european pharmacopoeia 5 with all supplements 1

Preface EUROPEAN PHARMACOPOEIA 5.0 I PREFACE The European Pharmacopoeia was inaugurated in 1964 through the Convention on the Elaboration of a European Pharmacopoeia The present Fifth Edition of the European Pharmacopoeia is therefore published at the time where the 40th Anniversary of the Pharmacopoeia can be celebrated The work on the Pharmacopoeia has gone through a remarkable development since the first difficult years Elaboration and approval of monographs and other texts proceed by an effective and smoothly running process producing public quality standards that keep pace with scientific progresses The work is remarkable because of its volume - the Fifth Edition presents close to 2000 monographs and other texts - and because all technical requirements have to be adopted by the European Pharmacopoeia Commission by unanimous decision The monographs of the Pharmacopoeia are legally enforced in the countries being signatories to the Convention on the Elaboration of a European Pharmacopoeia In addition to the 31 European countries and the European Union now being parties to the Convention, the work on the Pharmacopoeia is followed by 16 European and non-European countries and the WHO as observers The quality standards of the European Pharmacopoeia have, therefore, an impact on the quality of medicines, which goes far beyond the European region The Fifth Edition of the European Pharmacopoeia will become effective on 1st January 2005 Like the Fourth Edition, the present main volumes will be added to by three annual supplements implementing the decisions of each of the three annual Sessions of the European Pharmacopoeia Commission The presentation of the Pharmacopoeia in a main volume and three annual supplements was initiated by the publication of the Fourth Edition The intention was to increase the flexibility of the publication scheme and, in particular, to shorten the time span between adoption and enforcement The shortening of the time span, which has indeed been successful, is possible only thanks to a very flexible attitude by those countries that make national translations of the European Pharmacopoeia monographs A very low number of rapid revisions implemented in the past three years is another result of the new publication scheme The Fourth Edition is completed with the publication of Supplement 4.8 since it is impracticable to work with more than the eight supplements The Commission decided therefore to proceed to the Fifth Edition by consolidation of the Fourth Edition after three years, only The change from First to Second Edition was caused by major changes in the general methods, while the change from Second to Third Edition was due to the wish to consolidate the work achieved and to change the form of presentation from a loose-leaf format into a main volume followed by annual supplements The change from Fourth Edition to Fifth Edition continues the work of making the publication of the Pharmacopoeia as user-friendly as possible It is assumed that the publication of this Fifth Edition will proceed by publication of supplements over the next three years The eight founder countries of the Convention realised in 1964 that manufacturing and quality control standards for medicinal products on the European market had to be harmonised for reasons of public health and to facilitate the free movement of medicines Since 1964 the world has changed and the market for medicinal products has become global Accordingly, international harmonisation among the three major pharmacopoeias of the world, the European Pharmacopoeia, the Japanese Pharmacopoeia and the United States Pharmacopeia, has been in progress since 1990 when the Pharmacopoeial Discussion Group was set up to co-ordinate the harmonisation work In the first years, the work was focused on the harmonisation of monographs on widely used excipients In the absence of harmonised general methods this was a difficult work, which has now been speeded up by ‘harmonisation by attribute’ meaning that there may be tests that cannot be fully harmonised before the concerned general method is harmonised At the stage where the monographs are harmonised, detailed information will be provided in the monograph and in a chapter of the Pharmacopoeia devoted to information on international harmonisation In recent years, harmonisation of a wide range of general methods has been in progress, partly because of an impact from the International Conference on Harmonisation (ICH) Implementation in the Pharmacopoeia of harmonised general methods, for example for a dosage form specification, needs careful consideration because the specification must be met by products already on the market as well as new products submitted to the regulatory process The European Pharmacopoeia Commission supports strongly the international harmonisation It is not the harmonisation work itself that gives rise to the greatest problems, rather the implementation, which has to be decided in mutual agreement with the registration authorities The links between the European Pharmacopoeia Commission and European regulators have been steadily strengthened during the years, as have the links with the pharmaceutical manufacturers and their associations The new European Directives 2001/82/EC and 2001/83/EC on medicines for human use and veterinary use maintain the mandatory character of the European Pharmacopoeia monographs in the preparation of dossiers for marketing authorisation of medicines, which was instituted in the first directive 75/318/EEC in 1975 It means that the monographs of the European Pharmacopoeia must therefore be updated to keep pace with products on the market, with scientific progress, and with regulatory developments In the field of active pharmaceutical substances, the European Pharmacopoeia Commission decided at its March 2002 Session that the principles and terminology of the revised ICH Q3A impurity testing guideline Impurities in new drug substances should as far as possible be implemented in the monographs on active substances, both new and already published A change in terminology has been introduced in the Impurities section of monographs published in Supplement 4.6 and later where the term ‘specified impurities’ is used for impurities that have a defined individual acceptance criterion A revision of the general monograph Substances for pharmaceutical use (2034) was also presented in Supplement 4.6 to implement the threshold values for reporting, identification and qualification of organic impurities in active substances of the revised ICH guideline For the Fifth Edition a new chapter, 5.10 Control of impurities in substances for pharmaceutical use has been developed with great assistance by the chairs of the chemical Groups of Experts and other experts from the Commission, and by consultations of the Groups of Experts The next step will be revision of monographs to ensure that they contain related substances tests and lists on specified and other detectable impurities Monographs containing a related substances test based on TLC will be considered for revision Major revision work will thus proceed during the coming years Hopefully, these revisions can be completed with the publication of the Sixth Edition In the meantime, users of the Pharmacopoeia must consult the new Chapter 5.10 i Preface EUROPEAN PHARMACOPOEIA 5.0 these characteristics The section does not necessarily give acceptance criteria for the concerned properties ; this is usually left as an option for labelling by the manufacturers and where specified, the values are indicative only This is a new development which is in agreement with the policy of the European Pharmacopoeia Commission to make monographs and other texts appropriate to the needs The aim of the revision is to ensure that the related of regulatory authorities and manufacturers of starting substances test and impurity lists reflect the purity of materials and medicinal products The intention is to provide pharmaceutical substances being authorised for the manufacturers of excipient materials and manufacturers European market The goal cannot be met without of medicinal products a ’common language’, to facilitate close collaboration with the registration authorities and the establishment of product-specific specifications, and to consultations regarding the specifications for impurities provide regulators with data generated by methods that have A procedure for co-operation with the CPMP/CVMP been independently assessed Quality Working Party has been established It will It is the intention of the European Pharmacopoeia certainly contribute to ensure the validity of the European Pharmacopoeia monographs The Certification of Suitability Commission to continue the work by drafting sections on functionality-related characteristics in monographs of Monographs of the European Pharmacopoeia might on excipients available in more than one physical grade be a valuable source of information on the purity of Introduction of the concept of functionality-related pharmaceutical substances The procedure is, however, characteristics presupposes that the relevant general confidential and will be kept so In cases where a new impurity is present and calls for revision of the monograph, methods are available in the Pharmacopoeia The European Pharmacopoeia Commission has therefore established a this can be done only when the manufacturer provides the Working Party on synthetic polymers to investigate the concerned Group of Experts with the information required need for general methods for polymers and a Working for updating Party on powder characterisation methods The provision The growing number of monographs on pharmaceutical of the needed general methods, for example in the field of substances and the need to keep them updated means powder characterisation, is also included in international a great workload on the Groups of Experts In 2001, harmonisation among the pharmacopoeias the number of chemical groups was increased and some The achievements of the European Pharmacopoeia reallocations of experts between the groups took place Commission during the past three years would not have There is, however, still a need for more experts with access been possible without the participation of the great number to experimental facilities as permanent members of the of experts from industry, academia and national authorities, Groups of Experts or as members on an ad hoc basis In who have given their time and expertise to the work of addition to the reorganisation of the system of Groups of Groups of Experts and Working Parties The Commission Experts and Working Parties the working procedures for is indebted to all these experts whose work is given on a the elaboration of monographs have been expanded In voluntary basis The Commission is equally indebted to the addition to Procedure 1, the traditional elaboration by a Chairs of the Groups and Working Parties who have the Group of Experts, and Procedure 2, adaptation of national responsibility of guiding the work through and bringing it to monographs, which is now considered almost complete, term according to tight time limits The Chairs are thanked Procedures and have been established in recent years for their contributions within the Groups and also for their Procedure applies to substances produced by only one advice and counsel to the Commission itself manufacturer and which are close to patent expiry The manufacturer and the national pharmacopoeia authority The work of the European Pharmacopoeia Commission is of the country where the substance is produced carry out strongly dependent on an effective Secretariat The role of the preliminary drafting stages and check the requirements the Secretariat is to obtain and process all the information experimentally The draft is reviewed by the working party and reports needed for the Groups of Experts, Working also responsible for the adaptation procedure and then Parties and for the Commission, to undertake laboratory processed in the usual way by public inquiry Procedure work to support the experts and to ensure the availability of has proved successful The Commission decided in 2002 to all the reference standards needed to allow the requirements establish a modified version, Procedure This procedure in the monographs to be tested The prompt publication of implies collaboration between the manufacturer of the the Pharmacopoeia main volumes and Supplements and substance and the EDQM on the draft monograph and the on-line electronic version is possible, only, because of experimental checking by the EDQM laboratory and dedicated and hard work by the staff at the Secretariat laboratories of national pharmacopoeia authorities before Along with the growing volume of the European publication for public inquiry At present, Procedure is run Pharmacopoeia and its adjustment to the regulatory process, as a pilot project supervised by members of the European the use of the Pharmacopoeia and its interpretation has Pharmacopoeia Commission It is the aim of the Commission become rather complex The journal of the European to have a full coverage of monographs on substances no Pharmacopoeia, Pharmeuropa, is a valuable source of longer subject to a patent and being present on more than information General chapters for information will appear one European market It requires the collaboration with the in the Pharmacopoeia during the publication of the Fifth innovators and manufacturers of active substances, which Edition as a result of the international harmonisation, and has been established during recent years because the European Pharmacopoeia Commission has agreed on the elaboration of other chapters for information The Fifth Edition