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Farmacopeea Europeana Editia A 8-A, Volumul 1
Farmacopeea Europeana Editia A 8-A, Volumul 1
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general chapters found in the printed version. With the publication of each supplement the electronic version is replaced
by a new, fully updated, cumulative version.
PHARMEUROPA ONLINE
European Pharmacopoeia’s free online forum (http://pharmeuropa.edqm.eu)
Pharmeuropa Online contains preliminary drafts of all new and revised monographs proposed for inclusion in the
European Pharmacopoeia and gives an opportunity for all interested parties to comment on the specifications before they
are finalised. Pharmeuropa Online also contains information on the work programme or of general interest and articles
published in Pharmeuropa Bio & Scientific Notes (containing scientific articles on pharmacopoeial matters). Archives of
Pharmeuropa and Pharmeuropa Bio & Scientific Notes can be accessed via this website.
PHARMACOPOEIAL HARMONISATION
See the information given in general chapter 5.8. Pharmacopoeial harmonisation.
WEBSITE
www.edqm.eu
https://www.edqm.eu/store (for prices and orders)
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To send a question or to contact the EDQM, use the HelpDesk, accessible through the EDQM website
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KNOWLEDGE
Consult Knowledge, the free database, at www.edqm.eu to obtain information on the work programme of the European
Pharmacopoeia, the volume of Pharmeuropa and of the European Pharmacopoeia in which a text has been published,
trade names of the reagents (for example, chromatography columns) that were used at the time of the elaboration of the
monographs, the history of the revisions of a text since its publication in the 5th Edition, representative chromatograms,
the list of reference standards used, and the list of certificates granted.
COMBISTATS
CombiStats is a computer program for the statistical analysis of data from biological assays in agreement with chapter 5.3
of the 8th Edition of the European Pharmacopoeia. For more information, visit the website (www.edqm.eu/combistats).
KEY TO MONOGRAPHS
Version date of the text 01/2008:0884 of this solution to 10.0 mL with a mixture of 20 volumes
corrected 8.0 of acetonitrile R and 80 volumes of water R.
CARBIMAZOLE Reference solution (b). Dissolve 5.0 mg of thiamazole R in
Text reference a mixture of 20 volumes of acetonitrile R and 80 volumes
number of water R and dilute to 10.0 mL with the same mixture of
Carbimazolum solvents. Dilute 1.0 mL of this solution to 100.0 mL with
Modification to be a mixture of 20 volumes of acetonitrile R and 80 volumes
taken into account from of water R.
the publication date of Column:
volume 8.0
– size: l = 0.15 m, Ø = 3.9 mm,
C7H10N2O2S – stationary phase: octadecylsilyl silica gel for
CAS number [22232-54-8] Mr 186.2 chromatography R (5 µm).
Mobile phase: acetonitrile R, water R (10:90 V/V).
DEFINITION
Flow rate: 1 mL/min.
Chemical name Ethyl 3-methyl-2-thioxo-2,3-dihydro-1H-imidazole-1-
in accordance carboxylate. Detection: spectrophotometer at 254 nm.
with IUPAC Content: 98.0 per cent to 102.0 per cent (dried substance). Injection: 10 µL.
nomenclature rules Run time: 1.5 times the retention time of carbimazole.
CHARACTERS
Appearance: white or yellowish-white, crystalline powder. Retention time: carbimazole = about 6 min.
Solubility: slightly soluble in water, soluble in acetone and in System suitability: reference solution (a):
ethanol (96 per cent). – resolution: minimum 5.0 between the peaks due to
Application of the impurity A and carbimazole.
first and second IDENTIFICATION
identification is First identification: B. Limits:
defined in the Second identification: A, C. – impurity A: not more than 0.5 times the area of the
N
General Notices principal peak in the chromatogram obtained with
A. Melting point (2.2.14): 122 °C to 125 °C. reference solution (b) (0.5 per cent),
E
(chapter 1)
B. Infrared absorption spectrophotometry (2.2.24). – unspecified impurities: for each impurity, not more
Preparation: discs.
I M
than 0.1 times the area of the principal peak in the
chromatogram obtained with reference solution (b)
C
Reference standard Comparison: carbimazole CRS. (0.10 per cent).
E
available from C. Thin-layer chromatography (2.2.27). Loss on drying (2.2.32): maximum 0.5 per cent,
SP
the Secretariat Test solution. Dissolve 10 mg of the substance to be determined on 1.000 g by drying in a desiccator over
(see www.edqm.eu) examined in methylene chloride R and dilute to 10 mL diphosphorus pentoxide R at a pressure not exceeding
with the same solvent. 0.7 kPa for 24 h.
Reference solution. Dissolve 10 mg of carbimazole CRS in Sulfated ash (2.4.14): maximum 0.1 per cent, determined
methylene chloride R and dilute to 10 mL with the same on 1.0 g.
solvent. ASSAY
Plate: TLC silica gel GF254 plate R. Dissolve 50.0 mg in water R and dilute to 500.0 mL
Reagent described Mobile phase: acetone R, methylene chloride R with the same solvent. To 10.0 mL add 10 mL of dilute
in chapter 4 (20:80 V/V). hydrochloric acid R and dilute to 100.0 mL with water R.
Measure the absorbance (2.2.25) at the absorption
Application: 10 µL. maximum at 291 nm.
Development: over a path of 15 cm. Calculate the content of C7H10N2O2S taking the specific
Further information absorbance to be 557.
Drying: in air for 30 min.
available on
Detection: examine in ultraviolet light at 254 nm. IMPURITIES
www.edqm.eu
(Knowledge) Results: the principal spot in the chromatogram obtained Specified impurities: A.
with the test solution is similar in position and size to Other detectable impurities (the following substances
the principal spot in the chromatogram obtained with would, if present at a sufficient level, be detected by one
the reference solution. or other of the tests in the monograph. They are limited
by the general acceptance criterion for other/unspecified
Reference to a TESTS impurities and/or by the general monograph Substances
general chapter Related substances. Liquid chromatography (2.2.29). for pharmaceutical use (2034). It is therefore not
necessary to identify these impurities for demonstration
Test solution. Dissolve 5.0 mg of the substance to be of compliance. See also 5.10. Control of impurities in
Line in the examined in 10.0 mL of a mixture of 20 volumes of substances for pharmaceutical use): B.
margin acetonitrile R and 80 volumes of water R. Use this solution
indicating within 5 min of preparation.
where part of Reference solution (a). Dissolve 5 mg of thiamazole R and
the text has 0.10 g of carbimazole CRS in a mixture of 20 volumes of
been modified acetonitrile R and 80 volumes of water R and dilute to
(technical 100.0 mL with the same mixture of solvents. Dilute 1.0 mL A. 1-methyl-1H-imidazole-2-thiol (thiamazole),
modification)
How to contact us
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ISBN: 978-92-871-7525-0
CONTENTS
VOLUME 1
I. PREFACE i
II. INTRODUCTION v
III. EUROPEAN PHARMACOPOEIA COMMISSION ix
IV. CONTENTS OF THE EIGHTH EDITION xxi
GENERAL CHAPTERS
1. General notices 1
2. Methods of analysis 11
2.1. Apparatus 13
2.2. Physical and physicochemical methods 19
2.3. Identification 117
2.4. Limit tests 125
2.5. Assays 153
2.6. Biological tests 173
2.7. Biological assays 227
2.8. Methods in pharmacognosy 269
2.9. Pharmaceutical technical procedures 283
3. Materials for containers and containers 371
3.1. Materials used for the manufacture of containers 373
3.2. Containers 407
4. Reagents 423
5. General texts 551
GENERAL MONOGRAPHS 739
MONOGRAPHS ON DOSAGE FORMS 777
MONOGRAPHS ON VACCINES FOR HUMAN USE 815
MONOGRAPHS ON VACCINES FOR VETERINARY USE 919
MONOGRAPHS ON IMMUNOSERA FOR HUMAN USE 1027
MONOGRAPHS ON IMMUNOSERA FOR VETERINARY USE 1035
MONOGRAPHS ON RADIOPHARMACEUTICAL PREPARATIONS AND STARTING MATERIALS FOR
RADIOPHARMACEUTICAL PREPARATIONS 1043
MONOGRAPHS ON SUTURES FOR HUMAN USE 1115
MONOGRAPHS ON SUTURES FOR VETERINARY USE 1125
MONOGRAPHS ON HERBAL DRUGS AND HERBAL DRUG PREPARATIONS 1133
MONOGRAPHS ON HOMOEOPATHIC PREPARATIONS 1427
VOLUME 2
MONOGRAPHS 1457
INDEX 3603
I. PREFACE
The European Pharmacopoeia was inaugurated in 1964 will now work on harmonising their policies and approaches
through the Convention on the Elaboration of a European towards monograph development by drafting what have been
Pharmacopoeia under the auspices of the Council of termed as a working title “Good Pharmacopoeial Practices”.
Europe. The 8th Edition will be published just before the Convergence in policies, e.g. with regard to control of
50th Anniversary of the European Pharmacopoeia. The impurities by applying ICH Q3 principles, will facilitate future
work of the European Pharmacopoeia has gone through a collaboration and harmonisation.
remarkable development since the first difficult years to its The implementation date for the 8th Edition is 1 January 2014
current strong position. The 3-year cycle of publication with and this edition will, over the next 3 years, be augmented with
thrice-yearly supplements has proven to be an efficient way to 8 supplements containing the texts adopted at the sessions
publish and update the results of the work of the European of the European Pharmacopoeia Commission. As ever, it
Pharmacopoeia Commission and its Expert Groups and is published in the 2 official languages of the Council of
Working Parties almost in real-time. Europe, i.e. English and French, both as a printed version and
The monographs of the Pharmacopoeia, both specific and electronically (online and on a USB key). It is noteworthy
general, together with other texts made mandatory by virtue of that certain member states undertake national or regional
reference to them in monographs, are applicable throughout translations, which they incorporate into their own national
the 37 member states, and the European Union, which is pharmacopoeias.
also a signatory party to the European Pharmacopoeia The work programme of the European Pharmacopoeia is
Convention. This means that in addition to applicability decided by the European Pharmacopoeia Commission, the
in all its member states, the European Pharmacopoeia has governing body of the Pharmacopoeia. Elaboration and
a special role in regulatory processes within the European approval of monographs and other texts proceed through an
Union. In addition to the 38 signatories of the European efficient and transparent process, which is based on scientific
Pharmacopoeia Convention, there are also a large number of co-operation between the members of the various Groups
observers, comprising the World Health Organization and of Experts and Working Parties set up by the European
23 countries, of which 16 are non-European. The quality Pharmacopoeia Commission. These experts give their time,
standards developed through the European Pharmacopoeia expertise and experience to produce public standards of the
therefore have an impact on the quality of medicinal products highest quality - standards that are continually revised in line
and substances far beyond the European region. Since the with scientific developments. The members of these groups
entry into force of the 7th edition, Ukraine has become a come from regulatory authorities, official medicines control
new member of the European Pharmacopoeia Convention laboratories, pharmaceutical and chemical manufacturers,
(in 2013), while the Republic of Guinea and Singapore have universities and research institutions. All monographs are
become new observers (in 2012). experimentally verified and submitted for public consultation
by online publication in Pharmeuropa, the forum of the
The 8 founder countries of the Convention realised in 1964 European Pharmacopoeia, before adoption and publication.
that manufacturing and quality control standards for medicinal
products on the European Market had to be harmonised for The growing number of monographs and the need to keep
reasons of public health and to facilitate free movement of them updated represents an increased workload and an
these products. Since then, the pharmaceutical world has increased need for experts with access to experimental
become globalised and international harmonisation among the facilities. The working procedures for the elaboration of
3 major pharmacopoeias (European Pharmacopoeia, Japanese monographs are:
Pharmacopoeia and United States Pharmacopeia) became a – Procedure 1: traditional elaboration by Groups of Experts
logical development. Harmonisation activities among these and Working Parties.
3 pharmacopoeias started in 1989 when the Pharmacopoeial – Procedure 2: adaptation of national monographs. (This
Discussion Group (PDG) was set up. The PDG has been procedure is no longer used since the work has been
working on monographs on widely-used excipients and 62 are completed.)
included in its work programme. Soon after the PDG began – Procedure 3: elaboration of monographs on chemical
work, it was recognised that the absence of harmonised substances produced only by one manufacturer and
general methods represented a significant obstacle. A wide typically close to patent expiry. The manufacturer and
range of general methods (35) have since been added to the national pharmacopoeia authority in the country where
work programme, including those from the work of the the substance is produced elaborate preliminary drafts
International Conference on Harmonisation (ICH) and, in and check the requirements experimentally. This results
particular, its guideline on setting specifications (Q6A). To in a draft that is then reviewed by a Group of Experts
date, 28 of the 35 general methods and 43 of 62 excipient or Working Party and processed in the usual way by
monographs have been harmonised. Detailed information on public enquiry. (This procedure has been integrated into
the work programme of the PDG is published regularly in Procedure 4.)
Pharmeuropa and in General Chapter 5.8. Pharmacopoeial – Procedure 4 (P4): a modified version of Procedure 3
harmonisation. for substances still under patent, which was introduced
However, it is evident that harmonisation between the by the European Pharmacopoeia Commission in 2002.
3 ICH regions is not enough in today’s world, where a The P4 procedure involves collaboration between the
high percentage of Active Pharmaceutical Ingredients manufacturer of the substance and a Working Party
(APIs) come from outside Europe, Japan and the USA. In solely composed of representatives of authorities and
early 2012, the WHO took the initiative and convened the the EDQM. Together they prepare a draft monograph
pharmacopoeias of the world for their first international with experimental verification by the EDQM laboratory
meeting in Geneva. The discussions at this level clearly and/or by national pharmacopoeia authorities or Official
identified the need to strengthen collaboration among Medicines Control Laboratories before publication for
pharmacopoeias worldwide. Based on the experience and public enquiry.
challenges with existing harmonisation initiatives such as – Procedure 5: applies to monographs on raw materials and
PDG that focus on retrospective harmonisation, it was stocks for homoeopathic preparations authorised for use
decided to take a different approach. World pharmacopoeias in the member states. The work is co-ordinated by the
i
Preface EUROPEAN PHARMACOPOEIA 8.0
EDQM and overseen by the HOM Working Party. This promote reduction and refinement of animal use, e.g. serology
procedure was introduced by the European Pharmacopoeia assays or single dilution assays for diphtheria, tetanus,
Commission in 2011. acellular pertussis and rabies (veterinary/human) vaccines.
Work under the P4 procedure has successfully continued A number of important European Pharmacopoeia activities
during the elaboration of the 7th Edition. Already have been initiated over the last few years, such as the
59 P4 monographs for chemical substances have been adopted establishment of a PAT (Process Analytical Technology)
by the European Pharmacopoeia Commission. Under the Working Party based on a request from the EMA. PAT tools
P4 procedure for chemical substances, a pilot project on make it possible to use additional information collected
bilateral prospective harmonisation of active substance throughout the production process, e.g. use of NIR
monographs with the USP was initiated and so far has resulted (near-infrared spectrophotometry) to determine tablet
in the adoption of 4 harmonised monographs. As the P4 content. Chapter 2.2.40 Near Infrared Spectrophotometry was
procedure for chemical substances has been such a success, revised to introduce PAT-related concepts such as in-line and
the European Pharmacopoeia Commission decided in 2009 on-line measurements. This was done in close consultation
to initiate a similar process for biological substances. The with the EMA’s CVMP/CHMP Quality Working Party so that
so-called P4-BIO procedure takes account of the increasing it would be aligned with the on-going revision of the EMA’s
number and importance of biologically-derived active Note for guidance on NIR. The revised chapter was adopted by
substances and biosimilars on the European market. Two the European Pharmacopoeia Commission at its November
monographs elaborated by the P4-BIO procedure have already session in 2012 and it will be complemented by the revised
been adopted by the European Pharmacopoeia Commission. EMA Guideline on the use of near infrared spectroscopy by the
pharmaceutical industry and the data requirements for new
The work on controlling impurities, a particular strength of submissions and variations, which is expected to be finalised
the European Pharmacopoeia, has continued. Monographs in 2013. The General Notices will be updated to take account
are evaluated and approved by the Competent Authorities of of real-time release testing, which will be done once the
member states, and the impurity profiles covered by these EMA Guideline has been adopted. The alternative, optional
monographs reflect the existing, approved routes of synthesis. Chapter 2.9.47. Demonstration of Uniformity of Dosage
A revision mechanism is in place for newly-approved products Units (UDU) using large sample sizes that could be used to
(e.g. new sources, new routes). The analytical methods in replace conventional UDU testing has also been adopted. The
monographs are robust and validated and are based on PAT Working Party is now reflecting on the need for new
collaborative laboratory testing. The monographs reflect general chapters.
regulatory practice by applying ICH guideline Q3A R to the A Heavy Metals Working Party has been created to implement
pharmacopoeial substances. The guideline of the European the EMA’s Guideline on metal catalysts and metal reagent
Medicines Agency (EMA) concerning the control of genotoxic residues and the future ICH Q3D guideline. The terms of
impurities, which came into force in 2007, has also been taken reference for this working party are to draft a general chapter
into account and has resulted in a revision of the general to implement the guideline, to assess the capability of the
monograph on Substances for Pharmaceutical use (2034) and current Chapter 2.4.8. Heavy metals to appropriately limit the
adoption of 3 general methods for genotoxic impurities. priority metals mentioned in the guideline and to consider
the introduction of instrumental screening methods, whilst
The European Pharmacopoeia Commission is also continuing also allowing other means of ensuring compliance where
its efforts to reduce the number of animals needed to perform possible and justified. Since the ICH guideline has not yet
tests (implementation of the 3Rs principle, i.e. replacing, been published, it was decided to introduce a new General
refining and reducing the use of animals in tests). It has Chapter 5.20. Metal catalyst or metal reagent residues and a
aligned pharmacopoeial texts with VICH Guidelines 41 (test new General Method 2.4.20. Determination of metal catalyst
for reversion to virulence) and 44 (developmental safety or metal reagent residues. General Chapter 5.20 reproduces
tests), which came into force in 2008 and 2009, respectively, the EMA’s guideline on the specification limits for residues
and with Directive 2010/63/EU of the European Parliament of metal catalysts or metal reagents. It is applicable to all
and of the Council of 22 September 2010 on the protection excipients and APIs, except those for veterinary use only, but
of animals used for scientific purposes. Furthermore, to not to starting materials or herbals. General Method 2.4.20
ensure consistency with European regulations the European describes the general approach for the determination of
Pharmacopoeia Commission has harmonised all the metal catalyst or metal reagent residues in substances for
veterinary vaccine monographs, including monographs on pharmaceutical use. As the chemical composition of the
vaccines intended for species that were outside the scope of the substances and the specification limits for the metal(s) of
VICH Guidelines. As a consequence, the safety tests and the interest vary considerably, it is not possible to describe all
tests for increased virulence performed during development of suitable sample preparation and measurement methods.
the vaccines have been harmonised, which will greatly reduce Therefore, any method that fulfils the requirements described
the number of animals used for testing. in this chapter may be used. Both General Chapter 5.20 and
The European Pharmacopoeia Commission continuously General Method 2.4.20 have been published in European
revises general texts and monographs, re-evaluates Pharmacopoeia supplement 7.7. A cross-reference is to be
the relevance of animal tests mentioned in European introduced into the general monograph on Substances for
Pharmacopoeia texts and, if deemed appropriate, includes Pharmaceutical Use (2034) to make General Chapter 5.20
alternative methods. The general monograph on Vaccines for legally-binding.
veterinary use (0062) was revised to delete the TABST (target As a follow-up to the Workshop on the future of monographs
animal batch safety test), except in ‘particular circumstances’ in the field of biologicals organised by the EDQM in
to cover the need to perform, on an ad hoc basis, further February 2011, 2 new working parties have been created: (1)
testing and safety tests in particular. In the interest of the 3Rs, the Raw Materials for the Production of Cellular and Gene
the European Pharmacopoeia Commission also adopted the Transfer Products Working Party, which will elaborate texts on
deletion of the TABST from the European Pharmacopoeia raw materials such as antibodies, basal media (for cell culture),
for all veterinary vaccines. Currently, at the European serum/serum replacements, growth factors and cytokines, and
Pharmacopoeia level, animals are no longer used in the (2) the Host Cell Proteins Working Party, which will draft
testing of medicinal products derived from human blood and recommendations with regard to the development, validation
plasma. In many cases, in vivo testing has been replaced by and use of in-house or commercial kits or test methods
in vitro methods for human and veterinary vaccines. For the for the detection and quantification of host cell-derived
remaining in vivo assays, different strategies are being used to proteins. In addition, the scope of the P4-BIO pilot project
ii
EUROPEAN PHARMACOPOEIA 8.0 Preface
has been extended in order to elaborate monographs on and one multi-source product) allocated to it by the European
one monoclonal antibody, one hormone/enzyme and one Pharmacopoeia Commission, while addressing issues related
pegylated protein. The P4-BIO working party has also been to the elaboration of chemically-defined finished products
asked to elaborate one finished product monograph. The monographs in order to assess whether such monographs
terms of reference of the Cell Therapy Products Working Party should be elaborated by the European Pharmacopoeia in the
have also been extended in order to elaborate a general text future, and (2) the Second Identification Test Working Party,
dealing with microbiological control of organs and tissues for which will prepare a guidance document that defines the
human use, including preservation and other related media. criteria for inclusion of a second series of identification tests
As a consequence, the Working Party has been renamed the (solely intended to be carried out in pharmacies) in individual
Cell Therapy Products, Tissues and Organs Working Party. monographs and will review the methods and instrumentation
The production section of the monograph Human normal available in pharmacies for this purpose.
immunoglobulin for intravenous administration (0918) has Compliance with the EU REACH (Registration, Evaluation,
been revised due to experience with an immunoglobulin Authorisation and Restriction of Chemical substances)
preparation that caused an increased rate of thromboembolic Regulation has posed a significant challenge and this issue
complications. In light of concerns for public health associated has been high on the agenda of the current Presidium.
with these thromboembolic events, the revised monograph The European Pharmacopoeia Commission approved the
will be implemented by the accelerated procedure. request for the revision of 215 monographs as a consequence
Due to the increasing number of fraudulent activities and cases of the EU REACH Regulation and already several revised
of adulteration, the European Pharmacopoeia Commission monographs have been adopted.
has decided to add a new section, Potential Adulteration, During the past 3 years I have had the honour, pleasure and
under § 1.4. MONOGRAPHS of the General Notices. The privilege to serve the European Pharmacopoeia Commission
need to include this section in individual monographs will as its 16th elected Chair. The task has been challenging, but also
be decided by the European Pharmacopoeia Commission interesting and rewarding because of the insights it has given
on a case-by-case basis. The objective of this section is to me into the various aspects of the development work that goes
make relevant information available to users of the European into the drafting of the quality standards provided by the texts
Pharmacopoeia to ensure the proper quality of medicinal of the Pharmacopoeia. It has also given me an insight into the
products (i.e. active substances, excipients, intermediate many other important areas in which the EDQM is involved.
products, bulk products and finished products). The new I wish to thank all the members of the European
version of the General Notices was adopted by the European Pharmacopoeia Commission for the trust and support that
Pharmacopoeia Commission at its 140th session. In relation allowed us to make substantial progress on a host of topics.
to this issue of adulteration, the Council of Europe and
its EDQM have adopted a multi-level, anti-counterfeiting I would like to thank the Director of the EDQM, Dr Susanne
strategy comprising various aspects, such as legislative actions Keitel, my two vice-chairs, Prof. Jos Hoogmartens and
against pharmaceutical crime by means of the Medicrime Ms An Lê, the Secretary to the European Pharmacopoeia
Convention. This Convention is the first international treaty Commission, Ms Cathie Vielle, and her two deputies,
against counterfeit medical products and similar crimes Dr Emmanuelle Charton and Dr Michael Wierer, for their
involving threats to public health. In addition, the EDQM is excellent work and support during my time as Chair. Together
developing eTACT; an anti-counterfeiting traceability service as the Presidium, we have managed to work very effectively to
for medicines. The aim of eTACT is to ensure the traceability guide the work of the European Pharmacopoeia Commission.
of individual packs of medicines using mass serialisation. It Finally, I would like to thank all the chairs and experts
would allow each pack of medicine to be traced and verified involved in the development of the European Pharmacopoeia
by the different stakeholders in the legal supply chain. Patients and the staff of the EDQM for their support. Their availability,
would also be allowed to verify the authenticity of their good advice and high quality input have made our work
medication. Governance of the eTACT system would be the possible and a pleasure to do.
responsibility of the EDQM as a public, inter-governmental
organisation that is able to ensure the confidentiality of the Dr Marianne Ek,
data handled by the system.
Chair of the European Pharmacopoeia Commission
Two additional new working parties have also recently been
created: (1) the Finished Product Monographs Working Party,
which aims to draft 2 monographs (i.e. on one single-source February 2013
iii
EUROPEAN PHARMACOPOEIA 8.0 Introduction
II. INTRODUCTION
The European Pharmacopoeia is prepared under the auspices In accordance with the terms of the Convention, the
of the Council of Europe in accordance with the terms of the contracting parties undertake to take the necessary
Convention on the Elaboration of a European Pharmacopoeia measures to ensure that the monographs of the European
(European Treaty Series No. 50) (‘the Convention’) as amended Pharmacopoeia shall become the official standards applicable
by the protocol to the Convention (European Treaty Series within their respective territories.
No. 134), signed by the governments of 37 member states
(Austria, Belgium, Bosnia and Herzegovina, Bulgaria, Croatia, PURPOSE OF THE EUROPEAN PHARMACOPOEIA
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, The purpose of the European Pharmacopoeia is to promote
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, public health by the provision of recognised common
Lithuania, Luxembourg, Malta, Montenegro, Netherlands, standards for the quality of medicines and their components.
Norway, Poland, Portugal, Romania, Serbia, Slovak Republic, Such standards are to be appropriate as a basis for the safe
Slovenia, Spain, Sweden, Switzerland, ‘the former Yugoslav use of medicines by patients. In addition, their existence
Republic of Macedonia’, Turkey, Ukraine and United facilitates the free movement of medicinal products in Europe
Kingdom) and by the European Union. and beyond.
The preparation of the European Pharmacopoeia is the European Pharmacopoeia monographs and other texts are
responsibility of the European Pharmacopoeia Commission designed to be appropriate to the needs of :
(‘the Commission’), appointed in accordance with article 5 – regulatory authorities ;
of the Convention. It is composed of delegations appointed – those engaged in the quality control of medicinal products
by the contracting parties. Each delegation consists of not and their components ;
more than 3 members chosen for their competence in matters
– manufacturers of medicinal products and their components.
within the functions of the Commission.
The European Pharmacopoeia is widely used internationally.
Observers from non-member states and international As globalisation and expansion in international trade present
organisations are admitted to sessions of the Commission a growing need to develop global quality standards for
in accordance with the Rules of Procedure. Observers are at medicines, the Commission works closely with all users of the
present admitted from : Albania, Algeria, Argentina, Armenia, Pharmacopoeia worldwide.
Australia, Brazil, Canada, China, Georgia, Israel, Madagascar,
Malaysia, Moldova, Morocco, Republic of Belarus, Republic SEAT OF THE EUROPEAN PHARMACOPOEIA
of Guinea, Republic of Kazakhstan, Republic of Singapore, COMMISSION
Russian Federation, Senegal, Syria, Tunisia, United States of The seat of the European Pharmacopoeia Commission is
America and the World Health Organization. situated in Strasbourg, the headquarters of the Council of
The Convention is open for signature by European countries Europe.
and observer status can serve to familiarise European
countries intending to become signatories with the working GENERAL PRINCIPLES
methods of the Commission. The Commission recognises that General rules for interpretation of the texts of the European
relations with countries outside Europe are essential in view Pharmacopoeia are given in the General Notices. The
of the globalisation of the supply chain for pharmaceuticals. following information should also be noted.
Observer status for non-European countries helps to foster The general principles applied in the elaboration of
these relations by facilitating regulatory partnerships and the monographs of the European Pharmacopoeia are laid down
exchange of information and working documents. in procedures and in technical guides available on the EDQM
The functions of the Commission established by article 6 of website. The principles applied are revised from time to
the Convention as amended by the protocol are : time without complete retrospective application so that
monographs already published may not always follow the
Article 6 latest recommendations, but wherever an issue with an impact
“Subject to the provision of Article 4 of the present on public health is identified, monographs are revised.
Convention, the functions of the Commission shall be : It is recognised that general chapters are also used elsewhere
(a) to determine the general principles applicable to the than in the monographs of the European Pharmacopoeia ; in
elaboration of the European Pharmacopoeia ; these circumstances users are recommended to consult the
(b) to decide upon methods of analysis for that purpose ; relevant technical guide, which gives extensive information on
the application of many of the methods.
(c) to arrange for the preparation of and to adopt monographs
to be included in the European Pharmacopoeia and ; General and individual monographs. The standards of the
European Pharmacopoeia are represented by general and
(d) to recommend the fixing of the time limits within which individual monographs. The use of general monographs has
its decisions of a technical character relating to the European developed in recent years to provide standards that best fulfil
Pharmacopoeia shall be implemented within the territories of the aims stated above and meet the needs of users. From the
the contracting parties.” 4th Edition, the scope of general monographs was extended,
The European Directorate for the Quality of Medicines & except where otherwise stated, to cover products where there
HealthCare (EDQM) of the Council of Europe supports is no individual monograph. It is now usually necessary
the Commission in the elaboration and revision of texts of to apply one or more general monographs along with any
the European Pharmacopoeia by providing the scientific individual monograph. Where a substance is subject to the
secretariat. In addition, it is responsible for the establishment, provisions of both a general monograph and an individual
production, monitoring and distribution of reference monograph, the two are complementary. An individual
standards needed when applying the monographs. The monograph may, exceptionally, include an exemption from
EDQM is also active in a number of other areas related to one or more provisions of the general monograph.
the protection of public health, for example in certifying the Since it is not practically possible to include in each individual
quality of active pharmaceutical ingredients from specific monograph a cross-reference to applicable or potentially
sources and in biological standardisation. applicable general monographs, cross-referencing has been
v
Introduction EUROPEAN PHARMACOPOEIA 8.0
discontinued except where it is necessary to avoid ambiguity. the older monographs elaborated before the establishment
A list of general monographs is included in each new edition of this policy have been revised to introduce quantitative
and supplement to aid users in identifying those that are methods. Where a monograph does not conform to the
needed for use with an individual monograph. general policy, compliance with the general monograph
Use of animals. In accordance with the European Substances for pharmaceutical use (2034) implies that the
Convention for the Protection of Vertebrate Animals used individual monograph requirements need to be supplemented
for Experimental and other Scientific Purposes (European accordingly.
Treaty Series No. 123) as amended by the protocol to the
convention (European Treaty Series No. 170), elaborated Except where required for the application of the monograph,
under the auspices of the Council of Europe, the Commission in which case the name is followed by ‘CRS’, impurities are
is committed to the reduction of animal usage wherever not provided as reference standards nor can they be provided
possible in pharmacopoeial testing, and encourages those for experimental purposes.
associated with its work to seek alternative procedures. An Chromatographic columns. As an aid to users, information
animal test is included in a monograph only if it has clearly is made available, via the website (see also Knowledge
been demonstrated that it is necessary to achieve satisfactory database, below), on chromatographic columns that have been
control for pharmacopoeial purposes. found to be satisfactory during development of monographs
Hydrates. Where applicable, the degree of hydration of a and general methods. Information is also given on other
substance is indicated in the monograph title. For existing equipment and reagents where this is considered useful. This
monographs where this is not yet the case, the degree of information is given without warranty and does not imply that
hydration will be introduced into the title during the next other columns, equipment or reagents than those specified are
technical revision of the monograph (including publication in not suitable.
Pharmeuropa Online). If monographs are published for both Residual solvents. The requirements for residual solvents are
the anhydrous form and a hydrated form of a given substance, given in the general monograph Substances for pharmaceutical
‘anhydrous’ is included in the title of the relevant monograph. use (2034) and general chapter 5.4. Residual solvents. Thus all
Chiral substances. Monographs on chiral substances that active substances and excipients are subject to relevant control
describe a particular enantiomer have a test to confirm of residual solvents, even where no test is specified in the
enantiomeric purity, usually by measurement of optical individual monograph. The requirements have been aligned
rotation. According to the current policy, a test for racemic with the ICH guideline on this topic.
character using optical rotation is included only if there is
information on the specific optical rotation of the enantiomers
Heavy metals. Limits for residues of metal catalysts or metal
that indicates that such a test would be discriminating in
reagents as defined in the respective guideline of the European
terms of enantiomeric purity. If other techniques, such as
Medicines Agency are reproduced in general chapter 5.20.
circular dichroism, can serve the intended purpose, they will
The requirements laid down in this chapter are not legally
be prescribed instead of optical rotation.
binding for users of the European Pharmacopoeia as long as
Polymorphism. Where a substance shows polymorphism, the chapter is not cross-referenced in a monograph (e.g. in the
this is usually stated under Characters. In general, no general monograph Substances for pharmaceutical use (2034)).
particular crystalline form is required in monographs ; The Commission plans to replace this chapter once the new
exceptionally, in a few monographs, the crystalline form ICH guideline for metal impurities, which is currently being
required is specified, for example, via an infrared absorption drafted, becomes available. Meanwhile, the Commission has
spectrophotometric identification test where the spectrum is decided not to devote further resources to revising existing
required to be recorded using the substance in the solid state tests (using method C or D) or creating new tests using general
without recrystallisation, the chemical reference substance chapter 2.4.8. Heavy Metals.
provided being of the required crystalline form. However,
for substances other than these exceptional cases, depending Homoeopathic preparations. A monograph on methods
on the use of a given substance in a dosage form, it may of preparation of homoeopathic stocks and potentisation,
be necessary for a manufacturer to ensure that a particular general monographs on homoeopathic preparations, mother
crystalline form is used. The information given under tinctures for homoeopathic preparations and herbal drugs for
Characters is intended to alert users to the need to evaluate this homoeopathic preparations, and individual monographs on
aspect during the development of a dosage form. The general raw materials and stocks for homoeopathic preparations are
monograph Substances for pharmaceutical use (2034) and included in a separate section in Volume 1. It is understood
general chapter 5.9. Polymorphism should also be consulted. that when the same substance is used in both homoeopathic
and other preparations then the monograph in the main body
Specificity of assays. For the elaboration of monographs on of the European Pharmacopoeia applies.
chemical active substances, the approach generally preferred
by the Commission is to provide control of impurities Herbal drugs and herbal drug preparations (including
(process-related impurities and degradation products) via a traditional Chinese medicines). All relevant monographs
well-designed Tests section, with stability-indicating methods, are grouped together in a separate section in Volume 1.
rather than by the inclusion of an assay that is specific for the Functionality-related characteristics. Following a policy
active moiety. It is therefore the full set of requirements of a decision of the Commission to highlight the need for attention
monograph that is designed to ensure that the product is of to functionality-related characteristics of excipients and to
suitable quality throughout its period of use. foster harmonisation of methods for their evaluation, an
Impurities. Following a review of policy on control of informative section has been created in the monographs.
impurities, general chapter 5.10. Control of impurities in The contents of this section do not constitute mandatory
substances for pharmaceutical use was included as of the requirements but the characteristics may be relevant for a
5th Edition. Together with the general monograph Substances particular use of an excipient. The characteristics may be
for pharmaceutical use (2034), it describes the policy of presented in different ways :
controlling impurities in individual monographs and provides
explanations on how the limits in the related substances test – citing the name only ;
should be understood.
The current general policy of the Commission is to include – citing the name and a suitable test method, preferably one
quantitative tests for impurities in monographs. Most of included in the European Pharmacopoeia ;
vi
EUROPEAN PHARMACOPOEIA 8.0 Introduction
– citing the name, a suitable test method and typical values the work programme. Changes to the work programme are
or tolerances on the stated value ; these values or tolerances published on the EDQM website and in Pharmeuropa Online.
are used to define a suitable grade of an excipient for a Information is also provided to industry associations registered
particular use. with the secretariat and to manufacturers’ liaison contacts.
In all cases, the method and acceptance criteria are not Interested parties are invited to contact the secretariat for any
mandatory requirements but are given for guidance. The items where they wish to be involved in the work.
decision to control a functionality-related characteristic of an CERTIFICATION PROCEDURE
excipient remains with the pharmaceutical manufacturer and
is taken with knowledge of the formulation of the product A procedure for the certification of suitability of monographs
in which it is to be used ; the method of determination, of the European Pharmacopoeia with respect to quality
acceptance criteria and tolerances are determined on a control of a product from a given source has been established
contractual basis by the user and the supplier of the excipient. (see Public Health Committee (Partial Agreement) Resolution
AP-CSP (07) 1 or any subsequent revision, available from the
Patents. The description in the European Pharmacopoeia EDQM and on its website) as an aid to the use of monographs
of articles subject to protection by patent does not confer or in applications for marketing authorisation. The certification
imply any right to the use of such patents by any person or procedure also applies to herbal drugs, herbal drug
persons other than the proprietors of the patents concerned. preparations and transmissible spongiform encephalopathy
(TSE) risk. Certificates of suitability are issued by the EDQM
Chemical Abstracts Service (CAS) registry number. Since only for substances produced under a suitable quality system.
the 6th Edition, CAS registry numbers have been included Certificates are granted with respect to published monographs.
for information in monographs, where applicable, to provide Details of the operation of this scheme are available from the
convenient access to useful information for users. Previously secretariat and on the EDQM website. A daily updated list of
these numbers were given only for reagents, where they certificates granted is available online on the EDQM website,
are of use in locating suppliers. CAS Registry Number® is a including voided or suspended certificates.
registered trademark of the American Chemical Society.
Protected species. Monographs, notably those on herbal PUBLICATIONS
drugs, may cover material obtained from protected species. The official version of the European Pharmacopoeia is
Inclusion of these monographs is without prejudice to the available in English and in French, in the form of a book with
provisions for protection of these species by national and 3 supplements per year, and in electronic format (online,
international law. including a tablet version, and on USB stick).
Archives. The European Pharmacopoeia Archives contain the
MONOGRAPHS ON PHARMACEUTICAL PREPARATIONS
1st Edition to 7th Edition in PDF format. They are available to
Up to the 8th Edition, individual monographs on all European Pharmacopoeia subscribers with an up-to-date
pharmaceutical preparations have not been elaborated, with subscription (paper, online or USB stick) and a registered
a few exceptions, e.g. those on immunosera for human use, EPID code.
immunosera for veterinary use, some biological preparations
Pharmeuropa, the European Pharmacopoeia forum, is
such as insulin preparations, radiopharmaceutical
published 4 times per year as an aid for the elaboration
preparations, vaccines for human use and vaccines for
of monographs and as a vehicle for information on
veterinary use.
pharmacopoeial and related matters. Pharmeuropa Bio &
The general monograph Pharmaceutical preparations (2619) Scientific Notes, a publication indexed by bibliographic
was introduced in the 7th Edition. This monograph is services, includes scientific papers related to the establishment
intended to be a reference source of standards in the European of biological reference preparations and validation of biological
Pharmacopoeia on active substances, excipients and dosage methods within the Biological Standardisation Programme of
forms, which are to be applied in the manufacture/preparation the EDQM, and to various aspects of pharmaceutical analysis
of pharmaceuticals ; it is not intended to be a guide on how to and other subjects relevant to the European Pharmacopoeia.
manufacture, as there is specific guidance available covering Since 2012, both publications are only available online, free
methods of manufacture and associated controls. of charge, and individual drafts and scientific papers are
Harmonisation and standardisation for pharmaceutical published on an ongoing basis.
preparations have so far been dealt with via the drafting Website. Information on activities and many other aspects of
of general dosage form monographs setting out elements the European Pharmacopoeia is to be found on the EDQM
common to all preparations within the scope of the website.
monograph, and via the development of standard test
methods used for testing of finished products. The inclusion Knowledge database. The EDQM website provides access
of these general monographs and methods in the European to a database containing information of various sorts related
Pharmacopoeia gives a common basis for competent to monographs and intended to facilitate their proper use.
authorities and manufacturers in the preparation and Information is provided on :
evaluation of applications for marketing authorisation. – chromatography columns used in monograph development ;
However, during its 143rd session, the Commission decided – suppliers of reagents and equipment that may be difficult
to revisit its policy and initiate a pilot phase on individual to find for some users ;
pharmaceutical preparation monographs to investigate further – the status of monographs (in development, adopted,
their feasibility and usefulness. published, under revision) ;
Reference standards established for the assay of active – revisions of the monographs on a historical basis, beginning
substances and excipients may be suitable for use as assay from the 5th Edition ;
standards for preparations when the conditions stated in
general chapter 5.12. Reference standards are fulfilled. – other useful information.
HelpDesk. Many technical and other enquiries are addressed
WORK PROGRAMME to the EDQM by users. They should be submitted via the
The work programme (elaboration of new monographs or HelpDesk on the EDQM website. The EDQM will deal with
general chapters or revision of existing texts) is decided enquiries that are related to the use of monographs of the
by the Commission at one of the three annual sessions. European Pharmacopoeia. The HelpDesk has a section of
In general, whenever 2 member states express a wish to Frequently Asked Questions that should be consulted by users
elaborate a monograph, the Commission adds the item to before submission of an enquiry.
vii
Introduction EUROPEAN PHARMACOPOEIA 8.0
viii
EUROPEAN PHARMACOPOEIA 8.0 European Pharmacopoeia Commission
Estonia Signe LEITO Portugal José Manuel CORREIA NEVES SOUSA LOBO
Juhan RUUT Domingos DE CARVALHO FERREIRA
Maria Joao PORTELA
Finland Marjo-Riitta HELLE
Eija PELKONEN Romania Anca CRUPARIU
Piia SALO Daniele ENACHE
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EUROPEAN PHARMACOPOEIA 8.0 Contents of the 8th Edition
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Contents of the 8th Edition EUROPEAN PHARMACOPOEIA 8.0
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EUROPEAN PHARMACOPOEIA 8.0 Contents of the 8th Edition
xxiii
EUROPEAN PHARMACOPOEIA 8.0 1. General notices
activity or other direct effect in the diagnosis, cure, mitigation, Any solvent required in a test or assay in which an indicator is
treatment or prevention of disease, or to affect the structure to be used is previously neutralised to the indicator, unless a
and function of the body. blank test is prescribed.
Excipient (auxiliary substance). Any constituent of a medicinal Water-bath. The term ‘water-bath’ means a bath of boiling
product that is not an active substance. Adjuvants, stabilisers, water unless water at another temperature is indicated.
antimicrobial preservatives, diluents, antioxidants, for Other methods of heating may be substituted provided the
example, are excipients. temperature is near to but not higher than 100 °C or the
Interchangeable methods. Certain general chapters contain indicated temperature.
a statement that the text in question is harmonised with Drying and ignition to constant mass. The terms ‘dried
the corresponding text of the Japanese Pharmacopoeia to constant mass’ and ‘ignited to constant mass’ mean that
and/or the United States Pharmacopeia and that these texts 2 consecutive weighings do not differ by more than 0.5 mg,
are interchangeable. This implies that if a substance or the 2nd weighing following an additional period of drying or
preparation is found to comply with a requirement using an of ignition respectively appropriate to the nature and quantity
interchangeable method from one of these pharmacopoeias of the residue.
it complies with the requirements of the European Where drying is prescribed using one of the expressions ‘in a
Pharmacopoeia. In the event of doubt or dispute, the text of desiccator’ or ‘in vacuo’, it is carried out using the conditions
the European Pharmacopoeia is alone authoritative. described in chapter 2.2.32. Loss on drying.
References to regulatory documents. Monographs and Reagents. The proper conduct of the analytical procedures
general chapters may contain references to documents described in the Pharmacopoeia and the reliability of the
issued by regulatory authorities for medicines, for example results depend, in part, upon the quality of the reagents used.
directives and notes for guidance of the European Union. The reagents are described in general chapter 4. It is assumed
These references are provided for information for users for that reagents of analytical grade are used ; for some reagents,
the Pharmacopoeia. Inclusion of such a reference does not tests to determine suitability are included in the specifications.
modify the status of the documents referred to, which may be
mandatory or for guidance. Solvents. Where the name of the solvent is not stated, the
term ‘solution’ implies a solution in water.
1.2. OTHER PROVISIONS APPLYING TO GENERAL Where the use of water is specified or implied in the
CHAPTERS AND MONOGRAPHS analytical procedures described in the Pharmacopoeia or
Quantities. In tests with numerical limits and assays, the for the preparation of reagents, water complying with the
quantity stated to be taken for examination is approximate. requirements of the monograph Purified water (0008) is
The amount actually used, which may deviate by not more used, except that for many purposes the requirements for
than 10 per cent from that stated, is accurately weighed or bacterial endotoxins (Purified water in bulk) and microbial
measured and the result is calculated from this exact quantity. contamination (Purified water in containers) are not relevant.
In tests where the limit is not numerical, but usually depends The term ‘distilled water’ indicates purified water prepared
upon comparison with the behaviour of a reference substance by distillation.
in the same conditions, the stated quantity is taken for The term ‘ethanol’ without qualification means anhydrous
examination. Reagents are used in the prescribed amounts. ethanol. The term ‘alcohol’ without qualification means
Quantities are weighed or measured with an accuracy ethanol (96 per cent). Other dilutions of ethanol are indicated
commensurate with the indicated degree of precision. For by the term ‘ethanol’ or ‘alcohol’ followed by a statement of the
weighings, the precision corresponds to plus or minus 5 units percentage by volume of ethanol (C2H6O) required.
after the last figure stated (for example, 0.25 g is to be Expression of content. In defining content, the expression
interpreted as 0.245 g to 0.255 g). For the measurement of ‘per cent’ is used according to circumstances with one of 2
volumes, if the figure after the decimal point is a zero or ends meanings :
in a zero (for example, 10.0 mL or 0.50 mL), the volume is
measured using a pipette, a volumetric flask or a burette, as – per cent m/m (percentage, mass in mass) expresses the
appropriate ; otherwise, a graduated measuring cylinder or a number of grams of substance in 100 g of final product ;
graduated pipette may be used. Volumes stated in microlitres – per cent V/V (percentage, volume in volume) expresses
are measured using a micropipette or microsyringe. the number of millilitres of substance in 100 mL of final
It is recognised, however, that in certain cases the precision product.
with which quantities are stated does not correspond to the The expression ‘parts per million’ (or ppm) refers to mass in
number of significant figures stated in a specified numerical mass, unless otherwise specified.
limit. The weighings and measurements are then carried out
with a sufficiently improved accuracy. Temperature. Where an analytical procedure describes
temperature without a figure, the general terms used have the
Apparatus and procedures. Volumetric glassware complies following meaning :
with Class A requirements of the appropriate International
Standard issued by the International Organisation for – in a deep-freeze : below − 15 °C ;
Standardisation. – in a refrigerator : 2 °C to 8 °C ;
Unless otherwise prescribed, analytical procedures are carried – cold or cool : 8 °C to 15 °C ;
out at a temperature between 15 °C and 25 °C.
– room temperature : 15 °C to 25 °C.
Unless otherwise prescribed, comparative tests are carried out
using identical tubes of colourless, transparent, neutral glass
with a flat base ; the volumes of liquid prescribed are for use 1.3. GENERAL CHAPTERS
with tubes having an internal diameter of 16 mm, but tubes Containers. Materials used for containers are described
with a larger internal diameter may be used provided the in general chapter 3.1. General names used for materials,
volume of liquid used is adjusted (2.1.5). Equal volumes of particularly plastic materials, each cover a range of products
the liquids to be compared are examined down the vertical varying not only in the properties of the principal constituent
axis of the tubes against a white background, or if necessary but also in the additives used. The test methods and limits
against a black background. The examination is carried out in for materials depend on the formulation and are therefore
diffuse light. applicable only for materials whose formulation is covered by
the preamble to the specification. The use of materials with Choice of vaccine strain, Choice of vaccine composition.
different formulations, and the test methods and limits applied The Production section of a monograph may define the
to them, are subject to agreement by the competent authority. characteristics of a vaccine strain or vaccine composition.
Unless otherwise stated, test methods given for verification of
The specifications for containers in general chapter 3.2 these characteristics are provided for information as examples
have been developed for general application to containers of suitable methods. Subject to approval by the competent
of the stated category, but in view of the wide variety of authority, other test methods may be used without validation
containers available and possible new developments, the against the method shown in the monograph.
publication of a specification does not exclude the use, in
justified circumstances, of containers that comply with POTENTIAL ADULTERATION
other specifications, subject to agreement by the competent Due to the increasing number of fraudulent activities and
authority. cases of adulteration, information may be made available to
Ph. Eur. users to help detect adulterated materials (i.e. active
Reference may be made within the monographs of the substances, excipients, intermediate products, bulk products
Pharmacopoeia to the definitions and specifications for and finished products).
containers provided in chapter 3.2. Containers. The general To this purpose, a method for the detection of potential
monographs for pharmaceutical dosage forms may, under adulterants and relevant limits, together with a reminder that
the heading Definition/Production, require the use of certain all stages of production and sourcing are subjected to a suitable
types of container ; certain other monographs may, under quality system, may be included in this section of monographs
the heading Storage, indicate the type of container that is on substances for which an incident has occurred or that
recommended for use. present a risk of deliberate contamination. The frequency of
testing by manufacturers or by users (e.g. manufacturers of
1.4. MONOGRAPHS intermediate products, bulk products and finished products,
where relevant) depends on a risk assessment, taking into
TITLES account the level of knowledge of the whole supply chain and
Monograph titles are in English and French in the respective national requirements.
versions and there is a Latin subtitle. This section constitutes requirements for the whole supply
RELATIVE ATOMIC AND MOLECULAR MASSES chain, from manufacturers to users (e.g. manufacturers of
The relative atomic mass (Ar) or the relative molecular intermediate products, bulk products and finished products,
mass (Mr) is shown, as and where appropriate, at the beginning where relevant). The absence of this section does not imply
of each monograph. The relative atomic and molecular masses that attention to features such as those referred to above is
and the molecular and graphic formulae do not constitute not required.
analytical standards for the substances described. CHARACTERS
CHEMICAL ABSTRACTS SERVICE (CAS) REGISTRY The statements under the heading Characters are not to be
NUMBER interpreted in a strict sense and are not requirements.
CAS registry numbers are included for information in Solubility. In statements of solubility in the Characters
monographs, where applicable, to provide convenient access section, the terms used have the following significance,
to useful information for users. CAS Registry Number® is a referred to a temperature between 15 °C and 25 °C.
registered trademark of the American Chemical Society. Descriptive term Approximate volume of solvent in millilitres
DEFINITION per gram of solute
Statements under the heading Definition constitute an official Very soluble less than 1
definition of the substance, preparation or other article that is 1 to 10
Freely soluble from
the subject of the monograph.
Soluble from 10 to 30
Limits of content. Where limits of content are prescribed,
they are those determined by the method described under Sparingly soluble from 30 to 100
Assay. to
Slightly soluble from 100 1000
Herbal drugs. In monographs on herbal drugs, the definition
Very slightly soluble from 1000 to 10 000
indicates whether the subject of the monograph is, for
example, the whole drug or the drug in powdered form. Practically insoluble more than 10 000
Where a monograph applies to the drug in several states, for
example both to the whole drug and the drug in powdered The term ‘partly soluble’ is used to describe a mixture where
form, the definition states this. only some of the components dissolve. The term ‘miscible’ is
used to describe a liquid that is miscible in all proportions
PRODUCTION
with the stated solvent.
Statements under the heading Production draw attention
to particular aspects of the manufacturing process but are IDENTIFICATION
not necessarily comprehensive. They constitute mandatory Scope. The tests given in the Identification section are not
requirements for manufacturers, unless otherwise stated. designed to give a full confirmation of the chemical structure
They may relate, for example, to source materials ; to the or composition of the product ; they are intended to give
manufacturing process itself and its validation and control ; to confirmation, with an acceptable degree of assurance, that the
in-process testing ; or to testing that is to be carried out by the article conforms to the description on the label.
manufacturer on the final article, either on selected batches First and second identifications. Certain monographs
or on each batch prior to release. These statements cannot have subdivisions entitled ‘First identification’ and ‘Second
necessarily be verified on a sample of the final article by an identification’. The test or tests that constitute the ‘First
independent analyst. The competent authority may establish identification’ may be used in all circumstances. The test or
that the instructions have been followed, for example, by tests that constitute the ‘Second identification’ may be used
examination of data received from the manufacturer, by in pharmacies provided it can be demonstrated that the
inspection of manufacture or by testing appropriate samples. substance or preparation is fully traceable to a batch certified
The absence of a Production section does not imply that to comply with all the other requirements of the monograph.
attention to features such as those referred to above is not Certain monographs give two or more sets of tests for the
required. purpose of the first identification, which are equivalent
and may be used independently. One or more of these sets Culture media. The culture media described in monographs
usually contain a cross-reference to a test prescribed in the and general chapters have been found to be satisfactory for
Tests section of the monograph. It may be used to simplify the intended purpose. However, the components of media,
the work of the analyst carrying out the identification and particularly those of biological origin, are of variable quality,
the prescribed tests. For example, one identification set and it may be necessary for optimal performance to modulate
cross-refers to a test for enantiomeric purity while the other the concentration of some ingredients, notably :
set gives a test for specific optical rotation : the intended – peptones and meat or yeast extracts, with respect to their
purpose of the two is the same, that is, verification that the nutritive properties ;
correct enantiomer is present.
– buffering substances ;
Powdered herbal drugs. Monographs on herbal drugs may
contain schematic drawings of the powdered drug. These – bile salts, bile extract, deoxycholate, and colouring matter,
drawings complement the description given in the relevant depending on their selective properties ;
identification test. – antibiotics, with respect to their activity.
TESTS AND ASSAYS STORAGE
Scope. The requirements are not framed to take account of all The information and recommendations given under the
possible impurities. It is not to be presumed, for example, that heading Storage do not constitute a pharmacopoeial
an impurity that is not detectable by means of the prescribed requirement but the competent authority may specify
tests is tolerated if common sense and good pharmaceutical particular storage conditions that must be met.
practice require that it be absent. See also below under
The articles described in the Pharmacopoeia are stored
Impurities.
in such a way as to prevent contamination and, as far as
Calculation. Where the result of a test or assay is required possible, deterioration. Where special conditions of storage
to be calculated with reference to the dried or anhydrous are recommended, including the type of container (see section
substance or on some other specified basis, the determination 1.3. General chapters) and limits of temperature, they are
of loss on drying, water content or other property is carried stated in the monograph.
out by the method prescribed in the relevant test in the The following expressions are used in monographs under
monograph. The words ‘dried substance’ or ‘anhydrous Storage with the meaning shown.
substance’ etc. appear in parentheses after the result.
Where a quantitative determination of a residual solvent is In an airtight container means that the product is stored in an
carried out and a test for loss on drying is not carried out, airtight container (3.2). Care is to be taken when the container
the content of residual solvent is taken into account for the is opened in a damp atmosphere. A low moisture content
calculation of the assay content of the substance, the specific may be maintained, if necessary, by the use of a desiccant in
optical rotation and the specific absorbance. No further the container provided that direct contact with the product
indication is given in the specific monograph. is avoided.
Limits. The limits prescribed are based on data obtained Protected from light means that the product is stored either
in normal analytical practice ; they take account of normal in a container made of a material that absorbs actinic light
analytical errors, of acceptable variations in manufacture and sufficiently to protect the contents from change induced by
compounding and of deterioration to an extent considered such light, or in a container enclosed in an outer cover that
acceptable. No further tolerances are to be applied to the limits provides such protection, or is stored in a place from which all
prescribed to determine whether the article being examined such light is excluded.
complies with the requirements of the monograph. LABELLING
In determining compliance with a numerical limit, the In general, labelling of medicines is subject to supranational
calculated result of a test or assay is first rounded to the and national regulation and to international agreements. The
number of significant figures stated, unless otherwise statements under the heading Labelling are not therefore
prescribed. The limits, regardless of whether the values are comprehensive and, moreover, for the purposes of the
expressed as percentages or as absolute values, are considered Pharmacopoeia only those statements that are necessary
significant to the last digit shown (for example 140 indicates 3 to demonstrate compliance or non-compliance with the
significant figures). The last figure of the result is increased by monograph are mandatory. Any other labelling statements are
one when the part rejected is equal to or exceeds one half-unit, included as recommendations. When the term ‘label’ is used
whereas it is not modified when the part rejected is less than a in the Pharmacopoeia, the labelling statements may appear
half-unit. on the container, the package, a leaflet accompanying the
Indication of permitted limit of impurities. The acceptance package, or a certificate of analysis accompanying the article,
criteria for related substances are expressed in monographs as decided by the competent authority.
either in terms of comparison of peak areas (comparative tests) WARNINGS
or as numerical values. For comparative tests, the approximate Materials described in monographs and reagents specified
content of impurity tolerated, or the sum of impurities, may for use in the Pharmacopoeia may be injurious to health
be indicated in brackets for information only. Acceptance unless adequate precautions are taken. The principles of
or rejection is determined on the basis of compliance or good quality control laboratory practice and the provisions
non-compliance with the stated test. If the use of a reference of any appropriate regulations are to be observed at all
substance for the named impurity is not prescribed, this times. Attention is drawn to particular hazards in certain
content may be expressed as a nominal concentration of the monographs by means of a warning statement ; absence of such
substance used to prepare the reference solution specified in a statement is not to be taken to mean that no hazard exists.
the monograph, unless otherwise described.
IMPURITIES
Herbal drugs. For herbal drugs, the sulfated ash, total ash, A list of all known and potential impurities that have been
water-soluble matter, alcohol-soluble matter, water content, shown to be detected by the tests in a monograph may be
content of essential oil and content of active principle are given. See also chapter 5.10. Control of impurities in substances
calculated with reference to the drug that has not been for pharmaceutical use. The impurities are designated by a
specially dried, unless otherwise prescribed in the monograph. letter or letters of the alphabet. Where a letter appears to
Equivalents. Where an equivalent is given, for the purposes be missing, the impurity designated by this letter has been
of the Pharmacopoeia only the figures shown are to be used in deleted from the list during monograph development prior to
applying the requirements of the monograph. publication or during monograph revision.
FUNCTIONALITY-RELATED CHARACTERISTICS OF L+/10 dose The smallest quantity of a toxin that, in the
EXCIPIENTS conditions of the test, when mixed with
Monographs on excipients may have a section on 0.1 IU of antitoxin and administered by the
functionality-related characteristics. The characteristics, any specified route, causes the death of the test
test methods for determination and any tolerances are not animals within a given period
mandatory requirements ; they may nevertheless be relevant L+ dose The smallest quantity of a toxin that, in the
for use of the excipient and are given for information (see also conditions of the test, when mixed with
section 1.1. General statements). 1 IU of antitoxin and administered by the
REFERENCE STANDARDS specified route, causes the death of the test
Certain monographs require the use of reference standards animals within a given period
(chemical reference substances, herbal reference standards, lr/100 dose The smallest quantity of a toxin that, in
biological reference preparations, reference spectra). See the conditions of the test, when mixed
also chapter 5.12. Reference standards. The European with 0.01 IU of antitoxin and injected
Pharmacopoeia Commission establishes the official intracutaneously causes a characteristic
reference standards, which are alone authoritative in case reaction at the site of injection within a
of arbitration. These reference standards are available from given period
the European Directorate for the Quality of Medicines & Lp/10 dose The smallest quantity of toxin that, in the
HealthCare (EDQM). Information on the available reference conditions of the test, when mixed with
standards and a batch validity statement can be obtained via 0.1 IU of antitoxin and administered by the
the EDQM website. specified route, causes paralysis in the test
animals within a given period
Lo/10 dose The largest quantity of a toxin that, in the
1.5. ABBREVIATIONS AND SYMBOLS conditions of the test, when mixed with
A Absorbance 0.1 IU of antitoxin and administered by the
specified route, does not cause symptoms of
Specific absorbance toxicity in the test animals within a given
Ar Relative atomic mass period
Lf dose The quantity of toxin or toxoid that
Specific optical rotation flocculates in the shortest time with 1 IU of
bp Boiling point antitoxin
BRP Biological reference preparation CCID50 The statistically determined quantity of
virus that may be expected to infect 50 per
CRS Chemical reference substance cent of the cell cultures to which it is added
Relative density EID50 The statistically determined quantity of
λ Wavelength virus that may be expected to infect 50 per
cent of the fertilised eggs into which it is
HRS Herbal reference standard inoculated
IU International Unit ID50 The statistically determined quantity of
a virus that may be expected to infect
M Molarity 50 per cent of the animals into which it is
Mr Relative molecular mass inoculated
mp PD50 The statistically determined dose of a
Melting point
vaccine that, in the conditions of the test,
Refractive index may be expected to protect 50 per cent of
Ph. Eur. U. European Pharmacopoeia Unit the animals against a challenge dose of the
micro-organisms or toxins against which it
ppb Parts per billion (micrograms per kilogram) is active
ppm Parts per million (milligrams per kilogram) ED50 The statistically determined dose of a
vaccine that, in the conditions of the
R Substance or solution defined under test, may be expected to induce specific
4. Reagents antibodies in 50 per cent of the animals for
RF Retardation factor (see chapter 2.2.46) the relevant vaccine antigens
Rst Used in chromatography to indicate the PFU Pock-forming units or plaque-forming units
ratio of the distance travelled by a substance SPF Specified-pathogen-free
to the distance travelled by a reference
substance Collections of micro-organisms
RV Substance used as a primary standard in ATCC American Type Culture Collection
volumetric analysis (chapter 4.2.1)
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immunoglobulins, immunosera and vaccines C.I.P. Collection de Bactéries de l’Institut Pasteur
LD50 The statistically determined quantity of a B.P. 52, 25 rue du Docteur Roux
substance that, when administered by the
specified route, may be expected to cause 75724 Paris Cedex 15, France
the death of 50 per cent of the test animals IMI International Mycological Institute
within a given period Bakeham Lane
MLD Minimum lethal dose Surrey TW20 9TY, Great Britain
I.P. Collection Nationale de Culture de 1.6. UNITS OF THE INTERNATIONAL SYSTEM (SI) USED
Microorganismes (C.N.C.M.) IN THE PHARMACOPOEIA AND EQUIVALENCE WITH
Institut Pasteur OTHER UNITS
25, rue du Docteur Roux INTERNATIONAL SYSTEM OF UNITS (SI)
75724 Paris Cedex 15, France The International System of Units comprises 3 classes of units,
namely base units, derived units and supplementary units(1).
NCIMB National Collection of Industrial and The base units and their definitions are set out in Table 1.6-1.
Marine Bacteria Ltd
The derived units may be formed by combining the
23 St Machar Drive base units according to the algebraic relationships linking
Aberdeen AB2 1RY, Great Britain the corresponding quantities. Some of these derived units
NCPF National Collection of Pathogenic Fungi have special names and symbols. The SI units used in the
Pharmacopoeia are shown in Table 1.6-2.
London School of Hygiene and Tropical
Medicine Some important and widely used units outside the
International System are shown in Table 1.6-3.
Keppel Street
The prefixes shown in Table 1.6-4 are used to form the names
London WC1E 7HT, Great Britain and symbols of the decimal multiples and submultiples of
NCTC National Collection of Type Cultures SI units.
Central Public Health Laboratory NOTES
Colindale Avenue 1. In the Pharmacopoeia, the Celsius temperature is used
London NW9 5HT, Great Britain (symbol t). This is defined by the following equation :
NCYC National Collection of Yeast Cultures
AFRC Food Research Institute
Colney Lane where T0 = 273.15 K by definition. The Celsius or centigrade
Norwich NR4 7UA, Great Britain temperature is expressed in degrees Celsius (symbol °C).
The unit ‘degree Celsius’ is equal to the unit ‘kelvin’.
NITE Biological Resource Center
2. The practical expressions of concentrations used in the
Department of Biotechnology Pharmacopoeia are defined in the General Notices.
National Institute of Technology and 3. The radian is the plane angle between two radii of a circle
Evaluation that cut off on the circumference an arc equal in length
2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, to the radius.
292-0818 4. In the Pharmacopoeia, conditions of centrifugation are
Japan defined by reference to the acceleration due to gravity (g) :
S.S.I. Statens Serum Institut
·
80 Amager Boulevard, Copenhagen,
Denmark 5. Certain quantities without dimensions are used in the
Pharmacopoeia : relative density (2.2.5), absorbance
(2.2.25), specific absorbance (2.2.25) and refractive index
(2.2.6).
6. The microkatal is defined as the enzymic activity that,
under defined conditions, produces the transformation
(e.g. hydrolysis) of 1 micromole of the substrate per second.
Table 1.6.-1. – SI base units
Quantity Unit Definition
Name Symbol Name Symbol
The metre is the length of the path travelled by light in a vacuum during a time
Length l metre m
interval of 1/299 792 458 of a second.
Mass m kilogram kg The kilogram is equal to the mass of the international prototype of the kilogram.
The second is the duration of 9 192 631 770 periods of the radiation corresponding
Time t second s to the transition between the two hyperfine levels of the ground state of the
caesium-133 atom.
Electric current I ampere A The ampere is that constant current which, maintained in two straight parallel
conductors of infinite length, of negligible circular cross-section and placed 1 metre
apart in vacuum would produce between these conductors a force equal to 2 × 10− 7
newton per metre of length.
Thermodynamic T kelvin K The kelvin is the fraction 1/273.16 of the thermodynamic temperature of the triple
temperature point of water.
Amount of substance n mole mol The mole is the amount of substance of a system containing as many elementary
entities as there are atoms in 0.012 kilogram of carbon-12*.
Luminous intensity Iv candela cd The candela is the luminous intensity in a given direction of a source emitting
monochromatic radiation with a frequency of 540 × 1012 hertz and whose energy
intensity in that direction is 1/683 watt per steradian.
* When the mole is used, the elementary entities must be specified and may be atoms, molecules, ions, electrons, other particles or specified
groups of such particles.
(1) The definitions of the units used in the International System are given in the booklet ‘Le Système International d’Unités (SI)’, published by the Bureau International des Poids
et Mesures, Pavillon de Breteuil, F-92310 Sèvres.
Table 1.6.-2. – SI units used in the European Pharmacopoeia and equivalence with other units
Quantity Unit
Name Symbol Name Symbol Expression in SI Expression in Conversion of other units into SI units
base units other SI units
Wave number ν one per metre 1/m m− 1
Frequency ν hertz Hz s −1
Table 1.6.-3. – Units used with the International System Table 1.6.-4. – Decimal multiples and sub-multiples of units
Quantity Unit Value in SI units
Factor Prefix Symbol Factor Prefix Symbol
Name Symbol
10 18 exa E 10− 1 deci d
Time minute min 1 min = 60 s
10 15
peta P 10 −2
centi c
hour h 1 h = 60 min = 3600 s
10 12
tera T 10 −3
milli m
day d 1 d = 24 h = 86 400 s
109 giga G 10− 6 micro μ
Plane angle degree ° 1° = (π/180) rad
10 6 mega M 10 −9 nano n
Volume litre L 1 L = 1 dm3 = 10− 3 m3
10 3
kilo k 10 − 12
pico p
Mass tonne t 1 t = 103 kg
2 − 15
10 hecto h 10 femto f
Rotational revolution r/min 1 r/min = (1/60) s− 1
frequency per minute 101 deca da 10− 18 atto a
10 4 - 10 4f – 4
16 10 - 16 4 4 –
40 16 - 40 3 3 3
– 40 - 50 – – 2
100 40 - 100 2 2 –
– 100 - 120 – – 1
160 100 - 160 1 1 –
– 150 - 200 0 0 –
Special Uses
Diameters in micrometres
< 2.5 Bacteriological filtration
4 - 10 Ultra-fine filtration, separation of micro-organisms of large
diameter
10 - 40 Analytical filtration, very fine filtration of mercury, very fine
dispersion of gases
40 - 100 Fine filtration, filtration of mercury, fine dispersion of gases
100 - 160 Filtration of coarse materials, dispersion and washing of gases,
support for other filter materials
160 - 500 Filtration of very coarse materials, dispersion and washing of
gases.
01/2008:20103
2.1.6. GAS DETECTOR TUBES Figure 2.1.6.-1. – Apparatus for gas detector tubes
Carbon dioxide detector tube. Sealed glass tube containing
Gas detector tubes are cylindrical, sealed tubes consisting of adsorbent filters and suitable supports for hydrazine and
an inert transparent material and are constructed to allow crystal violet indicators. The minimum value indicated is
the passage of gas. They contain reagents adsorbed onto 100 ppm with a relative standard deviation of at most ± 15 per
inert substrates that are suitable for the visualisation of the cent.
substance to be detected and, if necessary, they also contain
preliminary layers and/or adsorbent filters to eliminate Sulfur dioxide detector tube. Sealed glass tube containing
substances that interfere with the substance to be detected. adsorbent filters and suitable supports for the iodine and
The layer of indicator contains either a single reagent for starch indicator. The minimum value indicated is 0.5 ppm
the detection of a given impurity or several reagents for the with a relative standard deviation of at most ± 15 per cent.
detection of several substances (monolayer tube or multilayer Oil detector tube. Sealed glass tube containing adsorbent
tube). filters and suitable supports for the sulfuric acid indicator.
The test is carried out by passing the required volume of the The minimum value indicated is 0.1 mg/m3 with a relative
gas to be examined through the indicator tube. The length standard deviation of at most ± 30 per cent.
of the coloured layer or the intensity of a colour change on a Nitrogen monoxide and nitrogen dioxide detector
graduated scale gives an indication of the impurities present. tube. Sealed glass tube containing adsorbent filters and
The calibration of the detector tubes is verified according to suitable supports for an oxidising layer (Cr(VI) salt) and the
the manufacturer’s instructions. diphenylbenzidine indicator. The minimum value indicated is
0.5 ppm with a relative standard deviation of at most ± 15 per
Operating conditions. Examine according to the manufacturer’s cent.
instructions or proceed as follows :
Carbon monoxide detector tube. Sealed glass tube
The gas supply is connected to a suitable pressure regulator containing adsorbent filters and suitable supports for di-iodine
and needle valve. Connect the flexible tubing fitted with a pentoxide, selenium dioxide and fuming sulfuric acid
Y-piece to the valve and adjust the flow of gas to be examined indicators. The minimum value indicated is 5 ppm or less,
to purge the tubing in order to obtain an appropriate flow with a relative standard deviation of at most ± 15 per cent.
(Figure 2.1.6.-1). Prepare the indicator tube and fit to the
metering pump, following the manufacturer’s instructions. Hydrogen sulfide detector tube. Sealed glass tube containing
Connect the open end of the indicator tube to the short leg of adsorbent filters and suitable supports for an appropriate lead
the tubing and operate the pump by the appropriate number salt indicator. The minimum value indicated is 1 ppm or less,
of strokes to pass a suitable volume of gas to be examined with a relative standard deviation of at most ± 10 per cent.
through the tube. Read the value corresponding to the length Water vapour detector tube. Sealed glass tube containing
of the coloured layer or the intensity of the colour on the adsorbent filters and suitable supports for the magnesium
graduated scale. If a negative result is achieved, indicator perchlorate indicator. The minimum value indicated is
tubes can be verified with a calibration gas containing the 67 ppm or less, with a relative standard deviation of at most
appropriate impurity. ± 20 per cent.
transmitted light. A linear relationship between turbidity and are also used, and are equivalent to NTU in low regions
concentration is obtained when the particle sizes are uniform (up to 40 NTU). These units are used in all 3 instrumental
and homogeneous in the suspension. This is true only in methods (nephelometry, turbidimetry and ratio
very dilute suspensions containing small particles. Linearity turbidimetry).
between turbidity and concentration must be established by – Measuring range : 0.01-1100 NTU.
constructing a calibration curve using at least 4 concentrations. – Resolution : 0.01 NTU within the range of 0-10 NTU,
RATIO TURBIDIMETRY 0.1 NTU within the range of 10-100 NTU, and 1 NTU for
In ratio turbidimetry the relationship of the transmission the range > 100 NTU. The instrument is calibrated and
measurement to the 90° scattered light measurement is controlled with reference standards of formazin.
determined. This procedure compensates for the light that – Accuracy : 0-10 NTU : ± (2 per cent of reading + 0.01) NTU.
is diminished by the colour of the sample. The influence 10-1000 NTU : ± 5 per cent.
of the colour of the sample may also be eliminated by – Repeatability : 0-10 NTU : ± 0.01 NTU. 10-1000 NTU : ± 2 per
using an infrared light-emitting diode (IR LED) at 860 nm cent of the measured value.
as the light source of the instrument. The instrument’s
photodiode detectors receive and measure scattered light at a – Calibration : with 4 reference suspensions of formazin in
90° angle from the sample as well as measuring the forward the range of interest. Reference suspensions described
scatter (light reflected) in front of the sample along with in this chapter or suitable reference standards calibrated
the measurement of light transmitted directly through the against the primary reference suspensions may be used.
sample. The measuring results are given in NTU(ratio) and – Stray light : this is a significant source of error in low level
are obtained by calculating the ratio of the 90° angle scattered turbidimetric measurement ; stray light reaches the detector
light measured to the sum of the components of forward of an optical system, but does not come from the sample ;
scattered and transmitted light values. In ratio turbidimetry < 0.15 NTU for the range 0-10 NTU, < 0.5 NTU for the
the influence of stray light becomes negligible. Nephelometers range 10-1000 NTU.
are used for measurements of the degree of opalescence of Instruments complying with the above characteristics and
colourless liquids. verified using the reference suspensions described under
Measurements of reference suspensions I-IV with a ratio Visual method may be used instead of visual examination for
turbidimeter show a linear relationship between the determination of compliance with monograph requirements.
concentrations and measured NTU values (see Table 2.2.1.-2). Instruments with range or resolution, accuracy and
Reference suspensions I-IV (Ph. Eur.) may be used as repeatability capabilities other than those mentioned above
calibrators for the instrument. may be used provided they are sufficiently validated and
are capable for the intended use. The test methodology for
Table 2.2.1.-2 the specific substance/product to be analysed must also
Formazin suspensions Opalescent values (NTU) be validated to demonstrate its analytical capability. The
Reference suspension I 3 instrument and methodology should be consistent with the
attributes of the product to be tested.
Reference suspension II 6
Reference suspension IV 30
01/2008:20202
Standard of opalescence 60
mixture. Titrate and adjust the solution to contain 45.0 mg of Table 2.2.2.-2. - Reference solutions B
FeCl3,6H2O per millilitre by adding the same acidic mixture.
Protect the solution from light. Volumes in millilitres
Titration. Place in a 250 mL conical flask fitted with a Reference Standard solution B Hydrochloric acid
solution (10 g/L HCl)
ground-glass stopper, 10.0 mL of the solution, 15 mL of
water R, 5 mL of hydrochloric acid R and 4 g of potassium B1 75.0 25.0
iodide R, close the flask, allow to stand in the dark for 15 min B2 50.0 50.0
and add 100 mL of water R. Titrate the liberated iodine with
0.1 M sodium thiosulfate, using 0.5 mL of starch solution R, B3 37.5 62.5
added towards the end of the titration, as indicator. B4 25.0 75.0
Titration. Place in a 250 mL conical flask fitted with a Table 2.2.2.-3. - Reference solutions BY
ground-glass stopper, 5.0 mL of the solution, 5 mL of dilute
hydrogen peroxide solution R and 10 mL of a 300 g/L solution
Volumes in millilitres
of sodium hydroxide R. Boil gently for 10 min, allow to cool
and add 60 mL of dilute sulfuric acid R and 2 g of potassium Reference Standard solution BY Hydrochloric acid
iodide R. Close the flask and dissolve the precipitate by solution (10 g/L HCl)
shaking gently. Titrate the liberated iodine with 0.1 M sodium BY1 100.0 0.0
thiosulfate, using 0.5 mL of starch solution R, added towards BY2 75.0 25.0
the end of the titration, as indicator. The end-point is reached
when the solution turns pink. BY3 50.0 50.0
BY4 25.0 75.0
1 mL of 0.1 M sodium thiosulfate is equivalent to 23.79 mg of
CoCl2,6H2O. BY5 12.5 87.5
BY6 5.0 95.0
Blue primary solution. Dissolve 63 g of copper sulfate R in
about 900 mL of a mixture of 25 mL of hydrochloric acid R BY7 2.5 97.5
and 975 mL of water R and dilute to 1000.0 mL with the same
mixture. Titrate and adjust the solution to contain 62.4 mg of
CuSO4,5H2O per millilitre by adding the same acidic mixture.
Table 2.2.2.-4. - Reference solutions Y
Titration. Place in a 250 mL conical flask fitted with a
ground-glass stopper, 10.0 mL of the solution, 50 mL of Volumes in millilitres
water R, 12 mL of dilute acetic acid R and 3 g of potassium Reference Standard solution Y Hydrochloric acid
iodide R. Titrate the liberated iodine with 0.1 M sodium solution (10 g/L HCl)
thiosulfate, using 0.5 mL of starch solution R, added towards Y1 100.0 0.0
the end of the titration, as indicator. The end-point is reached
when the solution shows a slight pale brown colour. Y2 75.0 25.0
Y3 50.0 50.0
1 mL of 0.1 M sodium thiosulfate is equivalent to 24.97 mg
of CuSO4,5H2O. Y4 25.0 75.0
Table 2.2.2.-1
Table 2.2.2.-5. - Reference solutions GY
Volume in millilitres
Standard solution Yellow Red Blue Hydrochloric acid Volumes in millilitres
solution solution solution (10 g/L HCl)
Reference Standard solution GY Hydrochloric acid
B (brown) 3.0 3.0 2.4 1.6 solution (10 g/L HCl)
BY (brownish-yellow) 2.4 1.0 0.4 6.2 GY1 25.0 75.0
Table 2.2.2.-6. - Reference solutions R Method. Unless otherwise prescribed in the monograph, all
measurements are made at the same temperature (20-25 °C).
Volumes in millilitres Table 2.2.3.-2 shows the variation of pH with respect to
Reference Standard solution R Hydrochloric acid temperature of a number of reference buffer solutions used for
solution (10 g/L HCl) calibration. For the temperature correction, when necessary,
R1 100.0 0.0 follow the manufacturer’s instructions. The apparatus is
calibrated with the buffer solution of potassium hydrogen
R2 75.0 25.0
phthalate (primary standard) and 1 other buffer solution of
R3 50.0 50.0 different pH (preferably one shown in Table 2.2.3.-2). The
pH of a third buffer solution of intermediate pH read off on
R4 37.5 62.5
the scale must not differ by more than 0.05 pH unit from the
R5 25.0 75.0 value corresponding to this solution. Immerse the electrodes
in the solution to be examined and take the reading in the
R6 12.5 87.5
same conditions as for the buffer solutions.
R7 5.0 95.0
When the apparatus is in frequent use, checks must be carried
Storage out regularly. If not, such checks should be carried out before
each measurement.
For Method I, the reference solutions may be stored in sealed
tubes of colourless, transparent, neutral glass of 12 mm All solutions to be examined and the reference buffer solutions
external diameter, protected from light. must be prepared using carbon dioxide-free water R.
For Method II, prepare the reference solutions immediately
before use from the standard solutions.
PREPARATION OF REFERENCE BUFFER SOLUTIONS
Potassium tetraoxalate 0.05 M. Dissolve 12.61 g of
C4H3KO8,2H2O in carbon dioxide-free water R and dilute to
1000.0 mL with the same solvent.
01/2008:20203 Potassium hydrogen tartrate, saturated at 25 °C. Shake an
excess of C4H5KO6 vigorously with carbon dioxide-free water R
at 25 °C. Filter or decant. Prepare immediately before use.
2.2.3. POTENTIOMETRIC Potassium dihydrogen citrate 0.05 M. Dissolve 11.41 g
DETERMINATION OF pH of C6H7KO7 in carbon dioxide-free water R and dilute to
1000.0 mL with the same solvent. Prepare immediately before
The pH is a number which represents conventionally the use.
hydrogen ion concentration of an aqueous solution. For Potassium hydrogen phthalate 0.05 M. Dissolve 10.13 g of
practical purposes, its definition is an experimental one. The C8H5KO4, previously dried for 1 h at 110 ± 2 °C, in carbon
pH of a solution to be examined is related to that of a reference dioxide-free water R and dilute to 1000.0 mL with the same
solution (pHs) by the following equation : solvent.
Potassium dihydrogen phosphate 0.025 M + disodium
hydrogen phosphate 0.025 M. Dissolve 3.39 g of KH2PO4 and
3.53 g of Na2HPO4, both previously dried for 2 h at 120 ± 2 °C,
in which E is the potential, expressed in volts, of the cell in carbon dioxide-free water R and dilute to 1000.0 mL with
containing the solution to be examined and Es is the potential, the same solvent.
expressed in volts, of the cell containing the solution of known Potassium dihydrogen phosphate 0.0087 M + disodium
pH (pHs), k is the change in potential per unit change in pH hydrogen phosphate 0.0303 M. Dissolve 1.18 g of KH2PO4
expressed in volts, and calculated from the Nernst equation. and 4.30 g of Na HPO , both previously dried for 2 h at
2 4
120 ± 2 °C, in carbon dioxide-free water R and dilute to
Table 2.2.3.-1. – Values of k at different temperatures 1000.0 mL with the same solvent.
Temperature (°C) k (V) Disodium tetraborate 0.01 M. Dissolve 3.80 g of
15 0.0572 Na2B4O7,10H2O in carbon dioxide-free water R and dilute
to 1000.0 mL with the same solvent. Store protected from
20 0.0582 atmospheric carbon dioxide.
25 0.0592 Sodium carbonate 0.025 M + sodium hydrogen carbonate
30 0.0601 0.025 M. Dissolve 2.64 g of Na2CO3 and 2.09 g of NaHCO3
in carbon dioxide-free water R and dilute to 1000.0 mL with
35 0.0611 the same solvent. Store protected from atmospheric carbon
dioxide.
The potentiometric determination of pH is made by measuring
the potential difference between 2 appropriate electrodes Calcium hydroxide, saturated at 25 °C. Shake an excess of
immersed in the solution to be examined : 1 of these electrodes calcium hydroxide R with carbon dioxide-free water R and
is sensitive to hydrogen ions (usually a glass electrode) and decant at 25 °C. Store protected from atmospheric carbon
the other is the reference electrode (for example, a saturated dioxide.
calomel electrode).
Apparatus. The measuring apparatus is a voltmeter with an
STORAGE
input resistance at least 100 times that of the electrodes used.
It is normally graduated in pH units and has a sensitivity such Store buffer solutions in suitable chemically resistant, tight
that discrimination of at least 0.05 pH unit or at least 0.003 V containers, such as type I glass bottles or plastic containers
may be achieved. suitable for aqueous solutions.
01/2008:20204 01/2008:20205
The resonant frequency (f) is a function of the spring When white light is used, the refractometer is provided with a
constant (c) and the mass (m) of the system : compensating system. The apparatus gives readings accurate
to at least the third decimal place and is provided with a means
of operation at the temperature prescribed. The thermometer
is graduated at intervals of 0.5 °C or less.
Hence :
01/2008:20207
· · ·
k = constant of the viscometer, expressed in square
α = angle of rotation in degrees read at 20 ± 0.5°C ; millimetres per second squared,
l = length in decimetres of the polarimeter tube ; ρ = density of the liquid to be examined expressed
ρ20 = density at 20 °C in grams per cubic centimetre. in milligrams per cubic millimetre, obtained by
For the purposes of the Pharmacopoeia, density is multiplying its relative density ( ) by 0.9982,
replaced by relative density (2.2.5). t = flow time, in seconds, of the liquid to be examined.
2.2.8. VISCOSITY The minimum flow time should be 350 s for size no. 1 and
200 s for all other sizes.
(1) The European Pharmacopoeia describes the system proposed by the International Organisation for Standardisation (ISO).
APPARATUS
The following types of instruments are most common.
CONCENTRIC CYLINDER VISCOMETERS (ABSOLUTE
VISCOMETERS)
In the concentric cylinder viscometer (coaxial double cylinder
viscometer or simply coaxial cylinder viscometer), the viscosity
is determined by placing the liquid in the gap between the
inner cylinder and the outer cylinder. Viscosity measurement
can be performed by rotating the inner cylinder (Searle type
viscometer) or the outer cylinder (Couette type viscometer),
as shown in Figures 2.2.10.-1 and 2.2.10.-2, respectively. For
laminar flow, the viscosity (or apparent viscosity) η expressed
in pascal-seconds is given by the following formula :
– for cone-plates :
01/2008:20210
Figure 2.2.10.-3
Figure 2.2.10.-1
Figure 2.2.10.-4
SPINDLE VISCOMETERS (RELATIVE VISCOMETERS)
In the spindle viscometer, the viscosity is determined by
rotating a spindle (for example, cylinder- or disc-shaped,
as shown in Figures 2.2.10.-5 and 2.2.10.-6, respectively)
immersed in the liquid. Relative values of viscosity (or
apparent viscosity) can be directly calculated using conversion
factors from the scale reading at a given rotational speed.
Figure 2.2.10.-2
CONE-PLATE VISCOMETERS (ABSOLUTE VISCOMETERS)
In the cone-plate viscometer, the liquid is introduced into the
gap between a flat disc and a cone forming a define angle.
Viscosity measurement can be performed by rotating the cone
or the flat disc, as shown in Figures 2.2.10.-3 and 2.2.10.-4,
respectively. For laminar flow, the viscosity (or apparent
viscosity) η expressed in pascal-seconds is given by the
following formula :
01/2008:20211
Correct the observed temperatures for barometric pressure by Method. Clean the receiving tube and the condenser of the
means of the formula : apparatus, thoroughly rinse with water, and dry.
Introduce 200 mL of toluene R and about 2 mL of water R into
the dry flask. Distil for 2 h, then allow to cool for about 30 min
and read the water volume to the nearest 0.05 mL. Place in the
t1 = the corrected temperature, flask a quantity of the substance, weighed with an accuracy of
t2 = the observed temperature, at the barometric 1 per cent, expected to give about 2 mL to 3 mL of water. If
pressure b, the substance has a pasty consistency, weigh it in a boat of
metal foil. Add a few pieces of porous material and heat the
k = the correction factor taken from Table 2.2.11.-1
flask gently for 15 min. When the toluene begins to boil, distil
unless the factor is given,
at the rate of about two drops per second until most of the
b = the barometric pressure, expressed in kilopascals, water has distilled over, then increase the rate of distillation to
during the distillation. about four drops per second. When the water has all distilled
over, rinse the inside of the condenser tube with toluene R.
Table 2.2.11.-1. – Temperature correction in relation to the Continue the distillation for 5 min, remove the heat, allow the
pressure receiving tube to cool to room temperature and dislodge any
Distillation temperature Correction factor k droplets of water which adhere to the walls of the receiving
tube. When the water and toluene have completely separated,
up to 100 °C 0.30
read the volume of water and calculate the content present in
above 100 °C up to 140 °C 0.34 the substance as millilitres per kilogram, using the formula :
above 140 °C up to 190 °C 0.38
01/2008:20213
01/2008:20214 Repeat the operation with the other 4 capillary tubes and
calculate the result as the mean of the 5 readings.
2.2.14. MELTING POINT - CAPILLARY
01/2008:20216
METHOD
The melting point determined by the capillary method is 2.2.16. MELTING POINT -
the temperature at which the last solid particle of a compact
column of a substance in a tube passes into the liquid phase.
INSTANTANEOUS METHOD
When prescribed in the monograph, the same apparatus and The instantaneous melting point is calculated using the
method are used for the determination of other factors, such expression :
as meniscus formation or melting range, that characterise the
melting behaviour of a substance.
Apparatus. The apparatus consists of :
in which t1 is the first temperature and t2 the second
– a suitable glass vessel containing a liquid bath (for example,
temperature read under the conditions stated below.
water, liquid paraffin or silicone oil) and fitted with a
suitable means of heating, Apparatus. The apparatus consists of a metal block resistant
to the substance to be examined, of good heat-conducting
– a suitable means of stirring, ensuring uniformity of capacity, such as brass, with a carefully polished plane upper
temperature within the bath, surface. The block is uniformly heated throughout its mass by
– a suitable thermometer with graduation at not more than means of a micro-adjustable gas heater or an electric heating
0.5 °C intervals and provided with an immersion mark. device with fine adjustment. The block has a cylindrical cavity,
The range of the thermometer is not more than 100 °C, wide enough to accomodate a thermometer, which should be
– alkali-free hard-glass capillary tubes of internal diameter maintained with the mercury column in the same position
0.9 mm to 1.1 mm with a wall 0.10 mm to 0.15 mm thick during the calibration of the apparatus and the determination
and sealed at one end. of the melting point of the substance to be examined. The
Method. Unless otherwise prescribed, dry the finely powdered cylindrical cavity is parallel to the upper polished surface of
substance in vacuo and over anhydrous silica gel R for 24 h. the block and about 3 mm from it. The apparatus is calibrated
Introduce a sufficient quantity into a capillary tube to give using appropriate substances of known melting point.
a compact column 4 mm to 6 mm in height. Raise the Method. Heat the block at a suitably rapid rate to a temperature
temperature of the bath to about 10 °C below the presumed about 10 °C below the presumed melting temperature, then
melting point and then adjust the rate of heating to about adjust the heating rate to about 1 °C/min. At regular intervals
1 °C/min. When the temperature is 5 °C below the presumed drop a few particles of powdered and, where appropriate, dried
melting point, correctly introduce the capillary tube into thesubstance, prepared as for the capillary tube method, onto the
instrument. For the apparatus described above, immerse the block in the vicinity of the thermometer bulb, cleaning the
surface after each test. Record the temperature t1 at which the
capillary tube so that the closed end is near the centre of the
bulb of the thermometer, the immersion mark of which is at substance melts instantaneously for the first time in contact
the level of the surface of the liquid. Record the temperaturewith the metal. Stop the heating. During cooling drop a few
at which the last particle passes into the liquid phase. particles of the substance at regular intervals on the block,
Calibration of the apparatus. The apparatus may be calibrated cleaning the surface after each test. Record the temperature t2
using melting point reference substances such as those of the at which the substance ceases to melt instantaneously when it
World Health Organization or other appropriate substances. comes in contact with the metal.
Calibration of the apparatus. The apparatus may be calibrated
using melting point reference substances such as those of the
01/2008:20215 World Health Organization or other appropriate substances.
with an external diameter of about 40 mm. It is fixed to the There is another, narrower cylindrical vertical hole in which
test-tube by means of a laterally grooved stopper through a temperature sensor sits. This is positioned level with the
which the thermometer passes. The opening of the cup is sample cup. The heating block is surrounded by an electrical
placed about 15 mm from the bottom of the test-tube. The heating element. Below the heating block a lamp is mounted
whole device is immersed in a beaker with a capacity of about such that a beam of light shines through the light slit in the
1 L, filled with water. The bottom of the test-tube is placed collector sleeve, and onto a photo-sensor mounted opposite.
about 25 mm from the bottom of the beaker. The water level The heating block is capable of being maintained at a precise,
reaches the upper part of sheath A. A stirrer is used to ensure pre-defined temperature by the heating element, and of being
that the temperature of the water remains uniform. heated at a slow and steady, pre-defined rate after an initial
isothermal period.
rod (diameter about 4.5 mm). Check that the opening is Apparatus. The apparatus (see Figure 2.2.18.-1) consists of a
completely filled. Fill the sample cup about half full and test-tube about 25 mm in diameter and 150 mm long placed
compact the sample with a rod (diameter about 9 mm). Fill inside a test-tube about 40 mm in diameter and 160 mm
the sample cup completely and compact, adding more sample long. The inner tube is closed by a stopper which carries a
and compacting again if necessary, until the sample cup is thermometer about 175 mm long and graduated in 0.2 °C
completely full. fixed so that the bulb is about 15 mm above the bottom of the
Temperature programme for benzoic acid : start tube. The stopper has a hole allowing the passage of the stem
temperature = 118.0 °C ; heating rate = 0.2 °C/min ; end of a stirrer made from a glass rod or other suitable material
temperature = 126.0 °C. After inserting the cup at 118 °C, a formed at one end into a loop of about 18 mm overall diameter
waiting time of 30 s is set before heating starts. at right angles to the rod. The inner tube with its jacket is
Temperature programme for benzophenone : start supported centrally in a 1 L beaker containing a suitable
temperature = 44.0 °C ; heating rate = 0.2 °C/min ; end cooling liquid to within 20 mm of the top. A thermometer is
temperature = 56.0 °C. After inserting the cup at 44 °C, a supported in the cooling bath.
waiting time of 30 s is set before heating starts. Method. Place in the inner tube sufficient quantity of the
Check the 3 single results : the test is valid if the 3 results are liquid or previously melted substance to be examined, to cover
within 0.3 °C of the mean value. the thermometer bulb and determine the approximate freezing
point by cooling rapidly. Place the inner tube in a bath about
Calculate the corrected mean temperature (T)2 using the 5 °C above the approximate freezing point until all but the last
following expression : traces of crystals are melted. Fill the beaker with water or a
saturated solution of sodium chloride, at a temperature about
5 °C lower than the expected freezing point, insert the inner
tube into the outer tube, ensuring that some seed crystals are
present, and stir thoroughly until solidification takes place.
Note the highest temperature observed during solidification.
T1 = mean drop point temperature of 3 samples, in °C ;
F = compensation for the difference in temperature 01/2008:20219
between the sample and the point in the heating
block where the temperature is measured ; this will 2.2.19. AMPEROMETRIC TITRATION
vary depending upon the design of the automatic
drop point instrument and is provided by the In amperometric titration the end-point is determined by
manufacturer. following the variation of the current measured between
2 electrodes (either one indicator electrode and one reference
Taking into account the drop point (T0) of the certified electrode or 2 indicator electrodes) immersed in the solution
reference material, the accuracy of the temperature scale is to be examined and maintained at a constant potential
satisfactory if |T2 − T0| is not greater than 0.3 °C. difference as a function of the quantity of titrant added.
The potential of the measuring electrode is sufficient to ensure
01/2008:20218 a diffusion current for the electroactive substance.
Apparatus. The apparatus comprises an adjustable voltage
2.2.18. FREEZING POINT source and a sensitive microammeter ; the detection system
The freezing point is the maximum temperature occurring generally consists of an indicator electrode (for example,
during the solidification of a supercooled liquid. a platinum electrode, a dropping-mercury electrode, a
rotating-disc electrode or a carbon electrode) and a reference
electrode (for example, a calomel electrode or a silver-silver
chloride electrode).
A three-electrode apparatus is sometimes used, consisting of
an indicator electrode, a reference electrode and a polarised
auxiliary electrode.
Method. Set the potential of the indicator electrode as
prescribed and plot a graph of the initial current and the
values obtained during the titration as functions of the
quantity of titrant added. Add the titrant in not fewer than
3 successive quantities equal to a total of about 80 per cent
of the theoretical volume corresponding to the presumed
equivalence point. The 3 values must fall on a straight line.
Continue adding the titrant beyond the presumed equivalence
point in not fewer than 3 successive quantities. The values
obtained must fall on a straight line. The point of intersection
of the 2 lines represents the end-point of the titration.
For amperometric titration with 2 indicator electrodes, the
whole titration curve is recorded and used to determine the
end-point.
07/2013:20220
The potential is usually measured at zero or practically zero cx = concentration of the solution to be examined,
current. cs = concentration of the standard solution,
Apparatus. The apparatus used (a simple potentiometer or
electronic device) comprises a voltmeter allowing readings Ix = intensity of the light emitted by the solution to be
to the nearest millivolt. examined,
Is = intensity of the light emitted by the standard
The indicator electrode to be used depends on the substance solution.
to be determined and may be a glass or metal electrode (for
example, platinum, gold, silver or mercury). The reference If the intensity of the fluorescence is not strictly proportional
electrode is generally a calomel or a silver-silver chloride to the concentration, the measurement may be effected using
electrode. a calibration curve.
For acid-base titrations and unless otherwise prescribed, In some cases, measurement can be made with reference
a glass-calomel or glass-silver-silver chloride electrode to a fixed standard (for example a fluorescent glass or a
combination is used. solution of another fluorescent substance). In such cases,
the concentration of the substance to be examined must be
Method. In potentiometric titrations of weak acids or determined using a previously drawn calibration curve under
bases using non-aqueous solvents, either carry out a blank the same conditions.
determination or pre-neutralise the solvent mixture, if
necessary, before dissolution of the substance to be examined.
Where it is impracticable to use potentiometric detection for 01/2008:20222
this purpose, the solvent mixture can be pre-neutralised by
titration using a suitable indicator. Some examples are given 2.2.22. ATOMIC EMISSION
below :
SPECTROMETRY
Titrant Indicator GENERAL PRINCIPLE
Perchloric acid Crystal violet solution R Atomic emission is a process that occurs when electromagnetic
3 g/L solution of thymol blue R radiation is emitted by excited atoms or ions. In atomic
Tetrabutylammonium hydroxide emission spectrometry the sample is subjected to temperatures
in methanol R
Ethanolic sodium hydroxide Thymolphthalein solution R high enough to cause not only dissociation into atoms, but
also to cause significant amounts of collisional excitation
Plot a graph of the variation of potential difference as a and ionisation of the sample atoms to take place. Once the
function of the quantity of the titrant added, continuing the atoms and ions are in the excited states, they can decay to
addition of the titrant beyond the presumed equivalence lower states through thermal or radiative (emission) energy
point. The end-point corresponds to a sharp variation of transitions and electromagnetic radiation is emitted. An
potential difference. emission spectrum of an element contains several more lines
than the corresponding absorption spectrum.
Atomic emission spectrometry is a technique for determining
the concentration of an element in a sample by measuring the
intensity of one of the emission lines of the atomic vapour of
01/2008:20221 the element generated from the sample. The determination is
carried out at the wavelength corresponding to this emission
line.
2.2.21. FLUORIMETRY In this chapter only atomisation in flame is dealt with. The
method of inductively coupled plasma-atomic emission
Fluorimetry is a procedure which uses the measurement of the spectrometry (ICP-AES) is described in a different general
intensity of the fluorescent light emitted by the substance to chapter.
be examined in relation to that emitted by a given standard.
Method. Dissolve the substance to be examined in the solvent APPARATUS
or mixture of solvents prescribed in the monograph, transfer This consists essentially of :
the solution to the cell or the tube of the fluorimeter and – a sample introduction and nebulisation system ;
illuminate it with an excitant light beam of the wavelength – a flame to generate the atoms to be determined ;
prescribed in the monograph and as near as possible
monochromatic. – a monochromator ;
– a detector ;
Measure the intensity of the emitted light at an angle of 90°
to the excitant beam, after passing it through a filter which – a data-acquisition unit.
transmits predominantly light of the wavelength of the Oxygen, air and a combustible gas such as hydrogen, acetylene,
fluorescence. Other types of apparatus may be used provided propane or butane may be used in flames. The atomisation
that the results obtained are identical. source is critical, since it must provide sufficient energy to
excite and atomise the atoms. The atomic spectra emitted
For quantitative determinations, first introduce into the from flames have the advantage of being simpler than those
apparatus the solvent or mixture of solvents used to dissolve emitted from other sources, the main limitation being that the
the substance to be examined and set the instrument to zero. flames are not powerful enough to cause emission for many
Introduce the standard solution and adjust the sensitivity elements allowing their determination. Acidified water is the
of the instrument so that the reading is greater than 50. If solvent of choice for preparing test and reference solutions,
the second adjustment is made by altering the width of the although organic solvents may also be used if precautions are
slits, a new zero setting must be made and the intensity of taken to ensure that the solvent does not interfere with the
the standard must be measured again. Finally introduce the stability of the flame.
solution of unknown concentration and read the result on the
instrument. Calculate the concentration cx of the substance in INTERFERENCES
the solution to be examined, using the formula : Spectral interference is reduced or eliminated by choosing an
appropriate emission line for measurement or by adjusting the
slit for spectral band-width. Physical interference is corrected
by diluting the sample solution, by matching the matrix or by VALIDATION OF THE METHOD
using the method of standard additions. Chemical interference Satisfactory performance of methods prescribed in
is reduced by using chemical modifiers or ionisation buffers. monographs is verified at suitable time intervals.
MEMORY EFFECT LINEARITY
Prepare and analyse not fewer than 4 reference solutions over
The memory effect caused by deposit of analyte in the
the calibration range and a blank solution. Perform not fewer
apparatus may be limited by thoroughly rinsing between runs,
than 5 replicates.
diluting the solutions to be measured if possible and thus
reducing their salt content, and by aspirating the solutions The calibration curve is calculated by least-square regression
through as swiftly as possible. from all measured data. The regression curve, the means, the
measured data and the confidence interval of the calibration
METHOD curve are plotted. The operating method is valid when :
Use of plastic labware is recommended wherever possible. – the correlation coefficient is at least 0.99,
Operate an atomic emission spectrometer in accordance with – the residuals of each calibration level are randomly
the manufacturer’s instructions at the prescribed wavelength. distributed around the calibration curve.
Optimise the experimental conditions (flame temperature, Calculate the mean and relative standard deviation for the
burner adjustment, use of an ionic buffer, concentration of lowest and highest calibration level.
solutions) for the specific element to be analysed and in respect When the ratio of the estimated standard deviation of the
of the sample matrix. Introduce a blank solution into the lowest and the highest calibration level is less than 0.5 or
atomic generator and adjust the instrument reading to zero or greater than 2.0, a more precise estimation of the calibration
to its blank value. Introduce the most concentrated reference curve may be obtained using weighted linear regression. Both
solution and adjust the sensitivity to obtain a suitable reading. linear and quadratic weighting functions are applied to the
It is preferable to use concentrations which fall within the data to find the most appropriate weighting function to be
linear part of the calibration curve. If this is not possible, the employed. If the means compared to the calibration curve
calibration plots may also be curved and are then to be applied show a deviation from linearity, two-dimensional linear
with appropriate calibration software. regression is used.
Determinations are made by comparison with reference ACCURACY
solutions with known concentrations of the element to Verify the accuracy preferably by using a certified reference
be determined either by the method of direct calibration material (CRM). Where this is not possible, perform a test
(Method I) or the method of standard additions (Method II). for recovery.
METHOD I - DIRECT CALIBRATION Recovery. For assay determinations a recovery of 90 per cent
For routine measurements 3 reference solutions of the element to 110 per cent is to be obtained. For other determinations,
to be determined and a blank are prepared and examined. for example for trace element determination, the test is not
Prepare the solution of the substance to be examined (test valid if recovery is outside of the range 80 per cent to 120 per
solution) as prescribed in the monograph. Prepare not fewer cent at the theoretical value. Recovery may be determined on
than 3 reference solutions of the element to be determined, a suitable reference solution (matrix solution) which is spiked
the concentrations of which span the expected value in the with a known quantity of analyte (middle concentration of
test solution. For assay purposes, optimal calibration levels the calibration range).
are between 0.7 and 1.3 times the expected content of the REPEATABILITY
element to be determined or the limit prescribed in the The repeatability is not greater than 3 per cent for an assay
monograph. For purity determination, calibration levels are and not greater than 5 per cent for an impurity test.
between the limit of detection and 1.2 times the limit specified LIMIT OF QUANTIFICATION
for the element to be determined. Any reagents used in the
preparation of the test solution are added to the reference Verify that the limit of quantification (for example, determined
solutions and to the blank solution at the same concentration. using the 10 σ approach) is below the value to be measured.
Introduce each of the solutions into the instrument using the
same number of replicates for each solution, to obtain a steady 01/2008:20223
reading.
Calculation. Prepare a calibration curve from the mean of
the readings obtained with the reference solutions by plotting
2.2.23. ATOMIC ABSORPTION
the means as a function of concentration. Determine the SPECTROMETRY
concentration of the element in the test solution from the GENERAL PRINCIPLE
curve obtained.
Atomic absorption is a process that occurs when a ground
METHOD II - STANDARD ADDITIONS state-atom absorbs electromagnetic radiation of a specific
Add to at least 3 similar volumetric flasks equal volumes of wavelength and is elevated to an excited state. The atoms in
the solution of the substance to be examined (test solution) the ground state absorb energy at their resonant frequency
prepared as prescribed. Add to all but 1 of the flasks and the electromagnetic radiation is attenuated due to
progressively larger volumes of a reference solution containing resonance absorption. The energy absorption is virtually a
a known concentration of the element to be determined to direct function of the number of atoms present.
produce a series of solutions containing steadily increasing This chapter provides general information and defines
concentrations of that element known to give responses in the the procedures used in element determinations by atomic
linear part of the curve, if at all possible. Dilute the contents absorption spectrometry, either atomisation by flame, by
of each flask to volume with solvent. electrothermal vaporisation in a graphite furnace, by hydride
Introduce each of the solutions into the instrument using the generation or by cold vapour technique for mercury.
same number of replicates for each solution, to obtain a steady Atomic absorption spectrometry is a technique for
reading. determining the concentration of an element in a sample by
Calculation. Calculate the linear equation of the graph using measuring the absorption of electromagnetic radiation by
a least-squares fit, and derive from it the concentration of the the atomic vapour of the element generated from the sample.
element to be determined in the test solution. The determination is carried out at the wavelength of one of
the absorption (resonance) lines of the element concerned. matching, physical interference due to high salt content or
The amount of radiation absorbed is, according to the viscosity is eliminated. Spectral interference results from the
Lambert-Beer law, proportional to the element concentration. overlapping of resonance lines and can be avoided by using
a different resonance line. The use of Zeeman background
APPARATUS correction also compensates for spectral interference and
This consists essentially of : interferences from molecular absorption, especially when
– a source of radiation ; using the electrothermal atomisation technique. The use of
multi-element hollow-cathode lamps may also cause spectral
– a sample introduction device ; interference. Specific or non-specific absorption is measured
– a sample atomiser ; in a spectral range defined by the band-width selected by the
– a monochromator or polychromator ; monochromator (0.2-2 nm).
– a detector ; BACKGROUND CORRECTION
– a data-acquisition unit. Scatter and background in the flame or the electrothermal
The apparatus is usually equipped with a background atomisation technique increase the measured absorbance
correction system. Hollow-cathode lamps and electrodeless values. Background absorption covers a large range of
discharge lamps (EDL) are used as radiation source. The wavelengths, whereas atomic absorption takes place in a very
emission of such lamps consists of a spectrum showing very narrow wavelength range of about 0.005-0.02 nm. Background
narrow lines with half-width of about 0.002 nm of the element absorption can in principle be corrected by using a blank
being determined. solution of exactly the same composition as the sample, but
There are 3 types of sample atomisers : without the specific element to be determined, although this
method is frequently impracticable. With the electrothermal
– Flame technique atomisation technique the pyrolysis temperature is to be
A flame atomiser is composed of a nebulisation system optimised to eliminate the matrix decomposition products
with a pneumatic aerosol production accessory, a gas-flow causing background absorption. Background correction
regulation and a burner. Fuel-oxidant mixtures are can also be made by using 2 different light sources, the
commonly used to produce a range of temperatures from hollow-cathode lamp that measures the total absorption
about 2000 K to 3000 K. Fuel gases include propane, (element + background) and a deuterium lamp with a
hydrogen and acetylene ; air and nitrous oxide are used as continuum emission from which the background absorption
oxidants. The configuration of the burner is adapted to is measured. Background is corrected by subtracting the
the gases used and the gas flow is adjustable. Samples are deuterium lamp signal from the hollow-cathode lamp signal.
nebulised, acidified water being the solvent of choice for This method is limited in the spectral range on account of
preparing test and reference solutions. Organic solvents the spectra emitted by a deuterium lamp from 190-400 nm.
may also be used if precautions are taken to ensure that the Background can also be measured by taking readings at
solvent does not interfere with the stability of the flame. a non-absorbing line near the resonance line and then
– Electrothermal atomisation technique subtracting the results from the measurement at the resonance
An electrothermal atomiser is generally composed of line. Another method for the correction of background
a graphite tube furnace and an electric power source. absorption is the Zeeman effect (based on the Zeeman
Electrothermal atomisation in a graphite tube furnace splitting of the absorption line in a magnetic field). This is
atomises the entire sample and retains the atomic vapour particularly useful when the background absorption shows
in the light path for an extended period. This improves fine structure. It permits an efficient background correction
the detection limit. Samples, liquid as well as solid, in the range of 185-900 nm.
are introduced directly into the graphite tube furnace, CHOICE OF THE OPERATING CONDITIONS
which is heated in a programmed series of steps to dry
the sample and remove major matrix components by After selecting the suitable wavelength and slit width for
pyrolysis and to then atomise all of the analyte. The the specific element, the need for the following has to be
furnace is cleaned using a final temperature higher than the ascertained :
atomisation temperature. The flow of an inert gas during – correction for non-specific background absorption,
the pyrolysis step in the graphite tube furnace allows a – chemical modifiers or ionisation buffers to be added to the
better performance of the subsequent atomisation process. sample as well as to blank and reference solutions,
– Cold vapour and hydride technique – dilution of the sample to minimise, for example, physical
The atomic vapour may also be generated outside the interferences,
spectrometer. This is notably the case for the cold-vapour – details of the temperature programme, preheating, drying,
method for mercury or for certain hydride-forming pyrolysis, atomisation, post-atomisation with ramp and
elements such as arsenic, antimony, bismuth, selenium hold times,
and tin. For mercury, atoms are generated by chemical – inert gas flow,
reduction with stannous chloride or sodium borohydride
– matrix modifiers for electrothermal atomisation (furnace),
and the atomic vapour is swept by a stream of an inert gas
into a cold quartz cell mounted in the optical path of the – chemical reducing reagents for measurements of mercury
instrument. Hydrides thus generated are swept by an inert or other hydride-forming elements along with cold vapour
gas into a heated cell in which they are dissociated into cell or heating cell temperature,
atoms. – specification of furnace design (tank, L’vov platform, etc).
INTERFERENCES METHOD
Chemical, physical, ionisation and spectral interferences are Use of plastic labware is recommended wherever possible.
encountered in atomic absorption measurements. Chemical The preparation of the sample may require a dissolution, a
interference is compensated by addition of matrix modifiers, digestion (mostly microwave-assisted), an ignition step or a
of releasing agents or by using high temperature produced by combination thereof in order to clear up the sample matrix
a nitrous oxide-acetylene flame ; the use of specific ionisation and/or to remove carbon-containing material. If operating in
buffers (for example, lanthanum and caesium) compensates an open system, the ignition temperature should not exceed
for ionisation interference ; by dilution of the sample, 600 °C, due to the volatility of some metals, unless otherwise
through the method of standard additions or by matrix stated in the monograph.
Operate an atomic absorption spectrometer in accordance The calibration curve is calculated by least-square regression
with the manufacturer’s instructions at the prescribed from all measured data. The regression curve, the means, the
wavelength. Introduce a blank solution into the atomic measured data and the confidence interval of the calibration
generator and adjust the instrument reading so that it indicates curve are plotted. The operating method is valid when :
maximum transmission. The blank value may be determined – the correlation coefficient is at least 0.99,
by using solvent to zero the apparatus. Introduce the most – the residuals of each calibration level are randomly
concentrated reference solution and adjust the sensitivity to distributed around the calibration curve.
obtain a maximum absorbance reading. Rinse in order to
avoid contamination and memory effects. After completing Calculate the mean and relative standard deviation for the
the analysis, rinse with water R or acidified water. lowest and highest calibration level.
When the ratio of the estimated standard deviation of the
If a solid sampling technique is applied, full details of the lowest and the highest calibration level is less than 0.5 or
procedure are provided in the monograph. greater than 2.0, a more precise estimation of the calibration
Ensure that the concentrations to be determined fall preferably curve may be obtained using weighted linear regression. Both
within the linear part of the calibration curve. If this is not linear and quadratic weighting functions are applied to the
possible, the calibration plots may also be curved and are then data to find the most appropriate weighting function to be
to be applied with appropriate calibration software. employed. If the means compared to the calibration curve
show a deviation from linearity, two-dimensional linear
Determinations are made by comparison with reference regression is used.
solutions with known concentrations of the element to
be determined either by the method of direct calibration ACCURACY
(Method I) or the method of standard additions (Method II). Verify the accuracy preferably by using a certified reference
material (CRM). Where this is not possible, perform a test
METHOD I - DIRECT CALIBRATION
for recovery.
For routine measurements 3 reference solutions and a blank
solution are prepared and examined. Recovery. For assay determinations a recovery of 90 per cent
to 110 per cent is to be obtained. For other determinations,
Prepare the solution of the substance to be examined (test for example, for trace element determination the test is not
solution) as prescribed in the monograph. Prepare not fewer valid if recovery is outside of the range 80 per cent to 120 per
than 3 reference solutions of the element to be determined, cent at the theoretical value. Recovery may be determined on
the concentrations of which span the expected value in the test a suitable reference solution (matrix solution) which is spiked
solution. For assay purposes, optimal calibration levels are with a known quantity of analyte (middle concentration of
between 0.7 and 1.3 times the expected content of the element the calibration range).
to be determined or the limit prescribed in the monograph.
For purity determination, calibration levels are the limit of REPEATABILITY
detection and 1.2 times the limit specified for the element to The repeatability is not greater than 3 per cent for an assay
be determined. Any reagents used in the preparation of the and not greater than 5 per cent for an impurity test.
test solution are added to the reference and blank solutions at LIMIT OF QUANTIFICATION
the same concentration. Verify that the limit of quantification (for example, determined
Introduce each of the solutions into the instrument using the using the 10 σ approach) is below the value to be measured.
same number of replicates for each of the solutions to obtain
a steady reading. 01/2008:20224
Calculation. Prepare a calibration curve from the mean of
the readings obtained with the reference solutions by plotting 2.2.24. ABSORPTION
the means as a function of concentration. Determine the
concentration of the element in the test solution from the
SPECTROPHOTOMETRY, INFRARED
curve obtained. Infrared spectrophotometers are used for recording spectra in
METHOD II - STANDARD ADDITIONS the region of 4000-650 cm− 1 (2.5-15.4 μm) or in some cases
Add to at least 3 similar volumetric flasks equal volumes of down to 200 cm− 1 (50 μm).
the solution of the substance to be examined (test solution) APPARATUS
prepared as prescribed. Add to all but 1 of the flasks
progressively larger volumes of a reference solution containing Spectrophotometers for recording spectra consist of a suitable
a known concentration of the element to be determined to light source, monochromator or interferometer and detector.
produce a series of solutions containing steadily increasing Fourier transform spectrophotometers use polychromatic
concentrations of that element known to give responses in the radiation and calculate the spectrum in the frequency
linear part of the curve, if possible. Dilute the contents of each domain from the original data by Fourier transformation.
flask to volume with solvent. Spectrophotometers fitted with an optical system capable of
producing monochromatic radiation in the measurement
Introduce each of the solutions into the instrument, using the region may also be used. Normally the spectrum is given as
same number of replicates for each of the solutions, to obtain a function of transmittance, the quotient of the intensity of
a steady reading. the transmitted radiation and the incident radiation. It may
Calculation. Calculate the linear equation of the graph using also be given in absorbance.
a least-squares fit and derive from it the concentration of the The absorbance (A) is defined as the logarithm to base 10 of
element to be determined in the test solution. the reciprocal of the transmittance (T) :
minimum at 1589 cm− 1 and the absorption maximum at acquired using suitable purged instruments or ensuring that
1583 cm− 1 is greater than 0.08. sample and background single beam spectra are acquired
under exactly the same conditions.
IMPURITIES IN GASES
For the analysis of impurities, use a cell transparent to infrared
radiation and of suitable optical path length (for example,
1-20 m). Fill the cell as prescribed under Gases. For detection
and quantification of the impurities, proceed as prescribed in
the monograph.
01/2008:20225
2.2.25. ABSORPTION
SPECTROPHOTOMETRY,
ULTRAVIOLET AND VISIBLE
Determination of absorbance. The absorbance (A) of a
solution is defined as the logarithm to base 10 of the reciprocal
of the transmittance (T) for monochromatic radiation :
T = I/I0 ;
Figure 2.2.24.-1. – Typical spectrum of polystyrene used to
verify the resolution performance I0 = intensity of incident monochromatic radiation ;
Verification of the wave-number scale. The wave-number I = intensity of transmitted monochromatic radiation.
scale may be verified using a polystyrene film, which has
transmission minima (absorption maxima) at the wave In the absence of other physico-chemical factors, the
numbers (in cm–1) shown in Table 2.2.24.-1. absorbance (A) is proportional to the path length (b) through
which the radiation passes and to the concentration (c) of the
Table 2.2.24.-1. – Transmission minima and acceptable substance in solution in accordance with the equation :
tolerances of a polystyrene film
Transmission Acceptable tolerance (cm− 1)
minima (cm− 1) ε = molar absorptivity, if b is expressed in centimetres
and c in moles per litre.
Monochromator Fourier-transform
instruments instruments The expression representing the specific absorbance
of a dissolved substance refers to the absorbance of a 10 g/L
3060.0 ± 1.5 ± 1.0
solution in a 1 cm cell and measured at a defined wavelength
2849.5 ± 2.0 ± 1.0 so that :
tolerance is ± 1 nm for the ultraviolet range and ± 3 nm for Cells. The tolerance on the path length of the cells used is
the visible range. Suitable certified reference materials may ± 0.005 cm. When filled with the same solvent, the cells
also be used. intended to contain the solution to be examined and the
compensation liquid must have the same transmittance. If this
is not the case, an appropriate correction must be applied.
Table 2.2.25.-1. – Absorption maxima for control of wavelength
scale The cells must be cleaned and handled with care.
Table 2.2.25.-2
and 268 nm, respectively, as shown in Figure 2.2.25.-1. Unless portions, allowing each to dry before the next application.
otherwise prescribed in the monograph, the ratio A/B (see When more than one chromatogram is to be run on the same
Figure 2.2.25.-1) is not less than 0.2. strip of paper, space the solutions along the pencil line at
Procedure. Prepare the solution of the substance to be points not less than 3 cm apart. Insert the paper in the tank,
examined, adjust the various instrument settings according close the lid, and allow to stand for 1 h 30 min. Introduce into
to the manufacturer’s instructions, and calculate the amount the solvent trough, through the hole in the lid, a sufficient
of the substance to be determined as prescribed in the quantity of the mobile phase, close the tank and allow elution
monograph. to proceed for the prescribed distance or time. Remove the
paper from the tank and allow to dry in air. The paper should
be protected from bright light during the elution process.
01/2008:20226
01/2008:20227
2.2.26. PAPER CHROMATOGRAPHY
ASCENDING PAPER CHROMATOGRAPHY
2.2.27. THIN-LAYER
Apparatus. The apparatus consists of a glass tank of suitable CHROMATOGRAPHY
size for the chromatographic paper used, ground at the top Thin-layer chromatography is a separation technique in
to take a closely fitting lid. In the top of the tank is a device which a stationary phase consisting of an appropriate material
which suspends the chromatographic paper and is capable of is spread in a uniform thin layer on a support (plate) of
being lowered without opening the chamber. In the bottom of glass, metal or plastic. Solutions of analytes are deposited
the tank is a dish to contain the mobile phase into which the on the plate prior to development. The separation is based
paper may be lowered. The chromatographic paper consists on adsorption, partition, ion-exchange or on combinations
of suitable filter paper, cut into strips of sufficient length and of these mechanisms and is carried out by migration
not less than 2.5 cm wide ; the paper is cut so that the mobile (development) of solutes (solutions of analytes) in a solvent
phase runs in the direction of the grain of the paper. or a suitable mixture of solvents (mobile phase) through the
Method. Place in the dish a layer 2.5 cm deep of the mobile thin-layer (stationary phase).
phase prescribed in the monograph. If prescribed in the
monograph, pour the stationary phase between the walls of APPARATUS
the tank and the dish. Close the tank and allow to stand for Plates. The chromatography is carried out using pre-coated
24 h at 20 °C to 25 °C. Maintain the tank at this temperature plates as described under Reagents (4.1.1).
throughout the subsequent procedure. Draw a fine pencil Pre-treatment of the plates. It may be necessary to wash the
line horizontally across the paper 3 cm from one end. Using plates prior to separation. This can be done by migration of
a micro pipette, apply to a spot on the pencil line the volume an appropriate solvent. The plates may also be impregnated
of the solution prescribed in the monograph. If the total by procedures such as development, immersion or spraying.
volume to be applied would produce a spot more than 10 mm At the time of use, the plates may be activated, if necessary, by
in diameter, apply the solution in portions allowing each heating in an oven at 120 °C for 20 min.
to dry before the next application. When more than one
chromatogram is to be run on the same strip of paper, space Chromatographic tank with a flat bottom or twin trough,
the solutions along the pencil line at points not less than 3 cm of inert, transparent material, of a size suitable for the plates
apart. Insert the paper into the tank, close the lid and allow to used and provided with a tightly fitting lid. For horizontal
stand for 1 h 30 min. Lower the paper into the mobile phase development the tank is provided with a trough for the mobile
and allow elution to proceed for the prescribed distance or phase and it additionally contains a device for directing the
time. Remove the paper from the tank and allow to dry in air. mobile phase to the stationary phase.
Protect the paper from bright light during the elution process. Micropipettes, microsyringes, calibrated disposable
capillaries or other application devices suitable for the proper
DESCENDING PAPER CHROMATOGRAPHY application of the solutions.
Apparatus. The apparatus consists of a glass tank of suitable Fluorescence detection device to measure direct fluorescence
size for the chromatographic paper used, ground at the top or the inhibition of fluorescence.
to take a closely fitting glass lid. The lid has a central hole
about 1.5 cm in diameter closed by a heavy glass plate or a Visualisation devices and reagents. Suitable devices are used
stopper. In the upper part of the tank is suspended a solvent for derivatisation to transfer to the plate reagents by spraying,
trough with a device for holding the chromatographic paper. immersion or exposure to vapour and, where applicable, to
On each side of the trough, parallel to and slightly above its facilitate heating for visualisation of separated components.
upper edges, are two glass guide rods to support the paper Documentation. A device may be used to provide
in such a manner that no part of it is in contact with the documentation of the visualised chromatogram, for example a
walls of the tank. The chromatographic paper consists of photograph or a computer file.
suitable filter paper, cut into strips of sufficient length, and of
any convenient width between 2.5 cm and the length of the METHOD
trough ; the paper is cut so that the mobile phase runs in the Sample application. Apply the prescribed volume of the
direction of the grain of the paper. solutions at a suitable distance from the lower edge and
Method. Place in the bottom of the tank a layer 2.5 cm deep of from the sides of the plate and on a line parallel to the
the solvent prescribed in the monograph, close the tank and lower edge ; allow an interval of at least 10 mm (5 mm on
allow to stand for 24 h at 20 °C to 25 °C. Maintain the tank high-performance plates) between the centres of circular
at this temperature throughout the subsequent procedure. spots and 5 mm (2 mm on high-performance plates) between
Draw a fine pencil line horizontally across the paper at such the edges of bands. Apply the solutions in sufficiently small
a distance from one end that when this end is secured in the portions to obtain circular spots 2-5 mm in diameter (1-2 mm
solvent trough and the remainder of the paper is hanging on high-performance plates) or bands 10-20 mm (5-10 mm
freely over the guide rod, the line is a few centimetres below on high-performance plates) by 1-2 mm.
the guide rod and parallel with it. Using a micro-pipette, apply In a monograph, where both normal and high-performance
on the pencil line the volume of the solution prescribed in the plates may be used, the working conditions for
monograph. If the total volume to be applied would produce high-performance plates are given in the brackets [ ] after
a spot more than 10 mm in diameter, apply the solution in those for normal plates.
Vertical development. Line the walls of the chromatographic Substances separated by thin-layer chromatography and
tank with filter paper. Pour into the chromatographic tank responding to UV-Vis irradiation can be determined directly
a sufficient quantity of the mobile phase for the size of the on the plate, using appropriate instrumentation. While
tank to give after impregnation of the filter paper a layer of moving the plate or the measuring device, examine the plate
appropriate depth related to the dimension of the plate to be by measuring the reflectance of the incident light. Similarly,
used. For saturation of the chromatographic tank, replace the fluorescence may be measured using an appropriate optical
lid and allow to stand at 20-25 °C for 1 h. Unless otherwise system. Substances containing radionuclides can be quantified
indicated in the monograph, the chromatographic separation in 3 ways : either directly by moving the plate alongside a
is performed in a saturated tank. Apply the prescribed suitable counter or vice versa (see Radiopharmaceutical
volume of solutions as described above. When the solvent preparations (0125)), by cutting the plates into strips and
has evaporated from the applied solutions, place the plate measuring the radioactivity on each individual strip using
in the chromatographic tank, ensuring that the plate is as a suitable counter or by scraping off the stationary phase,
vertical as possible and that the spots or bands are above the dissolving it in a suitable scintillation cocktail and measuring
surface of the mobile phase. Close the chromatographic tank, the radioactivity using a liquid scintillation counter.
maintain it at 20-25 °C and protect from sunlight. Remove the Apparatus. The apparatus for direct measurement on the
plate when the mobile phase has moved over the prescribed plate consists of :
distance, measured between the points of application and the
solvent front. Dry the plate and visualise the chromatograms – a device for exact positioning and reproducible dispensing
as prescribed. of the amount of substances onto the plate ;
For two-dimensional chromatography, dry the plates after the – a mechanical device to move the plate or the measuring
first development and carry out a second development in a device along the x-axis or the y-axis ;
direction perpendicular to that of the first development. – a recorder and a suitable integrator or a computer ;
Horizontal development. Apply the prescribed volume – for substances responding to UV-Vis irradiation : a
of the solutions as described above. When the solvent has photometer with a source of light, an optical device able to
evaporated from the applied solutions, introduce a sufficient generate monochromatic light and a photo cell of adequate
quantity of the mobile phase into the trough of the chamber sensitivity are used for the measurement of reflectance
using a syringe or pipette, place the plate in the chamber or transmittance ; if fluorescence is measured, a suitable
after verifying that the latter is horizontal and connect the filter is required to prevent light used for excitation from
mobile phase direction device according to the manufacturer’s reaching the detector while permitting emitted light or a
instructions. If prescribed, develop the plate starting specific portion thereof to pass ;
simultaneously at both ends. Close the chamber and maintain – for substances containing radionuclides : a suitable counter
it at 20-25 °C. Remove the plate when the mobile phase has for radioactivity. The linearity range of the counting device
moved over the distance prescribed in the monograph. Dry is to be verified.
the plate and visualise the chromatograms as prescribed. Method. Prepare the solution of the substance to be examined
For two-dimensional chromatography, dry the plates after the (test solution) as prescribed in the monograph and, if
first development and carry out a second development in a necessary, prepare the reference solutions of the substance to
direction perpendicular to that of the first development. be determined using the same solvent as in the test solution.
Apply the same volume of each solution to the plate and
VISUAL EVALUATION
develop.
Identification. The principal spot in the chromatogram Substances responding to UV-Vis irradiation. Prepare and
obtained with the test solution is visually compared to the apply not fewer than 3 reference solutions of the substance to
corresponding spot in the chromatogram obtained with the be examined, the concentrations of which span the expected
reference solution by comparing the colour, the size and the value in the test solution (about 80 per cent, 100 per cent and
retardation factor (RF) of both spots. 120 per cent). Treat with the prescribed reagent, if necessary,
The retardation factor (RF) is defined as the ratio of the and record the reflectance, the transmittance or fluorescence
distance from the point of application to the centre of the spot in the chromatograms obtained with the test and reference
and the distance travelled by the solvent front from the point solutions. Use the measured results for the calculation of the
of application. amount of substance in the test solution.
Verification of the separating power for identification. Substances containing radionuclides. Prepare and apply a
Normally the performance given by the suitability test test solution containing about 100 per cent of the expected
described in Reagents (4.1.1) is sufficient. Only in special value. Determine the radioactivity as a function of the path
cases an additional performance criterion is prescribed in the length and report the radioactivity in each resulting peak as a
monograph. percentage of the total amount of radioactivity.
Related substances test. The secondary spot(s) in the Criteria for assessing the suitability of the system are described
chromatogram obtained with the test solution is (are) in the chapter on Chromatographic separation techniques
visually compared to either the corresponding spot(s) in (2.2.46). The extent to which adjustments of parameters of the
the chromatogram obtained with the reference solution chromatographic system can be made to satisfy the criteria of
containing the impurity(ies) or the spot in the chromatogram system suitability are also given in this chapter.
obtained with the reference solution prepared from a dilution
of the test solution.
Verification of the separating power. The requirements for 01/2008:20228
the verification of the separating power are prescribed in the
monographs concerned. 2.2.28. GAS CHROMATOGRAPHY
Verification of the detecting power. The detecting power
is satisfactory if a spot or band is clearly visible in the Gas chromatography (GC) is a chromatographic separation
chromatogram obtained with the most dilute reference technique based on the difference in the distribution of species
solution. between two non-miscible phases in which the mobile phase
is a carrier gas moving through or passing the stationary
QUANTITATIVE MEASUREMENT phase contained in a column. It is applicable to substances or
The requirements for resolution and separation are prescribed their derivatives which are volatilised under the temperatures
in the monographs concerned. employed.
GC is based on mechanisms of adsorption, mass distribution Helium or nitrogen are usually employed as the carrier gas for
or size exclusion. packed columns, whereas commonly used carrier gases for
capillary columns are nitrogen, helium and hydrogen.
APPARATUS
DETECTORS
The apparatus consists of an injector, a chromatographic
column contained in an oven, a detector and a data acquisition Flame-ionisation detectors are usually employed but additional
system (or an integrator or a chart recorder). The carrier gas detectors which may be used include : electron-capture,
flows through the column at a controlled rate or pressure and nitrogen-phosphorus, mass spectrometric, thermal
then through the detector. conductivity, Fourier transform infrared spectrophotometric,
and others, depending on the purpose of the analysis.
The chromatography is carried out either at a constant
temperature or according to a given temperature programme. METHOD
INJECTORS Equilibrate the column, the injector and the detector at
Direct injections of solutions are the usual mode of injection, the temperatures and the gas flow rates specified in the
unless otherwise prescribed in the monograph. Injection may monograph until a stable baseline is achieved. Prepare the test
be carried out either directly at the head of the column using a solution(s) and the reference solution(s) as prescribed. The
syringe or an injection valve, or into a vaporisation chamber solutions must be free from solid particles.
which may be equipped with a stream splitter. Criteria for assessing the suitability of the system are described
Injections of vapour phase may be effected by static or dynamic in the chapter on Chromatographic separation techniques
head-space injection systems. (2.2.46). The extent to which adjustments of parameters of the
Dynamic head-space (purge and trap) injection systems chromatographic system can be made to satisfy the criteria of
include a sparging device by which volatile substances in system suitability are also given in this chapter.
solution are swept into an absorbent column maintained at a
low temperature. Retained substances are then desorbed into Static head-space gas chromatography
the mobile phase by rapid heating of the absorbent column.
Static head-space gas chromatography is a technique
Static head-space injection systems include a thermostatically particularly suitable for separating and determining volatile
controlled sample heating chamber in which closed vials compounds present in solid or liquid samples. The method
containing solid or liquid samples are placed for a fixed is based on the analysis of the vapour phase in equilibrium
period of time to allow the volatile components of the sample with the solid or liquid phase.
to reach equilibrium between the non-gaseous phase and
the vapour phase. After equilibrium has been established, a APPARATUS
predetermined amount of the head-space of the vial is flushed The apparatus consists of a gas chromatograph provided with
into the gas chromatograph. a device for introducing the sample that may be connected
STATIONARY PHASES to a module that automatically controls the pressure and the
Stationary phases are contained in columns which may be : temperature. If necessary, a device for eliminating solvents
– a capillary column of fused-silica whose wall is coated with can be added.
the stationary phase, The sample to be analysed is introduced into a container fitted
– a column packed with inert particles impregnated with the with a suitable stopper and a valve-system which permits
stationary phase, the passage of the carrier gas. The container is placed in
a thermostatically controlled chamber at a temperature set
– a column packed with solid stationary phase. according to the substance to be examined.
Capillary columns are 0.1 mm to 0.53 mm in internal diameter The sample is held at this temperature long enough to allow
(Ø) and 5 m to 60 m in length. The liquid or stationary phase, equilibrium to be established between the solid or liquid phase
which may be chemically bonded to the inner surface, is a and the vapour phase.
film 0.1 μm to 5.0 μm thick.
The carrier gas is introduced into the container and, after
Packed columns, made of glass or metal, are usually 1 m to the prescribed time, a suitable valve is opened so that the gas
3 m in length with an internal diameter (Ø) of 2 mm to 4 mm. expands towards the chromatographic column taking the
Stationary phases usually consist of porous polymers or solid volatilised compounds with it.
supports impregnated with liquid phase.
Instead of using a chromatograph specifically equipped for
Supports for analysis of polar compounds on columns packed the introduction of samples, it is also possible to use airtight
with low-capacity, low-polarity stationary phase must be inert syringes and a conventional chromatograph. Equilibration is
to avoid peak tailing. The reactivity of support materials can then carried out in a separate chamber and the vapour phase
be reduced by silanising prior to coating with liquid phase. is carried onto the column, taking the precautions necessary
Acid-washed, flux-calcinated diatomaceous earth is often to avoid any changes in the equilibrium.
used. Materials are available in various particle sizes, the
most commonly used particles are in the ranges of 150 μm to METHOD
180 μm and 125 μm to 150 μm. Using the reference preparations, determine suitable
MOBILE PHASES instrument settings to produce an adequate response.
Retention time and peak efficiency depend on the carrier gas DIRECT CALIBRATION
flow rate ; retention time is directly proportional to column Separately introduce into identical containers the preparation
length and resolution is proportional to the square root of the to be examined and each of the reference preparations, as
column length. For packed columns, the carrier gas flow rate prescribed in the monograph, avoiding contact between the
is usually expressed in millilitres per minute at atmospheric sampling device and the samples.
pressure and room temperature. Flow rate is measured at the
detector outlet, either with a calibrated mechanical device Close the containers hermetically and place in the
or with a bubble tube, while the column is at operating thermostatically controlled chamber set to the temperature
temperature. The linear velocity of the carrier gas through a and pressure prescribed in the monograph ; after equilibration,
packed column is inversely proportional to the square root of carry out the chromatography under the prescribed conditions.
the internal diameter of the column for a given flow volume. STANDARD ADDITIONS
Flow rates of 60 mL/min in a 4 mm internal diameter column Add to a set of identical suitable containers equal volumes
and 15 mL/min in a 2 mm internal diameter column, give of the preparation to be examined. Add to all but one of
identical linear velocities and thus similar retention times. the containers, suitable quantities of a reference preparation
containing a known concentration of the substance to – resins or polymers with acid or basic groups, used in
be determined so as to produce a series of preparations ion-exchange chromatography, where separation is based
containing steadily increasing concentrations of the substance. on competition between the ions to be separated and those
Close the containers hermetically and place in the in the mobile phase,
thermostatically controlled chamber set to the temperature – porous silica or polymers, used in size-exclusion
and pressure prescribed in the monograph ; after equilibration, chromatography, where separation is based on differences
carry out the chromatography under the prescribed conditions. between the volumes of the molecules, corresponding to
Calculate the linear equation of the graph using a least-squares steric exclusion,
fit, and derive from it the concentration of the substance to be– a variety of chemically modified supports prepared from
determined in the preparation to be examined. polymers, silica or porous graphite, used in reversed-phase
Alternatively, plot on a graph the mean of readings against the LC, where the separation is based principally on partition
added quantity of the substance to be determined. Extrapolate of the molecules between the mobile phase and the
the line joining the points on the graph until it meets the stationary phase,
concentration axis. The distance between this point and the – special chemically modified stationary phases, e.g.
intersection of the axes represents the concentration of the cellulose or amylose derivatives, proteins or peptides,
substance to be determined in the preparation to be examined. cyclodextrins etc., for the separation of enantiomers (chiral
SUCCESSIVE WITHDRAWALS (MULTIPLE HEAD-SPACE chromatography).
EXTRACTION) Most separations are based upon partition mechanisms
If prescribed, the successive withdrawal method is fully utilising chemically modified silica as the stationary phase
described in the monograph. and polar solvents as the mobile phase. The surface of the
support, e.g. the silanol groups of silica, is reacted with various
silane reagents to produce covalently bound silyl derivatives
01/2008:20229 covering a varying number of active sites on the surface of
the support. The nature of the bonded phase is an important
2.2.29. LIQUID CHROMATOGRAPHY parameter for determining the separation properties of the
chromatographic system.
Liquid chromatography (LC) is a method of chromatographic Commonly used bonded phases are shown below :
separation based on the difference in the distribution of octyl = Si-[CH2]7-CH3 C8
species between two non-miscible phases, in which the mobile
phase is a liquid which percolates through a stationary phase octadecyl = Si-[CH2]17-CH3 C18
contained in a column. phenyl = Si-[CH2]n-C6H5 C 6H 5
LC is mainly based on mechanisms of adsorption, mass
distribution, ion exchange, size exclusion or stereochemical cyanopropyl = Si-[CH2]3-CN CN
interaction. aminopropyl = Si-[CH2]3-NH2 NH 2
MOBILE PHASES Molecules small enough to penetrate all the pore spaces elute
For normal-phase chromatography, less polar solvents are at the total permeation volume (Vt). On the other hand,
employed. The presence of water in the mobile phase is molecules apparently larger than the maximum pore size of
to be strictly controlled to obtain reproducible results. In the packing material migrate along the column only through
reversed-phase LC, aqueous mobile phases, with or without the spaces between the particles of the packing material
organic modifiers, are employed. without being retained and elute at the exclusion volume
Components of the mobile phase are usually filtered to remove (V0 void volume). Separation according to molecular size
particles greater than 0.45 μm. Multicomponent mobile phases occurs between the exclusion volume and the total permeation
are prepared by measuring the required volumes (unless volume, with useful separation usually occurring in the first
masses are specified) of the individual components, followed two thirds of this range.
by mixing. Alternatively, the solvents may be delivered by
individual pumps controlled by proportioning valves by which Apparatus. The apparatus consists essentially of a
mixing is performed according to the desired proportion. chromatographic column of varying length and internal
Solvents are normally degassed before pumping by sparging diameter (Ø), if necessary temperature-controlled, packed
with helium, sonication or using on-line membrane/vacuum with a separation material that is capable of fractionation in
modules to avoid the creation of gas bubbles in the detector the appropriate range of molecular sizes and through which
cell. the eluent is passed at a constant rate. One end of the column
is usually fitted with a suitable device for applying the sample
Solvents for the preparation of the mobile phase are normally such as a flow adapter, a syringe through a septum or an
free of stabilisers and are transparent at the wavelength of injection valve and may also be connected to a suitable pump
detection, if an ultraviolet detector is employed. Solvents and for controlling the flow of the eluent. Alternatively the sample
other components employed are to be of appropriate quality. may be applied directly to the drained bed surface or, where
Adjustment of the pH, if necessary, is effected using only the the sample is denser than the eluent, it may be layered beneath
aqueous component of the mobile phase and not the mixture. the eluent. The outlet of the column is usually connected
If buffer solutions are used, adequate rinsing of the system is to a suitable detector fitted with an automatic recorder
carried out with a mixture of water and the organic modifier which enables the monitoring of the relative concentrations
of the mobile phase (5 per cent V/V) to prevent crystallisation of separated components of the sample. Detectors are
of salts after completion of the chromatography. usually based on photometric, refractometric or luminescent
Mobile phases may contain other components, e.g. a properties. An automatic fraction collector may be attached, if
counter-ion for ion-pair chromatography or a chiral selector necessary.
for chromatography using an achiral stationary phase.
DETECTORS The packing material may be a soft support such as a swollen
gel or a rigid support composed of a material such as glass,
Ultraviolet/visible (UV/Vis) spectrophotometers, including
silica or a solvent-compatible, cross-linked organic polymer.
diode array detectors, are the most commonly employed
Rigid supports usually require pressurised systems giving
detectors. Fluorescence spectrophotometers, differential
faster separations. The mobile phase is chosen according to
refractometers, electrochemical detectors, mass spectrometers,
sample type, separation medium and method of detection.
light scattering detectors, radioactivity detectors or other
Before carrying out the separation, the packing material
special detectors may also be used.
is treated, and the column is packed, as described in the
METHOD monograph, or according to the manufacturer’s instructions.
Equilibrate the column with the prescribed mobile phase Criteria for assessing the suitability of the system are described
and flow rate, at room temperature or at the temperature in the chapter on Chromatographic separation techniques
specified in the monograph, until a stable baseline is achieved. (2.2.46). The extent to which adjustments of parameters of the
Prepare the solution(s) of the substance to be examined and chromatographic system can be made to satisfy the criteria of
the reference solution(s) required. The solutions must be free system suitability are also given in this chapter.
from solid particles.
DETERMINATION OF RELATIVE COMPONENT
Criteria for assessing the suitability of the system are described
in the chapter on Chromatographic separation techniques COMPOSITION OF MIXTURES
(2.2.46). The extent to which adjustments of parameters of the Carry out the separation as stated in the monograph. If
chromatographic system can be made to satisfy the criteria of possible, monitor the elution of the components continuously
system suitability are also given in this chapter. and measure the corresponding peak areas. If the sample
is monitored by a physico-chemical property to which all
the components of interest exhibit equivalent responses (for
01/2008:20230 example if they have the same specific absorbance), calculate
the relative amount of each component by dividing the
2.2.30. SIZE-EXCLUSION respective peak area by the sum of the peak areas of all the
components of interest. If the responses to the property used
CHROMATOGRAPHY for detection of the components of interest are not equivalent,
calculate the content by means of calibration curves obtained
Size-exclusion chromatography is a chromatographic with the calibration standards prescribed in the monograph.
technique which separates molecules in solution according
to their size. With organic mobile phases, the technique is DETERMINATION OF MOLECULAR MASSES
known as gel-permeation chromatography and with aqueous Size-exclusion chromatography may be used to determine
mobile phases, the term gel-filtration chromatography has molecular masses by comparison with appropriate calibration
been used. The sample is introduced into a column, which is standards specified in the monograph. The retention
filled with a gel or a porous particle packing material, and is volumes of the calibration standards may be plotted against
carried by the mobile phase through the column. The size the logarithm of their molecular masses. The plot usually
separation takes place by repeated exchange of the solute approximates a straight line within the exclusion and total
molecules between the solvent of the mobile phase and the permeation limits for the separation medium used. From the
same solvent in the stagnant liquid phase (stationary phase) calibration curve, molecular masses may be estimated. The
within the pores of the packing material. The pore-size range molecular-mass calibration is valid only for the particular
of the packing material determines the molecular-size range macromolecular solute/solvent system used under the
within which separation can occur. specified experimental conditions.
Prepare the test and reference solutions containing the subunits and that minimise aggregation. Most commonly, the
prescribed marker dye and make them dense by dissolving strongly anionic detergent sodium dodecyl sulfate (SDS) is
in them sucrose R, for example. Apply the solutions to the used in combination with heat to dissociate the proteins before
surface of a gel using a different tube for each solution. Add they are loaded on the gel. The denatured polypeptides bind
the same buffer to the upper reservoir. Connect the electrodes to SDS, become negatively charged and exhibit a consistent
to the power supply and allow electrophoresis to proceed charge-to-mass ratio regardless of protein type. Because the
at the prescribed temperature and using the prescribed amount of SDS bound is almost always proportional to the
constant voltage or current. Switch off the power supply molecular mass of the polypeptide and is independent of
when the marker dye has migrated almost into the lower its sequence, SDS-polypeptide complexes migrate through
reservoir. Immediately remove each tube from the apparatus polyacrylamide gels with mobilities dependent on the size of
and extrude the gel. Locate the position of the bands in the the polypeptide.
electropherogram as prescribed.♦ The electrophoretic mobilities of the resultant detergent-
SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL polypeptide complexes all assume the same functional
ELECTROPHORESIS (SDS-PAGE) relationship to their molecular masses. Migration of SDS
complexes is toward the anode in a predictable manner, with
Scope. Polyacrylamide gel electrophoresis is used for low-molecular-mass complexes migrating faster than larger
the qualitative characterisation of proteins in biological ones. The molecular mass of a protein can therefore be
preparations, for control of purity and quantitative estimated from its relative mobility in calibrated SDS-PAGE
determinations. and the occurrence of a single band in such a gel is a criterion
Purpose. Analytical gel electrophoresis is an appropriate of purity.
method with which to identify and to assess the homogeneity Modifications to the polypeptide backbone, such as N- or
of proteins in pharmaceutical preparations. The method is O-linked glycosylation, however, have a significant impact
routinely used for the estimation of protein subunit molecular on the apparent molecular mass of a protein since SDS does
masses and for determining the subunit compositions of not bind to a carbohydrate moiety in a manner similar to a
purified proteins. polypeptide. Thus, a consistent charge-to-mass ratio is not
Ready-to-use gels and reagents are widely available on the maintained. The apparent molecular mass of proteins having
market and can be used instead of those described in this undergone post-translational modifications is not a true
text, provided that they give equivalent results and that they reflection of the mass of the polypeptide chain.
meet the validity requirements given below under Validation
of the test. Reducing conditions. Polypeptide subunits and
three-dimensional structure is often maintained in proteins
CHARACTERISTICS OF POLYACRYLAMIDE GELS by the presence of disulfide bonds. A goal of SDS-PAGE
The sieving properties of polyacrylamide gels are established analysis under reducing conditions is to disrupt this structure
by the three-dimensional network of fibres and pores which is by reducing disulfide bonds. Complete denaturation and
formed as the bifunctional bisacrylamide cross-links adjacent dissociation of proteins by treatment with 2-mercaptoethanol
polyacrylamide chains. Polymerisation is catalysed by a free or dithiothreitol (DTT) will result in unfolding of the
radical-generating system composed of ammonium persulfate polypeptide backbone and subsequent complexation with SDS.
and tetramethylethylenediamine. In these conditions, the molecular mass of the polypeptide
As the acrylamide concentration of a gel increases, its subunits can be calculated by linear regression in the presence
effective pore size decreases. The effective pore size of a gel of suitable molecular-mass standards.
is operationally defined by its sieving properties ; that is, by Non-reducing conditions. For some analyses, complete
the resistance it imparts to the migration of macromolecules. dissociation of the protein into subunit peptides is not
There are limits on the acrylamide concentrations that can be desirable. In the absence of treatment with reducing agents
used. At high acrylamide concentrations, gels break much such as 2-mercaptoethanol or DTT, disulfide covalent bonds
more easily and are difficult to handle. As the pore size of remain intact, preserving the oligomeric form of the protein.
a gel decreases, the migration rate of a protein through the Oligomeric SDS-protein complexes migrate more slowly than
gel decreases. By adjusting the pore size of a gel, through their SDS-polypeptide subunits. In addition, non-reduced
manipulating the acrylamide concentration, the resolution proteins may not be completely saturated with SDS and,
of the method can be optimised for a given protein product. hence, may not bind the detergent in a constant mass
Thus, a given gel is physically characterised by its respective ratio. This makes molecular-mass determinations of these
composition in acrylamide and bisacrylamide. molecules by SDS-PAGE less straightforward than analyses of
In addition to the composition of the gel, the state of the fully denatured polypeptides, since it is necessary that both
protein is an important component to the electrophoretic standards and unknown proteins be in similar configurations
mobility. In the case of proteins, the electrophoretic mobility for valid comparisons. However, the staining of a single band
is dependent on the pK value of the charged groups and the in such a gel is a criterion of purity.
size of the molecule. It is influenced by the type, concentration
CHARACTERISTICS OF DISCONTINUOUS BUFFER
and pH of the buffer, by the temperature and the field strength
SYSTEM GEL ELECTROPHORESIS
as well as by the nature of the support material.
The most popular electrophoretic method for the
DENATURING POLYACRYLAMIDE GEL ELECTROPHO- characterisation of complex mixtures of proteins involves
RESIS the use of a discontinuous buffer system consisting of two
The method cited as an example is limited to the analysis contiguous, but distinct gels : a resolving or separating (lower)
of monomeric polypeptides with a mass range of 14 000 gel and a stacking (upper) gel. The two gels are cast with
to 100 000 daltons. It is possible to extend this mass range different porosities, pH, and ionic strengths. In addition,
by various techniques (e.g. gradient gels, particular buffer different mobile ions are used in the gel and electrode buffers.
system) but those techniques are not discussed in this chapter. The buffer discontinuity acts to concentrate large volume
Denaturing polyacrylamide gel electrophoresis using sodium samples in the stacking gel, resulting in improved resolution.
dodecyl sulfate (SDS-PAGE) is the most common mode of When power is applied, a voltage drop develops across the
electrophoresis used in assessing the pharmaceutical quality of sample solution which drives the proteins into the stacking gel.
protein products and will be the focus of the example method. Glycinate ions from the electrode buffer follow the proteins
Typically, analytical electrophoresis of proteins is carried into the stacking gel. A moving boundary region is rapidly
out in polyacrylamide gels under conditions that ensure formed with the highly mobile chloride ions in the front
dissociation of the proteins into their individual polypeptide and the relatively slow glycinate ions in the rear. A localised
high-voltage gradient forms between the leading and trailing the desired concentration of acrylamide for the resolving
ion fronts, causing the SDS-protein complexes to form into gel, using the values given in Table 2.2.31.-1. Mix the
a thin zone (stack) and migrate between the chloride and components in the order shown. Where appropriate,
glycinate phases. Within broad limits, regardless of the height before adding the ammonium persulfate solution and the
of the applied sample, all SDS-proteins condense into a very tetramethylethylenediamine (TEMED), filter the solution
narrow region and enter the resolving gel as a well-defined, if necessary under vacuum through a cellulose acetate
thin zone of high protein density. The large-pore stacking gel membrane (pore diameter 0.45 μm) ; keep the solution under
does not retard the migration of most proteins and serves vacuum by swirling the filtration unit until no more bubbles
mainly as an anticonvective medium. At the interface of the are formed in the solution. Add appropriate amounts of
stacking and resolving gels, the proteins experience a sharp ammonium persulfate solution and TEMED as indicated
increase in retardation due to the restrictive pore size of the in Table 2.2.31.-1, swirl and pour immediately into the gap
resolving gel. Once in the resolving gel, proteins continue to between the two glass plates of the mould. Leave sufficient
be slowed by the sieving of the matrix. The glycinate ions space for the stacking gel (the length of the teeth of the comb
overtake the proteins, which then move in a space of uniform plus 1 cm). Using a tapered glass pipette, carefully overlay the
pH formed by the tris(hydroxymethyl)aminomethane and solution with water-saturated isobutanol. Leave the gel in a
glycine. Molecular sieving causes the SDS-polypeptide vertical position at room temperature to allow polymerisation.
complexes to separate on the basis of their molecular masses. Preparation of the stacking gel. After polymerisation is
PREPARING VERTICAL DISCONTINUOUS BUFFER SDS complete (about 30 min), pour off the isobutanol and wash
POLYACRYLAMIDE GELS the top of the gel several times with water to remove the
Assembling of the gel moulding cassette. Clean the two glass isobutanol overlay and any unpolymerised acrylamide. Drain
plates (size : e.g. 10 cm × 8 cm), the polytetrafluoroethylene as much fluid as possible from the top of the gel, and then
comb, the two spacers and the silicone rubber tubing remove any remaining water with the edge of a paper towel.
(diameter e.g. 0.6 mm × 35 cm) with mild detergent and In a conical flask, prepare the appropriate volume of solution
rinse extensively with water. Dry all the items with a paper containing the desired concentration of acrylamide, using
towel or tissue. Lubricate the spacers and the tubing with the values given in Table 2.2.31.-2. Mix the components
non-silicone grease. Apply the spacers along each of the two in the order shown. Where appropriate, before adding the
short sides of the glass plate 2 mm away from the edges and ammonium persulfate solution and the TEMED, filter the
2 mm away from the long side corresponding to the bottom of solution if necessary under vacuum through a cellulose acetate
the gel. Begin to lay the tubing on the glass plate by using one membrane (pore diameter : 0.45 μm) ; keep the solution under
spacer as a guide. Carefully twist the tubing at the bottom of vacuum by swirling the filtration unit until no more bubbles
the spacer and follow the long side of the glass plate. While are formed in the solution. Add appropriate amounts of
holding the tubing with one finger along the long side twist ammonium persulfate solution and TEMED as indicated
again the tubing and lay it on the second short side of the glass in Table 2.2.31.-2, swirl and pour immediately into the gap
plate, using the spacer as a guide. Place the second glass plate between the two glass plates of the mould directly onto the
in perfect alignment and hold the mould together by hand surface of the polymerised resolving gel. Immediately insert
pressure. Apply two clamps on each of the two short sides a clean polytetrafluoroethylene comb into the stacking gel
of the mould. Carefully apply four clamps on the longer side solution, being careful to avoid trapping air bubbles. Add
of the gel mould thus forming the bottom of the gel mould. more stacking gel solution to fill the spaces of the comb
Verify that the tubing is running along the edge of the glass completely. Leave the gel in a vertical position and allow to
plates and has not been extruded while placing the clamps. polymerise at room temperature.
The gel mould is now ready for pouring the gel.
Preparation of the gel. In a discontinuous buffer SDS
polyacrylamide gel, it is recommended to pour the resolving
gel, let the gel set, and then pour the stacking gel since the
composition of the two gels in acrylamide-bisacrylamide,
buffer and pH are different.
Preparation of the resolving gel. In a conical flask,
prepare the appropriate volume of solution containing
Table 2.2.31.-1. – Preparation of resolving gel
Solution components Component volumes (mL) per gel mould volume of
5 mL l0 mL 15 mL 20 mL 25 mL 30 mL 40 mL 50 mL
Acrylamide solution (1) 1.0 2.0 3.0 4.0 5.0 6.0 8.0 10.0
1.5 M Tris (pH 8.8) (2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5
(3)
100 g/L SDS 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5
100 g/L APS(4) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5
(5)
TEMED 0.004 0.008 0.012 0.016 0.02 0.024 0.032 0.04
The conductivity (formerly called specific conductance) of If the determination of the cell constant is carried out at a
a solution (κ) is, by definition, the reciprocal of resistivity different temperature than that indicated for the certified
(ρ). Resistivity is defined as the quotient of the electric field reference material, the conductivity value may be calculated
and the density of the current. The resistance R (in Ω) of a from the following expression :
conductor of cross-section S (in cm2) and length L (in cm) is
given by the expression :
κT = value of conductivity at the different temperature,
κTCRM = value of conductivity of the certified reference
material,
thus : or T = temperature set for calibration,
TCRM = temperature indicated for the certified reference
L/S corresponds to the ideal cell constant. material,
The unit of conductivity in the International System is α = temperature coefficient for the conductivity value
the siemens per metre (S·m− 1). In practice, the electrical of the certified reference material ; for potassium
conductivity of a solution is expressed in siemens per chloride α = 0.021.
centimetre (S·cm− 1) or in microsiemens per centimetre
(μS·cm− 1). The unit of resistivity in the International System is Determination of the conductivity of the solution to be
the ohm-metre (Ω·m). The resistivity of a solution is generally examined
expressed in ohm-centimetres (Ω·cm). Unless otherwise After calibrating the apparatus with a certified reference
prescribed, the reference temperature for the expression of material solution, rinse the conductivity cell several times with
conductivity or resistivity is 25 °C. distilled water R and at least twice with the aqueous solution to
be examined. Carry out successive measurements as described
The apparatus and operating procedure described below are in the monograph.
applicable to laboratory measurement of conductivity greater
than 10 μS·cm− 1. The measurement of conductivity of water is 01/2008:20239
dealt with in the relevant monographs.
2.2.39. MOLECULAR MASS
APPARATUS
DISTRIBUTION IN DEXTRANS
The apparatus used (conductivity meter or resistivity meter)
measures the resistance of the column of liquid between the Examine by size-exclusion chromatography (2.2.30).
electrodes of the immersed measuring device (conductivity Test solution. Dissolve 0.200 g of the substance to be examined
cell). The apparatus is supplied with alternating current to in the mobile phase and dilute to 10 mL with the mobile phase.
avoid the effects of electrode polarisation. It is equipped with Marker solution. Dissolve 5 mg of glucose R and 2 mg of
a temperature probe and a temperature compensation device. dextran V0 CRS in 1 mL of the mobile phase.
The conductivity cell contains 2 parallel platinum electrodes Calibration solutions. Dissolve separately in 1 mL of the
coated with platinum black, each with a surface area S, and mobile phase 15 mg of dextran 4 for calibration CRS, 15 mg
separated from the other by a distance L. Both are generally of dextran 10 for calibration CRS, 20 mg of dextran 40 for
protected by a glass tube. Other types of cells may also be used. calibration CRS, 20 mg of dextran 70 for calibration CRS and
20 mg of dextran 250 for calibration CRS.
OPERATING PROCEDURE System suitability solution. Dissolve either 20 mg of dextran 40
for performance test CRS (for dextran 40) or 20 mg of
Determination of the cell constant dextran 60/70 for performance test CRS (for dextran 60 and
Choose a conductivity cell that is appropriate for the dextran 70) in 1 mL of the mobile phase.
properties and conductivity of the solution to be examined. The chromatographic procedure may be carried out using :
The higher the expected conductivity, the higher the cell – a column 0.3 m long and 10 mm in internal diameter,
constant that must be chosen (low ρ). Commonly used packed with cross-linked agarose for chromatography R or
conductivity cells have cell constants of the order of 0.1 cm− 1, a series of columns, 0.3 m long and 10 mm in internal
−1 −1
1 cm and 10 cm . Use a certified reference material, for diameter, packed with polyether hydroxylated gel for
example a solution of potassium chloride, that is appropriate chromatography R,
for the measurement. The conductivity value of the certified
– as the mobile phase, at a flow rate of 0.5-1 mL/min, kept
reference material, should be near the expected conductivity
constant to ± 1 per cent per hour, a solution containing 7 g
value of the solution to be examined. Other certified reference
of anhydrous sodium sulfate R and 1 g of chlorobutanol R
materials may be used especially for cells having a constant of
in 1 L of water R,
0.1 cm . Rinse the cell several times with distilled water R
−1
and at least twice with the certified reference material used – as detector a differential refractometer,
for the determination of the cell constant of the conductivity – a 100 μL to 200 μL loop injector,
cell. Measure the resistance of the conductivity cell using the maintaining the system at a constant temperature (± 0.1 °C).
certified reference material at 25 ± 1 °C. The cell constant Kcell
(in cm− 1) depends on the geometry of the conductivity cell CALIBRATION OF THE CHROMATOGRAPHIC SYSTEM
and is given by the expression : Carry out replicate injections of the chosen volume of the
marker solution. The chromatogram shows 2 peaks, the first
of which corresponds to dextran V0 CRS and the second of
which corresponds to glucose R. From the elution volume
RCRM = measured resistance, expressed in mega-ohms, of the peak corresponding to dextran V0, calculate the void
κCRM = conductivity of the certified reference material volume V0 and from the peak corresponding to dextrose,
calculate the total volume Vt.
solution used, expressed in microsiemens per
centimetre. Inject the chosen volume of each of the calibration solutions.
Draw carefully the baseline of each of the chromatograms.
The measured constant Kcell of the conductivity cell must be Divide each chromatogram into p (at least 60) equal vertical
within 5 per cent of the value indicated. sections (corresponding to equal elution volumes). In each
(1)
SYSTEM SUITABILITY
(2)
Inject the chosen volume of the appropriate system suitability
solution.
Average molecular mass of dextran for performance
test CRS. Calculate the average molecular mass Mw as
(3) indicated under Calibration of the chromatographic system,
using either the plotted calibration curve or the values
obtained above for b1, b2, b3, b4 and b5. The test is not valid
p = number of sections dividing the chromatograms, unless Mw is :
yi = height of the chromatographic line above the
baseline in section i, – 41 000 to 47 000 (dextran 40 for performance test CRS),
Mi = molecular mass in section i. – 67 000 to 75 000 (dextran 60/70 for performance test CRS).
(4) An iterative method such as the Gauss-Newton method modified by Hartley is suitable (see O. Hartley, Tecnometrics, 3 (1961) and G. Nilsson and K. Nilsson, J. Chromat. 101, 137
(1974)). A curve-fitting programme for microcomputers, capable of non-linear regression, may be used.
(for example in diode array instruments). Silicon, lead The resulting spectrum can be presented directly in terms of
sulfide, and indium gallium arsenide are examples of detector transflectance (T*) and/or absorbance (A*) (y-axis) versus the
materials. Conventional cuvette sample holders, fibre-optic wavelength or wavenumber (x-axis).
probes, transmission dip cells, neutral borosilicate vials and
spinning or traversing sample holders are a few examples of
sampling devices. The selection is based on the intended
application, paying particular attention to the suitability of
the sampling system for the type of sample to be analysed. I = intensity of transflected radiation measured from
Suitable data processing and evaluation units (e.g. software the sample ;
and computer) are usually part of the system. IT = intensity of transflected radiation of the reference
It is common to express the wavelength (λ) in nanometres material as background ;
and the wavenumber (v) in reciprocal centimetres (cm-1),
depending on the measurement technique and apparatus.
Conversion between nm and cm− 1 is performed according
to the following expression :
SAMPLE PREPARATION/PRESENTATION
Sample preparation and presentation may vary according
to the measurement mode. The following requirements are
necessary for all sampling techniques :
MEASUREMENT METHODS – optimise the measuring time and number of scans to
optimise the signal-to-noise ratio ;
Transmission mode. Transmittance (T) is a measure of the
decrease in radiation intensity at given wavelengths when – find the best suitable measurement mode for the
radiation is passed through the sample. The sample is placed intended application (transmission, diffuse reflection or
in the optical beam between the source and the detector. transflection) ;
The arrangement is analogous to that in many conventional – find the best orientation of the sample (e.g. to minimise the
spectrometers. The resulting spectrum can be presented impact of debossing on tablets) ;
directly in terms of transmittance (T) and/or absorbance (A) – find the best suitable accessory (e.g. transmission cell or
(y-axis) versus the wavelength or wavenumber (x-axis). immersion probe) ;
– optimise pathlength in transmission and transflection
modes ;
– find a suitable spectroscopic background reference material ;
I0 = intensity of incident radiation ; – show that the background reference material is reliable
over time and that the measurement of the background is
I = intensity of transmitted radiation ; reproducible and stable over time ;
– when measuring moving materials or samples (for
process-related measurements) it is important to obtain a
representative spectrum (e.g. by adjusting the measuring
time, the number of scans, co-adding individual spectra, or
Diffuse reflection mode. The diffuse reflection mode gives increasing the beam size) ;
a measure of reflectance (R), the ratio of the intensity of – ensure there is no fouling of the sensor, for example with
light reflected from the sample (I) to that reflected from a build-up of material or contamination ;
background or reference reflective surface (Ir). Depending on – the measuring conditions (measuring time, beam size) in
the chemical composition and physical characteristics of the relation to the minimal sample size should be justified.
sample, NIR radiation can penetrate a more or less substantial In some process analysis situations it may be impossible to
distance into the sample, where it can be absorbed by the remove a probe for reference background data collection ;
overtones and combinations of the fundamental vibrations various options are therefore to be considered, including
of the analyte species present in the sample. Non-absorbed internal referencing, measurement of a background reference
radiation is partially reflected back from the sample to the using a 2nd detector, etc. Only spectra measured against a
detector. NIR reflectance spectra are typically obtained background possessing the same optical properties can be
by calculating and plotting log10 (1/R) (y-axis) versus the directly compared with one another.
wavelength or wavenumber (x-axis).
Transmission mode. The measurement of transmittance (T)
is dependent on a background transmittance spectrum for its
calculation. Examples of background references include air,
a polymeric disc, an empty cell, a solvent blank or in special
I = intensity of light diffusively reflected from the cases a reference sample. The method generally applies to
sample ; liquids (diluted or undiluted), dispersions, solutions and
solids (including tablets and capsules). For transmittance
Ir = intensity of light reflected from the background or measurements of solids, a suitable sample accessory is used.
reference reflective surface ; Liquid samples are examined in a cell of suitable pathlength
(typically 0.5-4 mm), transparent to NIR radiation, or
alternatively by immersion of a fibre-optic probe of a suitable
configuration.
Diffuse reflection mode. This mode generally applies to
Transflection mode. This mode is a combination of solids. The sample is examined directly, or in a suitable device
transmittance and reflectance. In the measurement of (for example a sample holder), or in direct contact with a
transflectance (T*), a mirror or a diffuse reflectance surface fibre-optic probe. For process monitoring, material can be
is used to reflect the transmitted radiation back through analysed through a polished window interface (e.g. sapphire),
the sample, thus doubling the pathlength. Non-absorbed or using an in-line fibre-optic probe. Care must be taken to
radiation is reflected back from the sample to the detector. ensure that the measuring conditions are as reproducible as
possible from one sample to another. The reflected radiation calibration model. The aim can be, for example, to reduce
of a background reference is scanned to obtain the baseline, baseline variations, to reduce the impact of known variations
and then the reflectance of one or more analytical samples is that are interfering in the subsequent mathematical models,
measured. Common reflectance references include ceramic, or to simplify data before use. In some cases spectra may
thermoplastic resins and gold. Other suitable materials may also be normalised or corrected for scatter, for example
be used. using standard normal variate (SNV) transformation.
Transflection mode. This mode generally applies to liquids, Spectral pre-treatment techniques may include, for example,
suspensions and clear plastic materials. A reflector is placed windowing and noise reduction and the numerical calculation
behind the sample so as to double the pathlength. This of the first- or second-order derivative of the spectrum.
configuration can be adopted to share the same instrument Higher-order derivatives are not recommended because of
geometry with reflectance and fibre-optic probe systems increased spectral noise.
where the source and the detector are on the same side of the CONTROL OF INSTRUMENT PERFORMANCE
sample. The sample is examined through a cell with a mirror
or a suitable diffusive reflector, made either of metal or of Use the apparatus according to the manufacturer’s instructions
an inert substance (for example, dried titanium dioxide) not and carry out the prescribed verification at regular intervals,
absorbing in the NIR region. Liquids can also be measured according to the use of the apparatus and the application. For
using in-line transflectance probes. in-line and on-line applications, the use of alternative means
of control of instrument performance must be scientifically
FACTORS AFFECTING SPECTRAL RESPONSE justified. For example, utilise the standards built into the
instrument or separate channels/probes to demonstrate
Environment. The environment temperature and humidity instrument performance (pending practicality).
must be taken into consideration before carrying out
measurements. System suitability tests may be required prior to sample
scanning, and the instrument attributes with potential
Sample presentation area. The sample presentation area or impact on suitability of the final measurement (typically
probe end must be clean of residue prior to measurement. photometric noise and wavelength accuracy) must be tested.
Similarly, the in-line or on-line interface to the sample should The frequency at which each performance test is carried out
not have significant product or contamination build-up, which must be risk-assessed depending on the instrument type and
would interfere with the desired measurement. its environment. For example, instruments placed in harsh
Sample temperature. This parameter is important for environments with variations in temperature and humidity
aqueous solutions and many liquids, where a difference of a may need frequent performance testing. Cases where the
few degrees can result in measurable spectral changes which measurement system cannot be removed such as an in-line
may have a significant effect on the analysis. Temperature probe or flow cell are also to be considered.
is also an important parameter for solids and powders Some accessories are custom designed and therefore require
containing water. adequate performance testing.
Moisture and solvent residues. Moisture and solvent residues Verification and calibration of the wavelength or wavenumber
present in the samples will contribute significant absorption scale (except for filter apparatus). Verify the wavelength
bands in the NIR region. scale employed, generally in the region between 780 nm and
Sample thickness. Sample thickness is a known source of 2500 nm ( 12 800 cm− 1 to 4000 cm− 1) or in the intended
spectral variability and must be assessed and/or controlled, spectral range using one or more suitable wavelength
particularly for tablet and capsule analysis in transmittance standards which have characteristic maxima or minima within
mode. For the measurement of compressed powders, an the wavelength range to be used. For example, methylene
infinite thickness is typically reached after 5 mm of sample chloride R, talc R, wavelength reference lamps or a mixture
depth (e.g. in a vial). of rare-earth oxides are suitable reference materials. Other
suitable standards may also be used. Obtain a spectrum and
Sample optical properties. In solids, both surface and measure the position of at least 3 peaks distributed over the
bulk scattering properties of samples must be taken into range used. For rare-earth oxides, the National Institute of
account. Spectra of physically, chemically or optically Standards and Technology (NIST) provides suitable reference
heterogeneous samples may require increasing the beam materials. Fourier transform (FT) instruments have a linear
size, or examining multiple samples or spinning the sample frequency range, therefore wavelength certification at a single
to obtain a representative spectrum of the sample. Certain frequency is sufficient.
factors such as differing degree of compaction or particle size
Verification and calibration of photometric linearity.
in powdered materials and surface finish can cause significant
The photometric linearity is demonstrated with a set of
spectral differences.
transmittance or reflectance standards with known percentage
Solid-state forms. Variations in solid-state forms values of transmittance or reflectance. For reflectance
(polymorphs, hydrates, solvates and amorphous forms) measurements, carbon-doped polymer standards are
influence vibrational spectra. Hence, different crystalline available. Ensure that the absorbance of the materials used is
forms as well as the amorphous form of a solid may be relevant to the intended linear working range of the method.
distinguished from one another on the basis of their NIR Subsequent verifications of photometric linearity can use the
spectra. Where multiple crystalline forms are present, care initial observed absorbance values as the reference values.
must be taken to ensure that the calibration samples have a Non-linear calibration models and hence non-linear responses
distribution of forms relevant to the intended application. are acceptable with understanding demonstrated by the user.
Age of samples. Samples may exhibit changes in their Spectra obtained from reflectance and transmittance
chemical, physical or optical properties over time. Depending standards are subject to variability due to the differences
on the storage conditions, solid samples may either absorb between the experimental conditions under which they
or desorb water, and portions of amorphous material may were factory-calibrated and those under which they are
crystallise. Materials used for NIR calibration should be subsequently put to use. Hence, the percentage reflectance
representative of future samples and their matrix variables. values supplied with a set of calibration standards may not
be useful in the attempt to establish an ‘absolute’ calibration
PRE-TREATMENT OF NIR SPECTRAL DATA for a given instrument. As long as the standards do not
In many cases, and particularly for reflection mode spectra, change chemically or physically and the same reference
some form of mathematical pre-treatment of the spectrum background is used as was used to obtain the certified values,
may be useful prior to the development of a classification or subsequent measurements of the same standards under
identical conditions, including precise sample positioning, Recommendations for the conditions used to control
give information on long-term stability of the photometric instrument performance for the various measurement modes
response. A tolerance of ± 2 per cent of the absorbance value are summarised in Table 2.2.40.-1.
is acceptable for long-term stability ; this verification is only
necessary if the spectra are used without pre-treatment.
Table 2.2.40.-1 – Control of instrument performance
Measurement mode Reflection Transflection Transmission
Bench/mobile Measure talc R via a suitable medium A suspension of 1.2 g of dry titanium Methylene chloride R may be used and has
instrument or by fibre-optic probe. Talc R has dioxide R in about 4 mL of methylene characteristic sharp bands at 1155 nm,
characteristic peaks at 948 nm, 1391 nm chloride R is used directly with a 1366 nm, 1417 nm, 1690 nm, 1838 nm,
and 2312 nm suitable for calibration. cell or a probe. Titanium dioxide 1894 nm, 2068 nm and 2245 nm. Choose
Alternatively, other suitable standards has no absorption in the NIR range. 3 peaks across the wavelength range for
may also be used that ensure wavelength Spectra are recorded with a maximum calibration. Other suitable standards may
accuracy in the region of working nominal instrument bandwidth also be used.
methodology. For example, measure of 10 nm at 2500 nm (16 cm− 1 at
an internal polystyrene standard if 4000 cm− 1). Methylene chloride has
built-in, or measure a NIST standard characteristic sharp bands at 1155 nm,
or other traceable material, and assess 1366 nm, 1417 nm, 1690 nm, 1838 nm,
3 peaks across the wavelength range for 1894 nm, 2068 nm and 2245 nm.
calibration. Choose 3 peaks across the wavelength
range for calibration. Other suitable
standards may also be used, such as
a liquid transflection standard mixed
with titanium dioxide or some other
reflective medium.
Process instrument If it is not practically possible to measure a traceable reference material at the point of sample measurement, use internal material
such as polystyrene, fibreglass or solvent and/or water vapour. Alternatively, adopt a second external channel/probe.
For FT instruments, the calibration of the wavenumber scale may be performed using a narrow, isolated water-vapour line, for
example, the line at 7306.74 cm− 1, or 7299.45 cm− 1, or 7299.81 cm− 1 or a narrow line from a certified reference material.
Verification The standard deviation of the wavelength is consistent with the specifications of the instrument manufacturer, or otherwise
of wavelength scientifically justified.
repeatability (except
for filter apparatus)
Bench/mobile Verify the wavelength repeatability using a suitable external or internal standard.
instrument
Process instrument Verify the wavelength repeatability using a suitable external or internal standard.
Verification of Measure 4 photometric standards across the working method absorbance range.
photometric linearity
and response stability(1)
Bench/mobile Analyse 4 reflectance standards, for Transflection measurements can Analyse 4 transmittance standards to cover
instrument example in the range of 10-99 per use appropriate reflectance or the absorbance values over the working
cent, including 10 per cent, 20 per transmittance standards and criteria. absorbance range of the modelled data.
cent, 40 per cent and 80 per cent. Evaluate the observed absorbance values
In some circumstances 2 per cent against the reference absorbance values,
may be appropriate. Evaluate the for example perform a linear regression.
observed absorbance values against the Acceptable tolerances are 1.00 ± 0.05 for
reference absorbance values, for example the slope and 0.00 ± 0.05 for the intercept
perform a linear regression. Acceptable for the 1st verification of photometric
tolerances are 1.00 ± 0.05 for the slope linearity of an instrument. Subsequent
and 0.00 ± 0.05 for the intercept for the verifications of photometric linearity can
1st verification of photometric linearity of use the initial observed absorbance values
an instrument. Subsequent verifications as the reference values.
of photometric linearity can use the
initial observed absorbance values as the
reference values.
Process instrument If photometric reflectance and transmittance standards cannot be measured at the point of sample measurement, use the
photometric standards built into the instrument.
Process instruments can use internal photometric standards for photometric linearity. Follow the manufacturer’s verified
tolerances in such cases.
Verification of Determine the photometric noise at a relevant photometric region of the spectrum using a suitable reflectance standard, for
photometric noise(1) example, white reflective ceramic tiles or carbon-doped polymer standards. Follow the manufacturer’s methodology and
specifications.
Bench/mobile Scan the reflectance low flux standard (e.g. 5 or 10 per cent, carbon-doped polymer Scan the transmittance high flux standard
instrument standard) over a suitable wavelength range in accordance with the manufacturer’s (e.g. 90 or 99 per cent, carbon-doped
recommendation and calculate the photometric noise as peak-to-peak noise. polymer standard) over a suitable
wavelength/wavenumber range in
accordance with the manufacturer’s
recommendation and calculate the
photometric noise as peak-to-peak noise.
Process instrument As above, or if not practically possible, use the standard built into the instrument As above, or if not practically possible,
for noise testing and manufacturer specifications. use the standard built into the instrument
for noise testing and manufacturer
specifications.
Verification of photometric linearity and Verification of photometric noise are not required for instruments using methods to perform simple
(1)
identifications which do not use the photometric absorbances as part of model strategy (for example, simple correlation with absorbing wavelengths).
QUALITATIVE ANALYSIS (IDENTIFICATION AND database on the basis of their mathematical correlation or
CHARACTERISATION) other suitable algorithms. A set of known reference mean
Establishment of a spectral reference library. Record spectra and the variability around this mean can be used
the spectra of a suitable number of representative samples with an algorithm for classification ; alternatively, this can
of the substance which have known, traceable identities, be achieved visually by overlaying spectral data if specificity
and that exhibit the variation typical for the substance to is inherent. There are different techniques available, such as
be analysed (for example, solid-state form, particle size, principal component analysis (PCA), cluster analysis, and
etc.). Libraries are built using representative samples under soft independent modelling by class analogy (SIMCA). The
appropriate environmental conditions. The set of spectra reliability of the technique chosen for a particular application
obtained represents the information which can be used for has to be validated according to the following :
identification of the sample to be analysed. Validation of the model. Identification methods using direct
The collection of spectra in the library may be represented in spectral comparison must be validated in accordance with
different ways defined by the mathematical technique used for identification method validation procedures.
identification. These may be: The validation parameters for qualitative methods are
– all individual spectra representing the substance ; robustness and specificity.
– a mean spectrum of the measured batches for each chemical LIMIT ANALYSIS
substance ;
Relative comparison of spectra. A calibration is not required
– if necessary, a description of the variability within the when comparing a set of spectra for limit analysis purposes,
substance spectra. such as the maximum or minimum absorbance at which an
The number of substances in the library depends on the analyte absorbs. Also, dryer end point control may use a
specific application. All spectra in the library used have the qualitative approach around a specific absorbing wavelength.
same : Appropriate spectral ranges and pre-treatments (if used) must
– spectral range and number of data points ; be shown to be fit for purpose.
– technique of measurement ; Specificity. The relative discriminatory power for a limit test
must be demonstrated. The extent of specificity testing is
– data pre-treatment. dependent on the application and the risks being controlled.
If sub-groups (sub-libraries) are created, the above criteria Variations in matrix concentrations within the operating
are applied independently for each group. Sub-libraries range of the method must not affect the measurement.
are individually validated. Original spectral data for the
preparation of the spectral library must be archived. Caution TREND ANALYSIS
must be exercised when performing any mathematical Relative comparison of spectra. A calibration is not
transformation, as artefacts can be introduced or essential necessarily required when comparing a set of spectra for
information (important with qualification methods) can trend analysis purposes, such as the moving block approach
be lost. The suitability of the algorithm used should be to estimate statistical parameters such as mean, median and
demonstrated by successful method validation and in all cases standard deviation. For example, blend uniformity monitoring
the rationale for the use of transform must be documented. using NIR spectroscopy has adopted such data analysis
Direct comparison of substance and reference spectra. approaches. Appropriate spectral ranges and algorithms must
Direct comparison of representative spectra of the substance be used for trend analyses.
to be examined and of a reference substance for qualitative Specificity. The relative discriminatory power for trend
chemical or physical identification purposes may not require analysis must be demonstrated. The extent of specificity
use of a reference spectral library where specificity permits. testing is dependent on the application and the risks being
Data evaluation. Direct comparison of the representative controlled. Variations in matrix concentrations within the
spectrum of the substance to be examined is made with the operating range of the method must not affect the trend
individual or mean reference spectra of all substances in the analysis.
The ratio Vac/Vc is proportional to the differential absorption – the particle density, which also includes the volume due to
ΔA which created the signal. The region of wavelengths intraparticulate pores ;
normally covered by a dichrograph is 170 nm to 800 nm. – the bulk density, which further includes the interparticulate
Calibration of the apparatus void volume formed in the powder bed.
Accuracy of absorbance scale. Dissolve 10.0 mg of
isoandrosterone R in dioxan R and dilute to 10.0 mL with the TRUE DENSITY
same solvent. Record the circular dichroism spectrum of The true density of a substance is the ratio of the mass to the
the solution between 280 nm and 360 nm. Measured at the volume of the unit cell, exclusive of all voids that are not a
maximum at 304 nm, Δε is + 3.3. fundamental part of the molecular packing arrangement. It
The solution of (1S)-(+)-10-camphorsulfonic acid R may also is an intrinsic property of the specified crystal structure of
be used. substance, and hence should be independent of the method of
determination. The true density is determined by calculation.
Linearity of modulation. Dissolve 10.0 mg of
(1S)-(+)-10-camphorsulfonic acid R in water R and It is obtained using crystallographic data (volume and
dilute to 10.0 mL with the same solvent. Determine the exact composition of the unit cell) from, for example, X-ray
concentration of camphorsulfonic acid in the solution by diffraction data, either on a single crystal or by refinement of
ultraviolet spectrophotometry (2.2.25), taking the specific the crystalline structure from X-ray powder diffraction data.
absorbance to be 1.49 at 285 nm.
PARTICLE DENSITY
Record the circular dichroism spectrum between 185 nm and
340 nm. Measured at the maximum at 290.5 nm, Δε is + 2.2 The particle density takes into account both the true density
to + 2.5. Measured at the maximum at 192.5 nm, Δε is − 4.3 and the intraparticulate porosity (sealed and/or experimentally
to − 5. non-accessible open pores). Thus, particle density depends on
(1S)-(+)- or antipodal (1R)-(−)-ammonium 10-camphorsulfo- the value of the volume determined, which in turn depends
nate R can also be used. on the method of measurement. The particle density can be
determined using one of the 2 following methods.
The gas pycnometric density is determined by measuring
01/2010:20242 the volume occupied by a known mass of powder, which is
equivalent to the volume of gas displaced by the powder using
2.2.42. DENSITY OF SOLIDS a gas displacement pycnometer (2.9.23). In gas pycnometric
density measurements, the volume determined excludes the
The density of solids corresponds to their average mass volume occupied by open pores ; however, it includes the
per unit volume and typically is expressed in grams per cubic volume occupied by sealed pores or pores inaccessible to
3
centimetre (g/cm ) although the International Unit is the the gas. Due to the high diffusivity of helium, which is the
kilogram per cubic metre (1 g/cm3 = 1000 kg/m3). preferred choice of gas, most open pores are accessible to
Unlike gases and liquids whose density depends only on the gas. Therefore, the gas pycnometric density of a finely
temperature and pressure, the density of a solid also depends milled powder is generally not very different from the true
on its assembly and therefore varies with the crystal structure density. Hence, this density is the best estimate of the true
and degree of crystallinity. density of an amorphous or partially crystalline sample and
When a solid is amorphous or partially amorphous, its is therefore widely applicable for processed pharmaceutical
density may further depend upon the history of preparation, powder samples.
treatment and storage. The mercury porosimeter density is also called granular density.
Therefore, unlike fluids, the densities of 2 chemically With this method the volume determined includes the volume
equivalent solids may be different, and this difference occupied by sealed pores or pores inaccessible to mercury ;
reflects a difference in solid-state structure. The density of however, it includes the volume only from open pores
constituent particles is an important physical characteristic smaller than some size limit. This pore-size limit or minimal
of pharmaceutical powders. access diameter depends on the maximal mercury intrusion
The density of a solid particle can assume different values pressure applied during the measurement, and under normal
depending on the method used to measure the volume of operating pressures the mercury does not penetrate the
the particle. It is useful to distinguish 3 levels of expression finest pores accessible to helium. Various granular densities
of density : can be obtained from one sample since, for each applied
– the true density, which only includes the solid fraction of mercury intrusion pressure, a density can be determined that
the material ; in case of crystalline material, the true density corresponds to the pore-size limit at that pressure.
is also called crystal density ;
BULK AND TAPPED DENSITY – particle-beam interface : the mobile phase, which may flow
The bulk density of a powder includes the contribution of at a rate of up to 0.6 mL/min, is nebulised in a desolvation
interparticulate void volume. Hence, the bulk density depends chamber such that only the analytes, in neutral form, reach
on both the density of powder particles and the spatial the ion source of the apparatus ; this technique is used
arrangement of particles in the powder bed. for compounds of relatively low polarity with molecular
masses of less than 1000 Da,
The bulk density of a powder is often very difficult to measure
with good reproducibility since the slightest disturbance of – moving-belt interface : the mobile phase, which may flow
the bed may result in a new density. Thus, it is essential in at a rate of up to 1 mL/min, is applied to the surface of a
reporting bulk density to specify how the determination was moving belt ; after the solvent evaporates, the components
made. to be analysed are successively carried to the ion source
The bulk density and the tapped density are determined as of the apparatus where they are ionised ; this technique is
mentioned in chapter 2.9.34. Bulk density and tapped density. rather poorly suited to very polar or heat-labile compounds.
Other types of coupling (electrospray, thermospray,
atmospheric-pressure chemical ionisation) are considered to
01/2008:20243 be ionisation techniques in their own right and are described
in the section on modes of ionisation.
2.2.43. MASS SPECTROMETRY Supercritical fluid chromatography/mass spectrometry.
The mobile phase, usually consisting of supercritical carbon
Mass spectrometry is based on the direct measurement of dioxide enters the gas state after passing a heated restrictor
the ratio of the mass to the number of positive or negative between the column and the ion source.
elementary charges of ions (m/z) in the gas phase obtained
from the substance to be analysed. This ratio is expressed in Capillary electrophoresis/mass spectrometry. The eluent
atomic mass units (1 a.m.u. = one twelfth the mass of 12C) or is introduced into the ion source, in some cases after adding
in daltons (1 Da = the mass of the hydrogen atom). another solvent so that flow rates of the order of a few
The ions, produced in the ion source of the apparatus, are microlitres per minute can be attained. This technique is
accelerated and then separated by the analyser before reaching limited by the small quantities of sample introduced and the
the detector. All of these operations take place in a chamber need to use volatile buffers.
where a pumping system maintains a vacuum of 10− 3 to
10− 6 Pa. MODES OF IONISATION
The resulting spectrum shows the relative abundance of the Electron impact. The sample, in the gas state, is ionised by
various ionic species present as a function of m/z. The signal a beam of electrons whose energy (usually 70 eV) is greater
corresponding to an ion will be represented by several peaks than the ionisation energy of the sample. In addition to the
corresponding to the statistical distribution of the various molecular ion M+, fragments characteristic of the molecular
isotopes of that ion. This pattern is called the isotopic profile structure are observed. This technique is limited mainly by the
and (at least for small molecules) the peak representing need to vaporise the sample. This makes it unsuited to polar,
the most abundant isotopes for each atom is called the heat-labile or high molecular mass compounds. Electron
monoisotopic peak. impact is compatible with the coupling of gas chromatography
Information obtained in mass spectrometry is essentially to mass spectrometry and sometimes with the use of liquid
qualitative (determination of the molecular mass, information chromatography.
on the structure from the fragments observed) or quantitative Chemical ionisation. This type of ionisation involves a
(using internal or external standards) with limits of detection reagent gas such as methane, ammonia, nitrogen oxide,
ranging from the picomole to the femtomole. nitrogen dioxide or oxygen. The spectrum is characterised
INTRODUCTION OF THE SAMPLE by ions of the (M + H)+ or (M − H)– types, or adduct ions
formed from the analyte and the gas used. Fewer fragments
The very first step of an analysis is the introduction of are produced than with electron impact. A variant of this
the sample into the apparatus without overly disturbing technique is used when the substance is heat-labile : the
the vacuum. In a common method, called direct liquid sample, applied to a filament, is very rapidly vaporised by the
introduction, the sample is placed on the end of a cylindrical Joule-Thomson effect (desorption chemical ionisation).
rod (in a quartz crucible, on a filament or on a metal surface).
This rod is introduced into the spectrometer after passing Fast-atom bombardment (FAB) or fast-ion bombardment
through a vacuum lock where a primary intermediate ionisation (liquid secondary-ion mass spectrometry
vacuum is maintained between atmospheric pressure and the LSIMS). The sample, dissolved in a viscous matrix such as
secondary vacuum of the apparatus. glycerol, is applied to a metal surface and ionised by a beam of
Other introduction systems allow the components of a neutral atoms such as argon or xenon or high-kinetic-energy
mixture to be analysed as they are separated by an appropriate caesium ions. Ions of the (M + H)+ or (M − H)– types or adduct
apparatus connected to the mass spectrometer. ions formed from the matrix or the sample are produced.
This type of ionisation, well suited to polar and heat-labile
Gas chromatography/mass spectrometry. The use of compounds, allows molecular masses of up to 10 000 Da to
suitable columns (capillary or semi-capillary) allows the end be obtained. The technique can be combined with liquid
of the column to be introduced directly into the source of the chromatography by adding 1 per cent to 2 per cent of glycerol
apparatus without using a separator. to the mobile phase ; however, the flow rates must be very low
Liquid chromatography/mass spectrometry. This (a few microlitres per minute). These ionisation techniques
combination is particularly useful for the analysis of polar also allow thin-layer chromatography plates to be analysed by
compounds, which are insufficiently volatile or too heat-labile applying a thin layer of matrix to the surface of these plates.
to be analysed by gas chromatography coupled with mass Field desorption and field ionisation. The sample is
spectrometry. This method is complicated by the difficulty of vaporised near a tungsten filament covered with microneedles
obtaining ions in the gas phase from a liquid phase, which (field ionisation) or applied to this filament (field desorption).
requires very special interfaces such as : A voltage of about 10 kV, applied between this filament and a
– direct liquid introduction : the mobile phase is nebulised, counter-electrode, ionises the sample. These two techniques
and the solvent is evaporated in front of the ion source of mainly produce molecular ions M+, and (M + H)+ ions and are
the apparatus, used for low polarity and/or heat-labile compounds.
Matrix-assisted laser desorption ionisation (MALDI). The energy filter and allows the resolving power of the instrument
sample, in a suitable matrix and deposited on a metal support, to be increased appreciably. The maximum resolving power
is ionised by a pulsed laser beam whose wavelength may range of such an instrument (double sector) ranges from 10 000 to
from UV to IR (impulses lasting from a picosecond to a few 150 000 and in most cases allows the value of m/z ratios to
nanoseconds). This mode of ionisation plays an essential role be calculated accurately enough to determine the elemental
in the analysis of very high molecular mass compounds (more composition of the corresponding ions. For monocharged
than 100 000 Da) but is limited to time-of flight analysers (see ions, the mass range is from 2000 Da to 15 000 Da. Some ions
below). may decompose spontaneously (metastable transitions) or by
Electrospray. This mode of ionisation is carried out at colliding with a gas (collision-activated dissociation (CAD))
atmospheric pressure. The samples, in solution, are introduced in field-free regions between the ion source and the detector.
into the source through a capillary tube, the end of which has Examination of these decompositions is very useful for the
a potential of the order of 5 kV. A gas can be used to facilitate determination of the structure as well as the characterisation
nebulisation. Desolvation of the resulting microdroplets of a specific compound in a mixture and involves tandem mass
produces singly or multiply charged ions in the gas phase. spectrometry. There are many such techniques depending on
The flow rates vary from a few microlitres per minute to the region where these decompositions occur :
1 mL/min. This technique is suited to polar compounds and – daughter-ion mode (determination of the decomposition
to the investigation of biomolecules with molecular masses of ions of a given parent ion) : B/E = constant, MIKES
up to 100 000 Da. It can be coupled to liquid chromatography (Mass-analysed Ion Kinetic Energy Spectroscopy),
or capillary electrophoresis. – parent-ion mode (determination of all ions which by
Atmospheric-pressure chemical ionisation (APCI). decomposition give an ion with a specific m/z ratio) :
Ionisation is carried out at atmospheric pressure by the B2/E = constant,
action of an electrode maintained at a potential of several – neutral-loss mode (detection of all the ions that lose the
kilovolts and placed in the path of the mobile phase, which is same fragment) :
nebulised both by thermal effects and by the use of a stream
B/E(1 − E/E0)1/2 = constant, where E0 is the basic voltage
of nitrogen. The resulting ions carry a single charge and are of
of the electric sector.
the (M + H)+ type in the positive mode and of the (M − H)–
type in the negative mode. The high flow rates that can be Quadrupoles. The analyser consists of four parallel metal
used with this mode of ionisation (up to 2 mL/min) make this rods, which are cylindrical or hyperbolic in cross-section.
an ideal technique for coupling to liquid chromatography. They are arranged symmetrically with respect to the trajectory
of the ions ; the pairs diagonally opposed about the axis of
Thermospray. The sample, in the mobile phase consisting
symmetry of rods are connected electrically. The potentials to
of water and organic modifiers and containing a volatile
the two pairs of rods are opposed. They are the resultant of a
electrolyte (generally ammonium acetate) is introduced in
constant component and an alternating component. The ions
nebulised form after having passed through a metal capillary
produced at the ion source are transmitted and separated by
tube at controlled temperature. Acceptable flow rates are of
varying the voltages applied to the rods so that the ratio of
the order of 1 mL/min to 2 mL/min. The ions of the electrolyte
continuous voltage to alternating voltage remains constant.
ionise the compounds to be analysed. This ionisation process
The quadrupoles usually have a mass range of 1 a.m.u. to
may be replaced or enhanced by an electrical discharge
2000 a.m.u., but some may range up to 4000 a.m.u. Although
of about 800 volts, notably when the solvents are entirely
they have a lower resolving power than magnetic sector
organic. This technique is compatible with the use of liquid
analysers, they nevertheless allow the monoisotopic profile of
chromatography coupled with mass spectrometry.
single charged ions to be obtained for the entire mass range. It
is possible to obtain spectra using three quadrupoles arranged
ANALYSERS
in series, Q1, Q2, Q3 (Q2 serves as a collision cell and is not
Differences in the performance of analysers depend mainly on really an analyser ; the most commonly used collision gas is
two parameters : argon).
– the range over which m/z ratios can be measured, ie, the The most common types of scans are the following :
mass range, – daughter-ion mode : Q1 selects an m/z ion whose fragments
– their resolving power characterised by the ability to separate obtained by collision in Q2 are analysed by Q3,
two ions of equal intensity with m/z ratios differing by ΔM, – parent-ion mode: Q3 filters only a specific m/z ratio, while
and whose overlap is expressed as a given percentage of Q1 scans a given mass range. Only the ions decomposing to
valley definition ; for example, a resolving power (M/ΔM) of give the ion selected by Q3 are detected,
1000 with 10 per cent valley definition allows the separation
of m/z ratios of 1000 and 1001 with the intensity returning – neutral loss mode : Q1 and Q3 scan a certain mass range
to 10 per cent above baseline. However, the resolving power but at an offset corresponding to the loss of a fragment
may in some cases (time-of-flight analysers, quadrupoles, characteristic of a product or family of compounds.
ion-trap analysers) be defined as the ratio between the It is also possible to obtain spectra by combining quadrupole
molecular mass and peak width at half height (50 per cent analysers with magnetic or electrostatic sector instruments ;
valley definition). such instruments are called hybrid mass spectrometers.
Magnetic and electrostatic analysers. The ions produced Ion-trap analyser. The principle is the same as for a
in the ion source are accelerated by a voltage V, and focused quadrupole, this time with the electric fields in three
towards a magnetic analyser (magnetic field B) or an dimensions. This type of analyser allows product-ion spectra
electrostatic analyser (electrostatic field E), depending on the over several generations (MSn) to be obtained.
configuration of the instrument. They follow a trajectory of Ion-cyclotron resonance analysers. Ions produced in a cell
radius r according to Laplace’s law : and subjected to a uniform, intense magnetic field move in
circular orbits at frequencies which can be directly correlated
to their m/z ratio by applying a Fourier transform algorithm.
This phenomenon is called ion-cyclotron resonance.
Two types of scans can be used to collect and measure the Analysers of this type consist of superconducting magnets and
various ions produced by the ion source : a scan of B holding are capable of very high resolving power (up to 1000 000 and
V fixed or a scan of V with constant B. The magnetic analyser more) as well as MSn spectra. However, very low pressures are
is usually followed by an electric sector that acts as a kinetic required (of the order of 10− 7 Pa).
Control solutions. In addition to the TOC water control, The composition, pressure (density), temperature and flow
prepare suitable blank solutions or other solutions needed rate of the prescribed mobile phase may either be constant
for establishing the baseline or for calibration adjustments throughout the whole chromatographic procedure (isocratic,
following the manufacturer’s instructions ; run the appropriate isodense, isothermic elution) or may vary according to a
blanks to zero the instrument. defined programme (gradient elution of the modifier, pressure
System suitability. Run the following solutions and record (density), temperature or flow rate).
the responses : TOC water (rw) ; standard solution (rs) ; system Detectors
suitability solution (rss). Calculate the percentage response Ultraviolet/visible (UV/Vis) spectrophotometers and flame
efficiency using the expression : ionisation detectors are the most commonly employed
detectors. Light scattering detectors, infrared absorption
spectrophotometers, thermal conductivity detectors or other
special detectors may be used.
The system is suitable if the response efficiency is not less than
85 per cent and not more than 115 per cent of the theoretical METHOD
response. Prepare the test solution(s) and the reference solution(s) as
Procedure. Run the test solution and record the response (ru). prescribed. The solutions must be free from solid particles.
The test solution complies with the test if ru is not greater than Criteria for assessing the suitability of the system are described
r s – rw . in the chapter on Chromatographic separation techniques
The method can also be applied using on-line instrumentation (2.2.46). The extent to which adjustments of parameters of the
that has been adequately calibrated and shown to have chromatographic system can be made to satisfy the criteria of
acceptable system suitability. The location of instrumentation system suitability are also given in this chapter.
must be chosen to ensure that the responses are representative
of the water used.
04/2009:20246
01/2008:20245
Figure 2.2.46.-1.
The peak may be defined by the peak area, or the peak Retention factor (k)
height (h) and the peak width at half-height (wh), or the The retention factor (also known as mass distribution ratio
peak height (h) and the peak width between the points of (Dm) or capacity factor (k′)) is defined as :
inflection (wi). In Gaussian peaks (Figure 2.2.46.-1) there is
the following relationship :
amount of component in stationary phase
amount of component in mobile phase
Figure 2.2.46.-2.
theoretical plates), using the following equation, the values of w0.05 = width of the peak at one-twentieth of the peak
tR and wh being expressed in the same units : height ;
d = distance between the perpendicular dropped from
the peak maximum and the leading edge of the
peak at one-twentieth of the peak height.
tR = retention time of the peak corresponding to the An As value of 1.0 signifies symmetry. When As > 1.0, the peak
component ; is tailing. When As < 1.0, the peak is fronting.
wh = width of the peak at half-height.
Figure 2.2.46.-4
Symmetry factor (As) Hp = height above the extrapolated baseline of the minor
The symmetry factor of a peak (Figure 2.2.46.-5) is calculated peak ;
using the following equation : Hv = height above the extrapolated baseline at the lowest
point of the curve separating the minor and major
peaks.
Figure 2.2.46.-7.
System repeatability
The repeatability of response is expressed as an estimated
percentage relative standard deviation (sr(%)) of a consecutive
series of measurements for not fewer than 3 injections or
applications of a reference solution, and is calculated using
Figure 2.2.46.-6 the following equation :
Relative retention (r)
Relative retention is calculated as an estimate using the
following equation :
yi = individual values expressed as peak area,
peak height, or ratio of areas by the internal
standardisation method ;
= mean of individual values ;
tRi = retention time of the peak of interest ;
n = number of individual values.
tRst = retention time of the reference peak (usually
the peak corresponding to the substance to be
examined) ; SYSTEM SUITABILITY
tM = hold-up time. The various components of the equipment employed must
be qualified and be capable of achieving the performance
The unadjusted relative retention (rG) is calculated using the required to conduct the test or assay.
following equation : The system suitability tests represent an integral part of the
method and are used to ensure adequate performance of the
chromatographic system. Apparent efficiency, retention factor
(mass distribution ratio), resolution, relative retention and
symmetry factor are the parameters that are usually employed
Unless otherwise indicated, values for relative retention stated in assessing the performance of the column. Factors that may
in monographs correspond to unadjusted relative retention. affect the chromatographic behaviour include :
In planar chromatography, the retardation factors RFst and RFi – the composition, ionic strength, temperature and apparent
are used instead of tRst and tRi. pH of the mobile phase ;
Signal-to-noise ratio (S/N) – flow rate, column dimensions, column temperature and
pressure ;
The short-term noise influences the precision of quantification. – stationary phase characteristics including type of
The signal-to-noise ratio is calculated using the following chromatographic support (particle-based or monolithic),
equation : particle or macropore size, porosity, specific surface area ;
– reversed-phase and other surface-modification of the
stationary phases, the extent of chemical modification (as
expressed by end-capping, carbon loading etc.).
H = height of the peak (Figure 2.2.46.-7) corresponding The following requirements and any supplementary
to the component concerned, in the chromatogram requirements given in the individual monograph are to be
obtained with the prescribed reference solution, fulfilled unless otherwise prescribed :
measured from the maximum of the peak to the – in a related substances test or assay, for a peak in the
extrapolated baseline of the signal observed over chromatogram obtained with a reference solution used for
a distance equal to at least 5 times the width at quantification, the symmetry factor is 0.8 to 1.5, unless
half-height ; otherwise prescribed ;
h = range of the noise in a chromatogram obtained – in an assay of an active substance where the value is 100 per
after injection or application of a blank, observed cent for a pure substance, the maximum permitted relative
over a distance equal to at least 5 times the width standard deviation (sr(%)max) for the defined limits is
at half-height of the peak in the chromatogram calculated for a series of injections of the reference solution
obtained with the prescribed reference solution using the following equation :
and, if possible, situated equally around the place
where this peak would be found.
K = constant (0.349), obtained from the expression relative adjustment allows a range of 7-13 per cent whereas
in which represents a 2 per cent absolute adjustment allows a range of 8-12 per
the required percentage relative standard cent, the relative value therefore being the larger ; for a minor
deviation after 6 injections for B = 1.0 ; component at 5 per cent of the mobile phase, a 30 per cent
relative adjustment allows a range of 3.5-6.5 per cent whereas
B = upper limit given in the definition of the a 2 per cent absolute adjustment allows a range of 3-7 per
individual monograph minus 100 per cent ; cent, the absolute value being the larger in this case ; no other
n = number of replicate injections of the reference component is altered by more than 10 per cent absolute.
solution (3 ≤ n ≤ 6) ; pH of the aqueous component of the mobile phase : ± 0.2 pH,
t90%,n−1 = Student’s t at the 90 per cent probability level unless otherwise prescribed, or ± 1.0 pH when non-ionisable
(double sided) with n−1 degrees of freedom. substances are to be examined.
Unless otherwise prescribed, the maximum permitted Concentration of salts in the buffer component of a mobile
relative standard deviation does not exceed the appropriate phase : ± 10 per cent.
value given in Table 2.2.46.-1. This requirement does not Application volume : 10-20 per cent of the prescribed volume
apply to tests for related substances. if using fine particle size plates (2-10 μm).
Table 2.2.46.-1. – Repeatability requirements Liquid chromatography : isocratic elution
Composition of the mobile phase : the amount of the minor
Number of individual injections
solvent component may be adjusted by ± 30 per cent relative
3 4 5 6 or ± 2 per cent absolute, whichever is the larger (see example
above) ; no other component is altered by more than 10 per
B (per cent) Maximum permitted relative standard deviation
cent absolute.
2.0 0.41 0.59 0.73 0.85 pH of the aqueous component of the mobile phase : ± 0.2 pH,
2.5 0.52 0.74 0.92 1.06 unless otherwise prescribed, or ± 1.0 pH when non-ionisable
substances are to be examined.
3.0 0.62 0.89 1.10 1.27
Concentration of salts in the buffer component of a mobile
– in a related substances test, the limit of quantification phase : ± 10 per cent.
(corresponding to a signal-to-noise ratio of 10) is equal to Flow rate : ± 50 per cent ; a larger adjustment is acceptable
or less than the disregard limit. when changing the column dimensions (see the formula
Compliance with the system suitability criteria is required below).
throughout the chromatographic procedure. Depending on Column parameters
various factors, such as the frequency of use of the procedure Stationary phase :
and experience with the chromatographic system, the analyst
chooses an appropriate verification scheme to monitor this. – no change of the identity of the substituent of the
stationary phase permitted (e.g. no replacement of C18
ADJUSTMENT OF CHROMATOGRAPHIC CONDITIONS by C8) ;
The extent to which the various parameters of a – particle size : maximum reduction of 50 per cent ; no
chromatographic test may be adjusted to satisfy the system increase permitted.
suitability criteria without fundamentally modifying the Column dimensions :
methods are listed below. Adjustment of conditions with
gradient elutions is more critical than with isocratic elutions, – length : ± 70 per cent ;
since it may lead to shifts in peaks to a different step of the – internal diameter : ± 25 per cent.
gradient, thus leading to the incorrect assignment of peaks, When column dimensions are changed, the flow rate may
and to the masking of peaks or a shift such that elution be adjusted as necessary using the following equation :
occurs beyond the prescribed elution time. Changes other
than those indicated require revalidation of the method. The
chromatographic conditions described have been validated
during the elaboration of the monograph.
The system suitability tests are included to verify that the F1 = flow rate indicated in the monograph, in
separation required for satisfactory performance of the test or millilitres per minute ;
assay is achieved. Nonetheless, since the stationary phases are
F2 = adjusted flow rate, in millilitres per minute ;
described in a general way and there is such a variety available
commercially, with differences in chromatographic behaviour, l1 = length of the column indicated in the
some adjustments of the chromatographic conditions may monograph, in millimetres ;
be necessary to achieve the prescribed system suitability l2 = length of the column used, in millimetres ;
requirements. With reversed-phase liquid chromatographic
methods in particular, adjustment of the various parameters d1 = internal diameter of the column indicated in the
will not always result in satisfactory chromatography. In that monograph, in millimetres ;
case, it may be necessary to replace the column with another d2 = internal diameter of the column used, in
of the same type (e.g. octadecylsilyl silica gel), which exhibits millimetres.
the desired chromatographic behaviour. The Knowledge
database on the EDQM website usually contains information Temperature : ± 10 °C, where the operating temperature is
on the column(s) used during monograph elaboration. specified, unless otherwise prescribed.
For critical parameters the adjustments are defined clearly in Detector wavelength : no adjustment permitted.
the monograph to ensure the system suitability. Injection volume : may be decreased, provided detection and
Thin-layer chromatography and paper chromatography repeatability of the peak(s) to be determined are satisfactory ;
no increase permitted.
Composition of the mobile phase : the amount of the minor
solvent component may be adjusted by ± 30 per cent relative Liquid chromatography : gradient elution
or ± 2 per cent absolute, whichever is the larger ; for a minor Adjustment of chromatographic conditions for gradient
component at 10 per cent of the mobile phase, a 30 per cent systems requires greater caution than for isocratic systems.
Composition of the mobile phase/gradient elution : minor l2 = length of the column used, in millimetres ;
adjustments of the composition of the mobile phase and the
gradient are acceptable provided that : d1 = internal diameter of the column indicated in the
monograph, in millimetres ;
– the system suitability requirements are fulfilled ; d2 = internal diameter of the column used, in
– the principal peak(s) elute(s) within ± 15 per cent of the millimetres.
indicated retention time(s) ; Temperature : ± 5 °C, where the operating temperature is
– the final composition of the mobile phase is not weaker in specified, unless otherwise prescribed.
elution power than the prescribed composition. Detector wavelength : no adjustment permitted.
Where compliance with the system suitability requirements Injection volume : may be decreased, provided detection and
cannot be achieved, it is often preferable to consider the dwell repeatability of the peak(s) to be determined are satisfactory ;
volume or to change the column. no increase permitted.
Dwell volume. The configuration of the equipment employed Gas chromatography
may significantly alter the resolution, retention time and Column parameters
relative retentions described. Should this occur, it may be due Stationary phase :
to excessive dwell volume. Monographs preferably include an
isocratic step before the start of the gradient programme so – particle size : maximum reduction of 50 per cent ; no
that an adaptation can be made to the gradient time points increase permitted (packed columns) ;
to take account of differences in dwell volume between the – film thickness : − 50 per cent to + 100 per cent (capillary
system used for method development and that actually used. columns).
It is the user’s responsibility to adapt the length of the isocratic Column dimensions :
step to the analytical equipment used. If the dwell volume
used during the elaboration of the monograph is given in the – length : ± 70 per cent ;
monograph, the time points (t min) stated in the gradient table – internal diameter : ± 50 per cent.
may be replaced by adapted time points (tc min), calculated Flow rate : ± 50 per cent.
using the following equation :
Temperature : ± 10 per cent.
Injection volume and split volume : may be adjusted, provided
detection and repeatability are satisfactory.
Supercritical fluid chromatography
D = dwell volume, in millilitres ;
Composition of the mobile phase : for packed columns, the
D0 = dwell volume used for development of the method, amount of the minor solvent component may be adjusted by
in millilitres ; ± 30 per cent relative or ± 2 per cent absolute, whichever is
F = flow rate, in millilitres per minute. the larger ; no adjustment is permitted for a capillary column
system.
The isocratic step introduced for this purpose may be omitted Detector wavelength : no adjustment permitted.
if validation data for application of the method without this
Column parameters
step is available.
Stationary phase :
pH of the aqueous component of the mobile phase : no
adjustment permitted. – particle size : maximum reduction of 50 per cent ; no
increase permitted (packed columns).
Concentration of salts in the buffer component of a mobile Column dimensions :
phase : no adjustment permitted.
– length : ± 70 per cent ;
Flow rate : adjustment is acceptable when changing the column – internal diameter :
dimensions (see the formula below).
± 25 per cent (packed columns) ;
Column parameters
± 50 per cent (capillary columns).
Stationary phase : Flow rate : ± 50 per cent.
– no change of the identity of the substituent of the Temperature : ± 5 °C, where the operating temperature is
stationary phase permitted (e.g. no replacement of C18 specified.
by C8) ; Injection volume : may be decreased, provided detection and
– particle size : no adjustment permitted. repeatability are satisfactory ; no increase permitted.
Column dimensions : QUANTIFICATION
– length : ± 70 per cent ; Peaks due to solvents and reagents or arising from the
mobile phase or the sample matrix are disregarded during
– internal diameter : ± 25 per cent. quantification.
