance. Bum 20 mg by the method Filter the suspension, check the pH of the filtrate and repeat
for oxygen-flask combustion, Appendix VHI C, using 10 mL of the washing procedure with 200 mL quantities of water until
0.05m sodium hydroxide as the absorbing liquid. When the
the pH of the filtrate is less than 8 [5 litres may be required].
process is complete shake the flask vigorously, allow to stand Dry the resin and funnel at 60° at a pressure of 2 kPa for at
with frequent shaking for about 40 minutes or until no least 16 hours, breaking up any aggregates with a spatula and
cloudiness is observed; add 20 mL of ethanol (96%) and store in a dessicator.
0.2 mL of nitric acid. Titrate the resulting solution with
0.05m silver nitrate FS, determining the end point
Place 1 g (w g) of the free base resin prepared above and add
100 mL of 0.2m hydrochloric acid FS in a stoppered flask and
potentiometrically using a silver-silver chloride electrode and
a glass reference electrode (FmL). Repeat the procedure shake for not less than 2.5 hours. Filter a portion of the
without the substance being examined adding 10 mL of suspension through glass wool. Titrate 8 mL of the filtrate
0.0075m sodium chloride to the solution in the flask (Vx mL).
with 0.2m sodium hydroxide FS, determining the end point
Add 10 mL of 0.0075m sodium chloride to a flask containing a potentiometrically (a mL). Carry out a blank titration on
5 mL of the 0.2m hydrochloric acid FS used to equilibrate the
mixture of 10 mL of water and 20 mL of ethanol (96%), add
free-base resin diluted with 5 mL of water (b mL). Calculate
0.2 mL of nitric acid and titrate with 0.05m silver nitrate FS,
determining the end point potentiometrically using a silver- the exchange capacity in milliequivalents per g from the
silver chloride electrode and a glass reference electrode expression:
(F 2 mL). Determine the volume of 0.05m silver nitrate FS (20/w)(b/5—a/8)
required by the substance being examined using the following
expression: STORAGE
V-(Vr V2)
Colestipol Hydrochloride should be kept in an airtight
container.
Each mL of 0.05m silver nitrate FS is equivalent to 1.773 mg
of Cl.
W ater absorption capacity
Each g of Colestipol Hydrochloride absorbs not less than Colestyramine *****
3.3 g and not more than 5.3 g of water when determined in
+*
the following manner. Transfer 5 g to a dry, plastic container (Ph. Eur. monograph 1775) *
and add 80 g of water. Cover the container and allow the 11041-12-6
resulting suspension to equilibrate for 72 hours. Filter the
resulting slurry through a medium-porosity fritted-glass Action and use
funnel (KLMAX 60 mL-40M is suitable) at a pressure of Lipid-regulating drug.
2kPa; collect the filtrate in a tared, plastic container, Preparation
disconnecting the vacuum 2 minutes after collection of the Colestyramine Oral Powder
last portion of the filtrate. Immediately weigh the container
and the filtrate and determine the weight, in g, of the filtrate. PhEur_____________________ :____________________________________
Calculate the weight of water absorbed per g from the
DEFINITION
difference between the weight of the filtrate and the original
Strongly basic anion-exchange resin in chloride form,
weight of water used in the test
consisting of styrene-divinylbenzene copolymer with
Cholate binding capacity quaternary ammonium groups.
Prepare a solution containing 1.0 % w/v of sodium cholate and
Nominal exchange capacity 1.8 g to 2.2 g of sodium
0.9% w/v of sodium chloride and adjust to pH 6.4 by the drop
glycocholate per gram (dried substance).
wise addition of hydrochloric acid (solution A). Transfer 1 g
1-648 Colestyramine 2016
CHARACTERS Column:
Appearance — sizer. I = 0.30 m, 0 = 3.9 mm,
White or almost white, fine powder, hygroscopic. — stationary phase: octadecylsQyl silica gelfor chromatography R
Solubility (10 pm) with a specific surface area of 330 m2/g and a
Insoluble in water, in methylene chloride and in ethanol pore size of 12.5 nm.
(96 per cent). Mobile phase acetomtrUe R, water R (50:50 VIV).
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24). Detection Spectrophotometer at 254 nm.
Comparison colestyramine CRS. Injection 20 |iL of test solution and reference solutions (a)
and (d).
B. Chloride (see Tests).
TESTS — resolution: minimum 1.5 between the peaks due to
pH (2.2.5) impurity A and toluene.
4.0 to 6.0. Limir.
Suspend 0.100 g in 10 mL of water R and allow to stand for — impurity A: not more than the area of the principal peak
10 min. in the chromatogram obtained with reference solution (a)
Dialysable quaternary amines (1 ppm).
Maximum 500 ppm, expressed as benzyltrimethylammonium Chloride
chloride. 13.0 per cent to 17.0 per cent (dried substance).
Test solution Place a 25 cm piece of cellulose dialysis tubing To 0.2 g add 100 mL of water R and 50 mg of potassium
having a molecular weight cut-off of 12 000-14 000 and an nitrate R. Add, with stirring, 2 mL of nitric add R and titrate
inflated diameter of 3-6 cm (flat width of 5-9 cm) in water R with 0.1 M silver nitrate, determining the end-point
to hydrate until pliable, appropriately sealing one end. potentiometrically (2.2.20).
Introduce 2.0 g of the substance to be examined into the 1 mL of 0.1 M silver nitrate is equivalent to 3.55 mg of CL
tube and add 10 mL of water R. Seal the tube and
completely immerse it in 100 mL of water R in a suitable Heavy metals (2.48)
vessel and stir the liquid for 16 h to effect dialysis. Use the Maximum 20 ppm.
dialysate as test solution. 1.0 g complies with test F. Prepare the reference solution
Reference solution Prepare the reference solution in a similar
manner but using 10 mL of a freshly prepared 0.1 g/L Loss on drying (2.2.32)
solution of benzyltrimethylammonium chloride R instead of the Maximum 12 per cent, determined on 1.000 g by drying in
substance to be examined. an oven at 70 °C over diphosphorus pentoxide R at a pressure
Transfer 5.0 mL of the test solution to a separating funnel not exceeding 7 kPa for 16 h.
and add 5 ml. of a 3.8 g/L solution of disodium tetraborate R, Sulfated ash (2.414)
1 mL of a solution containing 1.5 g/L of bromothymol blue R Maximum 0.1 per cent, determined on 1.0 g.
and 4.05 g/L of sodium carbonate R and 10 mL of ASSAY
chloroform R. Shake the mixture vigourously for 1 min, allow
Exchange capacity
the phases to separate and transfer the clear organic layer to
a 25 mL volumetric flask. Repeat the extraction with a
Solution A Dissolve 1.500 g of sodium glycocholate R in a
further 10 mL of chloroform R, combine the organic layers
and dilute to 25 mL with chloroform R. Measure die solution containing 4 g/L of potassium dihydrogen phosphate R
absorbance (2.2.25) of the solution at the absorption and 12 g/L of dtpotassiitm hydrogen phosphate R and dilute to
maximum at 420 nm, using as compensation liquid a 100.0 mL with die same solution.
solution prepared in the same manner but using 5.0 mL of Test solution Add 20.0 mL of solution A to a quantity of die
water R instead of the test solution. substance to be examined equivalent to about 0.100 g of the
Repeat the operation using 5.0 mL of the reference solution. dried substance. Shake mechanically for 2 h and centrifuge
for 15 min. Dilute 5.0 mL of the supernatant to 50.0 mL
The absorbance obtained with the test solution is not greater
with water R.
than that obtained with the reference solution.
Reference solution (a) Dilute 4.0 mL of solution A to
Im purity A 100.0 mL with water R.
Reference solution (b) Dissolve 60 mg of sodium glycocholate R
Test solution Shake 5.0 g with 10 mL of acetone R for 30 min.
and 30 mg of sodium taurodeoxychdate R in water R and
Centrifuge and use the supernatant. dilute to 100 mL with the same solvent Dilute 1 mL of the
Reference solution (a) Dissolve 5 mg of styrene R in acetone R solution to 10 mL with water R.
and dilute to 100.0 mL with the same solvent. Dilute 1.0 mL Column:
of the solution to 100.0 mL with acetone R. — size: I = 0.25 m, 0 = 4.6 mm,
Reference solution (b) Dissolve 0.35 mL of styrene R in — stationary phase: octadecylsüyl silica gelfor chromatography R
acetone R and dilute to 100.0 mL with the same solvent. (5 pm).
Dilute 1.0 mL of the solution to 100.0 mL with acetone R. Mobile phase Mix 35 volumes of acetonitrüe R and 65 volumes
Reference solution (c) Dissolve 0.35 mL of toluene R in of a 10.9 g/L solution of potassium dihydrogen phosphate R
acetone R and dilute to 100.0 mL with the same solvent. adjusted to pH 3.0 with phosphoric acid R.
Reference solution (d) Mix 1.0 mL of reference solution (b) Flow rate 1.5 mL/min.
and 1.0 mL of reference solution (c) with acetone R and Detection Spectrophotometer at 214 nm.
dilute to 100.0 mL with the same solvent.
2016 Colistimethate Sodium 1-649
Injection50 jiL. Evaporate to dryness on a water-bath and continue the

Run time Twice the retention time of glycocholate. heating until the hydrochloric add has evaporated. Dissolve
the residue in 0.5 mL of water R.
— resolution: m in im u m 1 .5 between the peaks due to Reference solution (a) Dissolve 20 mg of leucine R in water R
glycocholate and taurodeoxycholate. and dilute to 10 mL with the same solvent.
Calculate the nominal exchange capacity using the following Reference solution (b) Dissolve 20 mg of threonine R in water R
expression: and dilute to 10 mL with the same solvent.
Reference solution (c) Dissolve 20 mg of phenylalanine R in
(2.5 A i — A 2) x m i x 1.2 water R and dilute to 10 mL with the same solvent.
12.5 x A i x m 2 Reference solution (d) Dissolve 20 mg of serine R in water R
and dilute to 10 mL with the same solvent.
A\ = area of the peak due to glycocholate in the Plate TLC silica gel G plate R.
chromatogram obtained with reference solution
Cany out thefollowing procedures protectedfrom light.
(a),
A2 = area of the peak due to glycocholate in the Mobile phase water R, phenol R (25:75 VIV).
chromatogram obtained with the test solution, Application 5 |iL as bands of 10 mm, then place the plate in
mx = mass, in m illigrams, of sodium glycocholate R used the chromatographic tank so that it is not in contact with the
in the preparation of solution A, mobile phase, and allow it to become impregnated with the
m2 — mass, in milligrams, of the dried substance to be vapour of the mobile phase for at least 12 h.
exam in ed used in the preparation of the test Development Over a path of 12 cm using the same mobile
solution, phase.
1.2 = correction factor to convert the true exchange Drying At 100-105 °C.
capacity to die conventionally used nominal
Detection Spray with ninhydrin solution R l and heat at 110 °C
exchange capacity.
for 5 min.
STORAGE Results The chromatogram obtained with the test solution
In an airtight container. shows zones corresponding to those in the chromatograms
IMPURITIES obtained with reference solutions (a) and (b), but shows no
zones corresponding to those in the chromatograms obtained
with reference solutions (c) and (d); the chromatogram
A. styrene. obtained with the test solution also shows a zone with a very
__________________________________________________________ PhEur low .Revalue (2,4-diaminobutyric add).
B. Dissolve about 5 mg in 3 mL of water R. Add 3 mL of
dilute sodium hydroxide solution R. Shake and add 0.5 mL of a
10 g/L solution of copper sulfate R. A violet colour is
*****
Colistimethate Sodium ★ ★ produced.
* * * * * C. Dissolve about 50 mg in 1 mL of 1 M hydrochloric acid
and add 0.5 mL of 0.01 M iodine. The solution is
8068-28-8 decolourised and gives reaction (a) of sulfates (2.3.1).
Action and use D. It gives reaction (b) of sodium (2.3.1).
Antibacterial. TESTS
Preparations Appearance of solution
Colistimethate Injection The solution is clear (2.2.1).
Colistimethate Sodium Powder for Nebuliser Solution Dissolve 0.16 g in 10 mL of water R.
pH (2.2.3)
6.5to 8.5.
DEFINITION Dissolve 0.1 g in carbon dioxide-free water R and dilute to
Colistimethate sodium is prepared from colistin by the action 10 mL with the same solvent. Measure after 30 min.
of formaldehyde and sodium hydrogen sulfite.
Semi-synthetic product derived from a fermentation product. —46 to -51 (dried substance).
Content Dissolve 1.25 g in water R and dilute to 25.0 mL with the
M in im u m 11 500 IU/mg (dried substance). same solvent.
CHARACTERS Free colistin
Appearance Dissolve 80 mg in 3 mL of water R. Add 0.1 mL of a
White or almost white, hygroscopic powder. 100 g/L solution of sSicotungsdc acid R; 10-20 s after addition
Solubility of the reagent, the solution is not more opalescent than
Very soluble in water, slighdy soluble in ethanol reference suspension II (2.2.1).
(96 per cent), practically insoluble in acetone. Total sulfite
Work in a fume cupboard. Dissolve 0.100 g in 50 mL of
IDENTIFICATION
water R and add 5 mL of a 100 g/L solution of sodium
hydroxide R and 0.3 g of potassium cyanide R. Boil gently
for
Test solution Dissolve 5 mg of the substance to be examined 3 min and then cool. Neutralise with 0.5 M sulfuric acid using
in 1 mL of a mixture of equal volumes of hydrochloric acid R 0.2 mL of methyl orange solution R as indicator. Add an
and water R. Heat at 135 °C in a sealed tube for 5 h.
1-650 Colistin Sulfate 2016
excess of 0.5 mL of the add and 0.2 g of potassium iodide R. Solubility

Titrate with 0.05 M iodine using 1 mL of starch solution R as Freely soluble in water, practically insoluble in acetone and
indicator. The volume of 0.05 M iodine used in the titradon in ethanol (96 per cent).
is 5.5 mL to 7.0 mL. IDENTIFICATION
Loss on drying (2.2.32) First identification B, E
Maximum 5.0 per cent, determined on 1.000 g by drying at Second identification A, C, D, E
60 °C over diphosphorus pentoxide R at a pressure not A. Thin-layer chromatography (2.2.27).
exceeding 670 Pa for 3 h.
Sulfa ted ash (2.4.14) in 1 mL of a mixture of equal volumes of hydrochloric acid R
16 per cent to 21 per cent, determined on 0.50 g. and water R. Heat at 135 °C in a sealed tube for 5 h.
Pyrogens (2.6.8) Evaporate to dryness on a water-bath and continue the
If intended for use in the manufacture of parenteral hearing until moistened blue litmus paper R does not turn red.
preparations without a further appropriate procedure for Dissolve the residue in 0.5 mL of water R.
removal of pyrogens, it complies with the test Inject, per Reference solution (a) Dissolve 20 mg of leucine R in water R
kilogram of the rabbit's mass, 1 mL of a solution in waterfor and dilute to 10 mL with the same solvent.
injections R containing 2.5 mg of the substance to be
Reference solution (b) Dissolve 20 mg of threonine R in water R
examined per millilitre.
and dilute to 10 mL with the same solvent
ASSAY Reference solution (c) Dissolve 20 mg of phenylalanine R in
Carry out the microbiological assay of antibiotics (2.7.2). water R and dilute to 10 mL with the same solvent.
STORAGE Reference solution (d) Dissolve 20 mg of serine R in water R
In an airtight container, protected from light If the substance and dilute to 10 mL with the same solvent.
is sterile, store in a sterile, airtight, tamper-proof container. Plate TLC silica gel G plate R.
__________________________________________________________ Ph Bur Carry out thefollowing procedures protectedfrom light
Mobile phase water R, phenol R (25:75 VIV).
Application 5 pL as bands of 10 mm, then place the plate in
the chromatographic tank so that it is not in contact with the
Colistin Sulfate ★***★ mobile phase, and allow it to become impregnated with the
★ ★
vapour of the mobile phase for at least 12 h.
Colistin Sulphate *****
Development Over half of the plate.
(Ph. Eur. monograph 0320) Drying At 105 °C.
Detection Spray with ninhydrin solution R1 and heat at 110 °C
for 5 min.
- l-DAB - L-Thr —l-DAB —l-DAB -♦ l-DAB ■» X -»■L-Leu -i
R3 t- L-Thr * l-DAB - l-DAB -J
-R2 shows zones corresponding to those in the chromatograms
i X H2 SO4 obtained with reference solutions (a) and (b), but shows no
DAB = 2,4-diaminobutanoic add zones corresponding to those in the chromatograms obtained
with reference solutions (c) and (d); the chromatogram
polymyxin X R1 R2 R3 Mol. Formula Mr
obtained with the test solution also shows a zone with a very
E1 D-Leu ch3 ch3 H Cs3Hl00N16Ol3 1170
low R v alu e (2,4-diaminobutyric add).
E2 D-leu ch3 H H C52H98 N16O 13 1155
E3 D-Leu H ch3 H Cs2H9eN16O i3 1155
composition.
E1-I o-lle ch3 CHa H C^H-iooN-igO-ia 1170 Results The peaks due to polymyxin E l and polymyxin E2 in
E1-7MOA d-Lsu H ch3 ch3 C53HiooNieOi3 1170 the chromatogram obtained with the test solution are similar
in retention time to die corresponding peaks in the
chromatogram obtained with reference solution (a).
1264-72-8
C. Dissolve about 5 mg in 3 mL of water R. Add 3 mL of
dilute sodium hydroxide solution R. Shake and add 0.5 mL of a
Action and use
Antibacterial. 10 g/L solution of copper sulfate R. A violet colour is
produced.
Preparation D. Dissolve about 50 mg in 1 mL of 1 M hydrochloric acid
Colistin Tablets and add 0.5 mL of 0.01 M iodine. The solution remains
PhEir_____________ coloured.
E. It gives reaction (a) of sulfates (2.3.1).
DEFINITION
A mixture of the sulfates of polypeptides produced by certain TESTS
strains of Bacillus polymyxa var. coUsdnus or obtained by any pH (2.2.3)
other means. 4.0 to 6.0.
Content Dissolve 0.1 g in carbon dioxide-free water R and dilute to
Minimum 19 000 IU/mg (dried substance). 10 mL with the same solvent.
CHARACTERS —73 to —63 (dried substance).
Appearance Dissolve 1.25 g in water R and dilute to 25.0 mL with the
White or almost white, hygroscopic powder. same solvent.
2016 Copovidone 1-651
Composition Sulfate
Liquid chromatography (2.2.29): use the normalisation 16.0 per cent to 18.0 per cent (dried substance).
procedure. Dissolve 0.250 g in 100 mL of water R and adjust to pH 11
Test solution Dissolve 25.0 mg of the substance to be with concentrated ammonia R. Add 10.0 m l . of 0.1 M barium
examined in 40 mL of water R and dilute to 50.0 ml. with chloride and about 0.5 mg of phthalein purple R. Titrate with
acetomtrUe R l. 0.1 M sodium edetaxe, adding 50 mL of ethanol (96 per cent) R
Reference solution (a) Dissolve 25.0 mg of colistin sulfate CRS when the colour of the solution begins to change and
in 40 mL of water R and dilute to 50.0 mL with continuing the titration until the violet-blue colour
acetonitrUe R l. disappears.
Dilute 1.0 mL of reference solution (a)
Reference solution (b) 1 mL of 0.1 M barium chloride is equivalent to 9.606 mg
to 100.0 mL with a mixture of 20 volumes of acetonitrUe R l of S04.
and 80 volumes of water R. Loss on drying (2.2.52)
Column: M axim um 3.5 per cent, determined on 1.000 g by drying at
— size: I = 0.15 m, 0 = 4.6 mm; 60 °C over diphosphorus pentoxide R at a pressure not
— stationary phase: end-capped octadecylsUyl sUica gel for exceed in g 0.67 kPa for 3 h.
chromatography R (3.5 jun); Sulfated ash (2.4.14)
— temperature: 30 °C. Maximum 1.0 per cent, determined on 1.0 g.
Mobile phase Mix 22 volumes of acetomtrUe R l and ASSAY
78 volumes of a solution prepared as follows: dissolve 4.46 g Carry out the microbiological assay of antibiotics (2.7.2).
of anhydrous sodium sulfate R in 900 mL of water R, adjust to
STORAGE
pH 2.4 with dilute phosphoric add R and dilute to 1000 ml.
with water R. In an airtight container, protected from light
__________________________________________________________PhEur
Injection 20 jiL.
Copovidone ★ ★
Run time 1.5 times the retention time of polymyxin El. ★ ★
★ ★
Identification ofpeaks Use the chromatogram supplied with (Ph Eur monograph 0891) ***
colistin sulfate CRS to identify the peaks due to polymyxins
El, E2, E3, E l-I and E1-7MOA.
Relative retention With reference to polymyxin E l (retention
time = about 16 mm): polymyxin E2 = about 0.45;
polymyxin E3 = about 0.5; polymyxin El-I = about 0.8;
polymyxin E1-7MOA = about 1.1.
System suitabUity. reference solution (a):
— resolution: minimum 8.0 between the peaks due to (QHçNO)*, (C4H60 2)m Mt(lll.l) n + (86 . 1)„ 25086-89-9
polymyxin E2 and polymyxin El; minimum 6.0 between
the peaks due to polymyxin E2 and polymyxin El-I; Action and use
minimum 2.5 between the peaks due to polymyxin E l-I Excipient in pharmaceutical products.
and polymyxin E l; minimum 1.5 between the peaks due
PhEur.
to polymyxin E l and polymyxin E1-7MOA.
Limity. DEFINITION
— polymyxin E3: maximum 10.0 per cent (dried substance); Copovidone is a copolymer of 1-ethenylpyrrolidin-2 -one and
— polymyxin El-I: maximum 10.0 per cent (dried ethenyl acetate in the mass proportion 3:2.
substance); Content
— polymyxin E1-7MOA: maximum 10.0 per cent (dried — nitrogen (N; A r 14.01): 7.0 per cent to 8.0 per cent
substance); (dried substance),
— sum ofpolymyxins E l, E2, E3, E l-I and E1-7MOA: — ethenyl acetate CjHeO^ 86.10): 35.3 per cent to
minimum 77.0 per cent (dried substance); 42.0 per cent (dried substance).
— disregard limit. the area of the peak due to polymyxin E l
K-value 90.0 per cent to 110.0 per cent of the value stated
in the chromatogram obtained with reference solution (b) on the label.
( 1.0 per cent).
CHARACTERS
Related substances
Liquid chromatography (2.2.29) as described in the test for Aspect,white or yellowish-white hygroscopic powder or flakes.
composition with the following modifications. Use the Solubility
normalisation procedure. Freely soluble in water, in ethanol (96 per cent) and in
Injection Test solution and reference solution (b). methylene chloride.
Limits: IDENTIFICATION
— any impurity, maximum 4.0 per cent; First identification: A.
— total: maximum 23.0 per cent; Second identification B, C
— disregard limit, the area of the peak
due to polymyxin El A. Infrared absorption spectrophotometry (2.2.24).
in the chromatogram obtained with reference solution (b);
Comparison Ph. Eur. reference spectrum of copovidone.
disregard the peaks due to polymyxins E l, E2, E3, E l-I
and E1-7MOA. B. To 1 mL of solution S (see Tests) add 5 mL of water R
and 0.2 mL of 0.05 M iodine. A red colour appears.
1-652 Copovidone 2016
C. Dissolve 0.7 g of hydroxylamine hydrochloride R in 10 mL Ai = absorbance of the reference solution before the
of methanol R, add 20 mL of a 40 g/L solution of sodium addition of aldehyde dehydrogenase;
hydroxide R and filter if necessary. To 5 mL of the solution ■
A-t2 = absorbance of the reference solution after the
add 0.1 g of the substance to be examined and boil for addition of aldehyde dehydrogenase;
2 min. Transfer 50 pL to a filter paper and add 0.1 mL of a Abi = absorbance of the blank before the addition of
mixture of equal volumes offerric chloride solution R l and aldehyde dehydrogenase;
hydrochloric acid R. A violet colour appears. A/,2 = absorbance of the blank after the addition of
aldehyde dehydrogenase;
TESTS
m = mass of povidone, in grams, calculated with
Solution S
-reference to the dried substance;
C = concentration (mg/ml), of acetaldehyde in the
same solvent. Add the substance to be examined to the
reference solution, calculated from the weight of
water R in small portions with constant stirring.
the acetaldehyde ammonia trimer trihydrate with
Appearance o f solution the factor 0.72.
Solution S is not more opalescent than reference
suspension HI (2.2.1) and not more intensely coloured than Peroxides
reference solution B5, R 5 or BY5 (2.2.2, Method II). Maximum 400 ppm, expressed as H 20 2.
Dilute 10 mL of solution S to 25 mL with water R.
Viscosity, expressed as ft-vahie
Add 2 mL of titanium trichloride-sulfuric acid reagent R and
Dilute 5.0 mL of solution S to 50.0 mL with water R. Allow
allow to stand for 30 min. The absorbance (2.2.25) of the
to stand for 1 h and determine the viscosity (2.2.9) of the
solution, measured at 405 nm using a mixture of 25 mL of a
solution at 25 ± 0.1 °C, using a size n° 1 viscometer with a
40 g/L solution of the substance to be examined and 2 mL of
minimum flow time of 100 s. Calculate the iC-value using the
a 13 per cent V/V solution of sulfuric acid R as the
compensation liquid, is not greater than 0.35.
Hydrazine
1.51ogio V ~ 1 , \/300c l°gio r) + (c + l-5c l°gi° r?)2 Thin-layer chromatography (2.2.27). Usefreshly prepared
solutions.
0.15 + 0.003c + 0.15c + 0.003c2
Test solution To 25 mL of solution S add 0.5 mL of a 50 g/L
c = percentage concentration (g/100 mL) of the solution of salicylaldehyde R in methanol R, mix and heat in a
substance to be examined, calculated with reference water-bath at 60 °C for 15 min. Allow to cool, add 2.0 mL
to the dried substance; of xylene R, shake for 2 min and centrifuge. Use the clear
rj = viscosity of the solution relative to that of water. supernatant layer.
Reference solution Dissolve 9 mg of salicylaldehyde azine R in
Aldehydes
xylene R and dilute to 100 mL with the same solvent. Dilute
Maximum 500 ppm, expressed as acetaldehyde.
1 mL of the solution to 10 mL with xylene R.
Hate TLC sUamsed silica gel plate R.
in phosphate buffer solution p H 9.0 R and dilute to 100.0 mL
with the same solvent. Stopper the flask and heat at 60 °C Mobile phase water R, methanol R (20:80 VtV).
for 1 h. Allow to cool. Application 10 jiL.
Reference solution Dissolve 0.140 g of acetaldehyde ammonia Development Over 3/4 of the plate.
trimer trihydrate R in water R and dilute to 200.0 mL with the Drying In air.
same solvent. Dilute 1.0 mL of the solution to 100.0 mL Detection Examine in ultraviolet light at 365 nm.
with phosphate buffer solution pH 9.0 R. Limit:
Into 3 identical spectrophotometric cells with a path length — hydrazine: any spot due to salicylaldehyde azine is not
of 1 cm, introduce separately 0.5 mL of the test solution, more intense than the spot in the chromatogram obtained
0.5 mL of the reference solution and 0.5 mL of water R with the reference solution (1 ppm).
(blank). To each cell add 2.5 mL of phosphate buffer solution
pH 9.0 R and 0.2 mL of nicotmamide-adenme dinucleotide
Monomers
solution R. Mix and stopper tightly. Allow to stand at
22 ± 2 °C for 2-3 min and measure the absorbance (2.2.25) Dissolve 10.0 g in 30 mT. of methanol R and add slowly
of each solution at 340 nm, using water R as the 20.0 mL of iodine bromide solution R. Allow to stand for
compensation liquid. To each cell, add 0.05 mL of aldehyde 30 min protected from light with repeated shaking.
dehydrogenase solution R, mix and stopper tightly. Allow to Add 10 mL of a 100 g/L solution of potassium iodide R and
stand at 22 ± 2 °C for 5 min. Measure the absorbance of titrate with 0.1 M sodium tMosulfate until a yellow colour is
each solution at 340 nm using water R as compensation obtained. Continue titration dropwise until the solution
liquid. Determine the content of aldehydes using the becomes colourless. Carry out a blank titration. Not more
following expression: than 1.8 mL of 0.1 M sodium thiosulfate is used.
Im purity A
(At 2 — A t\) — (Ab2 — A\,x)100 000 x C Liquid chromatography (2.2.29).
(A, 2 —At \) — (Ab2 —A n ) m Test solution Dissolve 0.100 g of the substance to be
A ti = absorbance of the test solution before the addition solvent.
of aldehyde dehydrogenase; Reference solution Dissolve 0.100 g of 2-pyrrolidone R
A [2 = absorbance of the test solution after the addition (impurity A) in water R and dilute to 100 mL with the same
of aldehyde dehydrogenase; solvent. Dilute 1.0 ml. to 100.0 mL with water R.
2016 Copper Sulfate 1-653
Precolumn: criteria. In such cases, a cross-reference to the tests described in the

— size. I = 0.025 m, 0 = 4 nun; mandatory part is included in the Functionality-related
— stationary phase: end-capped octadecylsũyl silica gel for characteristics section. Control of the characteristics can contribute
chromatography R (5 pm). to the quality of a medicinal product by improving the consistency
Column: of the manvfactuxmg process and the performance of the medicinal
— sizer. I = 0.25 m, 0 = 4 mm; product during use. Where control methods are cited, they are
— stationary phase: spherical aminohexadecyhủyl sũica gelfor recognised as being suitable for the purpose, but other methods can
chromatography R (5 pm); also be used Wherever resultsfor a particular characteristic are
— temperature: 30 °c. reported, the control method must be indicated
Mobile phase water R adjusted to pH 2.4 with phosphoric The following characteristics may be relevantfor copovidone used
acid R. as binder in tablets and granules.
Flow rate 1 mL/min. - Viscosity (2.2.9)

Detection Spectrophotometer at 205 nm. A detector is placed Determine the dynamic viscosity usjpg a capillary viscometer
between the precolumn and the analytical column. A second on a 10 per cent solution (dried substance) or on a
detector is placed after the analytical column. 20 per cent solution (dried substance) at 25 °C. It is typically
about 8 mPa-s or about 23 mPa-s, respectively.
Injection 10 pL. When impurity A has left the precolumn
(after about 1.2 min) switch the flow directly from the pump Pardcle-size distribution (2.9.31 or 2.9.38).
to the analytical column. Before the next chromatogram is Bulk and tapped density (2.9.34)
run, wash die precolumn by reversed flow. The following characteristic may be relevantfor copovidone used
Limit. as film former in coated dosageforms and in aerosols.
— impurity A: not more than the area of the principal peak Viscosity (2.2.9)
in the chromatogram obtained with the reference solution See above.
(0.5 per cent).
__________________________________________________________ PhEur
Maximum 20 ppm.
12 mL of solution s complies with test A. Prepare die
reference solution using lead standard solution (2 ppm Pb) R. Anhydrous Copper Sulfate *****
Loss on drying c2.2.32) *★ ***
M axim u m 5.0 per cent, determined on 0.500 g by drying in Anhydrous Copper Sulphate
an oven at 105 °c. (Ph. Eur. monograph 0893)
Sulfated ash (2.4.14) CuS04 159.6 7758-98-7

Action and use
ASSAY Used in treatment of copper deficiency.
Ethenyi acetate
Determine the saponification value (2.5.6) on 2.00 g of tile PhEtr__________________________________________________________
substance to be examined- Multiply the result obtained by DEFINITION
0.1534 to obtain the percentage content of the ethenyl Content
acetate component. 99.0 per cent to 101.0 per cent (dried substance).
Nitrogen
CHARACTERS
Carry out die determination of nitrogen (2.5.9) using
30.0 mg of die substance to be examined and 1 g of a Appearance
Greenish-grey powder, very hygroscopic.
mixture of 3 parts of copper sulfate R and 997 parts of
dxpotassium sulfate R, heating until a clear, light green solution Solubility
is obtained and then for a further 45 min. Freely soluble in water, slightly soluble in methanol,
practically insoluble in ethanol (96 per cent).
STORAGE
In an airtight container. IDENTIFICATION
A. Add several drops of dilute ammonia R2 to 1 mL of
LABELLING solution S (see Tests). A blue precipitate is formed.
The label states the -fC-value. On further addition of dilute ammonia R2 the precipitate
IMPURITIES dissolves and a dark blue colour is produced.
B. Loss on drying (see Tests).
C. Dilute 1 mL of solution S to 5 mL with water R.
The solution gives reaction (a) of sulfates (2.3.1).
à r °
TESTS
A. pyrrolidin-2-one (2-pyrrolidone). Solution S
FUNCTIONALITY-RELATED CHARACTERISTICS solvent.
Solution S is clear (2.2.1).
functions of the substance when used, as an excipient (see chapter
5.15). Some of the characteristics described in the Functionality- Chlorides (2.44)
related characteristics section may also be present in the mandatory M axim u m 150 ppm.
part of the monograph since they also represent mandatory quality Dilute 10 mL of solution S to 15 mL with water R.
1-654 Copper Sulfate 2016
Iron Solubility
Maximum 150 ppm. Freely soluble in water, soluble in methanol, practically
Atomic absorption spectrometry (2.2.25, Method I). insoluble in ethanol (96 per cent).
Test solution Dissolve 0.32 g in 10 mL of water R, add IDENTIFICATION
2.5 mL of lead-free nitric acid R and dilute to 25.0 mL with A. Add several drops of dilute ammonia R2 to 1 mL of
water R. solution S (see Tests). A blue precipitate is formed.
Reference solutions Prepare the reference solutions using iron On further addition of dilute ammonia R2 the precipitate
standard solution (20 ppm Fe) R, adding 2.5 mL of lead-free dissolves and a dark blue colour is produced.
nitric add R and diluting to 25.0 mL with water R. B. Loss on drying (see Tests).
Source Iron hollow-cathode lamp. C. Dilute 1 mL of solution S to 5 mL with water R.
Wavelength 248.3 nm. The solution gives reaction (a) of sulfates (2.3.1).
Atomisation device Air-acetylene flame. TESTS
Copper may form explosive acetyUdes with acetylene. Therefore, Solution S
clean the burner thoroughly before any residues become dry. Dissolve 5 g in water R and dilute to 100 mL with the same
Lead solvent.
Maximum 80 ppm. Appearance of solution
Atomic absorption spectrometry (2.2.25, Method I). Solution S is clear (2.2.1).
Test solution Dissolve 1.6 g in 10 mL of water R, add 2.5 mL Chlorides (2.4.4)
of lead-free nitric add R and dilute to 25.0 mL with water R. M axim um 100 ppm.
Reference solutions Prepare the reference solutions using lead Dilute 10 mL of solution S to 15 m l . with water R.
standard solution (100 ppm Pb) R, adding 2.5 mL of lead-free Iron
nitric add R and diluting to 25.0 ml. with water R. Maximum 100 ppm.
Source Lead hollow-cathode lamp. Atomic absorption spectrometry (2.2.23, Method I).
Wavelength 217.0 nm. Test solution Dissolve 0.5 g in 10 mL of water R, add 2.5 mL
Atomisation device Air-acetylene flame. of lead-free nitric add R and dilute to 25.0 mL with water R.
Copper may form explosive acetyUdes with acetylene. Therefore, Reference solutions Prepare the reference solutions using iron
dean the burner thoroughly before any residues become dry. standard solution (20 ppm Fe) R, adding 2.5 mL of lead-free
nitric add R and diluting to 25.0 m l . with water R.
Maximum 1.0 per cent, determined on 0.500 g by drying in Source Iron hollow-cathode lamp.
an oven at 250 ± 10 °C. Wavelength 248.3 nm.
ASSAY Atomisation device Air-acetylene flame.

Dissolve 0.125 g in 50 mL of water R. Add 2 mL of sulfuric Copper may form explosive acetyUdes with acetylene. Therefore,
add R and 3 g of potassium iodide R. Titrate with 0.1 M clean the burner thoroughly before any residues become dry.
sodium tkiosulfate, using 1 ml. of starch solution R, added Lead
towards the end of the titration. Maximum 50 ppm.
1 mL of 0.1 M sodium tkiosulfate is equivalent to 15.96 mg of Atomic absorption spectrometry (2.2.23, Method I).
CuS04.
Test solution Dissolve 2.5 g in 10 m l . of water R, add 2.5 mL
STORAGE of lead-free nitric add R and dilute to 25.0 mL with water R.
In an airtight container. Reference solutions Prepare the reference solu tion s u sin g lead
__________________________________________________________ PhEur standard solution (100 ppm Pb) R, adding 2.5 m l . of lead-free
nitric add R and diluting to 25.0 mL with water R.
Copper Sulfate Pentahydrate ***** Atomisation device Air-acetylene flame.

*+ +* Copper may form explosive acetyUdes with acetylene. Therefore,
Copper Sulphate Pentahydrate * dean the burner thoroughly before any residues become dry.
CuS04 ,5H 20 249.7 7758-99-8 35.0 per cent to 36.5 per cent, determined on 0.500 g by
drying in an oven at 250 ± 10 °C.
Action and use
Used in treatment of copper deficiency. ASSAY
Dissolve 0.200 g in 50 mL of water R. Add 2 mL of sulfuric
PhEir__________________________________________________________ add R and 3 g of potassium iodide R. Titrate with 0.1 M
DEFINITION sodium tkiosulfate, adding 1 mL of starch solution R towards
Content the end of the titration.

99.0 per cent to 101.0 per cent. 1 mL 0.1 M sodium tkiosulfate is equivalent to 24.97 mg of
CuS04,5H20 .
CHARACTERS
------ ------ ------------------------------------------------- PhEur
Appearance
Blue, crystalline powder or transparent, blue crystals.
2016 Cortisone Acetate 1-655
***** Detection B Spray with alcoholic solution of sulfuric acid R. Heat

Cortisone Acetate ★ ★ at 120 °C for 10 min or until the spots appear. Allow to
***** cool. Examine in daylight and in ultraviolet light at 365 nm.
Results B The principal spot in the chromatogram obtained
with the test solution is similar in position, colour in daylight,
solution (a).
C23H30O5 402.5 50-04-4 C. Thin-layer chromatography (2.2.27).
Test solution (a) Dissolve 25 mg of the substance to be
Action and use examined in methanol R with gentle heating and dilute to
Corticosteroid. 5 mL with the same solvent (solution A). Dilute 2 mL of this
Preparation solution to 10 mL with methylene chloride R.
Cortisone Tablets Test solution (b) Transfer 2 mL of solution A to a 15 mL
glass tube with a ground-glass stopper or
PhEur______________
a polytetrafluoroethylene cap. Add 10 mL of saturated
DEFINITION methanotic potassium hydrogen carbonate solution R and
17-Hydroxy-3,l l,20-trioxopregn-4-en-21-yl acetate. immediately pass a stream of nitrogen R briskly through the
Content solution for 5 min. Stopper the tube. Heat in a water-bath at
97.0 per cent to 103.0 per cent (dried substance). 45 °C protected from light for 2.5 h. Allow to cool.
Reference solution (a) Dissolve 25 mg of cortisone acetate CRS
CHARACTERS
in methanol R with gentle heating and dilute to 5 mL with
Appearance
the same solvent (solution B). Dilute 2 mL of this solution to
10 mL with methylene chloride R.
Solubility Reference solution (b) Transfer 2 mL of solution B to a 15 mL
Practically insoluble in water, freely soluble in methylene glass tube with a ground-glass stopper or
chloride, soluble in dioxan, sparingly soluble in acetone, a polytetrafluoroethylene cap. Add 10 mL of saturated
slightly soluble in ethanol (96 per cent) and in methanol. methanolic potassium hydrogen carbonate solution R and
It shows polymorphism (5.9). immediately pass a stream of nitrogen R briskly through the
IDENTIFICATION solution for 5 min. Stopper the tube. Heat in a water-bath at
First identification: A , B. 45 °C protected from light for 2.5 h. Allow to cool.
C, D, E
Second identification Plate TLC silica gel F 254 plate R.
A. Infrared absorption spectrophotometry (2.2.24). Mobile phase Add a mixture of 1.2 volumes of water R and
Comparison cortisone acetate CRS.
ether R and 77 volumes of methylene chloride R.
Application 5 |iL.
record new spectra using 50 g/L solutions in methylene
chloride R in a 0.2 mm celL Development Over a path of 15 cm.
B. Thin-layer chromatography (2.2.27). Drying In air.
Solvent mixture methanol R, methylene chloride R (1:9 VIV). Detection A Examine in ultraviolet light at 254 nm.
Test solution Dissolve 10 mg of the substance to be examined Results A The principal spot in each of the chromatograms
in the solvent mixture and dilute to 10 mL with the solvent obtained with the test solutions is similar in position and size
mixture. to the principal spot in the chromatogram obtained with the
corresponding reference solution.
Reference solution (a) Dissolve 20 mg of cortisone acetate CRS
in the solvent mixture and dilute to 20 mL with the solvent Detection B Spray with alcoholic solution of sulfuric acid R and
mixture. heat at 120 °C for 10 min or until the spots appear. Allow to
cool. Examine in daylight and in ultraviolet light at 365 nm.
Reference solution (b) Dissolve 10 mg of hydrocortisone
acetate R in reference solution (a) and dilute to 10 mL with Results B The principal spot in each of the chromatograms
reference solution (a). obtained with the test solutions is similar in position, colour
in daylight, fluorescence in ultraviolet light at 365 nm and
Plate T LC silica gel F 254 plate R.
size to the principal spot in the chromatogram obtained with
Mobile phase Add a mixture of 1.2 volumes of water R and the corresponding reference solution. The principal spots in
8 volumes of methanol R to a mixture of 15 volumes of the chromatograms obtained with test solution (b) and
ether R and 77 volumes of methylene chloride R. reference solution (b) have an RP value distinctly lower than
Application 5 (iL. that of the principal spots in the chromatograms obtained
Development Over a path of 15 cm. with test solution (a) and reference solution (a).
Drying In. air. D. Add about 2 mg to 2 mL of sulfuric acid R and shake to
Detection A Examine in ultraviolet light at 254 nm. dissolve. Within 5 min, a faint yellow colour develops.
Add this solution to 10 mL of water R and mix. The colour
Results A The principal spot in the chromatogram obtained
is discharged and a clear solution remains.
principal spot in the chromatogram obtained with reference E. About 10 mg gives the reaction of acetyl (2.3.1).
solution (a).
1-656 Cottonseed Oil 2016
TESTS
Dissolve 0.250 g in dioxan R and dilute to 25.0 mL with the
same solvent.
Related substances
Liquid chromatography (2.2.29). Prepare the solutions A. ll|3,17-dihydroxy-3,20-dioxopregn-4-en-21-yl acetate
immediately before use. (hydrocortisone acetate).
Test solution Dissolve 25.0 mg of the substance to be _____________________________________________ PhEur
examined in acetomtrile R and dilute to 10.0 mL with the
same solvent.
Reference solution (a). Dissolve 2 mg of cortisone acetate CRS
and 2 mg of hydrocortisone acetate CRS (impurity A) in
acetomtrile R and dilute to 100.0 mL with the same solvent
Hydrogenated Cottonseed Oil *****
Reference solution (b) Dilute 1.0 mL of the test solution to (Ph. Eur. monograph 1305) *
100.0 mL with acetonitrUe R. PhEur_____________________________________________
Column:
— sizer. I = 0.25 m, 0 = 4.6 mm; DEFINITION
— stationary phase: octadecylsUyl sUica gel for chromatography R Product obtained by refining and hydrogenation of oil
(5 pm). obtained from seeds of cultivated plants of various varieties of
Gossypium hirsutum L. or of other species of Gossypium.
MobUe phase In a 1000 mL volumetric flask mix 400 mL of
The product consists mainly of triglycerides of palmitic and
acetomtrUe R with 550 mL of water R and allow to
stearic adds.
equilibrate; dilute to 1000 mL with water R and mix again.
Flow rate 1 mlVmin. CHARACTERS
Appearance
White or almost white mass or powder which melts to a
Equilibration With the mobile phase for about 30 min.
clear, pale yellow liquid when heated.
Injection 20 jiL; inject acetomtrUe R as a blank.
Solubility
Run time Twice the retention time of cortisone acetate. Practically insoluble in water, freely soluble in methylene
Retention time Impurity A = about 10 min; cortisone chloride and in toluene, very slightly soluble in ethanol
acetate = about 12 min. (96 per cent).
System suitability: reference solution (a): IDENTIFICATION
— resolution: m in im u m 4.2 between the peaks due to A. Mdting point (see Tests).
impurity A and cortisone acetate; if necessary, adjust the
concentration of acetomtrile in the mobile phase. B. Composition of fatty adds (see Tests).
Limits: TESTS
— impurity A: not more than 0.5 times the area of the Melting point (2.2.14)
principal peak in the chromatogram obtained with 57 °C to 70 °C.
reference solution (b) (0.5 per cent); A d d value (2.5.1)
— total: not more than 1.5 times the area of the principal Maximum 0.5.
peak in the chromatogram obtained with reference Dissolve 10.0 g in 50 mT. of a hot mixture of equal volumes
solution (b) (1.5 per cent); of ethanol (96 per cent) R and toluene R , previously neutralised
— disregard limit: 0.05 times the area of the principal peak in with 0.1 M potassium hydroxide using 0.5 mL of
the chromatogram obtained with reference solution (b) phenolphthalem solution R l as indicator. Titrate the solution
(0.05 per cent).
immediately while still hot.
Loss on drying (2.2.52) Peroxide value (2.5.5, Method A)
Maximum 0.5 per cent, determined on 0.500 g by drying in Maximum 5.0.
an oven at 105 °C.
Unsaponifiable m atter (2.5.7)
ASSAY Maximum 1.0 per cent, determined on 5.0 g.
Alkaline impurities
100.0 mL with the same solvent Dilute 2.0 mL of this
Dissolve by gentle heating 2.0 g in a mixture of 1.5 mL of
solution to 100.0 mL with ethanol (96 per cent) R. Measure
ethanol (96 per cent) R and 3 mL of toluene R. Add 0.05 mL
the absorbance (2.2.25) at the absorption maximum at
of a 0.4 g/L solution of bromophenol blue R in ethanol
237 nm.
(96 per cent) R. Not more than 0.4 mL of 0.01 M hydrochloric
Calculate the content of C23H 30O6 taking the specific add is required to change the colour to yellow.
Composition of fatty acids (2.422, Method A)
STORAGE Use the mixture of calibrating substances in Table 2.4.22.-3.
Protected from light. Column:
IMPURITIES — material: fused silica;
Specified impurities A — size. 1= 25 m, 0 = 0.25 mm;
— stationary phase: poly(tyanopropyl)sibxane R (film thickness
0.2 jxm).
Carrier gas hdium for chromatography R.
2016 Cresol 1-657
Flow rate 0.65 mL/min. B. Add bromine water. A pale yellow flocculent precipitate is
Split ratio 1:100. produced.
Temperature: TESTS
— column: 180 °C for 35 min; Acidity
— injection port and detector. 250 °C. A 2.0% w/v solution is neutral to bromocresolpurple solution.
Detection Flame ionisation. Distillation range
Composition of thefatty-acid fraction of the oU: Not more than 2% v/v distils below 188° and not less than
— saturatedfatty acids of chain length less than Crf. maximum 80% v/v distils between 195° and 205°, Appendix V C.
0.2 per cent; Weight p er mL
— myrisdc acid: maximum 1.0 per cent; 1.029 to 1.044 g, Appendix V G.
— palmitic acid: 19.0 per cent to 26.0 per cent;
— stearic acid: 68.0 per cent to 80.0 per cent; Hydrocarbons
— oleic acid and isomers: maximum 4.0 per cent; Place 5 0 mL in a 5 0 0 mL round-bottomed flask, add
150 mL of 5 m sodium hydroxide and 3 0 mL of water and mix
— linoleic acid and isomers: maximum 1.0 per cent;
— arackidic acid: maximum 1.0 per cent; thoroughly. Connect the flask to a splash-bulb and air-
— behenic acid: maximum 1.0 per cent; condenser about 6 0 cm long, with the end of the air-
—' Ugnoceric acid: maximum 0.5 per cent. condenser fitting closely into the neck of a 2 5 0 mL pear-
shaped separating funnel and passing well into the separating
Nickel funnel, which has a cylindrical graduated portion above the
Maximum 1 ppm. stopcock. Fill the graduated portion of the separating funnel
Atomic absorption spectrometry (2.2.23, Method II). with water. Distil rapidly until 75 mL of distillate has been
Test solution Introduce 5.0 g into a platinum or silica crucible collected, cooling the separating funnel in running water if
tared after ignition. Cautiously heat and introduce into the necessary. Allow the separating funnel to stand in a vertical
substance a wick formed from twisted ashless filter paper. position until separation is complete and draw off the
Ignite the wick. When the substance ignites, stop heating. aqueous liquid into a titration flask for use in the test for
After combustion, ignite in a muffle furnace at about Volatile bases.
600 ± 50 °C. Continue the incineration until white ash is Allow the separating funnel to stand for a few minutes,
obtained. After cooling, take up the residue with 2 quantities, measure the volume of hydrocarbon oil in the graduated
each of 2 mL, of dilute hydrochloric acid R and transfer into a portion and warm, if necessary, to keep the oil in the liquid
25 mL graduated flask. Add 0.3 mL of nitric acid R and state. Subtract the volume of volatile bases in the
dilute to 25.0 mL with distilled water R. hydrocarbon oil, as determined in the following test
Reference solutions Prepare 3 reference solutions by adding Not more than 0.15% v/v of hydrocarbon oil is present.
1.0 mL, 2.0 ml. and 4.0 ml. of nickel standard solution Volatile bases
(0.2 ppm Ni) R to 2.0 mL portions of the test solution, To the aqueous liquid reserved in the test for Hydrocarbons
diluting to 10.0 mL with distilled water R. add any aqueous liquid still remaining in the separating
Source Nickel hollow-cathode lamp. funnel and neutralise, if necessary, with 0 . 1 m hydrochloric acid
Wavelength 232 am. using phenolphthalein solution R l as indicator. Titrate with
1m hydrochloric acid FS using methyl orange solution as
indicator. Wash the oil from the separating funnel into the
Carrier gas argon R.
titration flask with water and again titrate with 1m hydrochloric
STORAGE acid VS. From the volume of additional 1m hydrochloric acid
Protected from light. FS, calculate the proportion of volatile bases in the
Ph Eur hydrocarbon oil. From the total volume of 1m hydrochloric
add VS used in both titrations calculate the volume of
volatile bases in the substance being examined. Each mL of
1m hydrochloric acid FS is equivalent to 0.080 mL of volatile
bases. Not more than 0.15% v/v of volatile bases is present.
Cresol Hydrocarbons and volatile bases
The sum of the contents of hydrocarbon oil and volatile
Action and use bases, as determined in the tests for Hydrocarbons and for
Antiseptic; antimicrobial preservative. Volatile bases, does not exceed 0.25% v/v.
DEFINITION Sulfur compounds
Cresol is a mixture of cresols and other phenols obtained Place 20 mL in a small conical flask and over the mouth of
from coal tar. the flask fix a piece of filter paper moistened with a 10% w/v
solution of lead(n) acetate. Heat the flask on a water bath for
CHARACTERISTICS 5 minutes. Not more than a light yellow colour is produced
An almost colourless to pale brownish yellow liquid. on the filter paper.
Almost completely soluble in 50 volumes of water, freely Non-volatile m atter
soluble in ethanol (96%), in ether and in fixed and volatile
When evaporated on a water bath and dried at 105°, leaves
oils. not more than 0 . 1% w/v of residue.
IDENTIFICATION STORAGE
Shake 0.5 mL with 300 mL of water and filter. The filtrate
Cresol should be protected from light. It darkens with age or
complies with the following tests.
on exposure to light.
A Add inm(m) chloride solution R l. A transient blue colour is
produced.
1-658 Crude Cresol 2016
>** ★
★ .★**
Crude Cresol ★ ★ Croscarmellôse Sodium *
* *
*
***** *****
(Ph. Eur. monograph 1628) (Ph. Eur. monograph 0985)
OH Action and use

h3c - Excipient.
PhEur.
OjUsO 108.1 DEFINITION

Cross-linked sodium carboxymethylcellulose.
Action and use Sodium salt of a cross-linked, partly O-carboxymethylated
Antiseptic. cellulose.
PhEur_____________ CHARACTERS
Appeareance
DEFINITION
White or greyish-white powder.
Mixture of 2-, 3- and 4-methylphenol.
Solubility
CHARACTERS Practically insoluble in acetone, in anhydrous ethanol and in
Appearance toluene.
Colourless or pale brown liquid.
IDENTIFICATION
Solubility A. Mix 1 g with 100 mL of a solution containing 4 ppm of
Sparingly soluble in water, miscible with alcohol and with methylene blue R, stir the mixture and allow it to settle.
methylene chloride. The substance to be examined absorbs the methylene blue
IDENTIFICATION and settles as a blue, fibrous mass.
A. To 0.5 mL add 300 mL of water R, mix and filter. B. Mix 1 g with 50 mL of water R. Transfer 1 mL of the
To 10 mL of the filtrate add 1 mL of ferric chloride mixture to a small test-tube and add 1 mL of water R and
solution R l. A blue colour is produced. 0.05 mL of a freshly prepared 40 g/L solution of
B. To 10 mL of the filtrate obtained in identification test A, a-naphthol R in methanol R. Incline the test-tube and carefully
add 1 mL of bromine water R. A pale yellow flocculent add 2 m l. of sulfuric add R down the side so that it forms a
precipitate is produced. lower layer. A reddish-violet colour develops at the interface.
C. Relative density (see Tests). C. The solution prepared from the sulfated ash in the test for
heavy metals (see Tests) gives reaction (a) of sodium (2.3.1).
TESTS
Solution S TESTS
To 2.5 g of the substance to be examined add 50 mL of pH (2.2.3)
water R, shake for 1 min and filter through a moistened filter. 5.0 to 7.0 for the suspension.
Acidity or alk alin ity Shake 1 g with 100 m l. of carbon dioxide-free water R for
To 10 mL of solution S add 0.1 m l. of methyl red solution R 5 min.
and 0.2 mL of 0.01 M sodium hydroxide. The solution is Sodium chloride and sodium glycollate
yellow. Add 0.3 mL of 0.01 M hydrochloric add. The solution Maximum 0.5 per cent (dried substance) for the sum of the
is red. percentage contents of sodium chloride and sodium
Relative density (2.2.5) glycollate.
1.029 to 1.044. Sodium chloride Place 5.00 g in a 250 mL conical flask, add
Distillation range (2.2.11) 50 mL of water R and 5 mL of strong hydrogen peroxide
A maximum of 2.0 per cent V/V distils below 188 °C and a solution R and heat on a water-bath for 20 min, stirring
minimum of 80 per cent V/V distils between 195 °C and occasionally to ensure total hydration. Cool, add 100 mL of
205 °C. water R and 10 m l. of nitric acid R. Titrate with 0.05 M silver
nitrate, determining the end-point potentiometrically (2.2.20)
Sulfur compounds using a silver indicator electrode and a double-junction
Place 20 mL in a small conical flask. Over the mouth of the reference electrode containing a 100 g/L solution of potassium
flask fix a piece of filter paper moistened with lead acetate nitrate R in the outer jacket and a standard filling solution in
solution R. Heat on a water-bath for 5 min. Not more than a
the inner jacket, and stirring constantly.
light yellow colour is produced on the filter paper.
1 mL of 0.05 M silver nitrate is equivalent to 2.922 mg of
Residue on evaporation NaCl.
Sodium gfycoBate Place a quantity of the substance to be
Evaporate 2.0 g to dryness on a water-bath and dry at examined equivalent to 0.500 g of the dried substance in a
100-105 °C for 1 h. The residue weighs not more than 2 mg. 100 mL beaker. Add 5 mL of glacial acetic add R and 5 mL
STORAGE of water R and stir to ensure total hydration (about 15 min).
Protected from light. Add 50 m l. of acetone R and 1 g of sodium chloride R. Stir for
several minutes to ensure complete precipitation of the
PhEur
carboxymethylcellulose. Filter through a fast filter paper
impregnated with acetone R into a volumetric flask, rinse the
beaker and the filter with 30 mL of acetone R and dilute the
filtrate to 100.0 mL with the same solvent. Allow to stand for
2016 Croscarmellose Sodium 1-659
24 h without shaking. Use the clear supernatant to prepare FUNCTIONALITY-RELATED CHARACTERISTICS