of the European Pharmacopoeia During the past two years, the staff at the EDQM have has a number of excipient monographs containing offered training courses to users of the Pharmacopoeia a non-mandatory section on functionality-related The Commission is grateful to the EDQM for having characteristics The aim is to provide users with a list taken this initiative, which also strengthens the role of of physical and physicochemical characteristics that are the Pharmacopoeia and the links to its users The links critical to the typical uses of the concerned excipient, to users of the Pharmacopoeia are also strengthened by and to provide the general methods required to assess on impurity control for the interpretation of monographs published in the past and therefore adapted to a style that has now been changed as described above Users can in addition find information on representative chromatograms, reagents and columns used in drafting the monographs on the EDQM web site ii Preface EUROPEAN PHARMACOPOEIA 5.0 the frequent workshops and conferences organised by the EDQM This activity is highly valued by the Commission as it gives the opportunity to Commission members to exchange viewpoints and to discuss new developments with experts from authorities, industry and academia The EDQM web site is another valuable source for information on the work programme and other activities of the Commission, its Groups and the EDQM During the past three years I have had the honour to serve the European Pharmacopoeia Commission as its elected chair The task has been challenging but, certainly, rewarding because of the insight it has given me into the many quality aspects of the development, manufacture and marketing of medicinal products I wish to thank members of the European Pharmacopoeia Commission for their support and collaborative spirit within and in between the Sessions of the Commission The two vice-chairs of the Commission are thanked for good collaboration and support during the years we have joined the Presidium I will also thank the staff at the EDQM, in particular the secretaries to the Groups, for their kindness, enthusiasm and hard work for the benefit of the Pharmacopoeia Finally, I wish to express warm thanks to the Director of EDQM, Dr Agnes Artiges, and her deputy as secretary to the European Pharmacopoeia Commission, Mr Peter Castle I have appreciated our collaboration during the three years and wish to express heartfelt thanks to both for their support to the chair and for the tremendous work they are doing to develop the European Pharmacopoeia and its role in the European regulatory system Professor, Dr Henning G Kristensen Chair of the European Pharmacopoeia Commission iii Introduction EUROPEAN PHARMACOPOEIA 5.0 II INTRODUCTION The European Pharmacopoeia is prepared under the auspices of the Council of Europe in accordance with the terms of the Convention on the elaboration of a European Pharmacopoeia (European Treaty Series No 50) as amended by the Protocol to the Convention (European Treaty Series No 134), signed by the Governments of Austria, Belgium, Bosnia and Herzegovina, Croatia, Cyprus, the Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Luxembourg, the Netherlands, Norway, Portugal, Romania, Serbia and Montenegro, Slovak Republic, Slovenia, Spain, Sweden, Switzerland, “the Former Yugoslav Republic of Macedonia”, Turkey, the United Kingdom, and by the European Community European Pharmacopoeia monographs and other texts are designed to be appropriate to the needs of: — regulatory authorities ; — those engaged in the control of quality ; — manufacturers of starting materials and medicinal products The European Pharmacopoeia is widely used internationally It is the intention of the Commission to work closely with users of the Pharmacopoeia in order to satisfy better their needs and facilitate their co-operation To this end improved procedures are being developed for obtaining advice on priorities for elaborating new monographs and enhancing the quality of the Pharmacopoeia The preparation of the Pharmacopoeia is the responsibility of the European Pharmacopoeia Commission (“the Commission”), appointed in accordance with Article of the above-mentioned Convention It is composed of delegations appointed by the Contracting Parties Each delegation consists of not more than members chosen for their competence in matters within the functions of the Commission TECHNICAL SECRETARIAT AND LABORATORY The European Pharmacopoeia Commission has a Technical Secretariat with scientific and administrative staff, situated in Strasbourg The European Pharmacopoeia Laboratory is situated within the Secretariat and, amongst other duties, is in charge of the establishment and monitoring of all reference substances, preparations and spectra needed for the monographs of the Pharmacopoeia The Technical Secretariat is an administrative division of the European Directorate for the Quality of Medicines (EDQM) of the Council of Europe Observers from non-Member States and international organisations are admitted to Sessions of the Commission in accordance with the Rules of Procedures Observers are at present admitted from : Albania, Algeria, Australia, Bulgaria, Canada, China, Georgia, Lithuania, Malaysia, Malta, Morocco, Poland, Senegal, Syria, Tunisia, Ukraine, and the World Health Organisation GENERAL PRINCIPLES General rules for interpretation of the texts of the Pharmacopoeia are given in the General Notices The The functions of the Commission established by Article of following information should also be noted the Convention as amended by the Protocol are : The general principles applied in the elaboration of monographs of the European Pharmacopoeia are laid Article down in technical guides The Technical Guide for the “Subject to the provision of Article of the present Elaboration of Monographs, which deals mainly with Convention, the functions of the Commission shall be : monographs on chemical substances, is available as a special issue of Pharmeuropa (see below under Publications) (a) to determine the general principles applicable to the Other technical guides are being prepared to deal with elaboration of the European Pharmacopoeia ; aspects specific to monographs on other groups of (b) to decide upon methods of analysis for that purpose ; products The principles applied are revised from time to (c) to arrange for the preparation of and to adopt monographs time without complete retrospective application so that to be included in the European Pharmacopoeia and ; monographs published already may not always follow the (d) to recommend the fixing of the time limits within which latest recommendations, but wherever an issue with impact on public health is identified, monographs are revised its decisions of a technical character relating to the The procedures for the tests and assays published in the European Pharmacopoeia shall be implemented within individual monographs have been validated, according to the territories of the Contracting Parties.” current practice at the time of their elaboration, for the In accordance with the terms of the Convention, the purpose for which they are intended Contracting Parties undertake to take the necessary It is recognised that general chapters are used elsewhere measures to ensure that the monographs of the European than in the monographs of the Pharmacopoeia ; in these Pharmacopoeia shall become the official standards circumstances users are recommended to consult the applicable within their respective territories Technical Guide which gives extensive information on the PURPOSE OF THE EUROPEAN PHARMACOPOEIA application of many of the methods The purpose of the European Pharmacopoeia is to promote General monographs The standards of the European Pharmacopoeia are represented by general and specific public health by the provision of recognised common monographs The use of general monographs has developed standards for use by health-care professionals and others in recent years to provide standards that best fulfil the concerned with the quality of medicines Such standards are to be of appropriate quality as a basis for the safe use of aims stated above and meet the needs of users It is now usually necessary to apply one or more general medicines by patients and consumers Their existence : monographs along with any specific monograph Since — facilitates the free movement of medicinal products in it is not practically possible to include in each specific Europe ; monograph a cross-reference to applicable or potientially — ensures the quality of medicinal products exported from applicable general monographs, cross-referencing has been Europe discontinued except where it is necessary to avoid ambiguity v Introduction A list of general monographs is included in each new edition and supplement to aid users in identifying those that are needed for use with a specific monograph Use of animals In accordance with the European Convention on the protection of animals used for experimental and other scientific purposes (1986), the Commission is committed to the reduction of animal usage, wherever possible, in pharmacopoeia testing and encourages those associated with its work to seek alternative procedures An alternative or modified method is adopted by the Commission once it has been clearly demonstrated that it offers satisfactory control for pharmacopoeial purposes Considerable progress was made in this area while the 4th Edition was in force and while the 5th Edition was being prepared Hydrates With the publication of the 4th Edition, the policy on monograph titles for hydrated forms was changed For all monographs published for the first time in the 4th Edition or subsequent editions, the degree of hydration, where applicable, is indicated in the monograph title In previous editions, the policy was to indicate the degree of hydration only where several forms exist If a monograph on both an anhydrous and a hydrated form of a given substance are published, then “anhydrous” will be included in the title of the relevant form In order to avoid placing an unnecessary burden on manufacturers for relabelling, this policy will not be applied retrospectively to monographs published already, unless there is reason to believe that this is justified as a public health measure, notably for safety reasons where the substance contains a large proportion of water Chiral substances Monographs on chiral substances that describe a particular enantiomer have a test to confirm enantiomeric purity, usually by measurement of optical rotation Monographs that describe racemates are, in this respect, heterogeneous because of changes of policy during the 3rd Edition Older monographs not always have a test to show racemic character During the course of the 3rd Edition, a test for racemic character was included in all new and revised monographs on racemates, using measurement of optical rotation When it was shown that in many cases a test for optical rotation, even with narrow limits around zero rotation, was not necessarily sufficiently discriminating because of the low specific optical rotation of the enantiomers, the Commission modified the policy applied A test for racemic character using optical rotation is now included only if there is information on the specific optical rotation of the enantiomers that indicates that such a test would be discriminating in terms of enantiomeric purity If other techniques, such as circular dichroism, can serve the intended purpose, they will be prescribed instead of optical rotation Polymorphism Where a substance shows polymorphism, this is usually stated under Characters In general, no particular crystalline form is required in monographs ; exceptionally, in a few monographs, the crystalline form required is specified, for example, via an infrared absorption spectrophotometric identification test where the spectrum is required to be recorded using the substance in the solid state without recrystallisation, the chemical reference substance provided being of the required crystalline form However, for substances other than these exceptional cases, depending on the use of a given substance in a dosage form, it may be necessary for a manufacturer to ensure that a particular crystalline form is used The information given under Characters is intended to alert users to the need to evaluate this aspect during the development of a dosage form The monograph on Substances for pharmaceutical use (2034) and 5.9 Polymorphism should also be consulted vi EUROPEAN PHARMACOPOEIA 5.0 Specificity of assays For the elaboration of monographs on chemical substances, the approach generally preferred by the Commission is to provide control of impurities via a well designed Tests section rather than by the inclusion of an assay that is specific for the active moiety It is therefore the full set of requirements of a monograph that is designed to ensure that the product is of suitable quality Impurities Following a review of policy on control of impurities, a new general chapter 5.10 Control of impurities in substances for pharmaceutical use has been included in the 5th Edition Together with the general monograph Substances for pharmaceutical use (2034), it describes the policy of controlling impurities in specific monographs and provides explanations on how the limits in the related substances test should be understood Currently the test is a limit test (comparison of peaks areas) In the future (next Edition) and in order to be in line with licensing practice and international collaboration, this test will progressively be changed to utilise a quantitative acceptance criterion At present, some of the current monographs already satisfy this approach Except where required for the application