When column dimensions are changed, the flow rate may – Detector sensitivity. The detector sensitivity is the signal
be adjusted as necessary using the following equation : output per unit concentration or unit mass of a substance
in the mobile phase entering the detector. The relative
detector response factor, commonly referred to as response
factor, expresses the sensitivity of a detector for a given
substance relative to a standard substance. The correction
F1 = flow rate indicated in the monograph, in factor is the reciprocal of the response factor.
millilitres per minute ; – External standard method. The concentration of the
F2 = adjusted flow rate, in millilitres per minute ; component(s) to be analysed is determined by comparing
the response(s) (peak(s)) obtained with the test solution
l1 = length of the column indicated in the to the response(s) (peak(s)) obtained with a reference
monograph, in millimetres ; solution.
– Internal standard method. Equal amounts of a component The migration velocity of an analyte under an electric field
that will be resolved from the substance to be examined of intensity E, is determined by the electrophoretic mobility
(the internal standard) are introduced into the test of the analyte and the electro-osmotic mobility of the buffer
solution and a reference solution. The internal standard inside the capillary. The electrophoretic mobility of a solute
is chosen such that it does not react with the substance (μep) depends on the characteristics of the solute (electric
to be examined, is stable and does not contain impurities charge, molecular size and shape) and those of the buffer in
with the same retention time as that of the substance to which the migration takes place (type and ionic strength of the
be examined. The concentration of the substance to be electrolyte, pH, viscosity and additives). The electrophoretic
examined is determined by comparing the ratio of the peak velocity (νep) of a solute, assuming a spherical shape, is given
areas or peak heights due to the substance to be examined by the equation :
and the internal standard in the test solution with the ratio
of the peak areas or peak heights due to the substance to
be examined and the internal standard in the reference
solution.
q = effective charge of the solute,
– Normalisation procedure. The percentage content of a η = viscosity of the electrolyte solution,
component of the substance to be examined is calculated
by determining the area of the corresponding peak as a r = Stoke’s radius of the solute,
percentage of the total area of all the peaks, excluding those V = applied voltage,
due to solvents or reagents or arising from the mobile
phase or the sample matrix, and those at or below the L = total length of the capillary.
disregard limit.
When an electric field is applied through the capillary filled
– Calibration procedure. The relationship between the with buffer, a flow of solvent is generated inside the capillary,
measured or evaluated signal (y) and the quantity called electro-osmotic flow. The velocity of the electro-osmotic
(concentration, mass, etc.) of substance (x) is determined flow depends on the electro-osmotic mobility (μeo) which in
and the calibration function is calculated. The analytical turn depends on the charge density on the capillary internal
results are calculated from the measured signal or evaluated wall and the buffer characteristics. The electro-osmotic
signal of the analyte by means of the inverse function. velocity (νeo) is given by the equation :
When the related substances test prescribes the total of The velocity of the solute (ν) is given by :
impurities or there is a quantitative determination of an
impurity, it is important to choose an appropriate threshold
setting and appropriate conditions for the integration of the The electrophoretic mobility of the analyte and the
peak areas. In such tests the disregard limit, i.e. the limit at electro-osmotic mobility may act in the same direction or in
or below which a peak is disregarded, is generally 0.05 per opposite directions, depending on the charge of the solute.
cent. Thus, the threshold setting of the data collection system In normal capillary electrophoresis, anions will migrate
corresponds to at least half of the disregard limit. Integration in the opposite direction to the electro-osmotic flow and
of the peak area of any impurity that is not completely their velocities will be smaller than the electro-osmotic
separated from the principal peak is preferably performed by velocity. Cations will migrate in the same direction as the
valley-to-valley extrapolation (tangential skim). electro-osmotic flow and their velocities will be greater than
the electro-osmotic velocity. Under conditions in which
there is a fast electro-osmotic velocity with respect to the
electrophoretic velocity of the solutes, both cations and anions
can be separated in the same run.
The time (t) taken by the solute to migrate the distance (l)
from the injection end of the capillary to the detection point
(capillary effective length) is given by the expression :
01/2010:20247
corrected 7.1
In general, uncoated fused-silica capillaries above pH 3 have
negative charge due to ionised silanol groups in the inner
2.2.47. CAPILLARY wall. Consequently, the electro-osmotic flow is from anode to
ELECTROPHORESIS(5) cathode. The electro-osmotic flow must remain constant from
run to run if good reproducibility is to be obtained in the
migration velocity of the solutes. For some applications, it may
be necessary to reduce or suppress the electro-osmotic flow by
GENERAL PRINCIPLES modifying the inner wall of the capillary or by changing the
concentration, composition and/or pH of the buffer solution.
Capillary electrophoresis is a physical method of analysis After the introduction of the sample into the capillary,
based on the migration, inside a capillary, of charged analytes each analyte ion of the sample migrates within the
dissolved in an electrolyte solution, under the influence of a background electrolyte as an independent zone, according
direct-current electric field. to its electrophoretic mobility. Zone dispersion, that is
(5) This chapter has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.
the spreading of each solute band, results from different and degassed to avoid bubble formation that could interfere
phenomena. Under ideal conditions the sole contribution with the detection system or interrupt the electrical contact
to the solute-zone broadening is molecular diffusion of the in the capillary during the separation run. A rigorous rinsing
solute along the capillary (longitudinal diffusion). In this ideal procedure should be developed for each analytical method to
case the efficiency of the zone, expressed as the number of achieve reproducible migration times of the solutes.
theoretical plates (N), is given by :
CAPILLARY ZONE ELECTROPHORESIS
PRINCIPLE
In capillary zone electrophoresis, analytes are separated in a
capillary containing only buffer without any anticonvective
D = molecular diffusion coefficient of the solute in the medium. With this technique, separation takes place because
buffer. the different components of the sample migrate as discrete
In practice, other phenomena such as heat dissipation, sample bands with different velocities. The velocity of each band
adsorption onto the capillary wall, mismatched conductivity depends on the electrophoretic mobility of the solute and the
between sample and buffer, length of the injection plug, electro-osmotic flow in the capillary (see General Principles).
detector cell size and unlevelled buffer reservoirs can also Coated capillaries can be used to increase the separation
significantly contribute to band dispersion. capacity of those substances adsorbing on fused-silica surfaces.
Separation between 2 bands (expressed as the resolution, Rs) Using this mode of capillary electrophoresis, the
can be obtained by modifying the electrophoretic mobility analysis of both small (Mr < 2000) and large molecules
of the analytes, the electro-osmotic mobility induced in the (2000 < Mr < 100 000) can be accomplished. Due to the
capillary and by increasing the efficiency for the band of each high efficiency achieved in capillary zone electrophoresis,
analyte, according to the equation : separation of molecules having only minute differences in
their charge-to-mass ratio can be effected. This separation
mode also allows the separation of chiral compounds by
addition of chiral selectors to the separation buffer.
OPTIMISATION
and = electrophoretic mobilities of the Optimisation of the separation is a complex process where
2 analytes separated, several separation parameters can play a major role. The main
= mean electrophoretic mobility of the factors to be considered in the development of separations are
2 analytes . instrumental and electrolytic solution parameters.
Instrumental parameters
APPARATUS Voltage. A Joule heating plot is useful in optimising the applied
An apparatus for capillary electrophoresis is composed of : voltage and capillary temperature. Separation time is inversely
– a high-voltage, controllable direct-current power supply ; proportional to applied voltage. However, an increase in the
voltage used can cause excessive heat production, giving rise
– 2 buffer reservoirs, held at the same level, containing the to temperature and, as a result thereof, viscosity gradients
prescribed anodic and cathodic solutions ; in the buffer inside the capillary. This effect causes band
– 2 electrode assemblies (the cathode and the anode), broadening and decreases resolution.
immersed in the buffer reservoirs and connected to the Polarity. Electrode polarity can be normal (anode at the inlet
power supply ; and cathode at the outlet) and the electro-osmotic flow will
– a separation capillary (usually made of fused-silica) which, move toward the cathode. If the electrode polarity is reversed,
when used with some specific types of detectors, has an the electro-osmotic flow is away from the outlet and only
optical viewing window aligned with the detector. The charged analytes with electrophoretic mobilities greater than
ends of the capillary are placed in the buffer reservoirs. the electro-osmotic flow will pass to the outlet.
The capillary is filled with the solution prescribed in the Temperature. The main effect of temperature is observed on
monograph ; buffer viscosity and electrical conductivity, and therefore on
– a suitable injection system ; migration velocity. In some cases, an increase in capillary
– a detector able to monitor the amount of substances of temperature can cause a conformational change in proteins,
interest passing through a segment of the separation modifying their migration time and the efficiency of the
capillary at a given time ; it is usually based on absorption separation.
spectrophotometry (UV and visible) or fluorimetry, but Capillary. The dimensions of the capillary (length and
conductimetric, amperometric or mass spectrometric internal diameter) contribute to analysis time, efficiency of
detection can be useful for specific applications ; indirect separations and load capacity. Increasing both effective length
detection is an alternative method used to detect and total length can decrease the electric fields (working
non-UV-absorbing and non-fluorescent compounds ; at constant voltage) which increases migration time. For a
– a thermostatic system able to maintain a constant given buffer and electric field, heat dissipation, and hence
temperature inside the capillary is recommended to obtain sample band-broadening, depend on the internal diameter
a good separation reproducibility ; of the capillary. The latter also affects the detection limit,
depending on the sample volume injected and the detection
– a recorder and a suitable integrator or a computer. system employed.
The definition of the injection process and its automation Since the adsorption of the sample components on the capillary
are critical for precise quantitative analysis. Modes of wall limits efficiency, methods to avoid these interactions
injection include gravity, pressure or vacuum injection should be considered in the development of a separation
and electrokinetic injection. The amount of each sample method. In the specific case of proteins, several strategies
component introduced electrokinetically depends on its have been devised to avoid adsorption on the capillary wall.
electrophoretic mobility, leading to possible discrimination Some of these strategies (use of extreme pH and adsorption of
using this injection mode. positively charged buffer additives) only require modification
Use the capillary, the buffer solutions, the preconditioning of the buffer composition to prevent protein adsorption. In
method, the sample solution and the migration conditions other strategies, the internal wall of the capillary is coated
prescribed in the monograph of the considered substance. The with a polymer, covalently bonded to the silica, that prevents
employed electrolytic solution is filtered to remove particles interaction between the proteins and the negatively charged
silica surface. For this purpose, ready-to-use capillaries dodecyl sulfate and the samples are denatured by heating in
with coatings consisting of neutral-hydrophilic, cationic and a mixture of sodium dodecyl sulfate and 2-mercaptoethanol
anionic polymers are available. or dithiothreitol before injection. When non-reducing
Electrolytic solution parameters conditions are used (for example, analysis of an intact
antibody), 2-mercaptoethanol and dithiothreitol are not used.
Buffer type and concentration. Suitable buffers for capillary Separation in cross-linked gels can be optimised by modifying
electrophoresis have an appropriate buffer capacity in the the separation buffer (as indicated in the capillary zone
pH range of choice and low mobility to minimise current electrophoresis section) and controlling the gel porosity during
generation. the gel preparation. For cross-linked polyacrylamide gels, the
Matching buffer-ion mobility to solute mobility, whenever porosity can be modified by changing the concentration of
possible, is important for minimising band distortion. The acrylamide and/or the proportion of cross-linker. As a rule,
type of sample solvent used is also important to achieve a decrease in the porosity of the gel leads to a decrease in the
on-column sample focusing, which increases separation mobility of the solutes. Due to the rigidity of these gels, only
efficiency and improves detection. electrokinetic injection can be used.
An increase in buffer concentration (for a given pH) decreases Dynamically coated gels are hydrophilic polymers, such as
electro-osmotic flow and solute velocity. linear polyacrylamide, cellulose derivatives, dextran, etc.,
Buffer pH. The pH of the buffer can affect separation by which can be dissolved in aqueous separation buffers giving
modifying the charge of the analyte or additives, and by rise to a separation medium that also acts as a molecular sieve.
changing the electro-osmotic flow. In protein and peptide These separation media are easier to prepare than cross-linked
separation, changing the pH of the buffer from above to polymers. They can be prepared in a vial and filled by pressure
below the isoelectric point (pI) changes the net charge of the in a wall-coated capillary (with no electro-osmotic flow).
solute from negative to positive. An increase in the buffer pH Replacing the gel before every injection generally improves
generally increases the electro-osmotic flow. the separation reproducibility. The porosity of the gels can be
increased by using polymers of higher molecular mass (at a
Organic solvents. Organic modifiers (methanol, acetonitrile, given polymer concentration) or by decreasing the polymer
etc.) may be added to the aqueous buffer to increase the concentration (for a given polymer molecular mass). A
solubility of the solute or other additives and/or to affect the reduction in the gel porosity leads to a decrease in the mobility
degree of ionisation of the sample components. The addition of the solute for the same buffer. Since the dissolution of these
of these organic modifiers to the buffer generally causes a polymers in the buffer gives low viscosity solutions, both
decrease in the electro-osmotic flow. hydrodynamic and electrokinetic injection techniques can be
Additives for chiral separations. For the separation of optical used.
isomers, a chiral selector is added to the separation buffer.
The most commonly used chiral selectors are cyclodextrins, CAPILLARY ISOELECTRIC FOCUSING
but crown ethers, polysaccharides and proteins may also be PRINCIPLE
used. Since chiral recognition is governed by the different
interactions between the chiral selector and each of the In isoelectric focusing, the molecules migrate under the
enantiomers, the resolution achieved for the chiral compounds influence of the electric field, so long as they are charged, in
depends largely on the type of chiral selector used. In this a pH gradient generated by ampholytes having pI values in
regard, for the development of a given separation it may be a wide range (poly-aminocarboxylic acids), dissolved in the
useful to test cyclodextrins having a different cavity size (α-,separation buffer.
β-, or γ-cyclodextrin) or modified cyclodextrins with neutral The three basic steps of isoelectric focusing are loading,
(methyl, ethyl, hydroxyalkyl, etc.) or ionisable (aminomethyl, focusing and mobilisation.
carboxymethyl, sulfobutyl ether, etc.) groups. When using Loading step. Two methods may be employed :
modified cyclodextrins, batch-to-batch variations in the
degree of substitution of the cyclodextrins must be taken – loading in one step : the sample is mixed with ampholytes
into account since it will influence the selectivity. Other and introduced into the capillary either by pressure or
factors controlling the resolution in chiral separations are vacuum ;
concentration of chiral selector, composition and pH of the – sequential loading : a leading buffer, then the ampholytes,
buffer and temperature. The use of organic additives, such as then the sample mixed with ampholytes, again ampholytes
methanol or urea can also modify the resolution achieved. alone and finally the terminating buffer are introduced
into the capillary. The volume of the sample must be small
CAPILLARY GEL ELECTROPHORESIS enough not to modify the pH gradient.
PRINCIPLE Focusing step. When the voltage is applied, ampholytes
In capillary gel electrophoresis, separation takes place inside migrate toward the cathode or the anode, according to their
a capillary filled with a gel that acts as a molecular sieve. net charge, thus creating a pH gradient from anode (lower pH)
Molecules with similar charge-to-mass ratios are separated to cathode (higher pH). During this step the components to be
according to molecular size since smaller molecules move separated migrate until they reach a pH corresponding to their
more freely through the network of the gel and therefore isoelectric point (pI) and the current drops to very low values.
migrate faster than larger molecules. Different biological Mobilisation step. If mobilisation is required for detection,
macromolecules (for example, proteins and DNA fragments), use one of the following methods.
which often have similar charge-to-mass ratios, can thus be
separated according to their molecular mass by capillary gel – in the first method, mobilisation is accomplished during
electrophoresis. the focusing step under the effect of the electro-osmotic
flow ; the electro-osmotic flow must be small enough to
CHARACTERISTICS OF GELS allow the focusing of the components ;
2 types of gels are used in capillary electrophoresis :
permanently coated gels and dynamically coated gels. – in the second method, mobilisation is accomplished by
Permanently coated gels, such as cross-linked polyacrylamide, applying positive pressure after the focusing step ;
are prepared inside the capillary by polymerisation of the – in the third method, mobilisation is achieved after the
monomers. They are usually bonded to the fused-silica wall focusing step by adding salts to the cathode reservoir or
and cannot be removed without destroying the capillary. the anode reservoir (depending on the direction chosen
If the gels are used for protein analysis under reducing for mobilisation) in order to alter the pH in the capillary
conditions, the separation buffer usually contains sodium when the voltage is applied. As the pH is changed, the
proteins and ampholytes are mobilised in the direction of analyte migration velocity will depend only on the partition
the reservoir which contains the added salts and pass the coefficient between the micelle and the aqueous buffer. In the
detector. electropherogram, the peaks corresponding to each uncharged
The separation achieved, expressed as ΔpI, depends on the solute are always between that of the electro-osmotic flow
pH gradient , the number of ampholytes having marker and that of the micelle (the time elapsed between these
different pI values, the molecular diffusion coefficient (D), two peaks is called the separation window). For electrically
the intensity of the electric field (E) and the variation of charged solutes, the migration velocity depends on both the
the electrophoretic mobility of the analyte with the pH partition coefficient of the solute between the micelle and the
: aqueous buffer, and on the electrophoretic mobility of the
solute in the absence of micelle.
Since the mechanism in MEKC of neutral and weakly ionised
solutes is essentially chromatographic, migration of the solute
and resolution can be rationalised in terms of the retention
OPTIMISATION factor of the solute (k′), also referred to as mass distribution
The main parameters to be considered in the development ratio (Dm), which is the ratio of the number of moles of solute
of separations are : in the micelle to those in the mobile phase. For a neutral
Voltage. Capillary isoelectric focusing utilises very high compound, k′ is given by :
electric fields, 300 V/cm to 1000 V/cm in the focusing step.
Capillary. The electro-osmotic flow must be reduced or
suppressed depending on the mobilisation strategy (see
above). Coated capillaries tend to reduce the electro-osmotic
flow. tR = migration time of the solute,
Solutions. The anode buffer reservoir is filled with a solution t0 = analysis time of an unretained solute (determined
with a pH lower than the pI of the most acidic ampholyte by injecting an electro-osmotic flow marker which
and the cathode reservoir is filled with a solution with a pH does not enter the micelle, for instance methanol),
higher than the pI of the most basic ampholyte. Phosphoric tmc = micelle migration time (measured by injecting a
acid for the anode and sodium hydroxide for the cathode are micelle marker, such as Sudan III, which migrates
frequently used. while continuously associated in the micelle),
Addition of a polymer, such as methylcellulose, in the K = partition coefficient of the solute,
ampholyte solution tends to suppress convective forces (if
any) and electro-osmotic flow by increasing the viscosity. VS = volume of the micellar phase,
Commercial ampholytes are available covering many pH VM = volume of the mobile phase.
ranges and may be mixed if necessary to obtain an expanded
pH range. Broad pH ranges are used to estimate the isoelectric Likewise, the resolution between 2 closely-migrating solutes
point whereas narrower ranges are employed to improve (Rs) is given by :
accuracy. Calibration can be done by correlating migration
time with isoelectric point for a series of protein markers.
During the focusing step precipitation of proteins at their
isoelectric point can be prevented, if necessary, using buffer
additives such as glycerol, surfactants, urea or zwitterionic
buffers. However, depending on the concentration, urea
denatures proteins. N = number of theoretical plates for one of
the solutes,
MICELLAR ELECTROKINETIC CHROMATOGRAPHY α = selectivity,
(MEKC)
k′a and k′b = retention factors for both solutes,
PRINCIPLE respectively (k′b > k′a).
In micellar electrokinetic chromatography, separation takes
place in an electrolyte solution which contains a surfactant Similar, but not identical, equations give k′ and Rs values for
at a concentration above the critical micellar concentration electrically charged solutes.
(cmc). The solute molecules are distributed between the OPTIMISATION
aqueous buffer and the pseudo-stationary phase composed of The main parameters to be considered in the development
micelles, according to the partition coefficient of the solute. of separations by MEKC are instrumental and electrolytic
The technique can therefore be considered as a hybrid of solution parameters.
electrophoresis and chromatography. It is a technique that
can be used for the separation of both neutral and charged Instrumental parameters
solutes, maintaining the efficiency, speed and instrumental Voltage. Separation time is inversely proportional to applied
suitability of capillary electrophoresis. One of the most widely voltage. However, an increase in voltage can cause excessive
used surfactants in MEKC is the anionic surfactant sodium heat production that gives rise to temperature gradients and
dodecyl sulfate, although other surfactants, for example viscosity gradients of the buffer in the cross-section of the
cationic surfactants such as cetyltrimethylammonium salts, capillary. This effect can be significant with high conductivity
are also used. buffers such as those containing micelles. Poor heat dissipation
The separation mechanism is as follows. At neutral and causes band broadening and decreases resolution.
alkaline pH, a strong electro-osmotic flow is generated Temperature. Variations in capillary temperature affect the
and moves the separation buffer ions in the direction of partition coefficient of the solute between the buffer and the
the cathode. If sodium dodecyl sulfate is employed as the micelles, the critical micellar concentration and the viscosity
surfactant, the electrophoretic migration of the anionic micelle of the buffer. These parameters contribute to the migration
is in the opposite direction, towards the anode. As a result, the time of the solutes. The use of a good cooling system improves
overall micelle migration velocity is slowed down compared to the reproducibility of the migration time for the solutes.
the bulk flow of the electrolytic solution. In the case of neutral Capillary. As in capillary zone electrophoresis, the dimensions
solutes, since the analyte can partition between the micelle and of the capillary (length and internal diameter) contribute to
the aqueous buffer, and has no electrophoretic mobility, the analysis time and efficiency of separations. Increasing both
effective length and total length can decrease the electric CALCULATIONS
fields (working at constant voltage), increase migration time From the values obtained, calculate the content of the
and improve the separation efficiency. The internal diameter component or components being examined. When prescribed,
controls heat dissipation (for a given buffer and electric field) the percentage content of one or more components of the
and consequently the sample band broadening. sample to be examined is calculated by determining the
Electrolytic solution parameters corrected area(s) of the peak(s) as a percentage of the total
of the corrected areas of all peaks, excluding those due to
Surfactant type and concentration. The type of surfactant, solvents or any added reagents (normalisation procedure).
in the same way as the stationary phase in chromatography, The use of an automatic integration system (integrator or data
affects the resolution since it modifies separation selectivity. acquisition and processing system) is recommended.
Also, the log k′ of a neutral compound increases linearly with
the concentration of surfactant in the mobile phase. Since SYSTEM SUITABILITY
resolution in MEKC reaches a maximum when k′ approaches In order to check the behaviour of the capillary electrophoresis
the value of , modifying the concentration of system, system suitability parameters are used. The choice
surfactant in the mobile phase changes the resolution of these parameters depends on the mode of capillary
obtained. electrophoresis used. They are : retention factor (k′) (only for
Buffer pH. Although pH does not modify the partition micellar electrokinetic chromatography), apparent number of
coefficient of non-ionised solutes, it can modify the theoretical plates (N), symmetry factor (As) and resolution
electro-osmotic flow in uncoated capillaries. A decrease in the (Rs). In previous sections, the theoretical expressions for N and
buffer pH decreases the electro-osmotic flow and therefore Rs have been described, but more practical equations that allow
increases the resolution of the neutral solutes in MEKC, these parameters to be calculated from the electropherograms
resulting in a longer analysis time. are given below.
Organic solvents. To improve MEKC separation of APPARENT NUMBER OF THEORETICAL PLATES
hydrophobic compounds, organic modifiers (methanol, The apparent number of theoretical plates (N) may be
propanol, acetonitrile, etc.) can be added to the electrolytic calculated using the expression :
solution. The addition of these modifiers usually decreases
migration time and the selectivity of the separation. Since
the addition of organic modifiers affects the critical micellar
concentration, a given surfactant concentration can be used
t = migration time or distance along the baseline from
only within a certain percentage of organic modifier before the R
micellisation is inhibited or adversely affected, resulting in the the point of injection to the perpendicular dropped
absence of micelles and, therefore, in the absence of partition. from the maximum of the peak corresponding to
The dissociation of micelles in the presence of a high content the component,
of organic solvent does not always mean that the separation wh = width of the peak at half-height.
will no longer be possible ; in some cases the hydrophobic
interaction between the ionic surfactant monomer and the RESOLUTION
neutral solutes forms solvophobic complexes that can be The resolution (Rs) between peaks of similar height of
separated electrophoretically. 2 components may be calculated using the expression :
Additives for chiral separations. For the separation of
enantiomers using MEKC, a chiral selector is included in the
micellar system, either covalently bound to the surfactant or
added to the micellar separation electrolyte. Micelles that
have a moiety with chiral discrimination properties include
salts of N-dodecanoyl-L-amino acids, bile salts, etc. Chiral tR1 and tR2 = migration times or distances along the
resolution can also be achieved using chiral discriminators, baseline from the point of injection to
such as cyclodextrins, added to the electrolytic solutions the perpendiculars dropped from the
which contain micellised achiral surfactants. maxima of two adjacent peaks,
w h1 and wh2 = peak widths at half-height.
Other additives. Several strategies can be carried out to modify
selectivity, by adding chemicals to the buffer. The addition of When appropriate, the resolution may be calculated by
several types of cyclodextrins to the buffer can also be used to measuring the height of the valley (Hv) between 2 partly
reduce the interaction of hydrophobic solutes with the micelle, resolved peaks in a standard preparation and the height of the
thus increasing the selectivity for this type of compound. smaller peak (Hp) and calculating the peak-to-valley ratio :
The addition of substances able to modify solute-micelle
interactions by adsorption on the latter, is used to improve the
selectivity of the separations in MEKC. These additives may
be a second surfactant (ionic or non-ionic) which gives rise SYMMETRY FACTOR
to mixed micelles or metallic cations which dissolve in the The symmetry factor (As) of a peak may be calculated using
micelle and form co-ordination complexes with the solutes. the expression :
QUANTIFICATION
Peak areas must be divided by the corresponding migration
w0.05 = width of the peak at one-twentieth of the peak
time to give the corrected area in order to :
height,
– compensate for the shift in migration time from run to run, d = distance between the perpendicular dropped from
thus reducing the variation of the response, the peak maximum and the leading edge of the
– compensate for the different responses of sample peak at one-twentieth of the peak height.
constituents with different migration times. Tests for area repeatability (standard deviation of areas
Where an internal standard is used, verify that no peak of the or of the area/migration-time ratio) and for migration
substance to be examined is masked by that of the internal time repeatability (standard deviation of migration time)
standard. are introduced as suitability parameters. Migration time
naphthalene) for which accurate wavenumber shifts have Method. Prepare and examine the sample in the same
been established (see Table 2.2.48.-1). The indene sample manner as for the establishment of the database. A suitable
can favourably be placed in an NMR tube, evacuated and mathematical transformation of the Raman spectrum may be
sealed under inert gas, and stored cool in the dark to avoid calculated to facilitate spectrum comparison or quantitative
degradation of the sample. prediction.
Table 2.2.48.-1. – Wavenumber shifts (and acceptable Comparison of the spectra or transforms of the spectra
tolerances) of cyclohexane, indene and naphthalene. or quantitative prediction of properties or amounts in
the material in question may involve the use of a suitable
cyclohexane A indene B
naphthalene A chemometric or statistical classification or calibration
3056.4 (± 1.5) technique.
2938.3 (± 1.5)
01/2008:20249
2923.8 (± 1.5)
2852.9 (± 1.5) 2.2.49. FALLING BALL VISCOMETER
1609.7 (± 1.0) 1576.6 (± 1.0)
METHOD
1444.4 (± 1.0) 1552.6 (± 1.0) The determination of dynamic viscosity of Newtonian liquids
1464.5 (± 1.0)
using a suitable falling ball viscometer is performed at
1266.4 (± 1.0) 1205.2 (± 1.0) 1382.2 (± 1.0) 20 ± 0.1 °C, unless otherwise prescribed in the monograph.
1157.6 (± 1.0) 1147.2 (± 1.0)
The time required for a test ball to fall in the liquid to be
examined from one ring mark to the other is determined. If
1028.3 (± 1.0) 1018.6 (± 1.0) 1021.6 (± 1.0) no stricter limit is defined for the equipment used the result
801.3 (± 1.0) 730.5 (± 1.0) 763.8 (± 1.0)
is valid only if 2 consecutive measures do not differ by more
than 1.5 per cent.
533.9 (± 1.0) 513.8 (± 1.0) Apparatus. The falling ball viscometer consists of : a glass
A
Standard guide for Raman shift standards for spectrometer calibration tube enclosed in a mantle, which allow precise control of
(American Society for Testing and Materials ASTM E 1840). temperature ; six balls made of glass, nickel-iron or steel with
B
D. A. Carter, W. R. Thompson, C. E. Taylor and J. E. Pemberton, different densities and diameters. The tube is fixed in such
Applied Spectroscopy, 1995, 49 (11), 1561-1576. a way that the axis is inclined by 10 ± 1° with regard to the
Verification of the response-intensity scale. The absolute and vertical. The tube has 2 ring marks which define the distance
relative intensities of the Raman bands are affected by several the ball has to roll. Commercially available apparatus is
factors including : supplied with tables giving the constants, the density of the
balls and the suitability of the different balls for the expected
– the state of polarisation of the irradiating light, range of viscosity.
– the state of polarisation of the collection optics, Method. Fill the clean, dry tube of the viscometer, previously
– the intensity of the irradiating light, brought to 20 ± 0.1 °C, with the liquid to be examined,
– differences in instrument response, avoiding bubbles. Add the ball suitable for the range of
viscosity of the liquid so as to obtain a falling time not less than
– differences in focus and geometry at sample, 30 s. Close the tube and maintain the solution at 20 ± 0.1 °C
– differences in packing density for solid samples. for at least 15 min. Let the ball run through the liquid between
Appropriate acceptance criteria will vary with the application the 2 ring marks once without measurement. Let it run again
but a day-to-day variation of ± 10 per cent in relative band and measure with a stop-watch, to the nearest one-fifth of a
intensities is achievable in most cases. second, the time required for the ball to roll from the upper to
the lower ring mark. Repeat the test run at least 3 times.
Establishment of a spectral reference library. Record the spectra
of a suitable number of materials which have been fully tested Calculate the dynamic viscosity η in millipascal seconds using
(e.g. as prescribed in a monograph) and which exhibit the the formula :
variation (manufacturer, batch, crystal modification, particle
size, etc.) typical of the material to be analysed. The set of
spectra represents the information that defines the similarity
border or quantitative limits, which may be used, e.g. to k = constant, expressed in millimeter squared per
identify the substance or control the amount formed in a second squared,
manufacturing process. The number of substances in the 1 = density of the ball used, expressed in grams per
database depends on the specific application. The collection of cubic centimetre,
spectra in the database may be represented in different ways 2 = density of the liquid to be examined, expressed
defined by the mathematical technique used for classification in grams per cubic centimetre, obtained by
or quantitation. multiplying its relative density by 0.9982,
The selectivity of the database which makes it possible t
to identify positively a given material and distinguish it = falling time of the ball, in seconds.
adequately from other materials in the database is to be
established during the validation procedure. This selectivity 01/2010:20254
must be challenged on a regular basis to ensure ongoing
validity of the database ; this is especially necessary after any
major change in a substance (e.g. change in supplier or in the
2.2.54. ISOELECTRIC FOCUSING(6)
manufacturing process of the material) or in the set-up of the GENERAL PRINCIPLES
Raman instrument (e.g. verification of the wavenumber and Isoelectric focusing (IEF) is a method of electrophoresis
response repeatability of the spectrometer). that separates proteins according to their isoelectric point.
This database is then valid for use only with the originating Separation is carried out in a slab of polyacrylamide or
instrument, or with a similar instrument, provided the agarose gel that contains a mixture of amphoteric electrolytes
transferred database has been demonstrated to remain valid. (ampholytes). When subjected to an electric field, the
(6) This chapter has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.
ampholytes migrate in the gel to create a pH gradient. In some when the density is measured using a densitometer or similar
cases gels containing an immobilised pH gradient, prepared instrumentation to determine the relative concentration of
by incorporating weak acids and bases to specific regions of protein in the bands subject to validation.
the gel network during the preparation of the gel, are used.
When the applied proteins reach the gel fraction that has a pH APPARATUS
that is the same as their isoelectric point (pI), their charge is An apparatus for IEF consists of :
neutralised and migration ceases. Gradients can be made over – a controllable generator for constant potential, current
various ranges of pH, according to the mixture of ampholytes and power ; potentials of 2500 V have been used and
chosen. are considered optimal under a given set of operating
conditions ; a supply of up to 30 W of constant power is
THEORETICAL ASPECTS recommended ;
When a protein is at the position of its isoelectric point, it – a rigid plastic IEF chamber that contains a cooled plate, of
has no net charge and cannot be moved in a gel matrix by suitable material, to support the gel ;
the electric field. It may, however, move from that position – a plastic cover with platinum electrodes that are connected
by diffusion. The pH gradient forces a protein to remain to the gel by means of paper wicks of suitable width, length
in its isoelectric point position, thus concentrating it ; this and thickness, impregnated with solutions of anodic and
concentrating effect is called "focusing". Increasing the cathodic electrolytes.
applied voltage or reducing the sample load result in improved
separation of bands. The applied voltage is limited by the ISOELECTRIC FOCUSING IN POLYACRYLAMIDE GELS :
heat generated, which must be dissipated. The use of thin gels DETAILED PROCEDURE
and an efficient cooling plate controlled by a thermostatic The following method is a detailed description of an IEF
circulator prevents the burning of the gel whilst allowing sharp procedure in thick polyacrylamide slab gels, which is used unless
focusing. The separation is estimated by determining the otherwise stated in the monograph.
minimum pI difference (ΔpI), which is necessary to separate
2 neighbouring bands : PREPARATION OF THE GELS
Mould. The mould (see Figure 2.2.54.-1) is composed of
a glass plate (A) on which a polyester film (B) is placed to
facilitate handling of the gel, one or more spacers (C), a second
glass plate (D) and clamps to hold the structure together.
D = diffusion coefficient of the protein,
= pH gradient,
length of the gel and impregnate them with the electrolyte – the use of gel additives such as non-ionic detergents (e.g.
solutions : acid for the anode and alkaline for the cathode. The octylglucoside) or zwitterionic detergents (e.g., CHAPS or
compositions of the anode and cathode solutions are given CHAPSO), and the addition of ampholyte to the sample, to
in the monograph. Apply these paper wicks to each side of prevent proteins from aggregating or precipitating.
the gel several millimetres from the edge. Fit the cover so
that the electrodes are in contact with the wicks (respecting POINTS TO CONSIDER
the anodic and cathodic poles). Proceed with the isoelectric Samples can be applied to any area on the gel, but to protect
focusing by applying the electrical parameters described in the proteins from extreme pH environments samples should
the monograph. Switch off the current when the migration of not be applied close to either electrode. During method
the mixture of standard proteins has stabilised. Using forceps, development the analyst can try applying the protein in
remove the sample application strips and the 2 electrode 3 positions on the gel (i.e. middle and both ends) ; the pattern
wicks. Immerse the gel in fixing solution for isoelectric focusing of a protein applied at opposite ends of the gel may not be
in polyacrylamide gel R. Incubate with gentle shaking at identical.
room temperature for 30 min. Drain off the solution and add A phenomenon known as cathodic drift, where the pH
200 mL of destaining solution R. Incubate with shaking for 1 h. gradient decays over time, may occur if a gel is focused too
Drain the gel, add coomassie staining solution R. Incubate for long. Although not well understood, electroendoosmosis
30 min. Destain the gel by passive diffusion with destaining and absorption of carbon dioxide may be factors that lead to
solution R until the bands are well visualised against a clear cathodic drift. Cathodic drift is observed as focused protein
background. Locate the position and intensity of the bands in migrating off the cathode end of the gel. Immobilised pH
the electropherogram as prescribed in the monograph. gradients may be used to address this problem.
VARIATIONS TO THE DETAILED PROCEDURE (SUBJECT Efficient cooling (approximately 4 °C) of the bed that the gel
TO VALIDATION) lies on during focusing is important. High field strengths used
during isoelectric focusing can lead to overheating and affect
Where reference to the general method on isoelectric focusing the quality of the focused gel.
is made, variations in methodology or procedure may be
made subject to validation. These include :
01/2010:20255
– the use of commercially available pre-cast gels and of
commercial staining and destaining kits,
– the use of immobilised pH gradients,
2.2.55. PEPTIDE MAPPING(7)
– the use of rod gels, Peptide mapping is an identity test for proteins, especially
– the use of gel cassettes of different dimensions, including those obtained by rDNA technology. It involves the chemical
ultra-thin (0.2 mm) gels, or enzymatic treatment of a protein resulting in the formation
of peptide fragments followed by separation and identification
– variations in the sample application procedure, including of these fragments in a reproducible manner. It is a powerful
different sample volumes or the use of sample application test that is capable of identifying almost any single amino acid
masks or wicks other than paper, changes resulting from events such as errors in the reading of
– the use of alternate running conditions, including variations complementary DNA (cDNA) sequences or point mutations.
in the electric field depending on gel dimensions and Peptide mapping is a comparative procedure because the
equipment, and the use of fixed migration times rather information obtained, compared to a reference substance
than subjective interpretation of band stability, similarly treated, confirms the primary structure of the
– the inclusion of a pre-focusing step, protein, is capable of detecting whether alterations in structure
– the use of automated instrumentation, have occurred, and demonstrates process consistency and
– the use of agarose gels. genetic stability. Each protein presents unique characteristics
which must be well understood so that the scientific and
VALIDATION OF ISO-ELECTRIC FOCUSING analytical approaches permit validated development of a
PROCEDURES peptide map that provides sufficient specificity.
Where alternative methods to the detailed procedure are This chapter provides detailed assistance in the application of
employed they must be validated. The following criteria may peptide mapping and its validation to characterise the desired
be used to validate the separation : protein, to evaluate the stability of the expression construct of
cells used for recombinant DNA products and to evaluate the
– formation of a stable pH gradient of desired characteristics,
consistency of the overall process, to assess product stability
assessed for example using coloured pH markers of known
as well as to ensure the identity of the protein, or to detect the
isoelectric points,
presence of protein variant.
– comparison with the electropherogram provided with the
Peptide mapping is not a general method, but involves
chemical reference substance for the preparation to be
developing specific maps for each unique protein. Although
examined,
the technology is evolving rapidly, there are certain methods
– any other validation criteria as prescribed in the that are generally accepted. Variations of these methods will
monograph. be indicated, when appropriate, in specific monographs.
SPECIFIED VARIATIONS TO THE GENERAL METHOD A peptide map may be viewed as a fingerprint of a protein
and is the end product of several chemical processes that
Variations to the general method required for the analysis of provide a comprehensive understanding of the protein being
specific substances may be specified in detail in monographs. analysed. 4 principal steps are necessary for the development
These include : of the procedure : isolation and purification of the protein, if
– the addition of urea in the gel (3 M concentration is often the protein is part of a formulation ; selective cleavage of the
satisfactory to keep protein in solution but up to 8 M can be peptide bonds ; chromatographic separation of the peptides ;
used) : some proteins precipitate at their isoelectric point ; and analysis and identification of the peptides. A test sample
in this case, urea is included in the gel formulation to keep is digested and assayed in parallel with a reference substance.
the protein in solution ; if urea is used, only fresh solutions Complete cleavage of peptide bonds is more likely to occur
should be used to prevent carbamylation of the protein ; when enzymes such as endoproteases (e.g., trypsin) are used,
– the use of alternative staining methods ; instead of chemical cleavage reagents. A map must contain
(7) This chapter has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.
enough peptides to be meaningful. On the other hand, if there To allow the enzyme to have full access to cleavage sites and
are too many fragments, the map might lose its specificity permit some unfolding of the protein, it is often necessary to
because many proteins will then have the same profiles. reduce and alkylate the disulfide bonds prior to digestion.
Digestion with trypsin can introduce ambiguities in the
peptide map due to side reactions occurring during the
ISOLATION AND PURIFICATION digestion reaction, such as non-specific cleavage, deamidation,
disulfide isomerisation, oxidation of methionine residues,
Isolation and purification are necessary for analysis of bulk or formation of pyroglutamic groups created from the
drugs or dosage forms containing interfering excipients and deamidation of glutamine at the N-terminal side of a peptide.
carrier proteins and, when required, will be specified in the Furthermore, peaks may be produced by autohydrolysis of
monograph. Quantitative recovery of protein from the dosage trypsin. Their intensities depend on the ratio of trypsin to
form must be validated. protein. To avoid autohydrolysis, solutions of proteases may be
prepared at a pH that is not optimal (e.g. at pH 5 for trypsin),
which would mean that the enzyme would not become active
SELECTIVE CLEAVAGE OF PEPTIDE BONDS until diluted with the digest buffer.
The selection of the approach used for the cleavage of peptide Establishment of optimal digestion conditions. Factors
bonds will depend on the protein under test. This selection that affect the completeness and effectiveness of digestion of
process involves determination of the type of cleavage to be proteins are those that could affect any chemical or enzymatic
employed, enzymatic or chemical, and the type of cleavage reactions.
agent within the chosen category. Several cleavage agents and pH of the reaction milieu. The pH of the digestion mixture
their specificity are shown in Table 2.2.55.-1. This list is not is empirically determined to ensure the optimisation of the
all-inclusive and will be expanded as other cleavage agents performance of the given cleavage agent. For example, when
are identified. using cyanogen bromide as a cleavage agent, a highly acidic
environment (e.g. pH 2, formic acid) is necessary ; however,
Pretreatment of sample. Depending on the size or the when using trypsin as a cleavage agent, a slightly alkaline
configuration of the protein, different approaches in the environment (pH 8) is optimal. As a general rule, the pH of
pretreatment of samples can be used. If trypsin is used as the reaction milieu must not alter the chemical integrity of the
a cleavage agent for proteins with a molecular mass greater protein during the digestion and must not change during the
than 100 000 Da, lysine residues must be protected by course of the fragmentation reaction.
citraconylation or maleylation ; otherwise, too many peptides
will be generated. Temperature. A temperature between 25 °C and 37 °C is
adequate for most digestions. The temperature used is
Pretreatment of the cleavage agent. Pretreatment of cleavage intended to minimise chemical side reactions. The type
agents, especially enzymatic agents, might be necessary of protein under test will dictate the temperature of the
for purification purposes to ensure reproducibility of the reaction milieu, because some proteins are more susceptible
map. For example, trypsin used as a cleavage agent will to denaturation as the temperature of the reaction increases.
have to be treated with tosyl-L-phenylalanine chloromethyl For example, digestion of recombinant bovine somatropin
ketone to inactivate chymotrypsin. Other methods, such is conducted at 4 °C, because at higher temperatures it will
as purification of trypsin by high performance liquid precipitate during digestion.
chromatography (HPLC) or immobilisation of enzyme on a Time. If sufficient sample is available, a time course study is
gel support, have been successfully used when only a small considered in order to determine the optimum time to obtain
amount of protein is available. a reproducible map and avoid incomplete digestion. Time of
Pretreatment of the protein. Under certain conditions, it digestion varies from 2 h to 30 h. The reaction is stopped by
might be necessary to concentrate the sample or to separate the addition of an acid which does not interfere in the map
the protein from excipients and stabilisers used in formulation or by freezing.
of the product, if these interfere with the mapping procedure. Amount of cleavage agent used. Although excessive amounts
Physical procedures used for pretreatment can include of cleavage agent are used to accomplish a reasonably rapid
ultrafiltration, column chromatography and lyophilization. digestion time (i.e. 6-20 hours), the amount of cleavage agent
Other pretreatments, such as the addition of chaotropic agents is minimised to avoid its contribution to the chromatographic
(e.g. urea) can be used to unfold the protein prior to mapping. map pattern. A protein to protease ratio between 20:1 and
Table 2.2.55.-1. – Examples of cleavage agents
Type Agent Specificity
Enzymatic Trypsin (EC 3.4.21.4) C-terminal side of Arg and Lys
Chymotrypsin (EC 3.4.21.1) C-terminal side of hydrophobic residues (e.g. Leu, Met,
Ala, aromatics)
Pepsin (EC 3.4.23.1 and 2) Non-specific digest
Lysyl endopeptidase (Lys-C endopeptidase) (EC 3.4.21.50) C-terminal side of Lys
Glutamyl endopeptidase (from S. aureus strain V8) C-terminal side of Glu and Asp
(EC 3.4.21.19)
Peptidyl-Asp metallo-endopeptidase (endoprotei- N-terminal side of Asp
nase Asp-N)
Clostripain (EC 3.4.22.8) C-terminal side of Arg
Chemical Cyanogen bromide C-terminal side of Met
2-Nitro-5-thio-cyanobenzoic acid N-terminal side of Cys
O-Iodosobenzoic acid C-terminal side of Trp and Tyr
Dilute acid Asp and Pro
BNPS-skatole Trp
200:1 is generally used. It is recommended that the cleavage Isocratic elution. Isocratic HPLC systems using a single
agent is added in 2 or more stages to optimise cleavage. mobile phase are used on the basis of their convenience of
Nonetheless, the final reaction volume remains small enough use and improved detector responses. Optimal composition
to facilitate the next step in peptide mapping, the separation of a mobile phase to obtain clear resolution of each peak is
step. To sort out digestion artifacts that might interfere with sometimes difficult to establish. Mobile phases for which
the subsequent analysis, a blank determination is performed, slight changes in component ratios or in pH significantly
using a digestion control with all the reagents, except the test affect retention times of peaks in peptide maps must not be
protein. used in isocratic HPLC systems.
CHROMATOGRAPHIC SEPARATION Other parameters. Temperature control of the column is
usually necessary to achieve good reproducibility. The flow
Many techniques are used to separate peptides for mapping. rates for the mobile phases range from 0.1-2.0 mL/min, and
The selection of a technique depends on the protein being the detection of peptides is performed with a UV detector
mapped. Techniques that have been successfully used for at 200-230 nm. Other methods of detection have been used
separation of peptides are shown in Table 2.2.55-2. In this (e.g. post-column derivatisation), but they are not as robust
section, a most widely used reversed-phase HPLC method or versatile as UV detection.
is described as one of the procedures of chromatographic
separation. Validation. This section provides an experimental means
The purity of solvents and mobile phases is a critical factor in for measuring the overall performance of the test method.
HPLC separation. HPLC-grade solvents and water that are The acceptance criteria for system suitability depend on
commercially available, are recommended for reversed-phase the identification of critical test parameters that affect data
HPLC. Dissolved gases present a problem in gradient systems interpretation and acceptance. These critical parameters
where the solubility of the gas in a solvent may be less in are also criteria that monitor peptide digestion and peptide
a mixture than in a single solvent. Vacuum degassing and analysis. An indicator that the desired digestion endpoint
agitation by sonication are often used as useful degassing has been achieved is shown by comparison with a reference
procedures. When solid particles in the solvents are drawn standard, which is treated in the same manner as the
into the HPLC system, they can damage the sealing of pump test protein. The use of a reference substance in parallel
valves or clog the top of the chromatographic column. Both with the test protein is critical in the development and
pre- and post-pump filtration is also recommended. establishment of system suitability limits. In addition, a
chromatogram is included with the reference substance
Table 2.2.55-2. – Techniques used for the separation of peptides for additional comparison purposes. Other indicators may
Reversed-phase high performance liquid chromatography (HPLC) include visual inspection of protein or peptide solubility, the
absence of intact protein, or measurement of responses of a
Ion-exchange chromatography (IEC)
digestion-dependent peptide. The critical system suitability
Hydrophobic interaction chromatography (HIC) parameters for peptide analysis will depend on the particular
mode of peptide separation and detection and on the data
Polyacrylamide gel electrophoresis (PAGE), non-denaturating
analysis requirements.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
When peptide mapping is used as an identification test, the
Capillary electrophoresis (CE) system suitability requirements for the identified peptides
Paper chromatography-high voltage (PCHV) cover selectivity and precision. In this case, as well as when
identification of variant protein is done, the identification
High voltage-paper electrophoresis (HVPE) of the primary structure of the peptide fragments in the
peptide map provides both a verification of the known
Chromatographic column. The selection of a primary structure and the identification of protein variants by
chromatographic column is empirically determined for each comparison with the peptide map of the reference substance
protein. Columns with 10 nm or 30 nm pore size with silica for the specified protein. The use of a digested reference
support can give optimal separation. For smaller peptides, substance for a given protein in the determination of peptide
octylsilyl silica gel for chromatography R (3-10 μm) and resolution is the method of choice. For an analysis of a variant
octadecylsilyl silica gel for chromatography R (3-10 μm) protein, a characterised mixture of a variant and a reference
column packings are more efficient than butylsilyl silica gel for substance can be used, especially if the variant peptide is
chromatography R (5-10 μm). located in a less-resolved region of the map. The index of
Solvent. The most commonly used solvent is water with pattern consistency can be simply the number of major
acetonitrile as the organic modifier to which not more than peptides detected. Peptide pattern consistency can be best
0.1 per cent trifluoroacetic acid is added. If necessary, add defined by the resolution of peptide peaks. Chromatographic
propyl alcohol or isopropyl alcohol to solubilise the digest parameters, such as peak-to-peak resolution, maximum peak
components, provided that the addition does not unduly width, peak area, peak tailing factors, and column efficiency,
increase the viscosity of the components. may be used to define peptide resolution. Depending on the
Mobile phase. Buffered mobile phases containing phosphate protein under test and the method of separation used, single
are used to provide some flexibility in the selection of pH peptide or multiple peptide resolution requirements may be
conditions, since shifts of pH in the 3.0-5.0 range enhance the necessary.
separation of peptides containing acidic residues (e.g. glutamic The replicate analysis of the digest of the reference substance
and aspartic acids). Sodium or potassium phosphates, for the protein under test yields measures of precision and
ammonium acetate, phosphoric acid at a pH between 2 quantitative recovery. Recovery of the identified peptides is
and 7 (or higher for polymer-based supports) have also been generally ascertained by the use of internal or external peptide
used with acetonitrile gradients. Acetonitrile containing standards. The precision is expressed as the relative standard
trifluoroacetic acid is used quite often. deviation (RSD). Differences in the recovery and precision
Gradient. Gradients can be linear, nonlinear, or include of the identified peptides are to be expected ; therefore, the
step functions. A shallow gradient is recommended in order system suitability limits will have to be established for both the
to separate complex mixtures. Gradients are optimised to recovery and the precision of the identified peptides. These
provide clear resolution of 1 or 2 peaks that will become limits are unique for a given protein and will be specified in
"marker" peaks for the test. the individual monograph.
Visual comparison of the relative retentions, the peak is no loss of resolution due to scale-up. Eluates corresponding
responses (the peak area or the peak height), the number of to specific peptide peaks are collected, vacuum-concentrated,
peaks, and the overall elution pattern is completed initially. It and chromatographed again, if necessary. Amino acid
is then complemented and supported by mathematical analysis analysis of fragments may be limited by the peptide size. If
of the peak response ratios and by the chromatographic profile the N-terminus is blocked, it may need to be cleared before
of a 1:1 (V/V) mixture of sample and reference substance sequencing. C-terminal sequencing of proteins in combination
digest. If all peaks in the sample digest and in the reference with carboxypeptidase and matrix-assisted laser desorption
substance digest have the same relative retentions and peak ionisation coupled to time-of-flight analyser (MALDI-TOF)
response ratios, then the identity of the sample under test is can also be used for characterisation purposes.
confirmed. The use of MS for characterisation of peptide fragments
If peaks that initially eluted with significantly different is by direct infusion of isolated peptides or by the use of
relative retentions are then observed as single peaks in the on-line LC-MS for structure analysis. In general, it includes
1:1 mixture, the initial difference would be an indication of electrospray and MALDI-TOF-MS, as well as fast-atom
system variability. However, if separate peaks are observed in bombardment (FAB). Tandem MS has also been used to
the 1:1 mixture, this would be evidence of the nonequivalence sequence a modified protein and to determine the type of
of the peptides in each peak. If a peak in the 1:1 mixture amino acid modification that has occurred. The comparison
is significantly broader than the corresponding peak in the of mass spectra of the digests before and after reduction
sample and reference substance digest, it may indicate the provides a method to assign the disulfide bonds to the various
presence of different peptides. The use of computer-aided sulfydryl-containing peptides.
pattern recognition software for the analysis of peptide If regions of the primary structure are not clearly demonstrated
mapping data has been proposed and applied, but issues by the peptide map, it might be necessary to develop a
related to the validation of the computer software preclude its secondary peptide map. The goal of a validated method of
use in a compendial test in the near future. Other automated characterisation of a protein through peptide mapping is to
approaches have been used that employ mathematical reconcile and account for at least 95 per cent of the theoretical
formulas, models, and pattern recognition. Such approaches composition of the protein structure.
are, for example, the automated identification of compounds
by IR spectroscopy and the application of diode-array UV
spectral analysis for identification of peptides. These methods 01/2010:20256
have limitations due to inadequate resolutions, co-elution of
fragments, or absolute peak response differences between
reference substance and sample digest fragments.
2.2.56. AMINO ACID ANALYSIS(8)
The numerical comparison of the peak retention times and Amino acid analysis refers to the methodology used to
peak areas or peak heights can be done for a selected group determine the amino acid composition or content of proteins,
of relevant peaks that have been correctly identified in the peptides, and other pharmaceutical preparations. Proteins and
peptide maps. Peak areas can be calculated using 1 peak peptides are macromolecules consisting of covalently bonded
showing relatively small variation as an internal reference, amino acid residues organised as a linear polymer. The
keeping in mind that peak area integration is sensitive to sequence of the amino acids in a protein or peptide determines
baseline variation and likely to introduce error in the analysis. the properties of the molecule. Proteins are considered large
Alternatively, the percentage of each peptide peak height molecules that commonly exist as folded structures with a
relative to the sum of all peak heights can be calculated for specific conformation, while peptides are smaller and may
the sample under test. The percentage is then compared to consist of only a few amino acids. Amino acid analysis can
that of the corresponding peak of the reference substance. be used to quantify proteins and peptides, to determine
The possibility of auto-hydrolysis of trypsin is monitored the identity of proteins or peptides based on their amino
by producing a blank peptide map, that is, the peptide map acid composition, to support protein and peptide structure
obtained when a blank solution is treated with trypsin. analysis, to evaluate fragmentation strategies for peptide
The minimum requirement for the qualification of peptide mapping, and to detect atypical amino acids that might be
mapping is an approved test procedure that includes system present in a protein or peptide. It is necessary to hydrolyse
suitability as a test control. In general, early in the regulatory a protein/peptide to its individual amino acid constituents
process, qualification of peptide mapping for a protein is before amino acid analysis. Following protein/peptide
sufficient. As the regulatory approval process for the protein hydrolysis, the amino acid analysis procedure can be the same
progresses, additional qualifications of the test can include as that practiced for free amino acids in other pharmaceutical
a partial validation of the analytical procedure to provide preparations. The amino acid constituents of the test sample
assurance that the method will perform as intended in the are typically derivatised for analysis.
development of a peptide map for the specified protein. APPARATUS
ANALYSIS AND IDENTIFICATION OF PEPTIDES Methods used for amino acid analysis are usually based on
a chromatographic separation of the amino acids present in
This section gives guidance on the use of peptide mapping the test sample. Current techniques take advantage of the
during development in support of regulatory applications. automated chromatographic instrumentation designed for
The use of a peptide map as a qualitative tool does not require analytical methodologies. An amino acid analysis instrument
the complete characterisation of the individual peptide will typically be a low-pressure or high-pressure liquid
peaks. However, validation of peptide mapping in support chromatograph capable of generating mobile phase gradients
of regulatory applications requires rigorous characterisation that separate the amino acid analytes on a chromatographic
of each of the individual peaks in the peptide map. Methods column. The instrument must have post-column derivatisation
to characterise peaks range from N-terminal sequencing of capability, unless the sample is analysed using precolumn
each peak followed by amino acid analysis to the use of mass derivatisation. The detector is usually an ultraviolet/visible
spectroscopy (MS). or fluorescence detector depending on the derivatisation
For characterisation purposes, when N-terminal sequencing method used. A recording device (e.g., integrator) is used for
and amino acids analysis are used, the analytical separation is transforming the analogue signal from the detector and for
scaled up. Since scale-up might affect the resolution of peptide quantitation. It is preferred that instrumentation be dedicated
peaks, it is necessary, using empirical data, to assure that there particularly for amino acid analysis.
(8) This chapter has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.
be linear with concentration, and it needs to elute with a (e.g., incomplete hydrolysis or destruction of labile amino
unique retention time without overlapping other amino acids. acids). Complete proteolysis, using a mixture of proteases,
Commonly used amino acid standards include norleucine, has been used but can be complicated, requires the proper
nitrotyrosine, and α-aminobutyric acid. controls, and is typically more applicable to peptides than
proteins.
PROTEIN HYDROLYSIS
During initial analyses of an unknown protein, experiments
Hydrolysis of protein and peptide samples is necessary for with various hydrolysis time and temperature conditions are
amino acid analysis of these molecules. The glassware used conducted to determine the optimal conditions.
for hydrolysis must be very clean to avoid erroneous results.
Glove powders and fingerprints on hydrolysis tubes may cause METHOD 1
contamination. To clean glass hydrolysis tubes, boil tubes for Acid hydrolysis using hydrochloric acid containing phenol
1 h in 1 M hydrochloric acid or soak tubes in concentrated is the most common procedure used for protein/peptide
nitric acid or in a mixture of equal volumes of concentrated hydrolysis preceding amino acid analysis. The addition of
hydrochloric acid and nitric acid. Clean hydrolysis tubes phenol to the reaction prevents the halogenation of tyrosine.
are rinsed with high-purity water followed by a rinse with Hydrolysis solution. 6 M hydrochloric acid containing
HPLC grade methanol, dried overnight in an oven, and stored 0.1 per cent to 1.0 per cent of phenol.
covered until use. Alternatively, pyrolysis of clean glassware at
500 °C for 4 h may also be used to eliminate contamination Procedure
from hydrolysis tubes. Adequate disposable laboratory Liquid phase hydrolysis. Place the protein or peptide sample in
material can also be used. a hydrolysis tube, and dry (the sample is dried so that water
Acid hydrolysis is the most common method for hydrolysing a in the sample will not dilute the acid used for the hydrolysis).
protein sample before amino acid analysis. The acid hydrolysis Add 200 μL of hydrolysis solution per 500 μg of lyophilised
technique can contribute to the variation of the analysis due protein. Freeze the sample tube in a dry ice-acetone bath,
to complete or partial destruction of several amino acids : and flame seal in vacuo. Samples are typically hydrolysed at
tryptophan is destroyed ; serine and threonine are partially 110 °C for 24 h in vacuo or in an inert atmosphere to prevent
destroyed ; methionine might undergo oxidation ; and cysteine oxidation. Longer hydrolysis times (e.g., 48 h and 72 h)
is typically recovered as cystine (but cystine recovery is are investigated if there is a concern that the protein is not
usually poor because of partial destruction or reduction to completely hydrolysed.
cysteine). Application of adequate vacuum (less than 200 μm Vapour phase hydrolysis. This is one of the most common acid
of mercury or 26.7 Pa) or introduction of an inert gas (argon) hydrolysis procedures, and it is preferred for microanalysis
in the headspace of the reaction vessel can reduce the level of when only small amounts of the sample are available.
oxidative destruction. In peptide bonds involving isoleucine Contamination of the sample from the acid reagent is also
and valine the amido bonds of Ile-Ile, Val-Val, Ile-Val, and minimised by using vapour phase hydrolysis. Place vials
Val-Ile are partially cleaved ; and asparagine and glutamine containing the dried samples in a vessel that contains an
are deamidated, resulting in aspartic acid and glutamic appropriate amount of hydrolysis solution. The hydrolysis
acid, respectively. The loss of tryptophan, asparagine, and solution does not come in contact with the test sample. Apply
glutamine during an acid hydrolysis limits quantitation to an inert atmosphere or vacuum (less than 200 μm of mercury
17 amino acids. Some of the hydrolysis techniques described or 26.7 Pa) to the headspace of the vessel, and heat to about
are used to address these concerns. Some of the hydrolysis 110 °C for a 24 h hydrolysis time. Acid vapour hydrolyses the
techniques described (i.e., Methods 4-11) may cause dried sample. Any condensation of the acid in the sample
modifications to other amino acids. Therefore, the benefits of vials is to be minimised. After hydrolysis, dry the test sample
using a given hydrolysis technique are weighed against the in vacuo to remove any residual acid.
concerns with the technique and are tested adequately before METHOD 2
employing a method other than acid hydrolysis.
Tryptophan oxidation during hydrolysis is decreased by using
A time-course study (i.e., amino acid analysis at acid mercaptoethanesulfonic acid as the reducing acid.
hydrolysis times of 24 h, 48 h and 72 h) is often employed
to analyse the starting concentration of amino acids that Hydrolysis solution. 2.5 M mercaptoethanesulfonic acid
are partially destroyed or slow to cleave. By plotting the solution.
observed concentration of labile amino acids (e.g., serine and Vapour phase hydrolysis. Dry about 1 μg to 100 μg of the
threonine) versus hydrolysis time, the line can be extrapolated protein/peptide under test in a hydrolysis tube. Place the
to the origin to determine the starting concentration of these hydrolysis tube in a larger tube with about 200 μL of the
amino acids. Time-course hydrolysis studies are also used hydrolysis solution. Seal the larger tube in vacuo (about 50 μm
with amino acids that are slow to cleave (e.g., isoleucine and of mercury or 6.7 Pa) to vaporise the hydrolysis solution.
valine). During the hydrolysis time course, the analyst will Heat the hydrolysis tube to 170-185 °C for about 12.5 min.
observe a plateau in these residues. The level of this plateau After hydrolysis, dry the hydrolysis tube in vacuo for 15 min
is taken as the residue concentration. If the hydrolysis time is to remove the residual acid.
too long, the residue concentration of the sample will begin to METHOD 3
decrease, indicating destruction by the hydrolysis conditions.
Tryptophan oxidation during hydrolysis is prevented by using
An acceptable alternative to the time-course study is to subject thioglycollic acid (TGA) as the reducing acid.
an amino acid calibration standard to the same hydrolysis
conditions as the test sample. The amino acid in free form Hydrolysis solution. 7 M hydrochloric acid containing 1 per
may not completely represent the rate of destruction of labile cent of phenol, 10 per cent of trifluoroacetic acid and 20 per
amino acids within a peptide or protein during the hydrolysis. cent of thioglycollic acid.
This is especially true for peptide bonds that are slow to cleave Vapour phase hydrolysis. Dry about 10 μg to 50 μg of the
(e.g., Ile-Val bonds). However, this technique will allow the protein/peptide under test in a sample tube. Place the sample
analyst to account for some residue destruction. Microwave tube in a larger tube with about 200 μL of the hydrolysis
acid hydrolysis has been used and is rapid but requires solution. Seal the larger tube in vacuo (about 50 μm of
special equipment as well as special precautions. The optimal mercury or 6.7 Pa) to vaporise the TGA. Heat the sample
conditions for microwave hydrolysis must be investigated tube to 166 °C for about 15-30 min. After hydrolysis, dry the
for each individual protein/peptide sample. The microwave sample tube in vacuo for 5 min to remove the residual acid.
hydrolysis technique typically requires only a few minutes, but Recovery of tryptophan by this method may be dependent on
even a deviation of one minute may give inadequate results the amount of sample present.
represented by Glx. Proteins/peptides can be reacted with analogue signal is visualised by means of a data acquisition
bis(1,1-trifluoroacetoxy)iodobenzene (BTI) to convert the system, and the peak areas are integrated for quantification
asparagine and glutamine residues to diaminopropionic acid purposes.
and diaminobutyric acid residues, respectively, upon acid METHOD 1 - POST-COLUMN NINHYDRIN
hydrolysis. These conversions allow the analyst to determine DERIVATISATION
the asparagine and glutamine content of a protein/peptide in
Ion-exchange chromatography with post-column ninhydrin
the presence of aspartic acid and glutamic acid residues.
derivatisation is one of the most common methods employed
Reducing solutions. Prepare and filter 3 solutions : a solution for quantitative amino acid analysis. As a rule, a lithium-based
of 10 mM trifluoroacetic acid (Solution A), a solution of cation-exchange system is employed for the analysis of
5 M guanidine hydrochloride and 10 mM trifluoroacetic the more complex physiological samples, and the faster
acid (Solution B), and a freshly prepared solution of sodium-based cation-exchange system is used for the
dimethylformamide containing 36 mg of BTI per millilitre more simplistic amino acid mixtures obtained with protein
(Solution C). hydrolysates (typically containing 17 amino acid components).
Procedure. In a clean hydrolysis tube, transfer about 200 μg Separation of the amino acids on an ion-exchange column is
of the test sample, and add 2 mL of Solution A or Solution B accomplished through a combination of changes in pH and
and 2 mL of Solution C. Seal the hydrolysis tube in vacuo. cation strength. A temperature gradient is often employed
Heat the sample at 60 °C for 4 h in the dark. The sample to enhance separation.
is then dialysed with water to remove the excess reagents. When the amino acid reacts with ninhydrin, the reactant has
Extract the dialysed sample 3 times with equal volumes of a characteristic purple or yellow colour. Amino acids, except
butyl acetate, and then lyophilise. The protein can then imino acid, give a purple colour, and show an absorption
be acid hydrolysed using previously described procedures. maximum at 570 nm. The imino acids such as proline give a
The α,β-diaminopropionic and α,γ-diaminobutyric acid yellow colour, and show an absorption maximum at 440 nm.
residues do not typically resolve from the lysine residues upon The post-column reaction between ninhydrin and amino acids
ion-exchange chromatography based on amino acid analysis. eluted from the column is monitored at 440 nm and 570 nm,
Therefore, when using ion-exchange as the mode of amino and the chromatogram obtained is used for the determination
acid separation, the asparagine and glutamine contents are the of amino acid composition.
quantitative difference in the aspartic acid and glutamic acid The detection limit is considered to be 10 pmol for most of the
content assayed with underivatised and BTI-derivatised acid amino acid derivatives, but 50 pmol for the proline derivative.
hydrolysis. The threonine, methionine, cysteine, tyrosine, and Response linearity is obtained in the range of 20-500 pmol
histidine assayed content can be altered by BTI derivatisation ; with correlation coefficients exceeding 0.999. To obtain good
a hydrolysis without BTI will have to be performed if the composition data, samples larger than 1 μg before hydrolysis
analyst is interested in the composition of these other amino are best suited for this amino acid analysis of protein/peptide.
acid residues of the protein/peptide.
METHOD 2 - POST-COLUMN OPA DERIVATISATION
o-Phthalaldehyde (OPA) reacts with primary amines in the
METHODOLOGIES OF AMINO ACID ANALYSIS :
presence of thiol compound, to form highly fluorescent
GENERAL PRINCIPLES
isoindole products. This reaction is used for the post-column
Many amino acid analysis techniques exist, and the choice derivatisation in analysis of amino acids by ion-exchange
of any one technique often depends on the sensitivity chromatography. The rule of the separation is the same
required from the assay. In general, about one-half of as Method 1.
the amino acid analysis techniques employed rely on Although OPA does not react with secondary amines (imino
the separation of the free amino acids by ion-exchange acids such as proline) to form fluorescent substances, the
chromatography followed by post-column derivatisation oxidation with sodium hypochlorite or chloramine T allows
(e.g., with ninhydrin or o-phthalaldehyde). Post-column secondary amines to react with OPA. The procedure employs a
derivatisation techniques can be used with samples that strongly acidic cation-exchange column for separation of free
contain small amounts of buffer components, (such as salts amino acids followed by post-column oxidation with sodium
and urea) and generally require between 5 μg and 10 μg hypochlorite or chloramine T and post-column derivatisation
of protein sample per analysis. The remaining amino acid using OPA and a thiol compound such as N-acetyl-L-cysteine
techniques typically involve pre-column derivatisation or 2-mercaptoethanol. The derivatisation of primary amino
of the free amino acids (e.g., phenyl isothiocyanate ; acids is not noticeably affected by the continuous supply of
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate or sodium hypochlorite or chloramine T.
o-phthalaldehyde ; (dimethylamino)azobenzenesulfonyl
chloride ; 9-fluorenylmethyl chloroformate ; and Separation of the amino acids on an ion-exchange column
7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole) followed by is accomplished through a combination of changes in pH
reversed-phase HPLC. Pre-column derivatisation techniques and cation strength. After post-column derivatisation of
are very sensitive and usually require between 0.5 μg and 1.0 μg eluted amino acids with OPA, the reactant passes through
of protein sample per analysis but may be influenced by buffer the fluorometric detector. Fluorescence intensity of
salts in the samples. Pre-column derivatisation techniques OPA-derivatised amino acids are monitored with an excitation
may also result in multiple derivatives of a given amino acid, wavelength of 348 nm and an emission wavelength of 450 nm.
which complicates the result interpretation. Post-column The detection limit is considered to be a few tens of picomole
derivatisation techniques are generally influenced less level for most of the OPA-derivatised amino acids. Response
by performance variation of the assay than pre-column linearity is obtained in the range of a few picomole level to a
derivatisation techniques. few tens of nanomole level. To obtain good compositional
data, samples larger than 500 ng of protein/peptide before
The following methods may be used for quantitative
hydrolysis are recommended.
amino acid analysis. Instruments and reagents for these
procedures are available commercially. Furthermore, many METHOD 3 - PRE-COLUMN PITC DERIVATISATION
modifications of these methodologies exist with different Phenylisothiocyanate (PITC) reacts with amino acids to form
reagent preparations, reaction procedures, chromatographic phenylthiocarbamyl (PTC) derivatives which can be detected
systems, etc. Specific parameters may vary according to the with high sensitivity at 254 nm. Therefore, pre-column
exact equipment and procedure used. Many laboratories will derivatisation of amino acids with PITC followed by a
use more than one amino acid analysis technique to exploit reversed-phase HPLC separation with UV detection is used
the advantages offered by each. In each of these methods, the to analyse the amino acid composition.
After the reagent is removed under vacuum, the derivatised Pre-column derivatisation of amino acids with OPA is
amino acids can be stored dry and frozen for several weeks followed by a reversed-phase HPLC separation. Because
with no significant degradation. If the solution for injection of the instability of the OPA-amino acid derivative, HPLC
is kept cold, no noticeable loss in chromatographic response separation and analysis are performed immediately following
occurs after 3 days. derivatisation. The liquid chromatograph is equipped with a
Separation of the PTC-amino acids on a reversed-phase HPLC fluorometric detector for the detection of derivatised amino
with an octadecylsilyl (ODS) column is accomplished through acids. Fluorescence intensity of OPA-derivatised amino acids
a combination of changes in concentrations of acetonitrile is monitored with an excitation wavelength of 348 nm and an
and buffer ionic strength. PTC-amino acids eluted from the emission wavelength of 450 nm.
column are monitored at 254 nm. Detection limits as low as 50 fmol via fluorescence have been
reported, although the practical limit of analysis remains at
The detection limit is considered to be 1 pmol for most of the
1 pmol.
PTC-amino acids. Response linearity is obtained in the range
of 20-500 pmol with correlation coefficients exceeding 0.999. METHOD 6 - PRE-COLUMN DABS-Cl DERIVATISATION
To obtain good compositional data, samples larger than 500 ng Pre-column derivatisation of amino acids with
of protein/peptide before hydrolysis are recommended. (dimethylamino)azobenzenesulfonyl chloride (DABS-Cl)
METHOD 4 - PRE-COLUMN AQC DERIVITISATION followed by reversed-phase HPLC separation with visible light
detection is used.
Pre-column derivatisation of amino acids with
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate DABS-Cl is a chromophoric reagent employed for the
(AQC) followed by reversed-phase HPLC separation with labelling of amino acids. Amino acids labelled with DABS-Cl
fluorometric detection is used. (DABS-amino acids) are highly stable and show an absorption
maximum at 436 nm.
AQC reacts with amino acids to form stable, fluorescent
unsymmetric urea derivatives (AQC-amino acids) which DABS-amino acids, all naturally occurring amino acid
are readily amenable to analysis by reversed-phase HPLC. derivatives, can be separated on an ODS column of a
Therefore, pre-column derivatisation of amino acids with reversed-phase HPLC by employing gradient systems
AQC followed by reversed-phase HPLC separation with consisting of acetonitrile and aqueous buffer mixture.
fluorimetric detection is used to analyse the amino acid Separated DABS-amino acids eluted from the column are
composition. detected at 436 nm in the visible region.
Separation of the AQC-amino acids on a reversed-phase HPLC This method can analyse the imino acids such as proline
with an ODS column is accomplished through a combination together with the amino acids at the same degree of
of changes in concentrations of acetonitrile and buffer ionic sensitivity, DABS-Cl derivatisation method permits the
strengh. Selective fluorescence detection of the derivatives simultaneous quantification of tryptophan residues by
with an excitation wavelength at 250 nm and an emission previous hydrolysis of the protein/peptide with sulfonic acids
wavelength at 395 nm allows for the direct injection of the such as mercaptoethanesulfonic acid, p-toluenesulfonic acid
reaction mixture with no significant interference from the or methanesulfonic acid described in Method 2 under Protein
only major fluorescent reagent by-product, 6-aminoquinoline. hydrolysis. The other acid-labile residues, asparagine and
Excess reagent is rapidly hydrolysed (t1/2<15 s) to yield glutamine, can also be analysed by previous conversion into
6-aminoquinoline, N-hydroxysuccinimide and carbon dioxide, diaminopropionic acid and diaminobutyric acid, respectively,
and after 1 min no further derivatisation can take place. by treatment of protein/peptide with BTI described in
Method 11 under Protein hydrolysis.
Peak areas for AQC-amino acids are essentially unchanged for
at least 1 week at room temperature. Therefore AQC-amino The non-proteinogenic amino acid norleucine cannot be used
acids have more than sufficient stability to allow for overnight as an internal standard in this method as this compound is
automated chromatographic analysis. eluted in a chromatographic region crowded with peaks of
primary amino acids. Nitrotyrosine can be used as an internal
The detection limit is considered to range from about standard because it is eluted in a clean region.
40 fmol to 320 fmol for each amino acid, except for cystein.
The detection limit for cystein is approximately 800 fmol. The detection limit of DABS-amino acid is about 1 pmol.
Response linearity is obtained in the range of 2.5-200 μM with As little as 2-5 pmol of an individual DABS-amino acid can
correlation coefficients exceeding 0.999. Good compositional be quantitatively analysed with reliability, and only 10-30 ng
data can be obtained from the analysis of derivatised protein of the dabsylated protein hydrolysate is required for each
hydrolysates derived from as little as 30 ng of protein/peptide. analysis.
METHOD 5 - PRE-COLUMN OPA DERIVATISATION METHOD 7 - PRE-COLUMN FMOC-Cl DERIVATISATION
Pre-column derivatisation of amino acids with Pre-column derivatisation of amino acids with
o-phthalaldehyde (OPA) followed by reversed-phase HPLC 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by
separation with fluorometric detection is used. This technique reversed-phase HPLC separation with fluorometric detection
does not detect amino acids that exist as secondary amines is used.
(e.g., proline). FMOC-Cl reacts with both primary and secondary amino
OPA in conjunction with a thiol reagent reacts with primary acids to form highly fluorescent products. The reaction
amine groups to form highly fluorescent isoindole products. proceeds under mild conditions in aqueous solution and is
2-Mercaptoethanol or 3-mercaptopropionic acid can be used completed in 30 s. The derivatives are stable, only the histidine
as the thiol. OPA itself does not fluoresce and consequently derivative showing any breakdown. Although FMOC-Cl
produces no interfering peaks. In addition, its solubility and is fluorescent itself, the reagent excess and fluorescent
stability in aqueous solution, along with the rapid kinetics for side-products can be eliminated without loss of FMOC-amino
the reaction, make it amenable to automated derivatisation acids.
and analysis using an autosampler to mix the sample with the FMOC-amino acids are separated by a reversed-phase HPLC
reagent. However, lack of reactivity with secondary amino using an ODS column. The separation is carried out by
acids has been a predominant drawback. This method does gradient elution varied linearly from a mixture of 10 volumes
not detect amino acids that exist as secondary amines (e.g., of acetonitrile, 40 volumes of methanol and 50 volumes of
proline). To compensate for this drawback, this technique may acetic acid buffer to a mixture of 50 volumes of acetonitrile
be combined with another technique described in Method 7 and 50 volumes of acetic acid buffer and 20 amino acid
or Method 8. derivatives are separated in 20 min. Each derivative eluted
from the column is monitored by a fluorometric detector in which m is the recovered quantity, in nanomoles, of the
set at an excitation wavelength of 260 nm and an emission amino acid under test ; and Mr is the average molecular mass
wavelength of 313 nm. for that amino acid, corrected for the mass of the water
The detection limit is in the low femtomole range. A linearity molecule that was eliminated during peptide bond formation.
range of 0.1-50 μM is obtained for most of the amino acids. The sum of the masses of the recovered amino acids will give
an estimate of the total mass of the protein analysed after
METHOD 8 - PRE-COLUMN NBD-F DERIVATISATION appropriate correction for partially and completely destroyed
Pre-column derivatisation of amino acids with amino acids. If the molecular mass of the unknown protein is
7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) followed by available (i.e., by SDS-PAGE analysis or mass spectroscopy),
reversed-phase HPLC separation with fluorometric detection the amino acid composition of the unknown protein can be
is used. predicted. Calculate the number of residues of each amino
NBD-F reacts with both primary and secondary amino acid using the formula :
acids to form highly fluorescent products. Amino acids are
derivatised with NBD-F by heating to 60 °C for 5 min.
NBD-amino acid derivatives are separated on an ODS
column of a reversed-phase HPLC by employing a gradient
elution system consisting of acetonitrile and aqueous buffer
mixture, and 17 amino acid derivatives are separated in in which m is the recovered quantity, in nanomoles, of the
35 min. ε-Aminocaproic acid can be used as an internal amino acid under test ; M is the total mass, in micrograms, of
standard, because it is eluted in a clean chromatographic the protein ; and Mrt is the molecular mass of the unknown
region. Each derivative eluted from the column is monitored protein.
by a fluorometric detector set at an excitation wavelength of
480 nm and an emission wavelength of 530 nm. Known protein samples. This data analysis technique can be
The sensitivity of this method is almost the same as for used to investigate the amino acid composition and protein
the pre-column OPA derivatisation method (Method 5), concentration of a protein sample of known molecular mass
excluding proline to which OPA is not reactive, and might be and amino acid composition using the amino acid analysis
advantageous for NBD-F against OPA. The detection limit data. When the composition of the protein being analysed
for each amino acid is about 10 fmol. Profile analysis can be is known, one can exploit the fact that some amino acids
achieved with about 1.5 mg of protein hydrolysates in the are recovered well, while other amino acid recoveries may
pre-column reaction mixture. be compromised because of complete or partial destruction
(e.g., tryptophan, cysteine, threonine, serine, methionine),
DATA CALCULATION AND ANALYSIS incomplete bond cleavage (i.e., for isoleucine and valine) and
When determining the amino acid content of a protein/peptide free amino acid contamination (i.e., by glycine and serine).
hydrolysate, it should be noted that the acid hydrolysis step
destroys tryptophan and cysteine. Serine and threonine are Because those amino acids that are recovered best represent
partially destroyed by acid hydrolysis, while isoleucine and the protein, these amino acids are chosen to quantify the
valine residues may be only partially cleaved. Methionine can amount of protein. Well-recovered amino acids are, typically,
undergo oxidation during acid hydrolysis, and some amino aspartate-asparagine, glutamate-glutamine, alanine, leucine,
acids (e.g., glycine and serine) are common contaminants. phenylalanine, lysine, and arginine. This list can be modified
Application of adequate vacuum (less than 200 μm of based on experience with one’s own analysis system. Divide the
mercury or 26.7 Pa) or introduction of inert gas (argon) in quantity, in nanomoles, of each of the well-recovered amino
the headspace of the reaction vessel during vapour phase acids by the expected number of residues for that amino acid
hydrolysis can reduce the level of oxidative destruction. to obtain the protein content based on each well-recovered
Therefore, the quantitative results obtained for cysteine, amino acid. Average the protein content results calculated.
tryptophan, threonine, isoleucine, valine, methionine, glycine, The protein content determined for each of the well-recovered
and serine from a protein/peptide hydrolysate may be variable amino acids should be evenly distributed about the mean.
and may warrant further investigation and consideration. Discard protein content values for those amino acids that
have an unacceptable deviation from the mean. Typically
Amino Acid Mole Percent. This is the number of specific greater than 5 per cent variation from the mean is considered
amino acid residues per 100 residues in a protein. This unacceptable. Recalculate the mean protein content from the
result may be useful for evaluating amino acid analysis data remaining values to obtain the protein content of the sample.
when the molecular mass of the protein under investigation Divide the content of each amino acid by the calculated mean
is unknown. This information can be used to corroborate protein content to determine the amino acid composition of
the identity of a protein/peptide and has other applications. the sample by analysis.
Carefully identify and integrate the peaks obtained as directed
for each procedure. Calculate the mole percent for each amino Calculate the relative compositional error, in percentage, using
acid present in the test sample using the formula : the formula :
are preferable. The availability of hydrofluoric acid-resistant linear and quadratic weighting functions are applied to the
(for example perfluoroalkoxy polymer) sample-introduction data to find the most appropriate weighting function to be
systems and torches also allows the use of hydrofluoric acid. In employed.
selecting a sample-introduction method, the requirements for If the means compared to the calibration curve show a
sensitivity, stability, speed, sample size, corrosion resistance deviation from linearity, two-dimensional linear regression
and resistance to clogging have to be considered. The use of is used.
a cross-flow nebuliser combined with a spray chamber and
torch is suitable for most requirements. The peristaltic pumps ACCURACY
used for ICP-AES usually deliver the standard and sample Verify the accuracy preferably by using a certified reference
solutions at a rate of 1 mL/min or less. material (CRM). Where this is not possible, perform a test
for recovery.
In the case of organic solvents being used, the introduction of
oxygen must be considered to avoid organic layers. Recovery. For assay determinations a recovery of 90 per
cent to 110 per cent is to be obtained. The test is not valid
CHOICE OF OPERATING CONDITIONS if recovery, for example for trace-element determination,
The standard operating conditions prescribed by the is outside of the range 80 per cent to 120 per cent of the
manufacturer are to be followed. Usually, different sets of theoretical value. Recovery may be determined on a suitable
operating conditions are used for aqueous solutions and for reference solution (matrix solution) spiked with a known
organic solvents. Suitable operating parameters are to be quantity of analyte (concentration range that is relevant to
properly chosen : the samples to be determined).
– wavelength selection ; REPEATABILITY
– support-gas flow rates (outer, intermediate and inner tubes The repeatability is not greater than 3 per cent for an assay
of the torch); and not greater than 5 per cent for an impurity test.
– RF power ; LIMIT OF QUANTIFICATION
– viewing position (radial or axial) ; Verify that the limit of quantification (for example, determined
– pump speed ; using the 10 σ approach) is below the value to be measured.
– conditions for the detector (gain/voltage for photomultiplier
tube detectors, others for array detectors) ; 01/2008:20258
– integration time (time set to measure the emission intensity
at each wavelength). 2.2.58. INDUCTIVELY COUPLED
CONTROL OF INSTRUMENT PERFORMANCE
PLASMA-MASS SPECTROMETRY
System suitability
The following tests may be carried out with a multi-element
Inductively coupled plasma-mass spectrometry (ICP-MS) is a
control solution to ensure the adequate performance of the
mass spectrometry method that uses an inductively coupled
ICP-AES system :
plasma (ICP) as the ionisation source. The basic principles of
– energy transfer (generator, torch, plasma) ; measurement of ICP formation are described in chapter 2.2.57 on inductively
the ratio Mg II (280.270 nm)/Mg I (285.213 nm) may be coupled plasma-atomic emission spectrometry (ICP-AES).
used ; ICP-MS utilises the ability of the ICP to generate charged
– sample transfer, by checking nebuliser efficiency and ions from the element species within a sample. These ions
stability ; are then directed into a mass spectrometer, which separates
– resolution (optical system), by measuring peak widths at them according to their mass-to-charge ratio (m/z). Most
half height, for example As (189.042 nm), Mn (257.610 nm), mass spectrometers have a quadrupole system or a magnetic
Cu (324.754 nm) or Ba (455.403 nm) ; sector. Ions are transported from the plasma through 2 cones
(sampler and skimmer cones, forming the interface region)
– analytical performance, by calculating detection limits of
to the ion optics. The ion optics consist of an electrostatic
selected elements over the wavelength range.
lens, which takes ions from an area at atmospheric pressure
VALIDATION OF THE METHOD to the mass filter at a vacuum of 10-8 Pa or less, maintained
with a turbomolecular pump. After their filtration, ions of the
Satisfactory performance of methods prescribed in selected mass/charge ratio are directed to a detector (channel
monographs is verified at suitable time intervals. electromultiplier, Faraday cup, dynodes), where ion currents
LINEARITY are converted into electrical signals. The element is quantified
Prepare and analyse not fewer than 4 reference solutions over according to the number of ions arriving and generating
the calibration range plus a blank. Perform not fewer than electrical pulses per unit time.
5 replicates. The sample-introduction system and data-handling techniques
The calibration curve is calculated by least-square regression of an ICP-AES system are also used in ICP-MS.
from all measured data of the calibration test. The regression APPARATUS
curve, the means, the measured data and the confidence
interval of the calibration curve are plotted. The operating The apparatus consists essentially of the following elements :
method is valid when : – sample-introduction system, consisting of a peristaltic
– the correlation coefficient is at least 0.99 ; pump delivering the solution at constant flow rate into a
nebuliser ;
– the residuals of each calibration level are randomly
– radio-frequency (RF) generator ;
distributed around the calibration curve.
– plasma torch ;
Calculate the mean and relative standard deviation for the
lowest and for the highest calibration level. – interface region including cones to transport ions to the
ion optics ;
When the ratio of the estimated standard deviations of the
lowest and the highest calibration level is less than 0.5 or – mass spectrometer ;
greater than 2.0, a more precise estimation of the calibration – detector ;
curve may be obtained using weighted linear regression. Both – data-acquisition unit.
After proteolysis of the glycoprotein, the following approaches Deglycosylation of the glycopeptides. Identification of the
can be chosen. different glycosylation sites of a glycoprotein is made
Direct analysis by MS (2.2.43). Care should be taken that the possible by comparing peptide maps obtained by proteolytic
glycopeptide signal is not suppressed due to the presence of digestion of the intact glycoprotein to those obtained
other peptides, where glycopeptides represent a minor portion when the glycoprotein is deglycosylated previously or
of the total peptide mixture and where signal intensities are following proteolytic digestion. The peptide mass gives
lower than those of non-glycosylated peptides. information about the glycosylation sites and by calculating
the mass difference between the intact glycopeptide
Separation prior to analysis by MS. This additional step and the deglycosylated glycopeptide, it is possible to
overcomes the problems raised above. Enrichment or obtain information about the attached glycans concerning
fractionation techniques can be used either in parallel with or composition and heterogeneity. Approaches to deglycosylation
sequentially to direct analysis. Separation techniques such of the protein backbone are given in section 2-3-1. A separation
as liquid chromatography (LC) (2.2.29) and CE (2.2.47) are step can be performed after or before deglycosylation.
suitable. These techniques may be interfaced with MS to allow
online MS measurements.
General Notices (1) apply to all monographs and other texts 101
2.2.59. Glycan analysis of glycoproteins EUROPEAN PHARMACOPOEIA 8.0
2-3. ANALYSIS OF RELEASED GLYCANS Analysis of glycans provides information on the various
Analysis of released glycans provides a convenient way to populations of glycans present on the protein (high-mannose,
obtain information on the various populations of glycans hybrid, complex). Information on the relative amounts
present on the protein (bi-, tri-, and tetra-antennary of branched structures might be obtained by analysis of
profile). The degree of sialylation can also be addressed desialylated glycans.
at this stage. Depending on the chosen method, prior A separation step may be required. It implies the use of LC
derivatisation/labelling may be needed to allow the detection (2.2.29) and CE (2.2.47) as intermediate techniques. LC
of the glycans. (2.2.29) can be used preparatively with individual fractions
Analysis of released glycans generally involves the release and being collected (usually labelling is required) or can be directly
purification of glycans from the reaction mixture, followed by coupled to MS (2.2.43).
the labelling/derivatisation of the glycans, where needed ; the 2-3-2-1. Analysis of unlabelled glycans
glycans are then profiled (fractionation or separation). Native glycans can be analysed by high-pH anion-exchange
2-3-1. Release of glycans chromatography with pulsed amperometric detection
The selection of the approach used for the release of glycans (HPAEC-PAD), porous graphite chromatography (PGC) and
will depend on the glycoprotein under test. The cleavage MS (2.2.43).
agent to be employed is chosen according to the type of HPAEC-PAD has high sensitivity and can also separate some
cleavage needed and level of information required. Enzymatic linkage isomers. Response factors of the different signals
or chemical cleavage may be used. Table 2.2.59.-1 gives a are not equal for the different oligosaccharide structures.
non-exhaustive list of enzymatic cleavage agents and their Absolute quantification of the glycan is not possible unless an
specificity. oligosaccharide reference library is available. Quantification
Digestion efficiency is generally dependent on the accessibility can be obtained by comparison with a well-characterised
of the glycans on the protein and hence the protein can be reference standard of the substance being tested, or by relating
denatured to maximise glycosylation site exposure, unless it is the peak area of each glycan to the total peak area of all
desirable to distinguish between surface and buried glycans. glycans in the map.
Chemical cleavage agents might also be used, using for PGC can also be used to separate native glycans because of its
example hydrazine or alkaline borohydride for β-elimination. higher selectivity compared to the conventional non-polar
columns. A PGC-electrospray-ionisation-MS approach can be
Table 2.2.59.-1. – Examples of enzymatic cleavage agents applied for direct glycan analysis.
Agents Specificity 2-3-2-2. Analysis of labelled glycans
N-linked glycans release Labelling of glycans
Hydrolysis of an N4-(acetyl-β- D- The type of derivatisation carried out will depend on the
glucosaminyl)asparagine residue method used to detect glycans : UV or fluorescent.
in which the glucosamine residue Derivatisation with fluorescent labels is the most commonly
Peptide-N4-(N-acetyl-β-
may be further glycosylated,
glucosaminyl)asparagine amidase used technique for labelling glycans at their reducing end by
to yield a (substituted)
(EC 3.5.1.52) reductive amination. One label can be attached to every single
N-acetyl-β- D-glucosaminylamine
and a peptide containing an mono- and oligo-saccharide, allowing the determination of
aspartate residue molar quantities. Table 2.2.59.-2 gives a non-exhaustive list
Release of N-glycan chain but no of commonly used fluorescent labels and suitable analytical
- Peptide N-glycosidase F (PNGase F) release of N-glycan chain containing techniques.
(α1-3)-linked core fucose
General Notices (1) apply to all monographs and other texts 103
2.2.59. Glycan analysis of glycoproteins EUROPEAN PHARMACOPOEIA 8.0
3-3-2. Quantitative expressions of separation profile Release and isolation of oligosaccharides. The approach
chosen for the release of glycans will depend on the protein
Profiles or distribution patterns may be expressed numerically under test and will be based on the types of glycosylation, i.e.
in a number of ways, including the normalisation procedure ; N- or O-linked glycosylation. Non-compendial approaches
the percentage content of each analytical target, e.g. glycan available for the release of glycans must be optimised in
entity, is calculated by determining the response of the glycan order to ascertain a quantitative profiling of all glycan
entity as a percentage of the total response of all the entities, entities. Factors that impact cleavage efficiency, such as
excluding those due to solvents or any added reagents, and enzyme-to-protein concentration ratio, temperature, reaction
those below the disregard limit. In addition, numerical time course, and denaturation of protein prior to digestion,
expressions such as the Z number, which are method- and must be optimised.
product-specific and defined in specific monographs, can be
used. It is noteworthy that the enzymatic/chemical reaction must
not alter the glycan composition, e.g. not destroy sialic acid
residues. Where there is more than one glycosylation site,
4. REFERENCE STANDARDS the enzymatic treatment should proportionally release all
oligosaccharide moieties attached to the protein, independent
Reference standards for glycan analysis serve 2 functions : of their structure and their individual position in the protein.
the verification of the suitability of the system and the Reproducible recovery of all glycan entities from the reaction
confirmation that the article under test complies with specified mixture must be confirmed.
requirements.
Derivatisation of released glycans. Derivatisation is
The reference standards used for system suitability may be : usually carried out according to non-compendial protocols.
Therefore, the reproducible derivatisation of all glycan entities
– a reference substance for the substance being tested ; must be verified. This may be achieved through optimisation
– glycan moities liberated from a fully characterised reference of the reaction conditions such as amount of the derivatisation
standard of the substance being tested ; reagent, reaction temperature and time. The derivatisation
reaction must not change the glycan composition, e.g. not
– well-characterised glycan moities liberated from destroy sialic acid residues.
glycoproteins (e.g. fetuin, IgG) ; Separation, identification and system suitability. The
methods employed for glycan analysis must be capable of
– glycan markers characterised for identity and purity.
detecting and separating different glycan moieties to ascertain
The reference standard used for compliance of the glycoprotein a reliable identification and quantification.
under test is a preparation of the substances being tested. It
is noted that glycan analysis procedures described in specific The acceptance criteria for system suitability, which also cover
monographs prescribe the use of a reference standard for glycan cleavage, recovery and analysis, depend on the critical
the substance being tested and for which the glycan analysis test parameters that affect the outcome of the result.
procedure has been validated.
A comparison between the glycan map of the substance
under test and that of a reference substance, being treated
5. POINTS TO CONSIDER IN METHOD DEVELOPMENT in the same conditions, is an indicator to evaluate the
performance of the analytical procedure. In order to
This section provides means for measuring the overall further confirm the obtained results, the analyses may be
performance of the method during development. The extent repeated with an orthogonal method. The use of a reference
of method development and analytical validation is selected on standard (e.g. reference substance of the product being
the basis of their suitability for a specific product. Depending examined, system suitability glycan marker) is essential in the
on the chosen approach, several steps are necessary for glycan establishment of system suitability parameters and validation
analysis, for example : of the analytical procedure.
– isolation and purification (or desalting) of the glycoprotein ; Reproducibility of quantitative expression (e.g. Z number
estimation) of glycan profiles must be verified.
– enzymatic (or chemical) treatment of the glycoprotein to
selectively release either N- or O-linked glycans from the Determination of site occupancy based on relative
protein backbone ; quantities of glycosylated and non-glycosylated peptides.
Where site occupancy is estimated by comparison of
– isolation and purification of the released glycans ; glycosylated and non-glycosylated peptides from an
enzymatically digested glycoprotein, reproducible cleavage of
– verification of released sialic acid and monosaccharide both forms of the peptide must be demonstrated.
residues ;
– chromophore labelling of the released glycans ;
– separation of the glycans, native or fluorescence labelled ; 6. GLYCAN ANALYSIS DECISION-MAKING FRAMEWORK
– glycan identification and quantification (e.g. determination This decision-making framework is given for information
of the Z number) ; and does not constitute a mandatory part of the European
Pharmacopoeia.
– determination of site occupancy based on relative quantities
of glycosylated and non-glycosylated peptides. The choice of procedures used to analyse glycans is established
Protein isolation and purification. Isolation and purification according to the level of information required to ensure
of the glycoprotein from its matrix may be necessary to the quality of the glycoprotein and is set up during the
remove all interfering substances (e.g. excipients, salts) and, development phase of the product.
when required, will be specified in the specific monograph.
This must be performed in a reproducible manner in order to Figure 2.2.59.-2 provides guidance in the choice of methods to
guarantee a quantitative recovery of the protein. be used when glycan analysis is required.
04/2008:20260 The temperature at which the sensor signal first leaves its
initial value is defined as the beginning of melting, and the
temperature at which the sensor signal reaches its final value
2.2.60. MELTING POINT - is defined as the end of melting, or the melting point.
INSTRUMENTAL METHOD Use glass capillary tubes that are open at one end, about
100 mm long, with an external diameter of 1.3-1.5 mm and
This chapter describes the measurement of melting point
an internal diameter of 0.8-1.3 mm. The wall thickness of the
by the capillary method using an instrumental method of
tube is 0.1-0.3 mm.
determination.
Some apparatuses allow for the determination of the melting
APPARATUS point on more than 1 capillary tube.
There are 2 modes of automatic observation arrangements :
METHOD
– mode A : by light transmission through the capillary tube
loaded with the sample ; Introduce into the capillary tube a sufficient amount of the
substance to be examined, previously treated as described in
– mode B : by light being reflected from the sample in the the monograph, to form in each tube a compact column about
capillary tube. 4 mm high, and allow the tubes to stand for the appropriate
In both modes, the capillary tube sits in a hollow of a time at the prescribed temperature.
metal block, which is heated electrically and controlled by a Proceed as follows or according to the manufacturer’s
temperature sensor placed in another hollow of the metal instructions. Heat the heating block until the temperature is
block. The heating block is capable of being maintained about 5 °C below the expected melting point.
accurately at a pre-defined temperature (± 0.1 °C) by the
heating element, and of being heated at a slow and steady rate Place the capillary tube in the heating block with the closed
of 1 °C/min, after an initial isothermal period. end downwards. Start the temperature programme. When
the substance starts melting, it changes its appearance in the
In mode A, a beam of light shines through a horizontal hollow capillary tube. As a result, the temperature of the heating
and crosses the capillary tube. A sensor detects the beam at block is recorded automatically following the signal changes
the end of the cylindrical hole after the capillary tube. from the photosensor due to light transmission (mode A,
In mode B, a beam of light illuminates the capillary tube from Figure 2.2.60.-1), or following image processing (mode B,
the front and the sensor records the image. Figure 2.2.60.-2).
Some apparatuses allow for the visual determination of the Carry out the test on 2 other samples and calculate the mean
melting point. value of the 3 results.
General Notices (1) apply to all monographs and other texts 105
2.2.61. Characterisation of crystalline solids by calorimetry EUROPEAN PHARMACOPOEIA 8.0
CALIBRATION
SYSTEM SUITABILITY
Prepare 3 capillary tubes. Carry out the test and calculate the
mean value of the 3 results.
01/2013:20261
2.2.61. CHARACTERISATION
OF CRYSTALLINE SOLIDS BY
MICROCALORIMETRY AND
SOLUTION CALORIMETRY
For the purpose of this chapter, crystalline material, partially
crystalline material and amorphous material are considered as
solids.
INTRODUCTION - THE CONCEPT OF CRYSTALLINITY
The perfectly ordered crystal lattice with every molecule
in its expected lattice position is an ideal that is seldom, if
ever, achieved. The other extreme is the amorphous state,
in which a solid contains the maximum possible density of
imperfections (defects of various dimensionalities), such that
all long-range order is lost while only the short-range order,
imposed by its nearest neighbours, remains. Real crystals lie
somewhere between these 2 extremes. A crystal’s position on
A. glass capillary tube D. temperature sensor a scale bounded by these 2 extremes is termed crystallinity.
All real crystals, even in the pure state, possess some
B. sample E. heating block lattice imperfections or defects, which increase both the
C. photosensor F. light source energy (enthalpy under conditions of constant atmospheric
pressure) and the disorder (expressed as the entropy) of
the crystal lattice. A crystal with a relatively low density
Figure 2.2.60.-1. – Mode A : transmission of imperfections is said to be highly crystalline and to
General Notices (1) apply to all monographs and other texts 107
2.2.61. Characterisation of crystalline solids by calorimetry EUROPEAN PHARMACOPOEIA 8.0
recrystallised parts and its condensation. Thus, the area under The enthalpy of solution is measured either by an isoperibol
this exothermic recrystallisation response is proportional to (constant perimeter, i.e. jacket) solution calorimeter or by
the heat of recrystallisation. an isothermal (constant temperature) solution calorimeter.
Typically, at least 3 measurements are made with each sample.
The mean of these values is then calculated. The exact
requirements will depend upon the equipment capability and
degree of accuracy needed.
ISOPERIBOL SOLUTION CALORIMETRY
In the isoperibol solution calorimeter, the heat change
during the solution process causes a corresponding change in
temperature of the solvent-solute system (i.e. solution). This
temperature change is measured by a temperature sensor,
which is wired to an electrical circuit that records an electrical
signal corresponding to the temperature change. Typically,
this temperature change in an electronic form is measured at
precisely defined time intervals to produce temperature-time
data that are collected, analysed by a computer, and then
plotted. A blank run without addition of the solid solute to
the solvent normally shows no discernible change in the slope
of the temperature-time plot.
For isoperibol solution calorimeters, response is fairly
rapid, but corrections must be made for any heat losses to
or heat gains from the bath. Therefore, isoperibol solution
calorimeters are more advantageous than isothermal solution
calorimeters when the solution process is relatively fast. For
all measurements of enthalpy of solution using isoperibol
Figure 2.2.61.-1. – Typical microcalorimetric output of power solution calorimeters, the choice of solvent is critical. The
(in μW) as a function of time (in hours) : amorphous collapse nature and mass of the solvent and the mass of sample allow
peak (I) and crystallisation peak (II) for mainly amorphous the total heat change, corresponding to total dissolution of the
lactose at 25 °C and 75 per cent relative humidity solid, to proceed to completion within 5 min under vigorous
stirring at a constant rotational speed within the range of
METHOD 2 - SOLUTION CALORIMETRY 400-600 r/min.
(DETERMINATION OF CRYSTALLINITY) The effective heat capacity of the calorimeter cell and its
Solution calorimetry provides a means of determining contents is determined for every calorimeter run. This
enthalpy of solution (i.e. heat of solution under constant determination is accomplished by electrical heating of the
atmospheric pressure) of a substance. Enthalpy of solution contents of the calorimeter cell. The effective heat capacity is
is defined as the enthalpy of the substance dissolved in the determined according to 1 of 2 protocols : either by making
solution to a defined concentration minus the enthalpy of the 1 determination after ampoule breakage or by making
original substance. The solvent for the dissolution process 1 determination before and a 2nd determination after ampoule
must be such that the mass of solid dissolves within a time breakage and then averaging the 2 results. The accuracy
frame that matches the response time of the calorimeter, as and reliability of the electrical heating are established by
discussed below. The enthalpy of solution is proportional the accuracy and reliability of the aforementioned chemical
to the amount of solid being dissolved. This amount may calibrations.
be defined as 1 mol for molar enthalpy or as 1 g for specific ISOTHERMAL SOLUTION CALORIMETRY
enthalpy. If the substance possesses adequate purity (as
In the isothermal (constant temperature) solution calorimeter,
determined by the degree of accuracy required) and if its
the heat change during the solution process is compensated
molecular mass is known, the molar enthalpy is preferred,
for by an equal but opposite energy change, such that the
otherwise the specific enthalpy must be used. The enthalpy of
temperature of the solvent-solute system (i.e. solution)
solution is weakly dependent on both the temperature, which
remains essentially constant. This equal but opposite energy
is usually 25.0 °C, and the final concentration of the dissolved
change is measured and, when its sign is reversed, provides the
solute.
enthalpy of solution. For isothermal calorimeters, response is
It is usually preferred to express the crystallinity, Pc, of a relatively slow, but the compensation process eliminates the
substance on a percentage scale. This procedure requires effects of heat losses to or heat gains from the bath. Therefore,
2 reference standards, namely a highly crystalline sample isothermal solution calorimeters are more advantageous than
assuming 100 per cent crystallinity and having a measured isoperibol solution calorimeters when the solution process
enthalpy of solution of , and an amorphous sample is relatively slow.
assuming 0 per cent crystallinity and having a measured
enthalpy of solution of . From these values and from the SOLUTION CALORIMETER CALIBRATION
measured enthalpy of solution, , of the solid under study,To ensure the accuracy of the calorimeter, chemical calibrations
the percentage crystallinity of the solid, Pc, may be calculated must be performed on a regular basis. For an endothermic
as follows : solution process, the calibration of the calorimeter is checked
by measuring the heat absorbed during the dissolution of
potassium chloride in distilled water at 298.15 K (25.0 °C).
The established enthalpy change in this endothermic process
Clearly, crystallinity expressed on a percentage scale depends is 235.5 J/g (17.56 kJ/mol). For an exothermic solution
on 3 measured values and the enthalpies of solution may process, the calorimeter is checked by measuring the heat
be replaced by other corresponding physical quantities evolved during the dissolution of 5 g per litre of tromethamine
that depend on crystallinity. The value of the percentage [tris(hydroxymethyl)aminomethane, THAM] in a 0.1 mol/L
crystallinity of a sample, however, depends not only on the aqueous hydrochloric acid solution at 298.15 K (25.0 °C). The
nature and method of preparation of the 2 reference standards, established heat for the aforementioned process is − 246.0 J/g
but also on the choice of the physical quantity that is measured. (− 29.80 kJ/mol).
SAMPLE HANDLING Sample size. Usually a few milligrams are used. If sample sizes
The chemical and physical stability of solids may decrease with are variable, the effects of this variation on the appearance
of the spectrum are validated.
decreasing crystallinity. In particular, solids of low crystallinity,
especially amorphous solids, tend to sorb water vapour from Sample preparation. The test and reference samples must
the atmosphere, leading to crystallisation and a corresponding be comparable in terms of concentration, pH and buffer
gain in crystallinity. For these reasons, anhydrous samples composition. Typically, samples in solution are lyophilised,
whose crystallinity is to be determined must be stored at zero and the dried samples dissolved in deuterated water or a
humidity or below critical humidity levels in sealed chambers buffer in deuterated water. It may be worthwhile to lyophilise
containing a desiccant, preferably containing an indicator of a solution in deuterated water one or more times (‘deuterium
effectiveness. If crystallinity-humidity studies are to be carried
exchange’) as this reduces the intensity of strong solvent
out, the sample is stored in a sealed chamber containing a signals ; volatile process impurities such as ethanol will also
saturated salt solution to provide a defined relative humidity. be lost. Use of buffer for the final sample preparation can
reduce aggregation and improve spectral reproducibility
by reducing batch-to-batch pH variation. Some probes are
07/2011:20264 intolerant to high salt concentrations, but ionic strengths up
to 200 mM sodium chloride are normally tolerated. High salt
concentrations tend to increase 90° pulse length.
2.2.64. PEPTIDE IDENTIFICATION BY
VERIFICATION OF IDENTITY
NUCLEAR MAGNETIC RESONANCE
Determination of key spectral factors. Use of a qualitative
SPECTROMETRY approach does not entail stringent requirements on spectral
This general chapter is to be used in conjunction with general parameters (for example, fast pulse repetition rates can
chapter 2.2.33. Nuclear magnetic resonance spectrometry in the be used, as full relaxation is not required). The use of
context of peptide identification. The approach to be followed short pulse widths (for example, a 30° pulse) and fast
is qualitative and consists of comparing the nuclear magnetic repetition rates will have no significantly deleterious effect
resonance (NMR) spectrum of a test sample with that of a on spectra, and will allow faster acquisition of acceptable
reference sample acquired under identical conditions. signal-to-noise ratios. Variation in the pulse width and
acquisition time within wide limits will not affect the ability
This general chapter mainly applies to the use of proton NMR to compare spectra. The number of scans collected must give
1
( H NMR) spectrometry, to confirm the identity of small appropriate signal-to-noise ratios for low intensity resonances
peptide products (up to approximately 15 amino acids). It and therefore a minimum signal-to-noise ratio of 50:1 is
13
is also applicable when using C NMR spectrometry with recommended.
some modifications. The scope is restricted to the use of
one-dimensional NMR spectrometry. Identification of characteristic resonances. It is possible
to compare either the complete spectrum or a portion of
GENERAL PRINCIPLES it. Comparison of spectra of relevant samples will highlight
Equipment. Unless otherwise specified, an apparatus with a regions of the spectrum that are distinctive, and comparison
field strength giving an operating frequency for proton NMR can be constrained to these regions. It is important to define
of at least 300 MHz. resonances from impurities, such as residual solvents, which
may be essentially irrelevant to product quality and which
Spectral acquisition conditions and their optimisation. may vary in intensity between batches.
After introduction into the magnet, the sample is allowed
Spectral comparison. See the provisions of general
to come to thermal equilibrium, especially if analysis is
chapter 2.2.33.
carried out at a temperature significantly different from room
temperature : monitoring the lock signal is often a valuable
visual guide to the progress of this process. 01/2013:20265
The spectral width must encompass the complete spectrum
of the peptide, with an empty spectral region at each side. 2.2.65. VOLTAMETRIC TITRATION
Typically, a spectral width of 12 ppm or 16 ppm is appropriate.
In voltametric titration the end-point of the titration is
The following parameters may be optimised to improve
determined by following the variation of the voltage measured
resolution of characteristic peaks : temperature and/or pH
between 2 electrodes (either 1 indicator electrode and
primarily, buffer and peptide concentrations. Control of
1 reference electrode or 2 indicator electrodes) immersed in
sample temperature is recommended but is not mandatory ;
the solution to be examined and maintained at a constant
if not used, the effect of small temperature changes on the
current as a function of the quantity of titrant added.
appearance of the spectrum is validated.
Apparatus. The apparatus comprises an adjustable current
The number of data points collected is such as to define peaks source and a voltmeter; the detection system generally
adequately. consists of an indicator electrode (for example, a platinum
Solvent suppression is not recommended but, if used, the electrode, a rotating-disc electrode or a carbon electrode)
intensities of peaks close to the solvent resonance may be and a 2nd electrode (for example, a platinum electrode, a
affected and this has to be validated when comparing spectra. rotating-disc electrode or a carbon electrode).
Chemical shift referencing. For samples in aqueous solution, Method. Set the current to the indicator electrode as
sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS), sodium prescribed in the monograph and plot a graph of the initial
3-(trimethylsilyl)propionate (TSP) or a deuterated analogue voltage and the values obtained during the titration as
(TSP-d4) are appropriate, and the chemical shift of the methyl functions of the quantity of titrant added. Add the titrant
signals is often set to 0 ppm. Either the reference material in not fewer than 3 successive quantities equal to a total of
is added at low amounts (10-100 ppm has been found to be about 80 per cent of the theoretical volume corresponding
appropriate) to the deuterated water used to dissolve the final to the presumed equivalence point. The 3 values must fall
sample, or an easily recognised internal resonance that is on a straight line. Continue adding the titrant beyond the
consistently present (such as acetate anion) can be used as presumed equivalence point in not fewer than 3 successive
a secondary reference. In this case, a validation spectrum quantities. The values obtained must fall on another straight
obtained under the same spectral conditions is used to define line. The point of intersection of the 2 lines represents the
the chemical shift of the secondary standard. end-point of the titration.
General Notices (1) apply to all monographs and other texts 109
2.2.66. Detection and measurement of radioactivity EUROPEAN PHARMACOPOEIA 8.0
Using titration systems for voltametric titration with content must be made with reference to a specified time zone.
2 indicator electrodes, the whole titration curve is recorded The radioactivity at other times may be calculated from the
and used to determine the end-point. exponential decay equation or from tables.
In general, a correct measurement of radioactivity requires
01/2014:20266 that consideration is given to some or all of the following :
Dead-time losses. Due to the finite resolving time (dead
2.2.66. DETECTION AND time) of the detector and its associated electronic equipment,
MEASUREMENT OF RADIOACTIVITY it may be necessary to correct for losses by coincidence. The
resolving time of a counter is the minimum time interval
INTRODUCTION required by the counter to resolve 2 single pulses. Incident
Within the context of the European Pharmacopoeia, the term radiation events at shorter intervals may not be detected or
‘radioactivity’ is used both to describe the phenomenon of may be detected as a single event with the summed energy.
radioactive decay and to express the physical quantity of this These losses are sometimes referred to as ‘dead-time losses’.
phenomenon. In the monographs on radiopharmaceutical For a counting system with a fixed dead time τ following each
preparations, the detection and measurement of radioactivity count, the true count rate, per second, is calculated using the
are performed for different purposes : verification of the following expression :
characters, identification, determination of radionuclidic
and radiochemical purity, as well as determination of the
radioactivity in a substance (assay).
Under these assumptions, the measurement can be qualitative,
quantitative or both, depending whether it is directed to the N1 = the observed count rate, per second ;
identification of the radionuclide or the determination of its τ = the dead time, in seconds.
activity (rate of decay) or both of them.
Radioactive sources can produce various types of emissions, With some equipment this correction is made automatically.
such as alpha particles, electrons, positrons, gamma- and Corrections for losses by coincidence must be made before
X-rays, according to the radionuclidic composition. the correction for background radiation.
Each radionuclide yields characteristic emissions, with Correction for decay during measurement. If the time
specific energies and relative intensities. Such radiations period of an individual measurement, tm, is not negligibly
can be detected as a result of their ionising properties in an short compared with the half-life of the radionuclide, T1/2,
ionisation chamber but without further characterisation ; the decay during this measurement time must be taken into
when they are detected and analysed using a spectrometer, an account. For example, there is a 5 per cent cumulative loss of
energy spectrum is obtained. A detailed spectrum analysis is counts due to decay during a counting period that is 15 per
typically used to identify radionuclides present in a sample. cent of the half-life of the radionuclide.
Spectrometry can also be used for quantitative determination
of the radioactivity in sources made of a single radionuclide After having corrected the instrument reading (count rate,
or radionuclide mixtures or of the individual radionuclides ionisation current, etc.) for background signals and, if
present. necessary, for losses due to electronic effects, the instrument
reading corrected to the beginning of the individual
A measurement of radioactivity is generally performed by
measurement is calculated using the following expression :
counting the number of detected decay events (emissions).
Therefore, the geometry of the sample during the measurement
of radioactivity and the acquisition time strongly influence
the result. In general, the measurement geometry must
correspond to a calibrated geometry and the acquisition time
must be long enough to reach sufficient counting statistics. R = instrument reading before decay correction, but
already corrected for background signal, etc. ;
A measurement of radioactivity can be done in a
stand-alone mode (e.g. using an ionisation chamber or λ = radionuclide decay constant (ln 2/T½) ;
a spectrometer) or in combination with a separation e = base of natural logarithm ;
technique (e.g. radiochromatography) to account for relative
contributions from different radioactive chemical species that tm = measurement duration.
may be present in a mixture.
Statistics of radioactivity measurement. The results of
MEASUREMENT OF RADIOACTIVITY determinations of radioactivity show variations that derive
A direct determination of the radioactivity of a given sample, mainly from the random nature of nuclear transformations.
in becquerel (Bq), may be carried out if the decay scheme of Counting for any finite time can yield only an estimate of
the radionuclide is known, but in practice many corrections the true rate of nuclear transformations. A sufficient number
are required to obtain accurate results. For this reason, it of counts must be registered in order to compensate for
is possible to carry out the measurement with the aid of a variations in the number of transformations per time. In the
primary standard source or by using measuring instruments case of measurement of radioactivity, the standard deviation
such as an ionisation chamber or a spectrometer calibrated of the recorded counts is the square root of the counts, so at
using suitable standards for the particular radionuclides. least 10 000 counts are necessary to obtain a relative standard
deviation of not more than 1 per cent.
A spectrometer is used when measuring the radioactivity of
radionuclides in a mixture, each radionuclide being identified Linearity. The linearity of an instrument is the range of
by its emissions and their characteristic energies. radioactivity for a particular radionuclide over which its
All measurements of radioactivity must be corrected for efficiency remains constant.
dead-time losses and by subtracting the background signal The linear range of a radioactivity measurement assembly can
due to radiation in the environment and to spurious signals be determined by repeatedly counting a radioactive sample in
generated in the equipment itself. a fixed geometry as it decays from an activity level that is above
The radioactivity of a preparation is stated at a given date. the linear range. After correction for the background signal,
If the half-life of the radionuclide is less than 70 days, the the natural logarithm of the count rate data is plotted against
time is also indicated. This statement of the radioactive the elapsed time after the first measurement (Figure 2.2.66.-1).
General Notices (1) apply to all monographs and other texts 111
2.2.66. Detection and measurement of radioactivity EUROPEAN PHARMACOPOEIA 8.0
The sensitivity of a NaI(Tl) detector is higher than that of a by using an attenuating material. Introduce the preparation to
germanium detector of the same size. In general, peaks in an be examined in a container into the instrument chamber and
energy spectrum are identified with an uncertainty depending record the spectrum after closing the shielding.
upon the full width of the peak at its half-maximum height Ensure that the container used for quantitative measurements
(FWHM). The energy resolution of a solid-state scintillation is of the same shape, dimensions, volume and material as that
detector is much poorer than that of a semiconductor detector of the calibration standard.
and hence peaks obtained with a semiconductor detector Ensure that the composition of the solution and the position
are much narrower than those obtained with a scintillation of the container in the measuring chamber is the same for
detector. Figure 2.2.66.-2 shows a comparison of the spectra the container for the quantitative measurement as for the
obtained from the same source with the 2 types of detector. calibration standard.
Radionuclide identification. Calibrate the spectrometer in
The different performances of NaI(Tl) and HPGe detectors relation to energy. Determination of the correspondence of the
may limit their use in some spectrometric analyses. energy of the peaks detected from the sample to the energies
prescribed by a monograph is a valid identification test.
For the identification of the radionuclide(s) in a preparation Radionuclidic purity. Calibrate the spectrometer in relation to
and determination of radionuclidic purity, a risk assessment efficiency and energy. Determine the LOQ and resolution of
on the process of radionuclide production must assess the the equipment and ensure that they are in line with the limits
potential presence of other radionuclides with photon energies of the radionuclides to be determined. Record the spectrum
in the same range (± 10 per cent) as that of the radionuclide(s) of the preparation.
present in the radiopharmaceutical. Identify the radionuclides present in the preparation to be
examined and determine their radioactivity with the aid of
In case radionuclidic impurities can be present that emit chapter 5.7. Table of physical characteristics of radionuclides.
gamma- or X-rays with an energy in the same range as that of Because the level of radionuclidic impurities, expressed as a
the photons emitted by the radionuclide in the preparation, a percentage of the total radioactivity, may increase or decrease
measured peak energy within a maximum interval of ± 2 keV with time, the measured activity of each impurity must be
or ± 2 per cent (whichever is the larger) with respect to the recalculated to the activity during the period of validity of the
nominal peak energy (see 5.7. Table of physical characteristics preparation. The activities of all radionuclidic impurities need
of radionuclides) is sufficient for peak identification. to be summed (taking into account the limit of quantification)
and related to the total radioactivity of the preparation.
In the case where such impurities are not expected to be The sample is placed close to the detector or within a
present, a maximum interval of ± 10 keV or ± 6 per cent well-type detector. All the events within a pre-set energy
(whichever is the larger) with respect to the nominal peak range are collected and displayed on a ratemeter as counts
energy is acceptable for peak identification. per second or accumulated over a pre-set period of time. If
there is sufficient difference in photon energies emitted by
Method. Ensure that the counting rate of the sample falls the radionuclide(s), a sodium iodide detector can be suitable,
within the linearity range of the equipment. For liquid given its high sensitivity. However, if there is a need to
samples this may be achieved by appropriate dilution ; for discriminate emissions of similar energy, a HPGe detector or
solid samples, by increasing the source-to-detector distance or another semiconductor detector is needed.
Figure 2.2.66.-2. – Comparative pulse-height spectra recorded using a thallium-activated sodium iodide scintillator (upper curve)
and a high-purity germanium semiconductor detector (lower curve). The source was gamma- and X-ray radiation from the
decay of iodine-131.
General Notices (1) apply to all monographs and other texts 113
2.2.66. Detection and measurement of radioactivity EUROPEAN PHARMACOPOEIA 8.0
Figure 2.2.66.-3. – Typical HPGe efficiency curve measured DETECTION AND MEASUREMENT OF RADIOACTIVITY
using a dedicated container set on top of the detector IN COMBINATION WITH A SEPARATION TECHNIQUE
BETA-PARTICLE SPECTROMETRY A radioactive preparation may contain the radionuclide
In the case of a beta-particle emitter, a beta-particle in different chemical forms other than the intended one.
spectrometer is necessary to determine the energy distribution Therefore it is necessary to separate the different substances
of the emitted beta particles. It is analogous to a gamma-ray containing the radionuclide and determine the percentage of
spectrometer, but frequently uses liquid scintillators to radioactivity due to the radionuclide concerned associated
convert the energy of the beta particles into detectable light, with the stated chemical form and the contribution to the
which can then be analysed. Beta-particle spectrometry is total radioactivity due to the radionuclide concerned coming
mostly achieved by dissolving or suspending the sample in from other substances. For this purpose, instruments for
a liquid scintillation cocktail in transparent or translucent the detection and measurement of radioactivity are used in
(glass or plastic) containers and subsequent counting of the combination with a physico-chemical separation technique.
electrical pulses generated by a photomultiplier from the In principle, any method of separation may be used.
emitted light. The pulse amplitude is related to the energy Monographs for radiopharmaceutical preparations may
of the absorbed radiation. A beta-particle spectrum can be include the combined use of radioactivity measurement with
produced by collecting a sufficient number of pulses. The paper chromatography (2.2.26), thin-layer chromatography
liquid scintillation cocktail is chosen in such a way that (2.2.27), gas chromatography (2.2.28), liquid chromatography
counting errors due to quenching, chemoluminescence, (2.2.29), size-exclusion chromatography (2.2.30) or
phosphorescence, etc., are minimised. Coincidence counting electrophoresis (2.2.31).
with 2 or more photomultipliers is also used to minimise
counts from background radiation, electronics, etc. In all cases the radioactivity of each analyte is measured after
To differentiate between alpha- and beta-particle emissions, the separation has been achieved using the stated method.
pulse-shape discrimination is commonly used.
Radioactivity measurement may be performed using
Radionuclide identification. Determination of the detectors mounted in series with other detectors in analytical
correspondence of the mean and/or maximum energies in the instruments, such as liquid chromatographs, making in-line
energy spectrum from the sample to the energies prescribed detection of analytes, or performed off-line, i.e. after the
by a monograph is a valid identification test. analytical separation has been completed, by measuring
Calibration. A common method of energy calibration is to use the radioactivity of eluate fractions obtained after liquid
an unquenched reference sample to determine the maximum chromatographic separation of equal volume or as the
energy of the beta particles emitted by the radionuclide of distribution of radioactivity on paper chromatography or
interest. thin-layer chromatography supports.
General Notices (1) apply to all monographs and other texts 115
2.2.66. Detection and measurement of radioactivity EUROPEAN PHARMACOPOEIA 8.0
is numbered starting from the origin side and counted Radioactivity may be measured by integration using an
separately. Alternatively, for well-characterised systems the automatic-plotting instrument or a digital counter.
support may be cut into 2 or more unequal portions, folded if The ratios of the areas under the peaks give the ratios
necessary to approximately equal geometry before counting. of the percentages of radioactivity due to the respective
An ionisation chamber or a scintillation counter can be
radiochemical substances.
used for this purpose, provided they are used within the
instrument’s linearity range and above its LOQ. When the strips are cut into portions, the ratios of
Autoradiography. This may also be used to acquire an image the quantities of radioactivity measured give the ratio
of the radioactivity distribution on the chromatographic of percentages of radioactivity due to the respective
support. In this case, the response of the system used for radiochemical species.
the acquisition of the image, such as a phosphor imager or a Calibration. It is important to demonstrate the limits of
photographic film, must be shown to be linear with respect detection and quantification, and the linearity of the detector
to the radioactivity in the chromatogram. Otherwise the throughout the range of activities to be measured and in all
system must be pre-calibrated or exposed at the same time to positions on the support of the chromatographic system.
a series of reference radioactive sources, obtained by dilution This may be done by applying samples covering a range of
from a calibrated standard solution, covering the expected activities from 0.1 per cent to 100 per cent of the expected
radioactivity range that may be present on the support. range. Prepare the samples by dilution and apply equal
Method. Deposit the required amount of sample at the origin volumes of each, with drying if necessary. After examining
of the chromatographic support, with drying if necessary the radioactivity profile using the equipment’s standard
to avoid spreading of the spot. Develop the chromatogram settings, the peak areas are integrated for comparison with the
according to the prescribed method. A carrier may be added calculated amount of radioactivity applied to each spot. Verify
when prescribed in a particular monograph. that the response of the detector over the complete length and
In paper and thin-layer chromatography, it is preferable not width of the detector path is the same, as the response may
to dilute the preparation to be examined but it is important vary with the detector position.
to avoid depositing such a quantity of radioactivity that The peak-resolving power is influenced by the size of the spot,
counting losses by coincidence (dead-time losses) occur the total radioactivity of the radionuclide and the detector
during measurement of the radioactivity. equipment. It can be checked by applying 5 μL spots separated
After development, the support is dried and the positions by distances increasing from 4 mm to 20 mm in 2 mm
of the radioactive areas are detected by measurement of increments. The approximate resolution of the detection
radioactivity over the length of the chromatogram, using a system can be determined from the radioactivity profile as
suitable collimated counter, by autoradiography, or by cutting the distance between the 2 spots where the baseline is only
the strips into portions and counting each portion. just clearly separated.
General Notices (1) apply to all monographs and other texts 119
2.3.1. Identification reactions of ions and functional groups EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 121
2.3.2. Identification of fatty oils by TLC EUROPEAN PHARMACOPOEIA 8.0
Thin-layer chromatography (2.2.27). Test solution. Unless otherwise prescribed, dissolve about
20 mg (1 drop) of the fatty oil in 3 mL of methylene chloride R.
Test solution. Unless otherwise prescribed, dissolve about
20 mg (1 drop) of the fatty oil in 3 mL of methylene chloride R. Reference solution. Dissolve about 20 mg (1 drop) of maize
oil R in 3 mL of methylene chloride R.
Reference solution. Dissolve about 20 mg (1 drop) of maize Plate : a suitable octadecylsilyl silica gel for high performance
oil R in 3 mL of methylene chloride R. thin-layer chromatography as the coating substance.
Plate : a suitable octadecylsilyl silica gel for high performance Mobile phase : methylene chloride R, glacial acetic acid R,
thin-layer chromatography as the coating substance. acetone R (20:40:50 V/V/V).
Mobile phase : Application : 1 μL as bands of 8 mm. A suitable automated
apparatus may be used.
– mobile phase A : ether R ;
Development : over a path of 7 cm.
– mobile phase B : methylene chloride R, glacial acetic acid R,
acetone R (20:40:50 V/V/V). Drying : in air.
Application : 1 μL. Detection : treat with a 100 g/L solution of phosphomolybdic
acid R in ethanol (96 per cent) R. Heat the plate at 120 °C for
Development : twice over a path of 0.5 cm with mobile phase A, 3 min and examine in daylight.
then twice over a path of 8 cm with mobile phase B.
The chromatogram obtained typically shows zones comparable
Drying : in air. to those in Figure 2.3.2.-2.
1. arachis oil 4. rapeseed oil 7. linseed oil 10. almond oil 13. evening primrose oil
2. sesame oil 5. soya-bean oil 8. olive oil 11. wheat-germ oil 14. safflower oil (type I)
3. maize oil 6. rapeseed oil (erucic acid-free) 9. sunflower oil 12. borage oil 15. safflower oil (type II)
1. arachis oil 5. soya-bean oil 9. sunflower oil 13. evening primrose oil
2. sesame oil 6. rapeseed oil (erucic 10. almond oil 14. safflower oil (type I)
acid-free)
3. maize oil 7. linseed oil 11. wheat-germ oil 15. safflower oil (type II)
4. rapeseed oil 8. olive oil 12. borage oil 16. hydrogenated arachis oil
General Notices (1) apply to all monographs and other texts 123
EUROPEAN PHARMACOPOEIA 8.0 2.4.4. Chlorides
2.4. LIMIT TESTS After not less than 2 h the stain produced on the mercuric
bromide paper in the test is not more intense than that in the
standard.
01/2008:20401
corrected 8.0 METHOD B
Introduce the prescribed quantity of the substance to be
2.4.1. AMMONIUM examined into a test-tube containing 4 mL of hydrochloric
acid R and about 5 mg of potassium iodide R and add 3 mL of
Unless otherwise prescribed, use method A. hypophosphorous reagent R. Heat the mixture on a water-bath
for 15 min, shaking occasionally. Prepare a standard in the
METHOD A same manner, using 0.5 mL of arsenic standard solution
Introduce the prescribed solution into a test-tube or dissolve (10 ppm As) R.
the prescribed quantity of the substance to be examined in After heating on the water-bath, any colour in the test solution
14 mL of water R in a test-tube. Make the solution alkaline is not more intense than that in the standard.
if necessary by the addition of dilute sodium hydroxide
solution R, dilute to 15 mL with water R and add 0.3 mL of
alkaline potassium tetraiodomercurate solution R. Prepare a
standard by mixing 10 mL of ammonium standard solution
(1 ppm NH4) R, 5 mL of water R and 0.3 mL of alkaline
potassium tetraiodomercurate solution R. Stopper the
test-tubes.
After 5 min, any yellow colour in the test solution is not more
intense than that in the standard.
METHOD B
In a 25 mL jar fitted with a cap, place the prescribed quantity
of the finely powdered substance to be examined and dissolve
or suspend in 1 mL of water R. Add 0.30 g of heavy magnesium
oxide R. Close immediately after placing a piece of silver
manganese paper R 5 mm square, wetted with a few drops
of water R, under the polyethylene cap. Swirl, avoiding
projections of liquid, and allow to stand at 40 °C for 30 min.
If the silver manganese paper shows a grey colour, it is not
more intense than that of a standard prepared at the same
time and in the same manner using the prescribed volume of
ammonium standard solution (1 ppm NH4) R, 1 mL of water R
and 0.30 g of heavy magnesium oxide R.
01/2008:20402
2.4.2. ARSENIC
Figure 2.4.2.-1. - Apparatus for limit test A for arsenic
METHOD A Dimensions in millimetres
The apparatus (see Figure 2.4.2.-1) consists of a 100 mL conical
flask closed with a ground-glass stopper through which passes 01/2008:20403
a glass tube about 200 mm long and of internal diameter corrected 8.0
5 mm. The lower part of the tube is drawn to an internal
diameter of 1.0 mm, and 15 mm from its tip is a lateral orifice
2 mm to 3 mm in diameter. When the tube is in position in
2.4.3. CALCIUM
the stopper, the lateral orifice should be at least 3 mm below All solutions used for this test are prepared with distilled
the lower surface of the stopper. The upper end of the tube water R.
has a perfectly flat, ground surface at right angles to the axis To 0.2 mL of alcoholic calcium standard solution
of the tube. A second glass tube of the same internal diameter (100 ppm Ca) R add 1 mL of ammonium oxalate solution R.
and 30 mm long, with a similar flat ground surface, is placed After 1 min add a mixture of 1 mL of dilute acetic acid R and
in contact with the first, and is held in position by two spiral 15 mL of the prescribed solution or of a solution containing
springs. Into the lower tube insert 50 mg to 60 mg of lead the prescribed quantity of the substance to be examined, and
acetate cotton R, loosely packed, or a small plug of cotton and shake. Prepare a standard in the same manner using a mixture
a rolled piece of lead acetate paper R weighing 50 mg to 60 mg. of 10 mL of aqueous calcium standard solution (10 ppm Ca) R,
Between the flat surfaces of the tubes place a disc or a small 1 mL of dilute acetic acid R and 5 mL of distilled water R.
square of mercuric bromide paper R large enough to cover the After 15 min, any opalescence in the test solution is not more
orifice of the tube (15 mm × 15 mm). intense than that in the standard.
In the conical flask dissolve the prescribed quantity of the
substance to be examined in 25 mL of water R, or in the case 01/2008:20404
of a solution adjust the prescribed volume to 25 mL with
water R. Add 15 mL of hydrochloric acid R, 0.1 mL of stannous 2.4.4. CHLORIDES
chloride solution R and 5 mL of potassium iodide solution R,
allow to stand for 15 min and introduce 5 g of activated zinc R. To 15 mL of the prescribed solution add 1 mL of dilute
Assemble the two parts of the apparatus immediately and nitric acid R and pour the mixture as a single addition into a
immerse the flask in a bath of water at a temperature such that test-tube containing 1 mL of silver nitrate solution R2. Prepare
a uniform evolution of gas is maintained. Prepare a standard a standard in the same manner using 10 mL of chloride
in the same manner, using 1 mL of arsenic standard solution standard solution (5 ppm Cl) R and 5 mL of water R. Examine
(1 ppm As) R, diluted to 25 mL with water R. the tubes laterally against a black background.
General Notices (1) apply to all monographs and other texts 127
2.4.5. Fluorides EUROPEAN PHARMACOPOEIA 8.0
After standing for 5 min protected from light, any opalescence 01/2008:20406
in the test solution is not more intense than that in the
standard. 2.4.6. MAGNESIUM
01/2008:20405 To 10 mL of the prescribed solution add 0.1 g of disodium
tetraborate R. Adjust the solution, if necessary, to pH 8.8
to pH 9.2 using dilute hydrochloric acid R or dilute sodium
2.4.5. FLUORIDES hydroxide solution R. Shake with 2 quantities, each of 5 mL,
Introduce into the inner tube of the apparatus (see of a 1 g/L solution of hydroxyquinoline R in chloroform R,
Figure 2.4.5.-1) the prescribed quantity of the substance to be for 1 min each time. Allow to stand. Separate and discard
examined, 0.1 g of acid-washed sand R and 20 mL of a mixture the organic layer. To the aqueous solution add 0.4 mL of
of equal volumes of sulfuric acid R and water R. Heat the butylamine R and 0.1 mL of triethanolamine R. Adjust the
jacket containing tetrachloroethane R maintained at its boiling solution, if necessary, to pH 10.5 to pH 11.5. Add 4 mL
point (146 °C). Heat the steam generator and distil, collecting of the solution of hydroxyquinoline in chloroform, shake
the distillate in a 100 mL volumetric flask containing 0.3 mL for 1 min, allow to stand and separate. Use the lower layer
of 0.1 M sodium hydroxide and 0.1 mL of phenolphthalein for comparison. Prepare a standard in the same manner
solution R. Maintain a constant volume (20 mL) in the tube using a mixture of 1 mL of magnesium standard solution
during distillation and ensure that the distillate remains (10 ppm Mg) R and 9 mL of water R.
alkaline, adding 0.1 M sodium hydroxide if necessary. Dilute Any colour in the solution obtained from the substance to be
the distillate to 100 mL with water R (test solution). Prepare examined is not more intense than that in the standard.
a standard in the same manner by distillation, using 5 mL
of fluoride standard solution (10 ppm F) R instead of the 01/2008:20407
substance to be examined. Into two glass-stoppered cylinders
introduce 20 mL of the test solution and 20 mL of the standard
and 5 mL of aminomethylalizarindiacetic acid reagent R.
2.4.7. MAGNESIUM AND
After 20 min, any blue colour in the test solution (originally ALKALINE-EARTH METALS
red) is not more intense than that in the standard. To 200 mL of water R add 0.1 g of hydroxylamine
hydrochloride R, 10 mL of ammonium chloride buffer solution
pH 10.0 R, 1 mL of 0.1 M zinc sulfate and about 15 mg of
mordant black 11 triturate R. Heat to about 40 °C. Titrate
with 0.01 M sodium edetate until the violet colour changes
to full blue. To the solution add the prescribed quantity of
the substance to be examined dissolved in 100 mL of water R
or use the prescribed solution. If the colour of the solution
changes to violet, titrate with 0.01 M sodium edetate until the
full blue colour is again obtained.
The volume of 0.01 M sodium edetate used in the second
titration does not exceed the prescribed quantity.
07/2010:20408
If the result is difficult to judge, filter the solutions through a – the monitor solution is at least as intense as the reference
suitable membrane filter (nominal pore size 0.45 μm). Carry solution.
out the filtration slowly and uniformly, applying moderate Result : any brown colour in the test solution is not more
and constant pressure to the piston. Compare the spots on the intense than that in the reference solution.
filters obtained with the different solutions. If the result is difficult to judge, filter the solutions through a
METHOD B suitable membrane filter (nominal pore size 0.45 μm). Carry
out the filtration slowly and uniformly, applying moderate
Test solution. 12 mL of the prescribed solution of the and constant pressure to the piston. Compare the spots on the
substance to be examined prepared using an organic solvent filters obtained with the different solutions.
containing a minimum percentage of water (for example,
dioxan containing 15 per cent of water or acetone containing METHOD D
15 per cent of water). Test solution. In a silica crucible, mix thoroughly the
Reference solution (standard). A mixture of 10 mL of lead prescribed quantity of the substance to be examined with
standard solution (1 or 2 ppm Pb), as prescribed, and 2 mL of 0.5 g of magnesium oxide R1. Ignite to dull redness until a
the prescribed solution of the substance to be examined in homogeneous white or greyish-white mass is obtained. If
an organic solvent. Prepare the lead standard solution (1 or after 30 min of ignition the mixture remains coloured, allow
2 ppm Pb) by dilution of lead standard solution (100 ppm Pb) R to cool, mix using a fine glass rod and repeat the ignition. If
with the solvent used for the substance to be examined. necessary repeat the operation. Heat at 800 °C for about 1 h.
Blank solution. A mixture of 10 mL of the solvent used for the Take up the residue in 2 quantities, each of 5 mL, of a mixture
substance to be examined and 2 mL of the prescribed solution of equal volumes of hydrochloric acid R1 and water R. Add
of the substance to be examined in an organic solvent. 0.1 mL of phenolphthalein solution R and then concentrated
ammonia R until a pink colour is obtained. Cool, add glacial
To each solution, add 2 mL of buffer solution pH 3.5 R. Mix and acetic acid R until the solution is decolorised and add 0.5 mL
add to 1.2 mL of thioacetamide reagent R. Mix immediately. in excess. Filter if necessary and wash the filter. Dilute to
Examine the solutions after 2 min. 20 mL with water R.
System suitability : the reference solution shows a slight brown Reference solution (standard). Prepare as described for the test
colour compared to the blank solution. solution using the prescribed volume of lead standard solution
Result : any brown colour in the test solution is not more (10 ppm Pb) R instead of the substance to be examined and
intense than that in the reference solution. drying in an oven at 100-105 °C. To 10 mL of the solution
If the result is difficult to judge, filter the solutions through aobtained add 2 mL of the test solution.
suitable membrane filter (nominal pore size 0.45 μm). Carry Monitor solution. Prepare as described for the test solution,
out the filtration slowly and uniformly, applying moderate adding to the substance to be examined the volume of lead
and constant pressure to the piston. Compare the spots on the standard solution (10 ppm Pb) R prescribed for preparation of
filters obtained with the different solutions. the reference solution and drying in an oven at 100-105 °C. To
10 mL of the solution obtained add 2 mL of the test solution.
METHOD C Blank solution. A mixture of 10 mL of water R and 2 mL of
Test solution. Place the prescribed quantity (not more than 2 g) the test solution.
of the substance to be examined in a silica crucible with 4 mL To 12 mL of each solution, add 2 mL of buffer solution
of a 250 g/L solution of magnesium sulfate R in dilute sulfuric pH 3.5 R. Mix and add to 1.2 mL of thioacetamide reagent R.
acid R. Mix using a fine glass rod. Heat cautiously. If the Mix immediately. Examine the solutions after 2 min.
mixture is liquid, evaporate gently to dryness on a water-bath. System suitability :
Progressively heat to ignition and continue heating until an
almost white or at most greyish residue is obtained. Carry out – the reference solution shows a slight brown colour
the ignition at a temperature not exceeding 800 °C. Allow to compared to the blank solution,
cool. Moisten the residue with a few drops of dilute sulfuric – the monitor solution is at least as intense as the reference
acid R. Evaporate, ignite again and allow to cool. The total solution.
period of ignition must not exceed 2 h. Take up the residue Result : any brown colour in the test solution is not more
in 2 quantities, each of 5 mL, of dilute hydrochloric acid R. intense than that in the reference solution.
Add 0.1 mL of phenolphthalein solution R, then concentrated If the result is difficult to judge, filter the solutions through a
ammonia R until a pink colour is obtained. Cool, add glacial suitable membrane filter (nominal pore size 0.45 μm). Carry
acetic acid R until the solution is decolorised and add 0.5 mL out the filtration slowly and uniformly, applying moderate
in excess. Filter if necessary and wash the filter. Dilute to and constant pressure to the piston. Compare the spots on the
20 mL with water R. filters obtained with the different solutions.
Reference solution (standard). Prepare as described for the test
solution, using the prescribed volume of lead standard solution METHOD E
(10 ppm Pb) R instead of the substance to be examined. To Test solution. Dissolve the prescribed quantity of the substance
10 mL of the solution obtained add 2 mL of the test solution. to be examined in 30 mL of water R or the prescribed volume.
Monitor solution. Prepare as described for the test solution, Reference solution (standard). Unless otherwise prescribed,
adding to the substance to be examined the volume of lead dilute the prescribed volume of lead standard solution
standard solution (10 ppm Pb) R prescribed for preparation (1 ppm Pb) R to the same volume as the test solution.
of the reference solution. To 10 mL of the solution obtained Prepare the filtration apparatus by adapting the barrel of a
add 2 mL of the test solution. 50 mL syringe without its piston to a support containing, on
Blank solution. A mixture of 10 mL of water R and 2 mL of the plate, a membrane filter (nominal pore size 3 μm) and
the test solution. above it a prefilter (Figure 2.4.8.-1).
To 12 mL of each solution, add 2 mL of buffer solution Transfer the test solution into the syringe barrel, put the piston
pH 3.5 R. Mix and add to 1.2 mL of thioacetamide reagent R. in place and then apply an even pressure on it until the whole
Mix immediately. Examine the solutions after 2 min. of the liquid has been filtered. In opening the support and
removing the prefilter, check that the membrane filter remains
System suitability : uncontaminated with impurities. If this is not the case replace
– the reference solution shows a slight brown colour it with another membrane filter and repeat the operation
compared to the blank solution, under the same conditions.
General Notices (1) apply to all monographs and other texts 129
2.4.8. Heavy metals EUROPEAN PHARMACOPOEIA 8.0
Test solution. Place the prescribed amount of the substance to solutions through a suitable membrane filter (nominal pore
be examined (not more than 0.5 g) in a suitable, clean beaker. size 0.45 μm). Compare the spots on the filters obtained with
Add successively 2.7 mL of sulfuric acid R, 3.3 mL of nitric the different solutions.
acid R and 2.0 mL of strong hydrogen peroxide solution R System suitability : the spot obtained with the reference
using a magnetic stirrer. Allow the substance to react with a solution shows a brownish-black colour compared to the spot
reagent before adding the next one. Transfer the mixture to obtained with the blank solution.
a dry high-pressure-resistant digestion vessel (fluoropolymer
or quartz glass). Result : the brownish-black colour of the spot obtained with
the test solution is not more intense than that of the spot
Reference solution (standard). Prepare as described for the test obtained with the reference solution.
solution, using the prescribed volume of lead standard solution
(10 ppm Pb) R instead of the substance to be examined.
Monitor solution. Prepare as prescribed for the test solution,
adding to the substance to be examined the volume of lead 01/2008:20409
standard solution (10 ppm Pb) R prescribed for the preparation
of the reference solution.
2.4.9. IRON
Blank solution. Prepare as described for the test solution,
omitting the substance to be examined. Dissolve the prescribed quantity of the substance to be
Close the vessels and place in a laboratory microwave oven. examined in water R and dilute to 10 mL with the same
Digest using a sequence of 2 separate suitable programmes. solvent or use 10 mL of the prescribed solution. Add 2 mL of
Design the programmes in several steps in order to control a 200 g/L solution of citric acid R and 0.1 mL of thioglycollic
the reaction, monitoring pressure, temperature or energy acid R. Mix, make alkaline with ammonia R and dilute to
depending on the type of microwave oven available. After the 20 mL with water R. Prepare a standard in the same manner,
first programme allow the digestion vessels to cool before using 10 mL of iron standard solution (1 ppm Fe) R.
opening. Add to each vessel 2.0 mL of strong hydrogen After 5 min, any pink colour in the test solution is not more
peroxide solution R and digest using the second programme. intense than that in the standard.
After the second programme allow the digestion vessels to
cool before opening. If necessary to obtain a clear solution,
repeat the addition of strong hydrogen peroxide solution R and
the second digestion programme. 01/2008:20410
Cool, dilute cautiously with water R and rinse into a flask,
ensuring that the total volume does not exceed 25 mL. 2.4.10. LEAD IN SUGARS
Using short-range pH indicator paper as external indicator,
adjust the solutions to pH 3.0-4.0 with concentrated Determine the lead by atomic absorption spectrometry
ammonia R1 (dilute ammonia R1 may be used as the specified (2.2.23, Method II).
range is approached). To avoid heating of the solutions use an Test solution. Dissolve 20.0 g of the substance to be examined
ice-bath and a magnetic stirrer. Dilute to 40 mL with water R in a mixture of equal volumes of dilute acetic acid R and
and mix. Add 2 mL of buffer solution pH 3.5 R. Mix and add water R and dilute to 100.0 mL with the same mixture of
to 1.2 mL of thioacetamide reagent R. Mix immediately. Dilute solvents. Add 2.0 mL of a clear 10 g/L solution of ammonium
to 50 mL with water R, mix and allow to stand for 2 min. pyrrolidinedithiocarbamate R and 10.0 mL of methyl isobutyl
Filter the solutions through a suitable membrane filter ketone R and then shake for 30 s protected from bright light.
(nominal pore size 0.45 μm). Carry out the filtration slowly Allow the layers to separate and use the methyl isobutyl
and uniformly, applying moderate and constant pressure to ketone layer.
the piston. Compare the spots on the filters obtained with the Reference solutions. Prepare 3 reference solutions in the same
different solutions. manner as the test solution but adding 0.5 mL, 1.0 mL and
System suitability : 1.5 mL respectively of lead standard solution (10 ppm Pb) R in
addition to the 20.0 g of the substance to be examined.
– the spot obtained with the reference solution shows a
brown colour compared to the spot obtained with the blank Set the zero of the instrument using methyl isobutyl ketone R
solution, treated as described for the test solution without the substance
to be examined. Measure the absorbance at 283.3 nm using
– the spot obtained with the monitor solution is at least as
a lead hollow-cathode lamp as source of radiation and an
intense as the spot obtained with the reference solution.
air-acetylene flame.
Result : the brown colour of the spot obtained with the test
The substance to be examined contains not more than 0.5 ppm
solution is not more intense than that of the spot obtained
of lead, unless otherwise prescribed.
with the reference solution.
METHOD H
Test solution. Dissolve the prescribed quantity of the substance 01/2008:20411
to be examined in 20 mL of the solvent or solvent mixture
prescribed.
Reference solution. Dilute the prescribed volume of lead 2.4.11. PHOSPHATES
standard solution (10 ppm Pb) R to 20 mL with the solvent or To 100 mL of the solution prepared and, if necessary,
solvent mixture prescribed. neutralised as prescribed add 4 mL of sulfomolybdic
Blank solution. 20 mL of the solvent or solvent mixture reagent R3. Shake and add 0.1 mL of stannous chloride
prescribed. solution R1. Prepare a standard in the same manner using
To each solution, add 2 mL of buffer solution pH 3.5 R. Mix. 2 mL of phosphate standard solution (5 ppm PO4) R and 98 mL
(In some cases precipitation occurs, in which case the specific of water R. After 10 min, compare the colours using 20 mL
monograph would describe re-dissolution in a defined volume of each solution.
of a given solvent.) Add to 1.2 mL of thioacetamide reagent R. Any colour in the test solution is not more intense than that
Mix immediately and allow to stand for 2 min. Filter the in the standard.
General Notices (1) apply to all monographs and other texts 131
2.4.12. Potassium EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 133
2.4.20. Determination of metal catalyst or metal reagent residues EUROPEAN PHARMACOPOEIA 8.0
test of the method using such glassware have experimentally physicochemical methods, the concentration of a given metal
demonstrated that the method is suitable for the intended in the sample can be calculated from the concentration of the
purpose. metal in the solution using the following expression :
CAUTION : when using high-pressure digestion vessels and
microwave laboratory equipment, the safety precautions and
operating instructions given by the manufacturer must be
followed. C = concentration of metal in the analysed sample, in
MEASUREMENT micrograms per gram ;
A = instrument reading of the concentration of the
Method. The choice of the techniques depends mainly on
the sample matrix and the characteristics and specification metal in the sample solution, in micrograms per
limits of the metal(s) of interest. Analyse according to the millilitre ;
instructions of the manufacturer of the apparatus regarding m = mass of the sample in the initial sample solution,
programme and wavelength. in grams ;
System suitability. A system suitability test must be carried V1 = volume of the initial sample preparation, in
out on the day of the analysis to ensure that the sample millilitres ;
preparation and measurement system are appropriate. V2 = total volume of any dilution performed, in
Acceptance criterion for preparation of sample solution : a clear millilitres ;
solution is obtained. V3 = volume of initial sample preparation used in any
Acceptance criterion for measurement system : the measured dilution performed, in millilitres.
concentration of a standard solution of the metal at a
concentration within the range of the used calibration curve VALIDATION REQUIREMENTS
does not differ from the actual concentration by more than Some validation requirements provided below may differ from
20 per cent. those provided in general chapters of the Ph. Eur. (e.g. 2.2.22
Calculation. The blank value of reagents must be taken into (AES), 2.2.23 (AAS), 2.2.57 (ICP-AES), 2.2.58 (ICP-MS)).
account for the calculation of the content. Upon completion of Before the initial use of the selected procedure, the analyst
the analysis, the concentration of a given metal in the sample must ensure that the sample preparation and measurement
is calculated by the software of the instrument from the method are appropriate for the metal(s), sample matrix and
concentration of the metal in the test solution. If no calculation instrument used. This is accomplished by following the
software is available or no indication for calculation is given in validation procedure before the initial use and the system
the corresponding general chapter in section 2.2. Physical and suitability test on the day of the analysis.
General Notices (1) apply to all monographs and other texts 135
2.4.21. Foreign oils in fatty oils by thin-layer chromatography EUROPEAN PHARMACOPOEIA 8.0
– resolution : minimum 1.8 between the peaks due to methyl Methyl laurate R 20
oleate and methyl stearate in the chromatogram obtained Methyl myristate R 40
with reference solution (a) ;
General Notices (1) apply to all monographs and other texts 137
2.4.22. Composition of fatty acids by GC EUROPEAN PHARMACOPOEIA 8.0
Table 2.4.22.-3. – Mixture of calibrating substances (for gas Fatty acid Equivalent chain length
chromatography with capillary column and split inlet system, Alpha-linolenic acid 19.2
it is recommended that the component with the longest chain
length of the mixture to be examined be added to the calibration Arachidic acid 20.0
mixture, when the qualitative analysis is done using calibration Eicosenoic acid (gondoic acid) 20.2
curves)
Arachidonic acid 21.2
Mixture of the following substances Composition (per cent m/m)
Behenic acid 22.0
Methyl myristate R 5
Erucic acid 22.2
Methyl palmitate R 10
12-Oxostearic acid 22.7
Methyl stearate R 15
Ricinoleic acid 23.9
Methyl arachidate R 20
12-Hydroxystearic acid 23.9
Methyl oleate R 20
Lignoceric acid 24.0
Methyl eicosenoate R 10
Nervonic acid 24.2
Methyl behenate R 10
Methyl lignocerate R 10
METHOD B
Measure the reduced retention time (t′ R) of each peak in the This method is not applicable to oils that contain glycerides
chromatogram obtained with reference solution (a). t′ R is the of fatty acids with an epoxy-, hydroepoxy-, hydroperoxy-,
retention time measured from the solvent peak and not from cyclopropyl or cyclopropenyl group or to oils with an acid value
the time of injection. Plot the straight line : greater than 2.0.
Test solution. Introduce 0.100 g of the substance to be
equivalent chain length examined into a 10 mL centrifuge tube with a screw cap.
Dissolve with 1 mL of heptane R and 1 mL of dimethyl
The logarithms of t′ R of unsaturated acids are situated on this carbonate R and mix vigorously under gentle heating
line at points corresponding to non-integer values of carbon (50-60 °C). Add, while still warm, 1 mL of a 12 g/L solution
atoms known as ‘equivalent chain lengths’ ; the equivalent of sodium R in anhydrous methanol R, prepared with the
chain length is the length of the theoretical saturated chain necessary precautions, and mix vigorously for about 5 min.
that would have the same t′ R as the fatty acid to be identified. Add 3 mL of distilled water R and mix vigorously for about
For example, linoleic acid has the same t′ R as the theoretical 30 s. Centrifuge for 15 min at 1500 g. Inject 1 μL of the
saturated fatty acid having 18.8 carbon atoms. organic phase.
Identify the peaks in the chromatogram obtained with the Reference solutions and assessment of chromatograms. Where
test solution by means of the straight line and the reduced there is no specific prescription in the individual monograph,
retention times. Equivalent chain lengths are given in proceed as described under Method A.
Table 2.4.22.-4.
Column :
Quantitative analysis. In general, the normalisation
procedure is used in which the sum of the areas of the peaks in – material : fused silica ;
the chromatogram, except that of the solvent, is set at 100 per – size : l = 30 m, Ø = 0.25 mm ;
cent. The content of a constituent is calculated by determining – stationary phase : macrogol 20 000 R (film thickness
the area of the corresponding peak as a percentage of the sum 0.25 μm).
of the areas of all the peaks. Disregard any peak with an area Carrier gas : helium for chromatography R.
less than 0.05 per cent of the total area.
Flow rate : 0.9 mL/min.
In certain cases, for example in the presence of fatty acids
Split ratio : 1:100.
with 12 or less carbon atoms, correction factors can be
prescribed in the individual monograph to convert peak areas Temperature :
in per cent m/m. Time Temperature
Table 2.4.22.-4. – Equivalent chain lengths (this value, which is (min) (°C)
to be calculated using calibration curves, is given as an example Column 0 - 15 100
for a column of macrogol 20 000 R) 15 - 36 100 → 225
Fatty acid Equivalent chain length 36 - 61 225
Caproic acid 6.0
Injection port 250
Caprylic acid 8.0
Detector 250
Capric acid 10.0
Detection : flame ionisation.
Lauric acid 12.0
Injection : 1 μL.
Myristic acid 14.0
METHOD C
Palmitic acid 16.0
This method is not applicable to oils that contain glycerides of
Palmitoleic acid 16.3 fatty acids with epoxy-, hydroepoxy-, hydroperoxy-, aldehyde,
ketone, cyclopropyl and cyclopropenyl groups, and conjugated
Margaric acid 17.0
polyunsaturated and acetylenic compounds because of partial
Stearic acid 18.0 or complete destruction of these groups.
Oleic acid 18.3 Test solution. Dissolve 0.10 g of the substance to be
examined in 2 mL of a 20 g/L solution of sodium hydroxide R
Linoleic acid 18.8 in methanol R in a 25 mL conical flask and boil under
Gamma-linolenic acid 19.0 a reflux condenser for 30 min. Add 2.0 mL of boron
trifluoride-methanol solution R through the condenser and
General Notices (1) apply to all monographs and other texts 139
2.4.23. Sterols in fatty oils EUROPEAN PHARMACOPOEIA 8.0
Table 2.4.23.-1. – Relative retentions of sterols with reference to Simultaneously prepare under the same conditions the
β-sitosterol for 2 different columns unsaponifiable matter of sunflower oil R. This will in particular
Poly(cyanopropyl)(7)- Poly[methyl(95)-
serve to locate the sterol fraction to be collected.
(phenyl)(7)- phenyl(5)]siloxane Separation of the sterol fraction (LC)
(methyl)(86)siloxane
Cholesterol 0.64 0.63 Liquid chromatography (2.2.29).
Test solution. Take up the residue with 3 quantities, each
Brassicasterol 0.70 0.71
of 4 mL, of the solvent used during the preparation of the
24-Methylenecholesterol 0.79 0.80 unsaponifiable matter (generally ether R or light petroleum R)
and transfer to a 15 mL tube. Evaporate to dryness under
Campesterol 0.82 0.81
a current of nitrogen R. Dissolve the residue in a volume
Campestanol 0.83 0.82 of mobile phase sufficient to obtain a solution with an
approximate concentration of 40 mg/mL. Add a few drops of
Stigmasterol 0.87 0.87
2-propanol R1 to improve the solubility (3 drops are normally
Δ7-Campesterol 0.93 0.92 sufficient to ensure complete solubilisation). Filter through a
membrane filter (nominal pore size 0.45 μm).
Δ5,23-Stigmastadienol 0.95 0.95
Reference solution. Proceed as described for the test solution
Clerosterol 0.96 0.96 with the unsaponifiable matter obtained with sunflower oil R.
β-Sitosterol 1 1 Precolumn :
Sitostanol 1.01 1.02 – size : l = 5 mm, Ø = 4.6 mm ;
Δ5-Avenasterol 1.03 1.03 – stationary phase : silica gel for chromatography R (5 μm)
with a pore size of 6 nm.
Δ5,24-Stigmastadienol 1.09 1.08
Column :
Δ7-Stigmastenol(1) 1.13 1.12
– size : l = 0.25 m, Ø = 4.6 mm ;
Δ7-Avenasterol 1.18 1.16
– stationary phase : silica gel for chromatography R (5 μm)
Betulin 1.4 1.4 with a pore size of 6 nm.
(1) This sterol may also be referred to as Δ7-stigmasterol in literature. Mobile phase : 2-propanol R1, hexane R (1:99 V/V).
Flow rate : 1 mL/min.
The peak due to the internal standard (betulin) must be clearly
separated from the peaks due to the sterols to be determined. Detection : spectrophotometer at 210 nm.
For the chromatogram obtained with the test solution, identify Injection : 50 μL.
the peaks and calculate the percentage content of each sterol Identification of the peaks due to sterols : the sterol fraction
in the sterol fraction of the substance to be examined using elutes at the end of the chromatogram. Locate the fraction
the following expression : to be collected using the chromatogram obtained with the
reference solution, which shows 2 principal peaks eluting
approximately between 23 min and 32 min. Collect the
fraction at the detector outlet in a 15 mL tube with a screw
cap. Evaporate the solvent under a current of nitrogen R.
A = area of the peak due to the component to be
determined ; Determination of the sterols (GC)
S = sum of the areas of the peaks due to the components Gas chromatography (2.2.28).
indicated in Table 2.4.23.-1; disregard the peak due Test solution. Dissolve the residue of the sterol fraction
to betulin. obtained with the test solution in the previous LC step in
If required in the monograph, calculate the content of each 0.2 mL of anhydrous pyridine R and 0.2 mL of a mixture
sterol in milligrams per 100 grams of the substance to be of 1 volume of chlorotrimethylsilane R and 99 volumes of
examined using the following expression : N,O-bis(trimethylsilyl)trifluoroacetamide R. Stopper the tube
tightly and heat at 80 °C for 20 min. Allow to cool and use
the liquid phase.
Reference solution. Dissolve the residue of the sterol fraction
obtained with the reference solution in the previous LC step
A = area of the peak due to the component to be in 0.2 mL of anhydrous pyridine R and 0.2 mL of a mixture
determined ; of 1 volume of chlorotrimethylsilane R and 99 volumes of
A′ = area of the peak due to betulin ; N,O-bis(trimethylsilyl)trifluoroacetamide R. Stopper the tube
m = mass of the sample of the substance to be examined, tightly and heat at 80 °C for 20 min. Allow to cool and use
the liquid phase.
in grams ;
A standard of cholesterol (cholesterol R) may also be used,
m′ = mass of betulin R added, in milligrams.
alone or as a mixture with the sterol fraction of sunflower oil.
Proceed with derivatisation as described for the test solution.
METHOD B
Column :
Preparation of the unsaponifiable matter
– material : fused silica ;
Prepare the unsaponifiable matter according to the method
– size : l = 30 m, Ø = 0.25 mm ;
stated in the test for unsaponifiable matter of the monograph
on the substance to be examined. Failing this, prepare the – stationary phase : poly[methyl(95)phenyl(5)]siloxane R (film
unsaponifiable matter according to the method described thickness 0.25 μm).
in chapter 2.5.7. Unsaponifiable matter. After the final Carrier gas : helium for chromatography R.
neutralisation step, evaporate the ethanol, then add 6 mL
of acetone R and evaporate the solvent. Dry the residue at Flow rate : 2.6 mL/min.
100-105 °C. It is not necessary to dry to constant mass. Split ratio : 1:25.
Temperature : PROCEDURE
Time Temperature Examine by gas chromatography with static head-space
(min) (°C) injection (2.2.28).
Column 0 – 38 260 Sample preparation 1. This is intended for the control of
38 – 44 260 →290 residual solvents in water-soluble substances.
44 - 49 290
Sample solution (1). Dissolve 0.200 g of the substance to be
Injection port 290
examined in water R and dilute to 20.0 mL with the same
Detector 290 solvent.
Sample preparation 2. This is intended for the control of
Detection : flame ionisation. residual solvents in water-insoluble substances.
Injection : 1-3 μL (depending on the expected amount of
Sample solution (2). Dissolve 0.200 g of the substance to
sterols in the substance to be examined).
be examined in dimethylformamide R (DMF) and dilute to
Identification of peaks : use the chromatogram obtained with 20.0 mL with the same solvent.
the reference solution to identify the peaks due to campesterol,
stigmasterol, β-sitosterol and Δ7-stigmastenol. Identify the Sample preparation 3. This is intended for the control of
peaks due to the sterols in the chromatogram obtained with N,N-dimethylacetamide and/or N,N-dimethylformamide,
the test solution using the chromatogram obtained with the when it is known or suspected that one or both of these
reference solution and the relative retentions with reference to substances are present in the substance to be examined.
β-sitosterol (main peak) given in Table 2.4.23.-1. Sample solution (3). Dissolve 0.200 g of the substance to be
System suitability: reference solution : examined in 1,3-dimethyl-2-imidazolidinone R (DMI) and
dilute to 20.0 mL with the same solvent.
– resolution : minimum 4.0 between the peaks due to
campesterol and stigmasterol. In some cases none of the above sample preparation
Calculate the percentage content of each sterol in the sterol procedures are appropriate, in which case the diluent to be
fraction of the substance to be examined using the following used for the preparation of the sample solution and the static
expression : head-space conditions to be employed must be demonstrated
to be suitable.
Solvent solution (a). To 1.0 mL of Class 1 residual solvent
solution CRS, add 9 mL of dimethyl sulfoxide R and dilute
to 100.0 mL with water R. Dilute 1.0 mL of this solution to
A = area of the peak due to the component to be 100 mL with water R. Dilute 1.0 mL of this solution to 10.0 mL
determined ; with water R.
S = sum of the areas of the peaks due to the components The reference solutions correspond to the following limits :
indicated in Table 2.4.23.-1, except betulin.
– benzene : 2 ppm,
– carbon tetrachloride : 4 ppm,
01/2008:20424
– 1,2-dichloroethane : 5 ppm,
corrected 7.2
– 1,1-dichloroethene : 8 ppm,
2.4.24. IDENTIFICATION AND – 1,1,1-trichloroethane : 10 ppm.
CONTROL OF RESIDUAL SOLVENTS Solvent solution (b). Dissolve appropriate quantities of the
Class 2 residual solvents in dimethyl sulfoxide R and dilute to
The test procedures described in this general method may 100.0 mL with water R. Dilute to give a concentration of 1/20
be used : of the limits stated in Table 2 (see 5.4. Residual solvents).
i. for the identification of the majority of Class 1 and Class 2 Solvent solution (c). Dissolve 1.00 g of the solvent or solvents
residual solvents in an active substance, excipient or medicinal present in the substance to be examined in dimethyl sulfoxide R
product when the residual solvents are unknown ; or water R, if appropriate, and dilute to 100.0 mL with water R.
ii. as a limit test for Class 1 and Class 2 solvents when present Dilute to give a concentration of 1/20 of the limit(s) stated in
in an active substance, excipient or medicinal product ; Table 1 or 2 (see 5.4. Residual solvents).
iii. for the quantification of Class 2 solvents when the limits are Blank solution. Prepare as described for solvent solution (c)
greater than 1000 ppm (0.1 per cent) or for the quantification but without the addition of solvent(s) (used to verify the
of Class 3 solvents when required. absence of interfering peaks).
Class 1, Class 2 and Class 3 residual solvents are listed in Test solution. Introduce 5.0 mL of the sample solution and
general chapter 5.4. Residual solvents. 1.0 mL of the blank solution into an injection vial.
Three diluents are described for sample preparation and Reference solution (a) (Class 1). Introduce 1.0 mL of solvent
the conditions to be applied for head-space injection of the solution (a) and 5.0 mL of the appropriate diluent into an
gaseous sample onto the chromatographic system. Two injection vial.
chromatographic systems are prescribed but System A Reference solution (a1) (Class 1). Introduce 5.0 mL of the
is preferred whilst System B is employed normally for sample solution and 1.0 mL of solvent solution (a) into an
confirmation of identity. The choice of sample preparation injection vial.
procedure depends on the solubility of the substance to be
examined and in certain cases the residual solvents to be Reference solution (b) (Class 2). Introduce 1.0 mL of solvent
controlled. solution (b) and 5.0 mL of the appropriate diluent into an
injection vial.
The following residual solvents are not readily detected by
the head-space injection conditions described : formamide, Reference solution (c). Introduce 5.0 mL of the sample solution
2-ethoxyethanol, 2-methoxyethanol, ethylene glycol, and 1.0 mL of solvent solution (c) into an injection vial.
N-methylpyrrolidone and sulfolane. Other appropriate Reference solution (d). Introduce 1.0 mL of the blank solution
procedures should be employed for the control of these and 5.0 mL of the appropriate diluent into an injection vial.
residual solvents. Close the vials with a tight rubber membrane stopper coated
When the test procedure is applied quantitatively to control with polytetrafluoroethylene and secure with an aluminium
residual solvents in a substance, then it must be validated. crimped cap. Shake to obtain a homogeneous solution.
General Notices (1) apply to all monographs and other texts 141
2.4.24. Identification and control of residual solvents EUROPEAN PHARMACOPOEIA 8.0
The following static head-space injection conditions may be determined. The system is suitable if the chromatogram
used : obtained resembles the chromatogram shown in
Figure 2.4.24.-2 and the resolution between acetonitrile and
Sample preparation methylene chloride is at least 1.0.
procedure
Operating parameters 1 2 3 Inject 1 mL of the gaseous phase of the test solution onto
the column described in System A. If in the chromatogram
Equilibration temperature (°C) 80 105 80 obtained, there is no peak which corresponds to one of
Equilibration time (min) 60 45 45 the residual solvent peaks in the chromatograms obtained
with reference solution (a) or (b), then the substance to be
Transfer-line temperature (°C) 85 110 105 examined meets the requirements of the test. If any peak in
Carrier gas : Nitrogen for chromatography R or Helium for chromatography R
the chromatogram obtained with the test solution corresponds
at an appropriate pressure to any of the residual solvent peaks obtained with reference
Pressurisation time (s) 30 30 30 solution (a) or (b) then System B is to be employed.
Injection volume (mL) 1 1 1 Inject 1 mL of the gaseous phase of reference solution (a)
onto the column described in System B and record the
The chromatographic procedure may be carried out using : chromatogram under such conditions that the signal-to-noise
ratio for benzene can be measured. The signal-to-noise ratio
SYSTEM A must be at least 5. A typical chromatogram is shown in
Figure 2.4.24.-3.
– a fused-silica capillary or wide-bore column 30 m long and
0.32 mm or 0.53 mm in internal diameter coated with Inject 1 mL of the gaseous phase of reference solution (a1)
cross-linked 6 per cent polycyanopropylphenylsiloxane and onto the column described in System B. The peaks due to the
94 per cent polydimethylsiloxane (film thickness : 1.8 μm Class I residual solvents are still detectable.
or 3 μm),
Inject 1 mL of the gaseous phase of reference solution (b)
– nitrogen for chromatography R or helium for onto the column described in System B and record the
chromatography R as the carrier gas, split ratio 1:5 with a chromatogram under such conditions that the resolution
linear velocity of about 35 cm/s, between acetonitrile and trichloroethene can be determined.
The system is suitable if the chromatogram obtained
– a flame-ionisation detector (a mass spectrometer may also resembles the chromatogram shown in Figure 2.4.24.-4 and
be used or an electron-capture detector for the chlorinated the resolution between acetonitrile and trichloroethene is at
residual solvents of Class 1), least 1.0.
maintaining the temperature of the column at 40 °C for
20 min, then raising the temperature at a rate of 10 °C Inject 1 mL of the gaseous phase of the test solution onto
per min to 240 °C and maintaining it at 240 °C for 20 min the column described in System B. If in the chromatogram
and maintaining the temperature of the injection port at obtained, there is no peak which corresponds to any of the
140 °C and that of the detector at 250 °C, or, where there is residual solvent peaks in the chromatogram obtained with
interference from the matrix, use : the reference solution (a) or (b), then the substance to be
SYSTEM B examined meets the requirements of the test. If any peak in
the chromatogram obtained with the test solution corresponds
– a fused-silica capillary or wide-bore column 30 m long and to any of the residual solvent peaks obtained with reference
0.32 mm or 0.53 mm in internal diameter coated with solution (a) or (b) and confirms the correspondence obtained
macrogol 20 000 R (film thickness : 0.25 μm), when using System A, then proceed as follows.
– nitrogen for chromatography R or helium for Inject 1 mL of the gaseous phase of reference solution (c) onto
chromatography R as the carrier gas, split ratio 1:5 with a the column described for System A or System B. If necessary,
linear velocity of about 35 cm/s. adjust the sensitivity of the system so that the height of the
peak corresponding to the identified residual solvent(s) is at
– a flame-ionisation detector (a mass spectrophotometer least 50 per cent of the full scale of the recorder.
may also be used or an electron-capture detector for the
chlorinated residual solvents of Class 1), Inject 1 mL of the gaseous phase of reference solution (d) onto
maintaining the temperature of the column at 50 °C for the column. No interfering peaks should be observed.
20 min, then raising the temperature at a rate of 6 °C per min
to 165 °C and maintaining it at 165 °C for 20 min and Inject 1 mL of the gaseous phase of the test solution and
maintaining the temperature of the injection port at 140 °C 1 mL of the gaseous phase of reference solution (c) on to the
and that of the detector at 250 °C. column. Repeat these injections twice more.
Inject 1 mL of the gaseous phase of reference solution (a) The mean area of the peak of the residual solvent(s) in the
onto the column described in System A and record the chromatograms obtained with the test solution is not greater
chromatogram under such conditions that the signal-to-noise than half the mean area of the peak of the corresponding
ratio for 1,1,1-trichloroethane can be measured. The residual solvent(s) in the chromatograms obtained with
signal-to-noise ratio must be at least 5. A typical reference solution (c). The test is not valid unless the relative
chromatogram is shown in Figure 2.4.24.-1. standard deviation of the differences in areas between the
analyte peaks obtained from 3 replicate paired injections of
Inject 1 mL of the gaseous phase of reference solution (a1) reference solution (c) and the test solution, is at most 15 per
onto the column described in System A. The peaks due to the cent.
Class 1 residual solvents are still detectable.
A flow diagram of the procedure is shown in Figure 2.4.24.-5.
Inject 1 mL of the gaseous phase of reference solution (b)
onto the column described in System A and record the When a residual solvent (Class 2 or Class 3) is present at a level
chromatogram under such conditions that the resolution of 0.1 per cent or greater then the content may be quantitatively
between acetonitrile and methylene chloride can be determined by the method of standard additions.
Figure 2.4.24.-1. – Typical chromatogram of class 1 solvents using the conditions described for System A and Procedure 1.
Flame-ionisation detector.
2. acetonitrile 6. nitromethane 10. 1,1,2-trichloroethene 14. toluene 17. xylene ortho, meta, para
Figure 2.4.24.-2. – Chromatogram of Class 2 solvents using the conditions described for System A and Procedure 1.
Flame-ionisation detector.
General Notices (1) apply to all monographs and other texts 143
2.4.24. Identification and control of residual solvents EUROPEAN PHARMACOPOEIA 8.0
Figure 2.4.24.-3. – Chromatogram of Class 1 residual solvents using the conditions described for System B and Procedure 1.
Flame-ionisation detector.
2. acetonitrile 6. nitromethane 10. 1,1,2-trichloroethene 14. toluene 17. xylene ortho, meta, para
3. dichloromethane 7. chloroform 11. methylcyclohexane 15. 2-hexanone 18. tetralin (tR = 28 min)
Figure 2.4.24.-4. – Typical chromatogram of class 2 residual solvents using the conditions described for System B and Procedure 1.
Flame-ionisation detector.
Figure 2.4.24.-5. – Diagram relating to the identification of residual solvents and the application of limit tests
01/2014:20425 Head-space gas chromatography (2.2.28).
A. For samples soluble in or miscible with water, the following
2.4.25. ETHYLENE OXIDE AND procedure may be used.
DIOXAN Test solution. Weigh 1.00 g (MT) of the substance to
be examined in a 10 mL vial (other sizes may be used
The test is intended for the determination of residual depending on the operating conditions) and add 1.0 mL of
ethylene oxide and dioxan in samples soluble in water or water R. Close and mix to obtain a homogeneous solution.
dimethylacetamide. For substances that are insoluble or Allow to stand at 70 °C for 45 min.
insufficiently soluble in these solvents, the preparation of the
sample solution and the head-space conditions to be employed
are given in the individual monograph.
General Notices (1) apply to all monographs and other texts 145
2.4.26. N,N-Dimethylaniline EUROPEAN PHARMACOPOEIA 8.0
Reference solution (a). Weigh 1.00 g (MR) of the substance – signal-to-noise ratio : minimum 5 for the peaks due to
to be examined into an identical 10 mL vial, add 0.10 mL ethylene oxide and dioxan.
of dioxan solution R1 and 0.50 mL of ethylene oxide Verification of precision
solution R3. Close and mix to obtain a homogeneous
solution. Allow to stand at 70 °C for 45 min. For each pair of injections, calculate for ethylene oxide and
for dioxan the difference in area between the peaks obtained
Reference solution (b). To 0.50 mL of ethylene oxide with the test solution and reference solution (a). The test is
solution R3 in a 10 mL vial add 0.1 mL of a freshly prepared not valid unless the relative standard deviation of the 3 values
10 mg/L solution of acetaldehyde R and 0.10 mL of dioxan obtained for ethylene oxide is not greater than 15 per cent and
solution R1. Close and mix to obtain a homogeneous the relative standard deviation of the 3 values obtained for
solution. Allow to stand at 70 °C for 45 min. dioxan is not greater than 15 per cent. If the weighings used
B. For samples soluble in or miscible with dimethylacetamide, for the test solution and reference solution (a) differ from
the following procedure may be used. 1.00 g by more than 0.5 per cent, the appropriate corrections
Test solution. Weigh 1.00 g (MT) of the substance to must be made.
be examined in a 10 mL vial (other sizes may be used Calculate the content of ethylene oxide or dioxan in parts per
depending on the operating conditions) and add 0.20 mL million from the following expressions :
of water R and 1.0 mL of dimethylacetamide R. Close and
mix to obtain a homogeneous solution. Allow to stand at
90 °C for 45 min.
Reference solution (a). Weigh 1.00 g (MR) of the substance
to be examined into an identical 10 mL vial, add 0.10 mL AT = area of the peak due to ethylene oxide in the
of dioxan solution R1, 0.10 mL of ethylene oxide solution R2 chromatogram obtained with the test solution ;
and 1.0 mL of dimethylacetamide R. Close and mix to AR = area of the peak due to ethylene oxide in
obtain a homogeneous solution. Allow to stand at 90 °C the chromatogram obtained with reference
for 45 min. solution (a) ;
Reference solution (b). To 0.10 mL of ethylene oxide MT = mass of the substance to be examined in the test
solution R2 in a 10 mL vial, add 0.1 mL of a freshly prepared solution, in grams ;
10 mg/L solution of acetaldehyde R and 0.10 mL of dioxan MR = mass of the substance to be examined in reference
solution R1. Close and mix to obtain a homogeneous
solution (a), in grams ;
solution. Allow to stand at 70 °C for 45 min.
Column : C = amount of ethylene oxide added to reference
solution (a), in micrograms.
– material : glass or fused silica ;
– size : l = 30 m, Ø = 0.32 mm ;
– stationary phase : poly(dimethyl)siloxane R (film thickness
1.0 μm).
Carrier gas : helium for chromatography R or nitrogen for DT = area of the peak due to dioxan in the chromatogram
chromatography R. obtained with the test solution ;
Linear velocity : 20 cm/s. DR = area of the peak due to dioxan in the chromatogram
Split ratio : 1:20. obtained with reference solution (a) ;
Static head-space conditions that may be used : C = amount of dioxan added to reference solution (a)
in micrograms.
– equilibration temperature : 70 °C (90 °C for solutions in
dimethylacetamide) ;
– equilibration time : 45 min ;
– transfer-line temperature : 75 °C (150 °C for solutions in 01/2008:20426
dimethylacetamide) ;
– carrier gas : helium for chromatography R ; 2.4.26. N,N-DIMETHYLANILINE
– pressurisation time : 1 min ;
METHOD A
– injection time : 12 s.
Examine by gas chromatography (2.2.28), using
Temperature : N,N-diethylaniline R as the internal standard.
Time Temperature Internal standard solution. Dissolve 50 mg of
(min) (°C) N,N-diethylaniline R in 4 mL of 0.1 M hydrochloric
Column 0-5 50 acid and dilute to 50 mL with water R. Dilute 1 mL of this
5 - 31 50 → 180 solution to 100 mL with water R.
Test solution. Dissolve in a ground-glass-stoppered tube 0.50 g
31 - 32.5 180 → 230
of the substance to be examined in 30.0 mL of water R. Add
32.5 - 37.5 230 1.0 mL of the internal standard solution. Adjust the solution
to a temperature of 26-28 °C. Add 1.0 mL of strong sodium
Injection port 150
hydroxide solution R and mix until completely dissolved. Add
Detector 250 2.0 mL of trimethylpentane R. Shake for 2 min and allow the
phases to separate. Use the upper layer.
Detection : flame ionisation. Reference solution. Dissolve 50.0 mg of N,N-dimethylaniline R
Injection : a suitable volume, for example 1.0 mL, of the in 4.0 mL of 0.1 M hydrochloric acid and dilute to 50.0 mL
gaseous phase of the test solution and of reference solutions (a) with water R. Dilute 1.0 mL of this solution to 100.0 mL
and (b). Repeat the procedure twice more. with water R. Dilute 1.0 mL of this solution to 30.0 mL with
System suitability : reference solution (b) : water R. Add 1.0 mL of the internal standard solution and
1.0 mL of strong sodium hydroxide solution R. Add 2.0 mL of
– resolution : minimum 2.0 between the peaks due to trimethylpentane R. Shake for 2 min and allow the phases to
acetaldehyde and ethylene oxide ; separate. Use the upper layer.
The chromatographic procedure may be carried out using : CAUTION : when using closed high-pressure digestion vessels
and microwave laboratory equipment, be familiar with the
– a fused-silica capillary column 25 m long and
safety and operating instructions given by the manufacturer.
0.32 mm in internal diameter coated with cross-linked
polymethylphenylsiloxane R (film thickness 0.52 μm),
– helium for chromatography R as the carrier gas with a split APPARATUS
ratio 1:20, a column head pressure of 50 kPa and a split The apparatus typically consists of the following :
vent of 20 mL/min,
– as digestion flasks, polytetrafluoroethylene flasks with a
– a flame-ionisation detector, volume of about 120 mL, fitted with an airtight closure,
– a split-liner consisting of a column about 1 cm a valve to adjust the pressure inside the container and a
long packed with diatomaceous earth for gas polytetrafluoroethylene tube to allow release of gas,
chromatography R impregnated with 10 per cent m/m of – a system to make flasks airtight, using the same torsional
poly(dimethyl)siloxane R, force for each of them,
maintaining the temperature of the column at 150 °C for
– a microwave oven, with a magnetron frequency of
5 min, then raising the temperature at a rate of 20 °C
2450 MHz, with a selectable output from 0 to 630 ± 70 W
per min to 275 °C and maintaining it at 275 °C for 3 min and
in 1 per cent increments, a programmable digital computer,
maintaining the temperature of the detector at 300 °C and that
a polytetrafluoroethylene-coated microwave cavity with
of the injection port at 220 °C.
a variable speed exhaust fan, a rotating turntable drive
The retention times are : N,N-dimethylaniline about 3.6 min, system and exhaust tubing to vent fumes,
N,N-diethylaniline about 5.0 min.
– an atomic absorption spectrometer, equipped with
Inject 1 μL of the test solution and 1 μL of the reference hollow-cathode lamps as source of radiation and a
solution. deuterium lamp as background corrector ; the system is
fitted with :
METHOD B (a) a graphite furnace as atomisation device for cadmium,
Examined by gas chromatography (2.2.28), using copper, iron, lead, nickel and zinc.
naphthalene R as the internal standard.
(b) an automated continuous-flow hydride vapour
Internal standard solution. Dissolve 50 mg of naphthalene R generation system for arsenic and mercury.
in cyclohexane R and dilute to 50 mL with the same solvent.
Dilute 5 mL of this solution to 100 mL with cyclohexane R.
METHOD
Test solution. To 1.00 g of the substance to be examined
in a ground-glass-stoppered tube add 5 mL of 1 M sodium In case alternative apparatus is used, an adjustment of the
hydroxide and 1.0 mL of the internal standard solution. instrument parameters may be necessary.
Stopper the tube and shake vigorously for 1 min. Centrifuge if Clean all the glassware and laboratory equipment with a
necessary and use the upper layer. 10 g/L solution of nitric acid R before use.
Reference solution. To 50.0 mg of N,N-dimethylaniline R add Test solution. In a digestion flask place the prescribed quantity
2 mL of hydrochloric acid R and 20 mL of water R, shake to of the substance to be examined (about 0.50 g of powdered
dissolve and dilute to 50.0 mL with water R. Dilute 5.0 mL drug (1400) (2.9.12) or 0.50 g of fatty oil). Add 6 mL of
of this solution to 250.0 mL with water R. To 1.0 mL of the heavy metal-free nitric acid R and 4 mL of heavy metal-free
latter solution in a ground-glass-stoppered tube add 5 mL of hydrochloric acid R. Make the flask airtight.
1 M sodium hydroxide and 1.0 mL of the internal standard
solution. Stopper the tube and shake vigorously for 1 min. Place the digestion flasks in the microwave oven. Carry out
Centrifuge if necessary and use the upper layer. the digestion in 3 steps according to the following programme,
The chromatographic procedure may be carried out using : used for 7 flasks each containing the test solution : 80 per cent
power for 15 min, 100 per cent power for 5 min, 80 per cent
– a glass column 2 m long and 2 mm in internal diameter power for 20 min.
packed with silanised diatomaceous earth for gas
chromatography R impregnated with 3 per cent m/m of At the end of the cycle allow the flasks to cool in air and to
polymethylphenylsiloxane R, each add 4 mL of heavy metal-free sulfuric acid R. Repeat
the digestion programme. After cooling in air, open each
– nitrogen for chromatography R as the carrier gas at a flow digestion flask and introduce the clear, colourless solution
rate of 30 mL/min, obtained into a 50 mL volumetric flask. Rinse each digestion
– a flame-ionisation detector, flask with 2 quantities, each of 15 mL, of water R and collect
the rinsings in the volumetric flask. Add 1.0 mL of a 10 g/L
maintaining the temperature of the column at 120 °C and that solution of magnesium nitrate R and 1.0 mL of a 100 g/L
of the injection port and of the detector at 150 °C. solution of ammonium dihydrogen phosphate R and dilute to
Inject 1 μL of the test solution and 1 μL of the reference 50.0 mL with water R.
solution. Blank solution. Mix 6 mL of heavy metal-free nitric acid R and
4 mL of heavy metal-free hydrochloric acid R in a digestion
flask. Carry out the digestion in the same manner as for the
test solution.
General Notices (1) apply to all monographs and other texts 147
2.4.28. 2-Ethylhexanoic acid EUROPEAN PHARMACOPOEIA 8.0
01/2008:20428
2.4.29. COMPOSITION OF FATTY
ACIDS IN OILS RICH IN OMEGA-3
2.4.28. 2-ETHYLHEXANOIC ACID ACIDS
Examine by gas chromatography (2.2.28), using The assay may be used for quantitative determination of the
3-cyclohexylpropionic acid R as the internal standard. EPA and DHA content in omega-3-containing products of
Internal standard solution. Dissolve 100 mg of fish oil in different concentrations. The method is applicable
3-cyclohexylpropionic acid R in cyclohexane R and dilute to to triglycerides or ethyl esters and the results are expressed as
100 mL with the same solvent. triglycerides or ethyl esters, respectively.
EPA AND DHA Reference solution (c). Into a 10 mL volumetric flask dissolve
Gas chromatography (2.2.28). Carry out the operations as a sample containing about 55.0 mg of docosahexaenoic acid
rapidly as possible, avoiding exposure to actinic light, oxidising methyl ester R and about 5.0 mg of tetracos-15-enoic acid
agents, oxidation catalysts (for example, copper and iron) and methyl ester R in a 50 mg/L solution of butylhydroxytoluene R
air. in trimethylpentane R and dilute to 10.0 mL with the same
The assay is carried out on the methyl or ethyl esters of solution.
(all-Z)-eicosa-5,8,11,14,17-pentaenoic acid (EPA ; 20:5 n-3) Column :
and (all-Z)-docosa-4,7,10,13,16,19-hexaenoic acid (DHA ; – material : fused silica ;
22:6 n-3) in the substance to be examined. – dimensions: l = at least 25 m, Ø = 0.25 mm ;
Internal standard. Methyl tricosanoate R. – stationary phase : bonded macrogol 20 000 R (film thickness
Test solution (a) 0.2 μm).
A. Dissolve the mass of sample to be examined according Carrier gas : hydrogen for chromatography R or helium for
to Table 2.4.29.-1 and about 70.0 mg of the internal chromatography R.
standard in a 50 mg/L solution of butylhydroxytoluene R in Flow rate : 1 mL/min.
trimethylpentane R and dilute to 10.0 mL with the same
Split ratio : 1:200, alternatively splitless with temperature
solution. Gentle heating (up to 60 °C) may be applied to
control (sample solutions need to be diluted 1/200
dissolve the internal standard.
with a 50 mg/L solution of butylhydroxytoluene R in
Table 2.4.29.-1. trimethylpentane R before injection).
Approximate sum EPA + DHA Mass of sample to be examined Temperature :
(per cent) (g) Time Temperature
30 - 50 0.4 - 0.5 (min) (°C)
50 - 70 0.3 Column 0-2 170
25.7 - 28 240
Ethyl esters are now ready for analysis. For triglycerides
continue as described in step B. Injection port 250
B. Introduce 2.0 mL of the solution obtained in step A into Detector 270
a quartz tube and evaporate the solvent with a gentle
current of nitrogen R. Add 1.5 mL of a 20 g/L solution of Detection : flame ionisation.
sodium hydroxide R in methanol R, cover with nitrogen R, Injection : 1 μL, twice.
cap tightly with a polytetrafluoroethylene-lined cap, mix
and heat on a water-bath for 7 min. Allow to cool. Add System suitability :
2 mL of boron trichloride-methanol solution R, cover with – in the chromatogram obtained with reference solution (b),
nitrogen R, cap tightly, mix and heat on a water-bath for the area per cent composition increases in the following
30 min. Cool to 40-50 °C, add 1 mL of trimethylpentane R, order : methyl palmitate, methyl stearate, methyl arachidate,
cap and shake vigorously for at least 30 s. Immediately methyl behenate ; the difference between the percentage
add 5 mL of a saturated sodium chloride solution R, cover area of methyl palmitate and that of methyl behenate is less
with nitrogen R, cap and shake thoroughly for at least 15 s. than 2.0 area per cent units ;
Transfer the upper layer to a separate tube. Shake the – resolution : minimum of 1.2 between the peaks due to
methanol layer once more with 1 mL of trimethylpentane R. docosahexaenoic acid methyl ester and to tetracos-15-enoic
Wash the combined trimethylpentane extracts with 2 acid methyl ester in the chromatogram obtained with
quantities, each of 1 mL, of water R and dry over anhydrous reference solution (c) ;
sodium sulfate R. Prepare 3 solutions for each sample. – in the chromatogram obtained with test solution (a),
Test solution (b). Dissolve 0.300 g of the sample to be the peaks due to methyl tricosanoate and any
examined in a 50 mg/L solution of butylhydroxytoluene R heneicosapentaenoic acid methyl ester or ethyl ester
in trimethylpentane R and dilute to 10.0 mL with the same (C21:5) present when compared with the chromatogram
solution. Proceed as described for test solution (a). obtained with test solution (b) are clearly separated (if not,
Reference solution (a1). Dissolve about 70.0 mg of the a correction factor has to be used).
internal standard and 90.0 mg of eicosapentaenoic acid ethyl Calculate the percentage content of EPA and DHA using the
ester CRS in a 50 mg/L solution of butylhydroxytoluene R following expression and taking into account the assigned
in trimethylpentane R and dilute to 10.0 mL with the same value of the reference substances :
solution. Gentle heating (up to 60 °C) may be applied to
dissolve the internal standard.
Reference solution (a2). Dissolve 60.0 mg of docosahexaenoic
acid ethyl ester CRS and about 70.0 mg of the internal
standard in a 50 mg/L solution of butylhydroxytoluene R m1 = mass of the internal standard in test solution (a),
in trimethylpentane R and dilute to 10.0 mL with the same in milligrams ;
solution. Gentle heating (up to 60 °C) may be applied to m2
dissolve the internal standard. = mass of the sample to be examined in test
solution (a), in milligrams ;
For both reference solution (a1) and reference solution (a2)
proceed as described for test solution (a) step A when mx,3 = mass of the internal standard in reference
analysing ethyl esters. For analysis of triglycerides, continue solution (a1) (EPA determination), or in
with step B in the same manner as for test solution (a) and reference solution (a2) (DHA determination),
prepare 3 solutions for each sample. in milligrams ;
Reference solution (b). Into a 10 mL volumetric flask dissolve mx,r
0.3 g of methyl arachidate R, 0.3 g of methyl behenate R, 0.3 g of = mass of eicosapentaenoic acid ethyl ester CRS
methyl palmitate R and 0.3 g of methyl stearate R in a 50 mg/L in reference solution (a1) or docosahexaenoic
solution of butylhydroxytoluene R in trimethylpentane R and acid ethyl ester CRS in reference solution (a2),
dilute to 10.0 mL with the same solution. in milligrams ;
General Notices (1) apply to all monographs and other texts 149
2.4.30. Ethylene glycol and diethylene glycol in ethoxylated substances EUROPEAN PHARMACOPOEIA 8.0
Ax = area of the peak due to eicosapentaenoic acid Reference solution (a). Mix 30.0 mg of ethylene glycol R with
ester or docosahexaenoic acid ester in the acetone R and dilute to 100.0 mL with the same solvent. Dilute
chromatogram obtained with test solution (a) ; 1.0 mL to 10.0 mL with the internal standard solution.
Reference solution (b). Prepare a solution of diethylene glycol R
Ax,r = area of the peak due to eicosapentaenoic acid with a concentration corresponding to the prescribed limit
ester in the chromatogram obtained with and using the same solvents as for the preparation of reference
reference solution (a1) or to docosahexaenoic solution (a).
acid ester in the chromatogram obtained with Column :
reference solution (a2) ;
– material : fused silica,
A1 = area of the peak due to the internal standard – size : l = 30 m, Ø = 0.53 mm,
in the chromatogram obtained with test
– stationary phase : macrogol 20 000 R (film thickness 1 μm).
solution (a) ;
Carrier gas : helium for chromatography R.
Ax,3 = area of the peak due to the internal standard Flow rate : 10 mL/min.
in the chromatogram obtained with reference
solution (a1) (EPA determination) or with Split ratio : 1:3.
reference solution (a2) (DHA determination) ; Temperature :
C Time Temperature
= conversion factor between ethyl ester and
(min) (°C)
triglycerides,
Column 0 - 40 80 → 200
C = 1.00 for ethyl esters, 40 - 45 200 → 230
C = 0.954 for EPA, 45 - 65 230
Detector 250
TOTAL OMEGA-3 ACIDS
From the assay for EPA and DHA, calculate the Detection : flame ionisation.
percentage content of the total omega-3 acids using the Injection : 2 μL.
following expression and identifying the peaks from the
chromatograms : Relative retention with reference to 1,2-pentanediol (retention
time = about 19 min) : ethylene glycol = about 0.7 ; diethylene
glycol = about 1.3.
Zero solution. In a volumetric flask, introduce 1.0 mL of a carefully a fixed number of times, e.g. 60 times. Discard the
10 g/L solution of magnesium nitrate R, 1.0 mL of a 100 g/L aqueous phase, add 5 mL of a 3 per cent solution of potassium
solution of ammonium dihydrogen phosphate R and 12.0 mL hydroxide R to the ether phase and mix carefully 20 times.
of nickel-free nitric acid R. Dilute to 50.0 mL with water for Discard the aqueous phase, add another 5 mL of distilled
chromatography R and mix. water R and mix carefully a further 20 times. Transfer the
Method. Determine the absorbance of each solution ether phase into a small centrifuge tube, avoiding any transfer
at 232.0 nm using a suitable graphite furnace atomic of water. If an emulsion forms during the process, add a small
absorption spectrometer equipped with a background amount of sodium chloride R to get a separation of the phases.
compensation system, a pyrolytically-coated tube, and a nickel Evaporate to dryness under a gentle stream of nitrogen
hollow-cathode lamp. Maintain the drying temperature of with careful heating. Dissolve the residue in 600 μL of ethyl
the furnace at 120 °C for 35 s after a 5 s ramp, the ashing acetate R.
temperature at 1100 °C for 10 s after a 30 s ramp, the cooling Depending on the expected cholesterol content in the oil, the
temperature at 800 °C for 5 s after a 5 s decrease, and the solution is further diluted as follows :
atomisation temperature at 2600 °C for 7 s. Use the zero – content less than 3 mg/g : dilute 200 μL of the solution to
solution to set the instrument to zero. Using the calibration 2.0 mL with ethyl acetate R ;
curve, determine the concentrations of the test solution and
the blank solution from the corresponding absorptions. If – content greater than or equal to 3 mg/g : dilute 20 μL of the
necessary, dilute with the zero solution to obtain a reading solution to 2.0 mL with ethyl acetate R.
within the calibrated absorbance range. Reference solution (a). Transfer 1.0 mL of the internal
Calculate the content of Ni in micrograms per gram (ppm) standard stock/working solution and 1.0 mL of the cholesterol
using the following expression : stock/working solution, depending on the expected cholesterol
content in the oil (see Table 2.4.32.-1), to a 15 mL quartz tube
and continue as described for the test solution, starting with
“Evaporate to dryness on a heating block...”.
Reference solution (b). Mix 1.0 mL of the internal standard
c = measured concentration of Ni, in nanograms per stock solution, 1.0 mL of the cholesterol stock solution
millilitre ; and 2.0 mL of the α-tocopherol solution in a suitable flask.
f = dilution factor of the test solution ; Evaporate to dryness under a gentle stream of nitrogen with
careful heating. Dissolve the residue in ethyl acetate R and
m = mass of the substance to be examined, in grams.
dilute to 50.0 mL with the same solvent. Dilute 1.0 mL of this
solution to 10.0 mL with ethyl acetate R. The solution may be
stored in a deep-freezer for up to 6 months.
07/2010:20432
Table 2.4.32.-1. – Preparation of the test and reference solutions
2.4.32. TOTAL CHOLESTEROL IN OILS Test solution
Reference
solution (a)
Reference
solution (b)
RICH IN OMEGA-3 ACIDS less greater less greater
than than or than than or
This method may be used for the quantitative determination of 3 mg/g equal to 3 mg/g equal to
the sum of free and esterified cholesterol in products of fish oils 3 mg/g 3 mg/g
rich in omega-3 acids (as ethyl esters or triglycerides). Internal
standard – + – + +
Gas chromatography (2.2.28). stock solution
Internal standard stock solution. Dissolve 0.15 g of Internal
(5α)-cholestane R in heptane R and dilute to 50.0 mL with the standard + – + – –
working
same solvent. solution
Internal standard working solution. Prepare the solution Cholesterol
immediately before use. Dilute 1.0 mL of the internal standard – – – + +
stock solution
stock solution to 10.0 mL with heptane R. Cholesterol
Cholesterol stock solution. Dissolve 30.0 mg of cholesterol R in working – – + – –
heptane R and dilute to 10.0 mL with the same solvent. The solution
solution is dispensed into gas chromatography vials and may α-Tocopherol
– – – – +
be stored in a deep-freezer for up to 6 months. solution
Cholesterol working solution. Prepare the solution immediately Column :
before use. Dilute 1.0 mL of the cholesterol stock solution to – size : l = 30 m, Ø = 0.25 mm (film thickness 0.25 μm) ;
10.0 mL with heptane R.
– stationary phase : poly(dimethyl)(diphenyl)siloxane R.
α-Tocopherol solution. Dilute 15.0 mg of α-tocopherol CRS to
10.0 mL with heptane R. Carrier gas : helium for chromatography R.
Test solution. Weigh 0.100 g of the substance to be examined Flow rate : 1.3 mL/min.
into a 15 mL quartz tube. Add 1.0 mL of the internal standard Temperature :
stock/working solution, depending on the expected cholesterol Time Temperature
content in the oil (see Table 2.4.32.-1). (min) (°C)
Evaporate to dryness on a heating block at 50 °C under a gentle Column 0-1 170
stream of nitrogen, while mixing. Add 0.5 mL of a 50 per 1 - 38 170 → 320
cent solution of potassium hydroxide R and 3.0 mL of ethanol
(96 per cent) R. Fill the tube with nitrogen R and cap. Further 38 - 40 320
heat on the heating block at 100 °C for 1 h with stirring. Cool Injection port 320
for about 10 min and add 6 mL of distilled water R. Extract
with 4 quantities, each of 2.5 mL, of ether R, mixing each time Detector 300
for 1 min using a vortex mixer. Transfer the ether phase to
a large centrifuge tube or a separating funnel and wash the Detection : flame ionisation.
combined extracts with 5 mL of distilled water R, mixing Injection : 1 μL.
General Notices (1) apply to all monographs and other texts 151
2.4.32. Total cholesterol in oils rich in omega-3 acids EUROPEAN PHARMACOPOEIA 8.0
System suitability : reference solution (b) : m2 = mass of (5α)-cholestane in the internal standard
– resolution : minimum 1.2 between the peaks due to stock solution, in grams ;
cholesterol and α-tocopherol. F = 20 for oils with an expected cholesterol content
Calculate the content of total cholesterol, expressed as greater than or equal to 3 mg/g ; 2 for oils
milligrams of cholesterol per gram of oil, using the following with an expected cholesterol content less than
expression : 3 mg/g ;
R = response factor.
Calculate the response factor R using the following expression :
A1 = area of the peak due to cholesterol in the
chromatogram obtained with the test solution ;
A2 = area of the peak due to (5α)-cholestane in the A3 = area of the peak due to cholesterol in the
chromatogram obtained with the test solution ; chromatogram obtained with reference
m1 = mass of the substance to be examined in the solution (a) ;
test solution, in grams ; A4 = area of the peak due to (5α)-cholestane in
the chromatogram obtained with reference
solution (a) ;
m3 = mass of cholesterol in the cholesterol stock
solution, in grams.
2.5. ASSAYS appears add sufficient pyridine R to clear it, noting the volume
added. Shake the flask and replace in the water-bath for
01/2008:20501 10 min. Withdraw the flask and allow to cool. Rinse the
condenser and the walls of the flask with 5 mL of alcohol R,
previously neutralised to phenolphthalein solution R1. Titrate
2.5.1. ACID VALUE with 0.5 M alcoholic potassium hydroxide using 0.2 mL of
The acid value IA is the number that expresses, in milligrams phenolphthalein solution R1 as indicator (n1 mL of 0.5 M
the quantity of potassium hydroxide required to neutralise the alcoholic potassium hydroxide). Carry out a blank test under
free acids present in 1 g of the substance. the same conditions (n2 mL of 0.5 M alcoholic potassium
Dissolve 10.00 g of the substance to be examined, or the hydroxide).
quantity prescribed, (m g), in 50 mL of a mixture of equal
volumes of ethanol (96 per cent) R and light petroleum R3,
previously neutralised with 0.1 M potassium hydroxide
or 0.1 M sodium hydroxide, unless otherwise specified, METHOD B
using 0.5 mL of phenolphthalein solution R1 as indicator. If Introduce the prescribed quantity of the substance to be
necessary, heat to about 90 °C to dissolve the substance to be examined (m g) into a perfectly dry 5 mL conical flask fitted
examined. When the substance to be examined has dissolved, with a ground-glass or suitable plastic stopper and add 2.0 mL
titrate with 0.1 M potassium hydroxide or 0.1 M sodium of propionic anhydride reagent R. Close the flask and shake
hydroxide until the pink colour persists for at least 15 s (n mL gently to dissolve the substance. Allow to stand for 2 h unless
of titrant). When heating has been applied to aid dissolution, otherwise prescribed. Remove the stopper and transfer the
maintain the temperature at about 90 °C during the titration. flask and its contents into a wide-mouthed 500 mL conical
flask containing 25.0 mL of a 9 g/L solution of aniline R in
cyclohexane R and 30 mL of glacial acetic acid R. Swirl the
contents of the flask, allow to stand for 5 min, add 0.05 mL
of crystal violet solution R and titrate with 0.1 M perchloric
01/2008:20502
acid until an emerald-green colour is obtained (n1 mL of
0.1 M perchloric acid). Carry out a blank test under the same
2.5.2. ESTER VALUE conditions (n2 mL of 0.1 M perchloric acid).
The ester value IE is the number that expresses in milligrams
the quantity of potassium hydroxide required to saponify the
esters present in 1 g of the substance. It is calculated from the
saponification value IS and the acid value IA : To take account of any water present, determine this (y per
cent) by the semi-micro determination of water (2.5.12).
The hydroxyl value is then given by the equation :
01/2008:20503
General Notices (1) apply to all monographs and other texts 155
2.5.5. Peroxide value EUROPEAN PHARMACOPOEIA 8.0
the 0.1 M sodium thiosulfate dropwise until the colour is When the monograph does not specify the method to be used,
discharged (n1 mL of 0.1 M sodium thiosulfate). Carry out a method A is applied. Any change from method A to method B
blank test under the same conditions (n2 mL of 0.1 M sodium is validated.
thiosulfate).
METHOD A
Place 5.00 g of the substance to be examined (m g) in a 250 mL
METHOD B conical flask fitted with a ground-glass stopper. Add 30 mL
Unless otherwise prescribed, use the following quantities of a mixture of 2 volumes of chloroform R and 3 volumes of
(Table 2.5.4.-2) for the determination. glacial acetic acid R. Shake to dissolve the substance and add
0.5 mL of saturated potassium iodide solution R. Shake for
Table 2.5.4.-2 exactly 1 min then add 30 mL of water R. Titrate with 0.01 M
Presumed Mass (g) Mass (g) Iodine sodium thiosulfate, adding the titrant slowly with continuous
value II (corresponding (corresponding chloride vigorous shaking, until the yellow colour is almost discharged.
to an excess of to an excess of solution (mL)
150 per cent 100 per cent ICl) Add 5 mL of starch solution R and continue the titration,
ICl) shaking vigorously, until the colour is discharged (n1 mL of
<3 10 10 25 0.01 M sodium thiosulfate). Carry out a blank test under the
same conditions (n2 mL of 0.01 M sodium thiosulfate). The
3 8.4613 10.5760 25
volume of 0.01 M sodium thiosulfate used in the blank titration
5 5.0770 6.3460 25 must not exceed 0.1 mL.
10 2.5384 3.1730 20
20 0.8461 1.5865 20
40 0.6346 0.7935 20
Carry out a blank determination (V0 mL). If the result of several times, each with 40 mL of water R, until the aqueous
the blank determination exceeds 0.1 mL of titration reagent, phase is no longer alkaline to phenolphthalein. Transfer the
replace the impure reagents and repeat the determination. ether phase to a tared flask, washing the separating funnel
with peroxide-free ether R.
Distil off the ether with suitable precautions and add 6 mL of
acetone R to the residue. Carefully remove the solvent in a
c = concentration of the sodium thiosulfate solution current of air. Dry to constant mass at 100-105 °C. Allow to
in moles, per litre. cool in a desiccator and weigh (a g).
01/2008:20506
Dissolve the residue in 20 mL of alcohol R, previously
neutralised to phenolphthalein solution R and titrate with
2.5.6. SAPONIFICATION VALUE 0.1 M ethanolic sodium hydroxide. If the volume of 0.1 M
The saponification value IS is the number that expresses in ethanolic sodium hydroxide used is greater than 0.2 mL, the
milligrams the quantity of potassium hydroxide required to separation of the layers has been incomplete ; the residue
neutralise the free acids and to saponify the esters present in weighed cannot be considered as “unsaponifiable matter”. In
1 g of the substance. case of doubt, the test must be repeated.
Unless otherwise prescribed, use the quantities indicated in
Table 2.5.6.-1 for the determination. 01/2008:20508
Table 2.5.6.-1
Presumed value IS Quantity of sample (g)
2.5.8. DETERMINATION OF PRIMARY
<3 20
AROMATIC AMINO-NITROGEN
3 to 10 12 to 15
Dissolve the prescribed quantity of the substance to be
examined in 50 mL of dilute hydrochloric acid R or in another
10 to 40 8 to 12 prescribed solvent and add 3 g of potassium bromide R. Cool
40 to 60 5 to 8
in ice-water and titrate by slowly adding 0.1 M sodium nitrite
with constant stirring.
60 to 100 3 to 5 Determine the end-point electrometrically or by the use of
100 to 200 2.5 to 3 the prescribed indicator.
200 to 300 1 to 2
01/2008:20509
300 to 400 0.5 to 1
General Notices (1) apply to all monographs and other texts 157
2.5.10. Oxygen-flask method EUROPEAN PHARMACOPOEIA 8.0
used, eliminate residual water from the measurement cell 0.05 mL of methyl orange solution R and neutralise with strong
or carry out a pre-titration. Introduce the substance to be sodium hydroxide solution R (6.5 mL to 7 mL). If a precipitate
examined rapidly and in a suitable state of division. Add an forms dissolve it by adding, dropwise, sufficient dilute sulfuric
accurately measured volume of the titrant, sufficient to give acid R. Transfer the solution to a 250 mL conical flask, rinsing
an excess of about 1 mL or the prescribed volume. Allow to the combustion flask with 25 mL of water R. Add 25.0 mL
stand protected from light for 1 min or the prescribed time, of 0.02 M sodium edetate, 10 mL of acetate buffer solution
with stirring. Titrate the excess of reagent using methanol R pH 4.4 R and a few glass beads and boil gently for 3 min. Add
or the prescribed solvent, containing an accurately known 0.1 mL of pyridylazonaphthol solution R and titrate the hot
quantity of water. solution with 0.02 M copper sulfate until the colour changes
Suitability. The accuracy of the determination with the to purplish-brown. Carry out a blank titration omitting the
chosen titrant must be verified for each combination of vaccine.
substance, titrant and solvent to be examined. The following 1 mL of 0.02 M sodium edetate is equivalent to 0.5396 mg of Al.
procedure, given as an example, is suitable for samples
containing 2.5-25 mg of water. 01/2008:20514
The water content of the substance to be examined is
determined using the reagent/solvent system chosen. 2.5.14. CALCIUM IN ADSORBED
Thereafter, in the same titration vessel, sequential known
amounts of water, corresponding to about 50-100 per cent
VACCINES
of the amount found in the substance to be examined, are All solutions used for this test must be prepared using water R.
added in an appropriate form (at least 5 additions) and the Determine the calcium by atomic emission spectrometry
water content is determined after each addition. Calculate the (2.2.22, Method I). Homogenise the preparation to be
percentage recovery (r) after each addition using the following examined. To 1.0 mL add 0.2 mL of dilute hydrochloric acid R
expression : and dilute to 3.0 mL with water R. Measure the absorbance
at 620 nm.
01/2008:20515
W1 = amount of water added, in milligrams ;
2.5.15. PHENOL IN IMMUNOSERA
W2 = amount of water found, in milligrams.
AND VACCINES
Calculate the mean percentage recovery ( ). The
reagent/solvent system is considered to be acceptable if is Homogenise the preparation to be examined. Dilute an
between 97.5 per cent and 102.5 per cent. appropriate volume with water R so as to obtain a solution
presumed to contain 15 μg of phenol per millilitre. Prepare
Calculate the regression line. The x-axis represents the a series of reference solutions with phenol R containing
cumulative water added whereas the y-axis represents the sum 5 μg, 10 μg, 15 μg, 20 μg and 30 μg of phenol per millilitre
of the initial water content determined for the substance (M) respectively. To 5 mL of the solution to be examined and to
and the cumulative water determined after each addition. 5 mL of each of the reference solutions respectively, add 5 mL
Calculate the slope (b), the intercept with the y-axis (a) and of buffer solution pH 9.0 R, 5 mL of aminopyrazolone solution R
the intercept of the extrapolated calibration line with the and 5 mL of potassium ferricyanide solution R. Allow to stand
x-axis (d). for 10 min and measure the intensity of colour at 546 nm.
Calculate the percentage errors (e1 and e2) using the following Plot the calibration curve and calculate the phenol content of
expressions : the preparation to be examined.