the test solution. This section provides information cm characteristics that are
Prepare the reference solutions as follows: in a 100 mL recognised as bdng relevant control parameters for one or more
volumetric flask, dissolve 0.100 g of gfycoUic add R, functions of the substance when used as an excipient (see chapter
previously dried in vacuo over diphosphorus pentoxide R at 5.15). This section ừ a non-mcmdatory part of the monograph
room temperature overnight, in water R and dilute to and ừ is not necessary to verify the characteristics to demonstrate
100.0 mL with the same solvent; use the solution within compliance. Control of these characteristics can however contribute
30 days; transfer 1.0 m l, 2.0 mT0 3.0 mL and 4.0 mL of the to the quality of a medicinal product by improving the consistency
solution to separate volumetric flasks, dilute the contents of of the manufacturing process and the performance of the medicinal
each flask to 5.0 mL with water R, add 5 mL of glacial acetic product during use. Where control methods are cited, they are
acid R, dilute to 100.0 mL with acetone R and mix. recognised as being suitablefor the purpose, but other methods can
Transfer 2.0 mL of the test solution and 2.0 mL of each of also be used. Wherever resultsfor a particular characteristic are
the reference solutions to separate 25 mL volumetric flasks. reported, the control method must be indicated.
Heat the uncovered flasks for 20 min on a water-bath to The following characteristics may be relevant for croscarmeũose
eliminate acetone. Allow to cool and add 5.0 mL of sodium used as dismtegrant
2,7-dihydroxynaphthalene solution R to each flask. Mix, add a Settling volume
further 15.0 mL of 2,7-dihydroxynaphthalene solution R and Place 75 mL of water R in a 100 mL graduated cylinder and
mix again. Close the flasks with aluminium foil and heat on a add 1.5 g of the substance to be examined in 0.5 g portions,
water-bath for 20 min. Cool and dilute to 25.0 mL with shaking vigorously after each addition. Dilute to 100.0 mL
suljuric add R. with water R and shake again until the substance is
Measure the absorbance (2.2.25) of each solution at 540 nm. homogeneously distributed. Allow to stand for 4 h.
Prepare a blank using 2.0 mL of a solution containing The settling volume is between 10.0 mL and 30.0 mT_
5 per cent VIV each of glacial acetic add R and water R in Degree of substitution
acetone R. Prepare a standard curve using the absorbances 0.60 to 0.85 (dried substance).
obtained with the reference solutions. From the standard
Place 1.000 g in a 500 mL conical flask, add 300 mL of a
curve and the absorbance of the test solution, determine the
100 g/L solution of sodium chloride R and 25.0 mL of 0.1 M
mass (a) of glycollic add in the substance to be examined, in
sodium hydroxide, stopper the flask and allow to stand for
milligrams, and calculate the content of sodium glycollate
5 min, shaking occasionally. Add 0.05 mL of m-cresolpurple
using the following egression:
solution R and about 15 mL of 0.1 M hydrochloric add from a
10 x 1.29 x a burette. Insert the stopper and shake. If the solution is violet,
add 0.1 M hydrochloric acid in 1 mL portions until the
(100 — b)m
solution becomes yellow, shaking after each addition. Titrate
1.29 = the factor converting glycollic acid to sodium with 0.1 M sodium hydroxide until the colour turns to violet.
glycollate; Calculate the number of milliequivalents (M) of base
b = loss on drying as a percentage; required to neutralise the equivalent of 1 g of dried
m — mass of the substance to be examined, in grams. substance.
W ater-soluble substances Calculate the degree of acid carboxymethyl substitution (A)
Maximum 10.0 per cent. using the following egression:
Disperse 10.00 g in 800.0 mL of water R and stir for 1 min
every 10 min during the first 30 min. Allow to stand for 1 h 1150AÍ
and centrifuge if necessary. Decant 200.0 mL of the (7102 - 412AÍ - 80Ơ)
supernatant onto a fast filter paper in a vacuum filtration
funnel, apply vacuum and collect 150.0 mL of the filtrate. c _= sulfated ash as a percentage.
Evaporate to dryness and dry the residue at 100-105 °C for
Calculate the degree of sodium carboxymethyl
4 h.
substitution (S) using the following expression:
Maximum 20 ppm.
(162 + 58i4) c
To the residue obtained in the determination of the sulfated (7102 - 80C)
ash add 1 mL of hydrochloric add R and evaporate on a
water-bath. Take up the residue in 20 mL of water R. 12 mL
of the solution complies with test A. Prepare the reference The degree of substitution is the sum of A and s.
solution using lead standard sdution (1 ppm Pb) JR. Particle size distribution (2.9.31)
Loss on drying (2.2.32) or 2.9.38).
Maximum 10.0 per cent, determined on 1.000 g by drying in Hausner ratio (2.9.36)
an oven at 105 °C for 6 h. ---------------------------------------------------------------------------------------- PhEur
Sulfated ash(2.4.14)
14.0 per cent to 28.0 per cent (dried substance), determined
on 1.0 g, using a mixture of equal volumes of sulfuric add R
and water R.
M icrobial contamination
TYMC: acceptance criterion 102 CFU/g (2.6.12).
Absence of Escherichia coli (2.6.13).
1-660 Crospovidone 2016
★ ★ TESTS
Crospovidone ★ ★ Peroxides
(Ph. Eux. monograph 0892) ***** Type A maximum 400 ppm expressed as H 2O 2; type B:
maximum 1000 ppm expressed as H 2O2.
Suspend 2.0 g in 50 mL of water R. To 25 mL of this
suspension add 2 mL of titanium trichloride-sulfuric add
reagent R. Allow to stand for 30 min and filter.
The absorbance (2.2.25) of the filtrate, measured at 405 nm
using a mixture of 25 mL of a filtered 40 g/L suspension of
the substance to be examined and 2 mL of a 13 per cent VIV
(CeHçNO)^ 9003-39-8 solution of sulfuric acid R as the compensation liquid, has a
maximum of 0.35.
Action and use For type B use 10 mL of the suspension and dilute to 25 mL
Excipient in pharmaceutical products. with water R for the test.
PhEir.
W ater-soluble substances
DEFINITION Place 25.0 g in a 400 mL beaker, add 200 mL of water R
Cross-linked homopolymer of 1-ethenylpyrrolidin-2-one. and stir for 1 h using a magnetic stirrer. Transfer the
Content suspension to a 250.0 mL volumetric flask, rinsing with
11.0 per cent to 12.8 per cent of N (AT 14.01) (dried water i?, and dilute to volume with the same solvent. Allow
substance). the bulk of the solids to setde. Filter about 100 mL of the
2 types of crospovidone are available, depending on the almost clear supernatant through a membrane filter (nominal
particle size: type A and type B. pore size 0.45 pm), protected by superimposing a membrane
filter (nominal pore size 3 pm). While filtering, stir the liquid
CHARACTERS
above the membrane filter manually or by means of a
Appearance mechanical stirrer, taking care not to damage the membrane
Hygroscopic, white or yellowish-white powder or flakes. filter. Transfer 50.0 mL of the clear filtrate to a tared
Solubility 100 mL beaker, evaporate to dryness and dry at 105-110 °C
Practically insoluble in water, in ethanol 96 per cent and in for 3 h. The residue weighs a maximum of 75 mg.
methylene chloride. Im purity A
IDENTIFICATION Liquid chromatography (2.2.29).
A. Infrared absorption spectrophotometry (2.2.24). Test solution Suspend 1.250 g in 50.0 mL of methanol R and
Comparison crospovidone CRS. shake for 60 min. Leave the bulk to settle and filter through
B. Suspend 1 g in 10 mL of water R , add 0.1 mL of 0.05 M a membrane filter (nominal pore size 0.2 pm).
iodine and shake for 30 s. Add 1 mL of starch solution R and Reference solution (a) Dissolve 50 mg of 1-vinylpyrrolidm-2-
shake. No blue colour develops within 30 s. one R (impurity A) in methanol R and dilute to 100.0 mL
C. To 10 mL of water R, add 0.1 g and shake. A suspension with the same solvent. Dilute 1.0 mL of the solution to
is formed and no clear solution is obtained within 15 min. 100.0 mL with methanol R. Dilute 5.0 mL of this solution to
D. The analytical sieves must be clean and dry. For this purpose
the sieves are washed in hot water and allowed to dry overnight in Reference solution (b) Dissolve 10 mg of l-vinylpyrrolidin-2-
a drying cabinet at 105 °C. one R (impurity A) and 0.50 g of vinyl acetate R in
methanol R and dilute to 100 mL with the same solvent.
Place 20 g (dried substance) in a 1000 mL conical flask, add
Dilute 1.0 mL of the solution to 100.0 mL with the mobile
500 mL of water R and shake the suspension for 30 min.
phase.
Pour the suspension through a 63 jam analytical sieve,
previously tared, and rinse the sieve with water R until the Precolumn:
filtrate is clear. Dry the sieve and sample residue at 105 °C — size. I = 0.025 m, 0 = 4 mm;
for 5 h in a drying cabinet without circulating air. Cool in a — stationary phase. octadecylsUyl silica gelfor chromatography R
desiccator for 30 min and weigh- (5 pm).
Column:
Calculate the percentage sieving residue (fraction of sample
particles having a diameter of more than 63 pm), using the — size. I = 0.25 m, 0 = 4 mm;
following expression: — stationary phase. octadecylsUyl silica gelfor chromatography R
(5 pm);
m i —m 3 — temperature. 40 °C.
x 100 Mobile phase acetomtrüe R , water R (10:90 VIV).
m2
Flow rate 1 mL/min.
m\ — mass of the sieve and sample residue, after drying
for 5 h, in grams; Injection 50 pL. After each injection of die test solution, wash
m2 = initial mass of the sample, in grains; the precolumn by passing the mobile phase backwards, at the
m3 = mass of the sieve, in grams. same flow rate as applied in the test, for 30 min.
If the sieving residue fraction is more than 15 per cent, the System suitability:
substance is classified as type A; if the sieving residue fraction — resolution: minimum 2.0 between the peaks due to
is less than or equal to 15 per cent, the substance is classified impurity A and vinyl acetate in the chromatogram
as type B. obtained with reference solution (b);
2016 Crotamiton 1-661
— repeatability: maximum relative standard deviation of criteria. In such cases, a cross-reference to the tests described in the
2.0 per cent after 6 injections of reference solution (a). mandatory part is included m the Functionality-related
Calculation ofpercentage content. characteristics section. Control of the characteristics can contribute
— for impurity A, use the concentration of impurity A in to the quality of a medicinal product by improving the consistency
reference solution (a). of the manufacturing process and the performance of the medicinal
Limit: product during use. Where control methods are cited, they are
— impurity A: maximum 10 ppm. recognised as being suitable for the purpose, but other methods can
also be used. Wherever resultsfor a particular characteristic are
Heavy m etals (2.4.8) reported, the control method must be indicated.
Maximum 10 ppm.
The following characteristics may be relevantfor crospovidone used
2.0 g complies with test D. Prepare the reference solution as disintegrant
Hydration capacity
Loss on drying (2.2.32) Introduce 2.0 g into a 100 mL centrifuge tube and add
Maximum 5.0 per cent, determined on 0.500 g by drying in 40 mL of water R. Shake vigorously until a suspension is
an oven at 105 °C. obtained. Shake again 5 min and 10 min later, then
Sulfated ash (2.4.14) centrifuge for 15 min at 750 g. Decant the supernatant and
M axim um 0.1 per cent, determined on 1.0 g. weigh the residue. The hydration capacity is the ratio of the
ASSAY mass of the residue to the initial mass of the sample. It is
Place 0.100 g of the substance to be examined (m mg) in a typically 3 to 9.
combustion flask and add 5 g of a mixture of 1 g of copper Particle-size distribution (2.9.31)
sulfate R, 1 g of titanium dioxide R and 33 g of dipotassium Powder flow (2.9.36)
sulfate R, and 3 glass beads. Wash any adhering particles The following characteristic may be relevant for crospovidone used
from the neck into the flask with a small quantity of water R. as suspension stabiliser.
Add 7 mL of sulfuric acid R, allowing it to run down the
Settling volume
inside wall of the flask. Gradually heat the flask until the
Introduce 10 g into a 100 mL graduated cylinder and add
solution has a clear, yellowish-green colour, and the inside
90 mL of water R. Shake vigorously. Dilute to 100 mL with
wall of the flask is free from carbonised material, and then
water R, washing the powder residues from the walls of the
heat for a further 45 min. After cooling, cautiously add
cylinder. Allow to stand for 24 h, then read the volume of
20 mL of water R, and connect the flask to the distillation
the sediment. It is typically greater than 60 m L
apparatus, which has been previously washed by passing
steam through it. To the absorption flask add 30 mL of a PhEur
40 g/L solution of boric acid R, 0.15 mL of bromocresol green-
methyl red solution R and sufficient water R to immerse the
lower end of the condenser tube. Add 30 mL of strong sodium
hydroxide solution R through a funnel, cautiously rinse the ★ ★.
Crotamiton ★ ★
funnel with 10 mL of water R, immediately close the clamp
attached to the rubber tube, then start the distillation with (Ph Eta", monograph 1194) *****
steam to obtain 80-100 mL of distillate. Remove the
absorption flask from the lower end of the condenser tube, H3c
rinsing the end part with a small quantity of water R, and 'I ?Ha
h3^ \ ^ N^ and (Z)-isomer
titrate the distillate with 0.025 M sulfuric acid until the colour
of the solution changes from green through pale greyish-blue
to pale greyish red-purple. Carry out a blank determination
and make any necessary correction. CjaHnNO 203.3 483-63-6
1 mL of 0.025 M sulfuric acid is equivalent to 0.700 mg of N.
Action and use
STORAGE
Acaridde.
Preparations
LABELLING Crotamiton Cream
The label states the type of crospovidone (type A or type B).
Crotamiton Lotion
IMPURITIES
PhEtr.
ch 2
r DEFINITION
JV-Ethyl-N-(2-methylphenyI)but-2-enamide.
C r ° Content
— sum of the (E)- and (Z)-isomers: 96.0 per cent to
102.0 per cent;
A. 1-ethenylpyrrolidin-2-one (1-vinylpyrrolidin-2-one).
— (Z)-isomer, m axim um 15.0 per cent.
FUNCTIONALITY-RELATED CHARACTERISTICS
CHARACTERS
Appearance
Colourless or pale yellow, oily liquid.
5.15). Some of the characteristics described in the Functionality- Solubility
related characteristics section may also be present in the mandatory Slightly soluble in water, misdble with ethanol (96 per cent).
part of the monograph since they also represent mandatory quality At low temperatures it may partly or completely solidify.
1-662 Crotamiton 2016
IDENTIFICATION hydrochloric acid, 5 mL of nitric acid R and diluting to 50 ml.

First identification B with water R.
Second identification A, C, D Related substances
(2.2.25). Test solution (a) Dissolve 50.0 mg of the substance to be
Test solution Dissolve 25.0 mg in cyclohexane R and dilute to examined in the mobile phase and dilute to 100.0 mL with
100.0 mL with the same solvent Dilute 1.0 mL of this the mobile phase.
solution to 10.0 mL with cyclohexane R. Test solution (b) Dilute 1.0 mL of test solution (a) to
Spectral range 220-300 nm. 20.0 mL with the mobile phase.
Absorption maximum At 242 nm. Reference solution (a) Dissolve 50.0 mg of crotamiton CRS in
Specific absorbance at the absorption maximum 300 to 330.
phase. Dilute 1.0 mL of this solution to 20.0 mL with the
B. Infrared absorption spectrophotometry (2.2.24). mobile phase.
Comparison crotamiton CRS.
Reference solution (b) Dissolve 15.0 mg of crotamiton
C. Thin-layer chromatography (2.2.27). impurity A CRS in the mobile phase and dilute to 20.0 mL
Test solution Dissolve 25 mg of the substance to be examined with the mobile phase. Dilute 1.0 mL of this solution to
in anhydrous ethanol R and dilute to 10 mL with the same 50.0 mL with the mobile phase.
solvent. Reference solution (c) Dilute 1.0 mL of test solution (a) to
Reference solution Dissolve 25 mg of crotamiton CRS in 100.0 mL with the mobile phase.
anhydrous ethanol R and dilute to 10 mL with the same Reference solution (d) Dissolve 15 mg of crotamiton
solvent. impurity A CRS in the mobile phase and dilute to 100.0 mL
Plate T L C silica gel F 2sa plate R. with the mobile phase. Dilute 1.0 mL of this solution to
Mobile phase Shake 98 volumes of methylene chloride R with 10.0 mL with test solution (a).
2 volumes of concentrated ammonia R, dry over anhydrous Column:
sodium sulfate R, filter and mix 97 volumes of the filtrate with — size. I = 0.25 m, 0 = 4 mm;
3 volumes of 2-propanol R. — stationary phase, silica gelfor chromatography R (5 pm).
Application 5 |iL. Mobile phase tetrahydrofuran R, cyclohexane R (8:92 VIV).
Development Over a 2/3 of the plate. Flow rate 1.0 mlVmin.
Drying In air. Detection Spectrophotometer at 242 nm.
Detection Examine in ultraviolet light at 254 nm. Injection 20 |iL of test solution (a) and reference
Results The principal spot in the chromatogram obtained with solutions (b), (c) and (d).
the test solution is similar in position and size to the principal Run time 2.5 times the retention time of the (J5)-isomer.
spot in the chromatogram obtained with the reference Relative retention With reference to the (JS)-isomen
solution. (Z)-isomer = about 0.5; impurity A = about 0.8.
D. To 10 mL of a saturated solution add a few drops of a System suitability Reference solution (d):
3 g/L solution of potassium permanganate R. A brown colour — resolution', minimum 4.5 between the peaks due to
is produced and a brown precipitate is formed on standing. impurity A and the (J5)-isomer.
TESTS Limits:
Relative density (2.2.5) — impurity A: not more than the area of the corresponding
1.006 to 1.011. peak in the chromatogram obtained with reference
Refractive index (2.2.6) — unspecified impurities: for each impurity, not more than
1.540 to 1.542. 0.1 times the sum of the areas of the peaks due to the
Free amines (2)- and (E)- isomers in the chromatogram obtained with
Maximum 500 ppm, expressed as ethylaminotoluene. reference solution (c) (0.10 per cent);
Dissolve 5.00 g in 16 mL of methylene chloride R and add — sum of impurities other than A: not more than the sum of
4.0 mL of glacial acetic acid R. Add 0.1 mL of metanUyellow the areas of the peaks due to the (2 )- and (J5)-isomers in
solution R and 1.0 mL of 0.02 M perchloric add. The solution the chromatogram obtained with reference solution (c)
is red-violet ( 1.0 per cent);
Chlorides — disregard limit. 0.02 times the sum of the areas of the
Maximum 100 ppm. peaks due to the (Z)- and (£)-isomers in the
Boil 5.0 g under a reflux condenser for 1 h with 25 mL of
(0.02 per cent).
ethanol (96 per cent) R and 5 mL of a 200 g/L solution of
sodium hydroxide R. Cool, add 5 mL of water R and shake Sulfated ash (2.414)
with 25 mL of ether R. Dilute the lower layer to 20 mL with Maximum 0.1 per cent, determined on 1.0 g.
water R-, add 5 mL of nitric acid R, dilute to 50 mL with ASSAY
water R and add 1 mL of a freshly prepared 50 g/L solution Liquid chromatography (2.2.29) as described in the test for
of silver nitrate R. Any opalescence in the solution is not more related substances with the following modification.
intense than that in a mixture of 1 mL of a freshly prepared Injection Test solution (b) and reference solution (a).
50 g/L solution of silver nitrate R and a solution prepared by
diluting 5 mL of a 200 g/L solution of sodium hydroxide R to Calculate the percentage content of C 13H 17NO from the sum
20 mL with water R and adding 1.5 mL of 0.01 M of the areas of the peaks due to the (2)- and (J5)-isomers in
the chromatograms obtained. Calculate the content of the
2016 Cyanocobalamin 1-663
(Z)-isomer, as a percentage of the total content of the (£)- IDENTIFICATION

and (Z)-isomers3 from the chromatogram obtained with test A. Ultraviolet and visible absorption spectrophotometry
solution (b). (2.2.25).
STORAGE Test solution Dissolve 2.5 mg in water R and dilute to
Protected from light. 100.0 mL with the same solvent.
IMPURITIES
Specified impurities A Absorption maxima At 278 nm, 361 nm and from 547 nm to
559 nm.
Absorbance ratio’.
— A 361 ! -^547-559 = 3.15 to 3.45;
— -^361 ! A 278 — 1.70 to 1.90.
B. Thin-layer chromatography (2.2.27). Carry out the test
protectedfrom light.
A. iV-ethyl-iV-(2-methylphenyl)but-3-enamide. Test solution Dissolve 2 mg of the substance to be examined
__________________________________________________________ PhEur in 1 mL of a mixture of equal volumes of ethanol
(96 per cent) R and water R.
Reference solution Dissolve 2 mg of cyanocobalamin CRS in
1 mL of a mixture of equal volumes of ethanol (96 per cent) R
and water R.
Cyanocobalamln ? * *\ Plate TLC silica gel G plate R.
' _ _ V*. .*
(Ph. Eur. monograph 0547) * Mobile phase dilute ammonia R l, methanol R, methylene
chloride R (9:30:45 VỈVỈV).
Application 10 |iL.
Development In an unsaturated tank, over 2/3 of the plate.
Drying In air.
Detection Examine in daylight.
reference solution.
TESTS
Related substances
the mobile phase. Use within 1 h.
Reference soỉutừm (ạ) Dilute 3.0 mL of the test solution to
100.0 mL with the mobile phase. Use vritkin 1 h.
C63H88CoNi4014P 1355 68-19-9 50.0 mL with the mobile phase. Dilute 1.0 mL of this
solution to 100.0 mLwith the mobile phase. Use within 1 h.
Action and use Reference solution (c) Dissolve 25 mg of the substance to be
Vitamin B12 analogue. examined in 10 mL of ‘w ater R, w arm ing if necessary. Allow
Preparation to cool and add 5 mL of a 1.0 g/L solution of chỉoramine R
Cyanocobalamin Oral Solution and 0.5 m ĩ, of 0.05 M hydrochloric acid, then dilute to 25 mL
Cyanocobalamin Tablets with water R. Shake and allow to stand for 5 min. Dilute
1 mL of this solution to 10 mL with the mobile phase and
PhEtr__________________________________________________________ inject immediately.
DEFINITION Column:
a-(5j6-Dimethylbenzimidazol-l-yl)cobamide cyanide. — sizer. I = 0.25 m} 0 = 4 mm;
— stationary phase: octyìsũyl silica gelfor chromatography R
Content
(5|im).
Mobile phase Mix 26.5 volumes of methanol R and
This monograph applies to cyanocobalamin produced by
73.5 volumes of a 10 g/L solution of disodium hydrogen
fermentation.
phosphate R adjusted to pH 3.5 with phosphoric add R and
CHARACTERS use within 2 days.
Appearance Flow rate 0.8 ml/min.
Dark red, crystalline powder or dark red crystals. Detection Spectrophotometer at 361 nm.
Solubility Injection 20 |jL.
Sparingly soluble in water and in ethanol (96 per cent),
Run time 3 times the retention time of cyanocobalamin.
practically insoluble in acetone.
The anhydrous substance is very hygroscopic.
1-664 Cyclizine 2016
System suitability: TESTS

— the chromatogram obtained with reference solution (c) Alkalinity
shows 2 principal peaks; Shake 1 g with 25 mL of carbon dioxide-free water for
— resolution: minimum 2.5 between the 2 principal peaks in 5 minutes and filter. The pH of the filtrate is 7.6 to 8.6,
the chromatogram obtained with reference solution (c); Appendix V L.
— signal-to-noise ratio: minimum 5 for the principal peak in Clarity of solution
the chromatogram obtained with reference solution (b). A 1.0% w/v solution in ether and a 1.0% w/v solution in
Limits: 2m hydrochloric add are dear, Appendix IV A.
— total:not more than the area of the principal peak in the
Chloride
Dissolve 0.20 g in 2 m l. of methanol and dilute to 30 mL
(3 per cent);
with 2m nitric add. 15 mL of the resulting solution complies
— disregard limir. the area of the principal peak in the
with the limit testfor chlorides, Appendix VH (500 ppm).
(0.1 per cent). Related substances
Carry out the method for gas chromatography,
Appendix ID B, using the following solutions in methanol
Maximum 12.0 per cent, determined on 40.00 mg by drying
prepared immediately before use.
ASSAY
(2) Dilute 1 volume of solution (1) to 100 volumes and
Dissolve 100.0 mg in water R and dilute to 500.0 mL with further dilute 1 volume of the resulting solution to
the same solvent. Dilute 25.0 mL of the solution to 10 volumes.
200.0 mL with water R. Measure the absorbance (2.2.25) at
the absorption maximum at 361 nm. (3) 0.0025% w/v of cyclizine hydrochloride BPCRS,
0.0025% w/v of 1-methylpiperazine BPCRS (impurity A) and
Calculate the content of C ^ H g sC o N n O ^ taking the 0.0025% w/v of diphenylmethanol BPCRS (impurity B).
specific absorbance to be 207.
C H R O M A T O G R A P H IC C O N D IT IO N S
STORAGE
(a) Use a fused silica column (25 m x 0.33 mm) coated
with a 0.5-|im film of pdy(dimethyl) (diphenyl)sUoxane (HP-5
___________________________________________________________PhEur is suitable).
(b) Use hdium as the carrier gas at a flow rate of 1 mL per
minute.
(c) Use the gradient conditions described below.
Cyclizine (d) Use a split injection ratio of 1:25.
(e) Use a flame ionisation detector at 290°.
NMe (f) Inject 1 pL of each solution.
(g) The peaks elute in the order methanol,
1-methylpiperazine, diphenylmethanol, cyclizine.
Time Temperature Comment

(minutes)
C18H22N2 266.4 82-92-8 0->14 100°->240° linear gradient
Action and use 14—>16 240°->270° linear gradient

Histamine Hi receptor antagonist? antihistamine. 16-»30 270° isocratic
Preparation
Cyclizine Injection
DEFINITION SY ST E M S U IT A B IL IT Y
Cyclizine is l-benzhydryl-4-methyipiperazine. It contains not Inject solution (3) 6 times. The rdative standard deviation of
less than 98.5% and not more than 101.0% of C 18H 22N 2, each of the areas of the 3 prinripal peaks is not more than
calculated with reference to the dried substance. 5.0%.
CHARACTERISTICS The test is not valid unless, in the chromatogram obtained
A white or creamy white, crystalline powder. with solution (3);
Practically insoluble in water. It dissolves in most organic the peak-to-vaUey ratio between methanol and
solvents and in dilute adds. 1-methylpiperazine (impurity A) is at least 50;
IDENTIFICATION the resolutionfactor between diphenylmethanol (impurity B)
A. The infrared absorption spectrum, Appendix II A, is and cyclizine is at least 18.
concordant with the reference spectrum of cyclizine (RS 075). L IM IT S
B. Melting point, about 107°, Appendix V A In the chromatogram obtained with solution (1):
the area of the peak corresponding to 1-methylpiperazine
(impurity A) is not greater than the peak corresponding to
1-methylpiperazine in solution (3) (0.5%);
2016 Cyclizine Hydrochloride 1-665
the area of the peak corresponding to diphenylmethanol Test solution (b)Dilute 10.0 mL of test solution (a) to
(impurity B) is not greater than the peak corresponding to 100.0 mL with a 5 g/L solution of sulfuric acid R.
diphenylmethanol in solution (3) (0.5%); Spectral range 240-350 nm for test solution (a); 210-240 nm
the area of any other secondary peak is not greater than the for test solution (b).
area of the principal peak in the chromatogram obtained with Resolution (2.2.25): minimum 1.7.
solution (2) (0.1%); Absorption maxima At 258 nm and 262 nm for test
the Siam of the areas of all secondary peaks is not greater than solution (a); at 225 nm for test solution (b).
10 times the area of the principal peak in the chromatogram Absorbance raâo A262/A258 = 1.0 to 1.1.
obtained with solution (2) (1.0%).
Specific absorbance at the absorption maximum at 225 nm
Disregard any peak with an area less than 0.5 times that of 370 to 410 for test solution (b).
solution (2) (0.05%). B. Infrared absorption spectrophotometry (2.2.24).
Comparison cyclizine hydroçjdoride CRS.
Loss on drying
When dried to constant weight at 80°, loses not more than C. Thin-layer chromatography (2.2.27).
1.0% of its weight. Use 1 g. Test solution Dissolve 10 mg of the substance to be examined
Sulfated ash in methanol R and dilute to 10 mL with the same solvent.
Not more than 0.1%, Appendix IX A. Reference solution Dissolve 10 mg of cyclizine
hydrochloride CRS in methanol R and dilute to 10 mL with the
ASSAY
same solvent.
Carry out Method I for non-aqueous titration,
Plate TLC silica gel GF254 plate R.
Appendix VIII A, using 0.1 g and determining the end point
potentiometrically. Each mL of 0.1m perchloric add P'S is Mobile phase concentrated ammonia R, methanol R, methylene
equivalent to 13.32 mg of C18H22N2. chloride R (2:13:85 V/V/V).
Application 20 |iL.
Drying In air for 30 min.
*****
Cyclizine Hydrochloride * *
Detection Expose to iodine vapour for 10 min,
*•+ ** Restdts The prindpal spot in the chromatogram obtained with
(Ph. Eur. monograph 1092) **+
reference solution.
D. Dissolve 0.5 g in 10 mL of ethanol (60 per cent VIV) R,
Ha heating if necessary. Cool in iced water. Add 1 mL of dilute
sodium hydroxide solution R and 10 mL of water R. Filter,
wash the predpitate with water R and dry at 60 °C at a
pressure not exceeding 0.7 kPa for 2 h. The melting point
(2.2.14) is 105 °C to 108 °C.
Ci8H23ClN2 302.8 305-25-3
E. It gives reaction (a) of chlorides (2.3.1).
Action and use TESTS
Histamine Hi receptor antagonist; antihistamine. pH (2.2.3)
Preparations 4.5 to 5.5.
Cyclizine Tablets Dissolve 0.5 g in a mixture of 40 volumes of ethanol
Dipipanone and Cyclizine Tablets (96 per cent) R and 60 volumes of carbon dioxide-free water R
PhEir______________________________________________________
Related substances
DEFINITION Gas chromatography (2.2.28). Prepare the solutions immediately
1-(Diphenylmethyl)-4-methylpiperazine hydrochloride. before use. _
Content Test solution Dissolve 0.250 g of the substance to be
98.5 per cent to 101.0 per cent (dried substance). examined in 4.0 mL of methanol R and dilute to 5.0 mL with
1 M sodium hydroxide.
CHARACTERS
Appearance Reference solution (a) Dissolve 25 mg of the substance to be
White or almost white, crystalline powder. examined in 10.0 mL of methanol R. Dilute 1.0 mL of this
solution to 50.0 mL with methanol R.
Solubility
Sligjhtly soluble in water and in ethanol (96 per cent).
examined, 5.0 mg of cyclizine impurity A CRS and 5.0 mg of
IDENTIFICATION cyclizine impurity B CRS in methanol R and dilute to 20.0 mL
First identification B, E. with the same solvent.
Second identification A , C, D, E. Column:
A. Ultraviolet and visible absorption spectrophotometry — material: fused silica;
(2.2.25). — size: I = 25 m, 0 = 0.33 mm;
Test solution (a) Dissolve 20.0 mg in a 5 g/L solution of — stationary phase: poly(dimethyl) (diphenyl)sUoxane R (film
sulfuric addR and dilute to 100.0 mT. with the same add thickness 0.50 pm).
solution. Carrier gas helium for chromatography R.
1-666 Cyclopenthiazide 2016

Split ratio 1:25.
Temperature'.
Time Temperature
(min) CC)
Column 0 -1 4 100 ->240 B. diphenylmethanol (benzhydrol).
___________________________________________________________PhEur
14-16 240 -» 270
16-30 270
Injection port 250
Detector 290 Cyclopenthiazide

0 0 0 0
Detection Flame ionisation. \\ // \\ ❖
Injection 1 pL.
Relative retendon With reference to cyclizine (retention
C 13H 18CIN3O4S2 379.9 742-20-1
— peak-to-vaUey ratio: minimum 50, where Hp = height
above the baseline of the peak due to impurity A and Action and use
H v = height above the baseline of the lowest point of the Thiazide-diuretic.
curve separating this peak from the peak due to methanol.
Preparation
Limits:
Cyclopenthiazide Tablets
— impurities A , B: for each impurity, not more than the area
of the corresponding peak in the chromatogram obtained DEFINITION
with reference solution (b) (0.5 per cent); Cyclopenthiazide is 6-chloro-3-cyclopentylmethyl-3,4-
— unspecified impurities: for each impurity, not more than the dihydro-1,2,4-benzothiadiazine-7-sulfonamide 1,1 -dioxide.
area of the principal peak in the chromatogram obtained It contains not less than 98.0% and not more than 102.0%
with reference solution (a) (0.10 per cent); of Ci 3H 18d N 30 4 S2, calculated with reference to the dried
— total: not more than 10 times the area of the principal substance.
solution (a) ( 1.0 per cent); CHARACTERISTICS
— disregard limit. 0.5 times the area of the principal peak in A white powder.
the chromatogram obtained with reference solution (a) Practically insoluble in water, soluble in acetone and in ethanol
(0.05 per cent). (96%)', very slightly soluble in ether.
Loss on drying ( 2.2.32) IDENTIFICATION

Maximum 1.0 per cent, determined on 1.000 g by drying in A. The infrared absorption spectrum, Appendix II A, is
an oven at 130 °C. concordant with the reference spectrum of cyclopenthiazide
Sulfated ash (2.4.14) (RS 077).
Maximum 0.1 per cent, determined on 1.0 g. B. The light absorption, Appendix II B, in the range 230 to
ASSAY 350 nm of a 0 .002 % w/v solution in 0.0 1m sodium hydroxide
exhibits two maxima, at 273 nm and 320 nm. The absorbance
In order to avoid overheating in the reaction medium, mix
thoroughly throughout and stop the titration immediately after the
at 273 nm is about 0.88 and at 320 nm is about 0.12.
end-point has been reached. C. Carry out the method for thin-layer chromatography,
Appendix HI A, using silica gel GF 254 as the coating
Dissolve 0.120 g in 15 mL of anhydrous formic acid R and
substance and ethyl acetate as the mobile phase. Apply
add 40 mL of acetic anhydride R. Titrate with 0.1 M perchloric
separately to the plate 5 jiL of each of two solutions in
acid, 'determining the end-point potentiometrically (2.2.20).
acetone containing ( 1) 0 . 1% w/v of the substance being
1 mL of 0.1 M perchloric add is equivalent to 15.14 mg examined and (2) 0.1% w/v of cyclopenthiazide BPCRS. After
of C 18H 23CIN2. removal of the plate, dry it in a current of air, examine under
STORAGE ultraviolet light (254 nm) and then reveal the spots by
Protected from light. Method I. By each method of visualisation the principal spot
in the chromatogram obtained with solution (1) corresponds
IMPURITIES
in colour and intensity to that in the chromatogram obtained
Specified impurities A, B.
with solution (2 ).
TESTS
Related substances
Carry out the method for thin-layer chromatography,
Appendix ID A, using silica gel G as the coating substance
A. 1-methylpiperazine, and ethyl acetate as the mobile phase. Apply separately to the
plate 5 jiL of each of two solutions of the substance being
examined in acetone containing (1) 0.50% w/v and (2)
2016 Cyclopentolate Hydrochloride 1-667
0.0050% w/v. After removal of the plate, dry it in a current Test solution Dissolve 10 mg of the substance to be examined
of air and reveal the spots by Method. I. Any secondary spot in in 5 mL of ethanol (96 per cent) R.
the chromatogram obtained with solution (1) is not more Reference solution Dissolve 10 mg of cyclopentolate
intense rhan the spot in the chromatogram obtained with hydrochloride CRS in ethanol (96 per cent) R and dilute to
solution (2). 5 mL with the same solvent.
Loss on drying Plate TLC silica gel plate R.
When dried to constant weight at 105°, loses not more than Mobile phase concentrated ammonia R, water R, butyl acetate R,
0.5% of its weight Use 1 g. 2-propanol R (5:15:30:50 VIVIV/V).
Sulfated ash Application 10 pL.
Not more than 0.1%, Appendix IX A.
ASSAY Drying In air.
Dissolve 0.5 g in 50 mL of butylamine and carry out Detection Spray with alcoholic solution of sulfuric acid R and
Method II for rum-aqueous titration, Appendix VHI A, using heat at 120 °C for 30 min; examine in ultraviolet light at
0.1m tetrabutylammonium hydroxide KS as titrant and 365 nm.
magneson solution as indicator; titrate to a pure blue end
Result The principal spot in the chromatogram obtained with
point. Each mL of 0.1m tetrabutylammonium hydroxide FS is
the test solution is similar in position, fluorescence and size
equivalent to 18.99 mg of C 13H 18CIN3O4S2.
to the principal spot in the chromatogram obtained with the
reference solution.
TESTS
Cyclopentolate Hydrochloride ***** pH (2.2.3)
*. *
(Ph. Eur. monograph 1093) * 4.5 to 5.5.
Related substances
inwater R and dilute to 20.0 mL with the same solvent
C17H26d N 0 3 327.8 5870-29-1 Reference solution (a) Dilute 1.0 mL of the test solution to
100.0 mL with water R. Dilute 5.0 mL of this solution to
Action and use 10.0 mL with water R.
Anticholinergic. < Reference solution (b) Dissolve 10 mg of cyclopentolatefor
Preparation system suitability CRS (con taining impurity C) in water R and
Cyclopentolate Eye Drops dilute to 10.0 mL with the same solvent.
Column:
PhEur__________________________________________________________
— size. I = 0.125 m, 0 = 4.0 mm;
DEFINITION — stationary phase, spherical end-capped hexylsUyl silica gelfor
2-(Dimethylamino)ethyl (2RS)- chromatography R (5 pm).
(1-hydroxycyclopentyl) (phenyl) acetate hydrochloride. Mobile phase Dissolve 0.66 g of ammonium phosphate R in
Content water R, adjust to pH 3.0 with phosphoric add R and dilute to
98.5 per cent to 101.5 per cent (dried substance). 1000 mL with water R; mix and filter; mix 55 volumes of this
solution and 45 volumes of acetonitrüe R l.
CHARACTERS
Appearance Flow rate 1.0 ml /min.
White or almost white, crystalline powder. Detection Spectrophotometer at 220 nm.
Solubility Injection 20 pi.
Very soluble in water, freely soluble in ethanol (96 per cent). Run time 2.5 times the retention time of cyclopentolate.
It shows polymorphism (5.9). Identification of impurities Use the chromatogram supplied
IDENTIFICATION with cyclopentolatefor system suitability CRS and the

identify the peak due to impurity C.
Second identification A , C, D.
Relative retention With reference to cyclopentolate (retention
A. Melting point (2.2.14): 135 °C to 141 °C. time = about 4 min): impurity C = about 0.9.
B. Infrared absorption spectrophotometry (2.2.24). System suitability: reference solution (b):
Preparation Discs of potassium chloride R. — peak-to-vaEey ratio: minimum 6, where Hp = height above
Comparison cyclopentolate hydrochloride CRS. the baseline of the peak due to impurity C and
substance to be examined and the reference substance curve separating this peak from the peak due to
separately in ethanol (96 per cent) R, evaporate to dryness and cyclopentolate.
1-668 Cyclophosphamide 2016
Limits:
★*★
Cyclophosphamide ★ ★
— correctionfactor, for the calculation of content, multiply the ★ ★
peak area of impurity C by 2.0; (Ph. Eur. monograph 0711) *****
— impurity C: not more than the area of the principal peak
in the chromatogram obtained with reference solution (a) Cl
(0.5 per cent);
— unspecified impurities: for each impurity, not more than and enantiomer , H20
(0.10 per cent);
— total: not more than twice the area of the principal peak in CyHjgCysr^PjHzO 279.1 6055-19-2
(1.0 per cent); Action and use
— disregard limit. 0.1 times the area of the principal peak in Cytotoxic alkylating agent.
the chromatogram obtained with reference solution (a) Preparations
(0.05 per cent). Cydophosphamide Injection
Loss on drying (2.2.32) Cyclophosphamide Oral Solution
Maximum 0.5 per cent, determined on 1.000 g by drying in Cydophosphamide Tablets
Sulfa ted ash (2.4.14) PhEur______________________________
Maximum 0.1 per cent, determined on 1.0 g. DEFINITION
ASSAY Cyclophosphamide contains not less than 98.0 per cent and
Dissolve 0.250 g in a mixture of 1.0 mL of 0.1 M hydrochloric not more than the equivalent of 102.0 per cent of (2RS)-
add and 50 mL of ethanol (96 per cent) R. Cany out a AyV-bis(2-chloroethyl)tetrahydro-2ii-l,3,2-oxa2aphosphorin-
potentiometric titration (2.2.20), using 0.1 M sodium 2-amine 2-oxide, calculated with reference to the anhydrous
hydroxide. Read the volume added between the 2 points of substance.
inflexion. CHARACTERS
1 mL of 0.1 M sodium hydroxide is equivalent to 32.79 mg of A white or almost white, crystalline powder, soluble in water,
C17H 26C1N03. freely soluble in alcohol.
Specified impurities C First identification B.
Other detectable impurities (the following substances would, if Second identification A, C, D.
present at a sufficient level, be detected by one or other of A. Determine the mdting point (2.2.14) of the substance to
the tests in the monograph. They are limited by the general be examined. Mix equal parts of the substance to be
acceptance criterion for other/unspecified impurities and/or examined and cyclophosphamide CRS and determine the
by the general monograph Substances for pharmaceutical use melting point of the mixture. The difference between the
(2034). It is therefore not necessary to identify these melting points (which are about 51 °C) is not greater than
impurities for demonstration of compliance. See also 5.10. 2 °C.
Control of impurities in substances for pharmaceutical use): A, B.
cydophosphamide CRS.
C. Examine the chromatograms obtained in the test for
rdated substances. The principal spot in the chromatogram
D. Dissolve 0.1 g in 10 m l. of water R and add 5 mL of
silver nitrate solution R l; the solution remains dear. Boil, a
white predpitate is formed which dissolves in concentrated
ammonia R and is repredpitated on the addition of dilute
nitric add R.
B. phenylacetic add, TESTS

Solution S
Solution S is clear (2.2.1) and not more intensdy coloured
C. 2-(dimethylamino)ethyl phenylacetate. than reference solution Y6 (2.2.2, Method II).
PhEur pH (2.2.3)
The pH of solution S is 4.0 to 6.0, determined immediately
after preparation of the solution.
2016 Cyproheptadine Hydrochloride 1-669
Related substances ★ *★
Cyproheptadine Hydrochloride ★ ★
Examine by thin-layer chromatography (2.2.27), using silica ★ ★
gel G R as the coating substance. (PA. Eur. monograph 0817) *****
examined in alcohol R and dilute to 5 mL with the same
solvent.
Test solution (b) Dilute 1 mL of test solution (a) to 10 mL . HCI , 1’/,H20
with alcohol R.
Reference solution (a) Dissolve 10 mg of cyclophosphamide CRS
in alcohol R and dilute to 5 mL with the same solvent.
10 ml, with alcohol R. CaHaaON.l&HaO 350.9 41354-29-4
Action and use
Histamine Hx receptor antagonist; antihistamine.
anhydrous formic acid R, 4 volumes of acetone R, 12 volumes
of water R and 80 volumes of methyl ethyl ketone R. Dry the Preparation
plate in a current of warm air and heat at 110 °C for 10 min. Cyproheptadine Tablets
At the bottom of a chromatographic tank, place an PhEir.
evaporating dish containing a 50 g/L solution of potassium
permanganate R and add an equal volume of hydrochloric DEFINITION
acid R. Place the plate whilst still hot in the tank and close 4-(5H-Dibenzo [a,d[ [7] annulen-5-ylidene)-1 -methylpiperidine
the tank. Leave the plate in contact with the chlorine gas for hydrochloride sesquihydrate.
2 min. Withdraw the plate and place it in a current of cold Content
air until the excess of chlorine is removed and an area of 98.5 per cent to 101.0 per cent (anhydrous substance).
coating below the points of application gives at most a very
faint blue colour with a drop of potassium iodide and starch CHARACTERS
solution R. Avoid prolonged exposure to cold air. Spray with Appearance
potassium iodide and starch solution R and allow to stand for White or slightly yellow, crystalline powder.
5 mm. Any spot in the chromatogram obtained with test Solubility
solution (a), apart from the principal spot, is not more Slightly soluble in water, freely soluble in methanol, sparingly
intense than the spot in the chromatogram obtained with soluble in ethanol (96 per cent).
reference solution (b) (1.0 per cent). Disregard any spot IDENTIFICATION
remaining at the point of application.
Chlorides (2.4.4) Comparison cyproheptadine hydrochloride CRS.
Dissolve 0.15 g in water R and dilute to 15 mL with the
same solvent. The freshly prepared solution complies with B. A saturated solution gives reaction (b) of chlorides (2.3.1).
the limit test for chlorides (330 ppm). TESTS

Phosphates (2.4.11) Acidity
Dissolve 0.10 g in water R and dilute to 100 mL with the Dissolve 0.10 g in water R and dilute to 25 mL with the
same solvent The solution complies with the limit test for same solvent. Add 0.1 mL of methyl red sdudon R. Not more
phosphates (100 ppm). than 0.15 mL of 0.01 M sodium hydroxide is required to
change the colour of the indicator.
1.0 g complies with test C for heavy metals (20 ppm). Related substances
Prepare the reference solution using 2 mL of lead standard Liquid chromatography (2.2.29).
solution (10 ppm Pb) R. Test solution Dissolve 40.0 mg of the substance to be
W ater (2.5.12) examined in mobile phase A and dilute to 20.0 ml, with
6.0 per cent to 7.0 per cent, determined on 0.300 g by the mobile phase A.
semi-micro determination of water. Reference solution (a) Dilute 1.0 ml, of the test solution to
ASSAY
Dissolve 0.100 g in 50 mL of a 1 g/L solution of sodium
Reference solution (b) Dissolve 2.0 mg of
hydroxide R in ethylene glycol R and boil under a reflux
dibenzocydoheptene CRS (impurity A), 2.0 mg of
condenser for 30 min. Allow to cool and rinse the condenser
dibenzosuberone CRS (impurity B) and 2.0 mg of
with 25 mL of water R. Add 75 mL of 2-propanol R, 15 mL
cyproheptadine impurity C CRS in mobile phase A, add
of dilute nitric acid R, 10.0 mL of 0.1 M silver nitrate and
2.0 mL of ferric ammonium sulfate solution R2 and titrate with 1.0 mL of the test solution and dilute to 100.0 mL with
0.1 M ammonium thiocyanate.
mobile phase A.
Reference solution (c) Dilute 1.0 mL of reference solution (b)
1 mL of 0.1 M silver nitrate is equivalent to 13.05 mg of
to 10.0 mL with mobile phase A.
C7H15C12N202P.
Column:
PhEur
— size: I = 0.25 m, 0 = 4.6 mm;
— stationary phase: octylsüyl silica gelfor chromatography R
(5 nm).
1-670 Cyproterone Acetate 2016
Mobile phase:
— mobile phase A: dissolve 6.12 g of potassium dihydrogen
phosphate R in 900 mL of water R, adjust to pH 4.5 with
phosphoric add R and dilute to 1000 mL with water R',
mix 60 volumes of this solution and 40 volumes of
acetonitrUe for chromatography R;
— mobile phase B: dissolve 6.12 g of potassium dihydrogen
A. 5H-dibenzo[ajii] [7]annulene (dibenzocycloheptene),
phosphate R in 900 mL of water R, adjust to pH 4.5 with
phosphoric add R and dilute to 1000 mL with water R\
mix 40 volumes of this solution and 60 volumes of
acetonitrile for chromatography R',

0 - 10.0 100 0
10.0 - 10.1 100 -»0 0 ^ 100 B. 10,11 -dihydro- 5//-dibenzo [a3d\[7] annul en-5-one
(dibenzosuberone),
10.1 - 35 0 100

Injection 10 |iL.
Relative retention With reference to cyproheptadine (retention
impurity B = about 2.6; impurity A = about 3.9. C. 5-(l-methylpiperidin-4-yl)-5H-dibenzo[a,d\ [7]annulen-5-
System suitability-, reference solution (b): ol.
— resolution: minimum 7.0 between the peaks due to ___________________________________________________________ PhEur
impurity C and cyproheptadine.
Limits:
— impurities A , B, C: for each impurity, not more than
1.5 times the area of the corresponding peak in the *** *
★
chromatogram obtained with reference solution (c) Cyproterone Acetate ★ ★
(0.15 per cent); (Ph Em monograph 1094) * * * * *
— unspecified impurities', for each impurity, not more than the

(0.5 per cent);
(0.05 per cent).
W ater (2.5.12) C24H 29CIO4 416.9 427-51-0
Sulfated ash (2.4.14) Action and use
Maximum 0.1 per cent, determined on 1.0 g. Antiandrogen.
ASSAY P reparation
Dissolve 0.250 g in a mixture of 5.0 mL of 0.01 M Cyproterone Tablets
hydrochloric acid and 50 mL of ethanol (96 per cent) R. Carry PhEur.
out a potentiometric titration (2.2.20), vising 0.1 M sodium
hydroxide. Read the volume added between the 2 points of DEFINITION
inflexion. 6-Chloro-3,20-dioxo-l P,2|5-dihydro-3 'H-
1 mL of 0.1 M sodium hydroxide is equivalent to 32.39 mg of cyclopropa [1,2] pregna-1,4,6-trien-17-yl acetate.
C 21H 22CIN. Content
STORAGE
Protected from light. CHARACTERS
Appearance
IMPURITIES
Spedfied impurities A, B, C
Solubility
Practically insoluble in water, very soluble in methylene
chloride, freely soluble in acetone, soluble in methanol,
sparingly soluble in anhydrous ethanol,
mp: about 210 °C.
2016 Cyproterone Acetate 1-671
IDENTIFICATION Identification of impurities Use the chromatogram supplied

First identification A with cyproterone impurity mixture CRS and the chromatogram
Second identification B, C, D, E obtained with reference solution (b) to identify the peaks due
to impurities F and I; use the chromatogram supplied with
cyproterone acetate for peak identification CRS and the
Comparison cyproterone acetate CRS.
B. Thin-layer chromatography (2.2.27). the peaks due to impurities B, C, E and G.
Test solution Dissolve 20 mg of the substance to be examined Relative retention With reference to cyproterone acetate
in methylene chloride R and dilute to 10 mL with the same (retention time = about 22 min): impurity E = about 0.27;
solvent. impurity G = about 0.3; impurity F = about 0.5;
Reference solution Dissolve 10 mg of cyproterone acetate CRS in impurity B = about 0.7; impurity I = about 0.9;
methylene chloride R and dilute to 5 mL with the same impurity C = about 1.5.
solvent. System suitabUity: reference solution (b):
Plate T L C silica gel F 254 plate R. — resolution: m in im um 1.5 between the peaks due to
Mobile phase cycbhexane R, ethyl acetate R (50:50 VIV). impurity I and cyproterone acetate.
Limits:
Application 5 (iL.
Development Twice over 3/4 of the plate; dry in air between
the 2 developments. corresponding correction factor: impurity C = 1 .8 ;
Drying In air. impurity E = 0.7;
Detection Examine in ultraviolet light at 254 nm. — impurity F: not more than 0.4 times the area of the
Results The principal spot in the chromatogram obtained with principal peak in the chromatogram obtained with
the test solution is similar in position and size to the principal reference solution (a) (0.4 per cent);
spot in the chromatogram obtained with the reference — impurity E: not more than 0.2 times the area of the
solution. principal peak in the chromatogram obtained with
C. To about 1 mg add 2 mL of sulfuric acid R and heat on a reference solution (a) (0.2 per cent);
water-bath for 2 min. A red colour develops. Cool. Add this — impurities B, C, G: for each impurity, not more than
solution cautiously to 4 mL of water R and shake.
The solution becomes violet chromatogram obtained with reference solution (a)
(0.15 per cent);
D. Incinerate about 30 mg with 0.3 g of anhydrous sodium — unspecified impurities: for each impurity, not more than
carbonate R over a naked flame for about 10 min. Cool and
dissolve the residue in 5 mL of dilute nitric acid R. Filter. chromatogram obtained with reference solution (a)
To 1 mL of the filtrate add 1 mL of water R. The solution (0.10 per cent);
gives reaction (a) of chlorides (2.3.1). — total: not more than 0.5 times the area of the principal
E. It gives the reaction of acetyl (2.3.1). peak in the chromatogram obtained with reference
TESTS solution (a) (0.5 per cent);
Specific optical rotation (2.2.7) — disregard limit: 0.05 times the area of the principal peak in
+ 152 to + 157 (dried substance). the chromatogram obtained with reference solution (a)
(0.05 per cent).
Dissolve 0.25 g in acetone R and dilute to 25.0 mL with the
same solvent. Loss on drying (2.2.32)
Maximum 0.5 per cent, determined on 1.000 g by drying at
Related substances
80 °C at a pressure not exceeding 0.7 kPa.
Sulfated ash (2.414)
in acetomtrile R and dilute to 10.0 mL with the same solvent Maximum 0.1 per cent, determined on 1.0 g.
Reference solution ( a) Dilute 1.0 mL of the test solution to ASSAY
100.0 ml. with acetonitrUe R. Dissolve 50.0 mg in methanol R and dilute to 50.0 mL with
Reference solution (b) Dissolve the contents of a vial of the same solvent. Dilute 1.0 mL of the solution to 100.0 mL
cyproterone impurity mixture CRS (impurities F and I) in
with methanol R. Measure the absorbance (2.2.25) at the
1.0 mL of the test solution. absorption maximum at 282 nm.
Reference solution (c) Dissolve 2 mg of cyproterone acetate for
Calculate the content of C24H 29CIO4 taking the specific
peak identification CRS (containing impurities B, C, E and G)
in 2.0 mL of acetonitrUe R. STORAGE
Column: Protected from light.
— sizer. I = 0.125 m, 0 = 4.6 mm;
IMPURITIES
Specified impurities B, C, E, F, G
Other detectable impurities (the followingsubstances would, if
Mobile phase acetonitrUe R, water R (40:60 VIV).
Flow rate 1.5 mL/min. the tests in the monograph. They are limited by the general
Detection Spectrophotometer at 254 nm. acceptance criterion for other/unspecified impurities and/or
Injection 20 (iL. by the general monograph Substances for pharmaceutical use
Run time Twice the retention time of cyproterone acetate.
1-672 Cysteine Hydrochloride 2016
Control of impurities in substancesfor pharmaceutical use): A, D,
G. 6 ß-chloro-7a-hydroxy-3,20-dioxo-1ß,2 ß-dihydro-3'H-
cyclopropa[1,2]pregna-1,4-dien-17-yl acetate,
A. 3,20-dioxo-1ß,2ß-dihydro-3 '/f-cydopropa [1,2]pregna-
l,4,6-trien-17-yl acetate.
H. 3,20-dioxopregna-l,4-dien-17-yl acetate,
B. 6-methoxy-3,20-dioxo-lß,2ß-dihydro-3'H-
cyclopropa [1,2] pregna-1,4,6-trien-17-yl acetate,
I. 6-chloro-3,20-dioxopregna-1,4,6-trien-17-yl acetate
(delmadinone acetate),
C. 6-chloro-1a-(chloromethyi)-3,20-dioxopregna-4,6-dien-17-
yl acetate,
J. 6oc,7a-epoxy-3,20-dioxo-lß,2ß-dihydro-3 'H-
cyclopropa[1,2]pregna-1,4-dien-17-yl acetate.
___________________________________________________________ PhEur
D. la-(chloromethyl)-3,6,20-trioxopregn-4-en-17-yl acetate,
Cysteine Hydrochloride ?**%