of the monograph, in which case the name is followed by “CRS”, impurities are not provided as reference substances nor can they be provided for experimental purposes Chromatographic columns As an aid to users, information is made available via the web site www.pheur.org on chromatographic columns that have been found satisfactory during development of monographs and general methods Information is also given on other equipment and reagents where this is considered useful This information is given without warranty and does not imply that other columns, equipment or reagents than those specified are not suitable Residual solvents The requirements for residual solvents are given in the monograph Substances for pharmaceutical use (2034) together with the general chapters 2.4.24 Identification and control of residual solvents and 5.4 Residual solvents Thus all active substances and excipients are subject to relevant control of residual solvents, even where no test is specified in the individual monograph The requirements have been aligned with the ICH guideline on this topic Reference substances, reference preparations and reference spectra Where necessary for application of a monograph, reference substances, reference preparations and reference spectra are established and provided to users They are chosen for their suitability for the purposes stated in the monograph and are not necessarily suitable for other uses Any necessary information for proper use is given, for example a declared content, but no complete certificate of analysis is provided since this is not relevant for the intended use No expiry date is attributed to reference substances and preparations, which are subjected to regular periodic monitoring to ensure their continued suitability Where an assigned value for a given attribute, for example chemical content, is provided, no uncertainty for the assigned value is indicated The reference substances, preparations and spectra are provided to enable the analyst to determine compliance or otherwise with a monograph The uncertainty of an assigned value is not to be taken into account when judging compliance, since the uncertainty is already allowed for in the prescribed limits Medical devices All editions of the Pharmacopoeia have contained monographs on articles that are regarded as medical devices For Member States of the European Union, a unified framework for standardisation of medical devices is now provided by a Directive (93/42/EEC) Following EUROPEAN PHARMACOPOEIA 5.0 an agreement between the various parties involved, the Commission has decided that the monographs on medical devices will be deleted once standards have been developed as foreseen by the Directive Specifications included in the section on containers will be adapted to take account of future standards developed within the framework of the Directive The monographs on surgical sutures remain in the Pharmacopoeia but they have been modified to conform to the requirements of the Directive and are now to be seen as standards of the type foreseen there This adaptation of the monographs has involved deletion of some monographs on specific types of sutures in favour of a more general approach Homoeopathic preparations A general monograph on homoeopathic preparations was added to the Pharmacopoeia during the 2nd Edition A number of monographs on substances used in homoeopathic preparations are now also included and further monographs are in preparation All of these texts have been grouped in a separate section It is understood that when the same substance is used in both homoeopathic and other preparations then the monograph in the main body of the Pharmacopoeia applies Patents The description in the Pharmacopoeia of articles subject to protection by patent does not confer or imply any right to the use of such patents by any person or persons other than the proprietors of the patents concerned Protected species Monographs, notably those on herbal drugs, may cover material obtained from protected species Inclusion of these monographs is without prejudice to the provisions for protection of these species by national and international law CERTIFICATION PROCEDURE A procedure for the certification of suitability of monographs of the Pharmacopoeia with respect to control of the purity of a product from a given source has been established [see Public Health Committee (Partial Agreement) Resolution AP-CSP (99) or any subsequent revision available from EDQM and on the web site (www.pheur.org)] as an aid to the use of monographs in applications for marketing authorisation The certification procedure also applies to herbal drugs, herbal drug preparations and transmissible spongiform encephalopathy (TSE) risk Certificates may be granted with respect to published monographs Details of the operation of this scheme are Introduction available from the Secretariat and on the EDQM web site A daily updated list of certificates granted is available on-line on the EDQM web site A list of voided or suspended certificates is also published in Pharmeuropa PUBLICATIONS The European Pharmacopoeia is available in English and French versions in the form of a book with supplements per year, and in electronic form (internet and CD-ROM) Pharmeuropa, the European Pharmacopoeia Forum, is published times per year as an aid in the elaboration of monographs and as a vehicle for information on pharmacopoeial and related matters It is available on subscription from EDQM Web site Information on activities and many other aspects of the European Pharmacopoeia is to be found on the EDQM web site (www.pheur.org) Implementation The date on which monographs are to be implemented is fixed by a resolution of the Public Health Committee (Partial Agreement) of the Council of Europe, following a recommendation by the Commission This date is usually about months after publication Where a monograph is to be implemented at a date earlier than the next publication date of the Pharmacopoeia or a supplement, a Resolution of the Public Health Committee gives the full text to be implemented The text is also published in Pharmeuropa for information and posted on the web site as part of the Resolution Revision programme Monographs and other texts of the Pharmacopoeia are revised as necessary following a decision of the Commission Revision proposals are published in Pharmeuropa INTERNATIONAL HARMONISATION The European Pharmacopoeia is engaged in a process of harmonisation with the Japanese Pharmacopoeia and the United States Pharmacopeia, within an informal structure referred to as the Pharmacopoeial Discussion Group (PDG) The activities are developed in co-ordination with those of the International Conference on Harmonisation (ICH) Information on the status of harmonised texts is given in chapter 5.8 Pharmacopoeial harmonisation Harmonised general chapters have a preliminary statement indicating interchangeability with the other two pharmacopoeias vii Members of the Commission EUROPEAN PHARMACOPOEIA 5.0 III EUROPEAN PHARMACOPOEIA COMMISSION COMPOSITION OF THE COMMISSION, LIST OF EXPERTS AND OF THE SECRETARIAT AS OF 30 NOVEMBER 2003 CHAIR AND VICE-CHAIRS OF THE COMMISSION Chair Henning G KRISTENSEN Vice-chairs Dries Liisa DE KASTE TURAKKA Hungary Hilda Jozsef J KÖSZEGI-SZALAI LIPTAK Iceland Gudrun Ingolf J BALDURSDOTTIR PETERSEN Ireland T.A Michael Joan McGUINN MORRIS O’RIORDAN MEMBERS OF THE COMMISSION Austria Kristof Andreas Christian LISZKA MAYRHOFER NOE Italy CIGNITTI FARINA OREFICI Belgium Luc Jos Paule ANGENOT HOOGMARTENS JACQMAIN Maurizio Anna Graziella Latvia Janis OZOLINS Aida MEHMEDAGIC Luxembourg Bosnia and Herzegovina Jacqueline Jean-Louis GENOUX-HAMES ROBERT Croatia Dragica Ivana Laila BEGIC STARESINIC-SERNHORST STEFANINI ORESIC Netherlands Dries Jan Willem Pieter H DE KASTE DORPEMA VREE Norway Cyprus Louis PANAYI Gunhild Valborg Randi BRUGAARD HOLTEN WINSNES Portugal José Manuel CORREIA NEVES SOUSA LOBO Rui MORGADO Romania Daniele ENACHE Marija Stana MASKOVIC MICIC Czech Republic Jiri M Denmark PORTYCH TRAVNICKOVA Kirsten BRØNNUM-HANSEN Steen Honoré HANSEN Eva SANDBERG Estonia Signe LEITO Serbia and Montenegro Finland Jussi Kaarina Liisa HOLMALAHTI SINIVUO TURAKKA Slovak Republic N Ruzena J France Jean-Paul An Alain FOURNIER LÊ NICOLAS Slovenia Martina Evgen Uros CVELBAR TOMAZIN URLEB Germany Ulrike Dietrich D HOLZGRABE KRÜGER SCHNÄDELBACH Spain Franco Jordi Alexandra FERNANDEZ GONZALEZ RUIZ COMBALIA VARDULAKI Greece Michael A Alexandra KOUPPARIS TSOKA Sweden Lennart Marianne Christina AKERBLOM EK GRAFFNER CHALABALA MARTINCOVA SLANY ix Alternate Members EUROPEAN PHARMACOPOEIA 5.0 Switzerland Werner Silvia Helena ERNI WEBER BRUNNER WINDEMANN “The Former Yugoslav Republic of Macedonia” Aneta Tatjana DIMITROVSKA PERUSEVSKA Turkey Orhan Yilmaz Hayriye Slovak Republic Daniel Ladislav GRANCAI SOVIK Slovenia Maja Barbara Ales LUSIN RAZINGER-MIHOVEC ROTAR Sweden Torbjörn ARVIDSSON CANBOLAT CAPAN MIHCAK Switzerland Andreas Uwe Eugen BRUTSCHE VÖLKER WACHBERGER United Kingdom Derek H Gerard A.David CALAM LEE WOOLFSON United Kingdom Aileen M.T John M European Commission Nicolas ROSSIGNOL EMEA Emer COOKE ALTERNATE MEMBERS Austria J Josef KURZ TRENKER Belgium Jacques Luc Arnold J DE BEER DELATTRE VLIETINCK Denmark Sven Lars J Lene FRØKJAER HUSAGER THOMSEN Estonia Juhan RUUT Finland Hannele SALOMIES France BOURQUIN Thierry Hendrick Jan DE JONG VILAIN Caroline Germany Gerhard Rainer Rainer FRANZ MOHR SEITZ Greece Evangelos A PETRODASKALAKIS TSANTILI-KAKOULIDOU Hungary Tamas L PAÀL Ireland Edward BOURKE Italy Massimo Agostino Loredana DI MUZIO MACRI NICOLETTI Luxembourg Mariette BACKES-LIES Netherlands DE ROOIJ-LAMME Ellen Peter M.J.M JONGEN Jan-Anton NORDER x LEE MIDGLEY EXPERTS Susan Maqbool Jean-Marc Lennart Ferhan Concepcion Hansruedy Julio Hanspeter Cyrille Luc Gunnar Astrid Jean-Claude Torbjưrn Wilfried Nataliya Nikolaevna Jérơme Sylvie Paolo Teresa Kenneth Elizabeth Ann K Hüsnü Can Michel Alain Franỗoise Denis Leandro David N Brita Kathrin Serge Pietro Jean-Pierre Hanno Mikael Johannes Giovanni Colin Nicole Thierry Bernard AGERHOLM AHMED AIACHE AKERBLOM AKTAN ALONSO VERDURAS ALTORFER ALVAREZ-BUILLA AMSTUTZ ANDRES ANGENOT ANTONI ARBIN ARGOUD ARVIDSSON ARZ ASMOLOVA AUCOUTURIER AUDOLY AURELI AZCONA LLANEZA BÄCKSTRÖM BAKER BASER BAUER BAYOL BEAUJEAN BELLENOT BELLENTANI BENTLEY BERGH BERNARD-SUMMERMATTER BESSET BIANCHINI BINDER BINDER BISRAT BLÜMEL BOCCARDI BOOTH BORNSTEIN BOURQUIN BOUYSSIERE Experts EUROPEAN PHARMACOPOEIA 5.0 Brigitte Harald Per O Einar M Adrian F Kirsten Lukas Peter Volker Marian Rosario Jörg Roger Peter R Derek H Kandemir Salvador Franỗois Gunnar Sergio A.J Richard Pierre Xavier Vivienne Maurizio Klaus Juan Giovanni Laurence Pierre-Albert Stéphane Giordano Bruno Klaus Gérard Jacques-Christian Alastair Jacqueline Jacques Josep M Hendrick Jan Dries Carmen Anna Ellen Paul Clemens Louis H.T Luc Reto Joseph Jan S Jan J Roland Johannes Eric Erik Milada Thomas Jan Willem BRAKE BREIVIK BREMER BREVIK BRISTOW BRØNNUM-HANSEN BRUCKNER BRUEGGER BUEHLER BUKOVSKY BULLIDO BUND BURGENER BYRON CALAM CANEFE CANIGUERAL CANO CARLIN CAROLI CAWS CAWTHORNE CHAMINADE CHENIVESSE CHRIST CIANFRIGLIA CICHUTEK CLARAMUNT CAMPANA COLLI COLLIERE COMPAGNON CORNEN CORSI CUSSLER DAMIEN DARBORD DAVIDSON DAYAN-KENIGSBERG DE BEER DE CIURANA i GAY DE JONG DE KASTE DE LA MORENA CRIADO DE PASQUALE DE ROOIJ-LAMME DECLERCK DECRISTOFORO DEDEREN DELATTRE DELLA CASA DEMEESTER DEN HARTIGH DENYER DIJKSTERHUIS DOBBELAER DODT DOELKER DOEVENDANS DOLEZALOVA DOLL DORPEMA Maria Helena Robert Anil Siegfried Erling Marianne Torben Ulrich Magnus Bengt Jean-Pierre Øystein Bernard M Charles J Gemma L.M Peter Rainer V’Iain Øystein Ton Ulf Lucien Isabelle Jean-Paul Bruno Gerhard Florence Nicola Rose E Maria Cristina Andreas Jesus Didier Andrea Olga Nicole Michel Chris T Marcel Christina Tatjana Daniel Marta Norbert Gerhard Peter Kjell-Olov Jean Louis Emanuel Giuseppe Michèle Robert Sylvie Klaus Lilian Franz-Josef Steen Honoré Paul Goetz Vassiliki Kaare DOS ANJOS RODRIGUES AMARAL DRILLIEN DUDANI EBEL EHRIN EK ELHAUGE ENGEL ERICKSON ERLANDSSON ETCHEGARAY EVENSEN EVERETT FALLAIS FEENSTRA-BIELDERS FEIGENWINTER FENDT FENTON-MAY FODSTAD FÖRCH FORSMAN FOSSE FOURASTÉ FOURNIER FRANK FRANZ FUCHS FUZZATI GAINES DAS GALLI GARDI GARICANO AISA GARONNAT GAZZANIGA GELDOF GIBELIN GIRARD GODDARD GOVERDE GRAFFNER GRAFNETTEROVA GRANCAI GRANSTRÖM GREIDZIAK GROHMANN GRONSKI GRÖNVIK GROSSIORD GUADAGNINO GUALANDI GUITTET GURNY GUYOMARD-DEVANLAY HABERER HAMILTON HAMMERSCHMIDT HANSEN HARGREAVES HARNISCHFEGER HARTOFYLAX HASLOV xi Experts Heribert Mary Alice Ingrid Irmtraud Keith Peter Jaakko-Juhani B Franỗois Rikke Valborg Ulrike Ronald Ernử Rolf Anthony R Ronny Lars J Herwig Marianne Miia M.B Jana Christa Mats E Edgar Robert Peter M.J.M Karl-Henrik Juan Ignacio Mats Jan Hans Ernst Lawrence Isabelle Damien Marylène Claus Brigitte Frans Hilda Katjusa Henning G Nikolaus G Burt H M Michael Ursula Harry V Francesco Fritz Reinhard Mervi Christophe Daniel Annie Maria Grazia John Joyce An xii EUROPEAN PHARMACOPOEIA 5.0 HÄUSLER HEFFORD HEGGER HELD HELLIWELL HENRYS HIMBERG HINSCH HIRSCH HOFF-JØRGENSEN HOLTEN HOLZGRABE HOOGERBRUGGE HORVATH HOVIK HUBBARD HÜBINETTE HUSAGER IGEL INZELT-KOVACS JAKAVA-VILJANEN JAMES JERABKOVA JERSCH JOHANSSON JOHN JOHNSON JONGEN JÖNSSON JORQUERA NIETO JOSEFSON KARLSEN KEEGSTRA KELLER KELLY KEMPF KERLOC’H KOBISCH KOCH KOPP KORSE KÖSZEGI-SZALAI KREFT KRISTENSEN KRIZ KROES KROON KRÜGER KUKRAL KUPPERS LA TORRE LACKNER LANGE LANKINEN LAROCHE LARZUL LASSERRE LAVAGGI LAVDIOTIS LAWRENCE LÊ Marie-Frédérique Aileen M.T Eva Ulla Franck Silvano Céline Patrick Heiner Bruce Antonio Warren C Georges Carla Ana Maria Andreas Alessandro D.L Jos H.A Andreas Bernard Geerd J Jacques John M Giovanni Marianne Michael Miquel Tony Birgitte Thomas Patrick