01/2008:20516
General Notices (1) apply to all monographs and other texts 159
2.5.17. Nucleic acids in polysaccharide vaccines EUROPEAN PHARMACOPOEIA 8.0
reagent R and water R, prepared immediately before use. 30 μg to 600 μg of acetylcholine chloride (O-acetyl). Introduce
Stopper the tubes, mix by inverting and allow to stand in 0.3 mL, 0.5 mL and 1.0 mL in duplicate into 6 tubes (3 reaction
the dark for 30 min. The blue colour is stable for 60 min. If solutions and 3 correction solutions).
necessary, centrifuge to obtain clear solutions. Reference solutions. Dissolve 0.150 g of acetylcholine chloride R
Measure the absorbance (2.2.25) of each solution at 760 nm in 10 mL of water R (stock solution containing 15 g of
using the blank as the compensation liquid. Draw a calibration acetylcholine chloride per litre). Immediately before use,
curve from the absorbances of the 6 reference solutions and dilute 1 mL of the stock solution to 50 mL with water R
the corresponding protein contents and read from the curve (working dilution 1 : 300 μg of acetylcholine chloride per
the content of protein in the test solution. millilitre). Immediately before use, dilute 1 mL of the stock
solution to 25 mL with water R (working dilution 2 : 600 μg
01/2008:20517 of acetylcholine chloride per millilitre). Introduce 0.1 mL
and 0.4 mL of working dilution 1 in duplicate (reaction and
2.5.17. NUCLEIC ACIDS IN correction solutions) in 4 tubes and 0.6 mL and 1.0 mL of
working dilution 2 in duplicate (reaction and correction
POLYSACCHARIDE VACCINES solutions) in another 4 tubes.
Test solution. Use a volumetric flask with a suitable volume for Prepare a blank using 1 mL of water R.
preparation of a solution containing about 5 mg per millilitre Make up the volume in each tube to 1 mL with water R. Add
of dry polysaccharide. Transfer the contents of a container 1.0 mL of 4 M hydrochloric acid to each of the correction
quantitatively to the flask and dilute to volume with water R. tubes and to the blank. Add 2.0 mL of alkaline hydroxylamine
Dilute the test solution if necessary to obtain an absorbance solution R to each tube. Allow the reaction to proceed for
value suitable for the instrument used. Measure the absorbance exactly 2 min and add 1.0 mL of 4 M hydrochloric acid to
(2.2.25) at 260 nm using water R as the compensation liquid. each of the reaction tubes. Add 1.0 mL of a 100 g/L solution
of ferric chloride R in 0.1 M hydrochloric acid to each tube,
The absorbance of a 1 g/L solution of nucleic acid at 260 nm is stopper the tubes and shake vigorously to remove bubbles.
20.
Measure the absorbance (2.2.25) of each solution at 540 nm
using the blank as the compensation liquid. For each reaction
01/2008:20518 solution, subtract the absorbance of the corresponding
correction solution. Draw a calibration curve from the
2.5.18. PHOSPHORUS IN corrected absorbances for the 4 reference solutions and the
POLYSACCHARIDE VACCINES corresponding content of acetylcholine chloride and read
from the curve the content of acetylcholine chloride in the
Test solution. Use a volumetric flask with a suitable volume for test solution for each volume tested. Calculate the mean of
preparation of a solution containing about 5 mg per millilitre the 3 values.
of dry polysaccharide. Transfer the contents of a container 1 mole of acetylcholine chloride (181.7 g) is equivalent to
quantitatively to the flask and dilute to volume with water R. 1 mole of O-acetyl (43.05 g).
Dilute the solution so that the volume used in the test (1 mL)
contains about 6 μg of phosphorus. Transfer 1.0 mL of the 01/2008:20520
solution to a 10 mL ignition tube.
Reference solutions. Dissolve 0.2194 g of potassium dihydrogen 2.5.20. HEXOSAMINES IN
phosphate R in 500 mL of water R to give a solution containing
the equivalent of 0.1 mg of phosphorus per millilitre. Dilute POLYSACCHARIDE VACCINES
5.0 mL of the solution to 100.0 mL with water R. Introduce Test solution. Use a volumetric flask with a suitable volume for
0.5 mL, 1.0 mL and 2.0 mL of the dilute solution into 3 preparation of a solution containing about 5 mg per millilitre
ignition tubes. of dry polysaccharide. Transfer the contents of a container
Prepare a blank solution using 2.0 mL of water R in an ignition quantitatively to the flask and dilute to volume with water R.
tube. Dilute the solution so that the volumes used in the test contain
To all the tubes add 0.2 mL of sulfuric acid R and heat in an 125 μg to 500 μg of glucosamine (hexosamine). Introduce
oil bath at 120 °C for 1 h and then at 160 °C until white fumes 1.0 mL of the diluted solution into a graduated tube.
appear (about 1 h). Add 0.1 mL of perchloric acid R and heat at Reference solutions. Dissolve 60 mg of glucosamine
160 °C until the solution is decolorised (about 90 min). Cool hydrochloride R in 100 mL of water R (stock solution
and add to each tube 4 mL of water R and 4 mL of ammonium containing 0.500 g of glucosamine per litre). Introduce
molybdate reagent R. Heat in a water-bath at 37 °C for 90 min 0.25 mL, 0.50 mL, 0.75 mL, and 1.0 mL of the working dilution
and cool. Adjust the volume to 10.0 mL with water R. The into 4 graduated tubes.
blue colour is stable for several hours. Prepare a blank using 1 mL of water R.
Measure the absorbance (2.2.25) of each solution at 820 nm Make up the volume in each tube to 1 mL with water R.
using the blank solution as the compensation liquid. Draw Add 1 mL of a solution of hydrochloric acid R (292 g/L) to
a calibration curve with the absorbances of the 3 reference each tube. Stopper the tubes and place in a water-bath for
solutions as a function of the quantity of phosphorus in the 1 h. Cool to room temperature. Add to each tube 0.05 mL
solutions and read from the curve the quantity of phosphorus of a 5 g/L solution of thymolphthalein R in alcohol R ; add a
in the test solution. solution of sodium hydroxide R (200 g/L) until a blue colour
is obtained and then 1 M hydrochloric acid until the solution
01/2008:20519 is colourless. Dilute the volume in each tube to 10 mL with
water R (neutralised hydrolysates).
2.5.19. O-ACETYL IN In a second series of 10 mL graduated tubes, place 1 mL of each
POLYSACCHARIDE VACCINES neutralised hydrolysate. Add 1 mL of acetylacetone reagent
(a mixture, prepared immediately before use, of 1 volume
Test solution. Use a volumetric flask with a suitable volume for of acetylacetone R and 50 volumes of a 53 g/L solution of
preparation of a solution containing about 5 mg per millilitre anhydrous sodium carbonate R) to each tube. Stopper the
of dry polysaccharide. Transfer the contents of a container tubes and place in a water-bath at 90 °C for 45 min. Cool to
quantitatively to the flask and dilute to volume with water R. room temperature. Add to each tube 2.5 mL of alcohol R and
Dilute the solution so that the volumes used in the test contain 1.0 mL of dimethylaminobenzaldehyde solution (immediately
before use dissolve 0.8 g of dimethylaminobenzaldehyde R in Make up the volume in each tube to 1 mL with water R. Place
15 mL of alcohol R and add 15 mL of hydrochloric acid R) the tubes in iced water and add dropwise and with continuous
and dilute the volume in each tube to 10 mL with alcohol R. stirring to each tube 5.0 mL of borate solution R. Stopper the
Stopper the tubes, mix by inverting and allow to stand in thetubes and place in a water-bath for 15 min. Cool to room
dark for 90 min. Measure the absorbance (2.2.25) of each temperature. Add 0.20 mL of a 1.25 g/L solution of carbazole R
in ethanol R to each tube. Stopper the tubes and place in a
solution at 530 nm using the blank as the compensation liquid.
Draw a calibration curve from the absorbances for the water-bath for 15 min. Cool to room temperature. Measure
4 reference solutions and the corresponding content of the absorbance (2.2.25) of each solution at 530 nm using the
hexosamine and read from the curve the quantity of blank as the compensation liquid.
hexosamine in the test solution. Draw a calibration curve from the absorbances for the
5 reference solutions and the corresponding content of
glucuronic acid and read from the curve the quantity of
01/2008:20521
glucuronic acid in the test solution for each volume tested.
Calculate the mean of the 3 values.
2.5.21. METHYLPENTOSES IN
POLYSACCHARIDE VACCINES 01/2008:20523
Test solution. Use a volumetric flask with a suitable volume 2.5.23. SIALIC ACID IN
for preparation of a solution containing about 5 mg per
millilitre of dry polysaccharide. Transfer the contents of a POLYSACCHARIDE VACCINES
container quantitatively to the flask and dilute to volume with Test solution. Transfer quantitatively the contents of one or
water R. Dilute the solution so that the volumes used in the several containers to a volumetric flask of a suitable volume
test contain 2 μg to 20 μg of rhamnose (methylpentoses). that will give a solution with a known concentration of about
Introduce 0.25 mL, 0.50 mL and 1.0 mL of the diluted solution 250 μg per millilitre of polysaccharide and dilute to volume
into 3 tubes. with water R. Using a syringe, transfer 4.0 mL of this solution
Reference solutions. Dissolve 0.100 g of rhamnose R in 100 mL to a 10 mL ultrafiltration cell suitable for the passage of
of water R (stock solution containing 1 g of methylpentose molecules of relative molecular mass less than 50 000. Rinse
per litre). Immediately before use, dilute 1 mL of the stock the syringe twice with water R and transfer the rinsings to the
solution to 50 mL with water R (working dilution : 20 mg of ultrafiltration cell. Carry out the ultrafiltration, with constant
methylpentose per litre). Introduce 0.10 mL, 0.25 mL, 0.50 mL, stirring, under nitrogen R at a pressure of about 150 kPa. Refill
0.75 mL and 1.0 mL of the working dilution into 5 tubes. the cell with water R each time the volume of liquid in it has
Prepare a blank using 1 mL of water R. decreased to 1 mL and continue until 200 mL has been filtered
Make up the volume in each tube to 1 mL with water R. Place and the remaining volume in the cell is about 2 mL. Using a
the tubes in iced water and add dropwise and with continuous syringe, transfer this residual liquid to a 10 mL volumetric
stirring to each tube 4.5 mL of a cooled mixture of 1 volume flask. Wash the cell with 3 quantities, each of 2 mL, of water R,
of water R and 6 volumes of sulfuric acid R. Warm the tubes transfer the washings to the flask and dilute to 10.0 mL with
to room temperature and place in a water-bath for a few water R (test solution). In each of 2 test-tubes place 2.0 mL of
minutes. Cool to room temperature. Add to each tube 0.10 mL the test solution.
of a 30 g/L solution of cysteine hydrochloride R, prepared Reference solutions. Use the reference solutions prescribed in
immediately before use. Shake and allow to stand for 2 h. the monograph.
Measure the absorbance (2.2.25) of each solution at 396 nm Prepare 2 series of 3 test-tubes, place in the tubes of each
and at 430 nm using the blank as compensation liquid. For series 0.5 mL, 1.0 mL and 1.5 mL respectively, of the reference
each solution, calculate the difference between the absorbance solution corresponding to the type of vaccine to be examined
measured at 396 nm and that measured at 430 nm. Draw and adjust the volume in each tube to 2.0 mL with water R.
a calibration curve from the absorbance differences for Prepare blank solutions using 2.0 mL of water R in each of
the 5 reference solutions and the corresponding content 2 test-tubes.
of methylpentose and read from the curve the quantity of To all the tubes add 5.0 mL of resorcinol reagent R. Heat at
methylpentose in the test solution for each volume tested. 105 °C for 15 min, cool in cold water and transfer the tubes
Calculate the mean of the 3 values. to a bath of iced water. To each tube add 5 mL of isoamyl
alcohol R and mix thoroughly. Place in the bath of iced
01/2008:20522 water for 15 min. Centrifuge the tubes and keep them in
the bath of iced water until the examination by absorption
2.5.22. URONIC ACIDS IN spectrophotometry. Measure the absorbance (2.2.25) of each
supernatant solution at 580 nm and 450 nm using isoamyl
POLYSACCHARIDE VACCINES alcohol R as the compensation liquid. For each wavelength,
calculate the absorbance as the mean of the values obtained
Test solution. Use a volumetric flask with a suitable volume for
with 2 identical solutions. Subtract the mean value for the
preparation of a solution containing about 5 mg per millilitre
blank solution from the mean values obtained for the other
of dry polysaccharide. Transfer the contents of a container
solutions.
quantitatively to the flask and dilute to volume with water R.
Dilute the solution so that the volumes used in the test contain Draw a graph showing the difference between the absorbances
4 μg to 40 μg of glucuronic acid (uronic acids). Introduce at 580 nm and 450 nm of the reference solutions as a function
0.25 mL, 0.50 mL and 1.0 mL of the diluted solution into 3 of the content of N-acetylneuraminic acid and read from the
tubes. graph the quantity of N-acetylneuraminic acid (sialic acid) in
the test solution.
Reference solutions. Dissolve 50 mg of sodium glucuronate R
in 100 mL of water R (stock solution containing 0.4 g of 01/2011:20524
glucuronic acid per litre). Immediately before use, dilute 5 mL
of the stock solution to 50 mL with water R (working dilution :
40 mg of glucuronic acid per litre). Introduce 0.10 mL, 2.5.24. CARBON DIOXIDE IN GASES
0.25 mL, 0.50 mL, 0.75 mL, and 1.0 mL of the working dilution Gases absorb light at one or more specific wavelengths. This
into 5 tubes. property is widely used to allow highly selective measurement
Prepare a blank using 1 mL of water R. of their concentrations.
General Notices (1) apply to all monographs and other texts 161
2.5.25. Carbon monoxide in gases EUROPEAN PHARMACOPOEIA 8.0
Description and principle of measurement. The – a U-tube (U2) containing pellets of potassium hydroxide R ;
concentration of carbon dioxide in other gases can be – a U-tube (U3) containing diphosphorus pentoxide R
determined using an infrared analyser. dispersed on previously granulated, fused pumice ;
The infrared analyser generally consists of a light source – a U-tube (U4) containing 30 g of recrystallised iodine
emitting broadband infrared radiation, an optical device, pentoxide R in granules, previously dried at 200 °C and kept
a sample cell and a detector. The optical device may be at a temperature of 120 °C (T) during the test ; the iodine
positioned either before or after the sample cell and it consists pentoxide is packed in the tube in 1 cm columns separated
of one or several optical filters, through which the broadband by 1 cm columns of glass wool to give an effective length of
radiation is passed. The optical device in this case is selected 5 cm ;
for carbon dioxide. The measurement light beam passes – a reaction tube (F2) containing 2.0 mL of potassium iodide
through the sample cell and may also pass through a reference solution R and 0.15 mL of starch solution R.
cell if the analyser integrates such a feature (some use an
electronic system instead of a reference cell). Method. Flush the apparatus with 5.0 L of argon R and, if
necessary, discharge the blue colour in the iodide solution by
When carbon dioxide is present in the sample cell, absorption adding the smallest necessary quantity of freshly prepared
of energy in the measurement light beam will occur according 0.002 M sodium thiosulfate. Continue flushing until not more
to the Beer-Lambert law and this produces a change in than 0.045 mL of 0.002 M sodium thiosulfate is required after
the detector signal. This measurement signal is compared passage of 5.0 L of argon R. Pass the gas to be examined from
to a reference signal to generate an output related to the the cylinder through the apparatus, using the prescribed
concentration of carbon dioxide. The generated signal is volume and the flow rate. Flush the last traces of liberated
linearised in order to obtain the carbon dioxide concentration. iodine into the reaction tube by passing through the apparatus
To prevent the entry of particles into the sensors, which could 1.0 L of argon R. Titrate the liberated iodine with 0.002 M
cause stray-light phenomena, the apparatus is fitted with a sodium thiosulfate. Carry out a blank test, using the prescribed
suitable filter. volume of argon R. The difference between the volumes of
Required technical specifications. When used for a limit 0.002 M sodium thiosulfate used in the titrations is not greater
test, the infrared analyser meets the following technical than the prescribed limit.
specifications :
METHOD II
– limit of detection : (generally defined as a signal-to-noise
ratio of 2) maximum 20 per cent of the maximum Gases absorb light at one or more specific wavelengths. This
admissible concentration ; property is widely used to allow highly selective measurement
of their concentrations.
– repeatability : maximum relative standard deviation of
10 per cent of the maximum admissible concentration, Description and principle of measurement. The
determined on 6 measurements ; concentration of carbon monoxide in other gases can be
– linearity : maximum 10 per cent of the maximum admissible determined using an infrared analyser.
concentration. The infrared analyser generally consists of a light source
The technical specifications must be met in the presence of emitting broadband infrared radiation, an optical device,
the other gas impurities in the sample. a sample cell and a detector. The optical device may be
positioned either before or after the sample cell ; it consists of
one or several optical filters, through which the broadband
01/2011:20525 radiation is passed. The optical device in this case is selected
for carbon monoxide. The measurement light beam passes
2.5.25. CARBON MONOXIDE IN GASES through the sample cell and may also pass through a reference
cell if the analyser integrates such a feature (some use an
METHOD I electronic system instead of a reference cell).
Apparatus. The apparatus (Figure 2.5.25.-1) consists of the When carbon monoxide is present in the sample cell,
following parts connected in series : absorption of energy in the measurement light beam will
– a U-tube (U1) containing anhydrous silica gel R impregnated occur according to the Beer-Lambert law and this produces
with chromium trioxide R ; a change in the detector signal. This measurement signal is
– a wash bottle (F1) containing 100 mL of a 400 g/L solution compared to a reference signal to generate an output related
of potassium hydroxide R ; to the concentration of carbon monoxide. The generated
General Notices (1) apply to all monographs and other texts 163
2.5.29. Sulfur dioxide EUROPEAN PHARMACOPOEIA 8.0
01/2008:20532
produced at the anode reacts immediately with the water and 01/2008:20533
the sulfur dioxide contained in the reaction cell. The amount corrected 6.0
of water in the substance is directly proportional to the
quantity of electricity up until the titration end-point. When 2.5.33. TOTAL PROTEIN
all of the water in the cell has been consumed, the end-point
is reached and thus an excess of iodine appears. 1 mole of Many of the assay methods described in this chapter can be
iodine corresponds to 1 mole of water, a quantity of electricity performed using kits from commercial sources.
of 10.71 C corresponds to 1 mg of water.
METHOD 1
Moisture is eliminated from the system by pre-electrolysis.
Individual determinations can be carried out successively in Protein in solution absorbs ultraviolet light at a wavelength
the same reagent solution, under the following conditions : of 280 nm, due to the presence of aromatic amino acids,
mainly tyrosine and tryptophan, in the protein structure. This
– each component of the test mixture is compatible with the property can be used for assay purposes. If the buffer used to
other components, dissolve the protein has a high absorbance relative to that of
– no other reactions take place, water, an interfering substance is present. This interference
may be obviated by using the buffer as compensation
– the volume and the water capacity of the electrolyte reagent liquid but if the interfering substance produces a high
are sufficient. absorbance, the results may nevertheless be compromised.
Coulometric titration is restricted to the quantitative At low concentrations, protein adsorbed onto the cell may
determination of small amounts of water, a range of 10 μg up significantly reduce the content in solution. This can be
to 10 mg of water is recommended. prevented by preparing samples at higher concentration or by
using a non-ionic detergent in the preparation.
Accuracy and precision of the method are predominantly
governed by the extent to which atmospheric moisture is Test solution. Dissolve a suitable quantity of the substance
excluded from the system. Control of the system must be to be examined in the prescribed buffer to obtain a solution
monitored by measuring the amount of baseline drift. having a protein concentration between 0.2 mg/mL and
2 mg/mL.
APPARATUS Reference solution. Prepare a solution of a suitable reference
substance for the protein to be determined, in the same buffer
The apparatus consists of a reaction cell, electrodes and and at the same protein concentration as the test solution.
magnetic stirrer. The reaction cell consists of a large anode
compartment and a smaller cathode compartment. Depending Procedure. Keep the test solution, the reference solution and
on the design of the electrode, both compartments can be the compensation liquid at the same temperature during the
separated by a diaphragm. Each compartment contains performance of this test. Determine the absorbances (2.2.25)
a platinum electrode. Liquid or solubilised samples are of the test solution and the reference solution in quartz cells
introduced through a septum, using a syringe. Alternatively, at 280 nm, using the prescribed buffer as the compensation
an evaporation technique may be used in which the sample liquid. The response must be linear in the range of protein
is heated in a tube (oven) and the water is evaporated and concentrations to be assayed to obtain accurate results.
carried into the cell by means of a stream of dry inert gas. The Light scattering. The accuracy of the determination of protein
introduction of solid samples into the cell should in general be can be diminished by the scattering of light by the test sample.
avoided. However, if it has to be done it is effected through a If the proteins in solution exist as particles comparable in size
sealable port ; appropriate precautions must be taken to avoid to the wavelength of the measuring light (250 nm to 300 nm),
the introduction of moisture from air, such as working in a scattering of the light beam results in an apparent increase in
glove box in an atmosphere of dry inert gas. The analytical absorbance of the test sample. To calculate the absorbance at
procedure is controlled by a suitable electronic device, which 280 nm due to light scattering, determine the absorbances of
also displays the results. the test solution at wavelengths of 320 nm, 325 nm, 330 nm,
335 nm, 340 nm, 345 nm and 350 nm. Plot the logarithm
METHOD of the observed absorbance against the logarithm of the
Fill the compartments of the reaction cell with electrolyte wavelength and determine the standard curve best fitting the
reagent for the micro determination of water R according to plotted points by linear regression. Extrapolate the curve to
the manufacturer’s instructions and perform the coulometric determine the logarithm of the absorbance at 280 nm. The
titration to a stable end-point. Introduce the prescribed antilogarithm of this value is the absorbance attributed to
amount of the substance to be examined into the reaction cell, light scattering. Correct the observed values by subtracting
stir for 30 s, if not otherwise indicated in the monograph, and the absorbance attributed to light scattering from the total
titrate again to a stable end-point. In case an oven is used, absorbance at 280 nm to obtain the absorbance value of the
the prescribed sample amount is introduced into the tube protein in solution. Filtration with a 0.2 μm filter that does
and heated. After evaporation of the water from the sample not adsorb protein or clarification by centrifugation may be
into the titration cell, the titration is started. Read the value performed to reduce the effect of light scattering, especially if
from the instrument’s output and calculate if necessary the the solution is noticeably turbid.
percentage or amount of water that is present in the substance. Calculations. Use corrected values for the calculations.
When appropriate to the type of sample and the sample Calculate the concentration of protein in the test solution (CU)
preparation, perform a blank titration. from the following equation :
General Notices (1) apply to all monographs and other texts 165
2.5.33. Total protein EUROPEAN PHARMACOPOEIA 8.0
results in an absorbance maximum at 750 nm. The a vortex mixer and allow to stand at room temperature for
phosphomolybdotungstic reagent reacts primarily with 10 min. Add 0.1 mL of a 720 g/L solution of trichloroacetic
tyrosine residues in the protein. Colour development reaches acid R and mix using a vortex mixer. Centrifuge at 3000 g for
a maximum in 20 min to 30 min at room temperature, 30 min, decant the liquid and remove any residual liquid with
after which there is a gradual loss of colour. Because the a pipette. Redissolve the protein pellet in 1 mL of alkaline
method is sensitive to interfering substances, a procedure copper reagent.
for precipitation of the protein from the test sample may be
used. Most interfering substances cause a lower colour yield ; METHOD 3
however, some detergents cause a slight increase in colour. This method (commonly referred to as the Bradford assay)
A high salt concentration may cause a precipitate to form. is based on the absorption shift from 470 nm to 595 nm
Because different protein species may give different colour observed when the acid blue 90 dye binds to protein. The acid
response intensities, the reference substance and test protein blue 90 dye binds most readily to arginine and lysine residues
must be the same. Where separation of interfering substances in the protein which can lead to variation in the response of
from the protein in the test sample is necessary, proceed as the assay to different proteins. The protein used as reference
directed below for interfering substances prior to preparation substance must therefore be the same as the protein to be
of the test solution. The effect of interfering substances may determined. There are relatively few interfering substances,
be minimised by dilution, provided the concentration of the but it is preferable to avoid detergents and ampholytes in the
test protein remains sufficient for accurate measurement. test sample. Highly alkaline samples may interfere with the
Use distilled water R to prepare all buffers and reagents used acidic reagent.
for this method. Use distilled water R to prepare all buffers and reagents used
Test solution. Dissolve a suitable quantity of the substance for this method.
to be examined in the prescribed buffer to obtain a solution Test solution. Dissolve a suitable quantity of the substance
having a concentration within the range of the standard curve. to be examined in the prescribed buffer to obtain a solution
A suitable buffer will produce a solution of pH 10.0 to 10.5. having a concentration within the range of the standard curve.
Reference solutions. Dissolve the reference substance for the Reference solutions. Dissolve the reference substance for the
protein to be determined in the prescribed buffer. Dilute protein to be determined in the prescribed buffer. Dilute
portions of this solution with the same buffer to obtain portions of this solution with the same buffer to obtain
not fewer than five reference solutions having protein not fewer than five reference solutions having protein
concentrations evenly spaced over a suitable range situated concentrations evenly spaced over a suitable range situated
between 5 μg/mL and 100 μg/mL. between 0.1 mg/mL and 1 mg/mL.
Blank. Use the buffer used to prepare the test solution and Blank. Use the buffer used to prepare the test solution and
the reference solutions. the reference solutions.
Copper sulfate reagent. Dissolve 100 mg of copper sulfate R Acid blue 90 reagent. Dissolve 0.10 g of acid blue 90 R in
and 0.2 g of sodium tartrate R in distilled water R and dilute 50 mL of alcohol R. Add 100 mL of phosphoric acid R, dilute
to 50 mL with the same solvent. Dissolve 10 g of anhydrous to 1000 mL with distilled water R and mix. Filter the solution
sodium carbonate R in distilled water R and dilute to 50 mL and store in an amber bottle at room temperature. Slow
with the same solvent. Slowly pour the sodium carbonate precipitation of the dye occurs during storage. Filter the
solution into the copper sulfate solution with mixing. Use reagent before using.
within 24 h. Procedure. Add 5 mL of acid blue 90 reagent to 0.100 mL of
Alkaline copper reagent. Mix 1 volume of copper sulfate each reference solution, of the test solution and of the blank.
reagent, 2 volumes of a 50 g/L solution of sodium dodecyl Mix by inversion. Avoid foaming, which will lead to poor
sulfate R and 1 volume of a 32 g/L solution of sodium reproducibility. Determine the absorbances (2.2.25) of the
hydroxide R. Store at room temperature and use within standard solutions and of the test solution at 595 nm, using
2 weeks. the blank as compensation liquid. Do not use quartz (silica)
Diluted phosphomolybdotungstic reagent. Mix 5 mL of spectrophotometer cells because the dye binds to this material.
phosphomolybdotungstic reagent R with 55 mL of distilled Calculations. The relationship of absorbance to protein
water R. Store in an amber bottle, at room temperature. concentration is non-linear ; however, if the range of
Procedure. To 1.0 mL of each reference solution, of the test concentrations used to prepare the standard curve is
solution and of the blank, add 1.0 mL of alkaline copper sufficiently small, the latter will approach linearity. Plot
reagent and mix. Allow to stand for 10 min. Add 0.5 mL of the absorbances of the reference solutions against protein
the diluted phosphomolybdotungstic reagent, mix and allow concentrations and use linear regression to establish the
to stand at room temperature for 30 min. Determine the standard curve. From the standard curve and the absorbance
absorbances (2.2.25) of the solutions at 750 nm, using the of the test solution, determine the concentration of protein in
solution from the blank as compensation liquid. the test solution.
Calculations. The relationship of absorbance to protein METHOD 4
concentration is non-linear ; however, if the range of This method (commonly referred to as the bicinchoninic acid
concentrations used to prepare the standard curve is or BCA assay) is based on reduction of the cupric (Cu2+) ion to
sufficiently small, the latter will approach linearity. Plot the cuprous (Cu1+) ion by protein. The bicinchoninic acid reagent
absorbances of the reference solutions against the protein is used to detect the cuprous ion. Few substances interfere
concentrations and use linear regression to establish the with the reaction. When interfering substances are present
standard curve. From the standard curve and the absorbance their effect may be minimised by dilution, provided that
of the test solution, determine the concentration of protein in the concentration of the protein to be determined remains
the test solution. sufficient for accurate measurement. Alternatively, the protein
Interfering substances. In the following procedure, precipitation procedure given in Method 2 may be used to
deoxycholate-trichloroacetic acid is added to a test sample remove interfering substances. Because different protein
to remove interfering substances by precipitation of proteins species may give different colour response intensities, the
before determination ; this technique can also be used to reference protein and protein to be determined must be the
concentrate proteins from a dilute solution. same.
Add 0.1 mL of a 1.5 g/L solution of sodium deoxycholate R to Use distilled water R to prepare all buffers and reagents used
1 mL of a solution of the substance to be examined. Mix using for this method.
Test solution. Dissolve a suitable quantity of the substance Biuret reagent. Dissolve 3.46 g of copper sulfate R in 10 mL of
to be examined in the prescribed buffer to obtain a solution hot distilled water R, and allow to cool (Solution A). Dissolve
having a concentration within the range of the concentrations 34.6 g of sodium citrate R and 20.0 g of anhydrous sodium
of the reference solutions. carbonate R in 80 mL of hot distilled water R, and allow to
Reference solutions. Dissolve the reference substance for the cool (Solution B). Mix solutions A and B and dilute to 200 mL
protein to be determined in the prescribed buffer. Dilute with distilled water R. Use within 6 months. Do not use the
portions of this solution with the same buffer to obtain reagent if it develops turbidity or contains any precipitate.
not fewer than five reference solutions having protein Procedure. To one volume of the test solution add an equal
concentrations evenly spaced over a suitable range situated volume of a 60 g/L solution of sodium hydroxide R and mix.
between 10 μg/mL and 1200 μg/mL. Immediately add biuret reagent equivalent to 0.4 volumes
Blank. Use the buffer used to prepare the test solution and of the test solution and mix rapidly. Allow to stand at a
the reference solutions. temperature between 15 °C and 25 °C for not less than 15 min.
Within 90 min of addition of the biuret reagent, determine
BCA reagent. Dissolve 10 g of disodium bicinchoninate R, the absorbances (2.2.25) of the reference solutions and of the
20 g of sodium carbonate monohydrate R, 1.6 g of sodium test solution at the maximum at 545 nm, using the blank as
tartrate R, 4 g of sodium hydroxide R, and 9.5 g of sodium compensation liquid. Any solution that develops turbidity
hydrogen carbonate R in distilled water R. Adjust, if necessary, or a precipitate is not acceptable for calculation of protein
to pH 11.25 with a solution of sodium hydroxide R or a concentration.
solution of sodium hydrogen carbonate R. Dilute to 1000 mL
with distilled water R and mix. Calculations. The relationship of absorbance to protein
concentration is approximately linear within the indicated
Copper-BCA reagent. Mix 1 mL of a 40 g/L solution of copper range of protein concentrations for the reference solutions.
sulfate R and 50 mL of BCA reagent. Plot the absorbances of the reference solutions against protein
Procedure. Mix 0.1 mL of each reference solution, of the concentrations and use linear regression to establish the
test solution and of the blank with 2 mL of the copper-BCA standard curve. Calculate the correlation coefficient for the
reagent. Incubate the solutions at 37 °C for 30 min, note the standard curve. A suitable system is one that yields a line
time and allow the mixtures to cool to room temperature. having a correlation coefficient not less than 0.99. From
Within 60 min of the end of incubation, determine the the standard curve and the absorbance of the test solution,
absorbances (2.2.25) of the reference solutions and of the determine the concentration of protein in the test solution.
test solution in quartz cells at 562 nm, using the blank as Interfering substances. To minimise the effect of interfering
compensation liquid. After the solutions have cooled to substances, the protein can be precipitated from the test
room temperature, the colour intensity continues to increase sample as follows : add 0.1 volumes of a 500 g/L solution of
gradually. trichloroacetic acid R to 1 volume of a solution of the test
Calculations. The relationship of absorbance to protein sample, withdraw the supernatant layer and dissolve the
concentration is non-linear ; however, if the range of precipitate in a small volume of 0.5 M sodium hydroxide. Use
concentrations used to prepare the standard curve is the solution obtained to prepare the test solution.
sufficiently small, the latter will approach linearity. Plot
the absorbances of the reference solutions against protein METHOD 6
concentrations and use linear regression to establish the
standard curve. From the standard curve and the absorbance This fluorimetric method is based on the derivatisation of
of the test solution, determine the concentration of protein in the protein with o-phthalaldehyde, which reacts with the
the test solution. primary amines of the protein (N-terminal amino acid and
the ε-amino group of lysine residues). The sensitivity of the
METHOD 5 assay can be increased by hydrolysing the protein before
adding o-phthalaldehyde. Hydrolysis makes the α-amino
This method (commonly referred to as the biuret assay) is
group of the constituent amino acids available for reaction
based on the interaction of cupric (Cu2+) ion with protein in
with the phthalaldehyde reagent. The method requires
alkaline solution and resultant development of absorbance
very small quantities of the protein. Primary amines, such
at 545 nm. This test shows minimal difference between
as tris(hydroxymethyl)aminomethane and amino acid
equivalent IgG and albumin samples. Addition of the sodium
buffers, react with phthalaldehyde and must be avoided
hydroxide and the biuret reagent as a combined reagent,
or removed. Ammonia at high concentrations reacts with
insufficient mixing after the addition of the sodium hydroxide,
phthalaldehyde. The fluorescence obtained when amine reacts
or an extended time between the addition of the sodium
with phthalaldehyde can be unstable. The use of automated
hydroxide solution and the addition of the biuret reagent will
procedures to standardise this procedure may improve the
give IgG samples a higher response than albumin samples.
accuracy and precision of the test.
The trichloroacetic acid method used to minimise the effects
of interfering substances also can be used to determine the Use distilled water R to prepare all buffers and reagents used
protein content in test samples at concentrations below for this method.
500 μg/mL. Test solution. Dissolve a suitable quantity of the substance to
Use distilled water R to prepare all buffers and reagents used be examined in a 9 g/L solution of sodium chloride R to obtain
for this method. a solution having a concentration within the range of the
Test solution. Dissolve a suitable quantity of the substance concentrations of the reference solutions. Adjust the solution
to be examined in a 9 g/L solution of sodium chloride R to to pH 8 to 10.5 before addition of the phthalaldehyde reagent.
obtain a solution having a concentration within the range of Reference solutions. Dissolve the reference substance for
the concentrations of the reference solutions. the protein to be determined in a 9 g/L solution of sodium
Reference solutions. Dissolve the reference substance for chloride R. Dilute portions of this solution with a 9 g/L solution
the protein to be determined in a 9 g/L solution of sodium of sodium chloride R to obtain not fewer than five reference
chloride R. Dilute portions of this solution with a 9 g/L solution solutions having protein concentrations evenly spaced over
of sodium chloride R to obtain not fewer than three reference a suitable range situated between 10 μg/mL and 200 μg/mL.
solutions having protein concentrations evenly spaced over a Adjust the solutions to pH 8 to 10.5 before addition of the
suitable range situated between 0.5 mg/mL and 10 mg/mL. phthalaldehyde reagent.
Blank. Use a 9 g/L solution of sodium chloride R. Blank solution. Use a 9 g/L solution of sodium chloride R.
General Notices (1) apply to all monographs and other texts 167
2.5.34. Acetic acid in synthetic peptides EUROPEAN PHARMACOPOEIA 8.0
Borate buffer solution. Dissolve 61.83 g of boric acid R in Test solution. Prepare as described in the monograph. The
distilled water R and adjust to pH 10.4 with a solution of concentration of peptide in the solution may be adapted,
potassium hydroxide R. Dilute to 1000 mL with distilled depending on the expected amount of acetic acid in the
water R and mix. sample.
Phthalaldehyde stock solution. Dissolve 1.20 g of Reference solution. Prepare a 0.10 g/L solution of glacial
phthalaldehyde R in 1.5 mL of methanol R, add 100 mL of acetic acid R in a mixture of 5 volumes of mobile phase B and
borate buffer solution and mix. Add 0.6 mL of a 300 g/L 95 volumes of mobile phase A.
solution of macrogol 23 lauryl ether R and mix. Store at room
The chromatographic procedure may be carried out using :
temperature and use within 3 weeks.
Phthalaldehyde reagent. To 5 mL of phthalaldehyde stock – a stainless steel column 0.25 m long and 4.6 mm in
solution add 15 μL of 2-mercaptoethanol R. Prepare at least internal diameter packed with octadecylsilyl silica gel for
30 min before use. Use within 24 h. chromatography R (5 μm),
Procedure. Mix 10 μL of the test solution and of each of the – as mobile phase at a flow rate of 1.2 mL/min :
reference solutions with 0.1 mL of phthalaldehyde reagent and Mobile phase A. Dilute 0.7 mL of phosphoric acid R to
allow to stand at room temperature for 15 min. Add 3 mL of 1000 mL with water R ; adjust the pH to 3.0 with strong
0.5 M sodium hydroxide and mix. Determine the fluorescent sodium hydroxide solution R,
intensities (2.2.21) of solutions from the reference solutions Mobile phase B. Methanol R2,
and from the test solution at an excitation wavelength of
340 nm and an emission wavelength between 440 and 455 nm. Time Mobile phase A Mobile phase B
Measure the fluorescent intensity of a given sample only once, (min) (per cent V/V) (per cent V/V)
since irradiation decreases the fluorescence intensity. 0-5 95 5
Calculations. The relationship of fluorescence to protein 5 - 10 95 → 50 5 → 50
concentration is linear. Plot the fluorescent intensities of
the reference solutions against protein concentrations and 10 - 20 50 50
use linear regression to establish the standard curve. From 20 - 22 50 → 95 50 → 5
the standard curve and the fluorescent intensity of the test
solution, determine the concentration of protein in the test 22 - 30 95 5
solution.
– as detector a spectrophotometer set at 210 nm.
METHOD 7
Inject 10 μL of the reference solution and 10 μL of the
This method is based on nitrogen analysis as a means of test solution. In the chromatograms obtained, the peak
protein determination. Interference caused by the presence of corresponding to acetic acid has a retention time of 3-4 min.
other nitrogen-containing substances in the test sample can The baseline presents a steep rise after the start of the linear
affect the determination of protein by this method. Nitrogen gradient, which corresponds to the elution of the peptide
analysis techniques destroy the test sample during the analysis from the column. Determine the content of acetic acid in the
but are not limited to protein presentation in an aqueous peptide.
environment.
Procedure A. Proceed as prescribed for the determination of
nitrogen by sulfuric acid digestion (2.5.9) or use commercial
instrumentation for Kjeldahl nitrogen assay. 01/2011:20535
Procedure B. Commercial instrumentation is available
for nitrogen analysis. Most nitrogen analysis instruments
use pyrolysis (i.e. combustion of the sample in oxygen at 2.5.35. NITROUS OXIDE IN GASES
temperatures approaching 1000 °C), which produces nitric
oxide (NO) and other oxides of nitrogen (NOx) from the Gases absorb light at one or more specific wavelengths. This
nitrogen present in the substance to be examined. Some property is widely used to allow highly selective measurement
of their concentrations.
instruments convert the nitric oxides to nitrogen gas, which
is quantified using a thermal-conductivity detector. OtherDescription and principle of measurement. The
instruments mix nitric oxide (NO) with ozone (O3) to produce
concentration of nitrous oxide in other gases can be
excited nitrogen dioxide (NO2*), which emits light when determined using an infrared analyser.
it decays and can be quantified with a chemiluminescence
The infrared analyser generally consists of a light source
detector. A protein reference material that is relatively pure
emitting broadband infrared radiation, an optical device,
and is similar in composition to the test proteins is used
a sample cell and a detector. The optical device may be
to optimise the injection and pyrolysis parameters and to
positioned either before or after the sample cell and it consists
evaluate consistency in the analysis.
of one or several optical filters, through which the broadband
Calculations. The protein concentration is calculated by radiation is passed. The optical device in this case is selected
dividing the nitrogen content of the sample by the known for nitrous oxide. The measurement light beam passes through
nitrogen content of the protein. The known nitrogen the sample cell and may also pass through a reference cell if
content of the protein can be determined from the chemical
the analyser integrates such a feature (some use an electronic
composition of the protein or by comparison with a suitable
system instead of a reference cell).
reference substance.
When nitrous oxide is present in the sample cell, absorption
of energy in the measurement light beam will occur according
to the Beer-Lambert law and this produces a change in
01/2008:20534 the detector signal. This measurement signal is compared
to a reference signal to generate an output related to the
concentration of nitrous oxide. The generated signal is
2.5.34. ACETIC ACID IN SYNTHETIC linearised in order to obtain the nitrous oxide concentration.
PEPTIDES To prevent the entry of particles into the sensors, which could
cause stray-light phenomena, the apparatus is fitted with a
Examine by liquid chromatography (2.2.29). suitable filter.
General Notices (1) apply to all monographs and other texts 169
2.5.38. MMS, EMS and IMS in active substances EUROPEAN PHARMACOPOEIA 8.0
W1 = mass of each ester used to prepare reference At the end of analysis the temperature of the column is raised
solution (a), in milligrams ; to 270 °C and maintained at this temperature for 8 min.
W2 = mass of the substance to be examined in the test Detection : mass spectrometer as described below ; adjust the
solution, in milligrams ; detector settings so as to comply with the system suitability
0.05 = dilution factor. criteria :
– quadrupole mass spectrometer equipped with an electron
04/2013:20539
impact ionisation mode (70 eV) ;
2.5.39. METHANESULFONYL – mass spectrometer parameters for the fragmentometric
CHLORIDE IN METHANESULFONIC mode (single-ion monitoring (SIM)) set as follows :
ACID m/z
Substance Duration of monitoring
The following method has been validated for the determination Methanesulfonyl
of methanesulfonyl chloride in methanesulfonic acid at 79 tR between 3.3 min and 6.0 min
chloride
concentrations in the range of 0.05 ppm to 50 ppm. Butyl methane- 56 tR between 6.0 min and 8.0 min
Gas chromatography (2.2.28) coupled with mass spectrometry sulfonate (BMS)
(2.2.43).
Internal standard solution. Dissolve 7 μL of butyl Injection : 5 μL of the test solution, reference solutions (b)
methanesulfonate CRS (BMS) in methylene chloride R and and (c), the internal standard solution and methylene
dilute to 10.0 mL with the same solvent. Dilute 5.0 mL of this chloride R.
solution to 50.0 mL with methylene chloride R.
Relative retention with reference to the internal standard
Test solution. To 5 mL of water R, add 7.4 g of the substance
(BMS) (retention time = about 7.2 min) : methanesulfonyl
to be examined and mix slowly. After cooling, add 5.0 mL
chloride = about 0.68.
of methylene chloride R and 100 μL of the internal standard
solution and shake. Allow to separate and transfer the System suitability :
organic layer to a vial containing 1 g of anhydrous sodium
sulfate R. Repeat the extraction twice with 5.0 mL of methylene – in the chromatogram obtained with the internal standard
chloride R each time, combine the organic layers and filter. solution, there is no peak with the same retention time as
Reference solution (a). Dissolve 50.0 mg of methanesulfonyl methanesulfonyl chloride ;
chloride R in methylene chloride R and dilute to 10.0 mL with
the same solvent. Dilute 1.0 mL of the solution to 10.0 mL – resolution : minimum 5.0 between the peaks due to
with methylene chloride R. Dilute 300 μL of this solution to methanesulfonyl chloride and BMS in the chromatogram
10.0 mL with methylene chloride R. obtained with reference solution (b) ;
Reference solution (b). Dilute 500 μL of reference solution (a) – signal-to-noise ratio : minimum 10 for the peak due to
and 100 μL of the internal standard solution to 15.0 mL with methanesulfonyl chloride in the chromatogram obtained
methylene chloride R. with reference solution (c).
Reference solution (c). Dilute 25 μL of reference solution (a)
and 100 μL of the internal standard solution to 15.0 mL with Calculate the content of methanesulfonyl chloride in parts per
methylene chloride R. million using the following expression :
Column :
– material : fused silica ;
– size : l = 15 m, Ø = 0.25 mm ;
– stationary phase : poly(dimethyl)siloxane R (film thickness
A1 = area of the peak due to methanesulfonyl chloride
1 μm).
in the chromatogram obtained with reference
Carrier gas : helium for chromatography R. solution (b);
Flow rate : 1 mL/min. A2 = area of the peak due to methanesulfonyl chloride in
Pulsed splitless: 60 kPa, 0.1 min. the chromatogram obtained with the test solution ;
Temperature : C = percentage content of methanesulfonyl chloride ;
Time Temperature
I1 = area of the peak due to BMS in the chromatogram
(min) (°C)
obtained with reference solution (b) ;
Column 0-4 40
I2 = area of the peak due to BMS in the chromatogram
4-8 40 → 200 obtained with the test solution ;
Injection port 240 W1 = mass of methanesulfonyl chloride used to prepare
Detector : 280
reference solution (a), in milligrams ;
transfer line
W2 = mass of the sample in the test solution, in
source 230
milligrams ;
analyser 150 1.5 = dilution factor.
General Notices (1) apply to all monographs and other texts 171
EUROPEAN PHARMACOPOEIA 8.0 2.6.1. Sterility
General Notices (1) apply to all monographs and other texts 175
2.6.1. Sterility EUROPEAN PHARMACOPOEIA 8.0
Table 2.6.1.-1. – Strains of the test micro-organisms suitable for use in the growth promotion test and the method suitability test
Aerobic bacteria
Staphylococcus aureus ATCC 6538, CIP 4.83, NCTC 10788, NCIMB 9518, NBRC 13276
Bacillus subtilis ATCC 6633, CIP 52.62, NCIMB 8054, NBRC 3134
Pseudomonas aeruginosa ATCC 9027, NCIMB 8626, CIP 82.118, NBRC 13275
Anaerobic bacterium
Clostridium sporogenes ATCC 19404, CIP 79.3, NCTC 532, ATCC 11437, NBRC 14293
Fungi
METHOD SUITABILITY TEST alcoholic solutions and cellulose acetate filters, for example,
Carry out a test as described below under Test for sterility offor strongly alcoholic solutions. Specially adapted filters may
the product to be examined using exactly the same methods be needed for certain products, e.g. for antibiotics.
except for the following modifications. The technique described below assumes that membranes
Membrane filtration. After transferring the contents of the about 50 mm in diameter will be used. If filters of a different
container or containers to be tested to the membrane add an diameter are used the volumes of the dilutions and the
inoculum of a small number of viable micro-organisms (not washings should be adjusted accordingly. The filtration
more than 100 CFU) to the final portion of sterile diluent apparatus and membrane are sterilised by appropriate means.
used to rinse the filter. The apparatus is designed so that the solution to be examined
can be introduced and filtered under aseptic conditions ; it
Direct inoculation. After transferring the content of the permits the aseptic removal of the membrane for transfer to
container or containers to be tested (for catgut and other the medium or it is suitable for carrying out the incubation
surgical sutures for veterinary use : strands) to the culture after adding the medium to the apparatus itself.
medium add an inoculum of a small number of viable
micro-organisms (not more than 100 CFU) to the medium. Aqueous solutions. If appropriate, transfer a small quantity
of a suitable, sterile diluent such as a 1 g/L neutral solution
In both cases use the same micro-organisms as those described of meat or casein peptone pH 7.1 ± 0.2 onto the membrane
above under Growth promotion test of aerobes, anaerobes and in the apparatus and filter. The diluent may contain suitable
fungi. Perform a growth promotion test as a positive control. neutralising substances and/or appropriate inactivating
Incubate all the containers containing medium for not more substances for example in the case of antibiotics.
than 5 days.
If clearly visible growth of micro-organisms is obtained after Transfer the contents of the container or containers to be
the incubation, visually comparable to that in the control tested to the membrane or membranes, if necessary after
vessel without product, either the product possesses no diluting to the volume used in the method suitability test
antimicrobial activity under the conditions of the test or suchwith the chosen sterile diluent but in any case using not less
than the quantities of the product to be examined prescribed
activity has been satisfactorily eliminated. The test for sterility
may then be carried out without further modification. in Table 2.6.1.-2. Filter immediately. If the product has
antimicrobial properties, wash the membrane not less than
If clearly visible growth is not obtained in the presence of the
3 times by filtering through it each time the volume of the
product to be tested, visually comparable to that in the control
chosen sterile diluent used in the method suitability test. Do
vessels without product, the product possesses antimicrobial not exceed a washing cycle of 5 times 100 mL per filter, even
activity that has not been satisfactorily eliminated under the if during the method suitability test it has been demonstrated
conditions of the test. Modify the conditions in order to that such a cycle does not fully eliminate the antimicrobial
eliminate the antimicrobial activity and repeat the method activity. Transfer the whole membrane to the culture medium
suitability test. or cut it aseptically into 2 equal parts and transfer one half
This method suitability test is performed : to each of 2 suitable media. Use the same volume of each
a) when the test for sterility has to be carried out on a new medium as in the method suitability test. Alternatively,
product ; transfer the medium onto the membrane in the apparatus.
b) whenever there is a change in the experimental conditions Incubate the media for not less than 14 days.
of the test. Soluble solids. Use for each medium not less than the quantity
The method suitability test may be performed simultaneously prescribed in Table 2.6.1.-2 of the product dissolved in
with the test for sterility of the product to be examined. a suitable solvent such as the solvent provided with the
preparation, water for injections, saline or a 1 g/L neutral
TEST FOR STERILITY OF THE PRODUCT TO BE solution of meat or casein peptone and proceed with the test
EXAMINED as described above for aqueous solutions using a membrane
The test may be carried out using the technique of membrane appropriate to the chosen solvent.
filtration or by direct inoculation of the culture media Oils and oily solutions. Use for each medium not less than
with the product to be examined. Appropriate negative the quantity of the product prescribed in Table 2.6.1.-2. Oils
controls are included. The technique of membrane filtration and oily solutions of sufficiently low viscosity may be filtered
is used whenever the nature of the product permits, that without dilution through a dry membrane. Viscous oils may
is, for filterable aqueous preparations, for alcoholic or oily be diluted as necessary with a suitable sterile diluent such as
preparations and for preparations miscible with or soluble in isopropyl myristate shown not to have antimicrobial activity
aqueous or oily solvents provided these solvents do not have in the conditions of the test. Allow the oil to penetrate the
an antimicrobial effect in the conditions of the test. membrane by its own weight then filter, applying the pressure
Membrane filtration. Use membrane filters having a nominal or suction gradually. Wash the membrane at least 3 times
pore size not greater than 0.45 μm whose effectiveness to by filtering through it each time about 100 mL of a suitable
retain micro-organisms has been established. Cellulose nitrate sterile solution such as 1 g/L neutral meat or casein peptone
filters, for example, are used for aqueous, oily and weakly containing a suitable emulsifying agent at a concentration
Solids
– less than 50 mg The whole contents of each container
– 50 mg or more but less than 300 mg Half the contents of each container but not less than 50 mg
– 300 mg to 5 g 150 mg
Catgut and other surgical sutures for veterinary use 3 sections of a strand (each 30 cm long)
shown to be appropriate in the method suitability test, for aseptic precautions and remove 3 sections of the strand for
example polysorbate 80 at a concentration of 10 g/L. Transfer each culture medium. Carry out the test on 3 sections, each
the membrane or membranes to the culture medium or media 30 cm long, cut off from the beginning, the centre and the
or vice versa as described above for aqueous solutions, and end of the strand. Use whole strands from freshly opened
incubate at the same temperatures and for the same times. cassette packs. Transfer each section of the strand to the
Ointments and creams. Use for each medium not less than selected medium. Use sufficient medium to cover adequately
the quantities of the product prescribed in Table 2.6.1.-2. the material to be tested (20 mL to 150 mL).
Ointments in a fatty base and emulsions of the water-in-oil
type may be diluted to 1 per cent in isopropyl myristate as OBSERVATION AND INTERPRETATION OF RESULTS
described above, by heating, if necessary, to not more than
40 °C. In exceptional cases it may be necessary to heat to not At intervals during the incubation period and at its conclusion,
more than 44 °C. Filter as rapidly as possible and proceed as examine the media for macroscopic evidence of microbial
described above for oils and oily solutions. growth. If the material being tested renders the medium
turbid so that the presence or absence of microbial growth
Direct inoculation of the culture medium. Transfer the cannot be readily determined by visual examination, 14 days
quantity of the preparation to be examined prescribed in after the beginning of incubation transfer portions (each not
Table 2.6.1.-2 directly into the culture medium so that the less than 1 mL) of the medium to fresh vessels of the same
volume of the product is not more than 10 per cent of the medium and then incubate the original and transfer vessels
volume of the medium, unless otherwise prescribed. for not less than 4 days.
If the product to be examined has antimicrobial activity, carry
out the test after neutralising this with a suitable neutralising If no evidence of microbial growth is found, the product to be
substance or by dilution in a sufficient quantity of culture examined complies with the test for sterility. If evidence of
medium. When it is necessary to use a large volume of the microbial growth is found the product to be examined does
product it may be preferable to use a concentrated culture not comply with the test for sterility, unless it can be clearly
medium prepared in such a way that it takes account of the demonstrated that the test was invalid for causes unrelated
subsequent dilution. Where appropriate, the concentrated to the product to be examined. The test may be considered
medium may be added directly to the product in its container. invalid only if one or more of the following conditions are
fulfilled :
Oily liquids. Use media to which have been added a suitable
emulsifying agent at a concentration shown to be appropriate a) the data of the microbiological monitoring of the sterility
in the method suitability test, for example polysorbate 80 at a testing facility show a fault ;
concentration of 10 g/L.
b) a review of the testing procedure used during the test in
Ointments and creams. Prepare by diluting to about 1 in 10 question reveals a fault ;
by emulsifying with the chosen emulsifying agent in a suitable
sterile diluent such as a 1 g/L neutral solution of meat or c) microbial growth is found in the negative controls ;
casein peptone. Transfer the diluted product to a medium not d) after determination of the identity of the micro-organisms
containing an emulsifying agent. isolated from the test, the growth of this species or these
Incubate the inoculated media for not less than 14 days. species may be ascribed unequivocally to faults with respect
Observe the cultures several times during the incubation to the material and/or the technique used in conducting the
period. Shake cultures containing oily products gently each sterility test procedure.
day. However when fluid thioglycollate medium is used for
the detection of anaerobic micro-organisms keep shaking If the test is declared to be invalid it is repeated with the same
or mixing to a minimum in order to maintain anaerobic number of units as in the original test.
conditions. If no evidence of microbial growth is found in the repeat
Catgut and other surgical sutures for veterinary use. Use for test the product examined complies with the test for sterility.
each medium not less than the quantities of the product If microbial growth is found in the repeat test the product
prescribed in Table 2.6.1.-2. Open the sealed package using examined does not comply with the test for sterility.
General Notices (1) apply to all monographs and other texts 177
2.6.2. Mycobacteria EUROPEAN PHARMACOPOEIA 8.0
* If the batch size is not known, use the maximum number of items prescribed.
**If the contents of one container are enough to inoculate the 2 media, this column gives the number of containers needed for both the media
together.
APPLICATION OF THE TEST TO PARENTERAL If at the end of the incubation time no growth of mycobacteria
PREPARATIONS, OPHTHALMIC AND OTHER occurs in any of the test media, the preparation complies with
NON-INJECTABLE PREPARATIONS REQUIRED TO the test.
COMPLY WITH THE TEST FOR STERILITY
When using the technique of membrane filtration, use,
whenever possible, the whole contents of the container, but 01/2008:20607
not less than the quantities indicated in Table 2.6.1.-2, diluting corrected 6.1
where necessary to about 100 mL with a suitable sterile
solution, such as 1 g/L neutral meat or casein peptone.
When using the technique of direct inoculation of media,
2.6.7. MYCOPLASMAS
use the quantities shown in Table 2.6.1.-2, unless otherwise Where the test for mycoplasmas is prescribed for a master
justified and authorised. The tests for bacterial and fungal cell bank, for a working cell bank, for a virus seed lot or for
sterility are carried out on the same sample of the product to control cells, both the culture method and the indicator cell
be examined. When the volume or the quantity in a single culture method are used. Where the test for mycoplasmas is
container is insufficient to carry out the tests, the contents of 2 prescribed for a virus harvest, for a bulk vaccine or for the
or more containers are used to inoculate the different media. final lot (batch), the culture method is used. The indicator
MINIMUM NUMBER OF ITEMS TO BE TESTED cell culture method may also be used, where necessary, for
screening of media.
The minimum number of items to be tested in relation to the
size of the batch is given in Table 2.6.1.-3. Nucleic acid amplification techniques (NAT) may be used
as an alternative to one or both of the other methods after
Guidelines on the test for sterility are given in general suitable validation.
chapter 5.1.9.
CULTURE METHOD
01/2008:20602 CHOICE OF CULTURE MEDIA
The test is carried out using a sufficient number of both solid
2.6.2. MYCOBACTERIA and liquid media to ensure growth in the chosen incubation
If the sample to be examined may be contaminated conditions of small numbers of mycoplasmas that may be
by micro-organisms other than mycobacteria, treat present in the product to be examined. Liquid media must
it with a suitable decontamination solution, such as contain phenol red. The range of media chosen is shown
acetylcysteine-sodium hydroxide solution or sodium to have satisfactory nutritive properties for at least the
laurilsulfate solution. micro-organisms shown below. The nutritive properties of
each new batch of medium are verified for the appropriate
Inoculate 0.2 mL of the sample in triplicate onto each of micro-organisms in the list. When testing for mycoplasmas in
2 suitable solid media (Löwenstein-Jensen medium and the product to be examined, at least 1 of the following species
Middlebrook 7H10 medium are considered suitable). will be included as a positive control :
Inoculate 0.5 mL in triplicate into a suitable liquid medium.
Incubate all media at 37 °C for 56 days. – Acholeplasma laidlawii (vaccines for human and veterinary
use where an antibiotic has been used during production) ;
Establish the fertility of the media in the presence of the
preparation to be examined by inoculation of a suitable strain – Mycoplasma gallisepticum (where avian material has been
of a Mycobacterium sp. such as BCG and if necessary use a used during production or where the vaccine is intended
suitable neutralising substance. for use in poultry) ;
If contaminating micro-organisms develop during the first – Mycoplasma hyorhinis (non-avian veterinary vaccines) ;
8 days of incubation, repeat the test and carry out at the same – Mycoplasma orale (vaccines for human and veterinary use) ;
time a bacteriological sterility test.
– Mycoplasma pneumoniae (vaccines for human use) or other TEST FOR MYCOPLASMAS IN THE PRODUCT TO BE
suitable species of D-glucose fermenter such as Mycoplasma EXAMINED
fermentans; Inoculate 10 mL of the product to be examined per 100 mL
– Mycoplasma synoviae (where avian material has been used of each liquid medium. If it has been found that a significant
during production or where the vaccine is intended for pH change occurs upon the addition of the product to be
use in poultry). examined, the liquid medium is restored to its original pH
value by the addition of a solution of either sodium hydroxide
The test strains are field isolates having undergone a limited or hydrochloric acid. Inoculate 0.2 mL of the product to
number of subcultures (not more than 15), and are stored be examined on each plate of each solid medium. Incubate
frozen or freeze-dried. After cloning, the strains are identified liquid media for 20-21 days. Incubate solid media for not less
as being of the required species by comparison with type than 14 days, except those corresponding to the 20-21 day
cultures, for example : subculture, which are incubated for 7 days. At the same time
A. laidlawii NCTC 10116 CIP 75.27 ATCC 23206 incubate an uninoculated 100 mL portion of each liquid
M. gallisepticum NCTC 10115 CIP 104967 ATCC 19610 medium and agar plates, as a negative control. On days
2-4 after inoculation, subculture each liquid medium by
M. fermentans NCTC 10117 CIP 105680 ATCC 19989 inoculating 0.2 mL on at least 1 plate of each solid medium.
M. hyorhinis NCTC 10130 CIP 104968 ATCC 17981 Repeat the procedure between the 6th and 8th days, again
between the 13th and 15th days and again between the 19th
M. orale NCTC 10112 CIP 104969 ATCC 23714 and 21st days of the test. Observe the liquid media every
M. pneumoniae NCTC 10119 CIP 103766 ATCC 15531 2 or 3 days and if a colour change occurs, subculture. If a
liquid medium shows bacterial or fungal contamination, the
M. synoviae NCTC 10124 CIP 104970 ATCC 25204 test is invalid. The test is valid if at least 1 plate per medium
and per inoculation day can be read. Include in the test
Acholeplasma laidlawii BRP, Mycoplasma fermentans BRP, positive controls prepared by inoculation of not more than
Mycoplasma hyorhinis BRP, Mycoplasma orale BRP and 100 CFU of at least 1 test micro-organism on agar medium
Mycoplasma synoviae BRP are suitable for use as low-passage or into broth medium. Where the test for mycoplasmas is
reference strains. carried out regularly and where possible, it is recommended
to use the test micro-organisms in regular rotation. The test
INCUBATION CONDITIONS micro-organisms used are those listed under Choice of culture
Incubate liquid media in tightly stoppered containers at media.
35-38 °C. Incubate solid media in microaerophilic conditions
(nitrogen containing 5-10 per cent of carbon dioxide and INTERPRETATION OF RESULTS
sufficient humidity to prevent desiccation of the agar surface) At the end of the prescribed incubation period, examine all
at 35-38 °C. inoculated solid media microscopically for the presence of
mycoplasma colonies. The product complies with the test if
NUTRITIVE PROPERTIES
growth of typical mycoplasma colonies has not occurred. The
Carry out the test for nutritive properties for each new product does not comply with the test if growth of typical
batch of medium. Inoculate the chosen media with the mycoplasma colonies has occurred on any of the solid media.
appropriate test micro-organisms ; use not more than 100 CFU The test is invalid if 1 or more of the positive controls do not
(colony-forming units) per 60 mm diameter plate containing show growth of mycoplasmas on at least 1 subculture plate.
9 mL of solid medium and per 100 mL container of liquid The test is invalid if 1 or more of the negative controls show
medium ; use a separate plate and container for each species growth of mycoplasmas. If suspect colonies are observed, a
of micro-organism. Incubate the media and make subcultures suitable validated method may be used to determine whether
from 0.2 mL of liquid medium to solid medium at the specified they are due to mycoplasmas.
intervals (see below under Test for mycoplasmas in the
product to be examined). The solid medium complies with the
test if adequate growth is found for each test micro-organism The following section is published for information.
(growth obtained does not differ by a factor greater than 5
from the value calculated with respect to the inoculum). The
liquid medium complies with the test if growth on agar plates RECOMMENDED MEDIA FOR THE CULTURE METHOD
subcultured from the broth is found for at least 1 subculture The following media are recommended. Other media may
for each test micro-organism. be used, provided that their ability to sustain the growth of
INHIBITORY SUBSTANCES mycoplasmas has been demonstrated on each batch in the
The test for inhibitory substances is carried out once for a presence and absence of the product to be examined.
given product and is repeated whenever there is a change HAYFLICK MEDIA (RECOMMENDED FOR THE GENERAL
in production method that may affect the detection of DETECTION OF MYCOPLASMAS)
mycoplasmas.
To demonstrate absence of inhibitory substances, carry out the Liquid medium
test for nutritive properties in the presence and absence of the Beef heart infusion broth (1) 90.0 mL
product to be examined. If growth of a test micro-organism 20.0 mL
Horse serum (unheated)
occurs more than 1 subculture sooner in the absence of
the product to be examined than in its presence, or if plates Yeast extract (250 g/L) 10.0 mL
directly inoculated with the product to be examined have 5.0 mL
Phenol red (0.6 g/L solution)
fewer than 1/5 of the number of colonies of those inoculated
without the product to be examined, inhibitory substances are Penicillin (20 000 IU/mL) 0.25 mL
present and they must be neutralised or their effect otherwise 1.2 mL
Deoxyribonucleic acid (2 g/L solution)
countered, for example by passage in substrates not containing
inhibitors or dilution in a larger volume of medium before
the test. If dilution is used, larger medium volumes may be Adjust to pH 7.8.
used or the inoculum volume may be divided among several Solid medium
100 mL flasks. The effectiveness of the neutralisation or
other process is checked by repeating the test for inhibitory Prepare as described above replacing beef heart infusion broth
substances after neutralisation. by beef heart infusion agar containing 15 g/L of agar.
General Notices (1) apply to all monographs and other texts 179
2.6.7. Mycoplasmas EUROPEAN PHARMACOPOEIA 8.0
Liquid medium
(4) Hanks’ balanced salt solution (modified)
Hanks’ balanced salt solution (modified) (4) 800 mL
Sodium chloride 6.4 g
Distilled water 67 mL
Potassium chloride 0.32 g
Brain heart infusion (5) 135 mL
Magnesium sulfate heptahydrate 0.08 g
PPLO Broth (6) 248 mL
Magnesium chloride hexahydrate 0.08 g
Yeast extract (170 g/L) 60 mL
Calcium chloride, anhydrous 0.112 g
Bacitracin 250 mg
Disodium hydrogen phosphate dihydrate 0.0596 g
Meticillin 250 mg
Potassium dihydrogen phosphate, anhydrous 0.048 g
Phenol red (5 g/L) 4.5 mL
Distilled water to 800 mL
Horse serum 165 mL
Swine serum 165 mL (5) Brain heart infusion
Calf-brain infusion 200 g
Adjust to pH 7.40-7.45.
Beef-heart infusion 250 g
Solid medium
Hanks’ balanced salt solution (modified) (4) 200 mL Proteose peptone 10 g
General Notices (1) apply to all monographs and other texts 181
2.6.7. Mycoplasmas EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 183
2.6.9. Abnormal toxicity EUROPEAN PHARMACOPOEIA 8.0
the product to be examined. The “maximum temperature” of The test must also be carried out on 2 healthy guinea-pigs
each rabbit is the highest temperature recorded for that rabbit weighing 250 g to 400 g. Inject intraperitoneally into each
in the 3 h after the injection. Record the temperature of each animal 1 human dose but not more than 5.0 mL. The human
rabbit at intervals of not more than 30 min, beginning at least dose is that stated on the label of the preparation to be
90 min before the injection of the product to be examined and examined or on the accompanying leaflet. Observe the
continuing 3 h after the injection. The difference between the animals for 7 days.
maximum temperature and the initial temperature of each The preparation passes the test if none of the animals shows
rabbit is taken to be its response. When this difference is signs of ill health. If more than one animal dies the preparation
negative, the result is counted as a zero response. fails the test. If one of the animals dies or shows signs of ill
Rabbits showing a temperature variation greater than 0.2 °C health, repeat the test. The preparation passes the test if none
between two successive readings in the determination of of the animals in the 2nd group die or shows signs of ill health
the initial temperature are withdrawn from the test. In any in the time interval specified.
one test, only rabbits having initial temperatures which do
not differ from one another by more than 1 °C are used. All
rabbits having an initial temperature higher than 39.8 °C or 01/2008:20610
less than 38.0 °C are withdrawn from the test.
Interpretation of results. Having carried out the test first on a 2.6.10. HISTAMINE
group of three rabbits, repeat if necessary on further groups
of three rabbits to a total of four groups, depending on the Euthanise a guinea-pig weighing 250 g to 350 g that has been
results obtained. If the summed response of the first group deprived of food for the preceding 24 h. Remove a portion of
does not exceed the figure given in the second column of the the distal small intestine 2 cm in length and empty the isolated
Table 2.6.8.-1, the substance passes the test. If the summed part by rinsing carefully with solution B described below using
response exceeds the figure given in the second column of the a syringe. Attach a fine thread to each end and make a small
table but does not exceed the figure given in the third column transverse incision in the middle of the piece of intestine.
of the table, repeat the test as indicated above. If the summed Place it in an organ bath with a capacity of 10 mL to 20 mL,
response exceeds the figure given in the third column of the containing solution B maintained at a constant temperature
table, the product fails the test. (34 °C to 36 °C) and pass through the solution a current of a
Table 2.6.8.-1 mixture of 95 parts of oxygen and 5 parts of carbon dioxide.
Attach one of the threads near to the bottom of the organ bath.
Number of rabbits Product passes if summed Product fails if summed Attach the other thread to an isotonic myograph and record
response does not exceed response exceeds
the contractions of the organ on a kymograph or other suitable
3 1.15 °C 2.65 °C means of giving a permanent record. If a lever is used, its
6 2.80 °C 4.30 °C length is such that the movements of the organ are amplified
about 20 times. The tension on the intestine should be about
9 4.45 °C 5.95 °C 9.8 mN (1 g) and it should be adjusted to the sensitivity
12 6.60 °C 6.60 °C of the organ. Flush out the organ bath with solution B.
Allow it to stand for 10 min. Flush 2 or 3 times more with
Rabbits used in a test for pyrogens where the mean rise in the solution B. Stimulate a series of contractions by the addition of
rabbits’ temperature has exceeded 1.2 °C are permanently measured volumes between 0.2 mL and 0.5 mL of a solution of
excluded. histamine dihydrochloride R having a strength which produces
reproducible submaximal responses. This dose is termed the
“high dose”. Flush the organ bath (preferably by overflow
01/2008:20609 without emptying the bath) 3 times with solution B before
each addition of histamine. The successive additions should
2.6.9. ABNORMAL TOXICITY be made at regular intervals allowing a complete relaxation
between additions (about 2 min). Add equal volumes of
GENERAL TEST a weaker dilution of histamine dihydrochloride R which
Inject intravenously into each of 5 healthy mice, weighing produces reproducible responses approximately half as great as
17 g to 24 g, the quantity of the substance to be examined the “high dose”. This dose is termed the “low dose”. Continue
prescribed in the monograph, dissolved in 0.5 mL of water for the regular additions of “high” and “low” doses of histamine
injections R or of a 9 g/L sterile solution of sodium chloride R. solution as indicated above, and alternate each addition with
Inject the solution over a period of 15 s to 30 s, unless an equal volume of a dilution of the solution to be examined,
otherwise prescribed. adjusting the dilution so that the contraction of the intestine,
The substance passes the test if none of the mice die within if any, is smaller than that due to the “high dose” of histamine.
24 h or within such time as is specified in the individual Determine whether the contraction, if any, is reproducible and
monograph. If more than one animal dies the preparation that the responses to the “high” and “low” doses of histamine
fails the test. If one of the animals dies, repeat the test. The are unchanged. Calculate the activity of the substance to
substance passes the test if none of the animals in the 2nd group be examined in terms of its equivalent in micrograms of
die within the time interval specified. histamine base from the dilution determined as above.
The quantity so determined does not exceed the quantity
IMMUNOSERA AND VACCINES FOR HUMAN USE prescribed in the monograph.
Unless otherwise prescribed, inject intraperitoneally 1 human If the solution to be examined does not produce a contraction,
dose but not more than 1.0 mL into each of 5 healthy prepare a fresh solution adding a quantity of histamine
mice, weighing 17 g to 24 g. The human dose is that stated corresponding to the maximum tolerated in the monograph
on the label of the preparation to be examined or on the and note whether the contractions produced by the
accompanying leaflet. Observe the animals for 7 days. preparation with the added histamine correspond to the
The preparation passes the test if none of the animals amount of histamine added. If this is not the case, or if the
shows signs of ill health. If more than one animal dies, the contractions caused by the substance to be examined are not
preparation fails the test. If one of the animals dies or shows reproducible or if subsequent responses to “high” and “low”
signs of ill health, repeat the test. The preparation passes the doses of histamine are diminished, the results of the tests are
test if none of the animals in the 2nd group die or shows signs invalid and the test for depressor substances (2.6.11) must be
of ill health in the time interval specified. carried out.
General Notices (1) apply to all monographs and other texts 185
2.6.12. Microbial enumeration tests EUROPEAN PHARMACOPOEIA 8.0
5 passages removed from the original master seed-lot. Grow For solid media, growth obtained must not differ by a factor
each of the bacterial and fungal test strains separately as greater than 2 from the calculated value for a standardised
described in Table 2.6.12.-1. inoculum. For a freshly prepared inoculum, growth of the
micro-organisms comparable to that previously obtained with
Use buffered sodium chloride-peptone solution pH 7.0 or a previously tested and approved batch of medium occurs.
phosphate buffer solution pH 7.2 to make test suspensions ; to Liquid media are suitable if clearly visible growth of the
suspend A. brasiliensis spores, 0.05 per cent of polysorbate 80 micro-organisms comparable to that previously obtained with
may be added to the buffer. Use the suspensions within a previously tested and approved batch of medium occurs.
2 h or within 24 h if stored at 2-8 °C. As an alternative to 4-5. SUITABILITY OF THE COUNTING METHOD IN THE
preparing and then diluting a fresh suspension of vegetative PRESENCE OF PRODUCT
cells of A. brasiliensis or B. subtilis, a stable spore suspension
is prepared and then an appropriate volume of the spore 4-5-1. Preparation of the sample. The method for sample
suspension is used for test inoculation. The stable spore preparation depends upon the physical characteristics of the
suspension may be maintained at 2-8 °C for a validated period product to be tested. If none of the procedures described
of time. below can be demonstrated to be satisfactory, an alternative
procedure must be developed.
4-3. NEGATIVE CONTROL Water-soluble products. Dissolve or dilute (usually a 1 in
To verify testing conditions, a negative control is performed 10 dilution is prepared) the product to be examined in
using the chosen diluent in place of the test preparation. There buffered sodium chloride-peptone solution pH 7.0, phosphate
must be no growth of micro-organisms. A negative control buffer solution pH 7.2 or casein soya bean digest broth.
is also performed when testing the products as described in If necessary, adjust to pH 6-8. Further dilutions, where
section 5. A failed negative control requires an investigation. necessary, are prepared with the same diluent.
4-4. GROWTH PROMOTION OF THE MEDIA Non-fatty products insoluble in water. Suspend the product
Test each batch of ready-prepared medium and each batch of to be examined (usually a 1 in 10 dilution is prepared) in
medium, prepared either from dehydrated medium or from buffered sodium chloride-peptone solution pH 7.0, phosphate
the ingredients described. buffer solution pH 7.2 or casein soya bean digest broth. A
surface-active agent such as 1 g/L of polysorbate 80 may be
Inoculate portions/plates of casein soya bean digest broth added to assist the suspension of poorly wettable substances.
and casein soya bean digest agar with a small number (not If necessary, adjust to pH 6-8. Further dilutions, where
more than 100 CFU) of the micro-organisms indicated in necessary, are prepared with the same diluent.
Table 2.6.12.-1, using a separate portion/plate of medium for Fatty products. Dissolve in isopropyl myristate, sterilised
each. Inoculate plates of Sabouraud-dextrose agar with a small by filtration or mix the product to be examined with the
number (not more than 100 CFU) of the micro-organisms minimum necessary quantity of sterile polysorbate 80 or
indicated in Table 2.6.12.-1, using a separate plate of another non-inhibitory sterile surface-active agent, heated if
medium for each. Incubate in the conditions described in necessary to not more than 40 °C, or in exceptional cases to not
Table 2.6.12.-1. more than 45 °C. Mix carefully and if necessary maintain the
Table 2.6.12.-1. – Preparation and use of test micro-organisms
Micro-organism Preparation of test Growth promotion Suitability of counting method in the
strain presence of the product
Total aerobic Total yeasts and Total aerobic Total yeasts and
microbial count moulds count microbial count moulds count
Staphylococcus aureus Casein soya bean Casein soya bean digest Casein soya bean digest
such as : digest agar or casein agar and casein soya agar/MPN casein soya
soya bean digest broth bean digest broth bean digest broth
ATCC 6538 - -
30-35 °C ≤ 100 CFU ≤ 100 CFU
NCIMB 9518
18-24 h 30-35 °C 30-35 °C
CIP 4.83
≤ 3 days ≤ 3 days
NBRC 13276
Pseudomonas Casein soya bean Casein soya bean digest Casein soya bean digest
aeruginosa digest agar or casein agar and casein soya agar/MPN casein soya
such as : soya bean digest broth bean digest broth bean digest broth
ATCC 9027 30-35 °C ≤ 100 CFU - ≤ 100 CFU -
NCIMB 8626 18-24 h 30-35 °C 30-35 °C
CIP 82.118 ≤ 3 days ≤ 3 days
NBRC 13275
Bacillus subtilis Casein soya bean Casein soya bean digest Casein soya bean digest
such as : digest agar or casein agar and casein soya agar/MPN casein soya
ATCC 6633 soya bean digest broth bean digest broth bean digest broth
30-35 °C ≤ 100 CFU - ≤ 100 CFU -
NCIMB 8054
CIP 52.62 18-24 h 30-35 °C 30-35 °C
NBRC 3134 ≤ 3 days ≤ 3 days
Candida albicans Sabouraud-dextrose Casein soya bean Sabouraud-dextrose Casein soya bean Sabouraud-dextrose
such as : agar or Sabouraud- digest agar agar digest agar agar
dextrose broth ≤ 100 CFU ≤ 100 CFU ≤ 100 CFU ≤ 100 CFU
ATCC 10231 20-25 °C
NCPF 3179 30-35 °C 20-25 °C 30-35 °C 20-25 °C
2-3 days
IP 48.72 ≤ 5 days ≤ 5 days ≤ 5 days ≤ 5 days
NBRC 1594 MPN : not applicable
Aspergillus brasiliensis Sabouraud-dextrose Casein soya bean Sabouraud-dextrose Casein soya bean Sabouraud-dextrose
such as : agar or potato-dextrose digest agar agar digest agar agar
agar ≤ 100 CFU ≤ 100 CFU ≤ 100 CFU ≤ 100 CFU
ATCC 16404
20-25 °C 30-35 °C 20-25 °C 30-35 °C 20-25 °C
IMI 149007
5-7 days, or until good ≤ 5 days ≤ 5 days ≤ 5 days ≤ 5 days
IP 1431.83 sporulation is achieved
NBRC 9455 MPN : not applicable
temperature in a water-bath. Add sufficient of the pre-warmed Table 2.6.12.-2. – Common neutralising agents for interfering
chosen diluent to make a 1 in 10 dilution of the original substances
product. Mix carefully whilst maintaining the temperature for Interfering substance Potential neutralising
the shortest time necessary for the formation of an emulsion. method
Further serial tenfold dilutions may be prepared using the Glutaraldehyde, mercurials Sodium hydrogensulfite
chosen diluent containing a suitable concentration of sterile (sodium bisulfite)
polysorbate 80 or another non-inhibitory sterile surface-active Phenolics, alcohol, aldehydes, sorbate Dilution
agent.
Aldehydes Glycine
Fluids or solids in aerosol form. Aseptically transfer the
product into a membrane filter apparatus or a sterile container Quaternary Ammonium Compounds Lecithin
for further sampling. Use either the total contents or a defined (QACs), parahydroxybenzoates (parabens),
bis-biguanides
number of metered doses from each of the containers tested.
QACs, iodine, parabens Polysorbate
Transdermal patches. Remove the protective cover sheets
(‘release liners’) of the transdermal patches and place them, Mercurials Thioglycollate
adhesive side upwards, on sterile glass or plastic trays. Cover Mercurials, halogens, aldehydes Thiosulfate
the adhesive surface with a sterile porous material, for example
sterile gauze, to prevent the patches from sticking together, EDTA (edetate) Mg2+ or Ca2+ ions
and transfer the patches to a suitable volume of the chosen
diluent containing inactivators such as polysorbate 80 and/or 4-5-4. Recovery of micro-organism in the presence of
lecithin. Shake the preparation vigorously for at least 30 min. product. For each of the micro-organisms listed, separate
tests are performed. Only micro-organisms of the added test
4-5-2. Inoculation and dilution. Add to the sample prepared strain are counted.
as described above (4-5-1) and to a control (with no test 4-5-4-1. Membrane filtration. Use membrane filters having
material included) a sufficient volume of the microbial a nominal pore size not greater than 0.45 μm. The type
suspension to obtain an inoculum of not more than 100 CFU. of filter material is chosen such that the bacteria-retaining
The volume of the suspension of the inoculum should not efficiency is not affected by the components of the sample to
exceed 1 per cent of the volume of diluted product. be investigated. For each of the micro-organisms listed, one
To demonstrate acceptable microbial recovery from the membrane filter is used.
product, the lowest possible dilution factor of the prepared Transfer a suitable amount of the sample prepared as described
sample must be used for the test. Where this is not possible under 4-5-1 to 4-5-3 (preferably representing 1 g of the
due to antimicrobial activity or poor solubility, further product, or less if large numbers of CFU are expected) to the
appropriate protocols must be developed. If inhibition of membrane filter, filter immediately and rinse the membrane
growth by the sample cannot otherwise be avoided, the aliquot filter with an appropriate volume of diluent.
of the microbial suspension may be added after neutralisation,
For the determination of total aerobic microbial count
dilution or filtration.
(TAMC), transfer the membrane filter to the surface of casein
4-5-3. Neutralisation/removal of antimicrobial activity. soya bean digest agar. For the determination of total combined
The number of micro-organisms recovered from the prepared yeasts/moulds count (TYMC), transfer the membrane to the
sample diluted as described in 4-5-2 and incubated following surface of Sabouraud-dextrose agar. Incubate the plates as
the procedure described in 4-5-4, is compared to the number indicated in Table 2.6.12.-1. Perform the counting.
of micro-organisms recovered from the control preparation. 4-5-4-2. Plate-count methods. Perform plate-count methods
If growth is inhibited (reduction by a factor greater than 2), at least in duplicate for each medium and use the mean count
then modify the procedure for the particular enumeration of the result.
test to ensure the validity of the results. Modification of the 4-5-4-2-1. Pour-plate method
procedure may include, for example, (1) an increase in the For Petri dishes 9 cm in diameter, add to the dish 1 mL
volume of the diluent or culture medium, (2) incorporation of the sample prepared as described under 4-5-1 to
of specific or general neutralising agents into the diluent, 4-5-3 and 15-20 mL of casein soya bean digest agar or
(3) membrane filtration, or (4) a combination of the above Sabouraud-dextrose agar, both media being at not more
measures. than 45 °C. If larger Petri dishes are used, the amount of
Neutralising agents. Neutralising agents may be used to agar medium is increased accordingly. For each of the
neutralise the activity of antimicrobial agents (Table 2.6.12.-2). micro-organisms listed in Table 2.6.12.-1, at least 2 Petri dishes
They may be added to the chosen diluent or the medium are used. Incubate the plates as indicated in Table 2.6.12.-1.
preferably before sterilisation. If used, their efficacy and their Take the arithmetic mean of the counts per medium and
absence of toxicity for micro-organisms must be demonstrated calculate the number of CFU in the original inoculum.
by carrying out a blank with neutraliser and without product. 4-5-4-2-2. Surface-spread method
If no suitable neutralising method can be found, it can be For Petri dishes 9 cm in diameter, add 15-20 mL of casein soya
assumed that the failure to isolate the inoculated organism is bean digest agar or Sabouraud-dextrose agar at about 45 °C
attributable to the microbicidal activity of the product. This to each Petri dish and allow to solidify. If larger Petri dishes
information serves to indicate that the product is not likely to are used, the volume of the agar is increased accordingly.
be contaminated with the given species of the micro-organism. Dry the plates, for example in a laminar-air-flow cabinet
However, it is possible that the product only inhibits some of or an incubator. For each of the micro-organisms listed
the micro-organisms specified herein, but does not inhibit in Table 2.6.12.-1, at least 2 Petri dishes are used. Spread
others not included amongst the test strains or for which the a measured volume of not less than 0.1 mL of the sample
latter are not representative. Then, perform the test with the prepared as described under 4-5-1 to 4-5-3 over the surface
highest dilution factor compatible with microbial growth and of the medium. Incubate and count as prescribed under
the specific acceptance criterion. 4-5-4-2-1.
General Notices (1) apply to all monographs and other texts 187
2.6.12. Microbial enumeration tests EUROPEAN PHARMACOPOEIA 8.0
Incubate all tubes at 30-35 °C for not more than 3 days. If 1 0 0 3.6 0.2-17
reading of the results is difficult or uncertain owing to the 1 0 1 7.2 1.2-17
nature of the product to be examined, subculture in the same
broth, or in casein soya bean digest agar, for 1-2 days at the 1 0 2 11 4-35
same temperature and use these results. Determine the most 1 1 0 7.4 1.3-20
probable number of micro-organisms per gram or millilitre of
the product to be examined from Table 2.6.12.-3. 1 1 1 11 4-35
If the above criteria cannot be met for one or more of the 2 1 1 20 5-38
organisms tested with any of the described methods, the 2 1 2 27 9-94
method and test conditions that come closest to the criteria
are used to test the product. 2 2 0 21 5-40
2 2 1 28 9-94
2 2 2 35 9-94
5. TESTING OF PRODUCTS
2 3 0 29 9-94
5-1. AMOUNT USED FOR THE TEST
2 3 1 36 9-94
Unless otherwise prescribed, use 10 g or 10 mL of the product
to be examined taken with the precautions referred to above. 3 0 0 23 5-94
For fluids or solids in aerosol form, sample 10 containers. For 3 0 1 38 9-104
transdermal patches, sample 10 patches.
3 0 2 64 16-181
The amount to be tested may be reduced for active substances 3 1 0 43 9-181
that will be formulated in the following conditions : the
amount per dosage unit (e.g. tablet, capsule, injection) is less 3 1 1 75 17-199
than or equal to 1 mg or the amount per gram or millilitre (for 3 1 2 120 30-360
preparations not presented in dose units) is less than 1 mg. In
these cases, the amount to be tested is not less than the amount 3 1 3 160 30-380
present in 10 dosage units or 10 g or 10 mL of the product. 3 2 0 93 18-360
For materials used as active substances where sample quantity 3 2 1 150 30-380
is limited or batch size is extremely small (i.e. less than 3 2 2 210 30-400
1000 mL or 1000 g), the amount tested shall be 1 per cent of
the batch unless a lesser amount is prescribed or justified and 3 2 3 290 90-990
authorised. 3 3 0 240 40-990
3 3 1 460 90-1980
For products where the total number of entities in a batch is
less than 200 (e.g. samples used in clinical trials), the sample 3 3 2 1100 200-4000
size may be reduced to 2 units, or 1 unit if the size is less than
3 3 3 > 1100
100.
5-2. EXAMINATION OF THE PRODUCT
Select the sample(s) at random from the bulk material or from
the available containers of the preparation. To obtain the 5-2-1. Membrane filtration
required quantity, mix the contents of a sufficient number of Use a filtration apparatus designed to allow the transfer of the
containers to provide the sample. filter to the medium. Prepare the sample using a method that
General Notices (1) apply to all monographs and other texts 189
2.6.13. Test for specified micro-organisms EUROPEAN PHARMACOPOEIA 8.0
Use the suspensions within 2 h or within 24 h if stored at more than the shortest period of time specified in the test.
2-8 °C. Growth of the micro-organism comparable to that previously
3-1-2. Clostridia. Use Clostridium sporogenes such as obtained with a previously tested and approved batch of
ATCC 11437 (NBRC 14293, NCIMB 12343, CIP 100651) medium occurs.
or ATCC 19404 (NCTC 532 or CIP 79.03) or NBRC 14293. Test for inhibitory properties, liquid or solid media : inoculate
Grow the clostridial test strain under anaerobic conditions in the appropriate medium with at least 100 CFU of the
reinforced medium for clostridia at 30-35 °C for 24-48 h. As appropriate micro-organism. Incubate at the specified
an alternative to preparing and then diluting down a fresh temperature for not less than the longest period of time
suspension of vegetative cells of Cl. sporogenes, a stable spore specified in the test. No growth of the test micro-organism
suspension is used for test inoculation. The stable spore occurs.
suspension may be maintained at 2-8 °C for a validated period.
Test for indicative properties : perform the surface-spread
3-2. NEGATIVE CONTROL method, inoculating each plate with a small number (not more
To verify testing conditions, a negative control is performed than 100 CFU) of the appropriate micro-organism. Incubate at
using the chosen diluent in place of the test preparation. There the specified temperature for a period of time within the range
must be no growth of micro-organisms. A negative control specified in the test. Colonies are comparable in appearance
is also performed when testing the products as described in and indication reactions to those previously obtained with a
section 4. A failed negative control requires an investigation. previously tested and approved batch of medium.
3-3. GROWTH PROMOTION AND INHIBITORY 3-4. SUITABILITY OF THE TEST METHOD
PROPERTIES OF THE MEDIA For each product to be tested, perform the sample preparation
Test each batch of ready-prepared medium and each batch of as described in the relevant paragraph in section 4. Add each
medium prepared either from dehydrated medium or from test strain at the time of mixing, in the prescribed growth
ingredients. medium. Inoculate the test strains individually. Use a number
Verify suitable properties of relevant media as described in of micro-organisms equivalent to not more than 100 CFU in
Table 2.6.13.-1. the inoculated test preparation.
Test for growth promoting properties, liquid media : inoculate Perform the test as described in the relevant paragraph in
a portion of the appropriate medium with a small number section 4 using the shortest incubation period prescribed.
(not more than 100 CFU) of the appropriate micro-organism. The specified micro-organisms must be detected with the
Incubate at the specified temperature for not more than the indication reactions as described in section 4.
shortest period of time specified in the test. Clearly visible
growth of the micro-organism comparable to that previously Any antimicrobial activity of the product necessitates a
obtained with a previously tested and approved batch of modification of the test procedure (see 4-5-3 of general
medium occurs. chapter 2.6.12).
Test for growth promoting properties, solid media : perform If for a given product the antimicrobial activity with respect
the surface-spread method, inoculating each plate with a to a micro-organism for which testing is prescribed cannot
small number (not more than 100 CFU) of the appropriate be neutralised, then it is to be assumed that the inhibited
micro-organism. Incubate at the specified temperature for not micro-organism will not be present in the product.
Xylose, lysine, deoxycholate agar Growth promoting + indicative Salmonella enterica subsp. enterica
serovar Typhimurium or Salmonella
enterica subsp. enterica serovar Abony
Test for Pseudomonas aeruginosa Cetrimide agar Growth promoting P. aeruginosa
Inhibitory E. coli
Test for Staphylococcus aureus Mannitol salt agar Growth promoting + indicative S. aureus
Inhibitory E. coli
Test for clostridia Reinforced medium for clostridia Growth promoting Cl. sporogenes
Columbia agar Growth promoting Cl. sporogenes
Test for Candida albicans Sabouraud dextrose broth Growth promoting C. albicans
Sabouraud dextrose agar Growth promoting + indicative C. albicans
General Notices (1) apply to all monographs and other texts 191
2.6.13. Test for specified micro-organisms EUROPEAN PHARMACOPOEIA 8.0
4-6-3. Interpretation. The occurrence of anaerobic growth of Casein soya bean digest agar
rods (with or without endospores) giving a negative catalase Pancreatic digest of casein 15.0 g
reaction indicates the presence of clostridia. This is confirmed
by identification tests. Papaic digest of soya bean 5.0 g
Buffered sodium chloride-peptone solution pH 7.0 Mixture of peptic digest of animal tissue and pancreatic 10.0 g
digest of casein (1:1)
Potassium dihydrogen phosphate 3.6 g
Purified water 1000 mL
Disodium hydrogen phosphate 7.2 g, equivalent to 0.067 M phosphate
dihydrate
Sodium chloride 4.3 g Adjust the pH so that after sterilisation it is 5.6 ± 0.2 at 25 °C.
Sterilise in an autoclave using a validated cycle.
Peptone (meat or casein) 1.0 g
Purified water 1000 mL Adjust the pH so that after heating it is 7.2 ± 0.2 at 25 °C. Heat
at 100 °C for 30 min and cool immediately.
Adjust the pH so that after sterilisation it is 7.3 ± 0.2 at 25 °C.
Sterilise in an autoclave using a validated cycle.
MacConkey broth
20.0 g
Cetrimide agar
Pancreatic digest of gelatin
Pancreatic digest of gelatin 20.0 g
Lactose monohydrate 10.0 g
Magnesium chloride 1.4 g
Dehydrated ox bile 5.0 g
Dipotassium sulfate 10.0 g
Bromocresol purple 10 mg
Cetrimide 0.3 g
Purified water 1000 mL
Agar 13.6 g
Adjust the pH so that after sterilisation it is 7.3 ± 0.2 at 25 °C. Purified water 1000 mL
Sterilise in an autoclave using a validated cycle.
Glycerol 10.0 mL
MacConkey agar
Pancreatic digest of gelatin 17.0 g
Heat to boiling for 1 min with shaking. Adjust the pH so
Peptones (meat and casein) 3.0 g that after sterilisation it is 7.2 ± 0.2 at 25 °C. Sterilise in an
autoclave using a validated cycle.
Lactose monohydrate 10.0 g
General Notices (1) apply to all monographs and other texts 193
2.6.14. Bacterial endotoxins EUROPEAN PHARMACOPOEIA 8.0
Hydrate the agar, dissolve by heating to boiling with pipette tips for automatic pipetters, use apparatus shown to
continuous stirring. If necessary, adjust the pH so that after be free of detectable endotoxin and which does not interfere
sterilisation it is 6.8 ± 0.2 at 25 °C. Sterilise in an autoclave in the test.
using a validated cycle. NOTE : in this chapter, the term ‘tube’ includes all types of
receptacles, for example microtitre plate wells.
Columbia agar 2. REAGENTS, TEST SOLUTIONS
Pancreatic digest of casein 10.0 g
(1) Amoebocyte lysate
Meat peptic digest 5.0 g Amoebocyte lysate is a lyophilised product obtained from
Heart pancreatic digest 3.0 g amoebocyte lysate from the horseshoe crab (Limulus
polyphemus or Tachypleus tridentatus). This reagent refers
Yeast extract 5.0 g only to a product manufactured in accordance with the
Maize starch 1.0 g regulations of the competent authority.
Sodium chloride 5.0 g NOTE : amoebocyte lysate reacts with some β-glucans in
addition to endotoxins. Amoebocyte lysate preparations which
Agar, according to gelling power 10.0-15.0 g do not react with glucans are available ; they are prepared by
Purified water 1000 mL removing from amoebocyte lysate the G factor, which reacts
with glucans, or by inhibiting the G factor reacting system
of amoebocyte lysate. These preparations may be used for
Hydrate the agar, dissolve by heating to boiling with
endotoxin testing in the presence of glucans.
continuous stirring. If necessary, adjust the pH so that after
sterilisation it is 7.3 ± 0.2 at 25 °C. Sterilise in an autoclave (2) Lysate solution
using a validated cycle. Allow to cool to 45-50 °C ; add, where Dissolve amoebocyte lysate in water for BET or in a buffer, as
necessary, gentamicin sulfate corresponding to 20 mg of recommended by the lysate manufacturer, by gentle stirring.
gentamicin base and pour into Petri dishes. Store the reconstituted lysate, refrigerated or frozen, as
indicated by the manufacturer.
(3) Water for BET (water for bacterial endotoxins test)
Water for injections R or water produced by other procedures
that shows no reaction with the lysate employed at the
detection limit of the reagent.
01/2010:20614
corrected 7.0 3. PREPARATION OF THE STANDARD ENDOTOXIN
STOCK SOLUTION
The standard endotoxin stock solution is prepared from
2.6.14. BACTERIAL ENDOTOXINS an endotoxin reference standard that has been calibrated
against the International Standard, for example endotoxin
The test for bacterial endotoxins (BET) is used to detect standard BRP.
or quantify endotoxins from gram-negative bacteria using Endotoxin is expressed in International Units (IU). The
amoebocyte lysate from the horseshoe crab (Limulus equivalence in IU of the International Standard is stated by
polyphemus or Tachypleus tridentatus). There are 3 techniques the World Health Organization.
for this test : the gel-clot technique, which is based on
gel formation ; the turbidimetric technique, based on the NOTE : one International Unit (IU) of endotoxin is equal to
development of turbidity after cleavage of an endogenous one Endotoxin Unit (E.U.).
substrate ; and the chromogenic technique, based on Follow the specifications in the package leaflet and on the
the development of colour after cleavage of a synthetic label for preparation and storage of the standard endotoxin
peptide-chromogen complex. stock solution.
The following 6 methods are described in the present chapter :
4. PREPARATION OF THE STANDARD ENDOTOXIN
Method A. Gel-clot method : limit test SOLUTIONS
Method B. Gel-clot method : quantitative test After vigorously mixing the standard endotoxin stock solution,
prepare appropriate serial dilutions of this solution using
Method C. Turbidimetric kinetic method water for BET.
Method D. Chromogenic kinetic method Use the solutions as soon as possible to avoid loss of activity
Method E. Chromogenic end-point method by adsorption.
Method F. Turbidimetric end-point method 5. PREPARATION OF THE TEST SOLUTIONS
Proceed by any of the 6 methods for the test. In the event Prepare the test solutions by dissolving or diluting active
of doubt or dispute, the final decision is made based upon substances or medicinal products using water for BET.
method A unless otherwise indicated in the monograph. Some substances or preparations may be more appropriately
dissolved or diluted in other aqueous solutions. If necessary,
The test is carried out in a manner that avoids endotoxin adjust the pH of the test solution (or dilution thereof) so that
contamination. the pH of the mixture of the lysate and test solution falls within
the pH range specified by the lysate manufacturer, usually
1. APPARATUS 6.0 to 8.0. The pH may be adjusted by the use of acid, base or
a suitable buffer, as recommended by the lysate manufacturer.
Depyrogenate all glassware and other heat-stable apparatus in Acids and bases may be prepared from concentrates or solids
a hot-air oven using a validated process. A commonly used with water for BET in containers free of detectable endotoxin.
minimum time and temperature is 30 minutes at 250 °C. If Buffers must be validated to be free of detectable endotoxin
employing plastic apparatus, such as microtitre plates and and interfering factors.
6. DETERMINATION OF THE MAXIMUM VALID Confirmation of the lysate sensitivity is carried out when a
DILUTION new lot of lysate is used or when there is any change in the test
The Maximum Valid Dilution (MVD) is the maximum conditions which may affect the outcome of the test.
allowable dilution of a sample at which the endotoxin limit Prepare standard solutions of at least 4 concentrations
can be determined. Determine the MVD using the following equivalent to 2λ, λ, 0.5λ and 0.25λ by diluting the standard
formulae : endotoxin stock solution with water for BET.
Mix a volume of the lysate solution with an equal volume
of 1 of the standard solutions (such as 0.1 mL aliquots) in
each tube. When single test vials or ampoules containing
Endotoxin limit : the endotoxin limit for active substances lyophilised lysate are employed, add solutions of standards
administered parenterally, defined on the basis of dose, is directly to the vial or ampoule. Incubate the reaction mixture
equal to : for a constant period according to the recommendations of
the lysate manufacturer (usually at 37 ± 1 °C for 60 ± 2 min),
avoiding vibration. Test the integrity of the gel : for tubes, take
each tube in turn directly from the incubator and invert it
through approximately 180° in one smooth motion. If a firm
K = threshold pyrogenic dose of endotoxin per gel has formed that remains in place upon inversion, record
kilogram of body mass, the result as positive. A result is negative if an intact gel is not
M = maximum recommended bolus dose of product formed.
per kilogram of body mass. The test is considered valid when the lowest concentration of
When the product is to be injected at frequent intervals the standard solutions shows a negative result in all replicate
or infused continuously, M is the maximum total dose tests.
administered in a single hour period.
The end-point is the lowest concentration in the series
The endotoxin limit for active substances administered of decreasing concentrations of standard endotoxin that
parenterally is specified in units such as IU/mL, IU/mg, IU/Unit clots the lysate. Determine the geometric mean end-point
of biological activity, etc., in monographs. concentration by calculting the mean of the logarithms of
Concentration of test solution : the end-point concentrations of the 4 dilution series, take
– mg/mL if the endotoxin limit is specified by mass (IU/mg), the antilogarithm of this value, as indicated by the following
– Units/mL if the endotoxin limit is specified by unit of expression :
biological activity (IU/Unit),
– ml/mL if the endotoxin limit is specified by volume Geometric mean end-point concentration =
(IU/mL).
= sum of the log10 end-point concentrations of the
λ = the labelled lysate sensitivity in the gel-clot dilution series used,
technique (IU/mL) or the lowest concentration
used in the standard curve of the turbidimetric or f = number of replicates.
chromogenic techniques.
The geometric mean end-point concentration is the measured
7. GEL-CLOT TECHNIQUE (METHODS A AND B) sensitivity of the lysate solution (IU/mL). If this is not less
than 0.5λ and not more than 2λ, the labelled sensitivity is
The gel-clot technique allows detection or quantification confirmed and is used in the tests performed with this lysate.
of endotoxins and is based on clotting of the lysate in the
presence of endotoxins. The minimum concentration of (ii) Test for interfering factors
endotoxins required to cause the lysate to clot under standard Prepare solutions A, B, C and D as shown in Table 2.6.14.-1,
conditions is the labelled lysate sensitivity. To ensure both and use the test solutions at a dilution less than the MVD, not
the precision and validity of the test, confirm the labelled containing any detectable endotoxins, operating as described
lysate sensitivity and perform the test for interfering factors as under 1. Preparatory testing, (i) Confirmation of the labelled
described under 1. Preparatory testing. lysate sensitivity.
1. PREPARATORY TESTING
The geometric mean end-point concentrations of solutions B
(i) Confirmation of the labelled lysate sensitivity and C are determined using the expression described in
Confirm in 4 replicates the labelled sensitivity λ, expressed 1. Preparatory testing, (i) Confirmation of the labelled lysate
in IU/mL, of the lysate solution prior to use in the test. sensitivity.
Table 2.6.14.-1
Solution Endotoxin concentration/Solution to Diluent Dilution factor Endotoxin Number of replicates
which endotoxin is added concentration
A None/Test solution - - - 4
B 2λ/Test solution Test solution 1 2λ 4
2 1λ 4
4 0.5λ 4
8 0.25λ 4
C 2λ/Water for BET Water for BET 1 2λ 2
2 1λ 2
4 0.5λ 2
8 0.25λ 2
D None/Water for BET - - - 2
Solution A = solution of the preparation being examined that is free of detectable endotoxins.
Solution B = test for interference.
Solution C = control of the labelled lysate sensitivity.
Solution D = negative control (water for BET).
General Notices (1) apply to all monographs and other texts 195
2.6.14. Bacterial endotoxins EUROPEAN PHARMACOPOEIA 8.0
The test for interfering factors must be repeated when any (ii) Interpretation
changes are made to the experimental conditions that are The test is considered valid when both replicates of solution B
likely to influence the result of the test. and C are positive and those of solution D are negative.
The test is considered valid when all replicates of solutions A When a negative result is found for both replicates of
and D show no reaction and the result of solution C confirms solution A, the preparation being examined complies with
the labelled lysate sensitivity. the test.
If the sensitivity of the lysate determined with solution B is When a positive result is found for both replicates of
not less than 0.5λ and not greater than 2λ, the test solution solution A, the preparation being examined does not comply
does not contain interfering factors under the experimental with the test.
conditions used. Otherwise, the test solution interferes with When a positive result is found for one replicate of solution A
the test. and a negative result is found for the other, repeat the test.
If the preparation being examined interferes with the test at In the repeat test, the preparation being examined complies
a dilution less than the MVD, repeat the test for interfering with the test if a negative result is found for both replicates of
factors using a greater dilution, not exceeding the MVD. The solution A. The preparation does not comply with the test if a
use of a more sensitive lysate permits a greater dilution of the positive result is found for one or both replicates of solution A.
preparation being examined and this may contribute to the However, if the preparation does not comply with the test at a
elimination of interference. dilution less than the MVD, the test may be repeated using a
greater dilution, not exceeding the MVD.
Interference may be overcome by suitable validated treatment,
such as filtration, neutralisation, dialysis or heat treatment. 3. QUANTITATIVE TEST (METHOD B)
To establish that the treatment chosen effectively eliminates (i) Procedure
interference without loss of endotoxins, repeat the test for The test quantifies bacterial endotoxins in the test solution by
interfering factors using the preparation being examined to titration to an end-point. Prepare solutions A, B, C and D as
which the standard endotoxin has been added and which has shown in Table 2.6.14.-3, and test these solutions according
then been submitted to the chosen treatment. to the procedure described under 1. Preparatory testing, (i)
2. LIMIT TEST (METHOD A) Confirmation of the labelled lysate sensitivity.
(i) Procedure (ii) Calculation and interpretation
Prepare solutions A, B, C and D as shown in Table 2.6.14.-2, The test is considered valid when the following 3 conditions
and perform the test on these solutions following the procedure are met :
described under 1. Preparatory testing, (i) Confirmation of (a) both replicates of solution D (negative control) are negative,
the labelled lysate sensitivity. (b) both replicates of solution B (positive product control)
are positive,
Table 2.6.14.-2
(c) the geometric mean end-point concentration of solution C
Solution Endotoxin concentration/Solution Number of replicates is in the range of 0.5λ to 2λ.
to which endotoxin is added
A None/Diluted test solution 2
To determine the endotoxin concentration of solution A,
calculate the end-point concentration for each replicate, by
B 2λ/Diluted test solution 2 multiplying each end-point dilution factor by λ.
C 2λ/Water for BET 2 The endotoxin concentration in the test solution is the
end-point concentration of the replicates. If the test
D None/Water for BET 2 is conducted with a diluted test solution, calculate the
concentration of endotoxin in the original solution by
multiplying the result by the dilution factor.
Prepare solution A and solution B (positive product control) If none of the dilutions of the test solution is positive in a
using a dilution not greater than the MVD and treatments as valid test, report the endotoxin concentration as less than
described in 1. Preparatory testing, (ii) Test for interfering λ (or, if a diluted sample was tested, report as less than the
factors. Solutions B and C (positive controls) contain the lowest dilution factor of the sample × λ). If all dilutions are
standard endotoxin at a concentration corresponding to twice positive, the endotoxin concentration is reported as equal to
the labelled lysate sensitivity. Solution D (negative control) or greater than the largest dilution factor multiplied by λ (e.g.
consists of water for BET. in Table 2.6.14.-3, the initial dilution factor × 8 × λ).
Table 2.6.14.-3
Solution Endotoxin concentration/Solution to which Diluent Dilution factor Endotoxin Number of replicates
endotoxin is added concentration
A None/Test solution Water for BET 1 - 2
2 - 2
4 - 2
8 - 2
B 2λ/Test solution 1 2λ 2
Solution A = test solution at the dilution, not exceeding the MVD, with which the test for interfering factors was carried out. Subsequent dilution of
the test solution must not exceed the MVD. Use water for BET to make a dilution series of 4 tubes containing the test solution at concentrations of 1,
1/2, 1/4 and 1/8, relative to the dilution used in the test for interfering factors. Other dilutions up to the MVD may be used as appropriate.
Solution B = solution A containing standard endotoxin at a concentration of 2λ (positive product control).
Solution C = a dilution series of 4 tubes of water for BET containing the standard endotoxin at concentrations of 2λ, λ, 0.5λ and 0.25λ.
Solution D = water for BET (negative control).
General Notices (1) apply to all monographs and other texts 197
2.6.15. Prekallikrein activator EUROPEAN PHARMACOPOEIA 8.0
(3) the result obtained with solution D (negative control) Carry out all procedures from the beginning of the
does not exceed the limit of the blank value required in chromatography to freezing in portions during a single
the description of the lysate employed, or it is less than the working day.
endotoxin detection limit of the lysate reagent employed.
METHOD
(iii) Interpretation
The assay may be carried out using an automated enzyme
The preparation being examined complies with the test if the
analyser or a suitable microtitre plate system allowing kinetic
mean endotoxin concentration of the replicates of solution A,
measurements, with appropriate software for calculation of
after correction for dilution and concentration, is less than the
results. Standards, samples and prekallikrein substrate may be
endotoxin limit for the product.
diluted as necessary using buffer B.
Guidelines on the test for bacterial endotoxins are given in Incubate diluted standards or samples with prekallikrein
general chapter 5.1.10. substrate for 10 min such that the volume of the undiluted
sample does not exceed 1/10 of the total volume of the
incubation mixture to avoid errors caused by variation
01/2008:20615 in ionic strength and pH in the incubation mixture.
Incubate the mixture or a part thereof with at least an equal
2.6.15. PREKALLIKREIN ACTIVATOR volume of a solution of a suitable synthetic chromogenic
substrate, known to be specific for kallikrein (for example,
Prekallikrein activator (PKA) activates prekallikrein to N-benzoyl-L-prolyl-L-phenylalanyl-L-arginine 4-nitroanilide
kallikrein and may be assayed by its ability to cleave a acetate R or D-prolyl-L-phenylalanyl-L-arginine 4-nitroanilide
chromophore from a synthetic peptide substrate so that the dihydrochloride R), dissolved in buffer B. Record the rate
rate of cleavage can be measured spectrophotometrically and of change in absorbance per minute for 2-10 min at the
the concentration of PKA calculated by comparison with a wavelength specific for the substrate used. Prepare a blank for
reference preparation calibrated in International Units. each mixture of sample or standard using buffer B instead of
The International Unit is the activity of a stated amount of prekallikrein substrate.
the International Standard which consists of freeze-dried Depending on the method used, ΔA/min has to be corrected
prekallikrein activator. The equivalence in International Units by subtracting the value obtained for the corresponding
of the International Standard is stated by the World Health blank without the prekallikrein substrate. The results may be
Organization. calculated using a standard curve, a parallel-line or a slope
REAGENTS ratio assay or any other suitable statistical method. Plot
a calibration curve using the values thus obtained for the
Prekallikrein activator in albumin BRP is calibrated in
reference preparation and the respective concentrations ; use
International Units by comparison with the International
the curve to determine the PKA activity of the preparation to
Standard.
be examined.
Buffer A. Dissolve 6.055 g of tris(hydroxymethyl)aminome-
thane R, 1.17 g of sodium chloride R, 50 mg of hexadimethrine
bromide R and 0.100 g of sodium azide R in water R. Adjust
to pH 8.0 with 2 M hydrochloric acid R and dilute to 1000 mL 01/2011:20616
with water R.
Buffer B. Dissolve 6.055 g of tris(hydroxymethyl)aminome- 2.6.16. TESTS FOR EXTRANEOUS
thane R and 8.77 g of sodium chloride R in water R. Adjust to
pH 8.0 with 2 M hydrochloric acid R and dilute to 1000 mL AGENTS IN VIRAL VACCINES FOR
with water R. HUMAN USE
PREPARATION OF PREKALLIKREIN SUBSTRATE In those tests that require prior neutralisation of the virus,
To avoid coagulation activation, blood or plasma used for the use specific antibodies of non-human, non-simian origin ; if
preparation of prekallikrein must come into contact only with the virus has been propagated in avian tissues, the antibodies
plastics or silicone-treated glass surfaces. must also be of non-avian origin. To prepare antiserum, use
Draw 9 volumes of human blood into 1 volume of an immunising antigen produced in cell culture from a species
anticoagulant solution (ACD, CPD or 38 g/L solution of different from that used for the production of the vaccine
sodium citrate R) to which 1 mg/mL of hexadimethrine and free from extraneous agents. Where the use of SPF eggs
bromide R has been added. Centrifuge the mixture at 3600 g is prescribed, the eggs are obtained from a flock free from
for 5 min. Separate the plasma and centrifuge again at 6000 g specified pathogens (5.2.2).
for 20 min to sediment platelets. Separate the platelet-poor
plasma and dialyse against 10 volumes of buffer A for 20 h. VIRUS SEED LOT
Apply the dialysed plasma to a chromatography column Take samples of the virus seed lot at the time of harvesting and,
containing agarose-DEAE for ion-exchange chromatography R if they are not tested immediately, keep them at a temperature
which has been equilibrated in buffer A and is equal to below − 40 °C.
twice the volume of the plasma. Elute from the column with Adult mice. Inoculate each of not fewer than 10 adult mice,
buffer A at 20 mL/cm2/h. Collect the eluate in fractions and each weighing 15-20 g, intracerebrally with 0.03 mL and
record the absorbance at 280 nm (2.2.25). Pool the fractions intraperitoneally with 0.5 mL of the virus seed lot. Observe
containing the first protein peak so that the volume of the the mice for at least 21 days. Carry out an autopsy of all mice
pool is about 1.2 times the volume of the platelet-poor plasma. that die after the first 24 h of the test or that show signs of
Test the substrate pool for absence of kallikrein activity by illness, and examine for evidence of viral infection, both by
mixing 1 part with 20 parts of the pre-warmed chromogenic direct macroscopical observation and by subinoculation
substrate solution to be used in the assay and incubate at of appropriate tissue suspensions by the intracerebral and
37 °C for 2 min. The substrate is suitable if the increase in intraperitoneal routes into not fewer than 5 additional
absorbance is less than 0.001 per minute. Add to the pooled mice, which are observed for 21 days. The virus seed lot
solution 7 g/L of sodium chloride R and filter through a complies with the test if no mouse shows evidence of infection
membrane filter (nominal pore size 0.45 μm). Freeze the attributable to the seed lot. The test is not valid unless at
filtrate in portions and store at − 25 °C ; the substrate may be least 80 per cent of the original inoculated mice survive the
freeze-dried before storage. observation period.
Suckling mice. Inoculate each of not fewer than 20 mice, Insect viruses (only required for virus propagated in insect
each less than 24 h old, intracerebrally with 0.01 mL and cells). Neutralised samples equivalent, unless otherwise
intraperitoneally with at least 0.1 mL of the virus seed lot. prescribed, to 500 human doses of vaccine or 50 mL, whichever
Observe the mice daily for at least 14 days. Carry out an is the greater, are tested for the presence of extraneous agents
autopsy of all mice that die after the first 24 h of the test or by inoculation into at least one cell culture different from
that show signs of illness, and examine for evidence of viral that used in production and permissible to insect viruses,
infection, both by direct macroscopical observation and and that allows detection of human arboviruses. The choice
by subinoculation of appropriate tissue suspensions by the of cells is approved by the competent authority and takes
intracerebral and intraperitoneal routes into not fewer than into account the origin of the production cells and the likely
5 additional suckling mice, which are observed daily for contaminants that may be detected by the chosen cells. The
14 days. The virus seed lot passes the test if no mouse shows cells are incubated at 27 ± 1 °C and observed for a period
evidence of infection attributable to the seed lot. The test is of 14 days. The virus seed lot or harvest passes the tests if
not valid unless at least 80 per cent of the original inoculated none of the cell cultures show evidence of the presence of any
mice survive the observation period. extraneous agents. The test is not valid unless at least 80 per
Guinea-pigs. Inoculate intraperitoneally into each of not cent of the cell cultures remain viable.
fewer than 5 guinea pigs, each weighing 350-450 g, 5.0 mL of
PRODUCTION CELL CULTURE : CONTROL CELLS
the virus seed lot. Observe the animals for at least 42 days
for signs of disease. Carry out an autopsy of all guinea-pigs Examine the control cells microscopically for freedom from
that die after the first 24 h of the test or that show signs of any virus causing cytopathic degeneration throughout the
illness, and examine macroscopically ; examine the tissues time of incubation of the inoculated production cell cultures
both microscopically and culturally for evidence of infection. or for not less than 14 days beyond the time of inoculation of
Euthanise animals that survive the observation period and the production vessels, whichever is the longer. The test is
examine in a similar manner. The virus seed lot passes the test not valid unless at least 80 per cent of the control cell cultures
if no guinea-pig shows evidence of infection attributable to survive to the end of the observation period.
the seed lot. The test is not valid unless at least 80 per cent of At 14 days or at the time of the last virus harvest, whichever is
the guinea-pigs survive the observation period. the longer, carry out the tests described below.
Spiroplasmas. Virus seed lots produced in insect cells Haemadsorbing viruses. Examine not fewer than 25 per cent
are demonstrated by a validated method approved by the of the control cultures for the presence of haemadsorbing
competent authority to be free of spiroplasmas. viruses by the addition of guinea-pig red blood cells. If the test
for haemadsorbing viruses is not feasible, carry out a test for
haemagglutination viruses. If the guinea-pig red blood cells
VIRUS SEED LOT AND VIRUS HARVESTS have been stored, they shall have been stored at 5 ± 3 °C for not
more than 7 days. Read half of the cultures after incubation
Take samples at the time of harvesting and, if not tested at 5 ± 3 °C for 30 min and the other half after incubation at
immediately, keep them at a temperature below − 40 °C. 20-25 °C for 30 min. No evidence of haemadsorbing agents is
Bacterial and fungal sterility. A 10 mL sample complies with found.
the test for sterility (2.6.1). Tests in cell cultures for other extraneous agents. Pool the
Mycoplasmas. A 10 mL sample complies with the test for supernatant fluids from the control cells and examine for the
mycoplasmas (2.6.7). presence of extraneous agents by inoculation of simian kidney
and human cell cultures. If the virus is grown in a mammalian
Mycobacteria (2.6.2). A 5 mL sample is tested for the cell system other than simian or human, cells of that species,
presence of Mycobacterium spp. by culture methods known to but from a separate batch, are also inoculated. In each cell
be sensitive for the detection of these organisms. system, at least 5 mL is tested. Incubate the inoculated cultures
Test in cell culture for other extraneous agents. Neutralised at 36 ± 1 °C and observe for a period of 14 days. No evidence
samples equivalent, unless otherwise prescribed, to 500 human of extraneous agents is found.
doses of vaccine or 50 mL, whichever is the greater, are tested If the production cell culture is maintained at a temperature
for the presence of extraneous agents by inoculation into different from 36 ± 1 °C, a supplementary test for extraneous
continuous simian kidney and human cell cultures. If the agents is carried out at the production temperature using the
virus is grown in simian or human cells, the neutralised virus same type of cells as used for growth of the virus.
harvest is tested on a separate culture of these cells. If the
virus is grown in a mammalian or avian cell system other than If the virus is grown in insect cells the pooled supernatant is
simian or human, cells of that species, but from a separate also inoculated into at least one cell culture different from
batch, are also inoculated. The cells are incubated at 36 ± 1 °C that used in production and permissible to insect viruses,
and observed for a period of 14 days. The virus seed lot and that allows detection of human arboviruses. The cells are
or harvest passes the tests if none of the cell cultures show incubated at 27 ± 1 °C for 14 days. No evidence of extraneous
evidence of the presence of any extraneous agents. The test is agents is found.
not valid unless at least 80 per cent of the cell cultures remain Avian leucosis viruses (required only if the virus is
viable. propagated in primary avian tissues). Carry out a test for
Avian viruses (only required for virus seed lot propagated avian leucosis viruses using 5 mL of the supernatant fluid
in avian tissues and for virus harvest propagated in from the control cells.
primary avian tissues). Neutralise a sample equivalent to
100 human doses of vaccine or 10 mL, whichever is the CONTROL EGGS
greater. Using 0.5 mL per egg, inoculate a group of fertilised Haemagglutinating agents. Examine 0.25 mL of the allantoic
SPF eggs, 9-11 days old, by the allantoic route and a second fluid from each egg for haemagglutinating agents by mixing
group, 5-7 days old, into the yolk sac. Incubate for 7 days. directly with chicken red blood cells and after a passage in
The virus seed lot or harvest complies with the test if the SPF eggs carried out as follows : inoculate a 5 mL sample
allantoic and yolk sac fluids show no sign of the presence of the pooled amniotic fluids from the control eggs in
of any haemagglutinating agent and if all embryos and 0.5 mL volumes into the allantoic cavity and into the amniotic
chorio-allantoic membranes, examined for gross pathology, cavity of SPF eggs. The control eggs comply with the test if no
are normal. The test is not valid unless at least 80 per cent of evidence of the presence of haemagglutinating agents is found
the inoculated eggs survive for 7 days. in either test.
General Notices (1) apply to all monographs and other texts 199
2.6.17. Test for anticomplementary activity of immunoglobulin EUROPEAN PHARMACOPOEIA 8.0
A
2.6.17. TEST FOR = absorbance of the original suspension at 541 nm.
Incubate all tubes at 37 °C for 60 min and centrifuge at 1000 g Add 0.2 mL of sensitised sheep red blood cells to each tube,
for 5 min. Measure the absorbance (2.2.25) of the supernatants mix well and incubate at 37 °C for 60 min. Cool the tubes in
at 541 nm and calculate the percentage degree of haemolysis an ice-bath and centrifuge at 1000 g for 5 min. Measure the
in each tube using the following expression : absorbance of the supernatant at 541 nm and calculate the
degree of haemolysis (Y) using the following expression :
General Notices (1) apply to all monographs and other texts 201
2.6.18. Test for neurovirulence of live virus vaccines EUROPEAN PHARMACOPOEIA 8.0
activity of the preparation to be examined relative to the with the vaccine to be examined and the reference preparation.
complement control considered as 100 per cent, using the The animals are allocated randomly to treatment groups
following expression : and cages and their identity is coded so that the treatment
received by each animal is concealed from the observers
and the evaluators of the sections. The number of monkeys
inoculated is such that in the evaluation of both the vaccine
a and the reference preparation not fewer than eleven positive
= mean complement activity (CH50/mL) of monkeys are included for type 1 and type 2 virus and not
complement control ; fewer than eighteen positive monkeys for type 3 virus (positive
b = complement activity (CH50/mL) of tested sample. monkeys are those that show specific neuronal lesions of
poliovirus in the central nervous system). More than one
The test is not valid unless : batch of vaccine may be tested with the same homotypic
– the anticomplementary activities found for ACA negative reference. Monkeys from the same quarantine group are used
control and ACA positive control are within the limits stated wherever possible, otherwise monkeys from two groups are
in the leaflet accompanying the reference preparation ; used and equal numbers from each group are treated with the
– the mean complement activity of complement control (a) is vaccine and the reference preparation. If the test is carried out
in the range 80 CH50/mL to 120 CH50/mL. on two working days, an equal number of monkeys from each
group are inoculated on each day with the vaccine and the
01/2008:20618 homotypic reference preparation.
Virus content. The virus contents of the vaccine and
2.6.18. TEST FOR NEUROVIRULENCE the homotypic reference preparation are adjusted so as
OF LIVE VIRUS VACCINES to be as near as possible equal and between 105.5 and
106.5 CCID50/0.1 mL.
For each test, use not fewer than ten monkeys that are Observation. All monkeys are observed for 17 to 22 days
seronegative for the virus to be tested. For each monkey, for signs of poliomyelitis or other virus infection. Monkeys
inject not more than 0.5 mL of the material to be examined that survive the first 24 h but die before the 11th day after
into the thalamic region of each hemisphere, unless otherwise inoculation are autopsied to determine whether poliomyelitis
prescribed. The total amount of virus inoculated in each was the cause of death. Animals that die from causes
monkey must be not less than the amount contained in the other than poliomyelitis are excluded from the evaluation.
recommended single human dose of the vaccine. As a check Animals that become moribund or are severely paralysed are
against the introduction of wild neurovirulent virus, keep a euthanised and autopsied. All animals that survive until the
group of not fewer than four control monkeys as cage-mates or end of the observation period are autopsied. The test is not
in the immediate vicinity of the inoculated monkeys. Observe valid if more than 20 per cent of the animals show intercurrent
the inoculated monkeys for 17 to 21 days for symptoms of infection during the observation period.
paralysis and other evidence of neurological involvement ;
observe the control monkeys for the same period plus 10 days. Number of sections examined. The lumbar cord, the cervical
Animals that die within 48 h of injection are considered to cord, the lower and upper medulla oblongata, the midbrain,
have died from non-specific causes and may be replaced. The the thalamus and the motor cortex of each monkey, as a
test is not valid if : more than 20 per cent of the inoculated minimum, are subjected to histological examination. Sections
monkeys die from nonspecific causes ; serum samples taken are cut with a thickness of 15 μm and stained with gallocyanin.
from the control monkeys at the time of inoculation of the The minimum number of sections examined is as follows :
test animals and 10 days after the latter are euthanised show (a) 12 sections representative of the whole of the lumbar
evidence of infection by wild virus of the type to be tested enlargement,
or by measles virus. At the end of the observation period,
carry out autopsy and histopathological examinations of (b) 10 sections representative of the whole of the cervical
appropriate areas of the brain for evidence of central nervous enlargement,
system involvement. The material complies with the test if (c) 2 sections from the medulla oblongata,
there is no unexpected clinical or histopathological evidence (d) 1 section from the pons and cerebellum,
of involvement of the central nervous system attributable to
the inoculated virus. (e) 1 section from the midbrain,
(f) 1 section from the left and the right of the thalamus,
01/2008:20619 (g) 1 section from the left and the right motor cerebral cortex.
Scoring of virus activity. For the evaluation of virus activity
2.6.19. TEST FOR NEUROVIRULENCE in the hemisections of the spinal cord and brain-stem, a
OF POLIOMYELITIS VACCINE (ORAL) score system for the severity of lesions is used, differentiating
cellular infiltration and destruction of neurons as follows :
Monkeys used in the neurovirulence test comply with the
requirements given in the monograph on Poliomyelitis vaccine 1. Cellular infiltration only (the monkey is not counted as
oral (0215) and weigh not less than 1.5 kg. The pathogenicity positive),
for Macaca or Cercopithecus monkeys is tested in comparison 2. Cellular infiltration with minimal neuronal damage,
with that of a reference virus preparation for neurovirulence
testing by inoculation into the lumbar region of the central 3. Cellular infiltration with extensive neuronal damage,
nervous system after sedation with a suitable substance, for 4. Massive neuronal damage with or without cellular
example, ketamine hydrochloride. A sample of serum taken infiltration.
before the injection shall be shown not to contain neutralising The scores are recorded on a standard form(4). A monkey with
antibody at a dilution of 1:4 when tested against not more neuronal lesions in the sections but that shows no needle tract
than 1000 CCID50 of each of the three types of poliovirus. is counted as positive. A monkey showing a needle tract in the
Number of monkeys. The vaccine and the appropriate sections, but no neuronal lesions is not regarded as positive. A
homotypic reference virus are tested concurrently in the same section that shows damage from trauma but no specific virus
group of monkeys. Equal numbers of animals are inoculated lesions is not included in the score.
(4) A suitable form is shown in the Requirements for Poliomyelitis Vaccine (Oral) (Requirements for Biological Substances No. 7, World Health Organization).
Severity scores are based on hemisection readings of the The constants C1, C2 and C3 are calculated from the
lumbar (L), cervical (C) and brain (B) histological sections. expressions :
The lesion score (LS) for each positive monkey is calculated
as follows :
A mean lesion score is calculated for each group of positive N1 = number of positive monkeys per vaccine test,
monkeys.
N2 = number of positive monkeys in the two tests,
Evaluation. The comparison of the virus activity in the
vaccine and the reference preparation is based on the activity 2.3 = normal deviate at the 1 per cent level,
in the lumbar enlargement of the cord and the degree of 2.6 = normal deviate at the 0.5 per cent level,
spread of activity from this region to the cervical enlargement
and the brain. Acceptance or rejection is based on the total 1.6 = normal deviate at the 5 per cent level.
score of all the test animals. Individual animals showing A neurovirulence test in which the mean lesion score for the
evidence of unusually high activity, either in the lumbar region reference (Xref) is not compatible with previous experience
or as the result of spread from this region, are also taken into is not used for assessing a test vaccine. If the test is valid,
consideration in the final evaluation. The monovalent bulk the mean lesion score for the vaccine to be tested (Xtest) is
passes the test if the required number of animals is positive calculated and compared with that of the homotypic reference
and if none of the clinical and histopathological examinations vaccine.
shows a significant difference in pathogenicity between
the vaccine virus and the reference material. Criteria for
acceptance are given below.
Criteria. A suitable number of neurovirulence qualifying 07/2011:20620
tests (for example, four tests) is carried out on each reference
vaccine (types 1, 2 and 3) to provide data on the activity of 2.6.20. ANTI-A AND ANTI-B
such vaccines that will serve as the basis of the criteria for
vaccines to be tested. The overall mean lesion score (M) for
HAEMAGGLUTININS
the replicate tests on each reference virus is calculated together METHOD A : INDIRECT METHOD
with the pooled estimate of the within-test variance (s2) and Prepare in duplicate serial dilutions of the preparation to
the within-test deviation (s).
be examined in a 9 g/L solution of sodium chloride R. To
Validity criteria for the results of a test on a reference each dilution of 1 series add an equal volume of a 5 per
preparation are established on the basis of the cumulative cent V/V suspension of group A1 red blood cells previously
data from the qualifying tests. No generally applicable criteria washed 3 times with the sodium chloride solution. To each
can be given ; for laboratories with limited experience, the dilution of the other series add an equal volume of a 5 per
following empirical method for setting acceptable limits for cent V/V suspension of group B red blood cells previously
the mean lesion score for the reference preparation (Xref) may washed 3 times with the sodium chloride solution. Incubate
be helpful (see Table 2.6.19.-1) : the suspensions at 37 °C for 30 min then wash the cells
3 times with the sodium chloride solution. Leave the cells in
contact with a polyvalent anti-human globulin reagent for
Table 2.6.19.-1 30 min. Without centrifuging, examine each suspension for
agglutination under a microscope.
Lower limit Upper limit
Types 1 and 2 M− s M+ s METHOD B : DIRECT METHOD
Type 3 M − s/2 M+ s
MATERIALS
Phosphate-buffered saline (PBS). Dissolve 8.0 g of sodium
chloride R, 0.76 g of anhydrous disodium hydrogen phosphate R,
If the mean lesion score for the vaccine to be tested is Xtest and 0.2 g of potassium chloride R and 0.2 g of potassium dihydrogen
C1, C2 and C3 are constants determined as described below, phosphate R in water R and dilute to 1000 mL with the same
then : solvent. If the solution has to be kept for several days, 0.2 g
of sodium azide R may be added in order to avoid microbial
the vaccine is not acceptable if : contamination.
PBS-BSA solution. PBS containing 2 g/L of bovine albumin R
(Cohn Fraction V, for ELISA). Store the solution at 2-8 °C but
allow it to reach 19-25°C before use.
the vaccine may be retested once if : Papain solution. Use serological-grade papain from a
commercial source, the activity of which has been validated.
Red blood cells. Use pooled D-negative A1 (A1rr),
D-negative B (Brr) and D-negative O (Orr) red blood cells
from preferably 3 donors. When Immunoglobulin for anti-A
If the vaccine is retested, the means of the lesion scores for the and anti-B antibodies limit test BRP is used, 3 donors are to
vaccine to be tested and the reference vaccine are recalculated. be used. A red blood cells are not recommended as they give
2
The vaccine is not acceptable if : weaker reactions.
Wash the cells 4 times with PBS or until the supernatant is
clear. Each wash consists of suspending the cells in a minimum
of 2 volumes of PBS, centrifuging the cells at 1800 g for 5 min
General Notices (1) apply to all monographs and other texts 203
2.6.21. Nucleic acid amplification techniques EUROPEAN PHARMACOPOEIA 8.0
to pack, and discarding the supernatant. Treat the packed of the preparation to be examined, the positive control and
cells with papain solution according to the manufacturer’s the negative control. Mix by shaking the plate on a shaker for
instructions and wash the cells 4 times with PBS. 10 s (or until the cells are resuspended).
Red blood cells may be stored for not more than 1 week in a Centrifuge the plate at 80 g at room temperature for 1 min to
preservative solution at 2-8 °C. A preparation of the following pack the cells. Place the plate at an angle of approximately 70°.
composition is appropriate : Read after 4-5 min or when the negative controls (D-negative
Trisodium citrate 8 g/L O red blood cells and negative control solution) have streamed.
A cell button at the bottom of the well indicates a positive
D-glucose 20 g/L result. A stream of cells represents a negative result.
Citric acid 0.5 g/L Record the endpoint titre as the reciprocal of the highest
dilution that gives rise to a positive result.
Sodium chloride 4.2 g/L
The positive control has nominal anti-A and anti-B titres
Inosine 0.938 g/L of 32 (range 32-64 for anti-A ; range 16-32 for anti-B) and
Adenosine triphosphate (ATP) 0.4 g/L the negative controls (D-negative O red blood cells and
negative control solution) must not show agglutination at the
Chloramphenicol 0.34 g/L starting dilution of 1 in 2. Users must validate their own test
Neomycin sulfate 0.1 g/L conditions, and investigate their assay conditions and reagents
in the event of results being significantly different from
those expected. Failure to obtain negative reactions with the
If using stored cells, wash the cells at least twice in PBS or negative controls may indicate that, for example, insufficient
until the supernatant is clear before proceeding. time has elapsed for the cells to stream, or that reagents have
Microtitre plates. Use V-bottomed rigid microtitre plates. been used directly from cold storage.
Reference standards. Immunoglobulin (anti-A, anti-B If the anti-A or anti-B titre of the preparation to be examined
antibodies test positive control) BRP and Immunoglobulin is greater than the titre of the positive control when both
(anti-A, anti-B antibodies test negative control) BRP are preparations are titrated from 25 g/L, the test preparation is
suitable for use as the positive control and negative control, to be compared with Immunoglobulin for anti-A and anti-B
respectively, and should be used as guides for operators antibodies limit test BRP.
establishing and performing the direct method for anti-A and The maximum allowable titre is 64 when the preparations are
anti-B haemagglutinins. titrated from 25 g/L.
Immunoglobulin for anti-A and anti-B antibodies limit test BRP
defines the recommended maximum limits permissible for 07/2010:20621
batches of human immunoglobulin and must be used only
for comparison with batches of human immunoglobulin that
have higher titres than the positive control. 2.6.21. NUCLEIC ACID
METHOD AMPLIFICATION TECHNIQUES
The test described in this chapter is performed at room 1. INTRODUCTION
temperature on the positive control solutions, the negative
control solutions and the test solutions at the same time and Nucleic acid amplification techniques are based on 2 different
under identical conditions. Whenever necessary, a further approaches :
test is performed with Immunoglobulin for anti-A and anti-B 1. amplification of a target nucleic acid sequence using, for
antibodies limit test BRP. example, polymerase chain reaction (PCR), ligase chain
reaction (LCR), or isothermal ribonucleic acid (RNA)
Reference solutions. Reconstitute the positive control and amplification ;
the negative control according to the instructions. The
immunoglobulin G (IgG) concentration is 50 g/L in each of 2. amplification of a hybridisation signal using, for example,
the reconstituted preparations. Make a 2-fold dilution of each for deoxyribonucleic acid (DNA), the branched DNA
reconstituted preparation with PBS-BSA solution to obtain (bDNA) method ; in this case signal amplification is
solutions containing IgG at 25 g/L. Prepare 7 further serial achieved without subjecting the nucleic acid to repetitive
2-fold dilutions of each preparation using PBS-BSA solution cycles of amplification.
to extend the dilution range to 1/256 (0.195 g/L IgG). Add In this general chapter, the PCR method is described as the
20 μL of each dilution of each preparation in triplicate to the reference technique. Alternative methods may be used, if they
microtitre plate. comply with the quality requirements described below.
Test solutions. Dilute the preparation to be examined with 2. SCOPE
PBS-BSA solution to obtain a starting IgG concentration This section establishes the requirements for sample
of 25 g/L. For 50 g/L preparations, this is a 2-fold dilution ; preparation, in vitro amplification of DNA sequences and
adjust the dilution factor accordingly for preparations with detection of the specific PCR product. With the aid of PCR,
an IgG concentration other than 50 g/L to obtain a starting defined DNA sequences can be detected. RNA sequences can
concentration of 25 g/L for testing. This 25 g/L solution is also be detected following reverse transcription of the RNA to
assigned a nominal 2-fold dilution factor for comparison with complementary DNA (cDNA) and subsequent amplification.
the reference solutions, even if this does not reflect the true
dilution factor used to achieve 25 g/L. Prepare 7 further serial 3. PRINCIPLE OF THE METHOD
2-fold dilutions of the preparation using PBS-BSA solution to PCR is a procedure that allows specific in vitro amplification
extend the nominal dilution range to 1/256 (0.195 g/L IgG) for of segments of DNA or of RNA after reverse transcription
comparison with the reference preparations over the same IgG into cDNA.
concentration range. Add 20 μL of each dilution in triplicate
Following denaturation of double-stranded DNA into
to the microtitre plate.
single-stranded DNA, 2 synthetic oligonucleotide primers of
Prepare 3 per cent V/V suspensions of papain-treated opposite polarity anneal to their respective complementary
D-negative A1, B and O red blood cells in PBS/BSA solution. sequences in the DNA to be amplified. The short
Add 20 μL of D-negative A1, B and O red blood cells double-stranded regions that form as a result of specific base
respectively to the 1st, the 2nd and the 3rd dilution series of each pairing between the primers and the complementary DNA
sequence border the DNA segment to be amplified, and serve – the type of DNA polymerase, buffer composition and
as starting positions for in vitro DNA synthesis by means of a reaction volume used for the amplification ;
heat-stable DNA polymerase. – the type of thermocycler used and the thermal conductivity
Amplification of the DNA occurs in cycles consisting of : rate between the apparatus, reaction tube and reaction fluid.
– heat denaturation of the nucleic acid (target sequence) into 5.4. Detection
2 single strands ; The amplicon generated by PCR may be identified by
– specific annealing of the primers to the target sequence size, sequence, chemical modification or a combination
under suitable reaction conditions ; of these parameters. Detection and characterisation by
– extension of the primers, which are bound to both single size may be achieved by gel electrophoresis (using agarose
strands, by DNA polymerase at a suitable temperature or polyacrylamide slab gels or capillary electrophoresis)
(DNA synthesis). or column chromatography (for example, liquid
Repeated cycles of heat denaturation, primer annealing and chromatography). Detection and characterisation by sequence
DNA synthesis results in an exponential amplification of the composition may be achieved by the specific hybridisation
DNA segment limited by the primers. of probes having a sequence complementary to the target
The specific PCR product known as an amplicon can be sequence or by cleavage of the amplified material reflecting
detected by a variety of methods of appropriate specificity and target-specific restriction-enzyme sites. Detection and
sensitivity. characterisation by chemical modification may be achieved by,
for example, incorporation of a fluorophore into the amplicons
Multiplex PCR assays use several primer pairs designed for and subsequent detection of fluorescence following excitation.
simultaneous amplification of different targets in one reaction.
Detection of amplicons may also be achieved by using
4. TEST MATERIAL probes labelled to permit a subsequent radioisotopic or
Because of the high sensitivity of PCR, the samples must immuno-enzyme-coupled detection.
be protected against external contamination with target 6. EVALUATION AND INTERPRETATION OF RESULTS
sequences. Sampling, storage and transport of the test material
are performed under conditions that minimise degradation A valid result is obtained within a test only if the positive
of the target sequence. In the case of RNA target sequences, control(s) is unambiguously positive and the negative
special precautions are necessary since RNA is highly sensitive control(s) is unambiguously negative. Due to the very high
to degradation by ribonucleases. Care must be taken since sensitivity of the PCR method and the inherent risk of
some added reagents, such as anticoagulants or preservatives, contamination, it is necessary to confirm positive results by
may interfere with the test procedure. repeating the complete test procedure in duplicate, where
possible on a new aliquot of the sample. The sample is
5. TEST METHOD considered positive if at least one of the repeat tests gives a
5.1. Prevention of contamination positive result. As soon as a measurable target threshold is
defined, a quantitative test system is required.
The risk of contamination requires a strict segregation of the
areas depending on the material handled and the technology 7. QUALITY ASSURANCE
used. Points to consider include movement of personnel, 7.1. Validation of the PCR assay system
gowning, material flow and air supply and decontamination
procedures. The validation programme must include validation of
instrumentation and the PCR method employed. Reference
The system should be sub-divided into compartments such as : should be made to the ICH guidelines (topic Q2B) Validation
– master-mix area (area where exclusively template-free of Analytical Method : Methodology.
material is handled, e.g. primers, buffers, etc.) ; Appropriate official working reference preparations
– pre-PCR (area where reagents, samples and controls are or in-house reference preparations calibrated against
handled) ; International Standards for the target sequences for which the
– PCR amplification (amplified material is handled in a test system will be used are indispensable for validation of a
closed system) ; PCR test.
– post-PCR detection (the only area where the amplified 7.1.1. Determination of the positive cut-off point
material is handled in an open system). During validation of qualitative tests, the positive cut-off point
5.2. Sample preparation must be determined. The positive cut-off point is defined
When preparing samples, the target sequence to be amplified as the minimum number of target sequences per volume
needs to be efficiently extracted or liberated from the test sample that can be detected in 95 per cent of test runs. The
material in a reproducible manner and in such a way that positive cut-off point depends on interrelated factors such as
amplification under the selected reaction conditions is the volume of the sample extracted and the efficacy of the
possible. A variety of physico-chemical extraction procedures extraction methodology, the transcription of the target RNA
and/or enrichment procedures may be employed. into cDNA, the amplification process and the detection.
Additives present in test material may interfere with PCR. To define the detection limit of the assay system, reference
The procedures described under 7.3.2. must be used as a must be made to the positive cut-off point for each target
control for the presence of inhibitors originating from the sequence and the test performance above and below the
test material. positive cut-off point.
In the case of RNA-templates, care must be taken to avoid 7.1.2. Quantitative assay systems
ribonuclease activity. For a quantitative assay, the following parameters are
5.3. Amplification determined during validation : accuracy, precision, specificity,
quantitation limit, linearity, range and robustness.
PCR amplification of the target sequence is conducted
under defined cycling conditions (temperature profile 7.2. Quality control of reagents
for denaturation of double-stranded DNA, annealing All reagents crucial for the methodology used have to
and extension of primers ; incubation times at selected be controlled prior to use in routine applications. Their
temperatures ; ramp rates). These depend on various acceptance/withdrawal is based on pre-defined quality criteria.
parameters such as : Primers are a crucial component of the PCR assay and as
– the length and base composition of primer and target such their design, their purity and the validation of their use
sequences ; in a PCR assay require careful attention. Primers may be
General Notices (1) apply to all monographs and other texts 205
2.6.21. Nucleic acid amplification techniques EUROPEAN PHARMACOPOEIA 8.0
modified (for example, by conjugation with a fluorophore or user. Nevertheless, the performance of the kit with respect
antigen) in order to permit a specific method of detection to its intended use has to be demonstrated by the user (e.g.
of the amplicon, provided such modifications do not inhibit detection limit, robustness, cross-contamination).
accurate and efficient amplification of the target sequence.
2. SPECIFICITY
7.3. Run controls
Specificity is the ability to assess unequivocally nucleic acid
7.3.1. External controls in the presence of components that may be expected to be
In order to minimise the risk of contamination and to ensure present.
adequate sensitivity, the following external controls are The specificity of nucleic acid amplification analytical
included in each PCR assay : procedures is dependent on the choice of primers, the choice
– positive control : this contains a defined number of of probe (for analysis of the final product) and the stringency
target-sequence copies, the number being close to the of the test conditions (for both the amplification and the
positive cut-off value, and determined individually for each detection steps).
assay system and indicated as a multiple of the positive
When designing primers and probes, the specificity of
cut-off value of the assay system ;
the primers and probes to detect only HCV RNA should
– negative control : a sample of a suitable matrix already be investigated by comparing the chosen sequences with
proven to be free of the target sequences. sequences in published data banks. For HCV, primers
7.3.2. Internal control (and probes) will normally be chosen from areas of the 5’
Internal controls are defined nucleic acid sequences non-coding region of the HCV genome which are highly
containing, unless otherwise prescribed, the primer binding conserved for all genotypes.
sites. Internal controls must be amplified with defined efficacy, The amplified product should be unequivocally identified by
and the amplicons must be clearly discernible. Internal using one of a number of methods such as amplification with
controls must be of the same type of nucleic acid (DNA/RNA) nested primers, restriction enzyme analysis, sequencing, or
as the material to be tested. The internal control is preferably hybridisation with a specific probe.
added to the test material before isolating the nucleic acid In order to validate the specificity of the analytical procedure,
and therefore acts as an overall control (extraction, reverse at least 100 HCV RNA-negative plasma pools should be
transcription, amplification, detection). tested and shown to be non-reactive. Suitable samples of
7.3.3. Threshold control non-reactive pools are available from the European Directorate
The threshold control for quantitative assays is a test sample for the Quality of Medicines & HealthCare (EDQM).
with the analyte at a concentration that is defined as the The ability of the analytical procedure to detect all HCV
threshold not to be exceeded. It contains the analyte suitably genotypes will again depend on the choice of primers, probes
calibrated in International Units and is analysed in parallel in and method parameters. This ability should be demonstrated
each run of a quantitative assay. using characterised reference panels. However, in view of
7.4. External quality assessment the difficulty in obtaining samples of some genotypes (e.g.
Participation in external quality assessment programmes genotype 6), the most prevalent genotypes (e.g. genotypes 1
is an important PCR quality assurance procedure for each and 3 in Europe) should be detected at a suitable level.
laboratory and each operator. 3. DETECTION LIMIT
The following sections are published for information. The detection limit of an individual analytical procedure is the
lowest amount of nucleic acid in a sample that can be detected
Validation of nucleic acid amplification but not necessarily quantitated as an exact value.
techniques (NAT) for the detection of The nucleic acid amplification analytical procedure used for
hepatitis C virus (HCV) RNA in plasma the detection of HCV RNA in plasma pools usually yields
qualitative results. The number of possible results is limited
pools : guidelines to 2 : either positive or negative. Although the determination
of the detection limit is recommended, for practical purposes,
1. SCOPE a positive cut-off point should be determined for the nucleic
The majority of nucleic acid amplification analytical acid amplification analytical procedure. The positive cut-off
procedures are qualitative (quantal) tests for the presence of point (as defined in the general chapter 2.6.21) is the minimum
nucleic acid with some quantitative tests (either in-house or number of target sequences per volume sample that can be
commercial) being available. For the detection of HCV RNA detected in 95 per cent of test runs. This positive cut-off
contamination of plasma pools, qualitative tests are adequate point is influenced by the distribution of viral genomes in the
and may be considered to be a limit test for the control individual samples being tested and by factors such as enzyme
of impurities as described in the Pharmeuropa Technical efficiency, and can result in different 95 per cent cut-off values
Guide for the elaboration of monographs, December 1999, for individual analytical test runs.
Chapter III ‘Validation of analytical procedures’. These In order to determine the positive cut-off point, a dilution
guidelines describe methods to validate only qualitative series of a working reagent or of the hepatitis C virus BRP,
nucleic acid amplification analytical procedures for assessing which has been calibrated against the WHO HCV International
HCV RNA contamination of plasma pools. Therefore, the Standard 96/790, should be tested on different days to examine
2 characteristics regarded as the most important for validation variation between test runs. At least 3 independent dilution
of the analytical procedure are the specificity and the detection series should be tested with a sufficient number of replicates at
limit. In addition, the robustness of the analytical procedure each dilution to give a total number of 24 test results for each
should be evaluated. dilution, to enable a statistical analysis of the results.
However, this document may also be used as a basis for the For example, a laboratory could test 3 dilution series on
validation of nucleic acid amplification in general. different days with 8 replicates for each dilution, 4 dilution
For the purpose of this document, an analytical procedure is series on different days with 6 replicates for each dilution,
defined as the complete procedure from extraction of nucleic or 6 dilution series on different days with 4 replicates for
acid to detection of the amplified products. each dilution. In order to keep the number of dilutions at
Where commercial kits are used for part or all of the analytical a manageable level, a preliminary test (using, for example,
procedure, documented validation points already covered by log10 dilutions of the plasma pool sample) should be carried
the kit manufacturer can substitute for the validation by the out in order to obtain a preliminary value for the positive
cut-off point (i.e. the highest dilution giving a positive be documented by conducting a parallel test on 8 samples
signal). The range of dilutions can then be chosen around the of a plasma pool that is spiked with HCV RNA to a final
predetermined preliminary cut-off point (using, for example, concentration of 3 times the previously determined 95 per
a dilution factor of 0.5 log10 or less and a negative plasma pool cent cut-off value. All results should be positive.
for the dilution matrix). The concentration of HCV RNA Operator qualification. An appropriate qualification
that can be detected in 95 per cent of test runs can then be programme should be implemented for each operator involved
calculated using an appropriate statistical evaluation. in the testing. To confirm successful training each operator
These results may also serve to demonstrate the intra-assay should test at least 8 replicate samples of a plasma pool
variation and the day-to-day variation of the analytical spiked with HCV RNA to a final concentration of 3 times the
procedure. previously determined 95 per cent cut-off value. This test (8
replicate samples) should be repeated twice on 2 separate days,
4. ROBUSTNESS i.e. a total of 24 tests performed on 3 different days. All results
The robustness of an analytical procedure is a measure of should be positive.
its capacity to remain unaffected by small but deliberate
variations in method parameters and provides an indication
of its reliability during normal usage.
Validation of nucleic acid amplification
The evaluation of robustness should be considered during techniques (NAT) for the quantification of
the development phase. It should show the reliability of the B19 virus (B19V) DNA in plasma pools :
analytical procedure with respect to deliberate variations in guidelines
method parameters. For NAT, small variations in the method
parameters can be crucial. However, the robustness of the
method can be demonstrated during its development when 1. SCOPE
small variations in the concentrations of reagents (e.g. MgCl2, The European Pharmacopoeia requires that plasma pools used
primers or dNTP) are tested. To demonstrate robustness, at for manufacture of certain products are tested for the presence
least 20 HCV RNA negative plasma pools (selected at random) of B19 virus (B19V) DNA with a threshold concentration
spiked with HCV RNA to a final concentration of 3 times that must not be exceeded. In order to comply with these
the previously determined 95 per cent cut-off value should requirements, quantitative NAT tests are preferred. The
be tested and found positive. characteristics regarded as the most important for validation
Problems with robustness may also arise with methods that of the quantitative NAT procedure are accuracy, precision,
use an initial ultracentrifugation step prior to extraction of the specificity, quantitation limit, linearity and range. In addition,
viral RNA. Therefore, to test the robustness of such methods, the robustness of the analytical procedure should be evaluated.
at least 20 plasma pools containing varying levels of HCV This guideline describes methods to validate NAT analytical
RNA, but lacking HCV-specific antibodies, should be tested procedures for assessing B19V DNA contamination of plasma
and found positive. pools based on the ICH guidelines. However, this document
Cross-contamination prevention should be demonstrated may also be used as a basis for the validation of quantitative
by the accurate detection of a panel of at least 20 samples NAT in general.
consisting of alternate samples of negative plasma pools and For the purpose of this document, an analytical procedure is
negative plasma pools spiked with high concentrations of defined as the complete procedure from extraction of nucleic
HCV (at least 102 times the 95 per cent cut-off value or at acid to detection of the amplified products.
least 104 IU/mL).
Where commercial kits are used for part or all of the analytical
5. QUALITY ASSURANCE procedure, documented validation points already covered by
the kit manufacturer can substitute for the validation by the
For biological tests such as NAT, specific problems may arise user. Nevertheless, the performance of the kit with respect
that influence both the validation and the interpretation of to its intended use has to be demonstrated by the user (e.g.
results. The test procedures must be described precisely in the precision, accuracy, range, robustness).
form of standard operating procedures (SOPs). These should
cover :
2. ACCURACY
– the mode of sampling (type of container, etc.) ;
Accuracy expresses the closeness of agreement between the
– the preparation of mini-pools (where appropriate) ; value that is accepted as either a conventional true value
– the conditions of storage before analysis ; or an accepted reference value and the value found. The
accuracy of an assay is dependent on the calibration of
– the exact description of the test conditions, including the assay and on the variance of the different assay steps.
precautions taken to prevent cross-contamination or Though it is recommended to establish the accuracy across
destruction of the viral RNA, reagents and reference the specified range of the analytical procedure, the most
preparations used ; important assessment of accuracy is in the range of the
– the exact description of the apparatus used ; threshold concentration. In the case of B19V NAT assays for
– the detailed formulae for calculation of results, including investigation of plasma pools it is recommended to assess
statistical evaluation. the accuracy of the calibrated assay by assaying at least
5 concentrations (dilution factor of 0.5 log10) of B19 virus DNA
The use of a suitable run control (for example, an appropriate for NAT testing BRP or another material, suitably calibrated
dilution of hepatitis C virus BRP or plasma spiked with an in International Units against the actual WHO B19 DNA
HCV sample calibrated against the WHO HCV International International Standard, covering the range of the currently
Standard 96/790) can be considered a satisfactory recommended threshold concentration of 10.0 IU/μL B19V
system-suitability check and ensures that the reliability of the DNA (e.g. 105 IU/mL, 104.5 IU/mL, 104 IU/mL, 103.5 IU/mL
analytical procedure is maintained whenever used. and 103 IU/mL), with at least 3 replicates for each dilution.
Technical qualification. An appropriate installation and Accuracy should be reported for the different concentrations
operation qualification programme should be implemented in terms of percentage determined compared with the known
for each critical piece of the equipment used. For confirmation amount of B19V DNA. It should reflect the level of technology
of analytical procedure performance after a change of of the respective assays, which should also be defined, for
critical equipment (e.g. thermocyclers), the change should example in collaborative studies.
General Notices (1) apply to all monographs and other texts 207
2.6.21. Nucleic acid amplification techniques EUROPEAN PHARMACOPOEIA 8.0
for NAT testing BRP) is considered to be a satisfactory d) For a freeze-dried preparation, reconstitute using a suitable
system-suitability check and ensures that the reliability of the liquid. Unless otherwise stated or justified, the test substance
analytical procedure is maintained whenever used. must contain a quantity of virus equivalent to at least 10 doses
Technical qualification. An appropriate installation and of vaccine in 0.1 mL of inoculum.
operation qualification programme should be implemented e) If the virus of the seed lot would interfere with the
for each critical piece of the equipment used. For confirmation conduct and sensitivity of the test, neutralise the virus in the
of analytical procedure performance after a change of critical preparation with a monospecific antiserum.
equipment (e.g. thermocyclers), the change should be f) Monospecific antiserum and serum of avian origin used
documented by conducting a parallel test on 8 samples of a for cell culture or any other purpose, in any of these tests,
plasma pool that is spiked with a concentration of B19V DNA shall be free from antibodies against and free from inhibitory
around the threshold concentration. All results should be effects on the organisms listed hereafter under 7. Antibody
acceptable and reflect the features of the assay as determined specifications for sera used in extraneous agents testing.
during the validation phase. g) Where specified in a monograph or otherwise justified,
Operator qualification. An appropriate qualification if neutralisation of the virus of the seed lot is required but
programme should be implemented for each operator involved difficult to achieve, the in vitro tests described below are
in the testing. To confirm successful training, each operator adapted, as required, to provide the necessary guarantees of
should test, on 3 separate days, at least 8 replicate samples of a freedom from contamination with an extraneous agent.
plasma pool that is spiked with a concentration of B19V DNA h) Other types of tests than those indicated may be used
around the threshold concentration (i.e. a total of 24 samples). provided they are at least as sensitive as those indicated and of
All results should be acceptable and reflect the features of the appropriate specificity. Nucleic acid amplification techniques
assay as determined during the validation phase. (2.6.21) give specific detection for many agents and can be
used after validation for sensitivity and specificity.
01/2008:20622 1. TEST FOR EXTRANEOUS AGENTS USING
EMBRYONATED HENS’ EGGS
2.6.22. ACTIVATED COAGULATION Use a test substance, diluted if necessary, containing a quantity
FACTORS of neutralised virus equivalent to at least 10 doses of vaccine
in 0.2 mL of inoculum. Suitable antibiotics may be added.
Where applicable, determine the amount of heparin Inoculate the test substance into 3 groups of 10 embryonated
present (2.7.12) and neutralise the heparin, for example hens’ eggs as follows :
by addition of protamine sulfate R (10 μg of protamine – group 1 : 0.2 mL into the allantoic cavity of each
sulfate neutralises 1 IU of heparin). Prepare 1 to 10 and 1 9- to 11-day-old embryonated egg ;
to 100 dilutions of the preparation to be examined using
tris(hydroxymethyl)aminomethane buffer solution pH 7.5 R. – group 2 : 0.2 mL onto the chorio-allantoic membrane of
Place a series of polystyrene tubes in a water-bath at 37 °C each 9- to 11-day-old embryonated egg ;
and add to each tube 0.1 mL of platelet-poor plasma R and – group 3 : 0.2 mL into the yolk sac of each 5- to 6-day-old
0.1 mL of a suitable dilution of a phospholipid preparation to embryonated egg.
act as a platelet substitute. Allow to stand for 60 s. Add to Candle the eggs in groups 1 and 2 daily for 7 days and the
each tube either 0.1 mL of 1 of the dilutions or 0.1 mL of the eggs in group 3 daily for 12 days. Discard embryos that die
buffer solution (control tube). To each tube add immediately during the first 24 h as non-specific deaths ; the test is not
0.1 mL of a 3.7 g/L solution of calcium chloride R previously valid unless at least 6 embryos in each group survive beyond
heated to 37 °C, and measure, within 30 min of preparing the the first 24 h after inoculation. Examine macroscopically
original dilution, the time that elapses between addition of the for abnormalities all embryos that die more than 24 h after
calcium chloride solution and the formation of a clot. The inoculation, or that survive the incubation period. Examine
test is not valid unless the coagulation time measured for the also the chorio-allantoic membranes of these eggs for any
control tube is 200 s to 350 s. abnormality and test the allantoic fluids for the presence of
haemagglutinating agents.
Carry out a further embryo passage. Pool separately material
07/2009:20624 from live and from the dead and abnormal embryos. Inoculate
each pool into 10 eggs for each route as described above,
2.6.24. AVIAN VIRAL VACCINES : chorio-allantoic membrane material being inoculated onto
TESTS FOR EXTRANEOUS AGENTS IN chorio-allantoic membranes, allantoic fluids into the allantoic
cavity and embryo material into the yolk sac. For eggs
SEED LOTS inoculated by the allantoic and chorio-allantoic routes, candle
GENERAL PROVISIONS the eggs daily for 7 days, proceeding and examining the
material as described above. For eggs inoculated by the yolk
a) In the following tests, chickens and/or chicken material sac route, candle the eggs daily for 12 days, proceeding and
such as eggs and cell cultures shall be derived from chicken examining the material as described above.
flocks free from specified pathogens (SPF) (5.2.2).
The seed lot complies with the test if no test embryo shows
b) Cell cultures for the testing of extraneous agents comply macroscopic abnormalities or dies from causes attributable
with the requirements for the master cell seed of chapter 5.2.4. to the seed lot and if examination of the chorio-allantoic
Cell cultures for the production of veterinary vaccines, with the membranes and testing of the allantoic fluids show no
exception of the karyotype test and the tumorigenicity test, evidence of the presence of any extraneous agent.
which do not have to be carried out.
c) In tests using cell cultures, precise specifications are given 2. TEST IN CHICKEN KIDNEY CELLS
for the number of replicates, monolayer surface areas and Prepare 7 monolayers of chicken kidney cells, each monolayer
minimum survival rate of the cultures. Alternative numbers of having an area of about 25 cm2. Maintain 2 monolayers
replicates and cell surface areas are possible as well, provided as negative controls and treat these in the same way as the
that a minimum of 2 replicates are used, the total surface area 5 monolayers inoculated with the test substance, as described
and the total volume of test substance applied are not less below. Remove the culture medium when the cells reach
than that prescribed here and the survival rate requirements confluence. Inoculate 0.1 mL of the test substance onto each
are adapted accordingly. of the 5 monolayers. Allow adsorption for 1 h, add culture
General Notices (1) apply to all monographs and other texts 209
2.6.24. Avian viral vaccines : tests for extraneous agents in seed lots EUROPEAN PHARMACOPOEIA 8.0
medium and incubate the cultures for a total of at least The test is not valid if group-specific antigen is detected in
21 days, subculturing at 4- to 7-day intervals. Each passage is fewer than 5 of the 6 positive control replicate monolayers or
made with pooled cells and fluids from all 5 monolayers after if a positive result is obtained in any of the negative control
carrying out a freeze-thaw cycle. Inoculate 0.1 mL of pooled monolayers, or if the results for both of the 2 negative control
material onto each of 5 recently prepared monolayers of about monolayers are inconclusive. If the results for more than 1 of
25 cm2 each, at each passage. For the last passage, grow the the test replicate monolayers are inconclusive, then further
cells also on a suitable substrate so as to obtain an area of subcultures of reserved portions of the fibroblast monolayers
about 10 cm2 of cells from each of the monolayers for test A. shall be made and tested until an unequivocal result is
The test is not valid if less than 80 per cent of the monolayers obtained. If a positive result is obtained for any of the test
survive after any passage. monolayers, then the presence of avian leucosis virus in the
test substance has been detected.
Examine microscopically all the cell cultures frequently
throughout the entire incubation period for any signs The seed lot complies with the test if there is no evidence of
of cytopathic effect or other evidence of the presence of the presence of any avian leucosis virus.
contaminating agents in the test substance. At the end of the
total incubation period, carry out the following procedures. 4. TEST FOR AVIAN RETICULOENDOTHELIOSIS VIRUS
A. Fix and stain (with Giemsa or haematoxylin and Prepare 11 monolayers of primary or secondary chick
eosin) about 10 cm2 of confluent cells from each of the embryo fibroblasts from the tissues of 9- to 11-day old chick
5 monolayers. Examine the cells microscopically for any embryos or duck embryo fibroblasts from the tissues of
cytopathic effect, inclusion bodies, syncytial formation, 13- to 14-day-old embryos, each monolayer having an area
or other evidence of the presence of contaminating agents of about 25 cm2.
from the test substance. Remove the culture medium when the cells reach confluence.
2
B. Drain and wash about 25 cm of cells from each of the Inoculate 0.1 mL of the test substance onto each of 5
5 monolayers. Cover these cells with a 0.5 per cent of the monolayers. Allow adsorption for 1 h, and add
suspension of washed chicken erythrocytes (using at least culture medium. Inoculate 4 of the monolayers with avian
1 mL of suspension for each 5 cm2 of cells). Incubate the reticuloendotheliosis virus as positive controls (not more than
cells at 4 °C for 20 min and then wash gently in phosphate 10 CCID50 in 0.1 mL). Maintain 2 non-inoculated monolayers
buffered saline pH 7.4. Examine the cells microscopically as negative controls.
for haemadsorption attributable to the presence of a Incubate the cells for a total of at least 10 days, subculturing
haemadsorbing agent in the test substance. twice at 3- to 4-day intervals. The test is not valid if fewer
C. Test individual samples of the fluids from each cell than 3 of the 4 positive controls or fewer than 4 of the 5 test
culture using chicken erythrocytes for haemagglutination monolayers or neither of the 2 negative controls survive after
attributable to the presence of a haemagglutinating agent any passage.
in the test substance. For the last subculture, grow the fibroblasts on a suitable
2
The test is not valid if there are any signs of extraneous agents substrate so as to obtain an area of about 10 cm of
in the negative control cultures. The seed lot complies with the confluent fibroblasts from each of the original 11 monolayers
2
test if there is no evidence of the presence of any extraneous for the subsequent test : test about 10 cm of confluent
agent. fibroblasts derived from each of the original 11 monolayers by
immunostaining for the presence of avian reticuloendotheliosis
virus. The test is not valid if avian reticuloendotheliosis virus
3. TEST FOR AVIAN LEUCOSIS VIRUSES is detected in fewer than 3 of the 4 positive control monolayers
Prepare at least 13 replicate monolayers of either DF-1 cells or in any of the negative control monolayers, or if the results
or primary or secondary chick embryo fibroblasts from for both of the 2 negative control monolayers are inconclusive.
the tissues of 9- to 11-day-old embryos that are known to If the results for more than 1 of the test monolayers are
be genetically susceptible to subgroups A, B and J of avian inconclusive then further subcultures of reserved portions of
leucosis viruses and that support the growth of exogenous but the fibroblast monolayers shall be made and tested until an
not endogenous avian leucosis viruses (cells from C/E strain unequivocal result is obtained.
chickens are suitable). Each replicate shall have an area of The seed lot complies with the test if there is no evidence of
about 50 cm2. the presence of avian reticuloendotheliosis virus.
Remove the culture medium when the cells reach confluence.
Inoculate 0.1 mL of the test substance onto each of 5 of the 5. TEST FOR CHICKEN ANAEMIA VIRUS
replicate monolayers. Allow adsorption for 1 h, and add Prepare eleven 20 mL suspensions of the MDCC-MSBI cell
culture medium. Inoculate 2 of the replicate monolayers with line or another cell line of equivalent sensitivity in 25 cm2 cell
subgroup A avian leucosis virus (not more than 10 CCID50 in culture flasks containing about 5 × 105 cells/mL. Inoculate
0.1 mL), 2 with subgroup B avian leucosis virus (not more 0.1 mL of the test substance into each of 5 flasks. Inoculate
than 10 CCID50 in 0.1 mL) and 2 with subgroup J avian 4 of the suspensions with 10 CCID50 chicken anaemia virus as
leucosis virus (not more than 10 CCID50 in 0.1 mL) as positive positive controls. Maintain not fewer than 2 non-inoculated
controls. Maintain not fewer than 2 non-inoculated replicate suspensions. Maintain all the cell cultures for a total of at least
monolayers as negative controls. 24 days, subculturing 8 times at 3- to 4-day intervals. During
Incubate the cells for a total of at least 9 days, subculturing the subculturing the presence of chicken anaemia virus may
at 3- to 4-day intervals. Retain cells from each passage level be indicated by a metabolic colour change in the infected
and harvest the cells at the end of the total incubation period. cultures, the culture fluids becoming red in comparison with
Wash cells from each passage level from each replicate and the control cultures. Examine the cells microscopically for
resuspend the cells at 10 cells per millilitre in barbital-buffered cytopathic effect. At this time or at the end of the incubation
7
saline for subsequent testing by a Complement Fixation for period, centrifuge the cells from each flask at low speed
6
Avian Leucosis (COFAL) test or in phosphate buffered saline and resuspend at about 10 cells/mL and place 25 μL in
for testing by Enzyme-Linked Immunosorbent Assay (ELISA). each of 10 wells of a multi-well slide. Examine the cells by
Then, carry out 3 cycles of freezing and thawing to release any immunostaining.
group-specific antigen and perform a COFAL test or an ELISA The test is not valid if chicken anaemia virus is detected
test on each extract to detect group-specific avian leucosis in fewer than 3 of the 4 positive controls or in any of the
antigen if present. non-inoculated controls. If the results for more than 1 of the
test suspensions are inconclusive, then further subcultures of B. Additional tests for turkey extraneous agents
reserved portions of the test suspensions shall be made and If the seed virus is of turkey origin or was propagated in
tested until an unequivocal result is obtained. turkey substrates, tests for antibodies against the following
The seed lot complies with the test if there is no evidence of agents are also carried out.
the presence of chicken anaemia virus. Agent Type of test
Chlamydia spp. EIA
6. TEST FOR EXTRANEOUS AGENTS USING CHICKS Avian infectious haemorrhagic enteritis virus AGP
Inoculate each of at least 10 chicks with the equivalent of Avian paramyxovirus 3 HI
100 doses of vaccine by the intramuscular route and with
Avian infectious bursal disease virus type 2 SN
the equivalent of 10 doses by eye-drop. Chicks that are
2 weeks of age are used in the test except that if the seed A test for freedom from turkey lympho-proliferative
virus is pathogenic for birds of this age, older birds may disease virus is carried out by intraperitoneal inoculation
be used, if required and justified. In exceptional cases, for of twenty 4-week-old turkey poults. Observe the poults for
inactivated vaccines, the virus may be neutralised by specific 40 days. The test is not valid if more than 20 per cent of the
antiserum if the seed virus is pathogenic for birds at the poults die from non-specific causes. The seed lot complies
age of administration. Repeat these inoculations 2 weeks with the test if sections of spleen and thymus taken from
later. Observe the chicks for a period of 5 weeks from the 10 poults 2 weeks after inoculation show no macroscopic
day of the first inoculation. No antimicrobial agents shall be or microscopic lesions (other than those attributable to the
administered to the chicks during the test period. The test is seed lot virus) and no poult dies from causes attributable to
not valid if fewer than 80 per cent of the chicks survive to the the seed lot.
end of the test period.
C. Additional tests for duck extraneous agents
Collect serum from each chick at the end of the test period. If the seed virus is of duck origin or was propagated in
Test each serum sample for antibodies against each of the duck substrates, tests for antibodies against the following
agents listed below (with the exception of the virus type of agents are also carried out.
the seed lot) using one of the methods indicated for testing Agent Type of test
for the agent.
Chlamydia spp. EIA
Clinical signs of disease in the chicks during the test period
(other than signs attributable to the virus of the seed lot) and Duck and goose parvoviruses SN, EIA
the detection of antibodies in the chicks after inoculation Duck enteritis virus SN
(with the exception of antibodies to the virus of the seed lot),
are classed as evidence of the presence of an extraneous agent Duck hepatitis virus type I SN
in the seed lot.
The seed lot complies with the test if there is no evidence
It is recommended that sera from these birds is retained so that of the presence of any extraneous agent.
additional testing may be carried out if requirements change. D. Additional tests for goose extraneous agents
A. Standard tests If the seed virus is of goose origin or was prepared in goose
substrates, tests for the following agents are also carried out.
Agent Type of test Agent Type of test
Avian adenoviruses, group 1 SN, EIA, AGP Duck and goose parvovirus SN, EIA
Avian encephalomyelitis virus AGP, EIA Duck enteritis virus SN
Avian infectious bronchitis virus EIA, HI Goose haemorrhagic polyomavirus test in goslings shown below
or another suitable test
Avian infectious laryngotracheitis virus SN, EIA, IS
Inoculate subcutaneously the equivalent of at least 10 doses
Avian leucosis viruses SN, EIA to each of ten 1-day-old susceptible goslings. Observe the
Avian nephritis virus IS goslings for 28 days. The test is not valid if more than
20 per cent of the goslings die from non-specific causes.
Avian orthoreoviruses IS, EIA The seed virus complies with the test if no gosling dies
Avian reticuloendotheliosis virus AGP, IS, EIA from causes attributable to the seed lot.
Chicken anaemia virus IS, EIA, SN 7. ANTIBODY SPECIFICATIONS FOR SERA USED IN
EXTRANEOUS AGENTS TESTING
Egg drop syndrome virus HI, EIA
All batches of serum to be used in extraneous agents testing,
Avian infectious bursal disease virus Serotype 1 : AGP, EIA, SN either to neutralise the vaccine virus (seed lot or batch of
Serotype 2 : SN finished product) or as a supplement for culture media used
Influenza A virus AGP, EIA, HI for tissue culture propagation, shall be shown to be free from
Marek’s disease virus AGP
antibodies against and free from inhibitory effects on the
following micro-organisms by suitably sensitive tests.
Newcastle disease virus HI, EIA Avian adenoviruses
Turkey rhinotracheitis virus EIA Avian encephalomyelitis virus
Salmonella pullorum Agg Avian infectious bronchitis viruses
Avian infectious bursal disease virus types 1 and 2
Agg : agglutination
Avian infectious haemorrhagic enteritis virus
AGP : agar gel precipitation
EIA : enzyme immunoassay (e.g. ELISA) Avian infectious laryngotracheitis virus
HI : haemagglutination inhibition Avian leucosis viruses
IS : immunostaining (e.g. fluorescent antibody) Avian nephritis virus
SN : serum neutralisation Avian paramyxoviruses 1 to 9
General Notices (1) apply to all monographs and other texts 211
2.6.25. Avian live virus vaccines: extraneous agents in finished product EUROPEAN PHARMACOPOEIA 8.0
Avian orthoreoviruses from testing the batch of vaccine, as decribed under 6. Test
Avian reticuloendotheliosis virus for extraneous agents using chicks of chapter 2.6.24. Test for
extraneous agents in seed lots.
Chicken anaemia virus
Duck enteritis virus g) Monospecific antiserum and serum of avian origin used for
cell culture and any other purpose, in any of these tests, shall
Duck hepatitis virus type I be free of antibodies against and free from inhibitory effects
Egg drop syndrome virus on the organisms listed under 7. Antibody specifications for
Fowl pox virus sera used in extraneous agents testing (2.6.24).
Influenza viruses h) Other types of tests than those indicated may be used
Marek’s disease virus provided they are at least as sensitive as those indicated and of
appropriate specificity. Nucleic acid amplification techniques
Turkey herpesvirus (2.6.21) give specific detection for many agents and can be
Turkey rhinotracheitis virus used after validation for sensitivity and specificity.
Non-immune serum for addition to culture media can be
assumed to be free from antibodies against any of these 1. TEST FOR EXTRANEOUS AGENTS USING
viruses if the agent is known not to infect the species of origin EMBRYONATED HENS’ EGGS
of the serum and it is not necessary to test the serum for such Prepare the test vaccine, diluted if necessary, to contain
antibodies. Monospecific antisera for virus neutralisation can neutralised virus equivalent to 10 doses of vaccine in 0.2 mL
be assumed to be free from the antibodies against any of these of inoculum. Suitable antibiotics may be added. Inoculate
viruses if it can be shown that the immunising antigen could the test vaccine into 3 groups of 10 embryonated hens’ eggs
not have been contaminated with antigens derived from that as follows :
virus and if the virus is known not to infect the species of – group 1 : 0.2 mL into the allantoic cavity of each 9- to
origin of the serum ; it is not necessary to test the serum for 11-day-old embryonated egg,
such antibodies. It is not necessary to retest sera obtained – group 2 : 0.2 mL onto the chorio-allantoic membrane of
from birds from SPF chicken flocks (5.2.2). each 9- to 11-day-old embryonated egg,
Batches of sera prepared for neutralising the vaccine virus – group 3 : 0.2 mL into the yolk sac of each 5- to 6-day-old
must not be prepared from any passage level derived from embryonated egg.
the virus isolate used to prepare the master seed lot or from
an isolate cultured in the same cell line. Candle the eggs in groups 1 and 2 daily for 7 days and the
eggs in group 3 for 12 days. Discard embryos that die during
the first 24 h as non-specific deaths ; the test is not valid
01/2008:20625 unless at least 6 embryos in each group survive beyond the
first 24 h after inoculation. Examine macroscopically for
2.6.25. AVIAN LIVE VIRUS VACCINES : abnormalities all embryos which die more than 24 h after
inoculation, or which survive the incubation period. Examine
TESTS FOR EXTRANEOUS AGENTS IN also the chorio-allantoic membranes of these eggs for any
BATCHES OF FINISHED PRODUCT abnormality and test the allantoic fluids for the presence of
haemagglutinating agents.
GENERAL PROVISIONS Carry out a further embryo passage. Pool separately material
a) In the following tests, chickens and/or chicken material from live and from the dead and abnormal embryos. Inoculate
such as eggs and cell cultures shall be derived from chicken each pool into 10 eggs for each route as described above,
flocks free from specified pathogens (SPF) (5.2.2). chorio-allantoic membrane material being inoculated onto
b) Cell cultures for the testing of extraneous agents comply chorio-allantoic membranes, allantoic fluids into the allantoic
with the requirements for the master cell seed of chapter 5.2.4. cavity and embryo material into the yolk sac. For eggs
Cell cultures for the production of veterinary vaccines, with the inoculated by the allantoic and chorio-allantoic routes, candle
exception of the karyotype test and the tumorigenicity test, the eggs daily for 7 days, proceeding and examining the
which do not have to be carried out. material as described above. For eggs inoculated by the yolk
sac route, candle the eggs daily for 12 days, proceeding and
c) In tests using cell cultures, precise specifications are given examining the material as described above.
for the number of replicates, monolayer surface areas and
minimum survival rate of the cultures. Alternative numbers of The batch of vaccine complies with the test if no test
replicates and cell surface areas are possible as well, provided embryo shows macroscopic abnormalities or dies from
that a minimum of 2 replicates are used, the total surface area causes attributable to the vaccine and if examination of the
and the total volume of vaccine test applied are not less than chorio-allantoic membranes and testing of the allantoic fluids
that prescribed here and the survival rate requirements are show no evidence of the presence of extraneous agents.
adapted accordingly. 2. TEST IN CHICKEN EMBRYO FIBROBLAST CELLS
d) In these tests, use the liquid vaccine or reconstitute a
Prepare 7 monolayers of primary or secondary chicken
quantity of the freeze-dried preparation to be tested with the embryo fibroblasts, from the tissues of 9- to 11-day-old
liquid stated on the label or another suitable diluent such as embryos, each monolayer having an area of about 25 cm2.
water for injections. Unless otherwise stated or justified, the
Maintain 2 monolayers as negative controls and treat these
test substance contains the equivalent of 10 doses in 0.1 mL
in the same way as the 5 monolayers inoculated with the test
of inoculum.
vaccine, as described below. Remove the culture medium
e) If the vaccine virus would interfere with the conduct and when the cells reach confluence. Inoculate 0.1 mL of test
sensitivity of the test, neutralise the virus in the preparation vaccine onto each of 5 of the monolayers. Allow adsorption
with a monospecific antiserum. for 1 h and add culture medium. Incubate the cultures
f) Where specified in a monograph or otherwise justified, if for a total of at least 21 days, subculturing at 4- to 5-day
neutralisation of the vaccine virus is required but difficult intervals. Each passage is made with pooled cells and fluids
to achieve, the in vitro tests described below are adapted, as from all 5 monolayers after carrying out a freeze-thaw cycle.
required, to provide the necessary guarantees of freedom from Inoculate 0.1 mL of pooled material onto each of 5 recently
contamination with an extraneous agent. Alternatively, or in prepared monolayers of chicken embryo fibroblast cells, each
addition to in vitro tests conducted on the batch, a test for monolayer having an area of about 25 cm2 each as before. For
extraneous agents may be conducted on chick sera obtained the last passage, grow the cells also on a suitable substrate so
as to obtain an area of about 10 cm2 of cells from each of the The batch of vaccine complies with the test if there is no
monolayers, for test A. The test is not valid if less than 80 per evidence of the presence of egg drop syndrome virus or any
cent of the test monolayers, or neither of the 2 negative control other extraneous agent.
monolayers survive after any passage.
Examine microscopically all the cell cultures frequently 4. TEST FOR MAREK’S DISEASE VIRUS
throughout the entire incubation period for any signs Prepare 11 monolayers of primary or secondary chick embryo
of cytopathic effect or other evidence of the presence of fibroblasts from the tissues of 9- to 11-day-old embryos,
contaminating agents in the test vaccine. At the end of the each monolayer having an area of about 25 cm2. Remove the
total incubation period, carry out the following procedures. culture medium when the cells reach confluence. Inoculate
A. Fix and stain (with Giemsa or haematoxylin and eosin) 0.1 mL of test vaccine onto each of 5 of the monolayers (test
about 10 cm2 of confluent cells from each of the 5 original monolayers). Allow adsorption for 1 h, and add culture
monolayers. Examine the cells microscopically for any medium. Inoculate 4 of the monolayers with a suitable strain
cytopathic effect, inclusion bodies, syncytial formation, of Marek’s disease virus (not more than 10 CCID50 in 0.1 mL)
or any other evidence of the presence of a contaminating to serve as positive controls. Maintain 2 non-inoculated
agent from the test vaccine. monolayers as negative controls.
B. Drain and wash about 25 cm2 of cells from each of the Incubate the cultures for a total of at least 21 days, subculturing
5 monolayers. Cover these cells with a 0.5 per cent at 4- to 5-day intervals. Each passage is made as follows :
suspension of washed chicken red blood cells (using at least trypsinise the cells, prepare separate pools of the cells from
1 mL of suspension for each 5 cm2 of cells). Incubate the the test monolayers, from the positive control monolayers and
cells at 4 °C for 20 min and then wash gently in phosphate from the negative control monolayers. Mix an appropriate
buffered saline pH 7.4. Examine the cells microscopically quantity of each with a suspension of freshly prepared primary
for haemadsorption attributable to the presence of a or secondary chick embryo fibroblasts and prepare 5, 4 and
haemadsorbing agent in the test vaccine. 2 monolayers, as before. The test is not valid if fewer than 4 of
C. Test individually samples of the fluid from each cell culture the 5 test monolayers or fewer than 3 of the 4 positive controls
using chicken red blood cells for haemagglutination or neither of the 2 negative control monolayers survive after
attributable to the presence of a haemagglutinating agent any passage.
in the test vaccine. Examine microscopically all the cell cultures frequently
The test is not valid if there are any signs of extraneous agents throughout the entire incubation period for any signs of
in the negative control cultures. The batch of vaccine complies cytopathic effect or other evidence of the presence of a
with the test if there is no evidence of the presence of any contaminating agent in the test vaccine.
extraneous agent. For the last subculture, grow the cells on a suitable substrate
so as to obtain an area of about 10 cm2 of confluent cells from
3. TEST FOR EGG DROP SYNDROME VIRUS each of the original 11 monolayers for the subsequent test :
Prepare 11 monolayers of chicken embryo liver cells, from the test about 10 cm2 of confluent cells derived from each of the
tissues of 14- to 16-day-old embryos, each monolayer having original 11 monolayers by immunostaining for the presence of
an area of about 25 cm2. Remove the culture medium when Marek’s disease virus. The test is not valid if Marek’s disease
the cells reach confluence. Inoculate 0.1 mL of test vaccine virus is detected in fewer than 3 of the 4 positive control
onto each of 5 of the monolayers (test monolayers). Allow monolayers or in any of the negative control monolayers, or
adsorption for 1 h, add culture medium. Inoculate 4 of the if the results for both of the 2 negative control monolayers
monolayers with a suitable strain of egg drop syndrome virus are inconclusive.
(not more than 10 CCID50 in 0.1 mL) to serve as positive The batch of vaccine complies with the test if there is no
control monolayers. Maintain 2 non-inoculated monolayers evidence of the presence of Marek’s disease virus or any other
as negative control monolayers. extraneous agent.
Incubate the cells for a total of at least 21 days, subculturing
every 4-5 days. Each passage is made as follows : carry out 5. TESTS FOR TURKEY RHINOTRACHEITIS VIRUS
a freeze-thaw cycle ; prepare separate pools of the cells plus A. In chicken embryo fibroblasts
fluid from the test monolayers, from the positive control
monolayers and from the negative control monolayers ; NOTE : this test can be combined with Test 2 by using the
inoculate 0.1 mL of the pooled material onto each of 5, 4 and 2 same test monolayers and negative controls, for all stages up
recently prepared monolayers of chicken embryo liver cells, to the final specific test for turkey rhinotracheitis virus on
each monolayer having an area of about 25 cm2 as before. cells prepared from the last subculture.
The test is not valid if fewer than 4 of the 5 test monolayers Prepare 11 monolayers of primary or secondary chick
or fewer than 3 of the 4 positive controls or neither of the embryo fibroblasts from the tissues of 9- to 11-day-old
2 negative control monolayers survive after any passage. embryos, each monolayer having an area of about
Examine microscopically all the cell cultures at frequent 25 cm2. Remove the culture medium when the cells reach
intervals throughout the entire incubation period for any confluence. Inoculate 0.1 mL of test vaccine onto each of 5
signs of cytopathic effect or other evidence of the presence of a of the monolayers (test monolayers). Allow adsorption
contaminating agent in the test vaccine. At the end of the total for 1 h, and add culture medium. Inoculate 4 of the
incubation period, carry out the following procedure : test monolayers with a suitable strain of turkey rhinotracheitis
separately, cell culture fluid from the test monolayers, positive virus as positive controls (not more than 10 CCID50 in
control monolayers and negative control monolayers, using 0.1 mL). Maintain 2 non-inoculated monolayers as negative
chicken red blood cells, for haemagglutination attributable to controls.
the presence of haemagglutinating agents. Incubate the cultures for a total of at least 21 days,
The test is not valid if egg drop syndrome virus is detected subculturing at 4- to 5-day intervals. Each passage is made
in fewer than 3 of the 4 positive control monolayers or in as follows : carry out a freeze-thaw cycle ; prepare separate
any of the negative control monolayers, or if the results for pools of the cells plus fluid from the test monolayers,
both of the 2 negative control monolayers are inconclusive. from the positive control monolayers and from the
If the results for more than 1 of the test monolayers are negative control monolayers ; inoculate 0.1 mL of the
inconclusive then further subcultures of reserved portions of pooled material onto each of 5, 4 and 2 recently prepared
the monolayers shall be made and tested until an unequivocal monolayers of chicken embryo fibroblasts cells, each
result is obtained. monolayer having an area of about 25 cm2 as before. The
General Notices (1) apply to all monographs and other texts 213
2.6.25. Avian live virus vaccines: extraneous agents in finished product EUROPEAN PHARMACOPOEIA 8.0
test is not valid if fewer than 4 of the 5 test monolayers or cultures, the culture fluids becoming red in comparison with
fewer than 3 of the 4 positive controls or neither of the the control cultures. Examine the cells microscopically for
2 negative control monolayers survive after any passage. cytopathic effect. At this time or at the end of the incubation
For the last subculture, grow the cells on a suitable substrate period, centrifuge the cells from each flask at low speed,
so as to obtain an area of about 10 cm2 of confluent cells resuspend at about 106 cells per millilitre and place 25 μL in
from each of the original 11 monolayers for the subsequent each of 10 wells of a multi-well slide. Examine the cells by
test : test about 10 cm2 of confluent cells derived from each immunostaining.
of the original 11 monolayers by immunostaining for the The test is not valid if chicken anaemia virus is detected
presence of turkey rhinotracheitis virus. The test is not in fewer than 3 of the 4 positive controls or in any of the
valid if turkey rhinotracheitis virus is detected in fewer non-inoculated controls. If the results for more than 1 of the
than 3 of the 4 positive control monolayers or in any of the test suspensions are inconclusive then further subcultures of
negative control monolayers, or if the results for both of reserved portions of the test suspensions shall be made and
the 2 negative control monolayers are inconclusive. If the tested until an unequivocal result is obtained.
results for both of the 2 test monolayers are inconclusive The batch of vaccine complies with the test if there is no
then further subcultures of reserved portions of the evidence of the presence of chicken anaemia virus.
fibroblasts shall be made and tested until an unequivocal
result is obtained. 7. TEST FOR DUCK ENTERITIS VIRUS
The batch of vaccine complies with the test if there is no This test is carried out for vaccines prepared on duck or goose
evidence of the presence of turkey rhinotracheitis virus or substrates.
any other extraneous agent.
Prepare 11 monolayers of primary or secondary Muscovy
B. In Vero cells
duck embryo liver cells, from the tissues of 21- or 22-day-old
Prepare 11 monolayers of Vero cells, each monolayer having embryos, each monolayer having an area of about 25 cm2.
an area of about 25 cm2. Remove the culture medium when Remove the culture medium when the cells reach confluence.
the cells reach confluence. Inoculate 0.1 mL of test vaccine Inoculate 0.1 mL of test vaccine onto each of 5 of the
onto each of 5 of the monolayers (test monolayers). Allow monolayers (test monolayers). Allow adsorption for 1 h
adsorption for 1 h, and add culture medium. Inoculate and add culture medium. Inoculate 4 of the monolayers
4 of the monolayers with a suitable strain of turkey with a suitable strain of duck enteritis virus (not more than
rhinotracheitis virus (not more than 10 CCID50 in 0.1 mL) 10 CCID50 in 0.1 mL) to serve as positive controls. Maintain
to serve as positive controls. Maintain 2 non-inoculated 2 non-inoculated monolayers as negative controls.
monolayers as negative controls.
Incubate the cultures for a total of at least 21 days, subculturing
Incubate the cultures for a total of at least 21 days, at 4- to 5-day intervals. Each passage is made as follows :
subculturing at 4- to 5-day intervals. Each passage is made trypsinise the cells and prepare separate pools of the cells from
as follows : carry out a freeze-thaw cycle. Prepare separate the test monolayers, from the positive control monolayers
pools of the cells plus fluid from the test monolayers, and from the negative control monolayers. Mix a portion
from the positive control monolayers and from the of each with a suspension of freshly prepared primary or
negative control monolayers. Inoculate 0.1 mL of the secondary Muscovy duck embryo liver cells to prepare 5, 4 and
pooled material onto each of 5, 4 and 2 recently prepared 2 monolayers, as before. The test is not valid if fewer than 4 of
monolayers of Vero cells, each monolayer having an area of the 5 test monolayers or fewer than 3 of the 4 positive controls
about 25 cm2 as before. The test is not valid if fewer than 4 or neither of the 2 negative controls survive after any passage.
of the 5 test monolayers or fewer than 3 of the 4 positive
controls or neither of the 2 negative controls survive after For the last subculture, grow the cells on a suitable substrate
any passage. so as to obtain an area of about 10 cm2 of confluent cells from
each of the original 11 monolayers for the subsequent test :
For the last subculture, grow the cells on a suitable substrate test about 10 cm2 of confluent cells derived from each of the
so as to obtain an area of about 10 cm2 of confluent cells original 11 monolayers by immunostaining for the presence of
from each of the original 11 monolayers for the subsequent duck enteritis virus. The test is not valid if duck enteritis virus
test : test about 10 cm2 of confluent cells derived from each is detected in fewer than 3 of the 4 positive control monolayers
of the original 11 monolayers by immunostaining for the or in any of the negative control monolayers, or if the results
presence of turkey rhinotracheitis virus. The test is not for both of the 2 negative control monolayers are inconclusive.
valid if turkey rhinotracheitis virus is detected in fewer If the results for more than 1 of the test monolayers are
than 3 of the 4 positive control monolayers or in any of inconclusive then further subcultures of reserved portions of
the negative control monolayers, or if the results for both the monolayers shall be made and tested until an unequivocal
of the 2 negative control monolayers are inconclusive. result is obtained.
If the results for more than 1 of the test monolayers are
inconclusive then further subcultures of reserved portions The batch of vaccine complies with the test if there is no
of the monolayers shall be made and tested until an evidence of the presence of duck enteritis virus or any other
unequivocal result is obtained. extraneous agent.
The batch of vaccine complies with the test if there is no 8. TEST FOR DUCK AND GOOSE PARVOVIRUSES
evidence of the presence of turkey rhinotracheitis virus or
any other extraneous agent. This test is carried out for vaccines prepared on duck or goose
substrates.
6. TEST FOR CHICKEN ANAEMIA VIRUS Prepare a suspension of sufficient primary or secondary
Prepare eleven 20 mL suspensions of the MDCC-MSBI cell Muscovy duck embryo fibroblasts from the tissues of 16- to
line or another cell line of equivalent sensitivity in 25 cm2 18-day-old embryos, to obtain not fewer than 11 monolayers,
flasks containing about 5 × 105 cells/mL. Inoculate 0.1 mL each having an area of about 25 cm2. Inoculate 0.5 mL of test
of test vaccine into each of 5 of these flasks. Inoculate vaccine into an aliquot of cells for 5 monolayers and seed into
4 other suspensions with 10 CCID50 chicken anaemia virus as 5 replicate containers to form 5 test monolayers. Inoculate
positive controls. Maintain not fewer than 2 non-inoculated 0.4 mL of a suitable strain of duck parvovirus (not more than
suspensions. Maintain all the cell cultures for a total of at least 10 CCID50 in 0.1 mL) into an aliquot of cells for 4 monolayers
24 days, subculturing 8 times at 3- to 4-day intervals. During and seed into 4 replicate containers to form 4 positive control
the subculturing the presence of chicken anaemia virus may monolayers. Prepare 2 non-inoculated monolayers as negative
be indicated by a metabolic colour change in the infected controls.
Bacillus subtilis for example, ATCC 6633, CIP 52.62, V ≥ 10 1 per cent of total volume
NCIMB 8054
1 ≤ V < 10 100 μL
Pseudomonas aeruginosa for example, ATCC 9027, NCIMB 8626,
CIP 82.118 V<1 Not applicable
Candida albicans for example, ATCC 10231, IP 48.72, For haematopoietic products that require dilution before
NCPF 3179 freezing, the inoculum volume must be increased by the
Aspergillus brasiliensis for example, ATCC 16404, IP 1431.83, dilution factor. For other cellular products, suitable minimum
IMI 149007 amounts are defined in terms of volume or number of doses.
Anaerobic medium Analysis. Samples are inoculated into containers of culture
medium as soon as possible after collection and incubated
Clostridium sporogenes for example, ATCC 19404, CIP 79.3, at 35-37 °C for not less than 7 or 14 days, depending on the
NCTC 532 or ATCC 11437 detection system used. A suitable proportion of the inoculum
is added to the medium to be incubated in aerobic conditions
Bacteroides fragilis for example, ATCC 25285, CIP 77.16, and the remainder of the inoculum to the medium to be
NCTC 9343
incubated in anaerobic conditions.
General Notices (1) apply to all monographs and other texts 217
2.6.30. Monocyte-activation test EUROPEAN PHARMACOPOEIA 8.0
donors are to describe themselves as being in good health, 6-1. ASSURANCE OF CRITERIA FOR THE STANDARD
as not to be suffering from any bacterial or viral infections CURVE
and to have been free from the symptoms of any such Using the standard endotoxin solution, prepare at least
infection for a period of at least 1 week prior to the donation 4 endotoxin concentrations to generate the standard
of blood. Blood donors are not to have taken non-steroidal curve. Perform the test using at least 4 replicates of each
anti-inflammatory drugs during the 48 h prior to donating concentration of standard endotoxin.
blood and steroidal anti-inflammatory drugs during the 7 days
prior to donating blood. Individuals who have been prescribed The basal release of the chosen read-out (blank) in the absence
immunosuppressant or other drugs known to influence the of added standard endotoxin is to be optimised to be as low
production of the chosen readout are not to serve as blood as possible.
donors. Blood donation are to be tested for infection markers There are 2 acceptance criteria for the standard curve :
according to national requirements for transfusion medicine.
5-4. QUALIFICATION OF CELLS POOLED FROM A – the regression of responses (appropriately transformed
NUMBER OF DONORS if necessary) on log dose shall be statistically significant
(p < 0.01) ;
Pools (of whole blood or blood fractions, e.g. PBMC), must
consist of donations from a minimum of 4 individual donors – the regression of responses on log dose must not deviate
but preferably 8 or more donors, where practicable, taking significantly from linearity (p > 0.05). If analysis for
from each donation an approximately equal volume of blood, a 4-parameter logistic curve is performed, then the
or cells from an approximately equal volume of blood. For observed curve must not deviate significantly from the
the qualification of pooled cells proceed as follows : within theoretical curve as calculated by using the usual statistical
4 hours of collection of blood, generate dose-response methods (see chapter 5.3. Statistical analysis).
curves from the pool using standard endotoxin with at least
4 geometrically diluted endotoxin concentrations, e.g. in the 6-2. TEST FOR INTERFERING FACTORS
range of 0.01 IU/mL to 4 IU/mL. The dose-response curves To assure the validity of the test, preparatory tests are
are to meet the 2 criteria for the standard curve described conducted to assure that the test solution does not interfere
under section 6-1. with the test. Validation of the test method is required when
any changes are made to the experimental conditions that are
5-5. QUALIFICATION OF CRYO-PRESERVED CELLS likely to influence the result of the test. Using an appropriate
The cell source intended for use in a MAT, e.g. human whole diluent, dilute the preparation to be examined in geometric
blood, blood fractions, such as PBMC or monocytic cell steps, with all dilutions not exceeding the MVD. Make the
lines, may be cryo-preserved. Pools of cryo-preserved cells same dilutions of the preparation to be examined and add
are obtained by pooling before freezing, or by pooling single endotoxin at a concentration equal to or near the middle of
cryo-preserved donations immediately after thawing. Pools the standard curve (Method A) or equal to twice the LOD
must consist of donations from a minimum of 4 individual (Method B), or use a diluent containing added endotoxin at
donors but preferably 8 or more donors where practicable, a concentration equal to or near the middle of the standard
taking from each donation an approximately equal volume of curve (Method A) or equal to twice the LOD (Method B).
blood, or cells from an approximately equal volume of blood. Test these dilution series in parallel in the same experiment.
Qualification of cryo-preserved blood or cells is performed Use the standard curve to calculate the concentration of
immediately after thawing (and pooling if necessary): endotoxin-equivalents in each solution. Calculate the mean
dose-response curves for cryo-preserved blood or cells are to recovery of the added endotoxin by subtracting the mean
comply with the 2 criteria for the standard curve as described concentration of endotoxin equivalents in the solution (if any)
under section 6-1. from that in the solution containing the added endotoxin. The
test solution is considered free of interfering factors if, under
5-6. MONOCYTIC CONTINUOUS CELL LINES the conditions of the test, the measured endotoxin equivalents
A human monocytic cell line is continuously cultured in order in the test solution to which endotoxin is added is within
to warrant a sufficient supply for the MAT. To optimise the 50-200 per cent of the added concentration, after subtraction
method, clones derived from the cell line can be used. of any endotoxin equivalents detected in the solution without
added endotoxin. When this criterion is not met, Method C is
Cells must be maintained under aseptic conditions and to be preferred over Methods A and B.
regularly tested for the presence of mycoplasma contamination.
Additionally, cells must be regularly checked for identity (e.g. In Method C, a solution of the preparation being examined
doubling time, morphology, and function) and stability. The is tested at 3 dilutions : the highest concentration (lowest
functional stability of a cell line is assessed by monitoring its dilution) that stimulates the greatest release of the chosen
performance in relation to the number of passages during read-out and the 2-fold dilutions immediately below and
routine testing. Criteria for functional stability are to be above the chosen dilution. Since the concentration that
established and may include growth criteria, maximum signal stimulates the greatest release of the chosen read-out
obtained in the test, background noise and receptor expression. may be donor-dependent as well as batch-dependent, the
The receptor expression may be tested with specific ligands product-specific validation is to be performed in at least
e.g. lipopolysaccharide (LPS) for toll-like receptor 4 (TLR4), 3 independent tests, each using cells from different donors.
lipoteichoic acid (LTA) for toll-like receptor 2 (TLR2), The highest concentration (lowest dilution) that stimulates
synthetic bacterial lipoprotein for TLR2-TLR1 or synthetic the greatest release of the chosen read-out in the majority of
bacterial lipoprotein for TLR2-TLR6. donors, and the 2-fold dilutions immediately below and above
that dilution are deemed to be validated for further testing.
If undiluted test solution stimulates the greatest release of
the chosen read-out, subsequent testing is to be performed
6. PREPARATORY TESTING using undiluted test solution and also test solution diluted
in the ratios 1:2 and 1:4 before its addition to the PBMC.
To ensure both the precision and validity of the test, The 3 dilutions to be used in subsequent testing are not to
preparatory tests are conducted, to assure that the criteria for exceed the MVD ; the dilution factors for these 3 solutions
the standard curve are satisfied, that the solution does not are designated f1, f2 and f3. Following the product-specific
interfere with the test, that the test detects endotoxins and validation, the test is routinely performed with cells from
non-endotoxins contaminants and that the solution does not 4 individual donors or a single pool or with cells from
interfere in the detection system. 1 passage of a human monocytic cell line.
General Notices (1) apply to all monographs and other texts 219
2.6.30. Monocyte-activation test EUROPEAN PHARMACOPOEIA 8.0
Pyrogen-free
Standard MVD to overcome the interference because it contains or is
endotoxin at 4 believed to contain non-endotoxin contaminants. Responses
R2 saline or test
1 × LOD for the
diluent
test system to non-endotoxin contaminants may dilute out more rapidly
than responses to endotoxin, which makes it necessary to
Standard
Pyrogen-free
endotoxin at perform the test at a range of dilutions that include minimum
R3 saline or test 4 dilution. The test procedure is described below and includes
2 × LOD for the
diluent
test system an example for the comparison of a test lot with the reference
Standard lot.
Pyrogen-free
endotoxin at
R4 saline or test
4 × LOD for the
4 7-3-1. Test procedure
diluent
test system Using the validated test method, prepare the solutions shown
Solution A = solution of the preparation being examined at in Table 2.6.30.-3 and culture 4 replicates of each solution with
the dilution, here designated f, at which the test for interfering cells from each of 4 individual donors or a single pool or with
factors was completed. cells from 1 passage of a human monocytic cell line.
Solution B = solution of the preparation being examined at a Table 2.6.30.-3
dilution, here designated f1, not exceeding the MVD, chosen
after a review of data from the product-specific validation, e.g. Solution/dilution
Solution Number of replicates
factor
1:2 × MVD (i.e. a 2-fold dilution above the MVD).
Solution of reference 4
Solution C = solution of the preparation being examined at a A
lot/f1
dilution, here designated f2, not exceeding the MVD, chosen Solution of reference
after a review of data from the product-specific validation, B 4
lot/f2
e.g. MVD. Solution of reference
C 4
Solution D = solution A spiked with standard endotoxin at lot/f3
2 × LOD for the test system (as determined in preparatory Solution of test 4
D
testing). preparation/f1
Solution of test 4
Solution E = solution B spiked with standard endotoxin at E
preparation/f2
2 × LOD for the test system. Solution of test
F 4
Solution F = solution C spiked with standard endotoxin at preparation/f3
2 × LOD for the test system. G
Positive control 4
(standard endotoxin)
Solution R0 = negative control.
R0 Diluent (negative 4
Solution R1 = standard endotoxin at 0.5 × LOD for the test control)
system.
Solutions A, B and C are solutions of the reference lot diluted
Solution R2 = standard endotoxin at 1 × LOD for the test by the dilution factors, f1, f2 and f3, determined in the test for
system. interfering factors.
Solution R3 = standard endotoxin at 2 × LOD for the test Solutions D, E and F are solutions of the preparation
system. being examined diluted by the dilution factors, f1, f2 and f3,
Solution R4 = standard endotoxin at 4 × LOD for the test determined for the reference lot in the test for interfering
system. factors.
7-2-2. Calculation and interpretation Solution G is the positive test control for the viability of the
All data to be included in the data analysis are to relate to cells and is a standard endotoxin concentration that gives a
cells for which mean responses to solutions R0-R4 increase clear positive response.
progressively. The mean response to R0 may be equal to Solution R0 is the diluent used to dilute the preparation being
the mean response to R1. For each different cell source, examined and serves as the test blank.
e.g. individual donation, donor pool, or cell line, the mean
response to solution R2 is to be greater than a positive cut-off 7-3-2. Calculation and interpretation
value. Data below this cut-off value are considered negative. All data to be included in the data analysis are to relate to
If the mean response to R1 or R2 exceeds the cut-off value, cells for which solution G and at least one of solutions A, B
the response to the solution chosen for the pass/fail decision and C give a response that is greater than the basal release of
must be negative (= pass). For each negative solution of the the read-out (Solution R0). For each different cell source, e.g.
test preparation (A, B and C), the mean response to the individual donation, donor pool, or cell line, use the standard
corresponding spiked solution (D, E or F respectively) is curve for the read-out (a calibration curve in duplicate with
compared with the mean response to R3 to determine the a blank and at least 4 geometrically diluted concentrations
percentage spike recovery. The contaminant concentration of the standard for the chosen read-out) and calculate the
of the preparation being examined is less than the CLC for mean responses of the replicates of solutions A-F. Sum the
a given cell source if the solution of the test preparation mean responses to solutions A, B and C and sum the mean
designated for the pass/fail-decision and the dilutions below responses to solutions D, E and F. Divide the sum of the mean
all give negative results and the endotoxin spike recovery is responses to solutions D, E and F by the sum of the mean
within the range of 50-200 per cent. responses to solutions A, B and C. The preparation being
7-2-3. Pass/fail criteria of the preparation examined complies with the test for a given cell source if the
resulting value complies with a defined acceptance criterion
The criteria are the same as for method A (see 7-1-3). not exceeding 2.5.
7-3. METHOD C : REFERENCE LOT COMPARISON TEST 7-3-3. Pass/fail criteria of the preparation
Method C involves a comparison of the preparation being
examined with a validated reference lot of that preparation. The criteria are the same as for method A (see 7-1-3).
The reference lot is selected according to criteria that have To quantify more closely the level of contamination,
been justified and authorised. The test is intended to be Methods A, B and C may be performed using other dilutions
performed in cases where a preparation being examined of the solution of the preparation being examined not
shows marked interference but cannot be diluted within the exceeding the MVD.
The following section is published for information only. Where an endotoxin limit concentration (ELC) has been
specified for a product, the CLC is the same as the ELC,
Guidance notes unless otherwise prescribed. The CLC is expressed in terms
of endotoxin equivalents. The CLC is calculated using the
1. INTRODUCTION following expression :
The monocyte activation test (MAT) is primarily intended
to be used as an alternative method to the rabbit pyrogen
test. The MAT detects pyrogenic and pro-inflammatory
contaminants, including endotoxins from gram-negative K = threshold pyrogenic dose of endotoxin per
bacteria and ‘non-endotoxin’ contaminants, including kilogram of body mass ;
pathogen-associated molecular patterns (PAMPs), derived M = maximum recommended bolus dose of product
from gram-positive and gram-negative bacteria, viruses and per kilogram of body mass.
fungi, and product-related and process-related biological or When the product is to be injected at frequent intervals
chemical entities. or infused continuously, M is the maximum total dose
Since non-endotoxin contaminants are a physico-chemically administered in a single hour period.
diverse class of molecules, and usually the nature of The endotoxin limits depends on the product and its route of
the contaminant in a preparation being examined is administration and is stated in monographs.
unknown, the level of contamination is expressed either in
endotoxin-equivalent units, derived by comparison with Values for K are suggested in Table 2.6.30.-4.
responses to standard endotoxin, or by comparison with a Table 2.6.30.-4
reference lot of the test preparation. Route of administration K (IU of endotoxin per kilogram
of body mass)
In the MAT, responses to standard endotoxin usually dilute Intravenous 5.0
out over approximatively 1 log10 and responses to products
contaminated with non-endotoxin contaminants (alone Intravenous, for radiopharmaceuticals 2.5
or in combination with endotoxins) often show very steep 0.2
Intrathecal
dose-response curves, usually over only 1 or 2 dilution steps
when tested for their capability to stimulate monocytes.
For other routes, the acceptance criterion for bacterial
Frequently, the largest response to such contaminated
endotoxins is generally determined on the basis of results
products is obtained with undiluted solutions of preparations
obtained during the development of the preparation.
being examined or small dilutions of the preparations being
examined. For this reason test solutions of preparations 2-3. INFORMATION REGARDING CRYO-PROTECTANTS
being examined that contain or may contain non-endotoxin The influence of cryo-protectants, e.g. dimethyl
contaminants have to be tested at a range of dilutions that sulfoxide (DMSO), and their residues in thawed cells, is to be
includes minimum dilution. considered : DMSO is toxic to cells in culture and, even when
cells have been washed thoroughly, cryo-preservation may
2. METHODS have altered cell properties, e.g. cell membrane permeability.
2-1. INFORMATION REGARDING THE CHOICE OF 2-4. INTERFERENCE TESTING
METHODS Where practicable, interference testing is performed on at
Methods A, B and C, are not normally applied where a test least 3 different lots of the preparation being examined.
preparation has the intrinsic activity of stimulating the Preparations being examined that show marked batch-to-batch
release of the chosen read-out or where the test preparation variation, that effectively renders each batch unique for
is contaminated with the chosen read-out. In both cases, this the purposes of interference testing, are to be subjected
fact is to be addressed by modifying and validating the chosen to interference testing within each individual test, i.e.
method accordingly. The product-specific validation of the concomitant validation.
chosen method would be expected to identify the frequency Interference testing is preferably performed on batches of the
of non-responders to a particular product/contaminant(s) preparation being examined that are free of endotoxins and
combination and to identify steps to address this, e.g. other pyrogenic/pro-inflammatory contaminants and, where
screening of donors, increasing the number of donors per this is not practicable, none of the batches are to be heavily
test, and setting pass/fail criteria of appropriate stringency to contaminated. If only 1 batch is available the validation has to
maximise the likelihood of detecting contaminated batches. be performed on that batch in 3 independent tests. Precision
Methods A and B are appropriate when responses to dilutions parameters for reproducibility, e.g. ± 50 per cent, are to be
of a preparation being examined are broadly parallel to fulfilled.
responses to dilutions of standard endotoxin. Method B is a
semi-quantitative test that can also be applied when responses 3. REPLACEMENT OF THE RABBIT PYROGEN TEST BY
to dilutions of a test preparation are not parallel to responses THE MONOCYTE ACTIVATION TEST
to dilutions of standard endotoxin. As noted above, the monocyte activation test (MAT) is
Method C, the reference lot comparison test, was developed primarily intended to be used as an alternative method to the
to address extreme donor variability in responses to certain rabbit pyrogen test. Monographs on pharmaceutical products
product/contaminant(s) combinations. In this regard, intended for parenteral administration that may contain
it should be noted that, while monocytes from most pyrogenic contaminants require either a test for bacterial
donors respond in a broadly similar manner to bacterial endotoxins or a monocyte activation test.
endotoxin, responses of monocytes from different donors As a general policy :
to non-endotoxin contaminants can differ markedly, so – in any individual monograph, when a test is required, only
that it is possible to identify non-responders to certain 1 test is included, either that for bacterial endotoxins or
product/contaminant(s) combinations. the MAT. Before including the MAT in a monograph,
2-2. CALCULATION OF CONTAMINANT LIMIT evidence is required that 1 of the 3 methods described
CONCENTRATION in the MAT chapter can be applied satisfactorily to the
The acceptance criterion for a pass/fail decision is the product in question ;
contaminant limit concentration (CLC), which is expressed in – the necessary information is sought from manufacturers.
endotoxin equivalents per milligram or millilitre or in units Companies are invited to provide any validation data
of biological activity of the preparation being examined. that they have concerning the applicability of the MAT
General Notices (1) apply to all monographs and other texts 221
2.6.31. Microbiological examination of herbal products and extracts EUROPEAN PHARMACOPOEIA 8.0
to the substances and formulations of interest. Such data microbiological quality. When used for such purposes, follow
include details of sample preparation and of any procedures the instructions given below, including the number of samples
necessary to eliminate interfering factors. In addition, any to be taken, and interpret the results as stated below.
available parallel data for rabbit pyrogen testing that would Alternative microbiological procedures, including automated
contribute to an assurance that the replacement of a rabbit methods, may be used, provided that their equivalence to the
pyrogen test by the MAT is appropriate, is to be provided. Pharmacopoeia method has been demonstrated.