*★ ★*
(Cysteine Hydrochloride Monohydrate, *
H NHj
H S^ X ■ HCI , H p
C02H
E. 3,6,20-trioxo-1ß,2 ß-dihydro-3 '/f-cydopropa [1,2]pregna-

l,4-dien-17-yl acetate, C3H8CIN02S 3 20 175.6 7048-04-6
Action and use

Amino add.
PhEur___________________________________________ — ------------ —-—
DEFINITION
(2i?)-2-Ammo-3-sulfanylpropanoic add hydrochloride
monohydrate.
Fermentation product, extract or hydrolysate of protein.
F. 6-chloro-17-hydroxy-1ß, 2 ß-dihydro-3 'H- Content
cyclopropa[l,2]pregna-l,4,6-txiene-3,20-dione, 98.5 per cent to 101.0 per cent (dried substance).
2016 Cysteine Hydrochloride 1-673
CHARACTERS Ninhydrin-positive substances

Appearance Amino add analysis (2.2.56). For analysis, use Method 1.
White or almost white, crystalline powder or colourless Prepare the solutions immediately before use.
crystals. The concentrations of the test solution and the reference
Solubility solutions may be adapted according to the sensitivity of the
Freely soluble in water, slightly soluble in ethanol equipment used. The concentrations of all solutions are
(96 per cent). adjusted so that the system suitability requirements described
in general chapter 2.2.46 are fulfilled, keeping the ratios of
IDENTIFICATION
concentrations between all solutions as described.
First identification A, B, E
Solution A dilute hydrochloric add R1 or a sample preparation
buffer suitable for the apparatus used.
A. Specific optical rotation (see Tests). Test solution Dissolve 30.0 mg of the substance to be
B. Infrared absorption spectrophotometry (2.2.24). examined in solution A and dilute to 50.0 mL with
Comparison cysteine hydrochloride monohydrate CRS. solution A.
C. Thin-layer chromatography (2.2.27). Reference solution (a) Dilute 1.0 mL of the test solution to
Test solution Dissolve 20 mg of the substance to be examined 100.0 mL with solution A. Dilute 2.0 mL of this solution to
in water R and dilute to 10 mL with the same solvent. 10.0 mL with solution A.
Add 10 mL of a 40 g/L solution of N-ethylmakimide R in Reference solution (b) Dissolve 30.0 mg of L-cystine R
ethanol (96 per cent) R. Allow to stand for 5 min. Dilute (impurity A) in solution A and dilute to 100.0 mL with
2 mL of the solution to 10 mL with water R. solution A. Dilute 1.0 mL of the solution to 250.0 mL with
Reference solution Dissolve 20 mg of cysteine hydrochloride solution A.
monohydrate CRS in water R and dilute to 10 mL with the Reference solution (c) Dissolve 30.0 mg of proline R in
same solvent. Add 10 mL of a 40 g/L solution of N- solution A and dilute to 100.0 mL with solution A. Dilute
ethylmaleimide R in ethanol (96 per cent) R. Allow to stand for 1.0 mL of the solution to 250.0 mL with solution A.
5 min. Dilute 2 mL of the solution to 10 mL with water R. Reference solution (d) Dilute 6.0 mL of ammonium standard
Plate TLC silica gel plate R. solution (100 ppm N H J R to 50.0 mL with solution A. Dilute
Mobile phase glacial acetic add R, water R, butanol R 1.0 mL of this solution to 100.0 mL with solution A.
(20:20:60 VfV/V). Reference solution (e) Dissolve 30 mg of isoleudne R and
Application 5 |iL. 30 mg of leudrte R in solution A and dilute to 50.0 mL with
Development Over 2/3 of the plate. solution A. Dilute 1.0 mL of the solution to 200.0 mL with
solution A.
Drying At 80 °C for 30 min.
Detection Spray with ninhydrin solution R and heat at 105 °C
for 15 min. Inject suitable, equal amounts of the test, blank and reference
solutions into the amino add analyser. Run a program
Residts The principal spot in the chromatogram obtained with
suitable for the determ ination of physiological am ino adds.
System suitability: reference solution (e):
reference solution.
isoleucine and leucine.
D. Dissolve about 5 mg in 1 mL of dilute sodium hydroxide
solution R. Add 1 mL of a 30 g/L solution of sodium
— for impurity A, use the concentration of impurity A in
nitroprusside R. An intense violet colour develops which
reference solution (b);
becomes brownish-red and then orange. Add 1 mL of
— for any ninhydrin-positive substance detected at 570 nm,
hydrochloric add R. The solution becomes green.
use the concentration of cysteine in reference solution (a);
E. Dissolve about 50 mg in 5 mL of water R. Heat to about — for any ninhydrin-positive substance detected at 440 nm,
60 °C on a water-bath and carefully add, dropwise, 5 mT. of me the concentration of proline in reference solution (c);
strong hydrogen peroxide solution R. Heat the water-bath to if a peak is above the reporting threshold at both
boiling and maintain the sample on the water-bath for 1 h. wavelengths, use the result obtained at 570 nm for
After cooling to room temperature reconstitute the sample to quantification.
10 mL with water R. 2 mL of the solution gives reaction (a) Limits:
of chlorides (2.3.1). — impurity A at 570 nm: maximum 0.5 per cent;
TESTS — any ninhydrin-positive substance, for each impurity,
Solution S maximum 0.2 per cent;
Dissolve 2.5 g in distilled water R and dilute to 50 mL with — total: m axim um 1.0 per cent;
the same solvent. — reporting threshold: 0.05 per cent
Appearance o f solution The thresholds indicated under Related substances
The solution is clear (2.2.1) and not more intensely coloured (Table 2034.-1) in the general monograph Substancesfor
than reference solution BY6 (2.2.2, Method II). pharmaceutical use (2034) do not apply.
Dilute 10 mL of solution S to 20 mL with water R. Sulfates (2.4.13)

Specific optical rotation (2.2.7) Maximum 300 ppm.
+ 5.5 to + 7.0 (dried substance). Dilute 10 mL of solution S to 15 mL with distilled water R.
Dissolve 2.00 g in hydrochloric add R1 and dilute to 25.0 mL
with the same acid.
1-674 Cystine 2016
A m m o n iu m
Amino add analysis (2.2.56) as described in the test for
Cystine ;****,
★ ★
ninhydrin-positive substances with the following (Ph. Eur. monograph 0998) **
modifications.
Injection Test solution, reference solution (d) and blank H nh 2
solution. ho’c X - ' - s ' s ' ~ X co2h

Limit.: H NH2
— ammonium at 570 nm: not more than the area of the
reference solution (d) (0.02 per cent), taking into account C ^ N a O iS z 240.3 56-89-3
the peak due to ammonium in the chromatogram
Action and use
obtained with the blank solution.
Amino add.
Iron (2.4.9)
Maximum 20 ppm. PhEur_____ _____________________________________________________
In a separating funnel, dissolve 0.50 g in 10 mL of dilute DEFINITION

hydrochloric acid R. Shake with 3 quantities, each of 10 mL, Cystine contains not less than 98.5 per cent and not more
of methyl isobutyl ketone R l, shaking for 3 min each time. than the equivalent of 101.0 per cent of
To the combined organic layers add 10 mL of water R and 3,3'-disulfanediylbis[(2i?)-2-aminopropanoic acid], calculated
shake for 3 min. Use the aqueous layer. with reference to the dried substance.
Heavy metals (2.4.8) CHARACTERS
Maximum 10 ppm. A white or almost white, crystalline powder, practically
0.5 g complies with test G. Prepare the reference solution insoluble in water and in alcohol. It dissolves in dilute
using 0.5 ml. of lead standard solution (10 ppm Pb) R. solutions of alkali hydroxides.
Loss on drying (2.2.32) IDENTIFICATION
8.0 per cent to 12.0 per cent, determined on 1.000 g by First identification A , B
drying at a pressure not exceeding 0.7 kPa for 24 h.
Second identification A, C, D
Sulfa ted ash (2.4.14) A. Specific optical rotation (see Tests).
ASSAY (2.2.24), comparing with the spectrum obtained with
In a ground-glass stoppered flask dissolve 0.300 g of the cystine CRS. Examine the substances prepared as discs.
substance to be examined and 4 g of potassium iodide R in C. Examine the chromatograms obtained in the test for
20 mL of water R. Cool the solution in iced water and add ninhydrin-positive substances. The prindpal spot in the
3 mL of hydrochloric acid R l and 25.0 mL of 0.05 M iodine. chromatogram obtained with test solution (b) is similar in
Stopper the flask and allow to stand in the dark for 20 min. position, colour and size to the prindpal spot in the
Titrate with 0.1 M sodium thiosulfate using 3 mL of starch chromatogram obtained with reference solution (a).
solution R, added towards the end of the titration, as
D. To 0.1 g carefully add 1 mL of strong hydrogen peroxide
indicator. Carry out a blank titration.
solution R and 0.1 mL of ferric chloride solution R l. Allow to
1 mL of 0.05 M iodine is equivalent to 15.76 mg of cool. Add 1 mL of dilute hydrochloric acid R and 5 mL of
3 8C I N 02S .
C H
water R. Add 1 mL of barium chloride solution R l. Turbidity
STORAGE or a white predpitate develops within 3 min.
Protected from light. TESTS
IMPURITIES Appearance of solution
Specified impurities A Dissolve 1.0 g in dilute hydrochloric acid R and dilute to
Other detectable impurities (the following substances would, if 10 mL with the same add. The solution is dear (2.2.1) and
present at a sufficient level, be detected by one or other of not more intensely coloured than reference solution Y7
(2.2.2, Method II).
acceptance criterion for other/unspecified impurities. It is Specific optical rotation (2.2.7)
therefore not necessary to identify these impurities for Dissolve 0.50 g in 1 M hydrochloric add and dilute to
demonstration of compliance. See also 5.10. Control of 25.0 mL with the same add. The specific optical rotation is
impurities in substances for pharmaceutical use): B. -218 to -224, calculated with reference to the dried
substance.
H nh 2
Ninhydrin-positive substances
H NH2
TLC silica gel plate R
A. (2R,2'R)-3,3'-disulfanediylbis(2-arriinopropanoic add)
examined in 1 M hydrochloric acid and dilute to 10 mL with
(cystine),
the same add.
H NHj Test solution (b) Dilute 1 mL of test solution (a) to 50 mL
c o 2h with water R.
Reference solution (a) Dissolve 10 mg of cystine CRS in 1 mL
B. (25)-2-amino-3-hydroxypropanoic add (serine). of 1 M hydrochloric acid and dilute to 50 mL with water R.
PhEur
2016 Cytarabine 1-675
Reference solution (b) Dilute 2 mL of test solution (b) to

Cytarabine ★★* ★★
20 mL with water R. ★ ★
Reference solution (c) Dissolve 10 mg of cystine CRS and (Ph. Eur. monograph 0760) *****
10 mg of arginine hydrochloride CRS in 1 mL of 1 M
hydrochloric add and dilute to 25 mL with water R.
Apply separately to the plate 5 pL of each solution. Develop
concentrated ammonia R and 70 volumes of 2-propanol R.
Allow the plate to dry in air. Spray with nmhydrin solution R
and heat at 100 °C to 105 °C for 15 min. Any spot in the
chromatogram obtained with test solution (a), apart from the
principal spot, is not more intense than the spot in the
(0.2 per cent). The test is not valid unless the chromatogram C9H 13N3O5 243.2 147-94-4
separated spots. Action and use
Chlorides (2.4.4) Pyrimidine analogue; cytotoxic.
Dissolve 0.25 g in 5 mL of dilute nitric acid R and dilute to Preparation
15 mL with water R. The solution, without further addition Cytarabine Injection
of nitric add, complies with the limit test for chlorides
(200 ppm). PhEur.
Sulfates (2.4.13) DEFINITION

Dissolve 0.5 g in 5 mL of dilute hydrochloric acid R and dilute Cytarabine contains not less than 99.0 per cent and not more
to 15 mL with distilled water R. The solution complies with than the equivalent of 100.5 per cent of 4-amino- 1-ft-n-
the limit test for sulfates (300 ppm). arabinofuranosylpyrimidin-2 ( 1H)-one, calculated with
A m m o n iu m (2.4.1) reference to the dried substance.
0.10 g complies with limit test B for ammonium (200 ppm). CHARACTERS
Prepare the standard using 0.2 mL of ammonium standard A white or almost white, crystalline powder, freely soluble in
solution (100 ppm N H J R. water, very slightly soluble in alcohol and in methylene
Iron (2.4.9) chloride.
In a separating funnel, dissolve 1.0 g in 10 mL of dilute It mdts at about 215 °C.
hydrochloric add R. Shake with three quantities, each of
IDENTIFICATION
10 mT, of methyl isobutyl ketone R l, shaking for 3 min each
A. Dissolve 20.0 mg in 0.1 M hydrochloric acid and dilute to
time. To the combined organic layers add 10 mL of water R
100.0 mL with the same add. Dilute 5.0 mL of the solution
and shake for 3 min. The aqueous layer complies with the
to 100.0 mL with 0.1 M hydrochloric acid. Examined between
limit test for iron (10 ppm).
230 nm and 350 nm (2.2.25), the solution shows an
Heavy metals (2.4.8) absorption maximum at 281 nm. The specific absorbance at
2.0 g complies with test D for heavy metals (10 ppm). the maximum is 540 to 570.
Prepare the reference solution using 2 mL of lead standard
Loss on drying (2.2.32) cytarabine CRS. Examine the substances prepared as discs.
Not more than 0.5 per cent, determined on 1.000 g by C. Examine the chromatograms obtained in the test for
drying in an oven at 105 °C. rdated substances in ultraviolet light at 254 nm.
Sulfated ash (2.4.14) The principal spot in the chromatogram obtained with test
Not more than 0.1 per cent, determined on 1.0 g. solution (b) is similar in position and size to the prindpal
ASSAY spot in the chromatogram obtained with reference
In a flask with a ground-glass stopper, dissolve 0.100 g in a solution (a).
mixture of 2 mL of dilute sodium hydroxide solution R and TESTS
10 mL of water R. Add 10 mL of a 200 g/L solution of Appearance of solution
potassium bromide R, 50.0 mL of 0.0167 M potassium bromate Dissolve 1.0 g in water R and dilute to 10 mL with the same
and 15 mL of dilute hydrochloric add R. Stopper the flask and solvent. The solution is clear (2.2.1) and not more intensely
cool in iced water. Allow to stand in the dark for 10 min. coloured than reference solution Y5 (2.2.2, Method II).
Add 1.5 g of potassium iodide R. After 1 min, titrate with Specific optical rotation (2.2.7)
0.1 M sodium thiosulfate, using 2 mL of starch solution R,
added towards the end-point, as indicator. Carry out a blank same solvent The specific optical rotation is + 154 to + 160,
titration. calculated with reference to the dried substance.
1 mL of 0.0167Mpotassium bromate is equivalent to Related substances
2.403 mg of C 5HJ2N 2O4S2. Examine by thin-layer chromatography (2.2.27), using silica
STORAGE gel GF254 R as the coating substance.
Store protected from light. Test solution (a) Dissolve 0.25 g of the substance to be
______________________________________________________________ PhEur examined in water R and dilute to 5 mL with the same
solvent.
1-676 Dacarbazine 2016
Test solution (b) Dilute 2 mL of test solution (a) to 50 ml. ★ ★

with water R.
Dacarbazine ★ *
Reference solution (a) Dissolve 10 mg of cytarabine CRS in (Ph. Eur. monograph 1691) *****
water R and dilute to 5 mL with the same solvent.
Reference solution (c) Dissolve 20 mg of uridine R and 20 mg
of uracil arabinoside CRS in methanol R and dilute to 10 mL
C6H10N6O 182.2 4342-03-4
water R, 20 volumes of acetone R and 65 volumes of methyl
ethyl ketone R. Allow the plate to dry in air and examine in
Action and use
ultraviolet light at 254 nm. Any spot in the chromatogram Cytotoxic alkylating agent
obtained with test solution (a), apart from the principal spot,
is not more intense than the spot in the chromatogram
obtained with reference solution (b) (0.5 per cent). The test
DEFINITION
is not valid unless the chromatogram obtained with reference
5-[(l£)-3,3-Dimethyltriaz-1-enyl]-1//-imidazole-4-
solution (c) shows two clearly separated spots.
carboxamide.
Content
drying over diphosphorus pentoxide R at 60 °C at a pressure of
0.2 kPa to 0.7 kPa for 3 h. CHARACTERS
Sulfa ted ash (2.4.14) Appearance
Not more than 0.5 per cent, determined on 1.0 g. White or slightly yellowish, crystalline powder.
ASSAY Solubility
Slightly soluble in water and in anhydrous ethanol, practically
Dissolve 0.200 g in 60 mL of anhydrous acetic acid R,
wanning if necessary. Titrate with 0.1 M perchloric acid
determining the end-point potentiometrically (2.2.20). IDENTIFICATION
1 mL of 0.1 M perchloric acid is equivalent to 24.32 mg of First identification B
C9H 13N 3O5. Second identification A , C
STORAGE A. Ultraviolet and visible absorption spectrophotometry

Store in an airtight container, protected from light. (2.2.25).
Test solution Dissolve 15.0 mg in 100.0 mL of 0.1 M
IMPURITIES
hydrochloric acid. Dilute 5.0 mL of this solution to 100.0 mL
o with 0.1 M hydrochloric add.

Shoulder At 275 nm.
S pedfic absorbance a t the absorption maximum 1024 to 1131.
Comparison dacarbazine CRS.
A. R = OH, R' = H: 1-f^D-arabinofuranosylpyrimidine- Test solution Dissolve 2.0 mg of the substance to be examined
2,4(l//,3/i)-dione (uracil arabonoside), in methanol R and dilute to 5.0 ml. with the same solvent
B. R = OH, R ' = OH: 1-P-D-rabofuranosylpyrimidine- Reference solution Dissolve 2.0 mg of dacarbazine CRS in
2,4(1//,3H)-dione (uridine). methanol R and dilute to 5.0 mL with the same solvent.
_______________________________________________________________ PhEur Plate TLC silica gel F 254 plate R.

Mobile phase glacial acetic acid R, water R, butanol R
(1:2:5 V/V/V).
Application 10 jiL.
Drying In air.
solution.
2016 Dacarbazine 1-677
TESTS chromatogram obtained with reference solution (b)

Appearance of solution (0.10 per cent);
The solution is clear (2.2.1) and not more intensely coloured — total: not more than 5 times the area of the peak due
than reference solution BY6 (2.2.2, Method 11). to dacarbazine in the chromatogram obtained with
Dissolve 0.25 g in a 210 g/L solution of citric acid R and reference solution (b) (0.5 per cent);
dilute to 25.0 mL with the same solution. — disregard limit. 0.5 times the area of the peak due to
dacarbazine in the chromatogram obtained with
Related substances
reference solution (b) (0.05 per cent).
A. Liquid chromatography (2.2.29). Usefreshly prepared
solutions and protect themfrom light. Im purity D
Head-space gas chromatography (2.2.28).
examined and 75 mg of citric acid R in distilled water R and Test solution Introduce 0.200 g of the substance to be
dilute to 5.0 mL with the same solvent. examined into a 20 mL vial and firmly attach the septum
and cap. Using a 10 pL syringe, inject 5 pL of water R into
Reference solution (a) Dissolve 5.0 mg of dacarbazine
the vial.
impurity A CRS in distilled water R and dilute to 50.0 mL
with the same solvent. Dilute 5.0 mL of this solution to Reference solution (a) Dilute 2.5 mL of dimetkylamine
25.0 mL with distilled water R. sdution R (impurity D) to 100.0 mL with water R
(solution A). Firmly attach the septum and cap to a 20 mL
Reference solution (b) Dissolve 5.0 mg of dacarbazine
vial. Using a 10 pL syringe, inject 10 pL of solution A into
impurity B CRS in distilled water R, add 0.5 mL of the test
the vial.
solution and dilute to 10.0 mL with distilled water R. Dilute
1.0 mL of this solution to 50.0 mL with distilled water R. Reference solution (b) Firmly attach the septum and cap to a
Column'.
20 mL vial. Using a 10 pL syringe, inject 10 pL of
— size: I = 0.25 m, 0 = 4.6 mm; solution A and 10 pL of a 10 g/L solution of triethylamine R
— stationary phase: octadecylsUyl silica gelfor into the vial.
chromatography R (5 jam). Column:
Mobile phase 15.63 g/L solution of glacial acetic add R
— sizer. I = 30.0 m, 0 = 0.53 mm;
containing 2.33 g/L of sodium dioctyl sulfosucdnate R As the
— stationary phase: base-deactivated polyethylenegfycol R (film
mobile phase contains sodium dioctyl sulfosucdnate, it must
thickness 1.0 pm).
be freshly prepared every day, and the column must be
flushed with a mixture of equal volumes of methanol R and Carrier gas helium for chromatography R.
water R, after all tests have been completed or at the end of Flow rate 13 mL/min.
the day, for at least 2 h. Split ratio 1:1.
Flow rate 1.2 mL/min. Static head-space conditions that may be used:
Detection Spectrophotometer at 254 nm. — equilibration temperature: 60 °C;
Injection 25 pL of the test solution and reference solution (a).

— equilibration time: 10 mm;
— transfer-line temperature: 90 °C;
Run time 3 times the retention time of impurity A
— pressurisation time: 30 s.
Retention time Impurity A = about 3 min.
Temperature:
Limits:
— impurity A:not more than the area of the Time Temperature
corresponding peak in the chromatogram obtained (min) ec)
with reference solution (a) (0.2 per cent); Column 0 -3 35
— unspecified impurities eluting after impurity A: for each
3-11 3 5 -» 165
impurity, not more than 0.5 times the area of the
principal peak in the chromatogram obtained with Injection port 180
reference solution (a) (0.10 per cent). Detector 220
B. Liquid chromatography (2.2.29) as described in test A for
related substances with the following modifications. Detection Flame ionisation.
Irqection 1 mT-
glacial acetic add R containing 2.33 g/L of sodium dioctyl
sulfosucdnate R with 55 volumes of methanol R.
Injection 10 pL of the test solution and reference solution (b).
impurity D and triethylamine.
Run time Twice the retention time of dacarbazine. Lim it.
Relative retention With reference to dacarbazine (retention — impurity D: not more than the area of the corresponding
time = about 12 min): impurity B = about 0.7. peak in the chromatogram obtained with reference
System suitability: reference solution (b): solution (a) (0.05 per cent).
— resolution: minimum 1.5 between the peaks due to W ater (2.5.12)
impurity B and dacarbazine. Maximum 0.5 per cent, determined on 1.00 g.
Limits:
— not more than the area of the
impurity B:
corresponding peak in the chromatogram obtained
with reference solution (b) (0.1 per cent); ASSAY
— unspecified impurities, for each impurity, not more than Dissolve 0.150 g in 30 mL of anhydrous acetic add R. Titrate
the area of the peak due to dacarbazine in the with 0.1 M perchloric add, determining the end-point
1-678 Dalteparin Sodium 2016
1 mL of 0.1 M perchloric acid is equivalent to 18.22 mg PhEur________ __________________________________________________

of C6H 10N 6O. DEFINITION
STORAGE Dalteparin sodium is the sodium salt of a low-molecular-
At a temperature of 2 °C to 8 °C, protected from light. mass heparin that is obtained by nitrous add
IMPURITIES depolymerisation of heparin from porcine intestinal mucosa.
The majority of the components have a 2-O-sulfo-a-L-
Specified impurities A,B, D
idopyranosuronic add structure at the non-reducing end and
Other detectable impurities (the following substances would, if a 6-0-sulfo-2j5-anhydro-D-mannitol structure at the reducing
present at a sufficient level5 be detected by one or other of end of their chain.
Dalteparin sodium complies with the monograph Low-molecular-
mass heparins (0828) with the modifications and additional
by the general monograph Substances for pharmaceudcal use
requirements below.
impurities for demonstration of compliance. See also 5.10. The mass-average relative molecular mass ranges between
Control of impurities in substancesfor pharmaceutical use): C. 5600 and 6400, with a characteristic value of about 6000.
The degree of sulfatation is 2.0 to 2.5 per disaccharide unit.
/=*N The potency is not less than 110 IU and not more than
HN 1
210 IU of anti-factor Xa activity per milligram, calculated
Y V with reference to the dried substance. The anti-factor Ha
• nv nh activity is not less than 35 IU/mg and not more than
100 IU/mg, calculated with reference to the dried substance.
A. 3j7-dihydro-4//-imidazo [4,5-d] -1,2,3-triazin-4-one The ratio of anti-factor Xa activity to anti-factor Ha activity is
(2-azahypoxanthine), between 1.9 and 3.2.
/^ N PRODUCTION
Dalteparin sodium is produced by a validated manufacturing
VH2N y ~ and purification procedure under conditions designed to
o
minimise the presence of N-NO groups.
The manufacturing procedure must have been shown to
B. 5-amino-1H-imidazole-4-carboxamide,
reduce any contamination by N-NO groups to approved
/=*N limits using an appropriate, validated quantification method.
IDENTIFICATION
Carry out identification test A as described in the monograph
Low-molecular-mass heparins (0828) using dalteparin
sodium CRS.
C. 5-diazenyl- lH-imidazole-4-carboxarnide,
Carry out identification test C as described in the monograph
H Low-molecular-mass heparins (0828). The following
H3C ch3 requirements apply.
The mass-average relative molecular mass ranges
D. AT-methylmethanamme. between 5600 and 6400. The mass percentage of chains
___________________________________________________________PhEur lower than 3000 is not more than 13.0 per cent. The mass
percentage of chains higher than 8000 ranges between
15.0 per cent and 25.0 per cent.
TESTS
Dalteparin Sodium ***** Appearance of solution
** +* Dissolve 1 g in 10 mL of water R. The solution is dear
(Ph. Eur. monograph 1195) * ('2.2.1) and not more intensdy coloured than intensity 5 of
the range of reference solutions of the most appropriate
colour (2.2.2, Method IT).
Nitrite
Not more than 5 ppm. Examine by liquid chromatography
(2.2.29). Rinse all volumetricflasks at least three times with
water R before the preparation of the solutions.
solvent. Allow to stand for at least 30 min.
Reference solution (a) Dissolve 60.0 mg of sodium nitrite R in
water R and dilute to 1000.0 mL with the same solvent.
n = 3 to 20 , R = HotS 0 3Na , R' = S03NaorCO-CH3
R2 = H and R3 = COjNa or R2 = C02Na and R3 = H For the preparation of reference solution (b), use a pipette
previously rinsed with reference solution (a).
Action and use Reference solution (b) Dilute 1.00 mL of reference solution (a)
Low molecular weight heparin. to 50.0 mL with water R.
Before preparing reference solutions (c), (d) and (e), rinse all
Preparation
pipettes with reference solution (b).
Dalteparin Sodium Injection
2016 Danaparoid Sodium 1-679
Reference solution (c) Dilute 1.00 mL of reference solution (b) Reference solution (b) Prepare a 11.4 |ig/mL solution of boric
to 100.0 mT. with water R (corresponding to 1 ppm of nitrite acid R in a 1 per cent V/V solution of nitric acid R in waterfor
in the test sample). chromatography R (STD^d).
Reference solution (d) Dilute 3.00 mL of reference solution (b) Reference solution (c) Dissolve 0.2500 g of a reference
to 100.0 mL with water R (corresponding to 3 ppm of nitrite dalteparin sodium with no detectable boron in about 2 mL of
in the test sample). waterfor chromatography R, add 100 (iL of nitric acid R and
Reference solution (e) Dilute 5.00 mL of reference solution (b) dilute to 10.00 mL with the same solvent (STD0).
to 100.0 mL with water R (corresponding to 5 ppm of nitrite Reference solution (d) Dissolve 0.2500 g of a reference
in the test sample). dalteparin sodium with no boron detected in about 2 mL of
The chromatographic procedure may be carried out using: a 1 per cent V/V solution of nitric acid R in waterfor
— a column 0.125 m long and 4.3 mm in internal diameter chromatography R, add 10 (iL of a 5.7 mg/mL solution of
packed with a strong anion-exchange resin; boric add R and dilute to 10.00 mL with the same solvent
— as mobile phase at a flow rate of 1.0 mL/min a solution (STDi). This solution contains l pg/mL of boron.
consisting of 13.61 g of sodium acetate R dissolved in Calculate the content of boron in the substance to be
water R, adjusted to pH 4.3 with phosphoric acid R and examined, using the following correction factor
diluted to 1000 mL with water R'}
— as detector an appropriate electrochemical device with the j = (STDi - STDo) x 2
following characteristics and settings: a suitable working (STDcai —blank)
electrode, a detector potential of + 1.00 V versus
Ag/AgCl reference electrode and a detector sensitivity of Loss on drying (2.2.32)
0.1 jiA full scale. Not more than 5.0 per cent, determined on 1.000 g by
Inject 100 pL of reference solution (d). When the drying in an oven at 60 °C over diphosphorus pentoxide R at a
chromatograms are recorded in the prescribed conditions, the pressure not exceeding 670 Pa for 3 h.
retention time for nitrite is 3.3 to 4.0 min. The test is not __________________________________________________________ PhEur
valid unless:
— the number of theoretical plates calculated for the nitrite
peak is at least 7000 per metre per column (dalteparin
sodium will block the binding sites of the stationary ★ ★
phase, which will cause shorter retention times and lower Danaparoid Sodium ★ ★
★. ★
separation efficiency for the analyte; the initial (Ph. Eur. monograph 2090) *★*
performance of the column may be partially restored
using a 58 g/L solution of sodium chloride R at a flow rate Chondroitin sulfate and
Heparan sulfate family
derm atari sulfate family
of 1.0 ml ymin for 1 h; after regeneration the column is
rinsed with 200 mL to 400 mL of water R );
— the symmetry factor for the nitrite peak is less than 3;
— the relative standard deviation of the peak area for nitrite
obtained from 6 injections is less than 3.0 per cent.
Inject 100 pL each of reference solutions (c) and (e).
The test is not valid unless:
— the correlation factor for a linear relationship between HO
concentration and response for reference solutions (c), (d) HO
and (e) is at least 0.995; — n
— the signal-to-noise ratio for reference solution (c) is not
less than 5 (if the noise level is too high, electrode
ADi R1 R2 R3 ADiHS R1 R2 R3
recalibration is recommended);
-OS H H H -OS H Ac H
— a blank injection of water R does not give rise to spurious
-6S S03Na H H -6S S03Na Ac H
peaks. -4S H S 0 3Na H -NS H S 0 3Na H
Inject 100 pL of the test solution. Calculate the content of -US H H S 0 3Na -US H Ac S03Na
nitrite from the peak areas in the chromatogram obtained -(U,6)S SOsNa H S 0 3Na -(U,N)S H S03Na S 0 3Na
with reference solutions (c), (d) and (e). -<U.4)S H S 03Na S 0 3Na
-(6,N)S S03Na S03Na H
-(4t6)S S03Na S 0 3Na H
Boron -(U,N,6)S S03Na S 03Na S 0 3Na
-<U,4,6)S S03Na SOsNa S 0 3Na
Not more than 1 ppm, determined by inductively coupled
plasma atomic emission spectroscopy.
83513-48-8
Boron is determined by measurement of the emission from
an inductively coupled plasma (ICP) at a wavelength specific Action and use
to boron. The emission line at 249.733 nm is used. Use an Heparinoid; prevention of deep vein thrombosis.
appropriate apparatus, whose settings have been optimised as
directed by the manufacturer. PhEur.
Test solution Dissolve 0.2500 g of the substance to be DEFINITION

examined in about 2 mL of waterfor chromatography R, add Preparation containing the sodium salts of a mixture of
100 |xL of nitric acid R and dilute to 10.00 mL with the same sulfated glycosaminoglycans present in porcine tissues.
solvent. Danaparoid sodium is prepared from the intestinal mucosa of
Reference solution (a) Prepare a 1 per cent V/V solution of pigs. Its major constituents are heparan sulfate and dermatan
nitric acid R in waterfor chromatography R (blank). sulfate. On com plete hydrolysis it liberates D-glucosam ine,
D-galactosamine, D-glucuronic add, L-iduronic add, acetic
1-680 Danaparoid Sodium 2016
add and sulfuric add. It has the characteristic property of D etermine by selective enzymatic degradation.
enhancing the inactivation of activated factor X (factor Xa) Test solutions Dry the substance to be examined at 60 °C over
by antithrombin. It has a negligible effect on the inactivation diphosphorus pentoxide R at a pressure of about 670 Pa for
rate of thrombin by antithrombin. 3 h. Dissolve 0.200 g of the dried substance in 10.0 mL of
Potency water R. Dilute this solution as necessary to obtain 3 test
11.0 to 17.0 anti-factor Xa units per milligram (dried solutions containing 20 mg/mL, 10 mg/mL and 5 mg/mL of
substance). the dried substance to be examined in water R.
PRODUCTION Chondroitin sulfate reference solutions Dry chondroitin sulfate
The animals from which danaparoid sodium is derived must sodium CRS over diphosphorus pentoxide R at room
fulfil the requirements for the health of animals suitable for temperature at a pressure of about 670 Pa for 16 h. Prepare
human consumption. It is prepared using a process that solutions containing 1 mg/mL, 2 mg/mL and 3 mg/mL of
ensures that the relative proportion of active sulfated dried chondroitin sulfate sodium CRS in water R.
glycosaminoglycans is consistent. It is produced by methods Dermatan sulfate reference solutions Dry dermatan sulfate CRS
of manufacturing designed to minimise or eliminate over diphosphorus pentoxide R at room temperature at a
endotoxins and hypotensive substances. pressure of about 670 Pa for 16 h. Prepare solutions
CHARACTERS containing 1 mg/mL, 2 mg/mL and 3 mg/mL of dried
dermatan sulfate CRS in water R.
Appearance
White or almost white, hygroscopic powder. Chondroidnase A B C solution Dissolve chondroitinase A B C R in
tris-sodium acetate-sodium chloride buffer solution p H 8.0 R to
Solubility
Freely soluble in water. obtain an activity of 0.5-1.0 units per millilitre.
Chondroitinase A C solution Dissolve chondroitinase A C R in
IDENTIFICATION
tris-sodium acetate-sodium chloride buffer solution p H 7.4 R to
A. The ratio of anti-factor Xa activity to anti-factor Ha obtain an activity of 1.0-2.0 units per millilitre.
activity, determined as described under Assay and Tests
Procedure:
respectively, is not less than 22.
— Degradation with chondroidnase ABC: label 2 sets of
B. Molecular mass distribution (see Tests): the mass-average 10 tubes in triplicate: T l, T2 and T3 for the test
relative molecular mass ranges between 4000 and 7000. solutions; SD1, SD2 and SD3 for the dermatan sulfate
TESTS reference solutions; SCI, SC2 and SC3 for the
pH (2.2.3) chondroitin sulfate reference solutions; and B for the
5.5 to 7.0. blank (water R). To each tube add 1.25 mL of tris-sodium
Dissolve 0.5 g of the dried substance to be examined in acetate buffer solution p H 8.0 R and 150 pL of the test
carbon dioxide-free water R and dilute to 50 mL with the same solutions, dermatan sulfate reference solutions,
solvent. chondroitin sulfate reference solutions or water R.
A nti-factor Ila activity To each tube in 1 set of tubes add 75 jiL of
Maximum 0.5 units per milligram (dried substance). chondroitinase ABC solution. To determine the blank
response level, add 75 (iL of tris-sodium acetate-sodium
Test solutions Prepare 2 independent series of dilutions in
chloride buffer solution p H 8.0 R to each tube in the odaer
geometric progression of the substance to be examined in set of tubes. Mix the contents of the tubes using a vortex
phosphate buffer solution p H 6.5 R and in the concentration
mixer, cover with appropriate stoppers and incubate at
range of 0.0005 to 0.005 units of anti-factor Ha activity per 37 °C for at least 24 h.
millilitre.
— Degradation with chondroitinase AC: label 7 tubes in
Reference solutions Prepare 2 independent series of dilutions in triplicate: T l, T2 and T3 for the test solutions; SCI,
geometric progression of danaparoid sodium CRS in phosphate SC2 and SC3 for the chondroitin sulfate reference
buffer solution p H 6.5 R and in the concentration range of solutions; and B for the blank (water R). To each tube
0.0005 to 0.005 units of anti-factor Ha activity per millilitre. add 1.25 mL of tris-sodium acetate buffer solution p H 7.4 R
Transfer 50 jiL of each solution into the wells of a 96-well and 150 ^L of the test solutions,
microtitre plate. To each well add 50 jiL of antithrombin III chondroitin sulfate reference solutions or water R.
solution R3 and 50 |iL of human thrombin solution R l. Shake Add 75 jiL of chondroitinase AC solution to each tube.
the microtitre plate but do not allow bubbles to form. Mix the contents of the tubes using a vortex mixer, cover
Incubate for 75 min. To each well add 50 jiL of chromogenic with appropriate stoppers and incubate at 37 °C for at
substrate R4. Shake the microtitre plate. Measure the least 24 h. After the incubation period mix the contents
absorbances at 405 nm (2.2.25) using a suitable reading of the tubes using a vortex mixer and dilute to 12 times
device, exactly 4 min after the addition of the chromogenic with water R. Measure the absorbances (2.2.25) of the
substrate. The reaction may be stopped using 75 jiL of a diluted solutions at 234 nm against water R using a
20 per cent V/V solution of glacial acetic acid R. Determine suitable spectrophotometer.
the blank amidolytic activity in a similar manner, using Calculation Calculate the mean blank absorbance of each
phosphate buffer solution p H 6.5 R as the blank solution reference solution, i.e. the mean of die absorbances of the
(minimum 10 blanks per microtitre plate). Calculate the reference solutions to which no chondroitinase ABC has been
activity of the substance to be examined in units of anti added. Subtract the mean blank absorbance value from the
factor Ila activity per milligram using a suitable statistical individual absorbance of each reference solution. Calculate
method, for example the parallel-line assay. linear regression curves for the 2 chondroitin sulfate reference
Chondroitin sulfate and derm atan sulfate and the dermatan sulfate reference by plotting the blank-
Chondroitin sulfate: maximum 8.5 per cent (dried corrected absorbances against the concentrations.
substance); dermatan sulfate: 8.0 per cent to 16.0 per cent
(dried substance).
2016 Danaparoid Sodium 1-681
Calculate the average percentage content of dermatan sulfate determine the molecular mass distribution of the sample.
in the test solutions of all tested concentrations using the A calibration table is supplied with danaparoid sodium CRS.
following expression: Limits:
— chains with a relative molecular mass less than 2 0 0 0 .
a a (-^3 — A i — 1\) x B i T T maximum 13 per cent;
A2- Ai - ------------ =---- ------------h - h
— chains with a relative molecular mass less than 4000.
------------------ B % C ----------------- Xl0° maximum 39 per cent;
— chains with a relative molecular mass between 4000 and
Ai = blank absorbance of the test solution; 8000. minimum 50 per cent;
A2 = absorbance of the test solution with chondroitinase — chains with a relative molecular mass higher than 8000:
ABC; maximum 19 per cent;
A3 = absorbance of die test solution with chondroitinase — chains with a relative molecular mass higher than 1 0 000 '.
AC; maximum 11 per cent.
Bi = gradient of the curve obtained with the chondroitin Nitrogen (2.5.9)
sulfate reference solutions with chondroitinase AC; 2.4 per cent to 3.0 per cent (dried substance).
B2 = gradient of the curve obtained with the chondroitin Nucleic adds
sulfate reference solutions with chondroitinase ABC; Maximum 0.5 per cent (dried substance).
B3 = gradient of the curve obtained with the dermatan
Test solution Weigh about 50 mg of the dried substance to be
sulfate reference solutions with chondroitinase ABC;
C = concentration of the test solution, in milligrams per examined into a centrifuge tube and dissolve in 200 pL of
water R.
millilitre;
Ix = y-intercept of die curve obtained with the Reference solution Dissolve about 50 mg of ribonucleic
chondroitin sulfate reference solutions with acid CRS in 5 mL of 0.1 M sodium hydroxide and dilute to
chondroitinase AC; 20.0 mL with water R Transfer 200 pL of the solution into a
J2 = y-intercept of the curve obtained with the centrifuge tube.
chondroitin sulfate reference solutions with Add 4.0 mL of a 50 g/L solution of trichloroacetic acid R to
chondroitinase ABC; each tube and mix. Place all tubes in boiling water for
/3 = y-intercept of the curve obtained with the dermatan 30 min. Allow to cool to room temperature. Add again
sulfate reference solutions with chondroitinase ABC. 4.0 mL of a 50 g/L solution of trichloroacetic acid R to each
Calculate the average percentage content of chondroitin tube and mix. If any of the test solutions is not clear,
sulfate in the test solutions for all tested concentrations using sonicate all the tubes in an ultrasonic bath for 10 m in and
the following expression: centrifuge at 1500 g for 15 min. Dilute 1.0 mL of the clear
supernatant to 4.0 mL with water R. Measure the
(A 3 - A x - I x ) x 100 absorbances of the diluted reference and test solutions at
265 nm (2.2.25) against a blank solution prepared in the '
BxxC
same manner, and calculate the percentage nucleic acid 1
content of the sample.
M olecular mass distribution
Size-exclusion chromatography (2.2.30). Total protein (2.5.33, Method 2)
in 2 mL of the mobile phase. Dissolve the substance to be examined in water R. Use bovine
albumin R as the reference substance.
Reference solution Dissolve 10 mg of danaparoid sodium CRS in
2 mL of the mobile phase. Sodium
Column:
— size: I = 0.60 m, 0 = 7.5 mm; Atomic absorption spectrometry (2.2.23, Method I).
— stationary phase: hydropMHc silica gd for chromatography R Test solution Dissolve 0.125 g of the substance to be
(10 pm) with a fractionation range for proteins with a examined in 100.0 mL of a 1.27 mg/mL solution of caesium
relative molecular mass of approximately 5000-100 000; chloride R in 0.1 M hydrochloric acid.
— temperature: 30 °C. Reference solutions Prepare reference solutions containing
Mobile phase 28.4 g/L solution of anhydrous sodium sulfate R 50 ppm, 100 ppm and 150 ppm of Na by diluting sodium
adjusted to pH 5.0 with dilute sulfuric acid R standard solution (1000 ppm Na) R with a 1.27 mg/mL
Flow rate 0.9 ml 7min ± 2 per cent solution of caesium chloride R in 0.1 M hydrochloric add.
Detection Spectrophotometer at 210 nm. Source Sodium hollow-cathode lamp.
Irtjection 100 jiL. Wavelength 330.3 nm.
Run time For a period of time ensuring complete elution of Atomisation device Air-acetylene flame.
sample and solvent peaks (about 40 min). Loss on drying (2.2.32)
System suitability Inject the reference solution twice. Maximum 5.0 per cent, determined on 0.500 g by drying in
The difference between the retention times corresponding to an oven at 60 °C over diphosphorus pentoxide R at a pressure
the maxima of the peaks is not more than 5 s. of 670 Pa for 3 h.
Calibration Calibration is achieved by taking the relevant part Bacterial endotoxins (2.6.14)
of the chromatogram obtained with the reference solution, Less than 0.02 IU per unit of anti-factor Xa activity, if
i.e. excluding the sharp peak at the end of the intended for use in the manufacture of parenteral
chromatogram, and matching the chromatogram obtained preparations without a further appropriate procedure for the
with the test solution with the calibration table obtained with removal of bacterial endotoxins.
the reference solution. From the calibration curve obtained,
1-682 Dantrolene Sodium 2016
ASSAY CHARACTERISTICS
The anticoagulant activity of danaparoid sodium is A yellowish-orange to orange crystalline powder.
determined in vitro by an assay which determines its ability to Very slightly soluble in water, slightly soluble in ethanol
accelerate the inhibition of factor Xa by antithrombin HI (96%); sparingly soluble in methanol’, practically insoluble in
(anti-factor Xa assay). acetone.
Test solutions Prepare 2 independent series of dilutions in
IDENTIFICATION
geometric progression of the substance to be examined in
A. The infrared absorption spectrum, Appendix II A, is
tris(hydroxymethyl)aminomethane ED TA buffer solution
concordant with the reference spectrum of dantrolene sodium
p H 8.4 R and in the concentration range of 0.1 to 0.32 units
CRS 422).
of anti-factor Xa activity per millilitre.
B. In the Assay, the chromatogram obtained with solution
Reference solutions Prepare 2 independent series of dilutions in
(1) shows a peak with the same retention time as the
geometric progression of danaparoid sodium CRS in
tris(hydroxyrnethyl)aminomethane ED TA buffer solution
solution (2).
pH 8.4 R and in the concentration range of 0.08 to
0.35 units of anti-factor Xa activity per millilitre. C. To 0.1 g of the substance being examined add 20 mL of
water and 2 drops of acetic acid, shake well and filter.
Transfer 40 |iL of each solution into the wells of a 96-well
The filtrate yields the reactions characteristic of sodium salts,
microtitre plate. Add 40 jiL of antithrombin III solution R4 to
Appendix VI.
each well and shake the microtitre plate but do not allow
bubbles to form. Add 40 jiL of bovine factor X a solution R1 to TESTS
each well. Exactly 2 min after the addition of the factor Xa Alkalinity
solution, add 80 pL of chromogenic substrate R5. Measure the Shake 0.7 g in 10 mL of water for 5 minutes and centrifuge.
absorbance at 405 nm (2.2.25) using a suitable reading To 5 mL of die supernatant add 45 mL of water and 3 drops
device, exactly 4 min after the addition of the factor Xa of phenolphthalein solution R1 and 0.1 m l. of 0.1m hydrochloric
solution. The reaction may be stopped using 75 pL of a acid VS. A red colour is not produced.
20 per cent VIV solution of glacial acetic acid R. Determine Related substances
the blank amidolytic activity in the same manner, using Carry out the method for liquid chromatography,
tris(hydroxymethyl)aminomethane ED TA buffer solution Appendix IQ D, using the following solutions.
pH 8.4 R as the blank (minimum 8 blanks per microtitre
(1) Dissolve 50 mg of the substance being examined in
plate). Calculate the potency of the substance to be
20 mL of tetrahydrcfuran and 2 mL of glacial acetic acid and
examined in units of anti-factor Xa activity per milligram
dilute with sufficient absolute ethanol to produce 100 mL.
using a suitable statistical method, for example the parallel-
line assay. (2) Dilute 1 mL of solution (1) to 100 mL with absolute
ethanol.
STORAGE
(3) Dissolve 5 mg of dantrolene sodium BPCRS and 0.1 g of
In an airtight container. If the substance is sterile, store in a theophylline BPCRS in 20 mL of tetrahydrcfuran and 2 mL of
sterile, airtight, tamper-proof container. glacial acetic acid and dilute with sufficient absolute ethanol to
LABELLING produce 100 mT„ Further dilute 10 mL of this solution to
The label states the number of units of anti-factor Xa activity 100 m l. with absolute ethanol
per milligram. C H R O M A T O G R A P H IC C O N D IT IO N S
___________________________________________________________PhEtr (a) Use a stainless steel column (15 cm x 4.6 mm) packed
with silica gelfor chromatography (5 Jim) (Zorbax Sil is
suitable).
Dantrolene Sodium below.
(c) Adjust the flow rate of the mobile phase so that the
retention time of the peak corresponding to Dantrolene
Sodium is about 8 minutes.
(d) Use a column temperature of 30°.
NNa , 31/2 HzO
(f) Inject 10 |iL of each-solution.
(g) For solution (1) allow the chromatography to proceed for
at least twice the retention time of the principal peak.
C14H 9N4Na05 ,31/ 2H20 399.3 24868-20-0
MOBILE PHASE
Action and use 9 volumes of absolute ethanol, 10 volumes of glacial acetic acid
Skeletal muscle relaxant and 90 volumes of hexane.
Preparation SY ST E M S U IT A B IL IT Y
Dantrolene Oral Suspension The test is not valid unless, in the chromatogram obtained
with solution (3), the resolutionfactor between the peaks
DEFINITION
corresponding to theophylline and dantrolene is at least 6.
Dantrolene Sodium is l-(5-p-
nitrophenylfurfurylideneamino)hydantoin sodium. It contains L IM IT S
not less than 98.0% and not more than 102.0% of In the chromatogram obtained with solution (1):
Ci4H 9N 4Na 0 5 , calculated with reference to the anhydrous
substance.
2016 Dantron 1-683
the total area of all the secondary peaks is not greater than the CHARACTERISTICS
area of the principal peak in the chromatogram obtained with An orange, crystalline powder.
solution (2) (1%). Practically insoluble in water, slightly soluble in ether, very
Heavy metals slightly soluble in ethanol (96%). It dissolves in solutions of
1.0 g complies with limit test C for heavy metals, alkali hydroxides.
Appendix VII. Use 2 m l. of lead standard solution IDENTIFICATION
(10 ppm Pb) to prepare the standard (20 ppm).
A. The infrared absorption spectrum, Appendix II A, is
Water concordant with the reference spectrum of dantron (RS 083).
14.5 to 17.0% w/w, Appendix IX C. Use 0.2 g B. The light absorption, Appendix IIB , in the range 230 to
ASSAY 350 nm of a 0.001% w/v solution in dichbromethane exhibits
Carry out the method for liquid chromatography, maxima at 255 nm and 285 nm and a less well-defined
Appendix HI D, using the following solutions. maximum at 275 nm. The absorbance at the maximum at
(1) Dissolve 60 mg of the substance being examined in 255 nm is about 0.82 and at the maximum at 285 nm is
50 mL of dimethylformamide and dilute 1 volume of the about 0.48, each calculated with reference to the dried
resulting solution to 100 volumes with the mobile phase. substance.
(2) Dilute 1 volume of a 0.12% w/v solution of dantrolene C. Dissolve 5 mg in 5 m l. of 1m sodium hydroxide. A clear
sodium BPCRS in dimethylformamide to 100 volumes with the
red solution is produced immediately.
mobile phase. TESTS
C H R O M A T O G R A P H IC C O N D IT IO N S Mercury
(a) Use a stainless steel column (15 cm x 4.6 mm) packed To 0.50 g in a Kjeldahl flask add 2.5 mL of nitric acid and
with spherical particles of silica, 5 pm in diameter, the allow to stand until the initial vigorous reaction has subsided.
surface of which has been modified with chemically-bonded Add 2.5 mL of sulfuric acid and heat until dense white fumes
nitrile groups (Spherisorb CN is suitable). are evolved. Cool, add 2.5 mL of nitric add and heat until
fumes are again evolved. Repeat the procedure with a further
(b) Use isocratic elution and the mobile phase described 2.5 mL of nitric acid, cool, add 50 mL of water, boil the
below. solution until the volume has been reduced to about 25 mL
(c) Use a flow rate of 1 mL per minute. and cool. Transfer to a separating funnel using water, dilute
(d) Use an ambient column temperature. to about 50 mL with water and add 50 mL of 0.5m sulfuric
(e) Use a detection wavelength of 262 nm. add. Add 100 mL of water, 2 g of hydroxylamine
hydrochloride, 1 m l. of 0.05m disodium edetate, 1 ml. of glacial
(f) Inject 20 pL of each solution.
acetic add and 5 mL of dichbromethane, shake, allow to
MOBILE PHASE separate and discard the dichloromethane layer. Titrate the
15 volumes of acetonitrUe and 85 volumes of a phosphate aqueous layer with a 0.0008% w/v solution of dithizone in
buffer pH 6.8 prepared by dissolving 11.88 g of disodium dichbromethane, shaking vigorously after each addition,
hydrogen orthophosphate and 9.08 g of potassium dihydrogen allowing the layers to separate and discarding the
orthophosphate in 1000 m l. of water. dichloromethane layer, until the dichloromethane layer
D E T E R M IN A T IO N O F C O N T E N T
remains green. Repeat the operation using a solution
prepared by diluting 1 mL of mercury standard solution
Calculate the content of C 14H 9N 4Na 0 5 in the substance
(5 ppm Hg) to 100 mL with 0.5m sulfuric acid and beginning
being examined using the declared content of Ci4H9N4N a0 5
at the words ‘Add 100 mL of water ... ’. The volume of the
in dantrolene sodium BPCRS.
dithizone solution required by the substance being examined
does not exceed that required by the mercury standard
solution.
Related substances
Dantron Carry out the method for liquid chromatography,
Appendix EH D, using the following solutions.
OH O OH (1) Dissolve 50 mg of the substance being examined in
20 m l. of tetrahydrofuran and dilute to 100 mL with the
mobile phase.
(2) Dilute 1 volume of solution (1) to 50 volumes with the
O mobile phase.
(3) Dissolve 50 mg of dantron impurity standard BPCRS in
20 ml. of tetrahydrofuran and dilute to 100 mL with the
Ci4H80 4 240.2 117-10-2
mobile phase.
Action and use C H R O M A T O G R A P H IC C O N D IT IO N S
Anthraquinone stimulant laxative. (a) Use a stainless steel column (25 cm x 4.6 mm) packed
Preparation with octadecylsUyl silica gelfor chromatography (5 jim)
Co-danthrusate Capsules (Nucleosil C18 is suitable).
(b) Use an isocratic system using the mobile phase described
DEFINITION below.
Dantron is mainly 1,8-dihydroxyanthraquinone. It contains
(c) Use a flow rate of 1 mL per minute.
not less than 98.0% and not more than 102.0% of total
phenols, calculated as C 14H 804 and with reference to the (d) Use an ambient column temperature.
dried substance. (e) Use a detection wavelength of 254 nm.
1-684 Dapsone 2016
(f) Inject 20 nL of each solution. CHARACTERS