Manfred Jacques Laurence Carl Zdenka B.W Hartwig Michael Vera Philip S Ria Robin A.J Steven C Alain Loredana Vittorio Jochen Dagmar Ningur Werner Hok Liang Edgar Rose-Marie Bo Graziella Carsten Inge R.A A LE POTIER LEE LEMBERKOVICS LENNMARKER LEVEILLER LONARDI LORTEAU-SOURGEN LOUIS LUDWIG MADSEN MANES ARMENGOL MANN MANSVELD MARCHIORO MARQUES GONCALVES MARTI MARTINI MASSART MATHÔT MAYRHOFER MAZIERE MEYER MICHAUD MIDGLEY MIGLIACCIO MIKAELSSON MILCHARD MIR MOFFAT MØLLGAARD MONTAG-LESSING MONTENOISE MOOS MOREL MOUILLOT MROZ MRVOVA MÜLLER MÜLLER MUTZ MYSLIVCOVA NEWLANDS NIBBELING NICHOLAS NICHOLS NICOLAS NICOLETTI NISTRIO NORWIG NOVA NOYANALPAN OBEXER OEI OHST ÖLANDER OLSSON OREFICI OVERBALLE-PETERSEN OVERBY JENSEN PACKER PADILLA EUROPEAN PHARMACOPOEIA 5.0 Benzalkonium chloride solution ASSAY Dissolve 2.00 g in water R and dilute to 100.0 ml with the same solvent Transfer 25.0 ml of the solution to a separating funnel, add 25 ml of chloroform R, 10 ml of 0.1 M sodium hydroxide and 10.0 ml of a freshly prepared 50 g/l solution of potassium iodide R Shake well, allow to separate and discard the chloroform layer Shake the aqueous layer with three quantities, each of 10 ml, of chloroform R and discard IDENTIFICATION the chloroform layers To the aqueous layer add 40 ml of A Dissolve 80 mg in water R and dilute to 100 ml with the hydrochloric acid R, allow to cool and titrate with 0.05 M same solvent Examined between 220 nm and 350 nm potassium iodate until the deep-brown colour is almost (2.2.25), the solution shows three absorption maxima, at discharged Add ml of chloroform R and continue the 257 nm, 263 nm and 269 nm, and a shoulder at about titration, shaking vigorously, until the chloroform layer no 250 nm longer changes colour Carry out a blank titration on a B To ml of solution S (see Tests) add 0.1 ml of glacial acetic mixture of 10.0 ml of the freshly prepared 50 g/l solution acid R and, dropwise, ml of sodium tetraphenylborate of potassium iodide R, 20 ml of water R and 40 ml of solution R A white precipitate is formed Filter Dissolve hydrochloric acid R the precipitate in a mixture of ml of acetone R and ml of 0.05 M potassium iodate is equivalent to 35.4 mg ml of alcohol R, heating to not more than 70 °C Add of C22H40ClN water R dropwise to the warm solution until a slight opalescence forms Heat gently until the solution is clear and allow to cool White crystals separate Filter, wash 01/2005:0371 with three quantities, each of 10 ml, of water R and dry in vacuo over diphosphorus pentoxide R or anhydrous BENZALKONIUM CHLORIDE silica gel R at a temperature not exceeding 50 °C The crystals melt (2.2.14) at 127 °C to 133 °C SOLUTION C To ml of dilute sodium hydroxide solution R add 0.1 ml of bromophenol blue solution R1 and ml Benzalkonii chloridi solutio of chloroform R and shake The chloroform layer is DEFINITION colourless Add 0.1 ml of solution S and shake The chloroform layer becomes blue Benzalkonium chloride solution is an aqueous solution of a mixture of alkylbenzyldimethylammonium chlorides, the D To ml of solution S add ml of dilute nitric acid R alkyl groups having chain lengths of C8 to C18 Benzalkonium A white precipitate is formed which dissolves on the chloride solution contains not less than 475 g/l and not addition of ml of alcohol R The solution gives more than 525 g/l of alkylbenzyldimethylammonium reaction (a) of chlorides (2.3.1) chlorides, calculated as C22H40ClN (Mr 354.0) The solution may contain alcohol TESTS Solution S Dissolve 1.0 g in carbon dioxide-free water R CHARACTERS and dilute to 100 ml with the same solvent A clear, colourless or slightly yellowish liquid, miscible with Appearance of solution Solution S is clear (2.2.1) and not water and with alcohol It froths copiously when shaken more intensely coloured than reference solution Y6 (2.2.2, IDENTIFICATION Method II) A Dilute 0.3 ml to 100 ml with water R Examined between Acidity or alkalinity To 50 ml of solution S add 0.1 ml 220 nm and 350 nm (2.2.25), the solution shows three of bromocresol purple solution R Not more than 0.1 ml absorption maxima, at 257 nm, 263 nm and 269 nm, and of 0.1 M hydrochloric acid or 0.1 M sodium hydroxide is a shoulder at about 250 nm required to change the colour of the indicator B To 0.05 ml add ml of water R, 0.1 ml of glacial acetic Amines and amine salts Dissolve 5.0 g with heating in acid R and, dropwise, ml of sodium tetraphenylborate 20 ml of a mixture of volumes of M hydrochloric acid and solution R A white precipitate is formed Filter Dissolve 97 volumes of methanol R and add 100 ml of 2-propanol R the precipitate in a mixture of ml of acetone R and Pass a stream of nitrogen R slowly through the solution ml of alcohol R, heating to not more than 70 °C Add Gradually add 12.0 ml of 0.1 M tetrabutylammonium water R dropwise to the warm solution until a slight hydroxide and record the potentiometric titration curve opalescence forms Heat gently until the solution is clear (2.2.20) If the curve shows two points of inflexion, and allow to cool White crystals separate Filter, wash the volume of titrant added between the two points is not with three quantities, each of 10 ml, of water R and dry greater than 5.0 ml If the curve shows no point of inflexion, in vacuo over diphosphorus pentoxide R or anhydrous the substance to be examined does not comply with the test silica gel R at a temperature not exceeding 50 °C The If the curve shows one point of inflexion, repeat the test but crystals melt (2.2.14) at 127 °C to 133 °C add 3.0 ml of a 25.0 g/l solution of dimethyldecylamine R C To ml of dilute sodium hydroxide solution R add in 2-propanol R before the titration If the titration curve 0.1 ml of bromophenol blue solution R1 and ml after addition of 12.0 ml of the titrant shows only one point of chloroform R and shake The chloroform layer is of inflexion, the substance to be examined does not comply colourless Add 0.05 ml of the solution to be examined with the test and shake The chloroform layer becomes blue Water (2.5.12) Not more than 10 per cent, determined on D To 0.05 ml add ml of dilute nitric acid R A white 0.300 g by the semi-micro determination of water precipitate is formed which dissolves on the addition Sulphated ash (2.4.14) Not more than 0.1 per cent, of ml of alcohol R The solution gives reaction (a) of determined on 1.0 g chlorides (2.3.1) CHARACTERS A white or yellowish-white powder or gelatinous, yellowish-white fragments, hygroscopic, soapy to the touch, very soluble in water and in alcohol On heating it forms a clear molten mass An aqueous solution froths copiously when shaken General Notices (1) apply to all monographs and other texts 1069 Benzbromarone EUROPEAN PHARMACOPOEIA 5.0 TESTS Solution S Dilute 2.0 g to 100 ml with carbon dioxide-free water R Appearance of solution Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II) Acidity or alkalinity To 50 ml of solution S add 0.1 ml of bromocresol purple solution R Not more than 0.1 ml of 0.1 M hydrochloric acid or 0.1 M sodium hydroxide is required to change the colour of the indicator Amines and amine salts Mix 10.0 g, while heating, with 20 ml of a mixture of volumes of M hydrochloric acid and 97 volumes of methanol R and add 100 ml of 2-propanol R Pass a stream of nitrogen R slowly through the solution Gradually add 12.0 ml of 0.1 M tetrabutylammonium hydroxide and record the potentiometric titration curve (2.2.20) If the curve shows two 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 solution to be examined does not comply with the test If the curve shows one point of inflexion, repeat the test but add 3.0 ml of a 25.0 g/l 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 solution to be examined does not comply with the test Sulphated ash (2.4.14) Not more than 0.1 per cent, determined on 1.0 g ASSAY Determine the density (2.2.5) of the solution to be examined Dilute 4.00 g to 100.0 ml with water R Transfer 25.0 ml of the solution to a separating funnel, add 25 ml of chloroform R, 10 ml of 0.1 M sodium hydroxide and 10.0 ml of a freshly prepared 50 g/l solution of potassium iodide R Shake well, allow to separate and discard the chloroform layer Shake the aqueous layer with three quantities, each of 10 ml, of chloroform R and discard the chloroform layers To the aqueous layer add 40 ml of hydrochloric acid R, allow to cool and titrate with 0.05 M potassium iodate until the deep-brown colour is almost discharged Add ml of chloroform R and continue the titration, shaking vigorously, until the chloroform layer no longer changes colour Carry out a blank titration on a mixture of 10.0 ml of the freshly prepared 50 g/l solution of potassium iodide R, 20 ml of water R and 40 ml of hydrochloric acid R ml of 0.05 M potassium iodate is equivalent to 35.4 mg of C22H40ClN LABELLING The label states the content of alcohol, if any 01/2005:1393 BENZBROMARONE Benzbromaronum C17H12Br2O3 1070 Mr 424.1 DEFINITION Benzbromarone contains not less than 98.0 and not more than the equivalent of 101.0 per cent of (3,5-dibromo4-hydroxyphenyl)(2-ethylbenzofuran-3-yl)methanone, calculated with reference to the dried substance CHARACTERS A white or almost white, crystalline powder, practically insoluble in water, freely soluble in acetone and in methylene chloride, sparingly soluble in alcohol It melts at about 152 °C IDENTIFICATION A Examine by infrared absorption spectrophotometry (2.2.24), comparing with the Ph Eur reference spectrum of benzbromarone B By means of a copper wire, previously ignited, introduce a small amount of the substance into the non-illuminated part of a flame The colour of the flame becomes green TESTS Appearance of solution Dissolve 1.25 g in dimethylformamide R and dilute to 25 ml with the same solvent The solution is clear (2.2.1) and not more intensely coloured than reference solution Y5 (2.2.2, Method II) Acidity or alkalinity Shake 0.5 g with 10 ml of carbon dioxide-free water R for and filter To 2.0 ml of the filtrate add 0.1 ml of methyl red solution R and 0.1 ml of 0.01 M hydrochloric acid The solution is red Add 0.3 ml of 0.01 M sodium hydroxide The solution is yellow Related substances Examine by liquid chromatography (2.2.29) Test solution Dissolve 0.125 g of the substance in 30 ml of methanol R and dilute to 50.0 ml with the mobile phase Reference solution (a) Dilute 1.0 ml of the test solution to 100 ml with the mobile phase Dilute ml of this solution to 10 ml with the mobile phase Reference solution (b) Dissolve 10 mg of benzarone CRS in the mobile phase and dilute to 20 ml with the mobile phase Reference solution (c) To ml of reference solution (b) add ml of the test solution and dilute to 100 ml with the mobile phase The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), — as mobile phase at a flow rate of 1.5 ml/min a mixture of volumes of glacial acetic acid R, 25 volumes of acetonitrile R, 300 volumes of water R and 990 volumes of methanol R, — as detector a spectrophotometer set at 231 nm Inject 20 µl of reference solution (c) Adjust the sensitivity of the system so that the heights of the principal peaks in the chromatogram obtained are at least 50 per cent of the full scale of the recorder The test is not valid unless the resolution between the first peak (impurity C) and the second peak (benzbromarone) is at least 10.0 Inject 20 µl of the test solution and 20 µl of reference solution (a) Continue the chromatography of the test solution for 2.5 times the retention time of benzbromarone If peaks other than the principal peak are observed in the chromatogram obtained with the test solution, these may be due to impurity A or to impurity B When the See the information section on general monographs (cover pages) Benzethonium chloride EUROPEAN PHARMACOPOEIA 5.0 chromatograms are recorded in the prescribed conditions, the relative retention times are : impurity A about 0.6 and impurity B about In the chromatogram obtained with the test solution : the area of the peak corresponding to impurity A is not greater than four times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.4 per cent) ; the area of the peak corresponding to impurity B is not greater than ten times the area of the principal peak in the chromatogram obtained with reference solution (a) (1 per cent) ; the area of any peak, apart from the principal peak and the peaks corresponding to impurity A and impurity B, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent) ; the sum of the areas of any such peaks is not greater than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent) Disregard any peak with an area less than 0.2 times that of the principal peak in the chromatogram obtained with reference solution (a) Halides expressed as chlorides (2.4.4) Shake 1.25 g of the substance to be examined with a mixture of ml of dilute nitric acid R and 15 ml of water R Filter Rinse the filter with water R and dilute the filtrate to 25 ml with the same solvent Dilute 2.5 ml to 15 ml with water R The solution obtained complies with the limit test for chlorides (400 ppm) Iron (2.4.9) Moisten the residue obtained in the test for sulphated ash with ml of hydrochloric acid R and evaporate to dryness on a water-bath Add 0.05 ml of hydrochloric acid R and 10 ml of water R and heat until boiling for Allow to cool Rinse the crucible with water R, collect the rinsings and dilute to 25 ml with water R Dilute ml of this solution to 10 ml with water R The solution complies with the limit test for iron (125 ppm) Heavy metals (2.4.8) 0.5 g complies with limit test C for heavy metals (20 ppm) Prepare the standard using ml of lead standard solution (10 ppm Pb) R Loss on drying (2.2.32) Not more than 0.5 per cent, determined on 1.000 g by drying in an oven in vacuo at 50 °C for h Sulphated ash (2.4.14) Not more than 0.1 per