2-2. GENERAL PROCEDURES
4. VALIDATION OF ALTERNATIVE METHODS
The preparation of samples is carried out as described in
Replacement of a rabbit pyrogen test by a MAT, or replacement general chapter 2.6.12.
of a method for detecting pro-inflammatory/pyrogenic If the product to be examined has antimicrobial activity, this
contaminants by another method, is to be regarded as is as far as possible removed or neutralised as described in
the use of an alternative method in the replacement of a general chapter 2.6.12.
pharmacopoeial test, as described in the General Notices :
If surface-active substances are used for sample preparation,
‘The test and assays described are the official methods upon their absence of toxicity for micro-organisms and their
which the standards of the European Pharmacopoeia are compatibility with inactivators used must be demonstrated as
based. With the agreement of the competent authority, described in general chapter 2.6.12.
alternative methods of analysis may be used for control 2-3. GROWTH-PROMOTING AND INHIBITORY
purposes, provided that the methods used enable an PROPERTIES OF THE MEDIA, SUITABILITY OF THE TEST
unequivocal decision to be made as to whether compliance AND NEGATIVE CONTROLS
with the standards of the monographs would be achieved if the
official methods were used. In the event of doubt or dispute, The ability of the test to detect micro-organisms in the
the methods of analysis of the European Pharmacopoeia are presence of the product to be examined must be established.
alone authoritative.’ Suitability must be confirmed if a change in testing
performance, or the product, which may affect the outcome of
The following procedures are suggested for validating a the test is introduced.
method for the MAT other than the one indicated in the 2-3-1. PREPARATION OF TEST STRAINS
monograph :
Use standardised stable suspensions of test strains or prepare
– the procedure and the materials and reagents used in them as stated below. Seed lot culture maintenance techniques
the method should be validated as described for the test (seed-lot systems) are used so that the viable micro-organisms
concerned ; used for inoculation are not more than 5 passages removed
– the presence of interfering factors (and, if needed, the from the original master seed lot.
procedure for removing them) should be tested on samples 2-3-1-1. Aerobic micro-organisms. Grow each of the
of at least 3 production batches. bacterial test strains separately in casein soya bean digest broth
or on casein soya bean digest agar at 30-35 °C for 18-24 h.
MAT should be applied to all new products intended for
parenteral administration that have to be tested for the – Staphylococcus aureus such as ATCC 6538, NCIMB 9518,
presence of monocyte-activating contaminants according to CIP 4.83 or NBRC 13276 ;
the requirements of the European Pharmacopoeia. – Pseudomonas aeruginosa such as ATCC 9027, NCIMB 8626,
CIP 82.118 or NBRC 13275 ;
– Escherichia coli such as ATCC 8739, NCIMB 8545,
CIP 53.126 or NBRC 3972 ;
01/2014:20631 – Salmonella enterica subsp. enterica serovar Typhimurium
such as ATCC 14028 or, as an alternative, S. enterica subsp.
enterica serovar Abony such as NBRC 100797, NCTC 6017
2.6.31. MICROBIOLOGICAL or CIP 80.39 ;
EXAMINATION OF HERBAL – Bacillus subtilis such as ATCC 6633, NCIMB 8054,
MEDICINAL PRODUCTS FOR ORAL CIP 52.62 or NBRC 3134.
USE AND EXTRACTS USED IN THEIR Use buffered sodium chloride-peptone solution pH 7.0 or
phosphate buffer solution pH 7.2 to make test suspensions.
PREPARATION Use the suspensions within 2 h, or within 24 h if stored at
2-8 °C. As an alternative to preparing and then diluting a
1. MICROBIAL ENUMERATION TESTS fresh suspension of vegetative cells of B. subtilis, a stable spore
suspension is prepared and then an appropriate volume is
Total aerobic microbial count (TAMC). Perform as used for test inoculation. The stable spore suspension may be
described in general chapter 2.6.12. maintained at 2-8 °C for a validated period of time.
Total combined yeasts/moulds count (TYMC). Perform as 2-3-2. NEGATIVE CONTROL
described in general chapter 2.6.12. Due to the natural high
bioburden in the products covered by general chapter 5.1.8, To verify testing conditions, a negative control is performed
use of Sabouraud-dextrose agar containing antibiotics is using the chosen diluent in place of the test preparation. There
suitable. must be no growth of micro-organisms. A negative control
is also performed when testing the products as described in
section 2-4. A failed negative control requires an investigation.
2. TEST FOR SPECIFIED MICRO-ORGANISMS
2-3-3. GROWTH-PROMOTING AND INHIBITORY PROPERTIES
2-1. INTRODUCTION OF THE MEDIA
The tests described hereafter will allow determination of the
absence or limited occurrence of specified micro-organisms Test each batch of ready-prepared medium and each batch of
that may be detected under the conditions described. medium prepared either from dehydrated medium or from
The tests are designed primarily to determine whether a the ingredients described.
product, substance or preparation (hereinafter referred to as Verify suitable properties of relevant media as described in
‘the product’) complies with an established specification for Table 2.6.31.-1.
Table 2.6.31.-1. – Growth-promoting, inhibitory and indicative the prescribed growth medium (casein soya bean digest
properties of media broth or buffered peptone medium). For the enumeration
method for bile-tolerant gram-negative bacteria, inoculate
Medium Property Test strains
E. coli and P. aeruginosa individually. For the tests for E. coli
S. aureus and Salmonella, inoculate the specified micro-organism
Casein soya bean Growth
P. aeruginosa individually.
digest broth promoting
B. subtilis
Test for Growth E. coli Any antimicrobial activity of the product necessitates a
bile-tolerant Enterobacteria modification of the test procedure (see section 4-5-3 of general
enrichment
promoting P. aeruginosa
gram-negative chapter 2.6.12).
bacteria broth-Mossel Inhibitory S. aureus
If for a given product the antimicrobial activity with respect
Growth E. coli
Violet red bile
promoting to a micro-organism for which testing is prescribed cannot
glucose agar
+ indicative P. aeruginosa be neutralised, then it is to be assumed that the inhibited
S. aureus micro-organism will not be present in the product.
Casein soya bean Growth
P. aeruginosa
digest broth promoting 2-3-4-1. Test for absence. Use a number of micro-organisms
B. subtilis equivalent to not more than 100 CFU in the inoculated test
Growth
Test for promoting E. coli preparation. Perform the test as described in the relevant
Escherichia coli MacConkey broth paragraph in section 2-4 using the shortest incubation period
Inhibitory S. aureus
prescribed. The specified micro-organisms must be detected
Growth with the indication reactions as described in section 2-4.
MacConkey agar promoting E. coli
+ indicative 2-3-4-2. Enumeration test. Semi-quantitative test
S. enterica subsp. (probable-number method).
enterica serovar
Buffered peptone Growth Typhimurium or Use a number of micro-organisms equivalent to not more
medium promoting S. enterica subsp. than 100 CFU per gram or millilitre of product. Perform
enterica serovar the test as described in the relevant paragraph in section 2-4
Abony
using the shortest incubation period prescribed. The dilution
S. enterica subsp.
enterica serovar corresponding to 0.1 g or 0.1 mL of product must be positive.
Rappaport Growth Typhimurium 2-4. TESTING OF PRODUCTS
Vassiliadis promoting or S. enterica
Test for Salmonella Salmonella 2-4-1. BILE-TOLERANT GRAM-NEGATIVE BACTERIA
subsp. enterica
enrichment broth serovar Abony 2-4-1-1. Enumeration test. Semi-quantitative test
Inhibitory S. aureus (probable-number method).
S. enterica subsp. 2-4-1-1-1. Sample preparation and pre-incubation. Prepare
enterica serovar
Xylose, lysine, Growth Typhimurium
a sample using a 10-fold dilution of not less than 1 g of the
deoxycholate agar promoting or S. enterica product to be examined as described in general chapter 2.6.12,
+ indicative subsp. enterica but using casein soya bean digest broth as the chosen diluent,
serovar Abony mix and incubate at 20-25 °C for a time sufficient to resuscitate
Test for growth-promoting properties, liquid media : the bacteria but not sufficient to encourage multiplication of
inoculate a portion of the appropriate medium with a the organisms (2-3 h).
small number (not more than 100 CFU) of the appropriate 2-4-1-1-2. Selection and subculture. Inoculate suitable
micro-organism. Incubate at the specified temperature for quantities of enterobacteria enrichment broth-Mossel with
not more than the shortest period of time specified in the the preparation as described above and/or, depending on
test. Incubate the casein soya bean digest broth at 30-35 °C the limit applied for the particular product, with 3 of the 4
for not more than 3 days. Clearly visible growth of the dilutions of the preparation, which contain respectively 0.1 g,
micro-organism comparable to that obtained with a previously 0.01 g, 0.001 g and 0.0001 g (or 0.1 mL, 0.01 mL, 0.001 mL
tested and approved batch of medium occurs. and 0.0001 mL) of the product to be examined. Incubate at
30-35 °C for 24-48 h. Subculture each of the cultures on a plate
Test for growth-promoting properties, solid media : perform
of violet red bile glucose agar. Incubate at 30-35 °C for 18-24 h.
the surface-spread method, inoculating each plate with a
small number (not more than 100 CFU) of the appropriate 2-4-1-1-3. Interpretation. Growth of colonies constitutes a
micro-organism. Incubate at the specified temperature for not positive result. Note the smallest quantity of the product that
more than the shortest period of time specified in the test. gives a positive result and the largest quantity that gives a
Growth of the micro-organism comparable to that obtained negative result.
with a previously tested and approved batch of medium occurs. Determine from Table 2.6.31.-2 the probable number of
Test for inhibitory properties, liquid or solid media : bacteria.
inoculate the appropriate medium with at least 100 CFU of Table 2.6.31.-2. – Interpretation of results
the appropriate micro-organism. Incubate at the specified
temperature for not less than the longest period of time Results for each quantity of product Probable
number of
specified in the test. No growth of the test micro-organism bacteria
occurs. 0.1 g or 0.01 g or 0.001 g or 0.0001 g or per gram
0.1 mL 0.01 mL 0.001 mL 0.0001 mL or millilitre
Test for indicative properties : perform the surface-spread of product
method, inoculating each plate with a small number (not
more than 100 CFU) of the appropriate micro-organism. + + + + > 104
Incubate at the specified temperature for a period of time < 104 and
within the range specified in the test. Colonies are comparable + + + -
> 103
in appearance and indication reactions to those obtained with
a previously tested and approved batch of medium. + + - - < 103 and
> 102
2-3-4. SUITABILITY OF THE TEST METHOD
+ - - - < 102 and
For each product to be examined, perform the sample > 10
preparation as described in the relevant paragraph in - - - - < 10
section 2-4. Add each test strain at the time of mixing, in
General Notices (1) apply to all monographs and other texts 223
2.6.33. Residual pertussis toxin and irreversibility of pertussis toxoid EUROPEAN PHARMACOPOEIA 8.0
2-4-2. ESCHERICHIA COLI The product complies with the test if colonies of the types
2-4-2-1. Test for absence described are not present or if the identification tests are
negative.
2-4-2-1-1. Sample preparation and pre-incubation. Prepare
a sample using a 10-fold dilution of not less than 1 g of the The following section is given for information.
product to be examined as described in general chapter 2.6.12, RECOMMENDED SOLUTIONS AND CULTURE MEDIA
and use 10 mL or the quantity corresponding to 1 g or 1 mL The solutions and culture media mentioned in this chapter
to inoculate a suitable amount (determined as described in and described in general chapter 2.6.13 and the following
section 2-3-4) of casein soya bean digest broth, mix and buffered peptone medium have been found to be satisfactory
incubate at 30-35 °C for 18-24 h. for the purposes for which they are prescribed in this chapter.
2-4-2-1-2. Selection and subculture. Shake the container, Other media may be used provided that their suitability can
transfer 1 mL of casein soya bean digest broth to 100 mL be demonstrated.
of MacConkey broth and incubate at 42-44 °C for 24-48 h. Buffered peptone medium
Subculture on a plate of MacConkey agar at 30-35 °C for Potassium dihydrogen phosphate 1.5 g
18-72 h.
Disodium hydrogen phosphate dodecahydrate 9.0 g
2-4-2-1-3. Interpretation. Growth of colonies indicates the
possible presence of E. coli. This is confirmed by identification Sodium chloride 5.0 g
tests.
Peptone 10.0 g
The product complies with the test if no colonies are present
or if the identification tests are negative. Purified water 1000 mL
2-4-2-2. Enumeration test. Semi-quantitative test
(probable-number method). Adjust the pH so that after sterilisation it is 7.0 ± 0.2 at 25 °C.
Sterilise in an autoclave using a validated cycle.
2-4-2-2-1. Sample preparation and pre-incubation. Prepare
a sample using a 10-fold dilution of not less than 1 g of the
product to be examined as described in general chapter 2.6.12, 07/2013:20633
and use the quantities corresponding respectively to 0.1 g,
0.01 g and 0.001 g (or 0.1 mL, 0.01 mL and 0.001 mL) to 2.6.33. RESIDUAL PERTUSSIS TOXIN
inoculate a suitable amount (determined as described in
section 2-3-4) of casein soya bean digest broth, mix and
AND IRREVERSIBILITY OF PERTUSSIS
incubate at 30-35 °C for 18-24 h. TOXOID
2-4-2-2-2. Selection and subculture. Shake the container, This test is not necessary for the product obtained by genetic
transfer 1 mL of casein soya bean digest broth to 100 mL modification.
of MacConkey broth and incubate at 42-44 °C for 24-48 h. Pertussis toxin BRP or an in-house toxin preparation calibrated
Subculture on a plate of MacConkey agar at 30-35 °C for in International Units against the BRP are suitable for use as
18-72 h. a reference pertussis toxin preparation.
2-4-2-2-3. Interpretation. Growth of colonies indicates the
Mouse strain. A suitable mouse strain has a toxin LD50
possible presence of E. coli. This is confirmed by identification
between 6 IU and 50 IU.
tests.
Use equal groups of not fewer than 10 histamine-sensitive
Note the smallest quantity of the product that gives a positive
mice of suitable strain, age and weight. For the test for residual
result and the largest quantity that gives a negative result.
pertussis toxin, inject intraperitoneally into the 1st group
Determine from Table 2.6.31.-3 the probable number of between 1 and 2 human doses of the vaccine stored at 2-8 °C.
bacteria. For the test for irreversibility of pertussis toxoid, inject
Table 2.6.31.-3. – Interpretation of results intraperitoneally into the 2nd group between 1 and 2 human
Results for each quantity of product Probable
doses of the vaccine incubated at 37 °C for 4 weeks.
number of The histamine sensitivity criteria must be set during a
bacteria per validation study using multiple doses of a reference toxin ; a
0.01 g or 0.001 g or gram or
0.1 g or 0.1 mL significant dose-response must be demonstrated. A suitable
0.01 mL 0.001 mL millilitre of
product dose of the reference toxin, chosen in the linear region of the
dose-response curve and giving a positive response considered
+ + + > 103 appropriate by the competent authority, is subsequently
+ + - < 103 and > 102 included in each assay as the positive control to demonstrate
assay sensitivity.
+ - - < 102 and > 10
The positive control group of mice is injected with an
- - - < 10 equivalent volume of reference pertussis toxin preparation
(e.g. pertussis toxin BRP) at a dose that has been defined in
2-4-3. SALMONELLA the validation stage as demonstrating the assay sensitivity, for
2-4-3-1. Test for absence example a dose causing death in at least 30 per cent of the mice.
2-4-3-1-1. Sample preparation and pre-incubation. Use 25 g or The negative control group of mice is injected intraperitoneally
25 mL of the product to be examined to inoculate 225 mL of with an equivalent volume of diluent.
buffered peptone medium and mix (e.g. homogenise in a filter If a reference group of mice is used, it may be injected with a
bag by using a blender). Incubate at 30-35 °C for 18-24 h. reference pertussis toxin preparation (e.g. pertussis toxin BRP)
2-4-3-1-2. Selection and subculture. Transfer 0.1 mL of at a dose previously set as the allowable upper limit of pertussis
buffered peptone medium to 10 mL of Rappaport Vassiliadis toxin in the product according to historical safety data.
Salmonella enrichment broth and incubate at 30-35 °C Alternatively, a reference vaccine with established clinical
for 18-24 h. Subculture on plates of xylose, lysine and safety may be used instead of the reference toxin preparation.
deoxycholate agar. Incubate at 30-35 °C for 18-48 h. After 5 days, inject intraperitoneally into each mouse of each
2-4-3-1-3. Interpretation. The possible presence of Salmonella group the equivalent of 2 mg of histamine base in a volume not
is indicated by the growth of well-developed, red colonies, with exceeding 0.5 mL and observe for 24 h. The test is not valid if :
or without black centres. This is confirmed by identification – 1 or more mice in the negative control group die following
tests. histamine challenge ;
– the histamine sensitivity does not meet the defined limit If a reference group is included, the vaccine complies with the
(e.g. at least 30 per cent of the mice die in the positive test for residual pertussis toxin if the percentage of deaths in
control group). the 1st group is not greater than that in the reference group. If
a vaccine lot fails the test, the test may be repeated once with
If a reference group is not included in the assay, the vaccine the same number of mice or with a greater number and the
complies with the test for residual pertussis toxin if no mice results of the tests combined ; the vaccine complies with the
die in the 1st group. If a vaccine lot fails the test, the test may test if the percentage of deaths in the groups given the vaccine
be repeated once with the same number of mice or with a does not exceed the percentage of deaths in the reference
greater number and the results of the tests combined ; the groups. The vaccine complies with the test for irreversibility of
vaccine complies with the test if the percentage of deaths in pertussis toxoid if the 2nd group, given the vaccine incubated
the group given the vaccine does not exceed 5 per cent. The at 37 °C, also complies with these criteria.
vaccine complies with the test for irreversibility of pertussis Alternatively, after validation, a histamine-sensitisation test
toxoid if the 2nd group, given the vaccine incubated at 37 °C, based on body temperature measurements as end-points may
also complies with these criteria. be used instead of the lethal end-point test in mice.
General Notices (1) apply to all monographs and other texts 225
EUROPEAN PHARMACOPOEIA 8.0 2.7.1. Immunochemical methods
General Notices (1) apply to all monographs and other texts 229
2.7.2. Microbiological assay of antibiotics EUROPEAN PHARMACOPOEIA 8.0
arrange the solutions of the antibiotic to be examined and the In order that the validity of the assay may be assessed, use not
solutions of the reference substance in an alternate manner to fewer than 3 doses of the reference substance and 3 doses of the
avoid interaction of the more concentrated solutions. antibiotic to be examined having the same presumed activity
Incubate at a suitable temperature for about 18 h. A period as the doses of the reference substance. It is preferable to use
of diffusion prior to incubation, usually 1-4 h, at room a series of doses in geometric progression. In order to obtain
temperature or at about 4 °C, as appropriate, may be used the required linearity, it may be necessary to select from a
to minimise the effects of the variation in time between the large number 3 consecutive doses, using corresponding doses
application of the solutions and to improve the regression for the reference substance and the antibiotic to be examined.
slope.
Distribute an equal volume of each of the solutions into
Measure the diameters with a precision of at least 0.1 mm or identical test-tubes and add to each tube an equal volume of
the areas of the circular inhibition zones with a corresponding inoculated medium (for example, 1 mL of the solution and
precision and calculate the potency using appropriate 9 mL of the medium). For the assay of tyrothricin add 0.1 mL
statistical methods. of the solution to 9.9 mL of inoculated medium.
Use in each assay the number of replications per dose
sufficient to ensure the required precision. The assay may be Prepare at the same time 2 control tubes without antibiotic,
repeated and the results combined statistically to obtain the both containing the inoculated medium and to one of which is
required precision and to ascertain whether the potency of added immediately 0.5 mL of formaldehyde R. These tubes are
the antibiotic to be examined is not less than the minimum used to set the optical apparatus used to measure the growth.
required.
Place all the tubes, randomly distributed or in a Latin square
B. TURBIDIMETRIC METHOD or randomised block arrangement, in a water-bath or other
Inoculate a suitable medium with a suspension of the chosen suitable apparatus fitted with a means of bringing all the
micro-organism having a sensitivity to the antibiotic to be tubes rapidly to the appropriate incubation temperature
examined such that a sufficiently large inhibition of microbial and maintain them at that temperature for 3-4 h, taking
growth occurs in the conditions of the test. Use a known precautions to ensure uniformity of temperature and identical
quantity of the suspension chosen so as to obtain a readily incubation time.
measurable opacity after an incubation period of about 4 h.
After incubation, stop the growth of the micro-organisms
Use the inoculated medium immediately after its preparation. by adding 0.5 mL of formaldehyde R to each tube or by heat
Using the solvent and the buffer solution indicated in treatment and measure the opacity to 3 significant figures
Table 2.7.2.-2 prepare solutions of the reference substance and using suitable optical apparatus. Alternatively use a method
of the antibiotic to be examined having known concentrations which allows the opacity of each tube to be measured after
presumed to be of equal activity. exactly the same period of incubation.
Solvent to be used
Buffer solution Micro-organism Medium and final Incubation
Antibiotic Reference substance in preparing the
(pH) pH (± 0.1 pH unit) temperature
stock solution
Saccharomyces
Amphotericin B cerevisiae
Amphotericin B for microbiological Dimethyl sulfoxide R pH 10.5 (0.2 M) F - pH 6.1 35-37 °C
ATCC 9763
assay CRS
IP 1432-83
Micrococcus luteus
Bacitracin zinc Bacitracin zinc CRS 0.01 M hydrochloric NCTC 7743
pH 7.0 (0.05 M) A - pH 7.0 35-39 °C
acid CIP 53.160
ATCC 10240
Mycobacterium
Bleomycin sulfate Bleomycin sulfate CRS Water R pH 6.8 (0.1 M) smegmatis G - pH 7.0 35-37 °C
ATCC 607
Bordetella B - pH 7.3 35-39 °C
bronchiseptica
NCTC 8344
CIP 53.157
Colistimethate Colistimethate Water R ATCC 4617
pH 6.0 (0.05 M)
sodium sodium CRS
Escherichia coli B - pH 7.3 35-39 °C
NCIMB 8879
CIP 54.127
ATCC 10536
Bordetella B - pH 7.3 35-39 °C
bronchiseptica
NCTC 8344
CIP 53.157
Colistin sulfate for
Colistin sulfate microbiological Water R pH 6.0 (0.05 M) ATCC 4617
assay CRS Escherichia coli B - pH 7.3 35-39 °C
NCIMB 8879
CIP 54.127
ATCC 10536
General Notices (1) apply to all monographs and other texts 231
2.7.2. Microbiological assay of antibiotics EUROPEAN PHARMACOPOEIA 8.0
Solvent to be used
Buffer solution Micro-organism Medium and final Incubation
Antibiotic Reference substance in preparing the
(pH) pH (± 0.1 pH unit) temperature
stock solution
Bacillus subtilis E - pH 7.9 30-37 °C
NCTC 10400
CIP 52.62
Framycetin
Framycetin sulfate Water R pH 8.0 (0.05 M) ATCC 6633
sulfate CRS
Bacillus pumilus E - pH 7.9 30-37 °C
NCTC 8241
CIP 76.18
Bacillus pumilus A - pH 7.9 35-39 °C
NCTC 8241
CIP 76.18
Gentamicin Staphylococcus A - pH 7.9 35-39 °C
Gentamicin sulfate Water R pH 8.0 (0.05 M)
sulfate CRS epidermidis
NCIMB 8853
CIP 68.21
ATCC 12228
Bacillus subtilis
Josamycin Josamycin CRS Methanol R (see the pH 5.6 CIP 52.62
A - pH 6.6 35-37 °C
monograph) ATCC 6633
NCTC 10400
Bacillus subtilis
Josamycin Josamycin Methanol R (see the pH 5.6 CIP 52.62
A - pH 6.6 35-37 °C
propionate propionate CRS monograph) ATCC 6633
NCTC 10400
Bacillus subtilis A - pH 7.9 30-37 °C
Kanamycin NCTC 10400
monosulfate CIP 52.62
Kanamycin ATCC 6633
Water R pH 8.0 (0.05 M)
monosulfate CRS Staphylococcus aureus A - pH 7.9 35-39 °C
Kanamycin acid NCTC 7447
sulfate CIP 53.156
ATCC 6538 P
Bacillus pumilus E - pH 7.9 30-37 °C
NCTC 8241
CIP 76.18
Neomycin sulfate
Neomycin sulfate for microbiological Water R pH 8.0 (0.05 M) Bacillus subtilis E - pH 7.9 30-37 °C
assay CRS
NCTC 10400
CIP 52.62
ATCC 6633
Staphylococcus aureus
Netilmicin sulfate Netilmicin sulfate CRS Water R pH 8.0 ± 0.1 ATCC 6538 P A - pH 7.9 32-35 °C
CIP 53.156
Candida tropicalis F - pH 6.0 30-37 °C
CIP 1433-83
NCYC 1393
pH 6.0 (0.05 M)
Dimethylforma- containing 5 per Saccharomyces F - pH 6.0 30-32 °C
Nystatin Nystatin CRS
mide R cent V/V of dime- cerevisiae
thylformamide R
NCYC 87
CIP 1432-83
ATCC 9763
Micrococcus luteus
Rifamycin NCTC 8340
Rifamycin sodium Methanol R pH 7.0 (0.05 M) A - pH 6.6 35-39 °C
sodium CRS CIP 53.45
ATCC 9341
Bacillus subtilis
NCTC 10400
Spiramycin Spiramycin CRS Methanol R pH 8.0 (0.05 M) A - pH 7.9 30-32 °C
CIP 52.62
ATCC 6633
Solvent to be used
Buffer solution Micro-organism Medium and final Incubation
Antibiotic Reference substance in preparing the
(pH) pH (± 0.1 pH unit) temperature
stock solution
Bacillus subtilis A - pH 7.9 30-37 °C
NCTC 8236
CIP 1.83
Streptomycin
Streptomycin sulfate Water R pH 8.0 (0.05 M) Bacillus subtilis A - pH 7.9 30-37 °C
sulfate CRS
NCTC 10400
CIP 52.62
ATCC 6633
Bacillus subtilis
NCTC 10400
Teicoplanin Teicoplanin CRS pH 6.0 (0.05 M) pH 6.0 (0.05 M) H - pH 7.8-8.0 35-37 °C
CIP 52.62
ATCC 6633
Tylosin for 2.5 per cent V/V A mixture of
veterinary use 40 volumes of Micrococcus luteus
solution of
methanol R in 0.1 M methanol R and NCTC 8340
Tylosin CRS A - pH 8.0 32-35 °C
phosphate buffer 60 volumes of 0.1 M CIP 53.45
Tylosin tartrate for phosphate buffer
veterinary use solution pH 7.0 R ATCC 9341
solution pH 8.0 R
Bacillus subtilis
Vancomycin Vancomycin NCTC 10400
Water R pH 8.0 A - pH 8.0 37-39 °C
hydrochloride hydrochloride CRS CIP 52.62
ATCC 6633
Calculate the potency using appropriate statistical methods. two-point assay may be sufficient, subject to agreement by
Linearity of the dose-response relationship, transformed or the competent authority. However, in all cases of dispute, a
untransformed, is often obtained only over a very limited three-point assay must be applied.
range. It is this range which must be used in calculating the
activity and it must include at least 3 consecutive doses in Use in each assay the number of replications per dose
order to permit linearity to be verified. In routine assays when sufficient to ensure the required precision. The assay may be
the linearity of the system has been demonstrated over an repeated and the results combined statistically to obtain the
adequate number of experiments using a three-point assay, a required precision and to ascertain whether the potency of
the antibiotic to be examined is not less than the minimum
required.
Solvent to be used
Buffer solution Micro-organism Medium and final Incubation
Antibiotic Reference substance in preparing the
(pH) pH (± 0.1 pH unit) temperature
stock solution
Escherichia coli
Colistimethate Colistimethate Water R pH 7.0
NCIMB 8666
C - pH 7.0 35-37 °C
sodium sodium CRS CIP 2.83
ATCC 9637
Escherichia coli
Colistin sulfate for NCIMB 8666
Colistin sulfate microbiological Water R pH 7.0 C - pH 7.0 35-37 °C
assay CRS CIP 2.83
ATCC 9637
Staphylococcus aureus
Framycetin NCTC 7447
Framycetin sulfate Water R pH 8.0 C - pH 7.0 35-37 °C
sulfate CRS CIP 53.156
ATCC 6538 P
Staphylococcus aureus
Gentamicin NCTC 7447
Gentamicin sulfate Water R pH 7.0 C - pH 7.0 35-37 °C
sulfate CRS CIP 53.156
ATCC 6538 P
Enterococcus hirae
CIP 58.55
Gramicidin CRS Methanol R pH 7.0* ATCC 10541 C - pH 7.0 35-37 °C
Gramicidin Staphylococcus aureus
ATCC 6538 P
*Addition of a detergent may be necessary to avoid adsorption on the material during the dilutions, for example 0.1 mg/mL
of polysorbate 80 R
General Notices (1) apply to all monographs and other texts 233
2.7.2. Microbiological assay of antibiotics EUROPEAN PHARMACOPOEIA 8.0
Solvent to be used
Buffer solution Micro-organism Medium and final Incubation
Antibiotic Reference substance in preparing the
(pH) pH (± 0.1 pH unit) temperature
stock solution
Staphylococcus aureus
Josamycin Josamycin CRS Methanol R (see the pH 5.6 CIP 53.156
C - pH 8.0 35-37 °C
monograph) ATCC 6538 P
NCTC 7447
Staphylococcus aureus
Josamycin Josamycin Methanol R (see the pH 5.6 CIP 53.156
C - pH 8.0 35-37 °C
propionate propionate CRS monograph) ATCC 6538 P
NCTC 7447
Kanamycin Staphylococcus aureus
monosulfate
Kanamycin NCTC 7447
Water R pH 8.0 C - pH 7.0 35-37 °C
monosulfate CRS CIP 53.156
Kanamycin acid
sulfate ATCC 6538 P
Staphylococcus aureus
Neomycin sulfate NCTC 7447
Neomycin sulfate for microbiological Water R pH 8.0 C - pH 7.0 35-37 °C
assay CRS CIP 53.156
ATCC 6538 P
Escherichia coli
Rifamycin NCIMB 8879
Rifamycin sodium Methanol R pH 7.0 C - pH 7.0 35-37 °C
sodium CRS CIP 54.127
ATCC 10536
Staphylococcus aureus
NCTC 7447
Spiramycin Spiramycin CRS Methanol R pH 7.0 C - pH 7.0 35-37 °C
CIP 53.156
ATCC 6538 P
Klebsiella pneumoniae
Streptomycin NCTC 7427
Streptomycin sulfate Water R pH 8.0 C - pH 7.0 35-37 °C
sulfate CRS CIP 53.153
ATCC 10031
Tylosin for 2.5 per cent V/V Staphylococcus aureus
veterinary use solution of NCTC 6571
Tylosin CRS methanol R in 0.1 M pH 7.0 C - pH 7.0 37 °C
Tylosin tartrate for phosphate buffer ATCC 9144
veterinary use solution pH 7.0 R CIP 53.154
Enterococcus hirae
Tyrothricin Gramicidin CRS Alcohol R Alcohol R C - pH 7.0 37 °C
ATCC 10541
Staphylococcus aureus
Vancomycin Vancomycin
Water R pH 8.0 CIP 53.156 C - pH 7.0 37-39 °C
hydrochloride hydrochloride CRS
ATCC 6538 P
The following section is published for information. Alternatively, spore suspensions may be prepared by
cultivating the organisms in medium C at 26 °C for 4-6 days,
then adding, aseptically, sufficient manganese sulfate R to give
a concentration of 0.001 g/L and incubating for a further
Recommended micro-organisms 48 h. Examine the suspension microscopically to ensure
that adequate spore formation has taken place (about 80 per
cent) and centrifuge. Re-suspend the sediment in sterile
The following text details the recommended micro-organisms water R to give a concentration of 10 × 106 to 100 × 106 spores
and the conditions of use. Other micro-organisms may be per millilitre, and then heat to 70 °C for 30 min. Store the
used provided that they are shown to be sensitive to the suspension at a temperature not exceeding 4 °C.
antibiotic to be examined and are used in appropriate media
and appropriate conditions of temperature and pH. The Bordetella bronchiseptica. Grow the test organism on
concentrations of the solutions used should be chosen so as to medium B at 35-37 °C for 16-18 h. Wash off the bacterial
ensure that a linear relationship exists between the logarithm growth with sterile water R and dilute to a suitable opacity.
of the dose and the response in the conditions of the test.
Staphylococcus aureus ; Klebsiella pneumoniae; Escherichia
Preparation of inocula. Bacillus cereus var. mycoides ; Bacillus coli ; Micrococcus luteus ; Staphylococcus epidermidis. Prepare
subtilis ; Bacillus pumilus. Spore suspensions of the organisms as described above for B. bronchiseptica but using medium A
to be used as inocula are prepared as follows. and adjusting the opacity to one which has been shown to
produce a satisfactory dose-response relationship in the
Grow the organism at 35-37 °C for 7 days on the surface of turbidimetric assay, or to produce clearly defined zones of
a suitable medium to which has been added 0.001 g/L of inhibition of convenient diameter in the diffusion assay, as
manganese sulfate R. Using sterile water R, wash off the growth, appropriate.
which consists mainly of spores. Heat the suspension at 70 °C
for 30 min and dilute to give an appropriate concentration of Saccharomyces cerevisiae ; Candida tropicalis. Grow the test
spores, usually 10 × 106 to 100 × 106 per millilitre. The spore organism on medium F at 30-37 °C for 24 h. Wash off the
suspensions may be stored for long periods at a temperature growth with a sterile 9 g/L solution of sodium chloride R.
not exceeding 4 °C. Dilute to a suitable opacity with the same solution.
Water to 1000 mL
These buffer solutions are used for all microbiological assays
shown in Table 2.7.2.-1 with the exception of bleomycin Medium E
sulfate and amphotericin B. Peptone 5g
For bleomycin sulfate, prepare the buffer solution pH 6.8 as Meat extract 3g
follows : dissolve 6.4 g of potassium dihydrogen phosphate R
and 18.9 g of disodium hydrogen phosphate R in water R and Disodium hydrogen phosphate,12H2O 26.9 g
dilute to 1000 mL with water R. Agar 10 g
For amphotericin B, prepare the 0.2 M phosphate buffer Water to 1000 mL
solution pH 10.5 as follows : dissolve 35 g of dipotassium
hydrogen phosphate R in 900 mL of water R, add 20 mL of 1 M The disodium hydrogen phosphate is added as a sterile
sodium hydroxide and dilute to 1000.0 mL with water R. solution after sterilisation of the medium.
Culture media. The following media or equivalent media Medium F
may be used. Peptone 9.4 g
Agar 15 g
Medium G
Water to 1000 mL Glycerol 10 g
Peptone 10 g
Medium B
Meat extract 10 g
Pancreatic digest of casein 17 g
Sodium chloride 3g
Papaic digest of soya bean 3g
Agar 15 g
Sodium chloride 5g
Water to 1000 mL
Dipotassium hydrogen phosphate 2.5 g
General Notices (1) apply to all monographs and other texts 235
2.7.4. Assay of human coagulation factor VIII EUROPEAN PHARMACOPOEIA 8.0
01/2008:20704 the complete reagent are usually divided into at least 2 separate
reagents, each lacking the ability to generate factor Xa on
its own. One of the reagents contains calcium ions. After
2.7.4. ASSAY OF HUMAN reconstitution, the reagents may be combined provided
COAGULATION FACTOR VIII that no substantial amounts of factor Xa are generated in
the absence of factor VIII. In the final incubation mixture,
Human coagulation factor VIII is assayed by its biological factor VIII must be the only rate-limiting component.
activity as a cofactor in the activation of factor X by activated
factor IX (factor IXa) in the presence of calcium ions and The 2nd step comprises the quantification of the formed
phospholipid. The potency of a factor VIII preparation is factor Xa, employing a chromogenic substrate that is
estimated by comparing the quantity necessary to achieve a specific for factor Xa. Generally this consists of a derivatised
certain rate of factor Xa formation in a test mixture containing short peptide of between 3 and 5 amino acids, joined to a
the substances that take part in the activation of factor X, and chromophore group. On cleavage of this group from the
the quantity of the International Standard, or of a reference peptide substrate, its chromophoric properties shift to a
preparation calibrated in International Units, required to wavelength allowing its spectrophotometric quantification.
produce the same rate of factor Xa formation. The substrate must also contain appropriate inhibitors to
stop further factor Xa generation, e.g. chelating agents, and
The International Unit is the factor VIII activity of a stated to suppress thrombin activity.
amount of the International Standard, which consists of a
freeze-dried human coagulation factor VIII concentrate.
The equivalence in International Units of the International ASSAY PROCEDURE
Standard is stated by the World Health Organization. Reconstitute the entire contents of 1 ampoule of the reference
Human coagulation factor VIII BRP is calibrated in preparation and of the preparation to be examined ; use
International Units by comparison with the International immediately. Add sufficient prediluent to the reconstituted
Standard. preparations to produce solutions containing 0.5-2.0 IU/mL.
The chromogenic assay method consists of 2 consecutive The prediluent consists of haemophilia A plasma, or of an
steps : the factor VIII-dependent activation of factor X in a artificially prepared reagent that contains sufficient von
coagulation-factor reagent composed of purified components, Willebrand factor and that gives results that do not differ
and the enzymatic cleavage of a chromogenic factor Xa significantly from those obtained employing haemophilia
substrate to yield a chromophore that can be quantified plasma. The prediluted materials must be stable beyond the
spectrophotometrically. Under appropriate assay conditions, time required for the assay.
there is a linear relation between the rate of factor Xa
formation and the factor VIII concentration. The assay is Prepare further dilutions of the reference and test preparations
summarised by the following scheme. using a non-chelating, appropriately buffered solution, for
example, tris(hydroxymethyl)aminomethane or imidazole,
containing 1 per cent of human or bovine albumin. Prepare
at least 2 dilution series of at least 3 further dilutions for each
material. Prepare the dilutions such that the final factor VIII
concentration in the reaction mixture is preferably below
0.01 IU/mL, during the step of factor Xa generation.
Prepare a control solution that includes all components except
factor VIII.
Prepare all dilutions in plastic tubes and use immediately.
Step 1. Mix prewarmed dilutions of the factor VIII reference
Both steps employ reagents that may be obtained commercially preparation and of the preparation to be examined with an
from a variety of sources. Although the composition of appropriate volume of the prewarmed coagulation factor
individual reagents may be subject to some variation, their reagent or a combination of its separate constituents, and
essential features are described in the following specification. incubate the mixture in plastic tubes or microplate wells at
Deviations from this description may be permissible provided 37 °C. Allow the activation of factor X to proceed for a suitable
that it has been shown, using the International Standard time, terminating the reaction (step 2) when the factor Xa
for human blood coagulation factor VIII concentrate as the concentration has reached approximately 50 per cent of the
standard, that the results obtained do not differ significantly. maximal (plateau) level. Appropriate activation times are
usually between 2 min and 5 min.
It is important to demonstrate by validation the suitability of
the kit used, notably by checking the time course of factor Xa Step 2. Terminate the activation by addition of a prewarmed
generation in order to determine the time taken to reach reagent containing a chromogenic substrate. Quantify
50 per cent of the maximal factor Xa generation. the rate of substrate cleavage, which must be linear with
the concentration of factor Xa formed, by measuring the
REAGENTS absorbance change at an appropriate wavelength using
a spectrophotometer, either monitoring the absorbance
The coagulation factor reagent comprises purified proteins continuously, thus allowing the initial rate of substrate cleavage
derived from human or bovine sources. These include to be calculated, or terminating the hydrolysis reaction after a
factor X, factor IXa, and a factor VIII activator, usually suitable interval by lowering the pH by addition of a suitable
thrombin. These proteins are partly purified, preferably to at reagent, such as a 50 per cent V/V solution of acetic acid, or a
least 50 per cent, and do not contain impurities that interfere 1 M pH 3 citrate buffer solution. Adjust the hydrolysis time
with the activation of factor VIII or factor X. Thrombin to achieve a linear development of chromophore over time.
may be present in its precursor form prothrombin, provided Appropriate hydrolysis times are usually between 3 min and
that its activation in the reagent is sufficiently rapid to give 15 min, but deviations are permissible if better linearity of the
almost instantaneous activation of factor VIII in the assay. dose-response relationship is thus obtained.
Phospholipid may be obtained from natural sources or be
synthetically prepared, and must, to a substantial extent, Calculate the potency of the test preparation by the usual
consist of the species phosphatidylserine. The components of statistical methods (for example, 5.3).
01/2008:20705 Repeat the procedure using fresh dilutions and carrying out
the incubation in the order T1, T2, T3, S1, S2, S3. Calculate the
results by the usual statistical methods (5.3).
2.7.5. ASSAY OF HEPARIN Carry out not fewer than 3 independent assays. For each such
The anticoagulant activity of heparin is determined in vitro by assay prepare fresh solutions of the reference preparation
comparing its ability in given conditions to delay the clotting and the preparation to be examined and use another, freshly
of recalcified citrated sheep plasma with the same ability of a thawed portion of plasma substrate.
reference preparation of heparin calibrated in International Calculate the potency of the preparation to be examined,
Units. combining the results of these assays, by the usual statistical
The International Unit is the activity contained in a stated methods (5.3). When the variance due to differences between
amount of the International Standard, which consists assays is significant at P = 0.01, a combined estimate of
of a quantity of freeze-dried heparin sodium from pork potency may be obtained by calculating the non-weighted
intestinal mucosa. The equivalence in International Units mean of potency estimates.
of the International Standard is stated by the World Health
Organization. 01/2008:20706
Heparin sodium BRP is calibrated in International Units by corrected 6.0
comparison with the International Standard by means of the
assay given below. 2.7.6. ASSAY OF DIPHTHERIA
Carry out the assay using one of the following methods for VACCINE (ADSORBED)
determining the onset of clotting and using tubes and other
equipment appropriate to the chosen method : The potency of diphtheria vaccine is determined by
administration of the vaccine to guinea-pigs followed either
a) direct visual inspection, preferably using indirect by challenge with diphtheria toxin (method A or B) or by
illumination and viewing against a matt black background ; determination of the titre of antibodies against diphtheria
b) spectrophotometric recording of the change in optical toxin or toxoid in the serum of guinea-pigs (method C).
density at a wavelength of approximately 600 nm ; In both cases, the potency of the vaccine is calculated by
comparison with a reference preparation, calibrated in
c) visual detection of the change in fluidity on manual tilting
International Units.
of the tubes ;
The International Unit is the activity contained in a stated
d) mechanical recording of the change in fluidity on stirring, amount of the International Standard, which consists of
care being taken to cause the minimum disturbance of the a quantity of diphtheria toxoid adsorbed on aluminium
solution during the earliest phase of clotting. hydroxide. The equivalence in International Units of
the International Standard is stated by the World Health
ASSAY PROCEDURE Organization (WHO).
The volumes in the text are given as examples and may be Diphtheria vaccine (adsorbed) BRP is suitable for use as a
adapted to the apparatus used provided that the ratios between reference preparation.
the different volumes are respected. The method chosen for the assay of diphtheria vaccine
Dilute heparin sodium BRP with a 9 g/L solution of (adsorbed) depends on the intended purpose. Method A or
sodium chloride R to contain a precisely known number B is used :
of International Units per millilitre and prepare a similar 1. during development of a vaccine, to assay batches produced
solution of the preparation to be examined which is expected to validate the production ;
to have the same activity. Using a 9 g/L solution of sodium
2. wherever revalidation is needed following a significant
chloride R, prepare from each solution a series of dilutions in
change in the manufacturing process.
geometric progression such that the clotting time obtained
with the lowest concentration is not less than 1.5 times Method A or B may also be used for the routine assay of
the blank recalcification time, and that obtained with the batches of vaccine, but in the interests of animal welfare,
highest concentration is such as to give a satisfactory log method C is used wherever possible.
dose-response curve, as determined in a preliminary test. Method C may be used, except as specified under 1 and 2
Place 12 tubes in a bath of iced water, labelling them in above, after verification of the suitability of the method for
duplicate : T1, T2 and T3 for the dilutions of the preparation the product. For this purpose, a suitable number of batches
to be examined and S1, S2 and S3 for the dilutions of the (usually 3) are assayed by method C and method A or B.
reference preparation. To each tube add 1.0 mL of thawed Where different vaccines (monovalent or combinations) are
plasma substrate R1 and 1.0 mL of the appropriate dilution of prepared from diphtheria toxoid of the same origin, and with
the preparation to be examined or the reference preparation. comparable levels (expressed in Lf/mL) of the same diphtheria
After each addition, mix but do not allow bubbles to form. toxoid, suitability demonstrated for the combination with the
Treating the tubes in the order S1, S2, S3, T1, T2, T3, transfer highest number of components can be assumed to be valid
each tube to a water-bath at 37 °C, allow to equilibrate at 37 °C for combinations with fewer components and for monovalent
for about 15 min and add to each tube 1 mL of a suitable vaccines. Any combinations containing a whole-cell pertussis
APTT (Activated Partial Thromboplastin Time) reagent component or containing haemophilus type b conjugate
containing phospholipid and a contact activator, at a dilution vaccine with diphtheria toxoid or CRM 197 diphtheria protein
giving a suitable blank recalcification time not exceeding as carrier in the same vial must always be assessed separately.
60 s. After exactly 2 min add 1 mL of a 3.7 g/L solution of For combinations containing diphtheria and tetanus
calcium chloride R previously heated to 37 °C and record as components, the serological assay (method C) can be
the clotting time the interval in seconds between this last performed with the same group of animals used for the
addition and the onset of clotting determined by the chosen serological assay of the tetanus vaccine (adsorbed) (2.7.8)
technique. Determine the blank recalcification time at the when the common immunisation conditions for the diphtheria
beginning and at the end of the procedure in a similar manner, and the tetanus components (for example, doses, duration)
using 1 mL of a 9 g/L solution of sodium chloride R in place have been demonstrated to be valid for the combined vaccine.
of one of the heparin dilutions ; the 2 blank values obtained The design of the assays described below uses multiple
should not differ significantly. Transform the clotting times dilutions for the test and reference preparations. Once the
to logarithms, using the mean value for the duplicate tubes. analyst has sufficient experience with this method for a given
General Notices (1) apply to all monographs and other texts 237
2.7.6. Assay of diphtheria vaccine (adsorbed) EUROPEAN PHARMACOPOEIA 8.0
vaccine, it is possible to apply a simplified model such as DETERMINATION OF THE ACTIVITY OF THE
a single dilution for both test and reference preparations. CHALLENGE TOXIN
Such a model enables the analyst to determine whether If necessary, inject the unvaccinated control animals with
the potency of the test preparation is significantly higher dilutions containing 80, 40, 20, 10 and 5 × 10-6 Lf of the
than the minimum required, but does not give information challenge toxin.
on linearity, parallelism and the dose-response curve. The
READING AND INTERPRETATION OF RESULTS
simplified model allows for a considerable reduction in the
number of animals required and must be considered by each Examine all injection sites 48 h after injection of the challenge
analyst in accordance with the provisions of the European toxin and record the incidence of specific diphtheria erythema.
Convention for the Protection of Vertebrate Animals Used for Record also the number of sites free from such reactions as
Experimental and Other Scientific Purposes. the intra-dermal challenge score. Tabulate the intradermal
challenge scores for all the animals receiving the same dilution
Where a single-dilution assay is used, production and test of vaccine and use those data with a suitable transformation,
consistency over time are monitored via suitable indicators such as (score)2 or arcsin ((score/6)2), to obtain an estimate
and by carrying out a full multiple-dilution assay periodically, of the relative potency for each of the test preparations by
for example every 2 years. For serological assays, suitable parallel-line quantitative analysis.
indicators to monitor test consistency are :
– the mean and standard deviation of relative antitoxin REQUIREMENTS FOR A VALID ASSAY
titres or scores of the serum samples obtained after The test is not valid unless :
administration of a fixed dose of the vaccine reference – for both the vaccine to be examined and the reference
preparation ; preparation, the mean score obtained at the lowest dose
– the antitoxin titres or scores of run controls (positive and level is less than 3 and the mean score at the highest dose
negative serum samples) ; level is more than 3 ;
– the ratio of antitoxin titres or scores for the positive – where applicable, the toxin dilution that contains
serum control to the serum samples corresponding to the 40 × 10-6 Lf gives a positive erythema in at least 80 per
reference vaccine. cent of the control guinea-pigs and the dilution containing
20 × 10-6 Lf gives a positive erythema in less than 80 per
METHOD A : INTRADERMAL CHALLENGE TEST IN cent of the guinea-pigs (if these criteria are not met a
GUINEA-PIGS different toxin has to be selected) ;
SELECTION AND DISTRIBUTION OF THE TEST ANIMALS – the confidence limits (P = 0.95) are not less than 50 per cent
Use in the test healthy, white guinea-pigs from the same stock and not more than 200 per cent of the estimated potency ;
and of a size suitable for the prescribed number of challenge – the statistical analysis shows no deviation from linearity
sites, the difference in body mass between the heaviest and parallelism.
and the lightest animal being not greater than 100 g. Use
guinea-pigs of the same sex or with males and females equally The test may be repeated but when more than 1 test is
distributed between the groups. Distribute the guinea-pigs performed the results of all valid tests must be combined in
in not fewer than 6 equal groups ; use groups containing the estimate of potency.
a number of animals sufficient to obtain results that fulfil
the requirements for a valid assay prescribed below. If the METHOD B : LETHAL CHALLENGE TEST IN
challenge toxin to be used has not been shown to be stable or GUINEA-PIGS
has not been adequately standardised, include 5 guinea-pigs SELECTION AND DISTRIBUTION OF THE TEST ANIMALS
as unvaccinated controls. Use in the test healthy guinea-pigs from the same stock, each
SELECTION OF THE CHALLENGE TOXIN weighing 250-350 g. Use guinea-pigs of the same sex or with
Select a preparation of diphtheria toxin containing 67 to males and females equally distributed between the groups.
133 lr/100 in 1 Lf and 25 000 to 50 000 minimal reacting doses Distribute the guinea-pigs in not fewer than 6 equal groups ;
for guinea-pig skin in 1 Lf. If the challenge toxin preparation use groups containing a number of animals sufficient to obtain
has been shown to be stable, it is not necessary to verify the results that fulfil the requirements for a valid assay prescribed
activity for every assay. below. If the challenge toxin to be used has not been shown
to be stable or has not been adequately standardised, include
PREPARATION OF THE CHALLENGE TOXIN SOLUTION
4 further groups of 5 guinea-pigs as unvaccinated controls.
Immediately before use, dilute the challenge toxin with a
suitable diluent to obtain a challenge toxin solution containing SELECTION OF THE CHALLENGE TOXIN
about 0.0512 Lf in 0.2 mL. Prepare from this a further series of Select a preparation of diphtheria toxin containing not less
5 four-fold dilutions containing about 0.0128, 0.0032, 0.0008, than 100 LD50 per millilitre. If the challenge toxin preparation
0.0002 and 0.00005 Lf in 0.2 mL. has been shown to be stable, it is not necessary to verify the
DILUTION OF THE TEST AND REFERENCE lethal dose for every assay.
PREPARATIONS PREPARATION OF THE CHALLENGE TOXIN SOLUTION
Using a 9 g/L solution of sodium chloride R, prepare Immediately before use, dilute the challenge toxin with a
dilutions of the vaccine to be examined and of the reference suitable diluent to obtain a challenge toxin solution containing
preparation, such that for each, the dilutions form a series approximately 100 LD50 per millilitre. If necessary, use
differing by not more than 2.5-fold steps and in which the portions of the challenge toxin solution diluted 1 to 32, 1
intermediate dilutions, when injected subcutaneously at a to 100 and 1 to 320 with the same diluent.
dose of 1.0 mL per guinea-pig, will result in an intradermal DILUTION OF THE TEST AND REFERENCE
score of approximately 3 when the animals are challenged. PREPARATIONS
IMMUNISATION AND CHALLENGE Using a 9 g/L solution of sodium chloride R, prepare dilutions
Allocate the dilutions, 1 to each of the groups of guinea-pigs, of the vaccine to be examined and of the reference preparation,
and inject subcutaneously 1.0 mL of each dilution into each such that for each, the dilutions form a series differing by
guinea-pig in the group to which that dilution is allocated. not more than 2.5-fold steps and in which the intermediate
After 28 days, shave both flanks of each guinea-pig and inject dilutions, when injected subcutaneously at a dose of 1.0 mL
0.2 mL of each of the 6 toxin dilutions intradermally into per guinea-pig, protect approximately 50 per cent of the
6 separate sites on each of the vaccinated guinea-pigs in such animals from the lethal effects of the subcutaneous injection
a way as to minimise interference between adjacent sites. of the quantity of diphtheria toxin prescribed for this test.
General Notices (1) apply to all monographs and other texts 239
2.7.6. Assay of diphtheria vaccine (adsorbed) EUROPEAN PHARMACOPOEIA 8.0
Place 25 μL of medium B in each well except those of potassium dihydrogen phosphate R in water R, and dilute
column 1. Place 25 μL of the diphtheria guinea-pig antiserum to 1000.0 mL with the same solvent. Adjust the pH if
(for vaccines-human use) (positive control serum, working necessary. Autoclave at 120 °C for 15 min.
dilution in medium B of 0.40 IU/mL) in wells A1, A2 and A11. – Thiazolyl blue MTT solution. Dissolve 0.1 g of thiazolyl
Place 25 μL of guinea-pig serum samples in wells B-G of blue MTT in 20 mL of PBS. Sterilise by filtration (0.2 μm)
columns 1, 2 and 11. Place 25 μL of negative control serum and store in dark bottle.
in row H of columns 1, 2 and 11. Using a multichannel – pH adjuster solution. Mix 40 mL of acetic acid R with
micropipette, make twofold serial dilutions across the plate 1.25 mL of 1 M hydrochloric acid and 8.75 mL of water R.
(from column 2 up to column 10 for rows A-G and up
to column 8 for row H). Discard 25 μL from the wells in – Extraction buffer pH 4.7. Dissolve 10 g of sodium
column 10 in rows A-G, and from well H8. laurilsulfate R in water R and add 50 mL of
dimethylformamide R, and dilute to 100 mL with
Reconstitute the diphtheria toxin with saline solution to give water R. Adjust the pH with an appropriate volume of pH
a solution of 50 IU/mL. Prepare a 50-fold dilution of this adjuster solution.
diphtheria toxin dilution in medium B to obtain a working
solution of 1.0 IU/mL. Add 25 μL of this working solution Vero cells are cultured in tissue culture flasks (for example
to wells A12 and B12 (toxin control). Make twofold serial 75 cm2/250 mL) in an incubator at 36 ± 1 °C, 5 per cent CO2
dilutions by tranferring 25 μL from one well to the next, from and 90 per cent relative humidity. Vero cells are grown in
well B12 down to H12. Change the tip between each dilution. the complete culture medium. After 6-7 days of growth, a
Discard 25 μL from well H12. Add 25 μL of medium B to confluent monolayer is obtained, the culture supernatant
wells B12-H12. Then, place 25 μL of the working dilution of is discarded and the cell layer is washed 3 times with
the diphtheria toxin (1.0 IU/mL) in each well of rows A-H, trypsin-EDTA : gently pipette out the medium, add 0.5-1 mL
from column 1-10, except in wells H9 and H10 (cells only, of trypsin-EDTA, swirl the flask and tip the trypsin out. Do
without serum and without toxin). this twice, and the 3rd time, place the flask in the incubator
for 5 min until the cells start to break from the monolayer.
Cover the plates with lids or sealer and shake gently. Incubate Vigorously tap the side of the flask to make the cells fall.
the plates for at least 2 h in a humid container in a CO2 Resuspend the cells in 6-25 mL of fresh complete culture
incubator at 37 °C. Add 200 μL of cell suspension containing medium to obtain a homogeneous suspension. Prepare a
1 x 105 cells/mL to all the wells. Cover the plates with cell suspension in complete culture medium containing
sealer. Incubate at 37 °C for 5 days. Check for microbial approximately 4 × 105 cells/mL.
contamination by microscopic examination.
Place 50 μL of complete culture medium in each well except
Yellow wells are recorded as negative and red wells indicate those of column 1. Place 100 μL of diphtheria guinea pig
dead cells and are recorded as positive. A colour between antiserum (for vaccines-human use) (positive control serum,
yellow and red indicates a mixture of viable and dead cells working dilution in complete culture medium of 0.12 IU/mL)
and is recorded as positive/negative. The results based on the in well A1 and 50 μL in well A11. Place 100 μL of guinea
change in colour can be confirmed by reading viable and dead pig test serum samples, diluted if necessary, in wells B1-G1.
cells under the microscope. Add 50 μL of the same sample to wells B11-G11 in the
The potency of the guinea-pig antiserum samples is obtained corresponding row. Place 100 μL of negative control serum
by comparing the last well of the standard preparation in well H1 and 50 μL in well H11. Using a multi-channel
showing complete neutralisation of the toxin, with the last well micropipette, make twofold serial dilutions by transferring
of the sample demonstrating the same effect. For calculations 50 μL from one well to the next working across the plate (from
of potency, it must be remembered that the endpoint may be column 1-10 for rows A-G and from column 1-8 for row H).
between a negative well and a positive/negative well. Diphtheria toxin of known activity and Lf content is diluted
Method 2 : Thiazolyl blue MTT is reduced to a blue/black to a suitable working stock containing at least 4 minimum
formazan product by the mitochondrial dehydrogenase of cytopathic doses in complete culture medium. Add 50 μL of
viable cells, and thus serves as a quantitative measure of living the diluted toxin to each well except H9 and H10 (cell control),
cells present, indicating when the toxin has been neutralised A11-H11 (serum control) and A12-H12 (toxin control). Add
by the antitoxin. White or colourless wells indicate absence of 100 μL of diluted toxin to well A12 and make twofold serial
viable cells due to insufficient antitoxin to neutralise the toxin. dilutions by transferring 50 μL from one well to the next
working down the plate (from well A12-H12). Discard 50 μL
Reagents and equipment from well H12. Add 50 μL of complete medium to wells H9
– MEM (Minimal Essential Media). and H10.
– Newborn calf serum. Cover the plates with a lid or sealer and leave for 1 h at room
– Antibiotic solution (containing 10 000 units of penicillin, temperature to allow toxin neutralisation to occur. 50 μL of
10 mg of streptomycin and 25 μg of amphotericin B per cell suspension containing approximately 4 × 105 cells/mL
millilitre). is added to each well. The plates are sealed and incubated
at 37 °C for 6 days. Check for microbial contamination by
– L-glutamine 200mM solution. microscopic examination. 10 μL of thyazolyl blue MTT
– Trypsin-EDTA. solution is added to each well. The plates are incubated at
– Thiazolyl blue MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- 37 °C for a further 2-4 h. Then, the medium is removed and
diphenyltetrazolium bromide]. 100 μL of extraction buffer pH 4.7 is added to each well.
The plates are incubated at 37 °C and left overnight to aid
– 1 M HEPES buffer pH 8.1. Dissolve 18.75 g of HEPES in
the extraction process. Once extraction and solubilisation is
82.5 mL of water R and 30.0 mL of 2 M sodium hydroxide R.
complete, plates are visually examined or read at 570 nm.
– Glucose solution (10 per cent). Blue/black wells are recorded as negative (all the cells are alive,
– Complete culture medium. Mix 200 mL of MEM with toxin neutralisation by antitoxin) and white or colourless wells
10 mL of newborn calf serum, 3.0 mL of 1 M HEPES buffer indicate dead cells (no toxin neutralisation) and are recorded
pH 8.1, 2.0 mL of glucose solution (10 per cent), 2.0 mL as positive.
of antibiotic solution and 2.0 mL of L-glutamine 200mM The potency of the test antitoxin is obtained by comparing
solution. the last well of the reference antitoxin preparation showing
– Phosphate-buffered saline pH 7.4 (PBS). Dissolve 10.0 g neutralisation of the toxin, with the last well of the antitoxin
of sodium chloride R, 0.75 g of potassium chloride R, preparation demonstrating the same effect. The neutralising
1.44 g of disodium hydrogen phosphate R, and 0.125 g of antibody titre of the sample being examined can be calculated
General Notices (1) apply to all monographs and other texts 241
2.7.7. Assay of pertussis vaccine (whole cell) EUROPEAN PHARMACOPOEIA 8.0
by multiplication of the dilution factor with total number of potency of the vaccine to be examined relative to the potency
International Units per millilitre of the reference preparation of the reference preparation on the basis of the numbers of
at the end point. The test is valid if all the cells in the toxin animals surviving in each of the groups of not fewer than 16.
control are dead and reference antitoxin gives a neutralisation The test is not valid unless :
in at least the first 2 dilutions tested. – for both the vaccine to be examined and the reference
preparation, the 50 per cent protective dose lies between
the largest and the smallest doses given to the mice ;
07/2011:20707 – the number of animals that die in the 4 groups of 10 injected
with the challenge suspension and its dilutions indicates
2.7.7. ASSAY OF PERTUSSIS VACCINE that the challenge dose is approximately 100 LD50 ; and
(WHOLE CELL) – the statistical analysis shows no deviation from linearity
or parallelism.
The potency of pertussis vaccine (whole cell) is determined The test may be repeated but when more than one test is
by comparing the dose necessary to protect mice against the performed the results of all valid tests must be combined.
effects of a lethal dose of Bordetella pertussis, administered
intracerebrally, with the quantity of a reference preparation,
01/2008:20708
calibrated in International Units, needed to give the same
corrected 6.0
protection.
The International Unit is the activity contained in a stated
amount of the International Standard which consists of
2.7.8. ASSAY OF TETANUS VACCINE
a quantity of dried pertussis vaccine. The equivalence in (ADSORBED)
International Units of the International Standard is stated by
The potency of tetanus vaccine is determined by administration
the World Health Organization.
of the vaccine to animals (guinea-pigs or mice) followed
Selection and distribution of the test animals. Use in the either by challenge with tetanus toxin (method A or B) or
test healthy mice less than 5 weeks old of a suitable strain from by determination of the titre of antibodies against tetanus
the same stock, the difference in mass between the heaviest toxoid in the serum of the guinea-pigs (method C). In both
and the lightest being not greater than 5 g. Distribute the cases, the potency of the vaccine is calculated by comparison
mice in 6 groups of not fewer than 16 and 4 groups of 10. The with a reference vaccine, calibrated in International Units. For
mice must all be of the same sex or the males and females methods A and B, in countries where the paralysis method
distributed equally between the groups. is not obligatory, the LD50 method may be used. For the
Selection of the challenge strain and preparation of the LD50 method, the number of animals and the procedure are
challenge suspension. Select a suitable strain of B. pertussis identical to those described for the paralysis method, but the
capable of causing the death of mice within 14 days of end-point is the death of the animal rather than paralysis.
intracerebral injection. If more than 20 per cent of the mice The International Unit is the activity contained in a stated
die within 48 h of the injection the strain is not suitable. Make amount of the International Standard for tetanus toxoid
one subculture from the strain and suspend the harvested B. (adsorbed). The equivalence in International Units of
pertussis in a solution containing 10 g/L of casein hydrolysate R the International Standard is stated by the World Health
and 6 g/L of sodium chloride R and having a pH of 7.0 to Organization.
7.2 or in another suitable solution. Determine the opacity Tetanus vaccine (adsorbed) BRP is calibrated in International
of the suspension. Prepare a series of dilutions in the same Units with reference to the International Standard.
solution and allocate each dilution to a group of 10 mice. The method chosen for the assay of tetanus vaccine (adsorbed)
Inject intracerebrally into each mouse a dose (0.02 mL or depends on the intended purpose. Method A or B is used :
0.03 mL) of the dilution allocated to its group. After 14 days,
count the number of mice surviving in each group. From 1. during development of a vaccine, to assay batches produced
the results, calculate the expected opacity of a suspension to validate the production ;
containing 100 LD50 in each challenge dose. For the test of 2. wherever revalidation is needed following a significant
the vaccine to be examined make a fresh subculture from the change in the manufacturing process.
same strain of B. pertussis and prepare a suspension of the Method A or B may also be used for the routine assay of
harvested organisms with an opacity corresponding to about batches of vaccine, but in the interests of animal welfare,
100 LD50 in each challenge dose. Prepare 3 dilutions of the method C is used wherever possible.
challenge suspension. Method C may be used, except as specified under 1 and 2
Determination of potency. Prepare 3 serial dilutions of the above, after verification of the suitability of the method for
vaccine to be examined and 3 similar dilutions of the reference the product. For this purpose, a suitable number of batches
preparation such that in each the intermediate dilution (usually 3) are assayed by method C and method A or B.
may be expected to protect about 50 per cent of the mice Where different vaccines (monovalent or combinations) are
from the lethal effects of the challenge dose of B. pertussis. prepared from tetanus toxoid of the same origin and with
Suggested doses are 1/8, 1/40 and 1/200 of the human dose of comparable levels (expressed in Lf/mL) of the same tetanus
the vaccine to be examined and 0.5 IU, 0.1 IU and 0.02 IU toxoid, suitability demonstrated for the combination with the
of the reference preparation, each dose being contained in a highest number of components can be assumed to be valid
volume not exceeding 0.5 mL. Allocate the 6 dilutions, one for combinations with fewer components and for monovalent
to each of the groups of not fewer than 16 mice, and inject vaccines. Any combinations containing a whole-cell pertussis
intraperitoneally into each mouse one dose of the dilution component or containing haemophilus type b conjugate
allocated to its group. After 14 - 17 days inject intracerebrally vaccine with tetanus toxoid in the same vial must always be
into each animal in the groups of not fewer than 16, one assessed separately.
dose of the challenge suspension. Allocate the challenge For combinations containing diphtheria and tetanus
suspension and the 3 dilutions made from it, one to each of components, the serological assay (method C) can be
the groups of 10 mice, and inject intracerebrally one dose of performed with the same group of animals used for the
each suspension into each mouse in the group to which that serological assay of the diphtheria vaccine (adsorbed) (2.7.6)
suspension is allocated. Exclude from consideration any mice when the common immunisation conditions for the tetanus
that die within 48 h of challenge. Count the number of mice and the diphtheria components (for example, doses, duration)
surviving in each of the groups after 14 days. Calculate the have been demonstrated to be valid for the combined vaccine.
The design of the assays described below uses multiple After 28 days, inject subcutaneously into each animal 1.0 mL
dilutions for the test and reference preparations. Based on of the challenge toxin solution (containing 50 times the 50 per
the potency data obtained in multiple-dilution assays, it cent paralytic dose).
may be possible to reduce the number of animals needed to DETERMINATION OF THE ACTIVITY OF THE
obtain a statistically significant result by applying a simplified CHALLENGE TOXIN
model such as a single dilution for both test and reference
If necessary, allocate the 3 dilutions made from the challenge
preparations. Such a model enables the analyst to determine
toxin solution, 1 to each of the 3 groups of 5 guinea-pigs,
whether the potency of the test preparation is significantly
and inject subcutaneously 1.0 mL of each solution into each
higher than the minimum required, but does not give
guinea-pig in the group to which that solution is allocated.
information on the dose-response curves and their linearity,
The activity and stability of the challenge toxin are determined
parallelism and significant slope. The simplified model allows
by carrying out a suitable number of determinations of the
for a considerable reduction in the number of animals required
50 per cent paralytic dose. It is then not necessary to repeat
and must be considered by each analyst in accordance with
the determination for each assay.
the provisions of the European Convention for the Protection
of Vertebrate Animals Used for Experimental and Other READING AND INTERPRETATION OF RESULTS
Scientific Purposes. Examine the guinea-pigs twice daily. Remove and euthanise
Where a single-dilution assay is used, production and test all animals showing definite signs of tetanus paralysis. Count
consistency over time are monitored via suitable indicators the number of guinea-pigs without paralysis 5 days after
and by carrying out a full multiple-dilution assay periodically, injection of the challenge toxin. Calculate the potency of the
for example every 2 years. For serological assays, suitable vaccine to be examined relative to the potency of the reference
indicators to monitor test consistency are : preparation on the basis of the proportion of challenged
animals without paralysis in each group of vaccinated
– the mean and standard deviation of relative antitoxin guinea-pigs, using the usual statistical methods (for example,
titres or scores of the serum samples obtained after 5.3).
administration of a fixed dose of the vaccine reference
preparation ; REQUIREMENTS FOR A VALID ASSAY
The test is not valid unless :
– the antitoxin titres or scores of run controls (positive and
negative serum samples) ; – for both the vaccine to be examined and the reference
preparation, the 50 per cent protective dose lies between
– the ratio of antitoxin titres or scores for the positive
serum control to the serum samples corresponding to the the largest and smallest doses of the preparations given to
the guinea-pigs ;
reference vaccine.
– where applicable, the number of paralysed animals
METHOD A. CHALLENGE TEST IN GUINEA-PIGS in the 3 groups of 5 injected with the dilutions of the
SELECTION AND DISTRIBUTION OF THE TEST ANIMALS challenge toxin solution indicates that the challenge was
approximately 50 times the 50 per cent paralytic dose ;
Use in the test healthy guinea-pigs from the same stock,
each weighing 250-350 g. Use guinea-pigs of the same sex – the confidence limits (P = 0.95) are not less than 50 per cent
or with males and females equally distributed between the and not more than 200 per cent of the estimated potency ;
groups. Distribute the guinea-pigs in not fewer than 6 equal – the statistical analysis shows a significant slope and
groups ; use groups containing a number of animals sufficient no deviation from linearity and parallelism of the
to obtain results that fulfil the requirements for a valid assay dose-response curves (chapter 5.3 describes possible
prescribed below. If the activity of the challenge toxin has to alternatives if significant deviations are observed).
be determined, include 3 further groups of 5 guinea-pigs as The test may be repeated but when more than 1 test is
unvaccinated controls. performed the results of all valid tests must be combined in
SELECTION OF THE CHALLENGE TOXIN the estimate of potency.
Select a preparation of tetanus toxin containing not less than
50 times the 50 per cent paralytic dose per millilitre. If the METHOD B. CHALLENGE TEST IN MICE
challenge toxin preparation has been shown to be stable, it is SELECTION AND DISTRIBUTION OF THE TEST ANIMALS
not necessary to verify the paralytic dose for every assay.
Use in the test healthy mice from the same stock, about
PREPARATION OF THE CHALLENGE TOXIN SOLUTION 5 weeks old and from a strain shown to be suitable. Use mice
Immediately before use, dilute the challenge toxin with a of the same sex or with males and females equally distributed
suitable diluent (for example, peptone buffered saline solution between the groups. Distribute the mice in not fewer than
pH 7.4) to obtain a stable challenge toxin solution containing 6 equal groups ; use groups containing a number of animals
approximately 50 times the 50 per cent paralytic dose per sufficient to obtain results that fulfil the requirements for a
millilitre. If necessary, use portions of the challenge toxin valid assay prescribed below. If the challenge toxin to be used
solution diluted 1 to 16, 1 to 50 and 1 to 160 with the same has not been shown to be stable or has not been adequately
diluent to determine the activity of the toxin. standardised, include 3 further groups of not fewer than
DILUTION OF THE TEST AND REFERENCE 5 mice to serve as unvaccinated controls.
PREPARATIONS SELECTION OF THE CHALLENGE TOXIN
Using a 9 g/L solution of sodium chloride R, prepare dilutions Select a preparation of tetanus toxin containing not less than
of the vaccine to be examined and of the reference preparation, 100 times the 50 per cent paralytic dose per millilitre. If the
such that for each, the dilutions form a series differing by challenge toxin preparation has been shown to be stable, it is
not more than 2.5-fold steps and in which the intermediate not necessary to verify the paralytic dose for every assay.
dilutions, when injected subcutaneously at a dose of 1.0 mL per PREPARATION OF THE CHALLENGE TOXIN SOLUTION
guinea-pig, protect approximately 50 per cent of the animals
Immediately before use, dilute the challenge toxin with a
from the paralytic effects of the subcutaneous injection of the
suitable diluent (for example, peptone buffered saline solution
quantity of tetanus toxin prescribed for this test.
pH 7.4) to obtain a stable challenge toxin solution containing
IMMUNISATION AND CHALLENGE approximately 50 times the 50 per cent paralytic dose in
Allocate the dilutions, 1 to each of the groups of guinea-pigs, 0.5 mL. If necessary, use portions of the challenge toxin
and inject subcutaneously 1.0 mL of each dilution into each solution diluted 1 to 16, 1 to 50 and 1 to 160 with the same
guinea-pig in the group to which that dilution is allocated. diluent to determine the activity of the toxin.
General Notices (1) apply to all monographs and other texts 243
2.7.8. Assay of tetanus vaccine (adsorbed) EUROPEAN PHARMACOPOEIA 8.0
– T3 : paralysis of 1 forelimb. The animal moves reluctantly, phosphate dihydrate R and 2.0 g of potassium chloride R in
the body is often slightly banana-shaped owing to scoliosis ; 1000 mL of water R. Store at room temperature to prevent
– T4 : the forelimb is completely stiff and the toes are crystallisation. Dilute to 10 times its volume with water R
immovable. The muscular contraction of the forelimb is before use.
very pronounced and usually scoliosis is observed ; – Citric acid solution. Dissolve 10.51 g of citric acid R in
– T5 : tetanus seizures, continuous tonic spasm of muscles ; 1000 mL of water R and adjust the solution to pH 4.0 with
– D : death. a 400 g/L solution of sodium hydroxide R.
– Washing buffer. PBS containing 0.5 g/L of polysorbate 20 R.
METHOD B. CHALLENGE TEST IN MICE
– Diluent block buffer. PBS containing 0.5 g/L of
READING AND INTERPRETATION OF RESULTS polysorbate 20 R and 25 g/L of dried skimmed milk.
In order to minimise suffering in the test animals, it is – Peroxidase substrate. Shortly before use, dissolve 10 mg
recommended to note the degree of paralysis on a scale such as of diammonium 2,2′-azinobis(3-ethylbenzothiazoline-
that shown below. The scale gives typical signs when injection 6-sulfonate) R (ABTS) in 20 mL of citric acid solution.
of the challenge toxin is made in the dorsal region, close to Immediately before use add 5 μL of strong hydrogen
one of the hind legs. Grade T3 is taken as the end-point, but peroxide solution R.
with experience grade T2 can be used instead. Tetanus toxin
produces in the toxin-injected hind leg paresis followed by Method
paralysis that can be recognised at an early stage. The tetanus The description below is given as an example of a suitable
grades in mice are characterised by the following signs : plate layout but others may be used. Wells 1A-H are for
– T1 : slight stiffness of toxin-injected hind leg, only observed negative control serum and wells 2A-H and 12A-H are for
when the mouse is lifted by the tail ; positive control serum for assay monitoring. Wells 3-11A-H
are for test samples.
– T2 : paresis of the toxin-injected hind leg, which still can
function for walking ; Coat each well of the ELISA plates with 100 μL of tetanus
– T3 : paralysis of the toxin-injected hind leg, which does not toxoid solution (0.5 Lf/mL in carbonate coating buffer pH 9.6).
function for walking ; Allow to stand overnight at 4 °C in a humid atmosphere. To
avoid temperature gradient effects, do not stack more than
– T4 : the toxin-injected hind leg is completely stiff with 4 plates high. On the following day, wash the plates thoroughly
immovable toes ; with washing buffer. Block the plates by addition of 100 μL
– T5 : tetanus seizures, continuous tonic spasm of muscles ; of diluent block buffer to each well. Incubate in a humid
– D : death. atmosphere at 37 °C for 1 h. Wash the plates thoroughly
with washing buffer. Place 100 μL of diluent block buffer
METHOD C. DETERMINATION OF ANTIBODIES IN in each well of the plates, except those of row A. Prepare
GUINEA-PIGS suitable dilutions of negative control serum, positive control
PREPARATION OF SERUM SAMPLES serum (from about 0.01 IU/mL) and test sera. Allocate the
For the preparation of serum samples, the following technique negative control serum to column 1, positive control serum
has been found to be suitable. Invert the tubes containing to columns 2 and 12 and test sera to columns 3-11 and add
blood samples 6 times and allow to stand at 37 °C for 2 h, 100 μL of each serum to the first 2 wells of the column to
then at 4 °C for 2 h. Centrifuge at room temperature at 800 g which it is allocated. Using a multichannel micropipette, make
for 20 min. Transfer the serum to sterile tubes and store at twofold serial dilutions from row B down the plate to row H,
a temperature below − 20 °C. At least a 40 per cent yield of by transferring 100 μL from one well to the next. Discard
serum is obtained by this procedure. 100 μL from the last row so that all wells contain 100 μL.
DETERMINATION OF ANTIBODY TITRE Incubate at 37 °C for 2 h. Wash thoroughly with washing
buffer. Prepare a suitable dilution (a 2000-fold dilution has
The ELISA and ToBI tests shown below are given as examples
been found to be suitable) of peroxidase conjugate in diluent
of immunochemical methods that have been found to be
block buffer and add 100 μL to each well. Incubate at 37 °C in
suitable for the determination of antibody titre.
a humid atmosphere for 1 h. Wash the plates thoroughly with
Determination of antibody titre in guinea-pig serum by washing buffer. Add 100 μL of peroxidase substrate to each
enzyme-linked immunosorbent assay (ELISA). Dilutions of well. Allow to stand at room temperature, protected from
test and reference sera are made on ELISA plates coated with light, for 30 min. Read the plates at 405 nm in the same order
tetanus toxoid. A positive guinea-pig serum control and a as addition of substrate was made.
negative guinea-pig serum control are included on each plate
Determination of antibody titre in guinea-pig serum by
to monitor the assay performance. Peroxidase-conjugated
toxin- or toxoid-binding inhibition (ToBI). Tetanus toxin
rabbit or goat antibody directed against guinea-pig-IgG is
or toxoid is added to serial dilutions of test and reference
added, followed by a peroxidase substrate. Optical density is
sera ; the serum/antigen mixtures are incubated overnight.
measured and the relative antibody titre is calculated using the
To determine unbound toxin or toxoid, the mixtures are
usual statistical methods (for example, 5.3).
transferred to an ELISA plate coated with tetanus antitoxin.
Reagents and equipment Peroxidase-conjugated equine anti-tetanus IgG is added
– ELISA plates : 96 wells, columns 1-12, rows A-H. followed by a peroxidase substrate. Optical density is
– Clostridium tetani guinea-pig antiserum (for measured and the antibody titre is calculated using the usual
vaccines-human use) BRP (positive control serum). statistical methods (for example, 5.3). A positive control
– Peroxidase conjugate. Peroxidase-conjugated rabbit or goat serum and a negative control serum are included on each plate
antibody directed against guinea-pig IgG. to monitor assay performance.
– Tetanus toxoid. Reagents and equipment
– Carbonate coating buffer pH 9.6. Dissolve 1.59 g of – Round-bottomed, rigid polystyrene microtitre plates.
anhydrous sodium carbonate R and 2.93 g of sodium – Flat-bottomed ELISA plates.
hydrogen carbonate R in 1000 mL of water R. Distribute – Tetanus toxin or tetanus toxoid.
into 150 mL bottles and sterilise by autoclaving at 121 °C
for 15 min. – Clostridium tetani guinea-pig antiserum (for
vaccines-human use) BRP (positive control serum).
– Phosphate-buffered saline pH 7.4 (PBS). Dissolve with
stirring 80.0 g of sodium chloride R, 2.0 g of potassium – Equine anti-tetanus IgG.
dihydrogen phosphate R, 14.3 g of disodium hydrogen – Peroxidase-conjugated equine anti-tetanus IgG.
General Notices (1) apply to all monographs and other texts 245
2.7.9. Test for Fc function of immunoglobulin EUROPEAN PHARMACOPOEIA 8.0
using albumin barbital buffer solution, thereby obtaining the largest value for S serves as Sexp. In addition, determine the
final adjusted volume Vf = Vr × A of sensitised human red absorbance at the start of measurement (As) by extrapolating
blood cells and adjusting A to 1.0 ± 0.1 for a tenfold dilution. the curve, which is almost linear and parallel to the time axis
Antibody binding of antigen-coated tanned human red within the first few minutes. Correct Sexp using the expression :
blood cells. Prepare the following solutions in succession and
in duplicate, using for each solution a separate half-micro
cuvette (for example, disposable type) or test-tube.
(1) Test solutions. If necessary, adjust the immunoglobulin to Calculate the arithmetic mean of the values of S′ for each
be examined to pH 7. preparation (test and reference solution). Calculate the index
of Fc function (IFc) from the expression :
Where method A is performed, dilute volumes of the
preparation to be examined with albumin barbital buffer to
obtain 30 mg and 40 mg of immunoglobulin and adjust the
volume to 900 μL with albumin barbital buffer.
Where method B is performed, dilute volumes of the = arithmetic mean of the corrected slope for the
preparation to be examined with albumin barbital buffer to preparation to be examined ;
obtain 15 mg and 30 mg of immunoglobulin and adjust the
volume to 1200 μL with albumin barbital buffer. = arithmetic mean of the corrected slope for the
reference preparation ;
(2) Reference solutions. Prepare as for the test solutions using
human immunoglobulin (Fc function and molecular size) BRP. = arithmetic mean of the corrected slope for the
(3) Complement control. Albumin barbital buffer solution. complement control.
Where method A is performed, add to each cuvette/test-tube Calculate the index of Fc function for the preparation to be
100 μL of sensitised human red blood cells and mix well. examined : the value is not less than that stated in the leaflet
Allow to stand for 15 min, add 1000 μL of albumin barbital accompanying the reference preparation.
buffer solution, collect the cells by centrifugation (1000 g for
10 min) of the cuvette/test-tube and remove 1900 μL of the
supernatant. Replace the 1900 μL with albumin barbital buffer
solution and repeat the whole of the washing procedure, 01/2008:20710
finally leaving a volume of 200 μL. Test samples may be stored
in sealed cuvettes/test-tubes at 4 °C for not longer than 24 h. 2.7.10. ASSAY OF HUMAN
Where method B is performed, add to each test-tube
300 μL of sensitised human red blood cells and mix well
COAGULATION FACTOR VII
(the final immunoglobulin concentration is in the range Human coagulation factor VII is assayed by its biological
of 10-20 mg/mL). Allow to stand for 15 min, add 1500 μL activity as a factor VIIa-tissue factor complex in the activation
of albumin barbital buffer solution and stir gently until of factor X in the presence of calcium ions and phospholipids.
homogeneous. Collect the cells by centrifugation (1000 g The potency of a factor VII preparation is estimated by
for 10 min) of the test-tube, remove the supernatant and comparing the quantity necessary to achieve a certain rate of
add approximately 3 mL of albumin barbital buffer solution. factor Xa formation in a test mixture containing the substances
Repeat this operation up to 4 times in total, leaving a final that take part in the activation of factor X, and the quantity
volume of 300 μL. Test samples may be stored in sealed of the International Standard, or of a reference preparation
test-tubes at 4 °C for not longer than 24 h. calibrated in International Units, required to produce the
Complement-initiated haemolysis. same rate of factor Xa formation.
To measure haemolysis where method A is performed, add The International Unit is the factor VII activity of a stated
600 μL of albumin barbital buffer solution warmed to 37 °C amount of the International Standard, which consists of
to the test sample, resuspend the cells carefully by repeated freeze-dried plasma. The equivalence in International Units
pipetting (not fewer than 5 times) and place the cuvette in of the International Standard is stated by the World Health
the thermostatted cuvette holder of a spectrophotometer. Organization.
After 2 min, add 200 μL of diluted guinea-pig complement Human coagulation factor VII concentrate BRP is calibrated
(125-200 CH50/mL), mix thoroughly by pipetting twice in International Units by comparison with the International
and start immediately after the second pipetting the Standard.
time-dependent recording of absorbance at 541 nm, using
The chromogenic assay method consists of 2 consecutive
albumin barbital buffer solution as the compensation liquid.
steps : the factor VII-dependent activation of factor X reagent
Stop the measurement if absorbance as a function of time has
clearly passed the inflexion point. mixture containing tissue factor, phospholipids and calcium
ions, followed by enzymatic cleavage of a chromogenic
To measure haemolysis where method B is performed, add factor Xa substrate into a chromophore that can be quantified
900 μL of albumin barbital buffer solution warmed to 37 °C spectrophotometrically. Under appropriate assay conditions,
to each test-tube and resuspend the cells carefully by repeated there is a linear relation between the rate of factor Xa
pipetting (not fewer than 5 times). The microtitre plate must formation and the factor VII concentration. The assay is
be prewarmed to 37 °C before starting the test. Transfer 240 μL summarised in the following scheme.
of each solution into 4 microtitre plate wells then incubate
the microplate at 37 °C for 6 min, stirring gently every 10 s.
To each microtitre plate well add 60 μL of diluted guinea-pig
complement (150 CH50/mL). Mix for 10 s and immediately
start recording the absorbance at 541 nm at 37 °C, measuring
every 20 s. Stop the measurement if the absorbance as a
function of time has clearly passed the inflexion point.
Evaluation. For each cuvette/test-tube/well, determine the
slope (S) of the haemolysis curve at the approximate inflexion
point by segmenting the steepest section in suitable time
intervals (for example, Δt = 1 min), and calculate S between
adjacent intersection points, expressed as ΔA per minute. The
General Notices (1) apply to all monographs and other texts 247
EUROPEAN PHARMACOPOEIA 8.0
Both steps employ reagents that may be obtained commercially . Terminate the activation by the addition of a
from a variety of sources. Although the composition of prewarmed reagent containing a chromogenic substrate.
individual reagents may be subject to some variation, their Quantify the rate of substrate cleavage, which must be linear
essential features are described in the following specification. with the concentration of factor Xa formed, by measuring
the absorbance change at an appropriate wavelength using
REAGENTS a spectrophotometer, either monitoring the absorbance
The coagulation factor reagent comprises purified proteins continuously, thus allowing the initial rate of substrate cleavage
derived from human or bovine sources. These include to be calculated, or terminating the hydrolysis reaction after
factor X and thromboplastin tissue factor/phospholipid as a suitable interval by lowering the pH by the addition of a
factor VII activator. These proteins are partly purified and suitable reagent, such as acetic acid (500 g/L C2H4O2) or a
do not contain impurities that interfere with the activation citrate solution (1 mol/L) at pH 3. Adjust the hydrolysis time
of factor VII or factor X. Factor X is present in amounts to achieve a linear development of chromophore with time.
giving a final concentration during the first step of the assay Appropriate hydrolysis times are usually between 3 min and
of 10-350 nmol/L, preferably 14-70 nmol/L. Thromboplastin 15 min, but deviations are permissible if better linearity of the
from natural sources (bovine or rabbit brain) or synthetic dose-response relationship is thus obtained.
preparations may be used as the tissue factor/phospholipid Check the validity of the assay and calculate the potency of the
component. Thromboplastin suitable for use in prothrombin test preparation by the usual statistical methods (for example,
time determination is diluted 1:5 to 1:50 in buffer such that 5.3).
the final concentration of Ca2+ is 15-25 mmol/L. The final
factor Xa generation is performed in a solution containing
human or bovine albumin at a concentration such that
adsorption losses do not occur and which is appropriately
buffered at pH 7.3-8.0. In the final incubation mixture,
factor VII must be the only rate-limiting component and each
reagent component must lack the ability to generate factor Xa
on its own.
The second step comprises the quantification of the formed The principle of the assay is to measure the ability of a
factor Xa employing a chromogenic substrate that is specific factor IX preparation to reduce the prolonged coagulation
for factor Xa. Generally this consists of a short peptide of time of factor IX-deficient plasma. The reaction is accelerated
between three and five amino acids, bound to a chromophore by addition of a reagent containing phospholipid and a contact
group. On cleavage of this group from the peptide substrate, activator, e.g. kaolin, silica or ellagic acid. The potency
its absorption maximum shifts to a wavelength allowing its is assessed by comparing the dose-response curve of the
spectrophotometric quantification. The substrate is usually preparation to be examined to that of a reference preparation,
dissolved in water R and used at a final concentration of calibrated in International Units.
0.2-2 mmol/L. The substrate may also contain appropriate The International Unit is the factor IX activity of a stated
inhibitors to stop further factor Xa generation (addition of amount of the International Standard, which consists of a
edetate). freeze-dried concentrate of human coagulation factor IX.
The equivalence in International Units of the International
ASSAY PROCEDURE Standard is stated by the World Health Organization.
Reconstitute the entire contents of one ampoule of the Human coagulation factor IX concentrate BRP is calibrated
reference preparation and the preparation to be examined by in International Units by comparison with the International
adding the appropriate quantity of water R ; use within 1 h. Standard.
Add sufficient prediluent to the reconstituted preparations to Reconstitute separately the preparation to be examined
produce solutions containing between 0.5 IU and 2.0 IU of and the reference preparation as stated on the label and
factor VII per millilitre. use immediately. Where applicable, determine the amount
Prepare further dilutions of reference and test preparations of heparin present (2.7.12) and neutralise the heparin,
using an isotonic non-chelating buffer containing 1 per cent for example by addition of protamine sulfate R (10 μg of
of bovine or human albumin, buffered preferably between protamine sulfate neutralises 1 IU of heparin). Predilute the
pH 7.3 and 8.0. Prepare at least three separate, independent preparation to be examined and the reference preparation in
dilutions for each material, preferably in duplicate. Prepare factor IX-deficient plasma (for example plasma substrate R2)
the dilutions such that the final factor VII concentration is to produce solutions containing 0.5-2.0 IU/mL. Prepare at
below 0.005 IU/mL. least 3 dilutions for each material, preferably in duplicate,
Prepare a control solution that includes all components except using a suitable buffer solution (for example imidazole buffer
factor VII. solution pH 7.3 R) containing 10 g/L of bovine or human
albumin. Use these dilutions immediately.
Prepare all dilutions in plastic tubes and use within 1 h.
Use an apparatus suitable for measurement of coagulation
. Mix dilutions of the factor VII reference preparation times or carry out the assay with incubation tubes maintained
and the preparation to be examined with an appropriate in a water-bath at 37 °C. Place in each tube 0.1 mL of
volume of the prewarmed coagulation factor reagent or a factor IX-deficient plasma (for example plasma substrate R2)
combination of its separate constituents, and incubate the and 0.1 mL of one of the dilutions of the reference preparation
mixture in plastic tubes or microplate wells at 37 °C. The or of the preparation to be examined. Add to each tube 0.1 mL
concentrations of the various components during the factor Xa of a suitable Activated Partial Thromboplastin Time (APTT)
generation must be as specified above under the description of reagent containing phospholipid and contact activator and
the reagents. incubate the mixture for a recommended time at 37 °C.
Allow the activation of factor X to proceed for a suitable To each tube, add 0.1 mL of a 3.7 g/L solution of calcium
time, usually terminating the reaction before the factor Xa chloride R previously heated to 37 °C. Using a timer, measure
concentration has reached its maximal level in order to obtain the coagulation time, i.e. the interval between the moment of
a satisfactory linear dose-response relationship. The activation the addition of the calcium chloride and the first indication
time is also chosen to achieve linear production of factor Xa of the formation of fibrin. The volumes given above may be
in time. Appropriate activation times are usually between adapted to the APTT reagent and apparatus used. Calculate
2 min and 5 min, but deviations are permissible if acceptable the potency using the usual statistical methods (for example,
linearity of the dose-response relationship is thus obtained. 5.3).
01/2008:20712 01/2008:20713
corrected 7.5
General Notices (1) apply to all monographs and other texts 249
2.7.13. Assay of human anti-D immunoglobulin EUROPEAN PHARMACOPOEIA 8.0
MATERIALS 01/2011:20714
Reagents not specified are of analytical grade.
PBS. Dissolve 8.0 g of sodium chloride R, 0.76 g of disodium 2.7.14. ASSAY OF HEPATITIS A
hydrogen phosphate R, 0.2 g of potassium chloride R and 0.2 g
of potassium dihydrogen phosphate R in water R and dilute to VACCINE
1000 mL with the same solvent.
PBS-BSA solution. PBS containing 10.0 g/L of bovine The assay of hepatitis A vaccine is carried out either in vivo, by
albumin R. comparing in given conditions its capacity to induce specific
antibodies in mice with the same capacity of a reference
Red blood cells. Use D-positive red blood cells obtained from preparation, or in vitro, by an immunochemical determination
a group O R1R1 donor within 2 weeks of collection. Store if of antigen content.
necessary in an appropriate stabiliser at 4 °C. Wash the cells at
least twice with PBS-BSA solution and prepare a suspension
containing 1 × 104 cells per microlitre but not more than IN VIVO ASSAY
5 × 104 cells per microlitre in PBS-BSA solution. The test in mice shown below is given as an example of a
Use D-negative red blood cells obtained from a group O rr method that has been found suitable for a given vaccine ; other
donor and prepared similarly. validated methods may also be used.
Secondary antibody. Use a suitable fluorescent dye-conjugated Selection and distribution of the test animals. Use in the
anti-IgG antibody fragment specific for human IgG or parts of test healthy mice from the same stock, about 5 weeks old and
it. Store and use according to the manufacturer’s instructions. from a strain shown to be suitable. Use animals of the same
sex. Distribute the animals in at least 7 equal groups of a
Microtitres plates. Use flat-bottomed plates without surface
number suitable for the requirements of the assay.
treatment for enzyme immunoassays.
METHOD Determination of potency of the vaccine to be examined.
Using a 9 g/L solution of sodium chloride R containing the
Test solutions. For freeze-dried preparations, reconstitute as aluminium adjuvant used for the vaccine, prepare at least
stated on the label. Prepare at least 3 independent replicates 3 dilutions of the vaccine to be examined and matching
of at least 3 serial 1.5- or 2-fold dilutions starting with a dilutions of the reference preparation. Allocate the dilutions
concentration in the range of 1.2-0.15 IU/mL using PBS/BSA one to each of the groups of animals and inject subcutaneously
solution as diluent. If necessary, adjust the starting dilution not more than 1.0 mL of each dilution into each animal in the
to obtain responses falling in the linear portion of the group to which that dilution is allocated. Maintain a group of
dose-response curve. unvaccinated controls, injected subcutaneously with the same
Reference solutions. Reconstitute the reference preparation volume of diluent. After 28 to 32 days, anaesthetise and bleed
according to instructions. Prepare at least 3 independent all animals, keeping the individual sera separate. Assay the
replicates of at least 3 serial 1.5- or 2-fold dilutions starting individual sera for specific antibodies against hepatitis A virus
with a concentration in the range of 1.2-0.15 IU/mL using by a suitable immunochemical method (2.7.1).
PBS-BSA solution as diluent. If necessary, adjust the starting Calculations. Carry out the calculations by the usual
dilution to obtain responses falling in the linear portion of statistical methods for an assay with a quantal response (5.3).
the dose-response curve.
Distribute 50 μL of the D-positive red blood cells into each well From the distribution of reaction levels measured on all the
of a microtitre plate. Add 50 μL of each of the dilutions of the sera in the unvaccinated group, determine the maximum
test solution or reference solution to each of a series of wells. reaction level that can be expected to occur in an unvaccinated
Use 50 μL of PBS-BSA solution as negative control. Distribute animal for that particular assay. Any response in vaccinated
50 μL of the D-negative red blood cells into 4 wells of the animals that exceeds this level is by definition a seroconversion.
same microtitre plate and add 50 μL of the lowest dilution of Make a suitable transformation of the percentage of animals
the test preparation. To monitor spurious reactions, distribute showing seroconversion in each group (for example, a
50 μL of the D-positive red blood cells into 4 wells of the same probit transformation) and analyse the data according to a
microtitre plate and add 50 μL of PBS-BSA solution. Seal with parallel-line log dose-response model. Determine the potency
plastic film and incubate at 37 °C for 40 min. of the test preparation relative to the reference preparation.
Centrifuge the plates at 50 g for 3 min, discard the supernatant Validity conditions. The test is not valid unless :
and wash the cells with 200-250 μL of PBS-BSA solution.
Repeat this at least once. – for both the test and the reference vaccine, the ED50 lies
between the smallest and the largest doses given to the
Centrifuge the plates at 50 g for 3 min, discard the supernatant animals ;
and add 50 μL of the secondary antibody diluted with
PBS-BSA solution to a suitable protein concentration. Seal – the statistical analysis shows no significant deviation from
with plastic film and incubate, protected from light, at room linearity or parallelism ;
temperature for 20 min.
– the confidence limits (P = 0.95) are not less than 33 per cent
Centrifuge the plates at 50 g for 3 min, discard the supernatant and not more than 300 per cent of the estimated potency.
and wash the cells with 200-250 μL of PBS-BSA solution.
Repeat this at least once. Potency requirement. The upper confidence limit (P = 0.95)
of the estimated relative potency is not less than 1.0.
Centrifuge the plates at 50 g for 3 min, resuspend the cells
into 200-250 μL of PBS. Transfer the cell suspension into a
tube suitable for the flow-cytometry equipment available and IN VITRO ASSAY
further dilute by adding PBS to allow a suitable flow rate. Carry out an immunochemical determination (2.7.1) of
Proceed immediately with measurement of the median antigen content with acceptance criteria validated against the
fluorescence intensity in a flow cytometer. Record at least in vivo test. The acceptance criteria are approved for a given
10 000 events without gating but excluding debris. reference preparation by the competent authority in the light
of the validation data.
Use the median fluorescence intensity in the linear range of the
dose-response curve to estimate the potency of the preparation Hepatitis A vaccine (inactivated, non-adsorbed) BRP is suitable
to be examined by the usual statistical methods (5.3). for use as a reference preparation.
General Notices (1) apply to all monographs and other texts 251
2.7.15. Assay of hepatitis B vaccine (rDNA) EUROPEAN PHARMACOPOEIA 8.0
Reference antiserum. A reference antiserum of assigned Reference antiserum. An in-house guinea-pig reference
activity is used in the test and serves as the basis for calculation antiserum of assigned activity is used in the test and serves as
of the antibody levels in the test sera. Bordetella pertussis the basis for calculation of the antibody levels in the test sera.
mouse antiserum BRP is suitable for use as a reference Dilution of the test and reference preparations. Using a
antiserum. 9 g/L solution of sodium chloride R as diluent, prepare serial
The following test model is given as an example of a method dilutions of the vaccine to be examined and the reference
that has been found to be satisfactory. preparation ; series differing by 2.5- to 5-fold steps have been
Selection and distribution of the test animals. Use in the found to be suitable. Use not fewer than 3 dilutions within
test healthy mice (for example, CD1 strain) of the same stock, the range found to be suitable for all the components in the
about 5 weeks old. Distribute the animals in 6 groups of a vaccine to be examined. Use the dilutions for immunisation
number appropriate to the requirements of the assay. Use preferably within 1 h of preparation. Allocate 1 dilution to
3 dilutions of the vaccine to be examined and 3 dilutions of a each group of guinea-pigs.
reference preparation and attribute each dilution to a group Immunisation. Inject subcutaneously into each guinea-pig
of mice. Inject intraperitoneally or subcutaneously into each 1.0 mL of the dilution allocated to its group.
mouse 0.5 mL of the dilution attributed to its group. During
Blood sampling. 35-42 days (5-6 weeks) after immunisation,
validation studies a further group of mice may be used as a
take a blood sample from each vaccinated and negative
negative control by injecting the animals with diluent alone.
control guinea-pig using a suitable method. Store the sera at
Collection of serum samples. 4-5 weeks after vaccination, − 20 °C until used for antibody determination. Avoid frequent
bleed the mice individually under anaesthesia. Store the sera freezing and thawing of serum samples.
at − 20 °C until used for antibody determination.
Antibody determination. Assay the individual sera for
Antibody determination. Assay the individual sera for content of specific antibodies to each acellular pertussis
content of specific antibodies to each acellular pertussis antigen using a validated method such as the ELISA test
antigen using a validated method such as the ELISA test shown below.
shown below.
ELISA test. Suitable 96-well microtitre plates are coated with
ELISA test. Microtitre plates (poly(vinyl chloride) or the purified antigens (e.g. pertussis toxin (PT), pertactin
polystyrene as appropriate for the specific antigen) are coated (PRN), filamentous haemagglutinin (FHA) and/or fimbrial
with the purified antigen at a concentration of 100 ng per agglutinogens (Fim 2/3)) representing components in the
well. After washing, unreacted sites are blocked by incubating combined vaccine at a concentration of 200-400 ng per well.
the plates with a solution of bovine serum albumin and After washing, unreacted sites are blocked by incubating the
then washed. 2-fold dilutions of sera from individual mice plates with a suitable blocking buffer and then washed. 2-fold
immunised with test or reference vaccines are made on dilutions of sera from individual guinea-pigs immunised with
the plates. Reference antiserum is included on each plate. test or reference vaccines are made on the plates. Reference
After incubation at 22-25 °C for 1 h, the plates are washed. antiserum is included on each plate. After incubation at
A suitable solution of enzyme-conjugated anti-mouse IgG 37 °C for 1 h, the plates are washed. A suitable solution of
antibody is added to each well and incubated at 22-25 °C for enzyme-conjugated anti-guinea-pig IgG antibody is added
1 h. After washing, a chromogenic substrate is added from to each well and incubated at 37 °C for 1 h. After washing, a
which the bound enzyme conjugate liberates a chromophore chromogenic substrate is added from which the bound enzyme
that can be quantified by measurement of absorbance (2.2.25). conjugate liberates a chromophore that can be quantified by
Calculations. The antibody titres in the sera of mice measurement of absorbance (2.2.25).
immunised with reference and test vaccines are calculated for Calculations. The antibody titres in the sera of guinea-pigs
each acellular pertussis antigen using the reference antiserum, immunised with reference and test vaccines are calculated for
and from the values obtained the relative potency of the test each acellular pertussis antigen using the reference antiserum,
vaccine in relation to the reference vaccine is calculated by the and from the values obtained the relative potency of the test
usual statistical methods (5.3). vaccine in relation to the reference vaccine is calculated by the
The assay is not valid unless : usual statistical methods (5.3).
– the confidence limits (P = 0.95) are not less than 50 per The assay is not valid unless :
cent and not more than 200 per cent of the relative potency – the confidence limits (P = 0.95) are not less than 50 per
estimate for each acellular pertussis antigen ; cent and not more than 200 per cent of the relative potency
– the statistical analysis shows a significant slope and estimate for each acellular pertussis antigen ;
no deviation from linearity and parallelism of the – the statistical analysis shows a significant slope and
dose-response curves (chapter 5.3 describes possible no deviation from linearity and parallelism of the
alternatives if significant deviations are observed). dose-response curves (chapter 5.3 describes possible
alternatives if significant deviations are observed).
METHOD B. SEROLOGY IN GUINEA-PIGS
Selection and distribution of the test animals. Use in the The following section is published for information.
test healthy guinea-pigs from the same stock, each weighing
250-350 g. Use guinea-pigs of the same sex or with males and
females equally distributed between the groups. Distribute Assay of pertussis vaccine (acellular) :
the guinea-pigs in not fewer than 6 equal groups ; use groups guidelines
containing a number of animals sufficient to obtain results
that fulfil the requirements for a valid assay prescribed below. METHOD B. DETERMINATION OF ANTIBODIES IN
During validation studies a further group of guinea-pigs GUINEA-PIGS
is used as a negative control by injecting the animals with The ELISA shown below is given as an example of an
diluent alone. immunochemical method that has been found to be suitable.
Reference vaccine. A batch of vaccine shown to be effective Determination of antibody titre by ELISA method for
in clinical trials or a batch representative thereof is used as pertussis toxin (PT), filamentous haemagglutinin (FHA),
a reference vaccine. For the preparation of a representative fimbrial agglutinogens (Fim 2/3) and pertactin (PRN).
batch, strict adherence to the production process used for the 2-fold dilutions of sera from test and reference vaccines are
batch tested in clinical trials is necessary. The stability of the made on ELISA plates coated with acellular pertussis antigens
reference vaccine shall be monitored and documented. (PRN, PT, FHA or Fim 2/3). A guinea-pig reference antiserum
General Notices (1) apply to all monographs and other texts 253
2.7.17. Assay of human antithrombin III EUROPEAN PHARMACOPOEIA 8.0
REAGENTS
Viper venom specific factor II activator
Echis
carinatus
METHOD
Test solution
Dilution buffer
tris(hydroxymethyl)aminomethane R sodium
chloride R imidazole R hexadimethrine
Reference solution bromide R bovine albumin R human albumin R
hydrochloric acid R
Test solution
Reference solution
2.2.25
A
A
2.2.25
5.3
A
A
01/2008:20719 A
01/2008:20720
in vivo
General Notices (1) apply to all monographs and other texts 257
2.7.22. Assay of human coagulation factor XI EUROPEAN PHARMACOPOEIA 8.0
suspension or is directly introduced into the associated tubes of moderate antigen density can be distinguished ; PMT
by the manufacturer. voltages are reviewed and adjusted periodically according
The absolute count of the CD34/CD45+ cells per microlitre is to standardised laboratory procedures.
calculated using the following expression : – Compensation : this must be acceptable for the colour
spectra overlap (for example, FITC/PE) encountered in
cell-surface marker analysis ; colour compensation is
analysed and adjusted according to standardised laboratory
procedures.
A = number of CD34/CD45+ cells counted ;
– Flow rate : this must be consistent with routine cell-surface
B = number of fluorosphere singlets counted ; marker analysis.
C = known fluorosphere concentration. – Gating regions : the gating regions established for the
CD34/CD45 samples are maintained unaltered for the
Gating strategies analysis of the negative region.
The purpose of sequential gating is to select the population of Calculation of absolute number of CD34/CD45+ cells
interest and simultaneously minimise interference from debris The absolute number of CD34/CD45+ cells is calculated using
and mature cells to which antibodies can bind non-specifically. the following expression :
If using a commercial kit, apply the gating recommended by
the manufacturer. If using an in-house assay, it is preferable to
apply a currently recommended strategy. A gating strategy that
uses light scattering parameters and CD34/CD45 fluorescence n = total number of CD34/CD45+ cells per microlitre ;
will aid in the accurate identification and enumeration of
D = dilution factor ;
CD34/CD45+ cells.
Number of events analysed V = volume of the product to be tested, in microlitres.
A sufficient number of events are analysed to maintain Results are reported as both the percentage of
acceptable precision, for example not fewer than CD34/CD45+ cells and the absolute number per
100 CD34+ events and not fewer than 60 000 CD45+ events ; microlitre. They may also be reported as the absolute number
the total number of cells counted may be greater if the per kilogram of recipient body mass, where this is possible.
percentage of CD34 is 0.1 per cent or less.
Specimen collection 01/2008:20724
Acid citrate dextrose (ACD) formula A is the anticoagulant
used in apheresis procedures. This anticoagulant allows both 2.7.24. FLOW CYTOMETRY
an automated leucocyte count and flow cytometry evaluation Flow cytometry consists of a multiparametric analysis of
to be performed on the same specimen. Edetic acid (EDTA) is optical properties of individual particles in a fluidic system.
the anticoagulant of choice for peripheral blood sampling.
Cells or particles in suspension are individually distributed
Specimen transport into a linear array (stream), which flows through a detection
Transport conditions guarantee the physical and thermal device. Solid tissues have to be reduced to a single-cell
safety of samples. suspension to be analysed.
Specimen integrity and storage The spectrum of parameters measurable by flow cytometry
Fresh (less than 24 h old) apheresis products, whole blood includes volume and morphological complexity of cells
samples, umbilical cord blood specimens or bone marrow or cell-like structures, cell pigments, DNA content, RNA
samples can be processed. Old specimens (more than 24 h content, proteins, cell surface markers, intracellular markers,
old) and specimens that have been frozen and thawed are enzymatic activity, pH, membrane and fluidity.
stained with a viability dye. On receipt, the temperature It is possible to collect 2 morphological parameters plus 1 or
within the package is verified. more fluorescence signals per single cell. The multiparametric
analysis allows the definition of cell populations by their
TECHNIQUE phenotype.
Sample preparation APPARATUS
Ensure that the concentration of leucocytes is suitable prior to Focusing, magnifying, and choice of light source are
staining with monoclonal antibodies. If necessary, dilute the optimised to allow the automatic detection and measurement
sample with medium that is compatible with the product to be of morphological differences and staining patterns. Flow
tested and the lysing system. Record the dilution factor. It is cytofluorimetric analysis meets the following criteria :
recommended to perform the test with a negative control.
– choice of light source depending on the parameters to be
Flow cytometry analysis analysed ;
Autostandardisation – adjustment of instrument settings depending on the cell
For analysis of cells labelled with a commercially available type to be analysed (for example, cell cultures, leucocytes,
kit, manufacturers have developed some quality tools platelets, bacteria, spermatozoa, yeast) and the analysis
for setting the flow cytometer. These settings are then to be performed (for example, phenotyping, cell cycle,
automatically transferred on protocol analysis of samples. apoptosis, cytokines, membrane fluidity, fluorescent
Specific fluorospheres are used to set the photomultiplier tube protein).
(PMT) on target values, compensation is set and the system Flow cytometry is characterised by the automated
is checked using a control preparation. quantification of set parameters for a high number of
System settings single cells during each analysis session. For example,
– Discriminator/threshold : the forward angle light scatter 100 000 particles or more (practically unlimited) are analysed
threshold is set to exclude debris (low forward scatter) but one after the other, typically in about 1 min. The detection
not small lymphocytes from the light-scatter plot. limit is as low as 100 fluorescent molecules per cell.
– PMT high voltage settings : these must be consistent with A flow cytometer apparatus has 5 main components :
cell-surface marker analysis and established within each – a fluidic system and a flow cell ;
laboratory so that negative and positive cell populations – a light source ;
General Notices (1) apply to all monographs and other texts 259
EUROPEAN PHARMACOPOEIA 8.0
– a detection and Analogue to Digital Conversion (ADC) SIGNAL MANAGEMENT AND ANALOGUE TO DIGITAL
system ; CONVERSION
Scatter and fluorescence signals emitted by cells when passing
– an amplification system ;
across the laser beam are sorted and addressed to their
– a computer provided with software for analysis of the detectors using optical filters. The detectors are transducers
signals. (photomultiplier tubes (PMTs)) that convert light signals
radiated from the cells into voltage pulses.
FLUIDIC SYSTEM AND FLOW CELL
The single cell is exposed to the light source and detected in The process of counting each pulse in the appropriate channel
the flow cell. The fluidic system carries the suspended cells is known as Analogue to Digital Conversion (ADC). The
individually from the sample tube to the laser intercept point. process is finally shown as a frequency histogram.
To achieve this, the sample stream is drawn out to a very thin . Voltage pulses need to be amplified for
fluid thread by a sheath fluid in the flow cell (hydrodynamic optimal visualisation. The amplification process accentuates
focusing). The light beam is focused in an elliptical shape, by the differences between cell signals, and consequently
2 confocal lenses, into the flow cell channel through which the increases the resolution among cell populations of different
cells pass. The flow rate must be constant during routine cell characteristics (for example, the differentiation of viable
surface marker analysis and must ensure a suitable distance from non-viable cells, or non-specific fluorescence from
between the cells to allow counting. antigen-specific fluorescence after staining with a fluorescent
LIGHT SOURCES monoclonal antibody).
Commonly used light sources are : There are 2 methods of amplification : linear or logarithmic ;
the choice between the 2 types is made for every single signal
– lamps (mercury, xenon) ;
according to the morphological characteristics of the cells
– high power water-cooled lasers (argon, krypton, dye laser) ; and the staining reagents used (for example, fluorescent
monoclonal antibodies, nucleic acid dyes).
– low power air-cooled lasers (argon (488 nm),
red helium-neon (633 nm), green helium-neon, Linear amplification, which enhances the differences among
helium-cadmium (UV)) ; strong pulses, is used with those parameters that generate high
intensity signals, for example :
– diode lasers (blue, green, red, violet).
– cell scatters ;
SIGNAL DETECTION
When a particle passes across the light beam, it scatters some – fluorescence from nucleic acid dyes for cell cycle studies.
of the light in all directions. Fluorescent dyes, when added to Logarithmic amplification, in contrast, is for weak pulses and
the particle, give off their own light (fluorescence), which is parameters or analysis conditions that may generate both
also radiated in all directions. 2 types of signals may thereby weak and strong pulses, for example :
be generated :
– cell antigens ;
– scatter of light ;
– scatter from platelets, bacteria, yeast ;
– fluorescence emission. – fluorescence from nucleic acid dyes for apoptosis studies.
The instrument’s light detectors collect some of this . Each fluorescent
scattered and fluorescent light and produce electronic signals dye has an absorption wavelength spectrum and a higher
proportional to the amount of light collected. emission wavelength spectrum. When using 2 or more
. 2 parameters of light scattering are measured : fluorescent probes simultaneously for staining cells (for
example, 4-antigen immunophenotyping), the fluorochromes
– the amount scattered mainly forward (forward scatter (FS)) emission spectra may overlap. As a consequence, each
fluorescence detector will sense its own specific fluorescent
– the amount scattered at 90° from the direction of the light
light and a variable quantity of light emitted by the other
beam (side scatter (SS)).
fluorescent probes. This results in signal over-evaluation and
Forward scatter correlates with the cell volume while side poor separation of the cell populations.
scatter is influenced by parameters such as the shape of the The solution is in the use of an electronic matrix that allows
nucleus, the amount and type of cytoplasmic granules or the the selective subtraction of the interfering signals from
membrane roughness, and correlates with the morphological each fluorescence signal after detector sensing (fluorescence
complexity of the cell, so that the higher the SS intensity, the compensation).
higher the cell complexity. As a function of the morphological
characteristics of cells, scatter signals will always be generated Fluorescence compensation requires the use of fluorescence
during a flow analysis ; they are defined as intrinsic parameters. calibrators, preferably positive cell samples stained with the
fluorochromes of interest, combined in a manner equivalent
. Depending on the type and number of light to that for the antibody used for the analysis.
sources, when a cell passes through the sensing area, it will
emit fluorescent light. Fluorescence signals are generated from SIGNAL PLOTTING AND DISPLAY
fluorescent dyes naturally present in the cells (for example, After amplification and compensation, the signals are plotted
co-enzymes, chlorophyll, seaweed pigments) and/or from in 2 or 3 dimensions. Histograms show the signal intensities
fluorescent probes taken up by the cells when stained for the versus the cell counts for a given parameter. Cytograms, in
analysis of specific characteristics (for example, fluorescent which each dot represents a cell, result from the combination
antibodies, nucleic acid dyes, pH probes, calcium probes, of 2 signal intensities (dual-parameter dot plots). The type
fluorescent proteins). Nowadays, there is a large number and and number of plots and signal combinations are chosen on
a wide range of different types of fluorescent probes available. the basis of the specimens and dyes used. When analysing
The optical filters must be adapted to the fluorochromes acquired data, the flow cytometry software can also generate
used and changed if necessary. Each fluorescent probe is other kinds of graphs (such as overlays, surface plots,
characterised by its excitation spectrum and its emission tomograms, contour plots, density plots, overlay plots).
spectrum. They are chosen depending on the nature of the Statistical data such as mean fluorescent intensities (and their
excitation source and the detection system, and according to shifts in time or their dependence on cell function) can also
the specific purpose of the analysis. be used.
General Notices (1) apply to all monographs and other texts 261
2.7.28. CFC assay for human haematopoietic progenitor cells EUROPEAN PHARMACOPOEIA 8.0
equivalence point of toxin/toxoid and antitoxin, flocculation If there is no indication of the expected Lf value of the sample
occurs in 1 or more tubes. The first tube in which flocculation available, it is advisable to obtain a rough estimate by use
occurs is used to determine the Lf value of the sample. of a wider range of antitoxin content in the tubes before
The Lf value of a toxin or toxoid is determined by the number proceeding to the final test.
of units of antitoxin that, when mixed with the sample, Example
produces an optimally flocculating mixture (Ramon assay).
Tube A B C D E F
Practical experience has shown that the results of the
calibration of antitoxins in International Units (IU), for Antitoxin 20 30 45 70 100 150
content
example by comparison to international antitoxin standards, (Lf-eq.)
depends on the immunochemical method used. For this
reason, antitoxins used for the Ramon assay must be directly The level of toxin or toxoid and antitoxin concentration
calibrated against the international biological reference in the test may be varied, but this will markedly affect the
reagents for diphtheria or tetanus toxoid for flocculation tests, flocculation time, so that at very low levels the test will take too
using the principles described below. The concentration thus long, whilst at a high concentration the onset of flocculation
determined may be indicated in Lf-equivalents per millilitre may be so rapid as to make it difficult to distinguish the first
(Lf-eq./mL). and second tubes to flocculate.
By definition, 1 Lf is the quantity of toxin or toxoid that Assay of low concentrations by blend flocculation
flocculates in the shortest time with 1 Lf-eq. of specific For very low concentrations, it is preferable to measure toxin
antitoxin. or toxoid by the method of blend flocculation. This involves
A range of volumes of the reference standard of antitoxin comparison of the Lf value of a known toxin or toxoid and
adjusted to a concentration of 100 Lf-eq./mL is dispensed into that of a mixture of the sample with that toxin or toxoid.
a series of, for example, 7 cm × 1 cm flocculation tubes. A When a toxin or toxoid with a known Lf value and a toxin
sufficient quantity of a 9 g/L solution of sodium chloride R or toxoid with an unknown Lf value are flocculated together,
is added to each tube to give a constant total volume of, for the mixture will flocculate as the sum of their values if they
example, 1 mL. The test sample is diluted to give an expected are homogeneous. If non-homogenous toxins or toxoids
concentration of approximately 50 Lf/mL, and, for example, are mixed they will produce an aberrant pattern with
1 mL aliquots of this dilution are dispensed into each of the 2 flocculation maxima.
tubes containing antitoxin. The tubes are properly mixed by
shaking, then placed in a water-bath at a constant temperature
between 30 °C and 52 °C, and observed at regular intervals for
the first appearance of floccules. This may require the use of a 01/2008:20728
magnifying lens and strong illumination.
The first and the second mixtures to flocculate are recorded
as well as the time taken for the first flocculation to appear. 2.7.28. COLONY-FORMING
2 tubes may flocculate simultaneously. CELL ASSAY FOR HUMAN
The first tube to flocculate is the one that contains the amount HAEMATOPOIETIC PROGENITOR
of antitoxin closest in equivalence to the amount of antigen in
the sample. The antitoxin content of this tube can be used to
CELLS
calculate the Lf value of the sample. If 2 tubes flocculate at the The haematopoietic system represents a continuum of cells
same time, the mean from the tubes are given as the result. whose phenotype and properties change as they progress from
The time taken for the first tube to flocculate (Kf) is a stem cells to differentiated cells.
useful indicator of the quality of the antigen. If at a given Haematopoietic progenitor cells (HPCs) are capable of
temperature and concentration of toxoid and antitoxin the forming colonies or ‘cell clusters’ in cultures grown in
Kf value is increased compared with normal, this indicates semi-solid media and are said to be ‘clonogenic’. The
that the antigen has been damaged. The Kf value may also determination of the number of colony-forming cells (CFCs)
change with the quality of the antitoxin used. in a cellular product is an indicator of the functional capacity
Example of the progenitor cells and is a predictor of haematopoietic
reconstitution. The measured number of CFCs correlates with
Tube A B C D E F
the minimum number of progenitors present in the sample.
Antitoxin added 40 45 50 55 60 65
(Lf-eq.) CELL-SURFACE MARKERS
Antitoxin added 0.40 0.45 0.50 0.55 0.60 0.65 The capacity of colony-forming cells to give rise to
(mL) haematopoietic colonies in vitro and/or to reconstitute
Saline added 0.60 0.55 0.50 0.45 0.40 0.35 the haematopoietic system has been correlated with the
(mL) expression of specific cell-surface antigens. The expression of
Diluted sample 1.0 1.0 1.0 1.0 1.0 1.0 the membrane antigen CD34 is an accepted marker for most
added of the haematopoietic progenitors and stem cells.
If in this example the first tube to flocculate is tube C then the
Lf value of the diluted sample is 50 Lf/mL. However, if the first COLONY ASSAY SPECIFICITY
tube to flocculate is tube A or tube F this does not indicate Colony-forming cells are identified with a nomenclature
equivalence at that level. It would be necessary to perform a based on the lineages of mature cells present in the colony
repeat test using either a different dilution of test sample or (for example, CFU-Mix, CFU-GEMM, CFU-GM, CFU-G,
selecting a different range of doses of reference antitoxin. CFU-M, BFU-E, CFU-E, CFU-Meg) and are a population of
More precision can be obtained by making allowance for progenitors able to give rise to colonies containing one or
the sequence of flocculation after the first tube. Thus, in the more lineages of haematopoietic cells. No or low capacity for
example quoted, if the second tube to flocculate had been self-renewal has been ascribed to this population of human
tube D, the final value for the diluted sample would be 52, HPCs compared with the most immature stem cells.
whereas if the second tube to flocculate was tube B, the final The amount and type of growth factors supplied during the
value would be 48. The test may be performed in duplicate culture modulate the type and size of colonies that will be
with slightly different dilutions of the test sample. formed.
in vitro
in vitro
2.7.24
01/2008:20729
MATERIALS
Growth factors
The haemocytometer is used to quantify the number of cells membrane integrity but its results do not necessarily reflect
in a given solution by calculation of the cell concentration per cell functionality. Recently trypsinised or thawed viable cells
millilitre (C) using the following expression : may have leaky membranes, causing them to absorb the dye.
Dye. Trypan blue is the stain most commonly used to
distinguish between viable and non-viable cells, but other
a = number of cells counted ; suitable dyes such as erythrosin B or nigrosin may also be
used. It is an acid dye (Mr 961), an anion with 4 sulfonate
d = dilution factor (where applicable) ; groups that can easily bind to proteins ; therefore the protein
n = factor varying with the volume of the concentration of the preparation to be tested must be as low
haemocytometer chamber. as possible.
Test conditions. Dye fixation is strongly influenced by pH,
It is possible to distinguish between mixed cell populations within a range of 6.6 to 7.6. Fixation is optimal at pH 7.5.
provided they differ in size or pigmentation (for example, The other conditions, such as the dye concentration and the
leukocytes and erythrocytes). staining time are validated.
Preparation of the counting chamber and analysis. Mount the Storage conditions of the dye : Generally a 0.4 or 0.5 per cent
coverslip (slightly moistened on the edges) on the slide. Move trypan blue solution in sterile phosphate-buffered saline is
the coverslip back and forth over the slide, pressing slightly on used. Store protected from light and air.
the sides. Prepare a suitable dilution of the cell suspension in
isotonic buffer or in haemolysis buffer. Test preparation and analysis. Stain the cell suspension at
the required dilution (usually in phosphate-buffered saline)
Add an appropriate volume of the dilution to the counting with, for example, a trypan blue solution having a final
chamber. The liquid is added to the border of the coverslip and concentration of 0.1 to 0.2 per cent. Mix gently. Incubate for
is drained inside the chamber by capillarity. Carefully place not more than 2-4 min at room temperature. Mix gently and
the haemocytometer under the microscope and focus. Count place a suitable volume in a counting chamber. Count without
the cells in a zone of the grid. Calculate the cell concentration delay.
in the diluted and original samples.
Determine the percentage of viable cells from the ratio of the
To increase the accuracy of the measurement, it is important number of unstained cells to the total number of cells under
to respect the following basic precautions : a light microscope, considering all stained cells as dead cells.
– use only suitably thickened coverslips ; Viability (V) is calculated as a percentage using the following
– wherever possible, count more than 100 cells (if necessary, expression :
count more areas) ;
– where cell clustering is detected (i.e. the cell suspension
is not monocellular), resuspend the cells before sampling
and count again ; n = number of unstained (viable) cells ;
– avoid underfilling or overflowing the chamber, otherwise N = total number of cells (stained and unstained).
the volume will no longer be accurate.
AUTOMATED COUNTING METHODS It is essential that the incubation time be not more than 4 min
Particle counters based on conductivity variation. Electronic as the number of stained cells may increase significantly
particle counting devices measure the size and number of afterwards. For a new determination, it may therefore be
particles in a solution. necessary to prepare a new test.
Particle counters are calibrated before use with a solution AUTOMATED METHODS
of particles of known concentration and size. To allow the Flow cytometry
counting of larger particles, tubes fitted with differently Test principle. The test is based on the ability of certain dyes to
calibrated orifices are available. These apparatuses do not cross damaged membranes and bind to DNA by intercalating
allow the discrimination between dead and live cells. As between bases so that dead cells may fluoresce and be detected
cell debris may also generate pulses that may cause errors, by flow cytometry (2.7.24). Non-viable cells are evaluated and
counters are also fitted with a threshold control allowing only discriminated by focusing on positive staining whereas viable
larger particles to be counted. cells remain unstained. This analysis is generally performed
The apparatus must be qualified for the counting of cellular with 7-aminoactinomycin D (7-AAD) or propidium
products (in terms of linearity, accuracy, etc.). iodide (PI) but other suitable dyes may also be used.
Particle counters based on flow cytometry (2.7.24). The flow Dye. 7-AAD and PI are given as examples of
cytometer is calibrated with reference particles of known membrane-impermeants that may be used as viability
concentration and size to give an absolute cell number per dyes.
volume. However, a calibrating solution is no longer necessary 7-AAD is an analogue of actinomycin D that contains a
in instruments using 2 electrodes inserted in the sampling substituted amino group at position 7 of the chromophore.
chamber where the fixed size of the sampling chamber and It intercalates between cytosine and guanine DNA bases.
distance between the 2 electrodes allow the measurement of The spectral properties of 7-AAD make this molecule
the content of a fixed volume. This type of instrument rarely particularly suitable for flow-cytometry analysis. The
needs to be calibrated after the initial setting. maximum absorption of the 7-AAD/DNA complex is situated
VIABILITY in the green spectral region and is thus suitable for an argon
laser-equipped cytometer (excitation wavelength of 488 nm).
This section applies to cell staining by viability dyes and The deep red fluorescence emission of the 7-AAD viability dye
manual or automated analysis, under a light microscope or (635 nm to 675 nm) eases the use of the probe in combination
by flow cytometry, of a cell suspension in order to determine with fluorescein isothiocyanate (FITC) and phycoerythrin
the percentage of viable cells. (PE)-conjugated antibodies, because in contrast to PI, the
Depending on the type of cells and the method used, the 7-AAD/DNA complex shows minimal overlap with FITC and
results may differ. PE.
MANUAL DYE-EXCLUSION METHOD PI binds to double-stranded DNA by intercalating between
Test principle. This test is based on the exclusion of the dye bases with little or no sequence preference and with a
from viable cells whereas dead or damaged cells absorb the dye stoichiometry of 1 dye molecule per 4-5 DNA base pairs. Once
and are coloured. It provides information on the cytoplasmic the dye is bound to nucleic acids, its fluorescence is enhanced
20- to 30-fold, the fluorescence excitation maximum is shifted kits and the manufacturer’s instructions are followed. The
around 30-40 nm towards the red and the fluorescence essential features of the procedure are described in the
emission maximum (615 nm) is shifted around 15 nm towards following example of a microtitre plate end-point method.
the blue. Although its absorptivity is quite low, PI exhibits a
sufficiently large Stokes shift to allow simultaneous detection REAGENTS
of nucleic acids and fluorescein-labelled antibodies, provided Dilution buffer pH 8.4. Dissolve 6.055 g of tris(hydroxy-
that the suitable optical filters are used. methyl)aminomethane R and 16.84 g of caesium chloride R in
Storage conditions of nucleic acid dye solution : 5 ± 3 °C. water R and adjust the pH if necessary. Dilute to 1000.0 mL
with water R.
Test preparation and analysis. In the case of haematopoietic
cells, the dye may be added after CD45 labelling to obtain a Human protein C activator. Protein isolated from the venom
better separation of cells from debris and platelets with a side of the viper Agkistrodon contortrix contortrix that specifically
scatter (SS)/CD45+ gating region. The incubation conditions activates human protein C. Reconstitute and store according
of the cell suspension with the dye are validated previously. to the manufacturer’s instructions. Dilute to 0.25 U/mL with
Incubation is performed at room temperature protected from water R before use in the assay.
light. Where necessary, lysis of red blood cells is performed Activated protein C chromogenic substrate. Specific
using, for example, ammonium chloride. If not, add buffer chromogenic substrate for APC, for example
alone. L-pyroglutamyl-L-prolyl-L-arginyl-p-nitroaniline
Percentages of viable cells are directly given by the flow hydrochloride (pyroGlu-Pro-Arg-pNA.HCl). Reconstitute
cytometer and deduced from the analysis of positive cells with water R to give a concentration of 4.5 mmol/L. Further
(dead cells) in the SS/7-AAD or SS/PI cytogram (dot plots). dilute to 1.1 mmol/L with dilution buffer pH 8.4 before use in
the assay.
Positive controls may consist of stabilised cells (dead cells)
mixed with fresh viable cells at a target value. METHOD
Digital imaging of stained cells. Digital imaging allows the Reconstitute or thaw the preparation to be examined according
automation of dye-exclusion methods. The cell suspension to the manufacturer’s instructions. Dilute with water R to
and viability-dye solution are directly mixed by a machine. produce at least 3 separate dilutions for each preparation in
The system, which allows sample aspiration, reagent handling, the range 0.050-0.200 IU/mL, preferably in duplicate.
and subsequent instrument cleaning is fully automated. Once Step 1. Mix 0.025 mL of each dilution with 0.050 mL of the
the cellular suspension has been aspirated and mixed with the human protein C activator, both previously heated to 37 °C,
dye solution, it is pumped to the flow cell for imaging. The and leave at 37 °C for exactly 10 min. For each dilution,
stained cell suspension is aspirated through a chamber where prepare a blank in the same manner, using water R instead of
stroboscopic light allows a camera to photograph the flowing the human protein C activator.
cells. The images are digitalised and the number of dead or
live cells counted by the software. Step 2. Add 0.150 mL of diluted chromogenic substrate,
previously heated to 37 °C, to each mixture and leave at 37 °C
for exactly 10 min. The incubation time must be adjusted, if
07/2008:20730 necessary, to ensure a linear development of chromophore
corrected 7.0 with time. Terminate the reaction by adding 0.050 mL of a
50 per cent V/V solution of glacial acetic acid R.
2.7.30. ASSAY OF HUMAN PROTEIN C Cleavage of the chromogenic substrate by APC causes release
of the chromophore pNA, in proportion to the concentration
1. CHROMOGENIC ASSAY of human protein C in the preparation. Measure the optical
Human protein C is a vitamin K-dependent plasma protein density at a wavelength of 405 nm. Subtract the optical density
that, upon activation to activated protein C (APC), can inhibit of the blank from the optical density of the test sample. Check
blood coagulation through the proteolytic cleavage of factors the validity of the assay and calculate the potency of the
Va and VIIIa. Human protein C activity is estimated using preparation to be examined using the usual statistical methods
a two-step method : in the 1st step, human protein C in the (5.3).
preparation is activated by a specific activator from snake
venom ; in the 2nd step, APC cleaves a specific chromogenic 2. CLOTTING ASSAY
substrate to form a chromophore that can be quantified Human protein C activity is estimated following cleavage
spectrophotometrically. to APC by a specific activator extracted from the venom of
the viper Agkistrodon contortrix contortrix. The resulting
APC inactivates factors Va and VIIIa, and thus prolongs the
APTT (Activated Partial Thromboplastin Time) of a system
in which all the coagulation factors are present, constant and
in excess, except for human protein C, which is derived from
the preparation being tested. Prolongation of the clotting time
is proportional to the concentration of human protein C in
the preparation.
The potency of human protein C is estimated by comparing
the ability of the preparation to be examined to prolong the
The potency of human protein C is estimated by comparing clotting time with the same ability of a reference standard
the ability of the preparation to be examined to cleave a of human protein C calibrated in International Units. The
chromogenic substrate with the same ability of a reference International Unit is the activity of a stated amount of the
standard of human protein C calibrated in International Units. International Standard for human protein C. The equivalence
The International Unit is the activity of a stated amount of the in International Units of the International Standard is stated
International Standard for human protein C. The equivalence by the World Health Organization.
in International Units of the International Standard is stated Individual reagents may be obtained separately or in
by the World Health Organization. commercial kits. Procedures and reagents may vary between
Individual reagents may be obtained separately or in different kits and the manufacturer’s instructions are followed.
commercial kits. Both end-point and kinetic methods are The essential features of the procedure are described in the
available. Procedures and reagents may vary between different following example.
General Notices (1) apply to all monographs and other texts 265
EUROPEAN PHARMACOPOEIA 8.0
concentration with the tris-albumin buffer solution. Incubate 0.45 mg/mL. Immediately start measurement of the change in
for a defined period of time, 3-10 min, at room temperature. absorbance (2.2.25) at 405 nm using a microtitre plate reader,
Since the activity of the solutions of the different porcine continuing the measurement for at least 5 min. Calculate the
pancreatic elastases may vary, the concentration of elastase rate of change of absorbance (ΔA/min). Alternatively, an
can be adjusted by evaluation of blank values containing end-point assay may be used by stopping the reaction with
elastase but no human α-1-proteinase inhibitor, to exhibit a acetic acid and measuring the absorbance at 405 nm. If the
suitable change of absorbance at 405 nm under the actual assay is performed in test tubes using spectrophotometers for
assay conditions. monitoring the change in absorbance at 405 nm, the volumes
Add to each well 100 μL of a solution of chromogenic of reagent solutions are changed proportionally.
substrate N-succinyl-tri-L-alanyl 4-p-nitroanilide The rate of change of absorbance (ΔA/min) is inversely
(Suc-Ala-Ala-Ala-pNA), reconstituted in dimethyl sulfoxide R proportional to human α-1-proteinase inhibitor activity.
to give a solution containing 4.5 mg/mL, then further diluted Check the validity of the assay and calculate the potency of the
with the tris-albumin buffer solution to a concentration of test preparation by the usual statistical methods (5.3).
General Notices (1) apply to all monographs and other texts 267
EUROPEAN PHARMACOPOEIA 8.0 2.8.7. Fatty oils and resinified essential oils in essential oils
2.8. METHODS IN For each sample of leaf, make not fewer than 10 determinations
PHARMACOGNOSY
and calculate the mean.
01/2008:20801
01/2008:20802
STOMATA 01/2008:20805
There are several types of stomata (see Figure 2.8.3.-1),
distinguished by the form and arrangement of the surrounding 2.8.5. WATER IN ESSENTIAL OILS
cells :
(1) The anomocytic (irregular-celled) type : the stoma is Mix 10 drops of the essential oil with 1 mL of carbon
surrounded by a varying number of cells in no way differing disulfide R. The solution remains clear on standing.
from those of the epidermis generally,
(2) The anisocytic (unequal-celled) type : the stoma is usually 01/2008:20806
surrounded by 3 subsidiary cells, of which one is markedly
smaller than the others, 2.8.6. FOREIGN ESTERS IN ESSENTIAL
(3) The diacytic (cross-celled) type : the stoma is accompanied OILS
by 2 subsidiary cells, whose common wall is at right angles
to the guard cells, Heat 1 mL of the essential oil for 2 min on a water-bath with
(4) The paracytic (parallel-celled) type : the stoma has on each 3.0 mL of a freshly prepared 100 g/L solution of potassium
side one or more subsidiary cells parallel to the long axis of hydroxide R in alcohol R. No crystals are formed within
the pore and guard cells. 30 min, even after cooling.
General Notices (1) apply to all monographs and other texts 271
2.8.8. Odour and taste of essential oils EUROPEAN PHARMACOPOEIA 8.0
The residue on evaporation of an essential oil is the percentage An essential oil is said to be “soluble in n volumes of alcohol
by mass of the oil which remains after evaporation on a of given strength t, becoming cloudy when diluted” when the
water-bath under the conditions specified below. clear solution in n volumes becomes cloudy in n1 volumes
(n1 less than 20) and stays so after further gradual addition
Apparatus. The apparatus (see Figure 2.8.9.-1) consists of : of alcohol of the same strength up to a total of 20 volumes
– water-bath with a cover having holes of 70 mm diameter ; of alcohol.
– evaporating dish of heat-resistant glass which is inert to An essential oil is said to be “soluble in n volumes of alcohol of
the contents ; given strength t with cloudiness between n1 and n2 volumes”
– desiccator. when the clear solution in n volumes becomes cloudy in
n1 volumes (n1 less than 20) and stays so after further gradual
addition of alcohol of the same strength up to a total of
n2 volumes of alcohol and then becomes clear (n2 less than 20).
An essential oil is said to be “soluble with opalescence” when
the alcoholic solution shows a bluish tinge, similar to that of
a standard of opalescence freshly prepared as follows : mix
0.5 mL of silver nitrate solution R2 and 0.05 mL of nitric
acid R ; add 50 mL of a 12 mg/L solution of sodium chloride R ;
mix and allow to stand protected from light for 5 min.
01/2008:20811
Figure 2.8.9.-1
Dimensions in millimetres
2.8.11. ASSAY OF 1,8-CINEOLE IN
Method. Weigh the evaporating dish after having heated it on
the water-bath for 1 h and cooled it in the desiccator. Weigh
ESSENTIAL OILS
into the evaporating dish 5.00 g of the essential oil, unless
otherwise prescribed. Heat the oil on the vigorously boiling Weigh 3.00 g of the oil, recently dried with anhydrous sodium
water-bath in a draught-free atmosphere for the prescribed sulfate R, into a dry test-tube and add 2.10 g of melted cresol R.
time. Allow to cool in the desiccator and weigh. Place the tube in the apparatus for the determination of
During the test, the level of water in the bath is maintained freezing point (2.2.18) and allow to cool, stirring continuously.
about 50 mm beneath the level of the cover. When crystallisation takes place there is a small rise in
temperature. Note the highest temperature reached (t1).
Remelt the mixture on a water-bath at a temperature that
does not exceed t1 by more than 5 °C and place the tube in
the apparatus, maintained at a temperature 5 °C below t1.
01/2008:20810 When crystallisation takes place, or when the temperature of
the mixture has fallen 3 °C below t1, stir continuously. Note
2.8.10. SOLUBILITY IN ALCOHOL OF Repeat the operation until 2 highest values obtained for t2
the highest temperature at which the mixture crystallises (t2).
ESSENTIAL OILS do not differ by more than 0.2 °C. If supercooling occurs,
induce crystallisation by adding a small crystal of the complex
Place 1.0 mL of the essential oil in a 25 mL or 30 mL consisting of 3.00 g of cineole R and 2.10 g of melted cresol R. If
glass-stoppered cylinder. Place in a constant temperature t2 is below 27.4 °C, repeat the determination after the addition
device, maintained at a temperature of 20 ± 0.2 °C. Using of 5.10 g of the complex.
Table 2.8.11.-1
t2 cineole t2 cineole t2 cineole t2 cineole
°C per °C per °C per °C per
cent m/m cent m/m cent m/m cent m/m
24 45.5 32 56.0 40 67.0 48 82.0
01/2008:20812
corrected 6.0
Figure 2.8.12.-1. - Apparatus for the determination of essential
oils in herbal drugs
2.8.12. ESSENTIAL OILS IN HERBAL Dimensions in millimetres
DRUGS Method. Use a thoroughly cleaned apparatus. Carry out the
assay according to the nature of the drug to be examined.
Place the prescribed volume of distillation liquid in the flask,
The determination of essential oils in herbal drugs is add a few pieces of porous porcelain and attach the condenser
carried out by steam distillation in a special apparatus in the assembly. Introduce water R through the filling funnel N until
conditions described below. The distillate is collected in the it is at the level B. Remove the stopper K′ and introduce the
graduated tube, using xylene to take up the essential oil ; the prescribed quantity of xylene R, using a pipette with its tip at
aqueous phase is automatically returned to the distillation the bottom of the tube K. Replace the stopper K′ and ensure
flask. that the orifice coincides with the vent. Heat the liquid in the
Apparatus. The apparatus comprises the following parts : flask to boiling and adjust the distillation rate to 2-3 mL/min,
unless otherwise prescribed.
(a) a suitable round-bottomed flask with a short, ground-glass
neck having an internal diameter of about 29 mm at the wide To determine the rate of distillation, during distillation
end ; lower the level of the water by means of the three-way tap
until the meniscus is at the level of the lower mark (a) (see
(b) a condenser assembly (see Figure 2.8.12.-1) that closely fits Figure 2.8.12.-2). Close the tap and measure the time taken
the flask, the different parts being fused into one piece ; the for the liquid to reach the upper mark (b). Open the tap and
glass used has a low coefficient of expansion : continue the distillation, modifying the heat to regulate the
distillation rate. Distil for 30 min. Stop the heating and after
– the stopper K′ is vented and the tube K has an orifice of at least 10 min read off the volume of xylene in the graduated
diameter about 1 mm that coincides with the vent ; the wide tube.
end of the tube K is of ground-glass and has an internal
diameter of 10 mm ;
– a pear-shaped swelling, J, of 3 mL capacity ;
– the tube JL is graduated in 0.01 mL ;
– the bulb-shaped swelling L has a capacity of about 2 mL ;
– M is a three-way tap ;
– the junction B is at a level 20 mm higher than the uppermost
graduation ;
Figure 2.8.12.-2
(c) a suitable heating device, allowing a fine control ;
Introduce into the flask the prescribed quantity of the drug
(d) a vertical support with a horizontal ring covered with and continue the distillation as described above for the time
insulating material. and at the rate prescribed. Stop the heating and after 10 min
General Notices (1) apply to all monographs and other texts 273
2.8.13. Pesticide residues EUROPEAN PHARMACOPOEIA 8.0
read the volume of liquid collected in the graduated tube and MRLHD = maximum residue limit of the pesticide in the
subtract the volume of xylene previously noted. The difference herbal drug as given in Table 2.8.13.-1 or in
represents the quantity of essential oil in the mass of the drug EU texts or calculated using the expression
taken. Calculate the result as millilitres per kilogram of drug. mentioned above ;
When the essential oil is to be used for other analytical DER = drug/extract ratio, i.e. the ratio between the
purposes, the water-free mixture of xylene and essential oil quantity of herbal drug used in the manufacture
may be recovered as follows : remove the stopper K′ and of a herbal drug preparation and the quantity of
introduce 0.1 mL of a 1 g/L solution of sodium fluoresceinate R herbal drug preparation obtained ;
and 0.5 mL of water R. Lower the mixture of xylene and MDDHP = daily dose of the herbal drug preparation, in
essential oil into the bulb-shaped swelling L by means of the kilograms.
three-way tap, allow to stand for 5 min and lower the mixture
slowly until it just reaches the level of the tap M. Open the tap The competent authority may grant total or partial exemption
anti-clockwise so that the water flows out of the connecting from the test when the complete history (nature and
tube BM. Wash the tube with acetone R and with a little quantity of the pesticides used, date of each treatment during
toluene R introduced through the filling funnel N. Turn the cultivation and after the harvest) of the treatment of the batch
tap anti-clockwise in order to recover the mixture of xylene is known and can be checked precisely according to good
and essential oil in an appropriate flask. agricultural and collection practice (GACP).
Table 2.8.13.-1
Substance Limit
(mg/kg)
Acephate 0.1
07/2008:20813 Alachlor 0.05
Azinphos-methyl 1
Definition. For the purposes of the Pharmacopoeia, a
pesticide is any substance or mixture of substances intended Bromide, inorganic (calculated as bromide ion) 50
for preventing, destroying or controlling any pest, unwanted Bromophos-ethyl 0.05
species of plants or animals causing harm during or otherwise
interfering with the production, processing, storage, transport Bromophos-methyl 0.05
or marketing of herbal drugs. The item includes substances Brompropylate 3
intended for use as growth-regulators, defoliants or desiccants
and any substance applied to crops, either before or after Chlordane (sum of cis-, trans - and oxychlordane) 0.05
harvest, to protect the commodity from deterioration during Chlorfenvinphos 0.5
storage and transport. Pesticide residues can be present and
are controlled in herbal drugs and herbal drug preparations. Chlorpyriphos-ethyl 0.2
Diazinon 0.5
Dichlofluanid 0.1
ADI = acceptable daily intake, as published by
FAO-WHO, in milligrams per kilogram of Dichlorvos 1
body mass ; Dicofol 0.5
M = body mass in kilograms (60 kg) ;
Dimethoate and omethoate (sum of) 0.1
MDDHD = daily dose of the herbal drug, in kilograms.
Dithiocarbamates (expressed as CS2) 2
The limits for pesticides in herbal drug preparations are Endosulfan (sum of isomers and endosulfan sulfate) 3
calculated using the following expressions : 0.05
Endrin
Ethion 2
Fenpropathrin 0.03
If DER > 10:
Fensulfothion (sum of fensulfothion, fensulfothion-oxon, 0.05
fensulfothion-oxonsulfon and fensulfothion-sulfon)
General Notices (1) apply to all monographs and other texts 275
2.8.15. Bitterness value EUROPEAN PHARMACOPOEIA 8.0
Calculate the percentage content of tannins expressed as Starting with dilution C-4 each panel member determines
pyrogallol from the expression : the dilution which still has a bitter taste. This solution is
designated D. Note the DF of solution D is Y.
Starting with solution D prepare the following sequence of
dilutions :
m1 = mass of the sample to be examined, in grams ; Solution D (mL) 1.2 1.5 2.0 3.0 6.0 8.0
m2 = mass of pyrogallol, in grams. water R (mL) 8.8 8.5 8.0 7.0 4.0 2.0
Unless otherwise indicated in the monograph, herbal drugs tightly in aluminium foil and store it below 4 °C. Before use,
contain not more than 2 μg/kg of aflatoxin B1. The competent do not remove the aluminium foil until the contents have
authority may also require compliance with a limit of 4 μg/kg reached room temperature. If the solution has to be stored
for the sum of aflatoxins B1, B2, G1 and G2. for a long period (for example, 1 month), weigh the flask and
The method described below is cited as an example of a record the mass before and after each use of the solution.
method that has been shown to be suitable for devil’s claw root, Aflatoxin B1 standard solutions. Place the volumes of aflatoxin
ginger and senna pods. Its suitability for other herbal drugs secondary stock solution indicated in Table 2.8.18.-1 in
must be demonstrated or another validated method used. separate 250 mL volumetric flasks. Pass a stream of nitrogen
through at room temperature until the solvent has just
METHOD evaporated. To each flask, add 75 mL of methanol R, allow the
Liquid chromatography (2.2.29). aflatoxin B1 to dissolve and dilute to 250 mL with water R.
Aflatoxins are subject to light degradation. Carry out the Table 2.8.18.-1. – Aflatoxin B1 standard solutions
determination protected from daylight by using UV-absorbing
foil on windows in combination with subdued light, or curtains Final concentration
Volume of secondary
Standard solution of standard solution
or blinds in combination with artificial light (fluorescent tubes stock solution (μL)
(ng/mL)
are acceptable). Protect aflatoxin-containing solutions from 1 125 0.05
daylight.
Rinse glassware before use with a 10 per cent V/V solution of 2 250 0.1
sulfuric acid R and then rinse carefully with distilled water R 3 500 0.2
until no more acid is present.
4 750 0.3
Test solution. Use an immunoaffinity column containing
antibodies against aflatoxin B1 with a capacity of not less than 5 1000 0.4
100 ng of aflatoxin B1 and which gives a recovery of not less
than 80 per cent when a solution of 5 ng of aflatoxin B1 in a Calibration curve. Prepare the calibration curve using
mixture of 12.5 mL of methanol R and 87.5 mL of water R is aflatoxin B1 standard solutions 1 to 5, which cover a range
passed through. Allow the immunoaffinity column to reach equivalent to 1-8 μg/kg of aflatoxin B1 in the herbal drug.
room temperature. To 5.00 g of the powdered drug (500) Check the plot for linearity. If the content of aflatoxin B1 in
(2.9.12) add 100 mL of a mixture of 30 volumes of water R and the sample to be examined is outside of the calibration range,
70 volumes of methanol R and extract by sonication for 30 min. the test solution must be diluted to an aflatoxin content that is
Filter through folded filter paper. Pipette 10.0 mL of the clearappropriate for the established calibration curve.
filtrate into a 150 mL conical flask. Add 70 mL of water R. Column :
Pass 40 mL through the immunoaffinity column at a flow rate – size : l = 0.25 m, Ø = 4.6 mm ;
of 3 mL/min (not exceeding 5 mL/min). Wash the column
with 2 volumes, each of 10 mL, of water R at a flow rate not – stationary phase : octadecylsilyl silica gel for
exceeding 5 mL/min and dry by applying a slight vacuum for chromatography R (5 μm).
5-10 s or by passing air through the immunoaffinity column Mobile phase :
by means of a syringe for 10 s. Apply 0.5 mL of methanol R – mobile phase A (for post-column derivatisation with
to the column and allow to pass through by gravity. Collect photochemical reactor or pyridinium bromide) :
the eluate in a 5 mL volumetric flask. After 1 min, apply a acetonitrile R, methanol R, water R (2:3:6 V/V/V);
2nd portion of 0.5 mL of methanol R. After a further 1 min, – mobile phase B (for post-column derivatisation with
apply a 3rd portion of 0.5 mL of methanol R. Collect most of electrochemically derived bromine) : add 0.12 g of
the applied elution solvent by pressing air through or applying potassium bromide R and 350 μL of dilute nitric acid R1
vacuum to the column. Dilute to 5 mL with water R and shake per litre of mobile phase A.
well. If the solution is clear it can be used directly for analysis.
Otherwise, pass it through a disposable filter unit prior to Flow rate : 1 mL/min.
injection. Use a disposable filter unit (e.g. 0.45 μm pore size Detection : fluorescence detector with a 360 nm excitation
polytetrafluoroethylene filter) that has been shown not to filter and a 420 nm cut-off emission filter, or equivalent.
cause loss of aflatoxin by retention. Recommended settings for adjustable detectors are 365 nm
Aflatoxin B1 primary stock solution. Dissolve aflatoxin B1 R in(excitation wavelength) and 435 nm (emission wavelength).
a mixture of 2 volumes of acetonitrile R and 98 volumes of Injection : 500 μL.
toluene R to give a 10 μg/mL solution. To determine the exact Post-column derivatisation with pyridinium hydrobromide
concentration of aflatoxin B1 in the stock solution, record perbromide (PBPB) :
the absorption curve (2.2.25) between 330 nm and 370 nm – pulseless pump ;
in quartz cells.
– T-piece with zero dead volume ;
Calculate the aflatoxin B1 mass concentration, in micrograms
per millilitre, using the following expression : – polytetrafluoroethylene reaction tube, l = 0.45 m,
Ø = 0.5 mm ;
– mobile phase A ;
– post-column derivatisation reagent : dissolve 50 mg of
pyridinium hydrobromide perbromide R in 1000 mL of
A = absorbance determined at the maximum of the water R (store protected from light and use within 4 days) ;
absorption curve ;
– flow rate of the derivatisation reagent : 0.4 mL/min.
M = molar mass of aflatoxin B1 (312 g/mol) ;
Post-column derivatisation with photochemical reactor
ε = molar absorptivity of aflatoxin B1 in the (PHRED)
toluene-acetonitrile mixture (1930 m2/mol) ; – reactor unit with one 254 nm low pressure mercury UV
l = optical path length of the cell (1 cm). bulb (minimum 8 W) ;
Aflatoxin B1 secondary stock solution. Prepare a secondary – polished support plate ;
stock solution containing 100 ng/mL aflatoxin B1 by diluting – knitted reactor coil : polytetrafluoroethylene tubing knitted
aflatoxin B1 primary stock solution with a mixture of 2 volumes tightly around the UV bulb, l = 25 m, Ø = 0.25 mm,
of acetonitrile R and 98 volumes of toluene R. Wrap the flask nominal void volume 1.25 mL ;
General Notices (1) apply to all monographs and other texts 277
2.8.20. Herbal drugs : sampling and sample preparation EUROPEAN PHARMACOPOEIA 8.0
– exposure time : 2 min ; Take one sample from each container to be sampled. The
– mobile phase A. sample is taken from the upper, middle or lower section of the
Post-column derivatisation with electrochemically generated container, such that the samples taken are representative of
bromine (KOBRA) : different parts of the containers. In the case of large bales or
bags, samples must be taken from a depth of at least 10 cm.
– KOBRA-cell : electrochemical cell that generates a reactive The mass of the material taken from each container is such
form of bromine for derivatisation of aflatoxins, resulting in that the total mass of the bulk sample complies with the
enhanced fluorescence ; available from various commercial following values.
suppliers ;
– Derivation direct-current supply in series with the Minimum mass of samples as
Mass of herbal drug in the
KOBRA-cell, providing a constant current of about 100 μA ; a percentage of the mass of the
batch (kg)
batch of herbal drug
– polytetrafluoroethylene reaction tube, l = 0.12 m,
< 50 1.00*
Ø = 0.25 mm ;
– mobile phase B. 50 - 100 0.50
Elution order : aflatoxin G2, aflatoxin G1, aflatoxin B2, aflatoxin > 100 - 250 0.25
B 1.
> 250 - 500 0.20
Calculation : calculate the calibration curve y = ax + b, with
aflatoxin B1 concentration (ng/mL) on the x-axis and the > 500 - 1000 0.18
signal (S) on the y-axis. The aflatoxin B concentration (C) in > 1000 - 2500 0.15
a measured solution is equal to .
Calculate the aflatoxin B1 content of the herbal drug, in > 2500 - 5000 0.10
nanograms per gram, using the following expression : > 5000 - 10 000 0.08
In order to reduce the effect of sampling in qualitative Leaves, flowers, seeds, fruits 250 g or mass of whole sample if
bulk sample is less than 250 g
and quantitative analysis, it is necessary to ensure that the
Broken or fragmented drugs (where 125 g
composition of the sample used is representative of the batch average mass of the pieces is less
of material being examined. The following procedures are than 0.5 g)
the minimum considered applicable for herbal drugs. NOTE :
other procedures may be used if they can be demonstrated to NOTE : quartering consists of placing the bulk sample,
produce representative batch samples. thoroughly mixed, as a level and square-shaped heap and
dividing it diagonally into 4 equal parts. 2 opposite quarters
BULK SAMPLE are retained and carefully remixed. The process is repeated as
Where external examination of containers, markings and necessary until the required minimum mass is obtained for the
labels of a batch indicate that it can be considered to be test sample.
homogeneous, sample the number of randomly selected
Mill the test sample in a single pass through a 1 mm screen
containers indicated below. Where a batch cannot be
or the screen size specified in the monograph. The use of a
considered to be homogeneous, divide it into sub-batches that
milling machine is recommended.
are as homogeneous as possible, then sample each sub-batch
as a homogeneous batch using, as a minimum, the number of Pass the milled sample through a 1 mm standard sieve or the
randomly selected containers indicated below. sieve specified in the monograph. The residue retained on
the sieve must not be more than 10 per cent of the total mass
Number of containers
Number of containers in batch (N) of the milled sample, of which not more than 2 per cent of
to be sampled (n)
the total mass of the milled sample may be of a particle size
1-3 all greater than 1.5 mm or 1.5 times the specified particle size
>3 n* = in the monograph. If these conditions are met, the sample
and residue are to be well mixed to form the test sample for
* round n up to the next integer analysis.
Table 2.8.20.-1. – Operation of the sampling procedure in order to obtain the prescribed bulk sample
Mass of herbal
drug in container 0.5 1 5
(kg)
Total mass of No. of No. of Total mass No. of No. of Total mass No. of No. of Total mass
herbal drug in containers containers to of samples containers containers to of samples containers containers to of samples
the batch (kg) in batch be sampled (g) in batch be sampled (g) in batch be sampled (g)
0.5 1 1 125 – – – – – –
1 2 2 125 1 1 125 – – –
25 – – – 25 6 250 5 4 250
250 – – – – – – 50 9 625
Mass of herbal
drug in container 25 125 500
(kg)
Total mass of No. of No. of Total mass No. of No. of Total mass No. of No. of Total mass
herbal drug in containers containers to of samples containers containers to of samples containers containers to of samples
the batch (kg) in batch be sampled (g) in batch be sampled (g) in batch be sampled (g)
25 1 1 250 – – – – – –
100 4 3 500 – – – – – –
In those cases where these requirements are not met, the These methods are not designed for inclusion as assay methods
test sample for analysis is composed of the 2 parts measured in monographs on those drugs that produce aristolochic acids
separately. Therefore, the quantity required for each analysis as secondary metabolites ; for these, a more sensitive, validated
is derived by weighing proportional quantities of the powder method is required.
and the residue.
NOTE : for determination of microscopic characters, a portion METHOD A : SCREENING TEST FOR ARISTOLOCHIC
of the milled test sample is re-milled through a 0.355 mm screen. ACIDS
Thin-layer chromatography (2.2.27).
01/2011:20821
Solvent mixture : anhydrous formic acid R, water R, methanol R
(1:9:40 V/V/V).
2.8.21. TEST FOR ARISTOLOCHIC
Test solution. To 1.0 g of the powdered herbal drug (710)
ACIDS IN HERBAL DRUGS (2.9.12) add 10.0 mL of the solvent mixture, sonicate for
CAUTION : aristolochic acids are very toxic and carcinogenic. 10 min and centrifuge.
Perform manipulations in a fume cupboard whenever possible. Reference solution (a). Disperse an amount of aristolochia HRS
Take particular precautions, such as use of a glove box, when corresponding to 0.10 mg of aristolochic acid I in 20.0 mL of
the substance is in dry form because of its electrostatic properties the solvent mixture, sonicate for 10 min and centrifuge.
and the tendency to disperse through the working areas. Reference solution (b). Dilute 1.0 mL of reference solution (a)
Methods A and B are intended to be cross-referenced to 25.0 mL with methanol R.
in monographs on herbal drugs that, according to
chemotaxonomic knowledge, are expected to be free from Plate : TLC silica gel F254 plate R (2-10 μm).
aristolochic acids, but that may be subject to adulteration or Mobile phase : anhydrous formic acid R, water R, ethyl
substitution with plant material containing aristolochic acids. acetate R, toluene R (3:3:30:60 V/V/V/V) ; use the upper layer.
Methods A and B are intended to be used in the screening of Application : 20 μL as bands of 8 mm.
herbal drugs for aristolochic acids at the stated limits and will
usually be complemented by macroscopic and/or microscopic Development : over a path of 6 cm.
tests to exclude plant material containing aristolochic acids. Drying : in a current of cold air for 5 min.
Method C will not be used in specific monographs but is Detection : spray with a 100 g/L solution of stannous chloride R
provided as a method to confirm the presence of aristolochic in dilute hydrochloric acid R until the plate is slightly wet, heat
acid I at levels equal to or greater than 2 ppm. It may be at 100 °C for 1 min and examine in ultraviolet light at 365 nm.
applied if chromatographic data suggests the presence of
aristolochic acid I.
General Notices (1) apply to all monographs and other texts 279
2.8.21. Test for aristolochic acids in herbal drugs EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 281
2.8.23. Microscopic examination of herbal drugs EUROPEAN PHARMACOPOEIA 8.0
Time Mobile phase A Mobile phase B tissues, ruthenium red solution R, which is used to show the
(min) (per cent V/V) (per cent V/V) presence of mucilage in cells or glycerol R used to show the
0 - 30 80 → 40 20 → 60 presence of starch and inulin.
30 - 35 40 → 20 60 → 80 Examination under polarised light (between crossed nicol
prisms) is used to identify starch granules (black cross
35 - 37 20 80 phenomenon), calcium oxalate crystals (refringence) or
37 - 40 20 → 80 80 → 20 lignified structures.
MOUNTING IN CHLORAL HYDRATE SOLUTION
Flow rate : 0.8 mL/min.
Place 2-3 drops of chloral hydrate solution R on a glass
Detection : fluorescence detector ; recommended settings for
microscope slide. Disperse a very small quantity of the
adjustable detectors are 330 nm (excitation wavelength) and
powdered drug in the liquid and cover the preparation with
460 nm (emission wavelength).
a cover slip. Heat the preparation very gently to boiling on a
Injection : 20 μL. hot plate or a micro gas burner. Maintain gentle boiling for
Calculation : calculate the calibration curve y = ax + b, with a short time. Make sure that the quantity of mounting fluid
ochratoxin A concentration (in nanograms per millilitre) on is sufficient. If necessary, add more fluid using a tapered
the x-axis and the signal (S) on the y-axis. The ochratoxin A glass pipette. Allow to cool and then examine under a
concentration (C) in a measured solution is equal to . microscope. Repeat the heating until the starch granules and
Calculate the ochratoxin A content of the herbal drug, in the water-soluble contents of the cells are no longer visible.
nanograms per gram, using the following expression : Examine under a microscope.
Chloral hydrate tends to crystallise as long needles. To avoid
this, proceed as follows : after heating, remove the cover slip ;
to the preparation add 1 drop of a 10 per cent V/V mixture of
chloral hydrate solution R in glycerol R ; place a clean cover slip
m = mass of the herbal drug used to prepare the test on the preparation ; examine under a microscope.
solution, in grams ;
MOUNTING IN A 50 PER CENT V/V SOLUTION OF
V1 = volume of dilution, in millilitres ;
GLYCEROL
Vi = aliquot taken for immunoaffinity clean-up, in Place 2 drops of a 50 per cent V/V solution of glycerol R on a
millilitres ; glass microscope slide. Disperse a very small quantity of the
V2 = volume in which the residue is taken up, in powdered drug in the liquid and cover the preparation with a
millilitres ; cover slip. Examine under a microscope.
C = measured ochratoxin A concentration of the test MOUNTING IN A 10 PER CENT V/V ALCOHOLIC
solution, in nanograms per millilitre. SOLUTION OF PHLOROGLUCINOL AND
HYDROCHLORIC ACID
Appendix : Decontamination procedures for Place a very small quantity of the powdered drug on a glass
microscope slide. Add 1-2 drops of a 10 per cent V/V alcoholic
laboratory glassware solution of phloroglucinol R. Mix and allow the solvent to
Rinse glassware with methanol R and decontaminate by evaporate almost completely. Add 1-2 drops of hydrochloric
immersion in strong sodium hypochlorite solution R for at least acid R and cover the preparation with a cover slip. Examine
2 h, then wash thoroughly with water. immediately under a microscope. The red colour indicates
the presence of lignin.
MOUNTING IN LACTIC REAGENT
04/2010:20823 Place 2-3 drops of lactic reagent R on a glass microscope slide.
Disperse a very small quantity of the powdered drug in the
2.8.23. MICROSCOPIC EXAMINATION liquid and cover the preparation with a cover slip. Heat the
preparation very gently to boiling. Maintain gentle boiling for
OF HERBAL DRUGS a short time. Make sure that the quantity of mounting fluid is
sufficient. If necessary, add more fluid using a tapered glass
The microscopic examination of herbal drugs is carried out on pipette. Allow to cool and then examine under a microscope.
the powdered drug (355) (2.9.12) unless otherwise prescribed Lignified structures stain bright yellow ; structures containing
in the monograph. cellulose remain colourless. Starch granules stain more or less
Chloral hydrate solution R is the most commonly prescribed violet ; certain secretions (e.g., essential oils, resins, oleoresins)
reagent. However, certain features are not visible or not stain orange and cork stains red.
easily seen after mounting in this reagent. In this case, other
reagents are used, for example, a 50 per cent V/V solution of MOUNTING IN RUTHENIUM RED SOLUTION
glycerol R, which makes it possible to visualise starch granules. Place 2 drops of ruthenium red solution R on a glass
It may also be necessary to prescribe specific reagents in microscope slide. Disperse a very small quantity of the
a monograph, for example : lactic reagent R which is used powdered drug in the liquid and cover the preparation with
to show the presence of various features, 10 per cent V/V a cover slip. After about 1 minute, allow a drop of distilled
alcoholic solution of phloroglucinol R and hydrochloric acid R, water R to be taken up between the slide and the cover slip.
which are used to identify the presence of lignin in cells or Examine under a microscope. The mucilage stains violet red.
2.9. PHARMACEUTICAL holes extend between the ends of the cylinder. One of the
TECHNICAL PROCEDURES
holes is centered on the cylindrical axis. The other holes
are centered 6 ± 0.2 mm from the axis on imaginary lines
perpendicular to the axis and parallel to each other. 4 identical
trapezoidal-shaped planes are cut into the wall of the cylinder,
nearly perpendicular to the ends of the cylinder. The
04/2011:20901 trapezoidal shape is symmetrical ; its parallel sides coincide
with the ends of the cylinder and are parallel to an imaginary
line connecting the centres of 2 adjacent holes 6 mm from
2.9.1. DISINTEGRATION OF TABLETS the cylindrical axis. The parallel side of the trapezoid on the
AND CAPSULES(1) bottom of the cylinder has a length of 1.6 ± 0.1 mm and its
bottom edges lie at a depth of 1.5 mm to 1.8 mm from the
This test is provided to determine whether tablets or capsules cylinder’s circumference. The parallel side of the trapezoid
disintegrate within the prescribed time when placed in a liquid on the top of the cylinder has a length of 9.4 ± 0.2 mm and
medium under the experimental conditions presented below. its centre lies at a depth of 2.6 ± 0.1 mm from the cylinder’s
For the purposes of this test, disintegration does not circumference. All surfaces of the disc are smooth.
imply complete dissolution of the unit or even of its active If the use of discs is specified, add a disc to each tube and
constituent. Complete disintegration is defined as that state in operate the apparatus as directed under Procedure. The discs
which any residue of the unit, except fragments of insoluble conform to the dimensions shown in Figure 2.9.1.-1.
coating or capsule shell, remaining on the screen of the test
apparatus or adhering to the lower surface of the discs, if used, The use of automatic detection employing modified discs
is a soft mass having no palpably firm core. is permitted where the use of discs is specified or allowed.
Such discs must comply with the requirements of density and
♦Use apparatus A for tablets and capsules that are not
dimension given in this chapter.
greater than 18 mm long. For larger tablets or capsules use
apparatus B.♦ Procedure. Place 1 dosage unit in each of the 6 tubes of the
basket and, if prescribed, add a disc. Operate the apparatus
TEST A - TABLETS AND CAPSULES OF NORMAL SIZE using the specified medium, maintained at 37 ± 2 °C, as
the immersion fluid. At the end of the specified time, lift
Apparatus. The apparatus consists of a basket-rack assembly, the basket from the fluid and observe the dosage units :
a 1 L, low-form beaker, 149 ± 11 mm in height and having all of the dosage units have disintegrated completely. If
an inside diameter of 106 ± 9 mm for the immersion fluid, a 1 or 2 dosage units fail to disintegrate, repeat the test on
thermostatic arrangement for heating the fluid between 35 °C 12 additional dosage units. The requirements of the test are
and 39 °C, and a device for raising and lowering the basket in met if not less than 16 of the 18 dosage units tested have
the immersion fluid at a constant frequency rate between 29 disintegrated.
and 32 cycles per minute, through a distance of 55 ± 2 mm.
The volume of the fluid in the vessel is such that at the highest ♦
point of the upward stroke the wire mesh remains at least TEST B – LARGE TABLETS AND LARGE CAPSULES
15 mm below the surface of the fluid, and descends to not less Apparatus. The main part of the apparatus (Figure 2.9.1.-2.)
than 25 mm from the bottom of the vessel on the downward is a rigid basket-rack assembly supporting 3 cylindrical
stroke. At no time should the top of the basket-rack assembly transparent tubes 77.5 ± 2.5 mm long, 33.0 mm ± 0.5 mm in
become submerged. The time required for the upward stroke internal diameter, and with a wall thickness of 2.5 ± 0.5 mm.
is equal to the time required for the downward stroke, and the Each tube is provided with a cylindrical disc 31.4 ± 0.13 mm
change in stroke direction is a smooth transition, rather than in diameter and 15.3 ± 0.15 mm thick, made of transparent
an abrupt reversal of motion. The basket-rack assembly moves plastic with a relative density of 1.18-1.20. Each disc is pierced
vertically along its axis. There is no appreciable horizontal by 7 holes, each 3.15 ± 0.1 mm in diameter, 1 in the centre
motion or movement of the axis from the vertical. and the other 6 spaced equally on a circle of radius 4.2 mm
Basket-rack assembly. The basket-rack assembly consists of from the centre of the disc. The tubes are held vertically
6 open-ended transparent tubes, each 77.5 ± 2.5 mm long by 2 separate and superimposed rigid plastic plates 97 mm
and having an inside diameter of 21.85 ± 1.15 mm and a wall in diameter and 9 mm thick, with 3 holes. The holes are
1.9 ± 0.9 mm thick ; the tubes are held in a vertical position equidistant from the centre of the plate and equally spaced.
by 2 plates, each 90 ± 2 mm in diameter and 6.75 ± 1.75 mm Attached to the under side of the lower plate is a piece of
in thickness, with 6 holes, each 24 ± 2 mm in diameter, woven gauze made from stainless steel wire 0.63 ± 0.03 mm
equidistant from the centre of the plate and equally spaced in diameter and having mesh apertures of 2.0 ± 0.2 mm.
from one another. Attached to the under surface of the lower The plates are held rigidly in position and 77.5 mm apart
plate is a woven stainless steel wire cloth, which has a plain by vertical metal rods at the periphery. A metal rod is also
square weave with 2.0 ± 0.2 mm mesh apertures and with a fixed to the centre of the upper plate to enable the assembly
wire diameter of 0.615 ± 0.045 mm. The parts of the apparatus to be attached to a mechanical device capable of raising and
are assembled and rigidly held by means of 3 bolts passing lowering it smoothly at a constant frequency of between
through the 2 plates. A suitable means is provided to suspend 29 and 32 cycles per minute, through a distance of 55 ± 2 mm.
the basket-rack assembly from the raising and lowering device The assembly is suspended in the specified liquid medium
using a point on its axis. in a suitable vessel, preferably a 1 L beaker. The volume of
The design of the basket-rack assembly may be varied the liquid is such that when the assembly is in the highest
somewhat provided the specifications for the glass tubes and position the wire mesh is at least 15 mm below the surface of
the screen mesh size are maintained. The basket-rack assembly the liquid, and when the assembly is in the lowest position the
conforms to the dimensions shown in Figure 2.9.1.-1. wire mesh is at least 25 mm above the bottom of the beaker
and the upper open ends of the tubes remain above the surface
Discs. The use of discs is permitted only where specified
of the liquid. A suitable device maintains the temperature of
or allowed. Each tube is provided with a cylindrical disc
the liquid at 35-39 °C.
9.5 ± 0.15 mm thick and 20.7 ± 0.15 mm in diameter.
The disc is made of a suitable, transparent plastic material The design of the basket-rack assembly may be varied provided
having a specific gravity of 1.18-1.20. 5 parallel 2 ± 0.1 mm the specifications for the tubes and wire mesh are maintained.
(1) This chapter has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.
General Notices (1) apply to all monographs and other texts 285
2.9.1. Disintegration of tablets and capsules EUROPEAN PHARMACOPOEIA 8.0
Method. Test 6 tablets or capsules either by using 2 basket-rack the specified liquid. Operate the apparatus for the prescribed
assemblies in parallel or by repeating the procedure. In each period, withdraw the assembly and examine the state of the
of the 3 tubes, place 1 tablet or capsule and, if prescribed, tablets or capsules. To pass the test, all 6 of the tablets or
add a disc ; suspend the assembly in the beaker containing capsules must have disintegrated.♦
Dimensions in millimetres
General Notices (1) apply to all monographs and other texts 287
2.9.3. Dissolution test for solid dosage forms EUROPEAN PHARMACOPOEIA 8.0
Figure 2.9.2.-2.
01/2012:20903
APPARATUS
Apparatus 1 (Basket apparatus). The assembly consists
of the following : a vessel, which may be covered, made of
glass or other inert, transparent material(2) ; a motor ; a drive
shaft ; and a cylindrical basket (stirring element). The vessel is
partially immersed in a suitable water-bath of any convenient
size or heated by a suitable device such as a heating jacket.
The water-bath or heating device permits maintaining the
temperature inside the vessel at 37 ± 0.5 °C during the test
Figure 2.9.2.-1. – Apparatus for disintegration of suppositories and keeping the dissolution medium in constant, smooth
and pessaries motion. No part of the assembly, including the environment in
which the assembly is placed, contributes significant motion,
Dimensions in millimetres agitation, or vibration beyond that due to the smoothly
rotating stirring element. Apparatus that permits observation
of the preparation and stirring element during the test is
preferable. The vessel is cylindrical, with a hemispherical
METHOD OF OPERATION FOR VAGINAL TABLETS
bottom and a capacity of 1 L. Its height is 160-210 mm and its
Use the apparatus described above, arranged so as to rest on inside diameter is 98-106 mm. Its sides are flanged at the top.
(3)
the hooks (see Figure 2.9.2.-2). Place it in a beaker of suitable A fitted cover may be used to retard evaporation . The shaft
diameter containing water maintained at 36-37 °C with the is positioned so that its axis is not more than 2 mm at any
level just below the upper perforated disc. Using a pipette, point from the vertical axis of the vessel and rotates smoothly
adjust the level with water at 36-37 °C until a uniform film and without significant wobble that could affect the results. A
covers the perforations of the disc. Use three vaginal tablets. speed-regulating device is used that allows the shaft rotation
Place each one on the upper plate of an apparatus and cover speed to be selected and maintained at a specified rate, within
the latter with a glass plate to maintain appropriate conditions ± 4 per cent.
of humidity. Examine the state of the samples after the period Shaft and basket components of the stirring element are
prescribed in the monograph. To pass the test all the samples fabricated of stainless steel, type 316 or equivalent, to the
must have disintegrated. specifications shown in Figure 2.9.3.-1.
(2) The materials must not sorb, react, or interfere with the preparation to be tested.
(3) If a cover is used, it provides sufficient openings to allow ready insertion of the thermometer and withdrawal of samples.
A basket having a gold coating of about 2.5 μm (0.0001 inch) the test. The metallic or suitably inert, rigid blade and shaft
thick may be used. The dosage unit is placed in a dry basket comprise a single entity. A suitable two-part detachable design
at the beginning of each test. The distance between the may be used provided the assembly remains firmly engaged
inside bottom of the vessel and the bottom of the basket is during the test. The paddle blade and shaft may be coated with
maintained at 25 ± 2 mm during the test. a suitable coating so as to make them inert. The dosage unit
is allowed to sink to the bottom of the vessel before rotation
of the blade is started. A small, loose piece of non-reactive
material, such as not more than a few turns of wire helix, may
be attached to dosage units that would otherwise float. An
alternative sinker device is shown in Figure 2.9.3.-3. Other
validated sinker devices may be used.
Apparatus 3 (Reciprocating cylinder). The assembly consists
of a set of cylindrical, flat-bottomed glass vessels ; a set of
glass reciprocating cylinders ; inert fittings (stainless steel
type 316 or other suitable material) and screens that are made
of suitable nonsorbing and nonreactive material, and that
are designed to fit the tops and bottoms of the reciprocating
cylinders ; a motor and drive assembly to reciprocate the
cylinders vertically inside the vessels, and if desired, index
the reciprocating cylinders horizontally to a different row
of vessels. The vessels are partially immersed in a suitable
water-bath of any convenient size that permits holding the
temperature at 37 ± 0.5 °C during the test. No part of the
assembly, including the environment in which the assembly is
placed, contributes significant motion, agitation, or vibration
beyond that due to the smooth, vertically reciprocating
cylinder. A device is used that allows the reciprocation
rate to be selected and maintained at the specified dip rate,
within ± 5 per cent. An apparatus that permits observation of
the preparations and reciprocating cylinders is preferable. The
vessels are provided with an evaporation cap that remains in
place for the duration of the test. The components conform
to the dimensions shown in Figure 2.9.3.-4 unless otherwise
specified.
Apparatus 4 (Flow-through cell). The assembly consists
of a reservoir and a pump for the dissolution medium ; a
flow-through cell ; a water-bath that maintains the dissolution
medium at 37 ± 0.5 °C. Use the specified cell size.
The pump forces the dissolution medium upwards through
the flow-through cell. The pump has a delivery range
between 240 mL/h and 960 mL/h, with standard flow rates
of 4 mL/min, 8 mL/min, and 16 mL/min. It must deliver a
constant flow (± 5 per cent of the nominal flow rate) ; the flow
profile is sinusoidal with a pulsation of 120 ± 10 pulses/min.
A pump without pulsation may also be used. Dissolution test
procedures using the flow-through cell must be characterised
with respect to rate and any pulsation.
The flow-through cell (see Figures 2.9.3.-5 and 2.9.3.-6) of
transparent and inert material is mounted vertically, with a
filter system that prevents escape of undissolved particles
from the top of the cell ; standard cell diameters are 12 mm
and 22.6 mm ; the bottom cone is usually filled with small
1) Screen with welded seam : 0.22-0.31 mm wire
diameter with wire opening of 0.36-0.44 mm. glass beads of about 1 mm diameter, with 1 bead of about
After welding the screen may be slighty altered. 5 mm positioned at the apex to protect the fluid entry tube ; a
2) Maximum allowable runout at “A” is 1.0 mm tablet holder (see Figures 2.9.3.-5 and 2.9.3.-6) is available for
when the part is rotated on center line axis with positioning of special dosage forms. The cell is immersed in a
basket mounted. water-bath, and the temperature is maintained at 37 ± 0.5 °C.
Figure 2.9.3.-1. – Apparatus 1, Basket stirring element The apparatus uses a clamp mechanism and 2 O-rings for the
Dimensions in millimetres fixation of the cell assembly. The pump is separated from
the dissolution unit in order to shield the latter against any
Apparatus 2 (Paddle apparatus). Use the assembly from vibrations originating from the pump. The position of the
Apparatus 1, except that a paddle formed from a blade and a pump must not be on a level higher than the reservoir flasks.
shaft is used as the stirring element. The shaft is positioned so Tube connections are as short as possible. Use suitably inert
that its axis is not more than 2 mm from the vertical axis of the tubing, such as polytetrafluoroethylene, with a 1.6 mm inner
vessel, at any point, and rotates smoothly without significant diameter and inert flanged-end connections.
wobble that could affect the results. The vertical center line
of the blade passes through the axis of the shaft so that the Apparatus suitability. The determination of suitability of
bottom of the blade is flush with the bottom of the shaft. The the apparatus to perform dissolution testing must include
paddle conforms to the specifications shown in Figure 2.9.3.-2. conformance to the dimensions and tolerances of the
The distance of 25 ± 2 mm between the bottom of the blade apparatus as given above. In addition, critical test parameters
and the inside bottom of the vessel is maintained during that have to be monitored periodically during use include
General Notices (1) apply to all monographs and other texts 289
2.9.3. Dissolution test for solid dosage forms EUROPEAN PHARMACOPOEIA 8.0
A and B dimensions do not vary more than 0.5 mm when part is rotated on center line axis. Tolerances
are ± 1.0 mm unless otherwise stated.
Figure 2.9.3.-2. – Apparatus 2, Paddle stirring element
Dimensions in millimetres
volume and temperature of the dissolution medium, rotation PROCEDURE
speed (Apparatus 1 and 2), dip rate (Apparatus 3), and flow APPARATUS 1 AND 2
rate of medium (Apparatus 4).
Conventional-release solid dosage forms
Determine the acceptable performance of the dissolution test
assembly periodically. Procedure. Place the stated volume of the dissolution medium
(± 1 per cent) in the vessel of the specified apparatus.
Assemble the apparatus, equilibrate the dissolution medium
to 37 ± 0.5 °C, and remove the thermometer. The test may
also be carried out with the thermometer in place, provided
it is shown that results equivalent to those obtained without
the thermometer are obtained.
Place 1 dosage unit in the apparatus, taking care to exclude 1 dosage unit in the apparatus, cover the vessel and operate
air bubbles from the surface of the dosage unit. Operate the apparatus at the specified rate. After 2 h of operation
the apparatus at the specified rate. Within the time interval in 0.1 M hydrochloric acid, withdraw an aliquot of the fluid
specified, or at each of the times stated, withdraw a specimen and proceed immediately as directed under Buffer stage.
from a zone midway between the surface of the dissolution Perform an analysis of the aliquot using a suitable assay
medium and the top of the rotating basket or blade, not less method.
than 1 cm from the vessel wall. Where multiple sampling
times are specified, replace the aliquots withdrawn for analysis – Buffer stage. Complete the operations of adding the buffer
with equal volumes of fresh dissolution medium at 37 °C or, and adjusting the pH within 5 min. With the apparatus
where it can be shown that replacement of the medium is not operating at the rate specified, add to the fluid in the
necessary, correct for the volume change in the calculation. vessel 250 mL of a 0.20 M solution of trisodium phosphate
Keep the vessel covered for the duration of the test and verify dodecahydrate R that has been equilibrated to 37 ± 0.5 °C.
the temperature of the medium at suitable times. Perform the Adjust, if necessary, with 2 M hydrochloric acid R or 2 M
analysis using a suitable assay method(4). Repeat the test with sodium hydroxide R to a pH of 6.8 ± 0.05. Continue to
additional dosage units. operate the apparatus for 45 min, or for the specified time.
At the end of the time period, withdraw an aliquot of
If automated equipment is used for sampling or the apparatus the fluid and perform the analysis using a suitable assay
is otherwise modified, verification that the modified apparatus method.
will produce results equivalent to those obtained with the
apparatus described in this chapter, is necessary. Method B
Dissolution medium. A suitable dissolution medium is used. – Acid Stage. Place 1000 mL of 0.1 M hydrochloric acid in
The volume specified refers to measurements made between the vessel and assemble the apparatus. Allow the medium
20 °C and 25 °C. If the dissolution medium is a buffered to equilibrate to a temperature of 37 ± 0.5 °C. Place
solution, adjust the solution so that its pH is within 0.05 units 1 dosage unit in the apparatus, cover the vessel, and operate
of the specified pH. Dissolved gases can cause bubbles to the apparatus at the specified rate. After 2 h of operation in
form, which may change the results of the test. In such cases, 0.1 M hydrochloric acid, withdraw an aliquot of the fluid,
dissolved gases must be removed prior to testing(5). and proceed immediately as directed under Buffer stage.
Time. Where a single time specification is given, the test Perform an analysis of the aliquot using a suitable assay
may be concluded in a shorter period if the requirement method.
for minimum amount dissolved is met. Samples are to be – Buffer stage. For this stage of the procedure use buffer
withdrawn only at the stated times, within a tolerance of that has previously been equilibrated to a temperature of
± 2 per cent. 37 ± 0.5 °C. Drain the acid from the vessel and add 1000 mL
Prolonged-release solid dosage forms of pH 6.8 phosphate buffer, prepared by mixing 3 volumes
of 0.1 M hydrochloric acid with 1 volume of a 0.20 M
Procedure. Proceed as described for conventional-release
solution of trisodium phosphate dodecahydrate R and
dosage forms.
adjusting, if necessary, with 2 M hydrochloric acid R or 2 M
Dissolution medium. Proceed as described for sodium hydroxide R to a pH of 6.8 ± 0.05. This may also be
conventional-release dosage forms. accomplished by removing from the apparatus the vessel
Time. The test-time points, generally 3, are expressed in hours. containing the acid and replacing it with another vessel,
containing the buffer and transferring the dosage unit to
Delayed-release solid dosage forms the vessel containing the buffer. Continue to operate the
Procedure. Use Method A or Method B. apparatus for 45 min, or for the specified time. At the end
Method A of the time period, withdraw an aliquot of the fluid and
perform the analysis using a suitable assay method.
– Acid stage. Place 750 mL of 0.1 M hydrochloric acid in the
vessel, and assemble the apparatus. Allow the medium Time. All test times stated are to be observed within a
to equilibrate to a temperature of 37 ± 0.5 °C. Place tolerance of ± 2 per cent, unless otherwise specified.
(4) Test specimens are filtered immediately upon sampling unless filtration is demonstrated to be unnecessary. Use an inert filter that does not cause adsorption of the active substance or
contain extractable substances that would interfere with the analysis.
(5) A method of deaeration is as follows : heat the medium, while stirring gently, to about 41 °C, immediately filter under vacuum using a filter having a porosity of 0.45 μm or less, with
vigorous stirring, and continue stirring under vacuum for about 5 min. Other validated deaeration techniques for removal of dissolved gases may be used.
General Notices (1) apply to all monographs and other texts 291
2.9.3. Dissolution test for solid dosage forms EUROPEAN PHARMACOPOEIA 8.0
APPARATUS 3
Conventional-release solid dosage forms
Procedure. Place the stated volume of the dissolution medium
(± 1 per cent) in each vessel of the apparatus. Assemble the
apparatus, equilibrate the dissolution medium to 37 ± 0.5 °C,
and remove the thermometer. Place 1 dosage unit in each of
the reciprocating cylinders, taking care to exclude air bubbles
from the surface of each dosage unit, and immediately operate
the apparatus as specified. During the upward and downward
stroke, the reciprocating cylinder moves through a total
distance of 9.9-10.1 cm. Within the time interval specified, or
at each of the times stated, raise the reciprocating cylinders
and withdraw a portion of the medium from a zone midway
between the surface of the dissolution medium and the bottom
of each vessel. Perform the analysis as directed. If necessary,
repeat the test with additional dosage units.
Replace the aliquot withdrawn for analysis with equal volumes
of fresh dissolution medium at 37 °C or, where it can be shown
that replacement of the medium is not necessary, correct for
the volume change in the calculation. Keep the vessel covered
with the evaporation cap for the duration of the test and verify
the temperature of the medium at suitable times.
Dissolution medium. Proceed as described for
conventional-release dosage forms under Apparatus 1 and 2.
Time. Proceed as described for conventional-release dosage
forms under Apparatus 1 and 2.
Prolonged-release dosage forms
Procedure. Proceed as described for conventional-release
dosage forms under Apparatus 3.
Dissolution medium. Proceed as described for
prolonged-release dosage forms under Apparatus 1 and 2.
Time. Proceed as described for prolonged-release dosage
forms under Apparatus 1 and 2.
Delayed-release dosage forms
Procedure. Proceed as described for delayed-release dosage
forms, Method B, under Apparatus 1 and 2, using one row
of vessels for the acid stage media and the following row of
vessels for the buffer stage media, and using the volume of
medium specified (usually 300 mL).
Time. Proceed as directed for delayed-release dosage forms
under Apparatus 1 and 2.
APPARATUS 4
Conventional-release dosage forms
Procedure. Place the glass beads into the cell specified. Place
1 dosage unit on top of the beads or, if specified, on a wire
carrier. Assemble the filter head and fix the parts together
by means of a suitable clamping device. Introduce by the
pump the dissolution medium warmed to 37 ± 0.5 °C through
the bottom of the cell to obtain the flow rate specified and
measured with an accuracy of 5 per cent. Collect the eluate by
fractions at each of the times stated. Perform the analysis as
directed. Repeat the test with additional dosage units.
Dissolution medium. Proceed as described for
conventional-release dosage forms under Apparatus 1 and 2.
Time. Proceed as described for conventional-release dosage
forms under Apparatus 1 and 2.
Prolonged-release dosage forms
Procedure. Proceed as described for conventional-release
dosage forms under Apparatus 4.
Dissolution medium. Proceed as described for
conventional-release dosage forms under Apparatus 4.
Figure 2.9.3.-4. – Apparatus 3, glass vessel and reciprocating Time. Proceed as described for conventional-release dosage
cylinder forms under Apparatus 4.
Dimensions in millimetres unless otherwise specified Delayed-release dosage forms
Procedure. Proceed as described for delayed-release dosage
forms under Apparatus 1 and 2, using the specified media.
Figure 2.9.3.-5. – Apparatus 4, large cell for tablets and capsules (top), tablet holder for the large cell (bottom)
Dimensions in millimetres unless otherwise specified
Time. Proceed as described for delayed-release dosage forms tested conform to Table 2.9.3.-1. Continue testing through
under Apparatus 1 and 2. the 3 levels unless the results conform at either S1 or S2.
The quantity Q, is the specified amount of dissolved active
INTERPRETATION substance, expressed as a percentage of the labelled content ;
Conventional-release solid dosage forms the 5 per cent, 15 per cent, and 25 per cent values in the Table
are percentages of the labelled content so that these values
Unless otherwise specified, the requirements are met if the and Q are in the same terms.
quantities of active substance dissolved from the dosage units
General Notices (1) apply to all monographs and other texts 293
2.9.3. Dissolution test for solid dosage forms EUROPEAN PHARMACOPOEIA 8.0
Figure 2.9.3.-6. – Apparatus 4, small cell for tablets and capsules (top), tablet holder for the small cell (bottom)
Dimensions in millimetres unless otherwise specified
General Notices (1) apply to all monographs and other texts 295
2.9.4. Dissolution test for transdermal patches EUROPEAN PHARMACOPOEIA 8.0
not overlap the borders of the SSDA. For this purpose and Cover. The cover has a central opening with a diameter
provided that the preparation is homogeneous and uniformly selected according to the size of the patch to be examined.
spread on the outer covering, an appropriate and exactly The patch can thus be precisely centred, and its releasing
measured piece of the patch may be cut and used for testing surface limited. The following diameters may be used : 20 mm,
the dissolution rate. This procedure may also be necessary 32 mm, 40 mm, 50 mm corresponding to areas of 3.14 cm2,
to achieve appropriate sink conditions. This procedure must 8.03 cm2, 12.56 cm2, 19.63 cm2, respectively. The cover is
not be applied to membrane-type patches. Place the patch held in place by nuts screwed onto bolts projecting from the
mounted on the SSDA flat at the bottom of the vessel with support. The cover is sealed to the support by a rubber ring
the release surface facing upwards. Immediately rotate the set on the reservoir.
paddle at 100 r/min, for example. At predetermined intervals,
withdraw a sample from the zone midway between the surface Extraction cell. The cell holds the patch flat, with the release
of the dissolution medium and the top of the blade, not less surface uppermost and parallel to the bottom of the paddle
than 1 cm from the vessel wall. blade. A distance of 25 ± 2 mm is maintained between the
paddle blade and the surface of the patch (see Figure 2.9.4.-4).
Perform the assay on each sample, correcting for any volume The temperature is maintained at 32 ± 0.5 °C. The vessel may
losses, as necessary. Repeat the test with additional patches. be covered during the test to minimise evaporation.
2. CELL METHOD Procedure. Place the prescribed volume of the dissolution
medium in the vessel and equilibrate the medium to the
Equipment. Use the paddle and vessel assembly from the prescribed temperature. Precisely centre the patch in the
paddle apparatus described in the dissolution test for solid cell with the releasing surface uppermost. Close the cell, if
oral dosage forms (2.9.3) with the addition of the extraction necessary applying a hydrophobic substance (for example,
cell (cell). petrolatum) to the flat surfaces to ensure the seal, and ensure
The cell is made of chemically inert materials and consists of that the patch stays in place. Introduce the cell flat into
a support, a cover and, if necessary, a membrane placed on the bottom of the vessel with the cover facing upwards.
the patch to isolate it from the medium that may modify or Immediately rotate the paddle, at 100 r/min for example. At
adversely affect the physico-chemical properties of the patch predetermined intervals, withdraw a sample from the zone
(see Figure 2.9.4.-3). midway between the surface of the dissolution medium and
the top of the paddle blade, not less than 1 cm from the vessel
wall.
Perform the assay on each sample, correcting for any volume
losses, as necessary. Repeat the test with additional patches.
sides than the patch. Place the patch on a clean surface with 01/2008:20905
the membrane in contact with this surface. Two systems for
adhesion to the cylinder may be used :
– apply a suitable adhesive to the exposed membrane borders
and, if necessary, to the back of the patch, 2.9.5. UNIFORMITY OF MASS OF
– apply a double-sided adhesive tape to the external wall of SINGLE-DOSE PREPARATIONS
the cylinder.
Using gentle pressure, carefully apply the non-adhesive side Weigh individually 20 units taken at random or, for single-dose
of the patch to the cylinder, so that the release surface is in preparations presented in individual containers, the contents
contact with the dissolution medium and the long axis of the of 20 units, and determine the average mass. Not more than
patch fits around the circumference of the cylinder. 2 of the individual masses deviate from the average mass by
The system for adhesion used is previously tested for absence more than the percentage deviation shown in Table 2.9.5.-1
of interference with the assay and of adsorption of the active and none deviates by more than twice that percentage.
ingredient(s).
Place the cylinder in the apparatus, and immediately rotate the For capsules and powders for parenteral administration,
cylinder at 100 r/min, for example. At determined intervals, proceed as described below.
withdraw a sample of dissolution medium from a zone
midway between the surface of the dissolution medium and
the top of the rotating cylinder, and not less than 1 cm from
the vessel wall. CAPSULES
Perform the assay on each sample as directed in the individual Weigh an intact capsule. Open the capsule without losing any
monograph, correcting for any volume withdrawn, as part of the shell and remove the contents as completely as
necessary. Repeat the test with additional patches. possible. For soft shell capsules, wash the shell with a suitable
Interpretation. The requirements are met if the quantity of solvent and allow to stand until the odour of the solvent is no
active ingredient(s) released from the patch, expressed as the longer perceptible. Weigh the shell. The mass of the contents
amount per surface area per time unit, is within the prescribed is the difference between the weighings. Repeat the procedure
limits at the defined sampling times. with another 19 capsules.
General Notices (1) apply to all monographs and other texts 297
2.9.6. Uniformity of content of single-dose preparations EUROPEAN PHARMACOPOEIA 8.0
Effervescent tablets and chewable tablets may have different respect to the direction of application of the force. Carry out
specifications as far as friability is concerned. In the case of the measurement on 10 tablets, taking care that all fragments
hygroscopic tablets, a humidity-controlled environment is of tablets have been removed before each determination.
required for testing. This procedure does not apply when fully automated equipment
A drum with dual scooping projections, or apparatus with is used.
more than one drum, for the running of multiple samples at
one time, are also permitted. EXPRESSION OF RESULTS
Express the results as the mean, minimum and maximum
values of the forces measured, all expressed in newtons.
Indicate the type of apparatus and, where applicable, the
01/2008:20908 orientation of the tablets.
General Notices (1) apply to all monographs and other texts 299
2.9.9. Measurement of consistency by penetrometry EUROPEAN PHARMACOPOEIA 8.0
PROCEDURE
Prepare the test samples according to one of the following
procedures.
A. Carefully and completely fill 3 containers, without forming
air bubbles. Level if necessary to obtain a flat surface.
Store the samples at 25 ± 0.5 °C for 24 h, unless otherwise
prescribed.
B. Store 3 samples at 25 ± 0.5 °C for 24 h, unless otherwise
prescribed. Apply a suitable shear to the samples for 5 min.
Carefully and completely fill 3 containers, without forming
Figure 2.9.9.-1. – Penetrometer air bubbles, and level if necessary to obtain a flat surface.
A. Scale showing the depth of penetration, graduated in C. Melt 3 samples and carefully and completely fill
tenths of millimetres. 3 containers, without forming air bubbles. Store the
B. Vertical shaft to maintain and guide the penetrating object. samples at 25 ± 0.5 °C for 24 h, unless otherwise prescribed.
C. Device to retain and to release the penetrating object Determination of penetration. Place the test sample on
automatically and for a constant time. the base of the penetrometer. Verify that its surface is
D. Device to ensure that the penetrating object is vertical and perpendicular to the vertical axis of the penetrating object.
that the base is horizontal. Bring the temperature of the penetrating object to 25 ± 0.5 °C
E. Penetrating object (see Figures 2.9.9.-2 and 3). and then adjust its position such that its tip just touches the
surface of the sample. Release the penetrating object and hold
F. Container. it free for 5 s. Clamp the penetrating object and measure the
G. Horizontal base. depth of penetration. Repeat the test with the 2 remaining
H. Control for the horizontal base. containers.
Figure 2.9.9.-2. – Cone (m = 102.5 ± 0.05 g), suitable container (d = 102 mm or 75 mm ; h ≥ 62 mm)
and shaft (l = 162 mm ; m = 47.5 ± 0.05 g).
Dimensions in millimetres
Figure 2.9.9.-3 – Micro-cone (m = 7.0 g), suitable container and shaft (l = 116 mm ; m = 16.8 g)
Dimensions in millimetres
EXPRESSION OF THE RESULTS
The penetration is expressed in tenths of a millimetre
as the arithmetic mean of the 3 measurements. If any of
the individual results differ from the mean by more than
3 per cent, repeat the test and express the results of the 6
measurements as the mean and the relative standard deviation.
04/2013:20910
METHOD A
Where preparations contain dissolved substances, the
dissolved substances must be separated from the ethanol that Figure 2.9.10.-1. – Apparatus for the determination of ethanol
is to be determined by distillation. Where distillation would content
distil volatile substances other than ethanol and water, the Dimensions in millimetres
appropriate precautions are stated in the monograph. Apparatus. The apparatus (see Figure 2.9.10.-1) consists of a
round-bottomed flask (A) fitted with a distillation head (B)
The relation between the density at 20 ± 0.1 °C, the relative
with a steam trap and attached to a vertical condenser (C). The
density (corrected to vacuum) and the ethanol content of
latter is fitted at its lower part with a tube (D), which carries the
a mixture of water and ethanol is given in the tables of the
distillate into the lower part of a 100 mL or 250 mL volumetric
International Organisation for Legal Metrology (1972),
flask. The volumetric flask is immersed in a mixture of ice
International Recommendation No. 22.
General Notices (1) apply to all monographs and other texts 301
2.9.10. Ethanol content EUROPEAN PHARMACOPOEIA 8.0
and water (E) during the distillation. A disc having a circular ρ20 Relative density of the Ethanol content in
aperture 6 cm in diameter is placed under the flask (A) to (kg·m− 3) distillate measured in air per cent V/V
reduce the risk of charring of any dissolved substances. at 20 °C
Method 983.0 0.9848 11.44
983.5 0.9853 11.02
Pycnometer method/oscillating transducer density meter
method. Transfer 25.0 mL of the preparation to be examined, 984.0 0.9858 10.60
measured at 20 ± 0.1 °C, to the distillation flask. Dilute with
984.5 0.9863 10.18
100-150 mL of distilled water R and add a few pieces of
pumice. Attach the distillation head and condenser. Distil and 985.0 0.9868 9.76
collect not less than 90 mL of distillate in a 100 mL volumetric
985.5 0.9873 9.35
flask. Adjust the temperature to 20 ± 0.1 °C and dilute to
100.0 mL with distilled water R at 20 ± 0.1 °C. Determine 986.0 0.9878 8.94
the relative density at 20 ± 0.1 °C using a pycnometer or an
986.5 0.9883 8.53
oscillating transducer density meter.
987.0 0.9888 8.13
The values indicated in Table 2.9.10.-1, column 3, are
multiplied by 4 to obtain the percentage of ethanol by volume 987.5 0.9893 7.73
(V/V) contained in the preparation. After calculation of the 988.0 0.9898 7.34
ethanol content using the table, round off the result to 1
decimal place. 988.5 0.9903 6.95
989.0 0.9908 6.56
Table 2.9.10.-1. – Relationship between density, relative density
and ethanol content 989.5 0.9913 6.17
General Notices (1) apply to all monographs and other texts 303
2.9.11. Test for methanol and 2-propanol EUROPEAN PHARMACOPOEIA 8.0
04/2013:20911 Calculate the methanol content in per cent V/V using the
following expression :
Temperature : 01/2008:20912
Time Temperature
(min) (°C)
2.9.12. SIEVE TEST
Column 0 - 1.6 40
The degree of fineness of a powder may be expressed by
1.6 - 9.9 40 → 65
reference to sieves that comply with the specifications for
9.9 - 13.6 65 → 175 non-analytical sieves (2.1.4).
13.6 - 20 175 Where the degree of fineness of powders is determined by
Injection port 200
sieving, it is defined in relation to the sieve number(s) used
either by means of the following terms or, where such terms
Detector 200 cannot be used, by expressing the fineness of the powder as a
percentage m/m passing the sieve(s) used.
Detection : flame ionisation. The following terms are used in the description of powders :
Coarse powder. Not less than 95 per cent by mass passes
Injection : 1.0 μL of the test solution and reference solution (c), through a number 1400 sieve and not more than 40 per cent
at least 3 times. by mass passes through a number 355 sieve.
Moderately fine powder. Not less than 95 per cent by mass
Elution order : methanol, ethanol, 2-propanol, 1-propanol. passes through a number 355 sieve and not more than 40 per
cent by mass passes through a number 180 sieve.
Relative retention with reference to ethanol (retention Fine powder. Not less than 95 per cent by mass passes
time = about 5.3 min): methanol = about 0.8 ; through a number 180 sieve and not more than 40 per cent by
2-propanol = about 1.2 ; 1-propanol = about 1.6. mass passes through a number 125 sieve.
Very fine powder. Not less than 95 per cent by mass passes
System suitability: reference solution (c) : through a number 125 sieve and not more than 40 per cent by
mass passes through a number 90 sieve.
– resolution : minimum 5 between the peaks due to methanol
and ethanol. If a single sieve number is given, not less than 97 per cent of
the powder passes through the sieve of that number, unless
otherwise prescribed.
Calculate the methanol content in per cent V/V using the
following expression : Assemble the sieves and operate in a suitable manner until
sifting is practically complete. Weigh the separated fractions
of the powder.
APPARATUS
A3 = area of the peak due to 2-propanol in the The apparatus consists of the following parts :
chromatogram obtained with the test solution ;
(a) a permeability cell (see Figure 2.9.14.-1), which consists
A4 = area of the peak due to 2-propanol in the
of a cylinder with an inner diameter of 12.6 ± 0.1 mm (A),
chromatogram obtained with reference constructed of glass or non-corroding metal. The bottom
solution (c) ; of the cell forms an airtight connection (for example, via an
I1 = area of the peak due to the internal standard in the adapter) with the manometer (Figure 2.9.14.-2). A ledge
chromatogram obtained with the test solution ; 0.5 mm to 1 mm in width is located 50 ± 15 mm from the
I2 = area of the peak due to the internal standard top of the cell. It is an integral part of the cell or firmly fixed
in the chromatogram obtained with reference so as to be airtight. It supports a perforated metal disk (B),
solution (c). constructed of non-corroding metal. The disk has a thickness
of 0.9 ± 0.1 mm and is perforated with thirty to forty holes
1 mm in diameter evenly distributed over this area.
General Notices (1) apply to all monographs and other texts 305
2.9.14. Specific surface area by air permeability EUROPEAN PHARMACOPOEIA 8.0
(1) (2)
t = flow time in seconds, The density of mercury and the viscosity of air over a range of
η temperatures are shown in Table 2.9.14.-1.
= dynamic viscosity of air in millipascal seconds (see
Table 2.9.14.-1), Table 2.9.14.-1.
K = apparatus constant determined according to
Equation (4), Temperature Density of mercury Viscosity of air (η)
(°C) (g/mL) (mPa·s)
ρ = density of the substance to be examined in grams
16 13.56 0.01800 0.1342
per millilitre,
ε = porosity of the compacted bed of powder. 17 13.56 0.01805 0.1344
18 13.55 0.01810 0.1345
19 13.55 0.01815 0.1347
CALIBRATION OF THE APPARATUS
20 13.55 0.01819 0.1349
The bulk volume of the compacted bed of powder is
21 13.54 0.01824 0.1351
determined by the mercury displacement method as follows :
22 13.54 0.01829 0.1353
Place two filter paper disks in the permeability cell, pressing
down the edges with a rod slightly smaller than the cell 23 13.54 0.01834 0.1354
diameter until the filter disks lie flat on the perforated metal 24 13.54 0.01839 0.1356
disk ; fill the cell with mercury, removing any air bubbles
adhering to the wall of the cell and wipe away the excess to
create a plane surface of mercury at the top of the cell. If the
cell is made of material that will amalgamate, grease the cell
and the metal disk first with a thin layer of liquid paraffin.
Pour out the mercury into a tared beaker and determine the
mass (MA) and the temperature of the mercury. 01/2008:20916
Make a compacted bed using the reference powder and
again fill the cell with mercury with a planar surface at the
top of the cell. Pour out the mercury in a tared beaker and
2.9.16. FLOWABILITY
again determine the mass of the mercury (MB). Calculate the
bulk volume (V) of the compacted bed of powder from the The test for flowability is intended to determine the ability of
following expression : divided solids (for example, powders and granules) to flow
vertically under defined conditions.
(3)
APPARATUS
= difference between the determined masses According to the flow properties of the material to be tested,
of mercury in grams, funnels with or without stem, with different angles and
orifice diameters are used. Typical apparatuses are shown
ρHg = density of mercury at the determined in Figures 2.9.16.-1 and 2.9.16.-2. The funnel is maintained
temperature in grams per millilitre. upright by a suitable device. The assembly must be protected
from vibrations.
Repeat the procedure twice, changing the powder each time ;
the range of values for the calculated volume (V) is not greater
than 0.01 mL. Use the mean value of the three determined METHOD
volumes for the calculations.
Into a dry funnel, whose bottom opening has been blocked by
The apparatus constant K is determined using a reference suitable means, introduce without compacting a test sample
powder with known specific surface area and density as weighed with 0.5 per cent accuracy. The amount of the sample
follows : depends on the apparent volume and the apparatus used.
Unblock the bottom opening of the funnel and measure the
Calculate the required quantity of the reference powder to time needed for the entire sample to flow out of the funnel.
be used (Eq. 1) using the stated density and the determined Carry out three determinations.
volume of the compacted powder bed (Eq. 3).
Homogenise and loosen up the powder by shaking it for 2 min EXPRESSION OF RESULTS
in a 100 mL bottle. Prepare a compacted powder bed and
measure the flow time of air as previously described. Calculate The flowability is expressed in seconds and tenths of seconds,
the apparatus constant (K) from the following expression : related to 100 g of sample.
The results depend on the storage conditions of the material
(4) to be tested.
The results can be expressed as the following :
Ssp = stated specific surface area of the reference powder, a) the mean of the determinations, if none of the individual
ρ = density of the substance to be examined in grams values deviates from the mean value by more than 10 per
per millilitre, cent ;
ε = porosity of the compacted bed of powder, b) as a range, if the individual values deviate from the mean
value by more than 10 per cent ;
t = flow time in seconds,
η c) as a plot of the mass against the flow time ;
= dynamic viscosity of air in millipascal seconds (see
Table 2.9.14.-1). d) as an infinite time, if the entire sample fails to flow through.
General Notices (1) apply to all monographs and other texts 307
2.9.17. Test for extractable volume of parenteral preparations EUROPEAN PHARMACOPOEIA 8.0
04/2010:20917
This test is used to determine the fine particle characteristics – ground-glass inlet socket 29/32
of the aerosol clouds generated by preparations for inhalation.
– ground-glass outlet cone 24/29
Unless otherwise justified and authorised, one of the following C Neck Modified glass adapter :
apparatus and test procedures is used.
– ground-glass inlet socket 24/29
Stage mensuration is performed periodically together with
confirmation of other dimensions critical to the effective – ground-glass outlet cone 24/29
operation of the impactor. Lower outlet section of
Re-entrainment (for apparatus D and E). To ensure efficient precision-bore glass tubing:
particle capture, coat each plate with glycerol, silicone oil or – bore diameter 14
similar high viscosity liquid, typically deposited from a volatile Selected bore light-wall glass
solvent. Plate coating must be part of method validation and tubing :
may be omitted where justified and authorised. – external diameter 17
Mass balance. The total mass of the active substance is not less D Upper Modified round-bottomed flask 100 mL
than 75 per cent and not more than 125 per cent of the average
delivered dose determined during testing for uniformity of impingement – ground-glass inlet socket 24/29
delivered dose. This is not a test of the inhaler but it serves to chamber – ground-glass outlet cone 24/29
ensure that the results are valid.
E Coupling tube Medium-wall glass tubing :
The apparatus is shown in Figure 2.9.18.-1 (see also Bent section and upper vertical
Table 2.9.18.-1). section:
– external diameter 13
Glass screwthread :
– thread size 28
General Notices (1) apply to all monographs and other texts 309
2.9.18. Preparations for inhalation EUROPEAN PHARMACOPOEIA 8.0
Introduce the liquid preparation for inhalation into the the discharge sequence. The number of discharges should
reservoir of the nebuliser. Fit the mouthpiece and connect it be minimised and typically would not be greater than 10.
by means of an adapter to the device. Dismantle the apparatus.