(g) Allow the chromatography to proceed for 1.5 times the A white or slightly yellowish-white, crystalline powder, very
retention time of the principal peak. slightly soluble in water, freely soluble in acetone, sparingly
soluble in alcohol. It dissolves freely in dilute mineral adds.
MOBILE PHASE
A mixture of 2.5 volumes of glacial acetic add, 40 volumes of IDENTIFICATION
tetrahydrqfuran and 60 volumes of water. A. Melting point (2.2.14): 175 °C to 181 °C.
SY ST E M S U IT A B IL IT Y
B. Dissolve 50.0 mg in methanol R and dilute to 100.0 mL
with the same solvent. Dilute 1.0 mL of this solution to
The test is not valid unless, in the chromatogram obtained
100.0 mL with methanol R. Examined between 230 nm and
with solution (3):
350 nm (2.2.25), the solution shows 2 absorption maxima, at
— a peak due to 1-hydroxyanthraquinone appears
260 nm and 295 nm. The specific absorbances at these
immediately before the principal peak, as indicated in die
maxima are 700 to 760 and 1150 to 1250, respectively.
reference chromatogram supplied with danvron
impurity standard BPCRS; C. Examine the chromatograms obtained in the test for
— the height of the trough separating the two peaks is not related substances. The prinripal spot in the chromatogram
greater than one third of the height of the peak due to obtained with test solution (b) is similar in position, colour
1-hydroxyanthraquinone. and size to the prindpal spot in the chromatogram obtained
L IM IT S
TESTS
In the chromatogram obtained with solution (1):
— the area of any peak corresponding to Related substances
1-hydroxyanthraquinone is not greater than 2.5 times the Examine by thin-layer chromatography (2.2.27), using silica
gel G R as the coating substance.
with solution (2) (3.3% taking into account the correction Test solution (a) Dissolve 0.10 g of the substance to be
factor of the impurity); examined in methanol R and dilute to 10 mL with the same
— the sum of die areas of any other secondary peaks is not solvent.
greater than the area of the principal peak in the Test solution (b) Dilute 1 mL of test solution (a) to 10 mL
chromatogram obtained with solution (2) (2%); with methanol R.
— disregard any peak with a retention time less than one Reference solution (a) Dissolve 10 mg of dapsone CRS in
third of that of the principal peak. methanol R and dilute to 10 mL with the same solvent.
Loss on drying Reference solution (b) Dilute 1 mL of test solution (b) to
When dried to constant weight at 105°, loses not more than 10 mL with methanol R.
0.5% of its weight. Use 1 g. Reference solution (c) Dilute 2 mL of reference solution (b) to
ASSAY 10 mL with methanol R.
Dissolve 0.2 g in 50 mL of anhydrous pyridine and carry out Apply separately to the plate 1 jiL of test solution (b), 1 jiL
Method II for non-aqueous titration, Appendix VUI A, using of reference solution (a), 10 (iL of test solution (a), 10 (iL of
0.1m tetrabutylammonium hydroxide FS as titrant and reference solution (b) and 10 pL of reference solution (c).
determining the end point potentiometrically. Each ml. of Develop in an unsaturated tank over a path of 15 cm using a
0.1m tetrabutylammonium hydroxide VS is equivalent to mixture of 1 volume of concentrated ammonia R, 6 volumes of
24.02 mg of total phenols, calculated as C 14H 80 4 . methanol R, 20 volumes of ethyl acetate R and 20 volumes of
heptane R. Allow the plate to dry in air. Spray the plate with
a 1 g/L solution of 4-dimethylaminodnnamaldehyde R in a
mixture of 1 volume of hydrochloric add R and 99 volumes of
★ ★ alcohol R. Examine in daylight. Any spot in the
Dapsone ★ ★ chromatogram obtained with test solution (a), apart from the
(Ph. Eur. monograph 0077) ***** prindpal spot, is not more intense than the spot in the
h2n n h 2 (1.0 per cent) and not more Than 2 such spots are more
s
// \\
o o Loss on drying (2.2.32)
C12H 12N2O2S 248.3 80-08-0
Action and use Not more than 0.1 per cent, determined on 1.0 g.
Folic acid synthesis inhibitor; treatment of leprosy.
ASSAY
Preparation Dissolve 0.100 g in 50 mL of dilute hydrochloric add R. Carry
Dapsone Tablets out the determination of primary aromatic amino-nitrogen
(2.5.8).
PhEur.
1 mL of 0.1 M sodium nitrite is equivalent to 12.42 mg of
DEFINITION C 12H 12N 2O2S.
Dapsone contains not less than 99.0 per cent and not more
than the equivalent of 101.0 per cent of STORAGE
4,4 '-sulfonyldianiline, calculated with reference to the dried Store protected from light.
substance. PhEur
2016 Daunorubicin Hydrochloride 1-685
★ ★ phase. Dilute 1.0 mL of the solution to 10.0 mL with the

Daunorubicin Hydrochloride ★ ★ mobile phase.
★, ★
(Ph. Eur. monograph 0662) *★* Reference solution (c) Dissolve 5.0 mg of daunorubicinone CRS
and 5.0 mg of doxorubicin hydrochloride CRS in the mobile
OH phase and dilute to 100.0 mL with the mobile phase. Dilute
1.0 mL of the solution to 10.0 mL with the mobile phase.
Reference solution (d) Dilute 1.0 mL of reference solution (a)
Column:
— size. I = 0.25 m, 0 = 4.0 mm,
— stationary phase, end-capped octadecylsHyl silica gel for
nh2 chromatography R (5 |im).
Mobile phase Mixture of equal volumes of acetomtrUe R and a
solution containing 2.88 g/L of sodium laurUsulfate R and
C^HaoClNO^ 564.0 23541-50-6
2.25 g/L of phosphoric acid R.
Action and use Flow rate 1 mL/min.
Cytostatic; anthracycline antibacterial. Detection Spectrophotometer at 254 nm.
PhEir----------------------------------------------------------------------------------------- Injection 5 ]iL; inject the test solution and reference

DEFINITION
Run time Twice the retention time of daunorubicin.
(8S, 10S)-8-Acetyl-l 0-[(3-amino-2,3,6-trideoxy-a-L-/y!Xí>-
hexopyranosyl)oxy]-6,8, 1l-trihydroxy-l-methoxy-7,8,9,10- Relative retention With reference to daunorubicin (retention
tetrahydrotetracene-5, 12-dione hydrochloride. time = about 15 min): impurity A = about 0.4;
impurity D = about 0.5; epirubicin = about 0.6;
Substance produced by certain strains of Streptomyces impurity B = about 0.7.
coeruleorubidus or of Streptomyces peucettus or obtained by any
other means.
— resolution: m inim u m of 2.0 between the peaks due to
Content impurity D and epirubidn.
PRODUCTION — impurity A: not more than the area of the corresp on d in g
It is produced by methods of manufacture designed to peak in the chromatogram obtained with reference
eliminate or m inim ise the presence of histamine. solution (c) (0.5 per cent),
— impurity B: not more than 3 times the area of the
CHARACTERS
Appearance
reference solution (d) (1.5 per cent),
Crystalline, orange-red powder, hygroscopic.
— impurity D: not more than the area of the corresponding
Solubility peak in the chromatogram obtained with reference
Freely soluble in water and in methanol, slightly soluble in solution (c) (0.5 per cent),
alcohol, practically insoluble in acetone. — any other impurity: not more than the area of the principal
IDENTIFICATION peak in the chromatogram obtained with reference
A. Infrared absorption spectrophotometry (2.2.24). solution (d) (0.5 per cent),
— total of other impurities: not more than 5 times the area of
Comparison daunorubicin hydrochloride CRS.
B. Dissolve about 10 mg in 0.5 mL of nitric acid R, add reference solution (d) (2.5 per cent),
0.5 mL of water R and heat over a flame for 2 min. Allow to — disregard limit: 0.1 times the area of the principal peak in
cool and add 0.5 mL of silver nitrate solution R l. A white the chromatogram obtained with reference solution (d)
precipitate is formed. (0.05 per cent).
TESTS Butanol (2.4.24, System B)
pH (2.2.3) Maximum 1.0 per cent.
4.5 to 6.5. Water (2.5.12)
Dissolve 50 mg in carbon dioxide-free water R and dilute to Maximum 3.0 per cent, determined on 0.100 g.
Related substances Less than 4.3 IU/mg, if intended for use in the manufacture
Liquid chromatography (2.2.29). Prepare the solutions of parenteral preparations without a further appropriate
immediately before use. procedure for the removal of bacterial endotoxins.
ASSAY
the mobile phase.
related substances.
Reference solution (a) Dissolve 50.0 mg of daunorubicin
Irqection Test solution and reference solution (a).
hydrochloride CRS in the mobile phase and dilute to 50.0 mL
with the mobile phase. Calculate the percentage content of C27H 30CINO10.
Reference solution (b) Dissolve 10 mg of doxorubicin STORAGE
hydrochloride CRS and 10 mg of epirubicin hydrochloride CR S In an airtight container, protected from light. If the substance
in the mobile phase and dilute to 100.0 mL with the mobile is sterile, store in a sterile, airtight, tamper-proof container.
1-686 Debrisoquine Sulfate 2016
A. The light absorption, Appendix IIB , in the range 230 to
0 OH
350 nm of a 0.05% w/v solution in 0.05m sulfuric acid
exhibits two maxima, at 262 nm and 270 nm. The absorbance
at the m a x i m u m at 262 nm is about 0.69 and at the
m a x i m u m at 270 nm is about 0.51.
OCH3 O OH H OH B. Carry out the method for thin-layer chromatography,

Appendix III A, using the following solutions in water.
A. R = CO-CH3: (85}105)-8-acetyl-6j8jl0jll-tetrahydroxy-
l-methoxy-7,8,9,1O-tetrahydrotetracene-5,12-dione
(daunorubicin agiycone, daunorubicinone), (2) 0.25% w/v of debrisoquine sulfate BPCRS.
E. R = CHOH-CH3: C85j 105)-6j8310j 1l-tetrahydroxy-8- (3) A mixture of equal volumes of solutions (1) and (2).
[(Ii?5)-l-hydroxyethyl]-l-methoxy-7j8j9,10- C H R O M A T O G R A P H IC C O N D IT IO N S
tetrahydrotetracene-5,12-dione (13-dihydrodaunorubicinone), (a) Use as the coating silica gel G.

o OH (b) Use the mobile phase as described below.
(c) Apply 10 pL of each solution.
(e) After removal of the plate, dry in air and spray with a
solution prepared by adding 1 mL of sulfuric acid to 40 mL
of a freshly prepared mixture of equal volumes of a
0.135% w/v solution of chloroplatinic(IV) acid and a 1.1% w/v
solution of potassium iodide.
M OBILE PHASE
B. R = CHOH-CH3: (8S,,10«S)-10-[(3-amino-2,3,6-trideoxy- 15 volumes of glacial acetic acid, 25 volumes of water and
a-L-/joco-hexopyranosyl) oxyJ-6,8,11-trihydroxy-8- [( 1RS)-1- 60 volumes of butan-l-ol.
hydroxyethyl]- l-methoxy-7,8,9,10-tetrahydrotetracene-5,12-
C O N F IR M A T IO N
dione (daunorubicinol),
C. R = CH2-CO-CH3: (8S,10S)-10-[(3-amino-2,3,6- The principal spot in the chromatogram obtained with
trideoxy-a-L-(yxi>-hexopyranosyl) oxy] -6,8}11-trihydroxy-1- solution (1) corresponds to that in the chromatogram
methoxy-8-(2-oxopropyi)-7}8j9j 10-tetrahydrotetracene-5,12- obtained with solution (2). The principal spot in the
dione (feudomycin B)} chromatogram obtained with solution (3) appears as a single
compact spot.
D. R = CO-CH2-OH: doxorubicin,
C. Yields the reactions characteristic of sulfates, Appendix VI.
F. R = CO-CH2-CH3: (8S, 10.S)-10- [(3-amino-233,6-
trideoxy-a-L-iyxo-hexopyranosyl) oxy] -6,8,11-trihydroxy-1- TESTS
methoxy-8-propanoyl-7,8,9,10-tetrahydrotetracene-5,12- Acidity
dione (8-ethyldaunorubicin). pH of a 3% w/v solution, 5.3 to 6.8, Appendix V L.
__________________________________________________________ PhEur Related substances
Carry out the method for thin-layer chromatography,
Appendix m A, using the following solutions in water.
Debrisoquine Sulfate (2) 0.010% w/v of debrisoquine sulfate BPCRS.
Debrisoquine Sulphate C H R O M A T O G R A P H IC C O N D IT IO N S
(a) Use as the coating silica gel G.

NH (b) Use the mobile phase as described below.
(c) Apply 10 (iL of each solution.
(e) After removal of the plate, dry in air and spray with a
solution prepared by adding 1 mL of sulfuric acid to 40 mL
of a freshly prepared mixture of equal volumes of a
(CuHiaNaJaKfeSO« 448.5 581-88-4
0.135% w/v solution of chloroplanmc(IV) acid and a 1.1% w/v
Action and use solution of potassium iodide.
Adrenergic neuron blocker. M OBILE PHASE
15 volumes of glacial acetic acid, 25 volumes of water and
DEFINITION 60 volumes of butan-l-ol
Debrisoquine Sulfate is l,2,3,4-tetrahydroisoquinoline-2-
L IM IT S
carboxamidine sulfate. It contains not less than 99.0% and
not more than 101.0% of (CioH13N 3) 2,H 2S04 , calculated Any secondary spot in the chromatogram obtained with
with reference to the dried substance. solution (1) is not more intense than the spot in the
chromatogram obtained with solution (2).
CHARACTERISTICS
A white, crystalline powder. Loss on drying
When dried to constant weight at 105°, loses not more than
Sparingly soluble in water, very slightly soluble in ethanol
0.5% of its weight. Use 1 g.
(96%); practically insoluble in ether.
2016 Demeclocycline Hydrochloride 1-687
Sulfated ash
Not more than 0.1%, Appendix IX A.
Demeclocycline Hydrochloride *****
ASSAY (Ph. Eur. monograph 0176) *
Carry out Method I for rum-aqueous titration,
Appendix VIII A, using 1 g and determining the end point
potentiometrically. Each mL of 0 .1 m perchloric acid FS is
equivalent to 44.85 mg of (CioHi3N 3) 2jH 2S0 4 .
STORAGE
Debrisoquine Sulfate should be protected from light.
C2iH22Cl2N 20 8 501.3 64-73-3
Decyl Oleate ★ ★ Action and use

★ ★
Tetracycline antibacterial.
Preparation
Action and use Demeclocycline Capsules
Excipient. PhEur__________________________________________________________
PhEtr______________________ DEFINITION
DEFINITION (4S,4aSj5aS,6S,12aS)-7-Chloro-4-(dimethylamino)-
Mixture consisting of decyl esters of fatty acids, mainly oleic 3,6,10,12,12a-pentahydroxy-l,l l-dioxo-l,4,4a,5,5a,6,l 1,12a-
(as-9-octadecenoic) acid. octahydrotetracene-2-carboxamide hydrochloride.
A suitable antioxidant may be added. Substance produced by certain strains of Streptomyces
aureofaciens.
CHARACTERS
Content
Appearance
Clear, pale yellow or colourless liquid.
Solubility CHARACTERS
Practically insoluble in water, misdble with ethanol Appearance
(96 per cent), with methylene chloride and with light Yellow powder.
petroleum (bp: 40-60 °C). Solubility
Soluble or sparingly soluble in water, slighdy soluble in
IDENTIFICATION
alcohol, very slightly soluble in acetone. It dissolves in
A. Relative density (see Tests).
solutions of alkali hydroxides and carbonates.
IDENTIFICATION
C. Oleic add (see Tests).
TESTS Test solution Dissolve 5 mg of the substance to be examined
Relative density (2.2.5) in methanol R and dilute to 10 mL with the same solvent.
0.860 to 0.870. Reference solution (a) Dissolve 5 mg of demeclocycline
A dd value (2.5.1) hydrochloride CRS in methanol R and dilute to 10 mL with the
Maximum 1.0, determined on 10.0 g. same solvent.
Iodine value (2.5.4, Method A) Reference solution (b) Dissolve 5 mg of demeclocycline
55 to 70. hydrochloride CRS, 5 mg of chlortetracycHne hydrochloride R and
Peroxide value (2.5.5, Method A) 5 mg of tetracycline hydrochloride R in methanol R and dilute to
Maximum 10.0. 10 mL with the same solvent.
Saponification value (2.5.6) Plate T L C octadecylsilyl silica gel F 254 plate R.
130 to 140, determined on 2.0 g. Mobile phase Mix 20 volumes of acetonitrUe R, 20 volumes of
methanol R and 60 volumes of a 63 g/L solution of oxalic
Oleic a d d (2.4.22, Method A)
acid R previously adjusted to pH 2 with concentrated
Minimum 60.0 per cent in the fatty add. fraction of the
ammonia R.
substance.
Application 1 |oL.
Water (2.5.12)
Maximum 1.0 per cent, determined on 1.00 g. Development Over 3/4 of the plate.
Drying In air.
Total ash (2.4.16)
Maximum 0.1 per cent, determined on 2.0 g. Detection Examine in ultraviolet light at 254 nm.
System suitability The chromatogram obtained with reference
STORAGE
Results The prindpal spot in the chromatogram obtained with
______________________________________ ___________________ PhEur
the test solution is similar in position and size to the prindpal
solution (a).
1-688 Demeclocycline Hydrochloride 2016
B. To about 2 mg add 5 mL of sulfuric add R. A violet Calculation of percentage contents:

colour develops. Add the solution to 2.5 mL of water R. — for each impurity, use the concentration of
The colour becomes yellow. demeclocycline in reference solution (b).
C. It gives reaction (a) of chlorides (2.3.1). Limits:
— impurities A, B: for each impurity, maximum 5.0 per cent;
TESTS
— impurities C, G: for each impurity, maximum 0.3 per cent;
pH (2.2.3) — impurity E: maximum 0.2 per cent;
2.0 to 3.0.
— arty other impurity: for each impurity, maximum
Dissolve 0.1 g in carbon dioxide-free water R and dilute to 0.15 per cent;
10 mL with the same solvent. — total: maximum 10.0 per cent;
Related substances — reporting threshold: 0.05 per cent
Liquid chromatography (2.2.29). Prepare the solutions Heavy metals (2.4.8)
immediately before use. Maximum 50 ppm.
Buffer 1 22.2 g/L solution of sodium edetate R adjusted to 0.5 g complies with test C. Prepare the reference solution
pH 7.5 with a 40 g/L solution of sodium hydroxide R. using 2.5 mL of lead standard solution (10 ppm Pb) R.
Buffer 2 17.0 g/L solution of tetrapropylammonium hydrogen
W ater (2.5.12)
sulfate R adjusted to pH 7.5 with a 40 g/L solution of sodium Maximum 3.0 per cent, determined on 1.000 g.
hydroxide R.
examined in a 1.0 g/L solution of hydrochloric add R and
dilute to 50.0 mL with the same solution. ASSAY
Reference solution (a) Dissolve 25.0 mg of demeclocycline Liquid chromatography (2.2.29) as described in the test for
hydrochloride CRS in a 1.0 g/L solution of hydrochloric add R related substances with the following modification.
and dilute to 50.0 mL with the same solution. Injection 10 (iL of the test solution and reference solution (a).
Reference solution (b) Dilute 1.0 mL of the test solution to Calculate the percentage content of C21H 22CI2N 2O8 using
100.0 mL with a 1.0 g/L solution of hydrochloric add R. the chromatogram obtained with reference solution (a) and
Reference solution (c) Dissolve 5 mg of demeclocyclinefor system taking into account the assigned content of demedocydine
suitability CRS (containing impurities A, B, C, E and G) in a hydrochloride CRS.
1.0 g/L solution of hydrochloric add R and dilute to 10 mL STORAGE
with the same solution. Protected from light.
Column'.
IMPURITIES
— size. I = 0.075 m, 0 = 4.6 mm;
Spedfied impurities A, B, C, E, G
— stationary phase, end-capped octylsilyl silica gelfor
chromatography with polar incorporated groups R (3.5 jam); Other detectable impurities(the following substances would, if
— temperature'. 40 °C. present at a sufficient level, be detected by one or other of
Mobile phase'.
— mobile phase A: acetomtrUe R, water R, buffer 1, buffer 2 acceptance criterion for other/unspecified impurities and/or
(2:28:35:35 V/V/V/V); by the general monograph Substancesfor pharmaceutical use
— mobile phase B: acetomtrUe R, buffer 1, buffer 2
(30:35:35 V/V/V); impurities for demonstration of compliance. See also 5.10.
Control of impurities in substances for pharmaceutical use): D, F.
Time Mobile phase A Mobile phase B oh o ohoh° 0

0-5 83 17
5 - 15 83 -» 30 17 70
15-25 30 70 HO H H N-CH3
H3C
Flow rate 1 mlVmin. A. (45,4a5,5a5,6S, 12aS)-4-(dimethylamino)-3,6,10,12,12a-

Detection Spectrophotometer at 280 nm. pentahydroxy-1,1 l-dioxo-l,4,4a,5,5a,6,l 1,12a-
Injection 10 (iL of the test solution and reference solutions (b) octahydrotetracene-2-carboxamide (demethyltetracycline),
and (c).
oh 0 oh . 0 0
with demedocydine for system suitability CRS and the nh2
the peaks due to impurities A, B, C, E and G.
C| HO H H3C —n h
Relative retention With reference to demeclocycline (retention
ch 3
impurity A = about 0.7; impurity B = about 0.8;
impurity E = about 1.2; impurity G = about 1.6. B. (4R,4a5,5a5,65,12aS)-7-chloro-4-(dimethylamino)-
System suitabUity. reference solution (c):
3,6,10,12,12a-pentahydroxy-l,l 1-dioxo-
— resolution: minimum 2.5 between the peaks due to 1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide
impurities A and B. (4-epidemeclocycline),
2016 Deptropine Citrate 1-689
Deptropine Citrate ★ ★
★ ★
HO H H3C —N H
CH3
HO C02H
C. (42?j4a5j5a5j65j 12a5)-4-(dimethylamino)-3j6j 10,12,12a- > H02C ^ y ^ C 0 2H
pentahydroxy-1,1 l-dioxo-l,4,4a,5,5a,6,l 1,12a-
octahydrotetxacene-2-carboxamide
(4-epidemethyltetxacycline),
C29H 35NO8 525.6 2169-75-7
Action and use

Histamine Hi receptor antagonist; anticholinergic.
H3C—N H PhEur.
J \
CH,
DEFINITION
D. (42?,4a5,12a5)-4-(dimethylamino)-3,10jl 1,12a- Deptropine dtrate contains not less than 98.0 per cent and
tetrahydroxy-1,12-dioxo-1,4,4a, 5,12,12a- not more than the equivalent of 101.0 per cent of
hexahydrotetracene-2-carboxamide (li?,3r,5¿)-3-(10,l l-dihydro-5/í-dibenzo[a,í¿] [7]annulen-5-
(4-epianhydrodemethyltetracycline), yloxy)-8-methyi-8-azabicydo[3.2.1]octane dihydrogen dtrate,
CHARACTERS
A white or almost white, microcrystalline powder, very
slightly soluble in water and in ethanol, practically insoluble
in methylene chloride.
H n -c h 3 It mdts at about 170 °C, with decomposition.
h3c
IDENTIFICATION
E. (45,4a5,12a5)-4-(dimethylamino)-3,10,l 1,12a-
Second identification B, C, D, E.
tetrahydroxy-1,12-dioxo-l,4,4a,5,12,12a-
hexahydrotetracene-2-carboxamide A. Examine by infrared absorption spectrophotometry
(anhydrodemethyitetracycline), (2.2.24), comparing with the spectrum obtained with
deptropine citrate CRS.
rdated substances. The prindpal spot in the chromatogram
with reference solution (b).
C. To about 1 mg add 0.5 mL of sulfuric acid R A stable
red-orange colour develops.
F. (4Æ,4aS,12aS>7-chloro-4-(dimethylamino)-3,10,l 1,12a- D. Dissolve about 1 mg in 0.25 mL of perchloric acid R and
tetrahydroxy-1,12-dioxo-1,4,4a,5,12,12a- warm gently until the solution becomes turbid. Add 5 mL of
hexahydrotetracene-2-carboxamide glacial acetic acid R; a pink colour with an intense green
(4-epianhydrodemedocycline), fluorescence appears.
E. To about 5 mg add 1 mL of acetic anhydride R and 5 mL
of pyridine R. A purple colour develops.
TESTS
pH (2.2.3)
Suspend 0.25 g in carbon dioxide-free water R, dilute to 25 mL
with the same solvent and filter. The pH of the solution is
3.7 to 4.5.
Related substances
G. (4S,4aS,12aS>7-chloro-4-(dimethylamino)-3,10,l 1,12a- Examine by thin-layer chromatography (2.2.27), using as the
tetrahydroxy-1,12-dioxo-1,4,4a,5,12,12a-hexahydrotetracene- coating substance a suitable silica gel with a fluorescent
2-carboxamide (anhydrodemeclocycline). indicator having an optimal intensity at 254 nm.
_______________________________________________________________ PhEur
examined in methanol R and dilute to 10 mL with the same
solvent.
Test solution (b) Dilute 1 mL of test solution (a) to 10 mL
with methanol R.
1-690 Dequalinimn Chloride 2016
Reference solution (a) Dilute 1.0 mL of test solution (a) to

100.0 mL with methanol R.
Reference solution (b) Dissolve 20 mg of deptropine citrate CRS
Dilute 1 mL of the solution to 10 mL with methanol R.
Reference solution (c) Dissolve 5 mg of tropine CRS in
methanol R and dilute to 100.0 mL with the same solvent.
B. (li?j3sj5S)-3-(10jl l-dihydro-5/f-dibenzo[a,d\ [7]annulen-
Reference solution (d) Dissolve 10 mg of deptropine citrate CRS 5-yloxy)-8-methyl-8-azabicyclo[3.2.1] octane
and 10 mg of tropine CRS in methanol R and dilute to 25 mL (pseudodeptropine),
Apply to the plate 40 pL of each solution. Develop over a
path of 10 cm using a mixture of 8 volumes of concentrated
ammonia R and 92 volumes of butanol R. Dry the plate at
100 °C to 105 °C until the ammonia has completely
evaporated. Examine in ultraviolet light at 254 nm. Any spot
in the chromatogram obtained with test solution (a), apart
from the principal spot, is not more intense than the spot in
the chromatogram obtained with reference solution (a) C. 10,11 -dihydro-5/i-dibenzo [a,d\ [7] annul en-5-ol
(1 per cent). Spray with dilute potassium iodobismuthate (dibenzocycloheptadienol),
solution R and then with a 10 g/L solution of sodium nitrite R.
Expose the plate to iodine vapours. Examine in daylight and
in ultraviolet light at 254 nm. In the chromatogram obtained
with test solution (a): any spot corresponding to tropine is
not more intense than the spot in the chromatogram
obtained with reference solution (c) (0.5 per cent); any spot,
apart from the principal spot and any spot corresponding to
tropine, is not more intense than the spot in the
chromatogram obtained with reference solution (a) D. (li?,3r,56)-3-(10jl 1-dihydro-5/i-dibenzo[a,d\ [7]annulen-
(1 per cent). The test is not valid unless the chromatogram 5-yloxy)-8-azabicyclo [3.2.1] octane (demethyldeptropine).
obtained with reference solution (d) shows two clearly
PhEur
separated spots.
Prepare the reference solution using 2 mL of lead standard
solution (10 ppm Pb) R. Dequalinium Chloride ★ ★
★ ★
Loss on drying (2.2.32) (Ph. Eur. monograph 1413) *****
Not more than 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.400 g in 50 ml. of anhydrous acetic acid R. Titrate
1 mL of 0.1 M perchloric acid is equivalent to 52.56 mg of
C29H 35NO 8. C30H40CI2N 4 527.6 522-51-0
STORAGE
Action and use
Store protected from light.
Antiseptic.
IMPURITIES
PhEur.
DEFINITION
1,1 '-(Decane-1,10-diyl) bis (4-amino-2-methylquinolinium)
dichloride (dried substance).
Content
A. (1i?,3r,55)-8-methyl-8-azabicyclo[3.2.l]octan-3-ol CHARACTERS
(tropine), Appearance
White or yellowish-white powder, hygroscopic.
Solubility
Slightly soluble in water and in ethanol (96 per cent).
IDENTIFICATION
First identification: Bs E.
2016 Dequalinium Chloride 1-691
Second identification A , C, D, E. curve separating this peak from the peak due to
A. Ultraviolet and visible absorption spectrophotometry dequalinium chloride. If necessary, adjust the
(2.2.25). concentration of methanol in the mobile phase.
Test solution Dissolve
about 10 mg in water R and dilute to Limits:
100 mL with the same solvent. Dilute 10 mL of the solution — impurity A: not more than 0.5 times the area of the
to 100 mL with water R. principal peak in the chromatogram obtained with
reference solution (b) (1 per cent);
— total of impurities other than A: not more than 5 times the
Shoulder At 336 nm. with reference solution (b) (10 per cent);
Absorbance ratios: — disregard limit: 0.025 times the area of the principal peak
— A2WA326 = 1-56 to 1.80; in the chromatogram obtained with reference solution (b)
— A32<j/A33g = 1.12 to 1.30. (0.05 per cent).
B. Infrared absorption spectrophotometry (2.2.24). Readily carbonisable substances
Spectral range 600-2000 cm-1. Dissolve 20 mg in 2 mL of sulfuric add R. After 5 min the
Comparison dequalinium chloride CRS. solution is not more intensely coloured than reference
solution BY4 (2.2.2, Method I).
C. To 5 ml, of solution S (see Tests) add 5 mL of potassium
ferricyanide solution R. A yellow precipitate is formed. Loss on drying (2.2.32)
D. To 10 mL of solution S add 1 mL of dilute nitric add R. Maximum 7.0 per cent, determined on 1.000 g by drying at
A white precipitate is formed. Filter and reserve the filtrate 105 °C at a pressure not exceeding 0.7 kPa.
for identification test E. Sulfated ash (2.414)
E. The filtrate from identification test D gives reaction (a) of Maximum 0.1 per cent, determined on 1.0 g.
chlorides (2.3.1). ASSAY
TESTS In order to avoid overheating in the reaction medium, mix
Solution S thoroughly throughout and stop the titration immediately after the
Dissolve 0.2 g in 90 mL of carbon dioxide-five water R, end-point has been reached
heating if necessary, and dilute to 100 mL with the same Dissolve 0.200 g in 5 ml, of anhydrous formic add R and add
solvent. 50 mL of acetic anhydride R. Titrate with 0.1 M perchloric
add, determining the end-point potentiometrically (2.2.20).
Solution S is clear (2.2.1) and colourless (2.2.2, Method II). 1 mL of 0.1 M perchloric add is equivalent to 26.38 mg of
C 30H 40CI2N 4.
To 5 mL of solution S add 0.1 mL of bromothymol blue STORAGE
solution R l. Not more than 0.2 mL of 0.01 M hydrochloric In an airtight container.
acid or 0.01 M sodium hydroxide is required to change the
IMPURITIES
colour of the indicator. Specified impurities A.
Related substances Other detectable impurities (the following substances would, if
Liquid chromatography (2.2.29). present at a sufficient level, be detected by one or other of
Test solution Dissolve 10.0 mg of the substance to be the tests in the monograph. They are limited by the general
examined in the mobile phase and dihate to 10.0 mL with acceptance criterion for other/unspecified impurities and/or
the mobile phase. by the general monograph Substances for pharmaceutical use
Reference solution (a) Dissolve 10.0 mg of dequalinium chloride (2034). It is therefore not necessary to identify these
for performance test CRS in the mobile phase and dilute to impurities for demonstration of compliance. See also 5.10.
10.0 mL with the mobile phase. Control of impurities in substancesfor pharmaceutical use): B, C.
Reference solution (b) Dissolve 10.0 mg of dequalinium
chloride CRS in the mobile phase and dilute to 10.0 mL with
the mobile phase. Dilute 1.0 mL of the solution to 50.0 mL
Column:
— size. I = 0.25 m, 0 = 4.6 mm; ch 3
— stationary phase: end-capped octadecylsûyl silica gel for
chromatography R.
A. 2-methylquinolin-4-amine,
Mobile phase Dissolve 2 g of sodium hexanesidfonate R in
300 mL of water R~, adjust to pH 4.0 with acetic add R and
add 700 mL of methanol R.
Injection 10 |iL.
Run time 5 times the retention time of dequalinium chloride.
B. 4-amino-1- [10- [(2-methylquinolin-4-yl) amino] decyl] -2-
— peak-to-vaüey ratio: minimum 2.0, where H p = height
methylquinolinium chloride,
above the baseline of the peak due to impurity B and
H v = height above the baseline of the lowest point of the
1-692 3-0-Desacyl-4’-Monophosphoryl lip id A 2016
monophosphoryl lipid A used as adjuvant in the particular

vaccine of proven clinical efficacy and safety in man or a
batch representative thereof.
BACTERIAL SEED LOTS
The bacterial strain used for master seed lots shall be
identified by historical records that include information on its
origin and the tests used to characterise the strain, in
particular genotypic and phenotypic information. Only a
C. 1- [10-(4-amino-2-me±ylqiiinolinio) decyl] -4- [[10- working seed lot that complies with the following
(4-amino-2-methylquinolinio)decyI] amino] -2- requirements may be used.
methylquinolinium trichloride. Identification
_______________________________________________ :___________PhEur
The working seed lot is identified by suitable methods such
as Gram staining and fatty add profiling (5.1.6).
Microbial Purity
Each seed lot complies with the requirements for absence of
3- £>Desacyi-4’-Monophosphoryl f * ** contaminating organisms. Purity of bacterial cultures is
Lipid A ***** verified by methods of suitable sensitivity and specificity.
(Ph Eur monograph 2537) PROPAGATION AND HARVEST
PhEur__________________________________________________________ The bacteria is grown using a suitable liquid medium. At the
end of cultivation, the culture is tested for purity and yield.
DEFINITION The culture medium is separated from the bacterial mass by
3-0-Desacyl-4 '-monophosphoryl lipid A is a detoxified a suitable method, for example filtration. Only a harvest that
derivative of the lipopolysaccharide (LPS) of Salmonella is consistent with respect to the profiles for growth rate, pH,
minnesota, strain R595, which retains the immunostimulatory and 0 2-consumption may be used for the extraction of LPS.
activities of the parent LPS. It consists of a mixture of TRIETHYLAMINE SALT OF 3-0-DESACYL-4 -
congeners, all containing a backbone of (31'-» 6-linked
MONOPHOSPHORYL LIPID A
disaccharide of 2 -deoxy-2 -aminoglucose phosphorylated at
LPS is extracted from the bacterial cells by successive alcohol
the 4 '-position, but differing in the fatty acid substitutions at
and chloroform-methanol extractions and is then converted
the 2 , 2 ' and 3' positions. The immunostimulatory activities
to 3-0-desacyl-4'-monophosphoryl lipid A by hydrolysis,
of 3-O-desacyl-4 '-monophosphoryl lipid A combined with
then purified and salified by triethanolamine before freeze-
the vaccine include up-regulation of co-stimulatory molecules
drying. The freeze-dried triethylamine salt of 3-0-desacyl-4'-
on antigen-presenting cells and secretion of pro-inflammatory
monophosphoryl lipid A must comply with the following
cytokines, resulting in an enhanced immune response of the
requirements.
Thl-type against the antigens. 3-0-desacyl-4'-
monophosphoryl lipid A is a lyophilised powder or a sterile Appearance
liquid. A visual description of the particular preparation after freeze-
drying is established and approved by the competent
Requirements given in the sections up to and including the authority; each batch of freeze-dried triethylamine salt of
section Triethylamine salt of 3-0-desacyl-4'-monophosphoryl 3-O-desacy1-4'-monophosphoryl lipid A must comply with
lipid A also apply to formulations that do not proceed to the this description.
3-0-desacyl-4 '-monophosphoryl lipid A liquid bulk.
Protein
PRODUCTION Less than 0.5 per cent mlm, determined using a suitable
GENERAL PROVISIONS method, for example a reversed-phase HPLC method for
The production method shall have been shown to yield amino add analysis (2.2.56). The total amino acid content in
consistently a 3-Odesacyl-4'-monophosphoryl lipid A micrograms is calculated by comparison to amino acid
comparable in structure and function with a preparation of standards and is equal to the protein concentration.
3-0-desacyl-4'-monophosphoryl lipid A used as adjuvant in Nucleic acid
the particular vaccine of proven clinical efficacy and safety in Maximum 0.3 per cent mlm, determined using a suitable
man. method. For example, a fluorimetric method may be used
During development studies, and wherever revalidation is where nucleic acids are extracted from the freeze-dried
necessary, a test for residual endotoxin activity is carried out triethylamine salt of 3-0-desacyl-4'-monophosphoryl lipid A,
by injecting intravenously 12-day-old embryonated hens' eggs using a solution containing NH 4OH and a suitable non-ionic
with 0.1 mL of dilutions of the test sample (8 eggs per detergent, and stained by a suitable fluorescent dye.
dilution) of 3-0-desacyl-4 '-monophosphoryl lipid A. Eggs are The nucleic add content in die test sample is interpolated
candled and read for mortality at 18-24 hours post from a calibration curve.
inoculation and the chick embryo 50 per cent lethal dose Hexosaxnine (2.5.20)
(CELD5o) is calculated. The residual endotoxin activity of 1000 nmol/mg to 1450 nmol/mg.
the 3-Odesacyl-4'-monophosphoryl lipid A is acceptable if Phosphorus (2.5.18)
the CELD50 is more than 100 (xg. 0.5 pmol/mg to 0.8 (imol/mg.
An endotoxin standard of Salmonella typhimurium is prepared Congener distribution
and selected dilutions are injected into each group of 8 eggs. The relative amount of tetraacyl, pentaacyl, hexaacyl and
For a test to be valid, the CELD50 of the endotoxin standard heptaacyl congener groups are determined by a suitable
must not be more than 0.05 |ig. method, for example reversed-phase HPLC analysis (2.2.29).
Reference preparation A batch of 3-0-desacyl-4'- The relative amount of each congener group in the
monophosphoryl lipid A shown to be comparable in structure triethylamine salt of 3-0-desacyl-4 '-monophosphoryl lipid A
and function with a preparation- of 3-0-desacyl-4'- is:
2016 Desferrioxamine Mesilate 1-693
— tetraacyl: 15 per cent to 35 per cent; liquid bulk is within the limits approved for the particular
— pentaacyl: 35 per cent to 60 per cent; product.
— hexaacyl: 20 per cent to 40 per cent; Sterility (2.6.1)
— heptaacyl: less than 0.5 per cent. It complies with the test, carried out losing 10 mL for each
Triethylamine medium.
4.2 to 5.8 per cent m/m, determined by a suitable method, Congener distribution
for example gas chromatography (2.2.28). The relative amount of tetraacyl, pentaacyl, hexaacyl and
Water (2.5.12) heptaacyl congener groups are determined by a suitable
Maximum 6.7 per cent mlm. method, for example reversed-phase HPLC analysis (2.2.29).
Free fatty acids The relative amount of each congener group in the 3-0-
Maximum 2.6 per cent mlm, determined by a suitable desacyl-4'-monophosphoryl lipid A liquid bulk is:
method, for example reversed-phase HPLC analysis (2.2.29). — tetraacyl: 15 per cent to 35 per cent;
2-Keto-3-deoxyoctonate — pentaacyl: 35 per cent to 60 per cent;
Less than 0.5 per cent m/m, determined by a suitable — hexaacyl: 20 per cent to 40 per cent;
method. For example, a colorimetric method may be used — heptaacyl: less than 0.5 per cent.
where 2-keto-3-deoxyoctonate is released by hydrolysis (0.2 ASSAY
N H2S 0 4 at 100 °C for 30 min), oxidised by periodic acid, The 3-0-desacyl-4'-monophosphoryl lipid A content is
and reacted with sodium arsenite to yield P-formylpyruvic determined by a suitable method, for example gas
acid, which subsequently is coupled to thiobarbituric acid to chromatographic quantification (2.2.28) of trifluoroacetic
give a red coloured chromophore with absorption maximum anhydride derivatised fatty add methyl esters of the 3-0-
at 550 nm. The amount of 2-keto-3-deoxyoctonate is desacyl-4'-monophosphoryl lipid A fatty adds dodecanoic
interpolated from a calibration curve. add (C l2:0), tetradecanoic add (C l4:0), 3-hydroxy
Identity tetradecanoic add (3-OH-Cl4:0) and hexadecanoic add
The test for congener distribution also serves to identify the (C16:0) obtained by hydrolysis of 3-0-desacyl-4'-
product monophosphoryl lipid A in an aqueous/methanol (50:50 VIV)
solution, containing 5 per cent of sodium hydroxide. To the
test sample, a reference sample and the dilutions of the
TAMC: acceptance criterion 101 CFU/10 mg (2.6.12).
calibration curve, pentadecanoic add (C l5:0) is added as an
Pyrogens (2.6.8) internal standard. The temperature gradient applied must
The triethylamine salt of 3-0-desacyl-4 '-monophosphoryl allow the separation of the fatty add methyl esters in about
lipid A complies with the test for pyrogens. Inject into each 40 min.
rabbit per kilogram of body mass 3 mL of a solution
The siim of the ratios between the area for each individual
containing 2.5 ng of 3-0-desacyl-4'-monophosphoryl lipid A.
fatty add methyl ester (C12:0, C14:0, 3-OH-Cl4:0 and
3-0-DESACYL-4' -MONOPHOSPHORYL UPID A LIQUID C l6:0) and the area of the internal standard (ratio = area
BULK Qc / area C15:0) is calculated The 3-0-desacyl-4'-
The triethylamine salt of 3-0-desacyl-4'-monophosphoryl monophosphoryl lipid A quantity corresponding to the sum
lipid A is dispersed in a liquid suitable for the subsequent ratio value on the calibration curve, established with the
processing steps at a defined target concentration. If the salt dilutions of the 3-0-desacyl-4'-monophosphoryl lipid A
is not soluble in water a microfluidisation step is necessary to standard, is reported.
prepare a stable aqueous suspension. The content of 3-0-desacyl-4'-monophosphoryl lipid A is
The liquid bulk is sterilised by filtration through a bacteria- not less than 80 per cent and not greater than 120 per cent
retentive filter. of the estimated content.
Only a 3-0-desacyl-4 '-monophosphoryl lipid A liquid bulk PhEur
that complies with the requirements given below under
Identification, Tests and Assay and that is within the limits
approved for the particular product may be used for the
preparation of 3-0-desacyl-4'-monophosphoryl lipid A in the
final lots. Desferrioxamine Mesilate ★ ★
★ ★
CHARACTERS (Deferoxamine Mesilate, Ph Eitr monograph 0896) *****
When dispersed in an aqueous solution: slightly turbid
suspension. OH
When dissolved in an organic solvent: a description of its
appearance is established and approved by the competent S03H
authority; the 3-0-desacyl-4'-monophosphoryl lipid A liquid
ch 3
bulk complies with this description.
IDENTIFICATION
Congener distribution (see Tests).
TESTS C26H52N5O11S 657 138-14-7
Particle size
Where applicable, the particle size in the microfluidised Action and use
liquid bulk is determined by a suitable method, for example Chelating agent (iron).
dynamic light scattering. The particle size for each batch of Preparation
Desferrioxamine Injection
1-694 Desfemoxamine Mesilate 2016

DEFINITION
immediately before use, protectedfrom light.
N'~ [5- [[4- [[5-(Acetylhydroxyamino)pentyl] amino]-4-
oxobutanoyl] hydroxyamino] pentyl] -N-(5-aminopentyl)-N-
hydroxybutanediamide methanesulfonate. examined in the mobile phase and dilute to 50.0 mL with
the mobile phase.
Content
Reference solution (a) Dissolve 10.0 mg of deferoxamine
mesilate CRS in the mobile phase and dilute to 10.0 mL with
PRODUCTION the mobile phase.
It is considered that alkylsulfonate esters are genotoxic and Reference solution (b) Dilute 1.0 mL of the test solution to
are potential impurities in deferoxamine mesilate. 25.0 mL with the mobile phase.
The manufacturing process should be'developed taking into
Column'.
consideration the principles of quality risk management,
— size. I — 0.25 m, 0 = 4.6 mm;
together with considerations of the quality of starting
— stationary phase: octadecylsilyl silica gel for chromatography R
materials, process capability and validation. The general
(10 |im).
methods 2.5.37. Methyl, ethyl and isopropyl methanestdfonate in
methanesidfonic acid, 2.5.38. Methyl, ethyl and isopropyl Mobile phase Dissolve 1.32 g of ammonium phosphate R and
methanesulfonate in active substances and 2.5.39. 0.37 g of sodium edetate R in 950 mL of water R; adjust to
Methanesidfonyl chloride in methanesidfonic acid are available to pH 2.8 with phosphoric acid R (about 3-4 mL) and add
assist manufacturers. 55 mL of tetrahydrqfuran R.
Flow rate 2 mL/min.
CHARACTERS
Appearance Detection Spectrophotometer at 220 nm.
White or almost white powder. Injection 20 pL.
Solubility Run time 3 times the retention time of deferoxamine.

Freely soluble in water, slightly soluble in methanol, very System suitability: reference solution (a):
slightly soluble in ethanol (96 per cent). — resolution: m in im u m 1.0 between the peak with a relative
retention time of about 0.8 and the principal peak.
IDENTIFICATION
Limits:
— impurity A: not more than the area of the principal peak
A. Infrared absorption spectrophotometry (2.2.24). (4.0 per cent);
Preparation Discs. — total: not more than 1.75 times the area of the principal
Comparison deferoxamine mesilate CRS. peak in the chromatogram obtained with reference
If the spectra obtained show differences, dissolve the solution (b) (7.0 per cent);
substance to be examined and the reference substance
separately in ethanol (96 per cent) R, evaporate to dryness and the chromatogram obtained with reference solution (b)
record new spectra vising the residues. (0.08 per cent).
B. Dissolve about 5 mg in 5 mL of water R. Add 2 mL of a Chlorides (2.44)
M axim um 330 ppm.
5 g/L solution of trisodium phosphate dodecahydrate R and
0.5 mL of a 25 g/L solution of sodium Dilute 2 mL of solution S to 20 mL with water R.
naphthoqumcmesulfonate R. A brownish-black colour develops. Sulfates (2.413)
C. Solution A obtained in the assay is brownish-red. Maximum 400 ppm.
To 10 mL of solution A add 3 mL of ether R and shake. Dilute 5 mL of solution S to 20 mL with distilled water R.
The organic layer is colourless. To 10 mL of solution A add Heavy metals (2.48)
3 mL of benzyl alcohol R and shake. The organic layer is Maximum 10 ppm.
brownish-red.
D. Dissolve 0.1 g in 5 mL of dilute hydrochloric acid R.
vising 2 mL of lead standard solution (10 ppm Pb) R.
Add 1 mL of barium chloride solution R2. The solution is
clear. In a porcelain crucible, mix 0.1 g with 1 g of anhydrous W ater (2.5.12)
sodium carbonate R, heat and ignite over a naked flame. Allow Maximum 2:0 per cent, determined on 1.000 g.
to cool. Dissolve the residue in 10 mL of water R, heating if Sulfa ted ash (2.4.14)
necessary, and filter. The filtrate gives reaction (a) of sulfates Maximum 0.1 per cent, determined on 1.0 g.
(2.3.1). Bacterial endotoxins (2.6.14)
TESTS Less than 0.025 IU/mg, if intended for vise in the
Solution S manufacture of parenteral preparations without a further
Dissolve 2.5 g in carbon dioxide-free water R prepared from appropriate procedure for the removal of bacterial
distilled water R and dilute to 25 mL with the same solvent. endotoxins.
Appearance o f solution ASSAY
Solution S is clear (2.2.1) and not more intensely coloured Dissolve 0.500 g in 25 mL of water R. Add 4 mL of
than reference solution Y5 (2.2.2, Method II). 0.05 M sulfuric acid. Titrate with 0.1 M ferric ammonium
pH (2.2.3) sulfate. Towards the end of the titration, titrate uniform ly and
3.7 to 5.5 for freshly prepared solution S. at a rate of about 0.2 mL/min. Determine the end-point
potentiometrically (2.2.20) using a platin u m indicator
2016 Desipramine Hydrochloride 1-695
electrode and a calomel reference electrode. Retain the ID E N T IF IC A T IO N

titrated solution (solution A) for identification test C. First identification B, E
1 m L of 0.1 M ferric ammonium sulfate is equivalent to Second identification A, C, D, E
65.68 m g o f C ie i^ N e O n S . A. Dissolve 40.0 mg in 0.01 M hydrochloric acid and dilute to
STORAGE 100.0 m L with the same acid. Dilute 5.0 m L of the solution
Protected from light. I f the substance is sterile, store in a to 100.0 m L with 0.01 M hydrochloric add. Examined
sterile, airtight, tam per-proof container. between 230 nm and 350 nm (2.2.25), the solution shows an
absorption maximum at 251 nm and a shoulder at 270 nm .
IM P U R IT IE S
T he specific absorbance at the maximum is 255 to 285.
Other detectable impurities (the following substances would, if (2.2.24), comparing with the spectrum obtained with
present at a sufficient level, be detected by one or other of desipramine hydrochloride CRS.
C. Examine the' chromatograms obtained in the test for
related substances. T he principal spot in the chromatogram
Control of impurities in substances for pharmaceutical use): B.
D . Dissolve about 50 m g in 3 m L of water R and add
OH 0 0.05 m L of a 25 g/L solution of quinhydrone R in methanol R.
An intense pink colour develops within about 15 min.
E. T o 0.5 m L of solution S (see Tests) add 1.5 m L of
water R. T he solution gives reaction (a) of chlorides (2.3.1).
TESTS
S o lu tio n S
Dissolve 1.25 g in carbon dioxide-free water R, warming to not
more than 30 °C if necessary, and dilute to 25 m L with the
A. N '- [5- [ [4- [ [4-(acetylhydroxyamino)butyl] amino] -4- same solvent.
oxobutanoyl]hydroxyamino] pentyl] -N'-(5-aminopentyl)-N-
hydroxybutanediamide (desferrioxamine AO,
Solution S, examined immediately after preparation, is not
B. other desfemoxamines. more intensely coloured than reference solution BY 6 (2.2.2,
__________________________________________________________ PhEur Method II).
T o 10 m L of solution S add 0.1 m L of methyl red solution R
and 0.3 m L of 0.01 M sodium hydroxide. T he solution is
yellow. N ot more than 0.5 m L o f 0.01 M hydrochloric acid is
Desipramine Hydrochloride ******
required to change the colour of the indicator to red.
*★ **
(Ph. Eur. monograph 0481) * R e la te d su b stan ces
Carry out the test protectedfrom bright light. Examine by thin-
layer chromatography (2.2.27), using a TLC silica gel plate R.
examined in a mixture of equal volumes of ethanol R and
mixture of solvents. Prepare immediately before use.
with a mixture of equal volumes o f ethanol R and methylene
C 18H23C1N2 302.8 58-28-6 chloride R.
A ctio n a n d u se Reference solution (a) Dissolve 25 mg of desipramine
M onoam ine reuptake inhibitor; tricyclic antidepressant. hydrochloride CRS in a mixture of equal volumes o f ethanol R
and methylene chloride R and dilute to 25 m L with the same
P re p a r a tio n mixture of solvents. Prepare immediately before use.
Desipram ine Tablets
Reference solution (b) Dilute 1 m L of reference solution (a) to
PhEur__________________________________________________________ 50 m l. with a mixture o f equal volumes of ethanol R and
D E F IN IT IO N
Desipramine hydrochloride contains not less than Apply to the plate 5 (iL of each solution. Develop over a
99.0 per cent and not more than the equivalent of path of 7 cm using a mixture of 1 volume of water R,
10 volumes of anhydrous acetic acid R and 10 volumes of
101.0 per cent o f 3-(10,l l-dihydro-5H-dibenzo [bj\ azepin-5-
yl)-N-methylpropan - 1-amine hydrochloride, calculated with toluene R. Dry the plate in a current of air for 10 min, spray
with a 5 g/L solution of potassium dichromate R in a mixture
o f 1 volume of sulfuric add R and 4 volumes of water R and
CHA RACTERS examine immediately. Any spot in the chromatogram
A white o r almost white, crystalline powder, soluble in water obtained with test solution (a), apart from the principal spot,
and in alcohol. is not more intense than the spot in the chromatogram
It melts at about 214 °C. obtained with reference solution (b) (0.2 per cent).
1-696 Desflurane 2016
Heavy m etals (2.4.8) Related substances

2.0 g complies with test C for heavy metals (20 ppm). Gas chromatography (2.2.28).
Prepare the reference solution using 4 m L of lead standard Test solution T he substance to be examined.
solution (10 ppm Pb) R. Reference solution (a) Introduce 25 m L o f the substance to be
Loss on drying (2.2.32) examined into a 50 m L flask fitted with a septum , and add
N ot more than 0.5 p er cent, determined on 1.000 g by 0.50 m L o f desflurane impurity A CRS and 1.0 m L of
drying in an oven at 105 °C. isoflurane CRS (impurity B). Add 50 nL of acetone R
Sulfated ash (2.4.14) (impurity H ), 10 nL o f chloroform R (impurity F) and 50 nL
N ot more than 0.1 p er cent, determined on 1.0 g. o f methylene chloride R (impurity E) to the solution, using an
airtight syringe, and dilute to 50.0 m L with the substance to
ASSAY be examined. D ilute 5.0 m L of this solution to 50.0 m L with
Dissolve 0.2500 g in a mixture o f 5 m L o f 0.01 M the substance to be examined. Store at a tem perature below
hydrochloric acid and 50 m L o f alcohol R. Carry out a 10 °C.
potentiom etric titration (2.2.20), using 0.1 M sodium
hydroxide. Read the volume added between the two points of
to 50.0 m L with the substance to be examined. Store at a
in fle x io n .
tem perature below 10 °C.
Reference solution (c) Dilute 5.0 m L of reference solution (b)
Ci8H23ClN2.
to 25.0 m L with the substance to be examined. Store at a
STORAGE tem perature below 10 °C.
Store protected from light. Column:
__________________________________________________________ PhEur — material: fused silica;
— size: I = 105 m , 0 = 0.32 mm;
— stationary phase:
poly[methyl(trifluoropropylmeihyl)sUoxane] R (film thickness
*** 1.5 nm).
* ★
Desflurane ★ ★ Carrier gas helium for chromatography R.
(Ph. Eur. monograph 1666) ***** Flow rate 2.0 mlVmin.
Split ratio 1:25.
F'< H I Temperature:
JL and enantiomer — column: 30 °C;
f 3c o f
— injection port. 150 °C;
3 2 60
C H F 168.0 57041-67-5
PhEur_____
Injection 2.0 |xL.
DEFINITION Run time 35 min.
(2RS) -2-(Difluoromethoxy) -1 ,1 ,1,2-tetrafluoroethane. Relative retention W ith reference to desflurane (retention
CHARACTERS time = about 11.5 min): impurity C = about 1.06;
Appearance impurity D = about 1.09; impurity A = about 1.14;
Clear, colourless, mobile, heavy liquid. impurity G = about 1.39; impurity E = about 1.5;
impurity B = about 1.7; impurity F = about 2.2;
Solubility
impurity H = about 2.6.
Practically insoluble in water, miscible with anhydrous
ethanol. System suitability: reference solution (a):
— number of theoretical plates: minim um 20 000 , calculated
Relative density for the peak due to impurity A;
1.47, determ ined at 15 °C. — symmetry factor, m aximum 2.0 for the peak due to
bp impurity B.
A bout 22 °C. Limits:
IDENTIFICATION — impurity B: n o t more than the difference between the area
Infrared absorption spectrophotometry* (2.2. 24). o f the corresponding peak in the chromatogram obtained
with reference solution (a) and the area of the
Preparation Examine the substance in the gaseous state.
Comparison Ph. Eur. reference spectrum of desflurane. the test solution (0.2 per cent V!V)\
TESTS — impurity A: n o t m ore than the difference between the area
The substance to be examined must be cooled to a temperature o f the corresponding peak in the chromatogram obtained
below 10 °C and the tests must be carried out at a temperature with reference solution (a) and the area o f the
below 20 °C. corresponding peak in the chromatogram obtained with
the test solution (0.1 per cent VIV);
— impurities C, D, G: for each impurity, n o t more than the
T o 20 m L add 20 m L of carbon dioxide-free water R, shake
difference between the area o f the peak due to impurity A
for 3 min and allow to stand. Collect the upper layer and
add 0.2 m L o f bromocresol purple solution R. N o t more than
and the area o f the peak due to impurity A in the
0.1 m L of 0.01 M sodium hydroxide o r 0.6 m L o f 0.01 M
chromatogram obtained with the test solution
hydrochloric acid is required to change the colour of the
indicator. (0.01 per cent V!V)\
2016 Desflurane 1-697
— impurities E, H: for each impurity, not m ore than the Reference solutions T o each of 1.0 m T, 2.0 mT, 3.0 mT.,
difference between the area of the corresponding peak in 4.0 m L and 5.0 m L o f antimony standard solution (100 ppm
the chromatogram obtained with reference solution (a) Sb) R add 20 m L of the solvent mixture and dilute to
and the area o f th e corresponding peak in the 100.0 m L with water R.
chromatogram obtained with the test solution Source Antimony hollow-cathode lam p using a transmission
(0.01 per cent V/V)’, band of 0.2 nm and a 75 per cent lamp current.
— impurity F: not m ore than the difference between the area Wavelength 217.6 nm.
of the corresponding peak in the chromatogram obtained
with reference solution (a) and the area o f the
corresponding peak in the chromatogram obtained with N o n -v o latile m a tte r
the test solution (0.002 per cent V/V)', Maximum 100 mg/L.
— unspecified impurities: for each impurity, no t more than Evaporate 20.0 m L to dryness with the aid of a stream of
0.5 times the difference between the area o f the peak due nitrogen R. T h e residue weighs not more than 2.0 mg.
to impurity A in the chromatogram obtained with
STORA GE
reference solution (b) and the area o f the peak due to
In a glass botde fitted with a polyethylene-lined cap. Before
impurity A in the chromatogram obtained with the test
opening the botde, cool the contents to below 10 °C.
solution (0.005 per cent V/V)-,
— sum of impurities other than A, B, C, D, E, F, G and H: IM P U R IT IE S
not m ore than th e difference between the area of the peak Specified impurities A, B, C, D, E, F , G, H
due to impurity A in the chromatogram obtained with
reference solution (b) and the area of the peak due to F F
impurity A in th e chromatogram obtained with th e test
solution (0.01 p er cent V/V)-, F a C ^ 'O '^ 'C F j
— disregard Unde, the difference between the area of the peak
due to impurity A in the chromatogram obtained with A. 1,1 '-oxybis( 1,2,2,2-tetrafluoroethane),
reference solution (c) and the area o f the peak due to
impurity A in the chromatogram obtained with the test h a f
solution (0.002 per cent V/V). J. and enantiomer
f 3c o f
F lu o rid e s
M axim um 10 ppm .
B. (22?S)-2-chloro-2-(difluoromethoxy)-l,l,l-trifluoroethane
Potentiom etry (2.2.36, Method I).
(isofluorane),
Test solution T o 10.0 m L in a separating funnel, add 10 m L
o f a mixture o f 30.0 m L of dilute ammonia R2 and 70.0 m i. R R’
o f distilled water R. Shake for 1 min and collect the upper
layer. Repeat this extraction procedure twice, collecting the 0 ,^ 0 ,
upper layer each time. Adjust the combined upper layers to
p H 5.2 with dilute hydrochloric acid R. Add 5.0 m L o f fluoride C. R = H , R ' = F: dichlorofluoromethane,
standard solution (1 ppm F) R and dilute to 50.0 m L with D . R = Cl, R ' = F: trichlorofluoromethane,
distilled water R. T o 20.0 m L o f this solution add 20.0 m L of E. R = R ' = H : dichloromethane (methylene chloride),
totd-iomc-strength-adjustment buffer R and dilute to 50.0 m L
F. R = H , R ' = Cl: trichloromethane (chloroform),
w ith distilled water R.
Reference solutions T o each of 1.0 mL, 2.0 m L, 3.0 m L , ci ci
4.0 m L and 5.0 m L o f fluoride standard solution (10 ppm F) R
add 20.0 m L of total-ionic-strength-adjustment buffer R and
dilute to 50.0 m L w ith distilled water R. F F
Indicator electrode Fluoride selective.