cent, determined on 1.0 g B R1 = R2 = R3 = Br : (6-bromo-2-ethylbenzofuran-3-yl)(3,5dibromo-4-hydroxyphenyl)methanone, C R1 = R2 = R3 = H : (2-ethylbenzofuran-3-yl)(4hydroxyphenyl)methanone (benzarone) 01/2005:0974 BENZETHONIUM CHLORIDE Benzethonii chloridum C27H42ClNO2 Mr 448.1 DEFINITION Benzethonium chloride contains not less than 97.0 per cent and not more than the equivalent of 103.0 per cent of N-benzyl-N,N-dimethyl-2-[2-[4-(1,1,3,3tetramethylbutyl)phenoxy]ethoxy]ethanaminium chloride, calculated with reference to the dried substance CHARACTERS A white or yellowish-white powder, very soluble in water and in alcohol, freely soluble in methylene chloride An aqueous solution froths copiously when shaken IDENTIFICATION A Melting point (2.2.14) : 158 °C to 164 °C, after drying at 105 °C for h B Examine by thin-layer chromatography (2.2.27), using as the coating substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm Test solution Dissolve 25 mg of the substance to be examined in water R and dilute to ml with the same solvent Reference solution Dissolve 25 mg of benzethonium ASSAY chloride CRS in water R and dilute to ml with the same Dissolve 0.300 g in 60 ml of methanol R Stir until solvent completely dissolved and add 10 ml of water R Titrate Apply to the plate 20 µl of each solution Develop over a with 0.1 M sodium hydroxide, determining the end-point path of 12 cm using a mixture of volumes of water R, potentiometrically (2.2.20) volumes of glacial acetic acid R and 100 volumes of ml of 0.1 M sodium hydroxide is equivalent to 42.41 mg of methanol R Dry the plate in a current of warm air and C17H12Br2O3 examine in ultraviolet light at 254 nm The principal spot in the chromatogram obtained with the test solution is STORAGE similar in position and size to the principal spot in the Store protected from light chromatogram obtained with the reference solution C To ml of dilute sodium hydroxide solution R add 0.1 ml IMPURITIES of bromophenol blue solution R1 and ml of methylene chloride R and shake The lower layer is colourless Add 0.1 ml of solution S (see Tests) and shake A blue colour develops in the lower layer D To ml of solution S add ml of dilute nitric acid R A white precipitate is formed which dissolves upon addition of ml of alcohol R The solution gives reaction (a) of chlorides (2.3.1) A R1 = R2 = H, R3 = Br : (3-bromo-4-hydroxyphenyl)(2ethylbenzofuran-3-yl)methanone, General Notices (1) apply to all monographs and other texts TESTS Solution S Dissolve 5.0 g in carbon dioxide-free water R and dilute to 50 ml with the same solvent 1071 Benzocaine EUROPEAN PHARMACOPOEIA 5.0 Appearance of solution Solution S is clear (2.2.1) and not more intensely coloured than reference solution Y6 (2.2.2, Method II) Acidity or alkalinity To 25 ml of solution S add 0.1 ml of phenolphthalein solution R The solution is colourless Add 0.3 ml of 0.01 M sodium hydroxide The solution is pink Add 0.1 ml of methyl red solution R and 0.5 ml of 0.01 M hydrochloric acid The solution is orange-red Volatile bases and salts of volatile bases (2.4.1) 0.20 g complies with limit test B for ammonium (50 ppm) Prepare the standard using 0.1 ml of ammonium standard solution (100 ppm NH4) R Replace heavy magnesium oxide by 2.0 ml of strong sodium hydroxide solution R Loss on drying (2.2.32) Not more than 5.0 per cent, determined on 1.000 g by drying in an oven at 100 °C to 105 °C for h Sulphated ash (2.4.14) Not more than 0.1 per cent, determined on 1.0 g Second identification : A, C, D A Melting point (2.2.14) : 89 °C to 92 °C B Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with benzocaine CRS C To about 50 mg in a test-tube add 0.2 ml of a 500 g/l solution of chromium trioxide R Cover the mouth of the tube with a piece of filter paper moistened with a freshly prepared mixture of equal volumes of a 50 g/l solution of sodium nitroprusside R and a 200 g/l solution of piperazine hydrate R Boil gently for at least 30 s A blue colour develops on the filter paper D Dissolve about 50 mg in alcohol R and dilute to 100 ml with the same solvent ml of the solution gives the reaction of primary aromatic amines (2.3.1) STORAGE Store protected from light STORAGE Store protected from light TESTS Appearance of solution Dissolve 1.0 g in alcohol R and dilute to 20 ml with the same solvent The solution is clear (2.2.1) and colourless (2.2.2, Method II) ASSAY Dissolve 2.000 g in water R and dilute to 100.0 ml with Acidity or alkalinity Dissolve 0.5 g in 10 ml of alcohol R the same solvent Transfer 25.0 ml of the solution to a previously neutralised to 0.05 ml of phenolphthalein separating funnel, add 10 ml of a g/l solution of sodium solution R Add 10 ml of carbon dioxide-free water R The hydroxide R, 10.0 ml of a freshly prepared 50 g/l solution solution remains colourless and not more than 0.5 ml of of potassium iodide R and 25 ml of methylene chloride R 0.01 M sodium hydroxide is required to change the colour Shake vigorously, allow to separate and discard the lower of the indicator layer Shake the upper layer with three quantities, each of Loss on drying (2.2.32) Not more than 0.5 per cent, 10 ml, of methylene chloride R and discard the lower layers determined on 1.00 g by drying in vacuo To the upper layer add 40 ml of hydrochloric acid R, allow to cool and titrate with 0.05 M potassium iodate until the deep Sulphated ash (2.4.14) Not more than 0.1 per cent, determined on 1.0 g brown colour is almost discharged Add ml of methylene chloride R and continue the titration, shaking vigorously, ASSAY until the lower layer is no longer brown Carry out a blank Dissolve 0.400 g in a mixture of 25 ml of hydrochloric titration using a mixture of 10.0 ml of a freshly prepared 50 g/l solution of potassium iodide R, 20 ml of water R and acid R and 50 ml of water R Carry out the determination of primary aromatic amino-nitrogen (2.5.8) 40 ml of hydrochloric acid R ml of 0.1 M sodium nitrite is equivalent to 16.52 mg ml of 0.05 M potassium iodate is equivalent to 44.81 mg of C9H11NO2 of C27H42ClNO2 01/2005:0011 01/2005:0066 BENZOCAINE BENZOIC ACID Benzocainum C9H11NO2 Acidum benzoicum Mr 165.2 DEFINITION Benzocaine contains not less than 99.0 per cent and not more than the equivalent of 101.0 per cent of ethyl 4-aminobenzoate, calculated with reference to the dried substance C7 H O Mr 122.1 DEFINITION Benzoic acid contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent of benzenecarboxylic acid CHARACTERS A white, crystalline powder or colourless crystals, very slightly soluble in water, freely soluble in alcohol CHARACTERS A white, crystalline powder or colourless crystals, odourless or with a very slight characteristic odour, slightly soluble in water, soluble in boiling water, freely soluble in alcohol and in fatty oils IDENTIFICATION First identification : A, B IDENTIFICATION A Melting point (2.2.14) : 121 °C to 124 °C 1072 See the information section on general monographs (cover pages) Benzoyl peroxide, hydrous EUROPEAN PHARMACOPOEIA 5.0 01/2005:0704 B Solution S (see Tests) gives reaction (a) of benzoates (2.3.1) TESTS Solution S Dissolve 5.0 g in alcohol R and dilute to 100 ml with the same solvent BENZOYL PEROXIDE, HYDROUS Benzoylis peroxidum cum aqua Appearance of solution Solution S is clear (2.2.1) and colourless (2.2.2, Method II) Carbonisable substances Dissolve 0.5 g with shaking in ml of sulphuric acid R After min, the solution is not more intensely coloured than reference solution Y5 (2.2.2, Method I) Oxidisable substances Dissolve 0.2 g in 10 ml of boiling water R Cool, shake and filter To the filtrate add ml of dilute sulphuric acid R and 0.2 ml of 0.02 M potassium permanganate After min, the solution is still coloured pink Halogenated compounds and halides C14H10O4 Mr 242.2 DEFINITION Content : — dibenzoyl peroxide : 70.0 per cent to 77.0 per cent, — water : minimum 20.0 per cent CHARACTERS Appearance : white, amorphous or granular powder Solubility : practically insoluble in water, soluble in acetone, soluble in methylene chloride with the separation of water, Solution (a) Dissolve 6.7 g of the substance to be examined slightly soluble in alcohol in a mixture of 40 ml of M sodium hydroxide and 50 ml It loses water rapidly on exposure to air with a risk of of alcohol R and dilute to 100.0 ml with water R To 10.0 ml explosion of this solution add 7.5 ml of dilute sodium hydroxide Mix the entire sample thoroughly before carrying out the solution R and 0.125 g of nickel-aluminium alloy R and following tests heat on a water-bath for 10 Allow to cool to room temperature, filter into a 25 ml volumetric flask and wash IDENTIFICATION with three quantities, each of ml, of alcohol R Dilute the filtrate and washings to 25.0 ml with water R This solution First identification : B Second identification : A, C, D is used to prepare solution A Solution (b) In the same manner, prepare a similar solution A Dissolve 80.0 mg in alcohol R and dilute to 100.0 ml with the same solvent Dilute 10.0 ml of the solution without the substance to be examined This solution is used to 100.0 ml with alcohol R (solution A) Dilute 10.0 ml to prepare solution B of solution A to 100.0 ml with alcohol R (solution B) In four 25 ml volumetric flasks, place separately 10 ml Examined between 250 nm and 300 nm (2.2.25), of solution (a), 10 ml of solution (b), 10 ml of chloride solution A shows an absorption maximum at 274 nm and standard solution (8 ppm Cl) R (used to prepare solution C) a shoulder at about 282 nm Examined between 220 nm and 10 ml of water R To each flask add ml of ferric and 250 nm, solution B shows an absorption maximum at ammonium sulphate solution R5, mix and add dropwise 235 nm The ratio of the absorbance at the maximum at and with swirling ml of nitric acid R and ml of mercuric 235 nm (solution B) to that at the maximum at 274 nm thiocyanate solution R Shake Dilute the contents of each (solution A) is 1.17 to 1.21 flask to 25.0 ml with water R and allow the solutions to B Infrared absorption spectrophotometry (2.2.24) stand in a water-bath at 20 °C for 15 Measure at 460 nm Comparison : Ph Eur reference spectrum of hydrous the absorbance (2.2.25) of solution A using solution B as benzoyl peroxide the compensation liquid, and the absorbance of solution C C Dissolve about 25 mg in ml of acetone R Add ml of a using the solution obtained with 10 ml of water R as the 10 g/l solution of diethylphenylenediamine sulphate R compensation liquid The absorbance of solution A is not and mix A red colour develops which quickly darkens greater than that of solution C (300 ppm) and becomes dark violet within Heavy metals (2.4.8) 12 ml of solution S complies with limit test B for heavy metals (10 ppm) Prepare the standard using D To g add ml of alcohol R, ml of dilute sodium hydroxide solution R and 10 ml of water R Boil the a mixture of ml of lead standard solution (1 ppm Pb) R mixture under reflux for 20 Cool The solution gives and ml of alcohol R reaction (c) of benzoates (2.3.1) Sulphated ash (2.4.14) Not more than 0.1 per cent, determined on 1.0 g TESTS Acidity Dissolve a quantity of the substance to be examined ASSAY containing the equivalent of 1.0 g of dibenzoyl peroxide in Dissolve 0.200 g in 20 ml of alcohol R and titrate with 0.1 M 25 ml of acetone R, add 75 ml of water R and filter Wash sodium hydroxide, using 0.1 ml of phenol red solution R as the residue with two quantities, each of 10 ml, of water R indicator, until the colour changes from yellow to violet-red Combine the filtrate and the washings and add 0.25 ml of phenolphthalein solution R1 Not more than 1.25 ml of 0.1 M sodium hydroxide is required to change the colour of ml of 0.1 M sodium hydroxide is equivalent to 12.21 mg the indicator Carry out a blank test of C7H6O2 All glassware used must be chloride-free and may be prepared by soaking overnight in a 500 g/l solution of nitric acid R, rinsed with water R and stored full of water R It is recommended that glassware be reserved for this test General Notices (1) apply to all monographs and other texts 1073 Benzoyl peroxide, hydrous EUROPEAN PHARMACOPOEIA 5.0 Related substances Liquid chromatography (2.2.29) Prepare the solutions immediately before use Chlorides (2.4.4) : maximum 0.4 per cent Dissolve a quantity of the substance to be examined containing the equivalent of 0.5 g of dibenzoyl peroxide in Test solution Dissolve a quantity of the substance to be 15 ml of acetone R Add, while stirring, 50 ml of 0.05 M examined containing the equivalent of 0.10 g of dibenzoyl peroxide in acetonitrile R and dilute to 50 ml with the same nitric acid Allow to stand for 10 and filter Wash the residue with quantities, each of 10 ml, of 0.05 M nitric solvent acid Combine the filtrate and the washings and dilute to Reference solution (a) Dilute 1.0 ml of the test solution to 100 ml with 0.05 M nitric acid 2.5 ml of the solution diluted 100.0 ml with acetonitrile R Dilute 1.0 ml of this solution to to 15.0 ml with water R complies with the limit test for 10.0 ml with acetonitrile R chlorides Reference solution (b) Dissolve 30.0 mg of benzoic acid R ASSAY in the mobile phase and dilute to 100.0 ml with the mobile phase Dilute 1.0 ml of the solution to 10.0 ml with the Solution (a) Dissolve 2.500 g immediately before use in mobile phase 75 ml of dimethylformamide R and dilute to 100.0 ml with Reference solution (c) Dissolve 50.0 mg of ethyl benzoate R the same solvent Dibenzoyl peroxide To 5.0 ml of solution (a) add 20 ml in the mobile phase and dilute to 100.0 ml with the mobile of acetone R and ml of a 500 g/l solution of potassium phase Dilute 1.0 ml of the solution to 100.0 ml with the iodide R and mix Allow to stand for Titrate with mobile phase 0.1 M sodium thiosulphate using ml of starch solution R, Reference solution (d) Dissolve 50.0 mg