Switch on the pump of the apparatus and after 10 s switch Wash the inner surface of the inlet tube to the lower
on the nebuliser. impingement chamber and its outer surface that projects into
the chamber with a suitable solvent, collecting the washings
After 60 s, unless otherwise justified, switch off the nebuliser, in the lower impingement chamber. Determine the content
wait for about 5 s and then switch off the pump of the of active substance in this solution. Calculate the amount of
apparatus. Dismantle the apparatus and wash the inner
active substance collected in the lower impingement chamber
surface of the upper impingement chamber collecting the per discharge and express the results as a percentage of the
washings in a volumetric flask. Wash the inner surface of the dose stated on the label.
lower impingement chamber collecting the washings in a
second volumetric flask. Finally, wash the filter preceding the
pump and its connections to the lower impingement chamber Fine particle dose and particle size
and combine the washings with those obtained from the
lower impingement chamber. Determine the amount of active distribution
substance collected in each of the 2 flasks. Express the results
for each of the 2 parts of the apparatus as a percentage of the APPARATUS C - MULTI-STAGE LIQUID IMPINGER
total amount of active substance. The multi-stage liquid impinger consists of impaction stages 1
Procedure for pressurised inhalers (pre-separator), 2, 3 and 4 and an integral filter stage (stage 5),
see Figures 2.9.18.-4/6. An impaction stage comprises an
Place the actuator adapter in position at the end of the throat upper horizontal metal partition wall (B) through which
so that the mouthpiece end of the actuator, when inserted to a a metal inlet jet tube (A) with its impaction plate (D) is
depth of about 10 mm, lines up along the horizontal axis of protruding. A glass cylinder (E) with sampling port (F) forms
the throat and the open end of the actuator, which accepts the the vertical wall of the stage, and a lower horizontal metal
pressurised container, is uppermost and in the same vertical partition wall (G) through which the tube (H) connects to the
plane as the rest of the apparatus. next lower stage. The tube into stage 4 (U) ends in a multi-jet
Introduce 7 mL and 30 mL of a suitable solvent into the upper arrangement. The impaction plate (D) is secured in a metal
and lower impingement chambers, respectively. frame (J) which is fastened by 2 wires (K) to a sleeve (L)
secured on the jet tube. The horizontal face of the collection
Connect all the component parts. Ensure that the assembly plate is perpendicular to the axis of the jet tube and centrally
is vertical and adequately supported and that the lower aligned. The upper surface of the impaction plate is slightly
jet-spacer peg of the lower jet assembly just touches the raised above the edge of the metal frame. A recess around the
bottom of the lower impingement chamber. Connect a perimeter of the horizontal partition wall guides the position
suitable pump to the outlet of the apparatus. Adjust the air of the glass cylinder. The glass cylinders are sealed against
flow through the apparatus, as measured at the inlet to the the horizontal partition walls with gaskets (M) and clamped
throat, to 60 ± 5 L/min. together by 6 bolts (N). The sampling ports are sealed by
Prime the metering valve by shaking for 5 s and discharging stoppers. The bottom-side of the lower partition wall of
once to waste ; after not less than 5 s, shake and discharge stage 4 has a concentrical protrusion fitted with a rubber
again to waste. Repeat a further 3 times. O-ring (P) which seals against the edge of a filter placed in
the filter holder. The filter holder (R) is constructed as a
Shake for about 5 s, switch on the pump to the apparatus basin with a concentrical recess in which a perforated filter
and locate the mouthpiece end of the actuator in the adapter, support (S) is flush-fitted. The filter holder is dimensioned for
discharge once immediately. Remove the assembled inhaler 76 mm diameter filters. The assembly of impaction stages is
from the adapter, shake for not less than 5 s, relocate the clamped onto the filter holder by 2 snap-locks (T). Connect
mouthpiece end of the actuator in the adapter and discharge an induction port (see Figure 2.9.18.-7) onto the stage 1 inlet
again. Repeat the discharge sequence. The number of jet tube of the impinger. A rubber O-ring on the jet tube
discharges should be minimised and typically would not be provides an airtight connection to the induction port. A
greater than 10. After the final discharge wait for not less than suitable mouthpiece adapter is used to provide an airtight seal
5 s and then switch off the pump. Dismantle the apparatus. between the inhaler and the induction port. The front face
Wash the inner surface of the inlet tube to the lower of the inhaler mouthpiece must be flush with the front face
impingement chamber and its outer surface that projects into of the induction port.
the chamber with a suitable solvent, collecting the washings
in the lower impingement chamber. Determine the content Table 2.9.18.-2. – Component specification for apparatus C in
of active substance in this solution. Calculate the amount of Figures 2.9.18.-4/6
active substance collected in the lower impingement chamber Code* Item Description Dimen-
per discharge and express the results as a percentage of the sions**
dose stated on the label.
A,H Jet tube Metal tube screwed onto partition see Figure
Procedure for powder inhalers wall sealed by gasket (C), polished 2.9.18.-5
inner surface
Introduce 7 mL and 30 mL of a suitable solvent into the upper
and lower impingement chambers, respectively. B,G Partition Circular metal plate
wall
Connect all the component parts. Ensure that the assembly is
vertical and adequately supported and that the jet-spacer peg – diameter 120
of the lower jet assembly just touches the bottom of the lower – thickness see Figure
impingement chamber. Without the inhaler in place, connect 2.9.18.-5
a suitable pump to the outlet of the apparatus. Adjust the air
C Gasket e.g. PTFE to fit jet
flow through the apparatus, as measured at the inlet to the tube
throat, to 60 ± 5 L/min.
D Impaction Porosity 0 sintered-glass disk
Prepare the inhaler for use and locate the mouthpiece in the
apparatus by means of a suitable adapter. Switch on the pump plate – diameter see Figure
for 5 s. Switch off the pump and remove the inhaler. Repeat 2.9.18.-5
– height 4
– height 6
– thickness 5
General Notices (1) apply to all monographs and other texts 311
2.9.18. Preparations for inhalation EUROPEAN PHARMACOPOEIA 8.0
Figure 2.9.18.-5. – Apparatus C : details of jet tube and impaction plate. Inserts show end of multi-jet tube U leading to stage 4.
(Numbers and lowercase letters refer to Table 2.9.18.-3 and uppercase letters refer to Figure 2.9.18.-4).
walls and the collection plate of each of the 4 upper stages Type Code(2) Stage 1 Stage 2 Stage 3 Stage 4 Filter
of the apparatus into the solution in the respective stage by (stage 5)
carefully tilting and rotating the apparatus, observing that no Radius(4) r 16 22 27 28.5 0
liquid transfer occurs between the stages. s n.a.
Radius 46 46 46 46
Using a suitable method of analysis, determine the quantity of n.a.
Radius t 50 50 50 50
active substance contained in each of the aliquots of solvent.
Angle w 10° 53° 53° 53° 53°
Calculate the fine particle dose (see Calculations).
Angle u n.a. n.a. n.a. 45° n.a.
Table 2.9.18.-3. – Dimensions(1) of jet tube with impaction plate
v n.a. n.a. n.a. n.a.
of apparatus C Angle 60°
Type Code(2) Stage 1 Stage 2 Stage 3 Stage 4 Filter (1) Measures in millimetres with tolerances according to ISO 2768-m
unless otherwise stated
(stage 5)
n.a. (2) Refer to Figure 2.9.18.-5
Distance 1 9.5 5.5 4.0 6.0
(3) Including gasket
(-.0+.5) (-.0+.5) (-.0+.5) (-.0+.5)
(4) Relative centreline of stage compartment
Distance 2 26 31 33 30.5 0
n.a. = not applicable
Distance 3 8 5 5 5 5
Distance 4 3 3 3 3 n.a.
Distance 5 0 3 3 3 3
(3)
Distance 6 20 25 25 25 25
Distance 7 n.a. n.a. n.a. 8.5 n.a.
Diameter c 25 14 8.0 21 14
(± .1)
Diameter d 50 30 20 30 n.a.
Diameter f 31.75 22 14 31 22
(-.0+.5)
Diameter g 25.4 21 13 30 21
Diameter h n.a. n.a. n.a. 2.70 n.a.
(± .5) Figure 2.9.18.-6. – Apparatus C : details of the filter stage
Diameter j n.a. n.a. n.a. 6.3 n.a. (stage 5). Numbers refer to dimensions (Ø = diameter).
Uppercase letters refer to Table 2.9.18.-2.
Diameter k n.a. n.a. n.a. 12.6 n.a.
Dimensions in millimetres unless otherwise stated
Procedure for powder inhalers Adjust the flow control valve to achieve steady flow through
Place a suitable low resistance filter capable of quantitatively the system at the required rate, Qout (± 5 per cent). Switch off
collecting the active substance in stage 5 and assemble the the pump. Ensure that critical flow occurs in the flow control
apparatus. Connect the apparatus to a flow system according valve by the following procedure.
to the scheme specified in Figure 2.9.18.-8 and Table 2.9.18.-4. With the inhaler in place and the test flow rate established,
Unless otherwise defined, conduct the test at the flow rate, measure the absolute pressure on both sides of the control
Qout, used in the test for uniformity of delivered dose, drawing valve (pressure reading points P2 and P3 in Figure 2.9.18.-8).
4 L of air from the mouthpiece of the inhaler and through A ratio P3/P2 of less than or equal to 0.5 indicates critical flow.
the apparatus. Switch to a more powerful pump and re-measure the test flow
Connect a flowmeter to the induction port. Use a flowmeter rate if critical flow is not indicated.
calibrated for the volumetric flow leaving the meter, or Dispense 20 mL of a solvent, capable of dissolving the active
calculate the volumetric flow leaving the meter (Qout) using substance into each of the 4 upper stages of the apparatus and
the ideal gas law. For a meter calibrated for the entering replace the stoppers. Tilt the apparatus to wet the stoppers,
volumetric flow (Qin), use the following expression : thereby neutralising electrostatic charge. Place a suitable
mouthpiece adapter in position at the end of the induction
port.
P0 = atmospheric pressure,
ΔP = pressure drop over the meter.
General Notices (1) apply to all monographs and other texts 313
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Figure 2.9.18.-9. – Apparatus D : Andersen cascade impactor used for pressurised inhalers
Procedure for powder inhalers may be omitted, where justified and authorised. Stages 6
and 7 may also be omitted at high flow rates, if justified. The
The aerodynamic cut-off diameters of the individual stages of pre-separator may be coated in the same way as the plates
this apparatus are currently not well-established at flow rates or may contain 10 mL of a suitable solvent. Connect the
other than 28.3 L/min. Users must justify and validate the use apparatus to a flow system according to the scheme specified
of the impactor in the chosen conditions, when flow rates in Figure 2.9.18.-8 and Table 2.9.18.-4.
different from 28.3 L/min are selected.
Unless otherwise defined, conduct the test at the flow rate,
Assemble the Andersen impactor with the pre-separator and a Qout, used in the test for uniformity of delivered dose drawing
suitable filter in place and ensure that the system is airtight. 4 L of air from the mouthpiece of the inhaler and through
Depending on the product characteristics, the pre-separator the apparatus.
General Notices (1) apply to all monographs and other texts 315
2.9.18. Preparations for inhalation EUROPEAN PHARMACOPOEIA 8.0
Figure 2.9.18.-10. – Connection of the induction port to the preseparator of the Andersen cascade impactor
Dimensions in millimetres unless otherwise stated
Connect a flowmeter to the induction port. Use a flowmeter into an aliquot of the solvent. Extract the active substance
calibrated for the volumetric flow leaving the meter, or from the inner walls and the collection plate of each of the
calculate the volumetric flow leaving the meter (Qout) using stages of the apparatus into aliquots of solvent.
the ideal gas law. For a meter calibrated for the entering Using a suitable method of analysis, determine the quantity of
volumetric flow (Qin), use the following expression : active substance contained in each of the aliquots of solvent.
Calculate the fine particle dose (see Calculations).
APPARATUS E
P0 = atmospheric pressure,
Apparatus E is a cascade impactor with 7 stages and a
ΔP = pressure drop over the meter. micro-orifice collector (MOC). Over the flow rate range of
30 L/min to 100 L/min the 50 per cent-efficiency cut-off
Adjust the flow control valve to achieve steady flow through diameters (D values) range between 0.24 μm to 11.7 μm,
the system at the required rate, Qout (± 5 per cent). Ensure that evenly spaced50on a logarithmic scale. In this flow range, there
critical flow occurs in the flow control valve by the procedure are always at least 5 stages with D values between 0.5 μm
50
described for Apparatus C. Switch off the pump. and 6.5 μm. The collection efficiency curves for each stage are
Prepare the powder inhaler for use according to the patient sharp and minimise overlap between stages.
instructions. With the pump running and the 2-way solenoid
Material of construction may be aluminium, stainless steel or
valve closed, locate the mouthpiece of the inhaler in the
other suitable material.
mouthpiece adapter. Discharge the powder into the apparatus
by opening the valve for the required time, T (± 5 per cent). The impactor configuration has removable impaction cups
Repeat the discharge sequence. The number of discharges with all the cups in one plane (Figures 2.9.18.-11/14). There
should be minimised and typically would not be greater are 3 main sections to the impactor ; the bottom frame
than 10. The number of discharges is sufficient to ensure an that holds the impaction cups, the seal body that holds the
accurate and precise determination of fine particle dose. jets and the lid that contains the interstage passageways
Dismantle the apparatus. Carefully remove the filter and (Figures 2.9.18.-11/12). Multiple nozzles are used at all but
extract the active substance into an aliquot of the solvent. the first stage (Figure 2.9.18.-13). The flow passes through the
Remove the pre-separator, induction port and mouthpiece impactor in a saw-tooth pattern.
adapter from the apparatus and extract the active substance Critical dimensions are provided in Table 2.9.18.-6.
General Notices (1) apply to all monographs and other texts 317
2.9.18. Preparations for inhalation EUROPEAN PHARMACOPOEIA 8.0
in the same orientation as intended for use. Connect the Prepare the powder inhaler for use according to the patient
apparatus to a flow system according to the scheme specified instructions. With the pump running and the 2-way solenoid
in Figure 2.9.18.-8 and Table 2.9.18.-4. valve closed, locate the mouthpiece of the inhaler in the
mouthpiece adapter. Discharge the powder into the apparatus
Unless otherwise prescribed, conduct the test at the flow rate, by opening the valve for the required time, T (± 5 per cent).
Qout, used in the test for uniformity of delivered dose drawing Repeat the discharge sequence. The number of discharges
4 L of air from the mouthpiece of the inhaler and through should be minimised and typically would not be greater
the apparatus. Connect a flowmeter to the induction port. than 10. The number of discharges is sufficient to ensure an
Use a flowmeter calibrated for the volumetric flow leaving the accurate and precise determination of fine particle dose.
meter, or calculate the volumetric flow leaving the meter (Qout)
using the ideal gas law. For a meter calibrated for the entering Dismantle the apparatus and recover the active substance as
volumetric flow (Qin), use the following expression : follows : remove the induction port and mouthpiece adapter
from the pre-separator, when used, and recover the deposited
active substance into an aliquot of solvent. When used,
remove the pre-separator from the impactor, being careful to
avoid spilling the cup liquid into the impactor. Recover the
P0 = atmospheric pressure, active substance from the pre-separator.
Open the impactor by releasing the handle and lifting the lid.
ΔP = pressure drop over the meter.
Remove the cup tray, with the collection cups, and recover the
active substance in each cup into an aliquot of solvent.
Adjust the flow control valve to achieve steady flow through
the system at the required rate, Qout (± 5 per cent). Ensure that Using a suitable method of analysis, determine the quantity of
critical flow occurs in the flow control valve by the procedure active substance contained in each of the aliquots of solvent.
described for Apparatus C. Switch off the pump. Calculate the fine particle dose (see Calculations).
General Notices (1) apply to all monographs and other texts 319
2.9.18. Preparations for inhalation EUROPEAN PHARMACOPOEIA 8.0
Table 2.9.18.-7. – Calculations for Apparatus C. Use q = , where Q is the test flow rate in litres per minute (Qout for
powder inhalers)
Cut-off diameter Mass of active substance deposited Cumulative mass of active substance Cumulative fraction of active substance
(μm) per discharge deposited per discharge (per cent)
d4 = 1.7 × q mass from stage 5, m5* c4 = m5 f4 = (c4/c) × 100
d3 = 3.1 × q mass from stage 4, m4 c3 = c4 + m 4 f3 = (c3/c) × 100
d2 = 6.8 × q mass from stage 3, m3 c2 = c3 + m 3 f2 = (c2/c) × 100
mass from stage 2, m2 c = c 2 + m2 100
Table 2.9.18.-8. – Calculations for Apparatus D when used at a flow rate of 28.3 L/min
Cut-off diameter Mass of active substance deposited Cumulative mass of active substance Cumulative fraction of active
(μm) per discharge deposited per discharge substance (per cent)
d7 = 0.4 mass from stage 8, m8 c 7 = m8 f7 = (c7/c) × 100
d6 = 0.7 mass from stage 7, m7 c6 = c7 + m7 f6 = (c6/c) × 100
d5 = 1.1 mass from stage 6, m6 c5 = c6 + m6 f5 = (c5/c) × 100
d4 = 2.1 mass from stage 5, m5 c4 = c5 + m5 f4 = (c4/c) × 100
d3 = 3.3 mass from stage 4, m4 c3 = c4 + m4 f3 = (c3/c) × 100
d2 = 4.7 mass from stage 3, m3 c2 = c3 + m3 f2 = (c2/c) × 100
d1 = 5.8 mass from stage 2, m2 c1 = c2 + m2 f1 = (c1/c) × 100
d0 = 9.0 mass from stage 1, m1 c0 = c1 + m1 f0 = (c0/c) × 100
mass from stage 0, m0 c = c0 + m0 100
Table 2.9.18.-9. – Calculations for Apparatus E. Use q = (60/Q)x, where Q is the test flow rate in litres per minute, and x
is listed in the table
Cut-off diameter x Mass of active substance Cumulative mass of active substance Cumulative fraction of active
(μm) deposited per discharge deposited per discharge substance (per cent)
d7 = 0.34 × q 0.67 mass from MOC or terminal c 7 = m8 F7 = (c7/c) × 100
filter, m8
d6 = 0.55 × q 0.60 mass from stage 7, m7 c6 = c7 + m7 F6 = (c6/c) × 100
d5 = 0.94 × q 0.53 mass from stage 6, m6 c5 = c6 + m6 F5 = (c5/c) × 100
d4 = 1.66 × q 0.47 mass from stage 5, m5 c4 = c5 + m5 F4 = (c4/c) × 100
d3 = 2.82 × q 0.50 mass from stage 4, m4 c3 = c4 + m4 F3 = (c3/c) × 100
d2 = 4.46 × q 0.52 mass from stage 3, m3 c2 = c3 + m3 F2 = (c2/c) × 100
d1 = 8.06 × q 0.54 mass from stage 2, m2 c1 = c2 + m2 F1 = (c1/c) × 100
mass from stage 1, m1 c = c1 + m1 100
04/2011:20919 for the test are not sufficient. The preparatory steps must
be repeated until the environment, glassware and water are
2.9.19. PARTICULATE suitable for the test.
Method
CONTAMINATION : SUB-VISIBLE
Mix the contents of the sample by slowly inverting the
PARTICLES(8) container 20 times successively. If necessary, cautiously
Particulate contamination of injections and infusions consists remove the sealing closure. Clean the outer surfaces of the
of extraneous, mobile undissolved particles, other than gas container opening using a jet of particle-free water R and
bubbles, unintentionally present in the solutions. remove the closure, avoiding any contamination of the
contents. Eliminate gas bubbles by appropriate measures such
For the determination of particulate contamination as allowing to stand for 2 min or sonicating.
2 procedures, Method 1 (Light Obscuration Particle Count
Test) and Method 2 (Microscopic Particle Count Test), For large-volume parenterals, single units are tested. For
are specified hereinafter. When examining injections and small-volume parenterals less than 25 mL in volume, the
infusions for sub-visible particles, Method 1 is preferably contents of 10 or more units are combined in a cleaned
applied. However, it may be necessary to test some container to obtain a volume of not less than 25 mL ; where
preparations by the light obscuration particle count test justified and authorised, the test solution may be prepared by
followed by the microscopic particle count test to reach a mixing the contents of a suitable number of vials and diluting
conclusion on conformance to the requirements. to 25 mL with particle-free water R or with an appropriate
solvent without contamination of particles when particle-free
Not all parenteral preparations can be examined for sub-visible water R is not suitable. Small-volume parenterals having a
particles by one or both of these methods. When Method 1 volume of 25 mL or more may be tested individually.
is not applicable, e.g. in case of preparations having reduced
clarity or increased viscosity, the test is carried out according Powders for parenteral administration are reconstituted with
to Method 2. Emulsions, colloids, and liposomal preparations particle-free water R or with an appropriate solvent without
are examples. Similarly, products that produce air or gas contamination of particles when particle-free water R is not
bubbles when drawn into the sensor may also require suitable.
microscopic particle count testing. If the viscosity of the The number of test specimens must be adequate to provide a
preparation to be tested is sufficiently high so as to preclude statistically sound assessment. For large-volume parenterals
its examination by either test method, a quantitative dilution or for small-volume parenterals having a volume of 25 mL
with an appropriate diluent may be made to decrease viscosity, or more, fewer than 10 units may be tested, based on an
as necessary, to allow the analysis to be performed. appropriate sampling plan.
The results obtained in examining a discrete unit or group Remove 4 portions, each of not less than 5 mL, and count
of units for particulate contamination cannot be extrapolated the number of particles equal to or greater than 10 μm and
with certainty to other units that remain untested. Thus, 25 μm. Disregard the result obtained for the first portion, and
statistically sound sampling plans must be developed if valid calculate the mean number of particles for the preparation to
inferences are to be drawn from observed data to characterise be examined.
the level of particulate contamination in a large group of units. Evaluation
METHOD 1. LIGHT OBSCURATION PARTICLE COUNT For preparations supplied in containers with a nominal
TEST volume of more than 100 mL, apply the criteria of test 1.A.
Use a suitable apparatus based on the principle of light For preparations supplied in containers with a nominal
blockage which allows an automatic determination of the size volume of less than 100 mL, apply the criteria of test 1.B.
of particles and the number of particles according to size. ♦For preparations supplied in containers with a nominal
The apparatus is calibrated using suitable certified reference volume of 100 mL, apply the criteria of test 1.B.♦
materials consisting of dispersions of spherical particles of If the average number of particles exceeds the limits, test the
known sizes between 10 μm and 25 μm. These standard preparation by the microscopic particle count test.
particles are dispersed in particle-free water R. Care must be Test 1.A – Solutions for infusion or solutions for injection
taken to avoid aggregation of particles during dispersion. supplied in containers with a nominal content of more than
General precautions 100 mL
The test is carried out under conditions limiting particulate The preparation complies with the test if the average number
contamination, preferably in a laminar-flow cabinet. of particles present in the units tested does not exceed 25 per
Very carefully wash the glassware and filtration equipment millilitre equal to or greater than 10 μm and does not exceed
used, except for the membrane filters, with a warm detergent 3 per millilitre equal to or greater than 25 μm.
solution and rinse with abundant amounts of water to remove Test 1.B – Solutions for infusion or solutions for injection
all traces of detergent. Immediately before use, rinse the supplied in containers with a nominal content of less than
equipment from top to bottom, outside and then inside, with 100 mL
particle-free water R. The preparation complies with the test if the average number
Take care not to introduce air bubbles into the preparation to of particles present in the units tested does not exceed 6000 per
be examined, especially when fractions of the preparation are container equal to or greater than 10 μm and does not exceed
being transferred to the container in which the determination 600 per container equal to or greater than 25 μm.
is to be carried out.
METHOD 2. MICROSCOPIC PARTICLE COUNT TEST
In order to check that the environment is suitable for the
test, that the glassware is properly cleaned and that the water Use a suitable binocular microscope, filter assembly for
to be used is particle-free, the following test is carried out : retaining particulate contamination and membrane filter for
determine the particulate contamination of 5 samples of examination.
particle-free water R, each of 5 mL, according to the method The microscope is equipped with an ocular micrometer
described below. If the number of particles of 10 μm or greater calibrated with an objective micrometer, a mechanical stage
size exceeds 25 for the combined 25 mL, the precautions taken capable of holding and traversing the entire filtration area
(8) This chapter has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.
General Notices (1) apply to all monographs and other texts 321
2.9.19. Particulate contamination : sub-visible particles EUROPEAN PHARMACOPOEIA 8.0
of the membrane filter, 2 suitable illuminators to provide For large-volume parenterals, single units are tested. For
episcopic illumination in addition to oblique illumination, small-volume parenterals less than 25 mL in volume, the
and is adjusted to 100 ± 10 magnifications. contents of 10 or more units are combined in a cleaned
The ocular micrometer is a circular diameter graticule (see container ; where justified and authorised, the test solution
Figure 2.9.19.-1.) and consists of a large circle divided by may be prepared by mixing the contents of a suitable number
crosshairs into quadrants, transparent and black reference of vials and diluting to 25 mL with particle-free water R or
circles 10 μm and 25 μm in diameter at 100 magnifications, with an appropriate solvent without contamination of particles
and a linear scale graduated in 10 μm increments. It is when particle-free water R is not suitable. Small-volume
calibrated using a stage micrometer that is certified by either parenterals having a volume of 25 mL or more may be tested
a domestic or international standard institution. A relative individually.
error of the linear scale of the graticule within ± 2 per cent isPowders for parenteral administration are constituted with
acceptable. The large circle is designated the graticule field particle-free water R or with an appropriate solvent without
of view (GFOV). contamination of particles when particle-free water R is not
2 illuminators are required. One is an episcopic brightfield suitable.
illuminator internal to the microscope, the other is an external,The number of test specimens must be adequate to provide a
focusable auxiliary illuminator adjustable to give reflected statistically sound assessment. For large-volume parenterals
oblique illumination at an angle of 10-20°. or for small-volume parenterals having a volume of 25 mL
The filter assembly for retaining particulate contamination or more, fewer than 10 units may be tested, based on an
consists of a filter holder made of glass or other suitable appropriate sampling plan.
material, and is equipped with a vacuum source and a suitable Wet the inside of the filter holder fitted with the membrane
membrane filter. filter with several millilitres of particle-free water R. Transfer
The membrane filter is of suitable size, black or dark grey to the filtration funnel the total volume of a solution pool or
in colour, non-gridded or gridded, and 1.0 μm or finer in of a single unit, and apply vacuum. If needed, add stepwise
nominal pore size. a portion of the solution until the entire volume is filtered.
After the last addition of solution, begin rinsing the inner
walls of the filter holder by using a jet of particle-free water R.
Maintain the vacuum until the surface of the membrane filter
is free from liquid. Place the filter in a Petri dish and allow the
filter to air-dry with the cover slightly ajar. After the filter has
been dried, place the Petri dish on the stage of the microscope,
scan the entire membrane filter under the reflected light from
the illuminating device, and count the number of particles
that are equal to or greater than 10 μm and the number of
particles that are equal to or greater than 25 μm. Alternatively,
partial filter count and determination of the total filter count
by calculation is allowed. Calculate the mean number of
particles for the preparation to be examined.
The particle sizing process with the use of the circular diameter
graticule is carried out by transforming mentally the image of
each particle into a circle and then comparing it to the 10 μm
and 25 μm graticule reference circles. Thereby the particles
are not moved from their initial locations within the graticule
field of view and are not superimposed on the reference
Figure 2.9.19.-1. – Circular diameter graticule circles for comparison. The inner diameter of the transparent
graticule reference circles is used to size white and transparent
General precautions
particles, while dark particles are sized by using the outer
The test is carried out under conditions limiting particulate diameter of the black opaque graticule reference circles.
contamination, preferably in a laminar-flow cabinet.
In performing the microscopic particle count test do not
Very carefully wash the glassware and filter assembly used, attempt to size or enumerate amorphous, semi-liquid, or
except for the membrane filter, with a warm detergent solution otherwise morphologically indistinct materials that have
and rinse with abundant amounts of water to remove all traces the appearance of a stain or discoloration on the membrane
of detergent. Immediately before use, rinse both sides of filter. These materials show little or no surface relief and
the membrane filter and the equipment from top to bottom, present a gelatinous or film-like appearance. In such cases
outside and then inside, with particle-free water R. the interpretation of enumeration may be aided by testing a
In order to check that the environment is suitable for the sample of the solution by the light obscuration particle count
test, that the glassware and the membrane filter are properly test.
cleaned and that the water to be used is particle-free, the Evaluation
following test is carried out : determine the particulate
contamination of a 50 mL volume of particle-free water R For preparations supplied in containers with a nominal
according to the method described below. If more than volume of more than 100 mL, apply the criteria of test 2.A.
20 particles 10 μm or larger in size or if more than 5 particles For preparations supplied in containers with a nominal
25 μm or larger in size are present within the filtration area, volume of less than 100 mL, apply the criteria of test 2.B.
the precautions taken for the test are not sufficient. The ♦For preparations supplied in containers with a nominal
preparatory steps must be repeated until the environment, volume of 100 mL, apply the criteria of test 2.B.♦
glassware, membrane filter and water are suitable for the test. Test 2.A – Solutions for infusion or solutions for injection
Method supplied in containers with a nominal content of more than
Mix the contents of the samples by slowly inverting the 100 mL
container 20 times successively. If necessary, cautiously remove The preparation complies with the test if the average number
the sealing closure. Clean the outer surfaces of the container of particles present in the units tested does not exceed 12 per
opening using a jet of particle-free water R and remove the millilitre equal to or greater than 10 μm and does not exceed
closure, avoiding any contamination of the contents. 2 per millilitre equal to or greater than 25 μm.
General Notices (1) apply to all monographs and other texts 323
2.9.23. Gas pycnometric density of solids EUROPEAN PHARMACOPOEIA 8.0
Method. Pour 5 mL of water at 36.5 ± 0.5 °C into the inner The gas pycnometric density measurement is performed at a
tube (A), introduce a suppository with the tip downwards and temperature between 15 °C and 30 °C that does not vary by
onto that, place the inset (C1 or C2). Note the time which more than 2 °C during the course of measurement.
elapses between this moment and the moment when the The apparatus is calibrated, which means that the volumes Vc
lower, rimmed end of the glass rod (C1) or the steel rod (C2) and Vr are determined using a suitable calibration standard
reaches the narrowed part of the inner glass tube. Melting or whose volume is known to the nearest 0.001 cm3. The
dissolution is then considered as complete. procedure described below is followed in 2 runs, firstly with
an empty test cell, and secondly with the calibration standard
placed in the test cell. The volumes Vc and Vr are calculated
using the equation for the sample volume (Vs), taking into
account that Vs is zero in the first run.
A = valve ;
Vr = expansion volume, in cubic centimetres ;
Vc = cell volume, in cubic centimetres ;
Figure 2.9.22.-2. – Apparatus B for measuring the softening
time of lipophilic suppositories Vs = sample volume, in cubic centimetres ;
Dimensions in millimetres M = manometer.
Figure 2.9.23.-1. – Schematic diagram of a gas pycnometer
07/2008:20923 METHOD
2.9.23. GAS PYCNOMETRIC DENSITY Volatile contaminants in the powder are removed by degassing
the powder under a constant purge of helium prior to the
OF SOLIDS measurement. Occasionally, powders may have to be degassed
under vacuum. Because volatiles may be evolved during the
Gas pycnometric density is determined by measuring the measurement, weighing of the sample is carried out after the
volume occupied by a known mass of powder, which is pycnometric measurement of volume.
equivalent to the volume of gas displaced by the powder using
a gas displacement pycnometer. In gas pycnometric density Weigh the test cell of the pycnometer and record the mass. Fill
measurements, the volume determined excludes the volume the test cell with a given mass of powder of the substance to
occupied by open pores ; however, it includes the volume be examined. Seal the test cell in the pycnometer. Record the
occupied by sealed pores or pores inaccessible to the gas. system reference pressure (Pr) as indicated by the manometer
while the valve that connects the expansion cell with the test
Usually, helium is used as a test gas due to its high diffusivity cell is open. Close the valve to separate the expansion cell from
into small open pores. If gases other than helium are used, the test cell. Pressurise the test cell with the gas to an initial
different values would be obtained, since the penetration of pressure (Pi) and record the value obtained. Open the valve to
the gas is dependent on the size of the pore as well as the connect the expansion cell with the test cell. Record the final
cross-sectional area of the gas molecules. pressure (Pf). Repeat the measurement sequence for the same
The measured density is a volume-weighted average of the powder sample until consecutive measurements of the sample
densities of individual powder particles. It is called the particle volume (Vs) agree to within 0.2 per cent. Unload the test cell
density, distinct from the true density of a solid or the bulk and measure the final powder mass (m), expressed in grams.
density of a powder. The density of solids is expressed in grams If the pycnometer differs in operation or construction from
per cubic centimetre (g/cm3), although the International Unit the one shown in Figure 2.9.23.-1, follow the instructions of
is the kilogram per cubic metre (1 g/cm3 = 1000 kg/m3). the manufacturer of the pycnometer.
APPARATUS EXPRESSION OF THE RESULTS
The apparatus (see Figure 2.9.23.-1) consists of the following : The sample volume (Vs) is given by the equation :
– a sealed test cell, with empty cell volume Vc, connected
through a valve to an expansion cell, with volume Vr ;
– a system capable of pressurising the test cell with the
measurement gas until a defined pressure (P) indicated by
a manometer ; The density (ρ) is given by the equation :
– the system is connected to a source of measurement gas,
preferably helium, unless another gas is specified.
The sample conditioning is indicated with the results. For The funnel and guides are mounted on the central chamber.
example, indicate whether the sample was tested as is or driedThe O-rings are incorporated in the piston recess with the
under specific conditions such as those described for loss on sealing ring around it ; the sealing rings ensure that the
drying. chamber is watertight. The horizontal pistons are placed in
the chewing chamber through the guides.
The gum is artificially chewed by the horizontal pistons, and
the vertical piston ensures that the gum stays in the right
04/2012:20925 place between chews.
Machine speed is controlled to ensure a constant cycle. One
2.9.25. DISSOLUTION TEST FOR cycle (chew) is defined as follows : the horizontal pistons
MEDICATED CHEWING GUMS start from their outermost position, move to their innermost
position then return to their outermost position. Within one
PRINCIPLE cycle, the vertical piston moves from its lowest position to its
The test is used to determine the dissolution rate of active uppermost position and back to its lowest position.
substances in medicated chewing gums. This is done by Each horizontal piston has a stroke of 25.0 mm. The
applying a mechanical kneading procedure to a piece of gum maximum distance between these 2 pistons is 50 mm. The
placed in a small chamber designed to simulate the process minimum distance between the 2 horizontal pistons is 0.1 mm
of chewing. to 1.0 mm. The vertical piston has a stroke of 22.0 mm.
APPARATUS A Horizontal piston movement is controlled so that the 2 pistons
are at their innermost position at the same time. Vertical
Chewing apparatus A (Figure 2.9.25.-1) consists of : piston movement is controlled so that it does not conflict with
– 1 chewing chamber ; the movement of the horizontal pistons.
– 1 vertical piston ; If necessary, the machine can be constructed so that the
– 2 horizontal pistons with O-rings and sealing rings. horizontal pistons rotate around their own axes in opposite
The chewing chamber consists of 4 individual parts : direction to each other by the end of the chew in order to
obtain maximum chewing.
– 1 central chamber ;
All parts of the apparatus that may come into contact with the
– 1 funnel (Figure 2.9.25.-2) ; preparation or the dissolution medium are chemically inert
– 2 guides with bushes (Figure 2.9.25.-3). and do not adsorb, react with or interfere with the sample.
General Notices (1) apply to all monographs and other texts 325
2.9.25. Dissolution test for medicated chewing gums EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 327
2.9.25. Dissolution test for medicated chewing gums EUROPEAN PHARMACOPOEIA 8.0
M16x1 61.6
Ø14
M8
20
G1/8 (2x)
Ø64
Ø44
90
2.2
0.0
Ø37 -0.1
2.2
3
6
0.5
45°
M8
18
10
Ø8.5
7.5
Ø27
(3)
(1)
The specific surface area is then calculated from the value of
Vm by equation (2) given above.
P = partial vapour pressure of adsorbate gas in The single-point method may be employed directly for a series
equilibrium with the surface at 77.4 K (b.p. of of powder samples of a given material for which the material
liquid nitrogen), in pascals, constant C is much greater than unity. These circumstances
Po = saturated pressure of adsorbate gas, in pascals, may be verified by comparing values of specific surface area
Va = volume of gas adsorbed at standard temperature determined by the single-point method with that determined
and pressure (STP) [273.15 K and atmospheric by the multiple-point method for the series of powder
pressure (1.013 × 105 Pa)], in millilitres, samples. Close similarity between the single-point values and
Vm = volume of gas adsorbed at STP to produce an multiple-point values suggests that 1/C approaches zero.
apparent monolayer on the sample surface, in The single-point method may be employed indirectly for
millilitres, a series of very similar powder samples of a given material
C = dimensionless constant that is related to the for which the material constant C is not infinite but may be
enthalpy of adsorption of the adsorbate gas on the assumed to be invariant. Under these circumstances, the error
powder sample. associated with the single-point method can be reduced or
A value of Va is measured at each of not less than 3 values of eliminated by using the multiple-point method to evaluate C
P/Po. for one of the samples of the series from the BET plot, from
which C is calculated as (1 + slope/intercept). Then Vm is
Then the BET value calculated from the single value of Va measured at a single
value of P/Po by the equation :
(4)
is plotted against P/Po according to equation (1). This plot
should yield a straight line usually in the approximate relative
pressure range 0.05 to 0.3. The data are considered acceptable The specific surface area is calculated from Vm by equation (2)
if the correlation coefficient, r, of the linear regression is not given above.
less than 0.9975 ; that is, r2 is not less than 0.995. From the
resulting linear plot, the slope, which is equal to (C − 1)/VmC,
and the intercept, which is equal to 1/VmC, are evaluated EXPERIMENTAL TECHNIQUES
by linear regression analysis. From these values, Vm is This section describes the methods to be used for the sample
calculated as 1/(slope + intercept), while C is calculated as preparation, the dynamic flow gas adsorption technique
(slope/intercept) + 1. From the value of Vm so determined, the (Method I) and the volumetric gas adsorption technique
specific surface area, S, in m2·g–1, is calculated by the equation : (Method II).
(9) This chapter has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.
General Notices (1) apply to all monographs and other texts 329
2.9.26. Specific surface area by gas adsorption EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 331
2.9.29. Intrinsic dissolution EUROPEAN PHARMACOPOEIA 8.0
influenced by extrinsic factors (test conditions), such as the powder to be tested is placed. The punch is then inserted
hydrodynamics, temperature, viscosity, pH, buffer strength in the die cavity and the material is compressed, generally
and ionic strength of the dissolution medium. using a benchtop hydraulic press. A hole through the head of
The assessment of intrinsic dissolution rate of a solid the punch allows insertion of a metal rod to facilitate removal
substance involves the preparation of a compact. Assurance of from the die after the test. A compact is formed in the cavity
appropriate compaction properties of the powder to be tested with a single face of defined area exposed on the bottom of
is needed prior to performing the test. the die (Figure 2.9.29.-1). The bottom of the die cavity is
threaded so that at least 50-75 per cent of the compact can
The intrinsic dissolution rate is determined by exposing a
dissolve without falling out of the die. The top of the die has
constant area of the compacted substance to an appropriate
a threaded shoulder that allows it to be attached to a holder.
dissolution medium, while maintaining constant stirring rate, The holder is mounted on a laboratory stirring device, and the
temperature, ionic strength and pH.
entire die, with the compact still in place, is immersed in the
The intrinsic dissolution rate is expressed in terms of dissolved dissolution medium and rotated by the stirring device.
mass of substance per time per exposed area, typically in
milligrams per minute per square centimetre (mg·min− 1·cm− 2). PROCEDURE
Weigh the material onto a piece of weighing paper. Attach the
APPARATUS surface plate to the underside of the die, and secure it with the
A typical apparatus consists of a punch and die fabricated out 3 provided screws. Transfer the sample of powder tested into
of hardened steel. The base of the die has 3 threaded holes the die cavity. Place the punch into the chamber, and secure
for the attachment of a surface plate made of polished steel, the metal plate on the top of the assembly. Compress the
providing a mirror-smooth base for the compact. The die has powder using a hydraulic press by applying a suitable pressure
a 0.1-1.0 cm diameter cavity into which a measured amount of for a sufficient dwell time to ensure a stable compact with
A. Surface plate B. Die C. Neoprene gasket D. Punch E. Holder and shaft assembly F. Die underside
Figure 2.9.29.-1. – Typical apparatus used to obtain the compact for the determination of the intrinsic dissolution
Dimensions in millimetres
minimal porosity ; the disintegration of the compact has to be The technique cannot distinguish between scattering by single
prevented as far as possible, since it would cause an increase particles and scattering by clusters of primary particles, i.e.
in surface area and hence in dissolution rate. Detach the by agglomerates or aggregates. As most particulate samples
surface plate, and screw the die with punch still in place into contain agglomerates or aggregates and as the focus of interest
the holder. Tighten securely. Remove all loose powder from is generally on the size distribution of primary particles, the
the surface of the die by blowing compressed air or nitrogen clusters are usually dispersed into primary particles before
across the surface of the compact. measurement.
Slide the die-holder assembly into the dissolution test chuck For non-spherical particles, an equivalent sphere-size
and tighten. Position the shaft in the spindle so that when the distribution is obtained because the technique assumes
test head is lowered, the exposed surface of the compact will spherical particles in its optical model. The resulting
be 3.8 cm from the bottom of the vessel. The disc assembly is particle-size distribution may differ from those obtained
aligned to minimise wobble and air bubbles are not allowed by methods based on other physical principles (e.g.
to form as this could decrease the compact surface in contact sedimentation, sieving).
with the dissolution medium. If possible, sink conditions are This chapter provides guidance for the measurement
maintained throughout the test. However, in order to obtain of size distributions of particles in different dispersed
detectable concentrations of solute, the use of a relatively small systems, for example, powders, sprays, aerosols, suspensions,
volume of medium may be necessary as a consequence of the emulsions, and gas bubbles in liquids, through analysis of
limited surface available for dissolution. their angular light-scattering patterns. It does not address
Warm the dissolution medium to the temperature chosen for specific requirements of particle size measurement of specific
the test. Lower the test head into position before rotation. products.
Care should be taken to ensure that air bubbles are excluded
from the surface of the compact as this could decrease the PRINCIPLE
compact surface in contact with the dissolution medium. A representative sample, dispersed at an adequate
Operate the apparatus immediately at the speed of rotation concentration in a suitable liquid or gas, is passed through
chosen for the test. a beam of monochromatic light, usually a laser. The light
Collect samples at fixed time intervals and assay them by scattered by the particles at various angles is measured by a
means of an analytical method of suitable sensitivity and multi-element detector. Numerical values representing the
accuracy. scattering pattern are then recorded for subsequent analysis.
These scattering pattern values are then transformed, using an
ASSESSMENT OF THE RESULTS appropriate optical model and mathematical procedure, to
The data for the cumulative amount dissolved at each time yield the proportion of total volume to a discrete number of
point are corrected for sampling losses. To calculate the size classes, forming a volumetric particle-size distribution.
intrinsic dissolution rate, plot the cumulative amount of
sample dissolved per unit area of the compact against time. INSTRUMENT
The cumulative amount dissolved per unit area is given by the The instrument is located in an environment where it is
cumulative amount dissolved at each time point divided by the not affected by electrical noise, mechanical vibrations,
surface area exposed. Linear regression is then performed on temperature fluctuations, humidity or direct bright light.
the normalised experimental data relevant to an appropriate An example of a set-up of a laser light diffraction instrument
time interval preceding the possible disintegration of the is given in Figure 2.9.31.-1. Other equipment may be used.
compact. The intrinsic dissolution rate of the substance tested, The instrument comprises a laser light source, beam
expressed in milligrams per minute per square centimetre, processing optics, a sample measurement region (or cell), a
is determined from the slope of the regression line. The Fourier lens, and a multi-element detector for measuring the
result for intrinsic dissolution rate must be accompanied by scattered light pattern. A data system is also required for
a statement of the precise conditions of compact preparation deconvolution of the scattering data into a volumetric size
and test method (dissolution medium, volume of medium distribution and associated data analysis and reporting.
used, stirring rate, temperature etc.).
The particles can enter the laser beam in 2 positions. In
NOTE : when necessary and justified, an apparatus with a the conventional case the particles enter the parallel beam
different configuration may be used, such as a die holder that before the collecting lens and within its working distance. In
holds the compact in a fixed vertical position, with agitation so-called reversed Fourier optics the particles enter behind the
provided by a paddle positioned at a defined distance from the collecting lens and thus, in a converging beam. The advantage
surface of the compact. of the conventional set-up is that a reasonable path length for
the sample is allowed within the working distance of the lens.
The second set-up allows only small path lengths but enables
01/2010:20931 measurement of scattered light at larger angles, which is useful
when submicron particles are present.
2.9.31. PARTICLE SIZE ANALYSIS The interaction of the incident light beam and the ensemble of
BY LASER LIGHT DIFFRACTION dispersed particles results in a scattering pattern with different
light intensities at various angles. The total angular intensity
The method is based on the ISO standards 13320-1(1999) and distribution, consisting of both direct and scattered light,
9276-1(1998). is then focused onto a multi-element detector by a lens or a
series of lenses. These lenses create a scattering pattern that,
INTRODUCTION within limits, does not depend on the location of the particles
The laser light diffraction technique used for the determination in the light beam. Hence, the continuous angular intensity
of particle-size distribution is based on the analysis of the distribution is converted into a discrete spatial intensity
diffraction pattern produced when particles are exposed distribution on a set of detector elements.
to a beam of monochromatic light. Historically, the early It is assumed that the measured scattering pattern of the
laser diffraction instruments only used scattering at small particle ensemble is identical to the sum of the patterns from
angles. However, the technique has since been broadened to all individual single scattering particles presented in random
include laser light scattering in a wider angular range and relative positions. Note that only a limited angular range of
application of the Mie theory, in addition to the Fraunhofer scattered light is collected by the lens(es) and, therefore, by
approximation and anomalous diffraction. the detector.
General Notices (1) apply to all monographs and other texts 333
2.9.31. Particle size analysis by laser light diffraction EUROPEAN PHARMACOPOEIA 8.0
1. Obscuration detector 5. Scattered light not collected by lens (4) 9. Working distance of lens (4)
2. Scattered beam 6. Particle ensemble 10. Multi-element detector
3. Direct beam 7. Light source laser 11. Focal distance of lens (4)
4. Fourier lens 8. Beam processing unit
Optimisation of the gas dispersion. For sprays and dry depends mostly on the instrument hardware. Accuracy should
powder dispersions, a compressed gas free from oil, water be confirmed through an appropriate instrument qualification
and particles may be used. To remove such materials from and comparison with microscopy, while precision may be
the compressed gas, a dryer with a filter can be used. Any assessed by means of a repeatability determination.
vacuum unit should be located away from the measurement The attainable repeatability of the method mainly depends
zone, so that its output does not disturb the measurement. on the characteristics of the material (milled/not milled,
Determination of the concentration range. In order to robust/fragile, width of its size distribution, etc.), whereas
produce an acceptable signal-to-noise ratio in the detector, the required repeatability depends on the purpose of the
the particle concentration in the dispersion must exceed a measurement. Mandatory limits cannot be specified in this
minimum level. Likewise, it must be below a maximum level chapter, as repeatabilities (different sample preparations) may
in order to avoid multiple scattering. The concentration range vary appreciably from one substance to another. However, it
is influenced by the width of the laser beam, the path length of is good practice to aim at acceptance criteria for repeatability
the measurement zone, the optical properties of the particles, such as srel ≤ 10 per cent [n = 6] for any central value of the
and the sensitivity of the detector elements. distribution (e.g. for x50). Values at the sides of the distribution
In view of the above, measurements must be performed at (e.g. x10 and x90) are oriented towards less stringent acceptance
different particle concentrations to determine the appropriate criteria such as srel ≤ 15 per cent [n = 6]. Below 10 μm, these
concentration range for any typical sample of material. (Note : values must be doubled. Robustness may be tested during the
in different instruments, particle concentrations are usually selection and optimisation of the dispersion media and forces.
represented by differently scaled and differently named The change of the dispersing energy may be monitored by the
numbers, e.g. obscuration, optical concentration, proportional change in the particle-size distribution.
number of total mass).
MEASUREMENT
Determination of the measuring time. The time of
measurement, the reading time of the detector and the Precautions. The instructions given in the instrument manual
acquisition frequency are determined experimentally in are followed :
accordance with the required precision. Generally, the time – never look into the direct path of the laser beam or its
for measurement permits a large number of detector scans or reflections ;
sweeps at short time intervals.
– earth all instrument components to prevent ignition of
Selection of an appropriate optical model. Most instruments solvents or dust explosions ;
use either the Fraunhofer or the Mie theory, though other
approximation theories are sometimes applied for calculation – check the instrument set-up (e.g. warm-up, required
of the scattering matrix. The choice of the theoretical model measuring range and lens, appropriate working distance,
depends on the intended application and the different position of the detector, no direct bright daylight) ;
assumptions (size, absorbance, refractive index, roughness, – in the case of wet dispersions, avoid air bubbles,
crystal orientation, mixture, etc.) made for the test material. evaporation of liquid, schlieren or other inhomogeneities
If the refractive index values (real and imaginary parts in the dispersion ; similarly, avoid improper mass-flow
for the used wavelength) are not exactly known, then the from the disperser or turbulent air-flow in the case of dry
Fraunhofer approximation or the Mie theory with a realistic dispersions ; such effects can cause erroneous particle-size
estimate of the refractive index can be used. The former distributions.
has the advantages that it is simple and it does not need
refractive index values ; the latter usually provides less-biased Measurement of the light scattering of dispersed sample(s).
particle-size distributions for small particles. For instance, After proper alignment of the optical part of the instrument,
if the Fraunhofer model is used for samples containing a blank measurement of the particle-free dispersion medium
an appreciable amount of small, transparent particles, must be performed using the same method as that used for the
a significantly larger amount of small particles may be measurement of the sample. The background signal must be
calculated. In order to obtain traceable results, it is essential below an appropriate threshold. The detector data are saved
to document the refractive index values used, since small in order to substract them later from the data obtained with
differences in the values assumed for the real and imaginary the sample. The sample dispersion is measured according to
part of the complex refractive index may cause significant the developed method.
differences in the resulting particle-size distributions. Small For each detector element, an average signal is calculated,
values of the imaginary part of the refractive index (about sometimes together with its standard deviation. The
0.01-0.1 i) are often applied to allow the correction of the magnitude of the signal from each detector element depends
absorbance for the surface roughness of the particles. It upon the detection area, the light intensity and the quantum
should be noted, in general, that the optical properties of the efficiency. The co-ordinates (size and position) of the
substance to be tested, as well as the structure (e.g. shape, detector elements together with the focal distance of the lens
surface roughness and porosity), bear upon the final result. determine the range of scattering angles for each element.
Validation. Typically, the validity of a procedure may be Most instruments also measure the intensity of the central
assessed by the evaluation of its specificity, linearity, range, (unscattered) laser beam. The ratio of the intensity of a
accuracy, precision and robustness. In particle-size analysis dispersed sample to that in its absence (a blank measurement)
by laser light diffraction, specificity as defined by ICH is indicates the proportion of scattered light and hence the
not applicable as it is not possible to discriminate between particle concentration.
different components in a sample, nor is it possible to Conversion of scattering pattern into particle-size
discriminate agglomerates from dispersed particles unless distribution. This deconvolution step is the inverse of the
properly complemented by microscopic techniques. Exploring calculation of a scattering pattern for a given particle-size
a linear relationship between concentration and response, or distribution. The assumption of spherical particle shape
a mathematical model for interpolation, is not applicable to is particularly important as most algorithms use the
this procedure. Rather than evaluating linearity, this method mathematical solution for scattering from spherical particles.
requires the definition of a concentration range within which Furthermore, the measured data always contain some random
the result of the measurements does not vary significantly. and systematic errors, which may vitiate the size distributions.
Concentrations below that range produce an error due to a Several mathematical procedures have been developed
poor signal-to-noise ratio, while concentrations above that for use in the available instruments. They contain some
range produce an error due to multiple scattering. The range weighting of deviations between measured and calculated
General Notices (1) apply to all monographs and other texts 335
2.9.32. Porosity and pore-size distribution by mercury porosimetry EUROPEAN PHARMACOPOEIA 8.0
scattering patterns (e.g. least squares), some constraints agreed, detailed operating procedure. The use of reference
(e.g. non-negativity for amounts of particles), and/or some values from methods other than laser diffraction may
smoothing of the size distribution curve. cause a significant bias. The reason for this bias is that the
The algorithms used are specific to each make and model different principles inherent in the various methods may
of equipment, and are proprietary. The differences in the lead to different sphere-equivalent diameters for the same
algorithms between different instruments may give rise to non-spherical particle.
differences in the calculated particle-size distributions. Although the use of certified reference materials is preferred,
Replicates. The number of replicate measurements (with other well-defined reference materials may also be employed.
individual sample preparations) to be performed depends on They consist of substances of typical composition and
the required measurement precision. It is recommended to set particle-size distribution for a specified class of substances.
this number in a substance-specific method. Their particle-size distribution has proven to be stable over
time. The results must comply with previously determined
REPORTING OF RESULTS data, with the same precision and bias as for the certified
The particle-size distribution data are usually reported reference material.
as cumulative undersize distribution and/or as density Qualification of the system. In addition to the calibration,
distribution by volume. The symbol x is used to denote the the performance of the instrument must be qualified at regular
particle size, which in turn is defined as the diameter of a time intervals or as frequently as appropriate. This can be
volume-equivalent sphere. Q3(x) denotes the volume fraction undertaken using any suitable reference material as mentioned
undersize at the particle size x. In a graphical representation, in the previous paragraph.
x is plotted on the abscissa and the dependent variable Q3 The qualification of the system is based on the concept that
on the ordinate. Most common characteristic values are the equipment, electronics, software and analytical operations
calculated from the particle-size distribution by interpolation. constitute an integral system, which can be evaluated as an
The particle sizes at the undersize values of 10 per cent, 50 per entity. Thus the entire measurement procedure is examined,
cent, and 90 per cent (denoted as x10, x50, and x90 respectively) including sample collection, sample dispersion, sample
are frequently used. x50 is also known as the median particle transport through the measuring zone, and the measurement
size. It is recognised that the symbol d is also widely used to and deconvolution procedure. It is essential that the total
designate the particle size, thus the symbol x may be replaced operational procedure is fully described.
by d. In general, unless otherwise specified in the individual
Moreover, sufficient information must be documented about monograph, the response of a laser diffraction instrument
the sample, the sample preparation, the dispersion conditions, is considered to meet the requirements if the x50 value does
and the cell type. As the results depend on the particular not deviate by more than 10 per cent from the range of values
instrument, data analysis program, and optical model used, of the reference material. If optionally the values at the sides
these details must also be documented. of the distribution are evaluated (e.g. x10 and x90), then these
values must not deviate by more than 15 per cent from the
CONTROL OF THE INSTRUMENT PERFORMANCE certified range of values. Below 10 μm, these values must be
Use the instrument according to the manufacturer’s doubled.
instructions and carry out the prescribed qualifications at an NOTE : for calibration of the instrument, stricter requirements
appropriate frequency, according to the use of the instrument are laid down in the paragraph Calibration.
and substances to be tested.
Calibration. Laser diffraction systems, although assuming 07/2008:20932
idealised properties of the particles, are based on first
principles of laser light scattering. Thus, calibration in the
strict sense is not required. However, it is still necessary to 2.9.32. POROSITY AND PORE-SIZE
confirm that the instrument is operating correctly. This can DISTRIBUTION OF SOLIDS BY
be undertaken using any certified reference material that is
acceptable in industrial practice. The entire measurement
MERCURY POROSIMETRY
procedure is examined, including sample collection, sample INTRODUCTION
dispersion, sample transport through the measuring zone, In general, different types of pores may be pictured as
measurement, and the deconvolution procedure. It is essential apertures, channels or cavities within a solid body, or as space
that the total operational procedure is fully described. (i.e. interstices or voids) between solid particles in a bed,
The preferred certified reference materials consist of spherical compact or aggregate. Porosity is a term that is often used
particles of a known distribution. They must be certified to indicate the porous nature of solid material, and is more
as to the mass-percentage size distribution by an absolute precisely defined as the ratio of the volume of accessible pores
technique, if available, and used in conjunction with an agreed, and voids to the total volume occupied by a given amount
detailed operation procedure. It is essential that the real and of the solid. In addition to the accessible pores, a solid may
imaginary parts of the complex refractive index of the material contain closed pores, which are isolated from the external
are indicated if the Mie theory is applied in data analysis. The surface and into which fluids are not able to penetrate. The
representation of the particle-size distribution by volume characterisation of closed pores, i.e. cavities with no access to
will equal that of the distribution by mass, provided that the an external surface, is not covered in this chapter.
density of the particles is the same for all size fractions. Porous materials may take the form of fine or coarse
The response of a laser diffraction instrument is considered to powders, compacts, extrudates, sheets or monoliths. Their
meet the requirements if the mean value of x50 from at least characterisation usually involves the determination of the total
3 independent measurements does not deviate by more than pore volume or porosity as well as the pore-size distribution.
3 per cent from the certified range of values of the certified It is well established that the performance of a porous solid
reference material. The mean values for x10 and x90 must not (e.g. its strength, reactivity, permeability or adsorbent power)
deviate by more than 5 per cent from the certified range of is dependent upon its pore structure. Many different methods
values. Below 10 μm, these values must be doubled. have been developed for the characterisation of pore structure.
Although the use of materials consisting of spherical particles In view of the complexity of most porous solids, it is not
is preferable, non-spherical particles may also be employed. surprising to find that the results obtained are not always in
Preferably, these particles have certified or typical values agreement and that no single technique can be relied upon to
from laser diffraction analyses performed according to an provide a complete picture of the pore structure. The choice
of the most appropriate method depends on the application between the mercury column in the capillary tube and a metal
of the porous solid, its chemical and physical nature and the sleeve around the outside of the capillary tube. If precise
range of pore-size. measurements are required the expected total void and pore
This chapter provides guidance for measurement of porosity volume of the sample should be between 20 per cent and
and pore-size distribution by mercury porosimetry. It is a 90 per cent of the internal volume of the capillary tube. Since
comparative test, usually destructive, in which the volume of different materials exhibit a wide range of open porosities,
mercury penetrating a pore or void is determined as a function a number of penetrometers with different capillary tube
of an applied hydrostatic pressure, which can be related to diameters and sample volumes may be required. A typical
a pore diameter. Other information such as pore shape and set-up for a mercury porosimeter instrument is given in
inter-connectivity, the internal and external surface area, Figure 2.9.32.-1. The porosimeter may have separate ports
powder granulometry, bulk and tapped density could also be for high- and low-pressure operation, or the low-pressure
inferred from volume-pressure curves ; however, these aspects measurement may be carried out on a separate unit.
of the technique do not fall under the scope of this chapter. The pressure range is typically 4-300 kPa for low-pressure
Practical considerations presently limit the maximum applied operation and above 300 kPa for high-pressure operation,
absolute pressure reached by some equipment to about depending on the design of the particular apparatus and on
400 MPa, corresponding to a minimum equivalent pore the intended use.
diameter of approximately 0.003 μm. The maximum diameter
will be limited for samples having a significant depth due to METHOD
the difference in hydrostatic head of mercury from the top to Sample preparation
the bottom of the sample. For most purposes this limit may
be regarded as 400 μm. The sample is pre-treated to remove adsorbed material that
can obscure its accessible porosity, for example by heating
Inter-particle and intra-particle porosity can be determined, and/or evacuation, or by flowing inert gas. It may be possible
but the method does not distinguish between these porosities to passivate the surface of wettable or amalgam-forming
where they co-exist. solids, for example by producing a thin layer of oxide, or by
The method is suitable for the study of most porous materials. coating with stearate.
Samples that amalgamate with mercury, such as certain The sample of the pre-treated solid is weighed and transferred
metals, may be unsuitable for this technique or may require to the penetrometer. The pore system of the sample is then
a preliminary passivation. Other materials may deform or degassed in a vacuum to a maximum residual pressure of 7 Pa.
compact under the applied pressure. In some cases it may
be possible to apply sample-compressibility corrections and Filling the penetrometer with mercury
useful comparative data may still be obtained. The mercury used is of analytical quality. Overlay the sample
Mercury porosimetry is considered to be comparative, as with mercury under vacuum. The vacuum is required to
for most porous media a theory is not available to allow ensure the transfer of mercury from the reservoir to the
an absolute calculation of results of pore-size distribution. penetrometer. In a filled penetrometer the filling pressure
Therefore this technique is mainly recommended for comprises the applied pressure plus the pressure contribution
development studies. created by the head of mercury contacting the sample. A
typical filling pressure would be about 4 kPa. The hydrostatic
Mercury is toxic. Appropriate precautions must be observed pressure of the mercury over the sample may be minimised by
to safeguard the health of the operator and others working in filling the penetrometer in the horizontal position.
the area. Waste material must also be disposed of in a suitable
manner, according to local regulations. Low-pressure measurement
Admit air or nitrogen in a controlled manner to increase
PRINCIPLE the pressure either in stages corresponding to the particular
The technique is based on the measurement of the mercury pore sizes of interest, or continuously at a slow rate. The
volume intruded into a porous solid as a function of the concomitant change in the length of the mercury column in
applied pressure. The measurement includes only those pores the capillary tube is recorded. When the maximum required
into which mercury can penetrate at the pressure applied. pressure has been reached, return to atmospheric pressure.
A non-wetting liquid penetrates into a porous system only High-pressure measurement
under pressure. The pressure to be applied is in inverse After measurement at low pressure, the penetrometer filled
proportion to the inner diameter of the pore aperture. In with mercury is transferred to the high-pressure port or unit
the case of cylindrical pores, the correlation between pore of the instrument and overlaid with hydraulic fluid. Mercury
diameter and pressure is given by the Washburn equation : is intruded into the pore system via the hydraulic fluid.
Increase the pressure in the system to the maximum pressure
· reached in the low-pressure measurement and record the
intrusion volume at this pressure, since subsequent intrusion
volumes are calculated from this initial volume. Increase the
dp = pore diameter, in metres ; pressure either in stages corresponding to the particular pore
σ = surface tension, in newtons per metre ; sizes of interest, or continuously at a slow rate. The fall in the
mercury column is measured up to the maximum required
θ = contact angle of mercury on the sample, in degrees ; pressure. If required the pressure may be decreased either in
p = applied pressure, in pascals. stages or continuously at a slow rate to determine the mercury
extrusion curve.
APPARATUS Corrections are made to take account of changes in the volume
of the mercury, the penetrometer and other components of
The sample holder, referred to as penetrometer or dilatometer,
the volume detector system under elevated pressure. The
has a calibrated capillary tube, through which the sample
extent of the corrections may be determined by means of
can be evacuated and through which mercury can enter.
blank measurements under the same conditions.
The capillary tube is attached to a wider tube in which the
test sample is placed. The change in the volume of mercury An experimentally determined volume-pressure curve is
intruded is usually measured by the change in capacitance shown in Figure 2.9.32.-2.
General Notices (1) apply to all monographs and other texts 337
2.9.32. Porosity and pore-size distribution by mercury porosimetry EUROPEAN PHARMACOPOEIA 8.0
(10) There are many other applications of the X-ray powder diffraction technique that can be applied to crystalline pharmaceutical substances such as : determination of crystal structures,
refinement of crystal structures, determination of crystallographic purity of crystalline phases, characterisation of crystallographic texture, etc. These applications are not described in
this chapter.
(11) An ‘ideal’ powder for diffraction experiments consists of a large number of small, randomly oriented spherical crystallites (coherently diffracting crystalline domains). If this number is
sufficiently large, there are always enough crystallites in any diffracting orientation to give reproducible diffraction patterns.
General Notices (1) apply to all monographs and other texts 339
2.9.33. Characterisation of crystalline solids by XRPD EUROPEAN PHARMACOPOEIA 8.0
The intensity is dependent upon many factors such as Depending on the type of analysis to be performed
(phase identification, quantitative analysis, lattice
structure factor, temperature factor, crystallinity, polarisation
factor, multiplicity and Lorentz factor. parameters determination, etc.), different XRPD instrument
The main characteristics of diffraction line profiles configurations and performance levels are required. The
are 2θ position, peak height, peak area and shape simplest instruments used to measure XRPD patterns are
(characterised by, for example, peak width or asymmetry, powder cameras. The replacement of photographic film as
analytical function, empirical representation). An example the detection method by photon detectors has led to the
of the type of powder patterns obtained for 5 different solid design of diffractometers in which the geometric arrangement
phases of a substance are shown in Figure 2.9.33.-2. of the optics is not truly focusing but parafocusing, such
as in the Bragg-Brentano geometry. The Bragg-Brentano
In addition to the diffraction peaks, an X-ray diffraction parafocusing configuration is currently the most widely used
experiment also generates a more-or-less uniform background, and is therefore briefly described here.
upon which the peaks are superimposed. Besides specimen
preparation, other factors contribute to the background, A given instrument may provide a horizontal or vertical
for instance the sample holder, diffuse scattering from air θ/2θ geometry or a vertical θ/θ geometry. For both geometries,
and equipment, other instrumental parameters such as the incident X-ray beam forms an angle θ with the specimen
surface plane and the diffracted X-ray beam forms an angle 2θ
detector noise, general radiation from the X-ray tube, etc. The
peak-to-background ratio can be increased by minimising with the direction of the incident X-ray beam (an angle
background and by choosing prolonged exposure times. θ with the specimen surface plane). The basic geometric
arrangement is represented in Figure 2.9.33.-3. The divergent
INSTRUMENT beam of radiation from the X-ray tube (the so-called ‘primary
beam’) passes through the parallel plate collimators and a
Instrument set-up. X-ray diffraction experiments are usually divergence slit assembly and illuminates the flat surface of
performed using powder diffractometers or powder cameras. the specimen. All the rays diffracted by suitably oriented
A powder diffractometer generally comprises 5 main parts : crystallites in the specimen at an angle 2θ converge to a line
an X-ray source ; incident beam optics, which may perform at the receiving slit. A second set of parallel plate collimators
monochromatisation, filtering, collimation and/or focusing and a scatter slit may be placed either behind or before the
of the beam ; a goniometer ; diffraction beam optics, which receiving slit. The axes of the line focus and of the receiving
may perform monochromatisation, filtering, collimation slit are at equal distances from the axis of the goniometer. The
and focusing or parallelising of the beam ; and a detector. X-ray quanta are counted by a radiation detector, usually a
Data-collection and data-processing systems are also required scintillation counter, a sealed-gas proportional counter, or a
and are generally included in current diffraction measurement position-sensitive solid-state detector such as imaging plate or
equipment. CCD detector. The receiving slit assembly and the detector are
Figure 2.9.33.-2. – X-ray powder diffraction patterns collected for 5 different solid phases of a substance
(the intensities are normalised)
A. X-ray tube C. sample E. receiving slit G. detector receiving slit J. diffractometer circle
B. divergence slit D. anti-diffusion slit F. monochromator H. detector K. focusing circle
coupled together and move tangentially to the focusing circle. obtained using Kβ filters, i.e. metal filters selected as having an
For θ/2θ scans the goniometer rotates the specimen about the absorption edge between the Kα and Kβ wavelengths emitted
same axis as that of the detector, but at half the rotational by the tube.
speed, in a θ/2θ motion. The surface of the specimen thus Such a filter is usually inserted between the X-ray tube
remains tangential to the focusing circle. The parallel plate and the specimen. Another, more-and-more-commonly
collimator limits the axial divergence of the beam and hence used way to obtain a monochromatic X-ray beam is via
partially controls the shape of the diffracted line profile. a large monochromator crystal (usually referred to as a
A diffractometer may also be used in transmission mode. The ‘monochromator’). This crystal is placed before or behind
advantage with this technology is to lessen the effects due to the specimen and diffracts the different characteristic peaks
preferred orientation. A capillary of about 0.5-2 mm thickness of the X-ray beam (i.e. Kα and Kβ) at different angles, so
can also be used for small sample amounts. that only one of them may be selected to enter into the
X-ray radiation. In the laboratory, X-rays are obtained by detector. It is even possible to separate Kα1 and Kα2 radiations
bombarding a metal anode with electrons emitted by the by using a specialised monochromator. Unfortunately, the
thermionic effect and accelerated in a strong electric field gain in getting a monochromatic beam by using a filter or a
(using a high-voltage generator). Most of the kinetic energy of monochromator is counteracted by a loss in intensity. Another
the electrons is converted to heat, which limits the power of way of separating Kα and Kβ wavelengths is by using curved
the tubes and requires efficient anode cooling. A 20- to 30-fold X-rays mirrors that can simultaneously monochromate and
increase in brilliance can be obtained using rotating anodes focus or parallelise the X-ray beam.
and by using X-ray optics. Alternatively, X-ray photons may RADIATION PROTECTION. Exposure of any part of the
be produced in a large-scale facility (synchrotron). human body to X-rays can be injurious to health. It is therefore
The spectrum emitted by an X-ray tube operating at sufficient essential that whenever X-ray equipment is used, adequate
voltage consists of a continuous background of polychromatic precautions are taken to protect the operator and any other
radiation and additional characteristic radiation that depends person in the vicinity. Recommended practice for radiation
on the type of anode. Only this characteristic radiation is protection as well as limits for the levels of X-radiation exposure
used in X-ray diffraction experiments. The principal radiation are those established by national legislation in each country.
sources utilised for X-ray diffraction are vacuum tubes utilising If there are no official regulations or recommendations in
copper, molybdenum, iron, cobalt or chromium as anodes ; a country, the latest recommendations of the International
copper, molybdenum or cobalt X-rays are employed most Commission on Radiological Protection should be applied.
commonly for organic substances (the use of cobalt anodes
can be especially preferred to separate distinct X-ray lines). SPECIMEN PREPARATION AND MOUNTING
The choice of radiation to be used depends on the absorption The preparation of the powdered material and mounting
characteristics of the specimen and possible fluorescence by of the specimen in a suitable holder are critical steps in
atoms present in the specimen. The wavelengths used in many analytical methods, and are particularly so for XRPD
powder diffraction generally correspond to the Kα radiation analysis, since they can greatly affect the quality of the data to
from the anode. Consequently, it is advantageous to make be collected(12). The main sources of error due to specimen
the X-ray beam ‘monochromatic’ by eliminating all the other preparation and mounting are briefly discussed here for
components of the emission spectrum. This can be partly instruments in Bragg-Brentano parafocusing geometry.
(12) Similarly, changes in the specimen can occur during data collection in the case of a non-equilibrium specimen (temperature, humidity).
General Notices (1) apply to all monographs and other texts 341
2.9.33. Characterisation of crystalline solids by XRPD EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 343
2.9.34. Bulk density and tapped density of powders EUROPEAN PHARMACOPOEIA 8.0
Tapped density in increments of, for example, 1250 taps, until the difference
between successive measurements is less than or equal to
The tapped density is an increased bulk density attained after 2 mL. Fewer taps may be appropriate for some powders,
mechanically tapping a receptacle containing the powder when validated. Calculate the tapped density in grams per
sample. millilitre using the formula m/Vf (where Vf is the final tapped
The tapped density is obtained by mechanically tapping a volume). Generally, replicate determinations are desirable for
graduated measuring cylinder or vessel containing the powder the determination of this property. Specify the drop height
sample. After observing the initial powder volume or mass, with the results.
the measuring cylinder or vessel is mechanically tapped, If it is not possible to use a 100 g test sample, use a reduced
and volume or mass readings are taken until little further amount and a suitable 100 mL graduated cylinder (readable
volume or mass change is observed. The mechanical tapping to 1 mL) weighing 130 ± 16 g and mounted on a support
is achieved by raising the cylinder or vessel and allowing it weighing 240 ± 12 g. The modified test conditions are
to drop, under its own mass, a specified distance by one of specified in the expression of the results.
3 methods as described below. Devices that rotate the cylinder
or vessel during tapping may be preferred to minimise any METHOD 2
possible separation of the mass during tapping down. Procedure. Proceed as directed under Method 1 except that
the mechanical tester provides a fixed drop of 3 ± 0.2 mm at
METHOD 1
a nominal rate of 250 taps per minute.
Apparatus. The apparatus (Figure 2.9.34.-3) consists of the
following : METHOD 3
– a 250 mL graduated cylinder (readable to 2 mL) with a Procedure. Proceed as described under Method 3 for
mass of 220 ± 44 g ; measuring the bulk density, using the measuring vessel
– a settling apparatus capable of producing, per minute, equipped with the cap shown in Figure 2.9.34.-2. The
either nominally 250 ± 15 taps from a height of 3 ± 0.2 mm, measuring vessel with the cap is lifted 50-60 times per
or nominally 300 ± 15 taps from a height of 14 ± 2 mm. minute by the use of a suitable tapped density tester. Carry
The support for the graduated cylinder, with its holder, has out 200 taps, remove the cap and carefully scrape excess
a mass of 450 ± 10 g. powder from the top of the measuring vessel as described
under Method 3 for measuring the bulk density. Repeat
Procedure. Proceed as described above for the determination the procedure using 400 taps. If the difference between the
of the bulk volume (V0). Secure the cylinder in the support. 2 masses obtained after 200 and 400 taps exceeds 2 per cent,
Carry out 10, 500 and 1250 taps on the same powder sample repeat the test using 200 additional taps until the difference
and read the corresponding volumes V10, V500 and V1250 to the between successive measurements is less than 2 per cent.
nearest graduated unit. If the difference between V500 and Calculate the tapped density in grams per millilitre using
V1250 is less than or equal to 2 mL, V1250 is the tapped volume. the formula Mf/100 (where Mf is the mass of powder in the
If the difference between V500 and V1250 exceeds 2 mL, repeat measuring vessel). Record the average of 3 determinations
General Notices (1) apply to all monographs and other texts 345
2.9.35. Powder fineness EUROPEAN PHARMACOPOEIA 8.0
using 3 different powder samples. The test conditions, Qr(x) = cumulative distribution of particles with a dimension
including tapping height, are specified in the expression of less than or equal to x where the subscript r reflects the
the results. distribution type.
r Distribution type
Measures of powder compressibility
0 Number
Because the interparticulate interactions influencing the
bulking properties of a powder are also the interactions that 1 Length
interfere with powder flow, a comparison of the bulk and 2 Area
tapped densities can give a measure of the relative importance
of these interactions in a given powder. Such a comparison is 3 Volume
often used as an index of the ability of the powder to flow, for Therefore, by definition :
example the compressibility index or the Hausner ratio.
Qr(x) = 0.90 when x = x90
The compressibility index and Hausner ratio are measures
of the propensity of a powder to be compressed as described Qr(x) = 0.50 when x = x50
above. As such, they are measures of the powder’s ability Qr(x) = 0.10 when x = x10
to settle, and they permit an assessment of the relative An alternative but less informative method of classifying
importance of interparticulate interactions. In a free-flowing powder fineness is by use of the descriptive terms in
powder, such interactions are less significant, and the bulk Table 2.9.35.-1.
and tapped densities will be closer in value. For more-poorly
Table 2.9.35.-1.
flowing materials, there are frequently greater interparticulate
interactions, and a greater difference between the bulk and Classification of powders by fineness
tapped densities will be observed. These differences are
reflected in the compressibility index and the Hausner ratio. Cumulative
Descriptive term x50 (μm) distribution by
Compressibility index : volume basis, Q3(x)
01/2010:20936
Depending on the material, the compressibility index can be
determined using V10 instead of V0. If V10 is used, it is clearly
stated with the results. 2.9.36. POWDER FLOW(16)
The widespread use of powders in the pharmaceutical industry
07/2008:20935 has generated a variety of methods for characterising powder
flow. Not surprisingly, scores of references appear in the
2.9.35. POWDER FINENESS pharmaceutical literature, attempting to correlate the various
measures of powder flow to manufacturing properties. The
Particle-size distribution is estimated by analytical sieving development of such a variety of test methods was inevitable ;
(2.9.38) or by application of other suitable methods where powder behavior is multifaceted and thus complicates the
appropriate. A simple descriptive classification of powder effort to characterise powder flow.
fineness is provided in this chapter. For practical reasons, The purpose of this chapter is to review the methods for
sieves are commonly used to measure powder fineness. characterising powder flow that have appeared most frequently
Sieving is most suitable where a majority of the particles are in the pharmaceutical literature. In addition, while it is
larger than about 75 μm, although it can be used for some clear that no single and simple test method can adequately
powders having smaller particle sizes where the method can characterise the flow properties of pharmaceutical powders,
be validated. Light diffraction is also a widely used technique this chapter proposes the standardisation of test methods that
for measuring the size of a wide range of particles. may be valuable during pharmaceutical development.
Where the cumulative distribution has been determined by 4 commonly reported methods for testing powder flow are :
analytical sieving or by application of other methods, particle
size may be characterised in the following manner : – angle of repose,
x90 – compressibility index or Hausner ratio,
= particle size corresponding to 90 per cent of the
cumulative undersize distribution ; – flow rate through an orifice,
x50 – shear cell.
= median particle size (i.e. 50 per cent of the particles
are smaller and 50 per cent of the particles are In addition, numerous variations of each of these basic
larger) ; methods are available. Given the number of test methods
x10 and variations, standardising the test methodology, where
= particle size corresponding to 10 per cent of the possible, would be advantageous.
cumulative undersize distribution.
With this goal in mind, the most frequently used methods
It is recognised that the symbol d is also widely used to are discussed below. Important experimental considerations
designate these values. Therefore, the symbols d90, d50, d10 are identified and recommendations are made regarding
may be used. standardisation of the methods. In general, any method of
The following parameters may be defined based on the measuring powder flow must be practical, useful, reproducible
cumulative distribution. and sensitive, and must yield meaningful results. It bears
(16) This chapter has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.
repeating that no simple powder flow method will adequately Table 2.9.36.-1. – Flow properties and corresponding angles
or completely characterise the wide range of flow properties of repose(17)
experienced in the pharmaceutical industry. An appropriate
Flow property Angle of repose (degrees)
strategy may well be the use of multiple standardised test
methods to characterise the various aspects of powder flow as Excellent 25-30
needed by the pharmaceutical scientist.
Good 31-35
General Notices (1) apply to all monographs and other texts 347
2.9.36. Powder flow EUROPEAN PHARMACOPOEIA 8.0
compressibility index and the Hausner ratio are calculated The flow rate through an orifice is generally measured as
as follows : the mass per time flowing from any of a number of types of
containers (cylinders, funnels, hoppers). Measurement of the
flow rate can be in discrete increments or continuous.
Basic methods for flow through an orifice
There are a variety of methods described in the literature. The
most common for determining the flow rate through an orifice
Alternatively, the compressibility index and Hausner ratio may can be classified based on 3 important experimental variables :
be calculated using measured values of bulk density (ρbulk) and – the type of container used to contain the powder. Common
tapped density (ρtapped) as follows : containers are cylinders, funnels, and hoppers from
production equipment ;
– the size and shape of the orifice used. The orifice diameter
and shape are critical factors in determining powder flow
rate ;
– the method of measuring powder flow rate. Flow rate can
In a variation of these methods, the rate of consolidation is be measured continuously using an electronic balance
sometimes measured rather than, or in addition to, the change with some sort of recording device (strip chart recorder,
in volume that occurs on tapping. For the compressibility computer). It can also be measured in discrete samples
index and the Hausner ratio, the generally accepted scale of (for example, the time it takes for 100 g of powder to pass
flowability is given in Table 2.9.36.-2. through the orifice to the nearest tenth of a second or the
amount of powder passing through the orifice in 10 s to
Table 2.9.36.-2. – Scale of flowability(17) the nearest tenth of a gram).
Compressibility Flow character Hausner ratio Variations in methods for flow through an orifice
index (per cent)
Either mass flow rate or volume flow rate can be determined.
1-10 Excellent 1.00-1.11 Mass flow rate is the easier of the methods, but it biases the
11-15 Good 1.12-1.18 results in favour of high-density materials. Since die fill is
volumetric, determining volume flow rate may be preferable.
16-20 Fair 1.19-1.25 A vibrator is occasionally attached to facilitate flow from the
21-25 Passable 1.26-1.34 container, however, this appears to complicate interpretation
of results. A moving orifice device has been proposed to
26-31 Poor 1.35-1.45 more closely simulate rotary press conditions. The minimum
32-37 Very poor 1.46-1.59 diameter orifice through which powder flows can also be
identified.
> 38 Very, very poor > 1.60
General scale of flowability for flow through an orifice
Experimental considerations for the compressibility index No general scale is available because flow rate is critically
and Hausner ratio dependent on the method used to measure it. Comparison
Compressibility index and Hausner ratio are not intrinsic between published results is difficult.
properties of the powder, that is to say, they are dependent Experimental considerations for flow through an orifice
upon the methodology used. The existing literature points out Flow rate through an orifice is not an intrinsic property of the
several important considerations affecting the determination powder. It is very much dependent upon the methodology
of the unsettled apparent volume, V0, of the final tapped used. The existing literature points out several important
volume, Vf, of the bulk density, ρbulk, and of the tapped density, considerations affecting these methods :
ρtapped : – the diameter and shape of the orifice,
– the diameter of the cylinder used, – the type of container material (metal, glass, plastic),
– the number of times the powder is tapped to achieve the – the diameter and height of the powder bed.
tapped density,
Recommended procedure for flow through an orifice
– the mass of material used in the test,
Flow rate through an orifice can be used only for materials
– rotation of the sample during tapping. that have some capacity to flow. It is not useful for cohesive
Recommended procedure for compressibility index and materials. Provided that the height of the powder bed (the
Hausner ratio ‘head’ of powder) is much greater than the diameter of the
orifice, the flow rate is virtually independent of the powder
Use a 250 mL volumetric cylinder with a test sample mass
head. It is advisable to use a cylinder as the container, because
of 100 g. Smaller amounts and volumes may be used, but
the walls of the container must have little effect on flow. This
variations in the method must be described with the results.
configuration results in flow rate being determined by the
An average of 3 determinations is recommended.
movement of powder over powder, rather than powder along
FLOW THROUGH AN ORIFICE the wall of the container. Powder flow rate often increases
when the height of the powder column is less than twice the
The flow rate of a material depends upon many factors, some diameter of the column. The orifice must be circular and the
of which are particle-related and some related to the process. cylinder must be free of vibration. General guidelines for
Monitoring the rate of flow of material through an orifice dimensions of the cylinder are as follows :
has been proposed as a better measure of powder flowability.
– diameter of the opening greater than 6 times the diameter
Of particular significance is the utility of monitoring flow
continuously, since pulsating flow patterns have been observed of the particles,
even for free-flowing materials. Changes in flow rate as the – diameter of the cylinder greater than twice the diameter of
container empties can also be observed. Empirical equations the opening.
relating flow rate to the diameter of the opening, particle Use of a hopper as the container may be appropriate and
size, and particle density have been determined. However, representative of flow in a production situation. It is not
determining the flow rate through an orifice is useful only advisable to use a funnel, particularly one with a stem, because
with free-flowing materials. flow rate will be determined by the size and length of the stem
as well as the friction between the stem and the powder. A 01/2010:20937
truncated cone may be appropriate, but flow will be influenced
by the powder-wall friction coefficient, thus, selection of an
appropriate construction material is important.
2.9.37. OPTICAL MICROSCOPY(18)
Optical microscopy for particle characterisation can generally
For the opening in the cylinder, use a flat-faced bottom be applied to particles of 1 μm and greater. The lower limit
plate with the option to vary orifice diameter to provide is imposed by the resolving power of the microscope. The
maximum flexibility and better ensure a powder-over-powder upper limit is less definite and is determined by the increased
flow pattern. Rate measurement can be either discrete or difficulty associated with the characterisation of larger
continuous. Continuous measurement using an electronic particles. Various alternative techniques are available for
balance can more effectively detect momentary flow rate particle characterisation outside the applicable range of optical
variations. microscopy. Optical microscopy is particularly useful for
characterising particles that are not spherical. This method
may also serve as a base for the calibration of faster and more
routine methods that may be developed.
SHEAR CELL METHODS
Apparatus. Use a microscope that is stable and protected
In an effort to put powder flow studies and hopper design on a from vibration. The microscope magnification (product of the
more fundamental basis, a variety of powder shear testers and objective magnification, ocular magnification, and additional
methods that permit more thorough and precisely defined magnifying components) must be sufficient to allow adequate
assessment of powder flow properties have been developed. characterisation of the smallest particles to be classified in the
Shear cell methodology has been used extensively in the study test sample. The greatest numerical aperture of the objective
of pharmaceutical materials. From these methods, a wide is sought for each magnification range. Polarising filters may
variety of parameters can be obtained, including the yield loci be used in conjunction with suitable analysers and retardation
representing the shear stress-shear strain relationship, the plates. Colour filters of relatively narrow spectral transmission
angle of internal friction, the unconfined yield strength, the are used with achromatic objectives, and are preferable
tensile strength, and a variety of derived parameters such as with apochromats ; they are required for appropriate colour
the flow factor and other flowability indices. Because of the rendition in photomicrography. Condensers, corrected at least
ability to control experimental parameters more precisely, for spherical aberration are used in the microscope substage
flow properties can also be determined as a function of and with the lamp. The numerical aperture of the substage
consolidation load, time, and other environmental conditions. condenser matches that of the objective under the conditions
These methods have been successfully used to determine of use ; this is affected by the actual aperture of the condenser
critical hopper and bin parameters. diaphragm and the presence of immersion oils.
Basic methods for shear cell Adjustment. The precise alignment of all elements of
the optical system and proper focusing are essential. The
One type of shear cell is the cylindrical shear cell which is focusing of the elements is done in accordance with the
split horizontally, forming a shear plane between the lower recommendations of the microscope manufacturer. Critical
stationary base and the upper moveable portion of the shear axial alignment is recommended.
cell ring. After powder bed consolidation in the shear cell Illumination. A requirement for good illumination is a
(using a well-defined procedure), the force necessary to shear uniform and adjustable intensity of light over the entire field
the powder bed by moving the upper ring is determined. of view ; Köhler illumination is preferred. With coloured
Annular shear cell designs offer some advantages over the particles, choose the colour of the filters so as to control the
cylindrical shear cell design, including the need for less contrast and detail of the image.
material. A disadvantage, however, is that because of its
design, the powder bed is not sheared as uniformly because Visual characterisation. The magnification and numerical
material on the outside of the annulus is sheared more aperture must be sufficiently high to allow adequate resolution
than material in the inner region. A third type of shear cell of the images of the particles to be characterised. Determine
(plate-type) consists of a thin sandwich of powder between a the actual magnification using a calibrated stage micrometer
lower stationary rough surface and an upper rough surface to calibrate an ocular micrometer. Errors can be minimised if
that is moveable. the magnification is sufficient that the image of the particle is
at least 10 ocular divisions. Each objective must be calibrated
All of the shear cell methods have their advantages and separately. To calibrate the ocular scale, the stage micrometer
disadvantages, but a detailed review is beyond the scope of scale and the ocular scale must be aligned. In this way, a
this chapter. As with the other methods for characterising precise determination of the distance between ocular stage
powder flow, many variations are described in the literature. divisions can be made. Several different magnifications may
A significant advantage of shear cell methodology in general be necessary to characterise materials having a wide particle
is a greater degree of experimental control. The methodology size distribution.
generally is rather time-consuming and requires significant Photographic characterisation. If particle size is to be
amounts of material and a well-trained operator. determined by photographic methods, take care to ensure
Recommendations for shear cell that the object is sharply focused at the plane of the
photographic emulsion. Determine the actual magnification
The many existing shear cell configurations and test methods by photographing a calibrated stage micrometer, using
provide a wealth of data and can be used very effectively photographic film of sufficient speed, resolving power, and
to characterise powder flow. They are also helpful in the contrast. Exposure and processing must be identical for
design of equipment such as hoppers and bins. Because of the photographs of both the test sample and the determination
diversity of available equipment and experimental procedures, of magnification. The apparent size of a photographic image
no specific recommendations regarding methodology are is influenced by the exposure, development, and printing
presented in this chapter. It is recommended that the results processes as well as by the resolving power of the microscope.
of powder flow characterisation using shear cell methodology Preparation of the mount. The mounting medium will
include a complete description of equipment and methodology vary according to the physical properties of the test sample.
used. Sufficient, but not excessive, contrast between the sample and
(18) This chapter has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.
General Notices (1) apply to all monographs and other texts 349
2.9.37. Optical microscopy EUROPEAN PHARMACOPOEIA 8.0
the mounting medium is required to ensure adequate detail – Feret’s diameter : the distance between imaginary parallel
of the sample edge. The particles must rest in one plane and lines tangent to a randomly oriented particle and
be adequately dispersed to distinguish individual particles of perpendicular to the ocular scale,
interest. Furthermore, the particles must be representative
of the distribution of sizes in the material and must not be – Martin’s diameter : the diameter of the particle at the point
altered during preparation of the mount. Care must be taken that divides a randomly oriented particle into 2 equal
to ensure that this important requirement is met. Selection of projected areas,
the mounting medium must include a consideration of the
analyte solubility. – projected area diameter : the diameter of a circle that has
the same projected area as the particle,
Crystallinity characterisation. The crystallinity of a material
may be characterised to determine compliance with the – length : the longest dimension from edge to edge of a
crystallinity requirement where stated in the individual particle oriented parallel to the ocular scale,
monograph of a drug substance. Unless otherwise specified
in the individual monograph, mount a few particles of the – width : the longest dimension of the particle measured at
sample in mineral oil on a clean glass slide. Examine the right angles to the length.
mixture using a polarising microscope : the particles show Particle shape characterisation. For irregularly shaped
birefringence (interference colors) and extinction positions particles, characterisation of particle size must also include
when the microscope stage is revolved. information on particle shape. The homogeneity of the
Limit test of particle size by microscopy. Weigh a suitable powder must be checked using appropriate magnification.
quantity of the powder to be examined (for example, The following defines some commonly used descriptors of
10-100 mg), and suspend it in 10 mL of a suitable medium particle shape (see Figure 2.9.37.-2).
in which the powder does not dissolve, adding, if necessary,
a wetting agent. A homogeneous suspension of particles – acicular : slender, needle-like particle of similar width and
can be maintained by suspending the particles in a medium thickness,
of similar or matching density and by providing adequate
– columnar : long, thin particle with a width and thickness
agitation. Introduce a portion of the homogeneous suspension
that are greater than those of an acicular particle,
into a suitable counting cell, and scan under a microscope
an area corresponding to not less than 10 μg of the powder – flake : thin, flat particle of similar length and width,
to be examined. Count all the particles having a maximum
dimension greater than the prescribed size limit. The size – plate : flat particle of similar length and width but with
limit and the permitted number of particles exceeding the greater thickness than a flake particle,
limit are defined for each substance.
– lath : long, thin, blade-like particle,
Particle size characterisation. The measurement of particle
size varies in complexity depending on the shape of the – equant : particle of similar length, width, and thickness ;
particle, and the number of particles characterised must be both cubical and spherical particles are included.
sufficient to ensure an acceptable level of uncertainty in the
measured parameters. Additional information on particle size General observations. A particle is generally considered
measurement, sample size, and data analysis is available, for to be the smallest discrete unit. A particle may be a liquid
example, in ISO 9276. For spherical particles, size is defined by or semi-solid droplet ; a single crystal or polycrystalline ;
the diameter. For irregular particles, a variety of definitions of amorphous or an agglomerate. Particles may be associated.
particle size exist. In general, for irregularly shaped particles, This degree of association may be described by the following
characterisation of particle size must also include information terms :
on the type of diameter measured as well as information on
particle shape. Several commonly used measurements of – lamellar : stacked plates,
particle size are defined in Figure 2.9.37.-1. – aggregate : mass of adhered particles,
– agglomerate : fused or cemented particles,
– conglomerate : mixture of 2 or more types of particles,
– spherulite : radial cluster,
– drusy : particle covered with tiny particles.
Particle condition may be described by the following terms :
– edges : angular, rounded, smooth, sharp, fractured,
– optical : color (using proper color balancing filters),
transparent, translucent, opaque,
– defects : occlusions, inclusions.
Surface characteristics may be described as :
– cracked : partial split, break, or fissure,
– smooth : free of irregularities, roughness, or projections,
– porous : having openings or passageways,
– rough : bumpy, uneven, not smooth,
Figure 2.9.37.-1. – Commonly used measurements of particle
size – pitted : small indentations.
General Notices (1) apply to all monographs and other texts 351
2.9.38. Particle-size distribution estimation by analytical sieving EUROPEAN PHARMACOPOEIA 8.0
electrostatic charge, careful observation must be made to normal agitation mechanism. It requires sequential analyses
ensure that such charging does not influence the analysis. on individual sieves starting with the finest sieve to obtain a
An antistatic agent, such as colloidal silicon dioxide and/or particle-size distribution. Air-jet sieving often includes the
aluminum oxide, may be added at a 0.5 per cent (m/m) level use of finer test sieves than used in ordinary dry sieving. This
to minimise this effect. If both of the above effects cannot technique is more suitable where only oversize or undersize
be eliminated, an alternative particle-sizing technique must fractions are needed.
be selected. In the sonic-sifter method, a nest of sieves is used, and the
Agitation methods. Several different sieve and test sample is carried in a vertically oscillating column of air
powder-agitation devices are commercially available, all of that lifts the sample and then carries it back against the mesh
which may be used to perform sieve analyses. However, the openings at a given number of pulses per minute. It may
different methods of agitation may give different results for be necessary to lower the sample amount to 5 g when sonic
sieve analyses and endpoint determinations because of the sifting is employed.
different types and magnitudes of the forces acting on the The air-jet sieving and sonic-sifter sieving methods may be
individual particles under test. Methods using mechanical useful for powders or granules when the mechanical sieving
agitation or electromagnetic agitation, and that can induce techniques are incapable of giving a meaningful analysis.
either a vertical oscillation or a horizontal circular motion, These methods are highly dependent upon proper dispersion
or tapping or a combination of both tapping and horizontal
of the powder in the air current. This requirement may be
circular motion are available. Entrainment of the particles hard to achieve if the method is used at the lower end of the
in an air stream may also be used. The results must indicate sieving range (i.e. below 75 μm), when the particles tend
which agitation method was used and the agitation parameters
to be more cohesive, and especially if there is any tendency
used (if they can be varied), since changes in the agitation for the material to develop an electrostatic charge. For the
conditions will give different results for the sieve analysis and above reasons endpoint determination is particularly critical,
endpoint determination, and may be sufficiently different to
and it is very important to confirm that the oversize material
give a failing result under some circumstances. comprises single particles and is not composed of aggregates.
Endpoint determination. The test sieving analysis is complete
when the mass on any of the test sieves does not change by INTERPRETATION
more than 5 per cent or 0.1 g (10 per cent in the case of 76 mm The raw data must include the mass of the test sample, the
sieves) of the previous mass on that sieve. If less than 5 per total sieving time, the precise sieving methodology, and the set
cent of the total sample mass is present on a given sieve, the values for any variable parameters, in addition to the masses
endpoint for that sieve is increased to a mass change of not retained on the individual sieves and in the pan.
more than 20 per cent of the previous mass on that sieve.
It may be convenient to convert the raw data into a cumulative
If more than 50 per cent of the total sample mass is found mass distribution, and if it is desired to express the distribution
on any one sieve, unless this is indicated in the monograph, in terms of a cumulative mass undersize, the range of sieves
the test is repeated, but with the addition to the sieve nest of used must include a sieve through which all the material
a more coarse sieve intermediate between that carrying the passes. If there is evidence on any of the test sieves that the
excessive mass and the next coarsest sieve in the original nest, material remaining on it is composed of aggregates formed
i.e. addition of the ISO series sieve omitted from the nest of during the sieving process, the analysis is invalid.
sieves.
SIEVING METHODS
Mechanical agitation (Dry sieving method). Tare each test 04/2011:20939
sieve to the nearest 0.1 g. Place an accurately weighed quantity
of test sample on the top (coarsest) sieve, and replace the lid.
Agitate the nest of sieves for 5 min, then carefully remove
2.9.39. WATER-SOLID INTERACTIONS :
each sieve from the nest without loss of material. Reweigh DETERMINATION OF SORPTION-
each sieve, and determine the mass of material on each one. DESORPTION ISOTHERMS AND OF
Determine the mass of material in the collecting pan in a
similar manner. Re-assemble the nest of sieves, and agitate for WATER ACTIVITY
5 min. Remove and weigh each sieve as previously described. INTRODUCTION
Repeat these steps until the endpoint criteria are met (see
Endpoint determination under Test sieves). Upon completion Pharmaceutical solids as raw materials or as constituents
of the analysis, reconcile the masses of material. Total loss of dosage forms most often come in contact with water
must not exceed 5 per cent of the mass of the original test during processing and storage. This may occur (a) during
sample. crystallisation, lyophilisation, wet granulation, or spray
drying ; and (b) because of exposure upon handling and
Repeat the analysis with a fresh sample, but using a single storage to an atmosphere containing water vapour or exposure
sieving time equal to that of the combined times used above. to other materials in a dosage form that contain water capable
Confirm that this sieving time conforms to the requirements of distributing it to other ingredients. Some properties known
for endpoint determination. When this endpoint has been to be altered by the association of solids with water include
validated for a specific material, then a single fixed time rates of chemical degradation in the “solid-state”, crystal
of sieving may be used for future analyses, providing the growth and dissolution, dispersibility and wetting, powder
particle-size distribution falls within normal variation. flow, lubricity, powder compactibility, compact hardness and
If there is evidence that the particles retained on any sieve are microbial contamination.
aggregates rather than single particles, the use of mechanical Although precautions can be taken when water is perceived to
dry sieving is unlikely to give good reproducibility, and a be a problem, i.e. eliminating all moisture, reducing contact
different particle-size analysis method must be used. with the atmosphere, or controlling the relative humidity of
Air-entrainment methods (Air-jet and sonic-sifter sieving). the atmosphere, such precautions generally add expense to the
Different types of commercial equipment that use a moving process with no guarantee that during the life of the product
air current are available for sieving. A system that uses a single further problems associated with moisture will be avoided. It
sieve at a time is referred to as air-jet sieving. It uses the same is also important to recognise that there are many situations
general sieving methodology as that described under Dry where a certain level of water in a solid is required for proper
sieving method, but with a standardised air jet replacing the performance, e.g. powder compaction. It is essential for both
General Notices (1) apply to all monographs and other texts 353
2.9.39. Water-solid interactions EUROPEAN PHARMACOPOEIA 8.0
reasons, therefore, that as much as possible is known about the increasingly above the glass transition temperature, it is
effects of moisture on solids before strategies are developed not surprising that a number of important bulk properties
for their handling, storage and use. dependent on the rheology of the solid are affected by moisture
Some of the more critical pieces of required information content. Since amorphous solids are metastable relative to the
concerning water-solid interactions are : crystalline form of the material, with small-molecular-mass
– total amount of water present ; materials, it is possible for absorbed moisture to initiate
reversion of the solid to the crystalline form, particularly
– the extent to which adsorption and absorption occur ; if the solid is transformed by the sorbed water to a “fluid”
– whether or not hydrates form ; state. This is the basis of “cake collapse” often observed
– specific surface area of the solid, as well as such properties during the lyophilisation process. An additional phenomenon
as degree of crystallinity, degree of porosity, and glass noted specifically with water-soluble solids is their tendency
transition and melting temperature ; to deliquesce, i.e. to dissolve in their own sorbed water, at
– site of water interaction, the extent of binding, and the relative humidities, RHi, in excess of the relative humidity of
degree of molecular mobility ; a saturated solution of the solid, RH0. Deliquescence arises
because of the high water solubility of the solid and the
– effects of temperature and relative humidity ; significant effect it has on the colligative properties of water. It
– essentially irreversible hydration ; is a dynamic process that continues to occur as long as RHi is
– kinetics of moisture uptake ; greater than RH0.
– various factors that might influence the rate at which water The key to understanding the effects water can have on the
vapour can be taken up by a solid ; properties of solids, and vice versa, rests with an understanding
– for water-soluble solids capable of being dissolved by the of the location of the water molecule and its physical state.
sorbed water, under which conditions dissolution will take More specifically, water associated with solids can exist in a
place. state that is directly bound to the solid, as well as in a state of
mobility approaching that of bulk water. This difference in
PHYSICAL STATES OF SORBED WATER mobility has been observed through such measurements as
Water can physically interact with solids in different ways. It heats of sorption, freezing point, nuclear magnetic resonance,
can interact at the surface (adsorption) or it can penetrate the dielectric properties and diffusion. Such changes in mobility
bulk solid structure (absorption). When both adsorption and have been interpreted as arising because of changes in the
absorption occur, the term sorption is often used. Adsorption thermodynamic state of water as more and more water
is particularly critical in affecting the properties of solids is sorbed. Thus, water bound directly to a solid is often
when the specific surface area is large. Large values of specific thought as unavailable to affect the properties of the solid,
surface area are seen with solids having very small particles, whereas larger amounts of sorbed water may become more
as well as with solids having a high degree of intraparticle clustered and form water more like that exhibiting solvent
porosity. Absorption is characterised by an association of properties. In the case of crystal hydrates, the combination of
water per gram of solid that is much greater than that which intermolecular forces (hydrogen bonding) and crystal packing
can form a monomolecular layer on the available surface, can produce very strong water-solid interactions. Recognising
and an amount that is generally independent of the specific that the presence of water in an amorphous solid can affect
surface area. the glass transition temperature and hence the physical state
Most crystalline solids will not absorb water into their bulk of the solid, at low levels of water, most polar amorphous
structures because of the close packing and high degree solids are in a highly viscous glassy state because of their
of order of the crystal lattice. Indeed, it has been shown high values of Tg. Hence, water is “frozen” into the solid
that the degree of absorption into solids exhibiting partial structure and is rendered immobile by the high viscosity, e.g.
crystallinity and partial amorphous structure is often inversely 1013 Pa·s. As the amount of water sorbed increases and Tg
proportional to the degree of crystallinity. With some decreases, approaching ambient temperatures, the glassy state
crystalline solids, however, crystal hydrates may form. These approaches that of a “fluid” state and water mobility along
hydrates may exhibit a stoichiometric relationship, in terms with the mobility of the solid itself increases significantly.
of water molecules bound per solid molecule, or they may At high RH, the degree of water plasticisation of the solid
be non-stoichiometric. Upon dehydration, crystal hydrates can be sufficiently high so that water and the solid can now
may either retain their original crystal structure, or lose their achieve significant amounts of mobility. In general, therefore,
crystallinity and become amorphous, or transform into a new this picture of the nature of sorbed water helps to explain
anhydrous or less-hydrated crystal form. the rather significant effect moisture can have on a number
Amorphous or partially amorphous solids are capable of taking of bulk properties of solids such as chemical reactivity and
up significant amounts of water because there is sufficient mechanical deformation. It suggests strongly that methods of
molecular disorder in the solid to permit penetration, evaluating chemical and physical stability of solids and solid
swelling or dissolution. Such behaviour is observed with dosage forms take into account the effects water can have on
most amorphous polymers and with small-molecular-mass the solid when it is sorbed, particularly when it enters the
solids rendered amorphous during preparation, e.g. by solid structure and acts as a plasticiser.
lyophilisation, or after milling. The introduction of defects Rates of water uptake. The rate and extent to which solids
into highly crystalline solids will also produce this behaviour. exposed to the atmosphere might either sorb or desorb water
The greater the chemical affinity of water for the solid, the vapour can be a critical factor in the handling of solids. Even
greater the total amount that can be absorbed. When water is the simple act of weighing out samples of solid on an analytical
absorbed by amorphous solids, the bulk properties of the solid balance and the exposure, therefore, of a thin layer of powder
can be significantly altered. It is well established, for example, to the atmosphere for a few minutes can lead to significant
that amorphous solids, depending on the temperature, error in, for example, the estimation of loss on drying values.
can exist in at least one of 2 states, “glassy” or “fluid” ; the It is well established that water-soluble solids exposed to
temperature at which one state transforms into the other is the relative humidities above that exhibited by a saturated solution
glass transition temperature, Tg. of that solid will spontaneously dissolve via deliquescence
Water absorbed into the bulk solid structure, by virtue of its and continue to dissolve over a long time period. The rate of
effect on the free volume of the solid, can act as an efficient water uptake in general depends on a number of parameters
plasticiser and reduce the value of Tg. Since the rheological not found to be critical in equilibrium measurements because
properties of “fluid” and “glassy” states are quite different, rates of sorption are primarily mass-transfer controlled
i.e. the “fluid” state exhibits much less viscosity as one goes with some contributions from heat-transfer mechanisms.
Thus, factors such as vapour diffusion coefficients in air and Methods. Samples may be stored in chambers at various
in the solid, convective airflow, and the surface area and relative humidities (Figure 2.9.39.-1). The mass gained or lost
geometry of the solid bed and surrounding environment, for each sample is then measured. The major advantage of
can play an important role. Indeed, the method used to this method is convenience, while the major disadvantages are
make measurements can often be the rate-determining factor the slow rate of reaching constant mass, particularly at high
because of these environmental and geometric factors. relative humidities, and the error introduced in opening and
closing the chamber for weighing.
DETERMINATION OF SORPTION-DESORPTION
ISOTHERMS Dynamic gravimetric water sorption systems allow the
Principle. The tendency to take up water vapour is best on-line weighing of a sample in a controlled system to
assessed by measuring sorption or desorption as a function assess the interaction of the material with moisture at
of relative humidity, at constant temperature, and under various programmable levels of relative humidity at a
conditions where sorption or desorption is essentially constant temperature. The major benefit of a controlled
occurring independently of time, i.e. equilibrium. Relative system is that isothermal conditions can be more reliably
humidity, RH, is defined by the following expression : established and that the dynamic response of the sample to
changing conditions can be monitored. Data points for the
determination of the sorption isotherm (e.g. from 0 per cent
to approximately 95 per cent RH, non condensing) are only
taken after a sufficiently constant signal indicates that the
Pc = pressure of water vapour in the system ; sample has reached equilibrium at a given level of humidity.
P0 In some cases (e.g. deliquescence), the maximum time may be
= saturation pressure of water vapour under the same
restricted although the equilibrium level is not reached. The
conditions.
apparatus must adequately control the temperature to ensure
The ratio Pc/P0 is referred to as the relative pressure. Sorption a good baseline stability as well as accurate control of the
or water uptake is best assessed starting with dried samples relative humidity generation. The required relative humidities
and subjecting them to a known relative humidity. Desorption can be generated, e.g. by accurately mixing dry and saturated
is studied by beginning with a system already containing vapour gas with flow controllers. The electrostatic behaviour
sorbed water and reducing the relative humidity. As the name of the powder must also be considered. The verification of
indicates, the sorption-desorption isotherm is valid only for the temperature and the relative humidity (controlled with,
the reference temperature, hence a special isotherm exists for for example, a certified hygrometer, certified salt solutions
each temperature. Ordinarily, at equilibrium, moisture content or deliquescence points of certified salts over an adequate
at a particular relative humidity must be the same, whether range), must be consistent with the instrument specification.
determined from sorption or desorption measurements. The balance must provide a sufficient mass resolution and
However, it is common to see sorption-desorption hysteresis. long term stability.
Figure 2.9.39.-1. – Example of an apparatus for the determination of the water sorption (other designs are possible)
General Notices (1) apply to all monographs and other texts 355
2.9.39. Water-solid interactions EUROPEAN PHARMACOPOEIA 8.0
It is also possible to measure amounts of water uptake not appear more like that seen with adsorption processes. X-ray
detectable gravimetrically using volumetric techniques. In crystallographic analysis and thermal analysis are particularly
some cases, direct analysis of water content by different useful for the study of such systems.
methods such as determination of the boiling point, For situations where water vapour adsorption occurs
determination of water by distillation, loss on drying or predominantly, it is very helpful to measure the specific
gas chromatography may be advantageous. In the case of surface area of the solid by an independent method and to
adsorption, to improve sensitivity, one can increase the specific express adsorption as mass of water sorbed per unit area of
surface area of the sample by reducing particle size or by solid surface. This can be very useful in assessing the possible
using larger samples to increase the total area. It is important, importance of water sorption in affecting solid properties.
however, that such comminution of the solid does not alter For example, 0.5 per cent m/m uptake of water could hardly
the surface structure of the solid or render it more amorphous cover the bare surface of 100 m2/g, while for 1.0 m2/g this
or otherwise less ordered in crystallinity. For absorption, amounts to 100 times more surface coverage. In the case of
where water uptake is independent of specific surface area, pharmaceutical solids which have a specific surface area in the
only increasing sample size will help. Increasing sample size, range of 0.01 m2/g to 10 m2/g, what appears to be low water
however, will increase the time to establish some type of content could represent a significant amount of water for the
equilibrium. To establish accurate values, it is important to get available surface. Since the “dry surface area” is not a factor
desolvation of the sample as thoroughly as possible. Higher in absorption, sorption of water with amorphous or partially
temperatures and lower pressures (vacuum) facilitate this amorphous solids can be expressed on the basis of unit mass
process ; however, one must be aware of any adverse effects corrected for crystallinity, when the crystal form does not
this might have on the solid such as dehydration, chemical sorb significant amounts of water relative to the amorphous
degradation or sublimation. Using higher temperatures to regions.
induce desorption, as in a thermogravimetric apparatus,
likewise must be carefully carried out because of these possible DETERMINATION OF THE WATER ACTIVITY
pitfalls. Principle. Water activity, Aw, is the ratio of vapour pressure
Report and interpretation of the data. Sorption data are of water in the product (P) to saturation pressure of water
usually reported as a graph of the apparent mass change in per vapour (P0) at the same temperature. It is numerically equal
cent of the mass of the dry sample as a function of relative to 1/100 of the relative humidity (RH) generated by the
humidity or time. Sorption isotherms are reported both in product in a closed system. RH can be calculated from direct
tabular form and as a graph. The measurement method must measurements of partial vapour pressure or dew point, or
be traceable with the data. from indirect measurement by sensors whose physical or
electric characteristics are altered by the RH to which they
Adsorption-desorption hysteresis can be interpreted, for are exposed. Ignoring activity coefficients, the relationship
example, in terms of the porosity of the sample, its state between Aw and equilibrium relative humidity (ERH) are
of agglomeration (capillary condensation), the formation represented by the following equations :
of hydrates, polymorphic change, or liquefying of the
sample. Certain types of systems, particularly those with
microporous solids and amorphous solids, are capable of
sorbing large amounts of water vapour. Here, the amount
of water associated with the solid as relative humidity is
decreased, is greater than the amount that originally sorbed as
Method. The water activity is determined by placing the
the relative humidity was increased. For microporous solids,
sample in a small airtight cup inside which the equilibrium
vapour adsorption-desorption hysteresis is an equilibrium
between the water in the solid and the headspace can be
phenomenon associated with the process of capillary
established. The volume of the headspace must be small in
condensation. This takes place because of the high degree of
relation to the sample volume in order not to change the
irregular curvature of the micropores and the fact that they
sorption state of sample during the test. The equilibration as
“fill” (adsorption) and “empty” (desorption) under different
a thermodynamic process takes time but may be accelerated
equilibrium conditions. For non-porous solids capable of
by forced circulation within the cell. The acquired water
absorbing water, hysteresis occurs because of a change in the
activity value is only valid for the simultaneously determined
degree of vapour-solid interaction due to a change in the
temperature. This requires a precise temperature-measuring
equilibrium state of the solid, e.g. conformation of polymer
device as part of the equipment. Furthermore, the probe must
chains, or because the time scale for structural equilibrium is
be thermally insulated to guarantee a constant temperature
longer than the time scale for water desorption. In measuring
during the test. The sensor measuring the humidity of
sorption-desorption isotherms, it is therefore important to
the headspace air above the sample is a key component.
establish that something close to an equilibrium state has
Theoretically, all types of hygrometers can be used, but for
been reached. Particularly with hydrophilic polymers at high
analytical purposes miniaturisation and robustness are a
relative humidities, the establishment of water sorption or
precondition. The Aw measurement may be conducted using
desorption values independent of time is quite difficult, since
the dew point/chilled mirror method(20). A polished, chilled
one is usually dealing with a polymer plasticised into its “fluid”
mirror is used as a condensing surface. The cooling system is
state, where the solid is undergoing significant change.
electronically linked to a photoelectric cell into which light
In the case of crystal hydrate formation, the plot of water is reflected from the condensing mirror. An air stream, in
uptake versus pressure or relative humidity will in these equilibrium with the test sample, is directed at the mirror,
cases exhibit a sharp increase in uptake at a particular which cools until condensation occurs on the mirror. The
pressure and the amount of water taken up will usually temperature at which this condensation begins is the dew point
exhibit a stoichiometric mole:mole ratio of water to solid. from which the ERH is determined. Commercially available
In some cases, however, crystal hydrates will not appear to instruments using the dew point/chilled mirror method
undergo a phase change or the anhydrous form will appear or other technologies need to be evaluated for suitability,
amorphous. Consequently, water sorption or desorption may qualified, and calibrated when used to make water activity
determinations. These instruments are typically calibrated apply to each active substance being comprised in dosage units
over an adequate range, for example, using some saturated salt containing one or more active substances, unless otherwise
solutions at 25 °C such as those listed in Table 2.9.39.-1. specified elsewere in this Pharmacopoeia.
Table 2.9.39.-1. – Standard saturated salt solutions The uniformity of dosage units can be demonstrated by either
of 2 methods : content uniformity or mass variation (see
Saturated salts solutions ERH Table 2.9.40.-1).
Aw
at 25 °C (per cent) The test for content uniformity of preparations presented in
Potassium sulfate dosage units is based on the assay of the individual contents of
97.3 0.973 active substance(s) of a number of dosage units to determine
(K2SO4)
whether the individual contents are within the limits set. The
Barium chloride content uniformity method may be applied in all cases.
90.2 0.902
(BaCl2)
The test for mass variation is applicable for the following
Sodium chloride
75.3 0.753
dosage forms :
(NaCl) (1) solutions enclosed in single-dose containers and in soft
Magnesium nitrate capsules ;
52.9 0.529
(Mg(NO3)2) (2) solids (including powders, granules and sterile solids) that
Magnesium chloride
are packaged in single-dose containers and contain no added
(MgCl2)
32.8 0.328 active or inactive substances ;
(3) solids (including sterile solids) that are packaged in
Lithium chloride single-dose containers, with or without added active or
11.2 0.112
(LiCl) inactive substances, that have been prepared from true
solutions and freeze-dried in the final containers and are
labelled to indicate this method of preparation ;
(4) hard capsules, uncoated tablets, or film-coated tablets,
04/2012:20940 containing 25 mg or more of an active substance comprising
25 per cent or more, by mass, of the dosage unit or, in the case
of hard capsules, the capsule contents, except that uniformity
2.9.40. UNIFORMITY OF of other active substances present in lesser proportions is
DOSAGE UNITS demonstrated by meeting content uniformity requirements.
The test for content uniformity is required for all dosage forms
To ensure the consistency of dosage units, each unit in a batch not meeting the above conditions for the mass variation test.
should have an active substance content within a narrow Alternatively, products that do not meet the 25 mg/25 per cent
range around the label claim. Dosage units are defined as threshold limit may be tested for uniformity of dosage units
dosage forms containing a single dose or a part of a dose of an by mass variation instead of the content uniformity test
active substance in each dosage unit. Unless otherwise stated, on the following condition : the concentration Relative
the uniformity of dosage units specification is not intended Standard Deviation (RSD) of the active substance in the final
to apply to suspensions, emulsions or gels in single-dose dosage units is not more than 2 per cent, based on process
containers intended for cutaneous administration. The test validation data and development data, and if there has been
for content uniformity is not required for multivitamin and regulatory approval of such a change. The concentration RSD
trace-element preparations. is the RSD of the concentration per dosage unit (m/m or m/V),
The term ‘uniformity of dosage unit’ is defined as the degree where concentration per dosage unit equals the assay result
of uniformity in the amount of the active substance among per dosage unit divided by the individual dosage unit mass.
dosage units. Therefore, the requirements of this chapter See the RSD formula in Table 2.9.40.-2.
Table 2.9.40.-1. – Application of Content Uniformity (CU) and Mass Variation (MV) test for dosage forms
Dosage forms Type Sub-Type Dose and ratio of active substance
Tablets uncoated MV CU
coated film-coated MV CU
others CU CU
Capsules hard MV CU
solutions MV MV
Solids in single-dose MV MV
single component
containers
others CU CU
Solutions enclosed in MV MV
single-dose containers
Others CU CU
General Notices (1) apply to all monographs and other texts 357
2.9.40. Uniformity of dosage units EUROPEAN PHARMACOPOEIA 8.0
L2 Maximum allowed range for On the low side, no dosage unit L2 = 25.0 unless otherwise specified
deviation of each dosage unit tested result can be less than 0.75 M while
from the calculated value of M on the high side, no dosage unit
result can be greater than 1.25 M
(This is based on L2 value of 25.0)
percentage of label claim (see Calculation of Acceptance than (1 − L2 × 0.01)M or more than (1 + L2 × 0.01)M in
Value). Assume that the concentration (mass of active calculation of acceptance value under content uniformity or
substance per mass of dosage unit) is uniform. Select not under mass variation. Unless otherwise specified, L1 is 15.0
fewer than 30 dosage units, and proceed as follows for the and L2 is 25.0.
dosage form designated.
Uncoated or film-coated tablets. Accurately weigh 10 tablets 04/2012:20941
individually. Calculate the active substance content, expressed
as percentage of label claim, of each tablet from the mass of
the individual tablets and the result of the assay. Calculate the
2.9.41. FRIABILITY OF GRANULES
acceptance value. AND SPHEROIDS
Hard capsules. Accurately weigh 10 capsules individually, This chapter describes 2 methods for determination of the
taking care to preserve the identity of each capsule. Remove friability of granules and spheroids, which may be used during
the contents of each capsule by suitable means. Accurately development studies. It is recognised, however, that many
weigh the emptied shells individually, and calculate for each methods with equal suitability may be used.
capsule the net mass of its contents by subtracting the mass of This test is intended to determine, under defined conditions,
the shell from the respective gross mass. Calculate the active the friability of granules and spheroids. Friability is defined as
substance content in each capsule from the mass of product a reduction in the mass of the granules or spheroids or in the
removed from the individual capsules and the result of the formation of fragments of granules or spheroids, occurring
assay. Calculate the acceptance value. when the granules or spheroids are subjected to mechanical
Soft capsules. Accurately weigh 10 intact capsules individually strain during handling (tumbling, vibration, fluidisation, etc.).
to obtain their gross masses, taking care to preserve the Examples of changes are abrasion, breakage or deformation of
identity of each capsule. Then cut open the capsules by means granules or spheroids.
of a suitable clean, dry cutting instrument such as scissors or a
sharp open blade, and remove the contents by washing with a METHOD A
suitable solvent. Allow the occluded solvent to evaporate from Apparatus (fluidised-bed apparatus). The apparatus (see
the shells at room temperature over a period of about 30 min, Figure 2.9.41.-1) consists of a glass cylinder (A) with a
taking precautions to avoid uptake or loss of moisture. Weigh conical lower part. The cylinder is provided with a sieve
the individual shells, and calculate the net contents. Calculate lid (B) having an aperture size of 500 μm or any other
the active substance content in each capsule from the mass of suitable sieve. The conical end is connected to a U-shaped
product removed from the individual capsules and the result glass tube (C) that can be disconnected from the cylinder
of the assay. Calculate the acceptance value. for removal of the granules or spheroids. The U-tube is
Solid dosage forms other than tablets and capsules. Proceed attached to a T-coupling (D). One inlet of the T-coupling
as directed for hard capsules, treating each unit as described is joined by a silicone tube to a manometer for regulating
therein. Calculate the acceptance value. the compressed-air flow (use compressed air complying with
the test for water in the monograph Medicinal air (1238)),
Liquid or semi-solid dosage forms. Accurately weigh the the other one is connected via a silicone tube to a by-pass
amount of liquid or semi-solid that is removed from each of 10 flowmeter (E) (0.10-1.00 m3·h− 1).
individual containers in conditions of normal use. If necessary,
compute the equivalent volume after determining the density. Procedure. The following procedure is usually suitable.
Calculate the active substance content in each container from Remove the fine particles by sieving (sieve having an aperture
the mass of product removed from the individual containers size of 710 μm or any other suitable sieve). Introduce about
and the result of the assay. Calculate the acceptance value. 8.0 g (m1) of granules or spheroids into the cylinder (A). Close
the apparatus with the sieve lid (B). Adjust the flow rate of
Calculation of Acceptance Value. Calculate the acceptance the compressed air to 0.45 m3·h− 1. After 15 min, remove the
value (AV) as shown in content uniformity, except that granules or spheroids from the apparatus by disconnecting
the individual contents of the units are replaced with the the U-tube and weigh again (m2). Test 3 samples and calculate
individual estimated contents defined below. the mean value. It is recommended to spray the inside of the
x1, x2,..., xn = individual estimated contents of the apparatus with an antistatic agent every 3 determinations in
dosage units tested ; order to prevent electrostatic charging.
Loss on drying. Dry in an oven at 105 °C, unless otherwise
where
prescribed. Alternatively, other drying conditions as described
in general chapter 2.2.32 may be used.
Calculation
w1, w2,..., wn = individual masses of the dosage units
tested ;
A = content of active substance (percentage of
F = friability ;
label claim) obtained using an appropriate
analytical method (assay) ; T1 = percentage loss on drying before the test (mean of
= mean of individual masses (w1, w2,..., wn). 2 determinations) ;
T2 = percentage loss on drying after the test (mean of
CRITERIA 2 determinations) ;
Apply the following criteria, unless otherwise specified. m1 = mass of the granules or spheroids before the test,
in grams ;
Solid, semi-solid and liquid dosage forms. The requirements
m2 = mass of the granules or spheroids after the test, in
for dosage uniformity are met if the acceptance value of
the first 10 dosage units is less than or equal to L1 per grams.
cent. If the acceptance value is greater than L1 per cent,
test the next 20 dosage units and calculate the acceptance METHOD B
value. The requirements are met if the final acceptance Apparatus (oscillating apparatus). The apparatus (see
value of the 30 dosage units is less than or equal to L1 per Figure 2.9.41.-2) consists of a glass container, containing the
cent and no individual content of the dosage unit is less granules or spheroids to be examined, which is subjected
General Notices (1) apply to all monographs and other texts 359
2.9.41. Friability of granules and spheroids EUROPEAN PHARMACOPOEIA 8.0
to horizontal oscillations. The frequency and duration of or for 120 s at a lower frequency (e.g. 140 oscillations/min)
the oscillations can be varied continuously. The frequency for soft granules or spheroids. Sieve (355 μm, or the same
can be adjusted, using a scale, to a value in the range sieve as used previously) and weigh the granules or spheroids
0-400 oscillations/min. The duration can be set to a value in again (m2). Test 3 samples and calculate the mean value.
the range 0-9999 s.
Loss on drying. Dry in an oven at 105 °C, unless otherwise
Procedure. The following procedure is usually suitable. prescribed. Alternatively, other drying conditions as described
Remove the fine particles by sieving (sieve having an aperture in general chapter 2.2.32 may be used.
size of 355 μm or any other suitable sieve). In the glass
container, weigh about 10.00 g (m1) of the granules or Calculation
spheroids. Install the container in the apparatus. Shake for
240 s at the highest frequency for hard granules or spheroids,
01/2008:20943
Figure 2.9.42.-1. – Flow-through apparatus
corrected 6.1
Dissolution medium. If the dissolution medium is buffered, 2.9.43. APPARENT DISSOLUTION
adjust its pH to within ± 0.05 units of the prescribed value.
Remove any dissolved gases from the dissolution medium This method is mainly used to determine the apparent
before the test since they can cause the formation of bubbles dissolution rate of pure solid substances. It may also be used
that significantly affect the results. for the determination of the apparent dissolution rate of active
substances in preparations presented as powders or granules.
METHOD APPARATUS
Place 1 unit of the preparation to be examined in chamber A. All parts of the apparatus that may come into contact with the
Close the cell with the prepared filter assembly. At the sample or the dissolution medium are chemically inert and do
beginning of the test, chamber A requires air removal via not adsorb, react with, or interfere with the test sample. No
a small orifice connected to the filter assembly. Heat the part of the assembly or its environment contributes significant
dissolution medium to an appropriate temperature taking the motion, agitation or vibration beyond that resulting from the
melting point of the preparation into consideration. Using a flow-through system.
suitable pump, introduce the warmed dissolution medium Apparatus that permits observation of the sample is preferable.
through the bottom of the cell to obtain a suitable continuous
flow through an open or closed circuit at the prescribed The apparatus (see Figure 2.9.43.-1) consists of :
rate (± 5 per cent). When the dissolution medium reaches – a reservoir for the dissolution medium ;
the overflow, air starts to escape through the capillary and – a pump that forces the dissolution medium upwards
chamber B fills with the dissolution medium. The preparation through the flow-through cell ;
General Notices (1) apply to all monographs and other texts 361
2.9.43. Apparent dissolution EUROPEAN PHARMACOPOEIA 8.0
A. reservoir for dissolution medium B. pump C. thermostatically controlled flow-through cell and filter D. collecting vessels for analysis
– a flow-through cell, preferably of transparent material, contain substances extractable by the dissolution medium
mounted vertically with a filter system preventing escape of that would interfere with the prescribed analytical method.
undissolved particles ; Proceed with the analysis of the filtrate as prescribed.
– a water-bath that will maintain the dissolution medium at
the chosen temperature (generally 37 ± 0.5 °C). ASSESSMENT OF THE RESULTS
The flow-through cell shown in Figure 2.9.43.-2 consists of When the test is performed for batch release purposes, an
3 parts that fit into each other. The lower part supports a adequate number of replicates is carried out.
system of grids and filters on which the powder is placed. The The results are expressed as :
middle part, which fits onto the lower part, contains an insert
that sieves the sample when the dissolution medium flows – the amount of dissolved substance by time unit (if the
through the cell. This insert is made up of 2 parts : a conical dissolution is linear);
sieve that is placed on the sample and a clip placed midway – the dissolution time of the whole sample and at appropriate
down the middle part to hold the sieve in place when the intermediate stages.
dissolution medium passes through. A 2nd filtration assembly
(grid and filter) is placed on top of the middle part before
fitting the upper part through which the dissolution medium
flows out of the cell.
DISSOLUTION MEDIUM
If the dissolution medium is buffered, adjust its pH to within
± 0.05 units. Remove any dissolved gases from the dissolution
medium before the test, since they can cause the formation of
bubbles, which significantly affect the results.
METHOD
Place a bead of 5 ± 0.5 mm diameter at the bottom of the
cone of the lower part followed by glass beads of suitable
size, preferably of 1 ± 0.1 mm diameter. Place a sieve (with
0.2 mm apertures), a suitable filter and a 2nd sieve on top of
the lower part. Fit the middle part onto the lower part. Weigh
the assembly. Place the sample on the filtration assembly and
weigh the sample in the cell. Place the sieve of the insert,
cone upwards, on the sample, and position the clip midway
down the middle part. Place a sieve (with 0.2 mm apertures)
and a suitable filter on top of the middle part. Fit the upper
part. Heat the dissolution medium to the chosen temperature.
Using a suitable pump, introduce the dissolution medium
through the bottom of the cell to obtain a suitable continuous
flow through an open or closed circuit at the prescribed
rate ± 5 per cent.
SAMPLING
A. lower part C. clip E. middle part
Samples of dissolution medium are collected at the outlet of
the cell, irrespective of whether the circuit is opened or closed. B. sieve D. insert F. upper part
General Notices (1) apply to all monographs and other texts 363
2.9.44. Preparations for nebulisation : characterisation EUROPEAN PHARMACOPOEIA 8.0
unambiguously in terms of the mass of active substance as a the sampling period (T0) is the most effective way to avoid
function of aerodynamic diameter. Laser diffraction may be overloading in any given system, balancing overloading with
used if validated against a cascade impaction method. analytical sensitivity.
Apparatus E (see below under Apparatus), a cascade impactor, Re-entrainment. Droplet bounce and re-entrainment are
has been calibrated at 15 L/min specifically to meet the less likely with nebuliser-produced droplets than with solid
recommendation of the European standard, and is therefore particles from inhalers and for that reason coating would not
used for this test. Determining mass balance in the same normally be required.
way as for powder inhalers and metered-dose inhalers is METHOD
not straightforward, in that the dose is being captured as Pre-cool the assembled impactor and induction port in a
a continuous output, and hence is not included. As part refrigerator (set at about 5 °C) for not less than 90 min and
of method development, recovery experiments must be start the determination within about 5 min of removal of the
performed to validate the method. impactor from the refrigerator. Other methods that maintain
It is also recognised that the control of evaporation of the impactor at a constant temperature (for example, use of a
droplets produced by nebulisers may be critical to avoid cooling cabinet) can also be employed when validated.
bias in the droplet size assessment process. Evaporation can Set up the nebuliser with a supply of driving gas (usually
be minimised by cooling the impactor to a temperature of air or oxygen), or use a compressor, at the pressure and
about 5 °C, typically achieved by cooling the impactor in a flow rate specified by the manufacturer of the nebuliser.
refrigerator for about 90 min. Typically, at least after each Take precautions to ensure that the gas supply line does not
day of use, the apparatus must be fully cleaned, including the become detached from the nebuliser when under pressure.
inter-stage passageways, in view of the greater risk of corrosion Fill the nebuliser with the volume of the medicinal product as
caused by the condensation/accumulation of saline-containing specified in the patient instructions.
droplets on inter-stage metalwork associated with cooling Remove the impactor from the refrigerator. Attach the
the impactor. It is recommended to dry all surfaces of the induction port to the impactor, and connect the outlet of the
apparatus after each test, for example with compressed air. impactor/external filter to a vacuum source that is capable of
Note : the micro-orifice collector (MOC) should not be dried drawing air through the system at 15 L/min as specified in
with compressed air. Figure 2.9.44.-2. Turn on the flow through the impactor.
APPARATUS Connect a flow meter, calibrated for the volumetric flow
A detailed description of Apparatus E and the induction port leaving the meter, to the induction port. Adjust the flow
is contained in general chapter 2.9.18, and includes details control valve located between the impactor and the vacuum
of critical dimensions and the qualification process for the source to achieve a steady flow through the system at 15 L/min
impactor (stage mensuration). (± 5 per cent). Remove the flow meter.
Make sure the nebuliser is positioned in the same orientation
A back-up filter in addition to the micro-orifice as intended for use then attach the mouthpiece of the
collector (MOC) must be used to ensure quantitative recovery nebuliser to the induction port, using a mouthpiece adapter if
of active substance from the nebulised aerosol at the specified required, ensuring that connections are airtight. Switch on the
flow rate of 15 L/min. The filter is located below the MOC flow/compressor for the nebuliser. Sample for a predetermined
(internal filter option) or a filter in holder, external to the time (T0). Once determined, this time (T0) must be defined
impactor, is used to capture any fine droplets that pass beyond and used in the analytical method for a particular medicinal
the last size fractionating stage. product to ensure that mass fraction data can be compared.
A pre-separator is not used for testing nebuliser-generated At the end of the sampling period, switch off the driving gas
aerosols. flow/compressor to the nebuliser, remove the nebuliser from
the induction port and switch off the flow from the vacuum
METHOD VALIDATION source to the impactor.
Impactor stage overloading. During method development Dismantle the impactor and, using a suitable method of
and validation, it is important to confirm that the volume analysis, determine the mass of active substance collected
of liquid sampled from the nebuliser does not overload the in the induction port, on each stage and on the back-up
impactor. Visual inspection of the collection surfaces on filter/external filter as described for Apparatus E in general
stages collecting most of the droplets may reveal streaking chapter 2.9.18. Add the mass of active substance collected in
if overloading has occurred. This phenomenon is usually the MOC to that deposited on the back-up filter/external filter
also associated with an increase in mass of active substance and treat as a single sample for the purpose of subsequent
collected on the final stage and back-up filter. Reducing calculations.
A. nebuliser B. induction port C. impactor (apparatus E) D. flow control valve E. vacuum source
Figure 2.9.44.-2. – Apparatus E for measuring the size distribution of preparations for nebulisation
General Notices (1) apply to all monographs and other texts 365
2.9.45. Wettability of porous solids including powders EUROPEAN PHARMACOPOEIA 8.0
(2)
η = viscosity of the liquid ;
ρ = density of the liquid ;
γ = surface tension of the liquid ;
θ = contact angle between the solid and the liquid ;
c = material constant, dependent on the porous texture
of the solid.
(3)
Figure 2.9.45.-2. – Sessile drop determination with visual
inspection of the droplet In setting up a Washburn determination, a liquid with known
Under equilibrium conditions the contact angle of a sessile density (ρ), viscosity (η), and surface tension (γ) is used.
drop depends on 3 interrelated surface tensions and is Under these conditions, when the mass of liquid rising into
determined using Young’s equation (see Figure 2.9.45.-2, the porous solid is monitored as a function of time (such that
1st part) : capillary penetration rate ( ) is the experimental data),
2 unknowns remain according to equation (3) : the contact
angle (θ) of the liquid on the solid, and the solid material
γS = surface tension of the solid with air ; constant (c).
γSL = interfacial tension of the solid with the liquid ; Determination of the material constant (c). The material
constant for a porous solid is determined by the following
γL = surface tension of the liquid with air. equation, considering cylindrical pores :
PROCEDURE
Since powders are unable to form a completely flat surface, (4)
the powder is usually compacted as a disc in an attempt to
make the surface smoother. A liquid drop with a given volume r
is placed on the disc (see Figure 2.9.45.-2) allowing direct = average capillary radius within the porous solid ;
measurement of the contact angle using a goniometer fitted N = number of capillaries per volumetric unit.
with an eyepiece protractor, or by geometric construction
on a photomicrograph. Other physical and mathematical If a Washburn determination is performed with a liquid
procedures of data analysis may also be appropriate. The drop considered to have a contact angle of 0° (cos 0° = 1) on the
volume may influence the result. Several determinations of solid, then the solid material constant (c) is the only remaining
the contact angle (θ) (n = 6) are usually carried out and the unknown in equation (3) and can thus be determined.
average is calculated. n-Heptane is the liquid of choice for determining material
WASHBURN METHOD constants because of its low surface tension (20.14 mN·m− 1
The Washburn method is able to measure the contact angle of at 25 °C). n-Hexane may also be used (18.43 mN·m− 1 at
porous solids with a contact angle in the range of 0-90°. 25 °C) but is more volatile. If the powder dissolves too
quickly in these liquids, hexamethyldisiloxane may be used
The tested material is the combination of the sample, the instead (15.9 mN·m− 1 at 25 °C). Replicate determinations are
holder and the filter system. Therefore, an estimation or performed (n = 6) and the average value calculated.
determination of the true value is not possible and only
apparent values of the contact angle can be determined. Once the material constant (c) has been determined for the
However, the contact angle of the sample is the functional solid to be examined, a sample of the solid can be tested
property on which the result is significantly dependent. The for wettability by another liquid. The material constant
outcome of the test is a ranking order listing the wettability determined by the n-heptane test is used in the Washburn
of different substances or formulations characterised by an equation, in combination with the capillary penetration
apparent contact angle. rate ( ) data obtained while testing the substance to be
PRINCIPLE examined in the prescribed liquid. This allows calculation of
If a porous solid is brought into contact with a liquid, such the contact angle.
that the solid is not submerged in the liquid, but rather is just
touching the liquid surface, then the rise of liquid into the NOTE : if a series of liquids (at least 2 liquids in addition to
pores of the solid due to capillary action will be governed by the liquid used to determine the material constant) is tested
the following equations : against a given solid then the resultant contact angle data can
be used to calculate the surface energy of the porous solid.
(1) APPARATUS
Figure 2.9.45.-3 shows the principal components of the
m = mass of liquid sucked into the solid ; apparatus. The main device is an electronic balance with
t a suitable processor ensuring a suitable resolution in force
= time elapsed since the solid and the liquid were measurement and a suitable resolution in lifting up the
brought into contact ; immersion liquid towards the sample.
A = constant, dependent on the properties of the liquid
and the solid to be examined, calculated using the Table 2.9.45.-1 indicates parameters of the electronic balance
following equation : that are generally considered suitable.
General Notices (1) apply to all monographs and other texts 367
2.9.47. Uniformity of dosage units using large sample sizes EUROPEAN PHARMACOPOEIA 8.0
Table 2.9.47.-1. – Acceptability constant (k) and acceptable number of dosage units
with a content outside (1 ± L2 × 0.01)M (= c2) for a given sample size n
100 2.15 804 2.26 2480 2.29 23 4366 2.30 41 6252 2.31 59 8243 2.31 78
7
105 2.16 905 2.27 2585 2.29 24 4471 2.30 42 6357 2.31 60 8347 2.31 79
120 2.17 0 908 2.27 8 2690 2.29 25 4576 2.30 43 6462 2.31 61
8452 2.31 80
1013 2.27 9 2794 2.29 26 4680 2.30 44 6566 2.31 62
139 2.18
8557 2.31 81
1118 2.27 10 2899 2.29 27 4785 2.30 45 6671 2.31 63
161 2.19
8662 2.31 82
1223 2.27 3004 2.29 28 4890 2.30 46 6776 2.31 64
176 2.19
11 8767 2.31 83
1276 2.28 3109 2.29 4995 2.30 47 6881 2.31 65
189 2.20 29
1 8871 2.31 84
1328 2.28 12 3171 2.30 5099 2.30 48 6985 2.31 66
224 2.21
1432 2.28 13 3213 2.30 30 5204 2.30 49 7090 2.31 67 8976 2.31 85
270 2.22
1537 2.28 14 3318 2.30 31 5309 2.30 50 7195 2.31 68 9081 2.31 86
280 2.22
2 1642 2.28 15 3423 2.30 32 5414 2.30 51 7300 2.31 69 9186 2.31 87
328 2.23
1747 2.28 16 3528 2.30 33 5519 2.30 52 7404 2.31 70
9290 2.31 88
385 2.23
3 1851 2.28 3633 2.30 34 5623 2.30 53 7509 2.31 71
17 9395 2.31 89
407 2.24
1918 2.29 3737 2.30 35 5728 2.30 54 7614 2.31 72
9500 2.31 90
490 2.24
1956 2.29 18 3842 2.30 36 5833 2.30 55 7719 2.31 73
4
9605 2.31 91
516 2.25
2061 2.29 19 3947 2.30 37 5938 2.30 56 7824 2.31 74
699 2.26 6 2375 2.29 22 4261 2.30 40 6147 2.31 58 8138 2.31 77 9919 2.31 94
General Notices (1) apply to all monographs and other texts 369
2.9.47. Uniformity of dosage units using large sample sizes EUROPEAN PHARMACOPOEIA 8.0
Table 2.9.47.-2. – Acceptable number of individual dosage units with a content outside (1 ± L1 × 0.01)M (= c1) and
(1 ± L2 × 0.01)M (= c2) respectively, for a given sample size n
100 3 1432 35 2899 67 4366 98 5833 129 7300 160 8767 191
123 4 0 1476 36 13 2935 68 27 4377 99 41 5835 130 7304 161 8780 192 83
55 69
159 5 1521 37 2981 69 4424 100 5883 131 7351 162
8828 193
176 5 1537 37 3004 69 4471 101 5930 132 7399 163
8871 193
196 6 1566 38 14 3027 70 28 4518 102 42 5938 132 7404 163
1 8875 194
1611 39 3073 71 4565 103 5977 133 56 7447 164 70
234 7 84
1642 39 8923 195
273 8 3109 71 4576 103 6024 134 7494 165
1656 40 3120 72 6042 134 7509 165 8971 196
280 8 15 4612 104 43
29
1701 41 3166 73 6072 135 57 7542 166 71 8976 196
313 9 2 4658 105
1746 42 3212 74 6119 136 7589 167
353 10 4680 105 9019 197 85
1747 42 3213 74 6147 136 7614 167
385 10 4705 106 44 9066 198
1791 43 16
3259 75 30 6166 137 58 7637 168 72
394 11 4752 107 9081 198
3 1836 44
3305 76 6214 138 7684 169
434 12 4785 107 9114 199 86
1851 44
3318 76 6252 138 7719 169
476 13 4799 108 45
1882 45 17 9162 200
3351 77 31 6261 139 7732 170 73
490 13 4846 109 59
1927 46 9186 200
3398 78 6308 140 7779 171
517 14 4 4890 109
1956 46 9210 201 87
3423 78 6355 141 7824 171
559 15 4893 110
1972 47 18
3444 79 32 46 6357 141 7827 172 9257 202
594 15 2018 48 4940 111 74
3491 80 6403 142 60 7875 173 9290 202
601 16 2061 48 4987 112
5 3528 80 6450 143 7922 174
4995 112 9305 203 88
644 17 2063 49
19 3537 81 6462 143 7928 174
686 18 2109 50 33 5034 113 47 9353 204
3584 82 6498 144 61 7970 175 75
699 18 2154 51 5081 114 9395 204
3630 83 6545 145 8017 176
729 19 6 2166 51 5099 114 9401 205
3633 83 6566 145 8033 176 89
772 20 2200 52 20 5128 115 48 9449 206
3677 84 34 6592 146 62 8065 177 76
804 20 2246 53 5175 116
3723 85 6640 147 8113 178 9496 207
815 21 2270 53 5204 116
7 3737 85 6671 147 8138 178 9500 207
858 22 2291 54 21 5222 117 49
3770 86 35 6687 148 63 8160 179 77 9544 208 90
2337 55 5269 118
902 23
3817 87 6734 149 8208 180 9592 209
2375 55
908 23 5309 118
3842 87 6776 149 8243 180
2383 56 9605 209
945 24 8 22 5317 119
3863 88 36 50 6782 150 8256 181 78
2429 57 64 9640 210 91
989 25 5364 120
3910 89 6829 151 8303 182
2475 58
1013 25 5411 121 9688 211
3947 89 6877 152 8347 182
2480 58
1033 26 9 5414 121 9710 211
3956 90 6881 152 8351 183
2520 59 23
37 5458 122 51 79
1077 27 4003 91 6924 153 65 8399 184 9735 212 92
2566 60
1118 27 5505 123 9783 213
2585 60 4050 92 6972 154 8446 185
1121 28 5519 123
2612 61 24 4052 92 6985 154 8452 185 9814 213
10
1165 29 4097 93 38 5552 124 52 7019 155 66 8494 186 80
2658 62 9831 214 93
1209 30 4143 94 5599 125 7067 156 8542 187
2690 62 9879 215
1223 30 4156 94 5623 125 7090 156 8557 187
2704 63 25
9919 215
1253 31 11 4190 95 39 5647 126 53 7114 157 67 8589 188 81
2750 64
9927 216
1298 32 2794 64 4237 96 5694 127 7161 158 8637 189
5728 127 9975 217 94
1328 32 2796 65 4261 96 7195 158 8662 189
26
1342 33 12 2843 66 4284 97 40 5741 128 54 7209 159 68 8685 190 82 10023 218
1387 34 2889 67 4330 98 5788 129 7256 160 8732 191 10070 219
General Notices (1) apply to all monographs and other texts 375
3.1.1.1. Plasticised PVC materials for containers for blood EUROPEAN PHARMACOPOEIA 8.0
– one of the following epoxidised oils : maximum 10 per cent Solution S2. Place 25 g of the material to be examined in a
or 10 per cent of a mixture of the two : borosilicate-glass flask. Add 500 mL of water for injections R
– epoxidised soya oil (plastic additive 04), of which the and cover the neck of the flask with a borosilicate-glass beaker.
oxiran oxygen content is 6 per cent to 8 per cent and the Heat in an autoclave at 121 ± 2 °C for 20 min. Allow to cool
iodine value is not greater than 6 ; and decant the solution. Make the volume up to 500 mL.
– epoxidised linseed oil (plastic additive 05), of which the Appearance of solution S2. Solution S2 is clear (2.2.1) and
oxiran oxygen content is not greater than 10 per cent colourless (2.2.2, Method II).
and the iodine value is not greater than 7. Acidity or alkalinity. To 100 mL of solution S2, add 0.15 mL
Very low amounts of antioxidants added to the vinyl chloride of BRP indicator solution R. Not more than 1.5 mL of 0.01 M
monomer may be detected in the polymer. sodium hydroxide is required to change the colour of the
No antioxidant additive may be added to the polymer. indicator to blue. To 100 mL of solution S2 add 0.2 mL of
methyl orange solution R. Not more than 1.0 mL of 0.01 M
Ultramarine blue is the only colouring material permitted to hydrochloric acid is required to initiate the colour change of
be added. the indicator from yellow to orange.
The supplier of the material must be able to demonstrate
that the qualitative and quantitative composition of the type Absorbance (2.2.25). Evaporate 100.0 mL of solution S2 to
sample is satisfactory for each production batch. dryness. Dissolve the residue in 5.0 mL of hexane R. From
250 nm to 310 nm the absorbance is not greater than 0.25.
CHARACTERS Reducing substances. Carry out the test within 4 h of
Colourless or pale yellow powder, beads, granules or, after preparation of solution S2. To 20.0 mL of solution S2 add 1 mL
transformation, translucent sheets of varying thickness or of dilute sulfuric acid R and 20.0 mL of 0.002 M potassium
containers, with a slight odour. On combustion it gives off permanganate. Boil under a reflux condenser for 3 min and
dense, black smoke. cool immediately. Add 1 g of potassium iodide R and titrate
immediately with 0.01 M sodium thiosulfate, using 0.25 mL of
IDENTIFICATION starch solution R as indicator. Carry out a blank titration using
If necessary, before use, cut the samples of the material to be 20 mL of water for injections R. The difference between the
examined into pieces of maximum dimension on a side of not two titration volumes is not more than 2.0 mL.
greater than 1 cm. Primary aromatic amines : maximum 20 ppm.
To 2.0 g of the material to be examined add 200 mL of To 2.5 mL of solution A1 obtained during the identification,
peroxide-free ether R and heat under a reflux condenser for add 6 mL of water R and 4 mL of 0.1 M hydrochloric acid.
8 h. Separate the residue B and the solution A by filtration. Shake vigorously and discard the upper layer. To the aqueous
Evaporate solution A to dryness under reduced pressure in a layer add 0.4 mL of a freshly prepared 10 g/L solution of
water-bath at 30 °C. Dissolve the residue in 10 mL of toluene R sodium nitrite R. Mix and allow to stand for 1 min. Add
(solution A1). Dissolve the residue B in 60 mL of ethylene 0.8 mL of a 25 g/L solution of ammonium sulfamate R,
chloride R, heating on a water-bath under a reflux condenser. allow to stand for 1 min and add 2 mL of a 5 g/L solution of
Filter. Add the solution obtained dropwise and with vigorous naphthylethylenediamine dihydrochloride R. After 30 min,
shaking to 600 mL of heptane R heated almost to boiling. any colour in the solution is not more intense than that in
Separate the coagulum B1 and the organic solution by hot a standard prepared at the same time in the same manner
filtration. Allow the latter to cool ; separate the precipitate B2 replacing the aqueous layer with a mixture of 1 mL of a
that forms and filter through a tared sintered-glass filter (40) 0.01 g/L solution of naphthylamine R in 0.1 M hydrochloric
(2.1.2). acid, 5 mL of water R and 4 mL of 0.1 M hydrochloric acid
A. Infrared absorption spectrophotometry (2.2.24). instead of the aqueous layer.
Preparation. Dissolve the coagulum B1 in 30 mL of Plastic additives 01, 04 and 05. Thin-layer chromatography
tetrahydrofuran R and add, in small volumes with shaking, (2.2.27).
40 mL of anhydrous ethanol R. Separate the precipitate B3 Reference solutions. Prepare 0.1 mg/mL solutions of
by filtration and dry in vacuo at a temperature not exceeding plastic additive 01 CRS, plastic additive 04 CRS and plastic
50 °C over diphosphorus pentoxide R. Dissolve a few additive 05 CRS, respectively, in toluene R.
milligrams of precipitate B3 in 1 mL of tetrahydrofuran R,
place a few drops of the solution obtained on a sodium Plate : TLC silica gel GF254 plate R.
chloride plate and evaporate to dryness in an oven at Mobile phase : toluene R.
100-105 °C. Application : 0.5 mL of solution A1 obtained during the
Comparison : poly(vinyl chloride) CRS. identification as a band 30 mm by 3 mm and 5 μL of each
B. Infrared absorption spectrophotometry (2.2.24). reference solution.
Examine the residue C obtained in the test for plastic Development : over a path of 15 cm.
additives 01, 04 and 05. Drying : in air.
Comparison : plastic additive 01 CRS. Detection A : examine in ultraviolet light at 254 nm.
TESTS Locate the zone corresponding to plastic additive 01
If necessary, before use, cut the samples of the material to be (RF = about 0.4). Remove the area of silica gel corresponding
examined into pieces of maximum dimension on a side of not to this zone and shake with 40 mL of ether R for 1 min. Filter,
greater than 1 cm. rinse with two quantities, each of 10 mL of ether R, add the
rinsings to the filtrate and evaporate to dryness. The residue C
Solution S1. Place 5.0 g of the material to be examined in weighs not more than 40 mg.
a combustion flask. Add 30 mL of sulfuric acid R and heat
until a black, syrupy mass is obtained. Cool and add carefully Detection B : expose the plate to iodine vapour for 5 min.
10 mL of strong hydrogen peroxide solution R. Heat gently. Examine the chromatogram and locate the band corresponding
Allow to cool and add 1 mL of strong hydrogen peroxide to plastic additives 04 and 05 (RF = 0). Remove the area of
solution R ; repeat by alternating evaporation and addition silica gel corresponding to this zone. Similarly remove a
of hydrogen peroxide solution until a colourless liquid is corresponding area of silica gel as a blank reference. Separately
obtained. Reduce the volume to about 10 mL. Cool and dilute shake both samples for 15 min with 40 mL of methanol R.
to 50.0 mL with water R. Filter, rinse with 2 quantities, each of 10 mL of methanol R,
add the rinsings to the filtrate and evaporate to dryness. The Test solution. Dilute solution S1 100 times with 0.1 M
difference between the masses of both residues is not more hydrochloric acid.
than 10 mg. Reference solutions. Prepare the reference solutions using
Plastic additive 03. zinc standard solution (100 ppm Zn) R, diluting with 0.1 M
Wash precipitate B2 obtained during the identification and hydrochloric acid.
contained in the tared sintered-glass filter (40) (2.1.2) with Source : zinc hollow-cathode lamp.
anhydrous ethanol R. Dry to constant mass over diphosphorus Wavelength : 213.9 nm.
pentoxide R and weigh the filter. The residue weighs not more Atomisation device : air-acetylene flame.
than 20 mg. Verify the absence of zinc in the hydrochloric acid used.
Infrared absorption spectrophotometry (2.2.24). Heavy metals (2.4.8) : maximum 50 ppm.
Preparation : the residue obtained above. To 10 mL of solution S1 add 0.5 mL of phenolphthalein
Comparison : plastic additive 03 CRS. solution R and then strong sodium hydroxide solution R until
Barium : maximum 5 ppm. a pale pink colour is obtained. Dilute to 25 mL with water R.
12 mL of the solution complies with test A. Prepare the
Inductively coupled plasma-atomic emission spectrometry reference solution using lead standard solution (2 ppm Pb) R.
(2.2.57).
Water extractable substances : maximum 0.3 per cent.
Test solution. Ignite 1.0 g of the substance to be examined in a
silica crucible. Take up the residue with 10 mL of hydrochloric Evaporate 50 mL of solution S2 to dryness on a water-bath
acid R and evaporate to dryness on a water-bath. Take up the and dry in an oven at 100-105 °C to constant mass. Carry
residue with 20 mL of 0.1 M hydrochloric acid. out a blank test with 50.0 mL of water for injections R. The
residue weighs not more than 7.5 mg taking into account the
Reference solution. A solution containing 0.25 ppm of barium blank test.
prepared by dilution of barium standard solution (50 ppm
Ba) R with 0.1 M hydrochloric acid. ASSAY
Wavelength : use the emission of barium at 455.40 nm, the Carry out the oxygen-flask method (2.5.10) using 50.0 mg.
spectral background being taken at 455.30 nm. Absorb the combustion products in 20 mL of 1 M sodium
Verify the absence of barium in the hydrochloric acid used. hydroxide. To the solution obtained add 1 mL of dibutyl
phthalate R, 2.5 mL of nitric acid R, 5 mL of ferric ammonium
Cadmium : maximum 0.6 ppm. sulfate solution R2 and 10.0 mL of 0.1 M silver nitrate. Titrate
Atomic absorption spectrometry (2.2.23, Method I). with 0.05 M ammonium thiocyanate until a reddish-yellow
Test solution. Evaporate 10 mL of solution S1 to dryness. colour is obtained. Carry out a blank test.
Take up the residue using 5 mL of a 1 per cent V/V solution 1 mL of 0.1 M silver nitrate is equivalent to 6.25 mg of
of hydrochloric acid R, filter and dilute the filtrate to 10.0 mL poly(vinyl chloride).
with the same acid solution. In addition, the following tests are carried out on the sterile
Reference solutions. Prepare the reference solutions using and empty containers.
cadmium standard solution (0.1 per cent Cd) R, diluting with a Solution S3. If the container to be examined contains an
1 per cent V/V solution of hydrochloric acid R. anticoagulant solution, empty the container and wash the
Source : cadmium hollow-cathode lamp. inside with 250 mL of water for injections R at 20 ± 1 °C and
Wavelength : 228.8 nm. discard the washings before the preparation of solution S3.
Introduce into the container a volume of water for injections R
Atomisation device : air-acetylene flame. corresponding to the volume of solution. Close the container
Verify the absence of cadmium in the hydrochloric acid used. and heat in an autoclave so that the temperature of the liquid
Calcium : maximum 0.07 per cent. is maintained at 110 °C for 30 min. After cooling, fill the
container with water for injections R to its nominal volume
Inductively coupled plasma-atomic emission spectrometry and homogenise.
(2.2.57).
Reference solution. Heat water for injections R in a
Test solution. Use the test solution prepared for the borosilicate-glass flask in an autoclave at 110 °C for 30 min.
determination of barium.
Reducing substances. Immediately after preparation of
Reference solution. A solution containing 50.0 ppm of calcium solution S3, transfer to a borosilicate-glass flask a volume
prepared by dilution of calcium standard solution (400 ppm corresponding to 8 per cent of the nominal volume of the
Ca) R with 0.1 M hydrochloric acid. container. At the same time, prepare a blank using an equal
Wavelength : use the emission of calcium at 315.89 nm, the volume of the freshly prepared reference solution in another
spectral background being taken at 315.60 nm. borosilicate-glass flask. To each solution add 20.0 mL of
Verify the absence of calcium in the hydrochloric acid used. 0.002 M potassium permanganate and 1 mL of dilute sulfuric
acid R. Allow to stand protected from light for 15 min. To
Tin maximum 20 ppm. each solution add 0.1 g of potassium iodide R. Allow to stand
Inductively coupled plasma-atomic emission spectrometry protected from light for 5 min and titrate immediately with
(2.2.57). 0.01 M sodium thiosulfate, using 0.25 mL of starch solution R
Test solution. Dilute solution S1 10 times with water R as indicator. The difference between the two titrations is not
immediately before use. more than 2.0 mL.
Reference solution. Introduce 2 mL of tin standard solution Acidity or alkalinity. To a volume of solution S3
(5 ppm Sn) R into a 50 mL flask containing 5 mL of a 20 per corresponding to 4 per cent of the nominal capacity of the
cent V/V solution of sulfuric acid R and dilute to 50 mL with container add 0.1 mL of phenolphthalein solution R. The
water R immediately before use. solution remains colourless. Add 0.4 mL of 0.01 M sodium
Wavelength : use the emission of tin at 189.99 nm, the spectral hydroxide. The solution is pink. Add 0.8 mL of 0.01 M
background being taken at 190.10 nm. hydrochloric acid and 0.1 mL of methyl red solution R. The
solution is orange-red or red.
Verify the absence of tin in the sulfuric acid used.
Chlorides (2.4.4) : maximum 0.4 ppm, determined on solution
Zinc maximum 0.2 per cent. S3. Prepare the standard using a mixture of 1.2 mL of chloride
Atomic absorption spectrometry (2.2.23, Method I). standard solution (5 ppm Cl) R and 13.8 mL of water R.
General Notices (1) apply to all monographs and other texts 377
3.1.1.2. Plasticised PVC materials for transfusion of blood EUROPEAN PHARMACOPOEIA 8.0
Detection : flame ionisation. Limit : locate the zone corresponding to plastic additive 01.
Injection : 1 mL of the head space. Remove the area of silica gel corresponding to this zone and
Limit : shake with 40 mL of ether R. Filter without loss and evaporate
to dryness. The residue weighs not more than 40 mg.
– vinyl chloride : maximum 1 ppm.
The supplier of the material must be able to demonstrate Barium : maximum 5 ppm.
that the qualitative and quantitative composition of the type Inductively coupled plasma-atomic emission spectrometry
sample is satisfactory for each production batch. (2.2.57).
Test solution. Ignite 1.0 g of the substance to be examined in a
CHARACTERS silica crucible. Take up the residue with 10 mL of hydrochloric
Almost colourless or pale-yellow material in the form of acid R and evaporate to dryness on a water-bath. Take up the
powder, beads, granules or, after transformation, tubes with a residue with 20 mL of 0.1 M hydrochloric acid.
slight odour. Reference solution. A solution containing 0.25 ppm of barium
On combustion it gives off dense, black smoke. prepared by dilution of barium standard solution (50 ppm
IDENTIFICATION Ba) R with 0.1 M hydrochloric acid.
If necessary, cut the samples of the material to be examined Wavelength : use the emission of barium at 455.40 nm, the
into pieces with a maximum dimension on a side of not greater spectral background being taken at 455.30 nm.
than 1 cm. Verify the absence of barium in the hydrochloric acid used.
A. To 0.5 g add 30 mL of tetrahydrofuran R. Heat with stirring Cadmium : maximum 0.6 ppm.
on a water-bath in a fume cupboard for 10 min. The Atomic absorption spectrometry (2.2.23, Method I).
material dissolves completely. Add methanol R dropwise Test solution. Evaporate 10.0 mL of solution S1 to dryness.
with stirring. A granular precipitate is formed. Filter the Take up the residue using 5 mL of a 1 per cent V/V solution
precipitate and dry at 60 °C. Examine the precipitate by of hydrochloric acid R, filter and dilute the filtrate to 10.0 mL
infrared absorption spectrophotometry (2.2.24). Dissolve with the same acid.
50 mg in 2 mL of tetrahydrofuran R and pour on a glass
slide. Dry in an oven at 80 °C, remove the film and fix Reference solutions. Prepare the reference solutions using
on a suitable mount. Examine by infrared absorption cadmium standard solution (0.1 per cent Cd) R, diluting with a
spectrophotometry (2.2.24), comparing with the spectrum 1 per cent V/V solution of hydrochloric acid R.
obtained with poly(vinyl chloride) CRS. Source : cadmium hollow-cathode lamp.
B. Infrared absorption spectrophotometry (2.2.24). Examine Wavelength : 228.8 nm.
the residue obtained in the test plastic additive 01. Atomisation device : air-acetylene flame.
Comparison : plastic additive 01 CRS. Verify the absence of cadmium in the hydrochloric acid used.
TESTS Tin : maximum 20 ppm.
If necessary, cut the samples of the material to be examined Inductively coupled plasma-atomic emission spectrometry
into pieces with a maximum dimension on a side of not greater (2.2.57).
than 1 cm. Test solution. Dilute solution S1 10 times with water R
Solution S1. Place 5.0 g of the material to be examined in immediately before use.
a combustion flask. Add 30 mL of sulfuric acid R and heat Reference solution. Introduce 2 mL of tin standard solution
until a black, syrupy mass is obtained. Cool and add carefully (5 ppm Sn) R into a 50 mL flask containing 5 mL of a 20 per
10 mL of strong hydrogen peroxide solution R. Heat gently. cent V/V solution of sulfuric acid R and dilute to 50 mL with
Allow to cool and add 1 mL of strong hydrogen peroxide water R immediately before use.
solution R ; repeat by alternating evaporation and addition Wavelength : use the emission of tin at 189.99 nm, the spectral
of hydrogen peroxide solution until a colourless liquid is background being taken at 190.10 nm.
obtained. Reduce the volume to about 10 mL. Cool and dilute Verify the absence of tin in the sulfuric acid used.
to 50.0 mL with water R.
Heavy metals (2.4.8) : maximum 50 ppm.
Solution S2. Place 25 g of the material to be examined in a
borosilicate-glass flask. Add 500 mL of water R and cover the To 10 mL of solution S1 add 0.5 mL of phenolphthalein
neck of the flask with a borosilicate-glass beaker. Heat in an solution R and then strong sodium hydroxide solution R until
autoclave at 121 ± 2 °C for 20 min. Allow to cool then decant a pale pink colour is obtained. Dilute to 25 mL with water R.
the solution and make up to a volume of 500 mL. 12 mL of the solution complies with test A. Prepare the
reference solution using lead standard solution (2 ppm Pb) R.
Appearance of solution S2. Solution S2 is clear (2.2.1) and
colourless (2.2.2, Method II). ASSAY
Plastic additive 01. Thin-layer chromatography (2.2.27). To 0.500 g add 30 mL of tetrahydrofuran R and heat with
Test solution. To 2.0 g of the material to be examined add stirring on a water-bath in a fume cupboard for 10 min. The
200 mL of peroxide-free ether R and heat under a reflux material dissolves completely. Add 60 mL of methanol R
condenser for 8 h. Separate the residue and the solution by dropwise with stirring. A granular precipitate of poly(vinyl
filtration and evaporate the solution to dryness under reduced chloride) is formed. Allow to stand for a few minutes.
pressure in a water-bath at 30 °C. Dissolve the residue in Continue addition of methanol R until no further precipitation
10 mL of toluene R. is observed. Transfer to a sintered-glass filter (40) (2.1.2),
Reference solution. Dissolve 0.8 g of plastic additive 01 CRS in using three small quantities of methanol R to aid transfer and
toluene R and dilute to 10 mL with the same solvent. to wash the precipitate. Dry the filter and the precipitate to
constant mass at 60 °C and weigh.
Plate : TLC silica gel G plate R.
In addition, carry out the following tests on sterilised sets.
Mobile phase : toluene R.
Application : 0.5 mL of the test solution and 5 μL of the Solution S3. Make a closed circulation system from 3 sets
reference solution, as a band 30 mm by 3 mm. and a 300 mL borosilicate-glass vessel. Fit to the vessel a
suitable thermostat device that maintains the temperature
Development : over a path of 15 cm. of the liquid in the vessel at 37 ± 1 °C. Circulate 250 mL of
Drying : in air. water for injections R through the system in the direction
Detection : in ultraviolet light at 254 nm. used for transfusion for 2 h at a rate of 1 L/h (for example
General Notices (1) apply to all monographs and other texts 379
3.1.3. Polyolefins EUROPEAN PHARMACOPOEIA 8.0
– 4,4′,4″-(2,4,6-trimethylbenzene-1,3,5-triyltrismethy-
using a peristaltic pump applied to as short a piece of suitable
silicone elastomer tubing as possible). Collect the whole of lene)tris[2,6-bis(1,1-dimethylethyl)phenol] (plastic
the solution and allow to cool. additive 10) : maximum 0.3 per cent ;
Appearance of solution. Solution S3 is clear (2.2.1) and – 2,2′-bis(octadecyloxy)-5,5′-spirobi[1,3,2-dioxa-
colourless (2.2.2, Method II). phosphinane] (plastic additive 14): maximum 0.3 per cent ;
Acidity or alkalinity. To 25 mL of solution S3 add 0.15 mL – didodecyl 3,3′-thiodipropionate (plastic additive 16) :
of BRP indicator solution R. Not more than 0.5 mL of 0.01 M maximum 0.3 per cent ;
sodium hydroxide is required to change the colour of the – dioctadecyl 3,3′-thiodipropionate (plastic additive 17) :
indicator to blue. To 25 mL of solution S3 add 0.2 mL of maximum 0.3 per cent ;
methyl orange solution R. Not more than 0.5 mL of 0.01 M – tris[2,4-bis(1,1-dimethylethyl)phenyl] phosphite (plastic
hydrochloric acid is required to initiate the colour change of additive 12) : maximum 0.3 per cent ;
the indicator from yellow to orange. – plastic additive 18 : maximum 0.1 per cent ;
Absorbance (2.2.25) : maximum 0.30, determined between – copolymer of dimethyl succinate and (4-hydroxy-2,2,6,6-
wavelengths of 230 nm and 250 nm on solution S3 ; maximum tetramethylpiperidin-1-yl)ethanol (plastic additive 22) :
0.15, determined between wavelengths of 251 nm and 360 nm maximum 0.3 per cent.
on solution S3. The total of antioxidant additives listed above does not exceed
Reducing substances. Carry out the test within 4 h of 0.3 per cent.
preparation of solution S3. To 20.0 mL of solution S3 add 1 mL – hydrotalcite : maximum 0.5 per cent ;
of dilute sulfuric acid R and 20.0 mL of 0.002 M potassium – alkanamides : maximum 0.5 per cent ;
permanganate. Boil for 3 min and cool immediately. Add
1 g of potassium iodide R and titrate with 0.01 M sodium – alkenamides : maximum 0.5 per cent ;
thiosulfate using 0.25 mL of starch solution R as indicator. – sodium silico-aluminate : maximum 0.5 per cent ;
Carry out a blank test using 20 mL of water for injections R. – silica : maximum 0.5 per cent ;
The difference between the titration volumes is not greater – sodium benzoate : maximum 0.5 per cent ;
than 2.0 mL. – fatty acid esters or salts : maximum 0.5 per cent ;
Water extractable substances. Evaporate 50.0 mL of – trisodium phosphate : maximum 0.5 per cent ;
solution S3 to dryness on a water-bath and dry to constant
– liquid paraffin : maximum 0.5 per cent ;
mass in an oven at 100-105 °C. Carry out a blank test using
50.0 mL of water for injections R. The residue obtained with – zinc oxide : maximum 0.5 per cent ;
solution S3 is not greater than 1.5 mg, taking account of the – talc : maximum 0.5 per cent ;
blank test. – magnesium oxide : maximum 0.2 per cent ;
– calcium stearate or zinc stearate or a mixture of both :
01/2008:30103 maximum 0.5 per cent ;
corrected 7.5 – titanium dioxide : maximum 4 per cent.
The supplier of the material must be able to demonstrate
3.1.3. POLYOLEFINS that the qualitative and quantitative composition of the type
sample is satisfactory for each production batch.
DEFINITION
Polyolefins are obtained by polymerisation of ethylene or CHARACTERS
propylene or by copolymerisation of these substances with Appearance : powder, beads, granules or, after transformation,
not more than 25 per cent of higher homologues (C4 to C10) sheets of varying thickness or containers.
or of carboxylic acids or of esters. Certain materials may be Solubility : practically insoluble in water, soluble in hot
mixtures of polyolefins. aromatic hydrocarbons, practically insoluble in anhydrous
ethanol, in hexane and in methanol.
PRODUCTION
They soften at temperatures between 65 °C and 165 °C. They
A certain number of additives are added to the polymer in burn with a blue flame.
order to optimise their chemical, physical and mechanical
properties in order to adapt them for the intended use. All IDENTIFICATION
of these additives are chosen from the appended list which If necessary, cut the samples of the material to be examined
specifies for each product the maximum allowable content. into pieces of maximum dimension on a side of not greater
They may contain at most 3 antioxidants, 1 or several than 1 cm.
lubricants or antiblocking agents as well as titanium dioxide A. Infrared absorption spectrophotometry (2.2.24).
as an opacifying agent when the material must provide
Preparation : to 0.25 g add 10 mL of toluene R and boil
protection from light.
under a reflux condenser for about 15 min ; place a few
– butylhydroxytoluene (plastic additive 07) : drops of the solution obtained on a sodium chloride slide
maximum 0.125 per cent ; and evaporate the solvent in an oven at 80 °C.
– pentaerythrityl tetrakis[3-(3,5-di-tert-butyl-4- Absorption maxima : at 2920 cm− 1, 2850 cm− 1, 1475 cm− 1,
hydroxyphenyl)propionate] (plastic additive 09) : maximum 1465 cm− 1, 1380 cm− 1, 1170 cm− 1, 735 cm− 1 and 720 cm− 1.
0.3 per cent ;
The spectrum obtained is identical to the spectrum
– 1,3,5-tris(3,5-di-tert-butyl-4-hydroxybenzyl)-S- obtained with the material selected for the type sample. If
triazine-2,4,6(1H,3H,5H)-trione, (plastic additive 13) : the material to be examined is in the form of sheets, the
maximum 0.3 per cent ; identification may be determined directly on a cut piece
– octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate of suitable size.
(plastic additive 11) : maximum 0.3 per cent ; B. It complies with the supplementary tests corresponding to
– ethylene bis[3,3-bis[3-(1,1-dimethylethyl)-4- the additives present.
hydroxyphenyl]butanoate] (plastic additive 08) : maximum C. In a platinum crucible, mix about 20 mg with 1 g of
0.3 per cent ; potassium hydrogen sulfate R and heat until completely
– dioctadecyl disulfide (plastic additive 15) : maximum 0.3 per melted. Allow to cool and add 20 mL of dilute sulfuric
cent ; acid R. Heat gently. Filter the resulting solution. To the
filtrate add 1 mL of phosphoric acid R and 1 mL of strong Extractable titanium : maximum 1 ppm.
hydrogen peroxide solution R. If the substance is opacified Inductively coupled plasma-atomic emission spectrometry
with titanium dioxide, an orange-yellow colour develops. (2.2.57).
TESTS Test solution. Use solution S3.
If necessary, cut the samples of the material to be examined Reference solutions. Prepare the reference solutions using
into pieces of maximum dimension on a side of not greater titanium standard solution (100 ppm Ti) R, diluting with 0.1 M
than 1 cm. hydrochloric acid.
Solution S1. Use solution S1 within 4 h of preparation. Place Wavelength : use the emission of titanium at 336.12 nm, the
25 g in a borosilicate-glass flask with a ground-glass neck. spectral background being taken as 336.16 nm.
Add 500 mL of water for injections R and boil under a reflux Verify the absence of titanium in the hydrochloric acid used.
condenser for 5 h. Allow to cool and decant. Reserve a portion Extractable zinc : maximum 1 ppm.
of the solution for the test for appearance of solution S1 and Atomic absorption spectrometry (2.2.23, Method I).
filter the rest through a sintered-glass filter (16) (2.1.2).
Test solution. Use solution S3.
Solution S2. Place 2.0 g in a conical borosilicate-glass flask
with a ground-glass neck. Add 80 mL of toluene R and boil Reference solutions. Prepare the reference solutions using
under a reflux condenser with constant stirring for 90 min. zinc standard solution (10 ppm Zn) R, diluting with 0.1 M
Allow to cool to 60 °C and add with continued stirring 120 mL hydrochloric acid.
of methanol R. Filter the solution through a sintered-glass Source : zinc hollow-cathode lamp.
filter (16) (2.1.2). Rinse the flask and the filter with 25 mL of a Wavelength : 213.9 nm.
mixture of 40 mL of toluene R and 60 mL of methanol R, add Atomisation device : air-acetylene flame.
the rinsings to the filtrate and dilute to 250 mL with the same
mixture of solvents. Prepare a blank solution. Verify the absence of zinc in the hydrochloric acid used.
Solution S3. Place 100 g in a conical borosilicate-glass flask Extractable heavy metals (2.4.8) : maximum 2.5 ppm.
with a ground-glass neck. Add 250 mL of 0.1 M hydrochloric Evaporate 50 mL of solution S3 to about 5 mL on a water-bath
acid and boil under a reflux condenser with constant stirring and dilute to 20.0 mL with water R. 12 mL of the solution
for 1 h. Allow to cool and decant the solution. complies with test A. Prepare the reference solution using
2.5 mL of lead standard solution (10 ppm Pb) R.
Appearance of solution S1. Solution S1 is clear (2.2.1) and
colourless (2.2.2, Method II). Sulfated ash (2.4.14) : maximum 1.0 per cent, determined
on 5.0 g. This limit does not apply to material that has been
Acidity or alkalinity. To 100 mL of solution S1, add 0.15 mL opacified with titanium dioxide.
of BRP indicator solution R. Not more than 1.5 mL of 0.01 M
sodium hydroxide is required to change the colour of the SUPPLEMENTARY TESTS
indicator to blue. To 100 mL of solution S1 add 0.2 mL of These tests are to be carried out, in whole or in part, only if
methyl orange solution R. Not more than 1 mL of 0.01 M required by the stated composition or the use of the material.
hydrochloric acid is required to initiate the colour change of
the indicator from yellow to orange. Phenolic antioxidants. Liquid chromatography (2.2.29).
Absorbance (2.2.25) : maximum 0.2, determined between Solvent mixture : acetonitrile R, tetrahydrofuran R (50:50 V/V).
wavelengths of 220 nm and 340 nm on solution S1. Test solution S21. Evaporate 50 mL of solution S2 to dryness
in vacuo at 45 °C. Dissolve the residue in 5.0 mL of the solvent
Reducing substances. To 20 mL of solution S1 add 1 mL
mixture. Prepare a blank solution from the blank solution
of dilute sulfuric acid R and 20 mL of 0.002 M potassium
corresponding to solution S2.
permanganate. Boil under a reflux condenser for 3 min and
cool immediately. Add 1 g of potassium iodide R and titrate Test solution S22. Evaporate 50 mL of solution S2 to dryness in
immediately with 0.01 M sodium thiosulfate, using 0.25 mL vacuo at 45 °C. Dissolve the residue with 5.0 mL of methylene
of starch solution R as indicator. Carry out a blank titration. chloride R. Prepare a blank solution from the blank solution
The difference between the titration volumes is not more than corresponding to solution S2.
3.0 mL. Test solution S23. Evaporate 50 mL of solution S2 to dryness
Substances soluble in hexane. Place 10 g in a 250 mL conical in vacuo at 45 °C. Dissolve the residue in 5.0 mL of a mixture
borosilicate-glass flask with a ground-glass neck. Add 100 mL of equal volumes of acetonitrile R and a 10 g/L solution of
of hexane R and boil under a reflux condenser for 4 h, stirring tert-butylhydroperoxide R in tetrahydrofuran R. Close the flask
constantly. Cool in iced water and filter rapidly (the filtration and allow to stand for 1 h. Prepare a blank solution using the
time must be less than 5 min ; if necessary the filtration may blank of solution S2.
be accelerated by applying pressure to the solution) through Of the following reference solutions, prepare only those that are
a sintered-glass filter (16) (2.1.2) maintaining the solution necessary for the analysis of the phenolic antioxidants stated in
at about 0 °C. Evaporate 20 mL of the filtrate in a tared the composition of the substance to be examined.
borosilicate-glass dish on a water-bath. Dry the residue in an Reference solution (a). Dissolve 25.0 mg of butylhydroxy-
oven at 100-105 °C for 1 h. The mass of the residue obtained toluene CRS (plastic additive 07) and 60.0 mg of plastic
must be within 10 per cent of that of the residue obtained with additive 08 CRS in 10.0 mL of the solvent mixture. Dilute
the type sample and does not exceed 5 per cent. 2.0 mL of this solution to 50.0 mL with the solvent mixture.
Extractable aluminium : maximum 1 ppm. Reference solution (b). Dissolve 60.0 mg of plastic
Inductively coupled plasma-atomic emission spectrometry additive 09 CRS and 60.0 mg of plastic additive 10 CRS in
(2.2.57). 10.0 mL of the solvent mixture. Dilute 2.0 mL of this solution
to 50.0 mL with the solvent mixture.
Test solution. Use solution S3.
Reference solution (c). Dissolve 60.0 mg of plastic
Reference solutions. Prepare the reference solutions using additive 11 CRS and 60.0 mg of plastic additive 12 CRS in
aluminium standard solution (200 ppm Al) R, diluting with 10.0 mL of methylene chloride R. Dilute 2.0 mL of this solution
0.1 M hydrochloric acid. to 50.0 mL with methylene chloride R.
Wavelength : use the emission of aluminium at 396.15 nm, the Reference solution (d). Dissolve 25.0 mg of plastic
spectral background being taken as 396.25 nm. additive 07 CRS in 10.0 mL of the solvent mixture. Dilute
Verify the absence of aluminium in the hydrochloric acid used. 2.0 mL of this solution to 50.0 mL with the solvent mixture.
General Notices (1) apply to all monographs and other texts 381
3.1.3. Polyolefins EUROPEAN PHARMACOPOEIA 8.0
Reference solution (n). Dissolve 60 mg of plastic additive 16 CRS Reference solution (r). Dissolve 40 mg of oleamide (plastic
in 10 mL of methylene chloride R. Dilute 2 mL of this solution additive 20 CRS) in 20 mL of methylene chloride R.
to 10 mL with acidified methylene chloride R. Reference solution (s). Dissolve 40 mg of erucamide (plastic
Reference solution (o). Dissolve 60 mg of plastic additive 17 CRS additive 21 CRS) in 20 mL of methylene chloride R.
in 10 mL of methylene chloride R. Dilute 2 mL of this solution Plate : TLC silica gel GF254 plate R (2 plates).
to 10 mL with acidified methylene chloride R.
A. Mobile phase : anhydrous ethanol R, trimethylpentane R
Reference solution (p). Dissolve 60 mg of plastic additive 16 CRS (25:75 V/V).
and 60 mg of plastic additive 17 CRS in 10 mL of methylene
Application : 10 μL of the test solution S24 and reference
chloride R. Dilute 2 mL of this solution to 10 mL with acidified
solution (q).
methylene chloride R.
Development : over a path of 10 cm.
Plate : TLC silica gel GF254 plate R.
Drying : in air.
Mobile phase A : hexane R.
Detection : spray with a 2 g/L solution of
Mobile phase B : methylene chloride R.
dichlorophenolindophenol, sodium salt R in anhydrous
Application : 20 μL of the test solution S24, the reference ethanol R and heat in an oven at 120 °C for a few minutes
solution (p) and the reference solutions corresponding to all to intensify the spots.
the phenolic and non-phenolic antioxidants mentioned in the
type composition of the material to be examined. Limit : any spot corresponding to plastic additive 19 in the
chromatogram obtained with test solution S24 is identical
Development A : over a path of 18 cm with mobile phase A. in position to (RF = about 0.5) but not more intense than
Drying A : in air. the spot in the chromatogram obtained with reference
Development B : over a path of 17 cm with mobile phase B. solution (q).
Drying B : in air. B. Mobile phase A : hexane R.
Detection : examine in ultraviolet light at 254 nm ; spray with Mobile phase B : methanol R, methylene chloride R
alcoholic iodine solution R and examine in ultraviolet light at (5:95 V/V).
254 nm after 10-15 min. Application : 10 μL of the test solution S24 and the reference
System suitability : reference solution (p): solutions (r) and (s).
– the chromatogram shows 2 clearly separated spots. Development A : over a path of 13 cm with mobile phase A.
Limit : any spots in the chromatogram obtained with test Drying A : in air.
solution S24 are not more intense than the spots in the Development B : over a path of 10 cm with mobile phase B.
corresponding positions in the chromatograms obtained with Drying B : in air.
the reference solutions.
Detection : spray with a 40 g/L solution of phosphomolybdic
Plastic additive 22. Liquid chromatography (2.2.29). acid R in anhydrous ethanol R. Heat in an oven at 120 °C
Test solution. Evaporate 25 mL of solution S2 to dryness in until spots appear.
vacuo at 45 °C. Dissolve the residue in 10 mL of toluene R and Limit : any spots corresponding to plastic additive 20 or
10 mL of a 10 g/L solution of tetrabutylammonium hydroxide R plastic additive 21 in the chromatogram obtained with test
in a mixture of 35 volumes of toluene R and 65 volumes of solution S24 are identical in position to (RF = about 0.2)
anhydrous ethanol R. Boil under a reflux condenser for 3 h. but not more intense than the corresponding spots in
Allow to cool and filter if necessary. the chromatograms obtained with reference solutions (r)
Reference solution. Dissolve 30 mg of plastic additive 22 CRS and (s).
in 50 mL of toluene R. Add 1 mL of this solution to 25 mL
of blank solution S2 and evaporate to dryness in vacuo at
45 °C. Dissolve the residue in 10 mL of toluene R and 10 mL 01/2008:30104
of a 10 g/L solution of tetrabutylammonium hydroxide R corrected 6.0
in a mixture of 35 volumes of toluene R and 65 volumes of
anhydrous ethanol R. Boil under a reflux condenser for 3 h. 3.1.4. POLYETHYLENE WITHOUT
Allow to cool and filter if necessary.
ADDITIVES FOR CONTAINERS FOR
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
PARENTERAL PREPARATIONS AND
– stationary phase : aminopropylsilyl silica gel for FOR OPHTHALMIC PREPARATIONS
chromatography R (5 μm). DEFINITION
Mobile phase : anhydrous ethanol R, hexane R (11:89 V/V). Polyethylene without additives is obtained by the
Flow rate : 2 mL/min. polymerisation of ethylene under high pressure in the presence
Detection : spectrophotometer at 227 nm. of oxygen or free-radical-forming initiators as catalyst.
Injection : 20 μL. CHARACTERS
Run time : 10 min.
Appearance : beads, granules, powder or, after transformation,
System suitability : translucent sheets of varying thickness or containers.
– resolution : minimum 7 between the peaks due to the “diol” Solubility : practically insoluble in water, soluble in hot
component and to the diluent of the reference solution. aromatic hydrocarbons, practically insoluble in anhydrous
Limit : the area of the peak due to the “diol” component ethanol, in hexane and in methanol.
from plastic additive 22 in the chromatogram obtained with It softens at temperatures beginning at 65 °C.
the test solution is less than the corresponding peak in the
chromatogram obtained with the reference solution. Relative density : 0.910 to 0.937.
Amides and stearates. Thin-layer chromatography (2.2.27). IDENTIFICATION
Test solution. Use test solution S24 described in the test for If necessary, cut the samples of the material to be examined
non-phenolic antioxidants. into pieces of maximum dimension on a side of not greater
Reference solution (q). Dissolve 20 mg of stearic acid (plastic than 1 cm.
additive 19 CRS) in 10 mL of methylene chloride R. A. Infrared absorption spectrophotometry (2.2.24).
General Notices (1) apply to all monographs and other texts 383
3.1.5. Polyethylene with additives for containers EUROPEAN PHARMACOPOEIA 8.0
Preparation : to 0.25 g add 10 mL of toluene R and boil methylene chloride R. Prepare a blank solution from the blank
under a reflux condenser for about 15 min. Place a few solution corresponding to solution S2.
drops of the solution on a sodium chloride disc and Reference solution. Dissolve 20 mg of plastic additive 15 CRS
evaporate the solvent in an oven at 80 °C. and 20 mg of plastic additive 08 CRS in methylene chloride R
Absorption maxima : at 2920 cm− 1, 2850 cm− 1, 1465 cm− 1, and dilute to 10 mL with the same solvent.
730 cm− 1 and 720 cm− 1. Plate : TLC silica gel G plate R.
The spectrum obtained is identical to that obtained with Mobile phase A : hexane R.
the material selected for the type sample. If the material to Mobile phase B : methanol R, methylene chloride R (5:95 V/V).
be examined is in the form of sheets, the identification may
be performed directly on a cut piece of suitable size. Application : 10 μL.
B. Additives (see Tests). Development A : over a path of 13 cm using mobile phase A.
Drying A : in air.
TESTS Development B : over a path of 10 cm using mobile phase B.
If necessary, cut the samples of the material to be examined Drying B : in air.
into pieces of maximum dimension on a side of not greater
than 1 cm. Detection : spray with a 40 g/L solution of phosphomolybdic
acid R in ethanol (96 per cent) R and heat at 120 °C until the
Solution S1. Place 25 g in a borosilicate-glass flask with a spots appear in the chromatogram obtained with the reference
ground-glass neck. Add 500 mL of water for injections R and solution.
heat under a reflux condenser for 5 h. Allow to cool and
System suitability : reference solution :
decant. Keep part of the solution for the test for appearance
of solution. Filter the rest through a sintered glass filter (16) – the chromatogram shows 2 separated spots.
(2.1.2). Use solution S1 within 4 h of preparation. Limit : no spot appears in the chromatogram obtained with
Solution S2. Place 2.0 g in a conical borosilicate-glass flask the test solution, except for a spot which may be at the solvent
with a ground-glass neck. Add 80 mL of toluene R and boil front from the first development and which corresponds
under a reflux condenser with constant stirring for 1 h 30 min. to oligomers. Disregard any spots corresponding to those
Allow to cool to 60 °C and add with continued stirring 120 mL obtained in the chromatogram with the blank solution.
of methanol R. Filter the solution through a sintered-glass Extractable heavy metals (2.4.8) : maximum 2.5 ppm.
filter (16) (2.1.2). Rinse the flask and the filter with 25 mL of a Evaporate 50 mL of solution S3 to about 5 mL on a water-bath
mixture of 40 mL of toluene R and 60 mL of methanol R, add and dilute to 20 mL with water R. 12 mL of solution complies
the rinsings to the filtrate and dilute to 250 mL with the same with test A. Prepare the reference solution using 2.5 mL of
mixture of solvents. Prepare a blank solution. lead standard solution (10 ppm Pb) R.
Solution S3. Place 100 g in a conical borosilicate-glass flask Sulfated ash (2.4.14) : maximum 0.02 per cent, determined
with a ground-glass neck. Add 250 mL of 0.1 M hydrochloric on 5.0 g.
acid and boil under a reflux condenser with constant stirring
for 1 h. Allow to cool and decant the solution.
01/2008:30105
Appearance of solution. Solution S1 is clear (2.2.1) and
corrected 7.5
colourless (2.2.2, Method II).
Acidity or alkalinity. To 100 mL of solution S1 add 0.15 mL 3.1.5. POLYETHYLENE WITH
of BRP indicator solution R. Not more than 1.5 mL of 0.01 M
sodium hydroxide is required to change the colour of the ADDITIVES FOR CONTAINERS FOR
indicator to blue. To 100 mL of solution S1 add 0.2 mL of PARENTERAL PREPARATIONS AND
methyl orange solution R. Not more than 1.0 mL of 0.01 M
hydrochloric acid is required to reach the beginning of the FOR OPHTHALMIC PREPARATIONS
colour change of the indicator from yellow to orange. DEFINITION
Absorbance (2.2.25) : maximum 0.2, determined between Polyethylene with additives is obtained by the polymerisation
wavelengths of 220 nm and 340 nm on solution S1. of ethylene under pressure in the presence of a catalyst or by
Reducing substances. To 20 mL of solution S1 add 1 mL copolymerisation of ethylene with not more than 25 per cent
of dilute sulfuric acid R and 20 mL of 0.002 M potassium of higher alkene homologues (C3 to C10).
permanganate. Boil under a reflux condenser for 3 min and
cool immediately. Add l g of potassium iodide R and titrate PRODUCTION
immediately with 0.01 M sodium thiosulfate, using 0.25 mL A certain number of additives are added to the polymer in
of starch solution R as indicator. Carry out a blank titration. order to optimise their chemical, physical and mechanical
The difference between the titration volumes is not more than properties in order to adapt them for the intended use. All
0.5 mL. these additives are chosen from the appended list which
specifies for each product the maximum allowable content.
Substances soluble in hexane. Place 10 g in a 250 mL conical
borosilicate-glass flask with a ground-glass neck. Add 100 mL They may contain at most 3 antioxidants, 1 or several
of hexane R and boil under a reflux condenser for 4 h, stirring lubricants or antiblocking agents as well as titanium dioxide
constantly. Cool in iced water and filter rapidly through a as an opacifying agent when the material must provide
sintered-glass filter (16) (2.1.2) maintaining the solution at protection from light.
0 °C (the filtration time must be less than 5 min ; if necessary – butylhydroxytoluene (plastic additive 07) : maximum
the filtration may be accelerated by applying pressure to the 0.125 per cent ;
solution). Evaporate 20 mL of the filtrate in a tared glass dish – pentaerythrityl tetrakis[3-(3,5-di-tert-butyl-4-
on a water-bath. Dry the residue in an oven at 100-105 °C hydroxyphenyl)propionate] (plastic additive 09): maximum
for 1 h. The mass of the residue obtained is within 10 per 0.3 per cent ;
cent of the residue obtained with the type sample and does – 1,3,5-tris(3,5-di-tert-butyl-4-hydroxybenzyl)-S-triazine-
not exceed 5 per cent. 2,4,6(1H,3H,5H)-trione (plastic additive 13) : maximum
Additives. Thin-layer chromatography (2.2.27). 0.3 per cent ;
Test solution. Evaporate 50 mL of solution S2 to dryness in – octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate,
vacuo at 45 °C. Dissolve the evaporation residue with 5 mL of (plastic additive 11) : maximum 0.3 per cent ;
– ethylene bis[3,3-bis[3-(1,1-dimethylethyl)-4- acid R. Heat gently. Filter the resulting solution. To the
hydroxyphenyl]butanoate] (plastic additive 08) : maximum filtrate add 1 mL of phosphoric acid R and 1 mL of strong
0.3 per cent ; hydrogen peroxide solution R. If the substance is opacified
– dioctadecyl disulfide (plastic additive 15): maximum with titanium dioxide, an orange-yellow colour develops.
0.3 per cent ;
TESTS
– 4,4′,4″-(2,4,6-trimethylbenzene-1,3,5-triyltrismethylene)-
If necessary, cut the samples of the material to be examined
tris[2,6-bis(1,1-dimethylethyl)phenol] (plastic additive 10) :
into pieces of maximum dimension on a side of not greater
maximum 0.3 per cent ;
than 1 cm.
– 2,2′-bis(octadecyloxy)-5,5′-spirobi[1,3,2-dioxa-
phosphinane] (plastic additive 14) : maximum 0.3 per cent ; Solution S1. Place 25 g in a borosilicate-glass flask with a
ground-glass neck. Add 500 mL of water for injections R and
– didodecyl 3,3′-thiodipropionate (plastic additive 16) : boil under a reflux condenser for 5 h. Allow to cool and decant.
maximum 0.3 per cent ; Reserve a portion of the solution for the test for appearance of
– dioctadecyl 3,3′-thiodipropionate (plastic additive 17) : solution and filter the rest through a sintered-glass filter (16)
maximum 0.3 per cent ; (2.1.2). Use within 4 h of preparation.
– tris [2,4-bis(1,1-dimethylethyl)phenyl] phosphite (plastic Solution S2. Place 2.0 g in a conical borosilicate-glass flask
additive 12): maximum 0.3 per cent. with a ground-glass neck. Add 80 mL of toluene R and boil
The total of antioxidant additives listed above does not exceed under a reflux condenser with constant stirring for 90 min.
0.3 per cent. Allow to cool to 60 °C and add with continued stirring 120 mL
– hydrotalcite : maximum 0.5 per cent ; of methanol R. Filter the solution through a sintered-glass
– alkanamides : maximum 0.5 per cent ; filter (16) (2.1.2). Rinse the flask and the filter with 25 mL of
– alkenamides : maximum 0.5 per cent ; a mixture of 40 mL of toluene R and 60 mL of methanol R,
add the rinsings to the filtrate and dilute to 250.0 mL with the
– sodium silico-aluminate : maximum 0.5 per cent ; same mixture of solvents. Prepare a blank solution.
– silica : maximum 0.5 per cent ;
Solution S3. Place 100 g in a conical borosilicate-glass flask
– sodium benzoate : maximum 0.5 per cent ; with a ground-glass neck. Add 250 mL of 0.1 M hydrochloric
– fatty acid esters or salts : maximum 0.5 per cent ; acid and boil under a reflux condenser with constant stirring
– trisodium phosphate : maximum 0.5 per cent ; for 1 h. Allow to cool and decant the solution.
– liquid paraffin : maximum 0.5 per cent ; Appearance of solution. Solution S1 is clear (2.2.1) and
– zinc oxide : maximum 0.5 per cent ; colourless (2.2.2, Method II).
– magnesium oxide : maximum 0.2 per cent ; Acidity or alkalinity. To 100 mL of solution S1 add 0.15 mL
– calcium stearate or zinc stearate or a mixture of both : of BRP indicator solution R. Not more than 1.5 mL of 0.01 M
maximum 0.5 per cent ; sodium hydroxide is required to change the colour of the
indicator to blue. To 100 mL of solution S1 add 0.2 mL of
– titanium dioxide only for materials for containers for
methyl orange solution R. Not more than 1.0 mL of 0.01 M
ophthalmic use : maximum 4 per cent.
hydrochloric acid is required to reach the beginning of the
The supplier of the material must be able to demonstrate colour change of the indicator from yellow to orange.
that the qualitative and quantitative composition of the type
sample is satisfactory for each production batch. Absorbance (2.2.25) : maximum 0.2, determined between
wavelengths of 220 nm and 340 nm on solution S1.
CHARACTERS Reducing substances. To 20 mL of solution S1 add 1 mL
Appearance : powder, beads, granules or, after transformation, of dilute sulfuric acid R and 20 mL of 0.002 M potassium
translucent sheets of varying thicknesses or containers. permanganate. Boil under a reflux condenser for 3 min and
Solubility : practically insoluble in water, soluble in hot cool immediately. Add 1 g of potassium iodide R and titrate
aromatic hydrocarbons, practically insoluble in anhydrous immediately with 0.01 M sodium thiosulfate, using 0.25 mL
ethanol, in hexane and in methanol. of starch solution R as indicator. Carry out a blank titration.
It softens at temperatures between 70 °C and 140 °C. The difference between the titration volumes is not more than
0.5 mL.
Relative density : 0.890 to 0.965.
Substances soluble in hexane. Place 10 g in a 250 mL conical
IDENTIFICATION borosilicate-glass flask with a ground-glass neck. Add 100 mL
If necessary, cut the samples of the material to be examined of hexane R and boil under a reflux condenser for 4 h, stirring
into pieces of maximum dimension on a side of not greater constantly. Cool in iced water and filter rapidly through a
than 1 cm. sintered-glass filter (16) (2.1.2) maintaining the solution at
A. Infrared absorption spectrophotometry (2.2.24). 0 °C (the filtration time must be less than 5 min ; if necessary
the filtration may be accelerated by applying pressure to
Preparation : to 0.25 g add 10 mL of toluene R and boil the solution). Evaporate 20 mL of the filtrate in a tared
under a reflux condenser for about 15 min. Place a few borosilicate-glass dish on a water-bath. Dry the residue in an
drops of the solution on a sodium chloride disc and oven at 100-105 °C for 1 h. The mass of the residue obtained
evaporate the solvent in an oven at 80 °C. must be within 10 per cent of the residue obtained with the
Absorption maxima : at 2920 cm− 1, 2850 cm− 1, 1465 cm− 1, type sample and does not exceed 5 per cent.
1375 cm− 1, 1170 cm− 1, 730 cm− 1 and 720 cm− 1.
Extractable aluminium : maximum 1 ppm.
The spectrum obtained is identical to the spectrum
obtained with the material selected for the type sample. If Inductively coupled plasma-atomic emission spectrometry
the material to be examined is in the form of sheets, the (2.2.57).
identification may be performed directly on a cut piece of Test solution. Use solution S3.
suitable size. Reference solutions. Prepare the reference solutions using
B. It complies with the supplementary tests corresponding to aluminium standard solution (200 ppm Al) R, diluting with
the additives present (see Tests). 0.1 M hydrochloric acid.
C. In a platinum crucible, mix about 20 mg with 1 g of Wavelength : use the emission of aluminium at 396.15 nm, the
potassium hydrogen sulfate R and heat until completely spectral background being taken as 396.25 nm.
melted. Allow to cool and add 20 mL of dilute sulfuric Verify the absence of aluminium in the hydrochloric acid used.
General Notices (1) apply to all monographs and other texts 385
3.1.5. Polyethylene with additives for containers EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 387
3.1.6. Polypropylene for containers and closures EUROPEAN PHARMACOPOEIA 8.0
Solution S1. Use solution S1 within 4 h of preparation. Place Reference solutions. Prepare the reference solutions using
25 g in a borosilicate-glass flask with a ground-glass neck. chromium standard solution (100 ppm Cr) R, diluting with a
Add 500 mL of water for injections R and boil under a reflux mixture of 2 volumes of hydrochloric acid R and 8 volumes
condenser for 5 h. Allow to cool and decant. Reserve a portion of water R.
of the solution for the test for appearance of solution and filter Wavelength : use the emission of chromium at 205.55 nm, the
the rest through a sintered-glass filter (16) (2.1.2). spectral background being taken as 205.50 nm.
Solution S2. Place 2.0 g in a conical borosilicate-glass flask Verify the absence of chromium in the hydrochloric acid used.
with a ground-glass neck. Add 80 mL of toluene R and boil
under a reflux condenser with constant stirring for 1 h 30 min. Extractable titanium : maximum 1 ppm.
Allow to cool to 60 °C and add with continued stirring 120 mL Inductively coupled plasma-atomic emission spectrometry
of methanol R. Filter the solution through a sintered-glass (2.2.57).
filter (16) (2.1.2). Rinse the flask and the filter with 25 mL of Test solution. Use solution S3.
a mixture of 40 mL of toluene R and 60 mL of methanol R,
add the rinsings to the filtrate and dilute to 250.0 mL with the Reference solutions. Prepare the reference solutions using
same mixture of solvents. Prepare a blank solution. titanium standard solution (100 ppm Ti) R, diluting with 0.1 M
hydrochloric acid.
Solution S3. Place 100 g in a conical borosilicate-glass flask Wavelength : use the emission of titanium at 336.12 nm, the
with a ground-glass neck. Add 250 mL of 0.1 M hydrochloric spectral background being taken as 336.16 nm.
acid and boil under a reflux condenser with constant stirring
for 1 h. Allow to cool and decant the solution. Verify the absence of titanium in the hydrochloric acid used.
Appearance of solution. Solution S1 is not more opalescent Extractable vanadium : maximum 0.1 ppm.
than reference suspension II (2.2.1) and is colourless (2.2.2, Inductively coupled plasma-atomic emission spectrometry
Method II). (2.2.57).
Acidity or alkalinity. To 100 mL of solution S1 add 0.15 mL Test solution. Use solution S3.
of BRP indicator solution R. Not more than 1.5 mL of 0.01 M Reference solutions. Prepare the reference solutions using
sodium hydroxide is required to change the colour of the vanadium standard solution (1 g/L V) R, diluting with a
indicator to blue. To 100 mL of solution S1 add 0.2 mL of mixture of 2 volumes of hydrochloric acid R and 8 volumes
methyl orange solution R. Not more than 1.0 mL of 0.01 M of water R.
hydrochloric acid is required to reach the beginning of the
Wavelength : use the emission of vanadium at 292.40 nm, the
colour change of the indicator from yellow to orange.
spectral background being taken as 292.35 nm.
Absorbance (2.2.25) : maximum 0.2, determined between Verify the absence of vanadium in the hydrochloric acid used.
wavelengths of 220 nm to 340 nm on solution S1.
Extractable zinc : maximum 1 ppm.
Reducing substances. To 20 mL of solution S1 add 1 mL
of dilute sulfuric acid R and 20 mL of 0.002 M potassium Atomic absorption spectrometry (2.2.23, Method I).
permanganate. Boil under a reflux condenser for 3 min and Test solution. Use solution S3.
cool immediately. Add 1 g of potassium iodide R and titrate Reference solutions. Prepare the reference solutions using
immediately with 0.01 M sodium thiosulfate, using 0.25 mL zinc standard solution (10 ppm Zn) R, diluting with 0.1 M
of starch solution R as indicator. Carry out a blank titration. hydrochloric acid.
The difference between the titration volumes is not more than
0.5 mL. Source : zinc hollow-cathode lamp.
Wavelength : 213.9 nm.
Substances soluble in hexane. Place 10 g in a 250 mL conical
borosilicate-glass flask with a ground-glass neck. Add 100 mL Atomisation device : air-acetylene flame.
of hexane R and boil under a reflux condenser for 4 h, stirring Verify the absence of zinc in the hydrochloric acid used.
constantly. Cool in iced water and filter rapidly through a
Extractable heavy metals (2.4.8) : maximum 2.5 ppm.
sintered-glass filter (16) (2.1.2) maintaining the solution at
0 °C (the filtration time must be less than 5 min ; if necessary Concentrate 50 mL of solution S3 to about 5 mL on a
the filtration may be accelerated by applying pressure to the water-bath and dilute to 20.0 mL with water R. 12 mL of the
solution). Evaporate 20 mL of the filtrate in a tared glass dish solution complies with test A. Prepare the reference solution
on a water-bath. Dry the residue in an oven at 100-105 °C for using 2.5 mL of lead standard solution (10 ppm Pb) R.
1 h. The mass of the residue obtained must be within 10 per Sulfated ash (2.4.14) : maximum 1.0 per cent, determined
cent of the residue obtained with the type sample and does on 5.0 g. This limit does not apply to material that has been
not exceed 5 per cent. opacified with titanium dioxide.
Extractable aluminium : maximum 1 ppm.
SUPPLEMENTARY TESTS
Inductively coupled plasma-atomic emission spectrometry These tests are to be carried out, in whole or in part, only if
(2.2.57). required by the stated composition of the material.
Test solution. Use solution S3. Phenolic antioxidants. Liquid chromatography (2.2.29).
Reference solutions. Prepare the reference solutions using Solvent mixture : acetonitrile R, tetrahydrofuran R (50:50 V/V).
aluminium standard solution (200 ppm Al) R, diluting with Test solution S21. Evaporate 50 mL of solution S2 to dryness
0.1 M hydrochloric acid. in vacuo at 45 °C. Dissolve the residue with 5.0 mL of the
Wavelength : use the emission of aluminium at 396.15 nm, the solvent mixture. Prepare a blank solution from the blank
spectral background being taken as 396.25 nm. solution corresponding to solution S2.
Test solution S22. Evaporate 50 mL of solution S2 to dryness in
Verify the absence of aluminium in the hydrochloric acid used. vacuo at 45 °C. Dissolve the residue with 5.0 mL of methylene
Extractable chromium : maximum 0.05 ppm. chloride R. Prepare a blank solution from the blank solution
corresponding to solution S2.
Inductively coupled plasma-atomic emission spectrometry
(2.2.57). Of the following reference solutions, only prepare those that
are necessary for the analysis of the phenolic antioxidants
Test solution. Use solution S3. stated in the composition of the substance to be examined.
General Notices (1) apply to all monographs and other texts 389
3.1.6. Polypropylene for containers and closures EUROPEAN PHARMACOPOEIA 8.0
Reference solution (o). Dissolve 60 mg of plastic additive 16 CRS Limit : any spots corresponding to plastic additive 20 or
and 60 mg of plastic additive 17 CRS in methylene chloride R plastic additive 21 in the chromatogram obtained with
and dilute to 10 mL with the same solvent. Dilute 2 mL of the test solution S23 are identical in position (RF about 0.2)
solution to 10 mL with acidified methylene chloride R. but not more intense than the corresponding spots in
Plate : TLC silica gel GF254 plate R. the chromatograms obtained with reference solutions (q)
and (r).
Mobile phase A : hexane R.
Mobile phase B : methylene chloride R.
Application : 20 μL of test solution S23, reference solution (o) 01/2008:30107
and reference solutions corresponding to all the phenolic and
non-phenolic antioxidants mentioned in the type composition
of the material to be examined.
3.1.7. POLY(ETHYLENE - VINYL
Development A : over a path of 18 cm with mobile phase A. ACETATE) FOR CONTAINERS AND
Drying A : in air. TUBING FOR TOTAL PARENTERAL
Development B : over a path of 17 cm with mobile phase B. NUTRITION PREPARATIONS
Drying B : in air. DEFINITION
Detection : examine in ultraviolet light at 254 nm ; spray with Poly(ethylene - vinyl acetate), complying with the following
alcoholic iodine solution R and examine in ultraviolet light at requirements, is suitable for the manufacture of containers
254 nm after 10-15 min. and tubing for total parenteral nutrition preparations. It is
System suitability : reference solution (o) : obtained by copolymerisation of mixtures of ethylene and
– the chromatogram shows 2 clearly separated spots. vinyl acetate.
Content of vinyl acetate :
Limits : any spots in the chromatogram obtained with test
solution S23 are not more intense than the spots in the same – material used for containers: a defined quantity of not more
positions in the chromatograms obtained with the reference than 25 per cent ;
solutions. – material used for tubing : a defined quantity of not more
Amides and stearates. Thin-layer chromatography (2.2.27). than 30 per cent.
Test solution. Use solution S23 described in the test for PRODUCTION
non-phenolic antioxidants. A certain number of additives are added to the polymer in
Reference solution (p). Dissolve 20 mg of stearic acid CRS order to optimise their chemical, physical and mechanical
(plastic additive 19) in methylene chloride R and dilute to properties in order to adapt them for the intended use. All
10 mL with the same solvent. these additives are chosen from the appended list which
Reference solution (q). Dissolve 40 mg of plastic additive 20 CRS specifies for each product the maximum allowable content.
in methylene chloride R and dilute to 20 mL with the same Poly(ethylene - vinyl acetate) may contain not more than 3
solvent. of the following antioxidants :
Reference solution (r). Dissolve 40 mg of plastic additive 21 CRS – butylhydroxytoluene (plastic additive 07) : maximum
in methylene chloride R and dilute to 20 mL with the same 0.125 per cent ;
solvent. – pentaerythrityl tetrakis[3-(3,5-di-tert-butyl-4-
Plate : TLC silica gel GF254 plate R (2 plates). hydroxyphenyl)propionate] (plastic additive 09): maximum
0.2 per cent ;
A. Mobile phase : anhydrous ethanol R, trimethylpentane R
(25:75 V/V). – octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate
(plastic additive 11) : maximum 0.2 per cent ;
Application : 10 μL of solution S23 and reference – tris(2,4-di-tert-butylphenyl) phosphite (plastic additive 12) :
solution (p). maximum 0.2 per cent ;
Development : over a path of 10 cm. – 2,2′,2″,6,6′,6″-hexa-tert-butyl-4,4′,4″-[(2,4,6-trimethyl-
Drying : in air. 1,3,5-benzenetriyl)trismethylene]triphenol (plastic
Detection : spray with a 2 g/L solution of additive 10) : maximum 0.2 per cent.
dichlorophenolindophenol, sodium salt R in anhydrous It may also contain :
ethanol R and heat in an oven at 120 °C for a few minutes – oleamide (plastic additive 20) : maximum 0.5 per cent ;
to intensify the spots. – erucamide (plastic additive 21) : maximum 0.5 per cent ;
Limit : any spot corresponding to plastic additive 19 in the – calcium stearate or zinc stearate or a mixture of both :
chromatogram obtained with test solution S23 is identical maximum 0.5 per cent ;
in position (RF about 0.5) but not more intense than the
spot in the same position in the chromatogram obtained – calcium carbonate or potassium hydroxide : maximum
with reference solution (p). 0.5 per cent ;
– colloidal silica : maximum 0.2 per cent.
B. Mobile phase A : hexane R.
The supplier of the material must be able to demonstrate
Mobile phase B : methanol R, methylene chloride R that the qualitative and quantitative composition of the type
(5:95 V/V). sample is satisfactory for each production batch.
Application : 10 μL of solution S23 and reference
solutions (q) and (r). CHARACTERS
Development A : over a path of 13 cm with mobile phase A. Appearance : beads, granules or, after transformation,
translucent sheets or tubing of varying thickness or samples of
Drying A : in air. finished objects.
Development B : over a path of 10 cm with mobile phase B. Solubility : practically insoluble in water, soluble in hot
Drying B : in air. aromatic hydrocarbons, practically insoluble in anhydrous
Detection : spray with a 40 g/L solution of phosphomolybdic ethanol, in methanol and in hexane, which dissolves, however,
acid R in anhydrous ethanol R ; heat in an oven at 120 °C low molecular mass polymers.
until spots appear. It burns with a blue flame.
General Notices (1) apply to all monographs and other texts 391
3.1.7. Poly(ethylene - vinyl acetate) for containers and tubing EUROPEAN PHARMACOPOEIA 8.0
The temperature at which the substance softens changes with Reference solution (b). Dissolve 40 mg of plastic additive 20 CRS
the vinyl acetate content : from about 100 °C for contents of a in 10 mL of methylene chloride R. Dilute 1 mL of this solution
few per cent to about 70 °C for contents of 30 per cent. to 5 mL with methylene chloride R.
Reference solution (c). Dissolve 40 mg of plastic additive 21 CRS
IDENTIFICATION in 10 mL of methylene chloride R. Dilute 1 mL of this solution
If necessary, cut the samples of material to be examined into to 5 mL with methylene chloride R.
pieces of maximum dimension on a side of not greater than Plates : TLC silica gel GF254 plate R (2 plates).
1 cm. A. Mobile phase : anhydrous ethanol R, trimethylpentane R
Infrared absorption spectrophotometry (2.2.24). (25:75 V/V).
Preparation : to 0.25 g add 10 mL of toluene R and boil under Application : 10 μL.
a reflux condenser for about 15 min. Place a few drops of the Development : over a path of 10 cm.
solution obtained on a disc of sodium chloride and evaporate Drying : in air.
the solvent in an oven at 80 °C.
Detection : spray with a 2 g/L solution of
Absorption maxima due to vinyl acetate : at 1740 cm− 1, dichlorophenolindophenol, sodium salt R in anhydrous
−1 −1 −1 −1
1375 cm , 1240 cm , 1020 cm and 610 cm . ethanol R and heat in an oven at 120 °C for a few minutes
Absorption maxima due to ethylene : at 2920-2850 cm− 1, to intensify the spots.