G . l,l,2-trichloro-l,2,2-trifluoroethane,
Reference electrode Silver-silver chloride.
Carry out the measurements on 20 m L of each solution. O
Calculate the concentration of fluorides using the calibration x
curve, taking into account the addition o f fluoride to the test H3C CH3
solution.
A n tim o n y H . propanone (acetone).
M axim um 3 ppm . __________________________________________________________ PhEur
Solvent mixture hydrochloric acid R, nitric acid R (50:50 V/V).
Test solution T ransfer 10 g, cooled to below 10 °C, to a tared
flask containing 20 m L of water R cooled to below 5 °C.
A dd 1 m L of the solvent mixture and leave at room
tem perature until the desflurane has evaporated completely.
Subsequently, reduce the volume to about 8 m L on a hot
plate. Cool to room temperature and transfer to a volumetric
flask. A dd 1 m L o f the solvent mixture and adjust to
1-698 Deslanoside 2016
D . Dissolve about 5 mg in 5 m L o f glacial acetic add R and

Deslanoside t* * * %
add 0.05 m L o f ferric chloride solution R l. Cautiously add
*+ 2 m L of sulfuric acid R, avoiding mixing the two liquids.
Allow to stand; a brown b u t n o t reddish ring develops at the
interface and a greenish-yellow, then bluish-green colour
diffuses from it to the upper layer.
TESTS
S o lu tio n S
Dissolve 0.20 g in a mixture o f equal volumes o f chloroform R
and methanol R and dilute to 10 m L with the same mixture
of solvents.
Dissolve 0.200 g in anhydrous pyridine R and dilute to
10.0 m L with the same solvent. T h e specific optical rotation
is + 6.5 to + 8.5, calculated with reference to the dried
substance.
Test solution (a) Use solution S.
C47H 740 19 943 17598-65-1
A c tio n a n d u se with a mixture o f equal volumes o f chloroform R and
N a/K -A TPase inhibitor; cardiac glycoside. methanol R.
Reference solution (a) Dissolve 20 m g of deslanoside CRS in a
PhEir ___________________________________________________________ mixture o f equal volumes o f chloroform R and methanol R and
D E F IN IT IO N dilute to 10 m L with the same mixture of solvents.
Deslanoside contains not less than 95.0 per cent and not Reference solution (b) Dilute 2.5 m L of reference solution (a)
more than the equivalent of 105.0 per cent of 3|3-[(0-p-D- to 10 m L with a mixture of equal volumes of chloroform R
glucopyranosyl-(l -> 4)-0-2j6-dideoxy-(3-D-ri6o-hexopyranosyl- and methanol R.
(1 ->4)-0-2j6-dideoxy-(3-D-ri&o-hexopyranosyl-(l ->4)-2,6- Reference solution (c) Dilute 1 m L o f reference solution (a) to
dideoxy-P-D-ri&o-hexopyranosyl)oxy] - 12P, 14-dihydroxy- 10 m L with a mixture of equal volumes of chloroform R and
5|3,14|3-card-20(22)-enolide, calculated with reference to the methanol R.
dried substance. Apply separately to the plate as 10 m m bands 5 |iL o f each
CHARACTERS solution. Develop immediately over a path o f 15 cm using a
A white or alm ost white, crystalline or finely crystalline mixture o f 3 volumes of water R, 36 volumes o f methanol R
powder, hygroscopic, practically insoluble in water, very and 130 volumes o f methylene chloride R. Dry the plate in a
slightly soluble in alcohol. In an atmosphere o f low relative current of warm air, spray with a mixture o f 5 volumes of
humidity, it loses water. sulfuric add R and 95 volumes o f alcohol R and heat at
140 °C for 15 min. Examine in daylight. In the
IDENTIFICATION
chromatogram obtained with test solution (a)j any zone,
First identification A. apart from the principal zone, is not more intense than the
Second identification B, C, D. zone in the chromatogram obtained with reference
A. Examine by infrared absorption spectrophotometry solution (b) (2.5 per cent) and at m ost two such zones are
(2.2.24), comparing with the spectrum obtained with more intense than the zone in the chromatogram obtained
deslanoside CRS. W hen comparing the spectra, special with reference solution (c) ( 1.0 per cent).
attention is given to the absence of a distinct absorption L oss o n d ry in g (2.2.32)
maximum at about 1260 cm -1 and to the intensity o f the N o t more than 5.0 per cent, determined on 0.500 g by
absorption maximum at about 1740 cm-1. Examine the drying in vacuo at 105 °C.
substances in discs prepared by dissolving 1 mg of the
S u lfa te d a sh (2.414)
substance to be examined or 1 mg o f the reference substance
N ot more than 0.1 per cent, determined on the residue
in 0.3 m L o f methanol R and triturating with about 0.4 g of
obtained in the test for loss on drying.
dry, finely powdered potassium bromide R until the mixture is
uniform and completely dry. A SSAY
B. Examine the chromatograms obtained in the test for Dissolve 50.0 m g in alcohol R and dilute to 50.0 m L w ith the
related substances. T he principal zone in the chromatogram same solvent. Dilute 5.0 m L o f this solution to 100.0 m L
obtained with test solution (b) is similar in position, colour with alcohol R. Prepare a reference solution in the same
and size to the principal zone in the chromatogram obtained manner, using 50.0 mg o f deslanoside CRS (undried).
with reference solution (a). T o 5.0 m L o f each solution add 3.0 m L of alkaline sodium
C. Suspend about 0.5 mg in 0.2 m L of alcohol picrate solution R and allow to stand protected from bright
(60 per cent V/V) R. Add 0.1 m L o f dinitrobenzoic acid light in a water-bath at 20 ± 1 °C for 40 min. Measure the
solution R and 0.1 m L of dilute sodium hydroxide solution R. absorbance (2.2.25) o f each solution at the m aximum at
A violet colour develops. 484 nm, using as the compensation liquid a mixture of
2016 Desloratadine 1-699
3.0 m L of alkaline sodium picrate solution R and 5.0 m L of Reference solution (c) Dissolve 4 m g o f desloratadine for system
alcohol R prepared at the same time. suitability CRS (containing impurities A and B) in the mobile
Calculate the content o f C 47H 74019 from the absorbances phase and dilute to 5.0 m L with the mobile phase. Dilute
measured and th e concentrations of the solutions. 1.0 m L of the solution to 10.0 m L w ith the mobile phase.
STORAGE
Column:
— sizer. I = 0.25 m, 0 = 4.6 mm;
Store in an airtight, glass container, protected from light, at a
tem perature below 10 °C.
__________________________________________________________ PhEur — temperature: 35 °C.
Mobile phase Dissolve 0.865 g of sodium dodecyl sulfate R in
water R, add 0.5 m L o f trifliwroacetic acid R and dilute to
1000 m L with water R; mix 57 volumes of this solution and
Desloratadine ***** 43 volumes *of acetonitrile R.
Detection Spectrophotometer at 280 run.
Injection 100 (iL of the test solution and reference
Run time 2.5 times the retention time o f desloratadine.
with desloratadine for system suitability CRS and the
the peaks due to impurities A and B.
C 19H i 9C1N2 310.8 100643-71-8 Relative retention W ith reference to desloratadine (retention
A ctio n a n d u se impurity B = about 0.9.
H istamine H l5 receptor .antagonist; antihistamine. System suitability: reference solution (c):
PhEur__________________________________________________________
im purity B and desloratadine.
D E F IN IT IO N Calculation of percentage contents:
8-C hloro-l l-(piperidin-4-ylidene)-6,l l-dihydro-5//- — correction factors: multiply the peak areas o f the following
benzo [5,6] cyclohepta [ 1,2-6] pyridine. impurities by the corresponding correction factor:
C o n te n t impurity A = 1.6; impurity B = 1.6;
98.0 per cent to 102.0 per cent (anhydrous substance). — for each impurity, use the concentration of desloratadine
in reference solution (b).
CHARACTERS
Limits:
A p p e a ra n c e
Solubility — unspecified impurities: for each impurity, maximum
Very slightly soluble o r pratically insoluble in water, freely 0.10 per cent;
soluble in ethanol (96 per cent), slightly soluble o r very — total: m aximum 0.4 per cent;
slightly soluble in heptane. — reporting threshold: 0.05 per cent.
It shows polymorphism (5.9). H eav y m e ta ls (2.4.8)
ID E N T IF IC A T IO N M aximum 20 ppm.
Infrared absorption spectrophotometry (2.2.24). Solvent methanol R.
Comparison desloratadine CRS. 0.250 g complies with test H . Prepare the reference solution
If the spectra obtained in the solid state show differences, using 0.5 m L o f lead standard solution (10 ppm Pb) R.
dissolve the substance to be examined and the reference W a te r (2.5.32)
substance separately in methyl isobutyl ketone R, evaporate to M aximum 0.5 per cent, determined on 0.250 g.
diyness and record new spectra using the residues. S u lfa te d a sh (2.4.14)
TESTS M aximum 0.2 per cent, determined on 0.5 g.
R e la te d su b s ta n c e s ASSAY
Test solution Dissolve 20.0 mg o f the substance to be related substances with the following modifications.
the mobile phase. D ilute 5.0 m L of the solution to 50.0 m L
— symmetry factor. 0.5 to 1.5 for the peak due to
Reference solution (a) Dissolve 20.0 mg of desloratadine CRS in desloratadine.
Calculate the percentage content of Q 9H 19CIN 2 taking into
account the assigned content o f desloratadine CRS.
mobile phase.
Reference solution (b) Dilute 1.0 m L o f the test solution to IMPURITIES
100.0 m L with th e mobile phase. Dilute 1.0 .m L o f this Specified impurities A, B.
1-700 Desmopressin 2016
Other detectable impurities (the following substances would, if C o n te n t

present at a sufficient level, be detected by one or other of 95.0 per cent to 105.0 per cent (anhydrous and acetic a d d -
the tests in the monograph. T hey are limited by the general free substance).
acceptance criterion for other/unspecified impurities and/or CHARACTERS
by the general monograph Substances for pharmaceutical use A p p e a ra n c e
(2034). It is therefore no t necessary to identify these W hite or almost white, fluffy powder.
S o lu b ility
Soluble in water, in ethanol (96 per cent) and in glacial
acetic ad d .
A. Examine the chromatograms obtained in the assay.
Results T he retention time and size o f the prindpal peak in
the chromatogram obtained with the test solution are
approximately the same as those o f the principal peak in the
chromatogram obtained with the reference solution.
A. (1 li?5)-8-chloro-l 1-fluoro-l l-(piperidin-4-yl)-6,l 1- B. Amino a d d analysis (2.2.56). F or hydrolysis use M ethod 1
dihydro-5//-benzo [5,6] cyclohepta [ 1, 2- 6]pyridine, and for analysis use M ethod 1.
Express the content o f each amino a d d in moles. Calculate
the relative proportions o f the amino adds, taking 1/6 o f the
sum o f the num ber of moles o f aspartic a d d , glutamic ad d,
proline, glycine, arginine and phenylalanine as equal to 1.
T h e values fall within the following limits: aspartic add:
0.90 to 1.10; glutamic add: 0.90 to 1.10; proline: 0.90 to
1.10; glydne: 0.90 to 1.10; arginine: 0.90 to 1.10;
phenylalanine: 0.90 to 1.10; tyrosine: 0.70 to 1.05; half
B. (1 li?S)- 8-chloro-1 l-(l,2,3,6-tetrahydropyridin-4-yl)-6, 1 1-
cystine: 0.30 to 1.05. Lysine, isoleucine and leudne are
dihydro-5//-benzo [5,6] cyclohepta [ 1,2-6]pyridine,
absent; n o t more than traces o f other amino ad d s are
o present.
TESTS
—72 to —82 (anhydrous and acetic add-free substance).
Dissolve 10.0 m g in a 1 per cent V/V solution of glacial acetic
acid R and dilute to 5.0 m L with the same add.
C. ethyl 4-(8-chloro-5, 6-dihydro - 1IH- Liquid chromatography (2.2.29): use the normalisation
benzo [5,6] cyclohepta [ 1,2-6] pyridin-11 -ylidene)piperidine-1- procedure.
carboxylate (loratadine). Test solution Dissolve 1.0 m g o f the substance to be examined
PhEur in 2.0 m L o f water R.
Resolution solution Dissolve the contents o f a vial o f
oxytocin/desmopressin validation mixture CRS in 500 nL o f
water R.
Desmopressin *****
★ ★ Column:
***** — size. I = 0.12 m, 0 = 4.0 mm;
(5 nm).
Mobile phase:
Tyr - Phe - Gin- Asn-Cys - Pro- D-Arg—Gly- NH2
— mobile phase A: 0.067 M phosphate buffer solution pH 7.0 R;
filter and degas;
C46H 64N 14 0 12S2 1069 16679-58-6 — mobile phase B: acetonitrUe for chromatography R, mobile
phase A (50:50 VIV)\ filter and degas.
Vasopressin analogue; treatm ent o f diabetes insipidus;
nocturnal enuresis; haemophilia; von Willebrand’s disease.
0 -4 76 24
Desmopressin Injection 4 - 18 76-» 58 24 -»42
D esmopressin Intranasal Solution 18-35 5 8 -»48 42-» 52
Desmopressin Tablets 35 -4 0 48 -» 76 52-» 24
PhEur. 40 - 50 76 24
D E F IN IT IO N Flow rate 1.5 mT 7min.

(3-Sulfanylpropanoyl)-L-tyrosyl-L-phenylalanyl-L-glutaminyl-L- Detection Spectrophotom eter at 220 nm .
asparaginyl-L-cystemyl-L-prolyl-D-arginylglycinamide cyclic Injection 50 jxL.
(l->6)-disulfide. Retention time Desmopressin = about 16 min;
Synthetic cyclic nonapeptide, available as an acetate. oxytocin = about 17 min.
2016 Desogestrel 1-701
System suitability Resolution solution:

— resolution: minim um 1.5 between the peaks due to Tyr - Phe - Gin - Asn - Cys - Pro - D-Arg—Gly - R
desmopressin and oxytoxin. N>| N*\
R4 R5
Limits:
— unspecified impurities:, for each impurity, maximum C. R = O H , R4 = R5 = H: [9-glycine]desmopressin,
0.5 per cent;
E. R = N H 2, R4 = C H 2-N H -C O -C H 3, R5 = H : N 5'4-
[(acetylamino)methyl] desmopressin,
— disregard limit: 0.05 per cent.
F. R = N H 2, R4 = H , R5 = C H 2-N H -C O -C H 3: N4'5-
A cetic a c id (2.5.34) [(acetylamino)methyl] desmopressin,
G . R = N (C H 3) 2, R4 = R5 = H : N 1’9^ 19-
Test solution Dissolve 20.0 mg of the substance to be dimethyldesmopressin.
examined in a mixture o f 5 volumes of mobile phase B and
PhEur
95 volumes of mobile phase A and dilute to 10.0 m L with
the same mixture o f mobile phases.
W a te r (2.5.32)
Maximum 6.0 per cent, determined on 20.0 mg. ** ★ ★
★
Desogestrel ★ ★
Less than 500 IU/mgj if intended for use in the m anufacture (Ph. Eur. monograph 1717) *****
ASSAY
Reference solution Dissolve the contents of a vial o f
desmopressin CRS in water R to obtain a concentration of
0.5 mg/mL.
C ^H Ô 310.5 54024-22-5
Mobile phase M obile phase B, mobile phase A (40:60 ViV).
Flow rate 2.0 mL/min. Action and use
Retention time Desmopressin = about 5 min. Progestogen.
Calculate the content of desmopressin (C 46H 64N 14 0 i 2S2) Preparation

from the declared content of C 46H 64N 140 12S2 in Desogestrel Tablets
desmopressin CRS. PhEur________________
STORA GE
DEFINITION
13-Ethyl-11-methylidene-l 8 ,19-dinor-l 7a-pregn-4-en-20-yn-
tem perature of 2 °C to 8 °C. If the substance is sterile, store
17-ol.
in a sterile, airtight, tam per-proof container.
Content
L A B E LL IN G 98.0 per cent to 102.0 per cent (dried substance).
T he label states:
— the mass o f peptide per container; CHARACTERS
— where applicable, that the substance is suitable for use in Appearance
the m anufacture o f parenteral preparations. W hite o r almost white, crystalline powder.
IM P U R IT IE S
Solubility
Practically insoluble in water, very soluble in methanol, freely
soluble in anhydrous ethanol and in methylene chloride.
the tests in the monograph. They are limited by the general IDENTIFICATION
acceptance criterion for other/unspecified impurities and/or A. Infrared absorption spectrophotom etry (2.2.24).
by the general monograph Substances for pharmaceutical use Comparison desogestrel CRS.
(2034). It is therefore n o t necessary to identify these B. Specific optical rotation (see Tests).
Control of impurities in substances for pharmaceutical use): A , B, TESTS
C, D, E, F, G. Specific optical rotation (2.2.7)
T yr-P he— X— Y— C ys-Pro— Z—Gly-NH2 25.0 m L with the same solvent.
4 i 8
Related substances
A. X = Gin, Y = Asp, Z = D-Arg: [5-L-aspartic
a d d ] desmopressin, Test solution Dissolve 20.0 mg o f the substance to be
examined in 25 m L of acetomtrUe R1 an d dilute to 50.0 m L
B. X = Glu, Y = A sn, Z = D-Arg: [4-L-glutamic
with water R.
a d d ] desm opressin,
Reference solution (a) Dissolve 4 mg o f desogestrelfor system
D . X = Gin, Y = Asn, Z = L-Arg: [8-l-
suitability CRS (containing impurities A, B, C and D) in
arginine] desmopressin,
5 m L o f acetonitrüe R1 and dilute to 10.0 m L w ith water R.
1-702 Desogestrel 2016
Reference solution (b) D ilute 1.0 m l. o f the test solution to Calculate the percentage content o f C 22H 30O from the areas
100.0 m L with a mixture o f equal volumes of acetonitrile R1 of the peaks and the declared content o f desogestrel CRS.
and water R.
IM P U R IT IE S
Reference solution (c) Dilute 1.0 m L of reference solution (b) Specified impurities A, B, C, D
to 10.0 m L with a mixture of equal volumes of acetonitrik R l
and water R.
Reference solution (d) Dissolve 20.0 mg of desogestrel CRS in the tests in the monograph. T hey are limited by the general
25 m L of acetonitrik R l and dilute to 50.0 m L with water R. acceptance criterion for other/unspecified impurities and/or
Column: by the general monograph Substances for pharm aceutical use
— size. I = 0.25 m , 0 = 4.6 mm, (2034). It is therefore not necessary to identify these
— stationary phase: sterically protected octadecylsUyl silica gel impurities for demonstration of compliance. See also 5.10.
for chromatography R (5 fim), Control of impurities in substances for pharmaceutical use): E.
Mobile phase water R, acetonitrik R l (27:73 ViV). CH
Flow rate 1.0 m lAnin-

Injection 15 jiL of the test solution and reference
Run time 2.5 times the retention time of desogestrel.
with desogestrelfor system suitability CRS and the A. 13-ethyl-l l-m ethylidene-18,19-dinor-5a,17a-pregn-3-en-
chrom atogram obtained with reference solution (a) to 20-yn-17-ol (desogestrel A3-isomer),
identify the peaks due to impurities A, B, C and D.
Relative retention W ith reference to desogestrel (retention
im purity D = about 0.25; impurity B = about 0.7;
im purity A = about 0.95; impurity C = about 1.05.
— peak-to-vaEey ratio: minimum 2.0, 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 B. R l = C H 3j R2 = O H, R3 = C = C H , R4 = R5 = H:
curve separating this peak from the peak due to 11 -methylidene-19-nor-17a-pregn-4-en-20-yn-17-ol,
desogestrel. C. R l = C 2H 5, R2 + R3 = O, R4 = R5 = H : 13-ethyl-l 1-
Limits:. methylidenegon-4-en-17-one,
D. R l = C 2H 5, R2 = O H , R3 = C = C H , R4 + R5 = O:
the peak area of the following impurities by the
13-ethyl-17-hydroxy-11 -m ethyiidene-18,19-dinor-17a-pregn-
corresponding correction factor impurity A = 1.8,
4-en-20-yn-3-one,
impurity D = 1.5;
— impurities A , B, C: for each impurity, not more than twice
CH
obtained with reference solution (c) ( 0.2 per cent);
— impurity D: not more than the area o f the principal peak
(0.1 per cent);
— total: not more than 0.5 times the area of the principal E. 13-ethyl-11 -methyiidene-18,19-dinor-17a-pregn-4-en-20-
peak in the chromatogram obtained with reference yne-3(3,17-diol.
solution (b) (0.5 per cent); _______________________________________________________________ PhEur
the chrom atogram obtained with reference solution (c)
(0.05 per cent).
in vacuo at a pressure not exceeding 2 kPa.
A SSAY
Injection Test solution and reference solution (d).
2016 Desoxycortone Acetate 1-703
Detection B Spray with alcoholic solution of sulfuric acid R, heat

Desoxycortone Acetate ***** at 120 °C for 10 m in or until the spots appear, and allow to
★ ★
(Ph. Eur. monograph 0322) ** cool; examine in daylight and in ultraviolet light at 365 nm .
Results B T h e principal spot in the chromatogram obtained
fluorescence in ultraviolet light at 365 n m and size to the
solution (a).
D. Add about 2 m g to 2 m L of sulfuric add R and shake to
C 23H 32O4 372.5 5647-3 dissolve. W ithin 5 min, a yellow colour develops. Add this
solution to 2 m L o f water R and mix. T h e resulting solution
A ctio n a n d u se is dichroic, showing an intense blue colour by transparency,
Mineralocorticoid. and red fluorescence that is particularly intense in ultraviolet
light at 365 nm.
PhEur----------------------------------------------------------------------------------------
E. About 10 mg gives the reaction of acetyl (2.3.1).
D E F IN IT IO N TESTS
3,20-Dioxopregn-4-en-21-yl acetate. Specific o p tic a l ro ta tio n (2.2.7)
C o n te n t + 171 to + 179 (dried substance).
97.0 per cent to 103.0 per cent (dried substance). Dissolve 0.250 g in dioxan R and dilute to 25.0 m L with the
CHARACTERS same solvent.
A p p e a ra n c e R e la te d su b sta n c e s
W hite or almost white, crystalline powder or colourless Liquid chromatography (2.2.29).
crystals. Test solution Dissolve 25.0 mg of the substance to be
S olubility examined in the mobile phase and dilute to 10.0 m L with
Practically insoluble in water, freely soluble in methylene the mobile phase.
chloride, soluble in acetone, sparingly soluble in ethanol Reference solution (a) Dissolve 2 mg o f desoxycortone
(96 per cent), slightly soluble in propylene glycol and in fatty acetate CRS and 2 mg of betamethasone 17-valerate CRS in the
oils. mobile phase and dilute to 200.0 m L with the mobile phase.
ID E N T IF IC A T IO N Reference solution (b) Dilute 1.0 m L o f the test solution to
First identification B, C. 200.0 m L with the mobile phase.
Second identification A, C, D, E. Column:
A. Melting point (2.2.14): 157 °C to 161 °C. — sizer. I = 0.25 m , 0 = 4.6 mm;
(5 |im).
Comparison desoxycortone acetate CRS.
Mobile phase In a 1000 m L volumetric flask mix 350 m L of
C. Thin-layer chromatography (2.2.27). water R w ith 600 m L o f acetonitrik R and allow to
Solvent mixture methanol R, methylene chloride R (1:9 V/V). equilibrate; dilute to 1000 m L with water R and mix again.
Test solution Dissolve 10 m g o f the substance to be examined Flow rate 1 mL/min.
in the solvent mixture and dilute to 10 m L with the solvent Detection Spectrophotom eter at 254 nm .
mixture.
Equilibration W ith the mobile phase for about 30 min.
Reference solution (a) Dissolve 20 mg of desoxycortone
Injection 20 |xL.
acetate CRS in the solvent mixture and dilute to 20 m L with
the solvent mixture. Rim time 3 times the retention time o f desoxycortone acetate.
Reference solution (b) Dissolve 10 mg of cortisone acetate R in Retention time Betamethasone 17-valerate = about 7.5 min;
reference solution (a) and dilute to 10 m L with reference desoxycortone acetate = about 9.5 min.
solution (a). System suitability: reference solution (a):
Plate TLC silica gel F254 plate R.
betam ethasone 17-valerate and desoxycortone acetate;
Mobile phase Add a mixture of 1.2 volumes o f water R and if necessary, adjust the concentration o f acetonitrile in the
8 volumes o f methanol R to a mixture of 15 volumes of mobile phase.
Limits:
Application 5 |iL. — unspecified impurities: for each impurity, not more than
Development Over 2/3 o f the plate. 0.2 times the area of the principal peak in the
Drying In air. chromatogram obtained with reference solution (b)
Detection A Examine in ultraviolet light at 254 nm. ( 0.10 p er cent);
— total: n o t more than the area of the principal peak in the
Results A T he principal spot in the chromatogram obtained
w ith the test solution is similar in position and size to the
(0.5 p er cent);
solution (a).
(0.05 p er cent).
1-704 Dexamethasone 2016
L o ss o n d ry in g (2.2.32) Solvent mixture methanol R, methylene chloride R (1:9 V/V).

M axim um 0.5 per cent, determ ined on 0.500 g by drying in Test solution Dissolve 10 m g of the substance to be examined
an oven at 105 °C. in the solvent mixture and dilute to 10 m L with the solvent
ASSAY mixture.
Dissolve 0.100 g in ethanol (96 per cent) R and dilute to Reference solution (a) Dissolve 20 m g o f dexamethasone CRS in
100.0 m L with the sam e solvent. Dilute 2.0 m L o f this the solvent mixture and dilute to 20 m L with the solvent
solution to 100.0 m L with ethanol (96 per cent) R. M easure mixture.
the absorbance (2.2.25) at the absorption maximum at Reference solution (b) Dissolve 10 m g o f betamethasone CRS in
240 nm. reference solution (a) and dilute to 10 m L with reference
Calculate the content of C 23H 32O 4 taking the specific solution (a).
absorbance to be 450. Plate TLC silica gel F 254 plate R.
STORAGE Mobile phase butanol R saturated with water R, toluene R,
Protected from light. ether R (5:10:85 V/V/V).
___________________________________________________________ PhEur Application 5 jiL.
Drying In air.
Dexamethasone ? **% Results A T h e principal spot in the chrom atogram obtained
** +* with the test solution is similar in position and size to the
(Ph. Eur. monograph 0388) * principal spot in the chrom atogram obtained with reference
solution (a).
at 120 °C for 10 min or until the spots appear. Allow to
cool. Examine in daylight and in ultraviolet light at 365 nm.
Results B T h e principal spot in the chrom atogram obtained
C 22H 29FO 5 392.5 50-02-2 principal spot in the chrom atogram obtained with reference
solution (a).
Action and use System suitability: reference solution (b):
G lucocorticoid — the chromatogram shows 2 spots which may, however,
Preparations n o t be completely separated.
D examethasone Eye D rops, Suspension D . Add about 2 m g to 2 m L o f sulfuric acid R and shake to
D examethasone and N eomycin E ar Spray dissolve. W ithin 5 min, a faint reddish-brown colour
develops. A dd this solution to 10 m L o f water R and mix;
Dexam ethasone Tablets
the colour is discharged.
PhEur___________________________________________________________ E. M ix about 5 m g with 45 mg o f heavy magnesium oxide R
DEFINITION and ignite in a crucible until an alm ost white residue is
obtained (usually less than 5 m in). Allow to cool, add 1 m L
9-Fluoro- 11P, 17,21-trihydroxy-16a-m ethylpregna-1,4-
diene3j20-dione. o f water R, 0.05 m l. o f phenolphthaletn solution R1 and about
1 m L o f dilute hydrochloric add R to render the solution
Content colourless. Filter. T o a freshly prepared mixture o f 0.1 m L of
97.0 per cent to 103.0 per cent (dried substance). alizarin S solution R and 0.1 m L o f zirconyl nitrate solution R,
CHARACTERS add 1.0 m L o f the filtrate. Mix, allow to stand for 5 min and
Appearance compare the colour of the solution with that o f a blank
W hite or almost white, crystalline powder. prepared in the same manner. T h e test solution is yellow and
the blank solution is red.
Solubility
Practically insoluble in water, sparingly soluble in anhydrous TESTS
ethanol, slightly soluble in methylene chloride. Specific optical rotation (2.2.7)
IDENTIFICATION + 86 to + 92 (dried substance).
First identification B, C Dissolve 0.250 g in anhydrous ethanol R and dilute to
Second identification A , C, D, E
A. Dissolve 10.0 m g in anhydrous ethanol R and dilute to Related substances
Liquid chromatography (2.2.29). Cany out the test protected
100.0 m L with the same solvent. Place 2.0 m L o f this
solution in a stoppered test tube, add 10.0 m L of from light
phenylhydrazine-sulfuric acid solution R, mix and heat in a Test solution Dissolve 25 m g o f th e substance to be examined
w ater-bath at 60 °C for 20 min. Cool immediately. in 1.5 m L o f acetoratrile R and add 5 m L o f mobile phase A.
T h e absorbance (2.2.25) m easured at the absorption Sonicate until dissolution is complete and dilute to 10.0 m L
maxim um at 419 n m is not less than 0.4. with mobile phase A.
B. Infrared absorption spectrophotom etry (2.2.24). Reference solution (a) Dissolve 5 m g o f dexamethasone for
Comparison dexamethasone CRS. system suitability CRS (containing impurities B, F and G) in
0.5 m L o f acetonitrile R and add 1 m L o f mobile phase A.
2016 Dexamethasone 1-705
Sonicate until dissolution is complete and dilute to 2.0 m L L oss o n d ry in g (2.2.32)

with mobile phase A. M aximum 0.5 per cent, determined on 0.500 g by drying in
Reference solution (b) Dilute 1.0 m L of the test solution to an oven at 105 °C.
100.0 m L with mobile phase A. Dilute 1.0 m L of this ASSAY
solution to 10.0 m L with mobile phase A. Dissolve 0.100 g in ethanol (96 per cent) R and dilute to
Reference solution (c) Dissolve 5 mg of dexamethasone for peak 100.0 m L with the same solvent. Dilute 2.0 m L of this
identification CRS (containing impurities J and K) in 0.5 m L solution to 100.0 m L with ethanol (96 per cent) R. Measure
o f acetonitrile R and add 1 m L o f mobile phase A. Sonicate the absorbance (2.2.25) at the absorption maximum at
until dissolution is complete and dilute to 2.0 m L with 238.5 nm.
mobile phase A. Calculate the content of C 22H 29F O 5 taking the specific
Column: absorbance to be 394.
— size. I = 0.15 m , 0 = 4.6 mm;
STORA GE
chromatography R (5 jim);
— temperature. 45 °C. IM P U R IT IE S
Mobile phase. Specified impurities B, F, G, J, K
— mobile phase A: mix 250 m L of acetonitrile R with 700 m L Other detectable impurities (the following substances would, if
of water R and allow to equilibrate; dilute to 1000.0 m L present at a sufficient level, be detected by one or other of
with water R and mix again; the tests in the monograph. T hey are limited by the general
— mobile phase B: acetonitrile R; acceptance criterion for other/unspecified impurities and/or
Time Mobile phase A Mobile phase B (2034). It is therefore n o t necessary to identify these
(min) (per cent V/V) (per cent V/V) impurities for demonstration of compliance. See also 5.10.
0 - 15 100 0 Control of impurities in substances for pharmaceutical use): A , C,
100 ->0 0 -> 100
D, E, H, I.
15-40

Injection 20 jjL; inject mobile phase A as a blank.
with dexamethasone for system suitability CRS and the
identify the peaks due to impurities B, F and G; use the A. 14-fluoro-11 ß, 17,21 -trihydroxy-16a-methylpregna-1,4-
chromatogram supplied with dexamethasone for peak diene-3,20-dione,
reference solution (c) to identify the peaks due to impurities J
and K.
Relative retention W ith reference to dexamethasone (retention
time = about 15 min): impurity J = about 0.90;
impurity B = about 0.94; impurity K = about 1.3;
impurity F = about 1.5; impurity G = about 1.7.
B. 9-fluoro-llß,17,21-trihydroxy-16ß-methylpregna-l,4-
diene-3,20-dione (betamethasone),
above the baseline of the peak due to impurity B and
dexamethasone.
Limits:
— impurity G: n o t more than 3 times the area of the
— impurities B, F, J K . for each impurity, no t more than
1.5 times the area of the principal peak in the C. 9-fluoro- 1 lß , 17,21 -trihydroxy-16a-methylpregn-4-ene-
chrom atogram obtained with reference solution (b) 3,20-dione,
(0.15 p er cent);
(0.5 per cent);
the chrom atogram obtained with reference solution (b) D. 9ß, 11ß-epoxy-17,21-dihydroxy-16a-methylpregna-1,4-
(0.05 per cent). diene-3,20-dione,
1-706 Dexamethasone Acetate 2016
E. 17,21 -dihydroxy-16a-m ethylpregna-1,4,9 (11 )-triene-3,20- K. 17,21-dihydroxy-16a-m ethylpregna-l,4,7,9(l l)-tetraene-

dione, 3,20-dione.
___________________________________________________________ PhEur
Dexamethasone Acetate *****

* ★
F. 9-fluoro-l lß ,21-dihydroxy-16a-methylpregna-l,4-diene-
3,20-dione,
C 24H 31F 0 6 434.5 1177-87-3
Glucocorticoid.
G. 9-fluoro-l 1ß, 17-dihydroxy-16a-methyl-3,20-dioxopregna-
l,4-dien-21-yl acetate (dexamethasone acetate), PhEur__________________________________________________________
D E F IN IT IO N
9-Fluoro-l 1P, 17-dihydroxy-16a-methyl-3,20-dioxopregna-
l,4-dien-21-yl acetate.
C o n te n t
CHARACTERS
A p p e a ra n c e
H. 17-hydroxy-16a-m ethyl-3,20-dioxopregna-l,4,9(l l)-trien-
21-yl acetate, S o lu b ility
(96 per cent), slightly soluble in methylene chloride.
First identification B, C.
Second identification A , C, D, E, F.
100.0 m L with the same solvent. Place 2.0 m L of this
I. 9a,lla-epoxy-17,21-dihydroxy-16a-m ethylpregna-l,4- solution in a ground-glass-stoppered tube, add 10.0 m L o f
diene-3,20-dione, phenylhydrazine-sidfitric acid solution R, mix and heat in a
water-bath at 60 °C for 20 m in. Cool immediately.
T he absorbance (2.2.25) m easured at the absorption
m aximum at 419 nm is no t less than 0.35.
Comparison dexamethasone acetate CRS.
substance separately in methylene chloride R, evaporate to
J. 17,21-dihydroxy-16a-m ethylpregna-l,4-diene-3,l 1, 20 -
trione,
Solvent mixture methanol R, methylene chloride R (1:9 V/V).
in the solvent mixture and dilute to 10 m l. with the solvent
mixture.
2016 Dexamethasone Acetate 1-707
Reference solution (a) Dissolve 20 mg of dexamethasone Reference solution (b) Dilute 1.0 m L o f the test solution to
acetate CRS in the solvent mixture and dilute to 20 m l. with 100.0 m L with the mobile phase. Dilute 1.0 m L of this
the solvent mixture. solution to 10.0 m L with the mobile phase.
Reference solution (b) Dissolve 10 mg o f cortisone acetate R in Reference solution (c) Dissolve the contents of a vial of
reference solution (a) and dilute to 10 m L with reference dexamethasone acetate impurity E CRS in 1.0 m L o f the
solution (a). mobile phase.
Plate TLC silica gel F 254 plate R. Column:
Mobile phase Add a mixture of 1.2 volumes of water R and — size. I = 0.25 m , 0 = 4.6 mm;
8 volumes of methanol R to a mixture o f 15 volumes of — stationary phase: octadecylsilyl silica gel for chromatography R
ether R and 77 volumes of methylene chloride R. (5 |im).
Application 5 |iL. Mobile phase Mix 380 m L of acetonitrile R with 550 m L of
water R and allow to equilibrate; dilute to 1000.0 m L with
water R and mix again.
Drying In air.
Flow rate 1 mL/min.
Results A T he principal spot in the chromatogram obtained
Injection 20 |iL.
principal spot in the chromatogram obtained with reference Run time 2.5 times the retention time of dexamethasone
solution (a). acetate.
Detection B Spray with alcoholic solution of sulfuric add R, heat Identification of impurities Use the chromatogram obtained
at 120 °C for 10 min or until the spots appear, and allow to with reference solution (a) to identify the peaks due to
cool; examine in daylight and in ultraviolet light at 365 nm . impurities A and D; use the chromatogram obtained with
reference solution (c) to identify the peak due to impurity E.
Residts B T he principal spot in the chromatogram obtained
with the test solution is similar in position, colour in daylight, Relative retention W ith reference to dexamethasone ^cetate
fluorescence in ultraviolet light at 365 nm and size to the (retention time = about 22 min): impurity A = about 0.4;
principal spot in the chromatogram obtained with reference impurity D = about 0.9; impurity E = about 1.2.
solution (a). System suitability, reference solution (a):
System suitability, reference solution (b): — resolution: minimum 3.3 between die peaks due to
— the chromatogram shows 2 clearly separated spots. impurity D and dexamethasone acetate.
D . Add about 2 mg to 2 m L of sulfuric add R and shake to Limits:
dissolve. W ithin 5 min, a faint reddish-brown colour — impurity D: n o t more than 3 times the area o f the
develops. Add this solution to 10 m L o f water R and mix. principal peak in the chromatogram obtained with
T he colour is discharged and a clear solution remains. reference solution (b) (0.3 per cent);
— impurities A , E: for each impurity, not more than twice
E. Mix about 5 mg 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
of 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. T o a freshly prepared mixture of 0.1 m L of
— total: not m ore than 5 times the area of the principal peak
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
(0.5 per cent);
compare the colour o f the solution with that of a blank
prepared in the same manner. The test solution is yellow and
the blank is red.
(0.05 per cent).
F. About 10 mg gives the reaction o f acetyl (2.3.1).
TESTS M aximum 0.5 per cent, determined on 0.500 g by drying
S pecific o p tical ro ta tio n (2.2.7) in vacuo in an oven at 105 °C.
ASSAY
Dissolve 0.250 g in anhydrous ethanol R and dilute to Dissolve 0.100 g in ethanol (96 per cent) R and dilute to
25.0 m L with the same solvent. 100.0 m L with the same solvent. Dilute 2.0 m L o f this
R e la te d su b sta n c e s solution to 100.0 m L with ethanol (96 per cent) R. Measure
Liquid chromatography (2.2.29). Carry out the test protected the absorbance (2.2.25) at the absorption maximum at
from light. 238.5 nm.
Test solution Dissolve 25 mg of the substance to be examined Calculate the content o f C 24H 31F 0 6 taking the specific
in about 4 m L of acetonitrile R and dilute to 10.0 m L with absorbance to be 357.
water R. STORAGE
Reference solution (a) Dissolve 2 mg o f dexamethasone CRS Protected from light.
(impurity A) and 2 m g of betamethasone acetate CRS
(impurity D) in 100.0 mT. o f the mobile phase and sonicate IMPURITIES
for about 10 m in (solution A). Mix 6.0 m L o f the test Specified impurities A, D , E
solution and 1.0 mT. o f solution A and dilute to 10.0 m L Other detectable impurities (the following substances would, if
1-708 Dexamethasone Isonicotinate 2016

H CH3 f ' 0 -A .
F, G, H.
G. 9-fluoro-l ip-hydroxy-16a-methyl-3j20-dioxopregna-lj4-
dien- 21-yl acetate.
o o
W t-o h ch3
A. 9-fluoro-11 p, 17,21-trihydroxy-16a-m ethylpregna-l ,4-

diene-3,20-dione (dexamethasone).
H . 17-hydroxy-16a-m ethyl-3,20-dioxopregna-l,4,9(l l)-trien-

21-yl acetate.
___________________________________________________________PhEur
★* ★
★ ★
B. 14-fluoro-l ip,17-<iihydroxy-16a-methyl-3j20-dioxopregna- Dexamethasone Isonicotinate ★ ★
l }4-dien-21-yl acetate, *****
\ 0
H CH3)
■OH CHj
C. 9 -flu o ro -lip }17P-dihydroxy-16a-methjd-3,20-

dioxopregna-1,4-dien-21-yl acetate. C 28H 32FN O 6 497.6 2265-64-7
Action and use

Glucocorticoid.
PhEur_____________
D E F IN IT IO N
9-Fluoro-l 1P, 17-dihydroxy-l 6a-methyl-3,20-dioxopregna-
l,4-dien-21-yl pyridine-4-carboxylate.
D . 9-fluoro-llp,17-dihydroxy-16p-m ethyl-3,20-dioxopregna- Content
l,4-dien-21-yl acetate (betamethasone acetate), 99.0 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance
H CH \ v --(
-o h 0 ^ W hite or alm ost white crystalline powder.
CHj
Solubility
Practically insoluble in water, slighdy soluble in anhydrous
ethanol and in acetone.
E. 9-fluoro- 11 P, 17-dihydroxy-16a-methyl-3,20-dioxopregn-4- Infrared absorption spectrophotometry (2.2.24).
en- 21 -yl acetate. Comparison dexamethasone isonicotinate CRS.
TESTS
Suspend 0.200 g in 4.0 m l. o f ethyl acetate R and dilute to
20.0 m L w ith ethanol (96 per cent) R. T reat in an ultrasonic
bath until a clear solution is obtained.
Related substances
F. 17-hydroxy-16a-methyl-3,20-dioxo-9P,l ip-epoxypregna- Liquid chrom atography (2.2.29). Prepare solutions immediately
l,4-dien-21-yl acetate, before use.
2016 Dexamethasone Sodium Phosphate 1-709
Test solution Suspend 50.0 m g in 7 m L o f acetcmitrile R and Loss o n d ry in g ( 2.2.32)

dilute to 10.0 m L with water R. T reat in an ultrasonic bath M aximum 1.0 per cent, determined on 1.000 g by drying in
until a clear solution is obtained. an oven at 102 °C under high vacuum for 4 h.
Reference solution (a) Suspend 5.0 mg o f dexamethasone CRS ASSAY
and 5.0 m g of dexamethasone acetate CRS in 70 m L of Dissolve 0.400 g in a mixture of 5 m L of anhydrous formic
acetomtrile R, add 1.0 m L of the test solution and dilute to acid R and 50 m L of glacial acetic acid R. Titrate with 0.1 M
100.0 m L with water R. T reat in an ultrasonic bath until a perchloric acid, determining the end-point potentiometrically
clear solution is obtained. (2. 2. 20).
Reference solution (b) Dilute 1.0 m L of reference solution (a) 1 m L of 0.1 M perchloric acid is equivalent to 49.76 mg
to 10.0 m L with water R. of C 2&H32F N O 6.
Reference solution (c) Suspend 5 mg of dexamethasone
IM P U R IT IE S
isonicotinate for impurity C identification CRS in 0.7 m L of
acetomtrUe R and dilute to 1 mL with water R. T reat in an Specified impurities A , B, C.
ultrasonic bath until a clear solution is obtained.
Column:
— sizer. I = 0.125 m , 0 = 4.0 mm,
(5 pm).
Mobile phase:
— mobile phase A: water R,
— mobile phase B: acetonitrile R, A. 9-fluoro-l lß,17,21-trihydroxy-16a-methylpregna-l,4-
diene-3,20-dione (dexamethasone),
(min) (set cent V/V) (per cent V/V)
0 -2 68 32
2-20 68 ->50 32 ->50
20-25 50->68 5 0 -> 3 2
25-35 68 32
B. 9-fluoro-l 1ß, 17-dihydroxy-l6a-methyl-3,20-dioxopregna-
l,4-dien-21-yl acetate (dexamethasone acetate),
Injection 10 jjJL.
with dexamethasone isomcotinate for impurity C
reference solution (c) to identify the peak due to impurity C.
Relative retention W ith reference to dexamethasone C. 9-fluoro-llß , 17-dihydroxy-16a-methylpregna-l,4-diene-
isonicotinate (retention time = about 12 min):
3,20-dione (21-deoxydexamethasone).
impurity A = about 0.4; impurity C = about 0.6;
impurity B = about 0.8. _________________________________________________________ PhEur

impurity B and dexamethasone isonicotinate.
Limits'. Dexamethasone Sodium *****
— impurity A: not m ore than 5 times the area o f the Phosphate *****
corresponding peak in the chromatogram obtained with (Ph. Ettr. monograph 0549)
— impurity B: not m ore than 3 times the area o f the
— impurity C: not m ore than 3 times the area of the peak
due to dexamethasone isonicotinate in the chromatogram
obtained with reference solution (b) (0.3 per cent),
— unspecified impurities: for each impurity, n ot more than the
area o f the peak due to dexamethasone isomcotinate in
CfcHzsFNajOsP 516.4 2392-39-4
(0.1 per cent), A c tio n a n d u se
— total: n o t more th an 8 times the area of the peak due to Glucocorticoid.
dexamethasone isomcotinate in the chromatogram
obtained with reference solution (b) (0.8 p er cent), P re p a ra tio n s
Dexamethasone Sodium Phosphate Eye Drops
dexamethasone isonicotinate in the chromatogram Dexamethasone Sodium Phosphate Injection
obtained with reference solution (b) (0.05 per cent). Dexamethasone Sodium Phosphate Oral Solution
1-710 Dexamethasone Sodium Phosphate 2016
PhEir ___________________________________________________ ______ System suitability', reference solution (b):

— the chromatogram shows 2 spots which may, however,
D E F IN IT IO N
n o t be completely separated.
9-Fluoro- l i p , 17-dihydroxy-16cc-methyl-3,20-dioxopregna-
l }4-dien-21-yl disodium phosphate. D . A dd about 2 mg to 2 m L o f sulfuric acid R and shake to
dissolve. W ithin 5 min, a faint yellowish-brown colour
C o n te n t
develops. A dd this solution to 10 m L o f water R and mix.
T h e colour fades and a clear solution remains.
CHARACTERS E. M ix about 5 mg with 45 mg o f heavy magnesium oxide R
A p p e a ra n c e and ignite in a crucible until an alm ost white residue is
W hite or almost white, very hygroscopic powder. obtained (usually less than 5 min). Allow to cool, add 1 m L
S o lu b ility o f water R, 0.05 m L o f phenolphthalein solution R1 and about
Freely soluble in water, slightly soluble in ethanol 1 m L o f dilute hydrochloric acid R to render the solution
(96 per cent), practically insoluble in methylene chloride. colourless. Filter. T o a freshly prepared m ixture of 0.1 m L of
It shows polymorphism (5.9). alizarin S solution R and 0.1 m L o f zxrcortyl nitrate solution R,
add 1.0 m L o f the filtrate. Mix, allow to stand for 5 min and
ID E N T IF IC A T IO N compare the colour of the solution with that o f a blank
First identification B, G prepared in the same m anner. T h e test solution is yellow and
Second identification A , C, D, E, F the blank is red.
A. Dissolve 10.0 mg in 5 mT. of water R and dilute to F. T o 40 m g add 2 m L of sulfuric acid R and heat gently
100.0 m L with anhydrous ethanol R. Place 2.0 m L o f this until white fumes are evolved, add nitric acid R dropwise,
solution in a ground-glass-stoppered tube, add 10.0 m L of continue the heating until the solution is alm ost colourless
phenylhydrazine-sulfuric acid solution R, mix and heat in a and cool. A dd 2 m L o f water R, heat until white fumes are
w ater-bath at 60 °C for 20 min. Cool immediately. again evolved, cool, add 10 m L o f water R and neutralise to
T h e absorbance (2.2.25) m easured at the absorption red litmus paper R with dilute ammonia R l. T h e solution gives
m axim um at 419 nm is at least 0.20. reaction (a) o f sodium (2.3.1) and reaction (b) of phosphates
B. Infrared absorption spectrophotometry (2.2.24). (2.3.1).
Comparison dexamethasone sodium phosphate CRS. G. Examine the chromatograms obtained in the assay.
If the spectra obtained in the solid state show differences, Results T h e principal peak in the chrom atogram obtained
dissolve the substance to be examined and the reference with the test solution is similar in retention time and size to
substance separately in the minim um volume o f ethanol the principal peak in the chromatogram obtained with
(96 per cent) R, evaporate to dryness on a water-bath and reference solution (b).
record new spectra using the residues. TESTS
C. Thin-layer chrom atography (2.2.27). S o lu tio n S
Test solution Dissolve 10 mg o f the substance to be examined Dissolve 1.0 g in carbon dioxide-free water R and dilute to
in methanol R and dilute to 10 m L with the same solvent. 20 m L with the same solvent.
Reference solution (a) Dissolve 20 m g of dexamethasone sodium A p p e a ra n c e o f so lu tio n
phosphate CRS in methanol R and dilute to 20 m L with the Solution S is clear (2.2.1) and n o t more intensely coloured
sam e solvent. than reference solution B7 (2.2.2, Method II).
Reference solution (b) Dissolve 10 m g o f prednisolone sodium pH (2.2.3)
phosphate CRS in reference solution (a) and dilute to 10 m L 7.5 to 9.5.
with reference solution (a). Dilute 1 mT. o f solution S to 5 m L with carbon dioxide-free
Plate TLC silica gel F254 plate R. water R.
Mobile phase glacial acetic add R, water R, butanol R S p ecific o p tic a l r o ta tio n (2.2.7)
(20:20:60 VIVIV). + 75 to + 83 (anhydrous substance).
Application 5 (iL. Dissolve 0.250 g in water R and dilute to 25.0 m L with the
Development Over 3/4 o f the plate. same solvent.
Drying In air. R e la te d su b s ta n c e s
Detection A Examine in ultraviolet light at 254 nm. Liquid chrom atography (2.2.29).
Results A T h e principal spot in the chromatogram obtained Solution A Dissolve 7.0 g o f ammonium acetate R in 1000 m L
w ith the test solution is similar in position and size to the o f water R.
principal spot in the chrom atogram obtained with reference Test solution Dissolve 10 m g of the substance to be examined
solution (a). in mobile phase A and dilute to 10.0 m L with mobile
Detection B Spray w ith alcoholic solution of sulfuric acid R, heat phase A.
at 120 °C for 10 min or until the spots appear, and allow to Reference solution (a) Dissolve 2 m g o f betamethasone sodium
cool; examine in daylight and in ultraviolet light at 365 nm. phosphate CRS (impurity B) and 2 m g o f dexamethasone
Results B T h e principal spot in the chromatogram obtained sodium phosphate CRS in mobile phase A, then dilute to
w ith the test solution is similar in position, colour in daylight, 100.0 m L with mobile phase A.
fluorescence in ultraviolet light at 365 nm and size to the Reference solution (b) Dissolve 2 m g of dexamethasone sodium
principal spot in the chrom atogram obtained with reference phosphate for peak identification CRS (containing impurities A,
solution (a). C, D , E, F and G) in mobile phase A and dilute to 2.0 m L
with mobile phase A.
2016 Dexamethasone Sodium Phosphate 1-711
Reference solution (c) Dilute 1.0 mL of the test solution to Dissolve 50 mg in water R and dilute to 100 m L with the
100.0 m L with mobile phase A. D ilute 1.0 m L of this same solvent. T o 10 m L of this solution add 5 m L of
solution to 10.0 m L with mobile phase A. molybdovanadic reagent R, mix and allow to stand for 5 min.
Column: Any yellow colour in the solution is not m ore intense than
— sizer. I = 0.125 m, 0 = 4.6 mm; that in a standard prepared at the same time in the same
— stationary phase, end-capped, octylsifyl silica gel for m anner using 10 m L o f phosphate standard solution (5 ppm
chromatography R (5 pm); POJ R.
— temperature: 30 °C. E th a n o l (2.4.24, System A)
Mobile phase. M aximum 1.5 per cent.
— mobile phase A: mix 300 mT. of solution A and 350 mT. o f W a te r (2.5.12)
water R, adjust to p H 3.8 with acetic acid R, then add M aximum 10.0 per cent, determined on 0.200 g.
350 m L of methanol R;
— mobile phase B: adjust 300 m L of solution A to p H 4.0 A SSAY
with acetic acid R, then add 700 m L of methanol R; Liquid chromatography (2.2.29).
(min)
the mobile phase. Dilute 5.0 mL o f the solution to 50.0 m L
(per cent V/V) (per cent V/V)
0 -3 .5
90 10
Reference solution (a) Dissolve 2 m g of dexamethasone CRS
3.5-23.5 90 -»60 10 -» 40
(impurity A) and 2 mg of dexamethasone sodium
23.5 - 34.5 60 -»5 40 -» 95 phosphate CRS in 2 m L o f tetrahydrofuran R, then dilute to
34.5 - 50 5 95 100.0 m L with the mobile phase. Dilute 5.0 m L of this
Reference solution (b) Dissolve 30.0 mg of dexamethasone
Flow rate 1.0 mlVmin. sodium phosphate CRS in the mobile phase and dilute to
Detection Spectrophotometer at 254 nm . 50.0 m L with the mobile phase. Dilute 5.0 m L o f the
Injection 20 |±L. solution to 50.0 m L with the mobile phase.
Identification of impurities Use the chromatogram supplied Column:
with dexamethasone sodium phosphate for peak — size. I = 0.15 m , 0 = 4.6 mm;
identification CRS and the chromatogram obtained with — stationary phase: end-capped octadecylsUyl silica gel for
reference solution (b) to identify the peaks due to impurities chromatography R (7 Jim).
A, C, D , E , F and G; use the chromatogram obtained with Mobile phase Mix 520 m L of water R with 2 m L of phosphoric
reference solution (a) to identify the peak due to impurity B. add R. Adjust the temperature to 20 °C, then adjust to
Relative retention W ith reference to dexamethasone sodium p H 2.6 with sodium hydroxide R. M ix this solution with
phosphate (retention time = about 22 min): 36 m L o f tetrahydrofuran R and 364 mL of methanol R.
im purity C = about 0.5; impurity D = about 0 . 6; Flow rate 1.5 mlVmin.
impurity E = about 0.8; impurity F = about 0.92; Detection Spectrophotometer at 254 nm.
im purity B = about 0.95; impurity A = about 1.37; Injection 20 |±L.
impurity G = about 1.41.
Run time 3 times the retention tim e of dexamethasone
System suitability,: reference solution (a): sodium phosphate.
impurity B and dexamethasone sodium phosphate.
Limits'. impurity A.
peak area o f impurity A by 0.75;
Relative retention W ith reference to dexamethasone sodium
— impurity A: not more than 5 times the area o f the phosphate (retention time = about 8 min):
reference solution (c) (0.5 per cent); System suitability: reference solution (a):
— impurity G: n o t more than 3 times the area o f the — resolution: minimum 6.0 between the peaks due to
principal peak in the chromatogram obtained with dexamethasone sodium phosphate and im purity A.
reference solution (c) (0.3 per cent); Calculate the percentage content o f C22H28FNa20 gP using
— impurities B, C, D, E, F: for each impurity, no t m ore than the chromatogram obtained with reference solution (b) and
twice the area of the principal peak in the chromatogram taking into account the assigned content o f dexamethasone
obtained with reference solution (c) (0.2 per cent); sodium phosphate CRS.
— unspecified impurities: for each impurity, n o t more than the STORAGE
— total: n o t more than 10 times the area o f the principal IM P U R IT IE S
peak in the chromatogram obtained with reference Spedfied impurities A, B, C, D, E, F , G
solution (c) ( 1.0 per cent); Other detectable impurities (the following substances would, if
— disregard limit: 0.5 times the area o f the principal peak in present at a sufficient level, be detected by one or other of
the chromatogram obtained with reference solution (c) the tests in the monograph. They are limited by the general
(0.05 per cent). acceptance criterion for other/unspecified impurities and/or
In o rg a n ic p h o sp h a te s by the general monograph Substances for pharmaceutical use
M axim um 1 per cent. (2034). It is therefore n o t necessary to identify these
1-712 Dexamfetamine Sulfate 2016