of benzaldehyde R added towards the end of the titration, as indicator Carry in the mobile phase and dilute to 100.0 ml with the mobile out a blank titration phase Dilute 1.0 ml of the solution to 100.0 ml with the ml of 0.1 M sodium thiosulphate is equivalent to 12.11 mg mobile phase of C14H10O4 Reference solution (e) Dissolve 30.0 mg of benzoic acid R Water (2.5.12) Carry out the semi-micro determination and 30.0 mg of benzaldehyde R in the mobile phase and dilute to 100.0 ml with the mobile phase Dilute 1.0 ml of the of water, using 5.0 ml of solution (a) Use as the solvent a mixture of 20.0 ml of anhydrous methanol R and solution to 10.0 ml with the mobile phase 3.0 ml of a 100 g/l solution of potassium iodide R in Column : dimethylformamide R After adding solution (a), stir for before starting the titration Carry out a blank — size : l = 0.25 m, Ø = 4.6 mm, determination — stationary phase: octadecylsilyl silica gel for Calculate the percentage content of water using the chromatography R (10 µm), expression : Mobile phase : glacial acetic acid R, acetonitrile R, water R (1:500:500 V/V/V) Flow rate : ml/min Detection : spectrophotometer at 235 nm n1 = Injection : 20 µl loop injector number of millilitres of iodosulphurous reagent R used in the sample determination, n2 = number of millilitres of iodosulphurous reagent R used in the blank determination, w = water equivalent of iodosulphurous reagent R in milligrams of water per millilitre of reagent, m = mass of the substance to be examined used for the preparation of solution (a) in grams, Run time : times the retention time of dibenzoyl peroxide Relative retention with reference to dibenzoyl peroxide (retention time = about 28.4 min) : impurity B = about 0.15 ; impurity A = about 0.2 ; impurity C = about 0.4 System suitability : reference solution (e) : = percentage content of dibenzoyl peroxide — resolution : minimum between the peaks corresponding p to benzoic acid and benzaldehyde STORAGE Limits : In a container that has been treated to reduce static — impurity A : not more than the area of the principal peak discharge and that has a device for release of excess pressure, in the chromatogram obtained with reference solution (d) at a temperature of °C to °C, protected from light (0.25 per cent), — impurity B : not more than the area of the principal peak IMPURITIES in the chromatogram obtained with reference solution (b) (1.5 per cent), — impurity C : not more than the area of the principal peak in the chromatogram obtained with reference solution (c) (0.25 per cent), — any other impurity : not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.1 per cent), A R = H : benzaldehyde, — disregard limit : 0.2 times the area of the principal peak B R = OH : benzoic acid, in the chromatogram obtained with reference solution (a) C R = O-CH2-CH3 : ethyl benzoate (0.02 per cent) 1074 See the information section on general monographs (cover pages) Benzyl alcohol EUROPEAN PHARMACOPOEIA 5.0 BENZYL 01/2005:0256 — stationary phase : macrogol 20 000 R (film thickness corrected 0.5 µm) Carrier gas : helium for chromatography R ALCOHOL Linear velocity : 25 cm/s Temperature : Alcohol benzylicus Column C7H 8O Mr 108.1 DEFINITION Phenylmethanol Content : 98.0 per cent to 100.5 per cent Time (min) Temperature (°C) - 34 50 → 220 34 - 69 220 Injection port 200 Detector 310 Detection : flame ionisation Benzyl alcohol not intended for parenteral use Injection : without air-plug, 0.1 µl of the test solution and CHARACTERS 0.1 µl of reference solution (a) Appearance : clear, colourless, oily liquid Relative retention with reference to benzyl alcohol Solubility : soluble in water, miscible with alcohol and with (retention time = about 26 min) : ethylbenzene = about 0.28 ; fatty and essential oils dicyclohexyl = about 0.59 ; benzaldehyde = about 0.68 ; cyclohexylmethanol = about 0.71 Relative density : 1.043 to 1.049 System suitability : reference solution (a) : IDENTIFICATION — resolution : minimum 3.0 between the peaks Infrared absorption spectrophotometry (2.2.24) corresponding to benzaldehyde and to Comparison : Ph Eur reference spectrum of benzyl cyclohexylmethanol alcohol In the chromatogram obtained with the test solution, verify that there are no peaks with the same retention time as the TESTS standards Appearance of solution Shake 2.0 ml with 60 ml of water R Limits : It dissolves completely The solution is clear (2.2.1) and — benzaldehyde : not more than the difference between colourless (2.2.2, Method II) the area of the peak due to benzaldehyde in the Acidity To 10 ml add 10 ml of alcohol R and ml of chromatogram obtained with reference solution (a) phenolphthalein solution R Not more than ml of 0.1 M and the area of the peak due to benzaldehyde in the sodium hydroxide is required to change the colour of the chromatogram obtained with the test solution (0.15 per indicator to pink cent) Refractive index (2.2.6) : 1.538 to 1.541 — cyclohexylmethanol : not more than the difference Peroxide value (2.5.5) : maximum between the area of the peak due to cyclohexylmethanol in the chromatogram obtained with reference solution (a) Benzaldehyde and other related substances Gas and the area of the peak due to cyclohexylmethanol in the chromatography (2.2.28) chromatogram obtained with the test solution (0.10 per Test solution The substance to be examined cent) Standard solution (a) Dissolve 0.100 g of ethylbenzene R — total of other peaks with a relative retention less than in the test solution and dilute to 10.0 ml with the same that of benzyl alcohol : not more than times the area solution Dilute 2.0 ml of this solution to 20.0 ml with the of the peak due to ethylbenzene in the chromatogram test solution obtained with reference solution (a) (0.04 per cent) Standard solution (b) Dissolve 2.000 g of dicyclohexyl R — total of peaks with a relative retention greater than that in the test solution and dilute to 10.0 ml with the same of benzyl alcohol : not more than the area of the peak solution Dilute 2.0 ml of this solution to 20.0 ml with the due to dicyclohexyl in the chromatogram obtained with test solution reference solution (a) (0.3 per cent) Reference solution (a) Dissolve 0.750 g of benzaldehyde R — disregard limit : 0.01 times the area of the peak due and 0.500 g of cyclohexylmethanol R in the test solution to ethylbenzene in the chromatogram obtained with and dilute to 25.0 ml with the test solution Add 1.0 ml of reference solution (a) (0.0001 per cent) this solution to a mixture of 2.0 ml of standard solution (a) Benzyl alcohol intended for parenteral use and 3.0 ml of standard solution (b) and dilute to 20.0 ml with the test solution Injection : without air-plug, 0.1 µl of the test solution and Reference solution (b) Dissolve 0.250 g of benzaldehyde R 0.1 µl of reference solution (b) and 0.500 g of cyclohexylmethanol R in the test solution Relative retention with reference to benzyl alcohol and dilute to 25.0 ml with the test solution Add 1.0 ml of (retention time = about 26 min) : ethylbenzene = about 0.28 ; this solution to a mixture of 2.0 ml of standard solution (a) dicyclohexyl = about 0.59 ; benzaldehyde = about 0.68 ; and 2.0 ml of standard solution (b) and dilute to 20.0 ml cyclohexylmethanol = about 0.71 with the test solution System suitability : reference solution (b) : Column : — resolution : minimum 3.0 between the peaks — material : fused silica, corresponding to benzaldehyde and to cyclohexylmethanol — size : l = 30 m, Ø = 0.32 mm, General Notices (1) apply to all monographs and other texts 1075 Benzyl benzoate EUROPEAN PHARMACOPOEIA 5.0 01/2005:0705 In the chromatogram obtained with the test solution, verify that there are no peaks with the same retention time as the standards BENZYL BENZOATE Limits : Benzylis benzoas — benzaldehyde : not more than the difference between the area of the peak due to benzaldehyde in the chromatogram obtained with reference solution (b) and the area of the peak due to benzaldehyde in the chromatogram obtained with the test solution (0.05 per cent) — cyclohexylmethanol : not more than the difference between the area of the peak due to cyclohexylmethanol in the chromatogram obtained with reference solution (b) and the area of the peak due to cyclohexylmethanol in the chromatogram obtained with the test solution (0.10 per cent) — total of other peaks with a relative retention less than that of benzyl alcohol : not more than twice the area of the peak due to ethylbenzene in the chromatogram obtained with reference solution (b) (0.02 per cent) — total of peaks with a relative retention greater than that of benzyl alcohol: not more than the area of the peak due to dicyclohexyl in the chromatogram obtained with reference solution (b) (0.2 per cent) C14H12O2 Mr 212.2 DEFINITION Benzyl benzoate contains not less than 99.0 per cent and not more than the equivalent of 100.5 per cent of phenylmethyl benzoate CHARACTERS Colourless or almost colourless crystals or a colourless or almost colourless, oily liquid, practically insoluble in water, miscible with alcohol, with methylene chloride and with fatty and essential oils It boils at about 320 °C IDENTIFICATION First identification : A Second identification : B, C — disregard limit : 0.01 times the area of the peak due A Examine by infrared absorption spectrophotometry to ethylbenzene in the chromatogram obtained with (2.2.24), comparing with the Ph Eur reference spectrum reference solution (b) (0.0001 per cent) of benzyl benzoate Residue on evaporation : maximum 0.05 per cent B To g add 25 ml of alcoholic potassium hydroxide solution R and boil under a reflux condenser for h After ensuring that the substance to be examined complies Remove the ethanol on a water-bath, add 50 ml of water R with the test for peroxide value, evaporate 10.0 g to dryness and distil ; collect about 25 ml of distillate and use it for on a water-bath, dry at 100-105 °C for h and allow to cool identification test C Acidify the liquid remaining in the in a desiccator The residue weighs a maximum of mg distillation flask with dilute hydrochloric acid R ; a white precipitate is formed, which, when washed with water R and dried in vacuo, melts (2.2.14) at 121 °C to 124 °C C To the distillate obtained in identification test B add 2.5 g ASSAY of potassium permanganate R and ml of dilute sodium To 0.900 g (m g) add 15.0 ml of a freshly prepared mixture of hydroxide solution R Boil under a reflux condenser for volume of acetic anhydride R and volumes of pyridine R 15 min, cool and filter Acidify the filtrate with dilute and boil under a reflux condenser for 30 Cool and hydrochloric acid R ; a white precipitate is formed which, add 25 ml of water R Using 0.25 ml of phenolphthalein when washed with water R and dried in vacuo, melts solution R as indicator, titrate with M sodium hydroxide (2.2.14) at 121 °C to 124 °C (n1 ml) Carry out a blank titration (n2 ml) TESTS Calculate the percentage content of C7H8O from the Acidity Dissolve 2.0 g in alcohol R and dilute to 10 ml with expression : the same solvent Titrate with 0.1 M sodium hydroxide using phenolphthalein solution R as indicator Not more than 0.2 ml is required to change the colour of the indicator to pink Relative density (2.2.5) : 1.118 to 1.122 Refractive index (2.2.6) : 1.568 to 1.570 STORAGE Freezing point (2.2.18) Not less than 17.0 °C In an airtight container, under nitrogen, protected from light Sulphated ash (2.4.14) Not more than 0.1 per cent, at a temperature between °C and °C determined on 1.0 g LABELLING The label states, where applicable, that the substance is suitable for use in the manufacture of parenteral dosage forms 1076 ASSAY To 2.000 g add 50.0 ml of 0.5 M alcoholic potassium hydroxide and boil gently under a reflux condenser for h Titrate the hot solution with 0.5 M hydrochloric acid using ml of phenolphthalein solution R as indicator Carry out a blank determination See the information section on general monographs (cover pages) EUROPEAN PHARMACOPOEIA 5.0 Benzylpenicillin, benzathine C Place about mg in a test-tube about 150 mm long and 15 mm in diameter Moisten with 0.05 ml of water R and add ml of sulphuric acid-formaldehyde reagent R STORAGE Mix the contents of the tube by swirling ; the solution is practically colourless Place the test-tube on a water-bath Store in an airtight, well-filled container, protected from for ; a reddish-brown colour develops light D To 0.1 g add ml of M sodium hydroxide and shake for Shake the mixture with two quantities, each of ml, of ether R Evaporate the combined ether layers 01/2005:0373 to dryness and dissolve the residue in ml of alcohol (50 per cent V/V) R Add ml of picric acid solution R, BENZYLPENICILLIN, BENZATHINE heat at 90 °C for and allow to cool slowly Separate the crystals and recrystallise from alcohol (25 per cent V/V) R containing 10 g/l of picric acid R The Benzylpenicillinum benzathinum crystals melt (2.2.14) at about 214 °C ml of 0.5 M alcoholic potassium hydroxide is equivalent to 106.1 mg of C14H12O2 TESTS Acidity or alkalinity To 0.50 g add 100 ml of carbon dioxide-free water R and shake for Filter through a sintered-glass filter To 20 ml of the filtrate add 0.1 ml of bromothymol blue solution R1 The solution is green or yellow Not more than 0.2 ml of 0.02 M sodium hydroxide is required to change the colour of the indicator to blue C48H56N6O8S2 Mr 909 Related substances Liquid chromatography (2.2.29) Prepare the solutions immediately before use, using DEFINITION sonication (for about min) to dissolve the samples Avoid any over heating during the sample preparation Benzathine benzylpenicillin is N,N′-dibenzylethane-1,2diamine compound (1:2) with (2S,5R,6R)-3,3-dimethyl-7-oxo- Test solution Dissolve 70.0 mg of the substance to be 6-[(phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2examined