1470 cm− 1, 1460 cm− 1, 1375 cm− 1, 730 cm− 1 and 720 cm− 1. Limit : any spot corresponding to plastic additive 19 in the
The spectrum obtained is identical to the spectrum obtained chromatogram obtained with the test solution is not more
with the type sample provided by the manufacturer. If the intense than the spot in the chromatogram obtained with
material to be examined is in the form of sheets, the spectrum reference solution (a).
may be determined directly on a cut piece of suitable size. B. Mobile phase A : hexane R.
Mobile phase B : methanol R, methylene chloride R
TESTS (5:95 V/V).
If necessary, cut the samples of the material to be examined Application : 10 μL.
into pieces of maximum dimension on a side of not greater Development A : over a path of 13 cm with mobile phase A.
than 1 cm.
Drying A : in air.
Solution S1. Place 2.0 g in a borosilicate-glass flask with a
ground-glass neck. Add 80 mL of toluene R and heat under a Development B : over a path of 10 cm with mobile phase B.
reflux condenser with constant agitation for 90 min. Allow to Drying B : in air.
cool to 60 °C and add 120 mL of methanol R to the flask with Detection : spray with a 40 g/L solution of phosphomolybdic
constant stirring. Filter the solution through a sintered-glass acid R in anhydrous ethanol R and heat in an oven at 120 °C
filter (16) (2.1.2). Rinse the flask and the filter with 25 mL of a until spots appear.
mixture of 40 mL of toluene R and 60 mL of methanol R, add Limit : any spots corresponding to plastic additive 21 or
the rinsing mixture to the filtrate and dilute to 250 mL with plastic additive 20 in the chromatogram obtained with
the same mixture of solvents. the test solution are not more intense than the spots in
Solution S2. Use within 4 h of preparation. Place 25 g in a the chromatograms obtained with reference solutions (b)
borosilicate-glass flask with a ground-glass neck. Add 500 mL and (c) respectively.
of water for injections R and boil under a reflux condenser Phenolic antioxidants. Liquid chromatography (2.2.29).
for 5 h. Allow to cool and decant. Reserve a portion of the
Solvent mixture : acetonitrile R, tetrahydrofuran R (50:50 V/V).
solution for the test for appearance of solution S2 and filter
the rest through a sintered-glass filter (16) (2.1.2). Test solution (a). Evaporate 50 mL of solution S1 to dryness
in vacuo at 45 °C and dissolve the residue in 5.0 mL of the
Appearance of solution S2. Solution S2 is clear (2.2.1) and solvent mixture.
colourless (2.2.2, Method II).
Test solution (b). Evaporate 50 mL of solution S1 to dryness in
Acidity or alkalinity. To 100 mL of solution S2 add 0.15 mL vacuo at 45 °C and dissolve the residue in 5.0 mL of methylene
of BRP indicator solution R. Not more than 1.0 mL of 0.01 M chloride R.
sodium hydroxide is required to change the colour of the Reference solution (a). Dissolve 25 mg of butylhydroxy-
indicator to blue. To 100 mL of solution S2 add 0.2 mL of toluene CRS (plastic additive 07), 40 mg of plastic
methyl orange solution R. Not more than 1.5 mL of 0.01 M additive 10 CRS, 40 mg of plastic additive 09 CRS and 40 mg of
hydrochloric acid is required to reach the beginning of the plastic additive 11 CRS in 10 mL of the solvent mixture. Dilute
colour change of the indicator from yellow to orange. 2 mL of this solution to 50.0 mL with the solvent mixture.
Absorbance (2.2.25) : maximum 0.2 determined between Reference solution (b). Dissolve 40 mg of plastic additive 11 CRS
wavelengths of 220 nm and 340 nm on solution S2. and 40 mg of plastic additive 12 CRS in 10 mL of methylene
Reducing substances. To 20 mL of solution S2 add 1 mL chloride R. Dilute 2 mL of this solution to 50.0 mL with
of dilute sulfuric acid R and 20 mL of 0.002 M potassium methylene chloride R.
permanganate. Boil under a reflux condenser for 3 min and Column :
cool immediately. Add 1 g of potassium iodide R and titrate – size : l = 0.25 m ; Ø = 4.6 mm ;
immediately with 0.01 M sodium thiosulfate, using 0.25 mL
of starch solution R as indicator. Carry out a blank titration. – stationary phase : octadecylsilyl silica gel for
The difference between the titration volumes is not more than chromatography R (5 μm).
0.5 mL. Mobile phase : water R, tetrahydrofuran R, acetonitrile R
(10:30:60 V/V/V).
Amides and stearic acid. Thin-layer chromatography
(2.2.27). Flow rate : 1.5 mL/min.
Test solution. Evaporate 100 mL of solution S1 to dryness Detection : spectrophotometer at 280 nm.
in vacuo at 45 °C. Dissolve the residue in 2 mL of acidified Injection : 20 μL of test solution (a) and reference solution (a).
methylene chloride R. System suitability : reference solution (a) :
Reference solution (a). Dissolve 20 mg of stearic acid CRS – resolution : minimum 2.0 between the peaks due to plastic
(plastic additive 19) in 10 mL of methylene chloride R. additive 09 and plastic additive 10 ;
General Notices (1) apply to all monographs and other texts 393
3.1.9. Silicone elastomer for closures and tubing EUROPEAN PHARMACOPOEIA 8.0
standard solution (10 ppm Pb) R and 0.5 mL of the solvent Solution S. Place 25 g in a borosilicate-glass flask with a
mixture. Immediately shake each solution vigorously for ground-glass neck. Add 500 mL of water R and boil under a
1 min. Any red colour in the test solution is not more intense reflux condenser for 5 h. Allow to cool and decant the solution.
than that in the reference solution. Appearance of solution. Solution S is clear (2.2.1).
Volatile matter : maximum 2.0 per cent, determined on 2.00 g Acidity or alkalinity. To 100 mL of solution S add 0.15 mL
by heating in an oven at 150 °C for 24 h. Carry out the test of bromothymol blue solution R1. Not more than 2.5 mL of
using a dish 60 mm in diameter and 10 mm deep. 0.01 M sodium hydroxide is required to change the colour of
LABELLING the indicator to blue. To a further 100 mL of solution S, add
0.2 mL of methyl orange solution R. Not more than 1.0 mL of
The label states :
0.01 M hydrochloric acid is required to reach the beginning of
– the nominal viscosity by a number placed after the name of the colour change of the indicator from yellow to orange.
the product ;
Relative density (2.2.5) : 1.05 to 1.25, determined using a
– that the contents are to be used as a lubricant. density bottle with anhydrous ethanol R as the immersion
liquid.
01/2008:30109
Reducing substances. To 20 mL of solution S add 1 mL
of dilute sulfuric acid R and 20 mL of 0.002 M potassium
3.1.9. SILICONE ELASTOMER FOR permanganate. Allow to stand for 15 min. Add 1 g of
CLOSURES AND TUBING potassium iodide R and titrate immediately with 0.01 M sodium
thiosulfate using 0.25 mL of starch solution R as indicator.
DEFINITION Carry out a blank titration using 20 mL of water R instead of
Silicone elastomer complying with the following requirements solution S. The difference between the titration volumes is
is suitable for the manufacture of closures and tubing. not more than 1.0 mL.
Silicone elastomer is obtained by cross-linking a linear Substances soluble in hexane : maximum 3 per cent.
polysiloxane constructed mainly of dimethylsiloxy units with
small quantities of methylvinylsiloxy groups ; the chain ends Evaporate 25 mL of the solution obtained in the test for
are blocked by trimethylsiloxy or dimethylvinylsiloxy groups. phenylated compounds in a glass evaporating dish on a
water-bath and dry in an oven at 100-105 °C for 1 h. The
The general formula of the polysiloxane is : residue weighs not more than 15 mg.
Phenylated compounds. Place 2.0 g in a borosilicate-glass
flask with a ground-glass neck and add 100 mL of hexane R.
Boil under a reflux condenser for 4 h. Cool, then filter rapidly
through a sintered-glass filter (16) (2.1.2). Collect the filtrate
and close the container immediately to avoid evaporation. At
wavelengths from 250 nm to 340 nm, the absorbance (2.2.25)
is not greater than 0.4.
The cross-linking is carried out in the hot state : Mineral oils. Place 2 g in a 100 mL conical flask containing
– either with : 30 mL of a mixture of 5 volumes of ammonia R and 95 volumes
– 2,4-dichlorobenzoyl peroxide for extruded products ; or of pyridine R. Allow to stand for 2 h, shaking frequently.
Decant the pyridine solution and examine in ultraviolet light
– 2,4-dichlorobenzoyl peroxide or dicumyl peroxide or
at 365 nm. The fluorescence is not greater than that of a
OO-(1,1-dimethylethyl) O-isopropyl monoperoxy-
solution containing 1 ppm of quinine sulfate R in 0.005 M
carbonate or 2,5-bis[(1,1-dimethylethyl)dioxy]-2,5-
sulfuric acid examined in the same conditions.
dimethylhexane for moulded products ;
– or by hydrosilylation by means of polysiloxane with -SiH Volatile matter : maximum 0.5 per cent for silicone elastomer
groups using platinum as a catalyst. prepared using peroxides ; maximum 2.0 per cent for silicone
elastomer prepared using platinum.
In all cases, appropriate additives are used such as silica
and sometimes small quantities of organosilicon additives Weigh 10.0 g of the substance previously stored for 48 h in a
(α,ω-dihydroxypolydimethylsiloxane). desiccator over anhydrous calcium chloride R. Heat in an oven
at 200 °C for 4 h, allow to cool in a desiccator and weigh again.
CHARACTERS Silicone elastomer prepared using peroxides complies with the
Appearance : transparent or translucent material. following additional test :
Solubility : practically insoluble in organic solvents, some Residual peroxides : maximum 0.08 per cent calculated as
of which, for example cyclohexane, hexane and methylene dichlorobenzoyl peroxide.
chloride, cause a reversible swelling of the material.
Place 5 g in a borosilicate-glass flask, add 150 mL of methylene
IDENTIFICATION chloride R and close the flask. Stir with a mechanical stirrer
A. Infrared absorption spectrophotometry (2.2.24) by the for 16 h. Filter rapidly, collecting the filtrate in a flask with
multiple reflection method for solids. a ground-glass neck. Replace the air in the container with
oxygen-free nitrogen R, introduce 1 mL of a 200 g/L solution
Comparison : silicone elastomer CRS. of sodium iodide R in anhydrous acetic acid R, close the flask,
B. Heat 1.0 g in a test-tube over a small flame until white shake thoroughly and allow to stand protected from light for
fumes begin to appear. Invert the tube over a 2nd tube 30 min. Add 50 mL of water R and titrate immediately with
containing 1 mL of a 1 g/L solution of chromotropic acid, 0.01 M sodium thiosulfate, using 0.25 mL of starch solution R
sodium salt R in sulfuric acid R so that the fumes reach the as indicator. Carry out a blank titration. The difference
solution. Shake the 2nd tube for about 10 s and heat on a between the titration volumes is not greater than 2.0 mL.
water-bath for 5 min. The solution is violet. Silicone elastomer prepared using platinum complies with the
C. 50 mg of the residue of combustion gives the reaction of following additional test :
silicates (2.3.1).
Platinum : maximum 30 ppm.
TESTS In a quartz crucible, ignite 1.0 g of the material to be examined,
If necessary, cut the material into pieces of maximum dimension raising the temperature gradually until a white residue is
on a side of not greater than 1 cm. obtained. Transfer the residue to a graphite crucible. To the
quartz crucible add 10 mL of a freshly prepared mixture of Fill a 50 mL polyethylene or polypropylene syringe with
1 volume of nitric acid R and 3 volumes of hydrochloric acid R, gaseous vinyl chloride R, allow the gas to remain in contact
heat on a water-bath for 1-2 min and transfer to the graphite with the syringe for about 3 min, empty the syringe and fill
crucible. Add 5 mg of potassium chloride R and 5 mL of again with 50 mL of gaseous vinyl chloride R. Fit a hypodermic
hydrofluoric acid R and evaporate to dryness on a water-bath. needle to the syringe and reduce the volume of gas in the
Add 5 mL of hydrofluoric acid R and evaporate to dryness syringe from 50 mL to 25 mL. Inject these 25 mL of vinyl
again ; repeat this operation twice. Dissolve the residue in chloride slowly into the vial, shaking gently and avoiding
5 mL of 1 M hydrochloric acid, warming on a water-bath. contact between the liquid and the needle. Weigh the vial
Allow to cool and add the solution to 1 mL of a 250 g/L again ; the increase in mass is about 60 mg (1 μL of the solution
solution of stannous chloride R in 1 M hydrochloric acid, rinse thus obtained contains about 1.2 μg of vinyl chloride). Allow
the graphite crucible with a few millilitres of 1 M hydrochloric to stand for 2 h. Keep the primary solution in a refrigerator.
acid and dilute to 10.0 mL with the same acid. Vinyl chloride standard solution : vinyl chloride primary
Prepare simultaneously the reference solution as follows : solution, dimethylacetamide R (1:3 V/V).
to 1 mL of a 250 g/L solution of stannous chloride R in 1 M Reference solutions. Place 10.0 mL of the internal standard
hydrochloric acid, add 1.0 mL of platinum standard solution solution in each of six 50 mL vials. Close the vials and
(30 ppm Pt) R and dilute to 10.0 mL with 1 M hydrochloric secure the stoppers. Inject 1 μL, 2 μL, 3 μL, 5 μL and 10 μL,
acid. respectively, of the vinyl chloride standard solution into 5 of
The colour of the test solution is not more intense than that of the vials. The 6 solutions thus obtained contain respectively,
the standard. 0 μg, about 0.3 μg, 0.6 μg, 0.9 μg, 1.5 μg and 3 μg of vinyl
chloride. Shake, avoiding contact between the stopper and the
LABELLING liquid. Place the vials in a water-bath at 60 ± 1 °C for 2 h.
The label states whether the material was prepared using Column :
peroxides or platinum.
– material : stainless steel ;
01/2008:30110 – size : l = 3 m, Ø = 3 mm ;
corrected 7.5 – stationary phase : silanised diatomaceous earth for gas
chromatography R impregnated with 5 per cent m/m of
3.1.10. MATERIALS BASED ON dimethylstearamide R and 5 per cent m/m of macrogol 400 R.
NON-PLASTICISED POLY(VINYL Carrier gas : nitrogen for chromatography R.
CHLORIDE) FOR CONTAINERS Flow rate : 30 mL/min.
Temperature :
FOR NON-INJECTABLE, AQUEOUS
– column : 45 °C ;
SOLUTIONS – injection port : 100 °C ;
DEFINITION – detector : 150 °C.
Materials based on non-plasticised poly(vinyl chloride) that Detection : flame ionisation.
comply with the following specifications are suitable for Injection : 1 mL of the head space.
the manufacture of containers for non-injectable aqueous
solutions. They may also be used for solid forms for oral Limit :
administration and in some cases, subject to special studies – vinyl chloride : maximum 1 ppm.
on the compatibility of the container with its contents, these Additives
materials may be suitable for the preparation of containers
In order to obtain the required mechanical and stability
for suppositories. They consist of 1 or more poly(vinyl
chloride/vinyl acetate) or of a mixture of poly(vinyl chloride) characteristics, materials based on non-plasticised poly(vinyl
chloride) may contain :
and poly(vinyl acetate) or of poly(vinyl chloride).
The chlorine content expressed as poly(vinyl chloride) is not – epoxidised soya oil of which the oxiran oxygen content is
less than 80 per cent. 6 per cent to 8 per cent and the iodine value is not greater
than 6 : maximum 8 per cent ;
They may contain not more than 15 per cent of copolymers
based on acrylic and/or methacrylic acids and/or their esters, – calcium salt or zinc salts of aliphatic fatty acids with more
and/or on styrene and/or butadiene. than 7 carbon atoms : maximum 1.5 per cent or maximum
1.5 per cent of their mixture ;
PRODUCTION – liquid paraffin : maximum 1.5 per cent ;
Materials based on non-plasticised poly(vinyl chloride) are – waxes : maximum 1.5 per cent ;
produced by polymerisation methods that guarantee a residual
– hydrogenated oils or esters of aliphatic fatty acids :
vinyl chloride content of less than 1 ppm. The manufacturing
maximum 2 per cent ;
process is validated to demonstrate that the product complies
with the following test. – macrogol esters : maximum 1.5 per cent ;
Vinyl chloride. Head-space gas chromatography (2.2.28). – sorbitol : maximum 1.5 per cent ;
Internal standard solution. Using a microsyringe, inject – 2,4-dinonylphenyl phosphite, or di(4-nonylphenyl)
10 μL of ether R into 20.0 mL of dimethylacetamide R, phosphite or tris(nonylphenyl) phosphite : maximum 1 per
immersing the tip of the needle in the solvent. Immediately cent.
before use, dilute the solution to 1000 times its volume with They may contain one of the following groups of stabilisers :
dimethylacetamide R. – tin as di(isooctyl) 2,2′-[(dioctylstannylene)bis(thio)]-
Test solution. Place 1.000 g of the material to be examined in a diacetate containing about 27 per cent of tri(isooctyl)
50 mL vial and add 10.0 mL of the internal standard solution. 2,2′,2″-[(monooctylstannylidyne)tris(thio)]triacetate :
Close the vial and secure the stopper. Shake, avoiding contact maximum 0.25 per cent ;
between the stopper and the liquid. Place the vial in a – tin as a mixture containing not more than 76 per cent of
water-bath at 60 ± 1 °C for 2 h. di(isooctyl) 2,2′-[(dimethylstannylene)bis(thio)]diacetate
Vinyl chloride primary solution. Prepare in a fume cupboard. and not more than 85 per cent of tri(isooctyl)
Place 50.0 mL of dimethylacetamide R in a 50 mL vial, stopper 2,2′,2″-[(monomethylstannylidyne)tris(thio)]triacetate ;
the vial, secure the stopper and weigh to the nearest 0.1 mg. (isooctyl is e.g. 2-ethylhexyl) : maximum 0.25 per cent ;
General Notices (1) apply to all monographs and other texts 395
3.1.10. Non-plasticised PVC materials for non-injectable solutions EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 397
3.1.13. Plastic additives EUROPEAN PHARMACOPOEIA 8.0
They may contain a colorant or pigment and may be opacified To 0.10 mL of solution S2 in a test tube add 0.05 mL of 1 M
by titanium dioxide. hydrochloric acid, 0.5 mL of potassium iodide solution R and
The supplier of the material must be able to demonstrate 5 mL of ethanol (96 per cent) R. Mix thoroughly and wait for
that the qualitative and quantitative composition of the type 5 min. Add 9 mL of water R and 0.1 mL of a 5 g/L solution of
sample is satisfactory for each production batch. sodium sulfite R and mix thoroughly. Add 1.5 mL of dithizone
solution R freshly diluted 100-fold with methylene chloride R,
CHARACTERS shake for 15 s and allow to stand for 2 min. At the same time
prepare a reference solution in the same manner using 0.1 mL
Appearance : powder, beads, granules, sheets of varying of the tin standard solution.
thicknesses or samples taken from finished objects.
Any violet colour in the lower layer obtained with solution S2
Solubility : insoluble in water, soluble in tetrahydrofuran, is not more intense than that obtained with the reference
slightly soluble in methylene chloride, insoluble in anhydrous solution. The greenish-blue colour of dithizone solution turns
ethanol. pink in the presence of tin.
They burn with an orange-yellow flame edged with green,
Non-tin-stabilised materials : maximum 25 ppm of Sn.
giving off thick black smoke.
To 5 mL of solution S2 in a test tube add 0.05 mL of 1 M
IDENTIFICATION hydrochloric acid and 0.5 mL of potassium iodide solution R.
Infrared absorption spectrophotometry (2.2.24). Mix thoroughly and wait for 5 min. Add 9 mL of water R
and 0.1 mL of a 5 g/L solution of sodium sulfite R and mix
Preparation. Dissolve residue A (see Tests : solution S2) in thoroughly. If the solution obtained is not colourless, add the
5 mL of tetrahydrofuran R. Apply a few drops of the solution sodium sulfite solution in 0.05 mL fractions. Add 1.5 mL of
to a sodium chloride plate and evaporate to dryness in an dithizone solution R freshly diluted 100-fold with methylene
oven at 100-105 °C. chloride R, shake for 15 s and allow to stand for 2 min. At the
Absorption maxima : at 2975 cm− 1, 2910 cm− 1, 2865 cm− 1, same time prepare a reference solution in the same manner
1430 cm− 1, 1330 cm− 1, 1255 cm− 1, 690 cm− 1 and 615 cm− 1. using 0.05 mL of the tin standard solution (see test above).
The spectrum obtained is identical to that of the material Any violet colour in the lower layer obtained with solution S2
selected for the type sample. is not more intense than that obtained with the reference
solution.
TESTS
Extractable heavy metals (2.4.8) : maximum 20 ppm.
If necessary, cut the samples of the material to be examined
12 mL of solution S3 complies with test A. Prepare the
into pieces with a maximum dimension on a side of not greater
reference solution using 10 mL of lead standard solution
than 1 cm.
(1 ppm Pb) R.
Solution S1. Place 25 g in a borosilicate-glass flask. Add
Extractable zinc : maximum 100 ppm.
500 mL of water R and cover the neck of the flask with
aluminium foil or a borosilicate glass beaker. Heat in an Atomic absorption spectrometry (2.2.23, Method I).
autoclave for 121 ± 2 °C for 20 min. Allow to cool and allow Test solution. Solution S3 diluted 10-fold with water R.
the solids to settle.
Reference solution. A solution containing 0.50 ppm of zinc
Solution S2. Dissolve 5.0 g in 80 mL of tetrahydrofuran R prepared by dilution of zinc standard solution (5 mg/mL Zn) R
and dilute to 100 mL with the same solvent. Filter if necessary with 0.01 M hydrochloric acid.
(the solution may remain opalescent). To 20 mL of the
Verify the absence of zinc in the hydrochloric acid used.
solution add, dropwise and with gentle shaking, 70 mL of
ethanol (96 per cent) R. Cool in ice for 1 h. Filter or centrifuge Examined at 214.0 nm, the absorbance of the test solution is
(residue A). Wash residue A with ethanol (96 per cent) R, add not greater than that of the reference solution.
the washings to the filtrate or the centrifugation liquid and Sulfated ash (2.4.14) : maximum 1.0 per cent, determined on
dilute to 100 mL with ethanol (96 per cent) R. 1.0 g ; maximum 4.0 per cent when the materials are opacified
Solution S3. Place 5 g in a borosilicate-glass flask with a using titanium dioxide.
ground-glass neck. Add 100 mL of 0.1 M hydrochloric acid
and boil under a reflux condenser for 1 h. Allow to cool and ASSAY
allow the solids to settle. Carry out the oxygen-flask method (2.5.10) using 50.0 mg
Appearance of solution S1. Solution S1 is not more of the material to be examined. Absorb the combustion
opalescent than reference suspension II (2.2.1) and is products in 20 mL of 1 M sodium hydroxide. To the solution
colourless (2.2.2, Method II). obtained add 2.5 mL of nitric acid R, 10.0 mL of 0.1 M silver
nitrate, 5 mL of ferric ammonium sulfate solution R2 and
Absorbance of solution S1 (2.2.25). Evaporate 100 mL 1 mL of dibutyl phthalate R. Titrate with 0.05 M ammonium
of solution S1 to dryness. Dissolve the residue in 5 mL of thiocyanate until a reddish-yellow colour is obtained. Carry
hexane R. Filter if necessary through a filter previously rinsed out a blank titration.
with hexane R. At wavelengths from 250 nm to 310 nm, the
absorbance of the filtrate is not greater than 0.3. 1 mL of 0.1 M silver nitrate is equivalent to 6.25 mg of
poly(vinyl chloride).
Absorbance of solution S2 (2.2.25) : maximum 1.0,
determined between wavelengths of 250 nm and 330 nm
on solution S2 for material that does not contain
1-phenyleicosane-1,3-dione ; maximum 0.4, determined
between wavelengths of 250 nm and 330 nm on a 10-fold 01/2008:30113
dilution of solution S2 in ethanol (96 per cent) R for material corrected 6.2
that contains 1-phenyleicosane-1,3-dione.
Tin-stabilised materials : maximum 0.25 per cent of Sn. 3.1.13. PLASTIC ADDITIVES
Tin stock solution. Dilute 81 mg of plastic additive 23 CRS to NOTE : the nomenclature given first is according to the IUPAC
100.0 mL with tetrahydrofuran R. rules. The synonym given in bold corresponds to the name
Tin standard solution. Dilute 20 mL of the tin stock solution given in the texts of Chapter 3. The synonym corresponding to
to 100.0 mL with ethanol (96 per cent) R. the rules of the texts of “Chemical Abstracts” is also given.
methanetetryltetramethyl tetrakis[3-[3,5-bis(1,1-
zinc (2RS)-2-ethylhexanoate
dimethylethyl)-4-hydroxyphenyl]propanoate]
synonyms : – zinc octanoate,
synonyms : – pentaerythrityl tetrakis[3-(3,5-di-tert-
– 2-ethylhexanoic acid, zinc salt (2:1), butyl-4-hydroxyphenyl)propionate],
– zinc 2-ethylcaproate. – 2,2-bis[[[3-[3,5-bis(1,1-dimethylethyl)-4-
hydroxyphenyl]propanoyl]oxy]methyl]propane-
add03. [05518-18-3]/[00110-30-5]. PM RN 53440/53520. 1,3-diyl 3-[3,5-bis(1,1-dimethylethyl)-4-
hydroxyphenyl]propanoate,
– benzenepropanoic acid, 3,5-bis(1,1-dimethyl-
ethyl)-4-hydroxy-2,2-bis(hydroxymethyl)-
propane-1,3-diol ester (4:1),
N,N′-ethylenedialcanamide (with n and m = 14 or 16) – 2,2-bis(hydroxymethyl)propane-1,3-diol
synonyms : – N,N′-diacylethylenediamines, tetrakis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)-
propionate].
– N,N′-diacylethylenediamine (in this context
acyl means in particular palmitoyl and stearoyl). add10. C54H78O3. [1709-70-2]. PM RN 95200.
add04. [8013-07-8]. PM RN 88640.
epoxidised soya oil
add05. [8016-11-3]. PM RN 64240.
epoxidised linseed oil
add06. [57455-37-5](TSCA)/[101357-30-6]
(EINECS)/Pigment blue 29 (CI 77007)
ultramarine blue 4,4′,4″-[(2,4,6-trimethylbenzene-1,3,5-triyl)tris(methylene)]-
add07. C15H24O. [128-37-0] PM RN 46640. tris[2,6-bis(1,1-dimethylethyl)phenol]
synonyms : – 2,2′,2″,6,6′,6″-hexa-tert-butyl-
4,4′,4″-[(2,4,6-trimethyl-1,3,5-
benzenetriyl)trismethylene]triphenol,
– 1,3,5-tris[3,5-di-tert-butyl-4-hydroxybenzyl]-
2,4,6-trimethylbenzene,
– phenol, 4,4′,4″-[(2,4,6-trimethyl-1,3,5-
2,6-bis(1,1-dimethylethyl)-4-methylphenol benzenetriyl)tris(methylene)]tris[2,6-bis(1,1-
dimethylethyl)-.
synonyms : – butylhydroxytoluene,
add11. C35H62O3. [2082-79-3]. PM RN 68320.
– 2,6-bis(1,1-dimethylethyl)-4-methylphenol,
– 2,6-di-tert-butyl-4-methylphenol.
add08. C50H66O8. [32509-66-3]. PM RN 53670.
octadecyl 3-[3,5-bis(1,1-dimethylethyl)-4-
hydroxyphenyl]propanoate
synonyms : – octadecyl 3-(3,5-di-tert-butyl-4-
hydroxyphenyl)propionate,
– propanoic acid, 3-[3,5-bis(1,1-dimethylethyl)-
4-hydroxyphenyl]-, octadecyl ester.
General Notices (1) apply to all monographs and other texts 399
3.1.13. Plastic additives EUROPEAN PHARMACOPOEIA 8.0
dioctadecyl 3,3′-sulfanediyldipropanoate
synonyms : – dioctadecyl 3,3′-thiodipropionate,
– dioctadecyl 3,3′-sulfanediyldipropanoate,
– propanoic acid, 3,3′-thiobis-, octadecyl diester,
– stearyl thiodipropionate.
add18. [119345-01-6]. PM RN 92560.
tris[2,4-bis(1,1-dimethylethyl)phenyl] phosphite mixture of seven products corresponding to reaction
synonyms : – tris(2,4-di-tert-butylphenyl) phosphite, product of di-tert-butyl phosphonite with biphosphorous
trichloride, reaction products with biphenyl and
– phenol, 2,4-bis(1,1-dimethylethyl)-, 2,4-bis(1,1-dimethylethyl)phenol :
phosphite (3:1),
– 2,4-bis(1,1-dimethylethyl)phenyl, phosphite.
add13. C48H69N3O6. [27676-62-6]. PM RN 95360.
component I
2,4-bis(1,1-dimethylethyl)phenyl biphenyl-4,4′-
diyldiphosphonite
1,3,5-tris[3,5-bis(1,1-dimethylethyl)-4-hydroxybenzyl]-1,3,5- component II
triazine-2,4,6(1H,3H,5H)-trione
synonyms : – 1,3,5-tris(3,5-di-tert-butyl-4-
hydroxybenzyl)-s-triazine-2,4,6(1H,3H,5H)-
trione,
– 1,3,5-triazine-2,4,6(1H,3H,5H)-trione, 2,4-bis(1,1-dimethylethyl)phenyl biphenyl-3,4′-
1,3,5-tris[[3,5-bis(1,1-dimethylethyl)-4- diyldiphosphonite
hydroxyphenyl]methyl]-.
component III
add14. C41H82O6P2. [3806-34-6]. PM RN 50080.
3,9-bis(octadecyloxy)-2,4,8,10-tetraoxa-3,9-diphospha-
spiro[5.5]undecane 2,4-bis(1,1-dimethylethyl)phenyl biphenyl-3,3′-
synonyms : – 2,2′-bis(octadecyloxy)-5,5′-spirobi[1,3,2- diyldiphosphonite
dioxaphosphinane], component IV
– 2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]un-
decane, 3,9-bis(octadecyloxy)-.
add15. C36H74S2. [2500-88-1]. PM RN 49840. 2,4-bis(1,1-dimethylethyl)phenyl biphenyl-4-ylphosphonite
component V
1,1′-disulfanediyldioctadecane
synonyms : – dioctadecyl disulfide, 2,4-bis(1,1-dimethylethyl)phenyl phosphite
component VI
– octadecane, 1,1′-dithio-.
add16. C30H58O4S. [123-28-4]. PM RN 93120.
2,4-bis(1,1-dimethylethyl)phenyl 4′-[bis[2,4-bis(1,1-
dimethylethyl)phenoxy]phosphanyl]biphenyl-4-
ylphosphonate
didodecyl 3,3′-sulfanediyldipropanoate
component VII
synonyms : – didodecyl 3,3′-thiodipropionate, R-OH : 2,4-bis(1,1-dimethylethyl)phenol
– didodecyl 3,3′-sulfanediyldipropanoate, add19. C18H36O2. [57-11-4]. PM RN 24550.
– propanoic acid, 3,3′-thiobis-, dodecyl diester,
– lauryl thiodipropionate. octadecanoic acid
General Notices (1) apply to all monographs and other texts 401
3.1.14. Plasticised PVC materials for intravenous solutions EUROPEAN PHARMACOPOEIA 8.0
Application : 0.5 mL of solution A1 obtained during the Verify the absence of calcium in the hydrochloric acid used.
identification as a band 30 mm by 3 mm and 5 μL of each Tin : maximum 20 ppm.
reference solution.
Inductively coupled plasma-atomic emission spectrometry
Development : over a path of 15 cm. (2.2.57).
Drying : in air. Test solution. Dilute solution S1 10 times with water R
Detection A : examine in ultraviolet light at 254 nm. immediately before use.
Locate the zone corresponding to plastic additive 01 (RF about Reference solution. Introduce 2 mL of tin standard solution
0.4). Remove the area of silica gel corresponding to this zone (5 ppm Sn) R into a 50 mL flask containing 5 mL of a 20 per
and shake with 40 mL of ether R for 1 min. Filter, rinse with cent V/V solution of sulfuric acid R and dilute to 50 mL with
2 quantities, each of 10 mL of ether R, add the rinsings to the water R immediately before use.
filtrate and evaporate to dryness. The residue C weighs not Wavelength : use the emission of tin at 189.99 nm, the spectral
more than 40 mg. background being taken at 190.10 nm.
Detection B : expose the plate to iodine vapour for 5 min. Verify the absence of tin in the hydrochloric acid used.
Examine the chromatogram and locate the band corresponding Zinc : maximum 0.2 per cent.
to plastic additives 04 and 05 (RF = 0). Remove the area of Atomic absorption spectrometry (2.2.23, Method I).
silica gel corresponding to this zone. Similarly remove a
corresponding area of silica gel as a blank reference. Separately Test solution. Dilute solution S1 100 times with 0.1 M
shake both samples for 15 min with 40 mL of methanol R. hydrochloric acid.
Filter, rinse with 2 quantities, each of 10 mL of methanol R, Reference solutions. Prepare the reference solutions using
add the rinsings to the filtrate and evaporate to dryness. The zinc standard solution (100 ppm Zn) R, diluting with 0.1 M
difference between the masses of both residues is not more hydrochloric acid.
than 10 mg. Source : zinc hollow-cathode lamp.
Plastic additive 03. Infrared absorption spectrophotometry Wavelength : 213.9 nm.
(2.2.24). Atomisation device : air-acetylene flame.
Preparation. Wash precipitate B2 obtained during the Verify the absence of zinc in the hydrochloric acid used.
identification and contained in the tared sintered-glass filter Heavy metals (2.4.8) : maximum 50 ppm.
(40) (2.1.2) with anhydrous ethanol R. Dry to constant mass
over diphosphorus pentoxide R and weigh the filter. The To 10 mL of solution S1 add 0.5 mL of phenolphthalein
residue weighs not more than 20 mg. solution R and then strong sodium hydroxide solution R until
a pale pink colour is obtained. Dilute to 25 mL with water R.
Comparison : plastic additive 03 CRS. 12 mL of solution complies with test A. Prepare the reference
Barium : maximum 5 ppm. solution using lead standard solution (2 ppm Pb) R.
Inductively coupled plasma-atomic emission spectrometry Water extractable substances : maximum 0.3 per cent.
(2.2.57). Evaporate 50.0 mL of solution S2 to dryness on a water-bath
Test solution. Ignite 1.0 g of the substance to be examined in a and dry at 100-105 °C until constant mass. Carry out a blank
silica crucible. Take up the residue with 10 mL of hydrochloric titration with 50.0 mL of water for injections R. The residue
acid R and evaporate to dryness on a water-bath. Take up the weighs not more than 7.5 mg taking into account the blank
residue with 20 mL of 0.1 M hydrochloric acid. test.
Reference solution. A solution containing 0.25 ppm of barium ASSAY
prepared by dilution of barium standard solution (50 ppm
Ba) R with 0.1 M hydrochloric acid. Carry out the oxygen-flask method (2.5.10) using 50.0 mg.
Absorb the combustion products in 20 mL of 1 M sodium
Wavelength : use the emission of barium at 455.40 nm, the hydroxide. To the solution obtained add 1 mL of dibutyl
spectral background being taken at 455.30 nm. phthalate R, 2.5 mL of nitric acid R, 5 mL of ferric ammonium
Verify the absence of barium in the hydrochloric acid used. sulfate solution R2 and 10.0 mL of 0.1 M silver nitrate. Titrate
Cadmium : maximum 0.6 ppm. with 0.05 M ammonium thiocyanate until a reddish-yellow
Atomic absorption spectrometry (2.2.23, Method I). colour is obtained. Carry out a blank test.
Test solution. Evaporate 10 mL of solution S1 to dryness. 1 mL of 0.1 M silver nitrate is equivalent to 6.25 mg of
Take up the residue using 5 mL of a 1 per cent V/V solution poly(vinyl chloride).
of hydrochloric acid R, filter and dilute the filtrate to 10.0 mL
with the same acid. 01/2008:30115
corrected 7.5
Reference solutions. Prepare the reference solutions using
cadmium standard solution (0.1 per cent Cd) R, diluting with a
1 per cent V/V solution of hydrochloric acid R. 3.1.15. POLYETHYLENE
Source : cadmium hollow-cathode lamp. TEREPHTHALATE FOR CONTAINERS
Wavelength : 228.8 nm. FOR PREPARATIONS NOT FOR
Atomisation device : air-acetylene flame. PARENTERAL USE
Verify the absence of cadmium in the hydrochloric acid used.
Calcium : maximum 0.07 per cent.
Inductively coupled plasma-atomic emission spectrometry
(2.2.57).
Test solution. Use the test solution prepared for the
determination of barium.
Reference solution. A solution containing 50.0 ppm of calcium DEFINITION
prepared by dilution of calcium standard solution (400 ppm Polyethylene terephthalate is obtained from the
Ca) R with 0.1 M hydrochloric acid. polymerisation of terephthalic acid or dimethyl
Wavelength : use the emission of calcium at 315.89 nm, the terephthalate with ethylene glycol. Isophthalic acid,
spectral background being taken at 315.60 nm. dimethyl isophthalate, 1,4-bis(hydroxymethyl)cyclohexane
General Notices (1) apply to all monographs and other texts 403
3.1.15. Polyethylene terephthalate for containers EUROPEAN PHARMACOPOEIA 8.0
(cyclohexane-1,4-dimethanol) or diethylene glycol may be The solution turns yellow. Not more than 0.5 mL of 0.01 M
used in the polymerisation. It may contain not more than hydrochloric acid is required to reach the beginning of the
0.5 per cent of silica or silicates and colouring matter approved colour change of the indicator to orange.
by the competent authority. Absorbance of solution S1 (2.2.25) : maximum 0.20 between
220 nm and 340 nm. In addition, for coloured polyethylene
PRODUCTION terephthalate : maximum 0.05 between 400 nm to 800 nm.
The manufacturing process is validated to demonstrate that Absorbance of solution S2 (2.2.25) : maximum 0.05 between
the residual acetaldehyde content is not greater than 10 ppm 400 nm and 800 nm.
in the granules.
Reducing substances. Add 2 mL of 0.5 M sulfuric acid and
CHARACTERS 20.0 mL of 0.002 M potassium permanganate to 20.0 mL of
solution S1. Boil for 3 min. Cool immediately to ambient
Appearance : clear or opaque granules. temperature. Add 1 g of potassium iodide R, 0.25 mL of
Solubility : practically insoluble in water, in ethanol (96 per starch solution R as indicator and titrate with 0.01 M sodium
cent) and in methylene chloride. It is hydrolysed by strong thiosulfate. Perform a blank titration using 20.0 mL of water R.
bases. The difference in volume used in the 2 titrations is not greater
than 0.5 mL.
IDENTIFICATION Substances soluble in dioxan : maximum 3 per cent.
A. Place 0.10 g of the material to be examined into a Place 2 g of the material to be examined in a borosilicate glass
borosilicate glass flask with a ground-glass neck. Add flask with a ground-glass neck. Add 20 mL of dioxan R and
25 mL of a 200 g/L solution of potassium hydroxide R in a heat under reflux for 2 h. Evaporate 10 mL of the solution
50 per cent V/V solution of anhydrous ethanol R. Reflux for to dryness on a water-bath and then dry the residue at
30 min. Allow to cool and dilute to 100 mL with water R. 100-105 °C. The residue weighs a maximum of 30 mg.
Filter if necessary. Dilute 1.0 mL of the filtrate to 100 mL
with water R. Examined between 210 nm and 330 nm Extractable aluminium : maximum 1 ppm.
(2.2.25), the solution shows an absorption maximum at Inductively coupled plasma-atomic emission spectrometry
240 nm. (2.2.57).
B. Dissolve 0.05 g of the material to be examined in 2 mL Test solution. Solution S3.
of 1,1,1,3,3,3-hexafluoropropan-2-ol R. Apply to a glass Reference solutions. Prepare the reference solutions using
plate on a water-bath in a fume cupboard several drops of aluminium standard solution (200 ppm Al) R, diluting with
the solution to produce a film of about 15 mm by 15 mm. 0.1 M hydrochloric acid.
Allow the solvent to evaporate and remove the film using a
Wavelength : 396.15 nm, the spectral background being taken
stream of water and a scraper. Dry in an oven at 100-105 °C
at 396.25 nm.
for 1-2 h. Examine the film by infrared absorption
spectrophotometry (2.2.24). The spectrum of the material Verify the absence of aluminium in the 0.1 M hydrochloric
to be examined shows maxima in particular at 1725 cm− 1, acid used.
1410 cm− 1, 1265 cm− 1, 1120 cm− 1, 1100 cm− 1, 1020 cm− 1, Extractable antimony : maximum 1 ppm.
875 cm− 1, 725 cm− 1. The spectrum obtained, in addition, is
Inductively coupled plasma-atomic emission spectrometry
identical to that of the material selected for the type sample.
(2.2.57).
TESTS Test solution. Solution S4.
If necessary, cut out samples for testing to a maximum size of Reference solutions. Prepare the reference solutions using
1 cm per side. antimony standard solution (100 ppm Sb) R, diluting with
0.01 M sodium hydroxide.
Solution S1. Place 10.0 g of the material to be examined in a
borosilicate glass flask with a ground-glass neck. Add 200 mL Wavelength : 231.15 nm or 217.58 nm, the spectral background
of water R and heat at 50 °C for 5 h. Allow to cool and decant being taken at 231.05 nm.
the solution. Use solution S1 within 4 h of its preparation. Extractable barium : maximum 1 ppm.
Solution S2. Place 10 g of the material to be examined in a Inductively coupled plasma-atomic emission spectrometry
borosilicate glass flask with a ground-glass neck. Add 100 mL (2.2.57).
of ethanol (96 per cent) R and heat at 50 °C for 5 h. Allow to Test solution. Solution S3.
cool and decant the solution. Use solution S2 within 4 h of its
Reference solutions. Prepare the reference solutions using
preparation.
barium standard solution (50 ppm Ba) R, diluting with 0.1 M
Solution S3. Place 20 g of the material to be examined in a hydrochloric acid.
borosilicate glass flask with a ground-glass neck. Add 50 mL Wavelength : 455.40 nm, the spectral background being taken
of 0.1 M hydrochloric acid and heat at 50 °C for 5 h. Allow at 455.30 nm.
to cool and decant the solution. Use solution S3 within 4 h
of its preparation. Verify the absence of barium in the 0.1 M hydrochloric acid
used.
Solution S4. Place 20 g of the material to be examined into a
borosilicate glass flask with a ground-glass neck. Add 50 mL Extractable cobalt : maximum 1 ppm.
of 0.01 M sodium hydroxide and heat at 50 °C for 5 h. Allow to Inductively coupled plasma-atomic emission spectrometry
cool and decant. Use solution S4 within 4 h of its preparation. (2.2.57).
Appearance of solution S1. Solution S1 is clear (2.2.1). Test solution. Solution S3.
Appearance of solution S2. Solution S2 is clear (2.2.1) and Reference solutions. Prepare the reference solutions using
colourless (2.2.2, Method II). cobalt standard solution (100 ppm Co) R, diluting with 0.1 M
hydrochloric acid.
Acidity or alkalinity. To 50 mL of solution S1 add 0.15 mL
of BRP indicator solution R. The solution turns yellow. Not Wavelength : 228.62 nm, the spectral background being taken
more than 0.5 mL of 0.01 M sodium hydroxide is required at 228.50 nm.
to change the colour of the indicator to blue. To another Verify the absence of cobalt in the 0.1 M hydrochloric acid
50 mL of solution S1 add 0.2 mL of methyl orange solution R. used.
General Notices (1) apply to all monographs and other texts 405
EUROPEAN PHARMACOPOEIA 8.0 3.2.1. Glass containers for pharmaceutical use
General Notices (1) apply to all monographs and other texts 409
3.2.1. Glass containers for pharmaceutical use EUROPEAN PHARMACOPOEIA 8.0
HYDROLYTIC RESISTANCE the mean value. This volume, expressed to 1 decimal place, is
the filling volume for the particular ampoule lot. The filling
volume may also be determined by weighing.
Table 3.2.1.-1. – Types of glass
General Notices (1) apply to all monographs and other texts 411
3.2.1. Glass containers for pharmaceutical use EUROPEAN PHARMACOPOEIA 8.0
cool in the desiccator. Weigh 10.00 g of the cleaned and dried Distinction between type I and type II glass containers
grains into 2 separate conical flasks. Add 50 mL of water R1 The results obtained in Test C are compared to those obtained
into each by means of a pipette (test solutions). Pipette 50 mL in Test A. The interpretation of the result is shown in
of water R1 into a third conical flask which will serve as Table 3.2.1.-4.
a blank. Distribute the grains evenly over the flat bases of
the flasks by gentle shaking. Close the flasks with neutral Table 3.2.1.-4. – Distinction between Types I and II glass
glass dishes or aluminium foil rinsed with water R or with containers
inverted beakers so that the inner surface of the beakers fit Type I Type II
snugly down onto the top rims of the flasks. Place all 3 flasks
in the rack in the autoclave containing the water at ambient The values are closely similar The values greatly exceed those found
to those found in the test for in the test for surface hydrolytic
temperature, and ensure that they are held above the level of surface hydrolytic resistance for resistance and are similar but not
the water in the vessel. Carry out the autoclaving procedure in type I glass containers. larger than those for type III glass
a similar manner to that described under test A, but maintain containers.
the temperature of 121 ± 1 °C only for 30 ± 1 min. Do not
open the autoclave until it has cooled to 95 °C. Remove the ARSENIC
hot samples from the autoclave and cool the flasks in running The test applies to glass containers for aqueous parenteral
tap water as soon as possible, avoiding thermal shock. To each preparations.
of the 3 flasks add 0.05 mL of methyl red solution R. Titrate the
blank solution immediately with 0.02 M hydrochloric acid then Hydride generation atomic absorption spectrometry (2.2.23,
titrate the test solutions until the colour matches that obtained Method I).
with the blank solution. Subtract the titration volume for the Test solution. Use the extract solution obtained from
blank solution from that for the test solutions. containers of types I and II, after autoclaving at 121 °C for 1 h
NOTE : where necessary to obtain a sharp end-point, the clear as described under test A for surface hydrolytic resistance.
solution is to be decanted into a separate 250 mL flask. Rinse Transfer 10.0 mL to a 100 mL volumetric flask. Add 10 mL
the grains with 3 quantities, each of 15 mL, of water R1 by of hydrochloric acid R and 5 mL of a 200 g/L solution of
swirling and add the washings to the main solution. Add potassium iodide R. Heat on a water-bath at 80 °C for 20 min,
0.05 mL of the methyl red solution R. Titrate and calculate as allow to cool and dilute to 100.0 mL with water R.
described below. In this case also add 45 mL of water R1 and Reference solutions. Prepare the reference solutions using
0.05 mL of methyl red solution R to the blank solution. arsenic standard solution (1 ppm As) R. Add 10 mL of
Calculate the mean value of the results in millilitres of 0.02 M hydrochloric acid R and 5 mL of a 200 g/L solution of
hydrochloric acid per gram of the sample and if required its potassium iodide R. Heat on a water-bath at 80 °C for 20 min,
equivalent in alkali extracted, calculated as micrograms of allow to cool and dilute to 100.0 mL with water R. The
sodium oxide per gram of glass grains. concentration range of the reference solutions is typically
0.005 ppm to 0.015 ppm of As.
1 mL of 0.02 M hydrochloric acid is equivalent to 620 μg of
Acid reservoir. Hydrochloric acid R.
sodium oxide.
Reducing reservoir. Sodium tetrahydroborate reducing
Repeat the test if the highest and lowest observed values differ
solution R.
by more than 20 per cent.
Use a hydride generation device to introduce the test solution
Limits. Type I glass containers require not more than 0.1 mL into the cuvette of an atomic absorption spectrometer.
of 0.02 M hydrochloric acid per gram of glass, type II and Establish and standardise instrumental operating conditions
type III glass containers require not more than 0.85 mL of according to the manufacturer’s instructions, optimise the
0.02 M hydrochloric acid per gram of glass. uptake rate of the peristaltic pump tubings, then connect
TEST C. TO DETERMINE WHETHER THE CONTAINERS tubings to the acid reservoir, the reducing reservoir and the
HAVE BEEN SURFACE-TREATED (ETCHING TEST) test solution.
When it is necessary to determine if a container has been Source : hollow-cathode lamp.
surface-treated, and/or distinguish between type I and Wavelength : 193.7 nm.
type II glass containers, test C is used in addition to test A.
Atomisation device : air-acetylene flame.
Alternatively, test A and B may be used. Test C may be carried
out either on unused samples or on samples previously tested Limit : maximum 0.1 ppm of As.
for test A.
SPECTRAL TRANSMISSION FOR COLOURED GLASS
Vials and bottles. The volumes of test liquid required are CONTAINERS
shown in Table 3.2.1.-2.
Equipment. A UV-VIS spectrophotometer, equipped with a
Rinse the containers twice with water R and fill to the brimful photodiode detector or equipped with a photomultiplier tube
point with a mixture of 1 volume of hydrofluoric acid R and coupled with an integrating sphere.
9 volumes of hydrochloric acid R and allow to stand for 10 min.
Empty the containers and rinse carefully 5 times with water R. Preparation of the specimen. Break the glass container or
Immediately before the test, rinse once again with water R. cut it with a circular saw fitted with a wet abrasive wheel,
Submit the containers thus prepared to the same autoclaving such as a carborundum or a bonded-diamond wheel. Select
and determination procedure as described in test A for surface sections representative of the wall thickness and trim them as
hydrolytic resistance. If the results are considerably higher suitable for mounting in a spectrophotometer. If the specimen
than those obtained from the original surfaces (by about a is too small to cover the opening in the specimen holder, mask
factor of 5 to 10), the samples have been surface-treated. the uncovered portion with opaque paper or tape, provided
that the length of the specimen is greater than that of the slit.
Ampoules Before placing in the holder, wash, dry and wipe the specimen
NOTE : ampoules made from glass tubing are not normally with lens tissue. Mount the specimen with the aid of wax,
subjected to internal surface treatment because their high or by other convenient means, taking care to avoid leaving
chemical resistance is dependent upon the chemical composition fingerprints or other marks.
of the glass as a material. Method. Place the specimen in the spectrophotometer with
Apply the test method as described above for vials and bottles. its cylindrical axis parallel to the slit and in such a way that the
If the ampoules are not surface-treated, the new values are light beam is perpendicular to the surface of the section and
slightly lower than those obtained in previous tests. that the losses due to reflection are at a minimum. Measure
the transmission of the specimen with reference to air in the Instructions on determination of the filling volume, cleaning
spectral region of 290-450 nm, continuously or at intervals of the containers, filling and heating are given above under
of 20 nm. Hydrolytic resistance and Test A. Hydrolytic resistance of the
Limits. The observed spectral transmission for coloured inner surfaces of glass containers.
glass containers for preparations that are not for parenteral SOLUTIONS
administration does not exceed 10 per cent at any wavelength Spectrochemical buffer solution. Dissolve 80 g of caesium
in the range of 290 nm to 450 nm, irrespective of the type chloride R in about 300 mL of water R1, add 10 mL of
and the capacity of the glass container. The observed spectral 6 M hydrochloric acid R and transfer to a 1000 mL volumetric
transmission in coloured glass containers for parenteral flask. Dilute to volume with water R1 and mix.
preparations does not exceed the limits given in Table 3.2.1.-5. Stock solutions:
Table 3.2.1.-5. – Limits of spectral transmission for coloured – sodium oxide, c(Na2O) = 1 mg/mL ;
glass containers for parenteral preparations – potassium oxide, c(K2O) = 1 mg/mL ;
– calcium oxide, c(CaO) = 1 mg/mL.
Maximum percentage of spectral transmission
at any wavelength between 290 nm and 450 nm Commercially available stock solutions may also be used.
Nominal volume (mL) Flame-sealed Containers with
Standard solutions. Prepare standard solutions by diluting
containers closures the stock solutions with water R1 to obtain concentrations
suitable for establishing the reference solutions in appropriate
Up to 1 50 25 manner, e.g. with concentrations of 20 μg/mL of sodium
Above 1 and up to 2 45 20 oxide, potassium oxide and calcium oxide, respectively.
Commercially available standard solutions may also be used.
Above 2 and up to 5 40 15
Reference solutions. Prepare the reference solutions for
Above 5 and up to 10 35 13 establishing the calibration graph (set of calibration solutions)
30 12
by diluting suitable concentrated standard solutions with
Above 10 and up to 20
water R1, so that the normal working ranges of the specific
Above 20 25 10 elements are covered, taking into account the instrument used
for the measurement. Typical concentration ranges of the
reference solutions are :
Annex – test for surface hydrolytic resistance – for determination by atomic emission spectrometry of
– determination by flame atomic absorption sodium oxide and potassium oxide : up to 10 μg/mL ;
– for determination by atomic absorption spectrometry of
spectrometry (faas) sodium oxide and potassium oxide : up to 3 μg/mL ;
The surface hydrolytic resistance of glass of types I and II may – for determination by atomic absorption spectrometry of
be determined by analysis of the leaching solution by flame calcium oxide : up to 7 μg/mL.
atomic absorption spectrometry. A number of elements that, Use reference solutions containing 5 per cent V/V of the
when present as oxides in glass, contribute to the alkalinity spectrochemical buffer solution.
of the solution, are determined and used to express an alkali METHOD
equivalent. The spectrometric method has the advantage of Carry out preliminary measurements of the potassium oxide
allowing the use of a much smaller sample of extract so that and calcium oxide concentrations on one of the extraction
it can be applied to small individual containers. This enables solutions. If, for one container type, the concentration of
an evaluation of the uniformity of the containers in a given potassium oxide is less than 0.2 μg/mL and if the concentration
batch where this is critical. The results of this measurement are of calcium oxide is less than 0.1 μg/mL, the remaining
not equivalent to those of titrimetry and the 2 methods cannot extraction solutions of this container type need not be
be considered interchangeable. A correlation between the 2 is analysed for these ions. Aspirate the extraction solution
dependent on the type of glass and the size and shape of the from each sample directly into the flame of the atomic
container. The titrimetric method is the reference method of absorption or atomic emission instrument and determine the
the Pharmacopoeia ; the spectrometric method may be used in approximate concentrations of sodium oxide (and potassium
justified and authorised cases. oxide and calcium oxide, if present) by reference to calibration
A method suitable for this type of analysis is shown below. graphs produced from the reference solutions of suitable
concentration.
The determination is carried out on unused containers.
The number of containers to be examined is indicated FINAL DETERMINATION
in Table 3.2.1.-6. If dilution is unnecessary add to each container a volume of
the spectrochemical buffer solution equivalent to 5 per cent
Table 3.2.1.-6. - Number of containers to be examined for the of the filling volume, mix well and determine sodium oxide,
spectrometric method calcium oxide and potassium oxide, if present, by reference
to calibration graphs. For the determination of the calcium
Filling volume (mL) Number of containers Additional containers
to be measured for preliminary
oxide concentration by flame atomic spectrometry, the nitrous
separately measurements oxide/acetylene flame shall be used.
If dilution is necessary, determine sodium oxide, calcium oxide
Up to 2 20 2
and potassium oxide, if present, following the procedures
Above 2 and up to 5 15 2 as described above. The measuring solutions shall contain
5 per cent V/V of the spectrochemical buffer solution.
Above 5 and up to 30 10 2
Concentration values less than 1.0 μg/mL are expressed to
Above 30 and up to 100 5 1 2 decimal places, values greater than or equal to 1.0 μg/mL
to 1 decimal place. Correct the result for the buffer addition
Above 100 3 1
and for dilution, if any.
General Notices (1) apply to all monographs and other texts 413
3.2.2. Plastic containers and closures for pharmaceutical use EUROPEAN PHARMACOPOEIA 8.0
that may be in contact with the infusion are sterilised. The 01/2008:30203
containers are impermeable to micro-organisms after closure.
The containers are such that after filling they are resistant to 3.2.3. STERILE PLASTIC CONTAINERS
damage from accidental freezing which may occur during
transport of the final preparation. The containers are and FOR HUMAN BLOOD AND
remain sufficiently transparent to allow the appearance of the BLOOD COMPONENTS
contents to be examined at any time, unless otherwise justified
and authorised. Plastic containers for the collection, storage, processing and
administration of blood and its components are manufactured
The empty containers display no defects that may lead to from one or more polymers, if necessary with additives.
leakage and the filled and closed containers show no leakage. The composition and the conditions of manufacture of
the containers are registered by the appropriate competent
For satisfactory storage of some preparations, the container authorities in accordance with the relevant national legislation
has to be enclosed in a protective envelope. The initial and international agreements.
evaluation of storage has then to be carried out using the When the composition of the materials of the different parts
container enclosed in the envelope. of the containers correspond to the appropriate specifications,
their quality is controlled by the methods indicated in those
specifications (see 3.1. Materials used for the manufacture of
TESTS containers and subsections).
Solution S. Use solution S within 4 h of preparation. Fill a Materials other than those described in the Pharmacopoeia
container to its nominal capacity with water R and close it, may be used provided that their composition is authorised by
if possible using the usual means of closure ; otherwise close the competent authority and that the containers manufactured
using a sheet of pure aluminium. Heat in an autoclave so from them comply with the requirements prescribed for Sterile
that a temperature of 121 ± 2 °C is reached within 20 min Plastic Containers for Human Blood and Blood Components.
to 30 min and maintain at this temperature for 30 min. If In normal conditions of use the materials do not release
heating at 121 °C leads to deterioration of the container, heat monomers, or other substances, in amounts likely to be
at 100 °C for 2 h. harmful nor do they lead to any abnormal modifications of
Blank. Prepare a blank by heating water R in a borosilicate-glass the blood.
flask closed by a sheet of pure aluminium at the temperature The containers may contain anticoagulant solutions,
and for the time used for the preparation of solution S. depending on their intended use, and are supplied sterile.
Appearance of solution S. Solution S is clear (2.2.1) and Each container is fitted with attachments suitable for the
colourless (2.2.2, Method II). intended use. The container may be in the form of a single unit
or the collecting container may be connected by one or more
Acidity or alkalinity. To a volume of solution S corresponding tubes to one or more secondary containers to allow separation
to 4 per cent of the nominal capacity of the container add of the blood components to be effected within a closed system.
0.1 mL of phenolphthalein solution R. The solution is The outlets are of a shape and size allowing for adequate
colourless. Add 0.4 mL of 0.01 M sodium hydroxide. The connection of the container with the blood-giving equipment.
solution is pink. Add 0.8 mL of 0.01 M hydrochloric acid and The protective coverings on the blood-taking needle and on
0.1 mL of methyl red solution R. The solution is orange-red the appendages must be such as to ensure the maintenance
or red. of sterility. They must be easily removable but must be
Absorbance (2.2.25). Measure the absorbance of solution S tamper-proof.
from 230 nm to 360 nm, using the blank (see solution S) as the The capacity of the containers is related to the nominal
compensation liquid. At these wavelengths, the absorbance is capacity prescribed by the national authorities and to the
not greater than 0.20. appropriate volume of anticoagulant solution. The nominal
Reducing substances. To 20.0 mL of solution S add 1 mL capacity is the volume of blood to be collected in the container.
of dilute sulfuric acid R and 20.0 mL of 0.002 M potassium The containers are of a shape such that when filled they may
permanganate. Boil for 3 min. Cool immediately. Add 1 g be centrifuged.
of potassium iodide R and titrate immediately with 0.01 M The containers are fitted with a suitable device for suspending
sodium thiosulfate, using 0.25 mL of starch solution R as or fixing which does not hinder the collection, storage,
indicator. Carry out a titration using 20.0 mL of the blank. processing or administration of the blood.
The difference between the titration volumes is not greater The containers are enclosed in sealed, protective envelopes.
than 1.5 mL.
CHARACTERS
Transparency. Fill a container previously used for the
preparation of solution S with a volume equal to the nominal The container is sufficiently transparent to allow adequate
capacity of the primary opalescent suspension (2.2.1) visual examination of its contents before and after the taking
diluted 1 in 200 for a container made from polyethylene of the blood and is sufficiently flexible to offer minimum
or polypropylene and 1 in 400 for other containers. The resistance during filling and emptying under normal
cloudiness of the suspension is perceptible when viewed conditions of use. The container contains not more than 5 mL
through the container and compared with a similar container of air.
filled with water R. TESTS
Solution S1. Fill the container with 100 mL of a sterile,
LABELLING pyrogen-free 9 g/L solution of sodium chloride R. Close the
container and heat it in an autoclave so that the contents are
The label accompanying a batch of empty containers includes maintained at 110 °C for 30 min.
a statement of : If the container to be examined contains an anticoagulant
solution, first empty it, rinse the container with 250 mL of
– the name and address of the manufacturer, water for injections R at 20 ± 1 °C and discard the rinsings.
– a batch number which enables the history of the container Solution S2. Introduce into the container a volume of water
and of the plastic material of which it is manufactured to for injections R corresponding to the intended volume of
be traced. anticoagulant solution. Close the container and heat it in an
General Notices (1) apply to all monographs and other texts 415
3.2.3. Sterile plastic containers for human blood EUROPEAN PHARMACOPOEIA 8.0
autoclave so that the contents are maintained at 110 °C for Transparency. Fill the empty container with a volume equal
30 min. After cooling, add sufficient water for injections R to to its nominal capacity of the primary opalescent suspension
fill the container to its nominal capacity. (2.2.1) diluted so as to have an absorbance (2.2.25) at 640 nm
of 0.37 to 0.43 (dilution factor about 1 in 16). The cloudiness
If the container to be examined contains an anticoagulant of the suspension must be perceptible when viewed through
solution, first empty it and rinse it as indicated above. the bag, as compared with a similar container filled with
Resistance to centrifugation. Introduce into the container a water R.
volume of water R, acidified by the addition of 1 mL of dilute Extractable matter. Tests are carried out by methods designed
hydrochloric acid R, sufficient to fill it to its nominal capacity. to simulate as far as possible the conditions of contact between
Envelop the container with absorbent paper impregnated with the container and its contents which occur in conditions of
a 1 in 5 dilution of bromophenol blue solution R1 or other use.
suitable indicator and then dried. Centrifuge at 5000 g for The conditions of contact and the tests to be carried out on
10 min. No leakage perceptible on the indicator paper and no the eluates are prescribed, according to the nature of the
permanent distortion occur. constituent materials, in the particular requirements for each
Resistance to stretch. Introduce into the container a type of container.
volume of water R, acidified by the addition of 1 mL of Haemolytic effects in buffered systems
dilute hydrochloric acid R, sufficient to fill it to its nominal
capacity. Suspend the container by the suspending device at Stock buffer solution. Dissolve 90.0 g of sodium chloride R,
the opposite end from the blood-taking tube and apply along 34.6 g of disodium hydrogen phosphate R and 2.43 g of sodium
the axis of this tube an immediate force of 20 N (2.05 kgf). dihydrogen phosphate R in water R and dilute to 1000 mL with
Maintain the traction for 5 s. Repeat the test with the force the same solvent.
applied to each of the parts for filling and emptying. No break Buffer solution A0. To 30.0 mL of stock buffer solution add
and no deterioration occur. 10.0 mL of water R.
Leakage. Place the container which has been submitted to Buffer solution B0. To 30.0 mL of stock buffer solution add
the stretch test between two plates covered with absorbent 20.0 mL of water R.
paper impregnated with a 1 in 5 dilution of bromophenol Buffer solution C0. To 15.0 mL of stock buffer solution add
blue solution R1 or other suitable indicator and then dried. 85.0 mL of water R.
Progressively apply force to the plates to press the container
so that its internal pressure (i.e. the difference between the Introduce 1.4 mL of solution S2 into each of three centrifuge
applied pressure and atmospheric pressure) reaches 67 kPa tubes. To tube I add 0.1 mL of buffer solution A0, to tube II
within 1 min. Maintain the pressure for 10 min. No signs of add 0.1 mL of buffer solution B0 and to tube III add 0.1 mL
leakage are detectable on the indicator paper or at any point of of buffer solution C0. To each tube add 0.02 mL of fresh,
attachment (seals, joints, etc.). heparinised human blood, mix well and warm on a water-bath
at 30 ± 1 °C for 40 min. Use blood collected less than
Vapour permeability. For a container containing an 3 h previously or blood collected into an anticoagulant
anticoagulant solution, fill with a volume of a 9 g/L solution of citrate-phosphate-dextrose solution (CPD) less than 24 h
sodium chloride R equal to the volume of blood for which the previously.
container is intended.
Prepare three solutions containing, respectively :
For an empty container, fill with the same mixture of 3.0 mL of buffer solution A0 and 12.0 mL of water R
anticoagulant solution and sodium chloride solution. Close (solution A1),
the container, weigh it and store it at 5 ± 1 °C in an atmosphere 4.0 mL of buffer solution B0 and 11.0 mL of water R
with a relative humidity of (50 ± 5) per cent for 21 days. At (solution B1),
the end of this period the loss in mass is not greater than 1 per
cent. 4.75 mL of buffer solution B0 and 10.25 mL of water R
(solution C1).
Emptying under pressure. Fill the container with a volume
of water R at 5 ± 1 °C equal to the nominal capacity. Attach To tubes I, II and III add, respectively, 1.5 mL of solution A1,
a transfusion set without an intravenous cannula to one of 1.5 mL of solution B1 and 1.5 mL of solution C1. At the same
the connectors. Compress the container so as to maintain time and in the same manner, prepare three other tubes,
throughout the emptying an internal pressure (i.e the replacing solution S2 by water R. Centrifuge simultaneously
difference between the applied pressure and atmospheric the tubes to be examined and the control tubes at exactly
pressure) of 40 kPa. The container empties in less than 2 min. 2500 g in the same horizontal centrifuge for 5 min. After
centrifuging, measure the absorbances (2.2.25) of the liquids
Speed of filling. Attach the container by means of the at 540 nm using the stock buffer solution as compensation
blood-taking tube fitted with the needle to a reservoir liquid. Calculate the haemolytic value as a percentage from
containing a suitable solution having a viscosity equal to the expression :
that of blood, such as a 335 g/L solution of sucrose R at
37 °C. Maintain the internal pressure of the reservoir (i.e.
the difference between the applied pressure and atmospheric
pressure) at 9.3 kPa with the base of the reservoir and the
upper part of the container at the same level. The volume of A100 = absorbance of tube III,
liquid which flows into the container in 8 min is not less than
Aexp = absorbance of tube I or II or of the corresponding
the nominal capacity of the container.
control tubes.
Resistance to temperature variations. Place the container
in a suitable chamber having an initial temperature of The solution in tube I gives a haemolytic value not greater
20-23 °C. Cool it rapidly in a deep-freeze to − 80 °C and than 10 per cent and the haemolytic value of the solution in
maintain it at this temperature for 24 h. Raise the temperature tube II does not differ by more than 10 per cent from that of
to 50 °C and maintain for 12 h. Allow to cool to room the corresponding control tube.
temperature. The container complies with the tests for Sterility (2.6.1). The containers comply with the test for
resistance to centrifugation, resistance to stretch, leakage, sterility. Introduce aseptically into the container 100 mL of
vapour permeability emptying under pressure and speed of a sterile 9 g/L solution of sodium chloride and shake the
filling prescribed above. container to ensure that the internal surfaces have been
entirely wetted. Filter the contents of the container through a hydroxide. The solution is pink. Add 0.8 mL of 0.01 M
membrane filter and place the membrane in the appropriate hydrochloric acid and 0.1 mL of methyl red solution R. The
culture medium, as prescribed in the test for sterility. solution is orange-red or red.
Pyrogens (2.6.8). Solution S1 complies with the test for Absorbance (2.2.25) : maximum 0.30, determined between
pyrogens. Inject 10 mL of the solution per kilogram of the wavelengths of 230 nm and 250 nm on solution S2 ; maximum
rabbit’s mass. 0.10, determined between wavelengths of 251 nm and 360 nm
Abnormal toxicity (2.6.9). Solution S1 complies with the test on solution S2. Use the reference solution as the compensation
for abnormal toxicity. Inject 0.5 mL of the solution into each liquid.
mouse. Oxidisable substances. Immediately after preparation of
solution S2 (see 3.2.3), transfer to a borosilicate-glass flask a
PACKAGING quantity corresponding to 8 per cent of the nominal capacity
The containers are packed in protective envelopes. of the container. At the same time, prepare a blank using an
On removal from its protective envelope the container shows equal volume of the freshly prepared reference solution in
no leakage and no growth of micro-organisms. The protective another borosilicate-glass flask. To each solution add 20.0 mL
envelope is sufficiently robust to withstand normal handling. of 0.002 M potassium permanganate and 1 mL of dilute sulfuric
acid R. Allow to stand protected from light for 15 min. To
The protective envelope is sealed in such a manner that it each solution add 0.1 g of potassium iodide R. Allow to stand
cannot be opened and re-closed without leaving visible traces protected from light for 5 min and titrate immediately with
that the seal has been broken. 0.01 M sodium thiosulfate, using 0.25 mL of starch solution R
as indicator. The difference between the 2 titrations is not
LABELLING more than 2.0 mL.
The labelling complies with the relevant national legislation
Extractable di(2-ethylhexyl) phthalate. Extraction solvent,
and international agreements. The label states :
ethanol (96 per cent) R diluted with water R to have a
– the name and address of the manufacturer, relative density (2.2.5) of 0.9389 to 0.9395, measured with a
– a batch number which enables the history of the container pycnometer.
and of the plastic material of which it is manufactured to Stock solution. Dissolve 0.100 g of di(2-ethylhexyl) phthalate R
be traced. in the extraction solvent and dilute to 100.0 mL with the same
A part of the label is reserved for : solvent.
– the statement of the blood group, the reference number and Standard solutions. Into 5 separate 100 mL volumetric flasks,
all other information required by national legislation or introduce respectively 1.0 mL, 2.0 mL, 5.0 mL, 10.0 mL
international agreements, and an empty space is provided and 20.0 mL of stock solution and dilute to 100.0 mL with
for the insertion of supplementary labelling. extraction solvent.
The label of the protective envelope or the label on the Measure the absorbances (2.2.25) of the standard solutions
container, visible through the envelope, states : at the absorption maximum at 272 nm, using the extraction
solvent as compensation liquid and plot a curve of absorbance
– the expiry date, against the concentration of di(2-ethylhexyl) phthalate.
– that, once withdrawn from its protective envelope, the Extraction procedure. Using the donor tubing and the needle
container must be used within 10 days. or adaptor, fill the empty container with a volume equal
The ink or other substance used to print the labels or the to half the nominal volume with the extraction solvent,
writing must not diffuse into the plastic material of the previously heated to 37 °C in a well-stoppered flask. Expel the
container and must remain legible up to the time of use. air completely from the container and seal the donor tube.
Immerse the filled container in a horizontal position in a
water-bath maintained at 37 ± 1 °C for 60 ± 1 min without
shaking. Remove the container from the water-bath, invert
01/2008:30204 it gently 10 times and transfer the contents to a glass flask.
Immediately measure the absorbance at the maximum at
3.2.4. EMPTY STERILE CONTAINERS 272 nm, using the extraction solvent as compensation liquid.
Determine the concentration of di(2-ethylhexyl) phthalate in
OF PLASTICISED POLY(VINYL milligrams per 100 mL of extract from the calibration curve.
CHLORIDE) FOR HUMAN BLOOD The concentration does not exceed :
AND BLOOD COMPONENTS – 10 mg per 100 mL for containers of nominal volume greater
than 300 mL but not greater than 500 mL ;
Unless otherwise authorised as described under Sterile plastic – 13 mg per 100 mL for containers of nominal volume greater
containers for human blood and blood components (3.2.3), than 150 mL but not greater than 300 mL ;
the nature and composition of the material from which – 14 mg per 100 mL for containers of nominal volume up
the containers are made comply with the requirements for to 150 mL.
Materials based on plasticised poly(vinyl chloride) for containers
for human blood and blood components and for containers for Chlorides (2.4.4) : maximum 0.4 ppm, determined with
aqueous solutions for intravenous infusion (3.1.1). solution S2.
Prepare the standard using a mixture of 1.2 mL of chloride
TESTS standard solution (5 ppm Cl) R and 13.8 mL of water R.
They comply with the tests prescribed for Sterile plastic Ammonium (2.4.1) : maximum 2 ppm.
containers for human blood and blood components (3.2.3) and Dilute 5 mL of solution S2 to 14 mL with water R.
with the following tests to detect extractable matter.
Residue on evaporation. Evaporate to dryness 100 mL
Reference solution. Heat water for injections R in a of solution S2 in a borosilicate-glass beaker of appropriate
borosilicate-glass flask in an autoclave at 110 °C for 30 min. capacity, previously heated to 105 °C. Evaporate to dryness in
Acidity or alkalinity. To a volume of solution S2 the same conditions 100 mL of the reference solution (blank
corresponding to 4 per cent of the nominal capacity of the test). Dry to constant mass at 100-105 °C. The residue from
container add 0.1 mL of phenolphthalein solution R. The solution S2 weighs a maximum of 3 mg, allowing for the blank
solution remains colourless. Add 0.4 mL of 0.01 M sodium test.
General Notices (1) apply to all monographs and other texts 417
3.2.5. Containers of plasticised PVC with anticoagulant solution EUROPEAN PHARMACOPOEIA 8.0
PACKAGING 01/2008:30206
See Sterile plastic containers for human blood and blood
components (3.2.3). 3.2.6. SETS FOR THE TRANSFUSION OF
BLOOD AND BLOOD COMPONENTS
LABELLING DEFINITION
See Sterile plastic containers for human blood and blood Sets for the transfusion of blood and blood components
components (3.2.3). consist principally of plastic tubing to which are fitted the
parts necessary to enable the set to be used for transfusion
in the appropriate manner. Sets include a closure-piercing
device, a blood filter, a drip chamber, a flow regulator, a Luer
connector and, usually, a site that allows an injection to be
01/2008:30205 made at the time of use. When the sets are to be used with
containers requiring an air-filter, this may be incorporated
in the closure-piercing device or a separate air-inlet device
3.2.5. STERILE CONTAINERS may be used. The chamber enclosing the blood filter, the drip
chamber and the main tubing are transparent. The materials
OF PLASTICISED POLY(VINYL chosen and the design of the set are such as to ensure absence
CHLORIDE) FOR HUMAN BLOOD of haemolytic effects. The sets comply with current standards
regarding dimensions and performance.
CONTAINING ANTICOAGULANT All parts of the set that may be in contact with blood and
SOLUTION blood components are sterile and pyrogen-free. Each set is
presented in an individual package that maintains the sterility
Sterile plastic containers containing an anticoagulant solution of the contents. The sets are not to be re-sterilised or re-used.
complying with the monographAnticoagulant and preservative Sets for the transfusion of blood and blood components
solutions for human blood (0209) are used for the collection, are manufactured in accordance with the rules of good
storage and administration of blood. Before filling they manufacturing practice for medical devices and any relevant
comply with the description and characters given under Empty national regulations.
sterile containers of plasticised poly(vinyl chloride) for human
blood and blood components (3.2.4). TESTS
Unless otherwise authorised as described under Sterile plastic Carry out the tests on sterilised sets.
containers for human blood and blood components (3.2.3), Solution S. Make a closed circulation system from 3 sets
the nature and composition of the material from which the and a 300 mL borosilicate-glass vessel. Fit to the vessel a
containers are made should comply with the requirements suitable thermostat device that maintains the temperature
prescribed for Materials based on plasticised poly(vinyl of the liquid in the vessel at 37 ± 1 °C. Circulate 250 mL of
chloride) for containers for human blood and blood components water for injections R through the system in the direction
and for containers for aqueous solutions for intravenous used for transfusion for 2 h at a rate of 1 L/h (for example
infusion (3.1.1). using a peristaltic pump applied to as short a piece of suitable
silicone elastomer tubing as possible). Collect the whole of
TESTS the solution and allow to cool.
Appearance of solution. Solution S is clear (2.2.1) and
They comply with the tests prescribed for Sterile plastic
colourless (2.2.2, Method II).
containers for human blood and blood components (3.2.3)
and with the following tests to measure the volume of Acidity or alkalinity. To 25 mL of solution S add 0.15 mL of
anticoagulant solution and to detect extractable matter. BRP indicator solution R. Not more than 0.5 mL of 0.01 M
sodium hydroxide is required to change the colour of the
Volume of anticoagulant solution. Empty the container, indicator to blue. To 25 mL of solution S add 0.2 mL of
collecting the anticoagulant solution in a graduated cylinder. methyl orange solution R. Not more than 0.5 mL of 0.01 M
The volume does not differ by more than ± 10 per cent from hydrochloric acid is required to reach the beginning of the
the stated volume. colour change of the indicator.
Spectrophotometric examination (2.2.25). Measure the Absorbance (2.2.25) : maximum 0.30, determined between
absorbance of the anticoagulant solution from the container wavelengths of 230 nm and 250 nm on solution S ; maximum
between 250 nm and 350 nm, using as the compensation liquid 0.15, determined between wavelengths of 251 nm and 360 nm
an anticoagulant solution of the same composition that has on solution S.
not been in contact with a plastic material. The absorbance at
the maximum at 280 nm is not greater than 0.5. Reducing substances. Carry out the test within 4 h of
preparation of solution S. To 20.0 mL of solution S add 1 mL
Extractable di(2-ethylhexyl) phthalate. Carefully remove of dilute sulfuric acid R and 20.0 mL of 0.002 M potassium
the anticoagulant solution by means of the flexible transfer permanganate. Boil for 3 min and cool immediately. Add
tube. Using a funnel fitted to the tube, completely fill the 1 g of potassium iodide R and titrate with 0.01 M sodium
container with water R, leave in contact for 1 min squeezing thiosulfate using 0.25 mL of starch solution R as indicator.
the container gently, then empty completely. Repeat the Carry out a blank test using 20 mL of water for injections R.
rinsing. The difference between the titration volumes is not greater
The container, so emptied and rinsed, complies with the test than 2.0 mL.
for extractable di(2-ethylhexyl) phthalate prescribed for Empty Ethylene oxide. Gas chromatography (2.2.28).
sterile plastic containers of plasticised poly(vinyl chloride) for Column :
human blood and blood components (3.2.4).
– material : stainless steel ;
– size : l = 1.5 m, Ø = 6.4 mm ;
PACKAGING AND LABELLING
– stationary phase : silanised diatomaceous earth for gas
See Sterile plastic containers for human blood and blood chromatography R impregnated with macrogol 1500 R (3 g
components (3.2.3). per 10 g).
Carrier gas : helium for chromatography R. a tank of water at 20-23 °C. Apply progressively an excess
Flow rate : 20 mL/min. pressure of 100 kPa and maintain for 1 min. No air bubble
escapes from the set.
Temperature :
Transparency. Use as reference suspension the primary
– column : 40 °C ; opalescent suspension (2.2.1) diluted 1 in 8 for sets having
– injection port : 100 °C ; tubing with an external diameter less than 5 mm and diluted
– detector : 150 °C. 1 in 16 for sets having tubing with an external diameter of
5 mm or greater. Circulate the reference suspension through
Detection : flame ionisation.
the set and compare with a set from the same batch filled
Verify the absence of peaks interfering with the ethylene with water R. The opalescence and presence of bubbles are
oxide peak by carrying out the test using an unsterilised set discernible.
or using the same chromatographic system with the following
modifications. Residue on evaporation. Evaporate 50.0 mL of solution S
to dryness on a water-bath and dry to constant mass in an
Column : oven at 100-105 °C. Carry out a blank test using 50.0 mL of
– size : l = 3 m, Ø = 3.2 mm ; water for injections R. The difference between the masses of
– stationary phase : silanised diatomaceous earth the residues is not greater than 1.5 mg.
for gas chromatography R impregnated with Sterility (2.6.1). The sets comply with the test for sterility. If
triscyanoethoxypropane R (2 g per 10 g) ; the sets are stated to be sterile only internally, pass 50 mL of
– temperature : 60 °C. buffered sodium chloride-peptone solution pH 7.0 (2.6.12)
through the set and use to carry out the test by the membrane
Ethylene oxide solution. Prepare in a fume cupboard. Place filtration method.
50.0 mL of dimethylacetamide R in a 50 mL vial, stopper,
secure the stopper and weigh to the nearest 0.1 mg. Fill a If the sets are stated to be sterile both internally and externally,
50 mL polyethylene or polypropylene syringe with gaseous open the package with the necessary aseptic precautions and :
ethylene oxide R, allow the gas to remain in contact with the – for the direct inoculation method, place the set or its
syringe for about 3 min, empty the syringe and fill again with components in a suitable container containing a sufficient
50 mL of gaseous ethylene oxide R. Fit a hypodermic needle to quantity of the culture medium to ensure complete
the syringe and reduce the volume of gas in the syringe from immersion ;
50 mL to 25 mL. Inject these 25 mL of ethylene oxide slowly – for the membrane filtration method, place the set or its
into the vial, shaking gently and avoiding contact between components in a suitable container containing a sufficient
the needle and the liquid. Weigh the vial again : the increase quantity of buffered sodium chloride-peptone solution
in mass is 45 mg to 60 mg and is used to calculate the exact pH 7.0 (2.6.12) to allow total rinsing for 10 min.
concentration of the solution (about 1 g/L). Pyrogens (2.6.8). Connect together 5 sets and pass through
Test. Weigh the set after removing the package. Cut the set into the assembly at a flow rate not exceeding 10 mL/min 250 mL
pieces of maximum dimension 1 cm and place the pieces in a of a sterile, pyrogen-free 9 g/L solution of sodium chloride R.
250-500 mL vial containing 150 mL of dimethylacetamide R. Collect the solution aseptically in a pyrogen-free container.
Close the vial with a suitable stopper and secure the stopper. The solution complies with the test for pyrogens. Inject per
Place the vial in an oven at 70 ± 1 °C for 16 h. Remove 1 mL of kilogram of the rabbit’s mass, 10 mL of the solution.
the hot gas from the vial and inject it onto the column. From
the calibration curve and the height of the peak obtained, LABELLING
calculate the mass of ethylene oxide in the vial. The label states, where applicable, that the set has been
Calibration curve. In a series of 7 vials of the same type sterilised using ethylene oxide.
as that used for the test and each containing 150 mL of
dimethylacetamide R, place respectively 0 mL, 0.05 mL, 01/2008:30208
0.10 mL, 0.20 mL, 0.50 mL, 1.00 mL and 2.00 mL of the
ethylene oxide solution, i.e. about 0 μg, 50 μg, 100 μg, 200 μg,
500 μg, 1000 μg and 2000 μg of ethylene oxide. Stopper the 3.2.8. STERILE SINGLE-USE PLASTIC
vials, secure the stoppers and place the vials in an oven at SYRINGES
70 ± 1 °C for 16 h. Inject 1 mL of the hot gas from each vial
onto the column and draw a calibration curve from the heights DEFINITION
of the peaks and the mass of ethylene oxide in each flask. Sterile single-use plastic syringes are medical devices intended
Limit : if the label states that ethylene oxide has been used for for immediate use for the administration of injectable
sterilisation : preparations. They are supplied sterile and pyrogen-free
and are not to be re-sterilised or re-used. They consist of a
– ethylene oxide : maximum 10 ppm.
syringe barrel and a piston which may have an elastomer
Extraneous particles. Fill the set via the normal inlet with a sealing ring ; they may be fitted with a needle which may be
0.1 g/L solution of sodium laurilsulfate R, previously filtered non-detachable. Each syringe is presented with individual
through a sintered-glass filter (16) (2.1.2) and heated to 37 °C. protection for maintaining sterility.
Collect the liquid via the normal outlet. When examined The barrel of the syringe is sufficiently transparent to permit
under suitable conditions of visibility, the liquid is clear and dosages to be read without difficulty and allow air bubbles and
practically free from visible particles and filaments (it is foreign particles to be discerned.
assumed that particles and filaments with a diameter equal to
or greater than 50 μm are visible to the naked eye). The plastics and elastomer materials of which the barrel and
piston are made comply with the appropriate specification
Flow rate. Pass through a complete set with the flow regulator or with the requirements of the competent authority. The
fully open 50 mL of a solution having a viscosity of 3 mPa·s most commonly used materials are polypropylene and
(3 cP) (for example a 33 g/L solution of macrogol 4000 R at polyethylene. The syringes comply with current standards
20 °C) under a static head of 1 m. The time required for regarding dimensions and performance.
passage of 50 mL of the solution is not greater than 90 s. Silicone oil (3.1.8) may be applied to the internal wall of the
Resistance to pressure. Make tight the extremities of the set barrel to assist in the smooth operation of the syringe but there
and any air-inlet device. Connect the set to a compressed remains no excess capable of contaminating the contents at
air outlet fitted with a pressure regulator. Immerse the set in the time of use. The inks, glues and adhesives for the marking
General Notices (1) apply to all monographs and other texts 419
3.2.8. Sterile single-use plastic syringes EUROPEAN PHARMACOPOEIA 8.0
on the syringe or on the package and, where necessary, the Test. Weigh the syringe after removing the package. Cut the
assembly of the syringe and its package, do not migrate across syringe into pieces of maximum dimension 1 cm and place
the walls. the pieces in a 250 mL to 500 mL vial containing 150 mL of
dimethylacetamide R. Close the vial with a suitable stopper
TESTS and secure the stopper. Place the vial in an oven at 70 ± 1 °C
Solution S. Prepare the solution in a manner that avoids for 16 h. Remove 1 mL of the hot gas from the vial and inject
contamination by foreign particles. Using a sufficient number it onto the column. From the calibration curve and the height
of syringes to produce 50 mL of solution, fill the syringes to of the peak obtained, calculate the mass of ethylene oxide in
their nominal volume with water for injections R and maintain the vial.
at 37 °C for 24 h. Combine the contents of the syringes in a Limit : if the label states that ethylene oxide has been used for
suitable borosilicate-glass container. sterilisation :
Appearance of solution. Solution S is clear (2.2.1) and – ethylene oxide : maximum 10 ppm.
colourless (2.2.2, Method II) and is practically free from Silicone oil. Calculate the internal surface area of a syringe in
foreign solid particles. square centimetres using the following expression :
Acidity or alkalinity. To 20 mL of solution S add 0.1 mL
of bromothymol blue solution R1. Not more than 0.3 mL · ·
of 0.01 M sodium hydroxide or 0.01 M hydrochloric acid is
required to change the colour of the indicator. V = nominal volume of the syringe, in cubic
centimetres ;
Absorbance (2.2.25) : maximum 0.40, determined between
wavelengths of 220 nm and 360 nm on solution S. h = height of the graduation, in centimetres.
Ethylene oxide. Gas chromatography (2.2.28). Take a sufficient number of syringes to give an internal surface
Column : area of 100 cm2 to 200 cm2. Aspirate into each syringe a
volume of methylene chloride R equal to half the nominal
– material : stainless steel ; volume and make up to the nominal volume with air. Rinse
– size : l = 1.5 m, Ø = 6.4 mm ; the internal surface corresponding to the nominal volume with
– stationary phase : silanised diatomaceous earth for gas the solvent by inverting the syringe ten times in succession
chromatography R impregnated with macrogol 1500 R (3 g with the needle fitting closed by a finger covered by a plastic
per 10 g). film inert to methylene chloride. Expel the extracts into a
Carrier gas : helium for chromatography R. tared dish and repeat the operation. Evaporate the combined
extracts to dryness on a water-bath. Dry at 100-105 °C for
Flow rate : 20 mL/min. 1 h. The residue weighs not more than 0.25 mg per square
Temperature : centimetre of internal surface area.
– Column : 40 °C ; Examine the residue by infrared absorption spectrophotometry
– Injection port : 100 °C ; (2.2.24). It shows absorption bands typical of silicone oil at
805 cm− 1, 1020 cm− 1, 1095 cm− 1, 1260 cm− 1 and 2960 cm− 1.
– Detector : 150 °C.
Detection : flame ionisation. Reducing substances. To 20.0 mL of solution S add
2 mL of sulfuric acid R and 20.0 mL of 0.002 M potassium
Verify the absence of peaks interfering with the ethylene oxide permanganate. Boil for 3 min. Cool immediately. Add 1 g
peak, either by carrying out the test using an unsterilised of potassium iodide R and titrate immediately with 0.01 M
syringe or using the same chromatographic system with the sodium thiosulfate using 0.25 mL of starch solution R as
following modifications : indicator. Carry out a blank titration using 20.0 mL of water
Column : for injections R. The difference between the titration volumes
– size : l = 3 m, Ø = 3.2 mm ; is not greater than 3.0 mL.
– stationary phase : silanised diatomaceous earth Transparency. Fill a syringe with water R (blank) and
for gas chromatography R impregnated with fill another with a 1 in 10 dilution of primary opalescent
triscyanoethoxypropane R (2 g per 10 g) ; suspension (2.2.1). Use primary opalescent suspension that
– temperature : 60 °C. has been allowed to stand at 20 ± 2 °C for 24 h before use.
Compare with the naked eye in diffused light against a dark
Ethylene oxide solution. Prepare in a fume cupboard. Place background. The opalescence of the suspension is detectable
50.0 mL of dimethylacetamide R in a 50 mL vial, stopper, when compared with the blank.
secure the stopper and weigh to the nearest 0.1 mg. Fill a
50 mL polyethylene or polypropylene syringe with gaseous Sterility (2.6.1). Syringes stated to be sterile comply with the
ethylene oxide R, allow the gas to remain in contact with the test for sterility carried out as follows. Using aseptic technique,
syringe for about 3 min, empty the syringe and fill again with open the package, withdraw the syringe, separate the
50 mL of gaseous ethylene oxide R. Fit a hypodermic needle to components and place each in a suitable container containing
the syringe and reduce the volume of gas in the syringe from sufficient culture media to cover the part completely. Use both
50 mL to 25 mL. Inject these 25 mL of ethylene oxide slowly the recommended media (2.6.1).
into the vial, shaking gently and avoiding contact between Syringes stated to be sterile only internally comply with the test
the needle and the liquid. Weigh the vial again : the increase for sterility carried out as follows. Use 50 mL of inoculation
in mass is 45 mg to 60 mg and is used to calculate the exact medium for each test syringe. Using aseptic technique, remove
concentration of the solution (about 1 g/L). the needle protector and submerge the needle in the culture
Calibration curve. In a series of seven vials of the same medium. Flush the syringe five times by withdrawing the
type as that used for the test and each containing 150 mL plunger to its fullest extent.
of dimethylacetamide R, place respectively 0 mL, 0.05 mL, Pyrogens (2.6.8). Syringes with a nominal volume equal to or
0.10 mL, 0.20 mL, 0.50 mL, 1.00 mL and 2.00 mL of the greater than 15 mL comply with the test for pyrogens. Fill a
ethylene oxide solution, i.e. about 0 μg, 50 μg, 100 μg, 200 μg, minimum of three syringes to their nominal volume with a
500 μg, 1000 μg and 2000 μg of ethylene oxide. Stopper the pyrogen-free 9 g/L solution of sodium chloride R and maintain
vials, secure the stoppers and place the vials in an oven at at a temperature of 37 °C for 2 h. Combine the solutions
70 ± 1 °C for 16 h. Inject 1 mL of the hot gas from each vial aseptically in a pyrogen-free container and carry out the test
onto the column and draw a calibration curve from the heights immediately. Inject per kilogram of the rabbit’s mass 10 mL of
of the peaks and the mass of ethylene oxide in each flask. the solution.
requirements as severe as for type I closures because of their 5.0 < A0 ≤ 10 (A0 − 1.0) to (A0 + 1.0)
chemical composition.
A0 > 10 (A0 − 2.0) to (A0 + 2.0)
The closures chosen for use with a particular preparation are
such that : In addition to the use of platinum and silica crucibles
– the components of the preparation in contact with the described in general chapter 2.4.16, porcelain crucibles may
closures are not adsorbed onto the surface of the closures be used. The sample may be ignited using a microwave
and do not migrate into or through the closures to an oven instead of a muffle furnace.
extent sufficient to affect the preparation adversely ;
TESTS
– the closures do not release substances in quantities
sufficient to affect the stability of the preparation or to The samples to be analysed may be washed and sterilised before
present a risk of toxicity. use.
The closures are compatible with the preparation for which Solution S. Place a number of uncut closures with a total
they are used throughout its period of validity. surface area of about 100 cm2 in a suitable glass container,
cover with water R, boil for 5 min and rinse 5 times with cold
The manufacturer of the preparation must obtain from the water R. Place the washed closures in a wide-necked flask
supplier an assurance that the composition of the closure (type I glass, 3.2.1), add 200 mL of water R and weigh. Cover
does not vary and that it is identical to that of the closure the mouth of the flask with a borosilicate-glass beaker. Heat
used during compatibility testing. If the supplier informs the in an autoclave so that a temperature of 121 ± 2 °C is reached
manufacturer of the preparation that changes have been made within 20-30 min and maintain at this temperature for 30 min.
to the composition, compatibility testing must be repeated, Cool to room temperature over about 30 min. Make up to
totally or partly, depending on the nature of the changes. the original mass with water R. Shake and decant the solution
The closures are washed and may be sterilised before use. immediately. Shake solution S before each test.
If using a tightly closed flask (type I glass, 3.2.1) with an
CHARACTERS inert closure instead of a wide-necked flask covered with a
Rubber closures are elastic. They are translucent or opaque borosilicate-glass beaker, it is not necessary to make up to the
and have no characteristic colour, the latter depending original mass.
on the additives used. They are practically insoluble in Blank solution. Prepare a blank solution in the same manner
tetrahydrofuran, in which, however, a considerable reversible using 200 mL of water R.
General Notices (1) apply to all monographs and other texts 421
3.2.9. Rubber closures for containers EUROPEAN PHARMACOPOEIA 8.0
Appearance of solution S. For type I closures, solution S is Volatile sulfides. Place closures, cut if necessary, with a total
not more opalescent than reference suspension II (2.2.1) and surface area of 20 ± 2 cm2 in a 100 mL conical flask and add
for type II closures, solution S is not more opalescent than 50 mL of a 20 g/L solution of citric acid R. Place a piece of
reference suspension III. Solution S is not more intensely lead acetate paper R over the mouth of the flask and maintain
coloured than reference solution GY5 (2.2.2, Method II). the paper in position by placing over it an inverted weighing
Acidity or alkalinity. To 20 mL of solution S add 0.1 mL bottle. Heat in an autoclave at 121 ± 2 °C for 30 min. Any black
of bromothymol blue solution R1. Not more than 0.3 mL of stain on the paper is not more intense than that of a standard,
0.01 M sodium hydroxide or 0.8 mL of 0.01 M hydrochloric treated in the same manner, prepared by mixing 50 mL of a
acid is required to change the colour of the indicator to blue 20 g/L solution of citric acid R and 5.0 mL of a freshly prepared
or yellow, respectively. 0.0308 g/L solution of sodium sulfide R in water R.
For the tests for penetrability, fragmentation and self-sealing,
Absorbance. Carry out the test within 5 h of preparation treat the closures as described for the preparation of solution S
of solution S. Filter solution S through a membrane filter and allow to dry.
(nominal pore size 0.45 μm), rejecting the first few millilitres
of filtrate. Measure the absorbance (2.2.25) of the filtrate at Penetrability. For closures intended to be pierced by a
wavelengths from 220-360 nm using the blank (see solution S) hypodermic needle, carry out the following test. Fill 10
as compensation liquid. At these wavelengths, the absorbance suitable vials to the nominal volume with water R, fit the
does not exceed 0.2 for type I closures or 4.0 for type II closures to be examined and secure with a cap. Using for each
closures. If necessary, dilute the filtrate before measurement closure a new, lubricated, long-bevel(1) (bevel angle 12 ± 2°)
of the absorbance and correct the result for the dilution. hypodermic needle with an external diameter of 0.8 mm,
pierce the closures with the needle perpendicular to the
Reducing substances. Carry out the test within 4 h of surface. The force required for piercing, determined with an
preparation of solution S. To 20.0 mL of solution S add accuracy of ± 0.25 N, is not greater than 10 N for each closure.
1 mL of dilute sulfuric acid R and 20.0 mL of 0.002 M
potassium permanganate. Boil for 3 min. Cool. Add 1 g Fragmentation. For closures intended to be pierced by a
of potassium iodide R and titrate immediately with 0.01 M hypodermic needle, carry out the following test. If the closures
sodium thiosulfate, using 0.25 mL of starch solution R as are to be used for aqueous preparations, introduce in 12
indicator. Carry out a titration using 20.0 mL of the blank. clean vials a volume of water R corresponding to the nominal
The difference between the titration volumes is not greater volume minus 4 mL, close the vials with the closures to be
than 3.0 mL for type I closures and 7.0 mL for type II closures. examined, secure with a cap and allow to stand for 16 h. If the
closures are to be used with dry preparations, close 12 clean
Ammonium (2.4.1, Method A) : maximum 2 ppm. vials with the closures to be examined. Using a lubricated,
Dilute 5 mL of solution S to 14 mL with water R. long-bevel(1) (bevel angle 12 ± 2°) hypodermic needle with
an external diameter of 0.8 mm fitted to a clean syringe,
Extractable zinc : maximum of 5 μg of extractable Zn per inject into the vial 1 mL of water R and remove 1 mL of air ;
millilitre of solution S. carry out this operation 4 times for each closure, piercing the
Atomic absorption spectrometry (2.2.23, Method I). closure each time at a different site. Use a new needle for each
closure and check that the needle is not blunted during the
Test solution. Dilute 10.0 mL of solution S to 100 mL with test. Pass the liquid in the vials through a filter with a pore
0.1 M hydrochloric acid. size of 0.5 μm. Count the fragments of rubber visible to the
Reference solutions. Prepare the reference solutions using naked eye. The total number of fragments does not exceed 5.
zinc standard solution (10 ppm Zn) R diluted with 0.1 M This limit is based on the assumption that fragments with
hydrochloric acid. a diameter equal to or greater than 50 μm are visible to the
naked eye ; in cases of doubt or dispute, the fragments are
Source : zinc hollow-cathode lamp. examined with a microscope to verify their nature and size.
Wavelength : 213.9 nm. Self-sealing test. For closures intended to be used with
multidose containers, carry out the following test. Fill
Atomisation device : air-acetylene flame. 10 suitable vials to the nominal volume with water R, fit the
Extractable heavy metals (2.4.8) : maximum 2 ppm. closures to be examined and secure with a cap. Using for each
closure a new hypodermic needle with an external diameter of
Solution S complies with test A. Prepare the reference solution 0.8 mm, pierce each closure 10 times, piercing the closure each
using lead standard solution (2 ppm Pb) R. time at a different site. Immerse the vials upright in a 1 g/L
Residue on evaporation. Evaporate 50.0 mL of solution S to solution of methylene blue R and reduce the external pressure
dryness on a water-bath and dry at 100-105 °C. The residue by 27 kPa for 10 min. Restore atmospheric pressure and leave
weighs not more than 2.0 mg for type I closures and not more the vials immersed for 30 min. Rinse the outside of the vials.
than 4.0 mg for type II closures. None of the vials contains any trace of coloured solution.
(1) See ISO 7864 "Sterile hypodermic needles for single use".
General Notices (1) apply to all monographs and other texts 425
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Acetic anhydride solution R1. 1000501. N-Acetyl-ε-caprolactam. C8H13NO2. (Mr 155.2). 1102700.
Dissolve 25.0 mL of acetic anhydride R in anhydrous [1888-91-1]. N-Acetylhexane-6-lactam.
pyridine R and dilute to 100.0 mL with the same solvent. Colourless liquid, miscible with anhydrous ethanol.
Storage : protected from light and air. : about 1.100.
Acetic anhydride - sulfuric acid solution. 1000502. : about 1.489.
Carefully mix 5 mL of acetic anhydride R with 5 mL of bp : about 135 °C.
sulfuric acid R. Add dropwise and with cooling to 50 mL of Acetyl chloride. C H ClO. (M 78.5). 1000800. [75-36-5].
anhydrous ethanol R. 2 3 r
Clear, colourless liquid, flammable, decomposes in contact
Prepare immediately before use.
with water and with ethanol (96 per cent), miscible with
Acetone. 1000600. [67-64-1]. ethylene chloride.
See Acetone (0872). : about 1.10.
Acetonitrile. C2H3N. (Mr 41.05). 1000700. [75-05-8]. Methyl Distillation range (2.2.11). Not less than 95 per cent distils
cyanide. Ethanenitrile. between 49 °C and 53 °C.
Clear, colourless liquid, miscible with water, with acetone and Acetylcholine chloride. C7H16ClNO2. (Mr 181.7). 1001000.
with methanol. [60-31-1].
: about 0.78. Crystalline powder, very soluble in cold water and in ethanol
: about 1.344. (96 per cent). It decomposes in hot water and in alkalis.
A 100 g/L solution is neutral to litmus paper. Storage : at − 20 °C.
Distillation range (2.2.11). Not less than 95 per cent distils Acetyleugenol. C12H14O3. (Mr 206.2). 1100700. [93-28-7].
between 80 °C and 82 °C. 2-Methoxy-4-(2-propenyl)phenylacetate.
Acetonitrile used in spectrophotometry complies with the Yellow coloured, oily liquid, practically insoluble in water,
following additional test. freely soluble in ethanol (96 per cent).
Minimum transmittance (2.2.25) using water R as
: about 1.521.
compensation liquid : 98 per cent from 255 nm to 420 nm.
bp : 281 °C to 282 °C.
Acetonitrile for chromatography. 1000701. Acetyleugenol used in gas chromatography complies with the
See Acetonitrile R. following additional test.
Acetonitrile used in chromatography complies with the Assay. Gas chromatography (2.2.28) as prescribed in the
following additional tests. monograph Clove oil (1091).
Minimum transmittance (2.2.25) using water R as Test solution. The substance to be examined.
compensation liquid : 98 per cent from 240 nm. Content : minimum 98.0 per cent, calculated by the
Content (2.2.28) : minimum 99.8 per cent. normalisation procedure.
Acetonitrile R1. 1000702. N-Acetylglucosamine. C8H15NO6. (Mr 221.2). 1133600.
Complies with the requirements prescribed for acetonitrile R [7512-17-6]. 2-(Acetylamino)-2-deoxy-D-glucopyranose.
and with the following additional requirements. mp : about 202 °C.
Content : minimum 99.9 per cent.
Absorbance (2.2.25) : maximum 0.10, determined at 200 nm Acetyl-11-keto-β-boswellic acid. C32H48O5. (Mr 512.7).
using water R as the compensation liquid. 1167700. [67416-61-9]. 3α-(Acetyloxy)-11-oxours-12-en-24-
oic acid. (4β)-3α-(Acetyloxy)-11-oxours-12-en-23-oic acid.
Acetoxyvalerenic acid. C17H24O4. (Mr 292.4). 1165800. White or almost white powder, insoluble in water, soluble in
[81397-67-3]. (2E)-3-[(1RS,4S,7R,7aR)-1-(Acetyloxy)- acetone, in anhydrous ethanol and in methanol.
3,7-dimethyl-2,4,5,6,7,7a-hexahydro-1H-inden-4-yl]-2-
methylprop-2-enoic acid. mp : 271 °C to 274 °C.
Colourless or pale yellow viscous oil. Acetyl-11-keto-β-boswellic acid used in liquid chromatography
complies with the following additional test.
Absorbance (2.2.25). A solution in methanol R shows an
absorption maximum at about 216 nm. Assay. Liquid chromatography (2.2.29) as prescribed in the
monograph on Indian frankincense (2310).
Acetylacetamide. C4H7NO2. (Mr 101.1). 1102600. Content : minimum 90 per cent, calculated by the
[5977-14-0]. 3-Oxobutanamide. normalisation procedure.
mp : 53 °C to 56 °C.
N-Acetylneuraminic acid. C11H19NO9. (Mr 309.3). 1001100.
Acetylacetone. C5H8O2. (Mr 100.1). 1000900. [123-54-6]. [131-48-6]. O-Sialic acid.
2,4-Pentanedione. White or almost white acicular crystals, soluble in water and
Colourless or slightly yellow, easily flammable liquid, freely in methanol, slightly soluble in anhydrous ethanol, practically
soluble in water, miscible with acetone, with ethanol (96 per insoluble in acetone.
cent) and with glacial acetic acid. : about − 36, determined on a 10 g/L solution.
: 1.452 to 1.453. mp : about 186 °C, with decomposition.
bp : 138 °C to 140 °C.
N-Acetyltryptophan. C13H14N2O3. (Mr 246.3). 1102800.
Acetylacetone reagent R1. 1000901. [1218-34-4]. 2-Acetylamino-3-(indol-3-yl)propanoic acid.
To 100 mL of ammonium acetate solution R add 0.2 mL of White or almost white powder or colourless crystals, slightly
acetylacetone R. soluble in water. It dissolves in dilute solutions of alkali
Acetylacetone reagent R2. 1000902. hydroxides.
Dissolve 0.2 mL of acetylacetone R, 3 mL of glacial acetic mp : about 205 °C.
acid R and 25 g of ammonium acetate R in water R and Assay. Liquid chromatography (2.2.29) as prescribed in the
dilute to 100 mL with the same solvent. monograph Tryptophan (1272).
Test solution. Dissolve 10.0 mg in a mixture of 10 volumes α1-Acid-glycoprotein silica gel for chiral separation.
of acetonitrile R and 90 volumes of water R and dilute to 1148700.
100.0 mL with the same mixture of solvents. A very finely divided silica gel for chromatography consisting
Content : minimum 99.0 per cent, calculated by the of spherical particles coated with α1-acid glycoprotein. The
normalisation procedure. particle size is indicated after the name of the reagent in the
tests where it is used.
Acetyltyrosine ethyl ester. C13H17NO4,H2O. (Mr 269.3).
1001200. [36546-50-6]. N-Acetyl-L-tyrosine ethyl Acrylamide. C3H5NO. (Mr 71.1). 1001500. [79-06-1].
ester monohydrate. Ethyl (S)-2-acetamido-3-(4- Propenamide.
hydroxyphenyl)propionate monohydrate.
Colourless or white flakes or a white or almost white,
White or almost white, crystalline powder suitable for the crystalline powder, very soluble in water and in methanol,
assay of chymotrypsin. freely soluble in anhydrous ethanol.
: + 21 to + 25, determined on a 10 g/L solution in ethanol mp : about 84 °C.
(96 per cent) R.
: 60 to 68, determined at 278 nm in ethanol (96 per 30 per cent acrylamide/bisacrylamide (29:1) solution.
cent) R. 1001501.
Acetyltyrosine ethyl ester, 0.2 M. 1001201. Prepare a solution containing 290 g of acrylamide R and
10 g of methylenebisacrylamide R per litre of water R. Filter.
Dissolve 0.54 g of acetyltyrosine ethyl ester R in ethanol
(96 per cent) R and dilute to 10.0 mL with the same solvent. 30 per cent acrylamide/bisacrylamide (36.5:1) solution.
1001502.
Acid blue 83. C45H44N3NaO7S2. (Mr 826). 1012200.
[6104-59-2]. Prepare a solution containing 292 g of acrylamide R and 8 g
Colour Index No. 42660. of methylenebisacrylamide R per litre of water R. Filter.
Brilliant blue. Coomassie brilliant blue R 250. Acrylic acid. C3H4O2. (Mr 72.1). 1133700. [79-10-7].
Brown powder insoluble in cold water, slightly soluble in Prop-2-enoic acid. Vinylformic acid.
boiling water and in anhydrous ethanol, soluble in sulfuric
Content : minimum 99 per cent.
acid, glacial acetic acid and in dilute solutions of alkali
hydroxides. It is stabilised with 0.02 per cent of hydroquinone monomethyl
ether.
Acid blue 90. C47H48N3NaO7S2. (Mr 854). 1001300.
[6104-58-1]. Corrosive liquid, miscible with water and ethanol (96 per
cent). It polymerises readily in the presence of oxygen.
Colour Index No. 42655.
Sodium [4-[[4-[(4-ethoxyphenyl)amino]phenyl][[4-(ethyl)(3- : about 1.05.
sulfonatobenzyl)amino]phenyl]methylene]cyclo-hexa-2,5- : about 1.421.
dien-1-ylidene](ethyl)-(3-sulfonatobenzyl)ammonium. bp : about 141 °C.
A dark brown powder, with a violet sheen and some particles mp : 12 °C to 15 °C.
having a metallic lustre, soluble in water and in anhydrous
ethanol. Actein. C37H56O11. (Mr 677). 1181500. [18642-44-9].
: greater than 500, determined at 577 nm in a 0.01 g/L (23R,24R,25S,26S)-3β-(β-D-Xylopyranosyloxy)-
solution in buffer solution pH 7.0 and calculated with 16β,23:23,26:24,25-triepoxy-26-hydroxy-9,19-cyclolanostan-
reference to the dried substance. 12β-yl acetate.
Loss on drying (2.2.32) : maximum 5.0 per cent, determined
on 0.500 g by drying in an oven at 105 °C. Acteoside. C29H36O15. (Mr 624.6). 1145100.
[61276-17-3]. 2-(3,4-Dihydroxyphenyl)ethyl
Acid blue 92. C26H16N3Na3O10S3. (Mr 696). 1001400. 3-O-(6-deoxy-α-L-mannopyranosyl)-4-O-[(2E)-3-
[3861-73-2]. (3,4-dihydroxyphenyl)prop-2-enoyl]-β-D-glucopyranoside.
Colour Index No. 13390. Verbascoside.
Coomassie blue. Anazolene sodium. Trisodium 8-hydroxy-4′- Light yellowish powder, freely soluble in water and in
(phenylamino)azonaphthalene-3,5′,6-trisulfonate. methanol.
Dark blue crystals, soluble in water, in acetone and in ethylene mp : about 140 °C, with decomposition.
glycol monoethylether, slightly soluble in ethanol (96 per
cent). Adamantane. C10H16. (Mr 136.2). 1181600. [281-23-2].
Tricyclo[3.3.1.13,7]decane.
Acid blue 92 solution. 1001401.
mp : about 270 °C.
Dissolve 0.5 g of acid blue 92 R in a mixture of 10 mL of
glacial acetic acid R, 45 mL of ethanol (96 per cent) R and Adenine. 1172800. [73-24-5].
45 mL of water R.
See Adenine (0800).
Acid blue 93. C37H27N3Na2O9S3. (Mr 800). 1134200.
[28983-56-4]. Adenosine. C10H13N5O4. (Mr 267.2). 1001600. [58-61-7].
6-Amino-9-β-D-ribofuranosyl-9H-purine.
Colour Index No. 42780.
Methyl blue. Poirrier blue. White or almost white, crystalline powder, slightly soluble in
Mixture of triphenylrosaniline di- and trisulfonate and of water, practically insoluble in acetone and in ethanol (96 per
triphenylpararosaniline. cent). It dissolves in dilute solutions of acids.
Dark blue powder. mp : about 234 °C.
Colour change : pH 9.4 to pH 14.0. Adipic acid. C6H10O4. (Mr 146.1). 1095600. [124-04-9].
Acid blue 93 solution. 1134201. Prisms, freely soluble in methanol, soluble in acetone,
Dissolve 0.2 g of acid blue 93 R in water R and dilute to practically insoluble in light petroleum.
100 mL with the same solvent. mp : about 152 °C.
General Notices (1) apply to all monographs and other texts 427
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Adrenaline. C9H13NO3. (Mr 183.2). 1155000. [51-43-4]. Agarose for electrophoresis. 1002000. [9012-36-6].
(1R)-1-(3,4-Dihydroxyphenyl)-2-(methylamino)ethanol. A neutral, linear polysaccharide, the main component of
4-[(1R)-1-hydroxy-2-(methylamino)ethyl]benzene-1,2-diol. which is derived from agar.
White or almost white powder, gradually becoming brown on White or almost white powder, practically insoluble in cold
exposure to light and air, very slightly soluble in water and water, very slightly soluble in hot water.
in ethanol (96 per cent), insoluble in acetone. It dissolves in
dilute solutions of mineral acids and alkali hydroxides. Agnuside. C22H26O11. (Mr 466.4). 1162000.
mp : about 215 °C. [11027-63-7]. (1RS,4aSR,5RS,7aRS)-5-Hydroxy-
7-[[(4-hydroxybenzoyl)oxy]methyl]-1,4a,5,7a-
Adrenalone hydrochloride. C9H12ClNO3. (Mr 217.7). tetrahydrocyclopenta[c]pyran-1-yl β-D-glucopyranoside.
1155100. [62-13-5]. 1-(3,4-Dihydroxyphenyl)-2- White or almost white crystals.
(methylamino)ethanone hydrochloride. 3′,4′-Dihydroxy-2-
(methylamino)acetophenone hydrochloride. Alanine. 1102900. [56-41-7].
Pale yellow crystals, freely soluble in water, soluble in ethanol See Alanine (0752).
(96 per cent).
β-Alanine. 1004500. [107-95-9].
mp : about 244 °C.
See 3-aminopropionic acid R.
Aescin. 1001700. [6805-41-0].
Albumin, bovine. 1002300. [9048-46-8].
A mixture of related saponins obtained from the seeds of
Aesculus hippocastanum L. Bovine serum albumin containing about 96 per cent of protein.
Fine, almost white or slightly reddish or yellowish, amorphous White to light brownish-yellow powder.
powder. Water (2.5.12) : maximum 3.0 per cent, determined on 0.800 g.
Chromatography. Thin-layer chromatography (2.2.27) as Albumin, bovine R1. 1183500. [9048-46-8].
prescribed in the monograph Senega root (0202) : apply 20 μL Bovine serum albumin containing about 96 per cent of protein.
of the solution ; after spraying with anisaldehyde solution R
and heating, the chromatogram shows a principal band with White or light brownish-yellow powder.
an RF of about 0.4. Albumin, human. 1133800.
Aflatoxin B1. C17H12O6. (Mr 312.3). 1166000. Human serum albumin containing not less than 96 per cent
[1162-65-8]. (6aR,9aS)-4-Methoxy-2,3,6a,9a-tetrahydro- of albumin.
cyclopenta[c]furo[3′,2′:4,5]furo[2,3-h][1]benzopyran-1,11-
dione. Albumin solution, human. 1002400. [9048-46-8].
White or faint yellow crystals. See Human albumin solution (0255).
Alizarin S. C14H7NaO7S,H2O. (Mr 360.3). 1002600. [130-22-3]. Americium-243 spiking solution. 1167500.
Schultz No. 1145. Contains 50 Bq/L 243Am and a 134 g/L solution of lanthanum
Colour Index No. 58005. chloride heptahydrate R in a 103 g/L solution of hydrochloric
Sodium 1,2-dihydroxyanthraquinone-3-sulfonate acid R.
monohydrate. Sodium 3,4-dihydroxy-9,10-dioxo-9,10-
Amido black 10B. C22H14N6Na2O9S2. (Mr 617). 1003100.
dihydroanthracene-2-sulfonate monohydrate.
[1064-48-8].
Orange-yellow powder, freely soluble in water and in ethanol
(96 per cent). Schultz No. 299.
Colour Index No. 20470.
Alizarin S solution. 1002601. Disodium 5-amino-4-hydroxy-6-[(4-nitrophenyl)azo]-3-
A 1 g/L solution. (phenylazo)naphthalene-2,7-disulfonate.
Test for sensitivity. If alizarin S solution is used for the Dark-brown to black powder, sparingly soluble in water,
standardisation of 0.05 M barium perchlorate, it shows soluble in ethanol (96 per cent).
a colour change from yellow to orange-red when it is
tested according to the standardisation of 0.05 M barium Amido black 10B solution. 1003101.
perchlorate. A 5 g/L solution of amido black 10B R in a mixture of
Colour change : pH 3.7 (yellow) to pH 5.2 (violet). 10 volumes of acetic acid R and 90 volumes of methanol R.
Alovudine. C10H13FN2O4. (Mr 244.2). 1185400. [25526-93-6]. Aminoazobenzene. C12H11N3. (Mr 197.2). 1003200.
1-[(2R,4S,5R)-4-Fluoro-5-(hydroxymethyl)tetrahydrofuran- [60-09-3].
2-yl]-5-methylpyrimidine-2,4(1H,3H)-dione. Colour Index No. 11000.
Fluorodeoxythymidine. 3′-Deoxy-3′-fluorothymidine. 4-(Phenylazo)aniline.
Content : minimum 95 per cent. Brownish-yellow needles with a bluish tinge, slightly soluble
Colourless crystals. in water, freely soluble in ethanol (96 per cent).
mp : about 128 °C.
Aluminium. Al. (Ar 26.98). 1118200. [7429-90-5].
White or almost white, malleable, flexible, bluish metal, 2-Aminobenzoic acid. C7H7NO2. (Mr 137.1). 1003400.
available as bars, sheets, powder, strips or wire. In moist air an [118-92-3]. Anthranilic acid.
oxide film forms which protects the metal from corrosion. A white or pale-yellow, crystalline powder, sparingly soluble
Analytical grade. in cold water, freely soluble in hot water, in ethanol (96 per
cent) and in glycerol. Solutions in ethanol (96 per cent) or in
Aluminium chloride. AlCl3,6H2O. (Mr 241.4). 1002700. ether and, particularly, in glycerol show a violet fluorescence.
[7784-13-6]. Aluminium chloride hexahydrate.
mp : about 145 °C.
Content : minimum 98.0 per cent of AlCl3,6H2O.
White or slightly yellowish, crystalline powder, hygroscopic, 3-Aminobenzoic acid. C7H7NO2. (Mr 137.1). 1147400.
freely soluble in water and in ethanol (96 per cent). [99-05-8].
Storage : in an airtight container. White or almost white crystals. An aqueous solution turns
brown on standing in air.
Aluminium chloride reagent. 1002702.
mp : about 174 °C.
Dissolve 2.0 g of aluminium chloride R in 100 mL of a 5 per
cent V/V solution of glacial acetic acid R in methanol R. Storage : in an airtight container, protected from light.
Aluminium chloride solution. 1002701. 4-Aminobenzoic acid. C7H7NO2. (Mr 137.1). 1003300.
[150-13-0].
Dissolve 65.0 g of aluminium chloride R in water R and
dilute to 100 mL with the same solvent. Add 0.5 g of White or almost white, crystalline powder, slightly soluble
activated charcoal R, stir for 10 min, filter and add to the in water, freely soluble in ethanol (96 per cent), practically
filtrate, with continuous stirring, sufficient of a 10 g/L insoluble in light petroleum.
solution of sodium hydroxide R (about 60 mL) to adjust mp : about 187 °C.
the pH to about 1.5. Chromatography. Thin-layer chromatography (2.2.27) as
Aluminium nitrate. Al(NO3)3,9H2O. (Mr 375.1). 1002800. prescribed in the monograph Procaine hydrochloride (0050) ;
[7784-27-2]. Aluminium nitrate nonahydrate. the chromatogram shows only one principal spot.
Crystals, deliquescent, very soluble in water and ethanol Storage : protected from light.
(96 per cent), very slightly soluble in acetone. 4-Aminobenzoic acid solution. 1003301.
Storage : in an airtight container. Dissolve 1 g of 4-aminobenzoic acid R in a mixture of 18 mL
Aluminium oxide, anhydrous. 1002900. [1344-28-1]. of anhydrous acetic acid R, 20 mL of water R and 1 mL of
Aluminium oxide, consisting of γ-Al2O3, dehydrated and phosphoric acid R. Immediately before use, mix 2 volumes
activated by heat treatment. of the solution with 3 volumes of acetone R.
Particle size : 75 μm to 150 μm. N-(4-Aminobenzoyl)- L-glutamic acid. C12H14N2O5.
(Mr 266.3). 1141700. [4271-30-1]. ABGA. (2S)-2-[(4-
Aluminium oxide, basic. 1118300. Aminobenzoyl)amino]pentanedioic acid.
A basic grade of anhydrous aluminium oxide R suitable for White or almost white, crystalline powder.
column chromatography.
mp : about 175 °C, with decomposition.
pH (2.2.3). Shake 1 g with 10 mL of carbon dioxide-free
water R for 5 min. The pH of the suspension is 9 to 10. 4-Aminobutanoic acid. C4H9NO2. (Mr 103.1). 1123200.
Aluminium oxide, neutral. 1118400. [56-12-2]. γ-Aminobutyric acid. GABA.
See Aluminium oxide, hydrated (0311). Leaflets from methanol and ether, needles from water and
ethanol (96 per cent). Freely soluble in water, practically
Aluminium potassium sulfate. 1003000. [7784-24-9]. insoluble or slightly soluble in other solvents.
See Alum (0006). mp : about 202 °C (decreases on rapid heating).
General Notices (1) apply to all monographs and other texts 429
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
cis-Aminoindanol. C9H11NO. (Mr 149.2). 1168300. 2-Aminophenol. C6H7NO. (Mr 109.1). 1147500. [95-55-6].
[126456-43-7]. (1S,2R)-1-Amino-2,3-dihydro-1H-inden-2-ol. Pale yellowish-brown crystals which rapidly become brown,
(−)-cis-1-Aminoindan-2-ol. sparingly soluble in water, soluble in ethanol (96 per cent).
Content : minimum 98.0 per cent (sum of enantiomers, mp : about 172 °C.
determined by gas chromatography). Storage : in an airtight container, protected from light.
: − 69 to − 59, determined on a 2 g/L solution in 3-Aminophenol. C6H7NO. (Mr 109.1). 1147600. [591-27-5].
chloroform R. Pale yellowish-brown crystals, sparingly soluble in water.
mp : 118 °C to 122 °C. mp : about 122 °C.
4-Aminophenol. C6H7NO. (Mr 109.1). 1004300. [123-30-8]. Ammonia, dilute R1. 1004702.
Content : minimum 95 per cent. Content : 100 g/L to 104 g/L of NH3 (Mr 17.03).
White or slightly coloured, crystalline powder, becoming Dilute 41 g of concentrated ammonia R to 100 mL with
coloured on exposure to air and light, sparingly soluble in water R.
water, soluble in anhydrous ethanol. Ammonia, dilute R2. 1004703.
mp : about 186 °C, with decomposition. Content : 33 g/L to 35 g/L of NH3 (Mr 17.03).
Storage : protected from light. Dilute 14 g of concentrated ammonia R to 100 mL with
water R.
Aminopolyether. C18H36N2O6. (Mr 376.5). 1112500.
[23978-09-8]. 4,7,13,16,21,24-hexaoxa-1,10- Ammonia, dilute R3. 1004704.
diazabicyclo[8,8,8]hexacosane. Content : 1.6 g/L to 1.8 g/L of NH3 (Mr 17.03).
mp : 70 °C to 73 °C. Dilute 0.7 g of concentrated ammonia R to 100 mL with
water R.
3-Aminopropanol. C3H9NO. (Mr 75.1). 1004400. [156-87-6].
3-Aminopropan-1-ol. Propanolamine. Ammonia, dilute R4. 1004706.
Clear, colourless, viscous liquid. Content : 8.4 g/L to 8.6 g/L of NH3 (Mr 17.03).
: about 0.99. Dilute 3.5 g of concentrated ammonia R to 100 mL with
water R.
: about 1.461.
mp : about 11 °C. Ammonia, lead-free. 1004705.
Complies with the requirements prescribed for dilute
3-Aminopropionic acid. C3H7NO2. (Mr 89.1). 1004500. ammonia R1 with the following additional test : to 20 mL
[107-95-9]. β-Alanine. of lead-free ammonia, add 1 mL of lead-free potassium
Content : minimum 99 per cent. cyanide solution R, dilute to 50 mL with water R and add
0.10 mL of sodium sulfide solution R. The solution is not
White or almost white, crystalline powder, freely soluble in more intensely coloured than a reference solution prepared
water, slightly soluble in ethanol (96 per cent), practically without sodium sulfide.
insoluble in acetone.
mp : about 200 °C, with decomposition. Ammonia, concentrated R1. 1004800.
Content : minimum 32.0 per cent m/m of NH3 (Mr 17.03).
Aminopyrazolone. C11H13N3O. (Mr 203.2). 1004600. A clear, colourless liquid.
[83-07-8]. 4-Amino-2,3-dimethyl-1-phenylpyrazolin-5-one.
: 0.883 to 0.889.
Light-yellow needles or powder, sparingly soluble in water, Assay. Weigh accurately a ground-glass-stoppered flask
freely soluble in ethanol (96 per cent). containing 50.0 mL of 1 M hydrochloric acid. Introduce 2 mL
mp : about 108 °C. of the concentrated ammonia and weigh again. Titrate the
solution with 1 M sodium hydroxide, using 0.5 mL of methyl
Aminopyrazolone solution. 1004601. red mixed solution R as indicator.
A 1 g/L solution in buffer solution pH 9.0 R. 1 mL of 1 M hydrochloric acid is equivalent to 17.03 mg of
NH3.
3-Aminosalicylic acid. C7H7NO3. (Mr 153.1). 1183600.
[570-23-0]. 3-Amino-2-hydroxybenzoic acid. Storage : protected from atmospheric carbon dioxide, at a
temperature below 20 °C.
mp : about 240 °C.
Slightly soluble in water. Ammonium acetate. C2H7NO2. (Mr 77.1). 1004900.
[631-61-8].
4-Aminosalicylic acid. C7H7NO3. (Mr 153.1). 1183700. Colourless crystals, very deliquescent, very soluble in water
[65-49-6]. 4-Amino-2-hydroxybenzoic acid. and in ethanol (96 per cent).
White or almost white, bulky powder, slightly soluble in water, Storage : in an airtight container.
soluble in ethanol (96 per cent), in dilute nitric acid and in Ammonium acetate solution. 1004901.
sodium hydroxide. It darkens on exposure to air and light.
Dissolve 150 g of ammonium acetate R in water R. Add
mp : 135 °C to 145 °C. 3 mL of glacial acetic acid R and dilute to 1000 mL with
Storage : at a temperature not exceeding 30 °C, in an airtight water R.
container, protected from light. Storage : use within 1 week.
Ammonia, concentrated. 1004700. Ammonium and cerium nitrate. (NH4)2Ce(NO3)6.
See Concentrated ammonia solution (0877). (Mr 548.2). 1005000. [16774-21-3].
Orange-yellow, crystalline powder, or orange transparent
Ammonia. 1004701. crystals, soluble in water.
Content : 170 g/L to 180 g/L of NH3 (Mr 17.03). Ammonium and cerium sulfate. (NH4)4Ce(SO4)4,2H2O.
Dilute 67 g of concentrated ammonia R to 100 mL with (Mr 633). 1005100. [10378-47-9].
water R. Orange-yellow, crystalline powder or crystals, slowly soluble
: 0.931 to 0.934. in water.
When used in the test for iron, ammonia R complies with (1R)-(–)-Ammonium 10-camphorsulfonate. C10H19NO4S.
the following additional requirement. Evaporate 5 mL of (Mr 249.3). 1103200.
ammonia to dryness on a water-bath, add 10 mL of water R,
Content : minimum 97.0 per cent of (1R)-(–)-ammonium
2 mL of a 200 g/L solution of citric acid R and 0.1 mL of
10-camphorsulfonate.
thioglycollic acid R. Make alkaline by adding ammonia R
and dilute to 20 mL with water R. No pink colour develops. : − 18 ± 2, determined on a 50 g/L solution.
Storage : protected from atmospheric carbon dioxide, at a Ammonium carbamate. CH6N2O2. (Mr 78.1). 1168400.
temperature below 20 °C. [1111-78-0]. Carbamic acid ammonium salt.
General Notices (1) apply to all monographs and other texts 431
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Ammonium phosphate. (NH4)2HPO4. (Mr 132.1). 1006100. β-Amyrin. C30H50O. (Mr 426.7). 1141800. [559-70-6].
[7783-28-0]. Diammonium hydrogen phosphate. Olean-12-en-3β-ol.
White or almost white crystals or granules, hygroscopic, very White or almost white powder.
soluble in water, practically insoluble in ethanol (96 per cent). mp : 187 °C to 190 °C.
pH (2.2.3) : about 8 for a 200 g/L solution.
Storage : in an airtight container. Anethole. C10H12O. (Mr 148.2). 1006900. [4180-23-8].
1-Methoxy-4-(propen-1-yl)benzene.
Ammonium pyrrolidinedithiocarbamate. C5H12N2S2. White or almost white, crystalline mass up to 20 °C to 21 °C,
(Mr 164.3). 1006200. [5108-96-3]. Ammonium liquid above 23 °C, practically insoluble in water, freely soluble
1-pyrrolidinyl-dithioformate. in anhydrous ethanol, soluble in ethyl acetate and in light
White or pale yellow, crystalline powder, sparingly soluble in petroleum.
water, very slightly soluble in ethanol (96 per cent). : about 1.56.
Storage : in a bottle containing a piece of ammonium carbonate bp : about 230 °C.
in a muslin bag.
Anethole used in gas chromatography complies with the
Ammonium reineckate. NH4[Cr(NCS)4(NH3)2],H2O. following additional test.
(Mr 354.4). 1006300. [13573-16-5]. Ammonium Assay. Gas chromatography (2.2.28) as prescribed in the
diamine-tetrakis(isothiocyanato)chromate(III) monohydrate. monograph Anise oil (0804).
Red powder or crystals, sparingly soluble in cold water, soluble Test solution. The substance to be examined.
in hot water and in ethanol (96 per cent).
Content : minimum 99.0 per cent of trans-anethole (retention
Ammonium reineckate solution. 1006301. time : about 41 min), calculated by the normalisation
A 10 g/L solution. Prepare immediately before use. procedure.
Ammonium sulfamate. NH2SO3NH4. (Mr 114.1). 1006400. Aniline. C6H7N. (Mr 93.1). 1007100. [62-53-3].
[7773-06-0]. Benzeneamine.
White or almost white, crystalline powder or colourless Colourless or slightly yellowish liquid, soluble in water,
crystals, hygroscopic, very soluble in water, slightly soluble in miscible with ethanol (96 per cent).
ethanol (96 per cent). : about 1.02.
mp : about 130 °C. bp : 183 °C to 186 °C.
Storage : in an airtight container. Storage : protected from light.
Ammonium sulfate. (NH4)2SO4. (Mr 132.1). 1006500. Aniline hydrochloride. C6H8ClN. (Mr 129.6). 1147700.
[7783-20-2]. [142-04-1]. Benzenamine hydrochloride.
Colourless crystals or white or almost white granules, very Crystals.
soluble in water, practically insoluble in acetone and in ethanol It darkens on exposure to air and light.
(96 per cent).
mp : about 198 °C.
pH (2.2.3) : 4.5 to 6.0 for a 50 g/L solution in carbon
dioxide-free water R. Storage : protected from light.
Sulfated ash (2.4.14) : maximum 0.1 per cent. Content : minimum 97.0 per cent.
Ammonium sulfide solution. 1123300. Anion-exchange resin. 1007200.
Saturate 120 mL of dilute ammonia R1 with hydrogen sulfide R Resin in chlorinated form containing quaternary ammonium
and add 80 mL of dilute ammonia R1. Prepare immediately groups [CH2N+(CH3)3] attached to a polymer lattice consisting
before use. of polystyrene cross-linked with 2 per cent of divinylbenzene.
It is available as spherical beads and the particle size is
Ammonium thiocyanate. NH4SCN. (Mr 76.1). 1006700. specified in the monograph.
[1762-95-4].
Wash the resin with 1 M sodium hydroxide on a sintered-glass
Colourless crystals, deliquescent, very soluble in water, soluble filter (40) (2.1.2) until the washings are free from chloride,
in ethanol (96 per cent). then wash with water R until the washings are neutral.
Storage : in an airtight container. Suspend in freshly prepared ammonium-free water R and
protect from atmospheric carbon dioxide.
Ammonium thiocyanate solution. 1006701.
A 76 g/L solution. Anion-exchange resin R1. 1123400.
Ammonium vanadate. NH4VO3. (Mr 117.0). 1006800. Resin containing quaternary ammonium groups
[7803-55-6]. Ammonium trioxovanadate(V). [CH2N+(CH3)3] attached to a lattice consisting of methacrylate.
White or slightly yellowish, crystalline powder, slightly soluble Anion-exchange resin R2. 1141900.
in water, soluble in dilute ammonia R1. Conjugate of homogeneous 10 μm hydrophilic polyether
Ammonium vanadate solution. 1006801. particles, and a quaternary ammonium salt, providing a
Dissolve 1.2 g of ammonium vanadate R in 95 mL of matrix suitable for strong anion-exchange chromatography
water R and dilute to 100 mL with sulfuric acid R. of proteins.
General Notices (1) apply to all monographs and other texts 433
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Apigenin 7-glucoside. C21H20O10. (Mr 432.4). 1095900. Assay. Gas chromatography (2.2.28) as prescribed in the
[578-74-5]. Apigetrin. 7-(β-D-Glucopyranosyloxy)-5- monograph on Tea tree oil (1837).
hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one. Content : minimum 92 per cent, calculated by the
Light yellowish powder, practically insoluble in water, normalisation procedure.
sparingly soluble in ethanol (96 per cent).
Arsenious trioxide. As2O3. (Mr 197.8). 1008300. [1327-53-3].
mp : 198 °C to 201 °C.
Arsenious anhydride. Diarsenic trioxide.
Chromatography. Thin-layer chromatography (2.2.27) as
Crystalline powder or a white or almost white mass, slightly
prescribed in the monograph Roman chamomile flower (0380) :
soluble in water, soluble in boiling water.
apply 10 μL of a 0.25 g/L solution in methanol R ; the
chromatogram shows in the middle third a principal zone of Arsenite solution. 1008301.
yellowish fluorescence. Dissolve 0.50 g of arsenious trioxide R in 5 mL of dilute
Apigenin-7-glucoside used in liquid chromatography complies sodium hydroxide solution R, add 2.0 g of sodium hydrogen
with the following additional test. carbonate R and dilute to 100.0 mL with water R.
Assay. Liquid chromatography (2.2.29) as prescribed in the
monograph Matricaria flower (0404). Ascorbic acid. 1008400. [50-81-7].
Test solution. Dissolve 10.0 mg in methanol R and dilute to See Ascorbic acid (0253).
100.0 mL with the same solvent.
Ascorbic acid solution. 1008401.
Content : minimum 95.0 per cent, calculated by the
normalisation procedure. Dissolve 50 mg in 0.5 mL of water R and dilute to 50 mL
with dimethylformamide R.
Aprotinin. 1007900. [9087-70-1].
Asiaticoside. C48H78O19. (Mr 959). 1123500.
See Aprotinin (0580). [16830-15-2]. O-6-Deoxy-α-L-mannopyranosyl-
Arabinose. C5H10O5. (Mr 150.1). 1008000. [87-72-9]. (1→4)-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl
L-(+)-Arabinose. 2α,3β,23-trihydroxy-4α-urs-12-en-28-oate.
White or almost white, crystalline powder, freely soluble in White or almost white powder, hygroscopic, soluble in
water. methanol, slightly soluble in anhydrous ethanol, insoluble in
: + 103 to + 105, determined on a 50 g/L solution in acetonitrile.
water R containing about 0.05 per cent of NH3. mp : about 232 °C, with decomposition.
Water (2.5.12) : 6.0 per cent.
Arachidyl alcohol. C20H42O. (Mr 298.5). 1156300. [629-96-9].
1-Eicosanol. Asiaticoside used in liquid chromatography complies with the
following additional test.
mp : about 65 °C.
Assay. Liquid chromatography (2.2.29) as prescribed in the
Content : minimum 96 per cent of C20H42O. monograph Centella (1498).
Arbutin. C12H16O7. (Mr 272.3). 1008100. [497-76-7]. Content : minimum 97.0 per cent, calculated by the
Arbutoside. 4-Hydroxyphenyl-β-D-glucopyranoside. normalisation procedure.
Fine, white or almost white, shiny needles, freely soluble in Storage : protected from humidity.
water, very soluble in hot water, soluble in ethanol (96 per
cent). Aspartic acid. 1134100. [56-84-8].
Chromatography. Thin-layer chromatography (2.2.27) as See Aspartic acid (0797).
prescribed in the monograph Bearberry leaf (1054) ; the
L-Aspartyl- L-phenylalanine. C13H16N2O5. (Mr 280.3).
chromatogram shows only one principal spot.
1008500. [13433-09-5]. (S)-3-Amino-N-[(S)-1-carboxy-2-
Arginine. 1103600. [74-79-3]. phenylethyl]-succinamic acid.
See Arginine (0806). White or almost white powder.
mp : about 210 °C, with decomposition.
Argon. Ar. (Ar 39.95). 1008200. [7440-37-1].
Content : minimum 99.995 per cent V/V. Astragaloside IV. C41H68O14. (Mr 785). 1178200.
Carbon monoxide (2.5.25, Method I) : maximum 0.6 ppm V/V ; [84687-43-4]. (20R,24S)-20,24-Epoxy-16β,25-dihydroxy-
after passage of 10 L of argon R at a flow rate of 4 L/h, not 3β-(β-D-xylopyranosyloxy)-9,19-cyclolanostan-6α-yl
more than 0.05 mL of 0.002 M sodium thiosulfate is required β-D-glucopyranoside.
for the titration. Atropine sulfate. 1159000. [5908-99-6].
Argon R1. Ar. (Ar 39.95). 1176000. [7440-37-1]. See Atropine sulfate (0068).
Content : minimum 99.99990 per cent V/V. Aucubin. C15H22O9. (Mr 346.3 ). 1145200. [479-98-1].
Argon for chromatography. Ar. (Ar 39.95). 1166200. [1S,4aR,5S,7aS)-5-Hydroxy-7-(hydroxymethyl)-1,4a,5,7a-
[7440-37-1]. tetrahydrocyclopenta[c]pyran-1-yl β-D-glucopyranoside.
Content : minimum 99.95 per cent V/V. Crystals, soluble in water, in ethanol (96 per cent) and in
methanol, practically insoluble in light petroleum.
Aromadendrene. C15H24. (Mr 204.4 ). 1139100. [489-39-4].
: about − 163.
(1R,2S,4R,8R,11R)-3,3,11-Trimethyl-7-methylenetricyclo-
[6.3.0.02,4]undecane. mp : about 181 °C.
Clear, almost colourless liquid. Azomethine H. C17H12NNaO8S2. (Mr 445.4). 1008700.
: about 0.911. [5941-07-1]. Sodium hydrogeno-4-hydroxy-5-(2-
: about 1.497. hydroxybenzylideneamino)-2,7-naphthalenedisulfonate.
: about + 12. Azomethine H solution. 1008701.
bp : about 263 °C. Dissolve 0.45 g of azomethine H R and 1 g of ascorbic acid R
Aromadendrene used in gas chromatography complies with with gentle heating in water R and dilute to 100 mL with
the following additional test. the same solvent.
General Notices (1) apply to all monographs and other texts 435
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Baicalin. C21H18O11. (Mr 446.4). 1179200. [21967-41-9]. Content : minimum 98.0 per cent.
5,6-Dihydroxy-4-oxo-2-phenyl-4H-1-benzopyran-7-yl-β-D- bp : about 261 °C.
glucopyranosiduronic acid. mp : about 39 °C.
Barbaloin. C21H22O9,H2O. (Mr 436.4). 1008800.
Benzaldehyde. C7H6O. (Mr 106.1). 1009600. [100-52-7].
[1415-73-2]. Aloin. 1,8-Dihydroxy-3-hydroxymethyl-10-β-D-
glucopyranosyl-10H-anthracen-9-one. Colourless or slightly yellow liquid, slightly soluble in water,
Yellow to dark-yellow, crystalline powder, or yellow needles, miscible with ethanol (96 per cent).
darkening on exposure to air and light, sparingly soluble in : about 1.05.
water and in ethanol (96 per cent), soluble in acetone, in : about 1.545.
ammonia and in solutions of alkali hydroxides. Distillation range (2.2.11). Not less than 95 per cent distils
: about 192 at 269 nm, about 226 at 296.5 nm, about between 177 °C and 180 °C.
259 at 354 nm, determined on a solution in methanol R and Storage : protected from light.
calculated with reference to the anhydrous substance.
Chromatography. Thin-layer chromatography (2.2.27) as Benzene. C6H6. (Mr 78.1). 1009800. [71-43-2].
prescribed in the monograph Frangula bark (0025) ; the Clear, colourless, flammable liquid, practically insoluble in
chromatogram shows only one principal spot. water, miscible with ethanol (96 per cent).
Barbital. 1008900. [57-44-3]. bp : about 80 °C.
See Barbital (0170).
Benzene-1,2,4-triol. C6H6O3. (Mr 126.1). 1177500.
Barbital sodium. C8H11N2NaO3. (Mr 206.2). 1009000. [533-73-3]. Hydroxyhydroquinone. Hydroxyquinol.
[144-02-5]. Sodium derivative of 5,5-diethyl-1H,3H,5H- Freely soluble in water, in ethanol (96 per cent) and in ethyl
pyrimidine-2,4,6-trione. acetate.
Content : minimum 98.0 per cent. mp : about 140 °C.
A white or almost white, crystalline powder or colourless
crystals, freely soluble in water, slightly soluble in ethanol Benzethonium chloride. C27H42ClNO2,H2O. (Mr 466.1).
(96 per cent). 1009900. [121-54-0]. Benzyldimethyl[2-[2-[4-(1,1,3,3-
tetramethylbutyl)phenoxy]ethoxy]ethyl]ammonium chloride
Barbituric acid. C4H4N2O3. (Mr 128.1). 1009100. [67-52-7]. monohydrate.
1H,3H,5H-Pyrimidine-2,4,6-trione. Fine, white or almost white powder or colourless crystals,
White or almost white powder, slightly soluble in water, freely soluble in water and in ethanol (96 per cent).
soluble in boiling water and in dilute acids.
mp : about 163 °C.
mp : about 253 °C.
Storage : protected from light.
Barium acetate. C4H6BaO4. (Mr 255.4). 1162700. [543-80-6].
Barium diacetate. Benzidine. C12H12N2. (Mr 184.2). 1145300. [92-87-5].
White or almost white powder, soluble in water. Biphenyl-4,4′-diamine.
: 2.47. Content : minimum 95 per cent.
White or slightly yellowish or reddish powder, darkening on
Barium carbonate. BaCO3. (Mr 197.3). 1009200. [513-77-9]. exposure to air and light.
White or almost white powder or friable masses, practically mp : about 120 °C.
insoluble in water.
Storage : protected from light.
Barium chloride. BaCl2,2H2O. (Mr 244.3). 1009300.
[10326-27-9]. Barium dichloride. Benzil. C14H10O2. (Mr 210.2). 1117800. [134-81-6].
Colourless crystals, freely soluble in water, slightly soluble in Diphenylethanedione.
ethanol (96 per cent). Yellow, crystalline powder, practically insoluble in water,
soluble in ethanol (96 per cent), ethyl acetate and toluene.
Barium chloride solution R1. 1009301.
mp : 95 °C.
A 61 g/L solution.
Benzocaine. C9H11NO2. (Mr 165.2). 1123600. [94-09-7].
Barium chloride solution R2. 1009302.
A 36.5 g/L solution. See Benzocaine (0011).
Barium hydroxide. Ba(OH)2,8H2O. (Mr 315.5). 1009400. Benzoic acid. 1010100. [65-85-0].
[12230-71-6]. Barium dihydroxide. See Benzoic acid (0066).
Colourless crystals, soluble in water.
Benzoin. C14H12O2. (Mr 212.3). 1010200. [579-44-2].
Barium hydroxide solution. 1009401. 2-Hydroxy-1,2-diphenylethanone.
A 47.3 g/L solution. Slightly yellowish crystals, very slightly soluble in water, freely
soluble in acetone, soluble in hot ethanol (96 per cent).
Barium nitrate. Ba(NO3)2. (Mr 261.3). 1163800.
[10022-31-8]. mp : about 137 °C.
Crystals or crystalline powder, freely soluble in water, very Benzophenone. C13H10O. (Mr 182.2). 1010300. [119-61-9].
slightly soluble in ethanol (96 per cent) and in acetone. Diphenylmethanone.
mp : about 590 °C. Prismatic crystals, practically insoluble in water, freely soluble
Barium sulfate. 1009500. [7727-43-7]. in ethanol (96 per cent).
See Barium sulfate (0010). mp : about 48 °C.
Benzalacetone. C10H10O. (Mr 146.2). 1168500. [122-57-6]. 1,4-Benzoquinone. C6H4O2. (Mr 108.1). 1118500. [106-51-4].
(3E)-4-phenylbut-3-en-2-one. Cyclohexa-2,5-diene-1,4-dione.
White or pale yellow mass. Content : minimum 98.0 per cent.
General Notices (1) apply to all monographs and other texts 437
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Bisbenzimide stock solution. 1103801. Boric acid solution, saturated, cold. 1011801.
Dissolve 5 mg of bisbenzimide R in water R and dilute to To 3 g of boric acid R add 50 mL of water R and shake for
100 mL with the same solvent. 10 min. Place the solution for 2 h in the refrigerator.
Storage : in the dark. Borneol. C10H18O. (Mr 154.3). 1011900. [507-70-0].
Bisbenzimide working solution. 1103802. endo-1,7,7-Trimethylbicyclo[2.2.1]heptan-2-ol.
Immediately before use, dilute 100 μL of bisbenzimide Colourless crystals, readily sublimes, practically insoluble
stock solution R to 100 mL with phosphate buffered saline in water, freely soluble in ethanol (96 per cent) and in light
pH 7.4 R. petroleum.
mp : about 208 °C.
Bismuth nitrate pentahydrate. Bi(NO3)3,5H2O. (Mr 485.1). Chromatography. Thin-layer chromatography (2.2.27), using
1165600. [10035-06-0]. silica gel G R as the coating substance. Apply to the plate
mp : about 30 °C. 10 μL of a 1 g/L solution in toluene R. Develop over a path of
10 cm using chloroform R. Allow the plate to dry in air, spray
Bismuth subnitrate. 4BiNO3(OH)2,BiO(OH). (Mr 1462). with anisaldehyde solution R, using 10 mL for a plate 200 mm
1011500. [1304-85-4]. square, and heat at 100-105 °C for 10 min. The chromatogram
White or almost white powder, practically insoluble in water. obtained shows only one principal spot.
Bismuth subnitrate R1. 1011501. Bornyl acetate. C12H20O2. (Mr 196.3). 1012000. [5655-61-8].
Content : 71.5 per cent to 74.0 per cent of bismuth (Bi), endo-1,7,7-Trimethylbicyclo[2.2.1]hept-2-yl acetate.
and 14.5 per cent to 16.5 per cent of nitrate, calculated as Colourless crystals or a colourless liquid, very slightly soluble
nitrogen pentoxide (N2O5). in water, soluble in ethanol (96 per cent).
Bismuth subnitrate solution. 1011502. mp : about 28 °C.
Dissolve 5 g of bismuth subnitrate R1 in a mixture of 8.4 mL Chromatography. Thin-layer chromatography (2.2.27), using
of nitric acid R and 50 mL of water R and dilute to 250 mL silica gel G R as the coating substance. Apply to the plate
with water R. Filter if necessary. 10 μL of a 2 g/L solution in toluene R. Develop over a path of
10 cm using chloroform R. Allow the plate to dry in air, spray
Acidity. To 10 mL add 0.05 mL of methyl orange solution R. with anisaldehyde solution R, using 10 mL for a plate 200 mm
5.0 mL to 6.25 mL of 1 M sodium hydroxide is required to square, and heat at 100-105 °C for 10 min. The chromatogram
change the colour of the indicator. obtained shows only one principal spot.
Bis-tris propane. C11H26N2O6. (Mr 282.3). 1185500. Boron trichloride. BCl3. (Mr 117.2). 1112000. [10294-34-5].
[64431-96-5]. 2,2′-(Propane-1,3-diyldiimino)bis[2- Colourless gas. Reacts violently with water. Available as
(hydroxymethyl)-1,3-propanediol. solutions in suitable solvents (2-chloroethanol, methylene
Content : minimum 99.0 per cent. chloride, hexane, heptane, methanol).
Biuret. C2H5N3O2. (Mr 103.1). 1011600. [108-19-0]. : about 1.420.
White or almost white crystals, hygroscopic, soluble in water, bp : about 12.6 °C.
sparingly soluble in ethanol (96 per cent). Caution : toxic and corrosive.
mp : 188 °C to 190 °C, with decomposition. Boron trichloride-methanol solution. 1112001.
Storage : in an airtight container. A 120 g/L solution of BCl3 in methanol R.
Biuret reagent. 1011601. Storage : protected from light at − 20 °C, preferably in
sealed tubes.
Dissolve 1.5 g of copper sulfate R and 6.0 g of sodium
potassium tartrate R in 500 mL of water R. Add 300 mL of a Boron trifluoride. BF3. (Mr 67.8). 1012100. [7637-07-2].
carbonate-free 100 g/L solution of sodium hydroxide R, dilute Colourless gas.
to 1000 mL with the same solution and mix.
Boron trifluoride-methanol solution. 1012101.
Blocking solution. 1122400. A 140 g/L solution of boron trifluoride R in methanol R.
A 10 per cent V/V solution of acetic acid R.
Brilliant blue. 1012200. [6104-59-2].
Blue dextran 2000. 1011700. [9049-32-5]. See acid blue 83 R.
Prepared from dextran having an average relative molecular
mass of 2 × 106 by introduction of a polycyclic chromophore Bromelains. 1012300. [37189-34-7].
that colours the substance blue. The degree of substitution is Concentrate of proteolytic enzymes derived from Ananas
0.017. comosus Merr.
It is freeze-dried and dissolves rapidly and completely in water Dull-yellow powder.
and aqueous saline solutions. Activity. 1 g liberates about 1.2 g of amino-nitrogen from a
Absorbance (2.2.25). A 1 g/L solution in a phosphate buffer solution of gelatin R in 20 min at 45 °C and pH 4.5.
solution pH 7.0 R shows an absorption maximum at 280 nm. Bromelains solution. 1012301.
Boldine. C19H21NO4. (Mr 327.3). 1118800. [476-70-0]. A 10 g/L solution of bromelains R in a mixture of 1 volume
1,10-Dimethoxy-6aα-aporphine-2,9-diol. of phosphate buffer solution pH 5.5 R and 9 volumes of a
White or almost white crystalline powder, very slightly 9 g/L solution of sodium chloride R.
soluble in water, soluble in ethanol (96 per cent) and in dilute Bromine. Br2. (Mr 159.8). 1012400. [7726-95-6].
solutions of acids. Brownish-red fuming liquid, slightly soluble in water, soluble
: about + 127, determined on a 1 g/L solution in in ethanol (96 per cent).
anhydrous ethanol R. : about 3.1.
mp : about 163 °C.
Bromine solution. 1012401.
Boric acid. 1011800. [10043-35-3]. Dissolve 30 g of bromine R and 30 g of potassium bromide R
See Boric acid (0001). in water R and dilute to 100 mL with the same solvent.
General Notices (1) apply to all monographs and other texts 439
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Brucine. C23H26N2O4,2H2O. (Mr 430.5). 1013100. [357-57-3]. Butylboronic acid. C4H11BO2. (Mr 101.9). 1013700.
10,11-Dimethoxystrychnine. [4426-47-5].
Colourless crystals, slightly soluble in water, freely soluble in Content : minimum 98 per cent.
ethanol (96 per cent). mp : 90 °C to 92 °C.
mp : about 178 °C. tert-Butylhydroperoxide. C4H10O2. (Mr 90.1). 1118000.
Butanal. C4H8O. (Mr 72.1). 1134400. [123-72-8]. [75-91-2]. 1,1-Dimethylethylhydroperoxide.
Butyraldehyde. Flammable liquid, soluble in organic solvents.
: 0.806. : 0.898.
: 1.380. : 1.401.
bp : 75 °C. bp : 35 °C.
Butyl 4-hydroxybenzoate. 1103900. [94-26-8].
Butane-1,4-diol. HO(CH2)4OH. (Mr 90.12). 1174800.
[110-63-4]. See Butyl parahydroxybenzoate R.
Butylhydroxytoluene. 1013800. [128-37-0].
Butanol. C4H10O. (Mr 74.1). 1013200. [71-36-3]. Butan-1-ol.
See Butylhydroxytoluene (0581).
Clear, colourless liquid, miscible with ethanol (96 per cent).
: about 0.81. Butyl methacrylate. C8H14O2. (Mr 142.2). 1145400.
[97-88-1]. Butyl 2-methylpropenoate.
bp : 116 °C to 119 °C.
Clear, colourless solution.
2-Butanol R1. C4H10O. (Mr 74.1). 1013301. [78-92-2]. : about 0.894.
Butan-2-ol. sec-Butyl alcohol. : about 1.424.
Content : minimum 99.0 per cent. bp : about 163 °C.
Clear, colourless liquid, soluble in water, miscible with ethanol tert-Butyl methyl ether. 1013900. [1634-04-4].
(96 per cent).
See 1,1-dimethylethyl methyl ether R.
: about 0.81.
Distillation range (2.2.11). Not less than 95 per cent distils Butyl parahydroxybenzoate. 1103900. [94-26-8].
between 99 °C and 100 °C. See Butyl parahydroxybenzoate (0881).
Assay. Gas chromatography (2.2.28) as prescribed in the Butyric acid. C4H8O2. (Mr 88.1). 1014000. [107-92-6].
monograph Isopropyl alcohol (0970). Butanoic acid.
Butyl acetate. C6H12O2. (Mr 116.2). 1013400. [123-86-4]. Content : minimum 99.0 per cent.
Clear, colourless liquid, flammable, slightly soluble in water, Oily liquid, miscible with water and with ethanol (96 per cent).
miscible with ethanol (96 per cent). : about 0.96.
: about 0.88. : about 1.398.
: about 1.395. bp : about 163 °C.
Distillation range (2.2.11). Not less than 95 per cent distils Butyrolactone. C4H6O2. (Mr 86.1). 1104000. [96-48-0].
between 123 °C and 126 °C. Dihydro-2(3H)-furanone. γ-Butyrolactone.
Oily liquid, miscible with water, soluble in methanol.
Butyl acetate R1. 1013401.
: about 1.435.
Content : minimum 99.5 per cent, determined by gas
bp : about 204 °C.
chromatography.
Clear, colourless liquid, flammable, slightly soluble in Cadmium. Cd. (Ar 112.4). 1014100. [7440-43-9].
water, miscible with ethanol (96 per cent). Silvery-white, lustrous metal, practically insoluble in water,
: about 0.883. freely soluble in nitric acid and in hot hydrochloric acid.
: about 1.395. Cadmium nitrate tetrahydrate. Cd(NO3)2,4H2O. (Mr 308.5).
Butanol : maximum 0.2 per cent, determined by gas 1174900. [10022-68-1].
chromatography. Hygroscopic orthorhombic crystals, very soluble in water,
n-Butyl formate : maximum 0.1 per cent, determined by soluble in acetone and in ethanol (96 per cent).
gas chromatography. mp : about 59.5 °C.
n-Butyl propionate : maximum 0.1 per cent, determined by Caesium chloride. CsCl. (Mr 168.4). 1014200. [7647-17-8].
gas chromatography. White or almost white powder, very soluble in water, freely
Water : maximum 0.1 per cent. soluble in methanol, practically insoluble in acetone.
Butylamine. C4H11N. (Mr 73.1). 1013600. [109-73-9]. Caffeic acid. C9H8O4. (Mr 180.2). 1014300. [331-39-5].
Butan-1-amine. (E)-3-(3,4-Dihydroxyphenyl)propenoic acid.
Distil and use within one month. White or almost white crystals or plates, freely soluble in hot
Colourless liquid, miscible with water, with ethanol (96 per water and in ethanol (96 per cent), sparingly soluble in cold
cent). water.
: about 1.401. mp : about 225 °C, with decomposition.
Absorbance (2.2.25). A freshly prepared solution at pH 7.6
bp : about 78 °C. shows 2 absorption maxima at 293 nm and 329 nm.
tert-Butylamine. 1100900. [75-64-9]. Caffeine. 1014400. [58-08-2].
See 1,1-dimethylethylamine R. See Caffeine (0267).
Butylated hydroxytoluene. 1013800. [128-37-0]. Calcium carbonate. 1014500. [471-34-1].
See Butylhydroxytoluene R. See Calcium carbonate (0014).
General Notices (1) apply to all monographs and other texts 441
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
ε-Caprolactam. C6H11NO. (Mr 113.2). 1104200. [105-60-2]. Do not use at a temperature higher than 400 °C.
Hexane-6-lactam.
Carbon for chromatography, graphitised R1. 1153500.
Hygroscopic flakes, freely soluble in water, in anhydrous
ethanol and in methanol. Porous spherical carbon particles comprised of flat sheets of
hexagonally arranged carbon atoms.
mp : about 70 °C.
Particle size : 5 μm to 7 μm.
Caprylic acid. C8H16O2. (Mr 144.2). 1142200. [124-07-2]. Pore volume : 0.7 cm3/g.
Octanoic acid.
Slightly yellow, oily liquid. Carbon monoxide. CO. (Mr 28.01). 1016000. [630-08-0].
: about 0.910. Content : minimum 99.97 per cent V/V.
: about 1.428. Carbon monoxide R1. CO. (Mr 28.01). 1134600. [630-08-0].
bp : about 239.7 °C. Content : minimum 99 per cent V/V.
mp : about 16.7 °C. Carbon tetrachloride. CCl4. (Mr 153.8). 1016100. [56-23-5].
Caprylic acid used in the assay of total fatty acids in Saw Tetrachloromethane.
palmetto fruit (1848) complies with the following additional Clear, colourless liquid, practically insoluble in water, miscible
test. with ethanol (96 per cent).
Assay. Gas chromatography (2.2.28) as prescribed in the : 1.595 to 1.598.
monograph Saw palmetto fruit (1848).
bp : 76 °C to 77 °C.
Content : minimum 98 per cent, calculated by the
normalisation procedure. Carbophenothion. C11H16ClO2PS3. (Mr 342.9). 1016200.
[786-19-6]. O,O-Diethyl S-[[(4-chlorophenyl)thio]methyl]-
Capsaicin. C18H27NO3. (Mr 305.4). 1147900. [404-86-4]. phosphorodithioate.
(E)-N-[(4-Hydroxy-3-methoxyphenyl)methyl]-8-methylnon-
Yellowish liquid, practically insoluble in water, miscible with
6-enamide.
organic solvents.
White or almost white, crystalline powder, practically
: about 1.27.
insoluble in water, freely soluble in anhydrous ethanol.
For the monograph Wool Fat (0134), a suitable certified
mp : about 65 °C.
reference solution (10 ng/μL in iso-octane) may be used.
Capsaicin used in the assay in Capsicum (1859) complies with
the following additional test. Car-3-ene. C10H16. (Mr 136.2). 1124000. [498-15-7].
Assay. Liquid chromatography (2.2.29) as prescribed in the 3,7,7-Trimethylbicyclo[4.1.0]hept-3-ene. 4,7,7-Trimethyl-3-
monograph Capsicum (1859). norcarene.
Content : minimum 95.0 per cent, calculated by the Liquid with a pungent odour, slightly soluble in water, soluble
normalisation procedure. in organic solvents.
: about 0.864.
Carbazole. C12H9N. (Mr 167.2). 1015400. [86-74-8].
: 1.473 to 1.474.
Dibenzopyrrole.
: + 15 to + 17.
Crystals, practically insoluble in water, freely soluble in
acetone, slightly soluble in anhydrous ethanol. bp : 170 °C to 172 °C.
mp : about 245 °C. Car-3-ene used in gas chromatography complies with the
following additional test.
Carbomer. 1015500. [9007-20-9]. Assay. Gas chromatography (2.2.28) as prescribed in the
A cross-linked polymer of acrylic acid ; it contains a large monograph Nutmeg oil (1552).
proportion (56 per cent to 68 per cent) of carboxylic acid Content : minimum 95.0 per cent, calculated by the
(CO2H) groups after drying at 80 °C for 1 h. Average relative normalisation procedure.
molecular mass about 3 × 106.
pH (2.2.3) : about 3 for a 10 g/L suspension. Carminic acid. C22H20O13. (Mr 492.4). 1156700. [1260-17-9].
7-α-D-Glucopyranosyl-3,5,6,8-tetrahydroxy-1-methyl-9,10-
Carbon dioxide. 1015600. [124-38-9]. dioxo-9,10-dihydroanthracene-2-carboxylic acid.
See Carbon dioxide (0375). Dark red powder, very slightly soluble in water, soluble in
dimethyl sulfoxide, very slightly soluble in ethanol (96 per
Carbon dioxide R1. CO2. (Mr 44.01). 1015700. [124-38-9].
cent).
Content : minimum 99.995 per cent V/V.
Carbon monoxide : less than 5 ppm. Carob bean gum. 1104500.
Oxygen : less than 25 ppm. The ground endosperm of the fruit kernels of Ceratonia
siliqua L. Taub.
Nitric oxide : less than 1 ppm.
White or almost white powder containing 70 per cent to
Carbon dioxide R2. CO2. (Mr 44.01). 1134500. [124-38-9]. 80 per cent of a water-soluble gum consisting mainly of
Content : minimum 99 per cent V/V. galactomannoglycone.
Carbon disulfide. CS2. (Mr 76.1). 1015800. [75-15-0]. Carvacrol. C10H14O. (Mr 150.2). 1016400. [499-75-2].
Colourless or yellowish, flammable liquid, practically insoluble 5-Isopropyl-2-methylphenol.
in water, miscible with anhydrous ethanol. Brownish liquid, practically insoluble in water, very soluble in
: about 1.26. ethanol (96 per cent).
bp : 46 °C to 47 °C. : about 0.975.
: about 1.523.
Carbon for chromatography, graphitised. 1015900. bp : about 237 °C.
Carbon chains having a length greater than C9 . Carvacrol used in gas chromatography complies with the
Particle size : 400 μm to 850 μm. following additional test.
Relative density : 0.72. Assay. Gas chromatography (2.2.28) as prescribed in the
Surface area : 10 m2/g. monograph Peppermint oil (0405).
Test solution. Dissolve 0.1 g in about 10 mL of acetone R. Caryophyllene oxide. C15H24O. (Mr 220.4).
Content : minimum 95.0 per cent, calculated by the 1149000. [1139-30-6]. (-)-β-Caryophyllene epoxide.
normalisation procedure. (1R,4R,6R,10S)-4,12,12-Trimethyl-9-methylene-5-
oxatricyclo[8.2.0.04,6]dodecane.
Carveol. C10H16O. (Mr 152.2). 1160400. [99-48-9]. p-Mentha- Colourless, fine crystals with lumps.
1(6),8-dien-2-ol. 2-Methyl-5-(1-methylethenyl)cyclohex-2-
enol. mp : 62 °C to 63 °C.
The substance contains a variable content of trans- and Caryophyllene oxide used in gas chromatography complies with
cis-carveol. the following additional test.
Carveol used in gas chromatography complies with the following Assay. Gas chromatography (2.2.28) as prescribed in the
additional test. monograph Turpentine oil, Pinus pinaster type (1627).
Assay. Gas chromatography (2.2.28) as prescribed in the Content : minimum 99.0 per cent, calculated by the
test for chromatographic profile in the monograph Caraway normalisation procedure.
oil (1817).
Casein. 1016600. [9000-71-9].
Content : minimum 97 per cent, calculated by the
normalisation procedure. Mixture of related phosphoproteins obtained from milk.
White or almost white, amorphous powder or granules, very
Carvone. C10H14O. (Mr 150.2). 1016500. [2244-16-8]. slightly soluble in water and in non-polar organic solvents. It
(+)-p-Mentha-6,8-dien-2-one. (5S)-2-Methyl-5-(1- dissolves in concentrated hydrochloric acid giving a pale-violet
methylethenyl)-cyclohex-2-enone. solution. It forms salts with acids and bases. Its isoelectric
Liquid, practically insoluble in water, miscible with ethanol point is at about pH 4.7. Alkaline solutions are laevorotatory.
(96 per cent).
Casticin. C19H18O8. (Mr 374.3). 1162200. [479-91-4].
: about 0.965 5-Hydroxy-2-(3-hydroxy-4-methoxyphenyl)-3,6,7-
: about 1.500. trimethoxy-4H-1-benzopyran-4-one.
: about + 61. Yellow crystals.
bp : about 230 °C.
Catalpol. C15H22O10. (Mr 362.3). 1142300. [2415-24-9].
Carvone used in gas chromatography complies with the (1aS,1bS,2S,5aR,6S,6aS)-6-Hydroxy-1a-(hydroxymethyl)-
following additional test. 1a,1b,2,5a,6,6a-hexahydrooxireno[4,5]cyclopenta[1,2-
Assay. Gas chromatography (2.2.28) as prescribed in the c]pyran-2-yl β-D-glucopyranoside.
monograph Peppermint oil (0405) using the substance to be
mp : 203 °C to 205 °C.
examined as the test solution.
Content : minimum 98.0 per cent, calculated by the Catechin. C15H14O6,xH2O. (Mr 290.3 for the anhydrous
normalisation procedure. substance). 1119000. [154-23-4]. (+)-(2R,3S)-2-(3,4-
Dihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol.
Carvone R1. 1016501. Catechol. Cianidanol. Cyanidol.
Complies with the requirements prescribed for carvone R
with the following additional requirement. Cation-exchange resin. 1016700.
Assay. Gas chromatography (2.2.28) as prescribed in the A resin in protonated form with sulfonic acid groups attached
test for chiral purity in the monograph Caraway oil (1817). to a polymer lattice consisting of polystyrene cross-linked with
8 per cent of divinylbenzene. It is available as beads and the
Content : minimum 98 per cent.
particle size is specified after the name of the reagent in the
(−)-Carvone. C10H14O. (Mr 150.2). 1160500. tests where it is used.
[6485-40-1]. (–)-p-Mentha-1(6),8-dien-2-one.
(5R)-2-Methyl-5-(1-methylethenyl)cyclohex-2-enone. Cation-exchange resin R1. 1121900.
Liquid. A resin in protonated form with sulfonic acid groups attached
to a polymer lattice consisting of polystyrene cross-linked with
: about 0.965. 4 per cent of divinylbenzene. It is available as beads and the
: about 1.4988. particle size is specified after the name of the reagent in the
: about − 62. tests where it is used.
bp : about 230 °C. Cation-exchange resin, strong. 1156800.
Assay. Gas chromatography (2.2.28) as prescribed in the test Strong cation-exchange resin in protonated form with sulfonic
for chiral purity in the monograph Caraway oil (1817). acid groups attached to a polymer lattice consisting of
Content : minimum 99 per cent. polystyrene cross-linked with divinylbenzene. The particle
size is specified after the name of the reagent in the tests where
β-Caryophyllene. C15H24. (Mr 204.4). 1101000. it is used.
[87-44-5]. (E)-(1R,9S)-4,11,11-Trimethyl-8-methylene-
bicyclo[7.2.0]undec-4-ene. Cation-exchange resin (calcium form), strong. 1104600.
Oily liquid, practically insoluble in water, miscible with Resin in calcium form with sulfonic acid groups attached to
ethanol (96 per cent). a polymer lattice consisting of polystyrene cross-linked with
β-Caryophyllene used in gas chromatography complies with 8 per cent of divinylbenzene. The particle size is specified after
the following additional test. the name of the reagent in the tests where it is used.
Assay. Gas chromatography (2.2.28) as prescribed in the Cation-exchange resin (sodium form), strong. 1176100.
monograph Clove oil (1091).
Resin in sodium form with sulfonic acid groups attached to
Test solution. The substance to be examined. a polymer lattice consisting of polystyrene cross-linked with
Content : minimum 90.0 per cent, calculated by the divinylbenzene. The particle size is specified after the name of
normalisation procedure. the reagent in the tests where it is used.
General Notices (1) apply to all monographs and other texts 443
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Cellulose for chromatography. 1016800. [9004-34-6]. Assay. Gas chromatography (2.2.28) as prescribed in the
Fine, white or almost white, homogeneous powder with an monograph Matricaria oil (1836).
average particle size less than 30 μm. Test solution : a 4 g/L solution in cyclohexane R.
Preparation of a thin layer. Suspend 15 g in 100 mL of water R Content : minimum 95.0 per cent, calculated by the
and homogenise in an electric mixer for 60 s. Coat carefully normalisation procedure.
cleaned plates with a layer 0.1 mm thick using a spreading
device. Allow to dry in air. Charcoal, activated. 1017800. [64365-11-3].
See Activated charcoal (0313).
Cellulose for chromatography R1. 1016900.
Microcrystalline cellulose. A fine, white or almost white Chloral hydrate. 1017900. [302-17-0].
homogeneous powder with an average particle size less than See Choral hydrate (0265).
30 μm.
Chloral hydrate solution. 1017901.
Preparation of a thin layer. Suspend 25 g in 90 mL of water R
and homogenise in an electric mixer for 60 s. Coat carefully A solution of 80 g in 20 mL of water R.
cleaned plates with a layer 0.1 mm thick using a spreading Chloramine. 1018000. [7080-50-4].
device. Allow to dry in air. See Tosylchloramide sodium (0381).
Cellulose for chromatography F254. 1017000. Chloramine solution. 1018001.
Microcrystalline cellulose F254. A fine, white or almost white, A 20 g/L solution. Prepare immediately before use.
homogeneous powder with an average particle size less than
30 μm, containing a fluorescent indicator having an optimal Chloramine solution R1. 1018002.
intensity at 254 nm. A 0.1 g/L solution of chloramine R. Prepare immediately
Preparation of a thin layer. Suspend 25 g in 100 mL of before use.
water R and homogenise using an electric mixer for 60 s.
Coat carefully cleaned plates with a layer 0.1 mm thick using Chloramine solution R2. 1018003.
a spreading device. Allow to dry in air. A 0.2 g/L solution. Prepare immediately before use.
Cerium sulfate. Ce(SO4)2,4H2O. (Mr 404.3). 1017300. Chlordane. C10H6Cl8. (Mr 409.8). 1124100. [12789-03-6].
[10294-42-5]. Cerium(IV) sulfate tetrahydrate. Ceric sulfate. bp : about 175 °C.
Yellow or orange-yellow, crystalline powder or crystals, very mp : about 106 °C.
slightly soluble in water, slowly soluble in dilute acids. A suitable certified reference solution of technical grade
Cerous nitrate. Ce(NO3)3,6H2O. (Mr 434.3). 1017400. (10 ng/μL in iso-octane) may be used.
[10294-41-4]. Cerium trinitrate hexahydrate. Chlordiazepoxide. 1113200. [58-25-3].
Colourless or pale yellow, crystalline powder, freely soluble in See Chlordiazepoxide (0656).
water and in ethanol (96 per cent).
Chlorfenvinphos. C12H14Cl3O4P. (Mr 359.6). 1124200.
Cetostearyl alcohol. 1017500. [67762-27-0]. [470-90-6].
See Cetostearyl alcohol (0702).
A suitable certified reference solution (10 ng/μL in
Cetrimide. 1017600. [8044-71-1]. cyclohexane) may be used.
See Cetrimide (0378). Chloroacetanilide. C8H8ClNO. (Mr 169.6). 1018100.
Cetyl alcohol. C16H34O. (Mr 242.4). 1160600. [36653-82-4]. [539-03-7]. 4′-Chloroacetanilide.
Hexadecan-1-ol. Content : minimum 95 per cent.
Content : minimum 95.0 per cent. Crystalline powder, practically insoluble in water, soluble in
mp : about 48 °C. ethanol (96 per cent).
mp : about 178 °C.
Cetylpyridinium chloride monohydrate. C21H38ClN,H2O.
(Mr 358.0). 1162800. [6004-24-6]. 1-Hexadecylpyridinium Chloroacetic acid. C2H3ClO2. (Mr 94.5). 1018200. [79-11-8].
chloride monohydrate. Colourless or white or almost white crystals, deliquescent,
White or almost white powder, freely soluble in water and in very soluble in water, soluble in ethanol (96 per cent).
ethanol (96 per cent). Storage : in an airtight container.
mp : 80 °C to 83 °C.
Chloroaniline. C6H6ClN. (Mr 127.6). 1018300. [106-47-8].
Cetyltrimethylammonium bromide. C19H42BrN. 4-Chloroaniline.
(Mr 364.5). 1017700. [57-09-0]. Cetrimonium bromide. Crystals, soluble in hot water, freely soluble in ethanol (96 per
N-Hexadecyl-N,N,N-trimethylammonium bromide. cent).
White or almost white, crystalline powder, soluble in water, mp : about 71 °C.
freely soluble in ethanol (96 per cent).
mp : about 240 °C. 4-Chlorobenzenesulfonamide. C6H6ClNO2S. (Mr 191.6).
1097400. [98-64-6].
Chamazulene. C14H16. (Mr 184.3). 1148000. [529-05-5]. White or almost white powder.
7-Ethyl-1,4-dimethylazulene.
mp : about 145 °C.
Blue liquid, very slightly soluble in water, soluble in ethanol
(96 per cent), miscible with fatty oils, with essential oils and 2-Chlorobenzoic acid. C7H5ClO2. (Mr 156.6). 1139300.
with liquid paraffin, soluble with discolouration in phosphoric [118-91-2].
acid (85 per cent m/m) and sulfuric acid (50 per cent V/V). Soluble in water, slightly soluble in anhydrous ethanol.
Appearance of solution. 50 mg is soluble in 2.5 mL of hexane R. bp : about 285 °C.
The blue solution is clear in a thin-layer obtained by tilting mp : about 140 °C.
the test-tube.
Chamazulene used for gas chromatography complies with the Chlorobutanol. 1018400. [57-15-8].
following additional test. See Anhydrous chlorobutanol (0382).
2-Chloro-2-deoxy- D-glucose. C6H11ClO5. (Mr 198.6). Content : minimum 99.8 per cent of CHCl3, determined by gas
1134700. [14685-79-1]. chromatography.
White or almost white crystalline, very hygroscopic powder, Chlorogenic acid. C16H18O9. (Mr 354.3). 1104700. [327-97-9].
soluble in water and in dimethyl sulfoxide, practically (1S,3R,4R,5R)-3-[(3,4-Dihydroxycinnamoyl)oxy]-1,4,5-
insoluble in ethanol (96 per cent). trihydroxycyclohexanecarboxylic acid.
2-Chloroethanol. C2H5ClO. (Mr 80.5). 1097500. [107-07-3]. White or almost white, crystalline powder or needles, freely
Colourless liquid, soluble in ethanol (96 per cent). soluble in boiling water, in acetone and in ethanol (96 per
cent).
: about 1.197.
: about − 35.2.
: about 1.442.
mp : about 208 °C.
bp : about 130 °C.
Chromatography. Thin-layer chromatography (2.2.27) as
mp : about − 89 °C. prescribed on Identification A in the monograph Belladonna
leaf dry extract, standardised (1294) ; the chromatogram shows
2-Chloroethanol solution. 1097501.
only one principal zone.
Dissolve 125 mg of 2-chloroethanol R in 2-propanol R and
Chlorogenic acid used in liquid chromatography complies with
dilute to 50 mL with the same solvent. Dilute 5 mL of the
the following additional test.
solution to 50 mL with 2-propanol R.
Assay. Liquid chromatography (2.2.29) as prescribed in the
Chloroethylamine hydrochloride. C2H7Cl2N. (Mr 116.0). monograph Artichoke Leaf (1866).
1124300. [870-24-6]. 2-Chloroethanamine hydrochloride. Content : minimum 97.0 per cent.
mp : about 145 °C.
3-Chloro-2-methylaniline. C7H8ClN. (Mr 141.6). 1139400.
(2-Chloroethyl)diethylamine hydrochloride. C6H15Cl2N. [87-60-5]. 6-Chloro-2-toluidine.
(Mr 172.1). 1018500. [869-24-9]. Not miscible with water, slightly soluble in anhydrous ethanol.
White or almost white, crystalline powder, very soluble in : about 1.171.
water and in methanol, freely soluble in methylene chloride, : about 1.587.
practically insoluble in hexane.
bp : about 115 °C.
mp : about 211 °C. mp : about 2 °C.
Chloroform. CHCl3. (Mr 119.4). 1018600. [67-66-3]. 2-Chloro-N-(2,6-dimethylphenyl)acetamide. C10H12ClNO.
Trichloromethane. (Mr 197.7). 1168700. [1131-01-7].
Clear, colourless liquid, slightly soluble in water, miscible with
ethanol (96 per cent). 2-Chloronicotinic acid. C6H4ClNO2. (Mr 157.6). 1157300.
[2942-59-8]. 2-Chloropyridine-3-carboxylic acid.
: 1.475 to 1.481.
White or almost white powder.
bp : about 60 °C.
mp : about 177 °C.
Ethanol : 0.4 per cent m/m to 1.0 per cent m/m.
Content : minimum 95 per cent.
Introduce 1.00 g (m g) into a ground-glass-stoppered flask.
Add 15.0 mL of nitrochromic reagent R, close the flask, shake 2-Chloro-4-nitroaniline. C6H5ClN2O2. (Mr 172.6). 1018800.
vigorously for 2 min and allow to stand for 15 min. Add [121-87-9].
100 mL of water R and 5 mL of a 200 g/L solution of potassium Yellow, crystalline powder, freely soluble in methanol.
iodide R. After 2 min titrate with 0.1 M sodium thiosulfate, mp : about 107 °C.
using 1 mL of starch solution R as indicator, until a light
green colour is obtained (n1 mL of 0.1 M sodium thiosulfate). Storage : protected from light.
Carry out a blank assay (n2 mL of 0.1 M sodium thiosulfate). 2-Chloro-5-nitrobenzoic acid. C7H4ClNO4. (Mr 201.6).
Calculate the percentage of ethanol using the following 1183800. [2516-96-3].
expression :
mp : 165 °C to 168 °C.
Chlorophenol. C6H5ClO. (Mr 128.6). 1018900. [106-48-9].
4-Chlorophenol.
Chloroform, acidified. 1018601. Colourless or almost colourless crystals, slightly soluble in
water, very soluble in ethanol (96 per cent) and in solutions of
To 100 mL of chloroform R add 10 mL of hydrochloric
alkali hydroxides.
acid R. Shake, allow to stand and separate the 2 layers.
mp : about 42 °C.
Chloroform, ethanol-free. 1018602.
Chloroplatinic acid. H2Cl6Pt,6H2O. (Mr 517.9). 1019000.
Shake 200 mL of chloroform R with four quantities, each of [18497-13-7]. Hydrogen hexachloroplatinate(IV) hexahydrate.
100 mL, of water R. Dry over 20 g of anhydrous sodium
sulfate R for 24 h. Distil the filtrate over 10 g of anhydrous Content : minimum 37.0 per cent m/m of platinum (Ar 195.1).
sodium sulfate R. Discard the first 20 mL of distillate. Brownish-red crystals or a crystalline mass, very soluble in
Prepare immediately before use. water, soluble in ethanol (96 per cent).
Assay. Ignite 0.200 g to constant mass at 900 ± 50 °C and
Chloroform stabilised with amylene. CHCl3. (Mr 119.4). weigh the residue (platinum).
1018700.
Storage : protected from light.
Clear, colourless liquid, slightly soluble in water, miscible with
ethanol (96 per cent). 3-Chloropropane-1,2-diol. C3H7ClO2. (Mr 110.5). 1097600.
Water : maximum 0.05 per cent. [96-24-2].
Residue on evaporation : maximum 0.001 per cent. Colourless liquid, soluble in water and ethanol (96 per cent).
Minimum transmittance (2.2.25) using water R as : about 1.322.
compensation liquid : 50 per cent at 255 nm, 80 per cent at : about 1.480.
260 nm, 98 per cent at 300 nm. bp : about 213 °C.
General Notices (1) apply to all monographs and other texts 445
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 447
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
85 volumes of toluene R. Allow the plate to dry in air and Citropten. C11H10O4. (Mr 206.2). 1021300. [487-06-9].
examine in ultraviolet light at 254 nm. The chromatogram Limettin. 5,7-Dimethoxy-2H-1-benzopyran-2-one.
shows only one principal spot. Needle-shaped crystals, practically insoluble in water and
Citral used in gas chromatography complies with the following in light petroleum, freely soluble in acetone and in ethanol
additional test. (96 per cent).
Assay. Gas chromatography (2.2.28) as prescribed in the mp : about 145 °C.
monograph Citronella oil (1609). Chromatography. Thin-layer chromatography (2.2.27), using
Content of citral (neral + geranial) : minimum 95.0 per cent, silica gel GF254 R as the coating substance : apply to the plate
calculated by the normalisation procedure. 10 μL of a 1 g/L solution in toluene R. Develop over a path of
15 cm using a mixture of 15 volumes of ethyl acetate R and
Citrated rabbit plasma. 1020900. 85 volumes of toluene R. Allow the plate to dry in air and
Collect blood by intracardiac puncture from a rabbit kept examine in ultraviolet light at 254 nm. The chromatogram
fasting for 12 h, using a plastic syringe with a No. 1 needle obtained shows only one principal spot.
containing a suitable volume of 38 g/L solution of sodium
citrate R so that the final volume ratio of citrate solution to Clobetasol propionate. C25H32ClFO5. (Mr 467.0). 1097700.
blood is 1 : 9. Separate the plasma by centrifugation at 1500 g [25122-46-7]. 21-Chloro-9-fluoro-11β,17-dihydroxy-16β-
to 1800 g at 15 °C to 20 °C for 30 min. methylpregna-1,4-diene-3,20-dione 17-propionate.
Storage : at 0 °C to 6 °C ; use within 4 h of collection. White or almost white crystalline powder, insoluble in water,
soluble in ethanol (96 per cent) and in acetone.
Citric acid. 1021000. [5949-29-1]. : about + 104 (in dioxan).
See Citric acid monohydrate (0456). mp : about 196 °C.
When used in the test for iron, it complies with the following
additional requirement. Coagulation factor V solution. 1021400.
Dissolve 0.5 g in 10 mL of water R, add 0.1 mL of thioglycollic Coagulation factor V solution may be prepared by the
acid R, mix and make alkaline with ammonia R. Dilute to following method or by any other method which excludes
20 mL with water R. No pink colour appears in the solution. factor VIII.
Citric acid, anhydrous. 1021200. [77-92-9]. Prepare the factor V reagent from fresh oxalated bovine
plasma, by fractionation at 4 °C with a saturated solution of
See Anhydrous citric acid (0455). ammonium sulfate R prepared at 4 °C. Separate the fraction
Citronellal. C10H18O. (Mr 154.3). 1113300. [106-23-0]. which precipitates between 38 per cent and 50 per cent
3,7-Dimethyl-6-octenal. of saturation, which contains factor V without significant
Very slightly soluble in water, soluble in ethanol (96 per cent). contamination with factor VIII. Remove the ammonium
sulfate by dialysis and dilute the solution with a 9 g/L solution
: 0.848 to 0.856. of sodium chloride R to give a solution containing between
: about 1.446. 10 per cent and 20 per cent of the quantity of factor V present
Citronellal used in gas chromatography complies with the in fresh human normal plasma.
following additional test. Assay of factor V. Prepare two dilutions of the preparation
Assay. Gas chromatography (2.2.28) as prescribed in the of factor V in imidazole buffer solution pH 7.3 R containing
monograph Citronella oil (1609). 1 volume of the preparation in 10 volumes and in 20 volumes
Content : minimum 95.0 per cent, calculated by the of the buffer solution respectively. Test each dilution as
normalisation procedure. follows : mix 0.1 mL of plasma substrate deficient in factor
V R, 0.1 mL of the solution to be examined, 0.1 mL of
Citronellol. C10H20O. (Mr 156.3). 1134900. [106-22-9]. thromboplastin R and 0.1 mL of a 3.5 g/L solution of calcium
3,7-Dimethyloct-6-en-1-ol. chloride R and measure the coagulation times, i.e. the interval
Clear, colourless liquid, practically insoluble in water, miscible between the moment at which the calcium chloride solution is
with ethanol (96 per cent). added and the first indication of the formation of fibrin, which
: 0.857. may be observed visually or by means of a suitable apparatus.
: 1.456. In the same manner, determine the coagulation time (in
duplicate) of four dilutions of human normal plasma in
bp : 220 °C to 222 °C. imidazole buffer solution pH 7.3 R, containing respectively,
Citronellol used in gas chromatography complies with the 1 volume in 10 (equivalent to 100 per cent of factor V),
following additional test. 1 volume in 50 (20 per cent), 1 volume in 100 (10 per cent),
Assay. Gas chromatography (2.2.28) as prescribed in the and 1 volume in 1000 (1 per cent). Using two-way logarithmic
monograph Citronella oil (1609). paper plot the average coagulation times for each dilution of
Content : minimum 95.0 per cent, calculated by the human plasma against the equivalent percentage of factor V
normalisation procedure. and read the percentage of factor V for the two dilutions of the
Storage : in an airtight container, protected from light. factor V solution by interpolation. The mean of the two results
gives the percentage of factor V in the solution to be examined.
Citronellyl acetate. C12H22O2. (Mr 198.3). 1135000. Storage : in the frozen state at a temperature not higher than
[150-84-5]. 3,7-Dimethyl-6-octen-1-yl acetate. − 20 °C.
: 0.890.
Cobalt chloride. CoCl2,6H2O. (Mr 237.9). 1021600.
: 1.443. [7791-13-1].
bp : 229 °C. Red, crystalline powder or deep-red crystals, very soluble in
Citronellyl acetate used in gas chromatography complies with water, soluble in ethanol (96 per cent).
the following additional test.
Cobalt nitrate. Co(NO3)2,6H2O. (Mr 291.0). 1021700.
Assay. Gas chromatography (2.2.28) as prescribed in the [10026-22-9].
monograph Citronella oil (1609).
Small garnet-red crystals, very soluble in water.
Content : minimum 97.0 per cent, calculated by the
normalisation procedure. Codeine. 1021800. [6059-47-8].
Storage : in an airtight container, protected from light. See Codeine (0076).
Codeine phosphate. 1021900. [52-28-8]. the temperature below 20 °C, add dropwise with continuous
See Codeine phosphate hemihydrate (0074). shaking 30 mL of strong sodium hydroxide solution R.
Filter through a sintered-glass filter (40) (2.1.2), wash with
Congo red. C32H22N6Na2O6S2. (Mr 697). 1022000. [573-58-0]. water R until the filtrate is clear and take up the precipitate
Schultz No. 360. with 200 mL of concentrated ammonia R. Filter through a
Colour Index No. 22120. sintered-glass filter (2.1.2) and repeat the filtration to reduce
Disodium (biphenyl-4,4′-diyl-bis-2,2′-azo)bis(1-amino- the residue to a minimum.
naphthalene-4-sulfonate).
Cortisone. C21H28O5. (Mr 360.4). 1175000. [53-06-5].
Brownish-red powder, soluble in water.
Content : minimum 95.0 per cent.
Congo red paper. 1022002. mp : 223-228 °C.
Immerse strips of filter paper for a few minutes in congo
red solution R. Allow to dry. Cortisone acetate. 1097800. [50-04-4].
See Cortisone acetate (0321).
Congo red solution. 1022001.
Dissolve 0.1 g of congo red R in a mixture of 20 mL of Coumaphos. C14H16ClO5PS. (Mr 362.8). 1124800. [56-72-4].
ethanol (96 per cent) R and water R and dilute to 100 mL mp : 91 °C to 92 °C.
with water R. A suitable certified reference solution (10 ng/μL in iso-octane)
Test for sensitivity. To 0.2 mL of the congo red solution add may be used.
100 mL of carbon dioxide-free water R and 0.3 mL of 0.1 M
hydrochloric acid. The solution is blue. Not more than o-Coumaric acid. C9H8O3. (Mr 164.2). 1157400. [614-60-8].
0.3 mL of 0.1 M sodium hydroxide is required to change (E)-2-Hydroxycinnamic acid. (2E)-3-(2-Hydroxyphenyl)prop-
the colour to pink. 2-enoic acid.
Colour change : pH 3.0 (blue) to pH 5.0 (pink). White or almost white powder.
mp : about 217 °C.
Coomassie blue. 1001400. [3861-73-2].
See acid blue 92 R. p-Coumaric acid. C9H8O3. (Mr 164.2). 1157500. [7400-08-0].
4-Hydroxycinnamic acid. 3-(4-Hydroxyphenyl)-prop-2-enoic
Coomassie blue solution. 1001401. acid.
See acid blue 92 solution R. White or almost white needles, practically insoluble in water,
Coomassie staining solution. 1012201. soluble in acetone and in methanol.
A 1.25 g/L solution of acid blue 83 R in a mixture consisting of mp : 214 °C to 217 °C.
1 volume of glacial acetic acid R, 4 volumes of methanol R and p-Coumaric acid used in the assay in Nettle leaf (1897) complies
5 volumes of water R. Filter. with the following additional tests.
Coomassie staining solution R1. 1173000. Loss on drying (2.2.32) : maximum 5.0 per cent, determined
Dissolve 0.275 g of acid blue 83 R in 200 mL of methanol R. on 0.200 g by drying in an oven at 105 °C for 2 h.
Stir until complete dissolution of the crystals (for about 2 h). Assay. Liquid chromatography (2.2.29) as prescribed in the
Add 750 mL of water R and 50 mL of glacial acetic acid R. Stir monograph Nettle leaf (1897).
overnight (for at least 16 h); filter. Content : minimum 95 per cent, calculated by the
normalisation procedure.
Copper. Cu. (Ar 63.55). 1022100. [7440-50-8].
Cleaned foil, turnings, wire or powder of the pure metal of Coumarin. C9H6O2. (Mr 146.1). 1124900. [91-64-5].
electrolytic grade. 2H-Chromen-2-one. 2H-1-Benzopyran-2-one.
Copper acetate. C4H6CuO4,H2O. (Mr 199.7). 1022200. Colourless, crystalline powder or orthorhombic or rectangular
[142-71-2]. crystals, very soluble in boiling water, soluble in ethanol
(96 per cent). It dissolves in solutions of alkali hydroxides.
Blue-green crystals or powder, freely soluble in boiling water,
soluble in water and in ethanol (96 per cent), slightly soluble mp : 68 °C to 70 °C.
in glycerol (85 per cent). Coumarin used in gas chromatography complies with the
following additional test.
Copper edetate solution. 1022300.
Assay. Gas chromatography (2.2.28) as prescribed in the
To 2 mL of a 20 g/L solution of copper acetate R add 2 mL of monograph Cassia oil (1496).
0.1 M sodium edetate and dilute to 50 mL with water R.
Content : minimum 98.0 per cent, calculated by the
Copper nitrate. Cu(NO3)2,3H2O. (Mr 241.6). 1022400. normalisation procedure.
[10031-43-3]. Chloride dinitrate trihydrate.
Dark blue crystals, hygroscopic, very soluble in water giving a Cresol. C7H8O. (Mr 108.1). 1022700. [95-48-7]. o-Cresol.
strongly acid reaction, freely soluble in ethanol (96 per cent) 2-Methylphenol.
and in dilute nitric acid. Crystals or a super-cooled liquid becoming dark on exposure
Storage : in an airtight container. to light and air, miscible with anhydrous ethanol, soluble
in about 50 parts of water and soluble in solutions of alkali
Copper sulfate. CuSO4,5H2O. (Mr 249.7). 1022500. hydroxides.
[7758-99-8]. : about 1.05.
Blue powder or deep-blue crystals, slowly efflorescent, very : 1.540 to 1.550.
soluble in water, slightly soluble in ethanol (96 per cent).
bp : about 190 °C.
Copper sulfate solution. 1022501. Freezing point (2.2.18) : minimum 30.5 °C.
A 125 g/L solution. Residue on evaporation : maximum 0.1 per cent m/m,
Copper tetrammine, ammoniacal solution of. 1022600. determined by evaporating on a water-bath and drying in an
oven at 100-105 °C.
Dissolve 34.5 g of copper sulfate R in 100 mL of water R and,
whilst stirring, add dropwise concentrated ammonia R until Storage : protected from light, moisture and oxygen.
the precipitate which forms dissolves completely. Keeping Distil before use.
General Notices (1) apply to all monographs and other texts 449
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
α-Cyclodextrin. C36H60O30. (Mr 972). 1176200. p-Cymene. C10H14. (Mr 134.2). 1113400. [99-87-6].
[10016-20-3]. Cyclohexakis-(1→4)-(α-D-glucopyranosyl). 1-Isopropyl-4-methylbenzene.
Cyclomaltohexaose. Alfadex. Colourless liquid, practically insoluble in water, soluble in
ethanol (96 per cent).
β-Cyclodextrin. 1184000. [7585-39-9].
: about 0.858.
See Betadex (1070).
: about 1.4895.
β-Cyclodextrin for chiral chromatography, modified. bp : 175 °C to 178 °C.
1154600. p-Cymene used in gas chromatography complies with the
30 per cent of 2,3-di-O-ethyl-6-O-tert-butyldimethyl- following additional test.
silyl-β-cyclodextrin dissolved in poly(dimethyl)(85)(diphen- Assay. Gas chromatography (2.2.28) as prescribed in the
yl)(15)siloxane R. monograph Peppermint oil (0405).
β-Cyclodextrin for chiral chromatography, modified R1. Test solution. The substance to be examined.
1160700. Content : minimum 96.0 per cent, calculated by the
30 per cent of 2,3-di-O-acetyl-6-O-tert-butylsilyl-β-cyclo- normalisation procedure.
dextrin dissolved in poly(dimethyl)(85)(diphenyl)(15)silox-
ane R. Cynarin. C25H24O12. (Mr 516.4). 1159300. [30964-13-7].
(1α,3α,4α,5β)-1,3-Bis[[3-(3,4-Dihydroxyphenyl)-1-oxo-2-
Cyclohexane. C6H12. (Mr 84.2). 1023900. [110-82-7]. propenyl]oxy]-4,5-dihydroxycyclohexanecarboxylic acid.
Clear, colourless, flammable liquid, practically insoluble in White or almost white amorphous mass, odourless.
water, miscible with organic solvents.
: about 0.78. Cypermethrin. C22H19Cl2NO3. (Mr 416.3). 1125100.
[52315-07-8].
bp : about 80.5 °C.
bp : 170 °C to 195 °C.
Cyclohexane used in spectrophotometry complies with the
following additional test. mp : 60 °C to 80 °C.
Minimum transmittance (2.2.25) using water R as A suitable certified reference solution (10 ng/μL in
compensation liquid : 45 per cent at 220 nm, 70 per cent at cyclohexane) may be used.
235 nm, 90 per cent at 240 nm, 98 per cent at 250 nm. L-Cysteine. C3H7NO2S. (Mr 121.1). 1024200. [52-90-4].
Cyclohexane R1. 1023901. Powder, freely soluble in water, in ethanol (96 per cent) and in
Complies with the requirements prescribed for acetic acid, practically insoluble in acetone.
cyclohexane R with the following additional requirement. Cysteine hydrochloride. 1024300. [7048-04-6].
The fluorescence, measured at 460 nm, under illumination See Cysteine hydrochloride monohydrate (0895).
with an excitant light beam at 365 nm, is not more intense
than that of a solution containing 0.002 ppm of quinine R L-Cystine. C6H12N2O4S2. (Mr 240.3). 1024400. [56-89-3].
in 0.05 M sulfuric acid. White or almost white, crystalline powder, practically
Cyclohexylamine. C6H13N. (Mr 99.2). 1024000. [108-91-8]. insoluble in water and in ethanol (96 per cent). It dissolves in
Cyclohexanamine. dilute solutions of alkali hydroxides.
Colourless liquid, soluble in water, miscible with usual organic : − 218 to − 224, determined in 1 M hydrochloric acid.
solvents. mp : 250 °C, with decomposition.
: about 1.460. Cytosine. C4H5N3O. (Mr 111.1). 1160800. [71-30-7].
bp : 134 °C to 135 °C. Content : minimum 95.0 per cent.
Cyclohexylenedinitrilotetra-acetic acid. C14H22N2O8,H2O. Daidzein. C15H10O4. (Mr 254.2). 1178400. [486-66-8].
(Mr 364.4). 1024100. trans-Cyclohexylene-1,2-dinitrilo- 7-Hydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one.
N,N,N’,N’-tetra-acetic acid.
White or almost white, crystalline powder. Daidzin. C21H20O9. (Mr 416.4). 1178300. [552-66-9].
mp : about 204 °C. Daidzein-7-O-glucoside. 7-(β-D-Glucopyranosyloxy)-3-(4-
hydroxyphenyl)-4H-1-benzopyran-4-one.
Cyclohexylmethanol. C7H14O. (Mr 114.2). 1135200.
[100-49-2]. Cyclohexylcarbinol. Dantron. C14H8O4. (Mr 240.2). 1024500. [117-10-2].
Liquid with a slight odour of camphor, soluble in ethanol 1,8-Dihydroxyanthraquinone. 1,8-Dihydroxyanthracene-9,10-
(96 per cent). dione.
: about 1.464. Crystalline orange powder, practically insoluble in water,
slightly soluble in ethanol (96 per cent), soluble in solutions of
bp : about 185 °C. alkali hydroxides.
3-Cyclohexylpropionic acid. C9H16O2. (Mr 156.2). 1119200. mp : about 195 °C.
[701-97-3]. Dantron used in the sesquiterpenic acids assay in Valerian
Clear liquid. root (0453) complies with the following additional tests.
General Notices (1) apply to all monographs and other texts 451
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Decanal used in gas chromatography complies with the Degree of deuteration : minimum 99.5 per cent.
following additional test. Clear, colourless liquid, miscible with water, with
Assay. Gas chromatography (2.2.28) as prescribed in the dimethylformamide, with anhydrous ethanol and with
monograph Sweet orange oil (1811). methanol.
Content : minimum 97 per cent, calculated by the : about 0.87.
normalisation procedure. : about 1.357.
Decane. C10H22. (Mr 142.3). 1024600. [124-18-5]. bp : about 55 °C.
Colourless liquid, practically insoluble in water. Water and deuterium oxide. Not more than 0.1 per cent.
: about 1.411. Deuterated acetonitrile. C22H3N. (Mr 44.1). 1173100.
bp : about 174 °C. [2206-26-0].
Decanol. C10H22O. (Mr 158.3). 1024700. [112-30-1]. Degree of deuteration : minimum 99.8 per cent.
Decan-1-ol. Clear, colourless liquid, miscible with water, with acetone and
Viscous liquid, solidifying at about 6 °C, practically insoluble with methanol.
in water, soluble in ethanol (96 per cent). : about 0.78.
: about 1.436. : about 1.344.
bp : about 230 °C. Deuterated chloroform. C2HCl3. (Mr 120.4). 1025000.
Deltamethrin. C22H19Br2NO3. (Mr 505.2). 1125800. [865-49-6]. (2H)-Chloroform. Chloroform-d.
[52918-63-5]. Degree of deuteration : minimum 99.7 per cent.
bp : about 300 °C. Clear, colourless liquid, practically insoluble in water, miscible
mp : about 98 °C. with acetone and with ethanol (96 per cent). It may be
A suitable certified reference solution (10 ng/μL in stabilised over silver foil.
cyclohexane) may be used. : about 1.51.
General Notices (1) apply to all monographs and other texts 453
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Diazobenzenesulfonic acid solution R1. 1026500. Dichlorobenzene. C6H4Cl2. (Mr 147.0). 1027100. [95-50-1].
Dissolve 0.9 g of sulfanilic acid R in a mixture of 30 mL of 1,2-Dichlorobenzene.
dilute hydrochloric acid R and 70 mL of water R. To 3 mL of Colourless, oily liquid, practically insoluble in water, soluble
the solution add 3 mL of a 50 g/L solution of sodium nitrite R. in anhydrous ethanol.
Cool in an ice-bath for 5 min, add 12 mL of the sodium nitrite : about 1.31.
solution and cool again. Dilute to 100 mL with water R and
bp : about 180 °C.
keep the reagent in an ice-bath. Prepare extemporaneously
but allow to stand for 15 min before use. 2,4-Dichlorobenzoic acid. C7H4Cl2O2. (Mr 191.0). 1185700.
[50-84-0].
Dibutylamine. C8H19N. (Mr 129.3). 1126000. [111-92-2].
N-Butylbutan-1-amine. Faintly beige powder.
Colourless liquid. mp : about 160 °C.
: about 1.417. 2,3-Dichloro-5,6-dicyanobenzoquinone. C8Cl2N2O2.
bp : about 159 °C. (Mr 227.0). 1153600. [84-58-2]. 4,5-Dichloro-3,6-dioxo-
cyclohexa-1,4-diene-1,2-dicarbonitrile.
Dibutylammonium phosphate for ion-pairing. 1168800. Yellow or orange crystals, soluble in dioxan and in acetic acid,
A colourless solution of 10 per cent to 15 per cent V/V slightly soluble in methylene chloride. It decomposes in water.
of di-n-butylamine and 12 per cent to 17 per cent V/V of mp : about 214 °C.
phosphoric acid in water, suitable for ion-pairing in liquid
chromatography. Storage : at a temperature of 2 °C to 8 °C.
Detector 280
Dicyclohexylamine. C12H23N. (Mr 181.3). 1027500.
[101-83-7]. N,N-Dicyclohexylamine. Detection : flame-ionisation.
Colourless liquid, sparingly soluble in water, miscible with the Injection : 1.0 μL.
usual organic solvents.
Limit :
: about 1.484.
– ethanolamine : maximum 1.0 per cent.
bp : about 256 °C.
Freezing point (2.2.18): 0 °C to 1 °C. Diethoxytetrahydrofuran. C8H16O3. (Mr 160.2). 1027900.
[3320-90-9]. 2,5-Diethoxytetrahydrofuran. A mixture of the
Dicyclohexylurea. C13H24N2O. (Mr 224.4). 1027600. cis and trans isomers.
[2387-23-7]. 1,3-Dicyclohexylurea. Clear, colourless or slightly yellowish liquid, practically
White or almost white, crystalline powder. insoluble in water, soluble in ethanol (96 per cent) and in most
mp : about 232 °C. other organic solvents.
: about 0.98.
Didocosahexaenoin. C47H68O5. (Mr 713.0). 1142700.
[88315-12-2]. Diglyceride of docosahexaenoic acid (C22:6). : about 1.418.
Glycerol didocosahexaenoate. (all-Z)-Docosahexaenoic acid, Diethylamine. C4H11N. (Mr 73.1). 1028000. [109-89-7].
diester with propane-1,2,3-triol. Clear, colourless, flammable liquid, strongly alkaline, miscible
Didodecyl 3,3′-thiodipropionate. C30H58O4S. (Mr 514.8). with water and with ethanol (96 per cent).
1027700. [123-28-4]. : about 0.71.
White or almost white, crystalline powder, practically bp : about 55 °C.
insoluble in water, freely soluble in acetone and in light Diethylamine R1. C4H11N. (Mr 73.1). 1028001. [109-89-7].
petroleum, slightly soluble in ethanol (96 per cent). N-Ethylethanamine.
mp : about 39 °C. Content : minimum 99.5 per cent.
Dieldrin. C12H8Cl6O. (Mr 380.9). 1126200. [60-57-1]. Clear, colourless, flammable liquid, strongly alkaline,
bp : about 385 °C. miscible with water and with ethanol (96 per cent).
mp : about 176 °C. : about 0.71.
A suitable certified reference solution (10 ng/μL in bp : about 55 °C.
cyclohexane) may be used. Diethylaminoethyldextran. 1028200.
Diethanolamine. C4H11NO2. (Mr 105.1). 1027800. Anion-exchange resin presented as the hydrochloride.
[111-42-2]. 2,2′-Iminobisethanol. Powder forming gels with water.
Viscous, clear, slightly yellow liquid or deliquescent crystals N,N-Diethylaniline. C10H15N. (Mr 149.2). 1028400. [91-66-7].
melting at about 28 °C, very soluble in water, in acetone and
in methanol. : about 0.938.
: about 1.09. bp : about 217 °C.
pH (2.2.3) : 10.0 to 11.5 for a 50 g/L solution. mp : about − 38 °C.
Diethanolamine used in the test for alkaline phosphatase Diethylene glycol. C4H10O3. (Mr 106.1). 1028300. [111-46-6].
complies with the following additional test. 2,2′-Oxydiethanol.
Ethanolamine. Gas chromatography (2.2.28). Content : minimum 99.5 per cent m/m.
Internal standard solution. Dissolve 1.00 g of Clear, colourless liquid, hygroscopic, miscible with water, with
3-aminopropanol R in acetone R and dilute to 10.0 mL with acetone and with ethanol (96 per cent).
the same solvent. : about 1.118.
Test solution (a). Dissolve 5.00 g of the substance to be : about 1.447.
examined in acetone R and dilute to 10.0 mL with the same bp : 244 °C to 246 °C.
solvent. Storage : in an airtight container.
Test solution (b). Dissolve 5.00 g of the substance to be
examined in acetone R, add 1.0 mL of the internal standard N,N-Diethylethane-1,2-diamine. 1028500. [100-36-7].
solution and dilute to 10.0 mL with the same solvent. See N,N-diethylethylenediamine R.
General Notices (1) apply to all monographs and other texts 455
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
N,N-Diethylethylenediamine. C6H16N2. (Mr 116.2). 1028500. Content calculated by the normalisation procedure :
[100-36-7]. – major component (trans-dihydrocarvone) : minimum 70 per
Content : minimum 98.0 per cent. cent ;
Slightly oily liquid, colourless or slightly yellow, strong odour – sum of cis- and trans-dihydrocarvone : minimum 98 per
of ammonia, irritant to the skin, eyes and mucous membranes. cent.
: 0.827. 2,5-Dihydroxybenzoic acid. C7H6O4. (Mr 154.1). 1148200.
bp : 145 °C to 147 °C. [490-79-9]. Gentisic acid.
Water (2.5.12) : maximum 1.0 per cent, determined on 0.500 g. Light yellow crystals.
mp : about 200 °C.
Di(2-ethylhexyl) phthalate. C24H38O4. (Mr 390.5). 1028100.
Di(2-ethylhexyl) benzene-1,2-dicarboxylate. 5,7-Dihydroxy-4-methylcoumarin. C10H8O4. (Mr 192.2).
Colourless, oily liquid, practically insoluble in water, soluble 1149400. [2107-76-8]. 5,7-Dihydroxy-4-methyl-2H-1-
in organic solvents. benzopyran-2-one.
: about 0.98. Light yellowish powder, practically insoluble in water,
: about 1.486. sparingly soluble in ethanol (96 per cent).
Viscosity (2.2.9) : about 80 mPa·s. mp : 295 °C to 303 °C.
Dihydroxynaphthalene. 1029000. [132-86-5].
Diethylphenylenediamine sulfate. C10H18N2O4S. (Mr 262.3).
1028600. [6283-63-2]. N,N’-Diethyl-p-phenylenediamine See 1,3-dihydroxynaphthalene R.
sulfate. N,N’-Diethylbenzene-1,4-diamine sulfate. 1,3-Dihydroxynaphthalene. C10H8O2. (Mr 160.2). 1029000.
White or slightly yellow powder, soluble in water. [132-86-5]. Naphthalene-1,3-diol.
mp : about 185 °C, with decomposition. Crystalline, generally brownish-violet powder, freely soluble
Storage : protected from light. in water and in ethanol (96 per cent).
mp : about 125 °C.
Diethylphenylenediamine sulfate solution. 1028601.
To 250 mL of water R add 2 mL of sulfuric acid R and 2,7-Dihydroxynaphthalene. C10H8O2. (Mr 160.2). 1029100.
25 mL of 0.02 M sodium edetate. Dissolve in this solution [582-17-2]. Naphthalene-2,7-diol.
1.1 g of diethylphenylenediamine sulfate R and dilute to Needles, soluble in water and in ethanol (96 per cent).
1000 mL with water R. mp : about 190 °C.
Do not use if the solution is not colourless.
2,7-Dihydroxynaphthalene solution. 1029101.
Storage : protected from light and heat for 1 month. Dissolve 10 mg of 2,7-dihydroxynaphthalene R in 100 mL
Diflubenzuron. C14H9ClF2N2O2. (Mr 310.7). 1180000. of sulfuric acid R and allow to stand until decolorised.
[35367-38-5]. 1-(4-Chlorophenyl)-3-(2,6-difluoro- Storage : use within 2 days.
benzoyl)urea.
5,7-Diiodoquinolin-8-ol. C9H5I2NO. (Mr 397.0). 1157100.
Colourless or white or almost white crystals, practically [83-73-8]. 5,7-Diiodooxine.
insoluble in water, freely soluble in dimethyl sulfoxide, slightly
soluble in acetone. Yellowish-brown powder, sparingly soluble in acetone and in
ethanol (96 per cent).
mp : 230 to 232 °C.
Content : minimum 95.0 per cent.
Digitonin. C56H92O29. (Mr 1229). 1028700. [11024-24-1]. Di-isobutyl ketone. C9H18O. (Mr 142.2). 1029200. [108-83-8].
3β-[O-β-D-Glucopyranosyl-(1→3)-O-β-D-galactopyranosyl-
(1→2)-O-[β-D-xylopyranosyl-(1→3)]-O-β-D-galactopyranosyl- Clear, colourless liquid, slightly soluble in water, miscible with
(1→4)-O-β-D-galactopyranosyloxy]-(25R)-5α-spirostan- most organic solvents.
2α,15β-diol. : about 1.414
Crystals, practically insoluble in water, sparingly soluble in bp : about 168 °C.
anhydrous ethanol, slightly soluble in ethanol (96 per cent).
Di-isopropyl ether. C6H14O. (Mr 102.2). 1029300. [108-20-3].