Control of impurities in substances for pharmaceutical use): H.
Dexamfetamine Sulfate
Dexamfetamine Sulphate
C n a r^ O H
.HjSCU
A ’CHs tt Me
)
X (C 9H 13N ) 2,H 2S 0 4 368.5 51-63-8
A. 9 -flu o ro -lip , 17,21 -trihydroxy-16a-methylpregna-1,4-
diene-3,20-dione (dexamethasone),
Amfetamine.
P r e p a r a tio n
D e x a m f e t a m in e Tablets
D E F IN IT IO N
Dexamfetamine Sulfate is (5)-a-m ethylphenethylam ine
sulfate. It contains not less th an 9 9 .0 % and not m ore than
1 0 0 .5 % o f (C 9H 13N ) 2JH 2S 04 , calculated with reference to
B. 9-fluoro-l ip,17-dihydroxy-16p-methyl-3,20-dioxopregna-
l,4-dien-21-yl dihydrogen phosphate (betamethasone C H A R A C T E R IS T IC S
phosphate), A white or almost white, crystalline powder.
F red y soluble in water, slightly soluble in ethanol (96%) \
A. Dissolve 1 g in 5 0 m L of water, add 10 m L of 5m sodium
hydroxide and 0 .5 m L of benzoyl chloride and shake. Repeat
the addition o f benzoyl chloride in 0 .5 m L quantities until no
further predpitate is produced. T h e melting point o f the
predpitate, after recrystallising twice from ethanol (50%), is
about 157°, Appendix V A.
B. Dissolve 2 mg in 4 m L o f water, add 1 m L of
1m hydrochloric add, 2 m L o f diazotised nitroanHine solution,
4 m L of 1m sodium hydroxide and 2 m L o f butan-l-ol, shake
and allow to separate. A red colour is produced in the
butanol layer (distinction from m ethylam fetamine).
C, D , E, F. for each impurity, one or more C. Yields the reactions characteristic of sulfates, Appendix VI.
diastereoisomer(s) o f (9-fluoro-11(3,17a-dihydroxy-16-methyl-
3.17 -dioxo-D -hom o-androsta-1,4-dien-17 a-yl)methyl TESTS
dihydrogen phosphate (undefined stereochemistry at C-16 A c id ity o r alk alin ity
and C-17a), o r (9-fluoro-11 p, 17-dihydroxy-16a-methyl- Dissolve 0 .5 g in 10 m L o f water and titrate with
3.17 a-dioxo-D -hom o-androsta-1,4-dien-17-yl)methyl 0 .0 1 m hydrochloric add FS o r 0 .0 1 m sodium hydroxide VS
dihydrogen phosphate (undefined stereochemistry at C -17), using methyl red solution as indicator. N o t m ore than 0.1 m L
o f 0.01m hydrochloric add VS or 0.01m sodium hydroxide VS is
required to change the colour o f the solution.
S p ecific o p tic a l ro ta tio n
In an 8 .0 % w/V solution, + 1 9 .5 to + 2 2 .0 , calculated with
reference to the dried substance, Appendix V F.
L oss o n d ry in g
W hen dried to constant w dght at 105°, loses not m ore than
G. 9-fluoro- 11P, 17-dihydroxy-16a-m ethyl-3-oxoandrosta-1,4- 1 .0 % of its weight. Use 1 g.
diene-17P-carboxylic ad d , S u lfa te d a s h
N o t more than 0.1% , Appendix IX A.
A SSA Y
Dissolve 0 .4 g in 1 2 0 m L o f water, add 2 m L of 5m sodium
hydroxide and distil into 5 0 rnT. of 0 .1 m hydrochloric add KS,
continuing the distillation until only 5 m L o f liquid is left in
the distillation flask. T itrate the excess o f a d d with
0.1m sodium hydroxide KS using methyl red solution as
indicator. Each m l. o f 0.1m hydrochloric add KS is equivalent
H . 9-fluoro-l ip , 17-dihydroxy-16a-methyl-3,20-dioxopregn- to 1 8 .4 2 m g of (C 9H 13N ) 2,H 2S 0 4 .
4-en-21-yI dihydrogen phosphate.
PhEur
2016 Dexchlorphenirainine Maleate 1-713
★ ★ A p p e a ra n c e o f so lu tio n
Dexchlorpheniramine Maleate ★ ★ Solution S is clear (2.2.1)- and not more intensely coloured
★ . ★
(Ph. Eur. monograph 1196) *★* than reference solution BY 6 (2.2.2, Method II).
p H (2.2.3)
4.5 to 5.5.
c o 2h Dissolve 0.20 g in 20 m L of water R.
( c o 2h
+ 22 to + 23 (dried substance), determined on solution S.
2438-32-6 Test solution Dissolve 10.0 mg of the substance to be
examined in 1.0 m L of methylene chloride R.
A c tio n a n d u se Reference solution Dissolve 5.0 mg o f brompheniramine
Histam ine H I receptor antagonist; antihistamine. maleate CRS in 0.5 m L of methylene chloride R and add
0.5 m L of the test solution. Dilute 0.5 m L o f this solution to
PhEur.
50.0 m L with methylene chloride R.
D E F IN IT IO N Column:
(3S)-3-(4-Chlorophenyl)-N,N-dimethyl-3-(pyridin-2- — material: glass;
yl) propan- 1-amine (Z)-butenedioate. — sizer. I = 2.3 m , 0 = 2 mm;
C o n te n t — stationary phase, ad d - and base-washed silanised
98.0 per cent to 100.5 per cent (dried substance). diatomaceous earth for gas chromatography R (135-175 |im)
impregnated with 3 per cent mlm of a mixture of
CHARACTERS
50 per cent of poly(dimethyl)siloxane and 50 per cent of
A p p e a ra n c e
poly(diphenyl)siloxane.
S o lu b ility
Flow rate 20 mlVmin.
Very soluble in water, freely soluble in ethanol (96 per cent),
in m ethanol and in methylene chloride. Temperature:
ID E N T IF IC A T IO N — infection port and detector. 250 °C.
First identification A , C, E Detection Flame ionisation.
Second identification A , B, D, E Iiyection 1 jiL.
A. Specific optical rotation (see Tests). Run time 2.5 times the retention time of
B. Melting point (2.2.14): 110 °C to 115 °C. dexchlorpheniramine.
C . Infrared absorption spectrophotometry (2.2.24). System suitability: reference solution:
Preparation Discs of potassium bromide R. — resolution: minimum 1.5 between the peaks due to
Comparison dexchlorpheniramine maleate CRS. dexchlorpheniramine and brompheniramine.
D . Thin-layer chromatography (2.2.27). Limits:
— impurity A: n o t more than 0.8 times the area of the peak
due to dexchlorpheniramine in the chromatogram
obtained with the reference solution (0.4 per cent);
Reference solution Dissolve 56 mg o f maleic acid R in — total: not m ore than twice the area of the peak due to
methanol R and dilute to 10 m L with the same solvent. dexchlorpheniramine in the chromatogram obtained with
Plate TLC silica gel F2 5 4 plate R. the reference solution (1 per cent).
Mobile phase water R, anhydrous formic acid R, methanol R, di E n a n tio m e ric p u rity
isopropyl ether R (3:7:20:70 V/V/V/V). Liquid chromatography (2.2.29).
Application 5 jiL. Test solution Dissolve 10.0 mg of the substance to be
Development Over a path o f 12 cm. examined in 3 m L of water R. Add a few drops of
Drying In a current o f air for a few minutes. concentrated ammonia R until an alkaline reaction is produced.
Shake with 5 m L of methylene chloride R. Separate the layers.
Evaporate the lower, methylene chloride layer to an oily
Results T he chromatogram obtained with the test solution residue on a water-bath. Dissolve the oily residue in
shows 2 clearly separated spots. The upper spot is similar in 2-propanol R and dilute to 10.0 m L with the same solvent.
position and size to the spot in the chromatogram obtained
Reference solution (a) Dissolve 10.0 mg o f dexchlorpheniramine
w ith the reference solution.
maleate CRS in 3 m L o f water R. Add a few drops of
E. T o 0.15 g in a porcelain crucible add 0.5 g of anhydrous concentrated ammonia R until an alkaline reaction is produced.
sodium carbonate R. H eat over an open flame for 10 min. Shake with 5 m L of methylene chloride R. Separate the layers.
Allow to cool. Take up the residue with 10 m L of dilute nitric Evaporate the lower, methylene chloride layer to an oily
acid R and filter. T o 1 m L o f the filtrate add 1 m L of residue on a water-bath. Dissolve the oily residue in
water R. T he solution gives reaction (a) of chlorides (2.3.1). 2-propanol R and dilute to 10.0 m L with the same solvent.
TESTS Reference solution (b) Dissolve 10.0 mg o f chlorphenamine
S o lu tio n S maleate CRS in 3 m L of water R. Add a few drops of
Dissolve 2.0 g in water R and dilute to 20.0 m L with the concentrated ammonia R until an alkaline reaction is produced.
sam e solvent.
1-714 Dexpanthenol 2016
Shake with 5 m L of methylene chloride R. Separate the layers.

Evaporate the lower, methylene chloride layer to an oily
residue on a water-bath. Dissolve the oily residue in
2-propanol R and dilute to 10.0 m L with the same solvent.
50 m L with 2-propanol R.
Column:
— size. I — 0.25 m , 0 = 4.6 mm; B. (3i?)-3-(4-chlorophenyl)-NJAr-dimethyl-3-(pyridin-2-
— stationary phase: amylose derivative of silica gelfor yl)propan-l-am ine ( (R)-enantiom er).
chromatography R. PhEir
Mobile phase diethylamine R, 2-propanol R, hexane R

(3:20:980 ViV/V).
Flow rate 1 mL/min.
***
Detection Spectrophotom eter at 254 nm . ★ ★
Dexpanthenol ★ ★
Injection 10 (iL. *****
U nder these conditions the peak due to the (5)-isomer
appears first. h3C. ch, 0
System suitability:
— resolution: minim um 1.5 between the peaks due to the
CR)-enantiomer (impurity B) and the (5)-enantiom er in H 'OH H
— the retention times of the principal peaks in the 205.3 81-13-0
chromatograms obtained with the test solution and
Action and use
reference solution (a) are identical ((5)-enantiomer).
Vitamin B 5 analogue.
Limits:
— (R)-enantiomer (impurity B): not more than the area of PhEur.
DEFINITION
reference solution (c) (2 per cent);
D expanthenol contains no t less than 98.0 per cent and not
— unspecified impurities', for each impurity, not more than
more than the equivalent o f 101.0 per cent o f (2R)-2,4r-
dihydroxy-iV-(3-hydroxypropyl)-3,3-dimethyibutanamide,
calculated with reference to the anhydrous substance.
(0.5 per cent).
H e a v y m e ta ls (2.4.8) CHARACTERS
M axim um 20 ppm. A colourless or slightly yellowish, viscous hygroscopic liquid,
or a white or alm ost white, crystalline powder, very soluble in
water, freely soluble in ethanol (96 per cent).
IDENTIFICATION
M aximum 0.5 per cent, determ ined on 1.000 g by drying in First identification A, B.
an oven at 65 °C for 4 h. Second identification A, C, D.
S u lfa te d a s h (2.4.14) A. Specific optical rotation (see Tests).
Maximum 0.1 per cent, determined on 1.0 g. B. Examine by infrared absorption spectrophotometry
A SSA Y
dexpanthenol CRS. Examine the substances using discs
Dissolve 0.150 g in 25 m L o f anhydrous acetic add R. Titrate
prepared as follows: dissolve the substance to be examined
w ith 0.1 M perchloric add, determining the end-point
and the reference substance separately in 1.0 m L of
potentiometrically (2 . 2. 20).
anhydrous ethanol R to obtain a concentration o f 5 mg/mL.
1 m L of 0.1 M perchloric acid is equivalent to 19.54 mg Place dropwise 0.5 m L o f this solution on a disc o f potassium
o f C 20H 23CIN 2O 4. bromide R. Dry the disc at 100-105 °C for 15 m in.
STO RA G E C. Examine the chromatograms obtained in the test for
Protected from light. 3-am inopropanol. T he principal spot in the chromatogram
IM P U R IT IE S obtained with test solution (b) is similar in position, colour
Spedfied impurities A, B
D . T o 1 m L o f solution S (see Tests) add 1 m L o f dilute
sodium hydroxide solution R and 0.1 m L o f copper sulfate
solution R. A blue colour develops.
and enantiomer
TESTS
Solution S
A. (3i?5)-ATJN-dimethyl-3-phenyl-3-(pyridin-2-yl)propan-l-
amine, Appearance o f solution
Solution S is clear (2.2.1) and n o t m ore intensely coloured
than reference solution B 6 (2.2.2, Method II).
2016 Dextran 1 1-715
p H (2.2.3)
T he p H of solution S is not greater than 10.5.
Dextran 1 for Injection *****
+*
S p ecific o p tic a l ro ta tio n (2.2.7) (Ph. Eur. monograph 1506) *
T he specific optical rotation is + 29.0 to + 32.0, determined
on solution S and calculated with reference to the anhydrous A ctio n a n d u se
substance. Plasma substitute.
3 -A m in o p ro p an o l PhEur_________________________________________________________
D E F IN IT IO N
Low-molecular-weight fraction o f dextran, consisting of a
Test solution (a) Dissolve 0.25 g of the substance to be mixture o f isomaltooligosaccharides.
examined in anhydrous ethanol R and dilute to 5 m L with the
Average relative molecular mass About 1000.
same solvent.
Test solution (b) Dilute 1 m L of test solution (a) to 10 m L P R O D U C T IO N
with anhydrous ethanol R. It is obtained by hydrolysis and fractionation of dextrans
Reference solution (a) Dissolve the contents of a vial of produced by fermentation of sucrose using Leuconostoc
dexpanthenol CRS in 1.0 m L of anhydrous ethanol R to obtain mesenteroides strain N R R L B-512 = C IP 78.59 or substrains
a concentration of 5 mgfrnL. thereof (for example L. mesenteroides B-512 F = N C T C
10817).
Reference solution (b) Dissolve 25 m g o f 3-aminopropanol R in
anhydrous ethanol R and dilute to 100 m L with the same It is prepared in conditions designed to minimise the risk of
microbial contamination.
solvent.
Apply separately to the plate 10 pL of each solution. Develop CHARACTERS
over a path of 15 cm using a mixture of 20 volumes of A p p e a ra n ce
concentrated ammonia R, 25 volumes of methanol R and White or almost white hygroscopic powder.
55 volumes of butanol R. Allow the plate to dry in air, spray S olub ility
with a 100 g/L solution o f trichloroacetic acid R in methanol R Very soluble in water, very slightly soluble in ethanol
and heat at 150 °C for 10 min. Spray with a 1 g/L solution (96 per cent).
of ninkydrin R in methanol R and heat at 120 °C until a
colour appears. Any spot due to 3-am inopropanol in the
chromatogram obtained with test solution (a) is n o t more A. Dissolve 3.000 g in water R, heat on a water-bath and
intense than the spot in the chromatogram obtained with dilute to 100.0 m L with the same solvent. The specific
reference solution (b) (0.5 per cent). optical rotation (2.2.7) is + 148 to + 164, calculated with
reference to the dried substance. Dry an aliquot of the
H eav y m e ta ls (2.4.8) solution first on a water-bath and then to constant weight
12 m L o f solution S complies with limit test A for heavy in vacuo at 70 °C. Calculate the dextran content after
metals (20 ppm). Prepare the reference solution using lead correction for the content of sodium chloride.
standard solution (1 ppm Pb) R.
W a te r (2.5.12)
Preparation T o 1-2 m g add 1 or a few drops of water R.
N ot more than 1.0 per cent, determined on 1.000 g.
Grind in an agate m ortar for 1-2 min. Add about 300 m g of
S u lfa te d a sh (2.4.14) potassium bromide R and mix to a slurry b u t do not grind.
N ot m ore than 0.1 per cent, determined on 1.0 g. Dry in vacuo at 40 °C for 15 min. Crush the residue. If it is
A SSA Y not dry, dry for another 15 min. Prepare a disc using
T o 0.400 g add 50.0 m L of 0.1 Mperchloric acid. Boil under potassium bromide R.
a reflux condenser for 5 h protected from humidity. Allow to Comparison Repeat the operations using dextran 1 CRS.
cool. Add 50 m L of dioxan R by rinsing the condenser, Blank Run the infrared spectrum with a blank disc using
protected from hum idity. Add 0.2 m L o f naphtholbenzein potassium bromide R in the reference beam.
solution R and titrate with 0.1 M potassium hydrogen phthalate C. Molecular-mass distribution (see Tests).
until the colour changes from green to yellow. Carry out a
blank titration. TESTS
S o lu tio n S
Dissolve 7.5 g in carbon dioxide-free water R, heat on a water-
of C 9 H 19 NO 4 .
bath and dilute to 50 m L with the same solvent
STO RA G E
A b so rb an ce (2.2.25)
In an airtight container. M aximum 0.12, determined at 375 a m on solution S.
PhEur A cidity o r alk alin ity
T o 10 m L of solution S add 0.1 m L o f phenolphthalein
solution R. The solution is colourless. Add 0.2 m L of 0.01 M
sodium hydroxide. The solution is pink. Add 0.4 m L of
0.01 M hydrochloric acid. The solution is colourless.
Add 0.1 m L o f methyl red solution R. T h e solution is red or
orange.
N itro g e n -c o n ta in in g su b sta n c e s
M aximum 110 ppm o f N .
Carry out the determination o f nitrogen by sulfuric acid
digestion (2.5.9), using 0.200 g and heating for 2 h. Collect
1-716 Dextran 40 2016
the distillate in a m ixture of 0.5 m L o f bromocresol green Oligosaccharide i m,

solution R, 0.5 m L of methyl red solution R and 20 m L o f glucose 180
water R. T itrate with 0.01 M hydrochloric add. N ot m ore than
isomaltose 342
0.15 m L of 0.01 M hydrochloric add is required to change the
colour of the indicator. isomaltotriose 504
S o d iu m c h lo rid e isomaltotetraose 666
M axim um 1.5 per cent. isomaltopentaose 828
Accurately weigh 3-5 g and dissolve in 100 m L of water R.
isomaltohexaose 990
Add 0.3 m L o f potassium chromate solution R and titrate with
0.1 M silver nitrate until the yellowish-white colour changes to isomaltoheptaose 1152
reddish-brown. isomaltooctaose 1314
1 m L o f 0.1 M silver nitrate is equivalent to 5.844 m g o f isomaltononaose 1476
N aCl.
isomaltodecaose 1638
M o le c u la r-m a s s d is trib u tio n
isomaltoundecaose 1800
Size-exclusion chromatography (2.2.30).
isomaltododecaose 1962
Test solution Dissolve 6 .0-6.5 m g of the substance to be
examined in 1.0 m L o f the mobile phase. isomaltotridecaose 2124
Reference solution (a) Dissolve 6.0-6.5 m g of dextran 1 CRS in isomaltotetradecaose 2286
1.0 m L of the mobile phase. 2448
isomaltopentadecaose
Reference solution (b) Dissolve the content o f an ampoule of
isomaltohexadecaose 2610
isomaltooligosaccharide CRS in 1 m L of the mobile phase, and
mix. T his corresponds to approximately 45 jog of isomaltoheptadecaose 2772
isomaltotriose (3 glucose units), approximately 45 |ig o f isomaltooctadecaose 2934
isomaltononaose (9 glucose units), and approximately 60 \i%
isomaltononadecaose 3096
of sodium chloride per 100 ^L.
Column 2 columns coupled in series:
— size: I = 0.30 m, 0 = 10 mm; System suitability T h e values obtained for dextran 1 CRS are
— stationary phase: dextran covalently bound to highly cross- within the values stated on the label.
linked porous agarose beads, allowing resolution of Limits:
oligosaccharides in the molecular mass range o f 180 to — average molecular mass (M^J: 850 to 1150;
3000; — fraction with less than 3 glucose units: less than 15 per cent;
— temperature: 20-25 °C. — fraction with more than 9 glucose units: less than
Mobile phase 2.92 g/L solution of sodium chloride R. 20 per cent.
Flow rate 0.07-0.08 m L/m in maintained constant to H e a v y m e ta ls (2.4.8)
± 1 per cent. M aximum 10 ppm .
Detection Differential refractometer. D ilute 20 m L of solution S to 30 m L with water R. 12 m L of
Injection 100 ^L. solution complies with test A. Prepare the reference solution
Identification of peaks U se the chrom atogram obtained with
reference solution (b) to identify the peaks due to L oss o n d ry in g (2.2.32)
isomaltotriose, isomaltononaose and sodium chloride. Maximum 5.0 per cent, determined on 5.000 g by drying in
D eterm ine the peak areas. Disregard any peak due to sodium an oven at 105 °C for 5 h.
chloride. Calculate the average relative molecular mass M„ B a c te ria l e n d o to x in s (2.6.14)
and the am ount o f the fraction with less than 3 and more Less than 25 IU/g.
than 9 glucose units, o f dextran 1 CRS and o f the substance M ic ro b ia l c o n ta m in a tio n
to be examined, using th e following expression: TA M C : acceptance criterion 102 CFU /g (2.6.12).
___________________________________________________________PhEur
M w = ^ 2 Wi x m i
Mm = average molecular mass o f the dextran;

= molecular mass o f oligosaccharide t; Dextran 40 for Injection *****
Wi = weight proportion o f oligosaccharide i. -*■*
Use the following m,- values for the calculation:
Plasma substitute.
P re p a r a tio n
D extran 40 Infusion
PhEur_________________________________________________________ _
D E F IN IT IO N
M ixture of polysaccharides, principally of the a - l , 6-glucan
type.
Average relative molecular mass A bout 40 000.
2016 Dextran 60 1-717
P R O D U C T IO N Flow rate 25 mL/min.

It is obtained by hydrolysis and fractionation of dextrans Temperature:
produced by fermentation o f sucrose using Leuconostoc — column: 190 °C;
mesenterotdes strain N RR L B-512 = C IP 78.59 or substrains — injection port. 240 °C;
thereof (for example L. mesenterotdes — detector. 210 °C.
B-512F = N C T C 10817). Detection Flame ionisation.
It is prepared in conditions designed to minimise the risk of Injection T h e chosen volume of each solution.
microbial contamination.
Limits:
CHARACTERS — ethanol: n o t more than the area of the corresponding peak
A p p e a ra n c e in the chromatogram obtained with the reference solution
W hite or almost white powder. (0.5 per cent);
S o lu b ility — methanol: not more than the area of the corresponding
Very soluble in water, very slightly soluble in ethanol peak in the chromatogram obtained with the reference
(96 per cent). solution (0.05 per cent);
— sum of solvents other than ethanol, methanol and propanol:
ID E N T IF IC A T IO N not more than the area of the peak due to the internal
A. Specific optical rotation (2.2.7): + 195 to + 201 (dried standard (0.5 per cent, calculated as propanol).
substance).
M o le c u la r-m a ss d is trib u tio n (2.2.39)
Dissolve 1.0 g in water R, heating on a water-bath, and dilute T he average molecular mass (M„) is 35 000 to 45 000.
to 50.0 m L with the same solvent. T he average molecular mass of the 10 per cent high fraction
B. Infrared absorption spectrophotometry (2.2.24). is not greater than 110 000. The average molecular mass of
Comparison dextran CRS. the 10 per cent low fraction is not less than 7000.
C. Molecular-mass distribution (see Tests). H eavy m e ta ls (2.4.8)
M aximum 10 ppm.
TESTS
S o lu tio n S 12 m L of solution S complies with test A. Prepare the
Dissolve 5.0 g in distilled water R, heating on a water-bath, reference solution using lead standard solution (1 ppm Pb) R.
and dilute to 50 m L with the same solvent. Loss o n d ry in g (2.2.32)
A p p e a ra n c e o f solu tio n Maximum 7.0 per cent, determined on 0.200 g by heating in
Solution S is clear (2.2./) and colourless (2.2.2, Method II). an oven at 105 ± 2 °C for 5 h.
A cid ity o r alk alin ity S u lfa te d a sh (2.4.14)

T o 10 m L of solution S add 0.1 m L of phenolphthalein M aximum 0.3 per cent, determined on 0.50 g.
solution R. T he solution remains colourless. Add 0.2 m L of B a c te ria l en d o to x in s (2.6.14)
0.01 M sodium hydroxide. T he solution is red. Add 0.4 m L o f Less than 10 IU/g.
0.01 M hydrochloric acid. T he solution is colourless. M icro b ia l c o n ta m in a tio n
Add 0.1 m L of methyl red solution R. T he solution is red or TAM C: acceptance criterion 102 CFU /g (2.6.12).
orange.
---------------------------------------------------------------------------- PhBjr
N itro g e n -c o n ta in in g su b stan ces
M aximum 110 ppm N.
Carry out the determination o f nitrogen by sulfuric a d d
the distillate in a mixture of 0.5 m L of bromocresol green Dextran 60 for Injection *****
solution R, 0.5 m L of methyl red solution R and 20 m L of +. *
(Ph. Eitr. monograph 1000) *
water R. Titrate with 0.01 M hydrochloric acid N o t more than
0.15 m L of 0.01 M hydrochloric acid is required to change the A ction a n d use
colour of the indicator. Plasma substitute.
R e sid u a l solvents
PhEur_____ _____________________________________________ _
Internal standard propanol R. D E F IN IT IO N
Test solution Dissolve 5 g of the substance to be examined in M ixture o f polysaccharides, prindpally o f the a - l , 6-glucan
100 m L of water R and distil. Collect the first 45 m L of the type.
distillate, add 1 m L of a 25 g/L solution of propanol R and Average relative molecular mass About 60 000.
dilute to 50 m L with water R. P R O D U C T IO N
Reference solution Mix 0.5 m L o f a 25 g/L solution of It is obtained by hydrolysis and fractionation o f dextrans
anhydrous ethanol R, 0.5 m L o f a 25 g/L solution of produced by fermentation of sucrose using Leuconostoc
propanol R and 0.5 m L o f a 2.5 g/L solution o f methanol R mesenterotdes strain N R R L B-512 = C IP 78.59 or substrains
and dilute to 25.0 m L with water R. thereof (for example L. mesenterotdes B-512F = N C T C
Column: 10817).
— material: stainless steel; It is prepared in conditions designed to minimise the risk of
— size. I = 1.8 m, 0 = 2 mm; microbial contamination.
— stationary phase: ethylvinylbenzerie-divinylbeTizene
copolymer R (125-150 pm). C HA RACTERS
A p p e a ra n c e
1-718 Dextran 70 2016
Solubility — methanol: not m ore than the area o f the corresponding

Very soluble in water, very slightly soluble in ethanol peak in the chromatogram obtained w ith the reference
(96 per cent). solution (0.05 per cent);
— sum of solvents other than ethanol, methanol and propanol:
IDENTIFICATION
n o t m ore than the area of the peak due to the internal
A. Specific optical rotation (2.2.7): + 195 to + 201 (dried
standard (0.5 per cent, calculated as propanol).
substance).
M o le c u la r-m a s s d is trib u tio n (2.2.39)
Dissolve 1.0 g in water R, heating on a water-bath, and dilute
T h e average molecular mass (Mw) is 54 000 to 66 000.
to 50.0 m L with the same solvent.
T h e average molecular mass o f the 10 per cent high fraction
B. Infrared absorption spectrophotometry (2.2.24). is n o t greater than 180 000. T h e average molecular mass of
Comparison dextran CRS. . the 10 per cent low fraction is n o t less than 14 000 .
C. M olecular-mass distribution (see Tests). H e a v y m e ta ls (2.48)
TESTS M axim um 10 ppm.
Solution S 12 m L o f solution S complies with test A. Prepare the
Dissolve 5.0 g in distilled water R, heating on a water-bath, reference solution using lead standard solution (1 ppm Pb) R.
and dilute to 50 m L w ith the same solvent. L o ss o n d ry in g (2.2.32)
Appearance o f solution M axim um 7.0 per cent, determined on 0.200 g by heating in
Solution S is clear (2.2.1) and colourless (2.2.2, Method II). an oven at 105 ± 2 °C for 5 h.
Acidity or alkalinity S u lfa te d a s h (2.414)
T o 10 m L o f solution S add 0.1 m L o f phenolphthalein M axim um 0.3 per cent, determ ined on 0.50 g.
solution R. T h e solution remains colourless. Add 0.2 m L o f B a c te ria l e n d o to x in s (2.6.14)
0.01 M sodium hydroxide. T he solution is red. Add 0.4 m L of Less than 16 IU/g.
0.01 M hydrochloric acid. T he solution is colourless.
Add 0.1 m L o f methyl red solution R. T h e solution is red or
T A M C : acceptance criterion 102 CFU /g (2.6.12).
orange.
___________________________________________________________ PhEur
Nitrogen-containing substances
M aximum 110 ppm o f N.
Carry out the determ ination of nitrogen by sulfuric a d d
the distillate in a m ixture of 0.5 m L o f bromocresol green Dextran 70 for Injection *****
solution R, 0.5 m L o f methyl red solution R and 20 m l. of *+ +*
water R. T itrate with 0.01 M hydrochloric add. N o t m ore than
0.15 m L of 0.01 M hydrochloric acid is required to change the A c tio n a n d u se
colour o f the indicator. Plasma substitute.
Residual solvents P r e p a r a tio n
Gas chrom atography (2.2.28). D extran 70 Infusion
Internal standard propanol R.
PhEur___________________________________________________________
Test solution Dissolve 5 g of the substance to be examined in
100 m L o f water R an d distil. Collect the first 45 m L o f the D E F IN IT IO N
distillate, add 1 m L o f a 25 g/L solution o f propanol R and M ixture o f polysaccharides, principally o f the a - l , 6-ghican
dilute to 50 m L with w a te r R. type.
Reference solution M ix 0.5 m L o f a 25 g/L solution of Average relative molecular mass A bout 70 000.
anhydrous ethanol R, 0.5 m L o f a 25 g/L solution of P R O D U C T IO N
propanol R and 0.5 m L of a 2.5 g/L solution of methanol R It is obtained by hydrolysis and fractionation o f dextrans
and dilute to 25.0 m L with water R. produced by fermentation o f sucrose using Leuconostoc
Column: mesenteroides strain N R R L B-512 = C IP 78.59 or substrains
— material: stainless steel; thereof (for example L. mesenteroides B -512F = N C T C
— sizer. I = 1.8 m , 0 = 2 mm; 10817).
— stationary phase: ethylvinylbenzem-divinylbenzene It is prepared in conditions designed to minimise the risk of
copolymer R (125-150 pm). microbial contamination.
CHARACTERS
Flow rate 25 mlVmin.
A p p e a ra n c e
Temperature: W hite o r alm ost white powder.
S o lu b ility
— injection port: 240 °C;
Very soluble in water, very slightly soluble in ethanol
(96 p e r cent).
Injection T h e chosen volume o f each solution.
A. Specific optical rotation (2.2.7): + 195 to + 201 (dried
Limits'.
substance).
— ethanol: n o t m ore th an the area o f the corresponding peak
in the chrom atogram obtained with the reference solution Dissolve 1.0 g in water R, heating on a water-bath, and dilute
(0.5 per cent); to 50.0 m L with the same solvent.
2016 Dextranomer 1-719
Comparison dextran CRS. is not greater than 185 000. The average molecular mass of
C. Molecular-mass distribution (see Tests). the 10 per cent low fraction is not less than 15 000.
TESTS H eav y m e ta ls (2.4.8)

M axim um 10 ppm .
S o lu tio n S
Dissolve 5.0 g in distilled water R, heating on a water-bath, 12 m L o f solution S complies with test A. Prepare the
and dilute to 50 m L with the same solvent. reference solution using lead standard solution (1 ppm Pb) R.
A p p e a ra n c e o f so lu tio n Loss o n d ry in g (2.2.32)
Solution S is clear (2.2. 1) and colourless (2.2.2, Method II). M aximum 7.0 per cent, determined on 0.200 g by heating in
an oven at 105 ± 2 °C for 5 h.
T o 10 m L o f solution S add 0.1 m L o f phenolphthalein S u lfa te d a sh (2.4.14)
solution R. T h e solution remains colourless. A dd 0.2 m L of M aximum 0.3 per cent, determined on 0.50 g.
0.01 M sodium hydroxide. T he solution is red. Add 0.4 m L of B a c te ria l en d o to x in s (2.6.14)
0.01 M hydrochloric acid. The solution is colourless. Less than 16 IU/g.
Add 0.1 m L of methyl red solution R. T he solution is red or
orange.
TA M C: acceptance criterion 102 C FU /g (2.6.12).
N itro g e n -c o n ta in in g su b stan ces
—----- ------------------------------------------------------------------------------- PhEur
Maximum 110 p pm o fN .
Carry out the determ ination of nitrogen by sulfuric acid
the distillate in a mixture of 0.5 m L o f bromocresol green
solution R, 0.5 m L of methyl red solution R and 20 m L of Dextranomer *****
water R. Titrate with 0.01 M hydrochloric acid. N ot m ore than
0.15 m L o f 0.01 M hydrochloric acid is required to change the (Ph. Eur. monograph 2238) *
colour o f the indicator. 56087-11-7
R e sid u a l solvents
A ctio n a n d u se
Fluid absorber; treatm ent of bum s, wounds and skin ulcers;
Internal standard propanol R. preparation for skin grafting.
Test solution Dissolve 5 g o f the substance to be examined in
100 m L o f water R and distil. Collect the first 45 m L o f the PhEur________________________________________
distillate, add 1 m L o f a 25 g/L solution of propanol R and D E F IN IT IO N
dilute to 50 m L with water R. Three-dimensional network made of dextran chains
Reference solution M ix 0.5 m L of a 25 g/L solution of 0 , 0 '-cross-linked with 2-hydroxypropane-l,3-diyl bridges and
anhydrous ethanol R, 0.5 m L of a 25 g/L solution of O-substituted with 2,3-dihydroxypropyl and 2-hydroxy-1-
propanol R and 0.5 m l. of a 2.5 g/L solution o f methanol R (hydroxymethyl)ethyl groups.
and dilute to 25.0 m L with water R.
CHARACTERS
Column'. A p p e a ra n c e
— material: stainless steel; W hite or almost white, spherical beads.
— size: I = 1.8 m, 0 = 2 mm;
— stationary phase: ethylvinylberizem-dwinylbenzene S o lubility
copolymer R (125-150 |im). Practically insoluble in water. It swells in water and in
electrolyte solutions.
Flow rate 25 mL/min. P R O D U C T IO N
Temperature: T he absorption capacity is determined using a 9.0 g/L
solution of sodium chloride R containing 20 (iL/L of
polysorbate 20 R or another suitable solution, with a suitable,
— injection pore 240 °C;
validated method.
T he particle size is controlled to a minimum of 80 per cent
of the num ber of dry beads within 100-300 nm and a
Injection T h e chosen volume of each solution. maximum of 7 per cent of their num ber below 100 nm using
Limits: a suitable, validated method.
— ethanol: not m ore than the area of the corresponding peak
in the chromatogram obtained with the reference solution ID E N T IF IC A T IO N
(0.5 p er cent); A. T he substance to be examined is practically insoluble in
— methanol: not m ore than the area of the corresponding water R. It swells in water R.
peak in the chromatogram obtained with the reference B. Infrared absorption spectrophotometry (2.2.24).
solution (0.05 p er cent); Preparation G rind the substance to be examined in acetone R.
— sum of solvents other than ethanol, methanol and propanol: Evaporate the solvent at room tem perature and use the
not m ore than th e area of the peak due to the internal residue.
standard (0.5 p er cent, calculated as propanol). Comparison dextranomer CRS.
M o le c u la r-m a s s d is trib u tio n (2.2.39)
TESTS
T he average molecular mass (MJ) is 64 000 to 76 000.
p H (2.2.3)
T he average molecular mass of the 10 per cent high fraction
5.3 to 7.5.
1-720 Dextrin 2016
Introduce 0.50 g to 30 m L o f a freshly prepared 74.6 g/L hydroxide solution R and, dropwise with shaking, 0.5 m L o f
solution of potassium chloride R. Allow to stand for 2 min. copper sulfate solution R and boil. A red p redpitate is
Determ ine the p H on the mucilage obtained. produced.
B o ro n c . It is very soluble in boiling water R, forming a
M aximum 30 ppm . mucilaginous solution.
Inductively coupled plasma-atomic emission spectrometry TESTS
(ICP-AES) (2.2.57). p H (2.2.3)
Test solution Introduce 3.0 g into a platinum dish and 2.0 to 8 .0 .
m oisten with 5 m L o f a 32.1 g/L solution of magnesium Disperse 5.0 g in 100 m L o f carbon dioxide-free water R.
nitrate R ina, mixture o f equal volumes o f ethanol
C h lo rid e s
(96 per cent) R and distilled water R. Evaporate to dryness on
a water-bath. Ignite a t 550 °C for 5 h. Take up the residue
with 5 m L of 6 M hydrochloric acid R and transfer to a 50 m L Dissolve 2.5 g in 50 m L o f boiling water R, dilute to 100 m L
volumetric flask. A dd about 20 m L o f distilled water R and with water R and filter. Dilute 1 m L o f the filtrate to 15 mL,
allow to digest for 1 h on a w ater-bath. Allow to cool and add 1 m L o f dilute nitric add R, p our the mixture as a single
dilute to 50.0 m L with distilled water R. addition into 1 m L o f silver nitrate solution R2 and allow to
stand for 5 m in protected from light. W hen viewed
Reference solutions Prepare the reference solutions using a transversely against a black background any opalescence
solution o f boric acid R containing 10 ppm of boron. Proceed
produced is n o t more intense than th at obtained by treating a
as described for the test solution.
mixture o f 10 m L o f chloride standard solution (5 ppm Cl) R
Wavelength 249.773 nm. and 5 m L o f water R, prepared in the same m anner.
H eav y m e ta ls (2.4.8) R e d u c in g su g a rs
M axim um 30 ppm . Maximum 10 per cent, calculated as glucose C ôHÔ tì.
1.0 g complies with test F. Prepare the reference solution T o a quantity of dextrin equivalent to 2.0 g (dried substance)
using 3 m L o f lead standard solution (10 ppm Pb) R. add 100 m L o f water R, shake for 30 min, dilute to
L oss o n d ry in g C2.2.32) 200.0 m L with water R and filter. T o 10.0 m L o f alkaline
M axim um 10.0 per cent, determ ined on 1.000 g by drying in cupri-tartaric solution R add 20.0 m L o f the filtrate, mix, and
an oven at 105 °C for 15 h. heat on a h o t plate adjusted to bring the solution to boil
S u lfa te d a sh (2.4.14) within 3 m in. Boil for 2 min, and cool immediately.
M aximum 0.4 p er cent, determined on 1.0 g. Add 5 m L o f a 300 g/L solution o f potassium iodide R and
10 m L o f 1 M sulfuric add, mix, and titrate immediately with
M ic ro b ia l c o n ta m in a tio n 0.1 M sodium thiosulfate, using starch solution R, added
TA M C : acceptance criterion 10 2 C FU /g (2.6.12), towards the end of the titration, as indicator. R epeat the
determ ined using the pour-plate method. procedure beginning with “T o 10.0 m L o£..”, using, in place
___________________________________________________________ PhEur of the filtrate, 20.0 m L o f a 1 g/L solution of glucose R,
accurately prepared. Perform a blank titration. (Vb — VÙ) is
not greater than (VB — v$)i in which Vjs, Vu and Vs are the
num ber o f millilitres of 0.1 M sodium thiosulfate consum ed in
the titrations o f the blank, the dextrin and the glucose,
Dextrin ***** respectively.
(Ph. Eur. monograph 1507) * H eav y m e ta ls (2.4.8)
M axim um 20 ppm .
A ctio n a n d u se
1.0 g complies with test c. Prepare the reference solution
Excipient.
PhEur___________________________________________________________ L oss o n d ry in g (2.2.32)
M aximum 13.0 per cent, determined on 1.000 g by drying at
D E F IN IT IO N
130-135 ° c for 90 min.
M aize, potato or cassava starch partly hydrolysed and
modified by heating with or w ithout the presence o f adds, S u lfa te d a s h (2.4.14)
alkalis or pH -control agents. M axim um 0.5 per cent, determined on 1.0 g.
CHARACTERS 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
A p p e a ra n c e This section provides information on characteristics that are
W hite or alm ost white, free-flowing powder. recognised as bâng relevant control parameters for one or more
S o lu b ility
5.15). This section ừ a non-mandatory part of the monograph
Very soluble in boiling water forming a mucilaginous
and ừ is not necessary to verify the characteristics to demonstrate
solution, slowly soluble in cold water, practically insoluble in
ID E N T IF IC A T IO N of the manufacturing process and the performance of the medicinal
A. Suspend 1 g in 50 m L o f water R, boil for 1 min and product during use. Where control methods are cited, they are
cool. T o 1 m L o f the solution add 0.05 m L o f iodine recognised as being suitable for the purpose, but other methods can
solution R l. A dark blue or reddish-brown colour is also be used Wherever results for a particular characteristic are
produced, which disappears o n heating. reported, the control method must be indicated
B. Centrifuge 5 m L o f the mucilage obtained in identification The following characteristics may be relevant for dextrm used as
test A. T o the upper layer add 2 m L o f dilute sodium filler and binder, in tablets and capsules.
2016 Dextromethorphan Hydrobromide 1-721
P a rtic le -s iz e d istrib u tio n (2.9.31) Test solution Dissolve 25 mg of the substance to be examined
or 2.9.38). in methanol R and dilute to 10 m L with the same solvent.
P o w d e r flow (2.9.36) Reference solution Dissolve 25 mg o f dextromethorphan
The following characteristic may be relevant for dextrin used as hydrobromide CRS in methanol R and dilute to 10 m L with
viscosity-increasing agent. the same solvent.
A p p a re n t v iscosity (2.2.10) Plate TLC silica gel G plate R.
Typically 100 mPa-s to 350 mPa-s (dried substance), Mobile phase concentrated ammonia R, methylene chloride R,
depending on the grade of dextrin. methanol R, ethyl acetate R, toluene R
In a beaker, prepare a 10-50 per cent slurry so that the (2:10:13:20:55 VIVIV/VIV).
viscosity value ranges from 100 mPa-s to 350 mPa-s. Application 5 pL.
T he total mass of the sample plus water m ust be 600 g. Development Over 2/3 of the plate.
Mix with a plastic rod to obtain a homogeneous slurry. Place
Drying In air.
the beaker in a water-bath at 100 ± 1 °C. Introduce the
paddle of a stirrer into the beaker and close the beaker with a Detection Spray with potassium iodobismuthate solution R2.
lid. Start agitation at 250 r/min as rapidly as possible and Results T he principal spot in the chromatogram obtained with
carry on for exactly 30 min. Transfer the paste immediately the test solution is similar in position and size to the principal
to the beaker to be used for viscosity measurement, placed in spot in the chromatogram obtained with the reference
a water-bath at 40 ± 1 °C. Stir until the tem perature in the solution.
beaker is 40 ± 1 °C then measure the apparent viscosity D . It gives reaction (a) o f bromides (2.3.1).
using spindle no. 2 and a rotation speed of 100 r/min.
TESTS
__________________________________________________________ PhEur S o lu tio n S
Dissolve 1.0 g in ethanol (96per cent) R and dilute to 20 m l.
Dextromethorphan Hydrobromide ***** Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
★ * A cid ity o r alk alin ity
Dissolve 0.4 g in carbon dioxide-free water R with gentle
heating, cool and dilute to 20 m L with the same solvent.
/CH,* Add 0.1 m L of methyl red solution R and 0.2 m L of 0.01 M
sodium hydroxide. T he solution is yellow. N ot more than
0.4 m L of 0.01 M hydrochloric acid is required to change the
colour of the indicator to red.
Dissolve 0.200 g in 0.1 M hydrochloric acid and dilute to
Ci8H 26B rN 0 ^ i20 370.3 6700-34-1 10.0 m L with the same acid.
A ctio n a n d u se Liquid chromatography (2.2.29).
Opioid receptor agonist; cough suppressant.
PhEur__________________________________________________________ examined in the mobile phase and dilute to 10.0 m L with
the mobile phase.
D E F IN IT IO N
erzr-3-Methoxy-17 -methylinorphinan hydrobromide Reference solution (a) Dissolve 2 m g of dextromethorphan
monohydrate.
impurity A CRS in 2 m L of the test solution and dilute to
C o n te n t
CHARACTERS Column:
A p p e a ra n c e — size: I = 0.25 m , 0 = 4.6 mm;
A lmost white, crystalline powder. — stationary phase: octadecylsQyl silica gel for chromatography R
S o lu b ility (5 pm).
Sparingly soluble in water, freely soluble in ethanol Mobile phase Dissolve 3.11 g of docusate sodium R in a
(96 per cent). mixture of 400 m L of water R and 600 m L o f acetonitrile R,
mp add 0.56 g o f ammonium nitrate R and adjust to apparent
A bout 125 °C, with decomposition. p H 2.0 with glacial acetic acid R.
Flow rate 1.0 m lVmin.
First identification A, B, D Detection Spectrophotometer at 280 nm.
Second identification A , C, D Irgection 20 pL.
A. Specific optical rotation (see Tests). Run time Twice the retention time of dextromethorphan.
B. Infrared absorption spectrophotometry (2.2.24). Relative retention W ith reference to dextromethorphan
(retention time = about 22 min): impurity B = about 0.4;
Comparison dextromethorphan hydrobromide CRS.
im purity C = about 0.8; impurity D = about 0.9;
C. Thin-layer chromatography (2.2.27). impurity A •= about 1.1.
1-722 Dextromoramide Tartrate 2016
ch3
System suitability: reference solution (a): o h n
dextrom ethorphan and im purity A.
Limits:
peak area of im purity C by 0.2; h3c o
— impurities A , B, C, D: for each impurity, not more than
C. enr-3-methoxy-17-m ethylm orphinan-10-one,
obtained with reference solution (b) (0.5 per cent), and ch3
n o t more than 1 such peak has an area greater than H N

(0.25 per cent);
— unspecified impurities: for each impurity, no t more than
h3c o
chrom atogram obtained with reference solution (b) D . enr-(14S)-3-methoxy-l 7-methylmorphinan.
(0.10 per cent); PhEur
— total: not m ore than twice the area of the principal peak in
( 1.0 per cent);
— disregard limir. 0.1 times the area of the principal peak in ★ ★
the chrom atogram obtained with reference solution (b) Dextromoramide Tartrate * *
(0.05 per cent). (Ph. Eur. monograph 0021) *****
N, N - D im e th y la n ilin e
M aximum 10 ppm.
Dissolve 0.5 g with heating in 20 m L of water R. Allow to
cool, add 2 m L of dilute acetic acid R and 1 m L of a 10 g/L 0
H I H OH
solution of sodium nitrite R and dilute to 25 mT. with water R.
T h e solution is not m ore intensely coloured than a reference r \ .
solution prepared at the same time and in the same m anner ) — H OH
using 20 m L o f a 0.25 mg/L solution o f N,N-
dimethylamUne R.
W a te r (2.5.12)
4.0 per cent to 5.5 p er cent, determined on 0.200 g. 542.6 2922-44-3
C 29H 38N 208
M axim um 0.1 per cent, determined on 1.0 g. A ctio n a n d u se
A SSA Y Opioid receptor agonist; analgesic.
Dissolve 0.300 g in a mixture of 5.0 m L of 0.01 M P re p a r a tio n
hydrochloric acid and 20 m L o f ethanol (96 per cent) R. T itrate Dextromoramide Tablets
w ith 0.1 M sodium hydroxide, determining the end-point
PhEur_
potentiometrically (2.2.20). Read the volume added between
the 2 points o f inflexion. D E F IN IT IO N
1 m L of 0.1 M sodium hydroxide is equivalent to 35.23 mg Dextrom oram ide tartrate contains not less than 98.0 per cent
o f C 18H 26BrN O . and not more than the equivalent of 101.0 per cent of
l-[(35)-3-m ethyl-4-(m orpholin-4-yl)-2,2-
STORAGE
diphenylbutanoyl]pyrrolidine hydrogen (2R,3R)-2,3-
dihydroxybutanedioate, calculated with reference to the dried
IM P U R IT IE S substance.
Specified impurities A, B, C, D
CHARACTERS
A white or almost white, am orphous or crystalline powder,
soluble in water, sparingly soluble in alcohol.
It melts at about 190 °C, with slight decomposition.
A. Dissolve 75 m g in 1 M hydrochloric acid and dilute to
100.0 m L with the same acid. Examined between 230 nm
A. enr-3-methoxymorphinan, and 350 nm (2.2.25), the solution shows 3 absorption
ch. maxima, at 254 nm , 259 nm and 264 nm . T h e specific
H N absorbances at the maxima are about 6.9, 7.7 and 6.5,
respectively.
B. Dissolve about 50 mg in water R and dilute to 10 m L
with the same solvent. T o 2 m L o f the solution add 3 m L of
ammoniacal silver nitrate solution R and heat on a water-bath.
A grey or black precipitate is formed.
B. ent-17-methylmorphinan-3-ol, C. It gives reaction (b) of tartrates (2.3.1).
2016 Dextropropoxyphene Hydrochloride 1-723
TESTS CHA RACTERS

p H (2.2.3) A p p e a ra n c e
Dissolve 0.2 g in carbon dioxide-free water R and dilute to W hite or almost white, crystalline powder.
20 m L with the same solvent. T he pH o f the solution is S o lu b ility
3.0 to 4.0. Very soluble in water, freely soluble in ethanol (96 per cent),
Specific o p tica l ro ta tio n (2.2.7) mp
Dissolve 0.50 g in O.i M kydrocMoric add and dilute to A bout 165 °C.
10.0 m L with the same acid. The specific optical rotation is
+ 21 to + 23. ID E N T IF IC A T IO N
R e la te d su b sta n ce s
Examine by thin-layer chromatography (2.2.27), using silica B. Infrared absorption spectrophotometry (2.2.24).
gel G R as the coating substance. Comparison dextropropoxyphene hydrochloride CRS.
Test solution Dissolve 0.2 g of the substance to be examined C. Solution S (see Tests) gives reaction (a) of chlorides
in methanol R and dilute to 10 m L with the same solvent. (2.3.1).
Reference solution Dilute 1 m L of the test solution to 100 m L TESTS
with methanol R. S o lu tio n S
Apply separately to the plate 10 îL of each solution. Develop Dissolve 1.5 g in carbon dioxide-free water R and dilute to
over a path of 15 cm using methanol R. Allow the plate to dry 30 m L with the same solvent.
in air and spray with dilute potassium iodobismuthate solution R. A p p e a ra n c e o f so lu tio n
Any spot in the chromatogram obtained with the test Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
solution, apart from the principal spot, is not more intense
A cid ity o r alkalinity
than the spot in the chromatogram obtained with the
Dilute 10 m L of solution S to 25 m L with carbon dioxide-free
reference solution ( 1.0 per cent).
water R. T o 10 m L of this solution add 0.1 m L of methyl red
L oss o n d ry in g (2.2.32) solution R and 0.2 m L of 0.01 M sodium hydroxide.
N ot m ore than 0.5 per cent, determined on 1.00 g by drying T h e solution is yellow. Add 0.4 m L of 0.01 M hydrochloric
in an oven at 105 °C. add. T he solution is red.
S u lfa te d a sh (2.4.14) Specific o p tical ro ta tio n (2.2.7)
N ot m ore than 0.1 per cent, determined on 1.0 g. + 52 to + 57.
A SSA Y Dissolve 0.100 g in water R and dilute to 10.0 m L with the
Dissolve 0.250 g in 30 m L of anhydrous acetic add R. Titrate same solvent.
with 0.05 M perchloric add using 0.15 m L of naphtholbenzdn R e la te d su b stan ces
solution R as indicator. Liquid chromatography (2.2.29).
1 m L o f 0.05 M perchloric add is equivalent to 27.13 mg of Solvent mixture acetonitrUe R, methanol R (50:50 V/V).
C 29H 38N 2O 8. Test solution Dissolve 0.100 g of the substance to be
__________________________________________________________ PhEur examined in the solvent mixture and dilute to 50.0 m L with
Reference solution (a) D ilute 1.0 m L of the test solution to
★* ★ 50.0 m L with the solvent mixture. Dilute 1.0 m L of this
★ ★
Dextropropoxyphene ★ ★ solution to 20.0 m L with the solvent mixture.
Hydrochloride ***** Reference solution (b) Dissolve 2 mg of dextropropoxyphene for
(Ph. Eur. monograph 0713) system suitability CRS (containing impurities A, B, C and D)
in 1.0 m L of the solvent mixture.
h 3c v. ^ c h 3
N Reference solution (c) Dilute 1.0 m L of toluene R to 50.0 m L
with the solvent mixture. Dilute 1.0 m L of this solution to
HCI
Column:
— sizer. I = 0.15 m, 0 = 4.6 m m ;
— stationary phase. octylsUyl silica gel for chromatography R
(5 (im).
Mobile phase:
C22H 30CINO2 1639-60-7
— mobile phase A: dissolve 2.5 g of ammonium phosphate R in
A ctio n a n d u se water R, adjust to p H 5.6 with dilute phosphoric add R and
Opioid receptor agonist; analgesic. dilute to 1000 m L with the same solvent;
— mobile phase B: acetonitrUe R l.
P r e p a r a tio n
Co-proxamol Tablets
PhEur_______________________________ (min) (per cent V/V) (per cent V/V)
0 -2 85 15
D E F IN IT IO N
(1 S,2R) -1 -Benzyl-3-(dimethylamino)-2-methyl-1- 2 -7 85->75 15 ->25
phenylpropyl propanoate hydrochloride. 7 -2 4 75->50 25->50
C o n te n t 24-32 50->40 50 ->60
1-724 Dextropropoxyphene Napsilate 2016