in 25 ml of methanol R and dilute to 50.0 ml with carboxylic acid It contains not less than 96.0 per cent and a solution containing 6.8 g/l of potassium dihydrogen not more than the equivalent of 102.0 per cent of benzathine phosphate R and 1.02 g/l of disodium hydrogen benzylpenicillin and not less than 24.0 per cent and not phosphate R more than 27.0 per cent of N,N′-dibenzylethylenediamine Reference solution (a) Dissolve 70.0 mg of benzathine (benzathine C16H20N2 ; Mr 240.3), both calculated with benzylpenicillin CRS in 25 ml of methanol R and dilute reference to the anhydrous substance It contains a variable to 50.0 ml with a solution containing 6.8 g/l of potassium quantity of water Dispersing or suspending agents may be dihydrogen phosphate R and 1.02 g/l of disodium hydrogen added phosphate R Reference solution (b) Dilute 1.0 ml of reference solution (a) CHARACTERS A white powder, very slightly soluble in water, freely soluble to 100.0 ml with mobile phase A in dimethylformamide and in formamide, slightly soluble in Column : alcohol — size : l = 0.25 m, Ø = 4.0 mm, — stationary phase : end-capped octadecylsilyl silica gel IDENTIFICATION for chromatography R (5 µm), First identification : A — temperature : 40 °C Second identification : B, C, D Mobile phase : A Examine by infrared absorption spectrophotometry — mobile phase A : mix 10 volumes of a 34 g/l solution of (2.2.24), comparing with the spectrum obtained with potassium dihydrogen phosphate R adjusted to pH 3.5 benzathine benzylpenicillin CRS with phosphoric acid R, 30 volumes of methanol R and B Examine by thin-layer chromatography (2.2.27), using a 60 volumes of water R, TLC silanised silica gel plate R — mobile phase B : mix 10 volumes of a 34 g/l solution of Test solution Dissolve 25 mg of the substance to be potassium dihydrogen phosphate R adjusted to pH 3.5 examined in ml of methanol R with phosphoric acid R, 30 volumes of water R and Reference solution Dissolve 25 mg of benzathine 60 volumes of methanol R, benzylpenicillin CRS in ml of methanol R Time Mobile phase A Mobile phase B Apply to the plate µl of each solution Develop over a (min) (per cent V/V) (per cent V/V) path of 15 cm using a mixture of 30 volumes of acetone R - 10 75 25 and 70 volumes of a 154 g/l solution of ammonium 10 - 20 75 → 25 → 100 acetate R, the pH of which has been adjusted to 7.0 with ammonia R Allow the plate to dry in air and expose it to 20 - 55 100 iodine vapour until the spots appear Examine in daylight 55 - 70 75 25 The two principal spots in the chromatogram obtained with the test solution are similar in position, colour and Flow rate : ml/min size to the two principal spots in the chromatogram obtained with the reference solution The test is not valid Detection : spectrophotometer at 220 nm unless the chromatogram obtained with the reference Injection : 20 µl ; inject the test solution and the reference solution shows two clearly separated spots solutions General Notices (1) apply to all monographs and other texts 1077 Benzylpenicillin potassium EUROPEAN PHARMACOPOEIA 5.0 System suitability : reference solution (a) — relative retention with reference to benzylpenicillin : benzathine = 0.3 to 0.4 ; impurity C = about 2.4 If necessary, adjust the concentration of methanol in the mobile phase Limits : — impurity C : not more than twice the sum of the areas of the principal peaks in the chromatogram obtained with reference solution (b) (2 per cent), — any other impurity : not more than the sum of the areas C benzylpenicilloic acids benzathide, of the principal peaks in the chromatogram obtained with reference solution (b) (1 per cent), — disregard limit: 0.05 times the sum of the areas of the principal peaks in the chromatogram obtained with reference solution (b) (0.05 per cent) Water (2.5.12) : 5.0 per cent to 8.0 per cent, determined on 0.300 g by the semi-micro determination of water Bacterial endotoxins (2.6.14, Method E) Suspend 20 mg of the substance to be examined in 20 ml of a solution of 0.1 M D (3S,7R,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7asodium hydroxide diluted to 100, shake thoroughly and tetrahydroimidazo[5,1-b]thiazole-3,7-dicarboxylic centrifuge The supernatant contains less than 0.13 IU/ml, acid (penillic acid of benzylpenicillin), if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins ASSAY Liquid chromatography (2.2.29) as described in the test for related substances Mobile phase : phosphate buffer solution pH 3.5 R, methanol R, water R (10:35:55 V/V/V) Injection : 20 µl ; inject the test solution and reference solution (a) Calculate the percentage contents of benzathine and of benzathine benzylpenicillin Calculate the latter by multiplying the percentage content of benzylpenicillin by 1.36 STORAGE Store in an airtight container If the substance is sterile, store in a sterile, airtight, tamper-proof container LABELLING The label states : — where applicable, the name and quantity of any added dispersing or suspending agent, — where applicable, that the substance is free from bacterial endotoxins IMPURITIES E (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penicilloic acids of benzylpenicillin), F (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penilloic acids of benzylpenicillin) 01/2005:0113 BENZYLPENICILLIN POTASSIUM Benzylpenicillinum kalicum A monobenzylethylenediamine, C16H17KN2O4S B phenylacetic acid, DEFINITION Benzylpenicillin potassium is potassium (2S,5R,6R)3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-1azabicyclo[3.2.0]heptane-2-carboxylate, a substance produced by the growth of certain strains of Penicillium notatum or related organisms, or obtained by any other 1078 Mr 372.5 See the information section on general monographs (cover pages) Benzylpenicillin potassium EUROPEAN PHARMACOPOEIA 5.0 means It contains not less than 96.0 per cent and not more than the equivalent of 102.0 per cent of benzylpenicillin potassium, calculated with reference to the dried substance CHARACTERS A white or almost white, crystalline powder, very soluble in water, practically insoluble in fatty oils and in liquid paraffin IDENTIFICATION First identification : A, D phase Inject 20 µl of test solution (b) and start the elution isocratically Immediately after elution of the benzylpenicillin peak start the following linear gradient : Time (min) Mobile phase A (per cent V/V) Mobile phase B (per cent V/V) Comment - 20 70 → 30 → 100 linear gradient 20 - 35 100 isocratic 35 - 50 70 30 re-equilibration Inject water R and use the same elution pattern to obtain a blank In the chromatogram obtained with test solution (b), the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (d) (1 per cent) B Examine by thin-layer chromatography (2.2.27), using a Loss on drying (2.2.32) Not more than 1.0 per cent, TLC silanised silica gel plate R determined on 1.000 g by drying in an oven at 100-105 °C Test solution Dissolve 25 mg of the substance to be Bacterial endotoxins (2.6.14, Method E) : less than examined in ml of water R 0.16 IU/mg, if intended for use in the manufacture of Reference solution (a) Dissolve 25 mg of benzylpenicillin parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins potassium CRS in ml of water R Second identification : B, C, D A Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with benzylpenicillin potassium CRS Reference solution (b) Dissolve 25 mg of benzylpenicillin ASSAY potassium CRS and 25 mg of phenoxymethylpenicillin Examine by liquid chromatography (2.2.29) Prepare the potassium CRS in ml of water R solutions immediately before use Apply to the plate µl of each solution Develop over a Test solution (a) Dissolve 50.0 mg of the substance to be path of 15 cm using a mixture of 30 volumes of acetone R examined in water R and dilute to 50.0 ml with the same and 70 volumes of a 154 g/l solution of ammonium solvent acetate R, the pH of which has been adjusted to 5.0 with Test solution (b) Dissolve 80.0 mg of the substance to be glacial acetic acid R Allow the plate to dry in air and expose it to iodine vapour until the spots appear Examine examined in water R and dilute to 20.0 ml with the same solvent in daylight The principal spot in the chromatogram Reference solution (a) Dissolve 50.0 mg of benzylpenicillin obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram sodium CRS in water R and dilute to 50.0 ml with the same solvent obtained with reference solution (a) The test is not valid unless the chromatogram obtained with reference Reference solution (b) Dissolve 10 mg of benzylpenicillin solution (b) shows clearly separated spots sodium CRS and 10 mg of phenylacetic acid CRS in water R and dilute to 50 ml with the same solvent C Place about mg in a test-tube about 150 mm long and 15 mm in diameter Moisten with 0.05 ml of water R and Reference solution (c) Dilute 1.0 ml of reference solution (a) add ml of sulphuric acid-formaldehyde reagent R to 20.0 ml with water R Dilute 1.0 ml of the solution to Mix the contents of the tube by swirling ; the solution is 50.0 ml with water R practically colourless Place the test-tube on a water-bath Reference solution (d) Dilute 4.0 ml of reference solution (a) for ; a reddish-brown colour develops to 100.0 ml with water R D It gives reaction (a) of potassium (2.3.1) The chromatographic procedure may be carried out using : — a column 0.25 m long and 4.6 mm in internal TESTS diameter packed with octadecylsilyl silica gel for chromatography R (5 µm), pH (2.2.3) Dissolve 2.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent The pH of the — as mobile phase at a flow rate of 1.0 ml/min : solution is 5.5 to 7.5 Mobile phase A Mix 10 volumes of a 68 g/l solution of Specific optical rotation (2.2.7) Dissolve 0.500 g in carbon potassium dihydrogen phosphate R adjusted to pH 3.5 dioxide-free water R and dilute to 25.0 ml with the same with a 500 g/l solution of dilute phosphoric acid R, solvent The specific optical rotation is + 270 to + 300, 30 volumes of methanol R and 60 volumes of water R, calculated with reference to the dried substance Mobile phase B Mix 10 volumes of a 68 g/l solution of potassium dihydrogen phosphate R adjusted to pH 3.5 Absorbance (2.2.25) Dissolve 94.0 mg in water R and with a 500 g/l solution of dilute phosphoric acid R, dilute to 50.0 ml with the same solvent Measure the 40 volumes of water R and 50 volumes of methanol R, absorbance of the solution at 325 nm, 280 nm and at the maximum at 264 nm, diluting the solution, if necessary, for — as detector a spectrophotometer set at 225 nm the measurement at 264 nm The absorbances at 325 nm Equilibrate the column with a mobile phase ratio A:B and 280 nm not exceed 0.10 and that at the maximum of 70:30 Inject 20 µl of reference solution (b) The test at 264 nm is 0.80 to 0.88, calculated on the basis of the is not valid unless the resolution between the principal undiluted (1.88 g/l) solution Verify the resolution of the peaks is at least 6.0 (if necessary, adjust the ratio A:B of apparatus (2.2.25) ; the ratio of the absorbances is at least 1.7 the mobile phase) and the mass distribution ratio for the Related substances Examine by liquid chromatography second peak (benzylpenicillin) is 4.0 to 6.0 Inject 20 µl of (2.2.29) as described under Assay Inject 20 µl of reference reference solution (c) Adjust the system to obtain a peak solution (d) and elute isocratically with the chosen mobile with a signal-to-noise ratio of at least General Notices (1) apply to all monographs and other texts 1079 Benzylpenicillin, procaine EUROPEAN PHARMACOPOEIA 5.0 Calculate the percentage content of benzylpenicillin potassium by multiplying the percentage content of benzylpenicillin sodium by 1.045 STORAGE Store in an airtight container If the substance is sterile, store in a sterile, airtight, tamper-proof container LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins IMPURITIES F (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penilloic acids of benzylpenicillin) 01/2005:0115 BENZYLPENICILLIN, PROCAINE Benzylpenicillinum procainum A (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid (6-aminopenicillanic acid), C29H38N4O6S,H2O DEFINITION Procaine benzylpenicillin is the monohydrate of the (2S,5R,6R)-3,3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid compound with 2-(diethylamino)ethyl 4-aminobenzoate It contains not less than 96.0 per cent and not more than the equivalent of 102.0 per cent of procaine benzylpenicillin and not less than 39.0 per cent and not more than 42.0 per cent of procaine (C13H20N2O2 ; Mr 236.3), both calculated with reference to the anhydrous substance Dispersing or suspending agents (for example, lecithin and polysorbate 80) may be added B phenylacetic acid, CHARACTERS A white, crystalline powder, slightly soluble in water, sparingly soluble in alcohol C (2S,5R,6R)-6-[[(4-hydroxyphenyl)acetyl]amino]-3,3dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2carboxylic acid, D (3S,7R,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7atetrahydroimidazo[5,1-b]thiazole-3,7-dicarboxylic acid (penillic acid of benzylpenicillin), E (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penicilloic acids of benzylpenicillin), 1080 Mr 588.7 IDENTIFICATION First identification : A Second identification : B, C, D A Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with procaine benzylpenicillin CRS B Examine by thin-layer chromatography (2.2.27), using a TLC silanised silica gel plate R Test solution Dissolve 25 mg of the substance to be examined in ml of acetone R Reference solution Dissolve 25 mg of procaine benzylpenicillin CRS in ml of acetone R Apply to the plate µl of each solution Develop over a path of 15 cm using a mixture of 30 volumes of acetone R and 70 volumes of a 154 g/l solution of ammonium