Digitoxin. 1028800. [71-63-6]. Clear, colourless liquid, very slightly soluble in water, miscible
See Digitoxin (0078). with ethanol (96 per cent).
: 0.723 to 0.728.
Dihydrocapsaicin. C18H29NO3. (Mr 307.4). 1148100.
bp : 67 °C to 69 °C.
[19408-84-5]. N-[(4-Hydroxy-3-methoxyphenyl)methyl]-8-
methylnonanamide. Do not distil if the di-isopropyl ether does not comply with the
test for peroxides.
White or almost white, crystalline powder, practically
insoluble in cold water, freely soluble in anhydrous ethanol. Peroxides. Place 8 mL of potassium iodide and starch solution R
in a 12 mL ground-glass-stoppered cylinder about 1.5 cm in
10,11-Dihydrocarbamazepine. C15H14N2O. diameter. Fill completely with the substance to be examined,
(Mr 238.3). 1028900. [3564-73-6]. 10,11-Dihydro- shake vigorously and allow to stand protected from light for
5H-dibenzo[b,f]azepine-5-carboxamide. 30 min. No colour is produced.
mp : 205 °C to 210 °C. The name and concentration of any added stabiliser are stated
on the label.
Dihydrocarvone. C10H16O. (Mr 152.2). 1160900. Storage : protected from light.
[7764-50-3]. p-Menth-8-en-2-one. 2-Methyl-5-(1-
methylethenyl)cyclohexanone. N,N′-Diisopropylethylenediamine. C8H20N2. (Mr 144.3).
Dihydrocarvone used in gas chromatography complies with the 1140600. [4013-94-9]. N,N′-Bis(1-methylethyl)-1,2-
following additional test. ethanediamine.
Assay. Gas chromatography (2.2.28) as prescribed in the Colourless or yellowish, corrosive, flammable, hygroscopic
test for chromatographic profile in the monograph Caraway liquid.
oil (1817). : about 0.798.
General Notices (1) apply to all monographs and other texts 457
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
1,1-Dimethylethyl methyl ether R1. C5H12O. (Mr 88.1). N,N-Dimethyl- L-phenylalanine. C11H15NO2. (Mr 193.2).
1126400. [1634-04-4]. 2-Methoxy-2-methylpropane. 1164000. [17469-89-5]. (2S)-2-(Dimethylamino)-3-
tert-Butyl methyl ether. phenylpropanoic acid.
Content : minimum 99.5 per cent. mp : about 226 °C.
: about 0.741. Dimethylpiperazine. C6H14N2. (Mr 114.2). 1030700.
: about 1.369. [106-58-1]. 1,4-Dimethylpiperazine.
A colourless liquid, miscible with water and with ethanol
bp : about 55 °C.
(96 per cent).
Dimethylformamide. C3H7NO. (Mr 73.1). 1030300. : about 0.85.
[68-12-2]. : about 1.446.
Clear, colourless neutral liquid, miscible with water and with bp : about 131 °C.
ethanol (96 per cent).
Dimethylstearamide. C20H41NO. (Mr 311.6). 1030800.
: 0.949 to 0.952. N,N-Dimethylstearamide.
bp : about 153 °C. White or almost white solid mass, soluble in many organic
Water (2.5.12): maximum 0.1 per cent. solvents, including acetone.
mp : about 51 °C.
Dimethylformamide diethylacetal. C7H17NO2. (Mr 147.2).
1113600. [1188-33-6]. N,N-Dimethylformamide diethylacetal. Dimethylstearylamide. 1030800.
: about 1.40. See dimethylstearamide R.
bp : 128 °C to 130 °C. Dimethyl sulfone. C2H6O2S. (Mr 94.1). 1030900. [67-71-0].
N,N-Dimethylformamide dimethylacetal. C5H13NO2. White or almost white, crystalline powder, freely soluble in
(Mr 119.2). 1140700. [4637-24-5]. 1,1-Dimethoxytrimethyl- water, soluble in acetone and ethanol (96 per cent).
amine. mp : 108 °C to 110 °C.
Clear, colourless liquid. Dimethyl sulfoxide. 1029500. [67-68-5].
: about 0.896. See Dimethyl sulfoxide (0763).
: about 1.396. Dimethyl sulfoxide used in spectrophotometry complies with
bp : about 103 °C. the following additional test.
General Notices (1) apply to all monographs and other texts 459
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Diphenylamine. C12H11N. (Mr 169.2). 1032100. [122-39-4]. 2,2-Diphenylglycine. C14H13NO2. (Mr 227.26). 1174300.
White or almost white crystals, slightly soluble in water, [3060-50-2]. Amino(diphenyl)acetic acid.
soluble in ethanol (96 per cent). 1,2-Diphenylhydrazine. C12H12N2. (Mr 184.3). 1140800.
mp : about 55 °C. [122-66-7]. Hydrazobenzene. 1,2-Diphenyldiazane.
Storage : protected from light. Orange powder.
Diphenylamine solution. 1032101. mp : about 125 °C.
A 1 g/L solution in sulfuric acid R. Diphenylmethanol. C13H12O. (Mr 184.2). 1145700. [91-01-0].
Storage : protected from light. Benzhydrol.
Diphenylamine solution R1. 1032102. White or almost white, crystalline powder.
A 10 g/L solution in sulfuric acid R. The solution is mp : about 66 °C.
colourless. Diphenyloxazole. C15H11NO. (Mr 221.3). 1032700. [92-71-7].
Diphenylamine solution R2. 1032103. 2,5-Diphenyloxazole.
Dissolve 1 g of diphenylamine R in 100 mL of glacial acetic White or almost white powder, practically insoluble in water,
acid R and add 2.75 mL of sulfuric acid R. Use immediately. soluble in methanol, sparingly soluble in dioxan and in glacial
acetic acid.
Diphenylanthracene. C26H18. (Mr 330.4). 1032200. mp : about 70 °C.
[1499-10-1]. 9,10-Diphenylanthracene.
: about 1260 determined at 305 nm in methanol R.
Yellowish or yellow, crystalline powder, practically insoluble
in water. Diphenyloxazole used for liquid scintillation is of a suitable
mp : about 248 °C. analytical grade.
Disodium hydrogen phosphate. 1033300. [10039-32-4]. water R and 10 mL of a 200 g/L solution of hydroxylamine
See Disodium phosphate dodecahydrate (0118). hydrochloride R. Titrate with the dithizone solution ; after
each addition, shake the mixture twenty times and towards
Disodium hydrogen phosphate solution. 1033301. the end of the titration allow to separate and discard
A 90 g/L solution. the chloroform layer. Titrate until a bluish-green colour
is obtained. Calculate the equivalent in micrograms of
Disodium hydrogen phosphate, anhydrous. Na2HPO4. mercury per millilitre of the dithizone solution from the
(Mr 142.0). 1033400. [7558-79-4]. expression 20/V, where V is the volume in millilitres of the
Disodium hydrogen phosphate dihydrate. 1033500. dithizone solution used in the titration.
[10028-24-7]. Dithizone R1. C13H12N4S. (Mr 256.3). 1105500. [60-10-6].
See Disodium phosphate dihydrate (0602). 1,5-Diphenylthiocarbazone.
Disodium tetraborate. 1033600. [1303-96-4]. Content : minimum 98.0 per cent.
See Borax (0013). Bluish-black, brownish-black or black powder, practically
insoluble in water, soluble in ethanol (96 per cent).
Borate solution. 1033601.
Storage : protected from light.
Dissolve 9.55 g of disodium tetraborate R in sulfuric acid R,
heating on a water-bath, and dilute to 1 L with the same Divanadium pentoxide. V2O5. (Mr 181.9). 1034000.
acid. [1314-62-1]. Vanadic anhydride.
Ditalimphos. C12H14NO4PS. (Mr 299.3). 1126700. Content : minimum 98.5 per cent.
[5131-24-8]. O,O-Diethyl (1,3-dihydro-1,3-dioxo-2H- Yellow-brown or rust-brown powder, slightly soluble in water,
isoindol-2-yl)phosphonothioate. soluble in strong mineral acids and in solutions of alkali
Very slightly soluble in water, in ethyl acetate and in anhydrous hydroxides with formation of salts.
ethanol. Appearance of solution. Heat 1 g for 30 min with 10 mL of
A suitable certified reference solution may be used. sulfuric acid R. Allow to cool and dilute to 10 mL with the
same acid. The solution is clear (2.2.1).
5,5′-Dithiobis(2-nitrobenzoic acid). C14H8N2O8S2. Sensitivity to hydrogen peroxide. Dilute 1.0 mL of the solution
(Mr 396.4). 1097300. [69-78-3]. 3-Carboxy-4- prepared for the test for appearance of solution cautiously to
nitrophenyldisulfide. Ellman’s reagent. DTNB. 50.0 mL with water R. To 0.5 mL of the solution add 0.1 mL
Yellow powder sparingly soluble in ethanol (96 per cent). of a solution of hydrogen peroxide R (0.1 g/L of H2O2). The
mp : about 242 °C. solution has a distinct orange colour compared with a blank
prepared from 0.5 mL of the solution to be examined and
Dithiol. C7H8S2. (Mr 156.3). 1033800. [496-74-2]. 0.1 mL of water R. After the addition of 0.4 mL of hydrogen
Toluene-3,4-dithiol. 4-Methylbenzene-1,2-dithiol. peroxide solution (0.1 g/L H2O2), the orange solution becomes
White or almost white crystals, hygroscopic, soluble in orange-yellow.
methanol and in solutions of alkali hydroxides. Loss on ignition : maximum 1.0 per cent, determined on 1.00 g
mp : about 30 °C. at 700 ± 50 °C.
Storage : in an airtight container. Assay. Dissolve 0.200 g with heating in 20 mL of a 70 per
cent m/m solution of sulfuric acid R. Add 100 mL of water R
Dithiol reagent. 1033801. and 0.02 M potassium permanganate until a reddish colour is
To 1 g of dithiol R add 2 mL of thioglycollic acid R and dilute obtained. Decolorise the excess of potassium permanganate
to 250 mL with a 20 g/L solution of sodium hydroxide R. by the addition of a 30 g/L solution of sodium nitrite R. Add
Prepare immediately before use. 5 g of urea R and 80 mL of a 70 per cent m/m solution of
Dithiothreitol. C4H10O2S2. (Mr 154.2). 1098200. sulfuric acid R. Cool. Using 0.1 mL of ferroin R as indicator,
[27565-41-9]. threo-1,4-Dimercaptobutane-2,3-diol. titrate the solution immediately with 0.1 M ferrous sulfate
until a greenish-red colour is obtained.
Slightly hygroscopic needles, freely soluble in water, in acetone
and in anhydrous ethanol. 1 mL of 0.1 M ferrous sulfate is equivalent to 9.095 mg of V2O5.
Storage : in an airtight container. Divanadium pentoxide solution in sulfuric acid.
1034001.
Dithizone. C13H12N4S. (Mr 256.3). 1033900. [60-10-6].
1,5-Diphenylthiocarbazone. Dissolve 0.2 g of divanadium pentoxide R in 4 mL of
sulfuric acid R and dilute to 100 mL with water R.
A bluish-black, brownish-black or black powder, practically
insoluble in water, soluble in ethanol (96 per cent). Docosahexaenoic acid methyl ester. C23H34O2. (Mr 342.5).
Storage : protected from light. 1142800. [301-01-9]. DHA methyl ester. Cervonic acid
methyl ester. (all-Z)-Docosa-4,7,10,13,16,19-hexaenoic acid
Dithizone solution. 1033901. methyl ester.
A 0.5 g/L solution in chloroform R. Prepare immediately Content : minimum 90.0 per cent, determined by gas
before use. chromatography.
Dithizone solution R2. 1033903. Docusate sodium. 1034100. [577-11-7].
Dissolve 40.0 mg of dithizone R in chloroform R and dilute See Docusate sodium (1418).
to 1000.0 mL with the same solvent. Dilute 30.0 mL of the
solution to 100.0 mL with chloroform R. Dodecyltrimethylammonium bromide. C15H34BrN.
Assay. Dissolve a quantity of mercuric chloride R equivalent (Mr 308.4). 1135500. [1119-94-4]. N,N,N-Trimethyldodecan-
to 0.1354 g of HgCl2 in a mixture of equal volumes of dilute 1-aminium bromide.
sulfuric acid R and water R and dilute to 100.0 mL with the White or almost white crystals.
same mixture of solvents. Dilute 2.0 mL of this solution mp : about 246 °C.
to 100.0 mL with a mixture of equal volumes of dilute
sulfuric acid R and water R. (This solution contains 20 ppm D-Dopa. C9H11NO4. (Mr 197.2). 1164100. [5796-17-8].
of Hg). Transfer 1.0 mL of the solution to a separating (2R)-2-Amino-3-(3,4-dihydroxyphenyl)propanoic acid.
funnel and add 50 mL of dilute sulfuric acid R, 140 mL of 3-Hydroxy-D-tyrosine. 3,4-Dihydroxy-D-phenylalanine.
General Notices (1) apply to all monographs and other texts 461
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
: + 9.5 to + 11.5, determined on a 10 g/L solution in 1 M β-Endosulfan. C9H6Cl6O3S. (Mr 406.9). 1126900.
hydrochloric acid. [33213-65-9].
mp : about 277 °C. bp : about 390 °C.
mp : about 207 °C.
Dotriacontane. C32H66. (Mr 450.9). 1034200. [544-85-4].
n-Dotriacontane. A suitable certified reference solution (10 ng/μL in
cyclohexane) may be used.
White or almost white plates, practically insoluble in water,
sparingly soluble in hexane. Endrin. C12H8Cl6O. (Mr 380.9). 1127000. [72-20-8].
mp : about 69 °C. A suitable certified reference solution (10 ng/μL in
Impurities. Not more than 0.1 per cent of impurities with cyclohexane) may be used.
the same tR value as α-tocopherol acetate, determined by the Erucamide. C22H43NO. (Mr 337.6). 1034500. [112-84-5].
gas chromatographic method prescribed in the monograph (Z)-Docos-13-enoamide.
α-Tocopherol acetate (0439).
Yellowish or white powder or granules, practically insoluble
Doxycycline. 1145800. in water, very soluble in methylene chloride, soluble in
anhydrous ethanol.
See Doxycycline monohydrate (0820).
mp : about 70 °C.
Echinacoside. C35H46O20. (Mr 786.5). 1159400. [82854-37-3]. Erythritol. 1113800. [149-32-6].
β-(3′,4′-Dihydroxyphenyl)-ethyl-O-α-L-rhamnopyranosyl
(1→3)-O-β-D-[β-D-glucopyranosyl(1→6)]-(4-O-caffeoyl)- See Erythritol (1803).
glucopyranoside. Esculetin. C9H6O4. (Mr 178.1). 1185800. [305-01-1].
Pale yellow powder, odourless. 6,7-Dihydroxy-2H-1-benzopyran-2-one. Aesculetin.
Edotreotide. C65H92N14O18S2. (Mr 1422). 1182400. Esculin. C15H16O9,11/2H2O. (Mr 367.3). 1119400. [531-75-9].
[204318-14-9]. N-[[4,7,10-Tris(carboxymethyl)-1,4,7,10- 6-(β-D-Glucopyranosyloxy)-7-hydroxy-2H-chromen-2-one.
tetraazacyclododecan-1-yl]acetyl]-D-phenylalanyl-L-cysteinyl- White or almost white powder or colourless crystals, sparingly
L-tyrosyl-D-tryptophyl-L-lysyl-L-threonyl-N-[(1R,2R)-2- soluble in water and in ethanol (96 per cent), freely soluble in
hydroxy-1-(hydroxymethyl)propyl]-L-cysteinamide cyclic hot water and in hot ethanol (96 per cent).
(2→7)-disulfide. DOTATOC. DOTA-[Tyr3]-octreotide. Chromatography (2.2.27). Thin-layer chromatography (2.2.27)
White or almost white powder. as prescribed in the monograph Eleutherococcus (1419) ; the
Content : minimum 95.0 per cent. chromatogram shows only one principal spot.
Electrolyte reagent for the micro determination of water. Estradiol. C18H24O2. (Mr 272.4). 1135600. [50-28-2].
1113700. Estra-1,3,5(10)-triene-3,17β-diol. β-Estradiol.
Commercially available anhydrous reagent or a combination Prisms stable in air, practically insoluble in water, freely
of anhydrous reagents for the coulometric titration of water, soluble in ethanol (96 per cent), soluble in acetone and in
containing suitable organic bases, sulfur dioxide and iodide dioxane, sparingly soluble in vegetable oils.
dissolved in a suitable solvent. mp : 173 °C to 179 °C.
Elementary standard solution for atomic spectrometry 17α-Estradiol. C18H24O2. (Mr 272.4). 1034600. [57-91-0].
(1.000 g/L). 5004000. White or almost white, crystalline powder or colourless
This solution is prepared, generally in acid conditions, from crystals.
the element or a salt of the element whose minimum content mp : 220 °C to 223 °C.
is not less than 99.0 per cent. The quantity per litre of solution
is greater than 0.995 g throughout the guaranteed period, as Estragole. C10H12O. (Mr 148.2). 1034700. [140-67-0].
long as the vial has not been opened. The starting material 1-Methoxy-4-prop-2-enylbenzene.
(element or salt) and the characteristics of the final solvent Liquid, miscible with ethanol (96 per cent).
(nature and acidity, etc.) are mentioned on the label. : about 1.52.
bp : about 216 °C.
Emetine dihydrochloride. 1034300. [316-42-7].
Estragole used in gas chromatography complies with the
See Emetine hydrochloride pentahydrate (0081). following test.
Emodin. C15H10O5. (Mr 270.2). 1034400. [518-82-1]. Assay. Gas chromatography (2.2.28) as prescribed in the
1,3,8-Trihydroxy-6-methylanthraquinone. monograph Anise oil (0804).
Orange-red needles, practically insoluble in water, soluble in Test solution. The substance to be examined.
ethanol (96 per cent) and in solutions of alkali hydroxides. Content : minimum 98.0 per cent, calculated by the
Chromatography. Thin-layer chromatography (2.2.27) normalisation procedure.
as prescribed in the monograph Rhubarb (0291) ; the Ethanol. 1034800. [64-17-5].
chromatogram shows only one principal spot.
See Ethanol, anhydrous R.
Endoprotease LysC. 1173200. Ethanol, anhydrous. 1034800. [64-17-5].
Microbial extracellular proteolytic enzyme secreted by See Ethanol, anhydrous (1318).
Achromobacter lyticus. A lyophilised powder, free of salts.
Ethanol R1. 1034801.
α-Endosulfan. C9H6Cl6O3S. (Mr 406.9). 1126800. [959-98-8].
Complies with the requirements prescribed for the
bp : about 200 °C. monograph Ethanol, anhydrous (1318) with the following
mp : about 108 °C. additional requirement.
A suitable certified reference solution (10 ng/μL in Methanol. Gas chromatography (2.2.28).
cyclohexane) may be used. Test solution. The substance to be examined.
Reference solution. Dilute 0.50 mL of anhydrous methanol R Storage : in an airtight container, protected from light, at a
to 100.0 mL with the substance to be examined. Dilute temperature not exceeding 15 °C.
1.0 mL of this solution to 100.0 mL with the substance to
be examined. Ether, peroxide-free. 1035100.
Column : See Anaesthetic ether (0367).
– material : glass ; Ethion. C9H22O4P2S4. (Mr 384.5). 1127100. [563-12-2].
– size : l = 2 m, Ø = 2 mm ; mp : − 24 °C to − 25 °C.
– stationary phase : ethylvinylbenzene-divinylbenzene A suitable certified reference solution (10 ng/μL in
copolymer R (75-100 μm). cyclohexane) may be used.
Carrier gas : nitrogen for chromatography R.
Ethoxychrysoidine hydrochloride. C14H17ClN4O. (Mr 292.8).
Flow rate : 30 mL/min. 1035200. [2313-87-3]. 4-[(4-Ethoxyphenyl)diazenyl]phenyl-
Temperature : ene-1,3-diamine hydrochloride.
– column : 130 °C ; Reddish powder, soluble in ethanol (96 per cent).
– injection port : 150 °C ;
Ethoxychrysoidine solution. 1035201.
– detector : 200 °C.
A 1 g/L solution in ethanol (96 per cent) R.
Detection : flame-ionisation.
Test for sensitivity. To a mixture of 5 mL of dilute
Injection : 1 μL of the test solution and 1 μL of the reference hydrochloric acid R and 0.05 mL of the ethoxy-chrysoidine
solution, alternately, three times. solution add 0.05 mL of 0.0167 M bromide-bromate. The
After each chromatography, heat the column to 230 °C colour changes from red to light yellow within 2 min.
for 8 min. Integrate the methanol peak. Calculate
the percentage methanol content from the following Ethyl acetate. C4H8O2. (Mr 88.1). 1035300. [141-78-6].
expression : Clear, colourless liquid, soluble in water, miscible with ethanol
(96 per cent).
: 0.901 to 0.904.
bp : 76 °C to 78 °C.
a = percentage V/V content of methanol in the Ethyl acetate, treated. 1035301.
reference solution,
Disperse 200 g of sulfamic acid R in ethyl acetate R and
b = area of the methanol peak in the chromatogram make up to 1000 mL with the same solvent. Stir the
obtained with the test solution, suspension obtained for three days and filter through a
c = area of the methanol peak in the chromatogram filter paper.
obtained with the reference solution. Storage : use within 1 month.
Limit :
Ethyl acrylate. C5H8O2. (Mr 100.1). 1035400. [140-88-5].
– methanol : maximum 0.005 per cent V/V. Ethyl prop-2-enoate.
Ethanol (96 per cent). 1002500. [64-17-5]. Colourless liquid.
See Ethanol (96 per cent) (1317). : about 0.924.
Ethanol (x per cent V/V). 1002502. : about 1.406.
Mix appropriate volumes of water R and ethanol (96 per bp : about 99 °C.
cent) R, allowing for the effects of warming and volume mp : about − 71 °C.
contraction inherent to the preparation of such a mixture, 4-[(Ethylamino)methyl]pyridine. C8H12N2. (Mr 136.2).
to obtain a solution whose final content of ethanol 1101300. [33403-97-3].
corresponds to the value of x.
Pale yellow liquid.
Ethanolamine. C2H7NO. (Mr 61.1). 1034900. [141-43-5]. : about 0.98.
2-Aminoethanol. : about 1.516.
Clear, colourless, viscous, hygroscopic liquid, miscible with
bp : about 98 °C.
water and with methanol.
: about 1.04. Ethylbenzene. C8H10. (Mr 106.2). 1035800. [100-41-4].
: about 1.454. Content : minimum 99.5 per cent m/m, determined by gas
mp : about 11 °C. chromatography.
Storage : in an airtight container. Clear, colourless liquid, practically insoluble in water, soluble
in acetone, and in ethanol (96 per cent).
Ether. C4H10O. (Mr 74.1). 1035000. [60-29-7]. : about 0.87.
Clear, colourless, volatile and very mobile liquid, very : about 1.496.
flammable, hygroscopic, soluble in water, miscible with
ethanol (96 per cent). bp : about 135 °C.
: 0.713 to 0.715. Ethyl benzoate. C9H10O2. (Mr 150.2). 1135700. [93-89-0].
bp : 34 °C to 35 °C. A clear, colourless, refractive liquid, practically insoluble
Do not distil if the ether does not comply with the test for in water, miscible with ethanol (96 per cent) and with light
peroxides. petroleum.
Peroxides. Place 8 mL of potassium iodide and starch solution R : about 1.050.
in a 12 mL ground-glass-stoppered cylinder about 1.5 cm in : about 1.506.
diameter. Fill completely with the substance to be examined, bp : 211 °C to 213 °C.
shake vigorously and allow to stand in the dark for 30 min.
No colour is produced. Ethyl 5-bromovalerate. C7H13BrO2. (Mr 209.1). 1142900.
The name and concentration of any added stabilisers are [14660-52-7]. Ethyl 5-bromopentanoate.
stated on the label. Clear, colourless liquid.
General Notices (1) apply to all monographs and other texts 463
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
: about 1.321. Clear, colourless liquid, miscible with water, with acetone and
bp : 104 °C to 109 °C. with ethanol (96 per cent).
: about 0.97.
Ethyl cyanoacetate. C5H7NO2. (Mr 113.1). 1035500.
: about 1.403.
[105-56-6].
bp : about 125 °C.
Colourless or pale yellow liquid, slightly soluble in water,
miscible with ethanol (96 per cent). Ethylene oxide. C2H4O. (Mr 44.05). 1036400. [75-21-8].
bp : 205 °C to 209 °C, with decomposition. Oxirane.
Colourless, flammable gas, very soluble in water and in
Ethylene chloride. C2H4Cl2. (Mr 99.0). 1036000. [107-06-2]. anhydrous ethanol.
1,2-Dichloroethane.
Liquefaction point : about 12 °C.
Clear, colourless liquid, soluble in about 120 parts of water
and in 2 parts of ethanol (96 per cent). Ethylene oxide solution. 1036402.
: about 1.25. Weigh a quantity of cool ethylene oxide stock solution R
Distillation range (2.2.11). Not less than 95 per cent distils equivalent to 2.5 mg of ethylene oxide into a cool flask and
between 82 °C and 84 °C. dilute to 50.0 g with macrogol 200 R1. Mix well and dilute
2.5 g of this solution to 25.0 mL with macrogol 200 R1
Ethylenediamine. C2H8N2. (Mr 60.1). 1036500. [107-15-3]. (5 μg of ethylene oxide per gram of solution). Prepare
Ethane-1,2-diamine. immediately before use.
Clear, colourless, fuming liquid, strongly alkaline, miscible The solution can be prepared using commercially available
with water and with ethanol (96 per cent). reagents instead of ethylene oxide stock solution R, making
bp : about 116 °C. appropriate dilutions.
into 50 mL of macrogol 200 R1. Determine the absorbed Ethyl 4-hydroxybenzoate. 1035700. [120-47-8].
quantity of ethylene oxide by weighing before and after See Ethyl parahydroxybenzoate R.
absorption (Meo). Dilute to 100.0 mL with macrogol 200 R1.
Mix well before use. N-Ethylmaleimide. C6H7NO2. (Mr 125.1). 1036700.
Assay. To 10 mL of a 500 g/L suspension of magnesium [128-53-0]. 1-Ethyl-1H-pyrrole-2,5-dione.
chloride R in anhydrous ethanol R add 20.0 mL of 0.1 M Colourless crystals, sparingly soluble in water, freely soluble
alcoholic hydrochloric acid in a flask. Stopper and shake to in ethanol (96 per cent).
obtain a saturated solution and allow to stand overnight to mp : 41 °C to 45 °C.
equilibrate. Weigh 5.00 g of ethylene oxide stock solution Storage : at a temperature of 2 °C to 8 °C.
(2.5 g/L) into the flask and allow to stand for 30 min. Titrate
with 0.1 M alcoholic potassium hydroxide determining the Ethyl methanesulfonate. C3H8O3S. (Mr 124.2). 1179300.
end-point potentiometrically (2.2.20). [62-50-0].
Carry out a blank titration, replacing the substance to be Clear, colourless liquid.
examined with the same quantity of macrogol 200 R1. Content : minimum 99.0 per cent.
Ethylene oxide content in milligrams per gram is given by : Density : about 1.206 g/cm3 (20 °C).
: about 1.418.
bp : about 213 °C.
V0, V1 = volumes of 0.1 M alcoholic potassium Ethyl methyl ketone. 1054100. [78-93-3].
hydroxide used respectively for the blank See methyl ethyl ketone R.
titration and the assay,
2-Ethyl-2-methylsuccinic acid. C7H12O4. (Mr 160.2).
f = factor of the alcoholic potassium hydroxide 1036800. [631-31-2]. 2-Ethyl-2-methylbutanedioic acid.
solution,
mp : 104 °C to 107 °C.
m = mass of the sample taken (g).
Ethyl parahydroxybenzoate. 1035700. [120-47-8].
Ethylene oxide stock solution R1. 1036406. See Ethyl parahydroxybenzoate (0900).
A 50 g/L solution of ethylene oxide R in methanol R.
2-Ethylpyridine. C7H9N. (Mr 107.2). 1133400. [100-71-0].
Either use a commercially available reagent or prepare the
solution corresponding to the aforementioned composition. Colourless or brownish liquid.
: about 0.939.
Ethylene oxide stock solution R2. 1036408. : about 1.496.
A 50 g/L solution of ethylene oxide R in methylene bp : about 149 °C.
chloride R.
Either use a commercially available reagent or prepare the Ethylvinylbenzene-divinylbenzene copolymer. 1036900.
solution corresponding to the aforementioned composition. Porous, rigid, cross-linked polymer beads. Several grades are
available with different sizes of bead. The size range of the
Ethyl formate. C3H6O2. (Mr 74.1). 1035600. [109-94-4]. Ethyl beads is specified after the name of the reagent in the tests
methanoate. where it is used.
Clear, colourless, flammable liquid, freely soluble in water,
miscible with ethanol (96 per cent). Ethylvinylbenzene-divinylbenzene copolymer R1. 1036901.
: about 0.919. Porous, rigid, cross-linked polymer beads, with a nominal
: about 1.36. specific surface area of 500 m2/g to 600 m2/g and having pores
with a mean diameter of 7.5 nm. Several grades are available
bp : about 54 °C. with different sizes of beads. The size range of the beads is
2-Ethylhexane-1,3-diol. C8H18O2. (Mr 146.2). 1105900. specified after the name of the reagent in the tests where it
[94-96-2]. is used.
Slightly oily liquid, soluble in anhydrous ethanol, 2-propanol, Eugenol. C10H12O2. (Mr 164.2). 1037000. [97-53-0].
propylene glycol and castor oil. 4-Allyl-2-methoxyphenol.
: about 0.942. Colourless or pale yellow, oily liquid, darkening on exposure
: about 1.451. to air and light and becoming more viscous, practically
bp : about 244 °C. insoluble in water, miscible with ethanol (96 per cent) and
with fatty and essential oils.
2-Ethylhexanoic acid. C8H16O2. (Mr 144.2). 1036600. : about 1.07.
[149-57-5].
bp : about 250 °C.
Colourless liquid.
Eugenol used in gas chromatography complies with the
: about 0.91. following additional test.
: about 1.425. Assay. Gas chromatography (2.2.28) as prescribed in the
Related substances. Gas chromatography (2.2.28). monograph Clove oil (1091).
Injection : 1 μL of the test solution. Test solution. The substance to be examined.
Test solution : suspend 0.2 g of the 2-ethylhexanoic acid in Content : minimum 98.0 per cent, calculated by the
5 mL of water R, add 3 mL of dilute hydrochloric acid R and normalisation procedure.
5 mL of hexane R, shake for 1 min, allow the layers to separate Storage : protected from light.
and use the upper layer. Carry out the chromatographic
procedure as prescribed in the test for 2-ethylhexanoic acid in Euglobulins, bovine. 1037100.
the monograph on Amoxicillin sodium (0577). Use fresh bovine blood collected into an anticoagulant
Limit : the sum of the area of any peaks, apart from the solution (for example, sodium citrate solution). Discard any
principal peak and the peak due to the solvent, is not greater haemolysed blood. Centrifuge at 1500-1800 g at 15-20 °C to
than 2.5 per cent of the area of the principal peak. obtain a supernatant plasma poor in platelets.
General Notices (1) apply to all monographs and other texts 465
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
To 1 L of bovine plasma add 75 g of barium sulfate R and in a water-bath at 37 °C introduce 0.1 mL of a solution of a
shake for 30 min. Centrifuge at not less than 1500-1800 g at reference preparation of streptokinase containing 10 IU of
15-20 °C and draw off the clear supernatant. Add 10 mL of a streptokinase activity per millilitre and 0.1 mL of a solution of
0.2 mg/mL solution of aprotinin R and shake to ensure mixing. human thrombin R containing 20 IU/mL. Add rapidly 1 mL
In a container with a minimum capacity of 30 L in a chamber of a solution containing 10 mg of human euglobulins per
at 4 °C introduce 25 L of distilled water R at 4 °C and add about millilitre. A firm clot forms in less than 10 s. Note the time
500 g of solid carbon dioxide. Immediately add, while stirring, that elapses between the addition of the solution of human
the supernatant obtained from the plasma. A white precipitate euglobulins and the lysis of the clot. The lysis time does not
is formed. Allow to settle at 4 °C for 10-15 h. Remove the clear exceed 15 min.
supernatant solution by siphoning. Collect the precipitate by Storage : in an airtight container at 4 °C ; use within 1 year.
centrifuging at 4 °C. Suspend the precipitate by dispersing
mechanically in 500 mL of distilled water R at 4 °C, shake Factor VII-deficient plasma. 1185900.
for 5 min and collect the precipitate by centrifuging at 4 °C. Plasma that is deficient in factor VII.
Disperse the precipitate mechanically in 60 mL of a solution
containing 9 g/L of sodium chloride R and 0.9 g/L sodium Factor Xa, bovine, coagulation. 1037300. [9002-05-5].
citrate R and adjust to pH 7.2-7.4 by adding a 10 g/L solution An enzyme which converts prothrombin to thrombin. The
of sodium hydroxide R. Filter through a sintered glass filter semi-purified preparation is obtained from liquid bovine
(2.1.2) ; to facilitate the dissolution of the precipitate crush the plasma and it may be prepared by activation of the zymogen
particles of the precipitate with a suitable instrument. Wash factor X with a suitable activator such as Russell’s viper venom.
the filter and the instrument with 40 mL of the chloride-citrate Storage : freeze-dried preparation at − 20 °C and frozen
solution described above and dilute to 100 mL with the same solution at a temperature lower than − 20 °C.
solution. Freeze-dry the solution. The yields are generally 6 g
to 8 g of euglobulins per litre of bovine plasma. Factor Xa solution, bovine. 1037301.
Test for suitability. For this test, prepare the solutions using Reconstitute as directed by the manufacturer and dilute
phosphate buffer solution pH 7.4 R containing 30 g/L of bovine with tris(hydroxymethyl)aminomethane sodium chloride
albumin R. buffer solution pH 7.4 R.
Into a test-tube 8 mm in diameter placed in a water-bath at Any change in the absorbance of the solution, measured at
37 °C introduce 0.2 mL of a solution of a reference preparation 405 nm (2.2.25) against tris(hydroxymethyl)aminomethane
of urokinase containing 100 IU/mL and 0.1 mL of a solution of sodium chloride buffer solution pH 7.4 R and from which
human thrombin R containing 20 IU/mL. Add rapidly 0.5 mL the blank absorbance has been substracted, is not more
of a solution containing 10 mg of bovine euglobulins per than 0.20 per minute.
millilitre. A firm clot forms in less than 10 s. Note the time
that elapses between the addition of the solution of bovine Factor Xa solution, bovine R1. 1037302.
euglobulins and the lysis of the clot. The lysis time does not Reconstitute as directed by the manufacturer and dilute to
exceed 15 min. 1.4 nkat/mL with tris(hydroxymethyl)aminomethane-EDTA
Storage : protected from moisture at 4 °C ; use within 1 year. buffer solution pH 8.4 R.
Ferric chloride. FeCl3,6H2O. (Mr 270.3). 1037800. Ferrous sulfate. 1038300. [7782-63-0].
[10025-77-1]. Iron trichloride hexahydrate. See Ferrous sulfate heptahydrate (0083).
Yellowish-orange or brownish crystalline masses, deliquescent, Ferrous sulfate solution R2. 1038301.
very soluble in water, soluble in ethanol (96 per cent). On Dissolve 0.45 g of ferrous sulfate R in 50 mL of 0.1 M
exposure to light, ferric chloride and its solutions are partly hydrochloric acid and dilute to 100 mL with carbon
reduced. dioxide-free water R. Prepare immediately before use.
Storage : in an airtight container.
Ferulic acid. C10H10O4. (Mr 194.2). 1149500.
Ferric chloride solution R1. 1037801. [1135-24-6]. 4-Hydroxy-3-methoxycinnamic acid.
A 105 g/L solution. 3-(4-Hydroxy-3-methoxyphenyl)propenoic acid.
Faint yellow powder, freely soluble in methanol.
Ferric chloride solution R2. 1037802.
mp : 172.9 °C to 173.9 °C.
A 13 g/L solution.
Ferulic acid used in the assay of eleutherosides in
Ferric chloride solution R3. 1037803. Eleutherococcus (1419) complies with the following additional
test.
Dissolve 2.0 g of ferric chloride R in anhydrous ethanol R
and dilute to 100.0 mL with the same solvent. Assay. Liquid chromatography (2.2.29) as prescribed in the
monograph Eleutherococcus (1419).
Ferric chloride-ferricyanide-arsenite reagent. 1037805. Content : minimum 99 per cent, calculated by the
Immediately before use mix 10 mL of a 27 g/L solution normalisation procedure.
of ferric chloride R in dilute hydrochloric acid R, 7 mL of
potassium ferricyanide solution R, 3 mL of water R and Fibrin blue. 1101400.
10 mL of sodium arsenite solution R. Mix 1.5 g of fibrin with 30 mL of a 5 g/L solution of indigo
carmine R in 1 per cent V/V dilute hydrochloric acid R.
Ferric chloride-sulfamic acid reagent. 1037804. Heat the mixture to 80 °C and maintain at this temperature
A solution containing 10 g/L of ferric chloride R and 16 g/L whilst stirring for about 30 min. Allow to cool. Filter.
of sulfamic acid R. Wash extensively by resuspension in 1 per cent V/V dilute
hydrochloric acid R and mixing for about 30 min ; filter. Repeat
Ferric nitrate. Fe(NO3)3,9H2O. (Mr 404). 1106100. the washing operation three times. Dry at 50 °C. Grind.
[7782-61-8].
Fibrin congo red. 1038400.
Content : minimum 99.0 per cent m/m of Fe(NO3)3,9H2O.
Take 1.5 g of fibrin and leave overnight in 50 mL of a 20 g/L
Light-purple crystals or crystalline mass, very soluble in water. solution of congo red R in ethanol (90 per cent V/V) R. Filter,
Free acid : not more than 0.3 per cent (as HNO3). rinse the fibrin with water R and store under ether R.
General Notices (1) apply to all monographs and other texts 467
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
hydroxide solution R and add 1 mL to 2 mL in excess. Filter It loses its water of crystallisation at 120 °C.
the precipitate through a sintered-glass filter (40) (2.1.2) mp : about 260 °C, with decomposition.
and wash with water R. Dissolve the precipitate in 70 mL of Chromatography. Thin-layer chromatography (2.2.27) as
methanol R, previously heated to boiling, and add 300 mL of prescribed in the monograph Bearberry leaf (1054) ; the
water R at 80 °C. Allow to cool to room temperature, filter chromatogram shows only one principal spot.
and dry the crystals in vacuo.
Crystals with a greenish-bronze sheen, soluble in water and in Gallium (68Ga) chloride solution. 68GaCl3. (Mr 174.3).
ethanol (96 per cent). 1182500.
Storage : protected from light. Solution containing gallium-68 in the form of gallium chloride
in dilute hydrochloric acid R.
Fuchsin solution, decolorised. 1039401. Content : 90 per cent to 110 per cent of the declared gallium-68
Dissolve 0.1 g of basic fuchsin R in 60 mL of water R. Add radioactivity at the date and time stated on the label.
a solution containing 1 g of anhydrous sodium sulfite R or Gastric juice, artificial. 1039900.
2 g of sodium sulfite R in 10 mL of water R. Slowly and with
Dissolve 2.0 g of sodium chloride R and 3.2 g of pepsin
continuous shaking add 2 mL of hydrochloric acid R. Dilute
powder R in water R. Add 80 mL of 1 M hydrochloric acid and
to 100 mL with water R. Allow to stand protected from
dilute to 1000 mL with water R.
light for at least 12 h, decolorise with activated charcoal R
and filter. If the solution becomes cloudy, filter before use. GC concentrical column. 1135100.
If on standing the solution becomes violet, decolorise again A commercially available system consisting of 2 concentrically
by adding activated charcoal R. arranged tubes. The outer tube is packed with molecular
Test for sensitivity. To 1.0 mL add 1.0 mL of water R and sieves and the inner tube is packed with a porous polymer
0.1 mL of aldehyde-free alcohol R. Add 0.2 mL of a solution mixture. The main application is the separation of gases.
containing 0.1 g/L of formaldehyde (CH2O, Mr 30.0). A
pale-pink colour develops within 5 min. Gelatin. 1040000. [9000-70-8].
See Gelatin (0330).
Storage : protected from light.
Gelatin, hydrolysed. 1040100.
Fuchsin solution, decolorised R1. 1039402.
Dissolve 50 g of gelatin R in 1000 mL of water R. Autoclave in
To 1 g of basic fuchsin R add 100 mL of water R. Heat to saturated steam at 121 °C for 90 min and freeze dry.
50 °C and allow to cool with occasional shaking. Allow to
stand for 48 h, shake and filter. To 4 mL of the filtrate addGeraniol. C10H18O. (Mr 154.2). 1135900. [106-24-1].
6 mL of hydrochloric acid R, mix and dilute to 100 mL with (E)-3,7-Dimethylocta-2,6-dien-1-ol.
water R. Allow to stand for at least 1 h before use. Oily liquid, slight odour of rose, practically insoluble in water,
miscible with ethanol (96 per cent).
Fucose. C6H12O5. (Mr 164.2). 1039500. [6696-41-9]. Geraniol used in gas chromatography complies with the
6-Deoxy-L-galactose. following additional test.
White or almost white powder, soluble in water and in ethanol Assay. Gas chromatography (2.2.28) as prescribed in the
(96 per cent). monograph Citronella oil (1609).
: about − 76, determined on a 90 g/L solution 24 h after Content : minimum 98.5 per cent, calculated by the
dissolution. normalisation procedure.
mp : about 140 °C. Storage : in an airtight container, protected from light
Fumaric acid. C4H4O4. (Mr 116.1). 1153200. [110-17-8]. Geranyl acetate. C12H20O2. (Mr 196.3). 1106500. [105-87-3].
(E)-Butenedioic acid. (E)-3,7-Dimethylocta-2,6-dien-1-yl acetate.
White or almost white crystals, slightly soluble in water, Colourless or slightly yellow liquid, slight odour of rose and
soluble in ethanol (96 per cent), slightly soluble in acetone. lavender.
Geranyl acetate used in gas chromatography complies with the
mp : about 300 °C.
following additional test.
Furfural. C5H4O2. (Mr 96.1). 1039600. [98-01-1]. Assay. Gas chromatography (2.2.28) as prescribed in the
2-Furaldehyde. 2-Furanecarbaldehyde. monograph Bitter-orange-flower oil (1175).
Clear, colourless to brownish-yellow, oily liquid, miscible in Test solution. The substance to be examined.
11 parts of water, miscible with ethanol (96 per cent). Content : minimum 98.0 per cent, calculated by the
: 1.155 to 1.161. normalisation procedure.
Distillation range (2.2.11). Not less than 95 per cent distils Ginsenoside Rb1. C54H92O23,3H2O. (Mr 1163). 1127500.
between 159 °C and 163 °C. [41753-43-9]. (20S)-3β-Di-D-glucopyranosyl-20-di-D-
Storage : in a dark place. glucopyranosylprotopanaxadiol. (20S)-3β-[(2-O-β-D-
Glucopyranosyl-β-D-glucopyranosyl)oxy]-20-[(6-O-β-D-
Galactose. C6H12O6. (Mr 180.2). 1039700. [59-23-4]. glucopyranosyl-β-D-glucopyranosyl)oxy]-5α-dammar-
D-(+)-Galactose. 24-en-12β-ol. (20S)-3β-[(2-O-β-D-Glucopyranosyl-β-D-
glucopyranosyl)oxy]-20-[(6-O-β-D-glucopyranosyl-β-D-
White or almost white, crystalline powder, freely soluble in glucopyranosyl)oxy]-4,4,8,14-tetramethyl-18-nor-5α-cholest-
water. 24-en-12β-ol.
: + 79 to + 81, determined on a 100 g/L solution in A colourless solid, soluble in water, in anhydrous ethanol and
water R containing about 0.05 per cent of NH3. in methanol.
Gallic acid. C7H6O5,H2O. (Mr 188.1). 1039800. [5995-86-8]. : + 11.3 determined on a 10 g/L solution in methanol R.
3,4,5-Trihydroxybenzoic acid monohydrate. mp : about 199 °C.
Crystalline powder or long needles, colourless or slightly Water (2.5.12) : maximum 6.8 per cent.
yellow, soluble in water, freely soluble in hot water, in ethanol Assay. Liquid chromatography (2.2.29) as prescribed in the
(96 per cent) and in glycerol. monograph Ginseng (1523).
General Notices (1) apply to all monographs and other texts 469
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Test solution. Dissolve 3.0 mg, accurately weighted, of Shows mutarotation : : + 11.7 → + 36.3.
ginsenoside Rb1 in 10 mL of methanol R. Assay. Dissolve 0.150 g in 50 mL of anhydrous methanol R
Content : minimum 95.0 per cent, calculated by the while stirring under nitrogen. Titrate with 0.1 M
normalisation procedure. tetrabutylammonium hydroxide, protecting the solution from
atmospheric carbon dioxide throughout solubilisation and
Ginsenoside Re. C48H82O18. (Mr 947.2). 1157800. titration. Determine the end-point potentiometrically (2.2.20).
[52286-59-6]. (3β,6α,12β)-20-(β-D-Glucopyranosyloxy)-
3,12-dihydroxydammar-24-en-6-yl 2-O-(6-deoxy-α-L- 1 mL of 0.1 M tetrabutylammonium hydroxide is equivalent to
mannopyranosyl)-β-D-glucopyranoside. 19.41 mg of C6H10O7.
Colourless solid, soluble in water, in ethanol (96 per cent) and Glutamic acid. 1040400. [56-86-0].
in methanol. See Glutamic acid (0750).
Ginsenoside Rf. C42H72O14,2H2O. (Mr 837). 1127700.
Glutamyl endopeptidase for peptide mapping. 1173300.
[52286-58-5]. (20S)-6-O-[β-D-Glucopyranosyl-(1→2)-β-D-
[137010-42-5]. Endoproteinase Glu-C of high purity from
glycopyranoside]-dammar-24-ene-3β,6α,12β,20-tetrol.
Staphylococcus aureus strain V8 (EC 3.4.21.19).
A colourless solid, soluble in water, in anhydrous ethanol and
in methanol. L-γ-Glutamyl- L-cysteine. C8H14N2O5S. (Mr 250.3). 1157900.
: + 12.8 determined on a 10 g/L solution in methanol R. [636-58-8].
mp : about 198 °C. Glutaraldehyde. C5H8O2. (Mr 100.1). 1098300. [111-30-8].
Ginsenoside Rg1. C42H72O14,2H2O. (Mr 837). Oily liquid, soluble in water.
1127600. [22427-39-0]. (20S)-6β-D-Glucopyranosyl- : about 1.434.
D-glucopyranosylprotopanaxatriol. (20S)-6α,20-Bis(β-
bp : about 188 °C.
D-glucopyranosyloxy)-5α-dammar-24-ene-3β,12β-diol.
(20S)-6α,20-Bis(β-D-glucopyranosyloxy)-4,4,8,14- Glutaric acid. C5H8O4. (Mr 132.1). 1149700. [110-94-1].
tetramethyl-18-nor-5α-cholest-24-ene-3β,12β-diol. Pentanedioic acid.
A colourless solid, soluble in water, in anhydrous ethanol and White or almost white, crystalline powder.
in methanol.
: + 31.2 determined on a 10 g/L solution in methanol R. L-Glutathione, oxidised. C20H32N6O12S2. (Mr 612.6). 1158000.
[27025-41-8]. Bis(L-γ-glutamyl-L-cysteinylglycine) disulfide.
mp : 188 °C to 191 °C.
Water (2.5.12) : maximum 4.8 per cent. Glycerol. 1040500. [56-81-5].
Assay. Liquid chromatography (2.2.29) as prescribed in the See Glycerol (0496).
monograph Ginseng (1523).
Glycerol R1. 1040501.
Test solution. Dissolve 3.0 mg, accurately weighted, of
ginsenoside Rg1 in 10 mL of methanol R. Complies with the requirements prescribed for the
monograph Glycerol (0496) and free from diethylene glycol
Content : minimum 95.0 per cent, calculated by the
when examined as prescribed in the test for impurity A and
normalisation procedure.
related substances in that monograph.
Ginsenoside Rg2. C42H72O13. (Mr 785). 1182600.
[52286-74-5]. 3β,12β,20-Trihydroxydammar-24-en-6α-yl Glycerol (85 per cent). 1040600.
2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranoside. See Glycerol (85 per cent) (0497).
Gitoxin. C41H64O14. (Mr 781). 1040200. [4562-36-1]. Glycerol (85 per cent) R1. 1040601.
Glycoside of Digitalis purpurea L. 3β-(O-2,6-Dideoxy- Complies with the requirements prescribed for the
β-d-ribo-hexopyranosyl-(1→4)-O-2,6-dideoxy-β-d- monograph Glycerol 85 per cent (0497) and free from
ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-d-ribo- diethylene glycol when examined as prescribed in the test
hexopyranosyloxy)-14,16β-dihydroxy-5β,14β-card-20(22)- for impurity A and related substances in that monograph.
enolide.
A white or almost white, crystalline powder, practically Glycerol 1-decanoate. C13H26O4. (Mr 246.3). 1169400.
insoluble in water and in most common organic solvents, [2277-23-8]. (2RS)-2,3-Dihydroxypropyl decanoate.
soluble in pyridine. α-Monocaprin. 1-Monodecanoyl-rac-glycerol.
: + 20 to + 24, determined on a 5 g/L solution in a mixture Content : about 99 per cent.
of equal volumes of chloroform R and methanol R. Glycerol 1-octanoate. C11H22O4. (Mr 218.3). 1169500.
Chromatography. Thin-layer chromatography (2.2.27) as [502-54-5]. (2RS)-2,3-Dihydroxypropyl octanoate.
prescribed in the monograph Digitalis leaf (0117) ; the α-Monocaprylin. 1-Monooctanoyl-rac-glycerol.
chromatogram shows only one principal spot. Content : about 99 per cent.
Glucosamine hydrochloride. C6H14ClNO5. (Mr 215.6). Glycidol. C3H6O2. (Mr 74.1). 1127800. [556-52-5].
1040300. [66-84-2]. D-Glucosamine hydrochloride.
Slightly viscous liquid, miscible with water.
Crystals, soluble in water.
: about 1.115.
: + 100, decreasing to + 47.5 after 30 min, determined
on a 100 g/L solution. : about 1.432.
Glycyrrhetic acid. C30H46O4. (Mr 470.7). 1040900. Guaiazulene. C15H18. (Mr 198.3). 1041500. [489-84-9].
[471-53-4]. Glycyrrhetinic acid. 12,13-Didehydro-3β- 1,4-Dimethyl-7-isopropylazulene.
hydroxy-11-oxo-olean-30-oic acid. Dark-blue crystals or blue liquid, very slightly soluble in water,
A mixture of α- and β-glycyrrhetic acids in which the β-isomer miscible with fatty and essential oils and with liquid paraffin,
is predominant. sparingly soluble in ethanol (96 per cent), soluble in 500 g/L
sulfuric acid and 80 per cent m/m phosphoric acid, giving a
White or yellowish-brown powder, practically insoluble in colourless solution.
water, soluble in anhydrous ethanol and in glacial acetic acid.
mp : about 30 °C.
: + 145 to + 155, determined on a 10.0 g/L solution in Storage : protected from light and air.
anhydrous ethanol R.
Chromatography. Thin-layer chromatography (2.2.27) using Guanidine hydrochloride. CH5N3HCl. (Mr 95.5). 1098500.
silica gel GF254 R as the coating substance ; prepare the slurry [50-01-1].
using a 0.25 per cent V/V solution of phosphoric acid R. Crystalline powder, freely soluble in water and in ethanol
Apply to the plate 5 μL of a 5 g/L solution of the glycyrrhetic (96 per cent).
acid in a mixture of equal volumes of chloroform R and Guanine. C5H5N5O. (Mr 151.1). 1041600. [73-40-5].
methanol R. Develop over a path of 10 cm using a mixture 2-Amino-1,7-dihydro-6H-purin-6-one.
of 5 volumes of methanol R and 95 volumes of chloroform R.
Examine the chromatogram in ultraviolet light at 254 nm. The Amorphous white or almost white powder, practically
chromatogram shows a dark spot (RF about 0.3) corresponding insoluble in water, slightly soluble in ethanol (96 per cent).
to β-glycyrrhetic acid and a smaller spot (RF about 0.5) It dissolves in ammonia and in dilute solutions of alkali
corresponding to α-glycyrrhetic acid. Spray with anisaldehyde hydroxides.
solution R and heat at 100-105 °C for 10 min. Both spots are Haemoglobin. 1041700. [9008-02-0].
coloured bluish-violet. Between them a smaller bluish-violet Nitrogen : 15 per cent to 16 per cent.
spot may be present.
Iron : 0.2 per cent to 0.3 per cent.
18α-Glycyrrhetinic acid. C30H46O4. (Mr 470.7). 1127900. Loss on drying (2.2.32) : maximum 2 per cent.
[1449-05-4]. (20β)-3β-Hydroxy-11-oxo-18α-olean-12-en-29- Sulfated ash (2.4.14) : maximum 1.5 per cent.
oic acid.
Haemoglobin solution. 1041701.
White or almost white powder, practically insoluble in water,
Transfer 2 g of haemoglobin R to a 250 mL beaker and add
soluble in anhydrous ethanol, sparingly soluble in methylene
75 mL of dilute hydrochloric acid R2. Stir until solution is
chloride.
complete. Adjust the pH to 1.6 ± 0.1 using 1 M hydrochloric
Glyoxalhydroxyanil. C14H12N2O2. (Mr 240.3). 1041000. acid. Transfer to a 100 mL flask with the aid of dilute
[1149-16-2]. Glyoxal bis(2-hydroxyanil). hydrochloric acid R2. Add 25 mg of thiomersal R. Prepare
daily, store at 5 ± 3 °C and readjust to pH 1.6 before use.
White or almost white crystals, soluble in hot ethanol (96 per Storage : at 2 °C to 8 °C.
cent).
mp : about 200 °C. Harpagoside. C24H30O11. (Mr 494.5). 1098600.
White or almost white, crystalline powder, very hygroscopic,
Glyoxal solution. 1098400. [107-22-2]. soluble in water and in ethanol (96 per cent).
Contains about 40 per cent (m/m) glyoxal. mp : 117 °C to 121 °C.
Assay. In a ground-glass stoppered flask place 1.000 g of Storage : in an airtight container.
glyoxal solution, 20 mL of a 70 g/L solution of hydroxylamine Hederacoside C. C59H96O26. (Mr 1221). 1158100.
hydrochloride R and 50 mL of water R. Allow to stand for [14216-03-6]. O-6-Deoxy-α-L-mannopyranosyl-(1→4)-
30 min and add 1 mL of methyl red mixed solution R and O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl
titrate with 1 M sodium hydroxide until the colour changes (4R)-3β-[[2-O(-6-deoxy-α-L-mannopyranosyl)-α-L-
from red to green. Carry out a blank titration. arabinopyranosyl]oxy]-23-hydroxyolean-12-en-28-oate.
1 mL of 1 M sodium hydroxide is equivalent to 29.02 mg of Colourless crystals or white or almost white powder.
glyoxal (C2H2O2). mp : about 220 °C.
Hederacoside C used in liquid chromatography complies with
Gonadotrophin, chorionic. 1041100. [9002-61-3]. the following additional test.
See Chorionic gonadotrophin (0498). Assay. Liquid chromatography (2.2.29) as prescribed in the
monograph Ivy leaf (2148).
Gonadotrophin, serum. 1041200.
Test solution. Dissolve 5.0 mg of hederacoside C in 5.0 mL
See Equine serum gonadotrophin for veterinary use (0719). of methanol R.
Content : minimum 95 per cent, calculated by the
Guaiacol. C7H8O2. (Mr 124.1). 1148300. [90-05-1]. normalisation procedure.
2-Methoxyphenol. 1-Hydroxy-2-methoxybenzene.
Crystalline mass or colourless or yellowish liquid, hygroscopic, Hederagenin. C30H48O4. (Mr 472.7). 1184100.
slightly soluble in water, very soluble in methylene chloride, [465-99-6]. Astrantiagenin E. Caulosapogenin.
freely soluble in ethanol (96 per cent). 3β,23-Dihydroxy-4α-olean-12-en-28-oic acid.
bp : about 205 °C. α-Hederin. C41H66O12. (Mr 751.0). 1158200. [27013-91-8].
(+)-(4R)-3β-[[2-O-(6-Deoxy-α-L-mannopyranosyl)-α-L-
mp : about 28 °C.
arabinopyranosyl]oxy]-23-hydroxyolean-12-en-28-oic acid.
Guaiacum resin. 1041400. White or almost white powder.
Resin obtained from the heartwood of Guaiacum officinale L. mp : about 256 °C.
and Guaiacum sanctum L. Helium for chromatography. He. (Ar 4.003). 1041800.
Reddish-brown or greenish-brown, hard, glassy fragments ; [7440-59-7].
fracture shiny. Content : minimum 99.995 per cent V/V of He.
General Notices (1) apply to all monographs and other texts 471
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 473
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
alcoholic potassium hydroxide until a greenish-yellow Hypericin. C30H16O8. (Mr 504.4). 1149800.
colour is obtained. Dilute to 50.0 mL with ethanol (96 per [548-04-9]. 1,3,4,6,8,13-Hexahydroxy-10,11-
cent) R. dimethylphenanthro[1,10,9,8-opqra]perylene-7,14-dione.
Hydroxylamine solution, alcoholic. 1044301. Content : minimum 85 per cent.
Dissolve 3.5 g of hydroxylamine hydrochloride R in 95 mL Hyperoside. C21H20O12. (Mr 464.4). 1045000.
of ethanol (60 per cent V/V) R, add 0.5 mL of a 2 g/L 2-(3,4-Dihydroxyphenyl)-3-β-D-galactopyranosyloxy-
solution of methyl orange R in ethanol (60 per cent V/V) R 5,7-dihydroxychromen-4-one.
and sufficient 0.5 M potassium hydroxide in alcohol (60 per Faint yellow needles, soluble in methanol.
cent V/V) to give a pure yellow colour. Dilute to 100 mL
with ethanol (60 per cent V/V) R. mp : about 240 °C, with decomposition.
Absorbance (2.2.25). A solution in methanol R shows
Hydroxylamine solution, alkaline. 1044302. 2 absorption maxima at 259 nm and at 364 nm.
Immediately before use, mix equal volumes of a 139 g/L
solution of hydroxylamine hydrochloride R and a 150 g/L Hypophosphorous reagent. 1045200.
solution of sodium hydroxide R. Dissolve with the aid of gentle heat, 10 g of sodium
hypophosphite R in 20 mL of water R and dilute to 100 mL
Hydroxylamine solution, alkaline R1. 1044303. with hydrochloric acid R. Allow to settle and decant or filter
Solution A. Dissolve 12.5 g of hydroxylamine hydrochloride R through glass wool.
in methanol R and dilute to 100 mL with the same solvent.
Solution B. Dissolve 12.5 g of sodium hydroxide R in Hypoxanthine. C5H4N4O. (Mr 136.1). 1045300. [68-94-0].
methanol R and dilute to 100 mL with the same solvent. 1H-Purin-6-one.
Mix equal volumes of solution A and solution B White or almost white, crystalline powder, very slightly soluble
immediately before use. in water, sparingly soluble in boiling water, soluble in dilute
acids and in dilute alkali hydroxide solutions, decomposes
Hydroxymethylfurfural. C6H6O3. (Mr 126.1). 1044400. without melting at about 150 °C.
[67-47-0]. 5-Hydroxymethylfurfural. Chromatography. Thin-layer chromatography (2.2.27) as
Acicular crystals, freely soluble in water, in acetone and in prescribed in the monograph Mercaptopurine (0096) ; the
ethanol (96 per cent). chromatogram shows only one principal spot.
mp : about 32 °C. Imidazole. C H N . (M 68.1). 1045400. [288-32-4].
3 4 2 r
Hydroxynaphthol blue, sodium salt. C20H11N2Na3O11S3. White or almost white, crystalline powder, soluble in water
(Mr 620). 1044500. [63451-35-4]. Trisodium and in ethanol (96 per cent).
2,2′-dihydroxy-1,1′-azonaphthalene-3′,4,6′-trisulfonate. mp : about 90 °C.
2-Hydroxypropylbetadex for chromatography R. 1146000. Iminodibenzyl. C14H13N. (Mr 195.3). 1045500. [494-19-9].
Betacyclodextrin modified by the bonding of (R) or (RS) 10,11-Dihydrodibenz[b,f]azepine.
propylene oxide groups on the hydroxyl groups.
Pale yellow, crystalline powder, practically insoluble in water,
Hydroxypropyl-β-cyclodextrin. 1128600. [94035-02-6]. freely soluble in acetone.
See Hydroxypropylbetadex (1804). mp : about 106 °C.
pH (2.2.3): 5.0 to 7.5 for a 20 g/L solution. Imperatorin. C16H14O4. (Mr 270.3). 1180200. [482-44-0].
Hydroxyquinoline. C9H7NO. (Mr 145.2). 1044600. 9-[(3-Methylbut-2-enyl)oxy]-7H-furo[3,2-g][1]benzopyran-
[148-24-3]. 8-Hydroxyquinoline. Quinolin-8-ol. 7-one.
White or slightly yellowish, crystalline powder, slightly soluble 2-Indanamine hydrochloride. C9H12ClN. (Mr 169.7).
in water, freely soluble in acetone, in ethanol (96 per cent) 1175800. [2338-18-3]. 2-Aminoindane hydrochloride.
and in dilute mineral acids. 2,3-Dihydro-1H-inden-2-amine hydrochloride.
mp : about 75 °C.
Sulfated ash (2.4.14) : maximum 0.05 per cent. Indigo carmine. C16H8N2Na2O8S2. (Mr 466.3). 1045600.
[860-22-0].
12-Hydroxystearic acid. C18H36O3. (Mr 300.5). 1099000. Schultz No. 1309.
[106-14-9]. 12-Hydroxyoctadecanoic acid.
Colour Index No. 73015.
White or almost white powder. 3,3′-Dioxo-2,2′-bisindolylidene-5,5′-disulfonate disodium.
mp : 71 °C to 74 °C. E 132.
It usually contains sodium chloride.
5-Hydroxyuracil. C4H4N2O3. (Mr 128.1). 1044700.
[496-76-4]. Isobarbituric acid. Pyrimidine-2,4,5-triol. Blue or violet-blue powder or blue granules with a coppery
White or almost white, crystalline powder. lustre, sparingly soluble in water, practically insoluble in
ethanol (96 per cent). It is precipitated from an aqueous
mp : about 310 °C, with decomposition. solution by sodium chloride.
Chromatography. Thin-layer chromatography (2.2.27)
as prescribed in the monograph Fluorouracil (0611) ; the Indigo carmine solution. 1045601.
chromatogram shows a principal spot with an RF of about 0.3. To a mixture of 10 mL of hydrochloric acid R and 990 mL
Storage : in an airtight container. of 200 g/L nitrogen-free sulfuric acid R add 0.2 g of indigo
carmine R.
Hyoscine hydrobromide. 1044800. [6533-68-2]. The solution complies with the following test : add 10 mL
See Hyoscine hydrobromide (0106). to a solution of 1.0 mg of potassium nitrate R in 10 mL of
water R, rapidly add 20 mL of nitrogen-free sulfuric acid R
Hyoscyamine sulfate. 1044900. [620-61-1]. and heat to boiling. The blue colour is discharged within
See Hyoscyamine sulfate (0501). 1 min.
General Notices (1) apply to all monographs and other texts 475
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
2-Iodohippuric acid. C9H8INO3,2H2O. (Mr 341.1). 1046200. Preparation of a column. Unless otherwise prescribed, use a
[147-58-0]. 2-(2-Iodobenzamido)acetic acid. tube with a fused-in sintered glass disc having a length of
White or almost white, crystalline powder, sparingly soluble 400 mm, an internal diameter of 20 mm and a filling height
in water. of about 200 mm. Introduce the resin, mixing it with water R
mp : about 170 °C. and pouring the slurry into the tube, ensuring that no air
bubbles are trapped between the particles. When in use, the
Water (2.5.12) : 9 per cent to 13 per cent, determined on liquid must not be allowed to fall below the surface of the
1.000 g. resin. If the resin is in its protonated form, wash with water R
Chromatography. Thin-layer chromatography (2.2.27), using until 50 mL requires not more than 0.05 mL of 0.1 M sodium
cellulose for chromatography F254 R as the coating substance : hydroxide for neutralisation, using 0.1 mL of methyl orange
apply to the plate 20 μL of a solution of the 2-iodohippuric solution R as indicator.
acid, prepared by dissolving 40 mg in 4 mL of 0.1 M sodium If the resin is in its sodium form or if it requires regeneration,
hydroxide and diluting to 10 mL with water R. Develop over pass about 100 mL of a mixture of equal volumes of
a path of about 12 cm using as the mobile phase the upper hydrochloric acid R1 and water R slowly through the column
layer obtained by shaking together 20 volumes of water R, and then wash with water R as described above.
40 volumes of glacial acetic acid R and 40 volumes of toluene R.
Allow the plate to dry in air and examine in ultraviolet light at Irisflorentin. C20H18O8. (Mr 386.4). 1186300. [41743-73-1].
254 nm. The chromatogram shows only one principal spot. 9-Methoxy-7-(3,4,5-trimethoxyphenyl)-8H-1,3-dioxolo-
[4,5-g][1]benzopyran-8-one.
Iodoplatinate reagent. 1046300.
To 3 mL of a 100 g/L solution of chloroplatinic acid R add Iron. Fe. (Ar 55.85). 1046600. [7439-89-6].
97 mL of water R and 100 mL of a 60 g/L solution of potassium Grey powder or wire, soluble in dilute mineral acids.
iodide R.
Iron salicylate solution. 1046700.
Storage : protected from light.
Dissolve 0.1 g of ferric ammonium sulfate R in a mixture of
Iodoplatinate reagent R1. 1172200. 2 mL of dilute sulfuric acid R and 48 mL of water R and dilute
Mix 2.5 mL of a 50 g/L solution of chloroplatinic acid R, to 100 mL with water R. Add 50 mL of a 11.5 g/L solution of
22.5 mL of a 100 g/L solution of potassium iodide R and 50 mL sodium salicylate R, 10 mL of dilute acetic acid R, 80 mL of
of water R. a 136 g/L solution of sodium acetate R and dilute to 500 mL
Storage : protected from light, at a temperature of 2-8 °C. with water R. The solution should be recently prepared.
Storage : in an airtight container, protected from light.
Iodosulfurous reagent. 1046400.
The apparatus, which must be kept closed and dry during the Isatin. C8H5NO2. (Mr 147.1). 1046800. [91-56-5].
preparation, consists of a 3000 mL to 4000 mL round-bottomed Indoline-2,3-dione.
flask with three inlets for a stirrer and a thermometer and Small, yellowish-red crystals, slightly soluble in water, soluble
fitted with a drying tube. To 700 mL of anhydrous pyridine R in hot water and in ethanol (96 per cent), soluble in solutions
and 700 mL of ethylene glycol monomethyl ether R add, with of alkali hydroxides giving a violet colour becoming yellow
constant stirring, 220 g of finely powdered iodine R, previously on standing.
dried over diphosphorus pentoxide R. Continue stirring until mp : about 200 °C, with partial sublimation.
the iodine has completely dissolved (about 30 min). Cool to Sulfated ash (2.4.14) : maximum 0.2 per cent.
− 10 °C, and add quickly, still stirring, 190 g of sulfur dioxide R.
Do not allow the temperature to exceed 30 °C. Cool. Isatin reagent. 1046801.
Assay. Add about 20 mL of anhydrous methanol R to a titration Dissolve 6 mg of ferric sulfate R in 8 mL of water R and add
vessel and titrate to the end-point with the iodosulfurous cautiously 50 mL of sulfuric acid R. Add 6 mg of isatin R
reagent (2.5.12). Introduce in an appropriate form a suitable and stir until dissolved.
amount of water R, accurately weighed, and repeat the The reagent should be pale yellow, but not orange or red.
determination of water. Calculate the water equivalent in Isoamyl alcohol. C5H12O. (Mr 88.1). 1046900. [123-51-3].
milligrams per millilitre of iodosulfurous reagent. 3-Methylbutan-1-ol.
The minimum water equivalent is 3.5 mg of water per millilitre Colourless liquid, slightly soluble in water, miscible with
of reagent. ethanol (96 per cent).
Work protected from humidity. Standardise immediately bp : about 130 °C.
before use.
Storage : in a dry container. Isoamyl benzoate. C12H16O2. (Mr 192.3). 1164200. [94-46-2].
Isopentyl benzoate. 3-Methylbutyl benzoate.
5-Iodouracil. C4H3IN2O2. (Mr 238.0). 1046500. [696-07-1]. : about 1.494.
5-Iodo-1H,3H-pyrimidine-2,4-dione.
bp : about 261 °C.
mp : about 276 °C, with decomposition.
Colourless or pale yellow liquid.
Chromatography. Thin-layer chromatography (2.2.27) as
prescribed in the monograph Idoxuridine (0669) : apply 5 μL Isoandrosterone. C19H30O2. (Mr 290.4). 1107100. [481-29-8].
of a 0.25 g/L solution ; the chromatogram obtained shows only Epiandrosterone. 3β-Hydroxy-5α-androstan-17-one.
one principal spot. White or almost white powder, practically insoluble in water,
Ion-exclusion resin for chromatography. 1131000. soluble in organic solvents.
A resin with sulfonic acid groups attached to a polymer lattice : + 88, determined on 20 g/L solution in methanol R.
consisting of polystyrene cross-linked with divinylbenzene. mp : 172 °C to 174 °C.
ΔA (2.2.41) : 14.24 × 103, determined at 304 nm on a 1.25 g/L
Ion-exchange resin, strongly acidic. 1085400. solution.
Resin in protonated form with sulfonic acid groups attached to
a lattice consisting of polystyrene cross-linked with 8 per cent N-Isobutyldodecatetraenamide. C16H25NO. (Mr 247.4).
of divinylbenzene. It is available as spherical beads ; unless 1159500. [866602-52-0]. (2E,4E,8Z,10EZ)-N-2-
otherwise prescribed, the particle size is 0.3 mm to 1.2 mm. (Methylpropyl)dodeca-2,4,8,10-tetraenamide.
Capacity. 4.5 mmol to 5 mmol per gram, with a water content White or almost white or non-coloured crystals.
of 50 per cent to 60 per cent. mp : about 70 °C.
General Notices (1) apply to all monographs and other texts 477
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
11-Keto-β-boswellic acid. C30H46O4. (Mr 470.7). 1167600. Lactic acid. 1047800. [50-21-5].
[17019-92-0]. 3α-Hydroxy-11-oxours-12-en-24-oic acid. See Lactic acid (0458).
(4β)-3α-Hydroxy-11-oxours-12-en-23-oic acid.
White or almost white powder, insoluble in water, soluble in Lactic reagent. 1047801.
acetone, in anhydrous ethanol and in methanol. Solution A. To 60 mL of lactic acid R add 45 mL of
mp : 195 °C to 197 °C. previously filtered lactic acid R saturated without heating
with Sudan red G R ; as lactic acid saturates slowly without
11-Keto-β-boswellic acid used in liquid chromatography heating, an excess of colorant is always necessary.
complies with the following additional test.
Solution B. Prepare 10 mL of a saturated solution of
Assay. Liquid chromatography (2.2.29) as prescribed in the aniline R. Filter.
monograph Indian frankincense (2310).
Solution C. Dissolve 75 mg of potassium iodide R in water
Content : minimum 90 per cent, calculated by the and dilute to 70 mL with the same solvent. Add 10 mL of
normalisation procedure. ethanol (96 per cent) R and 0.1 g of iodine R. Shake.
Kieselguhr for chromatography. 1047500. Mix solutions A and B. Add solution C.
White or yellowish-white, light powder, practically insoluble
Lactobionic acid. C12H22O12. (Mr 358.3). 1101600. [96-82-2].
in water, in dilute acids and in organic solvents.
White or almost white, crystalline powder, freely soluble in
Filtration rate. Use a chromatography column 0.25 m long and
water, practically insoluble in ethanol (96 per cent).
10 mm in internal diameter with a sintered-glass (100) plate
and two marks at 0.10 m and 0.20 m above the plate. Place mp : about 115 °C.
sufficient of the substance to be examined in the column to Lactose. 1047900. [5989-81-1].
reach the first mark and fill to the second mark with water R.
When the first drops begin to flow from the column, fill to See Lactose (0187).
the second mark again with water R and measure the time β-Lactose. C12H22O11. (Mr 342.3). 1150100. [5965-66-2].
required for the first 5 mL to flow from the column. The flow β-D-Lactose.
rate is not less than 1 mL/min. White or slightly yellowish powder.
Appearance of the eluate. The eluate obtained in the test for Content : minimum 99 per cent.
filtration rate is colourless (2.2.2, Method I).
α-D-Lactose : not greater than 35 per cent.
Acidity or alkalinity. To 1.00 g add 10 mL of water R, shake
vigorously and allow to stand for 5 min. Filter the suspension Assay. Gas chromatography (2.2.28) : use the normalisation
on a filter previously washed with hot water R until the procedure.
washings are neutral. To 2.0 mL of the filtrate add 0.05 mL Column :
of methyl red solution R ; the solution is yellow. To 2.0 mL of – size : l = 30 m, Ø = 0.25 mm ;
the filtrate add 0.05 mL of phenolphthalein solution R1 ; the – stationary phase : poly[(cyanopropyl)(phenyl)][dimeth-
solution is at most slightly pink. yl]siloxane R (film thickness 1 μm).
Water-soluble substances. Place 10.0 g in a chromatography Carrier gas : helium for chromatography R.
column 0.25 m long and 10 mm in internal diameter and elute
with water R. Collect the first 20 mL of eluate, evaporate to Temperature :
dryness and dry the residue at 100 °C to 105 °C. The residue Time Temperature
weighs not more than 10 mg. (min) (°C)
Iron (2.4.9) : maximum 200 ppm. Column 0 - 32.5 20 → 280
To 0.50 g add 10 mL of a mixture of equal volumes of Injection port 250
hydrochloric acid R1 and water R, shake vigorously, allow to
stand for 5 min and filter. 1.0 mL of the filtrate complies with Detector 250
the test for iron.
Detection : flame ionisation.
Loss on ignition : maximum 0.5 per cent. During heating to
Injection : an appropriate derivatised sample.
red heat (600 ± 50 °C) the substance does not become brown
or black. α-Lactose monohydrate. C12H22O11,H2O. (Mr 360.3).
1150000. [5989-81-1]. α-D-Lactose monohydrate.
Kieselguhr G. 1047600.
White or almost white powder.
Consists of kieselguhr treated with hydrochloric acid and
calcined, to which is added about 15 per cent of calcium Content : minimum 97 per cent.
sulfate hemihydrate. β-D-Lactose : less than 3 per cent.
A fine greyish-white powder ; the grey colour becomes more Assay. Gas chromatography (2.2.28) : use the normalisation
pronounced on triturating with water. The average particle procedure.
size is 10-40 μm. Column :
Calcium sulfate content. Determine by the method prescribed – size : l = 30 m, Ø = 0.25 mm ;
for silica gel G R. – stationary phase : poly(dimethyl)siloxane R (film thickness
pH (2.2.3). Shake 1 g with 10 mL of carbon dioxide-free 1 μm).
water R for 5 min. The pH of the suspension is 7 to 8. Carrier gas : helium for chromatography R.
Chromatographic separation. Thin-layer chromatography Temperature :
(2.2.27). Prepare plates using a slurry of the kieselguhr G
with a 2.7 g/L solution of sodium acetate R. Apply 5 μL of a Time Temperature
solution containing 0.1 g/L of lactose, sucrose, glucose and (min) (°C)
fructose in pyridine R. Develop over a path of 14 cm using a Column 0 - 12.5 230 → 280
mixture of 12 volumes of water R, 23 volumes of 2-propanol R Injection port 250
and 65 volumes of ethyl acetate R. The migration time of
the solvent is about 40 min. Dry, spray onto the plate about Detector 280
10 mL of anisaldehyde solution R and heat for 5-10 min at
100-105 °C. The chromatogram shows four well-defined spots Detection : flame ionisation.
without tailing and well separated from each other. Injection : an appropriate derivatised sample.
General Notices (1) apply to all monographs and other texts 479
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Lanatoside C. C49H76O20. (Mr 985). 1163300. [17575-22-3]. Test solution. The substance to be examined.
3β-[(β-D-Glucopyranosyl-(1→4)-3-O-acetyl-2,6- Content : minimum 93.0 per cent, calculated by the
dideoxy-β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy- normalisation procedure.
β-D-ribo-hexopyranosyl-(1→4)-2,6-dideoxy-β-D-ribo-
hexopyranosyl)oxy]-12β,14-dihydroxy-5β-card-20(22)- Lead acetate. C4H6O4Pb,3H2O. (Mr 379.3). 1048100.
enolide. [6080-56-4]. Lead di-acetate.
Long, flat prisms obtained after recrystallisation in ethanol Colourless crystals, efflorescent, freely soluble in water, soluble
(96 per cent), freely soluble in pyridine and in dioxane. in ethanol (96 per cent).
Lanthanum chloride heptahydrate. LaCl3,7H2O. (Mr 371.4). Lead acetate cotton. 1048101.
1167200. Immerse absorbent cotton in a mixture of 1 volume
White or almost white powder or colourless crystals, freely of dilute acetic acid R and 10 volumes of lead acetate
soluble in water. solution R. Drain off the excess of liquid, without squeezing
the cotton, by placing it on several layers of filter paper.
Lanthanum chloride solution. 1114001. Allow to dry in air.
To 58.65 g of lanthanum trioxide R slowly add 100 mL of Storage : in an airtight container.
hydrochloric acid R. Heat to boiling. Allow to cool and dilute
to 1000.0 mL with water R. Lead acetate paper. 1048102.
Lanthanum nitrate. La(NO3)3,6H2O. (Mr 433.0). 1048000. Immerse filter paper weighing about 80 g/m2 in a mixture
[10277-43-7]. Lanthanum trinitrate hexahydrate. of 1 volume of dilute acetic acid R and 10 volumes of lead
acetate solution R. After drying, cut the paper into strips
Colourless crystals, deliquescent, freely soluble in water.
15 mm by 40 mm.
Storage : in an airtight container.
Lead acetate solution. 1048103.
Lanthanum nitrate solution. 1048001.
A 95 g/L solution in carbon dioxide-free water R.
A 50 g/L solution.
Lead dioxide. PbO2. (Mr 239.2). 1048200. [1309-60-0].
Lanthanum trioxide. La2O3. (Mr 325.8). 1114000.
[1312-81-8]. Dark brown powder, evolving oxygen when heated, practically
insoluble in water, soluble in hydrochloric acid with evolution
An almost white, amorphous powder, practically insoluble in of chlorine, soluble in dilute nitric acid in the presence of
water R. It dissolves in dilute solutions of mineral acids and hydrogen peroxide, oxalic acid or other reducing agents,
absorbs atmospheric carbon dioxide. soluble in hot, concentrated alkali hydroxide solutions.
Calcium : maximum 5 ppm.
Lead nitrate. Pb(NO3)2. (Mr 331.2). 1048300. [10099-74-8].
Lauric acid. C12H24O2. (Mr 200.3). 1143100. [143-07-7]. Lead dinitrate.
Dodecanoic acid. White or almost white, crystalline powder or colourless
White or almost white, crystalline powder, practically crystals, freely soluble in water.
insoluble in water, freely soluble in ethanol (96 per cent).
mp : about 44 °C. Lead nitrate solution. 1048301.
Lauric acid used in the assay of total fatty acids in Saw palmetto A 33 g/L solution.
fruit (1848) complies with the following additional test. Lead subacetate solution. 1048400. [1335-32-6]. Basic lead
Assay. Gas chromatography (2.2.28) as prescribed in the acetate solution.
monograph Saw palmetto fruit (1848). Content : 16.7 per cent m/m to 17.4 per cent m/m of Pb
Content : minimum 98 per cent, calculated by the (Ar 207.2) in a form corresponding approximately to the
normalisation procedure. formula C8H14O10Pb3.
Lauryl alcohol. C12H26O. (Mr 186.3). 1119900. [112-53-8]. Dissolve 40.0 g of lead acetate R in 90 mL of carbon dioxide-free
Dodecan-1-ol. water R. Adjust the pH to 7.5 with strong sodium hydroxide
: about 0.820. solution R. Centrifuge and use the clear colourless supernatant
solution.
mp : 24 °C to 27 °C.
The solution remains clear when stored in a well-closed
Content : minimum 98.0 per cent, determined by gas container.
chromatography.
Leiocarposide. C27H34O16. (Mr 614.5). 1150200.
Lavandulol. C10H18O. (Mr 154.2). 1114100. [498-16-8]. [71953-77-0]. 2-(β-D-Glucopyranosyloxy)benzyl
(R)-5-Methyl-2-(1-methylethenyl)-4-hexen-1-ol. 3-(β-D-glucopyranosyloxy)-6-hydroxy-2-methoxybenzoate.
Oily liquid with a characteristic odour. 2-[[[3-(β-D-Glucopyranosyloxy)-6-hydroxy-2-
Lavandulol used in gas chromatography complies with the methoxybenzoyl]oxy]methyl]phenyl-β-D-glucopyranoside.
following additional test. White or almost white powder, soluble in water, freely soluble
Assay. Gas chromatography (2.2.28) as prescribed in the in methanol, slightly soluble in ethanol (96 per cent).
monograph Lavender oil (1338). mp : 190 °C to 193 °C.
Test solution. The substance to be examined.
Lemon oil. 1101700.
Content : minimum 90.0 per cent, calculated by the
normalisation procedure. See Lemon oil (0620).
Lavandulyl acetate. C12H20O2. (Mr 196.3). 1114200. Leucine. 1048500. [61-90-5].
[25905-14-0]. 2-Isopropenyl-5-methylhex-4-en-1-yl acetate. See Leucine (0771).
Colourless liquid with a characteristic odour.
Levodopa. 1170000. [59-92-7].
Lavandulyl acetate used in gas chromatography complies with
the following additional test. See Levodopa (0038).
Assay. Gas chromatography (2.2.28) as prescribed in the (Z)-Ligustilide. C12H14O2. (Mr 190.2). 1180300. [81944-09-4].
monograph Lavender oil (1338). (3Z)-3-Butylidene-1,3,4,5-tetrahydroisobenzofuran-1-one.
Limonene. C10H16. (Mr 136.2). 1048600. [5989-27-5]. Linolenic acid. C18H30O2. (Mr 278.4). 1143300. [463-40-1].
D-Limonene. (+)-p-Mentha-1,8-diene. (R)-4-Isopropenyl-1- (9Z,12Z,15Z)-Octadeca-9,12,15-trienoic acid. α-Linolenic
methylcyclohex-1-ene. acid.
Colourless liquid, practically insoluble in water, soluble in Colourless liquid, practically insoluble in water, soluble in
ethanol (96 per cent). organic solvents.
: about 0.84. : about 0.915.
: 1.471 to 1.474. : about 1.480.
: about + 124. Linolenic acid used in the assay of total fatty acids in Saw
bp : 175 °C to 177 °C. palmetto fruit (1848) complies with the following additional
test.
Limonene used in gas chromatography complies with the
following additional test. Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Saw palmetto fruit (1848).
Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Peppermint oil (0405). Content : minimum 98 per cent, calculated by the
normalisation procedure.
Test solution. The substance to be examined.
Content : minimum 99.0 per cent, calculated by the Linolenyl alcohol. C18H32O. (Mr 264.4). 1156200.
normalisation procedure. [24149-05-1]. (9Z,12Z,15Z)-Octadeca-9,12,15-trien-1-ol.
α-Linolenyl alcohol.
Linalol. C10H18O. (Mr 154.2). 1048700. [78-70-6].
Content : minimum 96 per cent.
(RS)-3,7-Dimethylocta-1,6-dien-3-ol.
Mixture of two stereoisomers (licareol and coriandrol). Linoleyl alcohol. C18H34O. (Mr 266.5). 1155900. [506-43-4].
Liquid, practically insoluble in water. (9Z,12Z)-Octadeca-9,12-dien-1-ol.
: about 0.860. Relative density : 0.830.
: about 1.462. Content : minimum 85 per cent.
bp : about 200 °C. Linsidomine hydrochloride. C6H11ClN4O2. (Mr 206.6).
Linalol used in gas chromatography complies with the following 1171200. [16142-27-1]. 3-(Morpholin-4-yl)sydnonimine
test. hydrochloride. 3-(Morpholin-4-yl)-1,2,3-oxadiazol-3-ium-5-
Assay. Gas chromatography (2.2.28) as prescribed in the aminide hydrochloride.
monograph Anise oil (0804). White or almost white powder.
Test solution. The substance to be examined. Liquid scintillation cocktail. 1167300.
Content : minimum 98.0 per cent, calculated by the Commercially available solution for the determination of
normalisation procedure. radioactivity by liquid scintillation counting. It contains
Linalyl acetate. C12H20O2. (Mr 196.3). 1107200. [115-95-7]. one or more fluorescent agents and mostly one or more
(RS)-1,5-Dimethyl-1-vinylhex-4-enyl acetate. emulsifying agents in a suitable organic solvent or mixture of
organic solvents.
Colourless or slightly yellow liquid with a strong odour of
bergamot and lavender. Liquid scintillation cocktail R1. 1176800.
: 0.895 to 0.912. To 1000 mL of dioxan R, add 0.3 g of methylphenyloxazolylben-
: 1.448 to 1.451. zene R, 7 g of diphenyloxazole R and 100 g of naphthalene R.
bp : about 215 °C. Lithium. Li. (Ar 6.94). 1048800. [7439-93-2].
Linalyl acetate used in gas chromatography complies with the A soft metal whose freshly cut surface is silvery-grey. It rapidly
following additional test. tarnishes in contact with air. It reacts violently with water,
Assay. Gas chromatography (2.2.28) as prescribed in the yielding hydrogen and giving a solution of lithium hydroxide ;
monograph Bitter-orange-flower oil (1175). soluble in methanol, yielding hydrogen and a solution of
Test solution. The substance to be examined. lithium methoxide ; practically insoluble in light petroleum.
Content : minimum 95.0 per cent, calculated by the Storage : under light petroleum or liquid paraffin.
normalisation procedure.
Lithium carbonate. Li2CO3. (Mr 73.9). 1048900. [554-13-2].
Lindane. C6H6Cl6. (Mr 290.8). 1128900. [58-89-9]. Dilithium carbonate.
γ-Hexachlorocyclohexane. White or almost white, light powder, sparingly soluble
For the monograph Wool fat (0134), a suitable certified in water, very slightly soluble in ethanol (96 per cent). A
reference solution (10 ng/μL in cyclohexane) may be used. saturated solution at 20 °C contains about 13 g/L of Li2CO3.
Linoleic acid. C18H32O2. (Mr 280.5). 1143200. [60-33-3]. Lithium chloride. LiCl. (Mr 42.39). 1049000. [7447-41-8].
(9Z,12Z)-Octadeca-9,12-dienoic acid. Crystalline powder or granules or cubic crystals, deliquescent,
Colourless, oily liquid. freely soluble in water, soluble in acetone and in ethanol
(96 per cent). Aqueous solutions are neutral or slightly
: about 0.903.
alkaline.
: about 1.470.
Storage : in an airtight container.
Linoleic acid used in the assay of total fatty acids in Saw
palmetto fruit (1848) complies with the following additional Lithium hydroxide. LiOH,H2O. (Mr 41.96). 1049100.
test. [1310-66-3]. Lithium hydroxide monohydrate.
Assay. Gas chromatography (2.2.28) as prescribed in the White or almost white, granular powder, strongly alkaline, it
monograph Saw palmetto fruit (1848). rapidly absorbs water and carbon dioxide, soluble in water,
Content : minimum 98 per cent, calculated by the sparingly soluble in ethanol (96 per cent).
normalisation procedure. Storage : in an airtight container.
General Notices (1) apply to all monographs and other texts 481
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Lithium metaborate, anhydrous. LiBO2. (Mr 49.75). Absorbance (2.2.25). A solution in methanol R shows
1120000. [13453-69-5]. absorption maxima at 255 nm, 267 nm, 290 nm and 350 nm.
Lithium sulfate. Li2SO4,H2O. (Mr 128.0). 1049200. mp : about 247 °C.
[10102-25-7]. Dilithium sulfate monohydrate. Macrogol 23 lauryl ether. 1129000.
Colourless crystals, freely soluble in water, practically
See Macrogol lauryl ether (1124), the number of moles of
insoluble in ethanol (96 per cent).
ethylene oxide reacted per mole of lauryl alcohol being 23
Lithium trifluoromethanesulfonate. CF3LiO3S. (Mr 156.0). (nominal value).
1173400. [33454-82-9].
Macrogol 200. 1099200. [25322-68-3]. Polyethyleneglycol
Litmus. 1049300. [1393-92-6]. 200.
Schultz No. 1386. Clear, colourless or almost colourless viscous liquid, very
Indigo-blue fragments prepared from various species of soluble in acetone and in anhydrous ethanol, practically
Rocella, Lecanora or other lichens, soluble in water, practically insoluble in fatty oils.
insoluble in ethanol (96 per cent). : about 1.127.
Colour change : pH 5 (red) to pH 8 (blue). : about 1.450.
Litmus paper, blue. 1049301. Macrogol 200 R1. 1099201.
Boil 10 parts of coarsely powdered litmus R for 1 h with
Introduce 500 mL of macrogol 200 R into a 1000 mL round
100 parts of ethanol (96 per cent) R. Decant the alcohol
bottom flask. Using a rotation evaporator remove any
and add to the residue a mixture of 45 parts of ethanol
volatile components applying for 6 h a temperature of 60 °C
(96 per cent) R and 55 parts of water R. After 2 days decant
and a vacuum with a pressure of 1.5-2.5 kPa.
the clear liquid. Impregnate strips of filter paper with the
solution and allow to dry. Macrogol 300. 1067100. [25322-68-3]. Polyethyleneglycol
Test for sensitivity. Immerse a strip measuring 10 mm by 300.
60 mm in a mixture of 10 mL of 0.02 M hydrochloric acid See Macrogols (1444).
and 90 mL of water R. On shaking the paper turns red
within 45 s. Macrogol 400. 1067200. [25322-68-3]. Polyethyleneglycol
400.
Litmus paper, red. 1049302.
To the blue litmus extract, add dilute hydrochloric acid R See Macrogols (1444).
dropwise until the blue colour becomes red. Impregnate Macrogol 1000. 1067300. [25322-68-3]. Polyethyleneglycol
strips of filter paper with the solution and allow to dry. 1000.
Test for sensitivity. Immerse a strip measuring 10 mm by See Macrogols (1444).
60 mm in a mixture of 10 mL of 0.02 M sodium hydroxide
and 90 mL of water R. On shaking the paper turns blue Macrogol 1500. 1067400. [25322-68-3]. Polyethyleneglycol
within 45 s. 1500.
Loganin. C17H26O10. (Mr 390.4). 1136700. [18524-94-2]. See Macrogols (1444).
Methyl (1S,4aS,6S,7R,7aS)-1-(β-D-glucopyranosyloxy)-
6-hydroxy-7-methyl-1,4a,5,6,7,7a-hexahydro- Macrogol 20 000. 1067600. Polyethyleneglycol 20 000.
cyclopenta[c]pyran-4-carboxylate. See Macrogols (1444).
mp : 220 °C to 221 °C. Macrogol 20 000 2-nitroterephthalate. 1067601.
Longifolene. C15H24. (Mr 204.4). 1150300. [475-20-7]. Polyethyleneglycol 20 000 2-nitroterephthalate.
(1S,3aR,4S,8aS)-4,8,8-Trimethyl-9-methylenedecahydro-1,4- Macrogol 20 000 R modified by treating with
methanoazulene. 2-nitroterephthalate acid.
Oily, colourless liquid, practically insoluble in water, miscible A hard, white or almost white, waxy solid, soluble in
with ethanol (96 per cent). acetone.
: 0.9319.
Magnesium. Mg. (Ar 24.30). 1049500. [7439-95-4].
: 1.5050.
: + 42.7. Silver-white ribbon, turnings or wire, or a grey powder.
bp : 254 °C to 256 °C. Magnesium acetate. C4H6MgO4,4H2O. (Mr 214.5). 1049600.
Longifolene used in gas chromatography complies with the [16674-78-5]. Magnesium diacetate tetrahydrate.
following additional test. Colourless crystals, deliquescent, freely soluble in water and
Assay. Gas chromatography (2.2.28) as prescribed in the in ethanol (96 per cent).
monograph Turpentine oil, Pinus pinaster type (1627). Storage : in an airtight container.
Content : minimum 98.0 per cent, calculated by the
normalisation procedure. Magnesium chloride. 1049700. [7791-18-6].
See Magnesium chloride hexahydrate (0402).
Low-vapour-pressure hydrocarbons (type L). 1049400.
Unctuous mass, soluble in benzene and in toluene. Magnesium nitrate. Mg(NO3)2,6H2O. (Mr 256.4). 1049800.
[13446-18-9]. Magnesium nitrate hexahydrate.
Lumiflavine. C13H12N4O2. (Mr 256.3). 1141000. [1088-56-8].
7,8,10-Trimethylbenzo[g]pteridine-2,4(3H,10H)-dione. Colourless, clear crystals, deliquescent, very soluble in water,
freely soluble in ethanol (96 per cent).
Yellow powder or orange crystals, very slightly soluble in
water, freely soluble in methylene chloride. Storage : in an airtight container.
Luteolin-7-glucoside. C21H20O11. (Mr 448.4). 1163400. Magnesium nitrate solution. 1049801.
[5373-11-5]. 2-(3,4-Dihydroxyphenyl)-7-(β-D- Dissolve 17.3 g of magnesium nitrate R in 5 mL of water R
glucopyranosyloxy)-5-hydroxy-4H-1-benzopyran-4-one. warming gently and add 80 mL of ethanol (96 per cent) R.
Yellow powder. Cool and dilute to 100.0 mL with the same solvent.
Magnesium nitrate solution R1. 1049802. Maleic anhydride. C4H2O3. (Mr 98.1). 1050700. [108-31-6].
Dissolve 20 g of magnesium nitrate R (Mg(NO3)2,6H2O) Butenedioic anhydride. 2,5-Furandione.
in deionised distilled water R and dilute to 100 mL with White or almost white crystals, soluble in water forming
the same solvent. Immediately before use, dilute 10 mL to maleic acid, very soluble in acetone and in ethyl acetate, freely
100 mL with deionised distilled water R. A volume of 5 μL soluble in toluene, soluble in ethanol (96 per cent) with ester
will provide 0.06 mg of Mg (NO3)2. formation, very slightly soluble in light petroleum.
Magnesium oxide. 1049900. [1309-48-4]. mp : about 52 °C.
See Light magnesium oxide (0040). Any residue insoluble in toluene does not exceed 5 per cent
(maleic acid).
Magnesium oxide R1. 1049901.
Maleic anhydride solution. 1050701.
Complies with the requirements prescribed for magnesium
oxide R with the following modifications. Dissolve 5 g of maleic anhydride R in toluene R and dilute
to 100 mL with the same solvent. Use within one month.
Arsenic (2.4.2, Method A) : maximum 2 ppm. If the solution becomes turbid, filter.
Dissolve 0.5 g in a mixture of 5 mL of water R and 5 mL of
hydrochloric acid R1. Maltitol. 1136800. [585-88-6].
Heavy metals (2.4.8) : maximum 10 ppm. See Maltitol (1235).
Dissolve 1.0 g in a mixture of 3 mL of water R and 7 mL Maltotriose. C18H32O16. (Mr 504.4). 1176300. [1109-28-0].
of hydrochloric acid R1. Add 0.05 mL of phenolphthalein α-D-Glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→4)-D-
solution R and concentrated ammonia R until a pink colour glucose.
is obtained. Neutralise the excess of ammonia by the
addition of glacial acetic acid R. Add 0.5 mL in excess and White or almost white, crystalline powder, very soluble in
dilute to 20 mL with water R. Filter, if necessary. 12 mL of water.
the solution complies with test A. Prepare the reference mp : about 134 °C.
solution using a mixture of 5 mL of lead standard solution
(1 ppm Pb) R and 5 mL of water R. Mandelic acid. C8H8O3. (Mr 152.1). 1171300. [90-64-2].
2-Hydroxy-2-phenylacetic acid.
Iron (2.4.9) : maximum 50 ppm.
White crystalline flakes, soluble in water.
Dissolve 0.2 g in 6 mL of dilute hydrochloric acid R and
dilute to 10 mL with water R. mp : 118 to 121 °C.
Magnesium oxide, heavy. 1050000. [1309-48-4]. Manganese sulfate. MnSO4,H2O. (Mr 169.0). 1050900.
[10034-96-5]. Manganese sulfate monohydrate.
See Heavy magnesium oxide (0041).
Pale-pink, crystalline powder or crystals, freely soluble in
Magnesium silicate for pesticide residue analysis. 1129100. water, practically insoluble in ethanol (96 per cent).
[1343-88-0]. Loss on ignition : 10.0 per cent to 12.0 per cent, determined on
Magnesium silicate for chromatography (60-100 mesh). 1.000 g at 500 ± 50 °C.
Magnesium sulfate. 1050200. [10034-99-8]. Mannitol. 1051000. [69-65-8].
See Magnesium sulfate heptahydrate (0044). See Mannitol (0559).
Magnolol. C18H18O2. (Mr 266.3). 1182800. [528-43-8]. Mannose. C6H12O6. (Mr 180.2). 1051100. [3458-28-4].
5,5′-Di(prop-2-enyl)biphenyl-2,2′-diol. 5,5′-Diallyl-2,2′- D-(+)-Mannose.
dihydroxybiphenyl. 5,5′-Di-2-propenyl-[1,1′-biphenyl]-2,2′- white or almost white, crystalline powder or small crystals,
diol. very soluble in water, slightly soluble in anhydrous ethanol.
Maize oil. 1050400. : + 13.7 + 14.7, determined on a 200 g/L solution in
See Maize oil, refined (1342). water R containing about 0.05 per cent of NH3.
mp : about 132 °C, with decomposition.
Malachite green. C23H25ClN2. (Mr 364.9). 1050500.
[123333-61-9]. Marrubiin. C20H28O4. (Mr 332.4). 1158300. [465-92-9].
Schultz No. 754. (2aS,5aS,6R,7R,8aR,8bR)-6-[2-(Furan-3-yl)ethyl]-6-hydroxy-
2a,5a,7-trimethyldecahydro-2H-naphtho[1,8-bc]furan-2-one.
Colour Index No. 42000.
[4-[[4-(Dimethylamino)phenyl]phenylmethylene]cyclohexa- Colourless, microcrystalline powder.
2,5-dien-1-ylidene]dimethylammonium chloride. Marrubiin used in liquid chromatography complies with the
Green crystals with a metallic lustre, very soluble in water following additional test.
giving a bluish-green solution, soluble in ethanol (96 per cent) Assay. Liquid chromatography (2.2.29) as prescribed in the
and in methanol. monograph White horehound (1835).
Absorbance (2.2.25). A 0.01 g/L solution in ethanol (96 per Content : minimum 95.0 per cent, calculated by the
cent) R shows an absorption maximum at 617 nm. normalisation procedure.
Malachite green solution. 1050501. Meclozine dihydrochloride. 1051200. [1104-22-9].
A 5 g/L solution in anhydrous acetic acid R. See Meclozine dihydrochloride (0622).
Malathion. C10H19O6PS2. (Mr 330.3). 1129200. [121-75-5]. Melamine. C3H6N6. (Mr 126.1). 1051300. [108-78-1].
bp : about 156 °C. 1,3,5-Triazine-2,4,6-triamine.
A suitable certified reference solution (10 ng/μL in iso-octane) A white or almost white, amorphous powder, very slightly
may be used. soluble in water and in ethanol (96 per cent).
Maleic acid. 1050600. [110-16-7]. Menadione. 1051400. [58-27-5].
See Maleic acid (0365). See Menadione (0507).
General Notices (1) apply to all monographs and other texts 483
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Menthofuran. C10H14O. (Mr 150.2). 1051500. Mercuric acetate. C4H6HgO4. (Mr 318.7). 1052000.
[17957-94-7]. 3,9-Epoxy-p-mentha-3,8-diene. [1600-27-7]. Mercury diacetate.
3,6-Dimethyl-4,5,6,7-tetrahydro-benzofuran. White or almost white crystals, freely soluble in water, soluble
Slightly bluish liquid, very slightly soluble in water, soluble in in ethanol (96 per cent).
ethanol (96 per cent).
Mercuric acetate solution. 1052001.
: about 0.965.
Dissolve 3.19 g of mercuric acetate R in anhydrous acetic
: about 1.480. acid R and dilute to 100 mL with the same acid. If necessary,
: about + 93. neutralise the solution with 0.1 M perchloric acid using
bp : 196 °C. 0.05 mL of crystal violet solution R as indicator.
Menthofuran used in gas chromatography complies with the
Mercuric bromide. HgBr2. (Mr 360.4). 1052100. [7789-47-1].
following additional test. Mercury dibromide.
Assay. Gas chromatography (2.2.28) as prescribed in the
White or faintly yellow crystals or a crystalline powder, slightly
monograph Peppermint oil (0405).
soluble in water, soluble in ethanol (96 per cent).
Test solution. The substance to be examined.
Content : minimum 97.0 per cent, calculated by the Mercuric bromide paper. 1052101.
normalisation procedure. In a rectangular dish place a 50 g/L solution of mercuric
bromide R in anhydrous ethanol R and immerse in it pieces
Menthol. 1051600. [2216-51-5]. of white filter paper weighing 80 g per square metre (speed
See Levomenthol (0619) and Racemic menthol (0623). of filtration = filtration time expressed in seconds for
Menthol used in gas chromatography complies with the 100 mL of water at 20 °C with a filter surface of 10 cm2 and
following additional test. constant pressure of 6.7 kPa : 40 s to 60 s), each measuring
Assay. Gas chromatography (2.2.28) as prescribed in the 1.5 cm by 20 cm and folded in two. Allow the excess liquid
related substances test included in the monograph Racemic to drain and allow the paper to dry, protected from light,
menthol (0623). suspended over a non-metallic thread. Discard 1 cm from
Content : minimum 98.0 per cent, calculated by the each end of each strip and cut the remainder into 1.5 cm
normalisation procedure. squares or discs of 1.5 cm diameter.
Storage : in a glass-stoppered container wrapped with black
Menthone. C10H18O. (Mr 154.2). 1051700. [14073-97-3]. paper.
(2S,5R)-2-Isopropyl-5-methylcyclohexanone.
(–)-trans-p-Menthan-3-one. Mercuric chloride. 1052200. [7487-94-7].
Contains variable amounts of isomenthone. See Mercuric chloride (0120).
Colourless liquid, very slightly soluble in water, very soluble Mercuric chloride solution. 1052201.
in ethanol (96 per cent). A 54 g/L solution.
: about 0.897.
: about 1.450. Mercuric iodide. HgI2. (Mr 454.4). 1052300. [7774-29-0].
Mercury di-iodide.
Menthone used in gas chromatography complies with the
following additional test. Dense, scarlet, crystalline powder, slightly soluble in water,
sparingly soluble in acetone and in ethanol (96 per cent),
Assay. Gas chromatography (2.2.28) as prescribed in the
soluble in an excess of potassium iodide solution R.
monograph Peppermint oil (0405).
Storage : protected from light.
Test solution. The substance to be examined.
Content : minimum 90.0 per cent, calculated by the Mercuric nitrate. Hg(NO3)2,H2O. (Mr 342.6). 1052400.
normalisation procedure. [7783-34-8]. Mercury dinitrate monohydrate.
Menthyl acetate. C12H22O2. (Mr 198.3). 1051800. [2623-23-6]. Colourless or slightly coloured crystals, hygroscopic, soluble
2-Isopropyl-5-methylcyclohexyl acetate. in water in the presence of a small quantity of nitric acid.
Colourless liquid, slightly soluble in water, miscible with Storage : in an airtight container, protected from light.
ethanol (96 per cent). Mercuric oxide. HgO. (Mr 216.6). 1052500. [21908-53-2].
: about 0.92. Yellow mercuric oxide. Mercury oxide.
: about 1.447. A yellow to orange-yellow powder, practically insoluble in
bp : about 228 °C. water and in ethanol (96 per cent).
Menthyl acetate used in gas chromatography complies with the Storage : protected from light.
following additional test. Mercuric sulfate solution. 1052600. [7783-35-9].
Assay. Gas chromatography (2.2.28) as prescribed in the Dissolve 1 g of mercuric oxide R in a mixture of 20 mL of
monograph Peppermint oil (0405). water R and 4 mL of sulfuric acid R.
Test solution. The substance to be examined.
Content : minimum 97.0 per cent, calculated by the Mercuric thiocyanate. Hg(SCN)2. (Mr 316.7). 1052700.
normalisation procedure. [592-85-8]. Mercury di(thiocyanate).
White or almost white, crystalline powder, very slightly
2-Mercaptobenzimidazole. C7H6N2S. (Mr 150.2). 1170100. soluble in water, slightly soluble in ethanol (96 per cent),
[583-39-1]. 1H-benzimidazole-2-thiol. soluble in solutions of sodium chloride.
mp : about 302 °C.
Mercuric thiocyanate solution. 1052701.
2-Mercaptoethanol. C2H6OS. (Mr 78.1). 1099300. [60-24-2]. Dissolve 0.3 g of mercuric thiocyanate R in anhydrous
Liquid, miscible with water. ethanol R and dilute to 100 mL with the same solvent.
: about 1.116. Storage : use within 1 week.
bp : about 157 °C.
Mercury. Hg. (Ar 200.6). 1052800. [7439-97-6].
Mercaptopurine. 1051900. [6112-76-1]. Silver-white liquid, breaking into spherical globules which do
See Mercaptopurine (0096). not leave a metallic trace when rubbed on paper.
General Notices (1) apply to all monographs and other texts 485
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
trans-2-Methoxycinnamaldehyde. C10H10O2. (Mr 162.2). Content : minimum 99.5 per cent, determined by gas
1129500. [60125-24-8]. chromatography.
mp : 44 °C to 46 °C. Methyl 4-aminobenzoate. C8H9NO2. (Mr 151.2). 1175600.
trans-2-Methoxycinnamaldehyde used in gas chromatography [619-45-4].
complies with the following additional test. mp : 110 °C to 113 °C.
Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Cassia oil (1496). 4-Methylaminophenol sulfate. C14H20N2O6S. (Mr 344.4).
1053800. [55-55-0].
Content : minimum 96.0 per cent, calculated by the
normalisation procedure. Colourless crystals, very soluble in water, slightly soluble in
ethanol (96 per cent).
(1RS)-1-(6-Methoxynaphthalen-2-yl)ethanol. mp : about 260 °C.
C13H14O2. (Mr 202.3). 1159600. [77301-42-9].
6-Methoxy-α-methyl-2-naphthalenemethanol. 3-(Methylamino)-1-phenylpropan-1-ol. C10H15NO.
White or almost white powder. (Mr 165.2). 1186400. [42142-52-9].
mp : about 113 °C. White or almost white powder.
mp : 59 °C to 64 °C.
1-(6-Methoxynaphthalen-2-yl)ethanone. C13H12O2.
(Mr 200.2). 1159700. [3900-45-6]. 6′-Methoxy-2′- Methyl anthranilate. C8H9NO2. (Mr 151.2). 1107300.
acetonaphthone. [134-20-3]. Methyl 2-aminobenzoate.
White or almost white powder. Colourless crystals or a colourless or yellowish liquid, soluble
mp : about 108 °C. in water, freely soluble in ethanol (96 per cent).
bp : 134 °C to 136 °C.
6-Methoxy-2-naphthoic acid. C12H10O3. (Mr 202.2). mp : 24 °C to 25 °C.
1184200. [2471-70-7]. 6-Methoxynaphthalene-2-carboxylic
acid. Methyl anthranilate used in gas chromatography complies with
the following additional test.
White or almost white, crystalline powder.
Assay. Gas chromatography (2.2.28) as prescribed in the
mp : 201 °C to 206 °C. monograph Bitter-orange-flower oil (1175).
Methoxyphenylacetic acid. C9H10O3. (Mr 166.2). 1053600. Test solution. The substance to be examined.
[7021-09-2]. (RS)-2-Methoxy-2-phenylacetic acid. Content : minimum 95.0 per cent, calculated by the
White, crystalline powder or white or almost white crystals, normalisation procedure.
sparingly soluble in water, freely soluble in ethanol (96 per
Methyl arachidate. C21H42O2. (Mr 326.6). 1053900.
cent).
[1120-28-1]. Methyl eicosanoate.
mp : about 70 °C. Content : minimum 98.0 per cent, determined by gas
Methoxyphenylacetic reagent. 1053601. chromatography (2.4.22).
Dissolve 2.7 g of methoxyphenylacetic acid R in 6 mL of White or yellow, crystalline mass, soluble in ethanol (96 per
tetramethylammonium hydroxide solution R and add 20 mL cent) and in light petroleum.
of anhydrous ethanol R. mp : about 46 °C.
Storage : in a polyethylene container. Methyl behenate. C23H46O2. (Mr 354.6). 1107500. [929-77-1].
3-Methoxy- L-tyrosine. C10H13NO4H2O. (Mr 229.2). 1164400. Methyl docosanoate.
[200630-46-2]. mp : 54 °C to 55 °C.
Off-white or yellow powder. Methyl benzenesulfonate. C7H8O3S. (Mr 172.2). 1159800.
Methyl acetate. C3H6O2. (Mr 74.1). 1053700. [79-20-9]. [80-18-2].
Clear, colourless liquid, soluble in water, miscible with ethanol Clear, colourless liquid.
(96 per cent). bp : about 148 °C.
: about 0.933. Methyl benzoate. C8H8O2. (Mr 136.2). 1164500. [93-58-3].
: about 1.361. Benzoic acid, methyl ester.
bp : 56 °C to 58 °C. Colourless liquid.
: 1.088.
Methyl 4-acetylbenzoate. C10H10O3. (Mr 178.2). 1154100.
[3609-53-8]. bp : about 200 °C.
mp : about 94 °C. Methylbenzothiazolone hydrazone hydrochloride.
C8H10ClN3S,H2O. (Mr 233.7). 1055300. [38894-11-0].
Methyl 4-acetylbenzoate reagent. 1154101. 3-Methylbenzothiazol-2(3H)-one hydrazone hydrochloride
Dissolve 0.25 g of methyl 4-acetylbenzoate R in a mixture of monohydrate.
5 mL of sulfuric acid R and 85 mL of cooled methanol R. Almost white or yellowish, crystalline powder.
Methylal. C3H8O2. (Mr 76.1). 1173500. [109-87-5]. mp : about 270 °C.
Dimethoxymethane. Dioxapentane. Formaldehyde dimethyl Suitability for determination of aldehydes. To 2 mL of
acetal. Methylene dimethyl ether. aldehyde-free methanol R add 60 μL of a 1 g/L solution of
Clear, colourless, volatile, flammable liquid, soluble in water propionaldehyde R in aldehyde-free methanol R and 5 mL
and miscible with ethanol (96 per cent). of a 4 g/L solution of methylbenzothiazolone hydrazone
: about 0.860. hydrochloride. Mix. Allow to stand for 30 min. Prepare a
blank omitting the propionaldehyde solution. Add 25.0 mL of
: about 1.354.
a 2 g/L solution of ferric chloride R to the test solution and to
bp : about 41 °C. the blank, dilute to 100.0 mL with acetone R and mix. The
Methylal used in gas chromatography complies with the absorbance (2.2.25) of the test solution, measured at 660 nm
following additional test. using the blank as compensation liquid, is not less than 0.62.
(R)-(+)-α-Methylbenzyl isocyanate. C9H9NO. (Mr 147.2). Methyl decanoate. C11H22O2. (Mr 186.3). 1054000.
1171400. [33375-06-3]. (+)-(R)-α-Methylbenzyl isocyanate. [110-42-9].
(+)-[(1R)-1-Isocyanatoethyl]benzene. (+)-(1R)-1-Phenylethyl Content : minimum 99.0 per cent.
isocyanate. Clear, colourless or yellow liquid, soluble in light petroleum.
Content : minimum 99.0 per cent. : 0.871 to 0.876.
Colourless liquid. : 1.425 to 1.426.
: about 1.045. Foreign substances. Gas chromatography (2.2.28), injecting
: about 1.513. equal volumes of each of the following :
bp : 55 °C to 56 °C at 2.5 mm Hg. A 0.02 g/L solution of the substance to be examined in carbon
Enantiomeric purity : minimum 99.5. disulfide R (solution A), a 2 g/L solution of the substance to
Storage : at a temperature of 2 °C to 8 °C. be examined in carbon disulfide R (solution B), and carbon
disulfide R (solution C). Carry out the chromatographic
(S)-(−)-α-Methylbenzyl isocyanate. C9H9NO. (Mr 147.2). procedure under the conditions of the test for butylated
1170200. [14649-03-7]. (−)-(S)-α-Methylbenzyl isocyanate. hydroxytoluene prescribed in the monograph Wool fat (0134).
(−)-[(1S)-1-Isocyanatoethyl]benzene. (−)-(1S)-1-Phenylethyl The total area of any peaks, apart from the solvent peak
isocyanate. and the principal peak, in the chromatogram obtained with
Content : minimum 99.0 per cent. solution B is less than the area of the principal peak in the
Colourless liquid. chromatogram obtained with solution A.
: about 1.045. Methyldopa, racemic. C10H13NO4,1½H2O. (Mr 238.2).
: about 1.514. 1175100.
bp : 55 °C to 56 °C at 2.5 mm Hg. Mixture of equal volumes of (2S)- and (2R)-2-amino-3-(3,4-
Enantiomeric purity : minimum 99.5 per cent. dihydroxyphenyl)-2-methylpropanoic acids.
Storage : at a temperature of 2 °C to 8 °C. 3-O-Methyldopamine hydrochloride. C9H14ClNO2.
NOTE : do not use the reagent if it is coloured. (Mr 203.7). 1055600. [1477-68-5]. 4-(2-Aminoethyl)-2-
methoxyphenol hydrochloride.
2-Methylbutane. C5H12. (Mr 72.2). 1099500. [78-78-4].
Isopentane. mp : 213 °C to 215 °C.
Content : minimum 99.5 per cent of C5H12. 4-O-Methyldopamine hydrochloride. C9H14ClNO2.
Very flammable colourless liquid. (Mr 203.7). 1055700. [645-33-0]. 5-(2-Aminoethyl)-2-
: about 0.621. methoxyphenol hydrochloride.
: about 1.354. mp : 207 °C to 208 °C.
bp : about 29 °C. Methylenebisacrylamide. C7H10N2O2. (Mr 154.2). 1056000.
Water (2.5.12) : maximum 0.02 per cent. [110-26-9]. N,N′-Methylenebispropenamide.
Residue on evaporation : maximum 0.0003 per cent. Fine, white or almost white powder, slightly soluble in water,
Minimum transmittance (2.2.25) using water R as soluble in ethanol (96 per cent).
compensation liquid : 50 per cent at 210 nm, 85 per cent at mp : 300 °C, with decomposition.
220 nm, 98 per cent at 240 nm and at higher wavelengths. Methylene blue. C16H18ClN3S,xH2O. (Mr 319.9 for the
2-Methylbut-2-ene. C5H10. (Mr 70.1). 1055400. [513-35-9]. anhydrous substance). 1055800. [122965-43-9].
Very flammable liquid, practically insoluble in water, miscible Schultz No. 1038.
with ethanol (96 per cent). Colour Index No. 52015.
bp : 37.5 °C to 38.5 °C. 3,7-Dimethylaminophenothiazin-5-ium chloride.
It occurs in different hydrated forms and may contain up to
Methyl caprate. 1054000. 22 per cent of water.
See Methyl decanoate R. Dark-green or bronze, crystalline powder, freely soluble in
Methyl caproate. C7H14O2. (Mr 130.2). 1120300. [106-70-7]. water, soluble in ethanol (96 per cent).
Methyl hexanoate. Methylene chloride. CH2Cl2. (Mr 84.9). 1055900. [75-09-2].
: about 0.885. Dichloromethane.
: about 1.405. Colourless liquid, sparingly soluble in water, miscible with
bp : 150 °C to 151 °C. ethanol (96 per cent).
bp : 39 °C to 42 °C.
Methyl caprylate. C9H18O2. (Mr 158.2). 1120400. [111-11-5].
Methyl octanoate. Methylene chloride used in fluorimetry complies with the
following additional test.
: about 0.876.
Fluorescence. Under irradiation at 365 nm, the fluorescence
: about 1.417. (2.2.21) measured at 460 nm in a 1 cm cell is not more intense
bp : 193 °C to 194 °C. than that of a solution containing 0.002 ppm of quinine R in
Methylcellulose 450. 1055500. [9004-67-5]. 0.5 M sulfuric acid measured in the same conditions.
See Methylcellulose (0345). Methylene chloride, acidified. 1055901.
Nominal viscosity : 450 mPa·s. To 100 mL of methylene chloride R add 10 mL of
hydrochloric acid R, shake, allow to stand and separate the
Methyl cinnamate. C10H10O2. (Mr 162.2). 1099400. two layers. Use the lower layer.
[103-26-4].
Colourless crystals practically insoluble in water, soluble in Methyl eicosenoate. C21H40O2. (Mr 324.5). 1120500.
ethanol (96 per cent). [2390-09-2]. Methyl (11Z)-eicos-11-enoate.
: about 1.56. Methyl erucate. C23H44O2. (Mr 352.6). 1146100. [1120-34-9].
bp : about 260 °C. Methyl (13Z)-docos-13-enoate.
mp : 34 °C to 36 °C. : about 0.871.
General Notices (1) apply to all monographs and other texts 487
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 489
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine. C12H15N. Test for sensitivity. To 0.1 mL of the methyl red solution
(Mr 173.3). 1137100. [28289-54-5]. MPTP. add 100 mL of carbon dioxide-free water R and 0.05 mL of
White or almost white, crystalline powder, slightly soluble in 0.02 M hydrochloric acid. The solution is red. Not more
water. than 0.1 mL of 0.02 M sodium hydroxide is required to
mp : about 41 °C. change the colour to yellow.
Colour change : pH 4.4 (red) to pH 6.0 (yellow).
Methylpiperazine. C5H12N2. (Mr 100.2). 1056300. [109-01-3].
1-Methylpiperazine. Methyl salicylate. 1146200. [119-36-8].
Colourless liquid, miscible with water and with ethanol See Methyl salicylate (0230)
(96 per cent). Methyl stearate. C19H38O2. (Mr 298.5). 1055200. [112-61-8].
: about 0.90. Methyl octadecanoate.
: about 1.466. Content : minimum 98.0 per cent, determined by gas
bp : about 138 °C. chromatography (2.4.22).
4-(4-Methylpiperidin-1-yl)pyridine. C11H16N2. (Mr 176.3). White or yellow, crystalline mass, soluble in ethanol (96 per
1114400. [80965-30-6]. cent) and in light petroleum.
Clear liquid. mp : about 38 °C.
: about 1.565. Methylthymol blue. C37H40N2Na4O13S. (Mr 845). 1158500.
[1945-77-3]. Tetrasodium 2,2′,2″,2′″-[3H-2,1-benzoxathiol-
2-Methylpropanol. C4H10O. (Mr 74.1). 1056400. [78-83-1]. 3-ylidenebis[[6-hydroxy-2-methyl-5-(1-methylethyl)-3,1-
Isobutyl alcohol. 2-Methylpropan-1-ol. phenylene]methylenenitrilo]]tetraacetate S,S-dioxide.
Clear colourless liquid, soluble in water, miscible with ethanol Produces a blue colour with calcium in alkaline solution.
(96 per cent).
: about 0.80. Methylthymol blue mixture. 1158501.
: 1.397 to 1.399. A mixture of 1 part of methylthymol blue R and 100 parts
of potassium nitrate R.
bp : about 107 °C.
Distillation range (2.2.11). Not less than 96 per cent distils N-Methyl-m-toluidine. C8H11N. (Mr 121.2). 1175200.
between 107 °C and 109 °C. [696-44-6]. N,3-Dimethylaniline. N,3-Dimethylbenzenamine.
Methyl-m-tolylamine.
2-Methyl-2-propanol. C4H10O. (Mr 74.1). 1056500.
Content : minimum 97 per cent.
[75-65-0]. 1,1-Dimethyl ethyl alcohol. tert-Butyl alcohol.
Clear, colourless liquid or crystalline mass, soluble in water, Methyl tricosanoate. C24H48O2. (Mr 368.6). 1111500.
miscible with ethanol (96 per cent). [2433-97-8]. Tricosanoic acid methyl ester.
Freezing point (2.2.18) : about 25 °C. Content : minimum 99.0 per cent.
Distillation range (2.2.11). Not less than 95 per cent distils White or almost white crystals, practically insoluble in water,
between 81 °C and 83 °C. soluble in hexane.
mp : 55 °C to 56 °C.
(15R)-15-Methylprostaglandin F2α. C21H36O5. (Mr 368.5).
1159900. [35864-81-4]. (5Z)-7-[(1R,2R,3R,5S)-3,5- Methyl tridecanoate. C14H28O2. (Mr 228.4). 1121100.
Dihydroxy-2-[(1E)-(3R)-3-hydroxy-3-methyloct-1- [1731-88-0].
enyl]cyclopentyl]hept-5-enoic acid. Colourless or slightly yellow liquid, soluble in ethanol (96 per
Available as a 10 g/L solution in methyl acetate R. cent) and in light petroleum.
Storage : at a temperature below − 15 °C. : about 0.86.
N-Methylpyrrolidine. C5H11N. (Mr 85.2). 1164700. : about 1.441.
[120-94-5]. mp : about 6 °C.
Content : minimum 97.0 per cent. Methyl 3,4,5-trimethoxybenzoate. C11H14O5. (Mr 226.23).
bp : about 80 °C. 1177200. [1916-07-0].
N-Methylpyrrolidone. C5H9NO. (Mr 99.1). 1164800. N-Methyltrimethylsilyl-trifluoroacetamide.
[872-50-4]. 1-Methylpyrrolidin-2-one. C6H12F3NOSi. (Mr 199.3). 1129600. [24589-78-4].
: about 1.028. 2,2,2-Trifluoro-N-methyl-N-(trimethylsilyl)acetamide.
bp : about 202 °C. : about 1.380.
mp : about − 24 °C. bp : 130 °C to 132 °C.
Methyl red. C15H15N3O2. (Mr 269.3). 1055100. [493-52-7]. Minocycline hydrochloride. 1146300.
Schultz No. 250. See Minocycline hydrochloride (1030).
Colour Index No. 13020. Molecular sieve. 1056600.
2-(4-Dimethylamino-phenylazo)benzoic acid. Molecular sieve composed of sodium aluminosilicate. It
Dark-red powder or violet crystals, practically insoluble in is available as beads with a pore size of 0.4 nm and with a
water, soluble in ethanol (96 per cent). diameter of 2 mm.
Methyl red mixed solution. 1055101. Molecular sieve for chromatography. 1129700.
Dissolve 0.1 g of methyl red R and 50 mg of methylene Molecular sieve composed of sodium aluminosilicate. The
blue R in 100 mL of ethanol (96 per cent) R. pore size is indicated after the name of the reagent in the tests
Colour change : pH 5.2 (red-violet) to pH 5.6 (green). where it is used. If necessary, the particle size is also indicated.
Methyl red solution. 1055102. Molybdovanadic reagent. 1056700.
Dissolve 50 mg in a mixture of 1.86 mL of 0.1 M sodium In a 150 mL beaker, mix 4 g of finely powdered ammonium
hydroxide and 50 mL of ethanol (96 per cent) R and dilute molybdate R and 0.1 g of finely powdered ammonium
to 100 mL with water R. vanadate R. Add 70 mL of water R and grind the particles
using a glass rod. A clear solution is obtained within a few β-Myrcene. C10H16. (Mr 136.2). 1114500. [123-35-3].
minutes. Add 20 mL of nitric acid R and dilute to 100 mL 7-Methyl-3-methylenocta-1,6-diene.
with water R. Oily liquid with a pleasant odour, practically insoluble in
water, miscible with ethanol (96 per cent), soluble in glacial
Monodocosahexaenoin. C25H38O4. (Mr 402.6). 1143600.
acetic acid. It dissolves in solutions of alkali hydroxides.
[124516-13-8]. Monoglyceride of docosahexaenoic
acid (C22:6). Glycerol monodocosahexaenoate. : about 0.794.
)-Docosa-4,7,10,13,16,19-hexaenoic
(all-Z acid, monoester : about 1.470.
with propane-1,2,3-triol. β-Myrcene used in gas chromatography complies with the
following additional test.
Mordant black 11. C20H12N3NaO7S. (Mr 461.4). 1056800.
[1787-61-7]. Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Peppermint oil (0405).
Schultz No. 241.
Test solution. The substance to be examined.
Colour Index No. 14645.
Sodium 2-hydroxy-1-[(1-hydroxynaphth-2-yl)azo]-6- Content : minimum 90.0 per cent, calculated by the
nitronaph-thalene-4-sulfonate. Eriochrome black. normalisation procedure.
Brownish-black powder, soluble in water and in ethanol Myristic acid. C14H28O2. (Mr 228.4). 1143700. [544-63-8].
(96 per cent). Tetradecanoic acid.
Storage : in an airtight container, protected from light. Colourless or white or almost white flakes.
Mordant black 11 triturate. 1056801. mp : about 58.5 °C.
Mix 1 g of mordant black 11 R with 99 g of sodium Myristic acid used in the assay of total fatty acids in Saw
chloride R. palmetto fruit (1848) complies with the following additional
test.
Test for sensitivity. Dissolve 50 mg in 100 mL of water R.
Assay. Gas chromatography (2.2.28) as prescribed in the
The solution is brownish-violet. On addition of 0.3 mL
monograph Saw palmetto fruit (1848).
dilute of ammonia R1 the solution turns blue. On the
subsequent addition of 0.1 mL of a 10 g/L solution of Content : minimum 97 per cent, calculated by the
magnesium sulfate R, it turns violet. normalisation procedure.
Storage : in an airtight container, protected from light. Myristicine. C11H12O3. (Mr 192.2). 1099600. [607-91-0].
5-Allyl-1-methoxy-2,3-methylenedioxybenzene.
Mordant blackR1.11 triturate 1056802. 4-Methoxy-6-(prop-2-enyl)-1,3-benzodioxole.
Mix 1.0 g of mordant black 11 R, 0.4 g of methyl orange R Oily colourless liquid, practically insoluble in water, slightly
and 100 g of sodium chloride R. soluble in anhydrous ethanol, miscible with toluene and with
Morphine hydrochloride. 1056900. xylene.
See Morphine hydrochloride (0097). : about 1.144.
: about 1.540.
Morpholine. C4H9NO. (Mr 87.1). 1057000. [110-91-8]. bp : 276 °C to 277 °C.
Tetrahydro-1,4-oxazine.
mp : about 173 °C.
Colourless, hygroscopic liquid, flammable, soluble in water
and in ethanol (96 per cent). Chromatography. Thin-layer chromatography (2.2.27)
as prescribed in the monograph Star anise (1153) ; the
: about 1.01. chromatogram shows only one principal spot.
Distillation range (2.2.11). Not less than 95 per cent distils Myristicine used in gas chromatography complies with the
between 126 °C and 130 °C. following additional test.
Storage : in an airtight container. Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Nutmeg oil (1552).
Morpholine for chromatography. 1057001.
Content : minimum 95.0 per cent, calculated by the
Complies with the requirements prescribed for normalisation procedure.
morpholine R with the following additional requirement.
Storage : protected from light.
Content : minimum 99.5 per cent.
Myristyl alcohol. C14H30O. (Mr 214.4). 1121300. [112-72-1].
2-[N-Morpholino]ethanesulfonic acid. C6H13NO4S. Tetradecan-1-ol.
(Mr 195.2). 1186500. [4432-31-9]. 2-(Morpholin-4-yl)sulfonic
MES. acid. : about 0.823.
mp : 38 °C to 40 °C.
White or almost white, crystalline powder, soluble in water.
mp : about 300 °C. Myrtillin. C21H21ClO12. (Mr 500.8). 1172300. [6906-38-3].
Delphinidin 3-O-glucoside chloride.
Murexide. C8H8N6O6,H2O. (Mr 302.2). 1137200.
5,5′-Nitrilobis(pyrimidine-2,4,6(1H,3H,5H)-trione) Naphthalene. C10H8. (Mr 128.2). 1057100. [91-20-3].
monoammonium salt. White or almost white crystals, practically insoluble in water,
Brownish-red crystalline powder, sparingly soluble in cold soluble in ethanol (96 per cent).
water, soluble in hot water, practically insoluble in ethanol mp : about 80 °C.
(96 per cent), soluble in solutions of potassium hydroxide or Naphthalene used for liquid scintillation is of a suitable
sodium hydroxide giving a blue colour. analytical grade.
Myosmine. C9H10N2. (Mr 146.2). 1121200. [532-12-7]. Naphtharson. C16H11AsN2Na2O10S2. (Mr 576.3). 1121400.
3-(4,5-Dihydro-3H-pyrrol-2-yl)pyridine. [3688-92-4]. Thorin. Disodium 4-[(2-arsonophenyl)azo]-3-
Colourless crystals. hydroxynaphthalene-2,7-disulfonate.
mp : about 45 °C. Red powder, soluble in water.
General Notices (1) apply to all monographs and other texts 491
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Nickel chloride. NiCl2. (Mr 129.6). 1057900. [7718-54-9]. Ninhydrin solution R1. 1058304.
Nickel chloride, anhydrous. Dissolve 1.0 g of ninhydrin R in 50 mL of ethanol (96 per
cent) R and add 10 mL of glacial acetic acid R.
Yellow, crystalline powder, very soluble in water, soluble in
ethanol (96 per cent). It sublimes in the absence of air and Ninhydrin solution R2. 1058305.
readily absorbs ammonia. The aqueous solution is acid. Dissolve 3 g of ninhydrin R in 100 mL of a 45.5 g/L solution
of sodium metabisulfite R.
Nickel nitrate hexahydrate. Ni(NO3)2,6H2O. (Mr 290.8).
1175300. [13478-00-7]. Ninhydrin solution R3. 1058306.
A 4 g/L solution in a mixture of 5 volumes of anhydrous
Nickel sulfate. NiSO4,7H2O. (Mr 280.9). 1058000. acetic acid R and 95 volumes of butanol R.
[10101-98-1]. Nickel sulfate heptahydrate.
Green, crystalline powder or crystals, freely soluble in water, Ninhydrin solution R4. 1058307.
slightly soluble in ethanol (96 per cent). A 3 g/L solution of ninhydrin R in a mixture of 5 volumes
of glacial acetic acid R and 95 volumes of 2-propanol R.
Nicotinamide-adenine dinucleotide. C21H27N7O14P2.
(Mr 663). 1108100. [-84-9]. NAD+. Nitrazepam. 1143900. [146-22-5].
See Nitrazepam (0415).
White or almost white powder, very hygroscopic, freely
soluble in water. Nitric acid. HNO3. (Mr 63.0). 1058400. [7697-37-2].
Content : 63.0 per cent m/m to 70.0 per cent m/m.
Nicotinamide-adenine dinucleotide solution. 1108101.
Clear, colourless or almost colourless liquid, miscible with
Dissolve 40 mg of nicotinamide-adenine dinucleotide R in water.
water R and dilute to 10 mL with the same solvent. Prepare
: 1.384 to 1.416.
immediately before use.
A 10 g/L solution is strongly acid and gives the reaction of
Nicotinic acid. 1158600. [59-67-6]. nitrates (2.3.1).
See Nicotinic acid (0459). Appearance. Nitric acid is clear (2.2.1) and not more intensely
coloured than reference solution Y6 (2.2.2, Method II).
Nile blue A. C20H21N3O5S. (Mr 415.5). 1058200. [3625-57-8]. Chlorides (2.4.4) : maximum 0.5 ppm.
Schultz No. 1029. To 5 g add 10 mL of water R and 0.3 mL of silver nitrate
Colour Index No. 51180. solution R2 and allow to stand for 2 min protected from light.
5-Amino-9-(diethylamino)benzo[a]phenoxazinylium Any opalescence is not more intense than that of a standard
hydrogen sulfate. prepared in the same manner using 13 mL of water R, 0.5 mL
of nitric acid R, 0.5 mL of chloride standard solution (5 ppm
Green, crystalline powder with a bronze lustre, sparingly Cl) R and 0.3 mL of silver nitrate solution R2.
soluble in ethanol (96 per cent), in glacial acetic acid and in Sulfates (2.4.13) : maximum 2 ppm.
pyridine.
Evaporate 10 g to dryness with 0.2 g of sodium carbonate R.
Absorbance (2.2.25). A 0.005 g/L solution in ethanol (50 per Dissolve the residue in 15 mL of distilled water R. Prepare the
cent V/V) R shows an absorption maximum at 640 nm. standard using a mixture of 2 mL of sulfate standard solution
(10 ppm SO4) R and 13 mL of distilled water R.
Nile blue A solution. 1058201.
Arsenic (2.4.2, Method A) : maximum 0.02 ppm.
A 10 g/L solution in anhydrous acetic acid R.
Gently heat 50 g with 0.5 mL of sulfuric acid R until white
Test for sensitivity. To 50 mL of anhydrous acetic acid R fumes begin to evolve. To the residue add 1 mL of a 100 g/L
add 0.25 mL of the Nile blue A solution. The solution is solution of hydroxylamine hydrochloride R and dilute to 2 mL
blue. On the addition of 0.1 mL of 0.1 M perchloric acid, with water R. Prepare the standard using 1.0 mL of arsenic
the colour changes to blue-green. standard solution (1 ppm As) R.
Colour change : pH 9.0 (blue) to pH 13.0 (red). Iron (2.4.9) : maximum 1 ppm.
General Notices (1) apply to all monographs and other texts 493
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Dissolve the residue from the determination of sulfated ash in Source : lead hollow-cathode lamp.
1 mL of dilute hydrochloric acid R and dilute to 50 mL with Wavelength : 283.3 nm or 217.0 nm.
water R. Dilute 5 mL of this solution to 10 mL with water R. Atomisation device : air-acetylene flame.
Heavy metals (2.4.8) : maximum 2 ppm.
Dilute 10 mL of the solution prepared for the limit test for Nitric acid, lead-free R1. 1058405.
iron to 20 mL with water R. 12 mL of the solution complies Nitric acid R containing not more than 1 μg/kg of lead.
with test A. Prepare the reference solution using lead standard
Nitric acid, lead-free, dilute. 1058406.
solution (2 ppm Pb) R.
Dilute 5 g of lead-free nitric acid R1 to 100 mL with
Sulfated ash : maximum 0.001 per cent.
deionised distilled water R.
Carefully evaporate 100 g to dryness. Moisten the residue with
a few drops of sulfuric acid R and heat to dull red. Nitric acid, nickel-free. 1058408.
Assay. To 1.50 g add about 50 mL of water R and titrate with Complies with the requirements prescribed for nitric acid R
1 M sodium hydroxide, using 0.1 mL of methyl red solution R with the following additional requirement.
as indicator. Nickel : maximum 0.005 ppm.
1 mL of 1 M sodium hydroxide is equivalent to 63.0 mg of
HNO3. Nitric acid, fuming. 1058500. [52583-42-3].
Storage : protected from light. Clear, slightly yellowish liquid, fuming on contact with air.
: about 1.5.
Nitric acid, cadmium- and lead-free. 1058401.
Complies with the requirements prescribed for nitric acid R Nitrilotriacetic acid. C6H9NO6. (Mr 191.1). 1137400.
and with the following additional test. [139-13-9].
Test solution. To 100 g add 0.1 g of anhydrous sodium White or almost white crystalline powder, practically insoluble
carbonate R and evaporate to dryness. Dissolve the residue in water and in most organic solvents.
in water R heating slightly, and dilute to 50.0 mL with the mp : about 240 °C, with decomposition.
same solvent.
Nitroaniline. C6H6N2O2. (Mr 138.1). 1058600. [100-01-6].
Cadmium : maximum 0.1 ppm. 4-Nitroaniline.
Atomic absorption spectrometry (2.2.23, Method II). Bright yellow, crystalline powder, very slightly soluble in water,
Source : cadmium hollow-cathode lamp. sparingly soluble in boiling water, soluble in ethanol (96 per
Wavelength : 228.8 nm. cent), forms water-soluble salts with strong mineral acids.
Atomisation device : air-acetylene or air-propane flame. mp : about 147 °C.
Lead : maximum 0.1 ppm. Nitrobenzaldehyde. C7H5NO3. (Mr 151.1). 1058700.
Atomic absorption spectrometry (2.2.23, Method II). [552-89-6]. 2-Nitrobenzaldehyde.
Source : lead hollow-cathode lamp. Yellow needles, slightly soluble in water, freely soluble in
Wavelength : 283.3 nm or 217.0 nm. ethanol (96 per cent), volatile in steam.
Atomisation device : air-acetylene flame. mp : about 42 °C.
Nitric acid, dilute. 1058402. Nitrobenzaldehyde paper. 1058701.
Contains about 125 g/L of HNO3 (Mr 63.0). Dissolve 0.2 g of nitrobenzaldehyde R in 10 mL of a 200 g/L
Dilute 20 g of nitric acid R to 100 mL with water R. solution of sodium hydroxide R. Use the solution within 1 h.
Immerse the lower half of a slow filter paper strip 10 cm
Nitric acid, dilute R1. 1058407. long and 0.8-1 cm wide. Absorb the excess reagent between
Dilute 40 g of nitric acid R to 100 mL with water R. two sheets of filter paper. Use within a few minutes of
preparation.
Nitric acid, dilute R2. 1058409.
Nitrobenzaldehyde solution. 1058702.
Dilute 30 g of nitric acid R to 100 mL with water R.
Add 0.12 g of powdered nitrobenzaldehyde R to 10 mL
Nitric acid, heavy metal-free. 1058404. of dilute sodium hydroxide solution R ; allow to stand for
Complies with the requirements prescribed for nitric acid R 10 min shaking frequently and filter. Prepare immediately
with the following maximum contents of heavy metals. before use.
As : 0.005 ppm. Nitrobenzene. C6H5NO2. (Mr 123.1). 1058800. [98-95-3].
Cd : 0.005 ppm. Colourless or very slightly yellow liquid, practically insoluble
Cu : 0.001 ppm. in water, miscible with ethanol (96 per cent).
Fe : 0.02 ppm. bp : about 211 °C.
Hg : 0.002 ppm. Dinitrobenzene. To 0.1 mL add 5 mL of acetone R, 5 mL of
Ni : 0.005 ppm. water R and 5 mL of strong sodium hydroxide solution R. Shake
Pb : 0.001 ppm. and allow to stand. The upper layer is almost colourless.
Zn : 0.01 ppm. 4-Nitrobenzoic acid. C7H5NO4. (Mr 167.1). 1144000.
[62-23-7].
Nitric acid, lead-free. 1058403.
Yellow crystals.
Complies with the requirements prescribed for Nitric
acid R with the following additional test. mp : about 240 °C.
Lead : maximum 0.1 ppm. Nitrobenzoyl chloride. C7H4ClNO3. (Mr 185.6). 1058900.
Atomic absorption spectrometry (2.2.23, Method II). [122-04-3]. 4-Nitrobenzoyl chloride.
Test solution. To 100 g add 0.1 g of anhydrous sodium Yellow crystals or a crystalline mass, decomposing in moist
carbonate R and evaporate to dryness. Dissolve the residue air, completely soluble in sodium hydroxide solution giving a
in water R, heating slightly, and dilute to 50.0 mL with the yellowish-orange colour.
same solvent. mp : about 72 °C.
Nitrobenzyl chloride. C7H6ClNO2. (Mr 171.6). 1059000. 3-Nitrosalicylic acid. C7H5NO5. (Mr 183.1). 1184300.
[100-14-1]. 4-Nitrobenzyl chloride. [85-38-1]. 2-Hydroxy-3-nitrobenzoic acid.
Pale-yellow crystals, lachrymatory, practically insoluble in Yellowish crystals, slightly soluble in water, freely soluble in
water, very soluble in ethanol (96 per cent). ethanol (96 per cent).
4-(4-Nitrobenzyl)pyridine. C12H10N2O2. (Mr 214.2). mp : 142 °C to 147 °C.
1101900. [1083-48-3]. N-Nitrosodiethanolamine. C4H10N2O3. (Mr 134.1). 1129800.
Yellow powder. [1116-54-7]. 2,2′-(Nitrosoimino)diethanol.
mp : about 70 °C. Yellow liquid, miscible with anhydrous ethanol.
Nitrochromic reagent. 1059100. : about 1.485.
Dissolve 0.7 g of potassium dichromate R in nitric acid R and bp : about 125 °C.
dilute to 100 mL with the same acid. N-Nitrosodiisopropanolamine. C6H14N2O3. (Mr 162.2).
Nitroethane. C2H5NO2. (Mr 75.1). 1059200. [79-24-3]. 1176500. [53609-64-6]. 1,1′-(Nitrosoimino)bispropan-2-ol.
Clear, oily, colourless liquid. bp : 122-124 °C.
bp : about 114 °C. Nitrosodipropylamine. C6H14N2O. (Mr 130.2). 1099900.
[621-64-7]. Dipropylnitrosamine.
Nitrofurantoin. 1099700. [67-20-9].
Liquid, soluble in anhydrous ethanol and in strong acids.
See Nitrofurantoin (0101).
: about 0.915.
Nitrogen. N2. (Mr 28.01). 1059300. [7727-37-9]. bp : about 78 °C.
Nitrogen, washed and dried. Appropriate grade for chemiluminescence determination.
Nitrogen gas mixture. 1136900. Nitrosodipropylamine solution. 1099901.
Nitrogen R containing 1 per cent V/V of each of the Inject 78.62 g of anhydrous ethanol R through the septum
following gases : carbon dioxide R2, carbon monoxide R1 of a vial containing nitrosodipropylamine R. Dilute 1/100
and oxygen R1. in anhydrous ethanol R and place 0.5 mL aliquots in
Nitrogen, oxygen-free. 1059600. crimp-sealed vials.
Nitrogen R which has been freed from oxygen by passing it Storage : in the dark at 5 °C.
through alkaline pyrogallol solution R. Nitrotetrazolium blue. C40H30Cl2N10O6. (Mr 818). 1060000.
Nitrogen R1. N2. (Mr 28.01). 1059400. [7727-37-9]. [298-83-9]. 3,3′-(3,3′-Dimethoxy-4,4′-diphenylene)di[2-
(4-nitrophenyl)-5-phenyl-2H-tetrazolium] dichloride.
Content : minimum 99.999 per cent V/V. p-Nitro-tetrazolium blue.
Carbon monoxide : less than 5 ppm. Crystals, soluble in methanol, giving a clear, yellow solution.
Oxygen : less than 5 ppm.
mp : about 189 °C, with decomposition.
Nitrogen dioxide. NO2. (Mr 46.01). 1179600. [10102-44-0].
Nitrous oxide. N2O. (Mr 44.01). 1108500.
Content : minimum 98.0 per cent V/V.
Content : minimum 99.99 per cent V/V.
Nitrogen for chromatography. N2. (Mr 28.01). 1059500. Nitrogen monoxide : less than 1 ppm.
[7727-37-9]. Carbon monoxide : less than 1 ppm.
Content : minimum 99.95 per cent V/V.
Nonivamide. C17H27NO3. (Mr 293.4). 1148500. [2444-46-4].
Nitrogen monoxide. NO. (Mr 30.01). 1108300. N-[(4-Hydroxy-3-methoxyphenyl)methyl]nonanamide.
Content : minimum 98.0 per cent V/V. White or almost white, crystalline powder, practically
insoluble in cold water, freely soluble in anhydrous ethanol.
Nitromethane. CH3NO2. (Mr 61.0). 1059700. [75-52-5].
Nonivamide used in the test for nonivamide in the monograph
Clear, colourless, oily liquid, slightly soluble in water, miscible
Capsicum (1859) complies with the following additional test.
with ethanol (96 per cent).
Assay. Liquid chromatography (2.2.29) as prescribed in the
: 1.132 to 1.134.
monograph Capsicum (1859).
: 1.381 to 1.383.
Content : minimum 98.0 per cent, calculated by the
Distillation range (2.2.11). Not less than 95 per cent distils normalisation procedure.
between 100 °C and 103 °C.
Nonylamine. C9H21N. (Mr 143.3). 1139800. [112-20-9].
Nitro-molybdovanadic reagent. 1060100. Nonan-1-amine. 1-Aminononane.
Solution A. Dissolve 10 g of ammonium molybdate R in Corrosive, colourless, clear liquid.
water R, add 1 mL of ammonia R and dilute to 100 mL with
: about 0.788.
water R.
Solution B. Dissolve 2.5 g of ammonium vanadate R in hot : about 1.433.
water R, add 14 mL of nitric acid R and dilute to 500 mL with Nordazepam. C15H11ClN2O. (Mr 270.7). 1060200.
water R. [1088-11-5]. 7-Chloro-2,3-dihydro-5-phenyl-1H-1,4-
To 96 mL of nitric acid R add 100 mL of solution A and benzodiazepin-2-one.
100 mL of solution B and dilute to 500 mL with water R. White or pale yellow, crystalline powder, practically insoluble
4-Nitrophenol. C6H5NO3. (Mr 139.1). 1146400. [100-02-7]. in water, slightly soluble in ethanol (96 per cent).
p-Nitrophenol. mp : about 216 °C.
Content : minimum 95 per cent. DL-Norleucine. C6H13NO2. (Mr 131.2). 1060300. [616-06-8].
Colourless or slightly yellow powder, sparingly soluble in (RS)-2-Aminohexanoic acid.
water and in methanol. Shiny crystals, sparingly soluble in water and in ethanol
mp : about 114 °C. (96 per cent), soluble in acids.
General Notices (1) apply to all monographs and other texts 495
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
compounds, it is carefully end-capped to cover most of the Nitrogen and argon : less than 100 ppm.
remaining silanol groups. The particle size is indicated after Carbon dioxide : less than 10 ppm.
the name of the reagent in the tests where it is used. Carbon monoxide : less than 5 ppm.
Organosilica polymer, amorphous, polar-embedded Oxygen R1. O2. (Mr 32.00). 1137600.
octadecylsilyl, end-capped. 1150600.
Content : minimum 99 per cent V/V.
Synthetic, spherical hybrid particles containing both inorganic
(silica) and organic (organosiloxanes) components, chemically Oxytetracycline hydrochloride. 1146500.
modified at the surface by the bonding of polar embedded See Oxytetracycline hydrochloride (0198).
octadecylsilyl groups. To minimise any interaction with basic
compounds, it is carefully end-capped to cover most of the Palladium. Pd. (Ar 106.4). 1114700. [7440-05-3].
remaining silanol groups. The particle size is indicated after Grey white metal, soluble in hydrochloric acid.
the name of the reagent in the tests where it is used.
Palladium chloride. PdCl2. (Mr 177.3). 1061500. [7647-10-1].
Organosilica polymer, amorphous, propyl-2-phenylsilyl, Red crystals.
end-capped. 1178100. mp : 678 °C to 680 °C.
Synthetic, spherical hybrid particles containing both inorganic
(silica) and organic (organosiloxanes) components, chemically Palladium chloride solution. 1061501.
modified at the surface by the bonding of propyl-2-phenylsilyl Dissolve 1 g of palladium chloride R in 10 mL of warm
groups. To minimise any interaction with basic compounds, it hydrochloric acid R. Dilute the solution to 250 mL with a
is carefully end-capped to cover most of the remaining silanol mixture of equal volumes of dilute hydrochloric acid R and
groups. The particle size is indicated after the name of the water R. Dilute this solution immediately before use with
reagent in the tests where it is used. 2 volumes of water R.
Organosilica polymer for mass spectrometry, amorphous, Palmitic acid. C16H32O2. (Mr 256.4). 1061600. [57-10-3].
octadecylsilyl, end-capped. 1164900. Hexadecanoic acid.
Synthetic, spherical hybrid particles containing both inorganic White or almost white, crystalline scales, practically insoluble
(silica) and organic (organosiloxanes) components. To in water, freely soluble in hot ethanol (96 per cent).
minimise any interaction with basic compounds, it is carefully mp : about 63 °C.
end-capped to cover most of the remaining silanol groups. Chromatography. Thin-layer chromatography (2.2.27)
The particle size is indicated after the name of the reagent in as prescribed in the monograph Chloramphenicol
the tests where it is used. palmitate (0473) ; the chromatogram shows only one principal
Osmium tetroxide. OsO4. (Mr 254.2). 1061200. [20816-12-0]. spot.
Light-yellow needles or a yellow, crystalline mass, hygroscopic, Palmitic acid used in the assay of total fatty acids in the
light sensitive, soluble in water and in ethanol (96 per cent). monograph Saw palmetto fruit (1848) complies with the
following additional test.
Storage : in an airtight container.
Assay. Gas chromatography (2.2.28) as prescribed in the
Osmium tetroxide solution. 1061201. monograph Saw palmetto fruit (1848).
A 2.5 g/L solution in 0.05 M sulfuric acid. Content : minimum 98 per cent, calculated by the
normalisation procedure.
Osthole. C15H16O3. (Mr 244.3). 1180500. [484-12-8].
7-Methoxy-8-(3-methylbut-2-enyl)-2H-1-benzopyran-2-one. Palmitoleic acid. C16H30O2. (Mr 254.4). 1144400. [373-49-9].
7-Methoxy-8-isopentenylcoumarin. (9Z)-Hexadec-9-enoic acid.
Oxalic acid. C2H2O4,2H2O. (Mr 126.1). 1061400. [6153-56-6]. Clear, colourless liquid.
Ethanedioic acid dihydrate. bp : about 162 °C.
White or almost white crystals, soluble in water, freely soluble Palmitoleic acid used in the assay of total fatty acids in Saw
in ethanol (96 per cent). palmetto fruit (1848) complies with the following additional
test.
Oxalic acid and sulfuric acid solution. 1061401.
Assay. Gas chromatography (2.2.28) as prescribed in the
A 50 g/L solution of oxalic acid R in a cooled mixture of monograph Saw palmetto fruit (1848).
equal volumes of sulfuric acid R and water R.
Content : minimum 98 per cent, calculated by the
Oxazepam. 1144300. [604-75-1]. normalisation procedure.
See Oxazepam (0778). Palmityl alcohol. C16H34O. (Mr 242.4). 1156100.
Ox brain, acetone-dried. 1061300. [36653-82-4]. Hexadecan-1-ol. Cetyl alcohol.
Cut into small pieces a fresh ox brain previously freed mp : about 48 °C.
from vascular and connective tissue. Place in acetone R Content : minimum 96 per cent.
for preliminary dehydration. Complete the dehydration by Pancreas powder. 1061700.
pounding in a mortar 30 g of this material with successive
quantities, each of 75 mL, of acetone R until a dry powder See Pancreas powder (0350).
is obtained after filtration. Dry at 37 °C for 2 h or until the Papain. 1150700. [9001-73-4].
odour of acetone is no longer present. A proteolytic enzyme obtained from the latex of the green
2,2′-Oxybis(N,N-dimethylethylamine). C8H20N2O. fruit and leaves of Carica papaya L.
(Mr 160.3). 1141200. [3033-62-3]. Bis(2-dimethylaminoethyl) Papaverine hydrochloride. 1061800. [61-25-6].
ether.
See Papaverine hydrochloride (0102).
Colourless, corrosive liquid.
: about 0.85. Paper chromatography performance test solutions.
: about 1.430. 1150800.
Test solution (A). Sodium pertechnetate (99mTc) injection
Oxygen. O2. (Mr 32.00). 1108800. (fission) (0124) or Sodium pertechnetate (99mTc) injection
Content : minimum 99.99 per cent V/V. (non-fission) (0283).
General Notices (1) apply to all monographs and other texts 497
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Test solution (B). In a closed vial mix 100 μL of a 5 g/L Parthenolide. C15H20O3. (Mr 248.3). 1129900. [20554-84-1].
solution of stannous chloride R in 0.05 M hydrochloric acid (4E)-(1aR,7aS,10aS,10bS)-1a,5-Dimethyl-8-methylene-
and 100 MBq to 200 MBq of Sodium pertechnetate (99mTc) 2,3,6,7,7a,8,10a,10b-octahydro-oxireno[9,10]cyclodeca[1,2-
injection (fission) (0124) or Sodium pertechnetate (99mTc) b]furan-9(1aH)-one. (E)-(5S,6S)-4,5-Epoxygermacra-
injection (non-fission) (0283) in a volume not exceeding 2 mL. 1(10),11(13)-dieno-12(6)-lactone.
White or almost white, crystalline powder, very slightly
Paper for chromatography. 1150900.
soluble in water, very soluble in methylene chloride, soluble
Pure cellulose grade thin paper with a smooth surface and in methanol.
a thickness of about 0.2 mm.
: − 71.4, determined on a 2.2 g/L solution in methylene
Chromatographic separation. To 2 strips of paper for chloride R.
chromatography R apply separately 2-5 μL of test solution (a) mp : 115 °C to 116 °C.
and test solution (b) of paper chromatography performance
test solutions R. Develop over a pathlength of 3/4 of the paper Absorbance (2.2.25). A 0.01 g/L solution in ethanol (96 per
height, using a mixture of equal volumes of methanol R cent) R shows an absorption maximum at 214 nm.
and water R. Allow to dry and determine the distribution Assay. Liquid chromatography (2.2.29) as prescribed in the
of radioactivity using a suitable detector. The paper is not monograph Feverfew (1516), at the concentration of the
satisfactory, unless the chromatogram obtained with test reference solution.
solution (a) shows a single radioactivity spot with an RF value Content : minimum 90 per cent, calculated by the
in the range 0.8-1.0 and the chromatogram obtained with test normalisation procedure.
solution (b) shows a single radioactivity spot at the application
point (RF value in the range 0.0-0.1). Penicillinase solution. 1062300.
Paracetamol. 1061900. [103-90-2]. Dissolve 10 g of casein hydrolysate, 2.72 g of potassium
dihydrogen phosphate R and 5.88 g of sodium citrate R in
See Paracetamol (0049). 200 mL of water R, adjust to pH 7.2 with a 200 g/L solution
Paracetamol, 4-aminophenol-free. 1061901. of sodium hydroxide R and dilute to 1000 mL with water R.
Dissolve 0.41 g of magnesium sulfate R in 5 mL of water R
Recrystallise paracetamol R from water R and dry in vacuo and add 1 mL of a 1.6 g/L solution of ferrous ammonium
at 70 °C ; repeat the procedure until the product complies sulfate R and sufficient water R to produce 10 mL. Sterilise
with the following test : dissolve 5 g of the dried substance both solutions by heating in an autoclave, cool, mix, distribute
in a mixture of equal volumes of methanol R and water R in shallow layers in conical flasks and inoculate with Bacillus
and dilute to 100 mL with the same mixture of solvents. cereus (NCTC 9946). Allow the flasks to stand at 18 °C to
Add 1 mL of a freshly prepared solution containing 10 g/L 37 °C until growth is apparent and then maintain at 35 °C
of sodium nitroprusside R and 10 g/L of anhydrous sodium to 37 °C for 16 h, shaking constantly to ensure maximum
carbonate R, mix and allow to stand for 30 min protected aeration. Centrifuge and sterilise the supernatant by filtration
from light. No blue or green colour is produced. through a membrane filter. 1.0 mL of penicillinase solution
contains not less than 0.4 microkatals (corresponding to the
Paraffin, liquid. 1062000. [8042-47-5]. hydrolysis of not less than 500 mg of benzylpenicillin to
See Liquid paraffin (0239). benzylpenicilloic acid per hour) at 30 °C and pH 7, provided
that the concentration of benzylpenicillin does not fall below
Paraffin, white soft. 1062100. the level necessary for enzyme saturation.
A semi-liquid mixture of hydrocarbons obtained from The Michaelis constant for benzylpenicillin of the penicillinase
petroleum and bleached, practically insoluble in water and in penicillinase solution is approximately 12 μg/mL.
in ethanol (96 per cent), soluble in light petroleum R1, the
Sterility (2.6.1). It complies with the test for sterility.
solution sometimes showing a slight opalescence.
Storage : at a temperature between 0 °C and 2 °C for 2 to
Paraldehyde. 1151000. [123-63-7]. 3 days. When freeze-dried and kept in sealed ampoules, it
See Paraldehyde (0351). may be stored for several months.
Pentane. C5H12. (Mr 72.2). 1062500. [109-66-0]. Permethrin. C21H20Cl2O3. (Mr 391.3). 1130000. [52645-53-1].
Clear, colourless, flammable liquid, very slightly soluble in mp : 34 °C to 35 °C.
water, miscible with acetone and with anhydrous ethanol. A suitable certified reference solution (10 ng/μL in
: about 0.63. cyclohexane) may be used.
: about 1.359. Peroxide test strips. 1147800.
bp : about 36 °C. Use commercial test strips with a suitable scale in the range
Pentane used in spectrophotometry complies with the following from 0 ppm to 25 ppm peroxide.
additional test.
Minimum transmittance (2.2.25) using water R as Perylene. C20H12. (Mr 252.3). 1130100. [198-55-0].
compensation liquid : 20 per cent at 200 nm, 50 per cent at Dibenz[de,kl]anthracene.
210 nm, 85 per cent at 220 nm, 93 per cent at 230 nm, 98 per Orange powder.
cent at 240 nm. mp : about 279 °C.
1,2-Pentanediol. C5H12O2. (Mr 104.2). 1155800. [5343-92-0]. Petroleum, light. 1063100. [8032-32-4]. Petroleum ether
(2RS)-Pentane-1,2-diol. 50-70 °C.
: about 0.971. Clear, colourless, flammable liquid without fluorescence,
: about 1.439. practically insoluble in water, miscible with ethanol (96 per
bp : about 201 °C. cent).
: 0.661 to 0.664.
Pentanol. C5H12O. (Mr 88.1). 1062600. [71-41-0]. Distillation range (2.2.11) : 50 °C to 70 °C.
Pentan-1-ol.
Colourless liquid, sparingly soluble in water, miscible with Petroleum, light R1. 1063101. Petroleum ether 40-60 °C.
ethanol (96 per cent). Complies with the requirements prescribed for light
: about 1.410. petroleum R, with the following modifications.
bp : about 137 °C. : 0.630 to 0.656.
Distillation range (2.2.11) : 40 °C to 60 °C. It does not
3-Pentanone. C5H10O. (Mr 86.13). 1173600. [96-22-0]. become cloudy at 0 °C.
Pentan-3-one. Diethyl ketone.
Petroleum, light R2. 1063102. Petroleum ether 30-40 °C.
tert-Pentyl alcohol. C5H12O. (Mr 88.1). 1062700. [75-85-4].
tert-Amyl alcohol. 2-Methyl-2-butanol. Complies with the requirements prescribed for light
petroleum R, with the following modifications.
Volatile, flammable liquid, freely soluble in water, miscible
with ethanol (96 per cent) and with glycerol. : 0.620 to 0.630.
: about 0.81. Distillation range (2.2.11) : 30 °C to 40 °C. It does not
become cloudy at 0 °C.
Distillation range (2.2.11). Not less than 95 per cent distils
between 100 °C and 104 °C. Petroleum, light R3. 1063103. Petroleum ether 100-120 °C.
Storage : protected from light. Complies with the requirements prescribed for light
petroleum R, with the following modifications.
Pentetic acid. C14H23N3O10. (Mr 393.3). 1183100. [67-43-6].
[[(Carboxymethyl)imino]bis(ethylenenitrilo)]tetraacetic acid. : about 0.720.
White or almost white powder, slightly soluble in water. Distillation range (2.2.11) : 100 °C to 120 °C.
mp : 219 °C to 220 °C, with decomposition. Water (2.5.12) : maximum 0.03 per cent.
Pepsin powder. 1062800. [9001-75-6]. Petroleum, light R4. 1063104. Petroleum ether 80-100 °C.
See Pepsin powder (0682). Complies with the requirements prescribed for light
petroleum R, with the following modifications.
Peptide N-glycosidase F. 1186600. [83534-39-8].
: about 0.70.
Peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase
Distillation range (2.2.11) : 80 °C to 100 °C.
(EC 3.5.1.52). PNGase F.
Perchloric acid. HClO4. (Mr 100.5). 1062900. [7601-90-3]. pH indicator strip. 1178900.
Content : 70.0 per cent m/m to 73.0 per cent m/m. Plastic strip containing multiple segments of different
dye-impregnated papers allowing visual determination of pH
Clear, colourless liquid, miscible with water. in the prescribed range by comparison with a master chart.
: about 1.7.
Assay. To 2.50 g add 50 mL of water R and titrate with 1 M α-Phellandrene. C10H16. (Mr 136.2). 1130400.
sodium hydroxide, using 0.1 mL of methyl red solution R as [4221-98-1]. (R)-5-Isopropyl-2-methyl-cyclohexa-1,3-diene.
indicator. (–)-p-Mentha-1,5-diene.
1 mL of 1 M sodium hydroxide is equivalent to 100.5 mg of : about 1.471.
HClO4. bp : 171 °C to 174 °C.
α-Phellandrene used in gas chromatography complies with the
Perchloric acid solution. 1062901. following additional test.
Dilute 8.5 mL of perchloric acid R to 100 mL with water R. Assay. Gas chromatography (2.2.28) as prescribed in the
Periodic acetic acid solution. 1063000. monograph Eucalyptus oil (0390).
Dissolve 0.446 g of sodium periodate R in 2.5 mL of a 25 per Test solution. The substance to be examined.
cent V/V solution of sulfuric acid R. Dilute to 100.0 mL with Content : 95.0 per cent, calculated by the normalisation
glacial acetic acid R. procedure.
Periodic acid. H 5IO6. (Mr 227.9). 1108900. [10450-60-9]. Phenanthrene. C14H10. (Mr 178.2). 1063200. [85-01-8].
Crystals, freely soluble in water and soluble in ethanol (96 per White or almost white crystals, practically insoluble in water,
cent). sparingly soluble in ethanol (96 per cent).
mp : about 122 °C. mp : about 100 °C.
General Notices (1) apply to all monographs and other texts 499
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Phenanthroline hydrochloride. C12H9ClN2,H2O. (Mr 234.7). Phenoxyacetic acid. C8H8O3. (Mr 152.1). 1063800.
1063300. [3829-86-5]. 1,10-Phenanthroline hydrochloride [122-59-8]. 2-Phenoxyethanoic acid.
monohydrate. Almost white crystals, sparingly soluble in water, freely soluble
White or almost white, crystalline powder, freely soluble in in ethanol (96 per cent), and in glacial acetic acid.
water, soluble in ethanol (96 per cent). mp : about 98 °C.
mp : about 215 °C, with decomposition. Chromatography. Thin-layer chromatography (2.2.27) as
prescribed in the monograph Phenoxymethylpenicillin (0148) ;
Phenazone. 1063400. [60-80-0]. the chromatogram shows only one principal spot.
See Phenazone (0421). 2-Phenoxyaniline. C12H11NO. (Mr 185.2). 1165500.
[2688-84-8]. 2-Phenoxybenzenamine. 2-Aminophenyl phenyl
Phenol. 1063500. [108-95-2].
ether.
See Phenol (0631).
Phenoxybenzamine hydrochloride. C18H23Cl2NO.
Phenolphthalein. C20H14O4. (Mr 318.3). 1063700. [77-09-8]. (Mr 340.3). 1063900. N-(2-Chloroethyl)-N-(1-methyl-2-
3,3-Bis(4-hydroxyphenyl)-3H-isobenzofuran-1-one. phenoxyethyl)-benzylamine hydrochloride.
White or yellowish-white powder, practically insoluble in Content : 97.0 per cent to 103.0 per cent (dried substance).
water, soluble in ethanol (96 per cent). White or almost white, crystalline powder, sparingly soluble
in water, freely soluble in ethanol (96 per cent).
Phenolphthalein paper. 1063704. mp : about 138 °C.
Immerse strips of filter paper for a few minutes in Loss on drying (2.2.32) : maximum 0.5 per cent, determined
phenolphthalein solution R. Allow to dry. by drying over diphosphorus pentoxide R at a pressure not
exceeding 670 Pa for 24 h.
Phenolphthalein solution. 1063702.
Assay. Dissolve 0.500 g in 50.0 mL of ethanol-free chloroform R
Dissolve 0.1 g of phenolphthalein R in 80 mL of ethanol and extract with three quantities, each of 20 mL, of 0.01 M
(96 per cent) R and dilute to 100 mL with water R. hydrochloric acid. Discard the acid extracts, filter the
Test for sensitivity. To 0.1 mL of the phenolphthalein chloroform layer through cotton and dilute 5.0 mL of the
solution add 100 mL of carbon dioxide-free water R. The filtrate to 500.0 mL with ethanol-free chloroform R. Measure
solution is colourless. Not more than 0.2 mL of 0.02 M the absorbance of the resulting solution in a closed cell at the
sodium hydroxide is required to change the colour to pink. maximum at 272 nm. Calculate the content of C18H23Cl2NO,
taking the specific absorbance to be 56.3.
Colour change : pH 8.2 (colourless) to pH 10.0 (red).
Storage : protected from light.
Phenolphthalein solution R1. 1063703. Phenoxyethanol. C8H10O2. (Mr 138.2). 1064000. [122-99-6].
A 10 g/L solution in ethanol (96 per cent) R. 2-Phenoxyethanol.
Clear, colourless, oily liquid, slightly soluble in water, freely
Phenol red. 1063600. [143-74-8]. soluble in ethanol (96 per cent).
Bright red or dark red, crystalline powder, very slightly soluble : about 1.11.
in water, slightly soluble in ethanol (96 per cent).
: about 1.537.
Phenol red solution. 1063601. Freezing point (2.2.18) : minimum 12 °C.
Dissolve 0.1 g of phenol red R in a mixture of 2.82 mL of Phenylacetic acid. C8H8O2. (Mr 136.2). 1160000. [103-82-2].
0.1 M sodium hydroxide and 20 mL of ethanol (96 per White or almost white powder, soluble in water.
cent) R and dilute to 100 mL with water R.
bp : about 265 °C.
Test for sensitivity. Add 0.1 mL of the phenol red solution
mp : about 75 °C.
to 100 mL of carbon dioxide-free water R. The solution is
yellow. Not more than 0.1 mL of 0.02 M sodium hydroxide Phenylalanine. 1064100. [63-91-2].
is required to change the colour to reddish-violet. See Phenylalanine (0782).
Colour change : pH 6.8 (yellow) to pH 8.4 (reddish-violet).
p-Phenylenediamine dihydrochloride. C6H10Cl2N2.
Phenol red solution R2. 1063603. (Mr 181.1). 1064200. [615-28-1]. 1,4-Diaminobenzene
dihydrochloride.
Solution A. Dissolve 33 mg of phenol red R in 1.5 mL of
dilute sodium hydroxide solution R and dilute to 100 mL Crystalline powder or white or slightly coloured crystals,
with water R. turning reddish on exposure to air, freely soluble in water,
slightly soluble in ethanol (96 per cent).
Solution B. Dissolve 25 mg of ammonium sulfate R in
235 mL of water R ; add 105 mL of dilute sodium hydroxide α-Phenylglycine. C8H9NO2. (Mr 151.2). 1064300.
solution R and 135 mL of dilute acetic acid R. [2835-06-5]. (RS)-2-Amino-2-phenylacetic acid.
Add 25 mL of solution A to solution B. If necessary, adjust D-Phenylglycine. C8H9NO2. (Mr 151.2). 1144500. [875-74-1].
the pH of the mixture to 4.7. (2R)-2-Amino-2-phenylacetic acid.
Phenol red solution R3. 1063604. Content : minimum 99 per cent.
White or almost white, crystalline powder.
Solution A. Dissolve 33 mg of phenol red R in 1.5 mL of
dilute sodium hydroxide solution R and dilute to 50 mL Phenylhydrazine hydrochloride. C6H9ClN2. (Mr 144.6).
with water R. 1064500. [59-88-1].
Solution B. Dissolve 50 mg of ammonium sulfate R in White or almost white, crystalline powder, becoming brown
235 mL of water R ; add 105 mL of dilute sodium hydroxide on exposure to air, soluble in water and in ethanol (96 per
solution R and 135 mL of dilute acetic acid R. cent).
Add 25 mL of solution A to solution B ; if necessary, adjust mp : about 245 °C, with decomposition.
the pH of the mixture to 4.7. Storage : protected from light.
General Notices (1) apply to all monographs and other texts 501
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Phthalic anhydride. C8H4O3. (Mr 148.1). 1065700. [85-44-9]. Piperidine. C5H11N. (Mr 85.2). 1066000. [110-89-4].
Isobenzofuran-1,3-dione. Hexahydropyridine.
Content : minimum 99.0 per cent. Colourless to slightly yellow, alkaline liquid, miscible with
White or almost white flakes. water, with ethanol (96 per cent) and with light petroleum.
mp : 130 °C to 132 °C. bp : about 106 °C.
Assay. Dissolve 2.000 g in 100 mL of water R and boil under a Piperine. C17H19NO3. (Mr 285.3). 1183200. [94-62-2].
reflux condenser for 30 min. Cool and titrate with 1 M sodium (2E,4E)-1-(Piperidin-1-yl)-5-(1,3-benzodioxol-5-yl)penta-
hydroxide, using phenolphthalein solution R as indicator. 2,4-dien-1-one. 1-Piperoyl-piperidine. 1-[(2E,4E)-5-(3,4-
1 mL of 1 M sodium hydroxide is equivalent to 74.05 mg of Methylenedioxyphenyl)-1-oxo-2,4-pentadienyl]piperidine.
C 8H 4O 3.
Piperitone. C10H16O. (Mr 152.2). 1151200. [89-81-6].
Phthalic anhydride solution. 1065701. 6-Isopropyl-3-methyl-cyclohex-2-en-1-one.
Dissolve 42 g of phthalic anhydride R in 300 mL of
anhydrous pyridine R. Allow to stand for 16 h. Pirimiphos-ethyl. C13H24N3O3PS. (Mr 333.4). 1130300.
[23505-41-1].
Storage : protected from light ; use within 1 week.
mp : 15 °C to 18 °C.
Picein. C14H18O7. (Mr 298.3). 1130700. [530-14-3]. A suitable certified reference solution (10 ng/μL in
1-[4-(β-D-Glucopyranosyloxy)phenyl]ethanone. cyclohexane) may be used.
p-(Acetylphenyl)-β-D-glucopyranoside.
mp : 194 °C to 195 °C. Plasma, platelet-poor. 1066100.
Picric acid. C6H3N3O7. (Mr 229.1). 1065800. [88-89-1]. Withdraw 45 mL of human blood into a 50 mL plastic syringe
2,4,6-Trinitrophenol. containing 5 mL of a sterile 38 g/L solution of sodium citrate R.
Without delay, centrifuge at 1500 g at 4 °C for 30 min. Remove
Yellow prisms or plates, soluble in water and in ethanol (96 per the upper two-thirds of the supernatant plasma using a plastic
cent). syringe and without delay centrifuge at 3500 g at 4 °C for
Storage : moistened with water R. 30 min. Remove the upper two-thirds of the liquid and freeze
it rapidly in suitable amounts in plastic tubes at or below
Picric acid solution. 1065801. − 40 °C. Use plastic or silicone-treated equipment.
A 10 g/L solution.
Plasma substrate. 1066200.
Picric acid solution R1. 1065802.
Separate the plasma from human or bovine blood collected
Prepare 100 mL of a saturated solution of picric acid R and into one-ninth its volume of a 38 g/L solution of sodium
add 0.25 mL of strong sodium hydroxide solution R. citrate R, or into two-sevenths its volume of a solution
α-Pinene. C10H16. (Mr 136.2). 1130800. [7785-70-8]. containing 20 g/L of disodium hydrogen citrate R and 25 g/L
(1R,5R)-2,6,6-Trimethylbicyclo[3.1.1]hept-2-ene. of glucose R. With the former, prepare the substrate on the
day of collection of the blood. With the latter, prepare within
Liquid not miscible with water.
two days of collection of the blood.
: about 0.859.
Storage : at − 20 °C.
: about 1.466.
bp : 154 °C to 156 °C. Plasma substrate R1. 1066201.
α-Pinene used in gas chromatography complies with the Use water-repellent equipment (made from materials such
following additional test. as suitable plastics or suitably silicone-treated glass) for
Assay. Gas chromatography (2.2.28) as prescribed in the taking and handling blood.
monograph Bitter-orange-flower oil (1175). Collect a suitable volume of blood from each of at least five
Test solution. The substance to be examined. sheep ; a 285 mL volume of blood collected into 15 mL
of anticoagulant solution is suitable but smaller volumes
Content : minimum 99.0 per cent, calculated by the may be collected, taking the blood, either from a live
normalisation procedure. animal or at the time of slaughter, using a needle attached
β-Pinene. C10H16. (Mr 136.2). 1109000. [127-91-3]. to a suitable cannula which is long enough to reach the
6,6-Dimethyl-2-methylenebicyclo[3.1.1]heptane. bottom of the collecting vessel. Discarding the first few
Colourless, oily liquid, odour reminiscent of turpentine, millilitres and collecting only free-flowing blood, collect
practically insoluble in water, miscible with ethanol (96 per the blood in a sufficient quantity of an anticoagulant
cent). solution containing 8.7 g of sodium citrate R and 4 mg of
aprotinin R per 100 mL of water R to give a final ratio of
β-Pinene used in gas chromatography complies with the blood to anticoagulant solution of 19 to 1. During and
following additional test. immediately after collection, swirl the flask gently to ensure
Assay. Gas chromatography (2.2.28) as prescribed in the mixing but do not allow frothing to occur. When collection
monograph Bitter-orange-flower oil (1175). is complete, close the flask and cool to 10-15 °C. When
Test solution. The substance to be examined. cold, pool the contents of all the flasks with the exception
Content : minimum 95.0 per cent. of any that show obvious haemolysis or clots and keep the
pooled blood at 10-15 °C.
1,4-Piperazinediethanesulfonic acid. C8H18N2O6S2. As soon as possible and within 4 h of collection, centrifuge
(Mr 302.4). 1186700. [5625-37-6]. Piperazine-1,4- the pooled blood at 1000-2000 g at 10-15 °C for 30 min.
bis(2-ethanesulfonic acid). 2,2′-(Piperazine-1,4- Separate the supernatant and centrifuge it at 5000 g for
diyl)bis(ethanesulfonic acid). Piperazine-N,N′-bis(2- 30 min. (Faster centrifugation, for example 20 000 g for
ethanesulfonic acid). PIPES. 30 min, may be used if necessary to clarify the plasma,
Content : minimum 99 per cent. but filtration procedures should not be used.) Separate
White, crystalline powder. the supernatant and, without delay, mix thoroughly and
distribute the plasma substrate into small stoppered
Piperazine hydrate. 1065900. [142-63-2]. containers in portions sufficient for a complete heparin
See Piperazine hydrate (0425). assay (for example 10 mL to 30 mL). Without delay, rapidly
General Notices (1) apply to all monographs and other texts 503
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Potassium chlorate. KClO3. (Mr 122.6). 1069000. Potassium dihydrogen phosphate. 1069600. [7778-77-0].
[3811-04-9]. See Potassium dihydrogen phosphate (0920).
A white or almost white powder, granules or crystals, soluble
in water. Potassium dihydrogen phosphate, 0.2 M. 1069601.
A solution of potassium dihydrogen phosphate R containing
Potassium chloride. 1069100. [7447-40-7]. the equivalent of 27.22 g of KH2PO4 in 1000.0 mL.
See Potassium chloride (0185).
Potassium ferricyanide. K3[Fe(CN)6]. (Mr 329.3). 1069700.
Potassium chloride used for infrared absorption
[13746-66-2]. Potassium hexacyanoferrate(III).
spectrophotometry (2.2.24) also complies with the following
additional test. Red crystals, freely soluble in water.
A disc 2 mm thick, prepared from the substance previously Potassium ferricyanide solution. 1069701.
dried at 250 °C for 1 h, has a substantially flat baseline over
Wash 5 g of potassium ferricyanide R with a little water R,
the range 4000 cm− 1 to 620 cm− 1. It exhibits no maxima
dissolve and dilute to 100 mL with water R. Prepare
with absorbance greater than 0.02 above the baseline, except
immediately before use.
maxima for water at 3440 cm− 1 and 1630 cm− 1.
Potassium ferriperiodate solution. 1070801.
Potassium chloride, 0.1 M. 1069101.
Dissolve 1 g of potassium periodate R in 5 mL of a freshly
A solution of potassium chloride R containing the equivalent
prepared 120 g/L solution of potassium hydroxide R. Add
of 7.46 g of KCl in 1000.0 mL.
20 mL of water R and 1.5 mL of ferric chloride solution R1.
Potassium chromate. K2CrO4. (Mr 194.2). 1069200. Dilute to 50 mL with a freshly prepared 120 g/L solution of
[7789-00-6]. Dipotassium chromate. potassium hydroxide R.
Yellow crystals, freely soluble in water. Potassium ferrocyanide. K4[Fe(CN)6],3H2O. (Mr 422.4).
Potassium chromate solution. 1069201. 1069800. [14459-95-1]. Potassium hexacyanoferrate(II).
A 50 g/L solution. Transparent yellow crystals, freely soluble in water, practically
insoluble in ethanol (96 per cent).
Potassium citrate. 1069300. [6100-05-6].
Potassium ferrocyanide solution. 1069801.
See Potassium citrate (0400).
A 53 g/L solution.
Potassium cyanide. KCN. (Mr 65.1). 1069400. [151-50-8].
Potassium fluoride. KF. (Mr 58.1). 1137800. [7789-23-3].
White or almost white, crystalline powder or white or almost
white mass or granules, freely soluble in water, slightly soluble Colourless crystals or white or almost white crystalline
in ethanol (96 per cent). powder, deliquescent, soluble in water, practically insoluble in
ethanol (96 per cent).
Potassium cyanide solution. 1069401.
Potassium hydrogen carbonate. KHCO3. (Mr 100.1).
A 100 g/L solution. 1069900. [298-14-6]. Potassium bicarbonate.
Potassium cyanide solution, lead-free. 1069402. Transparent, colourless crystals, freely soluble in water,
Dissolve 10 g of potassium cyanide R in 90 mL of water R, practically insoluble in ethanol (96 per cent).
add 2 mL of strong hydrogen peroxide solution R diluted 1 Potassium hydrogen carbonate solution, saturated
to 5. Allow to stand for 24 h, dilute to 100 mL with water R methanolic. 1069901.
and filter.
Dissolve 0.1 g of potassium hydrogen carbonate R in 0.4 mL
The solution complies with the following test : take 10 mL of of water R, heating on water-bath. Add 25 mL of methanol R
the solution, add 10 mL of water R and 10 mL of hydrogen and swirl, keeping the solution on the water-bath until
sulfide solution R. No colour is evolved even after addition dissolution is complete. Use a freshly prepared solution.
of 5 mL of dilute hydrochloric acid R.
Potassium hydrogen phthalate. C8H5KO4. (Mr 204.2).
Potassium dichromate. K2Cr2O7. (Mr 294.2). 1069500.
1070000. [877-24-7]. Potassium hydrogen benzene-1,2-
[7778-50-9]. Dipotassium dichromate.
dicarboxylate.
Potassium dichromate used for the calibration of
White or almost white crystals, soluble in water, slightly
spectrophotometers (2.2.25) contains not less than 99.9 per
soluble in ethanol (96 per cent).
cent of K2Cr2O7, calculated with reference to the substance
dried at 130 °C. Potassium hydrogen phthalate, 0.2 M. 1070001.
Orange-red crystals, soluble in water, practically insoluble in A solution of potassium hydrogen phthalate R containing
ethanol (96 per cent). the equivalent of 40.84 g of C8H5KO4 in 1000.0 mL.
Assay. Dissolve 1.000 g in water R and dilute to 250.0 mL with
the same solvent. To 50.0 mL of this solution add a freshly Potassium hydrogen sulfate. KHSO4. (Mr 136.2). 1070100.
prepared solution of 4 g of potassium iodide R, 2 g of sodium [7646-93-7].
hydrogen carbonate R and 6 mL of hydrochloric acid R in Colourless, transparent, hygroscopic crystals, freely soluble in
100 mL of water R in a 500 mL flask. Stopper the flask and water giving a strongly acid solution.
allow to stand protected from light for 5 min. Titrate with Storage : in an airtight container.
0.1 M sodium thiosulfate, using 1 mL of iodide-free starch
solution R as indicator. Potassium hydrogen tartrate. C4H5KO6. (Mr 188.2).
1 mL of 0.1 M sodium thiosulfate is equivalent to 4.903 mg 1070200. [868-14-4]. Potassium hydrogen (2R,3R)-2,3-
of K2Cr2O7. dihydroxybutane-1,4-dioate.
White or almost white, crystalline powder or colourless,
Potassium dichromate solution. 1069501. slightly opaque crystals, slightly soluble in water, soluble in
A 106 g/L solution. boiling water, practically insoluble in ethanol (96 per cent).
Potassium dichromate solution R1. 1069502. Potassium hydroxide. 1070300. [1310-58-3].
A 5 g/L solution. See Potassium hydroxide (0840).
General Notices (1) apply to all monographs and other texts 505
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 507
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Pyruvic acid. C3H4O3. (Mr 88.1). 1109300. [127-17-3]. : about + 260, determined on a 10 g/L solution in
2-Oxopropanoic acid. anhydrous ethanol R.
Yellowish liquid, miscible with water and with anhydrous mp : about 172 °C.
ethanol. Storage : protected from light.
: about 1.267.
Quinidine sulfate. 1109500. [6591-63-5].
: about 1.413.
See Quinidine sulfate (0017).
bp : about 165 °C.
Quinine. C20H24N2O2. (Mr 324.4). 1074100. [130-95-0].
Quercetin dihydrate. C15H10O7,2H2O. (Mr 338.2). 1138100.
(R)-(6-Methoxyquinol-4-yl)[(2S,4S,5R)-5-vinylquinuclidin-2-
2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-
yl]methanol.
4-one.
White or almost white, microcrystalline powder, very slightly
Yellow crystals or yellowish powder, practically insoluble in
soluble in water, slightly soluble in boiling water, very soluble
water, soluble in acetone and in methanol.
in anhydrous ethanol.
Water (2.5.12) : maximum 12.0 per cent, determined on
0.100 g. : about − 167, determined on a 10 g/L solution in
anhydrous ethanol R.
Assay. Liquid chromatography (2.2.29) as prescribed in the
monograph Ginkgo leaf (1828). mp : about 175 °C.
Content :minimum 90 per cent (anhydrous substance) Storage : protected from light.
calculated by the normalisation procedure. Quinine hydrochloride. 1074200. [6119-47-7].
Storage : protected from light. See Quinine hydrochloride (0018).
Quercitrin. C21H20O11. (Mr 448.4). 1138200. Quinine sulfate. 1074300. [6119-70-6].
[522-12-3]. Quercetin 3-L-rhamnopyranoside.
3-[(6-Deoxy-α-L-mannopyranosyl)oxy]-2-(3,4- See Quinine sulfate (0019).
dihydroxyphenyl)-5,7-dihydroxy-4H-1-benzopyran-4-one. Rabbit erythrocyte suspension. 1074500.
Quercitroside.
Prepare a 1.6 per cent V/V suspension of rabbit erythrocytes
Yellow crystals, practically insoluble in cold water, soluble in as follows : defibrinate 15 mL of freshly drawn rabbit blood by
ethanol (96 per cent). shaking with glass beads, centrifuge at 2000 g for 10 min and
mp : 176 °C to 179 °C. wash the erythrocytes with three quantities, each of 30 mL,
Chromatography. Thin-layer chromatography (2.2.27) as of a 9 g/L solution of sodium chloride R. Dilute 1.6 mL of
prescribed in the monograph Goldenrod (1892) : apply 20 μL the suspension of erythrocytes to 100 mL with a mixture of
of the solution ; after spraying, the chromatogram shows a 1 volume of phosphate buffer solution pH 7.2 R and 9 volumes
yellowish-brown fluorescent zone with an RF of about 0.6. of a 9 g/L solution of sodium chloride R.
Storage : at a temperature of 2 °C to 8 °C. Raclopride tartrate. C19H26Cl2N2O9. (Mr 497.3). 1144700.
Quillaia saponins, purified. 1184500. [98185-20-7]. Raclopride L-tartrate.
A mixture of related saponins obtained from the bark of White or almost white solid, sensitive to light, soluble in water.
Quillaja saponaria Molina s.l. : + 0.3, determined on a 3 g/L solution.
Chromatography. Thin-layer chromatography (2.2.27) as mp : about 141 °C.
prescribed in the monograph Quillaia bark (1843) : apply 5 μL
of the solution ; after treating with a 10 per cent V/V solution Rapeseed oil. 1074600.
of sulfuric acid R in methanol R, heat at 120 °C for 5 min and See Rapeseed oil, refined (1369).
examine in daylight ; the chromatogram shows 3 principal
zones in the upper part of the middle third. Reducing mixture. 1074700.
Grind the substances added in the following order to obtain a
Quinaldine red. C21H23IN2. (Mr 430.3). 1073800. [117-92-0]. homogeneous mixture : 20 mg of potassium bromide R, 0.5 g
2-[2-[4-(Dimethylamino)phenyl]ethenyl]-1-ethylquinolinium of hydrazine sulfate R and 5 g of sodium chloride R.
iodide.
Dark bluish-black powder, sparingly soluble in water, freely Reichstein’s substance S. C21H30O4. (Mr 346.5). 1175400.
soluble in ethanol (96 per cent). [152-58-9].
Content : minimum 95.0 per cent.
Quinaldine red solution. 1073801.
mp : about 208 °C.
Dissolve 0.1 g of quinaldine red R in methanol R and dilute
to 100 mL with the same solvent. Resin for reversed-phase ion chromatography. 1131100.
Colour change : pH 1.4 (colourless) to pH 3.2 (red). A neutral, macroporous, high specific surface area with a
non-polar character resin consisting of polymer lattice of
Quinhydrone. C12H10O4. (Mr 218.2). 1073900. [106-34-3]. polystyrene cross-linked with divinylbenzene.
Equimolecular compound of 1,4-benzoquinone and
hydroquinone. Resin, weak cationic. 1096000.
Dark green, lustrous crystals or a crystalline powder, slightly See weak cationic resin R.
soluble in water, sparingly soluble in hot water, soluble in
ethanol (96 per cent) and in concentrated ammonia. Resorcinol. 1074800. [108-46-3].
mp : about 170 °C. See Resorcinol (0290).
General Notices (1) apply to all monographs and other texts 509
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Resveratrol. C14H12O3. (Mr 228.2). 1186900. Sabinene. C10H16. (Mr 136.2). 1109700. [3387-41-5]. Thuj-
[501-36-0]. 3,4′,5-Stilbenetriol. 5-[(E)-2-(4-Hydroxy- 4(10)-ene. 4-Methylene-1-isopropylbicyclo[3.1.0]hexane.
phenyl)ethenyl]benzene-1,3-diol. A colourless, oily liquid.
Rhamnose. C6H12O5,H2O. (Mr 182.2). 1074900. [6155-35-7]. Sabinene used in gas chromatography complies with the
L-(+)-Rhamnose. 6-Deoxy-L-mannose. following additional test.
White or almost white, crystalline powder, freely soluble in Assay. Gas chromatography (2.2.28) as prescribed in the
water. monograph Bitter-orange-flower oil (1175).
: + 7.8 to + 8.3, determined on a 50 g/L solution in Test solution. The substance to be examined.
water R containing about 0.05 per cent of NH3. Content : minimum 95.0 per cent, calculated by the
Rhaponticin. C21H24O9. (Mr 420.4). 1075000. [155-58-8]. normalisation procedure.
3-Hydroxy-5-[2-(3-hydroxy-4-methoxyphenyl)ethenyl]phenyl Saccharin sodium. 1131400. [128-44-9].
β-D-glucopyranoside. See Saccharin sodium (0787).
Yellowish-grey, crystalline powder, soluble in ethanol (96 per
cent) and in methanol. Safrole. C10H10O2. (Mr 162.2). 1131200. [94-59-7].
Chromatography. Thin-layer chromatography (2.2.27) 5-(Prop-2-enyl)-1,3-benzodioxole. 4-Allyl-1,2-
as prescribed in the monograph Rhubarb (0291) ; the (methylenedioxy)benzene.
chromatogram shows only one principal spot. Colourless or slightly yellow, oily liquid, with the odour of
sassafras, insoluble in water, very soluble in ethanol (96 per
Rhodamine 6 G. C28H31ClN2O3. (Mr 479.0). 1153300. cent), miscible with hexane.
[989-38-8].
: 1.095 to 1.096.
Colour Index No. 45160.
9-[2-(Ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7- : 1.537 to 1.538.
dimethylxanthenylium chloride. bp : 232 °C to 234 °C.
Brownish-red powder. Freezing point : about 11 °C.
Rhodamine B. C28H31ClN2O3. (Mr 479.0). 1075100. [81-88-9]. Safrole used in gas chromatography complies with the following
additional test.
Schultz No. 864.
Assay. Gas chromatography (2.2.28) as prescribed in the
Colour Index No. 45170. monograph Cinnamon bark oil, Ceylon (1501).
[9-(2-Carboxyphen-yl)-6-(diethylamino)-3H-xanthen-3-
ylidene]diethylammonium chloride. Content : minimum 96.0 per cent, calculated by the
normalisation procedure.
Green crystals or reddish-violet powder, very soluble in water
and in ethanol (96 per cent). Salicin. C13H18O7. (Mr 286.3). 1131300. [138-52-3].
2-(Hydroxymethyl)phenyl-β-D-glucopyranoside. Salicoside.
Ribose. C5H10O5. (Mr 150.1). 1109600. [50-69-1]. D-Ribose.
Soluble in water, slightly soluble in ethanol (96 per cent). : − 62.5 ± 2.
mp : 88 °C to 92 °C. mp : 199 °C to 201 °C.
Assay. Liquid chromatography (2.2.29) as prescribed in the
Ricinoleic acid. C18H34O3. (Mr 298.5). 1100100. [141-22-0]. monograph Willow bark (1583) at the concentration of the
(9Z,12R)-12-Hydroxyoctadec-9-enoic acid. 12-Hydroxyoleic reference solution.
acid. Content : minimum 99.0 per cent, calculated by the
Yellow or yellowish-brown viscous liquid, consisting of a normalisation procedure.
mixture of fatty acids obtained by the hydrolysis of castor
oil, practically insoluble in water, very soluble in anhydrous Salicylaldehyde. C7H6O2. (Mr 122.1). 1075400. [90-02-8].
ethanol. 2-Hydroxybenzaldehyde.
: about 0.942. Clear, colourless, oily liquid.
: about 1.472. : about 1.167.
mp : about 285 °C, with decomposition. : about 1.574.
Rosmarinic acid. C18H16O8. (Mr 360.3). 1138300. bp : about 196 °C.
[20283-92-5]. mp : about − 7 °C.
mp : 170 °C to 174 °C. Salicylaldehyde azine. C14H12N2O2. (Mr 240.3). 1075500.
Ruthenium red. [(NH3)5RuORu(NH3)4ORu(NH3)5]Cl6,4H2O. [959-36-4]. 2,2′-Azinodimethyldiphenol.
(Mr 858). 1075200. [11103-72-3]. Dissolve 0.30 g of hydrazine sulfate R in 5 mL of water R, add
Brownish-red powder, soluble in water. 1 mL of glacial acetic acid R and 2 mL of a freshly prepared
20 per cent V/V solution of salicylaldehyde R in 2-propanol R.
Ruthenium red solution. 1075201. Mix, allow to stand until a yellow precipate is formed. Shake
A 0.8 g/L solution in lead acetate solution R. with two quantities, each of 15 mL, of methylene chloride R.
Combine the organic layers and dry over anhydrous sodium
Rutin. C27H30O16,3H2O. (Mr 665). 1075300. [153-18-4]. sulfate R. Decant or filter the solution and evaporate to
Rutoside. 3-(O-6-Deoxy-α-L-mannopyranosyl-(1→6)-β-D- dryness. Recrystallise from a mixture of 40 volumes of
glucopyranosyloxy)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy- methanol R and 60 volumes of toluene R with cooling. Dry the
4H-chromen-4-one. crystals in vacuo.
Yellow, crystalline powder, darkening in light, very slightly
mp : about 213 °C.
soluble in water, soluble in about 400 parts of boiling water,
slightly soluble in ethanol (96 per cent), soluble in solutions of Chromatography. Thin-layer chromatography (2.2.27)
the alkali hydroxides and in ammonia. as prescribed in the test for hydrazine in the monograph
Povidone (0685) ; the chromatogram shows only one principal
mp : about 210 °C, with decomposition.
spot.
Absorbance (2.2.25). A solution in ethanol (96 per cent) R
shows two absorption maxima at 259 nm and 362 nm. Salicylic acid. 1075600. [69-72-7].
Storage : protected from light. See Salicylic acid (0366).
Salvianolic acid B. C36H30O16. (Mr 719). 1184600. SDS-PAGE sample buffer for reducing conditions
[121521-90-2]. (2R)-2-[[(2E)-3-[(2S,3S)-3-[[(1R)-1- (concentrated). 1122100.
Carboxy-2-(3,4-dihydroxyphenyl)ethoxy]carbonyl]-2-(3,4- Dissolve 3.78 g of tris(hydroxymethyl)aminomethane R, 10.0 g
dihydroxyphenyl)-7-hydroxy-2,3-dihydrobenzofuran-4- of sodium dodecyl sulfate R and 100 mg of bromophenol blue R
yl]prop-2-enoyl]oxy]-3-(3,4-dihydroxyphenyl)propanoic acid. in water R. Add 50.0 mL of glycerol R and dilute to 200 mL
Sand. 1075800. with water R. Add 25.0 mL of 2-mercaptoethanol R. Adjust
to pH 6.8 with hydrochloric acid R, and dilute to 250.0 mL
White or slightly greyish grains of silica with a particle size with water R.
between 150 μm and 300 μm.
Alternatively, dithiothreitol may be used as reducing
Sarafloxacin hydrochloride. C20H18ClF2N3O3. (Mr 421.8). agent instead of 2-mercaptoethanol. In this case
1181400. [91296-87-6]. 6-Fluoro-1-(4-fluorophenyl)-4-oxo- prepare the sample buffer as follows : dissolve 3.78 g of
7-piperazin-1-yl-1,4-dihydroquinoline-3-carboxylic acid tris(hydroxymethyl)aminomethane R, 10.0 g of sodium dodecyl
hydrochloride. sulfate R and 100 mg of bromophenol blue R in water R. Add
50.0 mL of glycerol R and dilute to 200 mL with water R. Adjust
Schisandrin. C24H32O7. (Mr 432.5). 1173800. to pH 6.8 with hydrochloric acid R, and dilute to 250.0 mL
[7432-28-2]. Schisandrol A. Wuweizichun A. with water R. Immediately before use, add dithiothreitol R to a
(6S,7S,12aRa)-5,6,7,8-Tetrahydro-1,2,3,10,11,12- final concentration of 100 mM.
hexamethoxy-6,7-dimethyldibenzo[a,c]cyclooctan-6-ol.
White or almost white, crystalline powder. Selenious acid. H2SeO3. (Mr 129.0). 1100200. [7783-00-8].
Schisandrin used in the assay in the monograph Schisandra Deliquescent crystals, freely soluble in water.
fruit (2428) complies with the following additional test. Storage : in an airtight container.
Assay. Liquid chromatography (2.2.29) as prescribed in the
monograph Schisandra fruit (2428). Selenium. Se. (Ar 79.0). 1075900. [7782-49-2].
Content : minimum 95 per cent, calculated by the Brown-red or black powder or granules, practically insoluble
normalisation procedure. in water and in ethanol (96 per cent), soluble in nitric acid.
Storage : in an airtight container, at − 20 °C or below. mp : about 220 °C.
γ-Schisandrin. C23H28O6. (Mr 400.5). 1173900. Serine. 1076000. [56-45-1].
[61281-37-6]. Schisandrin B. Wuweizisu B. rac- See Serine (0788).
(6R,7S,13aRa)-1,2,3,13-Tetramethoxy-6,7-dimethyl-5,6,7,8-
tetrahydrobenzo[3,4]cycloocta[1,2-f][1,3]benzodioxole. Sialic acid. 1001100. [131-48-6].
White or almost white, crystalline powder. See N-acetylneuraminic acid R.
Storage : in an airtight container, at − 20 °C or below.
Silibinin. C25H22O10. (Mr 482.4). 1151400. [22888-70-6].
Sclareol. C20H36O2. (Mr 308.5). 1139900. [515-03-7]. Silybin. (2R,3R)-3,5,7-Trihydroxy-2-[(2R,3R)-3-(4-hydroxy-
(1R,2R,4aS,8aS)-1-[(3R)-3-Hydroxy-3-methylpent-4-enyl]- 3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydro-1,4-
2,5,5,8a-tetramethyldecahydronaphthalen-2-ol. benzodioxin-6-yl]-2,3-dihydro-4H-1-benzopyran-4-one.
Odourless crystals. White or yellowish powder, practically insoluble in water,
: 6.7, determined with a solution in anhydrous ethanol. soluble in acetone and in methanol.
bp19 mm : 218 °C to 220 °C. Silibinin used in the assay of Milk thistle fruit (1860) complies
mp : 96 °C to 98 °C. with the following additional test.
Sclareol used in the chromatographic profile test in the Assay. Liquid chromatography (2.2.29) as prescribed in the
monograph Clary sage oil (1850) complies with the following monograph Milk thistle fruit (1860).
additional test. Test solution. Dissolve 5.0 mg of silibinin, dried in vacuo, in
Assay. Gas chromatography (2.2.28) as prescribed in the methanol R and dilute to 50.0 mL with the same solvent.
monograph Clary sage oil (1850). Silibinin A and silibinin B content : minimum 95.0 per cent,
Content : minimum 97 per cent, calculated by the calculated by the normalisation procedure.
normalisation procedure.
Silica gel π-acceptor/π-donor for chiral separations.
Scopoletin. C10H8O4. (Mr 192.2). 1158700. [92-61-5]. 1160100.
7-Hydroxy-6-methoxy-2H-1-benzopyran-2-one. A very finely divided silica gel for chromatography consisting
7-Hydroxy-6-methoxycoumarin. of spherical particles to which 1-(3,5-dinitrobenzamido)-
Faintly beige, fine crystals. 1,2,3,4-tetrahydrophenantrene has been covalently bound,
mp : 202 °C to 208 °C. showing both π-electron acceptor and π-electron donor
characteristics. The particle size and the configuration are
SDS-PAGE running buffer. 1114900. indicated after the name of the reagent in the tests where it
Dissolve 151.4 g of tris(hydroxymethyl)aminomethane R, is used.
721.0 g of glycine R and 50.0 g of sodium laurilsulfate R
Silica gel for chiral separation, amylose derivative of.
in water R and dilute to 5000 mL with the same solvent.
1171700.
Immediately before use, dilute to 10 times its volume with
water R and mix. Measure the pH (2.2.3) of the diluted A very finely divided silica gel for chromatography (5 μm)
solution. The pH is between 8.1 and 8.8. coated with the following derivative :
General Notices (1) apply to all monographs and other texts 511
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Silica gel for chiral separation, cellulose derivative of. Fine, white or almost white, homogeneous powder, practically
1110300. insoluble in water and in ethanol (96 per cent).
A very finely divided silica gel for chromatography (5 μm)
coated with the following derivative : Silica gel for chromatography, aminopropylmethylsilyl.
1102400.
Silica gel with a fine particle size (between 3 μm and 10 μm),
chemically modified by bonding aminopropylmethylsilyl
groups on the surface. The particle size is indicated after the
name of the reagent in the tests where it is used.
Fine, white or almost white, homogeneous powder, practically
insoluble in water and in ethanol (96 per cent).
Silica gel AD for chiral separation. 1171700.
See Amylose derivative of silica gel for chiral separation R. Silica gel for chromatography, aminopropylsilyl. 1077000.
Silica gel with a fine particle size (between 3 μm and 10 μm),
Silica gel AGP for chiral chromatography. 1148700. chemically modified by bonding aminopropylsilyl groups on
See α1-Acid-glycoprotein silica gel for chiral separation R. the surface. The particle size is indicated after the name of the
Silica gel, anhydrous. 1076100. [112926-00-8]. reagent in the tests where it is used.
Partly dehydrated polymerised, amorphous silicic acid, Fine, white or almost white, homogeneous powder, practically
absorbing at 20 °C about 30 per cent of its mass of water. insoluble in water and in ethanol (96 per cent).
Practically insoluble in water, partly soluble in solutions
of sodium hydroxide. It contains a suitable indicator for Silica gel for chromatography, aminopropylsilyl R1.
detection of the humidity status, for which the colour change 1077001.
from the hydrated to anhydrous form is given on the label. Silica gel with a particle size of about 55 μm, chemically
modified by bonding aminopropylsilyl groups on the surface.
Silica gel BC for chiral chromatography. 1161300.
A very finely divided silica gel for chromatography (5 μm) Silica gel for chromatography, amylose derivative of.
coated with β-cyclodextrin. Higher selectivity may be obtained 1109800.
when cyclodextrin has been derivatized with propylene oxide.
A very finely divided (10 μm) silica gel, chemically modified
Silica gel for chiral chromatography, urea type. 1181000. at the surface by the bonding of an amylose derivative. The
A very finely divided silica gel (5 μm) coated with the particle size is indicated after the name of the reagent in the
following derivative : test where it is used.
Fine, white or almost white, homogenous powder, practically
insoluble in water and in ethanol (96 per cent).
Silica gel for chromatography, di-isobutyloctadecylsilyl. Silica gel for chromatography, human albumin coated.
1140000. 1138500.
A very finely divided silica gel chemically modified at the A very finely divided (3-10 μm) silica gel, chemically modified
surface by the bonding of di-isobutyloctadecylsilyl groups. at the surface by the bonding of human albumin. The particle
The particle size is indicated after the name of the reagent in size is indicated after the name of the reagent in the tests
the tests where it is used. where it is used.
White or almost white, fine, homogeneous powder.
Silica gel for chromatography, diisopropylcyanopropylsilyl.
1168100. Silica gel for chromatography, hydrophilic. 1077200.
A very finely divided silica gel chemically modified at the A very finely divided (3-10 μm) silica gel whose surface has
surface by the bonding of diisopropylcyanopropylsilyl groups. been modified to provide hydrophilic characteristics. The
The particle size is indicated after the name of the reagent in particle size may be stated after the name of the reagent in the
which the test is used. tests where it is used.
Silica gel for chromatography, nitrile. 1077300.
Silica gel for chromatography, dimethyloctadecylsilyl.
A very finely divided silica gel, chemically modified at the
1115100.
surface by the bonding of cyanopropylsilyl groups. The
A very finely divided silica gel (3-10 μm), chemically modified particle size is indicated after the name of the reagent in the
at the surface by the bonding of dimethyloctadecylsilyl groups. test where it is used.
The particle size is indicated after the name of the reagent in Fine white or almost white, homogenous powder, practically
the tests where it is used. insoluble in water and in ethanol (96 per cent).
Fine, white or almost white, homogeneous powder, practically
insoluble in water and in ethanol (96 per cent). Irregular Silica gel for chromatography, nitrile R1. 1077400.
particle size. A very finely divided silica gel consisting of porous, spherical
particles with chemically bonded nitrile groups. The particle
Specific surface area : 300 m /g.
2
size is indicated after the name of the reagent in the test where
it is used.
Silica gel for chromatography, diol. 1110000.
Fine, white or almost white, homogeneous powder, practically
Spherical silica particles to which dihydroxypropyl groups are insoluble in water and in ethanol (96 per cent).
bonded. Pore size 10 nm.
Silica gel for chromatography, nitrile R2. 1119500.
Silica gel for chromatography, dodecylsilyl, end-capped. Ultrapure silica gel, chemically modified at the surface by the
1179700. introduction of cyanopropylsilyl groups. Less than 20 ppm
A very finely divided silica gel, chemically modified at of metals. The particle size is indicated after the name of the
the surface by the introduction of dodecylsilyl groups. To reagent in the tests where it is used.
minimise any interaction with basic compounds, it is carefully Fine white or almost white, homogenous powder, practically
end-capped to cover most of the remaining silanol groups. insoluble in water and in ethanol (96 per cent).
Silica gel for chromatography, hexadecylamidylsilyl. Silica gel for chromatography, nitrile, end-capped. 1174500.
1162500. A very finely divided silica gel, chemically modified at the
A very finely divided (5 μm) silica gel, chemically surface by the bonding of cyanopropylsilyl groups. To
modified at the surface by the introduction of minimise any interaction with basic components it is carefully
hexadecylcarboxamidopropyldimethylsilyl groups. end-capped to cover most of the remaining silanol groups.
The particle size is indicated after the name of the reagent in
Silica gel for chromatography, hexadecylamidylsilyl, the test where it is used.
end-capped. 1172400. A fine, white or almost white, homogenous powder, practically
insoluble in water and in anhydrous ethanol.
A very finely divided (5 μm) silica gel, chemically
modified at the surface by the introduction of Silica gel for chromatography, 4-nitrophenylcarbamidesilyl.
hexadecylcarboxamidopropyldimethylsilyl groups. To 1185200.
minimise any interaction with basic compounds it is carefully A very finely divided silica gel, chemically modified at the
end-capped to cover most of the remaining silanol groups. surface by bonding of 4-nitrophenylcarbamide groups. The
particle size is indicated after the name of the reagent in the
Silica gel for chromatography, hexylsilyl. 1077100. tests where it is used.
A very finely divided (3-10 μm) silica gel, chemically modified Fine, white or almost white, homogeneous powder, practically
at the surface by the bonding of hexylsilyl groups. The particle insoluble in water and in ethanol (96 per cent).
size is indicated after the name of the reagent in the tests
where it is used. Silica gel for chromatography, octadecanoylaminopropyl-
silyl. 1115200.
Fine, white or almost white, homogeneous powder, practically
insoluble in water and in ethanol (96 per cent). A very finely divided (3-10 μm) silica gel, chemically modified
at the surface by the bonding of aminopropylsilyl groups
Silica gel for chromatography, hexylsilyl, end-capped. which are acylated with octadecanoyl groups. The particle
1174400. size is indicated after the name of the reagent in the tests
where it is used.
A very finely divided (3-10 μm) silica gel, chemically modified
Fine, white or almost white, homogeneous powder, practically
at the surface by the bonding of hexylsilyl groups. To
insoluble in water and in ethanol (96 per cent).
minimise any interaction with basic compounds it is carefully
end-capped to cover most of the remaining silanol groups. Silica gel for chromatography, octadecylsilyl. 1077500.
The particle size is indicated after the name of the reagent in A very finely divided (3-10 μm) silica gel, chemically modified
the tests where it is used. at the surface by the bonding of octadecylsilyl groups. The
A fine, white or almost white, homogeneous powder, particle size is indicated after the name of the reagent in the
practically insoluble in water and in ethanol (96 per cent). tests where it is used.
General Notices (1) apply to all monographs and other texts 513
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Fine, white or almost white, homogeneous powder, practically carefully end-capped to cover most of the remaining silanol
insoluble in water and in ethanol (96 per cent). groups. The particle size is indicated after the name of the
reagent in the test where it is used.
Silica gel for chromatography, octadecylsilyl R1. 1110100.
Fine, white or almost white, homogeneous powder, practically
A very finely divided ultrapure silica gel, chemically modified insoluble in water and in ethanol (96 per cent).
at the surface by the bonding of octadecylsilyl groups. The
particle size, the pore size and the carbon loading are indicated Silica gel for chromatography, octadecylsilyl, monolithic.
after the name of the reagent in the tests where it is used. Less 1154500.
than 20 ppm of metals. Monolithic rods of highly porous (greater than 80 per cent)
Silica gel for chromatography, octadecylsilyl R2. 1115300. metal-free silica with a bimodal pore structure, modified at
the surface by the bonding of octadecylsilyl groups.
A very finely divided (15 nm pore size) ultrapure silica
gel, chemically modified at the surface by the bonding of Silica gel for chromatography, octadecylsilyl, with
octadecylsilyl groups (20 per cent carbon load), optimised embedded polar groups, end-capped. 1177900.
for the analysis of polycyclic aromatic hydrocarbons. The A very finely divided silica gel (3-10 μm). The particles are
particle size is indicated after the name of the reagent in the based on a mixture of silica chemically modified at the surface
tests where it is used. by the bonding of octadecylsilyl groups and silica chemically
Fine, white or almost white, homogeneous powder, practically modified with a reagent providing a surface with chains having
insoluble in water and in ethanol (96 per cent). embedded polar groups. Furthermore, the packing material is
end-capped. The particle size is indicated after the name of
Silica gel for chromatography, octadecylsilyl, the reagent in the tests where it is used.
base-deactivated. 1077600.