Injection 10 |xL.
with dextropropoxyphene for system suitability CRS and the
identify the peaks d u e to impurities A, B, C and D. Use the
A. R = H: (2<S,3i?)-4-(dimethylamino)-l,2-diphenyl-3-
the peak due to toluene.
methyl-butan- 2-ol (oxyphene),
Relative retention W ith reference to dextropropoxyphene
B. R = C O -C H 3: (IS , 2R )-\-benzyl-3-(dimethylamino)-2-
(retention time = ab o u t 18 min): impurity A = about 0.8;
methyl-l-phenylpropyl acetate (acetoxyphene),
impurity B = about 0.9; impurity D = about 1.1;
impurity C = about 1.2. C. R = C O -C H 2-C H 2-C H 3: (lS,2J?)-l-benzyl-3-
(dimethylamino)- 2-methyl- 1-phenylpropyl butanoate
(butyroxyphene),
— peak-to-valley ratio: minimum 5, where Hp = height above
the baseline of th e peak due to impurity D and
Hv = height above the baseline of the lowest point o f the
dextropropoxyphene.
ch.
Limits:
— impurities A , B: for each impurity, not more than 5 times
— impurities C, D: for each impurity, not more than twice
the area o f the principal peak in the chromatogram D . ( 15 ,2 5 )-l -benzyl-3-(dimethylamino)-2-methyl-l -
obtained with reference solution (a) (0.2 per cent); phenylpropyl propanoate (isopropoxyphene),
— unspecified impurities, for each impurity, not more than the
— total: not more th an 10 times the area of the principal H3C

solution (a) ( 1.0 per cent);
F . (2i?S>3-(dimethylainino)-2-methyi- 1-phenylpropan- 1-one.
the chrom atogram obtained with reference solution (a) ___________________________________________________________PhEur
(0.05 per cent); disregard any peak due to toluene
(relative retention = about 1.24).
M aximum 1.0 per cent, determined on 1.000 g by drying in Dextropropoxyphene Napsilate
S u lfa te d a s h (2.4.14) H Me
M aximum 0.1 per cent, determined on 1.0 g. NMe2 SO3 H
ASSAY Ph* OCOEt

Dissolve 0.270 g in 60 m L o f acetic anhydride R. T itrate with
0.1 M perchloric acid, determining the end-point
C^P^gNO^CioHgOsSjPÔ 565.8 26570-10-5
1 m L o f 0.1 M perchloric acid is equivalent to 37.59 m g of A ctio n a n d u se
C 22H 30CINO 2. Opioid receptor agonist; analgesic.
STO RA G E P re p a r a tio n
Protected from light. Dextropropoxyphene Capsules
IM P U R IT IE S
D E F IN IT IO N
Specified impurities A, B, C , D
Dextropropoxyphene Napsilate is ( 1S,2R ) - 1-benzyl-3-
Other detectable impurities (the following substances would, if dimethylamino- 2-methyl- 1-phenylpropyl propionate
present at a sufficient level, be detected by one or other of naphthalene-2-sulfonate monohydrate. It contains not less
the tests in the monograph. They are limited by the general than 98.0% and not more than 101.0% of
acceptance criterion for other/unspecified impurities and/or C 22H 29N 02 ,C ioH 80 3S, calculated with reference to the
by the general monograph Substances for pharmaceutical use anhydrous substance.
impurities for dem onstration of compliance. See also 5.10. C H A R A C T E R IS T IC S
Control of impurities in substances for pharmaceutical use): F. A white powder. It exhibits polymorphism.
Practically insoluble in water, soluble in ethanol (96%).
with the reference spectrum o f dextropropoxyphene napsilate
2016 Diacerein 1-725
(RS 092). I f the spectra are not concordant, dissolve a corresponding ratios in the chromatogram obtained with
sufficient quantity in the minimum volume of dichloromethane solution (3) (0.67% each).
IR, evaporate to dryness, dry the residue at 105° for 1 hour Sulfated ash
and prepare a new spectrum. N ot more than 0.1%, Appendix IX A.
TESTS Water
Specific optical rotation 3.0 to 5.0% w/w, Appendix IX C . Use 0.5 g.
In a 5% w/v solution in ethanol (96%), +26 to +31,
ASSAY
calculated with reference to the anhydrous substance,
T o 0.75 g add 50 m L of water, swirl to disperse, add 5 mT.
Appendix V F.
o f 5m sodium hydroxide and extract with five 25-m L quantities
Related substances o f dichloromethane, washing each extract with the same
Carry out the m ethod for gas chromatography, 20 m L o f water. Dry the combined extracts with anhydrous
Appendix IE B. Dissolve 10 mg o f triphenylamtne (internal sodium sulfate, evaporate to about 3 m L on a water bath in a
standard) in sufficient dichloromethane to produce 50 m l, current o f air and allow to evaporate to dryness at room
(solution A). tem perature. Carry out Method I for non-aqueous titration on
(1) Dissolve 0.3 g o f the substance being examined in 5 m L the residue, Appendix VUI A, using 1-naphtholbenzein solution
o f dichloromethane, add 10 m L o f water, 2 m L of as indicator. Each m l, o f 0.1m perchloric acid VS is equivalent
1.25m sodium hydroxide and 15 m L o f dichloromethane and to 54.78 mg o f C 22H 29N O 2JC j 0H 8O 3S .
shake. Extract the aqueous layer with two 20-m L quantities
o f dichloromethane. Shake the combined dichloromethane
extracts with 5 g of anhydrous sodium sulfate, filter and
evaporate to dryness at a tem perature not exceeding 40°
using a rotary evaporator. Dissolve the residue in 10 m L of Diacerein *****
★ ★
dichloromethane. * * * * *
(2) Prepare solution (2) in the same m anner as solution ( 1)
b u t add 5 m L o f solution A to the initial solution o f the
c o 2h
(3) A dd 5 m L of solution A, 10 m L o f water, 2 m L of
1.25m sodium hydroxide and 15 m L o f dichloromethane to
5 m L o f a solution in dichloromethane containing 0.022% w/v
o f (lS,2R)-l-benzyl-3-dimethylamino-2-methyl-l-phenylpropyl
acetate BPCRS and 0 .020% w/v of 4îmethylainino-3-metkyl-
l,2-diphenylbutan-2-ol hydrochloride BPCRS and shake. Extract
the aqueous layer with two 20-m L quantities of Ci9H120 8 368.3 13739-02-1
dichloromethane. Shake the combined dichloromethane
extracts with 5 g of anhydrous sodium sulfate, filter and Action and Use
evaporate to dryness at a tem perature not exceeding 40° Anti-inflammatory used in the treatm ent o f arthritis and
using a rotary evaporator. Dissolve the residue in 10 mT. of osteoarthritis.
dichloromethane.
PhEir.
(a) Use a glass column (60 cm x 3 mm) packed with acid- DEFINITION
washed, sUanised diatomaceous support (100 to 120 mesh) 4,5-Diacetoxy-9,10-dioxo-9, 10-dihydroanthracene- 2-
coated with 3% w/w of dimethyl silicone fluid (O V-lO l is carboxylic acid.
suitable). Content
(b) Use helium as the carrier gas at 60 m L per minute. 98.0 per cent to 102.0 per cent (dried substance).
(c) Use isothermal conditions maintained at 160°. CHARACTERS
(d) Use an inlet tem perature of 150°. Appearance
(e) Use a flame ionisation detector. Yellow, crystalline powder.
SYSTEM SUITABILITY Solubility

Practically insoluble in water, soluble in dimethylacetamide,
T he peaks, other than the solvent peak, in the chromatogram
slightly soluble in tetrahydrofuran, practically insoluble in
obtained w ith solution (3) elute in the following o rd e r the
anhydrous ethanol.
internal standard, (15,2i?)-l-benzyl-3-dimethylamino-2-
methyl-1-phenylpropyl acetate and 4-dimethylamino-3- IDENTIFICATION
m ethyl-1,2-diphenyIbutan-2-ol hydrochloride. Infrared absorption spectrophotometry (2.2.24).
LIM ITS Comparison diacerein CRS.
In the chrom atogram obtained with solution (2): TESTS
the ratio o f the area o f any peak corresponding to (l«S,2i?)-l- Impurities B and H
benzyl-3-dimethylamino- 2-m ethyl-l -phenylpropyl acetate to Liquid chromatography (2.2.29).
that o f the peak due to the internal standard and the ratio of Carry out the test protectedfrom light
the area of any peak corresponding to 4-dimethylamino-3- Solution A Dissolve 10 g of sodium hydroxide R in 500 m L of
m ethyl-1,2-diphenyIbutan-2-ol hydrochloride to th at o f the water R.
peak due to the internal standard are not greater than the
Solution B Dissolve 14.7 g of sodium chloride R and 18.8 g of
glycine R in 500 m L of water R.
1-726 Diacerein 2016
Solution C M ix 25.3 volumes o f solution A and 74.6 volumes Reference solution (c) Dissolve the contents of a vial of
of solution B. If necessary, adjust to p H 9.5 using dilute diacerein impurity mixture CRS (impurities C and F) in a
sodium hydroxide solution R o r dilute sulfuric add R. mixture of 0.5 m l. of tetrahydrofuran R and 0.5 m L of the
Solution D D ilute 5 m L of dilute sulfuric add R to 500 m L solvent mixture.
with water R. Column:
Test solution Dissolve 0.100 g o f the substance to be — size: I = 0.10 m, 0 = 4.6 mm;
examined in 30 m L of solution A, mix for 10 min. — stationary phase: end-capped polar-embedded octadecylsifyl
Add 70 m L o f solution B and adjust to p H 9.5 with dilute amorphous organosUica polymer R (5 nm);
sodium hydroxide solution R or dilute sulfuric add R, if — temperature: 30 °C.
necessary. Extract with 3 quantities, each of 25 mL, of Mobile phase:
methylene chloride R. Combine the methylene chloride extracts — mobile phase A: to 353 m L o f water R add 147 m L of
and wash w ith 2 quantities, each of 8 mL, of solution C and phosphoric add R and mix; dilute 2 m L of the solution to
then once with 10 m L of solution D . Evaporate the organic 1000 m L w ith water R;
layer to dryness at 33 °C, completing the drying procedure — mobile phase B: acetonitrUe Rm
,
using compressed air. Dissolve the residue in 2.0 m L o f the
mobile phase. Time Mobile phase A Mobile phase B
Reference solution (a) Dissolve 7.5 m g of diacerein (min) (per cent V/V) (per cent V/V)
impurity B CRS in tetrahydrofuran R and dilute to 25.0 m L 0 -3 80 20
with the same solvent. Sonicate for n o t more than 30 s. 20*40
3 - 13 8 0 * 60
Dilute 1.0 m L o f the solution to 100.0 m L with solution A.
Dilute 5.0 m L of this solution to 50.0 m L with solution A. 1 3-20 60 40
Mix 5.0 m L o f this solution with 25 m L o f solution A for
10 m in. Add 70 m L o f solution B and adjust to p H 9.5 with
dilute sodium hydroxide solution R or dilute sulfuric add R, if
necessary. Perform the extraction as described for the test Detection Spectrophotom eter at 254 nm.
solution. Care should be taken that the time between dissolution Injection 20 jiL.
of diacerdn impurity B in tetrahydrofuran and extraction does not Identification of impurities Use the chromatogram supplied
exceed 30 min. with diacerdn impurity mixture CRS and the chromatogram
Reference solution (b) Dilute 1.0 m L o f reference solution (a) obtained with reference solution (c) to identify the peaks due
to 5.0 m L with the mobile phase. to impurities C and F; use the chromatogram obtained with
Column: reference solution (b) to identify the peaks due to
— sizer. I = 0.125 m, 0 = 4.6 mm; impurities D and E.
— stationary phase: irregular octadecylsifyl silica gel for Relative retention W ith reference to diacerein (retention
chromatography R (5 pm); time = about 13.5 min): impurity D = about 1.1;
— temperature'. 1 6 + 1 °C. impurity E = about 1.15; impurity C = about 1.2;
Mobile phase tetrahydrofuran R, acetonitrUe R, 4 g/L solution of impurity F = about 1.3.
citric add R (8:27.5:64.5 VIV/V). System suitability:
Flow rate 1.0 mlVmin. — resolution: minimum 1.5 between the peaks due to
impurities D and E in the chrom atogram obtained with
reference solution (b);
Injection 100 jiL. — signal-to-noise ratio: minimum 100 for the principal peak
Run time 2.5 times the retention time o f impurity B. in the chromatogram obtained with reference solution (a).
Retention time Im purity B = about 11 min. Limits:
System suitability: reference solution (b): — correction factors: for the calculation of content, multiply
— signal-to-noise ratio: minimum 10 for the principal peak. the peak areas of the following impurities by the
Limit: corresponding correction factor: impurity C = 1.4;
— sum of impurities B and H: n o t more than the area o f the impurity D = 1.3; im purity E = 1.3; impurity F = 9.5;
peak due to impurity B in the chromatogram obtained — impurities D, E: for each impurity, not more than 5 times
w ith reference solution (a) (15 ppm). the area of the principal peak in the chromatogram
Related substances
— impurity C: n o t more than twice the area o f the principal
Liquid chrom atography (2.2.29). Cany out the test protected
from light.
Solvent mixture Mobile phase A, mobile phase B (50:50 V/V). — impurity F: n o t more than 1.5 times the area of the
Test solution Dissolve 0.100 g of the substance to be principal peak in the chrom atogram obtained with
examined in 50 m L of tetrahydrofuran R and dilute to reference solution (a) (0.15 per cent);
100.0 m L with the solvent mixture. — unspecified impurities: for each impurity, not more than the
Reference solution (a) Dilute 1.0 'm l. o f the test solution to area o f the principal peak in the chromatogram obtained
100.0 m L with tetrahydrofuran R. Dilute 1.0 m L of this with reference solution (a) (0.10 per cent);
solution to 10.0 m L w ith the solvent mixture. — total: not more than 20 times the area of the principal
Reference solution (b) In order to prepare impurities D and E peak in the chromatogram obtained with reference
in situ, add 10.0 m L o f 0.01 M sodium hydroxide to 0.100 g of solution (a) (2.0 per cent);
the substance to be examined. Add 40 m L of — disregard limit: 0.5 times the area of the principal peak in
tetrahydrofuran R and dilute to 100.0 m L with the solvent the chromatogram obtained with reference solution (a)
mixture. (0.05 per cent).
2016 Diacerein 1-727
C h ro m iu m
c o 2h
Maximum 10 ppm .
Test solution In a digestion bomb, dissolve 0.25 g o f the
oh o oh
substance to be examined in a mixture o f 2 m L of strong
hydrogen peroxide solution R and 6 mT. of nitric acid R.
C . 4,5-dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-
Mineralise using a microwave oven with a power-
carboxylic acid (rhein),
incrementing system. Transfer quantitatively to a volumetric
flask with wetter R and dilute to 50.0 m L with water R.
Centrifuge. Dilute 5.0 m L of the clear supernatant to
c o 2h
Blank solution Prepare as described for the test solution,
omitting the substance to be examined.
Stock solution Dilute 5.0 m L of chromium standard solution
(100 ppm Cr) R to 50.0 m L with water R. Dilute 5.0 m L of
this solution to 100.0 mT. with water R. Dilute 2.0 mT. o f this
solution to 100.0 m L with a 0.12 per cent VIV solution of D . 5-acetoxy-4-hydroxy-9,10-dioxo-9,10-dihydroanthracene-
dilute nitric acid R. 2-carboxylic acid (monoacetyl rhein isomer A),
Reference solutions Prepare the reference solutions using the
stock solution, diluting with die blank solution.
Source Chromium hollow-cathode lamp using a transmission c o 2h
band preferably o f 0.2 nm.
Atomisation device G raphite furnace.
an oven at 105 °C. E. 4-acetoxy-5-hydroxy-9,10-dioxo-9, 10-dihydroanthracene-
S u lfated a s h (2.4.14) 2-carboxylic a d d (monoacetyl rhein isomer B),
ASSAY
examined in tetrahydrofuran R and dilute to 50.0 m L with die
same solvent. D ilute 2.0 m L of the solution to 25.0 m L with Y
o
Reference solution Dissolve 60.0 mg o f diacerein CRS in
tetrahydrofuran R and dilute to 50.0 m L with the same
solvent. Dilute 2.0 m L of the solution to 25.0 m L with the
solvent mixture.
Calculate the percentage content o f C i 9H 120 8 taking into
account die assigned content of diacerein CRS.
F. (10S)-3-(acetoxymethyl)-10-(2,3,4,6-tetra-0-acetyl-P-r>-
STORA GE glucopyranosyl)-9-oxo-9,10-dihydroanthracene- 1, 8-diyl
In an airtight container, protected from light. diacetate (heptaacetyl aloin, heptaacetyi barbaloin),
IM P U R IT IE S
Specified impurities B, C, D , E, F, H
the tests in the m onograph. They are limited by die general
by the general monograph Substances for pharmaceutical use Y
0
Control of impurities in substances for pharmaceutical use): G.
G . 3-(acetoxymethyl)-10-(2,3,4,6-tetra-0-acetyl-P-i>
OH O OH glucopyranosyl) anthracene-1,8 ,9-triyl triacetate,
B. 1,8-dihydroxy-3-(hydroxymethyl)-anthracene-9, 10-dione
(aloe-emodin),
1-728 Diamorphine Hydrochloride 2016
(a) Use a stainless steel colum n (12.5 cm x 4.6 mm) packed
with base-deactivated octylsUyl silica gel for chromatography,
(5 |im) (Lichrospher RP-select B is suitable).
H3C O 0 o ch3
T0 T0 below.
(c) Use a flow rate o f 1 m L per minute.
H. 3-(acetoxymethyl)-9j 10-dioxo-9, 1O-dihydroanthracene-
I , 8-diyl diacetate (triacetyl aloe-emodin). (d) Use an am bient colum n temperature.
PhEur
(g) Allow die chromatography to proceed for twice the
retention time of the peak due to diamorphine hydrochloride.
Diamorphine Hydrochloride M O B IL E P H A S E
0.11 % w/v o f sodium octanesidfonate in a mixture of

10 volumes o f glacial acetic acid, 10 volumes o f methanol,
115 volumes o f acetomtrUe and 365 volumes o f water.
SYSTEM SUITABILITY
,HCI T he test is n o t valid unless:
the chromatogram obtained with solution (3) exhibits two
secondary peaks with retention times relative to the principal
peak o f about 0.23 (morphine) and 0.43 (6-O-acetyl-
morphine);
C 21H 23N 0 5,HC1,H20 423.9 1502-95-0 in the chromatogram obtained with solution (3), the resolution
factor between the peaks due to morphine and 6-O-acetyl-
A ctio n a n d u se morphine is at least 2 .0 .
LIMITS
In the chromatogram obtained with solution (1):
Bupivacaine and D iam orphine Injection
the area o f any peak corresponding to 6-O-acetylmorphine is
Diamorphine Tablets
not greater than the area o f the principal peak in the
D iamorphine Injection chromatogram obtained with solution (2) (2 %);
D E F IN IT IO N the sum o f the areas o f any other secondary peaks is not
D iamorphine Hydrochloride is 4,5-epoxy-17- greater than 0.25 times the area of the principal peak in the
m ethylm orphinan-3,6-diyl diacetate hydrochloride chromatogram obtained with solution (2) (0.5%).
monohydrate. It contains n o t less than 98.0% and n o t more Disregard any peak with an area less than the area o f the
than 102.0% o f C 2iH 23N 0 5iH Cl, calculated with reference principal peak in the chromatogram obtained with solution
to the dried substance. (4) (0.1%).
C H A R A C T E R IS T IC S L oss o n d ry in g
A white or almost white, crystalline powder. W hen dried to constant weight at 105°, loses 3.0 to 4.5% of
its weight. U se 1 g.
Freely soluble in water, soluble in ethanol (96%); practically
insoluble in ether. S u lfa te d a s h
N o t more than 0.1%, Appendix IX A.
A SSAY
A. Dissolve a sufficient quantity in the minimum volume o f
dichloromethane and evaporate to dryness. The infrared Dissolve 0.40 g in 50 m L o f ethanol (96%) and add 5.0 m L
absorption spectrum o f the residue. Appendix II A, is o f 0.01m hydrochloric acid F5. T itrate with 0.1m sodium
concordant with the reference spectrum of diamorphine hydroxide F 5 , determining the end point potentiometrically.
hydrochloride (RS 093). M easure the volum e of titrant required between the two
points of inflection. Each m L o f 0.1m sodium hydroxide F 5 is
B. Yields reaction A characteristic o f chlorides, Appendix VI.
equivalent to 40.59 mg o f C 2iH 23N 05 ,HCl.
TESTS
STO R A G E
A cid ity
Diamorphine Hydrochloride should be protected from light.
Dissolve 0.2 g in 10 m L of carbon dioxide-free water and
titrate with 0.02m sodium hydroxide VS using methyl red IM P U R IT IE S
solution as indicator. N o t m ore than 0.2 m L o f 0.02m sodium T h e im purity limited by this monograph is:
hydroxide KS is required.
Appendix IH D , using the following solutions.
(1) 0.5% w/v o f the substance being examined in water.
(2) Dilute 1 volume o f solution (1) to 50 volumes with water.
(3) A freshly prepared solution containing 0.1% w/v o f the
substance being examined in 0 .0 1 m sodium hydroxide.
(4) Dilute 1 volume o f solution (2 ) to 20 volumes with water. A. 6-O-acetylmorphine.
2016 Diazepam 1-729
★ ★ Identification of impurities Use the chromatogram supplied

Diazepam ★ ★ with diazepam for system suitability CRS and the
(Ph. Eur. monograph 0022) ***** chrom atogram obtained with reference solution (b) to
identify the peaks due to impurities A, B and E.
Relative retention W ith reference to diazepam (retention
impurity A = about 0.8; impurity B = about 1.3.
impurities E and A and minimum 6.0 between the peaks
due to impurity A and diazepam.
Limits:.
C 16H 13C1N20 284.7 439-14-5
A ctio n a n d u se the peak areas of the following impurities by the
Benzodiazepine. corresponding correction factor: impurity B = 1.3;
impurity E = 1.3;
Diazepam Injection — impurities A , B, E: for each impurity, not m ore than the
Diazepam Oral Solution
Diazepam Rectal Solution — unspecified impurities: for each impurity, not m ore than the
D iazepam Tablets area of the principal peak in the chromatogram obtained
PhEir_____________________________
D E F IN IT IO N the chromatogram obtained with reference solution (a)
7-Chloro - 1-methyl-5-phenyl-133-dihydro-2ii-1,4- (0.2 per cent);
benzodiazepin-2-one. — disregard limit. 0.5 times the area of the principal peak in
C o n te n t the chromatogram obtained with reference solution (a)
99.0 per cent to 101.0 per cent (dried substance). (0.05 per cent).
CHARACTERS H eav y m e ta ls (2.48)

Maximum 20 ppm .
A p p e a ra n c e
W hite or almost white, crystalline powder. 2.0 g complies with test C. Prepare the reference solution
S o lu b ility
Very slightly soluble in water, soluble in ethanol L oss o n d ry in g (2.2.32)
(96 per cent). Maximum 0.5 per cent, determined on 1.000 g by drying
Comparison: diazepam CRS.
ASSAY
TESTS Dissolve 0.200 g in 50 m L of acetic anhydride R. Titrate with
R e la te d su b stan ces 0.1 M perchloric acid, determining the end-point
Liquid chromatography (2.2.29). Prepare the solutions protected potentiometrically (2.2.20).
from bright light
Test solution Dissolve 25.0 mg of the substance to be o f C 16H 13C1N20 .
examined in 0.5 m L of acetomtrUe R and dilute to 50.0 m L
STORAGE
Reference solution (a) Dilute 1.0 mL o f the test solution to
100.0 m L with the mobile phase. D ilute 1.0 m L o f this IM P U R IT IE S
solution to 10.0 m L with the mobile phase. Specified impurities A, B, E
Reference solution (b) Dissolve the contents o f a vial o f Other detectable impurities (the following substances would, if
diazepam for system suitability CRS (containing present at a sufficient level, be detected by one o r other o f the
impurities A, B and E) in 1.0 m L o f the mobile phase. tests in the monograph. They are limited by the general
Column: acceptance criterion for other/unspecified impurities and/or by
— sizer. I = 0.15 m, 0 = 4.6 mm; the general monograph Substances for pharmaceutical m e
— stationary phase:, spherical end-capped octylsUyl silica gel for (2034). It is therefore not necessary to identify these impurities
chromatography R (5 pm); for demonstration o f compliance. See also 5.10. Control of
— temperature: 30 °C. impurities in substancesfor pharmaceutical use): C, D, F.
.0
Mobile phase Mix 22 volumes of acetomtrUe R, 34 volumes of
methanol R and 44 volumes o f a 3.4 g/L solution o f potassium
dilute sodium hydroxide solution R.
Injection 20 pL. A. 7-chloro-5-phenyl-1,3-dihydro-2fi- 1,4-benzodiazepin-2-
Run time About 4 times the retention time of diazepam. one (nordazepam)',
1-730 Diazoxide 2016
CHARACTERS
A white or almost white, fine or crystalline powder,
practically insoluble in water, freely soluble in
dimethylformamide, slightly soluble in alcohol. It is very
soluble in dilute solutions of the alkali hydroxides.
B. R = C O -C H 2-Cl: 2 -chloro-N-(4-chloro-2-benzoylphenyl)- Second identification A, C, D.
Af-methylacetaroide,
A. Dissolve 50.0 mg in 5 m L of 1 M sodium hydroxide and
D. R = H : [5-chloro-2- dilute to 50.0 m L with water R. D ilute 1.0 m L of this
(methylamino)phenyl] phenylmethanone. solution to 100.0 m L with 0 . 1 M sodium hydroxide. Examined
ch3 between 230 nm and 350 nm (2.2.25), the solution shows an
absorption maximum at 280 nm and a shoulder at 304 nm.
T he specific absorbance at the maximum is 570 to 610.
diazoxide CRS. Examine the substances prepared as discs
using potassium bromide R.
C. Examine the chromatograms obtained in the test for
C. 3-amino-6-chJoro - 1-methyl-4-phenylquinolin-2(lii)-one, related substances in ultraviolet light at 254 nm.
çh3 T he principal spot in the chromatogram obtained with test
solution (b) is similar in position and size to the principal
solution (b).
D . Dissolve about 20 mg in a mixture of 5 m L of hydrochloric
add R and 10 m L of water R. Add 0.1 g of zinc powder R.
Boil for 5 min, cool and filter. T o the filtrate add 2 m L of a
1 g/L solution o f sodium nitrite R and mix. Allow to stand for
E. 6-chloro-1-methyl-4-phenylquinazoIin-2( lH)-onc, 1 min and add 1 m L of a 5 g/L solution of
naphthylethylenediamine dihydrochloride R. A red or violet-red
colour develops.
TESTS
Ci A p p e a ra n c e o f so lu tion
Dissolve 0.4 g in 2 m l, of 1 M sodium hydroxide and dilute to
20 m L with water R. The solution is clear (2.2.1) and not
m ore intensely coloured than reference solution Y 7 (2.2.2,
F. 7-chloro-2-m ethoxy-5-phenyl-3/i- 1,4-benzodiazepine. Method II).
A c id ity o r alk alin ity
___ PhEur
T o 0.5 g of the powdered substance to be examined add
30 m L of carbon dioxide-free water R, shake for 2 m in and
filter. T o 10 m L o f the filtrate add 0.2 m L of 0.01 M sodium
★* ★ hydroxide and 0.15 mT. o f methyl red solution R. T he solution
Diazoxide ★ ★ is yellow. N o t more than 0.4 m L of 0.01 M hydrochloric add
★ ★
is required to change the colour o f the indicator to red.
(Ph. Etcr. monograph 0550) *****
gel GF2 5 4 R as the coating substance.
examined in a mixture of 0.5 m L of 1 M sodium hydroxide
and 1 m L o f methanol R and dilute to 5 m L with methanol R.
C8H7a N 20 2S 230.7 364-98-7
A c tio n a n d u se with a mixture of 1 volume of 1 M sodium hydroxide and
Vasodilator; T reatm ent of hypertension. 9 volumes of methanol R.
P re p a r a tio n s Reference solution (a) Dilute 0.5 m L o f test solution (a) to
Diazoxide Injection 100 m L with a mixture of 1 volume of 1 M sodium hydroxide
and 9 volumes of methanol R.
Diazoxide Tablets
Reference solution (b) Dissolve 20 m g of diazoxide CRS in a
PhEur________________________________ mixture o f 0.5 m l, of 1 M sodium hydroxide and 1 m L of
D E F IN IT IO N
methanol R and dilute to 5 m L with methanol R.
Diazoxide contains n o t less than 98.0 per cent and n o t more Apply separately to the plate 5 (jL o f each solution. Develop
than the equivalent o f 101.0 per cent of 7-chloro-3-methyl- over a path o f 15 cm using a mixture o f 7 volumes o f
2ii-l,2,4-benzothiadiazine 1, 1-dioxide, calculated with concentrated ammonia R, 25 volumes of methanol R and
reference to the dried substance.' 68 volumes of chloroform R. Allow the plate to dry in air and
2016 Dibrompropamidine Isetionate 1-731
examine in ultraviolet light at 254 nm. Any spot in the B. Mix 0.1 g w ith 0.5 g o f anhydrous sodium carbonate R,
chrom atogram obtained with test solution (a)3 apart from the ignite and take up the residue with 20 m L of water R. Filter
principal spot3 is not m ore intense than the spot in the and neutralise the filtrate to blue litmus paper R with nitric
chrom atogram obtained with reference solution (a) add R. T he filtrate gives reaction (a) of bromides (2.3.1).
(0.5 per cent).
TESTS
L oss o n d ry in g (2.2.32) p H (2.2.3)
N ot more than 0.5 per cent3 determined on 1.000 g by 5.0 to 6.0.
S u lfa te d a s h (2.4.14) 10 m L with the same solvent.
N ot more than 0.1 per cent3 determined on 1.0 g.
A SSA Y Liquid chromatography (2.2.29).
Dissolve 0.200 g with gentle heating in 50 m L of a mixture Solvent mixture anhydrous formic add R, methanol R, ethyl
of 1 volume o f water R and 2 volumes of acetate R (0.01:8:12 VIVIV).
dimethytformamide R. T itrate with 0.1 M sodium hydroxide, Test solution T o 8 m L o f methanol R add 20.0 m g of the
determining the end-point potentiometrically (2.2.20). Carry substance to be examined and dissolve with the aid of an
out a blank titration. ultrasonic bath. Add 11 m L of ethyl acetate R then 10 (iL of
1 m L of 0.1 M sodium hydroxide is equivalent to 23.07 m g of anhydrous formic acid R and mix. Dilute to 20.0 m L with ethyl
C 8H 7GIN 2O 2S. acetate R.
___________________________________________________________ PhEur Reference solution (a) Dilute 1.0 m L of the test solution to
100.0 m L with the solvent mixture. D ilute 1.0 m L of this
Reference solution (b) Dissolve 10 mg of dibrompropamidine for
system suitability CRS (containing impurities A and B) in
Dibrompropamidine Isetionate ***** 4 m L of methanol R using an ultrasonic bath. Add 5 m L of
*★ *
(Dibrompropamidine Dnsetionate, ** ethyl acetate R then 5 fiL of anhydrous formic acid R and mix.
Ph Eur monograph 2300) Dilute to 10.0 m L with ethyl acetate R.
Column'.
NH NH — sizer. I = 0.25 m, 0 = 4.6 mm 3
— stationary phase: strong cation-exchange silica gel for
Mobile phase Mix 4 volumes of a 25 g/L solution of
Br Br ammonium formate R in methanol R and 6 volumes of ethyl
9 ,/*\. ^ s o 3h acetate R.
’ 1 HO
Flow rate 1 mL/min.
C j i H ^ N ^ oSj 722 614-87-9 Detection Spectrophotometer at 254 nm.
Injection 40 (iL.
A ctio n a n d u se Run time 1.5 times the retention time of dibrompropamidine.
Antiseptic.
Ph Eur_______ ____ _____________________________________________ with dibrompropamidine for system suitability CRS and the
D E F IN IT IO N
identify the peaks due to impurities A and B.
3,3 '-D ibrom o-434 '-(propane-133-diylbisoxy) dibenzimidamide
Relative retention W ith reference to dibrompropamidine
bis( 2-hydroxyethanesulfonate).
C o n te n t impurity B = about 1.1.
P R O D U C T IO N — peak-to-vaUey ratio: minimum 1.5, where Hp = height
T he production m ethod m ust be evaluated to determine the above the baseline o f the peak due to impurity B and
potential for formation o f alkyl 2-hydroxyethanesulfonates, Hv = height above the baseline of the lowest point o f the
which is particularly likely to occur if the reaction medium curve separating this peak from the peak due to
contains lower alcohols. W here necessary, the production dibrompropamidine.
method is validated to demonstrate that alkyl Limits:
2-hydroxyethanesulfonates are not detectable in the final — impurity A: n o t more than 3 times the area o f the
product. principal peak in the chromatogram obtained with
CHA RACTERS reference solution (a) (0.3 per cent);
— impurity B: n o t more than 5 times the area of the
A p p e a ra n c e
S o lubility — unspecified impurities: for each impurity, not more than the
Freely soluble or soluble in water, slightly soluble in ethanol area of the principal peak in the chromatogram obtained
(96 per cent), practically insoluble in methylene chloride. with reference solution (a) (0.1 per cent);
ID E N T IF IC A T IO N — total: not more than 10 times the area of the principal
A. Infrared absorption spectrophotometry (2.2.24). peak in the chromatogram obtained with reference
Comparison dibrompropamidine diisetionate CRS. solution (a) ( 1.0 per cent);
1-732 Dibutyl Phthalate 2016
— disregard, limit. 0.5 times the area of the principal peak in B. Refractive index (2.2.6): 1.490 to 1.495.
the chromatogram obtained with reference solution (a) C. Infrared absorption spectrophotometry (2.2.24).
(0.05 per cent). Comparison dibutyl phthalate CRS.
L o ss on d ry in g ( 2.2.32) D . Thin-layer chromatography (2.2.27).
an oven at 105 °C.
in ether R and dilute to 10 m L with the same solvent.
S u lfa te d a s h (2.4.14) Reference solution Dissolve 50 m g of dibutyl phthalate CRS in
ether R and dilute to 10 m L with the same solvent.
A SSA Y Plate TLC silica gel GF2 5 4 plate R.
Dissolve 0.250 g in 50 m L o f dimethylformamide R. T itrate Mobile phase heptane R, ether R (30:70 V/V).
w ith 0.1 M tetrabutylammonium hydroxide under a current o f
Application 10 pL.
nitrogen R, determining the end-point
potentiometrically (2.2.20). Development Over a path of 15 cm.
1 m L of 0.1 M tetrabutylammonium hydroxide is equivalent to Drying In air.
36.12 mg of C 2iH 3oBr2N 4OioS 2. Detection Examine in ultraviolet light at 254 nm.
IM P U R IT IE S Results T he principal spot in the chromatogram obtained with
Specified impurities A, B. the test solution is similar in position and size to the principal
nh x solution.
E. T o about 0.1 m L add 0.25 m L o f sulfuric add R and
50 m g of resordnol R. H eat in a water-bath for 5 min. Allow
to cool. Add 10 m L of water R and 1 m L o f strong spdium
R Br hydroxide solution R The solution becomes yellow of
brownish-yellow and shows a green fluorescence.
A. R = Br, X = O: 3-brom o-4-[3-(2-bromo-4- TESTS
carbamimidoyiphenoxy)propoxy]benzamide, Appearance
B. R = H, X = N H : 3-bromo-4-[3- T h e substance to be examined is clear (2.2.1) and not more
(4-carbamimidoylphenoxy)propoxy] benzimidamide. intensely coloured than reference solution Y 6 (2.2.2,
PhEur Method II).
Acidity
Dissolve 20.0 g in 50 m l. of ethanol (96per cent) R previously
neutralised to phenolphthaldn solution R l. Add 0.2 m L of
★ ★ phenolphthalein solution R l. N o t more than 0.50 m L of 0.1 M
Dibutyl Phthalate ★ ★ sodium hydroxide is required to change the colour of the
(Ph. Eur. monograph 0762) ***** indicator.
Related substances
Internal standard solution Dissolve 60 mg of bibenzyl R in
O i: methylene chloride R and dilute to 20 m L with the same

solvent.
o Test solution (a) Dissolve 1.0 g of the substance to be
examined in methylene chloride R and dilute to 20.0 m L with
C 16H 22O4 278.3 84-74-2 the same solvent.
Test solution (b) Dissolve 1.0 g of the substance to be
examined in methylene chloride R, add 2.0 m L o f the internal
Insect repellent.
standard solution and dilute to 20.0 m l. with methylene
PhEur__________________________________________________________ chloride R.
Reference solution T o 1.0 m L o f test solution (a) add 10.0 m L
D E F IN IT IO N
o f the internal standard solution and dilute to 100.0 m L with
Dibutyl benzene- 1,2-dicarboxylate.
C o n te n t Column:
99.0 per cent mlm to 101.0 per cent mlm. — material: glass;
CHARACTERS — sizer. I = 1.5 m , 0 = 4 mm;
A p p e a ra n c e — stationary phase: sUanised diatomaceous earth for gas
Clear, oily liquid, colourless or very slightly yellow. chromatography R (150-180 pm) impregnated with
3 per cent mlm of polymethylphenylsiloxane R.
S o lu b ility
Practically insoluble in water, miscible with ethanol Carrier gas nitrogen for chromatography R.
(96 per cent). Flow rate 30 mL/min.
ID E N T IF IC A T IO N Temperature:
First identification B, C. — column: 190 °C;
Second identification A, D, E.
A. Relative density (2.2.5): 1.043 to 1.048.
2016 Dichlorobenzyl 1-733
Irgecdon 1 |iL. mp
Run time 3 times the retention time o f dibutyl phthalate. A bout 59 °C.
Elution order Bibenzylj dibutyl phthalate. ID E N T IF IC A T IO N
Retention time Dibutyl phthalate = about 12 min. Infrared absorption spectrophotometry (2.2.24).
System suitability: Comparison 2,4-dichlorobenzyl alcohol CRS.
— resolution: minimum 12 between the peaks due to bibenzyl TESTS
and dibutyl phthalate in the chromatogram obtained with
Related substances
the reference solution;
— in the chromatogram obtained with test solution (a), there
is no peak with the same retention time as the internal Solvent mixture acetonitrUe R l, water R (50:50 V/V).
standard. Buffer solution Dissolve 0.68 g of potassium dihydrogen
Limit: phosphate R in 900 m L of water R, adjust to pH 3.0 with
— total: calculate the ratio (R) of the area o f the peak due to phosphoric add R and dilute to 1000.0 m L with water R.
dibutyl phthalate to the area of the peak due to the Test solution (a) Dissolve 0.100 g o f the substance to be
internal standard from the chromatogram obtained with examined in 10.0 m L of acetonitrUe R l and dilute to 50.0 m L
the reference solution; from the chromatogram obtained with the solvent mixture.
with test solution (b), calculate the ratio of the sum of the Test solution (b) Dilute 5.0 m L of test solution (a) to
areas of any peaks, apart from the principal peak and the 50.0 m L with the solvent mixture.
peak due to the internal standard, to the area of the peak Reference solution (a) Dilute 1.0 m L of test solution (a) to
due to the internal standard: this ratio is not greater than 100.0 m L with the solvent mixture. Dilute 1.0 m L of this
R (1.0 per cent). solution to 10.0 m L with the solvent mixture.
Water (2.5.12) Reference solution (b) Dissolve 20.0 m g of 2¡4-dichlorobenzyl
Maximum 0.2 per cent, determined on 10.00 g. alcohol impurity A CRS and 20.0 m g of 2, 4-dichlorobenzyl
S u lfa te d a sh (.2.4.14) alcohol impurity C CRS in 100.0 m L of acetomtrüe R l. Dilute
M aximum 0.1 per cent, determined on 1.0 g. 1.0 m L o f the solution to 100.0 m L with the solvent mixture.
ASSAY Reference solution (c) Dissolve 0.100 g of 2,4-dichlorobenzyl
Introduce 0.750 g into a 250 m L borosilicate glass flask. alcohol CRS in 10.0 m L of acetomtrüe R l and dilute to
Add 25.0 m L of 0.5 M alcoholic potassium hydroxide and a few 50.0 m L with the solvent mixture. Dilute 5.0 m L of the
glass beads. H eat in a water-bath under a reflux condenser solution to 50.0 m L with the solvent mixture.
for 1 h. Add 1 m i. o f phenolphthalein solution R1 and titrate Column:
immediately with 0.5 M hydrochloric acid until the colour — size. I — 0.15 m , 0 = 4.6 mm;
changes from red to colourless. Carry out a blank titration. — stationary phase, end-capped octadecylsüyl silica gel for
Calculate the volume o f potassium hydroxide used in the chromatography R (5 |im);
saponification. — temperature. 30 °C.
1 m L o f 0.5 M alcoholic potassium hydroxide is equivalent to Mobile phase.
69.59 m g of CKSH22O 4. — mobile phase A: methanol R2, acetonitrUe R l, buffer solution
(20:30:50 V/V/V);
STORA GE — mobile phase B: acetonitrUe R l ;
__________________________________________________________ PhEur
0-7 100 0
7 - 17 100*20 0 * 80
2,4-Dichlorobenzyl Alcohol ***** 17 -3 0 20 80
Irtjection 10 jiL o f test solution (a) and reference solutions (a)
and (b).
CvHeClaO 177.0 1777-82-8 Relative retention W ith reference to 2,4-dichlorobenzyl alcohol
PhEur__________________________________________________________ (retention time = about 7 min): impurity C = about 0.87;
D E F IN IT IO N
(2,4-EHchlorophenyl)methanol. System suitability, reference solution (b):
— peak-to-vaUey ratio: minimum 4, where Hp = height above
C o n te n t the baseline of the peak due to impurity C and
98.0 p er cent to 102.0 per cent (anhydrous substance). Hv = height above the baseline of the lowest point o f the
CHARACTERS curve separating this peak from the peak due to
A p p e a ra n c e impurity A.
W hite or almost white, crystalline powder. Calculation of percentage contents:
S o lu b ility — for each impurity, use the concentration of
Very slightly soluble in water, very soluble in ethanol 2,4-dichlorobenzyl alcohol in reference solution (a).
(96 per cent).
1-734 Dichloromethane 2016
Limits:
0.10 per cent;
— total: maximum 0 .3 per cent;
— reporting threshold: 0.05 p er cen t F. 2,4-dichlorobenzaldehyde,
W a te r (2.5.32)
M axim um 0.2 per cent, determined on 0.500 g using the
evaporation technique:
— temperature: 120 °C .
S u lfa te d a s h (2.4.14) G. 1,1 /-(oxydimethylene)bis(2,4-dichlorobenzene).
PhEur
A SSAY
Calculate the percentage content of CyHôCÔ taking into
Dich loromethane / *
account the assigned content of 2,4-dichlorobenzyl (Methylene Chloride, Ph. Eur. monograph 0932) *
alcohol CRS.
CH 2C12 84.9 75-09-2
IM P U R IT IE S
Other detectable impurities (the following substances would, if A ctio n a n d u se
present at a sufficient level, be detected by one or other of Excipient.
PhEur__________ :_______________________________________________
acceptance criterion fo r other/unspecified impurities and/or
by the general m onograph Substances for pharmaceutical use D E F IN IT IO N
(2034). It is therefore not necessary to identify these D ichloromethane.
impurities for dem onstration of compliance. See also 5.10. It may contain maximum 2.0 per cent V/V of anhydrous
Control of impurities in substances for pharmaceutical use): A, B, ethanol and/or maximum 0.03 per cent V/V of 2-methylbut-
C, D, E, F, G. 2-ene as stabiliser.
CHARACTERS
A p p e a ra n c e
Clear, colourless, volatile liquid.
S o lu b ility
Sparingly soluble in water, m isdble with ethanol
A. (2,5-dichlorophenyl)methanol,
(96 per cent).
ci ID E N T IF IC A T IO N
First identification B, C.
Second identification A, D, E.
A. Relative density (see Tests).
B. Refractive index (see Tests).
B, (2, 6-dichlorophenyl) methanol,
Preparation Films.
Comparison methylene chloride CRS.
D . H eat 2 m L with 2 g o f potassium hydroxide R and 20 m L
ci of ethanol (96 per cent) R under a reflux condenser for
30 min. Allow to cool. Add 15 m L of dilute sulfuric acid R
C. (3,4-dichlorophenyl)methanol, and filter. T o 1 m L of the filtrate add 1 m L of a 15 g/L
solution o f chromotropic acid, sodium salt R, 2 m L of water R
o and 8 m L o f sulfuric acid R. A violet colour is produced.
E. 2 m L o f the filtrate obtained in identification test D gives
TESTS
A p p e a ra n c e
D. 2,4-dichlorobenzyl acetate, It is clear (2.2.1) and colourless (2.2.2, Method IT).
A cid ity
T o 50 m L o f methanol R previously neutralised to 0.1 m L of
bromothymol blue solution R l, add 50 g of the substance to be
examined. N o t more than 0.15 m L o f 0.1 M sodium hydroxide
is required to change the colour o f the indicator to blue.
E. 2,4-dichlorobenzoic ad d , R elativ e d e n sity (2.2.5)
1.320 to 1.332.
2016 Dichloromethane 1-735
R e fra c tiv e in d ex (2.2.6) — sum of impurities other than ethanol and 2-methyBmt-2-ene:
1.423 to 1.425. maximum 0.1 per cent F/F;
E th a n o l, 2 -m eth y Ib u t-2 -en e a n d v o latile im p u ritie s — reporting threshold: 50 ppm F/F; the reporting threshold
Gas chromatography (2.2.28). does n o t apply to impurity A.
Test solution T he substance to be examined. F re e c h lo rin e

Place 5 m L in a ground-glass-stoppered tube. Add 5 m L of a
Reference solution (a) Dilute 100 |iL o f carbon tetrachloride R
100 g/L solution o f potassium iodide R and 0.2 g of soluble
(impurity A), 500 pL of chloroform R (impurity B), 3.0 m L of
starch R. Shake for 30 s and allow to stand for 5 m in.
2-methylbut-2-ene R and 5.0 m l. o f methanol R (impurity D)
N o blue colour develops.
to 100.0 m L with the test solution.
Reference solution (b) Dilute 2.0 m L o f anhydrous ethanol R H eavy m e ta ls (2.4.8)
and 1.0 m L of reference solution (a) to 100.0 m L with the M aximum 1 ppm .
test solution. T o the residue obtained in the test for residue on evaporation
Column: add 1 m L of hydrochloric acid R and evaporate again. Dissolve
— material’, fused silica; the residue in 2 m L o f acetic acid R and dilute to 50 m L with
— size. I = 30 m, 0 = 0.32 mm; water R. 12 m L o f the solution complies with test A. Prepare
— stationary the reference solution using 10 m L of lead standard solution
phase: poly[(cyanopropyl) (phenyl)][dimethyl]sUoxane R (film (1 ppm Pb) R.
thickness 1.8 pm). R esid u e o n e v a p o ra tio n
Carrier gas nitrogen for chromatography R. Maximum 20 ppm.
Flow rate 1.0 m lVmin, constant flow. Evaporate 50.0 g to dryness on a water-bath and dry at
100-105 °C for 30 min. T he residue weighs a maximum of
Split ratio 1:40.
1 mg.
Temperature:
W a te r (2.5.32)
Temperature
M aximum 0.02 per cent m/m, determined on 10.00 g.
Tune
(min) CC) STORAGE
Column 0 -5 40 In an airtight container, protected from light.
5 -12.5 40-» 55 LA B E LL IN G
125 - 18 55 -» 100 T he label states the name and concentration of any
100
stabilisers.
18-20
260 IM P U R IT IE S
Injection port
Specified impurities A , B
Detector 300
Detection Flame ionisation; make-up gas flow rate: the tests in the monograph. They are limited by the general
25 mT -/min. acceptance criterion for other/unspecified impurities and/or
Injection 2 pL of the test solution and reference solution (b). by the general monograph Substances for pharmaceutical use
Relative retention W ith reference to methylene chloride (2034). It is therefore not necessary to identify these
(retention time = about 7 min): impurity D = about 0.6; impurities for demonstration of compliance. See also 5.10.
ethanol = about 0.8; 2-methyIbut-2-ene = about 0.9; Control of impurities in substances for pharmaceutical use): D.
impurity B = about 1.7; impurity A = about 1.8.
ci ci
— resolution: minimum 3.0 between the peaks due to ethanol
and 2-methylbut- 2-ene;
— signal-to-noise rado: minimum 5 for the peak due to A. carbon tetrachloride,
impurity A.
Calculation ofpercentage contents: H ci
— for ethanol, 2-methylbut-2-ene and impurities A and B,
a ^ a
use the respective concentration of these substances in
reference solution (b); correct the areas o f the peaks in
the chromatogram obtained w ith reference solution (b) by B. trichloromethane (chloroform),
subtracting the area of the corresponding peak in the
chrom atogram obtained with the test solution; h 3c - o h
— for any other impurity, use the concentration of
im purity D in reference solution (b); correct the area of D . methanol.
the peak due to impurity D in the chromatogram
__________________________________________________________ PhEur
obtained with reference solution (b) by subtracting the
area o f the corresponding peak in the chromatogram
obtained with the test solution.
Limits:
— ethanol: maximum 2.0 per cent F/F;
— 2-methylbut-2-ene: maximum 300 ppm F/F;
— impurity A: maximum 10 ppm F/F;
— impurity B: maximum 50 ppm F/F;
1-736 Dichlorophen 2016
o f 1.5 m L per m inute a mixture o f 25 volumes of water and