acetate R, the pH of which has been adjusted to 7.0 with ammonia R Allow the plate to dry in air and expose it to iodine vapour until the spots appear Examine in daylight The principal spots in the chromatogram obtained with the test solution are similar in position, colour and size to the principal spots in the chromatogram obtained with the reference solution The test is not valid unless the chromatogram obtained with the reference solution shows clearly separated spots See the information section on general monographs (cover pages) EUROPEAN PHARMACOPOEIA 5.0 Benzylpenicillin, procaine — as mobile phase at a flow rate of 1.75 ml/min a mixture prepared as follows : mix 250 ml of acetonitrile R, 250 ml of water R and 500 ml of a solution containing 14 g/l of potassium dihydrogen phosphate R and 6.5 g/l of tetrabutylammonium hydroxide solution (400 g/l) R adjusted to pH 7.0 with M potassium hydroxide ; adjust the mixture to pH 7.2 with dilute phosphoric acid R, if necessary, — as detector a spectrophotometer set at 225 nm Inject 10 µl of reference solution (b) When the chromatogram TESTS is recorded in the prescribed conditions, the substances pH (2.2.3) Dissolve 50 mg in carbon dioxide-free water R elute in the following order : 4-aminobenzoic acid, procaine, and dilute to 15 ml with the same solvent Shake until benzylpenicillin Adjust the sensitivity of the system so that dissolution is complete The pH of the solution is 5.0 to 7.5 the height of the peak corresponding to 4-aminobenzoic Specific optical rotation (2.2.7) Dissolve 0.250 g in a acid is at least 50 per cent of the full scale of the recorder mixture of volumes of water R and volumes of acetone R The test is not valid unless, the resolution between the first and dilute to 25.0 ml with the same mixture of solvents The peak (4-aminobenzoic acid) and the second peak (procaine) specific optical rotation is + 165 to + 180, calculated with is at least 2.0 If necessary, adjust the concentration of reference to the anhydrous substance acetonitrile in the mobile phase Inject reference solution (a) times The test is not valid unless the relative standard Related substances Examine by liquid chromatography (2.2.29) as prescribed under Assay Inject 10 µl of reference deviation for the areas of the peaks is at most 1.0 per cent Inject alternately test solution (b) and reference solution (a) solution (c) Adjust the sensitivity of the system so that Calculate the percentage contents of procaine and procaine the height of the peak due to benzylpenicillin is at least benzylpenicillin 50 per cent of the full scale of the recorder Inject 10 µl of test solution (a) and continue the chromatography for STORAGE 1.5 times the retention time of the benzylpenicillin peak In the chromatogram obtained with test solution (a) : Store in an airtight container If the substance is sterile, the area of any peak corresponding to 4-aminobenzoic store in a sterile, airtight, tamper-proof container acid is not greater than the area of the corresponding peak in the chromatogram obtained with reference LABELLING solution (c) (0.024 per cent) ; the area of any peak, apart The label states : from the principal peaks and any peak corresponding to — where applicable, the name and quantity of any added 4-aminobenzoic acid, is not greater than the area of the dispersing or suspending agents, peak corresponding to benzylpenicillin in the chromatogram obtained with reference solution (c) (1 per cent) — where applicable, that the substance is free from bacterial endotoxins Water (2.5.12) 2.8 per cent to 4.2 per cent, determined on 0.500 g by the semi-micro determination of water IMPURITIES Bacterial endotoxins (2.6.14, Method E) : less than 0.10 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins C Place about mg in a test-tube about 150 mm long and 15 mm in diameter Moisten with 0.05 ml of water R and add ml of sulphuric acid-formaldehyde reagent R Mix the contents of the tube by swirling ; the solution is practically colourless Place the test-tube on a water-bath for ; a reddish-brown colour develops D Dissolve 0.1 g in ml of dilute hydrochloric acid R and use the solution which may be turbid The solution gives the reaction of primary aromatic amines (2.3.1) ASSAY Examine by liquid chromatography (2.2.29) Prepare the A 4-aminobenzoic acid, solutions immediately before use Test solution (a) Dissolve 70.0 mg of the substance to be examined in the mobile phase and dilute to 50.0 ml with the mobile phase Test solution (b) Dissolve 70.0 mg of the substance to be examined in the mobile phase and dilute to 100.0 ml with the mobile phase Reference solution (a) Dissolve 70.0 mg of procaine benzylpenicillin CRS in the mobile phase and dilute to B (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5100.0 ml with the mobile phase dimethylthiazolidine-4-carboxylic acid (penicilloic acids Reference solution (b) Dissolve mg of 4-aminobenzoic of benzylpenicillin), acid R in reference solution (a) and dilute to 25 ml with the same solution Reference solution (c) Dissolve 16.8 mg of 4-aminobenzoic acid R in water R and dilute to 50.0 ml with the same solvent Dilute 1.0 ml of the solution to 10.0 ml with water R To 1.0 ml of this solution, add 1.0 ml of test solution (a) and dilute to 100.0 ml with the mobile phase The chromatographic procedure may be carried out using : — a stainless steel column 0.25 m long and 4.6 mm in C (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5internal diameter packed with octadecylsilyl silica gel for dimethylthiazolidine-4-carboxylic acid (penilloic acids of chromatography R (5 µm), benzylpenicillin), General Notices (1) apply to all monographs and other texts 1081 Benzylpenicillin sodium EUROPEAN PHARMACOPOEIA 5.0 in daylight The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with reference solution (a) The test is not valid unless the chromatogram obtained with reference solution (b) shows clearly separated spots C Place about mg in a test-tube about 150 mm long and 15 mm in diameter Moisten with 0.05 ml of water R and add ml of sulphuric acid-formaldehyde reagent R Mix the contents of the tube by swirling ; the solution is practically colourless Place the test-tube on a water-bath for ; a reddish-brown colour develops D It gives reaction (a) of sodium (2.3.1) D (3S,7R,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7atetrahydroimidazo[5,1-b]thiazole-3,7-dicarboxylic acid (penillic acid of benzylpenicillin), TESTS pH (2.2.3) Dissolve 2.0 g in carbon dioxide-free water R and dilute to 20 ml with the same solvent The pH of the 01/2005:0114 solution is 5.5 to 7.5 Specific optical rotation (2.2.7) Dissolve 0.500 g in carbon dioxide-free water R and dilute to 25.0 ml with the same BENZYLPENICILLIN SODIUM solvent The specific optical rotation is + 285 to + 310, calculated with reference to the dried substance Benzylpenicillinum natricum Absorbance (2.2.25) Dissolve 90.0 mg in water R and dilute to 50.0 ml with the same solvent Measure the absorbance of the solution at 325 nm, at 280 nm and at the maximum at 264 nm, diluting the solution, if necessary, for the measurement at 264 nm The absorbances at 325 nm and 280 nm are not greater than 0.10 and the absorbance at the maximum at 264 nm is 0.80 to 0.88, calculated on the basis of the undiluted (1.80 g/l) solution Verify the resolution C16H17N2NaO4S Mr 356.4 of the apparatus (2.2.25) ; the ratio of the absorbances is at least 1.7 DEFINITION Related substances Liquid chromatography (2.2.29) as Benzylpenicillin sodium is sodium (2S,5R,6R)-3, described under Assay Inject 20 µl of reference solution (d) 3-dimethyl-7-oxo-6-[(phenylacetyl)amino]-4-thia-1and elute isocratically with the chosen mobile phase Inject azabicyclo[3.2.0]heptane-2-carboxylate, a substance 20 µl of test solution (b) and start the elution isocratically produced by the growth of certain strains of Penicillium Immediately after elution of the benzylpenicillin peak start notatum or related organisms, or obtained by any other means It contains not less than 96.0 per cent and not more the following linear gradient : than the equivalent of 102.0 per cent of benzylpenicillin Time Mobile phase A Mobile phase B Comment sodium, calculated with reference to the dried substance (min) (per cent V/V) (per cent V/V) E phenylacetic acid CHARACTERS A white or almost white, crystalline powder, very soluble in water, practically insoluble in fatty oils and in liquid paraffin IDENTIFICATION First identification : A, D Second identification : B, C, D A Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with benzylpenicillin sodium CRS B Examine by thin-layer chromatography (2.2.27), using a TLC silanised silica gel plate R Test solution Dissolve 25 mg of the substance to be examined in ml of water R Reference solution (a) Dissolve 25 mg of benzylpenicillin sodium CRS in ml of water R Reference solution (b) Dissolve 25 mg of benzylpenicillin sodium CRS and 25 mg of phenoxymethylpenicillin potassium CRS in ml of water R Apply to the plate µl of each solution Develop over a path of 15 cm using a mixture of 30 volumes of acetone R and 70 volumes of a 154 g/l solution of ammonium acetate R, the pH of which has been adjusted to 5.0 with glacial acetic acid R Allow the plate to dry in air and expose it to iodine vapour until the spots appear Examine 1082 - 20 70 → 30 → 100 linear gradient 20 - 35 100 isocratic 35 - 50 70 30 re-equilibration Inject water R and use the same elution pattern to obtain a blank In the chromatogram obtained with test solution (b), the area of any peak, apart from the principal peak, is not greater than the area of the principal peak in the chromatogram obtained with reference solution (d) (1 per cent) 2-Ethylhexanoic acid (2.4.28) Not more than 0.5 per cent m/m Loss on drying (2.2.32) Not more than 1.0 per cent, determined on 1.000 g by drying in an oven at 100-105 °C Bacterial endotoxins (2.6.14, Method E) : less than 0.16 IU/mg, if intended for use in the manufacture of parenteral dosage forms without a further appropriate procedure for the removal of bacterial endotoxins ASSAY Liquid chromatography (2.2.29) Prepare the solutions immediately before use Test solution (a) Dissolve 50.0 mg of the substance to be examined in water R and dilute to 50.0 ml with the same solvent See the information section on general monographs (cover pages) Betacarotene EUROPEAN PHARMACOPOEIA 5.0 Test solution (b) Dissolve 80.0 mg of the substance to be examined in water R and dilute to 20.0 ml with the same solvent Reference solution (a) Dissolve 50.0 mg of benzylpenicillin sodium CRS in water R and dilute to 50.0 ml with the same solvent Reference solution (b) Dissolve 10 mg of benzylpenicillin sodium CRS and 10 mg of phenylacetic acid CRS in water R C (2S,5R,6R)-6-[[(4-hydroxyphenyl)acetyl]amino]-3,3dimethyl-7-oxo-4-thia- 1-azabicyclo[3.2.0]heptane-2and dilute to 50 ml with the same solvent carboxylic acid, Reference solution (c) Dilute 1.0 ml of reference solution (a) to 20.0 ml with water R Dilute 1.0 ml of the solution to 50.0 ml with water R Reference solution (d) Dilute 4.0 ml of reference solution (a) to 100.0 ml with water R The chromatographic procedure may be carried out using : — a column 0.25 m long and 4.6 mm in internal diameter packed with octadecylsilyl silica gel for D (3S,7R,7aR)-5-benzyl-2,2-dimethyl-2,3,7,7achromatography R (5 µm), tetrahydroimidazo[5,1-b]thiazole-3,7-dicarboxylic — as mobile phase at a flow rate of 1.0 ml/min : acid (penillic acid of benzylpenicillin), Mobile phase A Mix 10 volumes of a 68 g/l solution of potassium dihydrogen phosphate R adjusted to pH 3.5 with a 500 g/l solution of dilute phosphoric acid R, 30 volumes of methanol R and 60 volumes of water R Mobile phase B Mix 10 volumes of a 68 g/l solution of potassium dihydrogen phosphate R adjusted to pH 3.5 with a 500 g/l solution of dilute phosphoric acid R, 40 volumes of water R and 50 volumes of methanol R E (4S)-2-[carboxy[(phenylacetyl)amino]methyl]-5,5— as detector a spectrophotometer set at 225 nm dimethylthiazolidine-4-carboxylic acid (penicilloic acids of benzylpenicillin), Equilibrate the column with a mobile phase ratio A:B of 70:30 Inject 20 µl of reference solution (b) The test is not valid unless the resolution between the principal peaks is at least 6.0 (if necessary, adjust the ratio A:B of the mobile phase) and the mass distribution ratio for the second peak (benzylpenicillin) is 4.0 to 6.0 Inject 20 µl of reference solution (c) Adjust the system to obtain a peak with a signal-to-noise ratio of at least STORAGE Store in an airtight container If the substance is sterile, store in a sterile, airtight, tamper-proof container F (2RS,4S)-2-[[(phenylacetyl)amino]methyl]-5,5dimethylthiazolidine-4-carboxylic acid (penilloic acids of benzylpenicillin) LABELLING The label states, where applicable, that the substance is free from bacterial endotoxins 01/2005:1069 BETACAROTENE IMPURITIES Betacarotenum A (2S,5R,6R)-6-amino-3,3-dimethyl-7-oxo-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid (6-aminopenicillanic acid), B phenylacetic acid, General Notices (1) apply to all monographs and other texts C40H56 Mr 536.9 1083 ... 400 18 0 50 11 0 710 820 600 10 00 14 0 30 90 56 0 640 480 710 11 2 25 69 450 52 0 380 50 0 89 18 54 3 15 360 270 355 72 13 43 224 260 19 0 250 58 9.9 34 16 0 19 0 13 0 18 0 47 7.6 27 1 25 15 0 10 6 1 25 38 5. 8... dmax dmin 11 200 770 350 56 0 250 0 2900 210 0 8000 600 250 430 2000 2300 17 00 600 470 18 0 320 16 00 19 00 13 00 4000 370 13 0 250 14 00 17 00 12 00 800 290 90 19 0 11 20 13 00 950 2000 230 70 15 0 900 10 40 770... Prefix Symbol 10 deci d P 10 −2 centi c T 10 − milli m 10 −6 micro µ −9 nano n exa E 15 peta 10 12 tera 10 10 Factor 1 Symbol 18 10 giga G 10 mega M 10 10 kilo k 10 − 12 pico p hecto h 10 − 15 femto f