Dichlorophen 1 volume o f glacial acetic add and sufficient methanol to
produce a chromatogram with solution ( 1) closely resembling
the reference chromatogram supplied with the
im purity standard (75 volumes o f methanol is usually suitable)
and (c) a detection wavelength o f 280 nm . R ecord the
chromatograms until all o f the peaks nam ed on the reference
chrom atogram have em erged
In the chrom atogram obtained with solution (2) the area of
any peak corresponding to 4-chlorophenol is n o t greater than
C 13H 10Cl2O 2 269.1 97-23-4 the area o f the principal peak in the chrom atogram obtained
with solution (3) (0.1%). T h e content o f 4 ,4 /-dichloro-2,2/-
A ctio n a n d u se
(2-hydroxy-4-chloro-m-xylene-a,a/-diyl)diphenol in the
Antihelminthic.
substance being examined does not exceed 8 .0 % w/w and the
P r e p a r a tio n sum o f the contents o f any other impurities, excluding
D ichlorophen Tablets 4-chlorophenol, is not greater than 2.0% w/w calculated
using the declared content of 4,4 '-dichloro-2,2/-(2-hydroxy-
D E F IN IT IO N 4-chloro-m-xylene-a,a/-diyl)diphenol in dichlorophen
D ichlorophen is 4 ,4 /-dichloro-2,2/-methylenediphenol. impurity standard BPCRS.
It contains n o t less than 97.0% and not more than 101.0%
L o ss on d ry in g
of C 13H 10CI2O 23 calculated with reference to the dried
W hen dried to constant weight at 105°, loses n o t more than
substance.
1.0% of its w eight Use 1 g.
C H A R A C T E R IS T IC S S u lfa te d a sh
A white or n o t more than slightly cream powder. N o t more than 0.1%, Appendix IX A.
Practically insoluble in water, very soluble in ether, freely ASSAY
soluble in ethanol (96%). Dissolve 0.5 g in 20 m L o f propan-2-ol and carry out
ID E N T IF IC A T IO N M ethod II for non-aqueous titration, Appendix V m A, using
A. T he light absorption, Appendix II B, in the range 220 to 0.1m tetrabutylammonium hydroxide FS as titrant and
350 nm of a 0.002% w/v solution in 0.1m sodium hydroxide determining the end point potentiometrically. Each m L o f
exhibits two maxima, at 245 nm and 304 nm. The 0.1m tetrabutylammonium hydroxide FS is equivalent to
absorbances at the maxima are about 1.3 and about 0.54, 26.91 mg o f C 13H 10CI2O 2.
respectively.
B. Dissolve 0.2 g in a mixture of 5 m L of water and 5 m L of
5m sodium hydroxide, cool in ice and add a solution prepared
by mixing 1 m L of sodium nitrite solution with a cold solution
Diclofenac Diethylamine
containing 0.15 m L o f aniline in a mixture o f 4 m L o f water
and 1 m L o f hydrochloric acid. A reddish brown precipitate is
produced.
C. Fuse 0.5 g with 2 g of anhydrous sodium carbonate, cool, ^ nh° ^ Ch3
extract the residue with water and filter. T he filtrate yields c i\ A ^ ci
reaction A characteristic of chlorides, Appendix VI.
D. Melting point, about 175°, Appendix V A. u
TESTS C18H22C12N202 369.29 78213-16-8
C h lo rid e
Dissolve 1.0 g in 2 m L o f ethanol (96%), dilute to 100 m L A c tio n a n d u se
with water, allow to stand for 5 minutes and filter through a Cyclo-oxygenase inhibitor; analgesic; anti-inflammatory.
slow filter paper (W hatman No. 42 is suitable). 15 m L o f the P r e p a r a tio n
filtrate complies with the limit test for chlorides, Appendix VII Diclofenac Gel
(350 ppm).
D E F IN IT IO N
Sulfate
Diclofenac Diethylamine is diethylammonium 2-[(2,6-
Shake 0.8 g with 16 m L o f water for 2 minutes, filter and
dichloroanilino)phenyI] acetate. It contains no t less than
dilute 5 m L o f the filtrate to 15 m L with water. T he solution
99.0% and not m ore than 101.0% o f C 18H 22CI2N 2O 2,
complies w ith the limit test for sulfates, Appendix VII
(600 ppm ).
Carry out the m ethod for liquid chromatography, A white to light beige, crystalline powder.
Appendix HI D , using three solutions in the mobile phase Sparingly soluble in water and in acetone', freely soluble in
containing ( 1) 1. 0 % w/v o f dichlorophen ethanol (96%) and in methanol; practically insoluble in
impurity standard BPCRS, (2) 1.0% w/v of the substance 1m sodium hydroxide.
being examined and (3) 0.0010% w/v o f 4-chlorophenol. It melts at about 154°, with decomposition.
T he chrom atographic procedure may be carried out using ID E N T IF IC A T IO N
(a) a stainless steel colum n (20 cm x 5 mm) packed with A. T he infrared absorption spectrum, Appendix II A, is
octadecylsUyl silica gel for chromatography (10 |im) (Spherisorb concordant with the reference spectrum o f diclofenac
ODS 1 is suitable), (b) as the mobile phase with a flow rate diethylamine (RS 371).
2016 Diclofenac Potassium 1-737
B. Carry out the m ethod for thin-layer chromatography, are about 25 minutes for diclofenac and about 12 minutes
Appendix HI A, using the following solutions in methanol for diclofenac impurity A.
(1) 5.0% w/v o f the substance being examined. MOBILE PHASE
(2) 5.0% w/v of diclofenac diethylamine BPCRS. 34 volumes of a mixture o f equal volumes of a 0.1% w/v
CHROMATOGRAPHIC CONDITIONS solution o f orthophosphoric acid and a 0.16% w/v solution of
sodium dihydrogen orthophosphate adjusted to p H 2.5 and
(a) Use a silica gel precoated plate (Macherey Nagel SIL
66 volumes of methanol
G -25 H R or silica gel 6OF254 H P T L C plates are suitable).
(b) Use the mobile phase as described below. SYSTEM SUITABILITY
(c) Apply 2 (jL o f each solution. T he test is not valid unless, in the chromatogram obtained
with solution (3), the resolution factor betw een the peaks
corresponding to diclofenac and diclofenac im purity A is at
(e) After removal of the plate, dry it in a stream of warm air least 6.5.
for 10 minutes. Spray with nmhydrin solution and heat at 110°
LIMITS
for 15 minutes.
In the chromatogram obtained with solution ( 1):
MOBILE PHASE
the area of any secondary peak is n o t greater than the area of
1 volume of hydrochloric acid, 1 volume of water, 6 volumes of
glacial acetic acid and 11 volumes of ethyl acetate.
solution (2 ) (0 .2 %);
SYSTEM SUITABILITY the sum o f the areas of any secondary peaks is not greater than
T h e test is not valid unless the chromatogram obtained with 2.5 times the area of the principal peak in the chromatogram
solution (2) shows two clearly separated spots. obtained with solution (2) (0.5%).
CONFIRMATION Disregard any peak with an area less than 0.25 times the area
T he two principal spots in the chromatogram obtained with of the principal peak in the chromatogram obtained with
solution ( 1) are similar in position, colour and size to the solution (2) (0.05%).
corresponding spots in the chromatogram obtained with Loss o n d ry in g
solution ( 2). W hen dried at a pressure not exceeding 1 kPa for 24 hours,
TESTS loses not more than 0.5% of its weight. Use 1 g.
Acidity or alkalinity S u lfa te d a sh
pH of a 1% w/v solution in ethanol (10%), 6.4 to 8.4, N o t more than 0.1%, Appendix IX A, M ethod IL Use 1 g.
A ppendix V L. A SSAY
Clarity and colour o f solution Dissolve 0.5 g in 30 m L of anhydrous acetic acid and carry
A 5% w/v solution in methanol is dear, Appendix IV A. T h e out M ethod I for rum-aqueous titration, Appendix V IE A,
absorbance o f the solution measured at 440 nm is n o t greater determining the end point potentiometrically. E ach m L o f
than 0.05, Appendix II B. 0.1m perchloric add KS is equivalent to 36.93 mg of
Heavy metals C 1 3 H 2 2 C I2 N 2 O 2 .
2 g complies with limit test C for heavy metals, Appendix VH. STO R A G E
U se 2 m L of lead standard solution (10 ppm Pb) to prepare the Diclofenac Diethylamine should be kept in an airtight
standard (10 ppm). container and protected from light.
Related substances IM P U R IT IE S
T h e impurities limited by the requirem ents of this
Appendix HI D , using the following solutions in the mobile
monograph include those listed under Diclofenac Sodium.
phase.
( 1) 0 . 10% w/v o f the substance being examined.
(2) D ilute 2 volumes of solution (1) to 100 volumes and
dilute 1 volume of this solution to 10 volumes.
(3) Dissolve 1 m g of diclofenac impurity A BPCRS add 1 m L
Diclofenac Potassium *****
*+ +*
o f solution (1) and dilute to 200 mL. (Ph. Eur. monograph 1508) *
(a) Use a stainless steel column (25 cm x 4.6 m m ) packed
with end-capped octylsilyl silica gd for chromatography (5 Jim)
(end-capped Zorbax C 8 is suitable).
(b) U se isocratic elution and the mobile phase described
below.
(c) Use a flow rate of 1 m L per minute.
(d) Use an am bient column temperature.
C 14H 10CI2KNO 2 334.2 15307-81-0
(f) Inject 20 jiL of each solution. A c tio n a n d u se
(g) Allow the chromatography to proceed for 1.5 times the Cyclo-oxygenase inhibitor, analgesic; anti-inflammatory.
retention time o f diclofenac. PhEur__________________________________________________________
Inject 20 (iL o f solution (3). W hen the chromatograms are
D E F IN IT IO N
recorded under the prescribed conditions, the retention times
Potassium [2-[(2,6-dichlorophenyl)amino]phenyl] acetate.
1-738 Diclofenac Potassium 2016
Content Reference solution (a) Dilute 2.0 m L of the test solution to

99.0 per cent to 101.0 per cent (dried substance). 100.0 m L with the mobile phase. Dilute 1.0 m L of this
CHARACTERS
Appearance Reference solution (b) Dissolve the contents o f a vial of
W hite or slightly yellowish, slighdy hygroscopic, crystalline diclofenac for system suitability CRS (containing impurities A
powder. and F) in 1.0 m L of the mobile phase.
Solubility Column:
— size: I = 0.25 m, 0 = 4.6 mm;
in ethanol (96 per cent), slighdy soluble in acetone.
IDENTIFICATION Mobile phase Mix 34 volumes of a solution containing 0.5 g/L
First identification: A, D. of phosphoric acid R and 0.8 g/L of sodium dihydrogen
Second identification B, C, D phosphate R, previously adjusted to p H 2.5 with phosphoric
A. Infrared absorption spectrophotometry (2.2.24). add R, and 66 volumes o f methanol R.
Comparison diclofenac potassium CRS. Flow rate 1.0 mL/min.
B. Thin-layer chromatography (2.2.27). Detection Spectrophotom eter at 254 nm.
Test solution Dissolve 25 mg o f the substance to be examined Injection 20 pL.
in methanol R and dilute to 5 m L with the same solvent. Run time 1.6 times the retention time of diclofenac.
Reference solution (a) Dissolve 25 mg of diclofenac Identification of impurities Use the chromatogram supplied
potassium CRS in methanol R and dilute to 5 m L with the with diclofenac for system suitability CRS and the
same solvent. chromatogram obtained with reference solution (b) to
Reference solution (b) Dissolve 10 mg o f indometacin R in identify the peaks due to impurities A and F.
reference solution (a) and dilute to 2 m L with the same Relative retendon W ith reference to diclofenac
solution. (retention time = about 25 min): impurity A = about 0.4;
Plate TLC silica gel GF2 5 4 plate R. impurity F = about 0.8.
Mobile phase concentrated ammonia R, methanol R, ethyl System suitability, reference solution (b):
acetate R (10:10:80 VIVIV). — resolution: minimum 4.0 between the peaks due to
Application 5 pL. im purity F and diclofenac.
Development Over 1/2 o f the plate. Calculation of percentage contents:

— correction factors: multiply the peak areas o f the following
Drying In air. impurities by the corresponding correction factor,
Detection Examine in ultraviolet light at 254 nm. im purity A = 0.7; impurity F = 0.3;
System suitability, reference solution (b): — for each impurity, use the concentration o f diclofenac in
— the chrom atogram shows 2 clearly separated spots. reference solution (a).
Results T he principal spot in the chromatogram obtained with Limits:
the test solution is similar in position and size to the principal — impurities A , F: for each impurity, maximum
spot in the chrom atogram obtained with reference 0.15 p er cent;
solution (a). — unspecified impurities: for each impurity, maximum
C. Dissolve about 10 m g in 10 m L o f ethanol (96 per cent) R. 0.10 per cent;
T o 1 m L of this solution add 0.2 m L of a mixture, prepared — total: maximum 0.4 per cent;
immediately before use, of equal volumes of a 6 g/L solution — reporting threshold: 0.05 per cent.
of potassium ferricyanide R and a 9 g/L solution o f ferric H eav y m e ta ls (2.48)
chloride R. Allow to stand protected from light for 5 min. Maximum 10 ppm.
Add 3 m L o f a 10 g/L solution of hydrochloric add R. Allow Dissolve 2.0 g in 20 m L of methanol R. T h e solution
to stand protected from light for 15 min. A blue colour complies with test H . Prepare the reference solution using
develops and a precipitate is formed. lead standard solution (1 ppm Pb) obtained by diluting lead
D . Suspend 0.5 g in 10 m L o f water R. Stir and add water R standard solution (100 ppm Pb) R with methanol R.
until the substance is dissolved. Add 2 m L of hydrochloric L oss o n <hying (2.2.32)
add R l, stir for 1 h and filter with the aid of vacuum. M axim um 0.5 per cent, determined on 1.000 g by drying in
Neutralise with sodium hydroxide solution R. T h e solution an oven a t 105 °C for 3 h.
gives reaction (b) o f potassium (2.3.1).
A SSAY
TESTS
Dissolve 0.250 g in 60 m L of anhydrous acetic add R. Titrate
Appearance o f solution with 0.1 M perchloric add, determining the end-point
T he solution is clear (2.2.1) and its absorbance (2.2.25) at potentiometrically (2.2.2(f).
1 m L o f 0.1 M perchloric add is equivalent to 33.42 mg
Dissolve 1.25 g in methanol R and dilute to 25.0 m L with the
o f C 14H 10Cl2K N O 2.
same solvent.
STORA GE
Related substances
Test solution Dissolve 50.0 m g o f the substance to be IM P U R IT IE S
examined in the mobile phase and dilute to 50.0 m L with Specified impurities A, F
the mobile phase. Other detectable impurities (the following substances would, if
2016 Diclofenac Sodium 1-739

Diclofenac Sodium *****
by the general monograph Substances for pharmaceutical use ** +*
D ,E .
C 14H 10Cl2NNaO 2 318.1 15307-79-6
A ctio n a n d u se
Cyclo-oxygenase inhibitor; analgesiq anti-inflammatory.
A. 1-(2 j 6-dichlorophenyl)-1,3-dihydro-2/i-indol-2-onej P re p a ra tio n s
Prolonged-release Diclofenac Capsules
Gastro-resistant Diclofenac Tablets
Prolonged-release Diclofenac Tablets
PhEur__________________________________________________________
D E F IN IT IO N
Sodium [2- [(2,6-dichlorophenyl) amino] phenyl] acetate.
B. 2- [(2 j 6-dichlorophenyl) amino] benzaldehyde, C o n te n t

CHA RACTERS
A p p e a ra n c e
W hite or slightly yellowish, slightly hygroscopic, crystalline
powder.
S olu b ility
in ethanol (96 per cent), slightly soluble in acetone.
C . [2- [(2, 6-dichlorophenyl)amino] phenyl] methanol.
mp
First identification: A, D.
Comparison diclofenac sodium CRS.
D . [2- [(2-brom o- 6-chlorophenyl) amino]phenyl] acetic acid,
Reference solution (a) Dissolve 25 mg of diclofenac sodium CRS
Reference solution (b) Dissolve 10 mg of indometadn R in
reference solution (a) and dilute to 2 m L with reference
E. l,3-dihydro-2ii-indol-2-one,
solution (a).
Plate TLC silica gel GF 254 plate R.
acetate R (10:10:80 VIVIV).
Application 5 (iL.
Drying In air.
F. N -(4-chlorophenyl)- 2-( 2, 6-dichlorophenyl)acetamide.
PhEur
solution (a).
1-740 Diclofenac Sodium 2016
C. Dissolve about 10 mg in 10 m L o f ethanol (96 per cent) R. H eav y m e ta ls (2.4.8)

T o 1 m L of this solution add 0.2 m L of a mixture, prepared M axim um 10 ppm.
immediately before use, of equal volumes of a 6 g/L solution Dissolve 2.0 g in 20 mT. o f methanol R. T h e solution
of potassium ferricyanide R and a 9 g/L solution of ferric complies with test H . Prepare the reference solution using
chloride R. Allow to stand protected from light for 5 min. lead standard solution (1 ppm Pb) obtained by diluting lead
A dd 3 m L of a 10 g/L solution o f hydrochloric acid R. Allow standard solution (100 ppm Pb) R with methanol R.
to stand, protected from light, for 15 min. A blue colour
develops and a precipitate is form ed
D . Dissolve 60 mg in 0.5 m l. of methanol R and add 0.5 m L an oven at 105 °C for 3 h.
of water R. T he solution gives reaction (b) of sodium (2.3.1).
A SSAY
TESTS Dissolve 0.250 g in 60 m L of anhydrous acetic acid R. Titrate
Appearance of solution with 0.1 M perchloric acid, determining the end-point
T he solution is clear (2.2.1) and its absorbance (2.2.25) at potentiometrically (2.2.20).
Dissolve 1.25 g in methanol R and dilute to 25.0 m L with the of C i 4H 10Cl2N N aO 2.
same solvent.
STO RA G E
Related substances
Test solution Dissolve 50.0 m g of the substance to be IM P U R IT IE S
examined in the mobile phase and dilute to 50.0 m L with Specified impurities A, F
the mobile phase. Other detectable impurities (the following substances would, if
Reference solution (a) Dilute 2.0 mT, o f the test solution to present at a sufficient level, be detected by one or other o f
100.0 m L with the mobile phase. Dilute 1.0 m L o f this the tests in the monograph. They are limited by the general
solution to 10.0 m L with the mobile phase. acceptance criterion for other/unspecified impurities and/or
diclofenac for system suitability CRS (containing impurities A
and F) in 1.0 m L of the mobile phase.
Column:
D, E.
— size. I = 0.25 m, 0 = 4.6 mm;
Mobile phase M ix 34 volumes of a solution containing 0.5 g/L
of phosphoric acid R and 0.8 g/L of sodium dihydrogen
phosphate R, previously adjusted to p H 2.5 with phosphoric
acid R, and 66 volumes of methanol R.
Detection Spectrophotom eter at 254 nm. A. 1-(2, 6-dichlorophenyl)-1,3-dihydro-2//-indol-2-one,
Injection 20 jjL.
Run time 1.6 times the retention time of diclofenac.
with diclofenac for system suitability CRS and the
identify the peaks due to impurities A and F.
Relative retention W ith reference to diclofenac (retention
B . 2- [(2 , 6-dichlorophenyl) amino] benzaldehyde,
im purity F and diclofenac.
— correction factors: multiply the peak areas of the following
impurities by the corresponding correction factor
impurity A = 0.7; impurity F = 0.3;
— for each impurity, use the concentration of diclofenac in C. [2-[( 2, 6-dichlorophenyl)amino]phenyl]methanol,
Limits:
— impurity F: maximum 0.15 per cent;
CC~
0.10 per cent;
— reporting threshold: 0.05 per c en t
D . [2-[( 2-brom o- 6-chlorophenyl)amino]phenyl]acetic acid.
2016 Dicloxacillin Sodium 1-741
Reference solution (b) Dissolve 25 mg of cloxacillin

sodium CRS, 25 m g of dicloxacillin sodium CRS and 25 mg of
flucloxacMin sodium CRS in 5 m L of water R.
Plate TLC sUanised silica gel plate R.
Mobile phase Mix 30 volumes of acetone R and 70 volumes of
a 154 g/L solution of ammonium acetate R adjusted to pH 5.0
with glacial acetic acid R.
Application 1 |iL.
Drying In air.
F. N-(4-chlorophenyl)-2-(236-dichlorophenyl)acetamide. Results T h e principal spot in the chromatogram obtained with
_____________________________________________ PhEur the test solution is similar in position, colour and size to the
solution (a).
C. Place about 2 mg in a test-tube about 150 mm long and
about 15 mm in diameter. Moisten with 0.05 m L of water R
Dicloxacillin Sodium ****** and add 2 mT. o f sulfuric acid-formaldehyde reagent R Mix the
** +* contents o f the tube by swirling; the solution is slightly
greenish-yellow. Place the test-tube in a water-bath for
1 min; a yellow colour develops.
D . It gives reaction (a) of sodium (2.3.1).
TESTS
S o lu tio n S
CH3 o 25.0 m L with the same solvent.
C 19H 16Cl2N3Na0 5S,H20 510.3 13412-64-1 Solution S is clear (2.2.1) and its absorbance (2.2.25) at
A ctio n a n d use p H (2.2.3)
Penicillin antibacterial. 5.0 to 7.0 for solution S.
PhEur__________________________________________________________ Specific o p tic a l ro ta tio n (2.2.7)
D E F IN IT IO N
Sodium (2S,5i?,6i?)-6-[[[3-(2,6-dichlorophenyl)-5-
same solvent.
methylisoxazol-4-yl] carbonyl] amino] -3 j3-dimethyl-7 -oxo-4-
thia-l-azabicyclo[3.2.0]heptane-2-carboxylate monohydrate. R e la te d su b stan ces
Semi-synthetic product derived from a fermentation p ro d u ct Liquid chromatography (2.2.29).
C o n te n t
the mobile phase.
CHARACTERS Test solution (b) Dilute 5.0 mL of test solution (a) to
A p p e a ra n c e 50.0 m L with the mobile phase.
W hite or almost white, hygroscopic, crystalline powder.
Reference solution (a) Dissolve 50.0 mg of dicloxacillin.
S o lu b ility sodium CRS in the mobile phase and dilute to 50.0 mL with
Freely soluble in water, soluble in ethanol (96 per cent) and the mobile phase. Dilute 5.0 m L o f this solution to 50.0 m L
in methanol. with the mobile phase.
ID E N T IF IC A T IO N Reference solution (b) Dilute 5.0 m L of test solution (b) to
First identification A, D 50.0 m L with the mobile phase.
Second identification B, C, D Reference solution (c) Dissolve 5 m g of flucloxadllin
A. Infrared absorption spectrophotometry (2.2.24). sodium CRS and 5 mg of dicloxacillin sodium CRS in the
mobile phase, then dilute to 50.0 m L with the mobile phase.
Preparation Discs.
Column:
Comparison dicloxacillin sodium CRS. — sizer. I = 0.25 m , 0 = 4 mm;
B. Thin-layer chromatography (2.2.27). — stationary phase: octadecylsüyl silica gel for chromatography R
Test solution Dissolve 25 mg o f the substance to be examined (5 nm).
in 5 m L o f water R. Mobile phase M ix 25 volumes of acetonitrüe R and 75 volumes
Reference solution (a) Dissolve 25 m g of dicloxacillin of a 2.7 g/L solution of potassium dihydrogen phosphate R
sodium CRS in 5 m L of water R. adjusted to pH 5.0 with dilute sodium hydroxide solution R.
1-742 Dicycloverine Hydrochloride 2016

Detection Spectrophotom eter at 225 run.
Injection 20 |iL of test solution (a) and reference solutions (b)
H H
and (c).
Run time 5 times the retention time o f dicloxadllin. C . (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia-1 -
Retention time Dicloxadllin = about 10 min. azabicydo[3.2.0]heptane-2-carboxylic a d d
System suitability: reference solution (c): (6-aminopenicillanic ad d ),
flucloxacillin ( 1st peak) and dicloxadllin (2nd peak).
Limits:
— any impurity: for each impurity, not more than the area of
the prindpal peak in the chromatogram obtained with
reference solution (b) (1 per cent);
— total: not more than 5 times the area of the prindpal peak
(5 per cent); D . 3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carboxyiic
— disregard limir. 0.05 times the area of the principal peak in ad d .
the chrom atogram obtained with reference solution (b) PhEw
(0.05 per cent).
N, N -D im e th y la n ilin e (2.4.26, Method B)
M axim um 20 ppm.
★ **
★ ★
2 -E th y lliex an o ic a c id (2.4.28) Dicycloverine Hydrochloride ★ ★
M axim um 0.8 per cent mlm.
W a te r (2.5.12)
3.0 per cent to 4.5 p er cent, determined on 0.300 g. ch3
P y ro g e n s (2.6.8) r -CH,
If intended for use in the manufacture o f parenteral , HCI
removal of pyrogens, it complies with the test for pyrogens.
Inject per kilogram o f the rabbit's mass 1 m L o f a solution in
water for injections R containing 20 mg of the substance to be
examined p er millilitre.
A SSA Y A ctio n a n d u se
Liquid chrom atography (2.2.29) as described in the test for Anticholinergic.
related substances w ith the following modifications. P re p a ra tio n s
Injection T est solution (b) and reference solution (a). Dicycloverine Oral Solution
System suitability: reference solution (a): Dicycloverine Tablets
— repeatability, m aximum relative standard deviation of
PhEur.
STORAGE D E F IN IT IO N
In an airtight container, at a tem perature not exceeding Dicycloverine hydrochloride contains not less than
25 °C. If the substance is sterile, store in a sterile, airtight, 99.0 per cent and not more than the equivalent of
tam per-proof container. 101.0 per cent o f 2-(diethylamino)ethyl bicyclohexyl-1-
carboxylate hydrochloride, calculated with reference to the
IM P U R IT IE S dried substance.
CHARACTERS
A white or almost white, crystalline powder, soluble in water,
fredy soluble in alcohol and in methylene chloride.
First identification A , D
A. R = C 0 2H : (45)-2-[carboxy[[[3-(2,6-dichlorophenyl)-5- A. Examine by infrared absorption spectrophotometry
methylisoxazol-4-yl] carbonyl] amino]methyl]-5,5- (2.2.24), comparing with the spectrum obtained with
dimeth.ylthiazolidine-4-carboxylic a d d (penicilloic ad d s of dicycloverine hydrochloride CRS. Examine the substances
dicloxadllin), prepared as discs using potassium chloride R. If the spectra
B. R = H: (2J?S,4S)-2-[[[[3-(2,6-dichlorophenyl)-5- obtained show differences, dissolve the substance to be
methylisoxazol-4-yl] carbonyl] amino]methyl]-5,5- examined and the reference substance separately in acetone R,
dimethylthiazolidine-4-caiboxylic a d d (penilloic ad d s of evaporate to dryness and record new spectra using the
dicloxadllin), residues.
related substances. T he prin d p al spot in the chromatogram
2016 Didanosine 1-743

Didanosine *****
with reference solution (b). ** +*
C. T o 3 m L of a 1.0 g/L solution of sodium laurUsulfate R add
5 m L of methylene chloride R and 0.05 m L of a 2.5 g/L O
solution of methylene blue R, mix gently and allow to stand;
the lower layer is blue. Add 2 mL of a 20 g/L solution of the
substance to be examined, mix gently and allow to stand;
the upper layer is blue and the lower layer is colourless.
TESTS
p H (2.2.3) C ioH 12N40 3 236.2 69655-05-6
solvent. T h e pH of the solution is 5.0 to 5.5. A ctio n a n d use
R elated su b sta n c e s Nucleoside reverse transcriptase inhibitor; antiviral (HTV).
Examine by thin-layer chromatography (2.2.27), using a PhEur__________________________________________________________
suitable silica gel as the coating substance.
D E F IN IT IO N
Test solution (a) Dissolve 0.25 g to the substance to be
9-(2,3-Dideoxy-(^D-^yc£ro-pentofuranosyl)-l,9-dihydro-6H-
examined in methanol R and dilute to 5 m L with the same
solvent.
purin-6-one (2/,3/-dideoxyinosine).
Test solution (b) D ilute 1 m L of test solution (a) to 50 m L C o n te n t
with methanol R. 98.5 per cent to 101.0 per cent (anhydrous substance).
Reference solution (a) Dilute 1 mL of test solution (b) to CHARACTERS
10 m L with methanol R. A p p e a ra n c e
Reference solution (b) Dissolve 10 mg of dicycloverine White or almost white, crystalline powder.
hydrochloride CRS in methanol R and dilute to 10 m L with the S olubility
same solvent Sparingly soluble in water, freely soluble in dimethyl
Reference solution (c) Dissolve 5 mg o f tropicamide CRS in sulfoxide, slightly soluble in methanol and in ethanol
reference solution (b) and dilute to 5 m L with the same (96 per cent).
solution. ID E N T IF IC A T IO N
Apply separately to the plate 10 pL o f each solution. Develop A. Infrared absorption spectrophotometry (2.2.24).
over a path of 15 cm using a mixture of 5 volumes of Comparison didanosine CRS.
concentrated ammonia R, 10 volumes of ethyl acetate R,
B. Specific optical rotation (2.2.7): -2 8 .2 to -2 4 .2
10 volumes of water R and 75 volumes of propanol R. Dry the
plate in a current o f warm air. Spray with dilute potassium
iodobismuthate solution R. Any spot in the chromatogram Dissolve 0.100 g in water R and dilute to 10.0 m L with the
obtained with test solution (a), apart from the principal spot, same solvent.
is not m ore intense than the spot in the chromatogram TESTS
obtained with reference solution (a) (0.2 per cent). T h e test R ela te d su b stan ces
is not valid unless the chromatogram obtained with reference Liquid chromatography (2.2.29). Prepare the solutions
solution (c) shows two clearly separated spots. immediately before use.
Loss o n d ry in g (.2.2.32) Solvent mixture Mix 8 volumes of mobile phase B and
N ot more than 1.0 per cent, determined on 1.000 g by 92 volumes o f mobile phase A.
drying in an oven at 105 °C. Test solution Dissolve 25.0 mg of th e substance to be
Sulfa ted ash (2.4.14) examined in 50.0 m L o f the solvent mixture.
N ot more than 0.1 per cent, determined on 1.0 g. Reference solution (a) Dilute 1.0 m L of the test solution to
A SSAY 100.0 m L with the solvent mixture. Dilute 1.0 m L of this
Dissolve 0.300 g in a mixture of 5.0 m L o f 0.01 M solution to 10.0 m L with the solvent mixture.
hydrochloric acid and 50 m l. of alcohol R. Carry out a Reference solution (b) Dissolve 5.0 m g of didanosine
potentiometric titration (2.2.20), using 0.1 M sodium impurity A CRS in the solvent mixture and dilute to
hydroxide. Read the volume added between the two points of 100.0 m L with the solvent mixture. Dilute 1.0 m L to
inflexion. 20.0 m L with the solvent mixture.
1 m L of 0.1 M sodium hydroxide is equivalent to 34.60 mg of Reference solution (c) Dissolve 5 m g o f didanosine for system
C 19H 36C 1N 02. suitability CRS (containing impurities A to F) in the solvent
mixture and dilute to 10 mL with the solvent mixture.
IM P U R IT IE S
Reference solution (d) Dissolve 5 m g o f didanosine
/ V co2h impurity G CRS in the solvent mixture and dilute to 100 m L
with the solvent mixture. Dilute 1 m L to 20 m L with the
solvent mixture.
Column:
— size. I = 0.25 m, 0 = 4.6 mm ;
A. bicyclohexyl-l-carboxylic add. — stationary phase: base-deactivated octadecylsifyl silica gelfor
PhEur chromatography R (5 pm).
1-744 Didanosine 2016
Mobile phase: 1 m L o f 0.1 M perchloric acid is equivalent to 23.62 mg

— mobile phase A: mix 8 volumes o f methanol R and Of C 10H 12N 4O 3.
92 volumes o f a 3.86 g/L solution o f ammonium acetate R
IM P U R IT IE S
adjusted to p H 8.0 with concentrated ammonia R;
Specified impurities A, B, C, D , E, F , G
— mobile phase B: mix 30 volumes o f methanol R and
70 volumes o f a 3.86 g/L solution of ammonium acetate R Other detectable impurities (the following substances would, if
adjusted to p H 8.0 with concentrated ammonia R; present at a sufficient level, be detected by one or other of
0 - 18 100 0
1 8-25 100*0 0 -»■ 100 Control of impurities in substances for pharmaceutical use): H, I.
2 5 -4 5 0 100
O
45 - 50 0 -» 100 100 -»0
5 0 -6 0 100 0

A. l,7-dihydro-6H-purin-6-one (hypoxanthine),
Injection 20 |iL.
o
with didanosine for system suitability CRS and the
the peaks due to impurities A to F and use the
chrom atogram obtained with reference solution (d) to
identify the peak due to impurity G.
Relative retention W ith reference to didanosine (retention
time = about 13-15 m in): impurity A = about 0.3;
impurity B = about 0.4; im purity C = about 0.44; B. R = R' = O H : 9-P-o-ribofuranosyl-1,9-dihydro-6//-purin-
im purity D = about 0.48; im purity E = about 0.5; 6-one (inosine),
im purity F = about 0.8; impurity G = about 1. 6 . C. R = H , R' = OH: 9-(2-d.to^-f>-i>-erytkro-
System suitability: reference solution (c): pentofuranosyl)-l ,9-dihydro-6/i-purin-6-one
— resolution: minimum 2.5 between the peaks due to (2 '-deoxyinosine),
impurity C and impurity D. D . R = O H , R' = H: 9-(3-deoxy~P-D-erythro-
Limits: pentofuranosyl)-l ,9-dihydro-6fi-purin-6-one
— impurity A: not m ore than the area o f the principal peak (3 '-deoxyinosine),
in the chrom atogram obtained with reference solution (b) E. R + R' = O: 9-(2,3-anhydro-P-D-ribofuranosyl)-l39-
(0.5 per cent); dihydro-6H-purin-6-one (2 ',3 '-anhydroinosine).
— impurities B, C, D, E, F, G: for each impurity, not more
than twice the area o f the principal peak in the o
chrom atogram obtained w ith reference solution (a)
(0.2 per cent);
w ith reference solution (a) (0.10 per cent);
— total: not m ore than 10 times the area of the principal
solution (a) ( 1.0 p er cent); F. 9-(2j3-dideoxy-|3-D-£/ycen>-pent-2-enofuranosyl)-l,9-
— disregard limir. 0.5 times the area o f the principal peak in dihydro-6if-purin-6-one (2 ',3 '-d id eo x y -2 ',3 '-
the chromatogram obtained with reference solution (a) didehydroinosine),
(0.05 per cent).
H eav y m e ta ls (2.4.8) NH,
M aximum 20 ppm.
W a te r (2.5.12)
M axim um 0.1 per cent, determined on 1.0 g. G. R = OH: 9-(2,3-dideoxy-P-D-giycm>-pentofuranosyl)-9f/-
purin-6-am ine (2',3'-dideoxyadenosine),
A SSA Y
H . R = H: 9-(2,3,5-trideoxy-P-D-g/ycCTt>-pentofuranosyl)-9/i-
Dissolve 0.200 g in 50 m L o f glacial acetic acid R. T itrate
purin- 6-amine (2',3',5'-trideoxyadenosine),
2016 Diethyl Phthalate 1-745
E. T o about 0.1 m L add 0.25 m L o f sulfuric acid R and

50 mg of resorcinol R. H eat on a water-bath for 5 min. Allow
to cool. Add 10 m L of water R and 1 m L o f strong sodium
hydroxide solution R. T he solution becomes yellow or
brownish-yellow and shows green fluorescence.
TESTS
I. 9-(2j3-dideoxy-P-D-fiycm>-pent-2-enofuranosyl)-9ii-purin- Appearance
T he substance to be examined is clear (2.2.1) and n o t more
6-amine (2 ',3 '-dideoxy-2 ',3 '-didehydroadenosine).
intensely coloured than reference solution Y6 (2.2.2,
__________________________________________________________PhEur
Method II).
Acidity
Dissolve 20.0 g in 50 m L of ethanol (96 per cent) R previously
neutralised to phenolphthalem solution R l. Add 0.2 mT. of
Diethyl Phthalate ***** phenolphthalein solution R l. Not m ore than 0.1 m L o f 0.1 M
(Ph. Eur. monograph 0897) * sodium hydroxide is required to change the colour o f the
indicator to pink.
O Related substances
Internal standard solution Dissolve 60 mg o f naphthalene R in
o solvent.
C 12H 140 4 222.2 84-66-2 examined in methylene chloride R and dilute to 20.0 m L with
the same solvent.
A ctio n a n d u se
Excipient. Test solution (b) Dissolve 1.0 g o f the substance to be
examined in methylene chloride R, add 2.0 m L of the internal
PhEir ___________________________________:_______________________ standard solution and dilute to 20.0 m L with methylene
D E F IN IT IO N
chloride R.
Diethyl benzene-lj2-dicarboxylate. Reference solution T o 1.0 m L of test solution (a) a d d 10.0 m L
o f the internal standard solution and dilute to 100.0 m L with
C o n te n t
99.0 per cent m/m to 101.0 per cent m/m.
Column:
CHARACTERS — material: glass;
A p p e a ra n c e — sizer. I = 2 m , 0 = 2 mm;
Clear, colourless or very slightly yellow, oily liquid. — stationary phase: sUanised diatomaceous earth for gas
Solubility chromatography R (150-180 pm) impregnated with
Practically insoluble in water, miscible with ethanol 3 per cent m/m of polymethylphenylsUoxane R.
(96 per cent). Carrier gas nitrogen for chromatography R.
ID E N T IF IC A T IO N Flow rate 30 mL/min.
First identification B, C. Temperature:
Second idenafication A, D, E. — column: 150 °C;
A. Relative density (2.2.5): 1.117 to 1.121.
B. Refractive index (2.2.6): 1.500 to 1.505.
Injection 1 pL.
Rim time 3 times the retention time of diethyl phthalate.
Preparation T hin films.
Elution order Naphthalene, diethyl phtalate.
Comparison diethyl phthalate CRS.
System suitability:
D . Thin-layer chromatography (2.2.27). — resolution: minimum 10 between the peaks due to
Test solution Dissolve 50 mg of the substance to be examined naphthalene and diethyl phthalate in the chromatogram
in ether R and dilute to 10 m L with the same solvent. obtained with the reference solution;
Reference solution Dissolve 50 mg o f diethyl phthalate CRS in — in the chromatogram obtained with test solution (a), there
ether R and dilute to 10 m L with the same solvent. is no peak with the same retention time as the internal
Plate TLC silica gel GF2 5 4 plate R. standard.
Mobile phase heptane R, ether R (30:70 V/V). Limit.
— total: calculate the ratio (R) o f the area of the peak due to
Application 10 pL.
diethyl phthalate to the area o f the peak due to the
Development Over 2/3 o f the plate. internal standard from the chromatogram obtained with
Drying In air. the reference solution; from the chromatogram obtained
Detection Examine in ultraviolet light at 254 nm. with test solution (b), calculate the ratio of th e sum o f the
Results T h e principal spot in the chromatogram obtained with areas of any peaks, apart from the principal peak and the
the test solution is similar in position and size to the principal peak due to the internal standard, to the area o f the peak
spot in the chromatogram obtained with the reference due to the internal standard: this ratio is not greater than
solution. R (1.0 per cent).
1-746 Diethylamine Salicylate 2016
W a te r (;2.5.12) H ea v y m e ta ls
M axim um 0.2 per cent, determ ined on 10.0 g. 12 m L o f a 10.0% w/v solution complies w ith limit test A for
S u lfa te d a sh (2.4.14) heavy metals, Appendix VII. Use lead standard solution
M axim um 0.1 per cent, determ ined on 1.0 g. (1 ppm Pb) to prepare the standard (10 ppm ).
S u lfa te
A SSA Y
0.6 g complies with the limit test for sulfates, Appendix VII
Introduce 0.750 g into a 250 m L borosilicate glass flask.
(250 ppm ).
A dd 25.0 m L o f 0.5 M alcoholic potassium hydroxide and a few
glass beads. Boil in a water-bath under a reflux condenser for L oss o n d ry in g
1 h. Add 1 m L of phenolphthalein solution R l and titrate W hen dried at 60° for 3 hours, loses not m ore than 0.1% of
immediately with 0.5 M hydrochloric acid. Carry out a blank its w eight Use 1 g.
titration. Calculate the volume of 0.5 M alcoholic potassium A SSA Y
hydroxide used in the saponification. Carry out M ethod I for non-aqueous titration,
1 m L of 0.5 M alcoholic potassium hydroxide is equivalent to A ppendix VHI A, using 0.4 g and 1-naphtholbenzein solution
55.56 mg of C 12H 14O 4. as indicator. Each m L o f 0.1m perchloric acid KS is equivalent
ST O R A G E to 21.13 m g o f C 11H 17N O 3.
In an airtight container. STORAGE
___________________________________________________________PhEur Diethylamine Salicylate should be protected from lig h t
It should n o t be allowed to come into contact with iron or
iron salts.
Diethylamine Salicylate
COOH
Diethylcarbamazine Citrate *****
** +*
(PL Eur. monograph 0271) *
o
ho c o 2h
V CHa , HOaC^JX^COjH
Cn H 17N 0 3 211.3 4419-92-5
H3C" ch3
C ounter-irritant. C 16H 29N30 8 391.4 1642-54-2
P re p a r a tio n
Diethylamine Salicylate Cream A c tio n a n d u se
Antihelminthic.
D E F IN IT IO N P r e p a r a tio n
Diethylamine Salicylate contains n o t less than 99.0% and n o t Diethylcarbamazine Tablets
m ore than 101 .0 % o f C 11H 17N O 3.
PhEur__________________________________________________________
W hite or alm ost white crystals. D E F IN IT IO N
Very soluble in water, freely soluble in ethanol (96%). NjiV-Diethyl-4-methylpiperazine-1 -carboxamide dihydrogen
2-hydroxypropane- 1,2,3-tricarboxylate.
C o n te n t
concordant with the reference spectrum o f diethylamine
salicylate (RS 099). CHARACTERS
B. T o 0.2 g add 5 m L of 1m sodium hydroxide, heat to boiling A p p e a ra n c e
point, cool and acidify with 2 m hydrochloric add; a white W hite or alm ost white, crystalline powder, slighdy
precipitate is produced. T he melting point of the precipitate, hygroscopic.
after recrystallisation from water and drying at 105°, is about S o lu b ility
160°, Appendix V A. Very soluble in water, soluble in ethanol (96 per cent),
TESTS practically insoluble in acetone.
A cid ity mp: about 138 °C, with decomposition.
Dissolve 2 g in 25 m L o f water and titrate with 0.1m sodium ID E N T IF IC A T IO N
hydroxide VS using phenol red solution as indicator. N ot more First identification A , C
than 0.2 m L o f 0.1m sodium hydroxide KS is required to
change the colour o f the solution.
A 50% w/v solution is dear, A ppendix IV A, and not more
Comparison diethylcarbamazine citrate CRS.
intensely coloured than reference solution BYS) Appendix IV B, B. Examine the chromatograms obtained in the test for
M ethod II. impurities A and B.
M e ltin g p o in t Results T h e principal spot in the chromatogram obtained with
100° to 102°, A ppendix V A. the test solution is similar in position, colour and size to the
2016 Diethylcarbamazine Citrate 1-747
principal spot in the chromatogram obtained with reference Reference solution (d) Dissolve 5.0 mg o f diethykarbamazme
solution (a). citrate CRS in solution A and dilute to 50.0 m L with
C. Dissolve 0.1 g in 5 m L o f water R. T he solution gives the solution A.
reaction of citrates (2.3.1). Column:
— size: I = 0.15 m, 0 = 3.9 mm;
TESTS
S o lu tio n S
Shake 2.5 g with water R until dissolved and dilute to 25 m L
with the same solvent. Mobile phase M ix 100 volumes o f methanol R2 and
900 volumes of a 10 g/L solution of potassium dihydrogen
A p p e a ra n c e o f so lu tio n phosphate R.
Solution S is n o t more opalescent than reference
suspension II (2.2.1) and n o t more intensely coloured than
reference solution BY6 (2.2.2, Method II). Detection Spectrophotometer at 220 nm.
Im p u ritie s A a n d B Injection 20 pL o f test solution (a) and reference solutions (a),
Thin-layer chromatography (2.2.27). (b) and (c).
Test solution Dissolve 0.5 g of the substance to be examined Run time Twice the retention time of diethylcarbamazine.
in methanol R and dilute to 10 m L with the same solvent. Identification of impurities Use the chromatogram obtained
Reference solution (a) Dissolve 0.1 g o f diethykarbamazme with reference solution (b) to identify the peak due to the
citrate CRS in methanol R and dilute to 2.0 m L with the same citrate.
solvent. Relative retention W ith reference to diethylcarbamazine
Reference solution (b) Dissolve 10 mg of methylpiperaztne R (retention time = about 7 min): citrate = about 0.2;
(impurity A) in methanol R and dilute to 100 m L w ith the degradation product = about 1.6.
same solvent. System suitability: reference solution (c):
Reference solution (c) Dissolve 10 mg o f dtmethylpiperazine R — resolution: minimum 5 between the -peaks due to
(impurity B) in methanol R and dilute to 100 m L w ith the diethylcarbamazine and the degradation product.
same solvent. Limits:
Mobile phase concentrated ammonia R, methyl ethyl ketone R, with reference solution (a) (0.10 per cent);
methanol R (5:30:65 V/VIV). — total: n o t more than 5 times the area o f the principal peak
Application 10 pL. in the chromatogram obtained with reference solution (a)
Development Over 2/3 of the plate. (0.5 per cent);
Drying A t 100-105 °C. — disregard limit. 0.5 times the area of the principal peak in
Detection Expose to iodine vapour for 30 min.
(0.05 per cent); disregard the peak due to the citrate.
Retardation factors Impurity A = about 0.2;
impurity B = about 0.5. H eav y m e ta ls (2.4.8)
M aximum 20 ppm.
Limits:
— impurity A: any spot due to impurity A is not more 12 m L of solution S complies with test A. Prepare the
intense than the corresponding spot in the chromatogram reference solution using 10 m L o f lead standard solution
obtained with reference solution (b) (0.2 per cent); (2 ppm Pb) R.
— impurity B: any spot due to impurity B is not m ore L oss o n d ry in g (2.2.32)
intense than the corresponding spot in the chromatogram Maximum 0.5 per cent, determined on 1.000 g by drying
obtained with reference solution (c) (0.2 per cent). in vacuo at 60 °C for 4 h.
R elated su b sta n c e s S u lfated a s h (2.4.14)
Liquid chromatography (2.2.29). Maximum 0.1 per cent, determined on 1.0 g.
Solution A Dissolve 31.2 g o f potassium dihydrogen phosphate R ASSAY
in water R and dilute to 1000 m L with the same solvent. Liquid chromatography (2.2.29) as described in the test for
Test solution (a) Suspend 0.30 g of the substance to be related substances with the following modification.
examined in solution A and dilute to 100 m L with Injection 20 pL o f test solution (b) and reference solution (d).
solution A. Filter or centrifuge and use the clear filtrate or
Calculate the percentage content o f C 16H 29N30 8 from the
supernatant.
declared content of diethylcarbamazine citrate CRS.
examined in solution A and dilute to 100.0 m L with STORAGE
solution A. In an airtight container.
Reference solution (a) Dilute 1.0 m L of test solution (a) to IM P U R IT IE S
100.0 m L with solution A. Dilute 1.0 m L of this solution to Specified impurities A, B
10.0 m L with solution A.
Reference solution (b) Dissolve 10 mg o f citric add R in
solution A and dilute to 10 m L with solution A.
H3C 'Nv'" ^
Reference solution (c) T o 3 m L o f test solution (a) add 0.5 m L
of strong hydrogen peroxide solution R and maintain at 80 °C A. R = H: 1-methylpiperazine,
for 3 h. Dilute to 100 m L with solution A.
B. R = C H 3: 1,4-dimethylpiperazine.
PhEur
1-748 Diethylene Glycol Monoethyl Ether 2016
— stationary phase:
Diethylene Glycol Monoethyl Ether *****
pdy(oyanopropyl) (7) (phenyl) (7) ( methyl) (86)sUoxane R
(Ph. Eur. monograph 1198) * (film thickness 1 pm).
Carrier gas nitrogen for chromatography R o r helium for
chromatography R.
Q H 14O 3 134.2 111-90-0 Split ratio 1:80.
Temperature:
Excipient.
Time Temperature
PftEir __________________________________________________________ (min) CO
Column 0 -1 120
D E F IN IT IO N
2-(2-Ethoxyethoxy)ethanol, produced by condensation of 1-10 120 -» 225
ethylene oxide and alcohol, followed by distillation. 10-12 225
CHARACTERS Injection port 275
A p p e a ra n c e Detector 250
Clear, colourless, hygroscopic liquid.
S o lu b ility
Miscible with water, w ith acetone and with alcohol, m isable Detection Flam e ionisation.
in certain proportions with vegetable oils, n o t miscible with Injection 0.5 pL.
mineral oils. Relative retentions W ith reference to diethylene glycol
R elativ e d e n sity monoethyl ether (retention time = about 4 m in): ethylene
A bout 0.991. glycol monom ethyl ether = about 0.4; ethylene glycol
monoethyl ether = about 0.5; ethylene glycol = about 0.55;
diethylene glycol = about 1. 1.
A. Refractive index (2.2.6): 1.426 to 1.428.
System suitability:
B. Infrared absorption spectrophotometry (2.2.24). — resolution: minim um 3.0 between the peaks due to
Comparison Ph. Eur. reference spectrum of diethylene glycol ethylene glycol monoethyl ether and to ethylene glycol in
monoethyl ether. the chromatogram obtained with reference solution (b),
TESTS — signal-to-noise ratio: minim um 3.0 for the peak due to
A cid v alu e (2.5.1) ethylene glycol monomethyl ether in th e chrom atogram
M axim um 0.1. obtained with reference solution (a),
Mix 30.0 m L with 30 m L o f alcohol R previously neutralised Limits (take into account the impurity/internal standard peak
with 0.1 M potassium hydroxide using phenolpkthalein area ratio):
solution R as indicator. T itrate with 0.01 M alcoholic potassium — ethylene glycol monomethyl ether, not m ore than the area of
hydroxide. the corresponding peak in the chrom atogram obtained
with reference solution (a) (50 ppm ),
P e ro x id e v a lu e (2.5.5)
— ethylene glycol monoethyl ether, n o t m ore th an the area of
M aximum 8.0, determ ined on 2.00 g.
the corresponding peak in the chrom atogram obtained
R e la te d su b s ta n c e s with reference solution (a) (160 ppm ),
Gas chromatography (2.2.28). — ethylene glycol: not more than the area o f the
Internal standard solution Dilute 1.00 g of decane R to corresponding peak in the chrom atogram obtained with
100.0 m L w ith methanol R. reference solution (a) (620 ppm ),
Test solution T o 5.00 g of the substance to be examined, add — diethylene glycol: not m ore than the area o f the
0.1 m L of the internal standard solution and dilute to corresponding peak in the chrom atogram obtained with
10.0 m L with methanol R. reference solution (a) (250 ppm),
— total: no t more than the area of the principal peak in the
Reference solution (a) D ilute 25.0 mg o f ethylene glycol
monomethyl ether R, 80.0 m g of ethylene glycol monoethyl
(0.2 per cent).
ether R, 0.310 g of ethylene glycol R and 0.125 g o f diethylene
glycol R to 100.0 m L w ith methanol R. T o 1.0 m L of this E th y le n e ox id e
solution add 0.1 m L o f the internal standard solution and H ead-space gas chromatography (2.2.28).
dilute to 10.0 m L with methanol R. Test solution T o 1.00 g o f the substance to be examined in a
Reference solution (b) D ilute 25.0 mg of ethylene glycol vial, add 50 pL of water R.
monoethyl ether R and 25.0 m g of ethylene glycol R to Reference solution T o 1.00 g o f the substance to be examined
100.0 m L w ith methanol R. Dilute 1.0 m l. o f this solution to in a vial, add 50 pL of ethylene oxide solution R4 and close
5.0 m L with methanol R. tighdy.
Reference solution (c) D ilute 1.00 g of the substance to be Column:
examined to 100.0 m L with methanol R. T o 1.0 m L o f this — material: fused silica,
solution add 0.1 m l. o f the internal standard solution and — size. I = 30 m , 0 = 0.32 mm,
dilute to 10.0 m L with methanol R. — stationary phase:
Column: poly(cyanopropyl) (7) (phenyl) (7) ( methyl) (86)sUoxane R
— material: fused silica, (film thickness 1 pm).
— size. I = 30 m , 0 = 0.32 mm, Carrier gas helium for chromatography R.
2016 Diethylene Glycol Palmitostearate 1-749
Flow rate 1.1 mL/min. CHARACTERS

Static head-space conditions which may be used: Appearance
— equilibration temperature: 80 °C, W hite or almost white, waxy solid.
— equilibration time: 45 min, Solubility
— transfer Une temperature: 110 °C, Practically insoluble in water, soluble in acetone and in hot
— pressurisation time: 2 min, ethanol (96 per cent).
— injection time: 12 s.
IDENTIFICATION
Temperature:
A. Melting point (see Tests).
Time Temperature B. Composition of fatty adds (see Tests).
(min) (°C) C . It complies with the limit of the assay (monoesters
Column 0 -5 40 content).
5-18 40 -> 200
TESTS
Injection port 150 M elting point (2.2.15)
Detector 250 43 °C to 50 °C.
Acid value (2.5.1)
Detection Flame ionisation. M aximum 4.0.
Injection 1.0 mL. Iodine value (2.5.4, Method A)
T he peak due to ethylene oxide is identified by injecting M aximum 3.0.
solutions of ethylene oxide of increasing concentration. Saponification value (2.5.6)
Determine the content of ethylene oxide (ppm) in the 155 to 180, determined on 2.0 g.
substance to be examined using the following expression: Composition of fatty adds (2.4.22, Method A)
Sx = area o f the peak due to ethylene oxide in the Use the mixture of calibrating substances in Table 2.4.22.-1.
chromatogram obtained with the test solution, Composition of the fatty acidfraction of the substance:
5s = area o f the peak due to ethylene oxide in the — stearic acid: 40.0 per cent to 60.0 per cent;
chromatogram obtained with the reference — sum of contents ofpalmitic acid and stearic add: minimum
solution, 90.0 per c e n t
Mp = mass of the substance to be examined in the test
Free diethylene glycol
solution, in grams,
M aximum 2.5 per cent, determined as described in the assay.
Ms = mass o f the substance to be examined in the
reference solution, in grams, Total ash (2.4.16)
C = mass o f added ethylene oxide in the reference Maximum 0.1 per c e n t
solution, in micrograms. ASSAY
Limit. Size-exclusion chromatography (2.2.30).
— ethylene oxide: maximum 1 ppm. Test solution Into a 15 m L flask, weigh 0.200 g (m).
W a te r (2.5.12) A dd 5.0 m L o f tetrahydrqfuran R and shake to dissolve. H eat
Maximum 0.1 p er cent, determined on 10.0 g. gently, if necessary. Reweigh the flask and calculate die total
mass of solvent and substance (M).
ST O R A G E
U nder an inert gas, in an airtight container.
Reference solutions Into four 15 m L flasks, weigh, 2.5 mg,
5.0 mg, 10.0 m g and 20.0 mg respectively of diethyiene
L A B ELLIN G glycol R. Add 5.0 m L o f tetrahydrqfuran R. Weigh the flasks
T he label states th at the substance is stored under an inert again and calculate the concentration of diethylene glycol in
gas. milligrams per gram for each reference solution.
__________________________________________________________ PhEur Column:
— size. I = 0.6 m, 0 = 7 m m ,
— stationary phase, styrene-dxvinylbenzene copolymer R (5 |im)
with a pore size of 10 nm.
★★*★★
Diethyiene Glycol Palmitostearate ★ ★ Mobile phase tetrahydrqfuran R.
(Ph. Eur. monograph 1415) ***** Flow rate 1 mL/min.
Detection Differential refractometer.
Action and use Injection 40 |iL.
Excipient. Relative retention With reference to diethylene glycol:
PhEir.
diesters = about 0.78; monoesters = about 0.84.
Calculations:
D E F IN IT IO N
— free diethyiene glycol: from the calibration curve obtained
M ixture of diethylene glycol mono- and diesters o f stearic with the reference solutions, determine the
(octadecanoic) and palmitic (hexadecanoic) acids. concentration (Q o f diethyiene glycol in milligrams per
It is produced by esterification of diethylene glycol and gram in the test solution and calculate the percentage
stearic acid 50 (see Stearic acid (1474)) of vegetable or content of free diethyiene glycol in the substance to be
animal origin. examined using the following expression:
Content
— monoesters: 45.0 per cent to 60.0 per cent;
— diesters: 35.0 per cent to 55.0 per c en t
1-750 Diethylstilbestrol

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British Pharmacopoeia 2016

Effective date: 1 January 2016

London: The Stationery Office

THE DEPARTMENT OF HEALTH, SOCIAL SERVICES AND

ISBN 978 Oil 3230 006

Professor Sir Michael Rawlins

Monographs of the European Pharmacopoeia are distinguished by a chaplet

The British Pharmacopoeia Commission has caused this British

The British Pharmacopoeia Commission is appointed, on behalf of the

Members of Expert Advisory Groups, Panels of Experts and Working

Members of the British Pharmacopoeia Commission and its supporting

Professor John Miller MSc PhD MRSC CChem

The Commission appointed the following Expert Advisory Groups, Panels

EXPERT ADVISORY GROUPS

Secretariat M VaUender (Editor-in-Chief)

M Barrett, H Corns, P Crowley, A Evans, J Francomb, A Gibb, P Holland,

K Courtney (Laboratory Manager)

J Hook (MD Public Sector EMEA)

Triennial Review of the British Pharmacopoeia Commission

The BP 2016 comprises six volumes as follows.

Volumes I and II Medicinal Substances

National monographs omitted from this or earlier editions of the British

Likewise, the British Pharmacopoeia Commission has not assessed the

Revisions A significant number (141 comprising 87 technical revisions and 54

As a consequence of the conclusion by CHM, the Medicines and

Supplementary One new Supplementary Chapter to harmonise with the European

Supplementary Chapter VII C: Monographs on Herbal Drugs (Ph. Eur.

European Co-operation Agreement

It is also important to note that no requirement of the Pharmacopoeia can

Websites B ritish Pharm acopoeia Websites

International Therapeutic Goods A dm inistration, A ustralia The British

Medicines, Biological Products and Active Pharmaceutical Ingredients and

Acknowledgements The British Pharmacopoeia Commission is greatly indebted to die members

the importance of the work of the European Pharmacopoeia (Ph. Eur.)

* denotes a monograph of the European Pharmacopoeia

Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal

Formulated Preparations: Specific Monographs

2 Monograph suppressed by European Pharmacopoeia Commission on-1 April 2015.

Doxorubicin Injection Related substances

Phenytoin Capsules Non-interchangeability statement

Herbal Drugs, Herbal Drug Preparations and Herbal Medicinal

BRITISH PH A RM A CO PO EIA BRITISH PHARM A CO PO EIA

Medicinal and Pharm aceutical Substances

Herbal Drugs, H erbal D rug Preparations and Herbal Medicinal

CONTENTS OF THE GENERAL NOTICES

Part I Implementation of Pharmacopoeial Methods

European Monographs of the European Pharmacopoeia are reproduced in this edition

monograph. Any statement containing the word ‘should5 constitutes

Expression of Where the standard for the content of a substance described in a

Where the result of an assay or test is reqmred to be calculated with

Temperature The Celsius thermometric scale is used in expressing temperatures.

When the concentration of a solution is expressed in molarity designated

titles. A cumulative list of such Approved Synonyms is provided in

Chemical Formulae When the chemical composition of an official substance is known or

Me -C H 3 Bu* -CH(CH 3)CH 2CH 3

Definition Statements given under the heading Definition constitute an official

Additional statements concerning the definition of formulated

Manufacture of Attention is drawn to the need to observe adequate hygienic precautions in

( 1) compliance with all of the requirements stated in the monograph;

Methods of The methods of sterilisation used in preparing the sterile materials

Excipients Where an excipient for which there is a pharmacopoeial monograph is used

Colouring Agents If in a monograph for a formulated preparation defined by means of a full

Characteristics Statements given under the heading Characteristics are not to be

Descriptive term Approximate volume of