A very finely divided (3-10 μm) silica gel, pretreated before Silica gel for chromatography, octadecylsilyl, with polar
the bonding of octadecylsilyl groups by careful washing incorporated groups, end-capped. 1165100.
and hydrolysing most of the superficial siloxane bridges to A very finely divided silica gel (3-10 μm). The particles are
minimise the interaction with basic components. The particle based on silica, chemically modified with a reagent providing
size is indicated after the name of the reagent in the tests a surface with chains having polar incorporated groups and
where it is used. terminating octadecyl groups. Furthermore, the packing
Fine, white or almost white, homogeneous powder, practically material is end-capped. The particle size is indicated after the
insoluble in water and in ethanol (96 per cent). name of the reagent in the tests where it is used.
Fine, white or almost white, homogeneous powder.
Silica gel for chromatography, octadecylsilyl, end-capped.
1115400. Silica gel for chromatography, octylsilyl. 1077700.
A very finely divided (3-10 μm) silica gel, chemically modified A very finely divided (3-10 μm) silica gel, chemically modified
at the surface by the bonding of octadecylsilyl groups. To at the surface by the bonding of octylsilyl groups. The particle
minimise any interaction with basic compounds it is carefully size is indicated after the name of the reagent in the tests
end-capped to cover most of the remaining silanol groups. where it is used.
The particle size is indicated after the name of the reagent in
the tests where it is used. Fine, white or almost white, homogeneous powder, practically
insoluble in water and in ethanol (96 per cent).
Fine, white or almost white, homogenous powder, practically
insoluble in water and in ethanol (96 per cent). Silica gel for chromatography, octylsilyl R1. 1077701.
Silica gel for chromatography, octadecylsilyl, A very finely divided (3-10 μm) silica gel, chemically modified
end-capped R1. 1115401. at the surface by the bonding of octylsilyl and methyl groups
(double bonded phase). The particle size is indicated after the
A very finely divided (10 nm pore size) ultrapure silica name of the reagent in the tests where it is used.
gel, chemically modified at the surface by the bonding
of octadecylsilyl groups (19 per cent carbon load). To Fine, white or almost white, homogeneous powder, practically
minimise any interaction with basic compounds it is carefully insoluble in water and in ethanol (96 per cent).
end-capped to cover most of the remaining silanol groups. The
Silica gel for chromatography, octylsilyl R2. 1077702.
particle size is indicated after the name of the reagent in the
tests where it is used. It contains less than 20 ppm of metals. Ultrapure very finely divided (10 nm pore size) silica gel,
chemically modified at the surface by the bonding of octylsilyl
Silica gel for chromatography, octadecylsilyl, end-capped, groups (19 per cent carbon load). Less than 20 ppm of metals.
base-deactivated. 1108600.
A very finely divided (3-10 μm) silica gel with a pore size Silica gel for chromatography, octylsilyl R3. 1155200.
of 10 nm and a carbon loading of 16 per cent, pre-treated A very finely divided ultrapure silica gel, chemically modified
before the bonding of octadecylsilyl groups by washing and at the surface by the bonding of octylsilyl groups and sterically
hydrolysing most of the superficial siloxane bridges. To protected with branched hydrocarbons at the silanes. The
further minimise any interaction with basic compounds it is particle size is indicated after the name of the reagent in the
carefully end-capped to cover most of the remaining silanol tests where it is used.
groups. The particle size is indicated after the name of the
reagent in the test where it is used. Silica gel for chromatography, octylsilyl, base-deactivated.
1131600.
Fine, white or almost white, homogeneous powder, practically
insoluble in water and in ethanol (96 per cent). A very finely divided (3-10 μm) silica gel, pretreated before
the bonding of octylsilyl groups by careful washing and
Silica gel for chromatography, octadecylsilyl, end-capped, hydrolysing most of the superficial siloxane bridges to
base-deactivated R1. 1162600. minimise the interaction with basic components. The particle
A very finely divided (3-10 μm) silica gel pre-treated size is indicated after the name of the reagent in the tests
before the bonding of octadecylsilyl groups by washing and where it is used.
hydrolysing most of the superficial siloxane bridges. To Fine, white or almost white, homogeneous powder, practically
further minimise any interaction with basic compounds it is insoluble in water and in ethanol (96 per cent).
Silica gel for chromatography, octylsilyl, end-capped. Silica gel for chromatography, phenylsilyl R1. 1075700.
1119600. A very finely divided silica gel (5 μm), chemically modified at
A very finely divided (3-10 μm) silica gel, chemically modified the surface by the bonding of phenyl groups. The particle size
at the surface by the bonding of octylsilyl groups. To is indicated after the name of the reagent in the tests where
minimise any interaction with basic compounds, it is carefully it is used.
end-capped to cover most of the remaining silanol groups. Fine, white or almost white, homogeneous powder, practically
The particle size is indicated after the name of the reagent in insoluble in water, in ethanol (96 per cent) and in methylene
the tests where it is used. chloride.
Fine, white or almost white, homogeneous powder, practically Spheroidal silica : 8 nm.
insoluble in water and in ethanol (96 per cent). Specific surface area : 180 m2/g.
Carbon loading : 5.5 per cent.
Silica gel for chromatography, octylsilyl, end-capped,
base-deactivated. 1148800. Silica gel for chromatography, phenylsilyl, end-capped.
A very finely divided (3-10 μm) silica gel, pre-treated 1154900.
before the bonding of octylsilyl groups by washing and A very finely divided (5-10 μm) silica gel, chemically
hydrolysing most of the superficial siloxane bridges. To modified at the surface by the bounding of phenyl groups. To
further minimise any interaction with basic compounds it is minimise any interaction with basic compounds it is carefully
carefully end-capped to cover most of the remaining silanol end-capped to cover most of the remaining silanol groups.
groups. The particle size is indicated after the name of the The particle size is indicated after the name of the reagent in
reagent in the test where it is used. the tests where it is used.
Fine, white or almost white, homogeneous powder, practically Silica gel for chromatography, propoxybenzene,
insoluble in water and in ethanol (96 per cent). end-capped. 1174600.
A very finely divided (3-10 μm) silica gel, chemically modified
Silica gel for chromatography, octylsilyl, with polar at the surface by the bonding of propoxybenzene groups. The
incorporated groups, end-capped. 1152600. particle size is indicated after the name of the reagent in the
A very finely divided silica gel (3-10 μm). The particles are test where it is used.
based on silica, chemically modified with a reagent providing
a surface with chains having polar incorporated groups and Silica gel for chromatography, propylsilyl. 1170700.
terminating octyl groups. Furthermore, the packing material A very finely divided silica gel (3-10 μm), chemically modified
is end-capped. The particle size is indicated after the name of at the surface by the bonding of propylsilyl groups. The
the reagent in the tests where it is used. particle size is indicated after the name of the reagent in the
test where it is used.
Fine, white or almost white, homogeneous powder.
Silica gel for chromatography, strong-anion-exchange.
Silica gel for chromatography, oxypropionitrilsilyl. 1077800.
1184700. A very finely divided (3-10 μm) silica gel, chemically modified
A very finely divided silica gel chemically modified at the at the surface by the bonding of quaternary ammonium
surface by the bonding of oxypropionitrilsilyl groups. The groups. The particle size is indicated after the name of the
particle size is indicated after the name of the reagent in the reagent in the tests where it is used.
tests where it is used. Fine, white or almost white, homogeneous powder, practically
insoluble in water and in ethanol (96 per cent).
Silica gel for chromatography, palmitamidopropylsilyl,
end-capped. 1161900. pH limit of use : 2 to 8.
A very finely divided (3-10 μm) silica gel, chemically modified Silica gel for chromatography, strong cation-exchange.
at the surface by the bonding of palmitamidopropyl groups 1161400.
and end-capped with acetamidopropyl groups. The particle A very finely divided (5-10 μm) silica gel, chemically modified
size is indicated after the name of the reagent in the tests at the surface by the bonding of sulfonic acid groups. The
where it is used. particle size is specified after the name of the reagent in the
Fine, white or almost white, homogeneous powder, practically tests where it is used.
insoluble in water and in ethanol (96 per cent). Silica gel for chromatography, trimethylsilyl. 1115500.
Silica gel for chromatography, phenylhexylsilyl. 1153900. A very finely divided (3-10 μm) silica gel, chemically modified
at the surface by the bonding of trimethylsilyl groups. The
A very finely divided silica gel, chemically modified at the particle size is indicated after the name of the reagent in the
surface by the bonding of phenylhexyl groups. The particle tests where it is used.
size is indicated after the name of the reagent in the tests Fine, white or almost white, homogeneous powder, practically
where it is used. insoluble in water and in ethanol (96 per cent).
Silica gel for chromatography, phenylhexylsilyl, Silica gel for size-exclusion chromatography. 1077900.
end-capped. 1170600. A very finely divided silica gel (10 μm) with a very hydrophilic
A very finely divided silica gel (3 μm), chemically modified surface. The average diameter of the pores is about 30 nm.
at the surface by the bonding of phenylhexylsilyl groups. To It is compatible with aqueous solutions between pH 2 and 8
minimise any interaction with basic compounds, it is carefully and with organic solvents. It is suitable for the separation of
end-capped to cover most of the remaining silanol groups. proteins with relative molecular masses of 1 × 103 to 3 × 105.
The particle size is indicated after the name of the reagent in
the tests where it is used. Silica gel G. 1076300. [112926-00-8].
Contains about 13 per cent of calcium sulfate hemihydrate.
Silica gel for chromatography, phenylsilyl. 1110200. Fine, white or almost white, homogeneous powder with a
A very finely divided silica gel, chemically modified at the particle size of about 15 μm.
surface by the bonding of phenyl groups. The particle size Calcium sulfate content. Place 0.25 g in a ground-glass
is indicated after the name of the reagent in the tests where stoppered flask, add 3 mL of dilute hydrochloric acid R
it is used. and 100 mL of water R and shake vigorously for 30 min.
General Notices (1) apply to all monographs and other texts 515
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Filter through a sintered-glass filter (2.1.2) and wash the solution to a separating funnel by means of 100 mL of water R,
residue. Carry out on the combined filtrate and washings the acidify (pH 2 to 3) with dilute hydrochloric acid R and shake
complexometric assay of calcium (2.5.11). with three quantities, each of 10 mL of chloroform R. Dry
1 mL of 0.1 M sodium edetate is equivalent to 14.51 mg of the combined chloroform extracts over anhydrous sodium
CaSO4,1/2H2O. sulfate R, filter and evaporate to dryness on a water-bath.
Dissolve the residue in 50 mL of chloroform R. Examine by
pH (2.2.3). Shake 1 g for 5 min with 10 mL of carbon thin-layer chromatography (2.2.27), using silanised silica
dioxide-free water R. The pH of the suspension is about 7. gel HF254 as the coating substance. Apply to the plate at each
Silica gel GF254. 1076400. [112926-00-8]. of three separate points 10 μL of the chloroformic solution.
Develop over a path of 14 cm with a mixture of 10 volumes of
Contains about 13 per cent of calcium sulfate hemihydrate glacial acetic acid R, 25 volumes of water R and 65 volumes of
and about 1.5 per cent of a fluorescent indicator having an dioxan R. Dry the plate at 120 °C for 30 min. Allow to cool,
optimal intensity at 254 nm. spray with a 35 g/L solution of phosphomolybdic acid R in
Fine, white or almost white, homogeneous powder with a 2-propanol R and heat at 150 °C until the spots become visible.
particle size of about 15 μm. Treat the plate with ammonia vapour until the background
Calcium sulfate content. Determine by the method prescribed is white. The chromatograms show four clearly separated,
for silica gel G R. well-defined spots.
pH (2.2.3). Complies with the test prescribed for silica gel G R. Silica gel OC for chiral separations. 1146800.
Fluorescence. Thin-layer chromatography (2.2.27) using A very finely divided silica gel for chromatography (5 μm)
silica gel GF254 R as the coating substance. Apply separately coated with the following derivative :
to the plate at ten points increasing volumes from 1 μL to
10 μL of a 1 g/L solution of benzoic acid R in a mixture of
10 volumes of anhydrous formic acid R and 90 volumes of
2-propanol R. Develop over a path of 10 cm with the same
mixture of solvents. After evaporating the solvents examine
the chromatogram in ultraviolet light at 254 nm. The benzoic
acid appears as dark spots on a fluorescent background in the
upper third of the chromatogram for quantities of 2 μg and
greater. Silica gel OD for chiral separations. 1110300.
See Cellulose derivative of silica gel for chiral separation R.
Silica gel H. 1076500. [112926-00-8].
Fine, white or almost white, homogeneous powder with a Silica gel OJ for chiral separations. 1179800.
particle size of about 15 μm. A very finely divided silica gel for chromatography
pH (2.2.3). Complies with the test prescribed for silica gel G R. consisting of spherical particles coated with cellulose
tris(4-methylbenzoate). The particle size is indicated after the
Silica gel H, silanised. 1076600. name of the reagent in the test where it is used.
Preparation of a thin layer. See silanised silica gel HF254 R.
Silicotungstic acid. H4SiW12O40,xH2O. 1078000.
A fine, white or almost white homogeneous powder which, [11130-20-4].
after being shaken with water, floats on the surface because of
White or yellowish-white crystals, deliquescent, very soluble
its water-repellent properties.
in water and in ethanol (96 per cent).
Chromatographic separation. Complies with the test Storage : in an airtight container.
prescribed for silanised silica gel HF254 R.
Silicristin. C25H22O10. (Mr 482.4). 1151500. [33889-69-9].
Silica gel HF254. 1076700. (2R,3R)-3,5,7-Trihydroxy-2-[(2R,3S)-7-hydroxy-2-(4-
Contains about 1.5 per cent of a fluorescent indicator having hydroxy-3-methoxyphenyl)-3-hydroxymethyl-2,3-dihydro-1-
an optimal intensity at 254 nm. benzofuran-5-yl]chroman-4-one.
Fine, white or almost white, homogeneous powder with a White or yellowish powder, practically insoluble in water,
particle size of about 15 μm. soluble in acetone and in methanol.
pH. Complies with the test prescribed for silica gel G R.
Silidianin. C25H22O10. (Mr 482.4). 1151600. [29782-68-1].
Fluorescence. Complies with the test prescribed for silica (3R,3aR,6R,7aR,8R)-7a-Hydroxy-8-(4-hydroxy-3-
gel GF254 R. methoxyphenyl)-4-[(2R, 3R)-3,5,7-trihydroxy-4-oxochroman-
2-yl]-2,3,3a,7a-tetrahydro-3,6-methano-1-benzofuran-
Silica gel HF254, silanised. 1076800. 7(6aH)-one.
Contains about 1.5 per cent of a fluorescent indicator having White or yellowish powder, practically insoluble in water,
an optimal intensity at 254 nm. soluble in acetone and in methanol.
Fine, white or almost white, homogeneous powder which,
after shaking with water, floats on the surface because of its Silver diethyldithiocarbamate. C5H10AgNS2. (Mr 256.1).
water-repellent properties. 1110400. [1470-61-7].
Preparation of a thin layer. Vigorously shake 30 g for 2 min Pale-yellow or greyish-yellow powder, practically insoluble
with 60 mL of a mixture of 1 volume of methanol R and in water, soluble in pyridine.
2 volumes of water R. Coat carefully cleaned plates with a It may be prepared as follows. Dissolve 1.7 g of silver nitrate R
layer 0.25 mm thick using a spreading device. Allow the in 100 mL of water R. Separately dissolve 2.3 g of sodium
coated plates to dry in air and then heat in an oven at 100 °C diethyldithiocarbamate R in 100 mL of water R. Cool both
to 105 °C for 30 min. solutions to 10 °C, then mix and while stirring collect the
Chromatographic separation. Introduce 0.1 g each of methyl yellow precipitate on a sintered-glass filter (2.1.2) and wash
laurate R, methyl myristate R, methyl palmitate R and methyl with 200 mL of cold water R. Dry the precipitate in vacuo for
stearate R into a 250 mL conical flask. Add 40 mL of alcoholic 2-3 h.
potassium hydroxide solution R and heat under a reflux Silver diethyldithiocarbamate may be used provided it has not
condenser on a water-bath for 1 h. Allow to cool, transfer the changed in colour or developed a strong odour.
Silver manganese paper. 1078200. into a suitable 3 mL flask and evaporate to dryness under
Immerse strips of slow filter paper into a solution containing nitrogen R. To the residue add 100 μL of a freshly prepared
8.5 g/L of manganese sulfate R and 8.5 g/L of silver nitrate R. mixture of 50 μL of 1-methylimidazole R and 1.0 mL of
Maintain for a few minutes and allow to dry over diphosphorus heptafluoro-N-methyl-N-(trimethylsilyl)butanamide R. Close
pentoxide R protected from acid and alkaline vapours. the flask tightly and heat at 100 °C for 15 min. Allow to cool.
Injection : 1 μL of the test solution.
Silver nitrate. 1078300. [7761-88-8].
See Silver nitrate (0009). Sodium. Na. (Ar 22.99). 1078500. [7440-23-5].
A metal whose freshly cut surface is bright silver-grey. It
Silver nitrate reagent. 1078305. rapidly tarnishes in contact with air and is oxidised completely
To a mixture of 3 mL of concentrated ammonia R and to sodium hydroxide and converted to sodium carbonate. It
40 mL of 1 M sodium hydroxide, add 8 mL of a 200 g/L reacts violently with water, yielding hydrogen and a solution
solution of silver nitrate R, dropwise, with stirring. Dilute of sodium hydroxide ; soluble in anhydrous methanol, yielding
to 200 mL with water R. hydrogen and a solution of sodium methoxide ; practically
insoluble in light petroleum.
Silver nitrate solution R1. 1078301. Storage : under light petroleum or liquid paraffin.
A 42.5 g/L solution.
Storage : protected from light. Sodium acetate. 1078600. [6131-90-4].
See Sodium acetate trihydrate (0411).
Silver nitrate solution R2. 1078302.
A 17 g/L solution. Sodium acetate, anhydrous. C2H3NaO2. (Mr 82.0). 1078700.
[127-09-3].
Storage : protected from light.
Colourless crystals or granules, very soluble in water, sparingly
Silver nitrate solution, ammoniacal. 1078303. soluble in ethanol (96 per cent).
Dissolve 2.5 g of silver nitrate R in 80 mL of water R and Loss on drying (2.2.32). Not more than 2.0 per cent,
add dilute ammonia R1 dropwise until the precipitate determined by drying in an oven at 105 °C.
has dissolved. Dilute to 100 mL with water R. Prepare
Sodium arsenite. NaAsO2. (Mr 129.9). 1165900. [7784-46-5].
immediately before use.
Sodium metaarsenite.
Silver nitrate solution in pyridine. 1078304.
Sodium arsenite solution. 1165901.
An 85 g/L solution in pyridine R.
Dissolve 5.0 g of sodium arsenite R in 30 mL of 1 M sodium
Storage : protected from light. hydroxide. Cool to 0 °C and add, while stirring, 65 mL of
dilute hydrochloric acid R.
Silver oxide. Ag2O. (Mr 231.7). 1078400. [20667-12-3].
Disilver oxide. Sodium ascorbate solution. 1078800. [134-03-2].
Brownish-black powder, practically insoluble in water and in Dissolve 3.5 g of ascorbic acid R in 20 mL of 1 M sodium
ethanol (96 per cent), freely soluble in dilute nitric acid and hydroxide. Prepare immediately before use.
in ammonia.
Storage : protected from light. Sodium azide. NaN3. (Mr 65.0). 1078900. [26628-22-8].
White or almost white, crystalline powder or crystals, freely
Sinensetin. C20H20O7. (Mr 372.4). 1110500. [2306-27-6]. soluble in water, slightly soluble in ethanol (96 per cent).
3′,4′,5,6,7-Pentamethoxyflavone.
White or almost white, crystalline powder, practically Sodium bicarbonate. 1081300. [144-55-8].
insoluble in water, soluble in ethanol (96 per cent). See sodium hydrogen carbonate R.
mp : about 177 °C. Sodium bismuthate. NaBiO3. (Mr 280.0). 1079000.
Absorbance (2.2.25). A solution in methanol R shows [12232-99-4].
3 absorption maxima, at 243 nm, 268 nm and 330 nm. Content : minimum 85.0 per cent.
Assay. Liquid chromatography (2.2.29) as prescribed in the Yellow or yellowish-brown powder, slowly decomposing when
monograph Java tea (1229). moist or at a high temperature, practically insoluble in cold
Content : minimum 95 per cent, calculated by the water.
normalisation procedure. Assay. Suspend 0.200 g in 10 mL of a 200 g/L solution of
potassium iodide R and add 20 mL of dilute sulfuric acid R.
Sinomenine. C19H23NO4. (Mr 329.4). 1183400. [115-53-7].
Using 1 mL of starch solution R as indicator, titrate with 0.1 M
7,8-Didehydro-4-hydroxy-3,7-dimethoxy-17-methyl- sodium thiosulfate until an orange colour is obtained.
9α,13α,14α-morphinan-6-one. Cucoline.
1 mL of 0.1 M sodium thiosulfate is equivalent to 14.00 mg
Sitostanol. C29H52O. (Mr 416.7). 1140100. [19466-47-8]. of NaBiO3.
Dihydro-β-sitosterol.
Sodium bromide. 1154300. [7647-15-6].
Content : minimum 95.0 per cent.
See Sodium bromide (0190).
β-Sitosterol. C29H50O. (Mr 414.7). 1140200. [83-46-5].
Stigmast-5-en-3β-ol. 22,23-Dihydrostigmasterol. Sodium butanesulfonate. C4H9NaO3S. (Mr 160.2). 1115600.
[2386-54-1].
White or almost white powder, practically insoluble in water,
sparingly soluble in tetrahydrofuran. White or almost white, crystalline powder, soluble in water.
Content : minimum 75.0 per cent m/m (dried substance). mp : greater than 300 °C.
Assay. Gas chromatography (2.2.28), as prescribed in the Sodium calcium edetate. 1174000. [62-33-9].
monograph Phytosterol (1911). See sodium calcium edetate (0231).
Test solution. Dissolve 0.100 g of the substance to be
examined in tetrahydrofuran R and dilute to 10.0 mL Sodium carbonate. 1079200. [6132-02-1].
with the same solvent. Introduce 100 μL of this solution See Sodium carbonate decahydrate (0191).
General Notices (1) apply to all monographs and other texts 517
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Sodium carbonate, anhydrous. Na2CO3. (Mr 106.0). supernatant, measured at 260 nm using a mixture of 1 mL of
1079300. [497-19-8]. Disodium carbonate. imidazole buffer solution pH 6.5 R and 3 mL of perchloric acid
White or almost white powder, hygroscopic, freely soluble in (25 g/L HClO4) as compensation liquid, is not greater than 0.3.
water. In each of two tubes, place 0.5 mL of solution A and 0.5 mL
When heated to about 300 °C it loses not more than 1 per of a solution of a reference preparation of streptodornase
cent of its mass. containing 10 IU/mL in imidazole buffer solution pH 6.5 R.
To one tube add immediately 3 mL of perchloric acid (25 g/L
Storage : in an airtight container. HClO4). A precipitate is formed. Centrifuge and collect
Sodium carbonate solution. 1079301. supernatant A. Heat the other tube at 37 °C for 15 min and add
A 106 g/L solution of anhydrous sodium carbonate R. 3 mL of perchloric acid (25 g/L HClO4). Centrifuge and collect
supernatant B. The absorbance of supernatant B, measured at
Sodium carbonate solution R1. 1079302. 260 nm with reference to supernatant A is not less than 0.15.
A 20 g/L solution of anhydrous sodium carbonate R in Sodium diethyldithiocarbamate. C5H10NNaS2,3H2O.
0.1 M sodium hydroxide. (Mr 225.3). 1080000. [20624-25-3].
Sodium carbonate solution R2. 1079303. White or almost white or colourless crystals, freely soluble in
water, soluble in ethanol (96 per cent). The aqueous solution
A 40 g/L solution of anhydrous sodium carbonate R in
is colourless.
0.2 M sodium hydroxide.
Sodium dihydrogen phosphate. 1080100. [13472-35-0].
Sodium carbonate monohydrate. 1131700. [5968-11-6].
See Sodium dihydrogen phosphate dihydrate (0194).
See Sodium carbonate monohydrate (0192).
Sodium dihydrogen phosphate, anhydrous. NaH2PO4.
Sodium cetostearyl sulfate. 1079400. (Mr 120.0). 1080200. [7558-80-7].
See Sodium cetostearyl sulfate (0847). White or almost white powder, hygroscopic.
Sodium chloride. 1079500. [7647-14-5]. Storage : in an airtight container.
See Sodium chloride (0193). Sodium dihydrogen phosphate monohydrate.
NaH2PO4,H2O. (Mr 138.0). 1080300. [10049-21-5].
Sodium chloride solution. 1079502.
White or almost white, slightly deliquescent crystals or
A 20 per cent m/m solution. granules, freely soluble in water, practically insoluble in
Sodium chloride solution, saturated. 1079503. ethanol (96 per cent).
Mix 1 part of sodium chloride R with 2 parts of water R, Storage : in an airtight container.
shake from time to time and allow to stand. Before use, Sodium dioctyl sulfosuccinate. C20H37NaO7S. (Mr 444.6).
decant the solution from any undissolved substance and 1170800. [577-11-7]. Sodium 1,4-bis[(2-ethylhexyl)oxy]-
filter, if necessary. 1,4-dioxobutane-2-sulfonate. 1,4-Bis(2-ethylhexyl)
Sodium citrate. 1079600. [6132-04-3]. sulfobutanedioate sodium salt.
See Sodium citrate (0412). White or almost white, waxy solid.
Sodium dithionite. Na2S2O4. (Mr 174.1). 1080400.
Sodium cobaltinitrite. Na3[Co(NO2)6]. (Mr 403.9). 1079700.
[7775-14-6].
[13600-98-1]. Trisodium hexanitrocobaltate(III).
White or greyish-white, crystalline powder, oxidises in air,
Orange-yellow powder, freely soluble in water, slightly soluble
very soluble in water, slightly soluble in ethanol (96 per cent).
in ethanol (96 per cent).
Storage : in an airtight container.
Sodium cobaltinitrite solution. 1079701.
Sodium dodecyl sulfate. 1080500. [151-21-3].
A 100 g/L solution. Prepare immediately before use.
See Sodium laurilsulfate (0098).
Sodium decanesulfonate. C10H21NaO3S. (Mr 244.3). 1079800. Content : minimum 99.0 per cent.
[13419-61-9].
Sodium edetate. 1080600. [6381-92-6].
Crystalline powder or flakes, white or almost white, freely
soluble in water, soluble in methanol. See Disodium edetate (0232).
Sodium decyl sulfate. C10H21NaO4S. (Mr 260.3). 1138600. Sodium fluoresceinate. C20H10Na2O5. (Mr 376.3). 1080700.
[142-87-0]. [518-47-8].
Schultz No. 880.
Content : minimum 95.0 per cent.
Colour Index No. 45350.
White or almost white powder, freely soluble in water.
Fluorescein sodium. Disodium 2-(3-oxo-6-oxido-3H-
Sodium deoxycholate. C24H39NaO4. (Mr 414.6). 1131800. xanthen-9-yl)benzoate.
[302-95-4]. Sodium 3α,12α-dihydroxy-5β-cholan-24-oate. Orange-red powder, freely soluble in water. Aqueous solutions
display an intense yellowish-green fluorescence.
Sodium deoxyribonucleate. (About 85 per cent has a relative
molecular mass of 2 × 107 or greater). 1079900. [73049-39-5]. Sodium fluoride. 1080800. [7681-49-4].
White or almost white, fibrous preparation obtained from calf See Sodium fluoride (0514).
thymus.
Sodium formate. CHNaO2. (Mr 68.0). 1122200. [141-53-7].
Test for suitability. Dissolve 10 mg in imidazole buffer solution Sodium methanoate.
pH 6.5 R and dilute to 10.0 mL with the same buffer solution White or almost white, crystalline powder or deliquescent
(solution A). Dilute 2.0 mL of solution A to 50.0 mL with granules, soluble in water and in glycerol, slightly soluble in
imidazole buffer solution pH 6.5 R. The absorbance (2.2.25) of ethanol (96 per cent).
the solution, measured at 260 nm, is 0.4 to 0.8.
mp : about 253 °C.
To 0.5 mL of solution A add 0.5 mL of imidazole buffer
solution pH 6.5 R and 3 mL of perchloric acid (25 g/L HClO4). Sodium glucuronate. C6H9NaO7,H2O. (Mr 234.1). 1080900.
A precipitate is formed. Centrifuge. The absorbance of the Sodium D-glucuronate monohydrate.
: about + 21.5, determined on a 20 g/L solution. Sodium hydroxide solution, carbonate-free. 1081406.
Dissolve sodium hydroxide R in carbon dioxide-free water R
Sodium glycocholate. C26H42NNaO6,2H2O. to give a concentration of 500 g/L and allow to stand.
(Mr 523.6). 1155500. [207300-80-9]. Sodium Decant the clear supernatant, taking precautions to avoid
[(3,7,12-trihydroxy-5-cholan-24-oyl)amino]acetate dihydrate. the introduction of carbon dioxide.
N-[(3,5,7,12)-3,7,12-Trihydroxy-24-oxocholan-24-yl]glycine
monosodium salt dihydrate. Sodium hydroxide solution, dilute. 1081402.
Content : minimum 97 per cent of C26H42NNaO6,2H2O. Dissolve 8.5 g of sodium hydroxide R in water R and dilute
to 100 mL with the same solvent.
Sodium heptanesulfonate. C7H15NaO3S. (Mr 202.3). 1081000.
[22767-50-6]. Sodium hydroxide solution, methanolic. 1081403.
White or almost white, crystalline mass, freely soluble in Dissolve 40 mg of sodium hydroxide R in 50 mL of water R.
water, soluble in methanol. Cool and add 50 mL of methanol R.
Sodium hydroxide solution, methanolic R1. 1081405.
Sodium heptanesulfonate monohydrate. C7H15NaO3S,H2O.
(Mr 220.3). 1081100. Dissolve 200 mg of sodium hydroxide R in 50 mL of water R.
Cool and add 50 mL of methanol R.
Content : minimum 96 per cent (anhydrous substance).
White or almost white, crystalline powder, soluble in water, Sodium hydroxide solution, strong. 1081404.
very slightly soluble in anhydrous ethanol. Dissolve 42 g of sodium hydroxide R in water R and dilute
Water (2.5.12) : maximum 8 per cent, determined on 0.300 g. to 100 mL with the same solvent.
Assay. Dissolve 0.150 g in 50 mL of anhydrous acetic acid R. Sodium 2-hydroxybutyrate. C4H7NaO3. (Mr 126.1). 1158800.
Titrate with 0.1 M perchloric acid, determining the end-point [19054-57-0]. Sodium (2RS)-2-hydroxybutanoate.
potentiometrically (2.2.20).
Sodium hypobromite solution. 1081500.
1 mL of 0.1 M perchloric acid is equivalent to 20.22 mg of In a bath of iced water mix 20 mL of strong sodium hydroxide
C7H15NaO3S. solution R and 500 mL of water R, add 5 mL of bromine
Sodium hexanesulfonate. C6H13NaO3S. (Mr 188.2). 1081200. solution R and stir gently until solution is complete. Prepare
[2832-45-3]. immediately before use.
White or almost white powder, freely soluble in water. Sodium hypochlorite solution, strong. 1081600.
Content : 25 g/L to 30 g/L of active chlorine.
Sodium hexanesulfonate monohydrate. C6H13NaO3S,H2O.
(Mr 206.2). 1161500. [207300-91-2]. Yellowish liquid with an alkaline reaction.
Assay. Introduce into a flask, successively, 50 mL of water R,
White or almost white powder, soluble in water.
1 g of potassium iodide R and 12.5 mL of dilute acetic acid R.
Sodium hexanesulfonate monohydrate for ion-pair Dilute 10.0 mL of the substance to be examined to 100.0 mL
chromatography. C6H13NaO3S,H2O. (Mr 206.2). 1182300. with water R. Introduce 10.0 mL of this solution into the flask
[207300-91-2]. and titrate with 0.1 M sodium thiosulfate, using 1 mL of starch
solution R as indicator.
Content : minimum 99.0 per cent.
1 mL of 0.1 M sodium thiosulfate is equivalent to 3.546 mg
Sodium hydrogen carbonate. 1081300. [144-55-8]. of active chlorine.
See Sodium hydrogen carbonate (0195). Storage : protected from light.
Sodium hydrogen carbonate solution. 1081301. Sodium hypophosphite. NaH2PO2,H2O. (Mr 106.0). 1081700.
[10039-56-2]. Sodium phosphinate monohydrate.
A 42 g/L solution.
White or almost white, crystalline powder or colourless
Sodium hydrogen sulfate. NaHSO4. (Mr 120.1). 1131900. crystals, hygroscopic, freely soluble in water, soluble in ethanol
[7681-38-1]. Sodium bisulfate. (96 per cent).
Freely soluble in water, very soluble in boiling water. It Storage : in an airtight container.
decomposes in ethanol (96 per cent) into sodium sulfate and Sodium iodide. 1081800. [7681-82-5].
free sulfuric acid. See Sodium iodide (0196).
mp : about 315 °C.
Sodium laurilsulfate. 1081900. [151-21-3].
Sodium hydrogensulfite. NaHO3S. (Mr 104.1). 1115700. See Sodium laurilsulfate (0098).
[7631-90-5].
Sodium lauryl sulfate. 1081900. [151-21-3].
White or almost white, crystalline powder, freely soluble in
water, sparingly soluble in ethanol (96 per cent). See Sodium laurilsulfate R.
On exposure to air, some sulfur dioxide is lost and the Sodium laurylsulfonate for chromatography. C12H25NaO3S.
substance is gradually oxidated to sulfate. (Mr 272.4). 1132000. [2386-53-0].
White or almost white powder or crystals, freely soluble in
Sodium hydroxide. 1081400. [1310-73-2]. water.
See Sodium hydroxide (0677). Absorbance (2.2.25), determined in water R : about 0.05
2 M Sodium hydroxide. 3009800. at 210 nm ; about 0.03 at 220 nm ; about 0.02 at 230 nm ;
about 0.02 at 500 nm.
Dissolve 84 g of sodium hydroxide R in carbon dioxide-free
water R and dilute to 1000.0 mL with the same solvent. Sodium metabisulfite. 1082000. [7681-57-4].
See Sodium metabisulfite (0849).
Sodium hydroxide solution. 1081401.
Dissolve 20.0 g of sodium hydroxide R in water R and dilute Sodium methanesulfonate. CH3SO3Na. (Mr 118.1). 1082100.
to 100.0 mL with the same solvent. Verify the concentration [2386-57-4].
by titration with 1 M hydrochloric acid, using methyl orange White or almost white, crystalline powder, hygroscopic.
solution R as indicator, and adjust if necessary to 200 g/L. Storage : in an airtight container.
General Notices (1) apply to all monographs and other texts 519
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Sodium molybdate. Na2MoO4,2H2O. (Mr 242.0). 1082200. Sodium perchlorate. NaClO4,H2O. (Mr 140.5). 1083100.
[10102-40-6]. Disodium molybdate dihydrate. [7791-07-3].
White or almost white, crystalline powder or colourless Content : minimum 99.0 per cent of NaClO4,H2O.
crystals, freely soluble in water. White or almost white, deliquescent crystals, very soluble in
Sodium naphthoquinonesulfonate. C10H5NaO5S. (Mr 260.2). water.
1082300. [521-24-4]. Sodium 1,2-naphthoquinone-4- Storage : in a well-closed container.
sulfonate.
Sodium periodate. NaIO4. (Mr 213.9). 1083200. [7790-28-5].
Yellow or orange-yellow, crystalline powder, freely soluble in Sodium metaperiodate.
water, practically insoluble in ethanol (96 per cent).
Content : minimum 99.0 per cent.
Sodium nitrate. NaNO3. (Mr 85.0). 1082400. [7631-99-4]. White or almost white, crystalline powder or crystals, soluble
White or almost white powder or granules or colourless, in water and in mineral acids.
transparent crystals, deliquescent in moist air, freely soluble in
water, slightly soluble in ethanol (96 per cent). Sodium periodate solution. 1083201.
Storage : in an airtight container. Dissolve 1.07 g of sodium periodate R in water R, add
5 mL of dilute sulfuric acid R and dilute to 100.0 mL with
Sodium nitrite. NaNO2. (Mr 69.0). 1082500. [7632-00-0]. water R. Use a freshly prepared solution.
Content : minimum 97.0 per cent.
Sodium phosphite pentahydrate. Na2HPO3,5H2O.
White or almost white, granular powder or a slightly yellow, (Mr 216.0). 1132200. [13517-23-2].
crystalline powder, freely soluble in water.
White or almost white, crystalline powder, hygroscopic, freely
Assay. Dissolve 0.100 g in 50 mL of water R. Add 50.0 mL of soluble in water.
0.02 M potassium permanganate and 15 mL of dilute sulfuric
acid R. Add 3 g of potassium iodide R. Titrate with 0.1 M Storage : in an airtight container.
sodium thiosulfate, using 1.0 mL of starch solution R added Sodium picrate solution, alkaline. 1083300.
towards the end of the titration as indicator.
Mix 20 mL of picric acid solution R and 10 mL of a 50 g/L
1 mL of 0.02 M potassium permanganate is equivalent to solution of sodium hydroxide R and dilute to 100 mL with
3.450 mg of NaNO2. water R.
Sodium nitrite solution. 1082501. Storage : use within 2 days.
A 100 g/L solution. Prepare immediately before use.
Sodium potassium tartrate. C4H4KNaO6,4H2O. (Mr 282.2).
Sodium nitroprusside. Na2[Fe(CN)5(NO)],2H2O. 1083500. [6381-59-5].
(Mr 298.0). 1082600. [13755-38-9]. Sodium Colourless, prismatic crystals, very soluble in water.
pentacyano-nitrosylferrate(III) dihydrate.
Reddish-brown powder or crystals, freely soluble in water, Sodium pyrophosphate. Na4P2O7,10H2O. (Mr 446.1).
slightly soluble in ethanol (96 per cent). 1083600. [13472-36-1]. Tetrasodium diphosphate
decahydrate.
Sodium octanesulfonate. C8H17NaO3S. (Mr 216.3). 1082700. Colourless, slightly efflorescent crystals, freely soluble in water.
[5324-84-5].
Content : minimum 98.0 per cent. Sodium rhodizonate. C6Na2O6. (Mr 214.0). 1122300.
[523-21-7]. [(3,4,5,6-Tetraoxocyclohex-1-en-1,2-
White or almost white, crystalline powder or flakes, freely ylene)dioxy]disodium.
soluble in water, soluble in methanol.
Violet crystals, soluble in water with an orange-yellow colour.
Absorbance (2.2.25) : maximum 0.10, determined at 200 nm
and maximum 0.01, determined at 250 nm using a 54 g/L Solutions are unstable and must be prepared on the day of use.
solution. Sodium salicylate. 1083700. [54-21-7].
Sodium octanesulfonate monohydrate. C8H17NaO3S,H2O. See Sodium salicylate (0413).
(Mr 234.3). 1176700. [207596-29-0].
Sodium sulfate, anhydrous. 1083800. [7757-82-6].
White or almost white powder.
Ignite at 600 °C to 700 °C anhydrous sodium sulfate complying
Sodium octyl sulfate. C8H17NaO4S. (Mr 232.3). 1082800. with the requirements prescribed in the monograph on
[142-31-4]. Anhydrous sodium sulfate (0099).
White or almost white, crystalline powder or flakes, freely Loss on drying (2.2.32) : maximum 0.5 per cent, determined by
soluble in water, soluble in methanol. drying in an oven at 130 °C.
Sodium oxalate. C2Na2O4. (Mr 134.0). 1082900. [62-76-0]. Sodium sulfate, anhydrous R1. 1083801.
White or almost white, crystalline powder, soluble in water, Complies with the requirements prescribed for anhydrous
practically insoluble in ethanol (96 per cent). sodium sulfate R with the following maximum contents.
Sodium pentanesulfonate. C5H11NaO3S. (Mr 174.2). 1083000. Cl : 20 ppm.
[22767-49-3]. Pb : 10 ppm.
White or almost white, crystalline solid, soluble in water. As : 3 ppm.
Sodium pentanesulfonate monohydrate. C5H11NaO3S,H2O. Ca : 50 ppm.
(Mr 192.2). 1132100. [207605-40-1]. Fe : 10 ppm.
White or almost white crystalline solid, soluble in water. Mg : 10 ppm.
Sodium pentanesulfonate monohydrate R1. Sodium sulfate decahydrate. Na2SO4,10H2O. (Mr 322.2).
C5H11NaO3S,H2O. (Mr 192.2). 1172500. [207605-40-1]. 1132300. [7727-73-3].
Content : minimum 99 per cent of C5H11NaO3S,H2O. See Sodium sulfate decahydrate (0100).
Sodium sulfide. Na2S,9H2O. (Mr 240.2). 1083900. Sodium thioglycollate. C2H3NaO2S. (Mr 114.1). 1084500.
[1313-84-4]. Disodium sulfide nonahydrate. [367-51-1]. Sodium mercaptoacetate.
Colourless, rapidly yellowing crystals, deliquescent, very White or almost white, granular powder or crystals,
soluble in water. hygroscopic, freely soluble in water and in methanol, slightly
Storage : in an airtight container. soluble in ethanol (96 per cent).
Storage : in an airtight container.
Sodium sulfide solution. 1083901.
Sodium thiosulfate. 1084600. [10102-17-7].
Dissolve 12 g of sodium sulfide R with heating in 45 mL
of a mixture of 10 volumes of water R and 29 volumes of See Sodium thiosulfate (0414).
glycerol (85 per cent) R, allow to cool and dilute to 100 mL Sodium thiosulfate, anhydrous. Na2S2O3. (Mr 158.1).
with the same mixture of solvents. 1180700. [7772-98-7]. Disodium thiosulfate.
The solution should be colourless. Content : minimum 98.0 per cent.
Sodium sulfide solution R1. 1083902. Sodium tungstate. Na2WO4,2H2O. (Mr 329.9). 1084700.
Prepare by one of the following methods. [10213-10-2]. Disodium tungstate dihydrate.
– Dissolve 5 g of sodium sulfide R in a mixture of 10 mL of White or almost white, crystalline powder or colourless
water R and 30 mL of glycerol R. crystals, freely soluble in water forming a clear solution,
practically insoluble in ethanol (96 per cent).
– Dissolve 5 g of sodium hydroxide R in a mixture of 30 mL
of water R and 90 mL of glycerol R. Divide the solution Sorbitol. 1084800. [50-70-4].
into 2 equal portions. Saturate 1 portion with hydrogen See Sorbitol (0435).
sulfide R, with cooling. Mix the 2 portions.
Storage : in a well-filled container, protected from light ; use Squalane. C30H62. (Mr 422.8). 1084900. [111-01-3].
within 3 months. 2,6,10,15,19,23-Hexamethyltetracosane.
Colourless, oily liquid, freely soluble in fatty oils, slightly
Sodium sulfite. 1084000. [10102-15-5]. soluble in acetone, in ethanol (96 per cent), in glacial acetic
See Sodium sulfite heptahydrate (0776). acid and in methanol.
: 0.811 to 0.813.
Sodium sulfite, anhydrous. 1084100. [7757-83-7]. : 1.451 to 1.453.
See Anhydrous sodium sulfite (0775).
Stannous chloride. SnCl2,2H2O. (Mr 225.6). 1085000.
Sodium tartrate. C4H4Na2O6,2H2O. (Mr 230.1). 1084200. [10025-69-1]. Tin dichloride dihydrate.
[6106-24-7]. Disodium (2R,3R)-2,3-dihydroxybutanedioate Content : minimum 97.0 per cent of SnCl2,2H2O.
dihydrate. Colourless crystals, very soluble in water, freely soluble in
White or almost white crystals or granules, very soluble in ethanol (96 per cent), in glacial acetic acid and in dilute and
water, practically insoluble in ethanol (96 per cent). concentrated hydrochloric acid.
Assay. Dissolve 0.500 g in 15 mL of hydrochloric acid R in a
Sodium taurodeoxycholate. C26H44NNaO6S,H2O.
ground-glass-stoppered flask. Add 10 mL of water R and 5 mL
(Mr 539.7). 1155600. [110026-03-4]. Sodium
of chloroform R. Titrate rapidly with 0.05 M potassium iodate
2-[(3,12-dihydroxy-5-cholan-24-oyl)amino]ethanesulfonate
until the chloroform layer is colourless.
monohydrate. 2-[[(3,5,12)-3,12-Dihydroxy-24-oxocholan-24-
yl]amino]ethanesulfonic acid monosodium salt monohydrate. 1 mL of 0.05 M potassium iodate is equivalent to 22.56 mg of
SnCl2,2H2O.
Content : minimum 94 per cent of C26H44NNaO6S,H2O.
Stannous chloride solution. 1085001.
Sodium tetrahydroborate. NaBH4. (Mr 37.8). 1146900.
[16940-66-2]. Sodium borohydride. Heat 20 g of tin R with 85 mL of hydrochloric acid R until
no more hydrogen is released. Allow to cool.
Colourless, hygroscopic crystals, freely soluble in water, Storage : over an excess of tin R, protected from air.
soluble in anhydrous ethanol, decomposing at higher
temperature or in the presence of acids or certain metal salts Stannous chloride solution R1. 1085002.
forming borax and hydrogen. Immediately before use, dilute 1 volume of stannous
Storage : in an airtight container. chloride solution R with 10 volumes of dilute hydrochloric
acid R.
Sodium tetrahydroborate reducing solution. 1146901.
Introduce about 100 mL of water R into a 500 mL Stannous chloride solution R2. 1085003.
volumetric flask containing a stirring bar. Add 5.0 g To 8 g of stannous chloride R add 100 mL of a 20 per
of sodium hydroxide R in pellets and 2.5 g of sodium cent V/V solution of hydrochloric acid R. Shake until
tetrahydroborate R. Stir until complete dissolution, dilute dissolved, heating, if necessary, on a water-bath at
to 500.0 mL with water R and mix. Prepare immediately 50 °C. Pass a current of nitrogen R for 15 min. Prepare
before use. immediately before use.
Sodium tetraphenylborate. NaB(C6H5)4. (Mr 342.2). Stanolone. C19H30O2. (Mr 290.4). 1154400. [521-18-6].
1084400. [143-66-8]. 17β-Hydroxy-5α-androstan-3-one.
White or slightly yellowish, bulky powder, freely soluble in White or almost white powder.
water and in acetone. mp : about 180 °C.
Sodium tetraphenylborate solution. 1084401. Standard solution for the micro determination of water.
1147300.
Filter before use if necessary.
Commercially available standard solution for the coulometric
A 10 g/L solution. titration of water, containing a certified content of water in a
Storage : use within 1 week. suitable solvent.
General Notices (1) apply to all monographs and other texts 521
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Staphylococcus aureus strain V8 protease, type XVII-B. Stearic acid. C18H36O2. (Mr 284.5). 1085200. [57-11-4].
1115800. [66676-43-5]. Octadecanoic acid.
Microbial extracellular proteolytic enzyme. A lyophilised White or almost white powder or flakes, greasy to the touch,
powder containing 500 units to 1000 units per milligram of practically insoluble in water, soluble in hot ethanol (96 per
solid. cent).
mp : about 70 °C.
Starch, soluble. 1085100. [9005-84-9].
Stearic acid used in the assay of total fatty acids in Saw palmetto
White or almost white powder. fruit (1848) complies with the following additional test.
Prepare a 20 g/L solution in hot water R. The solution is at Assay. Gas chromatography (2.2.28) as prescribed in the
most slightly opalescent and remains fluid on cooling. monograph Saw palmetto fruit (1848).
Content : minimum 98 per cent, calculated by the
Starch iodate paper. 1085101. normalisation procedure.
Immerse strips of filter paper in 100 mL of iodide-free
starch solution R containing 0.1 g of potassium iodate R. Stearyl alcohol. C18H38O. (Mr 270.5). 1156400. [112-92-5].
Drain and allow to dry protected from light. Octadecan-1-ol.
mp : about 60 °C.
Starch iodide paper. 1085106. Content : minimum 95 per cent.
Immerse strips of filter paper in 100 mL of starch solution R
containing 0.5 g of potassium iodide R. Drain and allow Stigmasterol. C29H48O. (Mr 412.7). 1141400. [83-48-7].
to dry protected from light. (22E)-Stigmasta-5,22-dien-3β-ol. (22E)-24-Ethylcholesta-
5,22-dien-3β-ol.
Test for sensitivity. Mix 0.05 mL of 0.1 M sodium nitrite
with 4 mL of hydrochloric acid R and dilute to 100 mL with White or almost white powder, insoluble in water.
water R. Apply one drop of the solution to starch iodide mp : about 170 °C.
paper ; a blue spot appears. : about – 51, determined with a 20 g/L solution in
chloroform R.
Starch solution. 1085103.
Triturate 1.0 g of soluble starch R with 5 mL of water R and Streptomycin sulfate. 1085300. [3810-74-0].
whilst stirring pour the mixture into 100 mL of boiling See Streptomycin sulfate (0053).
water R containing 10 mg of mercuric iodide R.
Strongly acidic ion-exchange resin. 1085400.
Carry out the test for sensitivity each time the reagent is
used. See ion-exchange resin, strongly acidic R.
Test for sensitivity. To a mixture of 1 mL of the starch Strontium carbonate. SrCO3. (Mr 147.6). 1122700.
solution and 20 mL of water R, add about 50 mg of [1633-05-2].
potassium iodide R and 0.05 mL of iodine solution R1. The White or almost white, crystalline powder.
solution is blue.
Content : minimum 99.5 per cent.
Starch solution, iodide-free. 1085104. Strontium chloride hexahydrate. SrCl2,6H2O. (Mr 266.6).
Prepare the solution as prescribed for starch solution R 1167000. [10025-70-4].
omitting the mercuric iodide. Prepare immediately before White or almost white crystals, very soluble in water.
use.
mp : about 115 °C (loss of water) and 872 °C.
Starch solution R1. 1085105.
Strontium selective extraction resin. 1167100.
Mix 1 g of soluble starch R and a small amount of cold Commercially available resin prepared by loading a suspension
water R. Add this mixture, while stirring, to 200 mL of of 4,4′(5′)-di-tert-butylcyclohexano-18-crown-6 (crown ether)
boiling water R. Add 0.25 g of salicylic acid R and boil for in octanol onto an inert chromatographic support. The bed
3 min. Immediately remove from the heat and cool. density of this resin is approximately 0.35 g/mL.
Storage : long storage is required, the solution shall be
stored at 4 °C to 10 °C. A fresh starch solution shall be Strontium-85 spiking solution. 1166800.
prepared when the end-point of the titration from blue to Dilute strontium-85 standard solution R to a radioactivity
colourless fails to be sharp. If stored under refrigeration, concentration of approximately 10 kBq/mL with a 0.27 g/L
the starch solution is stable for about 2 to 3 weeks. solution of strontium chloride hexahydrate R in a 1.03 g/L
Test for sensitivity. A mixture of 2 mL of starch solution R1, solution of hydrochloric acid R.
20 mL of water R, about 50 mg of potassium iodide R and Strontium-85 standard solution. 1166900.
0.05 mL of iodine solution R1 is blue.
A solution of strontium-85 in the form of Sr2+ ions in a 51.5 g/L
Starch solution R2. 1085107. solution of hydrochloric acid R.
Triturate 1.0 g of soluble starch R with 5 mL of water R and Styrene. C8H8. (Mr 104.2). 1151700. [100-42-5].
whilst stirring pour the mixture into 100 mL of boiling Ethenylbenzene.
water R. Use a freshly prepared solution. bp : about 145 °C.
Test for sensitivity. To a mixture of 1 mL of the starch Colourless, oily liquid, very slightly soluble in water.
solution and 20 mL of water R, add about 50 mg of
potassium iodide R and 0.05 mL of iodine solution R1. The Styrene-divinylbenzene copolymer. 1085500.
solution is blue. Porous, rigid, cross-linked polymer beads. Several grades are
available with different sizes of beads. The size range of the
Stavudine. 1187000. [3056-17-5]. beads is specified after the name of the reagent in the tests
See Stavudine (2130). where it is used.
Succinic acid. C4H6O4. (Mr 118.1). 1085600. [110-15-6]. Sulfanilic acid solution, diazotised. 1086202.
Butanedioic acid. Dissolve, with warming, 0.9 g of sulfanilic acid R in 9 mL
White or almost white, crystalline powder or colourless of hydrochloric acid R, and dilute to 100 mL with water R.
crystals, soluble in water and in ethanol (96 per cent). Cool 10 mL of this solution in iced water and add 10 mL
mp : 184 °C to 187 °C. of an ice-cold 45 g/L solution of sodium nitrite R. Allow to
stand at 0 °C for 15 min (if stored at this temperature, the
Sucrose. 1085700. [57-50-1]. solution is stable for 3 days) and immediately before use
See Sucrose (0204). add 20 mL of a 100 g/L solution of sodium carbonate R.
Sudan orange. C16H12N2O. (Mr 248.3). 1110700. [842-07-9]. Sulfomolybdic reagent R2. 1086400.
Colour Index No. 12055. Dissolve about 50 mg of ammonium molybdate R in 10 mL
1-(Phenylazo)naphthalen-2-ol. Sudan I. of sulfuric acid R.
Orange-red powder, practically insoluble in water, soluble in Sulfomolybdic reagent R3. 1086500.
methylene chloride.
Dissolve with heating 2.5 g of ammonium molybdate R in
mp : about 131 °C. 20 mL of water R. Dilute 28 mL of sulfuric acid R in 50 mL
Sudan red G. C17H14N2O2. (Mr 278.3). 1085800. of water R, then cool. Mix the two solutions and dilute to
100 mL with water R.
Schultz No. 149.
Storage : in a polyethylene container.
Colour Index No. 12150.
Solvent Red 1. 1-[(2-Methoxyphenyl)azo]naphtalen-2-ol. Sulfosalicylic acid. C7H6O6S,2H2O. (Mr 254.2). 1086600.
Reddish-brown powder, practically insoluble in water. [5965-83-3]. 2-Hydroxy-5-sulfobenzoic acid.
Chromatography. Thin-layer chromatography (2.2.27) using White or almost white, crystalline powder or crystals, very
silica gel G R as the coating substance : apply 10 μL of a 0.1 g/L soluble in water and in ethanol (96 per cent).
solution in methylene chloride R and develop over a path of mp : about 109 °C.
10 cm with the same solvent ; the chromatogram shows only
one principal spot. Sulfur. 1110800. [7704-34-9].
See Sulfur for external use (0953).
Sulfanilamide. C6H8N2O2S. (Mr 172.2). 1086100. [63-74-1].
4-Aminobenzenesulfonamide. Sulfur dioxide. SO2. (Mr 64.1). 1086700. [7446-09-5].
White or almost white powder, slightly soluble in water, freely Sulfurous anhydride.
soluble in boiling water, in acetone, in dilute acids and in A colourless gas. When compressed it is a colourless liquid.
solutions of the alkali hydroxides, sparingly soluble in ethanol
(96 per cent) and in light petroleum. Sulfur dioxide R1. SO2. (Mr 64.1). 1110900. [7446-09-5].
mp : about 165 °C. Content : minimum 99.9 per cent V/V.
Sulfathiazole. C9H9N3O2S2. (Mr 255.3). 1086300. [72-14-0]. Sulfuric acid. H2SO4. (Mr 98.1). 1086800. [7664-93-9].
4-Amino-N-(thiazol-2-yl)benzenesulfonamide. Content : 95.0 per cent m/m to 97.0 per cent m/m.
White or yellowish-white powder or crystals, very slightly Colourless, caustic liquid with an oily consistency, highly
soluble in water, soluble in acetone, slightly soluble in ethanolhygroscopic, miscible with water and with ethanol (96 per
(96 per cent). It dissolves in dilute mineral acids and in cent) producing intense heat.
solutions of alkali hydroxides and carbonates. : 1.834 to 1.837.
mp : about 200 °C. A 10 g/L solution is strongly acid and gives the reactions of
sulfates (2.3.1).
Sulfamic acid. H3NO3S. (Mr 97.1). 1085900. [5329-14-6].
Appearance. It is clear (2.2.1) and colourless (2.2.2, Method II).
White or almost white crystalline powder or crystals, freely
soluble in water, sparingly soluble in acetone, in ethanol Oxidisable substances. Pour 20 g cautiously, with cooling,
(96 per cent) and in methanol. into 40 mL of water R. Add 0.5 mL of 0.002 M potassium
permanganate. The violet colour persists for at least 5 min.
mp : about 205 °C, with decomposition.
Chlorides : maximum 0.5 ppm.
Sulfan blue. C27H31N2NaO6S2. (Mr 566.6). 1086000. Pour 10 g, carefully and while cooling, into 10 mL of water R
[129-17-9]. and after cooling dilute to 20 mL with the same solvent.
Schultz No. 769. Add 0.5 mL of silver nitrate solution R2. Allow to stand for
Colour Index No. 42045. 2 min protected from bright light. The solution is not more
Acid Blue 1. Patent Blue VF. Disulfine blue. Blue VS. opalescent than a standard prepared at the same time using a
Sodium [[[(4-diethylamino)phenyl](2,4-disulfonatophenyl)- mixture of 1 mL of chloride standard solution (5 ppm Cl) R,
methylene]cyclohexa-2,5-dien-1-ylidene]diethylammonium. 19 mL of water R and 0.5 mL of silver nitrate solution R2.
Violet powder, soluble in water. Dilute solutions are blue and Nitrates : maximum 0.5 ppm.
turn yellow on the addition of concentrated hydrochloric acid. Pour 50 g or 27.2 mL, carefully and while cooling, into 15 mL
of water R. Add 0.2 mL of a freshly prepared 50 g/L solution
Sulfanilic acid. C6H7NO3S. (Mr 173.2). 1086200. [121-57-3]. of brucine R in glacial acetic acid R. After 5 min any colour
4-Aminobenzenesulfonic acid. is less intense than that of a reference mixture prepared in
Colourless crystals, sparingly soluble in water, practically the same manner and containing 12.5 mL of water R, 50 g
insoluble in ethanol (96 per cent). of nitrogen-free sulfuric acid R, 2.5 mL of nitrate standard
Sulfanilic acid solution. 1086203. solution (10 ppm NO3) R and 0.2 mL of a 50 g/L solution of
brucine R in glacial acetic acid R.
Dissolve 0.33 g of sulfanilic acid R in 75 mL of water R
heating gently if necessary and dilute to 100 mL with glacial Ammonium : maximum 2 ppm.
acetic acid R. Pour 2.5 g, carefully and while cooling, into water R and dilute
to 20 mL with the same solvent. Cool, and add dropwise
Sulfanilic acid solution R1. 1086201. 10 mL of a 200 g/L solution of sodium hydroxide R, followed by
Dissolve 0.5 g of sulfanilic acid R in a mixture of 75 mL of 1 mL of alkaline potassium tetraiodomercurate solution R. The
dilute acetic acid R and 75 mL of water R. colour of the solution is less intense than that of a mixture of
General Notices (1) apply to all monographs and other texts 523
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Sulfuric acid, alcoholic solution of. 1086803. Tartaric acid. 1087200. [87-69-4].
Carefully and with constant cooling, stir 20 mL of sulfuric See Tartaric acid (0460).
acid R into 60 mL of ethanol (96 per cent) R. Allow to cool Taxifolin. C15H12O7. (Mr 304.3). 1151800. [480-18-2].
and dilute to 100 mL with ethanol (96 per cent) R. Prepare (2R,3R)-2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-2,3-
immediately before use. dihydro-4H-1-benzopyran-4-one.
Sulfuric acid, dilute. 1086804. White or almost white powder, slightly soluble in anhydrous
Contains 98 g/L of H2SO4. ethanol.
Add 5.5 mL of sulfuric acid R to 60 mL of water R, allow to Absorbance (2.2.25). A solution in anhydrous ethanol R shows
cool and dilute to 100 mL with the same solvent. an absorption maximum at 290 nm.
Assay. Into a ground-glass-stoppered flask containing Tecnazene. C6HCl4NO2. (Mr 260.9). 1132400. [117-18-0].
30 mL of water R, introduce 10.0 mL of the dilute sulfuric bp : about 304 °C.
acid. Titrate with 1 M sodium hydroxide, using 0.1 mL of
methyl red solution R as indicator. mp : 99 °C to 100 °C.
1 mL of 1 M sodium hydroxide is equivalent to 49.04 mg A suitable certified reference solution (10 ng/μL in
of H2SO4. cyclohexane) may be used.
α-Terpinene. C10H16. ( Mr 136.2). 1140300. [99-86-5].
Sulfuric acid-formaldehyde reagent. 1086805.
1-Isopropyl-4-methylcyclohexa-1,3-diene.
Mix 2 mL of formaldehyde solution R with 100 mL of
Clear, almost colourless liquid.
sulfuric acid R.
: about 0.837.
Sulfuric acid, heavy metal-free. 1086807. : about 1.478.
Complies with the requirements prescribed for sulfuric bp : about 174 °C.
acid R with the following maximum contents of heavy
metals. α-Terpinene used in gas chromatography complies with the
following additional test.
As : 0.005 ppm.
Assay. Gas chromatography (2.2.28) as prescribed in the
Cd : 0.002 ppm. monograph Tea tree oil (1837).
Cu : 0.001 ppm. Content : minimum 90 per cent, calculated by the
Fe : 0.05 ppm. normalisation procedure.
General Notices (1) apply to all monographs and other texts 525
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Tetrachloroethane. C2H2Cl4. (Mr 167.9). 1088000. [79-34-5]. Tetrahexylammonium hydrogen sulfate. C24H53NO4S.
1,1,2,2-Tetrachloroethane. (Mr 451.8). 1116300. [32503-34-7]. N,N,N-Trihexylhexan-
Clear, colourless liquid, slightly soluble in water, miscible with 1-aminium hydrogen sulfate.
ethanol (96 per cent). White or almost white crystals.
: about 1.59. mp : 100 °C to 102 °C.
: about 1.495.
Tetrahydrofuran. C4H8O. (Mr 72.1). 1088500. [109-99-9].
Distillation range (2.2.11). Not less than 95 per cent distils Tetramethylene oxide.
between 145 °C and 147 °C.
Clear, colourless, flammable liquid, miscible with water, with
Tetrachlorvinphos. C10H9Cl4O4P. (Mr 366.0). 1132500. ethanol (96 per cent).
[22248-79-9]. : about 0.89.
mp : about 95 °C. Do not distil if the tetrahydrofuran does not comply with the
A suitable certified reference solution (10 ng/μL in iso-octane) test for peroxides.
may be used. Peroxides. Place 8 mL of potassium iodide and starch solution R
Tetracos-15-enoic acid methyl ester. C25H48O2. (Mr 380.7). in a 12 mL ground-glass-stoppered cylinder about 1.5 cm in
1144800. [2733-88-2]. 15-Tetracosaenoic acid methyl ester. diameter. Fill completely with the substance to be examined,
Methyl tetracos-15-enoate. Nervonic acid methyl ester. shake vigorously and allow to stand protected from light for
30 min. No colour is produced.
Content : minimum 99.0 per cent, determined by gas
chromatography. Tetrahydrofuran used in spectrophotometry complies with the
following additional test.
Liquid.
Minimum transmittance (2.2.25) using water R as
Tetracycline hydrochloride. 1147000. compensation liquid : 20 per cent at 255 nm, 80 per cent at
See Tetracycline hydrochloride (0210). 270 nm, 98 per cent at 310 nm.
Tetradecane. C14H30. (Mr 198.4). 1088200. [629-59-4]. Tetrahydrofuran for chromatography R. 1147100.
n-Tetradecane. Complies with the requirements prescribed for
Content : minimum 99.5 per cent m/m. tetrahydrofuran R with the following additional requirements :
A colourless liquid. = 0.8892.
: about 0.76. bp : about 66 °C.
: about 1.429. Content : minimum 99.8 per cent of C4H8O.
bp : about 252 °C.
α-Tetralone. C10H10O. (Mr 146.2). 1171800. [529-34-0].
mp : about − 5 °C. 1-Oxotetraline. 3,4-Dihydronaphthalen-1(2H)-one.
Tetradecylammonium bromide. C40H84BrN. (Mr 659). bp : about 115 °C.
1088300. [14937-42-9]. Tetrakis(decyl)ammonium bromide. mp : about 5 °C.
White or slightly coloured, crystalline powder or crystals.
mp : 88 °C to 89 °C. Tetramethylammonium bromide. C4H12BrN. (Mr 154.1).
1156600. [64-20-0]. N,N,N-Trimethylmethanaminium
Tetraethylammonium hydrogen sulfate. C8H21NO4S. bromide.
(Mr 227.3). 1116200. [16873-13-5]. White or slightly yellow crystals, freely soluble in water.
Hygroscopic powder. mp : about 285 °C, with decomposition.
mp : about 245 °C.
Tetramethylammonium chloride. C4H12ClN. (Mr 109.6).
Tetraethylammonium hydroxide solution. C8H21NO. 1100400. [75-57-0].
(Mr 147.3). 1100300. [77-98-5]. Colourless crystals, soluble in water and in ethanol (96 per
A 200 g/L solution. cent).
Colourless liquid, strongly alkaline. mp : about 300 °C, with decomposition.
: about 1.01.
Tetramethylammonium hydrogen sulfate. C4H13NO4S.
: about 1.372. (Mr 171.2). 1116400. [80526-82-5].
HPLC grade.
Hygroscopic powder.
Tetraethylene pentamine. C8H23N5. (Mr 189.3). 1102000. mp : about 295 °C.
[112-57-2]. 3,6,9-Triazaundecan-1,11-diamine.
Colourless liquid, soluble in acetone. Tetramethylammonium hydroxide. C4H13NO,5H2O.
(Mr 181.2). 1122800. [10424-65-4]. Tetramethylammonium
: about1.506. hydroxide pentahydrate.
Storage : protected from humidity and heat. Suitable grade for HPLC.
Tetraheptylammonium bromide. C28H60BrN. (Mr 490.7).
Tetramethylammonium hydroxide solution. 1088600.
1088400. [4368-51-8].
[75-59-2].
White or slightly coloured, crystalline powder or crystals.
Content : minimum 10.0 per cent m/m of C4H13NO. (Mr 91.2).
mp : 89 °C to 91 °C.
Clear, colourless or very pale yellow liquid, miscible with
Tetrahexylammonium bromide. C24H52BrN. (Mr 434.6). water and with ethanol (96 per cent).
1152500. [4328-13-6]. N,N,N-Trihexylhexan-1-aminium Assay. To 1.000 g add 50 mL of water R and titrate with 0.05 M
bromide. sulfuric acid, using 0.1 mL of methyl red solution R as indicator.
White or almost white, crystalline powder, hygroscopic. 1 mL of 0.05 M sulfuric acid is equivalent to 9.12 mg of
mp : about 100 °C. C4H13NO.
Tetramethylammonium hydroxide solution, dilute. Tetrazolium blue. C40H32Cl2N8O2. (Mr 728). 1089000.
1088601. [1871-22-3]. 3,3′-(3,3′-Dimethoxy[1,1′-biphenyl]-4,4′-
Dilute 10 mL of tetramethylammonium hydroxide diyl)bis[2,5-diphenyl-2H-tetrazolium] dichloride.
solution R to 100 mL with aldehyde-free alcohol R. Prepare Yellow crystals, slightly soluble in water, freely soluble in
immediately before use. ethanol (96 per cent) and in methanol, practically insoluble
in acetone.
Tetramethylbenzidine. C16H20N2. (Mr 240.3). 1132600. mp : about 245 °C, with decomposition.
[54827-17-7]. 3,3′,5,5′-Tetramethylbiphenyl-4,4′-diamine.
Powder, practically insoluble in water, very soluble in Tetrazolium bromide. C18H16BrN5S. (Mr 414.3).
methanol. 1152700. [298-93-1]. 3-(4,5-Dimethylthiazol-2-yl)-2,5-
mp : about 169 °C. diphenyltetrazolium bromide. MTT.
Tetrazolium salt. C20H17N5O6S2. (Mr 487.5). 1174200.
1,1,3,3-Tetramethylbutylamine. C8H19N. (Mr 129.3).
[138169-43-4]. 5-(3-Carboxymethoxyphenyl)-3-(4,5-
1141500. [107-45-9]. 2-Amino-2,4,4-trimethylpentane.
dimethylthiazol-2-yl)-2-(4-sulfophenyl)-2H-tetrazolium,
Clear, colourless liquid. inner salt. MTS.
: about 0.805.
Thallous sulfate. Tl2SO4. (Mr 504.8). 1089100. [7446-18-6].
: about 1.424. Dithallium sulfate.
bp : about 140 °C. White or almost white, rhomboid prisms, slightly soluble in
Tetramethyldiaminodiphenylmethane. C17H22N2. water, practically insoluble in ethanol (96 per cent).
(Mr 254.4). 1088700. [101-61-1]. 4,4′-Methylenebis-(N,N- Thebaine. C19H21NO3. (Mr 311.4). 1089200. [115-37-7].
dimethylaniline). (5R,9R,13S)-4,5-Epoxy-3,6-dimethoxy-9a-methylmorphina-
White or bluish-white crystals or leaflets, practically insoluble 6,8-diene.
in water, slightly soluble in ethanol (96 per cent), soluble in White or pale yellow, crystalline powder, very slightly soluble
mineral acids. in water, soluble in hot anhydrous ethanol and in toluene.
mp : about 90 °C. mp : about 193 °C.
Tetramethyldiaminodiphenylmethane reagent. 1088701. Chromatography (2.2.27). Thin-layer chromatography (2.2.27)
as prescribed in identification test B in the monograph Raw
Solution A. Dissolve 2.5 g of tetramethyldiaminodiphenyl- opium (0777) : apply to the plate as a band (20 mm × 3 mm)
methane R in 10 mL of glacial acetic acid R and add 50 mL 20 μL of a 0.5 g/L solution ; the chromatogram shows an
of water R. orange-red or red principal band with an RF of about 0.5.
Solution B. Dissolve 5 g of potassium iodide R in 100 mL
of water R. Theobromine. 1138800. [83-67-0].
Solution C. Dissolve 0.30 g of ninhydrin R in 10 mL of See Theobromine (0298).
glacial acetic acid R and add 90 mL of water R. Theophylline. 1089300. [58-55-9].
Mix solution A, solution B and 1.5 mL of solution C. See Theophylline (0299).
Tetramethylethylenediamine. C6H16N2. (Mr 116.2). 1088800. Thiamazole. C4H6N2S. (Mr 114.2). 1089400. [60-56-0].
[110-18-9]. N,N,N’,N’-Tetramethylethylenediamine. Methimazole. 1-Methyl-1H-imidazole-2-thiol.
Colourless liquid, miscible with water and with ethanol White or almost white, crystalline powder, freely soluble
(96 per cent). in water, soluble in ethanol (96 per cent) and in methylene
: about 0.78. chloride.
: about 1.418. mp : about 145 °C.
bp : about 121 °C. 2-(2-Thienyl)acetic acid. C6H6O2S. (Mr 142.1). 1089500.
[1918-77-0].
Tetramethylsilane. C4H12Si. (Mr 88.2). 1088900. [75-76-3].
TMS. Brown powder.
Clear, colourless liquid, very slightly soluble in water, soluble mp : about 65 °C.
in acetone and in ethanol (96 per cent). Thioacetamide. C2H5NS. (Mr 75.1). 1089600. [62-55-5].
: about 0.64. Crystalline powder or colourless crystals, freely soluble in
: about 1.358. water and in ethanol (96 per cent).
bp : about 26 °C. mp : about 113 °C.
Tetramethylsilane used in nuclear magnetic resonance Thioacetamide reagent. 1089601.
spectrometry complies with the following additional test.
To 0.2 mL of thioacetamide solution R add 1 mL of a
In the nuclear magnetic resonance spectrum of mixture of 5 mL of water R, 15 mL of 1 M sodium hydroxide
an approximately 10 per cent V/V solution of the and 20 mL of glycerol (85 per cent) R. Heat in a water-bath
tetramethylsilane in deuterated chloroform R, the intensity of for 20 s. Prepare immediately before use.
any foreign signal, excluding those due to spinning side bands
and to chloroform, is not greater than the intensity of the C-13 Thioacetamide solution. 1089602.
satellite signals located at a distance of 59.1 Hz on each side of A 40 g/L solution.
the principal signal of tetramethylsilane.
Thiobarbituric acid. C4H4N2O2S. (Mr 144.2). 1111200.
Tetrandrine. C38H42N2O6. (Mr 623). 1178500. [518-34-3]. [504-17-6]. 4,6-Dihydroxy-2-sulfanylpyrimidine.
Tetrapropylammonium chloride. C12H28ClN. (Mr 221.8). Thiodiethylene glycol. C4H10O2S. (Mr 122.2). 1122900.
1151900. [5810-42-4]. [111-48-8]. Di(2-hydroxyethyl) sulfide.
White or almost white, crystalline powder, sparingly soluble Colourless or yellow, viscous liquid.
in water. Content : minimum 99.0 per cent.
mp : about 241 °C. : about 1.18.
General Notices (1) apply to all monographs and other texts 527
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Thioglycollic acid. C2H4O2S. (Mr 92.1). 1089700. [68-11-1]. Thymine. C5H6N2O2. (Mr 126.1). 1090400. [65-71-4].
2-Mercaptoacetic acid. 5-Methylpyrimidine-2,4(1H,3H)-dione.
Colourless liquid, miscible with water, soluble in ethanol Short needles or plates, slightly soluble in cold water, soluble
(96 per cent). in hot water. It dissolves in dilute solution of alkali hydroxides.
Thiomalic acid. C4H6O4S. (Mr 150.2). 1161600. [70-49-5]. Thymol. 1090500. [89-83-8]. See Thymol (0791).
(2RS)-2-Sulfanylbutanedioic acid. Thymol used in gas chromatography complies with the following
mp : 150 °C to 152 °C. additional test.
Assay. Gas chromatography (2.2.28) as prescribed in the
Thiomersal. C9H9HgNaO2S. (Mr 404.8). 1089800. monograph Peppermint oil (0405).
[54-64-8]. Sodium mercurothiolate. Sodium Test solution. Dissolve 0.1 g in about 10 mL of acetone R.
2-[(ethylmercurio)thio]benzoate.
Content : minimum 95.0 per cent, calculated by the
Light, yellowish-white, crystalline powder, very soluble in normalisation procedure.
water, freely soluble in ethanol (96 per cent).
Thymol blue. C27H30O5S. (Mr 466.6). 1090600. [76-61-9].
Thiourea. CH4N2S. (Mr 76.1). 1089900. [62-56-6]. Thymolsulfonphthalein. 4,4′-(3H-2,1-Benzoxathiol-3-
White or almost white, crystalline powder or crystals, soluble ylidene)bis(2-isopropyl-5-methylphenol) S,S-dioxide.
in water and in ethanol (96 per cent). Brownish-green or greenish-blue, crystalline powder, slightly
mp : about 178 °C. soluble in water, soluble in ethanol (96 per cent) and in dilute
solutions of alkali hydroxides.
Threonine. 1090000. [72-19-5]. Thymol blue solution. 1090601.
See Threonine (1049). Dissolve 0.1 g of thymol blue R in a mixture of 2.15 mL
of 0.1 M sodium hydroxide and 20 mL of ethanol (96 per
Thrombin, bovine. 1090200. [9002-04-4].
cent) R and dilute to 100 mL with water R.
A preparation of the enzyme, obtained from bovine plasma, Test for sensitivity. To 0.1 mL of the thymol blue solution
that converts fibrinogen into fibrin. add 100 mL of carbon dioxide-free water R and 0.2 mL of
A yellowish-white powder. 0.02 M sodium hydroxide. The solution is blue. Not more
Storage : at a temperature below 0 °C. than 0.15 mL of 0.02 M hydrochloric acid is required to
change the colour to yellow.
Thrombin, human. 1090100. [9002-04-4]. Colour change : pH 1.2 (red) to pH 2.8 (yellow) ; pH 8.0
Dried human thrombin. A preparation of the enzyme which (olive-green) to pH 9.6 (blue).
converts human fibrinogen into fibrin. It is obtained from
Thymolphthalein. C28H30O4. (Mr 430.5). 1090700.
liquid human plasma and may be prepared by precipitation
[125-20-2]. 3,3-Bis(4-hydroxy-5-isopropyl-2-methylphenyl)-
with suitable salts and organic solvents under controlled
3H-isobenzo-furan-1-one.
conditions of pH, ionic strength and temperature.
White or yellowish-white powder, practically insoluble in
Yellowish-white powder, freely soluble in a 9 g/L solution of water, soluble in ethanol (96 per cent) and in dilute solutions
sodium chloride forming a cloudy, pale yellow solution. of alkali hydroxides.
Storage : in a sealed, sterile container under nitrogen, protected
from light, at a temperature below 25 °C. Thymolphthalein solution. 1090701.
A 1 g/L solution in ethanol (96 per cent) R.
Thrombin solution, human. 1090101. Test for sensitivity. To 0.2 mL of the thymolphthalein
Reconstitute human thrombin R as directed by solution add 100 mL of carbon dioxide-free water R. The
the manufacturer and dilute to 5 IU/mL with solution is colourless. Not more than 0.05 mL of 0.1 M
tris(hydroxymethyl)aminomethane sodium chloride buffer sodium hydroxide is required to change the colour to blue.
solution pH 7.4 R. Colour change: pH 9.3 (colourless) to pH 10.5 (blue).
Thrombin solution, human R1. 1090102. Tin. Sn. (Ar 118.7). 1090800. [7440-31-5].
Reconstitute human thrombin R as directed by the Silvery-white granules, soluble in hydrochloric acid with
manufacturer and dilute to 2.5 IU/mL with phosphate release of hydrogen.
buffer solution pH 6.5 R. Arsenic (2.4.2, Method A): maximum 10 ppm, determined
on 0.1 g.
Thromboplastin. 1090300.
A preparation containing the membrane glycoprotein tissue Titan yellow. C28H19N5Na2O6S4. (Mr 696). 1090900.
factor and phospholipid, either purified from animal brain [1829-00-1].
(usually rabbit) or human placenta or manufactured using Schultz No. 280.
recombinant DNA technology with added phospholipid. The Colour Index No. 19540.
preparation is formulated for routine use in the prothrombin Thiazol yellow. Disodium 2,2′-[(1-triazene-1,3-diyl)di-4,1-
time test and may contain calcium. phenylene]bis-[6-methylbenzothiazole-7-sulfonate].
Thujone. C10H16O. (Mr 152.2). 1116500. [76231-76-0]. A yellowish-brown powder, freely soluble in water and in
4-Methyl-1-(1-methylethyl)bicyclo[3.1.0]hexan-3-one. ethanol (96 per cent).
Colourless or almost colourless liquid, practically insoluble Titan yellow paper. 1090901.
in water, soluble in ethanol (96 per cent) and in many other Immerse strips of filter paper in titan yellow solution R and
organic solvents. leave for a few minutes. Allow to dry at room temperature.
Thymidine. C10H14N2O5. (Mr 242.2). 1158900. 1-(2-Deoxy-β- Titan yellow solution. 1090902.
D-erythro-pentofuranosyl)-5-methylpyrimidine-2,4(1H,3H)- A 0.5 g/L solution.
dione. Test for sensitivity. To 0.1 mL of the titan yellow solution
Needles, soluble in water, in hot ethanol (96 per cent) and in add 10 mL of water R, 0.2 mL of magnesium standard
glacial acetic acid. solution (10 ppm Mg) R and 1.0 mL of 1 M sodium
hydroxide. A distinct pink colour is visible by comparison spot of bromocresol green with an RF value less than 0.15, the
with a reference solution prepared in a similar manner spot of methyl orange with an RF value in the range of 0.1 to
omitting the magnesium. 0.25, the spot of methyl red with an RF value in the range of
0.35 to 0.55 and the spot of Sudan red G with an RF value in
Titanium. Ti. (Ar 47.88). 1091000. [7440-32-6]. the range of 0.75 to 0.98.
Content : minimum 99 per cent.
Metal powder, fine wire (diameter not more than 0.5 mm), TLC silica gel F254 plate. 1116800.
sponge. Complies with the requirements prescribed for TLC silica gel
plate R with the following modification.
mp : about 1668 °C.
It contains a fluorescent indicator having a maximum
Density : about 4.507 g/cm3.
absorbance at 254 nm.
Titanium dioxide. 1117900. [13463-67-7]. Fluorescence suppression. Apply separately to the plate at five
See Titanium dioxide (0150). points increasing volumes (1 μL to 10 μL for normal TLC
plates and 0.2 μL to 2 μL for fine particle size plates) of a
Titanium trichloride. TiCl3. (Mr 154.3). 1091200. 1 g/L solution of benzoic acid R in a mixture of 15 volumes
[7705-07-9]. Titanium(III) chloride. of anhydrous ethanol R and 85 volumes of cyclohexane R.
Reddish-violet crystals, deliquescent, soluble in water and in Develop over a pathlength half of the plate height with the
ethanol (96 per cent). same mixture of solvents. After evaporating the solvents
mp : about 440 °C. examine the chromatogram in ultraviolet light at 254 nm. For
Storage : in an airtight container. normal TLC plates the benzoic acid appears as dark spots on
a fluorescent background approximately in the middle of the
Titanium trichloride solution. 1091201. chromatogram for quantities of 2 μg and greater. For fine
: about 1.19. particle size plates the benzoic acid appears as dark spots on
A 150 g/L solution in hydrochloric acid (100 g/L HCl). a fluorescent background approximately in the middle of the
chromatogram for quantities of 0.2 μg and greater.
Titanium trichloride-sulfuric acid reagent. 1091202.
TLC silica gel F254, silanised plate. 1117200.
Carefully mix 20 mL of titanium trichloride solution R with
13 mL of sulfuric acid R. Add sufficient strong hydrogen It complies with the requirements prescribed for TLC silanised
peroxide solution R to give a yellow colour. Heat until white silica gel plate R with the following modification.
fumes are evolved. Allow to cool. Dilute with water R It contains a fluorescent indicator having a maximum
and repeat the evaporation and addition of water R until absorbance at 254 nm.
a colourless solution is obtained. Dilute to 100 mL with TLC silica gel G plate. 1116900.
water R.
Complies with the requirements prescribed for TLC silica gel
TLC aluminium oxide G plate. 1165200. plate R with the following modification.
Support of metal, glass or plastic, coated with a layer of It contains calcium sulfate hemihydrate as binder.
aluminium oxide (particle size 5-40 μm) containing about TLC silica gel GF254 plate. 1117000.
10 per cent of calcium sulfate hemihydrate as a binder.
Complies with the requirements prescribed for TLC silica gel
TLC octadecylsilyl silica gel plate. 1148600. plate R with the following modifications.
Support of glass, metal or plastic coated with a layer of It contains calcium sulfate hemihydrate as binder and a
octadecylsilyl silica gel. The plate may contain an organic fluorescent indicator having a maximum absorbance at
binder. 254 nm.
TLC octadecylsilyl silica gel F254 plate R. 1146600. Fluorescence suppression. Complies with the test prescribed
for TLC silica gel F254 plate R.
Support of glass, metal or plastic coated with a layer of
octadecylsilyl silica gel. TLC silica gel plate for aminopolyether test. 1172700.
It contains a fluorescent indicator having a maximum Immerse a TLC silica gel plate R in iodoplatinate reagent R1 for
absorbance in ultraviolet light at 254 nm. 5-10 s. Dry at room temperature for 12 h, protected from light.
TLC performance test solution. 1116600. Storage : protected from light, in an open container ; use within
30 days after preparation.
Prepare a mixture of 1.0 mL of each of the following solutions
and dilute to 10.0 mL with acetone R : a 0.5 g/L solution of TLC silica gel plate for chiral separations, octadecylsilyl.
Sudan red G R in toluene R, a 0.5 g/L solution of methyl 1137700.
orange R in ethanol R prepared immediately before use, a Support of glass, metal or plastic, coated with a layer of
0.5 g/L solution of bromocresol green R in acetone R and a octadecylsilyl silica gel, impregnated with Cu2+ ions and
0.25 g/L solution of methyl red R in acetone R. enantiomerically pure hydroxyproline. The plate may contain
an organic binder.
TLC silica gel plate. 1116700.
Support of glass, metal or plastic, coated with a layer of silica TLC silica gel, silanised plate. 1117100.
gel of a suitable thickness and particle size (usually 2 μm to Support of glass, metal or plastic, coated with a layer of
10 μm for fine particle size [High Performance Thin-Layer silanised silica gel of a suitable thickness and particle size
Chromatography, HPTLC] plates and 5 μm to 40 μm for (usually 2 μm to 10 μm for fine particle size [High Performance
normal TLC plates). If necessary, the particle size is indicated Thin-Layer Chromatography, HPTLC] plates and 5 μm to
after the name of the reagent in the tests where it is used. 40 μm for normal TLC plates). If necessary, the particle size
The plate may contain an organic binder. is indicated after the name of the reagent in the tests where
Chromatographic separation. Apply to the plate an appropriate it is used.
volume (10 μL for a normal TLC plate and 1 μL to 2 μL The plate may contain an organic binder.
for a fine particle size plate) of TLC performance test Chromatographic separation. Introduce 0.1 g each of methyl
solution R. Develop over a pathlength two-thirds of the plate laurate R, methyl myristate R, methyl palmitate R and methyl
height, using a mixture of 20 volumes of methanol R and stearate R into a 250 mL conical flask. Add 40 mL of alcoholic
80 volumes of toluene R. The plate is not satisfactory, unless potassium hydroxide solution R and heat under a reflux
the chromatogram shows four clearly separated spots, the condenser on a water-bath for 1 h. Allow to cool, transfer
General Notices (1) apply to all monographs and other texts 529
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
: − 85 to − 89, determined on a 10 g/L solution in ethanol 1,1,1-Trichloroethane. C2H3Cl3. (Mr 133.4). 1092600.
(96 per cent) R. [71-55-6]. Methylchloroform.
mp : about 105 °C. Non-flammable liquid, practically insoluble in water, soluble
: 290 to 320, determined at 228.5 nm in ethanol (96 per in acetone and in methanol.
cent) R. : about 1.34.
Toxaphene. 1132800. [8001-35-2]. : about 1.438.
A mixture of polychloro derivatives. bp : about 74 °C.
mp : 65 °C to 90 °C. Trichloroethylene. C2HCl3. (Mr 131.4). 1102100. [79-01-6].
A suitable certified reference solution (10 ng/μL in iso-octane) Colourless liquid, practically insoluble in water, miscible with
may be used. ethanol (96 per cent).
Tragacanth. 1092300. [9000-65-1]. : about 1.46.
See Tragacanth (0532). : about 1.477.
Tanshinone IIA. C19H18O3. (Mr 294.3). 1184800. [568-72-9]. Trichlorotrifluoroethane. C2Cl3F3. (Mr 187.4). 1092700.
1,6,6-Trimethyl-6,7,8,9-tetrahydrophenanthro[1,2-b]furan- [76-13-1]. 1,1,2-Trichloro-1,2,2-trifluoroethane.
10,11-dione. Colourless, volatile liquid, practically insoluble in water,
miscible with acetone.
Triacetin. C9H14O6. (Mr 218.2). 1092400. [102-76-1].
Propane-1,2,3-triyl triacetate. Glycerol triacetate. : about 1.58.
Almost clear, colourless to yellowish liquid, soluble in water, Distillation range (2.2.11). Not less than 98 per cent distils
miscible with ethanol (96 per cent). between 47 °C and 48 °C.
: about 1.16. Tricine. C6H13NO5. (Mr 179.2). 1138900. [5704–04–1].
: about 1.43. N-[2-Hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
bp : about 260 °C. Use electrophoresis-grade reagent.
Triamcinolone. C21H27FO6. (Mr 394.4). 1111300. [124-94-7]. mp : about 183 °C.
9-Fluoro-11β,16α,17,21-tetrahydroxypregna-1,4-diene-3,20- Tricosane. C23H48. (Mr 324.6). 1092800. [638-67-5].
dione.
White or almost white crystals, practically insoluble in water,
A crystalline powder.
soluble in hexane.
mp : 262 °C to 263 °C.
mp : about 48 °C.
Triamcinolone acetonide. 1133100. [76-25-5].
Tridocosahexaenoin. C69H98O6. (Mr 1023.5). 1144900.
See Triamcinolone acetonide (0533). [124596-98-1]. Triglyceride of docosahexaenoic acid
Tribromophenol. C6H3Br3O. (Mr 330.8). 1165300. [118-79-6]. (C22:6). Glycerol tridocosahexaenoate. Propane-1,2,3-triyl
2,4,6-Tribromophenol. tri-(all-Z)-docosa-4,7,10,13,16,19-hexaenoate.
The reagent from Nu-Chek Prep, Inc. has been found suitable.
Tributyl citrate. C18H32O7. (Mr 360.4). 1152800. [77-94-1].
Tributyl 2-hydroxypropane-1,2,3-tricarboxylate. Triethanolamine. 1092900. [102-71-6].
: about 1.043. See Trolamine (1577).
: about 1.445.
Triethylamine. C6H15N. (Mr 101.2). 1093000. [121-44-8].
Tributyl phosphate. C12H27O4P. (Mr 266.3). 1179900. N,N-Diethylethanamine.
[126-73-8]. Tributoxyphosphine oxide. Tributoxyphosphane Colourless liquid, slightly soluble in water at a temperature
oxide. below 18.7 °C, miscible with ethanol (96 per cent).
Colourless liquid, slightly soluble in water, soluble in the usual : about 0.727.
organic solvents.
: about 1.401.
: about 0.976.
bp : about 90 °C.
: about 1.422.
bp : about 289 °C, with decomposition. Triethylamine R1. C6H15N. (Mr 101.2). 1093001.
[121-44-8]. N,N-Diethylethanamine.
Tributylphosphine. C12H27P. (Mr 202.3). 1187100. [998-40-3].
Complies with the requirements prescribed for
Clear, colourless liquid. triethylamine R with the following additional requirements.
bp : about 240 °C. Content : minimum 99.5 per cent, determined by gas
mp : about − 60 °C. chromatography.
Trichlorethylene. 1102100. Water : maximum 0.1 per cent.
See Trichloroethylene R. Use freshly distilled or from a freshly opened container.
Trichloroacetic acid. C2HCl3O2. (Mr 163.4). 1092500. Triethylamine R2. C6H15N. (Mr 101.2). 1093002.
[76-03-9]. [121-44-8]. N,N-Diethylethanamine.
Colourless crystals or a crystalline mass, very deliquescent, Complies with the requirements prescribed for
very soluble in water and in ethanol (96 per cent). triethylamine R and with the following additional
Storage : in an airtight container. requirements.
Content : minimum 99.5 per cent, determined by gas
Trichloroacetic acid solution. 1092501. chromatography.
Dissolve 40.0 g of trichloroacetic acid R in water R and
Water : maximum 0.2 per cent.
dilute to 1000.0 mL with the same solvent. Verify the
concentration by titration with 0.1 M sodium hydroxide It is suitable for gradient elution in liquid chromatography.
and adjust if necessary to 40 ± 1 g/L. Use freshly distilled or from a freshly opened container.
General Notices (1) apply to all monographs and other texts 531
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Triethylenediamine. C6H12N2. (Mr 112.2). 1093100. Absorbance (2.2.25). Not more than 0.07 from 220 nm to
1,4-Diazabicyclo[2.2.2]octane. 360 nm, determined using water R as the compensation
Crystals, very hygroscopic, sublimes readily at room liquid.
temperature, freely soluble in water, in acetone and in Trimethylpentane for chromatography. 1093402.
anhydrous ethanol.
Complies with the requirements prescribed for
bp : about 174 °C. trimethylpentane R with the following additional
mp : about 158 °C. requirement.
Storage : in an airtight container. Residue on evaporation : maximum 2 mg/L.
Triethyl phosphonoformate. C7H15O5P. (Mr 210.2). 1132900. N,O-bis(Trimethylsilyl)acetamide. C8H21NOSi2. (Mr 203.4).
[1474-78-8]. Ethyl (diethoxyphosphoryl)formate. 1093600. [10416-59-8].
Colourless liquid. Colourless liquid.
bp12 mm : about 135 °C. : about 0.83.
Triflumuron. C15H10ClF3N2O3. (Mr 358.7). 1180800. N-Trimethylsilylimidazole. C6H12N2Si. (Mr 140.3). 1100500.
[64628-44-0]. 1-(2-Chlorobenzoyl)-3-(4-triflumoromethoxy- [18156-74-6]. 1-Trimethylsilylimidazole.
phenyl)urea. Colourless, hygroscopic liquid.
White or almost white crystalline powder, practically insoluble : about 0.96.
in water, sparingly soluble in acetone and in methylene
: about 1.48.
chloride.
Storage : in an airtight container.
Trifluoroacetic acid. C2HF3O2. (Mr 114.0). 1093200.
[76-05-1]. N,O-bis(Trimethylsilyl)trifluoroacetamide. C8H18F3NOSi2.
(Mr 257.4). 1133200. [25561-30-2]. BSTFA.
Content : minimum 99 per cent.
Colourless liquid.
Liquid, miscible with acetone and with ethanol (96 per cent).
: about 0.97.
: about 1.53.
: about 1.38.
bp : about 72 °C.
bp12mm : about 40 °C
Use a grade suitable for protein sequencing.
Storage : in an airtight container. Trimethylsulfonium hydroxide. C3H10OS. (Mr 94.2).
1145000. [17287-03-5].
Trifluoroacetic anhydride. C4F6O3. (Mr 210.0). 1093300. : about 0.81.
[407-25-0].
Colourless liquid. Trimethyltin chloride. C3H9ClSn. (Mr 199.3). 1170900.
: about 1.5. [1066-45-1]. Chlorotrimethylstannane.
2,4,6-Trinitrobenzene sulfonic acid. C6H3N3O9S,3H2O.
3-Trifluoromethylaniline. C7H6F3N. (Mr 161.1). 1171900.
(Mr 347.2). 1117500. [2508-19-2].
[98-16-8]. 3-(Trifluoromethyl)aniline. α,α,α-Trifluoro-m-
toluidine. 3-(Trifluoromethyl)benzenamide. White or almost white, crystalline powder, soluble in water.
Colourless liquid. mp : 190 °C to 195 °C.
Density : 1.30 g/cm3 (20 °C). Triolein. C57H104O6. (Mr 885.4). 1168200. [122-32-7].
Propane-1,2,3-triyl tris[(9Z)-octadec-9-enoate]. sn-Glyceryl
4-Trifluoromethylphenol. C7H5F3O. (Mr 162.1). 1161700. trioleate. Glycerol trioleate. Oleyl triglyceride.
[402-45-9].
Content : minimum 99.0 per cent.
White or light yellow, crystalline solid or powder.
mp : about 46 °C. Triphenylmethanol. C19H16O. (Mr 260.3). 1093700.
[76-84-6]. Triphenylcarbinol.
Trigonelline hydrochloride. C7H8ClNO2. (Mr 173.6). Colourless crystals, practically insoluble in water, freely
1117400. [6138-41-6]. 3-Carboxy-1-methylpyridinium soluble in ethanol (96 per cent).
chloride. Nicotinic acid N-methylbetaine hydrochloride.
Crystalline powder, very soluble in water, soluble in ethanol Triphenyltetrazolium chloride. C19H15ClN4. (Mr 334.8).
(96 per cent). 1093800. [298-96-4]. 2,3,5-Triphenyl-2H-tetrazolium
mp : about 258 °C. chloride.
Content : minimum 98.0 per cent of C19H15ClN4.
Trimethylpentane. C8H18. (Mr 114.2). 1093400. [540-84-1]. Pale or dull-yellow powder, soluble in water, in acetone and in
Iso-octane. 2,2,4-Trimethylpentane. ethanol (96 per cent).
Colourless, flammable liquid, practically insoluble in water, mp : about 240 °C, with decomposition.
soluble in anhydrous ethanol.
Assay. Dissolve 1.000 g in a mixture of 5 mL of dilute nitric
: 0.691 to 0.696. acid R and 45 mL of water R. Add 50.0 mL of 0.1 M silver
: 1.391 to 1.393. nitrate and heat to boiling. Allow to cool, add 3 mL of
Distillation range (2.2.11). Not less than 95 per cent distils dibutyl phthalate R, shake vigorously and titrate with 0.1 M
between 98 °C and 100 °C. ammonium thiocyanate, using 2 mL of ferric ammonium
Trimethylpentane used in spectrophotometry complies with the sulfate solution R2 as indicator.
following additional test. 1 mL of 0.1 M silver nitrate is equivalent to 33.48 mg of
Minimum transmittance (2.2.25) using water R as C19H15ClN4.
compensation liquid : 98 per cent from 250 nm to 420 nm. Storage : protected from light.
Trimethylpentane R1. 1093401. Triphenyltetrazolium chloride solution. 1093801.
Complies with the requirements prescribed for A 5 g/L solution in aldehyde-free alcohol R.
trimethylpentane R with the following modification. Storage : protected from light.
Triscyanoethoxypropane. C12H17N3O3. (Mr 251.3). 1093900. Tyrosine. C9H11NO3. (Mr 181.2). 1094800. [60-18-4].
1,2,3-Tris(2-cyanoethoxy)propane. 2-Amino-3-(4-hydroxyphenyl)propionic acid.
Viscous, brown-yellow liquid, soluble in methanol. Used as a White or almost white, crystalline powder, or colourless
stationary phase in gas chromatography. or white or almost white crystals, slightly soluble in water,
: about 1.11. practically insoluble in acetone and in anhydrous ethanol,
Viscosity (2.2.9): about 172 mPa·s. soluble in dilute hydrochloric acid and in solutions of alkali
hydroxides.
1,3,5-Tris[3,5-di(1,1-dimethylethyl)-4-hydroxybenzyl]-
1,3,5-triazine-2,4,6(1H,3H,5H)-trione. C48H69O6N3. Umbelliferone. C9H6O3. (Mr 162.1). 1137500. [93-35-6].
(Mr 784.1). 1094000. [27676-62-6]. 7-Hydroxycoumarin. 7-Hydroxy-2H-1-benzopyran-2-one.
White or almost white, crystalline powder. Needles from water.
mp : 218 °C to 222 °C. mp : 225 °C to 228 °C.
Tris[2,4-di(1,1-dimethylethyl)phenyl] phosphite. Uracil. C4H4N2O2. (Mr 112.1). 1161800. [66-22-8].
C42H63O3P. (Mr 647). 1094100. [31570-04-4]. Content : minimum 95.0 per cent.
White or almost white powder.
Urea. 1095000. [57-13-6].
mp : 182 °C to 186 °C.
See Urea (0743).
Tris(hydroxymethyl)aminomethane. 1094200. [77-86-1].
See Trometamol (1053). Uridine. C9H12N2O6. (Mr 244.2). 1095100. [58-96-8].
1-β-D-Ribofuranosyluracil.
Tris(hydroxymethyl)aminomethane solution. 1094201. White or almost white, crystalline powder, soluble in water.
A solution containing the equivalent of 24.22 g of C4H11NO3 mp : about 165 °C.
in 1000.0 mL.
Ursolic acid. C30H48O3. (Mr 456.7). 1141600. [77-52-1].
Tris(hydroxymethyl)aminomethane solution R1. 3β-Hydroxyurs-12-en-28-oic acid.
1094202.
White or almost white powder, practically insoluble in water,
Dissolve 60.6 mg of tris(hydroxymethyl)aminomethane R sparingly soluble in methanol, slightly soluble in ethanol
and 0.234 g of sodium chloride R in water R and dilute to (96 per cent).
100 mL with the same solvent.
Storage : at 2 °C to 8 °C ; use within 3 days. : about 67.50, determined on a 10 g/L solution in a
56.1 g/L solution of potassium hydroxide R in ethanol (96 per
Tripotassium phosphate trihydrate. K3PO4,3H2O. (Mr 266.3). cent) R.
1155300. [22763-03-7]. mp : 285 °C to 288 °C.
White or almost white crystalline powder, freely soluble in
water. Valencene. C15H24. (Mr 204.4). 1152100. [4630-07-3].
4βH,5α-Eremophila-1(10),11-diene. (1R,7R,8aS)-
Trisodium phosphate dodecahydrate. Na3PO4,12H2O. 1,8a-Dimethyl-7-(1-methylethenyl)-1,2,3,5,6,7,8,8a-
(Mr 380.1). 1094300. [10101-89-0]. octahydronaphthalene.
Colourless or white or almost white crystals, freely soluble in Oily, colourless or pale yellow liquid, with a characteristic
water. odour, practically insoluble in water, soluble in ethanol (96 per
cent).
Tropic acid. C9H10O3. (Mr 166.17). 1172000. [529-64-6].
(2RS)-3-Hydroxy-2-phenylpropanoic acid. : about 0.918.
: about 1.508.
Troxerutin. C33H42O19. (Mr 743). 1160300. [7085-55-4].
Trihydroxyethylrutin. 3′,4′,7-Tris[O-(2-hydroxyethyl)]rutin. bp : about 123 °C.
2-[3,4-Bis(2-hydroxyethoxy)phenyl]-3-[[6-O-(6-deoxy-α-L- Valencene used in gas chromatography complies with the
mannopyranosyl)-β-D-glucopyranosyl]oxy]-5-hydroxy-7-(2- following additional test.
hydroxyethoxy)-4H-1-benzopyran-4-one. Assay. Gas chromatography (2.2.28) as prescribed in the
mp : 168 °C to 176 °C. monograph Sweet orange oil (1811).
Trypsin. 1094500. [9002-07-7]. Content : minimum 80 per cent, calculated by the
normalisation procedure.
A proteolytic enzyme obtained by activation of trypsinogen
extracted from the pancreas of beef (Bos taurus L.). Valerenic acid. C15H22O2. (Mr 234.3). 1165700. [3569-10-6].
White or almost white, crystalline or amorphous powder, (2E)-3-[(4S,7R,7aR)-3,7-Dimethyl-2,4,5,6,7,7a-hexahydro-1H-
sparingly soluble in water. inden-4-yl]-2-methylprop-2-enoic acid.
Trypsin for peptide mapping. 1094600. [9002-07-7]. mp : 134 °C to 138 °C.
Trypsin of high purity treated to eliminate chymotryptic Valeric acid. C5H10O2. (Mr 102.1). 1095200. [109-52-4].
activity. Pentanoic acid.
Tryptophan. C11H12N2O2. (Mr 204.2). 1094700. [73-22-3]. Colourless liquid, soluble in water, freely soluble in ethanol
(96 per cent).
White or yellowish-white, crystalline powder or colourless
crystals, slightly soluble in water, very slightly soluble in : about 0.94.
ethanol (96 per cent). : about 1.409.
: about − 30, determined on a 10 g/L solution. bp : about 186 °C.
Tyramine. C8H11NO. (Mr 137.2). 1117600. [51-67-2]. Valine. 1185300. [72-18-4].
4-(2-Aminoethyl)phenol. See Valine (0796).
Crystals, sparingly soluble in water, soluble in boiling
anhydrous ethanol. Vanillin. 1095300. [121-33-5].
mp : 164 °C to 165 °C. See Vanillin (0747).
General Notices (1) apply to all monographs and other texts 533
4.1.1. Reagents EUROPEAN PHARMACOPOEIA 8.0
Vanillin reagent. 1095301. Assay. Gas chromatography (2.2.28) : use the normalisation
Carefully add, dropwise, 2 mL of sulfuric acid R to 100 mL procedure.
of a 10 g/L solution of vanillin R in ethanol (96 per cent) R. Column :
Storage : use within 48 h. – material : fused-silica ;
– size : l = 30 m, Ø = 0.5 mm ;
Vanillin solution, phosphoric. 1095302.
– stationary phase : macrogol 20 000 R.
Dissolve 1.0 g of vanillin R in 25 mL of ethanol (96 per
cent) R. Add 25 mL of water R and 35 mL of phosphoric Carrier gas : helium for chromatography R.
acid R. Temperature :
Veratrole. C8H10O2. (Mr 138.2). 1165400. [91-16-7]. Time Temperature
1,2-Dimethoxybenzene. (min) (°C)
Column 0-1 80
: 1.085.
: 1.534. 1 - 12 80 → 190
General Notices (1) apply to all monographs and other texts 535
4.1.2. Standard solutions for limit tests EUROPEAN PHARMACOPOEIA 8.0
Zinc chloride solution, iodinated. 1096602. Aluminium standard solution (200 ppm Al). 5000200.
Dissolve 20 g of zinc chloride R and 6.5 g of potassium Dissolve in water R a quantity of aluminium potassium
iodide R in 10.5 mL of water R. Add 0.5 g of iodine R and sulfate R equivalent to 0.352 g of AlK(SO4)2,12H2O. Add 10 mL
shake for 15 min. Filter if necessary. of dilute sulfuric acid R and dilute to 100.0 mL with water R.
Storage : protected from light.
Aluminium standard solution (100 ppm Al). 5000203.
Zinc iodide and starch solution. 1096502. Immediately before use, dilute with water R to 10 times its
To a solution of 2 g of zinc chloride R in 10 mL of water R volume a solution containing 8.947 g of aluminium chloride R
add 0.4 g of soluble starch R and heat until the starch has in 1000.0 mL of water R.
dissolved. After cooling to room temperature add 1.0 mL of
a colourless solution containing 0.10 g zinc R as filings and Aluminium standard solution (10 ppm Al). 5000201.
0.2 g of iodine R in water R. Dilute the solution to 100 mL Immediately before use, dilute with water R to 100 times
with water R and filter. its volume in a solution containing aluminium nitrate R
Storage : protected from light. equivalent to 1.39 g of Al(NO3)3,9H2O in 100.0 mL.
Test for sensitivity. Dilute 0.05 mL of sodium nitrite solution R Aluminium standard solution (2 ppm Al). 5000202.
to 50 mL with water R. To 5 mL of this solution add 0.1 mL of
dilute sulfuric acid R and 0.05 mL of the zinc iodide and starch Immediately before use, dilute with water R to 100 times its
solution and mix. The solution becomes blue. volume a solution containing aluminium potassium sulfate R
equivalent to 0.352 g of AlK(SO4)2,12H2O and 10 mL of dilute
Zinc oxide. 1096700. [1314-13-2]. sulfuric acid R in 100.0 mL.
See Zinc oxide (0252). Ammonium standard solution (100 ppm NH4). 5000300.
Zinc powder. Zn. (Ar 65.4). 1096800. [7440-66-6]. Immediately before use, dilute to 25 mL with water R 10 mL
Content : minimum 90.0 per cent. of a solution containing ammonium chloride R equivalent to
Very fine, grey powder, soluble in dilute hydrochloric acid R. 0.741 g of NH4Cl in 1000 mL.
Zinc sulfate. 1097000. [7446-20-0]. Ammonium standard solution (3 ppm NH4). 5006100.
See Zinc sulfate (0111). Immediately before use, dilute with water R to 100 times its
volume a solution containing ammonium chloride R equivalent
Zirconyl chloride. A basic salt corresponding approximately to 0.889 g of NH4Cl in 1000.0 mL.
to the formula ZrCl2O, 8H2O. 1097100. [15461-27-5].
Content : minimum 96.0 per cent of ZrCl2O,8H2O. Ammonium standard solution (2.5 ppm NH4). 5000301.
White or almost white, crystalline powder or crystals, freely Immediately before use, dilute with water R to 100 times its
soluble in water and in ethanol (96 per cent). volume a solution containing ammonium chloride R equivalent
to 0.741 g of NH4Cl in 1000.0 mL.
Assay. Dissolve 0.600 g in a mixture of 5 mL of nitric acid R
and 50 mL of water R. Add 50.0 mL of 0.1 M silver nitrate Ammonium standard solution (1 ppm NH4). 5000302.
and 3 mL of dibutyl phthalate R and shake. Using 2 mL of Immediately before use, dilute ammonium standard solution
ferric ammonium sulfate solution R2 as indicator, titrate with (2.5 ppm NH4) R to 2.5 times its volume with water R.
0.1 M ammonium thiocyanate until a reddish-yellow colour is
obtained. Antimony standard solution (100 ppm Sb). 5000401.
1 mL of 0.1 M silver nitrate is equivalent to 16.11 mg of Dissolve antimony potassium tartrate R equivalent to 0.274 g
ZrCl2O,8H2O. of C4H4KO7 Sb,½H2O in 500 mL of 1 M hydrochloric acid and
Zirconyl nitrate. A basic salt corresponding approximately to dilute the clear solution to 1000 mL with water R.
the formula ZrO(NO3)2,2H2O. 1097200. [14985-18-3]. Antimony standard solution (1 ppm Sb). 5000400.
A white or almost white powder or crystals, hygroscopic, Dissolve antimony potassium tartrate R equivalent to 0.274 g
soluble in water. The aqueous solution is a clear or at most of C4H4KO7Sb,1/2H2O in 20 mL of hydrochloric acid R1 and
slightly opalescent liquid. dilute the clear solution to 100.0 mL with water R. To 10.0 mL
Storage : in an airtight container. of this solution add 200 mL of hydrochloric acid R1 and dilute
to 1000.0 mL with water R. To 100.0 mL of this solution add
Zirconyl nitrate solution. 1097201.
300 mL of hydrochloric acid R1 and dilute to 1000.0 mL with
A 1 g/L solution in a mixture of 40 mL of water R and water R. Prepare the dilute solutions immediately before use.
60 mL of hydrochloric acid R.
Arsenic standard solution (10 ppm As). 5000500.
Immediately before use, dilute with water R to 100 times its
04/2010:40102 volume a solution prepared by dissolving arsenious trioxide R
equivalent to 0.330 g of As2O3 in 5 mL of dilute sodium
4.1.2. STANDARD SOLUTIONS FOR hydroxide solution R and diluting to 250.0 mL with water R.
LIMIT TESTS Arsenic standard solution (1 ppm As). 5000501.
Acetaldehyde standard solution (100 ppm C2H4O). 5000100. Immediately before use, dilute arsenic standard solution
Dissolve 1.0 g of acetaldehyde R in 2-propanol R and dilute to (10 ppm As) R to 10 times its volume with water R.
100.0 mL with the same solvent. Dilute 5.0 mL of the solution
to 500.0 mL with 2-propanol R. Prepare immediately before Arsenic standard solution (0.1 ppm As). 5000502.
use. Immediately before use, dilute arsenic standard solution
(1 ppm As) R to 10 times its volume with water R.
Acetaldehyde standard solution (100 ppm C2H4O) R1.
5000101. Barium standard solution (0.1 per cent Ba). 5000601.
Dissolve 1.0 g of acetaldehyde R in water R and dilute to Dissolve barium chloride R equivalent to 0.178 g of BaCl2,2H2O
100.0 mL with the same solvent. Dilute 5.0 mL of the solution in distilled water R and dilute to 100.0 mL with the same
to 500.0 mL with water R. Prepare immediately before use. solvent.
Barium standard solution (50 ppm Ba). 5000600. Chromium liposoluble standard solution (1000 ppm Cr).
Immediately before use, dilute with distilled water R to 5004600.
20 times its volume a solution in distilled water R containing A chromium (metal) organic compound in an oil.
barium chloride R equivalent to 0.178 g of BaCl2,2H2O in
100.0 mL. Chromium standard solution (0.1 per cent Cr). 5001002.
Dissolve potassium dichromate R equivalent to 2.83 g of
Barium standard solution (2 ppm Ba). 5005600. K2Cr2O7 in water R and dilute to 1000.0 mL with the same
Immediately before use, dilute barium standard solution solvent.
(50 ppm Ba) R to 25 times its volume with distilled water R.
Chromium standard solution (100 ppm Cr). 5001000.
Bismuth standard solution (100 ppm Bi). 5005300. Dissolve potassium dichromate R equivalent to 0.283 g of
Dissolve bismuth R equivalent to 0.500 g of Bi in 50 mL of K2Cr2O7 in water R and dilute to 1000.0 mL with the same
nitric acid R and dilute to 500.0 mL with water R. Dilute solvent.
the solution to 10 times its volume with dilute nitric acid R
immediately before use. Chromium standard solution (0.1 ppm Cr). 5001001.
Immediately before use, dilute chromium standard solution
Cadmium standard solution (0.1 per cent Cd). 5000700.
(100 ppm Cr) R to 1000 times its volume with water R.
Dissolve cadmium R equivalent to 0.100 g of Cd in the
smallest necessary amount of a mixture of equal volumes of Cobalt standard solution (100 ppm Co). 5004300.
hydrochloric acid R and water R and dilute to 100.0 mL with a Dissolve cobalt nitrate R equivalent to 0.494 g of
1 per cent V/V solution of hydrochloric acid R. Co(NO3)2,6H2O in 500 mL of 1 M nitric acid and dilute the
Cadmium standard solution (10 ppm Cd) . 5000701. clear solution to 1000 mL with water R.
Immediately before use, dilute cadmium standard solution Copper liposoluble standard solution (1000 ppm Cu).
(0.1 per cent Cd) R to 100 times its volume with a 1 per 5004700.
cent V/V solution of hydrochloric acid R. A copper (metal) organic compound in an oil.
Calcium standard solution (400 ppm Ca). 5000800. Copper standard solution (0.1 per cent Cu). 5001100.
Immediately before use, dilute with distilled water R to Dissolve copper sulfate R equivalent to 0.393 g of CuSO4,5H2O
10 times its volume a solution in distilled water R containing in water R and dilute to 100.0 mL with the same solvent.
calcium carbonate R equivalent to 1.000 g of CaCO3 and 23 mL
of 1 M hydrochloric acid in 100.0 mL. Copper standard solution (10 ppm Cu). 5001101.
Calcium standard solution (100 ppm Ca). 5000801. Immediately before use, dilute copper standard solution
(0.1 per cent Cu) R to 100 times its volume with water R.
Immediately before use, dilute with distilled water R to
10 times its volume a solution in distilled water R containing Copper standard solution (0.1 ppm Cu). 5001102.
calcium carbonate R equivalent to 0.624 g of CaCO3 and 3 mL
of acetic acid R in 250.0 mL. Immediately before use, dilute copper standard solution
(10 ppm Cu) R to 100 times its volume with water R.
Calcium standard solution (100 ppm Ca) R1. 5000804.
Ferrocyanide standard solution (100 ppm Fe(CN)6).
Immediately before use, dilute with water R to 10 times its 5001200.
volume a solution containing anhydrous calcium chloride R
equivalent to 2.769 g of CaCl2 in 1000.0 mL of dilute Immediately before use, dilute with water R to 10 times
hydrochloric acid R. its volume a solution containing potassium ferrocyanide R
equivalent to 0.20 g of K4Fe(CN)6,3H2O in 100.0 mL.
Calcium standard solution (100 ppm Ca), alcoholic.
5000802. Ferricyanide standard solution (50 ppm Fe(CN)6). 5001300.
Immediately before use, dilute with ethanol (96 per cent) R to Immediately before use, dilute with water R to 100 times
10 times its volume a solution in distilled water R containing its volume a solution containing potassium ferricyanide R
calcium carbonate R equivalent to 2.50 g of CaCO3 and 12 mL equivalent to 0.78 g of K3Fe(CN)6 in 100.0 mL.
of acetic acid R in 1000.0 mL.
Fluoride standard solution (10 ppm F). 5001400.
Calcium standard solution (10 ppm Ca). 5000803. Dissolve in water R sodium fluoride R previously dried at
Immediately before use, dilute with distilled water R to 300 °C for 12 h, equivalent to 0.442 g of NaF, and dilute to
100 times its volume a solution in distilled water R containing 1000.0 mL with the same solvent (1 mL = 0.2 mg F). Store in
calcium carbonate R equivalent to 0.624 g of CaCO3 and 3 mL a polyethylene container. Immediately before use, dilute the
of acetic acid R in 250.0 mL. solution to 20 times its volume with water R.
Chloride standard solution (50 ppm Cl). 5004100. Fluoride standard solution (1 ppm F). 5001401.
Immediately before use, dilute with water R to 10 times its Immediately before use, dilute fluoride standard solution
volume a solution containing sodium chloride R equivalent to (10 ppm F) R to 10 times its volume with water R.
0.824 g of NaCl in 1000.0 mL.
Formaldehyde standard solution (5 ppm CH2O). 5001500.
Chloride standard solution (8 ppm Cl). 5000900. Immediately before use, dilute with water R to 200 times its
Immediately before use, dilute with water R to 100 times its volume a solution containing 1.0 g of CH2O per litre prepared
volume a solution containing sodium chloride R equivalent to from formaldehyde solution R.
1.32 g of NaCl in 1000.0 mL.
Germanium standard solution (100 ppm Ge). 5004400.
Chloride standard solution (5 ppm Cl). 5000901. Dissolve ammonium hexafluorogermanate(IV) R equivalent
Immediately before use, dilute with water R to 100 times its to 0.307 g of (NH4)2GeF6 in a 0.01 per cent V/V solution of
volume a solution containing sodium chloride R equivalent to hydrofluoric acid R. Dilute the clear solution to 1000 mL with
0.824 g of NaCl in 1000.0 mL. water R.
General Notices (1) apply to all monographs and other texts 537
4.1.2. Standard solutions for limit tests EUROPEAN PHARMACOPOEIA 8.0
Glyoxal standard solution (20 ppm C2H2O2). 5003700. Lead standard solution (10 ppm Pb). 5001702.
In a 100 mL graduated flask weigh a quantity of glyoxal Immediately before use, dilute lead standard solution (100 ppm
solution R corresponding to 0.200 g of C2H2O2 and make up Pb) R to 10 times its volume with water R.
to volume with anhydrous ethanol R. Immediately before use
dilute the solution to 100 times its volume with the same Lead standard solution (10 ppm Pb) R1. 5001706.
solvent. Immediately before use, dilute with water R to 10 times its
volume a solution containing 0.160 g of lead nitrate R in
Glyoxal standard solution (2 ppm C2H2O2). 5003701. 100 mL of water R, to which is added 1 mL of lead-free nitric
Immediately before use, dilute glyoxal standard solution acid R and dilute to 1000.0 mL.
(20 ppm C2H2O2) R to 10 times its volume with anhydrous
ethanol R. Lead standard solution (10 ppm Pb) R2. 5005401.
Dilute lead standard solution (0.1 per cent Pb) R1 to 100 times
Hydrogen peroxide standard solution (10 ppm H2O2). its volume with dilute lead-free nitric acid R. Use within
5005200. 1 week.
Dilute 10.0 mL of dilute hydrogen peroxide solution R to
300.0 mL with water R. Dilute 10.0 mL of this solution to Lead standard solution (2 ppm Pb). 5001703.
1000.0 mL with water R. Prepare immediately before use. Immediately before use, dilute lead standard solution (10 ppm
Pb) R to 5 times its volume with water R.
Iodide standard solution (10 ppm I). 5003800.
Immediately before use, dilute with water R to 100 times its Lead standard solution (1 ppm Pb). 5001704.
volume a solution containing potassium iodide R equivalent Immediately before use, dilute lead standard solution (10 ppm
to 0.131 g of KI in 100.0 mL. Pb) R to 10 times its volume with water R.
Iron standard solution (0.1 per cent Fe). 5001605. Lead standard solution (0.5 ppm Pb). 5005402.
Dissolve 0.100 g of Fe in the smallest amount necessary of a Dilute lead standard solution (10 ppm Pb) R2 to 20 times its
mixture of equal volumes of hydrochloric acid R and water R volume with dilute lead-free nitric acid R. Use within 1 day.
and dilute to 100.0 mL with water R.
Lead standard solution (0.25 ppm Pb). 5006000.
Iron standard solution (250 ppm Fe). 5001606. Immediately before use, dilute lead standard solution (1 ppm
Immediately before use, dilute with water R to 40 times its Pb) R to 4 times its volume with water R.
volume a solution containing 4.840 g of ferric chloride R in a
Lead standard solution (0.1 ppm Pb). 5001705.
150 g/L solution of hydrochloric acid R diluted to 100.0 mL.
Immediately before use, dilute lead standard solution (1 ppm
Iron standard solution (20 ppm Fe). 5001600. Pb) R to 10 times its volume with water R.
Immediately before use, dilute with water R to 10 times its
Magnesium standard solution (0.1 per cent Mg). 5001803.
volume a solution containing ferric ammonium sulfate R
equivalent to 0.863 g of FeNH4(SO4)2,12H2O and 25 mL of Dissolve magnesium sulfate R equivalent to 1.010 g of
dilute sulfuric acid R in 500.0 mL. MgSO4,7H2O in distilled water R and dilute to 100.0 mL with
the same solvent.
Iron standard solution (10 ppm Fe). 5001601.
Magnesium standard solution (1000 ppm Mg). 5006200.
Immediately before use, dilute with water R to 100 times its
volume a solution containing ferrous ammonium sulfate R Dissolve 5.275 g of magnesium nitrate R in 16 mL of dilute
equivalent to 7.022 g of Fe(NH4)2(SO4)2,6H2O and 25 mL of nitric acid R and dilute to 500.0 mL with water R.
dilute sulfuric acid R in 1000.0 mL. Standardisation : carry out the determination of magnesium
by complexometry (2.5.11).
Iron standard solution (8 ppm Fe). 5001602.
Immediately before use, dilute with water R to 10 times its Magnesium standard solution (100 ppm Mg). 5001800.
volume a solution containing 80 mg of iron R and 50 mL of Immediately before use, dilute with water R to 10 times its
hydrochloric acid R (220 g/L HCl) in 1000.0 mL. volume a solution containing magnesium sulfate R equivalent
to 1.010 g of MgSO4,7H2O in 100.0 mL.
Iron standard solution (2 ppm Fe). 5001603.
Immediately before use, dilute iron standard solution (20 ppm Magnesium standard solution (10 ppm Mg). 5001801.
Fe) R to 10 times its volume with water R. Immediately before use, dilute magnesium standard solution
(100 ppm Mg) R to 10 times its volume with water R.
Iron standard solution (1 ppm Fe). 5001604.
Immediately before use, dilute iron standard solution (20 ppm Magnesium standard solution (10 ppm Mg) R1. 5001802.
Fe) R to 20 times its volume with water R. Immediately before use, dilute with water R to 100 times its
volume a solution containing 8.365 g of magnesium chloride R
Lead liposoluble standard solution (1000 ppm Pb). in 1000.0 mL of dilute hydrochloric acid R.
5004800.
A lead (metal) organic compound in an oil. Manganese standard solution (1000 ppm Mn). 5005800.
Dissolve manganese sulfate R equivalent to 3.08 g of
Lead standard solution (0.1 per cent Pb). 5001700. MnSO4,H2O in 500 mL of 1 M nitric acid and dilute the
Dissolve lead nitrate R equivalent to 0.400 g of Pb(NO3)2 in solution to 1000 mL with water R.
water R and dilute to 250.0 mL with the same solvent.
Manganese standard solution (100 ppm Mn). 5004500.
Lead standard solution (0.1 per cent Pb) R1. 5005400. Dissolve manganese sulfate R equivalent to 0.308 g of
Dissolve in dilute lead-free nitric acid R a quantity of lead MnSO4,H2O in 500 mL of 1 M nitric acid and dilute the clear
nitrate R equivalent to 0.400 g of Pb (NO3)2 and dilute to solution to 1000 mL with water R.
250.0 mL with the same solvent.
Mercury standard solution (1000 ppm Hg). 5001900.
Lead standard solution (100 ppm Pb). 5001701. Dissolve mercuric chloride R equivalent to 1.354 g of HgCl2
Immediately before use, dilute lead standard solution (0.1 per in 50 mL of dilute nitric acid R and dilute to 1000.0 mL with
cent Pb) R to 10 times its volume with water R. water R.
Mercury standard solution (10 ppm Hg). 5001901. Potassium standard solution (600 ppm K). 5005100.
Immediately before use, dilute with water to 100 times its Immediately before use, dilute with water R to 20 times its
volume a solution containing mercuric chloride R equivalent volume a solution containing dipotassium sulfate R equivalent
to 0.338 g of HgCl2 in 250.0 mL. to 2.676 g of K2SO4 in 100.0 mL.
Nickel liposoluble standard solution (1000 ppm Ni). Potassium standard solution (100 ppm K). 5002400.
5004900. Immediately before use, dilute with water R to 20 times its
A nickel (metal) organic compound in an oil. volume a solution containing dipotassium sulfate R equivalent
to 0.446 g of K2SO4 in 100.0 mL.
Nickel standard solution (10 ppm Ni). 5002000.
Potassium standard solution (20 ppm K). 5002401.
Immediately before use, dilute with water R to 100 times its
volume a solution containing nickel sulfate R equivalent to Immediately before use, dilute potassium standard solution
4.78 g of NiSO4,7H2O in 1000.0 mL. (100 ppm K) R to 5 times its volume with water R.
Nickel standard solution (5 ppm Ni). 5005900. Selenium standard solution (100 ppm Se). 5002500.
Dissolve 0.100 g of selenium R in 2 mL of nitric acid R.
Immediately before use dilute nickel standard solution (10 ppm
Evaporate to dryness. Take up the residue in 2 mL of water R
Ni) R to twice its volume with water for chromatography R.
and evaporate to dryness ; carry out three times. Dissolve the
Nickel standard solution (0.2 ppm Ni). 5002002. residue in 50 mL of dilute hydrochloric acid R and dilute to
1000.0 mL with the same acid.
Immediately before use, dilute nickel standard solution
(10 ppm Ni) R to 50 times its volume with water R. Selenium standard solution (1 ppm Se). 5002501.
Nickel standard solution (0.1 ppm Ni). 5002001. Immediately before use, dilute with water R to 40 times its
volume a solution containing selenious acid R equivalent to
Immediately before use, dilute nickel standard solution 6.54 mg of H2SeO3 in 100.0 mL.
(10 ppm Ni) R to 100 times its volume with water R.
Silver standard solution (5 ppm Ag). 5002600.
Nitrate standard solution (100 ppm NO3). 5002100. Immediately before use, dilute with water R to 100 times its
Immediately before use, dilute with water R to 10 times its volume a solution containing silver nitrate R equivalent to
volume a solution containing potassium nitrate R equivalent 0.790 g of AgNO3 in 1000.0 mL.
to 0.815 g of KNO3 in 500.0 mL.
Sodium standard solution (1000 ppm Na). 5005700.
Nitrate standard solution (10 ppm NO3). 5002101. Dissolve a quantity of anhydrous sodium carbonate R
Immediately before use, dilute nitrate standard solution equivalent to 2.305 g of Na2CO3 in a mixture of 25 mL of
(100 ppm NO3) R to 10 times its volume with water R. water R and 25 mL of nitric acid R and dilute to 1000.0 mL
with water R.
Nitrate standard solution (2 ppm NO3). 5002102.
Immediately before use, dilute nitrate standard solution Sodium standard solution (200 ppm Na). 5002700.
(10 ppm NO3) R to 5 times its volume with water R. Immediately before use, dilute with water R to 10 times its
volume a solution containing sodium chloride R equivalent to
Palladium standard solution (500 ppm Pd). 5003600. 0.509 g of NaCl in 100.0 mL.
Dissolve 50.0 mg of palladium R in 9 mL of hydrochloric
acid R and dilute to 100.0 mL with water R. Sodium standard solution (50 ppm Na). 5002701.
Dilute the sodium standard solution (200 ppm Na) R to four
Palladium standard solution (20 ppm Pd). 5003602. times its volume with water R.
Dissolve 0.333 g of palladium chloride R in 2 mL of warm Strontium standard solution (1.0 per cent Sr). 5003900.
hydrochloric acid R. Dilute the solution to 1000.0 mL with a
mixture of equal volumes of dilute hydrochloric acid R and Cover with water R, strontium carbonate R equivalent to
water R. Immediately before use dilute to 10 times its volume 1.6849 g of SrCO3. Cautiously add hydrochloric acid R until
with water R. all the solid has dissolved and there is no sign of further
effervescence. Dilute to 100.0 mL with water R.
Palladium standard solution (0.5 ppm Pd). 5003601.
Sulfate standard solution (100 ppm SO4). 5002802.
Dilute 1 mL of palladium standard solution (500 ppm Pd) R Immediately before use, dilute with distilled water R to
to 1000 mL with a mixture of 0.3 volumes of nitric acid R and 10 times its volume a solution in distilled water R containing
99.7 volumes of water R. dipotassium sulfate R equivalent to 0.181 g of K SO in
2 4
Phosphate standard solution (200 ppm PO4). 5004200. 100.0 mL.
Dissolve potassium dihydrogen phosphate R equivalent to Sulfate standard solution (10 ppm SO4). 5002800.
0.286 g of KH2PO4 in water R and dilute to 1000.0 mL with Immediately before use, dilute with distilled water R to
the same solvent. 100 times its volume a solution in distilled water R containing
Phosphate standard solution (5 ppm PO4). 5002200. dipotassium sulfate R equivalent to 0.181 g of K2SO4 in
100.0 mL.
Immediately before use, dilute with water R to 100 times
its volume a solution containing potassium dihydrogen Sulfate standard solution (10 ppm SO4) R1. 5002801.
phosphate R equivalent to 0.716 g of KH2PO4 in 1000.0 mL. Immediately before use, dilute with ethanol (30 per cent V/V) R
to 100 times its volume a solution containing dipotassium
Platinum standard solution (30 ppm Pt). 5002300. sulfate R equivalent to 0.181 g of K2SO4 in 100.0 mL of ethanol
Immediately before use, dilute with 1 M hydrochloric acid (30 per cent V/V) R.
to 10 times its volume a solution containing 80 mg of
chloroplatinic acid R in 100.0 mL of 1 M hydrochloric acid. Sulfite standard solution (80 ppm SO2). 5005500.
Dissolve 3.150 g of anhydrous sodium sulfite R in freshly
Potassium standard solution (0.2 per cent K). 5002402. prepared distilled water R and dilute to 100.0 mL with the
Dissolve dipotassium sulfate R equivalent to 0.446 g of K2SO4 in same solvent. Dilute 0.5 mL to 100.0 mL with freshly prepared
distilled water R and dilute to 100.0 mL with the same solvent. distilled water R.
General Notices (1) apply to all monographs and other texts 539
4.1.3. Buffer solutions EUROPEAN PHARMACOPOEIA 8.0
Sulfite standard solution (1.5 ppm SO2). 5002900. Phosphate buffer solution pH 2.0. 4007900.
Dissolve sodium metabisulfite R equivalent to 0.152 g of Dissolve 8.95 g of disodium hydrogen phosphate R and 3.40 g
Na2S2O5 in water R and dilute to 100.0 mL with the same of potassium dihydrogen phosphate R in water R and dilute to
solvent. Dilute 5.0 mL of this solution to 100.0 mL with 1000.0 mL with the same solvent. If necessary adjust the pH
water R. To 3.0 mL of the resulting solution, add 4.0 mL of with phosphoric acid R.
0.1 M sodium hydroxide and dilute to 100.0 mL with water R.
Sulfate buffer solution pH 2.0. 4008900.
Thallium standard solution (10 ppm Tl). 5003000. Dissolve 132.1 g of ammonium sulfate R in water R and dilute
Dissolve thallous sulfate R equivalent to 0.1235 g of Tl2SO4 in to 500.0 mL with the same solvent (Solution A). Carefully and
a 9 g/L solution of sodium chloride R and dilute to 1000.0 mL with constant cooling stir 14 mL of sulfuric acid R into about
with the same solution. Dilute 10.0 mL of the solution to 400 mL of water R ; allow to cool and dilute to 500.0 mL with
100.0 mL with the 9 g/L solution of sodium chloride R. water R (Solution B). Mix equal volumes of solutions A and B.
Adjust the pH if necessary.
Tin liposoluble standard solution (1000 ppm Sn). 5005000.
A tin (metal) organic compound in an oil. Buffer solution pH 2.2. 4010500.
Mix 6.7 mL of phosphoric acid R with 55.0 mL of a 40 g/L
Tin standard solution (5 ppm Sn). 5003100. solution of sodium hydroxide R and dilute to 1000.0 mL with
Dissolve tin R equivalent to 0.500 g of Sn in a mixture of 5 mL water R.
of water R and 25 mL of hydrochloric acid R and dilute to
1000.0 mL with water R. Dilute the solution to 100 times its Buffer solution pH 2.5. 4000300.
volume with a 2.5 per cent V/V solution of hydrochloric acid R Dissolve 100 g of potassium dihydrogen phosphate R in 800 mL
immediately before use. of water R ; adjust to pH 2.5 with hydrochloric acid R and
dilute to 1000.0 mL with water R.
Tin standard solution (0.1 ppm Sn). 5003101.
Immediately before use, dilute tin standard solution (5 ppm Buffer solution pH 2.5 R1. 4000400.
Sn) R to 50 times its volume with water R. To 4.9 g of dilute phosphoric acid R add 250 mL of water R.
Adjust the pH with dilute sodium hydroxide solution R and
Titanium standard solution (100 ppm Ti). 5003200. dilute to 500.0 mL with water R.
Dissolve 100.0 mg of titanium R in 100 mL of hydrochloric
acid R diluted to 150 mL with water R, heating if necessary. 0.2 M Phosphate buffer solution pH 2.5. 4014100.
Allow to cool and dilute to 1000 mL with water R. Dissolve 27.2 g of potassium dihydrogen phosphate R in about
900 mL of water R, adjust to pH 2.5 with phosphoric acid R
Vanadium standard solution (1 g/L V). 5003300. and dilute to 1.0 L with water R.
Dissolve in water R ammonium vanadate R equivalent to
0.230 g of NH4VO3 and dilute to 100.0 mL with the same Phosphate buffer solution pH 2.8. 4010600.
solvent. Dissolve 7.8 g of sodium dihydrogen phosphate R in 900 mL of
water R, adjust to pH 2.8 with phosphoric acid R and dilute to
Zinc standard solution (5 mg/mL Zn). 5003400. 1000 mL with the same solvent.
Dissolve 3.15 g of zinc oxide R in 15 mL of hydrochloric acid R
and dilute to 500.0 mL with water R. Buffer solution pH 3.0. 4008000.
Dissolve 21.0 g of citric acid R in 200 mL of 1 M sodium
Zinc standard solution (100 ppm Zn). 5003401. hydroxide and dilute to 1000 mL with water R. Dilute 40.3 mL
Immediately before use, dilute with water R to 10 times its of this solution to 100.0 mL with 0.1 M hydrochloric acid.
volume a solution containing zinc sulfate R equivalent to
0.440 g of ZnSO4,7H2O and 1 mL of acetic acid R in 100.0 mL. 0.25 M Citrate buffer solution pH 3.0. 4012600.
Dissolve 5.3 g of citric acid R in 80 mL of water R. Adjust the
Zinc standard solution (10 ppm Zn). 5003402. pH with 1 M sodium hydroxide and dilute to 100.0 mL with
Immediately before use, dilute zinc standard solution (100 ppm water R.
Zn) R to 10 times its volume with water R.
0.1 M Phosphate buffer solution pH 3.0. 4011500.
Zinc standard solution (5 ppm Zn). 5003403. Dissolve 12.0 g of anhydrous sodium dihydrogen phosphate R
Immediately before use, dilute zinc standard solution (100 ppm in water R, adjust the pH with dilute phosphoric acid R1 and
Zn) R to 20 times its volume with water R. dilute to 1000 mL with water R.
Zirconium standard solution (1 g/L Zr). 5003500. Phosphate buffer solution pH 3.0. 4000500.
Dissolve zirconyl nitrate R equivalent to 0.293 g of Mix 0.7 mL of phosphoric acid R with 100 mL of water R.
ZrO(NO3)2,2H2O in a mixture of 2 volumes of hydrochloric Dilute to 900 mL with the same solvent. Adjust to pH 3.0 with
acid R and 8 volumes of water R and dilute to 100.0 mL with strong sodium hydroxide solution R and dilute to 1000 mL
the same mixture of solvents. with water R.
Phosphate buffer solution pH 3.0 R1. 4010000.
01/2014:40103 Dissolve 3.40 g of potassium dihydrogen phosphate R in
900 mL of water R. Adjust to pH 3.0 with phosphoric acid R
4.1.3. BUFFER SOLUTIONS and dilute to 1000.0 mL with water R.
Buffered acetone solution. 4000100. Phosphate buffer solution pH 3.2. 4008100.
Dissolve 8.15 g of sodium acetate R and 42 g of sodium To 900 mL of a 4 g/L solution of sodium dihydrogen
chloride R in water R, add 68 mL of 0.1 M hydrochloric acid phosphate R, add 100 mL of a 2.5 g/L solution of phosphoric
and 150 mL of acetone R and dilute to 500 mL with water R. acid R. Adjust the pH if necessary.
Buffer solution pH 2.0. 4000200. Phosphate buffer solution pH 3.2 R1. 4008500.
Dissolve 6.57 g of potassium chloride R in water R and add Adjust a 35.8 g/L solution of disodium hydrogen phosphate R
119.0 mL of 0.1 M hydrochloric acid. Dilute to 1000.0 mL to pH 3.2 with dilute phosphoric acid R. Dilute 100.0 mL of
with water R. the solution to 2000.0 mL with water R.
Buffer solution pH 3.5. 4000600. dithizone R in chloroform R. Shake with carbon tetrachloride R
Dissolve 25.0 g of ammonium acetate R in 25 mL of water R until the extract is colourless. Filter the aqueous layer to
and add 38.0 mL of hydrochloric acid R1. Adjust the remove traces of carbon tetrachloride.
pH if necessary with dilute hydrochloric acid R or dilute Acetate buffer solution pH 4.7 R1. 4013600.
ammonia R1. Dilute to 100.0 mL with water R.
Dissolve 136.1 g of sodium acetate R in 500 mL of water R. Mix
Phosphate buffer solution pH 3.5. 4000700. 250 mL of this solution with 250 mL of dilute acetic acid R.
Dissolve 68.0 g of potassium dihydrogen phosphate R in Acetate buffer solution pH 5.0. 4009100.
water R and dilute to 1000.0 mL with the same solvent. Adjust
the pH with phosphoric acid R. To 120 mL of a 6 g/L solution of glacial acetic acid R add
100 mL of 0.1 M potassium hydroxide and about 250 mL of
Buffer solution pH 3.6. 4000800. water R. Mix. Adjust the pH to 5.0 with a 6 g/L solution of
To 250.0 mL of 0.2 M potassium hydrogen phthalate R add acetic acid R or with 0.1 M potassium hydroxide and dilute to
11.94 mL of 0.2 M hydrochloric acid. Dilute to 1000.0 mL 1000.0 mL with water R.
with water R. Citrate buffer solution pH 5.0. 4010700.
Buffer solution pH 3.7. 4000900. Prepare a solution containing 20.1 g/L of citric acid R and
To 15.0 mL of acetic acid R add 60 mL of ethanol (96 per 8.0 g/L of sodium hydroxide R. Adjust the pH with dilute
cent) R and 20 mL of water R. Adjust to pH 3.7 by the addition hydrochloric acid R.
of ammonia R. Dilute to 100.0 mL with water R. 0.2 M Deuterated sodium phosphate buffer solution
Buffered copper sulfate solution pH 4.0. 4001000. pH 5.0. 4013900.
Dissolve 0.25 g of copper sulfate R and 4.5 g of ammonium Dissolve 2.76 g of sodium dihydrogen phosphate monohydrate R
acetate R in dilute acetic acid R and dilute to 100.0 mL with in 90 mL of deuterium oxide R, adjust the pH with a deuterated
the same solvent. solution of phosphoric acid R or a deuterated 1 M solution of
sodium hydroxide R, dilute to 100 mL with deuterium oxide R
0.1 M Sodium acetate buffer solution pH 4.0. 4013800. and mix.
Dissolve 822 mg of sodium acetate R in 100 mL of water R
(solution A). Dilute 1.44 mL of glacial acetic acid R in 250 mL Phosphate buffer solution pH 5.0. 4011300.
of water R (solution B). Titrate 100 mL of solution B using Dissolve 2.72 g of potassium dihydrogen phosphate R in 800 mL
about 20 mL of solution A. of water R. Adjust the pH with 1 M potassium hydroxide and
dilute to 1000 mL with water R.
Acetate buffer solution pH 4.4. 4001100.
Dissolve 136 g of sodium acetate R and 77 g of ammonium Buffer solution pH 5.2. 4001700.
acetate R in water R and dilute to 1000.0 mL with the same Dissolve 1.02 g of potassium hydrogen phthalate R in 30.0 mL
solvent ; add 250.0 mL of glacial acetic acid R and mix. of 0.1 M sodium hydroxide. Dilute to 100.0 mL with water R.
Phthalate buffer solution pH 4.4. 4001200. 0.067 M Phosphate buffer solution pH 5.4. 4012000.
Dissolve 2.042 g of potassium hydrogen phthalate R in 50 mL Mix appropriate volumes of a 23.99 g/L solution of disodium
of water R, add 7.5 mL of 0.2 M sodium hydroxide and dilute hydrogen phosphate R with a 9.12 g/L solution of sodium
to 200.0 mL with water R. dihydrogen phosphate monohydrate R to obtain pH 5.4.
Acetate buffer solution pH 4.5. 4012500. Acetate-edetate buffer solution pH 5.5. 4001900.
Dissolve 77.1 g of ammonium acetate R in water R. Add 70 mL Dissolve 250 g of ammonium acetate R and 15 g sodium
of glacial acetic acid R and dilute to 1000.0 mL with water R. edetate R in 400 mL of water R and add 125 mL of glacial
acetic acid R.
0.5 M Ammonium acetate buffer solution pH 4.5. 4014200.
Mix 14.3 mL of glacial acetic acid R and 470 mL of water R Buffer solution pH 5.5. 4001800.
and adjust to pH 4.5 with concentrated ammonia R. Dilute to Dissolve 54.4 g of sodium acetate R in 50 mL of water R,
500.0 mL with water R. heating to 35 °C if necessary. After cooling, slowly add 10 mL
of anhydrous acetic acid R. Shake and dilute to 100.0 mL with
0.05 M Phosphate buffer solution pH 4.5. 4009000. water R.
Dissolve 6.80 g of potassium dihydrogen phosphate R in
1000.0 mL of water R. The pH of the solution is 4.5. Phosphate buffer solution pH 5.5. 4002000.
Dissolve 13.61 g of potassium dihydrogen phosphate R in
Sodium acetate buffer solution pH 4.5. 4010100. water R and dilute to 1000.0 mL with the same solvent
Dissolve 63 g of anhydrous sodium acetate R in water R, (solution A). Dissolve 35.81 g of disodium hydrogen
add 90 mL acetic acid R and adjust to pH 4.5, and dilute to phosphate R in water R and dilute to 1000.0 mL with the same
1000 mL with water R. solvent (solution B). Mix 96.4 mL of solution A and 3.6 mL
of solution B.
Acetate buffer solution pH 4.6. 4001400.
Dissolve 5.4 g of sodium acetate R in 50 mL of water R, add Phosphate-citrate buffer solution pH 5.5. 4008700.
2.4 g of glacial acetic acid R and dilute to 100.0 mL with Mix 56.85 mL of a 28.4 g/L solution of anhydrous disodium
water R. Adjust the pH if necessary. hydrogen phosphate R and 43.15 mL of a 21 g/L solution of
citric acid R.
Succinate buffer solution pH 4.6. 4001500.
Disssolve 11.8 g of succinic acid R in a mixture of 600 mL of Phosphate buffer solution pH 5.6. 4011200.
water R and 82 mL of 1 M sodium hydroxide and dilute to Dissolve 0.908 g of potassium dihydrogen phosphate R
1000.0 mL with water R. in water R and dilute to 100.0 mL with the same solvent
(solution A). Dissolve 1.161 g of dipotassium hydrogen
Acetate buffer solution pH 4.7. 4001600. phosphate R in water R and dilute to 100.0 mL with the same
Dissolve 136.1 g of sodium acetate R in 500 mL of water R. Mix solvent (solution B). Mix 94.4 mL of solution A and 5.6 mL
250 mL of this solution with 250 mL of dilute acetic acid R. of solution B. If necessary, adjust to pH 5.6 using solution A
Shake twice with a freshly prepared, filtered, 0.1 g/L solution of or solution B.
General Notices (1) apply to all monographs and other texts 541
4.1.3. Buffer solutions EUROPEAN PHARMACOPOEIA 8.0
Phosphate buffer solution pH 5.8. 4002100. 0.1 M Phosphate buffer solution pH 6.7. 4014300.
Dissolve 1.19 g of disodium hydrogen phosphate dihydrate R Dissolve 15.6 g of sodium dihydrogen phosphate R in water R
and 8.25 g of potassium dihydrogen phosphate R in water R and dilute to 1.0 L with the same solvent. Dissolve 17.8 g of
and dilute to 1000.0 mL with the same solvent. disodium hydrogen phosphate dihydrate R in water R and
dilute to 1.0 L with the same solvent. Mix the solutions, check
Acetate buffer solution pH 6.0. 4002200. the pH and if necessary adjust to pH 6.7.
Dissolve 100 g of ammonium acetate R in 300 mL of water R,
add 4.1 mL of glacial acetic acid R, adjust the pH if necessary Phosphate buffered saline pH 6.8. 4003200.
using ammonia R or acetic acid R and dilute to 500.0 mL with Dissolve 1.0 g of potassium dihydrogen phosphate R, 2.0 g
water R. of dipotassium hydrogen phosphate R and 8.5 g of sodium
chloride R in 900 mL of water R, adjust the pH if necessary
Diethylammonium phosphate buffer solution pH 6.0. and dilute to 1000.0 mL with the same solvent.
4002300.
Dilute 68 mL of phosphoric acid R to 500 mL with water R. Phosphate buffer solution pH 6.8. 4003300.
To 25 mL of this solution add 450 mL of water R and 6 mL Mix 77.3 mL of a 71.5 g/L solution of disodium hydrogen
of diethylamine R, adjust to pH 6 ± 0.05, if necessary, using phosphate R with 22.7 mL of a 21 g/L solution of citric acid R.
diethylamine R or phosphoric acid R and dilute to 500.0 mL
with water R. Phosphate buffer solution pH 6.8 R1. 4003400.
To 51.0 mL of a 27.2 g/L solution of potassium dihydrogen
Phosphate buffer solution pH 6.0. 4002400. phosphate R add 49.0 mL of a 71.6 g/L solution of disodium
Mix 63.2 mL of a 71.5 g/L solution of disodium hydrogen hydrogen phosphate R. Adjust the pH if necessary.
phosphate R and 36.8 mL of a 21 g/L solution of citric acid R. Storage : at 2 °C to 8 °C.
Phosphate buffer solution pH 6.0 R1. 4002500.
1 M tris-hydrochloride buffer solution pH 6.8. 4009300.
Dissolve 6.8 g of sodium dihydrogen phosphate R in water R
Dissolve 60.6 g of tris(hydroxymethyl)aminomethane R in
and dilute to 1000.0 mL with water R. Adjust the pH with
400 mL of water R. Adjust the pH with hydrochloric acid R
strong sodium hydroxide solution R.
and dilute to 500.0 mL with water R.
Phosphate buffer solution pH 6.0 R2. 4002600.
Buffer solution pH 7.0. 4003500.
To 250.0 mL of 0.2 M potassium dihydrogen phosphate R add
To 1000 mL of a solution containing 18 g/L of disodium
28.5 mL of 0.2 M sodium hydroxide and dilute to 1000.0 mL
hydrogen phosphate R and 23 g/L of sodium chloride R add
with water R.
sufficient (about 280 mL) of a solution containing 7.8 g/L
Phosphate buffer solution pH 6.4. 4002800. of sodium dihydrogen phosphate R and 23 g/L of sodium
chloride R to adjust the pH. Dissolve in the solution sufficient
Dissolve 2.5 g of disodium hydrogen phosphate R, 2.5 g of sodium azide R to give a 0.2 g/L solution.
sodium dihydrogen phosphate R and 8.2 g of sodium chloride R
in 950 mL of water R. Adjust the pH of the solution to 6.4 with Maleate buffer solution pH 7.0. 4003600.
1 M sodium hydroxide or 1 M hydrochloric acid, if necessary.
Dilute to 1000.0 mL with water R. Dissolve 10.0 g of sodium chloride R, 6.06 g of
tris(hydroxymethyl)aminomethane R and 4.90 g of maleic
0.5 M Phthalate buffer solution pH 6.4. 4009200. anhydride R in 900 mL of water R. Adjust the pH using a
170 g/L solution of sodium hydroxide R. Dilute to 1000.0 mL
Dissolve 100 g of potassium hydrogen phthalate R in water R
with water R.
and dilute to 1000.0 mL with the same solvent. Adjust the pH
if necessary, using strong sodium hydroxide solution R. Storage : at 2 °C to 8 °C ; use within 3 days.
Buffer solution pH 6.5. 4002900. 0.025 M Phosphate buffer solution pH 7.0. 4009400.
Dissolve 60.5 g of disodium hydrogen phosphate R and 46 g of Mix 1 volume of 0.063 M phosphate buffer solution pH 7.0 R
potassium dihydrogen phosphate R in water R. Add 100 mL of with 1.5 volumes of water R.
0.02 M sodium edetate and 20 mg of mercuric chloride R and
dilute to 1000.0 mL with water R. 0.03 M Phosphate buffer solution pH 7.0. 4010300.
Dissolve 5.2 g of dipotassium hydrogen phosphate R in
Imidazole buffer solution pH 6.5. 4003000. 900 mL of water for chromatography R. Adjust the solution to
Dissolve 6.81 g of imidazole R, 1.23 g of magnesium sulfate R pH 7.0 ± 0.1 using phosphoric acid R and dilute to 1000 mL
and 0.73 g of calcium sulfate R in 752 mL of 0.1 M hydrochloric with water for chromatography R.
acid. Adjust the pH if necessary and dilute to 1000.0 mL with
water R. 0.05 M Phosphate buffer solution pH 7.0. 4012400.
Mix 34 mL of water R and 100 mL of 0.067 M phosphate buffer
0.1 M phosphate buffer solution pH 6.5. 4010800. solution pH 7.0 R.
Dissolve 13.80 g of sodium dihydrogen phosphate
monohydrate R in 900 mL of distilled water R. Adjust the pH 0.063 M Phosphate buffer solution pH 7.0. 4009500.
using a 400 g/L solution of sodium hydroxide R. Dilute to Dissolve 5.18 g of anhydrous disodium hydrogen phosphate R
1000 mL with distilled water R. and 3.65 g of sodium dihydrogen phosphate monohydrate R in
950 mL of water R and adjust the pH with phosphoric acid R ;
Phosphate buffer solution pH 6.5. 4012800. dilute to 1000.0 mL with water R.
Dissolve 2.75 g of sodium dihydrogen phosphate R and 4.5 g of
sodium chloride R in 500 mL of water R. Adjust the pH with 0.067 M Phosphate buffer solution pH 7.0. 4003800.
phosphate buffer solution pH 8.5 R. Dissolve 0.908 g of potassium dihydrogen phosphate R
in water R and dilute to 100.0 mL with the same solvent
Buffer solution pH 6.6. 4003100. (solution A). Dissolve 2.38 g of disodium hydrogen phosphate R
To 250.0 mL of 0.2 M potassium dihydrogen phosphate R add in water R and dilute to 100.0 mL with the same solvent
89.0 mL of 0.2 M sodium hydroxide. Dilute to 1000.0 mL with (solution B). Mix 38.9 mL of solution A and 61.1 mL of
water R. solution B. Adjust the pH if necessary.
0.1 M Phosphate buffer solution pH 7.0. 4008200. Imidazole buffer solution pH 7.3. 4004500.
Dissolve 1.361 g of potassium dihydrogen phosphate R in Dissolve 3.4 g of imidazole R and 5.8 g of sodium chloride R in
water R and dilute to 100.0 mL with the same solvent. water R, add 18.6 mL of 1 M hydrochloric acid and dilute to
Adjust the pH using a 35 g/L solution of disodium hydrogen 1000.0 mL with water R. Adjust the pH if necessary.
phosphate R.
Barbital buffer solution pH 7.4. 4004700.
Phosphate buffer solution pH 7.0. 4003700. Mix 50 mL of a solution in water R containing 19.44 g/L of
Mix 82.4 mL of a 71.5 g/L solution of disodium hydrogen sodium acetate R and 29.46 g/L of barbital sodium R with
phosphate R with 17.6 mL of a 21 g/L solution of citric acid R. 50.5 mL of 0.1 M hydrochloric acid, add 20 mL of an 85 g/L of
sodium chloride R and dilute to 250 mL with water R.
Phosphate buffer solution pH 7.0 R1. 4003900. Buffer solution pH 7.4. 4004600.
Mix 250.0 mL of 0.2 M potassium dihydrogen phosphate R and Dissolve 0.6 g of potassium dihydrogen phosphate R, 6.4 g
148.2 mL of a 8 g/L solution of sodium hydroxide R, adjust the of disodium hydrogen phosphate R and 5.85 g of sodium
pH if necessary. Dilute to 1000.0 mL with water R. chloride R in water R, and dilute to 1000.0 mL with the same
Phosphate buffer solution pH 7.0 R2. 4004000. solvent. Adjust the pH if necessary.
Mix 50.0 mL of a 136 g/L solution of potassium dihydrogen Phosphate buffered saline pH 7.4. 4005000.
phosphate R with 29.5 mL of 1 M sodium hydroxide and dilute Dissolve 2.38 g of disodium hydrogen phosphate R, 0.19 g
to 100.0 mL with water R. Adjust the pH to 7.0 ± 0.1. of potassium dihydrogen phosphate R and 8.0 g of sodium
chloride R in water. Dilute to 1000.0 mL with the same solvent.
Phosphate buffer solution pH 7.0 R3. 4008600. Adjust the pH if necessary.
Dissolve 5 g of potassium dihydrogen phosphate R and 11 g
of dipotassium hydrogen phosphate R in 900 mL of water R. Phosphate buffer solution pH 7.4. 4004800.
Adjust to pH 7.0 with dilute phosphoric acid R or dilute sodium Add 250.0 mL of 0.2 M potassium dihydrogen phosphate R to
hydroxide solution R. Dilute to 1000 mL with water R and mix. 393.4 mL of 0.1 M sodium hydroxide.
Phosphate buffer solution pH 7.0 R4. 4010200. Tris(hydroxymethyl)aminomethane buffer solution pH 7.4.
4012100.
Dissolve 28.4 g of anhydrous disodium hydrogen phosphate R
and 18.2 g of potassium dihydrogen phosphate R in water R Dissolve 30.3 g of tris(hydroxymethyl)aminomethane R in
and dilute to 500 mL with the same solvent. approximately 200 mL of water R. Add 183 mL of 1 M
hydrochloric acid. Dilute to 500.0 mL with water R. Note :
Phosphate buffer solution pH 7.0 R5. 4011400. the pH is 7.7-7.8 at room temperature and 7.4 at 37 °C. This
solution is stable for several months at 4 °C.
Dissolve 28.4 g of anhydrous disodium hydrogen phosphate R
in 800 mL of water R. Adjust the pH using a 30 per cent m/m Tris(hydroxymethyl)aminomethane sodium chloride
solution of phosphoric acid R and dilute to 1000 mL with buffer solution pH 7.4. 4004900.
water R. Dissolve 6.08 g of tris(hydroxymethyl)aminomethane R, 8.77 g
of sodium chloride R in 500 mL of distilled water R. Add 10.0 g
Tetrabutylammonium buffer solution pH 7.0. 4010900. of bovine albumin R. Adjust the pH using hydrochloric acid R.
Dissolve 6.16 g of ammonium acetate R in a mixture of 15 mL Dilute to 1000.0 mL with distilled water R.
of tetrabutylammonium hydroxide solution (400 g/L) R and
185 mL of water R. Adjust the pH with nitric acid R. Tris(hydroxymethyl)aminomethane sodium chloride
buffer solution pH 7.4 R1. 4012200.
Buffered salt solution pH 7.2. 4004300. Dissolve 0.1 g of bovine albumin R in a mixture containing
Dissolve in water R 8.0 g of sodium chloride R, 0.2 g of 2 mL of tris(hydroxymethyl)aminomethane buffer solution
potassium chloride R, 0.1 g of anhydrous calcium chloride R, pH 7.4 R and 50 mL of a 5.84 mg/mL solution of sodium
0.1 g of magnesium chloride R, 3.18 g of disodium hydrogen chloride R. Dilute to 100.0 mL with water R.
phosphate R and 0.2 g of potassium dihydrogen phosphate R
Tris-sodium acetate buffer solution pH 7.4. 4012900.
and dilute to 1000.0 mL with water R.
Dissolve 6.3 g of tris(hydroxymethyl)aminomethane R and
Buffer solution pH 7.2. 4004100. 4.9 g of anhydrous sodium acetate R in 900 mL of water R.
To 250.0 mL of 0.2 M potassium dihydrogen phosphate R add Adjust to pH 7.4 with sulfuric acid R and dilute to 1000 mL
175.0 mL of 0.2 M sodium hydroxide. Dilute to 1000.0 mL with water R.
with water R. Adjust the pH if necessary. Tris-sodium acetate-sodium chloride buffer solution
pH 7.4. 4013000.
Phosphate-albumin buffered saline pH 7.2. 4004400.
Dissolve 30.0 g of tris(hydroxymethyl)aminomethane R, 14.5 g
Dissolve 10.75 g of disodium hydrogen phosphate R, 7.6 g of of anhydrous sodium acetate R and 14.6 g of sodium chloride R
sodium chloride R and 10 g of bovine albumin R in water R and in 900 mL of water R. Add 0.50 g of bovine albumin R. Adjust
dilute to 1000.0 mL with the same solvent. Immediately before to pH 7.4 with sulfuric acid R and dilute to 1000 mL with
use adjust the pH using dilute sodium hydroxide solution R water R.
or dilute phosphoric acid R.
Borate buffer solution pH 7.5. 4005200.
Phosphate-albumin buffered saline pH 7.2 R1. 4009600. Dissolve 2.5 g of sodium chloride R, 2.85 g of disodium
Dissolve 10.75 g of disodium hydrogen phosphate R, 7.6 g of tetraborate R and 10.5 g of boric acid R in water R and dilute to
sodium chloride R and 1 g of bovine albumin R in water R and 1000.0 mL with the same solvent. Adjust the pH if necessary.
dilute to 1000.0 mL with the same solvent. Immediately before Storage : at 2 °C to 8 °C.
use adjust the pH using dilute sodium hydroxide solution R
or dilute phosphoric acid R. Buffer (HEPES) solution pH 7.5. 4009700.
Dissolve 2.38 g of 2-[4-(2-hydroxyethyl)piperazin-1-
Phosphate buffer solution pH 7.2. 4004200. yl]ethanesulfonic acid R in about 90 mL of water R. Adjust the
Mix 87.0 mL of a 71.5 g/L solution of disodium hydrogen pH to 7.5 with sodium hydroxide solution R. Dilute to 100 mL
phosphate R with 13.0 mL of a 21 g/L solution of citric acid R. with water R.
General Notices (1) apply to all monographs and other texts 543
4.1.3. Buffer solutions EUROPEAN PHARMACOPOEIA 8.0
0.05 M Phosphate buffer solution pH 7.5. 4014400. 1 M Tris-hydrochloride buffer solution pH 8.0. 4012700.
Dissolve 0.89 g of disodium hydrogen phosphate dihydrate R Dissolve 121.1 g of tris(hydroxymethyl)aminomethane R and
in about 80 mL of water R. Adjust to pH 7.5 with an 8.5 per 1.47 g of calcium chloride R in 900 mL of water R. Adjust the
cent V/V solution of phosphoric acid R and dilute to 100.0 mL pH with hydrochloric acid R and dilute to 1000.0 mL with
with water R. water R.
0.2 M Phosphate buffer solution pH 7.5. 4005400. Tris-hydrochloride buffer solution pH 8.0. 4012300.
Dissolve 27.22 g of potassium dihydrogen phosphate R in Dissolve 1.21 g of tris(hydroxymethyl)aminomethane R and
930 mL of water R, adjust to pH 7.5 with a 300 g/L solution of 29.4 mg of calcium chloride R in water R. Adjust the pH with
potassium hydroxide R and dilute to 1000.0 mL with water R. 1 M hydrochloric acid and dilute to 100.0 mL with water R.
0.33 M Phosphate buffer solution pH 7.5. 4005300. Tris-sodium acetate buffer solution pH 8.0. 4013100.
Dissolve 119.31 g of disodium hydrogen phosphate R in water R Dissolve 6.3 g of tris(hydroxymethyl)aminomethane R and
and dilute to 1000.0 mL with the same solvent (solution A). 4.9 g of anhydrous sodium acetate R in 900 mL of water R.
Dissolve 45.36 g of potassium dihydrogen phosphate R in Adjust to pH 8.0 with sulfuric acid R and dilute to 1000 mL
water R and dilute to 1000.0 mL with the same solvent with water R.
(solution B). Mix 85 mL of solution A and 15 mL of solution B.
Adjust the pH if necessary. Tris-sodium acetate-sodium chloride buffer solution
pH 8.0. 4013200.
0.05 M Tris-hydrochloride buffer solution pH 7.5. 4005600.
Dissolve 30.0 g of tris(hydroxymethyl)aminomethane R, 14.5 g
Dissolve 6.057 g of tris(hydroxymethyl)aminomethane R in
of anhydrous sodium acetate R and 14.6 g of sodium chloride R
water R and adjust the pH with hydrochloric acid R. Dilute to
in 900 mL of water R. Add 0.50 g of bovine albumin R. Adjust
1000.0 mL with water R.
to pH 8.0 with sulfuric acid R and dilute to 1000 mL with
1 M Tris-hydrochloride buffer solution pH 7.5. 4014500. water R.
Dissolve 12.11 g of tris(hydroxymethyl)aminomethane R in Tris(hydroxymethyl)aminomethane buffer solution pH 8.1.
90 mL of water R, adjust to pH 7.5 with hydrochloric acid R 4006200.
and dilute to 100.0 mL with water R.
Dissolve 0.294 g of calcium chloride R in 40 mL of
Tris(hydroxymethyl)aminomethane buffer solution pH 7.5. tris(hydroxymethyl)aminomethane solution R and adjust the
4005500. pH with 1 M hydrochloric acid. Dilute to 100.0 mL with
Dissolve 7.27 g of tris(hydroxymethyl)aminomethane R and water R.
5.27 g of sodium chloride R in water R, and adjust the pH if
Tris-glycine buffer solution pH 8.3. 4006300.
necessary. Dilute to 1000.0 mL with water R.
Dissolve 6.0 g of tris(hydroxymethyl)aminomethane R and
Sodium citrate buffer solution pH 7.8 (0.034 M sodium 28.8 g of glycine R in water R and dilute to 1000.0 mL with the
citrate, 0.101 M sodium chloride). 4009800. same solvent. Dilute 1 volume to 10 volumes with water R
Dissolve 10.0 g of sodium citrate R and 5.90 g of sodium immediately before use.
chloride R in 900 mL of water R. Adjust the pH by addition of
hydrochloric acid R and dilute to 1000 mL with water R. Tris-hydrochloride buffer solution pH 8.3. 4011800.
Dissolve 9.0 g of tris(hydroxymethyl)aminomethane R in 2.9 L
0.0015 M Borate buffer solution pH 8.0. 4006000. of water R. Adjust the pH with 1 M hydrochloric acid. Adjust
Dissolve 0.572 g of disodium tetraborate R and 2.94 g of the volume to 3 L with water R.
calcium chloride R in 800 mL of water R. Adjust the pH with
1 M hydrochloric acid. Dilute to 1000.0 mL with water R. 0.05 M Tris-hydrochloride buffer solution pH 9.0. 4013500.
Dissolve 0.605 g of tris(hydroxymethyl)aminomethane R in
Buffer solution pH 8.0. 4005900. water R. Adjust the pH with 1 M hydrochloric acid and dilute
To 50.0 mL of 0.2 M potassium dihydrogen phosphate R add to 100.0 mL with water R.
46.8 mL of 0.2 M sodium hydroxide. Dilute to 200.0 mL with
water R. Barbital buffer solution pH 8.4. 4006400.
Buffer solution pH 8.0 R1. 4010400. Dissolve 8.25 g of barbital sodium R in water R and dilute to
1000.0 mL with the same solvent.
Dissolve 20 g of dipotassium hydrogen phosphate R in 900 mL
of water R. Adjust the pH with phosphoric acid R. Dilute to Tris-EDTA BSA buffer solution pH 8.4. 4006500.
1000 mL with water R. Dissolve 6.1 g of tris(hydroxymethyl)aminomethane R, 2.8 g
0.02 M Phosphate buffer solution pH 8.0. 4006100. of sodium edetate R, 10.2 g of sodium chloride R and 10 g
To 50.0 mL of 0.2 M potassium dihydrogen phosphate R add of bovine albumin R in water R, adjust to pH 8.4 using 1 M
46.8 mL of 0.2 M sodium hydroxide. Dilute to 500.0 mL with hydrochloric acid and dilute to 1000.0 mL with water R.
water R. Tris(hydroxymethyl)aminomethane-EDTA buffer solution
0.02 M Sodium phosphate buffer solution pH 8.0. 4013700. pH 8.4. 4006600.
Dissolve 0.31 g of sodium dihydrogen phosphate R in 70 mL of Dissolve 5.12 g of sodium chloride R, 3.03 g of
water R and adjust to pH 8.0 with 1 M sodium hydroxide, then tris(hydroxymethyl)aminomethane R and 1.40 g of sodium
dilute to 100 mL with water R. edetate R in 250 mL of distilled water R. Adjust the pH to 8.4
using hydrochloric acid R. Dilute to 500.0 mL with distilled
0.1 M Phosphate buffer solution pH 8.0. 4008400. water R.
Dissolve 0.523 g of potassium dihydrogen phosphate R and
16.73 g of dipotassium hydrogen phosphate R in water R and Guanidine-tris(hydroxymethyl)aminomethane-EDTA
dilute to 1000.0 mL with the same solvent. buffer solution pH 8.5. 4014600.
Dissolve 1.0 g of sodium edetate R, 12.1 g of
1 M Phosphate buffer solution pH 8.0. 4007800. tris(hydroxymethyl)aminomethane R and 57.0 g of
Dissolve 136.1 g of potassium dihydrogen phosphate R in guanidine hydrochloride R in 35 mL of water R. Adjust to
water R, adjust the pH with 1 M sodium hydroxide. Dilute to pH 8.5 with hydrochloric acid R and dilute to 100 mL with
1000.0 mL with water R. water R.
Ammonium chloride buffer solution pH 10.4. 4011000. Potassium hydrogen phthalate. C8H5KO4. (Mr 204.2).
Dissolve 70 g of ammonium chloride R in 200 mL of water R, 2000400. [877-24-7].
add 330 mL of concentrated ammonia R and dilute to Recrystallise potassium hydrogen phthalate R from boiling
1000.0 mL with water R. If necessary, adjust to pH 10.4 with water R, collect the crystals at a temperature above 35 °C and
ammonia R. dry to constant mass at 110 °C.
Borate buffer solution pH 10.4. 4011100. Sodium carbonate. Na2CO3 . (Mr 106.0). 2000500.
Dissolve 24.64 g of boric acid R in 900 mL of distilled water R. [497-19-8].
Adjust the pH using a 400 g/L solution of sodium hydroxide R. Filter at room temperature a saturated solution of sodium
Dilute to 1000 mL with distilled water R. carbonate R. Introduce slowly into the filtrate a stream of
carbon dioxide R with constant cooling and stirring. After
Ammonium chloride buffer solution pH 10.7. 4013400. about 2 h, collect the precipitate on a sintered-glass filter
Dissolve 67.5 g of ammonium chloride R in water R, add (2.1.2). Wash the filter with iced water R containing carbon
570 mL of concentrated ammonia R and dilute to 1000.0 mL dioxide. After drying at 100 °C to 105 °C, heat to constant
with water R. mass at 270-300 °C, stirring from time to time.
General Notices (1) apply to all monographs and other texts 545
4.2.2. Volumetric solutions EUROPEAN PHARMACOPOEIA 8.0
Sodium chloride. NaCl. (Mr 58.44). 2000600. [7647-14-5]. Standardisation. To 25.0 mL of the ammonium and cerium
To 1 volume of the saturated sodium chloride solution R add sulfate solution add 2.0 g of potassium iodide R and 150 mL
2 volumes of hydrochloric acid R. Collect the crystals formed of water R. Titrate immediately with 0.1 M sodium thiosulfate,
and wash with hydrochloric acid R1. Remove the hydrochloric using 1 mL of starch solution R as indicator.
acid by heating on a water-bath and dry the crystals to
constant mass at 300 °C. 0.01 M Ammonium and cerium sulfate. 3000400.
To 100.0 mL of 0.1 M ammonium and cerium sulfate add, with
Sulfanilic acid. C6H7NO3S. (Mr 173.2). 2000700. [121-57-3]. cooling, 30 mL of sulfuric acid R and dilute to 1000.0 mL
Recrystallise sulfanilic acid R from boiling water R. Filter and with water R.
dry to constant mass at 100-105 °C.
0.1 M Ammonium thiocyanate. 3000500.
Zinc. Zn. (Mr 65.4). 2000800. [7440-66-6].
Dissolve 7.612 g of ammonium thiocyanate R in water R and
Content : minimum 99.9 per cent. dilute to 1000.0 mL with the same solvent.
Standardisation. To 20.0 mL of 0.1 M silver nitrate add 25 mL
04/2010:40202 of water R, 2 mL of dilute nitric acid R and 2 mL of ferric
ammonium sulfate solution R2. Titrate with the ammonium
4.2.2. VOLUMETRIC SOLUTIONS thiocyanate solution until a reddish-yellow colour is obtained.
Volumetric solutions are prepared according to the usual 0.1 M Barium chloride. 3000600.
chemical analytical methods. The accuracy of the apparatus
used is verified to ensure that it is appropriate for the intended Dissolve 24.4 g of barium chloride R in water R and dilute to
use. 1000.0 mL with the same solvent.
The concentration of volumetric solutions is indicated in terms Standardisation. To 10.0 mL of the barium chloride solution
of molarity. Molarity expresses, as the number of moles, the add 60 mL of water R, 3 mL of concentrated ammonia R and
amount of substance dissolved in 1 L of solution. A solution 0.5-1 mg of phthalein purple R. Titrate with 0.1 M sodium
which contains x moles of substance per litre is said to be x M. edetate. When the solution begins to decolorise, add 50 mL
of ethanol (96 per cent) R and continue the titration until the
Volumetric solutions do not differ from the prescribed blue-violet colour disappears.
strength by more than 10 per cent. The molarity of the
volumetric solutions is determined by an appropriate number 0.05 M Barium perchlorate. 3000700.
of titrations. The repeatability does not exceed 0.2 per cent
(relative standard deviation). Dissolve 15.8 g of barium hydroxide R in a mixture of 7.5 mL
of perchloric acid R and 75 mL of water R, adjust the solution
Volumetric solutions are standardised by the methods to pH 3 by adding perchloric acid R and filter if necessary. Add
described below. When a volumetric solution is to be used 150 mL of ethanol (96 per cent) R and dilute to 250 mL with
in an assay in which the end-point is determined by an water R. Dilute to 1000.0 mL with buffer solution pH 3.7 R.
electrochemical process (for example, amperometry or
potentiometry) the solution is standardised by the same Standardisation. To 5.0 mL of 0.05 M sulfuric acid add 5 mL
method. The composition of the medium in which a of water R, 50 mL of buffer solution pH 3.7 R and 0.5 mL of
volumetric solution is standardised should be the same as that alizarin S solution R. Titrate with the barium perchlorate
in which it is to be used. solution until an orange-red colour appears. Standardise
immediately before use.
Solutions more dilute than those described are obtained
by dilution with carbon dioxide-free water R of the 0.025 M Barium perchlorate. 3009600.
least-concentrated solution that describes a standardisation.
The correction factors of these solutions are the same as those Dilute 500.0 mL of 0.05 M barium perchlorate to 1000.0 mL
from which the dilutions were prepared. with buffer solution pH 3.7 R.
0.1 M Cerium sulfate. 3001100. Standardisation. To 20.0 mL of the iodine solution add 1 mL
Dissolve 40.4 g of cerium sulfate R in a mixture of 500 mL of of dilute acetic acid R and 30 mL of water R. Titrate with 0.1 M
water R and 50 mL of sulfuric acid R. Allow to cool and dilute sodium thiosulfate, using starch solution R as indicator.
to 1000.0 mL with water R. Storage : protected from light.
Standardisation. To 20.0 mL of the cerium sulfate solution, 0.01 M Iodine. 3002900.
add 1.6 g of potassium iodide R, 100 mL of water R and 40 mL
of dilute sulfuric acid R. Titrate immediately with 0.1 M sodium Add 0.3 g of potassium iodide R to 20.0 mL of 0.05 M iodine
thiosulfate using 0.8 mL of starch solution R as indicator. and dilute to 100.0 mL with water R.
General Notices (1) apply to all monographs and other texts 547
4.2.2. Volumetric solutions EUROPEAN PHARMACOPOEIA 8.0
Standardisation. Dissolve 0.350 g of potassium hydrogen 0.5 M Potassium hydroxide in alcohol (60 per cent V/V).
phthalate RV in 50 mL of anhydrous acetic acid R, warming 3004900.
gently if necessary. Allow to cool protected from the air, and Dissolve 3 g of potassium hydroxide R in aldehyde-free
titrate with the perchloric acid solution, using 0.05 mL of alcohol R (60 per cent V/V) and dilute to 100.0 mL with the
crystal violet solution R as indicator. Note the temperature of same solvent.
the perchloric acid solution at the time of the titration. If the
temperature at which an assay is carried out is different from Standardisation. Titrate 20.0 mL of the alcoholic potassium
that at which the 0.1 M perchloric acid has been standardised, hydroxide solution (60 per cent V/V) with 0.5 M hydrochloric
the volume used in the assay becomes : acid, using 0.5 mL of phenolphthalein solution R as indicator.
0.5 M Potassium hydroxide, alcoholic. 3005000.
Dissolve 3 g of potassium hydroxide R in 5 mL of water R and
t1 = temperature during standardisation, dilute to 100.0 mL with aldehyde-free alcohol R.
t2 = temperature during the assay, Standardisation. Titrate 20.0 mL of the alcoholic potassium
hydroxide solution with 0.5 M hydrochloric acid, using 0.5 mL
Vc = corrected volume, of phenolphthalein solution R as indicator.
V = observed volume. 0.1 M Potassium hydroxide, alcoholic. 3005100.
1 mL of 0.1 M perchloric acid is equivalent to 20.42 mg Dilute 20.0 mL of 0.5 M alcoholic potassium hydroxide to
of C8H5KO4. 100.0 mL with aldehyde-free alcohol R.
0.05 M Perchloric acid. 3004000. 0.01 M Potassium hydroxide, alcoholic. 3009000.
Dilute 50.0 mL of 0.1 M perchloric acid to 100.0 mL with Dilute 2.0 mL of 0.5 M alcoholic potassium hydroxide to
anhydrous acetic acid R. 100.0 mL with aldehyde-free alcohol R.
0.02 M Perchloric acid. 3009900. 0.05 M Potassium iodate. 3005200.
Dilute 20.0 mL of 0.1 M perchloric acid to 100.0 mL with Dissolve 10.70 g of potassium iodate R in water R and dilute to
anhydrous acetic acid R. 1000.0 mL with the same solvent.
0.033 M Potassium bromate. 3004200. Standardisation. Dilute 25.0 mL of the potassium iodate
solution to 100.0 mL with water R. To 20.0 mL of this solution
Dissolve 5.5670 g of potassium bromate RV in water R and add 2 g of potassium iodide R and 10 mL of dilute sulfuric
dilute to 1000.0 mL with the same solvent. acid R. Titrate with 0.1 M sodium thiosulfate, using 1 mL of
0.02 M Potassium bromate. 3004300. starch solution R, added towards the end of the titration, as
indicator.
Dissolve 3.340 g of potassium bromate RV in water R and
dilute to 1000.0 mL with the same solvent. 0.001 M Potassium iodide. 3009200.
0.0167 M Potassium bromate. 3004400. Dilute 10.0 mL of potassium iodide solution R to 100.0 mL
with water R. Dilute 5.0 mL of this solution to 500.0 mL with
Prepare by diluting 0.033 M Potassium bromate. water R.
0.0083 M Potassium bromate. 3004500. 0.02 M Potassium permanganate. 3005300.
Prepare by diluting 0.033 M Potassium bromate. Dissolve 3.2 g of potassium permanganate R in water R and
0.0167 M Potassium dichromate. 3004600. dilute to 1000.0 mL with the same solvent. Heat the solution
for 1 h on a water-bath, allow to cool and filter through a
Dissolve 4.90 g of potassium dichromate R in water R and sintered-glass filter (2.1.2).
dilute to 1000.0 mL with the same solvent.
Standardisation. To 20.0 mL of the potassium permanganate
Standardisation. To 20.0 mL of the potassium dichromate solution, add 2 g of potassium iodide R and 10 mL of dilute
solution add 1 g of potassium iodide R and 7 mL of dilute sulfuric acid R. Titrate with 0.1 M sodium thiosulfate, using
hydrochloric acid R. Add 250 mL of water R and titrate with 1 mL of starch solution R, added towards the end of the
0.1 M sodium thiosulfate, using 3 mL of starch solution R as titration, as indicator. Standardise immediately before use.
indicator, until the colour changes from blue to light green.
Storage : protected from light.
0.1 M Potassium hydrogen phthalate. 3004700.
0.1 M Silver nitrate. 3005600.
In a conical flask containing about 800 mL of anhydrous acetic
acid R, dissolve 20.42 g of potassium hydrogen phthalate RV. Dissolve 17.0 g of silver nitrate R in water R and dilute to
Heat on a water-bath until completely dissolved, protected 1000.0 mL with the same solvent.
from humidity. Cool to 20 °C and dilute to 1000.0 mL with Standardisation. Dissolve 0.100 g of sodium chloride RV in
anhydrous acetic acid R. 30 mL of water R. Titrate with the silver nitrate solution,
determining the end-point potentiometrically (2.2.20).
1 M Potassium hydroxide. 3009100. 1 mL of 0.1 M silver nitrate is equivalent to 5.844 mg of NaCl.
Dissolve 60 g of potassium hydroxide R in carbon dioxide-free
Storage : protected from light.
water R and dilute to 1000.0 mL with the same solvent.
Standardisation. Titrate 20.0 mL of the potassium hydroxide 0.001 M Silver nitrate. 3009300.
solution with 1 M hydrochloric acid, using 0.5 mL of Dilute 5.0 mL of silver nitrate 0.1 M to 500.0 mL with water R.
phenolphthalein solution R as indicator.
0.1 M Sodium arsenite. 3005800.
0.1 M Potassium hydroxide. 3004800.
Dissolve arsenious trioxide RV equivalent to 4.946 g of As2O3
Dissolve 6 g of potassium hydroxide R in carbon dioxide-free in a mixture of 20 mL of strong sodium hydroxide solution R
water R and dilute to 1000.0 mL with the same solvent. and 20 mL of water R, dilute to 400 mL with water R and add
Standardisation. Titrate 20.0 mL of the potassium hydroxide dilute hydrochloric acid R until the solution is neutral to litmus
solution with 0.1 M hydrochloric acid, using 0.5 mL of paper R. Dissolve 2 g of sodium hydrogen carbonate R in the
phenolphthalein solution R as indicator. solution and dilute to 500.0 mL with water R.
General Notices (1) apply to all monographs and other texts 549
4.2.2. Volumetric solutions EUROPEAN PHARMACOPOEIA 8.0
0.05 M Sulfuric acid. 3008000. blue colour is again obtained. Protect the solution from
Dilute 100.0 mL of 0.5 M sulfuric acid to 1000.0 mL with atmospheric carbon dioxide throughout the titration. From
water R. the volume of titrant used in the second titration ascertain the
exact strength of the tetrabutylammonium hydroxide solution.
Standardisation. Carry out the titration described for 0.5 M Standardise immediately before use.
sulfuric acid, using 0.100 g of sodium carbonate RV, dissolved
in 20 mL of water R. 1 mL of 0.1 M tetrabutylammonium hydroxide is equivalent
to 12.21 mg of C7H6O2.
1 mL of 0.05 M sulfuric acid is equivalent to 5.30 mg of
Na2CO3. 0.1 M Tetrabutylammonium hydroxide in 2-propanol.
3008400.
0.1 M Tetrabutylammonium hydroxide. 3008300. Prepare as described for 0.1 M tetrabutylammonium hydroxide
Dissolve 40 g of tetrabutylammonium iodide R in 90 mL of using 2-propanol R instead of toluene R and standardise as
anhydrous methanol R, add 20 g of finely powdered silver described.
oxide R and shake vigorously for 1 h. Centrifuge a few
millilitres of the mixture and test the supernatant for iodides. 0.05 M Zinc chloride. 3008500.
If a positive reaction is obtained, add an additional 2 g of silver Dissolve 6.82 g of zinc chloride R, weighed with appropriate
oxide R and shake for a further 30 min. Repeat this procedure precautions, in water R. If necessary, add dropwise dilute
until the liquid is free from iodides, filter the mixture through hydrochloric acid R until the opalescence disappears. Dilute to
a fine sintered-glass filter (2.1.2) and rinse the reaction vessel 1000.0 mL with water R.
and filter with three quantities, each of 50 mL, of toluene R. Standardisation. To 20.0 mL of the zinc chloride solution add
Add the washings to the filtrate and dilute to 1000.0 mL with 5 mL of dilute acetic acid R and carry out the determination of
toluene R. Pass dry carbon dioxide-free nitrogen through the zinc by complexometry (2.5.11).
solution for 5 min.
Standardisation. To 10 mL of dimethylformamide R add 0.1 M Zinc sulfate. 3008600.
0.05 mL of a 3 g/L solution of thymol blue R in methanol R Dissolve 29 g of zinc sulfate R in water R and dilute to
and titrate with the tetrabutylammonium hydroxide solution 1000.0 mL with the same solvent.
until a pure blue colour is obtained. Immediately add 0.200 g Standardisation. To 20.0 mL of the zinc sulfate solution add
of benzoic acid RV. Stir to effect solution, and titrate with 5 mL of dilute acetic acid R and carry out the determination of
the tetrabutylammonium hydroxide solution until the pure zinc by complexometry (2.5.11).
may be used provided that it has been satisfactorily satisfactory by means of a microbial challenge test using a
demonstrated that the process chosen delivers an adequate and suitable test micro-organism. A suspension of Pseudomonas
reproducible level of lethality when operated routinely within diminuta (ATCC 19146, NCIMB 11091 or CIP 103020) may
the established tolerances. The procedures and precautions be suitable. It is recommended that a challenge of at least
employed are such as to give an SAL of 10− 6 or better. 107 CFU per cm2 of active filter surface is used and that the
Dry heat sterilisation is carried out in an oven equipped suspension is prepared in tryptone soya broth which, after
with forced air circulation or other equipment specially passage throug the filter, is collected aseptically and incubated
designed for the purpose. The steriliser is loaded in such aerobically at 32 °C. Such products need special precautions.
a way that a uniform temperature is achieved throughout The production process and environment are designed to
the load. Knowledge of the temperature within the steriliser minimise microbial contamination and are regularly subjected
during the sterilisation procedure is usually obtained to appropriate monitoring procedures. The equipment,
by means of temperature-sensing elements inserted into containers and closures and, wherever possible, the ingredients
representative containers together with additional elements at are subjected to an appropriate sterilisation process. It is
the previously established coolest part of the loaded steriliser. recommended that the filtration process is carried out as
The temperature throughout each cycle is suitably recorded. close as possible to the filling point. The operations following
Where a biological assessment is carried out, this is obtained filtration are carried out under aseptic conditions.
using a suitable biological indicator (5.1.2). Solutions are passed through a bacteria-retentive membrane
Dry heat at temperatures greater than 220 °C is frequently with a nominal pore size of 0.22 μm or less or any other
used for sterilisation and depyrogenation of glassware. In this type of filter known to have equivalent properties of bacteria
case demonstration of a 3 log10 reduction in heat resistant retention. Appropriate measures are taken to avoid loss of
endotoxin can be used as a replacement for biological solute by adsorption on to the filter and to avoid the release
indicators (5.1.2). of contaminants from the filter. Attention is given to the
bioburden prior to filtration, filter capacity, batch size and
Ionising radiation sterilisation. Sterilisation by this method duration of filtration. The filter is not used for a longer period
is achieved by exposure of the product to ionising radiation than has been approved by validation of the combination of
in the form of gamma radiation from a suitable radioisotopic the filter and the product in question.
source (such as cobalt 60) or of a beam of electrons energised
The integrity of an assembled sterilising filter is verified before
by a suitable electron accelerator.
use and confirmed after use by carrying out tests appropriate
In some countries there are regulations that lay down rules to the type of filter used and the stage of testing, for example
for the use of ionising radiation for sterilisation purposes, for bubble-point, pressure hold or diffusion rate tests.
example, in the appropriate European Community Notes for
Due to the potential additional risks of the filtration method
Guidance.
as compared with other sterilisation processes, a prefiltration
For this method of terminal sterilisation the reference through a bacteria-retentative filter may be advisable in cases
absorbed dose is 25 kGy. Other doses may be used provided where a low bioburden cannot be ensured by other means.
that it has satisfactorily been demonstrated that the dose
chosen delivers an adequate and reproducible level of lethality ASEPTIC PREPARATION
when the process is operated routinely within the established The objective of aseptic processing is to maintain the sterility
tolerances. The procedures and precautions employed are of a product that is assembled from components, each of
such as to give an SAL of 10− 6 or better. which has been sterilised by one of the above methods. This is
During the sterilisation procedure the radiation absorbed by achieved by using conditions and facilities designed to prevent
the product is monitored regularly by means of established microbial contamination. Aseptic processing may include
dosimetry procedures that are independent of dose rate. aseptic filling of products into container/closure systems,
Dosimeters are calibrated against a standard source at a aseptic blending of formulations followed by aseptic filling
reference radiation plant on receipt from the supplier and at and aseptic packaging.
suitable intervals of not longer than one year thereafter. In order to maintain the sterility of the components and the
Where a biological assessment is carried out, this is obtained product during processing, careful attention needs to be given
using a suitable biological indicator (5.1.2). to :
– environment,
Gas sterilisation. This method of sterilisation is only to be
used where there is no suitable alternative. It is essential – personnel,
that penetration by gas and moisture into the material to – critical surfaces,
be sterilised is ensured and that it is followed by a process – container/closure sterilisation and transfer procedures,
of elimination of the gas under conditions that have been – maximum holding period of the product before filling into
previously established to ensure that any residue of gas or its the final container.
transformation products in the sterilised product is below the Process validation includes appropriate checks on all the
concentration that could give rise to toxic effects during use of
above and checks on the process are regularly carried out by
the product. Guidance on this aspect with respect to the use means of process simulation tests using microbial growth
of ethylene oxide is provided, for example, in the appropriate media which are then incubated and examined for microbial
European Community Notes for Guidance.
contamination (media fill tests). In addition, a suitable sample
Wherever possible, the gas concentration, relative humidity, of each batch of any product that is sterilised by filtration
temperature and duration of the process are measured and/or aseptically processed is tested for sterility (2.6.1) before
and recorded. Measurements are made where sterilisation the batch is released.
conditions are least likely to be achieved, as determined at
validation.
01/2011:50102
The effectiveness of the process applied to each sterilisation
load is checked using a suitable biological indicator (5.1.2).
A suitable sample of each batch is tested for sterility (2.6.1)
5.1.2. BIOLOGICAL INDICATORS OF
before the batch is released. STERILISATION
FILTRATION Biological indicators are standardised preparations of
Certain active ingredients and products that cannot be selected micro-organisms used to assess the effectiveness
terminally sterilised may be subjected to a filtration procedure of a sterilisation procedure. They usually consist of a
using a filter of a type that has been demonstrated to be population of bacterial spores placed on a suitable inert
carrier. The inoculated carrier is covered in such a way that it radiation energy, especially in the case of accelerated electron
is protected from any deterioration or contamination, while sterilisation. The spores of Bacillus pumilus (for example,
allowing the sterilising agent to enter into contact with the ATCC 27142, NCTC 10327, NCIMB 10692 or CIP 77.25)
micro-organisms. Spore suspensions may be presented in or other strains of micro-organisms having demonstrated
sealed ampoules. Biological indicators are prepared in such equivalent performance are recommended. The number of
a way that they can be stored under defined conditions ; an viable spores exceeds 1 × 107 per carrier. The D-value is not
expiry date is set. less than 1.9 kGy. It is verified that there is no growth of the
Micro-organisms of the same bacterial species as the bacteria reference micro-organisms after the biological indicators have
used to manufacture the biological indicators may be been exposed to 25 kGy (minimum absorbed dose).
inoculated directly into a liquid product to be sterilised or Gas sterilisation. The use of biological indicators is necessary
into a liquid product similar to that to be sterilised. In this for all gas sterilisation procedures, both for the validation
case, it must be demonstrated that the liquid product has no of the cycles and for routine operations. Gas sterilisation
inhibiting effect on the spores used, especially as regards their is widely used for medical devices, isolators, chambers, etc.
germination. Use for such purposes is outside the scope of the European
A biological indicator is characterised by the name of the Pharmacopoeia. The use of spores of Bacillus atrophaeus (for
species of bacterium used as the reference micro-organism, example, ATCC 9372, NCIMB 8058 or CIP 77.18) or other
the number of the strain in the original collection, the number strains of micro-organisms having demonstrated equivalent
of viable spores per carrier and the D-value. The D-value is performance is recommended for ethylene oxide. The number
the value of a parameter of sterilisation (duration or absorbed of viable spores exceeds 1 × 106 per carrier. The parameters
dose) required to reduce the number of viable organisms to of resistance are the following : the D-value is not less than
10 per cent of the original number. It is of significance only 2.5 min for a test cycle involving 600 mg/L of ethylene oxide,
under precisely defined experimental conditions. Only the at 54 °C and at 60 per cent relative humidity. It is verified that
stated micro-organisms are present. Biological indicators there is no growth of the reference micro-organisms after
consisting of more than one species of bacteria on the same the biological indicators have been exposed to the test cycle
carrier may be used. Information on the culture medium and described above for 25 min and that exposing the indicators
the incubation conditions is supplied. to a reduced temperature cycle (600 mg/L, 30 °C and 60 per
It is recommended that the indicator organisms are placed cent relative humidity) for 50 min leaves revivable spores.
at the locations presumed, or wherever possible, found by
previous physical measurement to be least accessible to the
sterilising agent. After exposure to the sterilising agent,
aseptic technique is used to transfer carriers of spores to the 01/2011:50103
culture media, so that no contamination is present at the time
of examination. Biological indicators that include an ampoule 5.1.3. EFFICACY OF ANTIMICROBIAL
of culture medium placed directly in the packaging protecting
the inoculated carrier may be used.
PRESERVATION
A choice of indicator organisms is made such that : If a pharmaceutical preparation does not itself have adequate
a) the resistance of the test strain is suitable for the particular antimicrobial activity, antimicrobial preservatives may be
sterilisation method and is great compared to the resistance of added, particularly to aqueous preparations, to prevent
micro-organisms potentially contaminating the product ; proliferation or to limit microbial contamination which,
b) the test strain is non-pathogenic ; during normal conditions of storage and use, particularly for
multidose containers, could occur in a product and present
c) the test strain is easy to culture. a hazard to the patient from infection and spoilage of the
After incubation, growth of the reference micro-organisms preparation. Antimicrobial preservatives must not be used as
subjected to a sterilisation procedure indicates that the a substitute for good manufacturing practice.
procedure has been unsatisfactory. The efficacy of an antimicrobial preservative may be enhanced
Steam sterilisation. The use of biological indicators intended or diminished by the active constituent of the preparation
for steam sterilisation is recommended for the validation of or by the formulation in which it is incorporated or by the
sterilisation cycles. Spores of Geobacillus stearothermophilus container and closure used. The antimicrobial activity of the
(for example, ATCC 7953, NCTC 10007, NCIMB 8157 preparation in its final container is investigated over the period
or CIP 52.81) or other strains of micro-organisms having of validity to ensure that such activity has not been impaired
demonstrated equivalent performance are recommended. by storage. Such investigations may be carried out on samples
The number of viable spores exceeds 5 × 105 per carrier. The removed from the final container immediately prior to testing.
D-value at 121 °C is not less than 1.5 min. It is verified that During development of a pharmaceutical preparation, it
exposing the biological indicators to steam at 121 ± 1 °C for shall be demonstrated that the antimicrobial activity of the
6 min leaves revivable spores, and that there is no growth of preparation as such or, if necessary, with the addition of
the reference micro-organisms after the biological indicators a suitable preservative or preservatives provides adequate
have been exposed to steam at 121 ± 1 °C for 15 min. protection from adverse effects that may arise from microbial
Dry-heat sterilisation. Spores of Bacillus atrophaeus (for contamination or proliferation during storage and use of the
example, ATCC 9372, NCIMB 8058 or CIP 77.18) or other preparation.
strains of micro-organisms having demonstrated equivalent The efficacy of the antimicrobial activity may be demonstrated
performance are recommended for the preparation of by the test described below. The test is not intended to be used
biological indicators. The number of viable spores exceeds for routine control purposes.
1 × 106 per carrier and the D-value at 160 °C is not less
than 2.5 min. Dry heat at temperatures greater than 220 °C TEST FOR EFFICACY OF ANTIMICROBIAL
is frequently used for sterilisation and depyrogenation of PRESERVATION
glassware. In this case, demonstration of a 3 log10 reduction in The test consists of challenging the preparation, wherever
heat-resistant bacterial endotoxin can be used as a replacement possible in its final container, with a prescribed inoculum of
for biological indicators. suitable micro-organisms, storing the inoculated preparation
Ionising radiation sterilisation. Biological indicators may at a prescribed temperature, withdrawing samples from the
be used to monitor routine operations, as an additional container at specified intervals of time and counting the
possibility to assess the effectiveness of the set dose of organisms in the samples so removed.
General Notices (1) apply to all monographs and other texts 557
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The preservative properties of the preparation are adequate if, filtration (2.6.12). Ensure that any residual antimicrobial
in the conditions of the test, there is a significant fall or no activity of the product is eliminated by dilution, by filtration or
increase, as appropriate, in the number of micro-organisms by the use of a specific inactivator. When dilution procedures
in the inoculated preparation after the times and at the are used, due allowance is made for the reduced sensitivity
temperatures prescribed. The acceptance criteria, in terms of in the recovery of small numbers of viable micro-organisms.
decrease in the number of micro-organisms with time, vary When a specific inactivator is used, the ability of the system to
for different types of preparations according to the degree of support the growth of the test organisms is confirmed by the
protection intended (see Tables 5.1.3.-1/2/3). use of appropriate controls.
Test micro-organisms The procedure is validated to verify its ability to demonstrate
the required reduction in count of viable micro-organisms.
Pseudomonas aeruginosa ATCC 9027 ; NCIMB 8626 ; CIP 82.118.
01/2014:50104 If it has been shown that none of the prescribed tests will
allow valid enumeration of micro-organisms at the level
5.1.4. MICROBIOLOGICAL QUALITY prescribed, a validated method with a limit of detection as
OF NON-STERILE PHARMACEUTICAL close as possible to the indicated acceptance criterion is used.
In addition to the micro-organisms listed in Table 5.1.4.-1, the
PREPARATIONS AND SUBSTANCES significance of other micro-organisms recovered is evaluated
FOR PHARMACEUTICAL USE(1) in terms of :
The presence of certain micro-organisms in non-sterile – use of the product : hazard varies according to the route of
preparations may have the potential to reduce or even administration (eye, nose, respiratory tract) ;
inactivate the therapeutic activity of the product and has – nature of the product : its ability to support growth, the
a potential to adversely affect the health of the patient. presence of adequate antimicrobial preservation ;
Manufacturers therefore have to ensure a low bioburden of – method of application ;
finished dosage forms by implementing current guidelines
– intended recipient : risk may differ for neonates, infants,
on Good Manufacturing Practice during the manufacture,
the debilitated ;
storage and distribution of pharmaceutical preparations.
Microbial examination of non-sterile products is performed – use of immunosuppressive agents, corticosteroids ;
according to the methods given in general chapters 2.6.12 and – presence of disease, wounds, organ damage.
2.6.13. Acceptance criteria for non-sterile pharmaceutical Where warranted, a risk-based assessment of the relevant
products based upon the total aerobic microbial count factors is conducted by personnel with specialised training in
(TAMC) and the total combined yeasts/moulds count (TYMC) microbiology and the interpretation of microbiological data.
are given in Tables 5.1.4.-1 and 5.1.4.-2. Acceptance criteria For raw materials, the assessment takes account of processing
are based on individual results or on the average of replicate to which the product is subjected, the current technology of
counts when replicate counts are performed (e.g. direct testing and the availability of materials of the desired quality.
plating methods).
When an acceptance criterion for microbiological quality is Table 5.1.4.-2. – Acceptance criteria for microbiological quality
prescribed it is interpreted as follows: of non-sterile substances for pharmaceutical use
1
– 10 CFU : maximum acceptable count = 20 ; TAMC TYMC
– 102 CFU : maximum acceptable count = 200 ; (CFU/g or CFU/mL) (CFU/g or CFU/mL)
– 103 CFU : maximum acceptable count = 2000, and so forth.
Substances for 103 102
Table 5.1.4.-1 includes a list of specified micro-organisms for pharmaceutical use
which acceptance criteria are set. The list is not necessarily
exhaustive and for a given preparation it may be necessary to ♦Recommended acceptance criteria for microbiological quality
test for other micro-organisms depending on the nature of the of herbal medicinal products for oral use and extracts used in
starting materials and the manufacturing process. their preparation are given in general chapter 5.1.8.♦
Table 5.1.4.-1. – Acceptance criteria for microbiological quality of non-sterile dosage forms
TAMC TYMC
Route of administration (CFU/g or (CFU/g or Specified micro-organisms
CFU/mL) CFU/mL)
Non-aqueous preparations for oral use 103 102 Absence of Escherichia coli (1 g or 1 mL)
(1) This chapter has undergone pharmacopoeial harmonisation. See chapter 5.8. Pharmacopoeial harmonisation.
General Notices (1) apply to all monographs and other texts 559
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(bioluminescence in tube or microtitre plate), total ability to detect the presence of specific enzymes using suitable
viable aerobic count (bioluminescence on membrane), substrates has led to the development of a large number of
environmental and water monitoring. The method finds methods for the identification of micro-organisms employing
applications in filterable and non-filterable products. manual or automated methods. The incorporation of such
2-1-1-5. Microcalorimetry substrates into a selective or non-selective primary isolation
Principles of measurement. Microbial catabolism generates medium can eliminate the need for further subculture
heat which can be accurately measured by microcalorimetry. and biochemical tests to identify certain micro-organisms.
Heat production can be detected by placing the contaminated Consequently, chromogenic liquid or solid culture media are
sample in a sealed ampoule containing a growth medium designed to produce specific enzymatic activities for detection
and enclosing within a calorimeter. Using sensitive and differentiation of micro-organisms. In these particular
instrumentation microbial growth curves can be established. media, defined substrates are introduced into the formulation
High bioburdens may be detectable by flow calorimetry. and are hydrolysed by the specific cell enzyme of a given
bacteria or fungi during growth. These substrates are chosen
Critical aspects. Theoretically, this method does not require according to the diagnostic enzymatic activity sought and are
microbial growth but simply catabolic activity. Nevertheless, linked to coloured indicators.
a minimum number of micro-organisms are required to give
heat output measures above base-line and this is usually Critical aspects. The use of innovative media presents several
achieved by use of an enrichment method. advantages : improved discrimination of colonies in mixed
culture, ease of use and ease of interpretation. In addition,
Potential uses. Test for efficacy of antimicrobial preservation. response times are shorter because growth and identification
2-1-1-6. Turbidimetry of the micro-organism are simultaneous. However, validation
Principles of measurement. Microbial growth will lead to of the media must be undertaken carefully to ensure a
detectable changes in medium opacity. This can be accurately combination of specificity, selectivity and robustness. The
quantified by optical density measurement at a specified quality of the signal is based not only on the careful choice
wavelength. In its simplest form such measurements are of the enzymes used as the basis of detection, as these
performed in a standard spectrophotometer over a wavelength enzymes may be present in different genera, but also on
range generally of 420-615 nm. Alternative automated systems physico-chemical characteristics of the medium such as pH.
employ microtitre plate readers offering continuous readout Potential uses. Detection of specified micro-organisms such as
with early detection of optical density change. E. coli, coliforms, Salmonella, Staphylococcus and Streptococcus
Critical aspects. Attempts have been made to extrapolate the spp. ; particular benefit may be found in presence/absence
initial bioburden from the time for detection but this may be testing. Yeasts can also be detected using chromogenic culture
limited to healthy micro-organisms with reproducible growth media.
characteristics. The methods cannot distinguish between 2-2. DIRECT MEASUREMENT
viable and non-viable micro-organisms.
Potential uses. By means of calibration graphs, determination 2-2-1. Solid phase cytometry
of the inoculum size of microbial suspensions for use in Principles of measurement. A membrane filter is used to
pharmacopoeial tests. In automated mode, establishment of retain microbial contaminants. Micro-organisms are stained
the preservative sensitivity of test micro-organisms recovered by labelling using a fluorophore as a viability indicator,
from formulated products. either before or after filtration. The fluorophore is initially
2-1-1-7. Phage-based methods a non-fluorogenic, conjugated substrate that requires
intracellular enzymatic activity to cleave the substrate and
Principles of measurement. Bacterial viruses (bacteriophage,
release the fluorescent moiety. An intact cellular membrane
phage) can infect host cells causing either lysis or incorporation
is required to retain fluorophore within the cytoplasm. Laser
of their genetic material and expression of novel proteins.
excitation and automated scanning allows the detection of
Their high level of host specificity can be employed in
single, viable fluorescent micro-organisms. Appropriate
detection methods which exploit the consequences of infection
software permits differentiation of viable micro-organisms
as an end-point. Such end-points include : plaque formation
from auto-fluorescent particles. The high sensitivity and
on a solid lawn of reporter bacteria ; detection of intracellular
rapidity of detection permits near-real-time detection of
contents released from lysed bacteria (possibly by colorimetric
microbial contaminants. Total cell counts can be obtained
method) ; or phage-induced effects such as ice nucleation or
using a permanently fluorescing stain.
bioluminescence following infection by genetically modified
phage. Fluorescently labelled coliphages can be used for the Critical aspects. Metabolically active, fastidious and viable
selective detection of viable E. coli in combination with DEFT non-culturable micro-organisms can be detected. This may
(see 2-3-3.). result in reappraisal of the microbial limits established for
Critical aspects. Phage-based detection can be used in both the samples under evaluation. Spores require initiation of
single and mixed cultures where host specificity allows germination to enable detection. Single cell detection may
both detection and identification. Detectable end-points be achievable, but identification is not currently part of the
often require a minimum number of target cells to ensure a routine test protocol. The use of fluorescent antibody may
measurable signal, necessitating enrichment in situations of offer a route to selective detection. False positives may occur
low bioburden. The viral infection process can be adversely from auto-fluorescent particles, which can be difficult to
affected by sample composition. In most cases there is a differentiate from micro-organisms.
narrow host range which makes it difficult to detect a broad Potential uses. Rapid and sensitive method for the non-specific
spectrum of microbial contaminants. evaluation of bioburden. It has found applications in testing
Potential uses. These methods are used mainly for research pharmaceutical-grade waters.
purposes with commercial development aimed principally 2-2-2. Flow cytometry
towards uses in clinical and food microbiology. These Principles of measurement. Fluorophore-labelled
methods are likely to be employed for presence/absence micro-organisms can be detected in suspension as they pass
determinations of specified micro-organisms. through a flow cell cytometer. Where a viability-indicating
2-1-2. Media development to improve detection fluorophore substrate is employed, viable micro-organisms
Principles of measurement. Culture media have existed for can be differentiated from non-viable particles (see 2-2-1.).
many years and have been constantly improved. A recent Critical aspects. Flow cytometry may be applied for the
innovation is the appearance of chromogenic substrates which microbiological analysis of both filterable and non-filterable
are increasingly used in clinical and food microbiology. The materials. Flow cytometric analysis gives near-real-time
detection, but it is not as sensitive as solid phase cytometry. 2-3-1-2. Fatty acid profiles
To increase sensitivity for use in the pharmaceutical field, it Principles of measurement. The fatty acid composition of
often becomes necessary to add an incubation step in culture micro-organisms is stable, well conserved and shows a high
media and in that case the method becomes a growth-based degree of homogeneity within different taxonomic groups.
method. Analysis of non-filterable samples may require serial The isolate is grown on a standard medium and harvested.
dilution to optimise performance, and particulate size can The fatty acids are saponified, methylated and extracted and
have a significant effect on performance. With the exception the occurrence and amount of the resulting fatty acid methyl
of filterability, similar considerations apply to those of solid esters are measured by high resolution gas chromatography.
phase cytometry. Clumping of bacteria can be a problem (e.g. The fatty acid composition of an unknown isolate is compared
S. aureus). with a database of known isolates for a possible match and
Potential uses. In contrast with solid phase cytometry, this identification.
method offers the potential to detect and enumerate the Critical aspects. The use of fatty acid profiles for microbial
microbial bioburden in materials containing significant levels identification requires a high degree of standardisation. It
of particulate matter. If a pre-incubation step is needed, the is critical for the fatty acid composition of microbial cells
method becomes a qualitative determination. that isolates are grown using standard media and standard
2-2-3. Direct epifluorescent filtration technique (DEFT) incubation conditions. Standard conditions for operation
Principles of measurement. This technique may be considered of the gas chromatograph must be employed, with frequent
to be a forerunner of solid phase cytometry. Micro-organisms runs of calibration standards and known isolates being very
concentrated by filtration from the sample are stained with important.
a fluorescent dye, formerly acridine orange and now more Potential uses. Identification or characterisation of
commonly 4′,6-diamidino-2-phenylindole (DAPI), that may environmental and product flora for contaminant tracing and
be detected by epifluorescent illumination. Fluorescent vital detection of specified micro-organisms.
staining techniques as employed in solid phase cytometry
2-3-1-3. Fourier transform infrared (FTIR) spectroscopy
(see 2-2-1.) are amenable to DEFT and fluorescent redox dyes
such as 5-cyano-2,3-ditolyltetrazolium chloride (CTC) can be Principles of measurement. A Fourier transformation of the
used to highlight respiring cells. Coupled with microscopy, infrared spectrum of whole micro-organisms gives a stable,
the method allows rapid detection of micro-organisms, recognisable pattern typical of the taxonomic groups of
the absolute sensitivity depending on the volume of micro-organisms. The analysis of the FTIR pattern can be
product filtered and the number of fields of view examined. performed in instruments available on the market. The isolate
Semi-automated auto-focusing systems coupled to image is grown on a standard medium and harvested. Cell mass is
analysis have served to improve the utility of this method. transferred to a carrier, and the infrared spectrum is recorded.
A modification to the principle employs sampling using an The Fourier transformation is calculated and the pattern is
adhesive sheet which permits collection of cells from surfaces, compared with a database of known isolates for a possible
staining on the sheet and subsequent direct observation under match and identification.
the epifluorescence microscope. Critical aspects. The use of FTIR-patterns for microbial
Critical aspects. The distribution of micro-organisms on identification requires a high degree of standardisation. It is
the membrane affects method robustness. The intensity critical for the FTIR-pattern of microbial cells that isolates
of fluorescence can be influenced by the staining process are grown using standard media and standard incubation
and the metabolic status of the micro-organisms. A brief conditions. The cells must be in the same state of the growth
period of culture on the filter surface prior to staining cycle when analysed. Particular attention needs to be paid to
allows microcolony formation ; these microcolonies stain the validation process.
readily, can be easily enumerated and are demonstrable Potential uses. Identification or characterisation of
evidence of viability. Developments using fluorescence in environmental and product flora for contaminant tracing and
situ hybridisation (FISH) arising from the complementary detection of specified micro-organisms.
interaction of a fluorescently-labelled oligonucleotide probe
with a specific rRNA sequence offer a route to selective 2-3-1-4. Mass spectrometry
detection. Principles of measurement. Gaseous breakdown products
Potential uses. DEFT is generally limited to low viscosity released by heating microbial isolates in a vacuum can be
fluids although pre-dilution or pre-filtration has occasionally analysed by mass spectrometry, providing characteristic
been applied to viscous or particulate products. Bioburden spectra. Similarly, intact microbial cells, when subject to
monitoring has been successfully achieved in aqueous intense ionisation under matrix-assisted laser desorption
pharmaceuticals. ionisation-time of flight (MALDI-TOF) mass spectrometry,
release a distinctive pattern of charged species. Such spectra
2-3. CELL COMPONENT ANALYSIS can be compared with known profiles as a rapid aid to
2-3-1. Phenotypic identification.
2-3-1-1. Immunological methods Critical aspects. Isolates require culture prior to analysis.
Principles of measurement. Antibody-antigen reactions can be Potential uses. Identification or characterisation of
employed to detect unique cellular determinants of specific environmental and product flora for contaminant tracing and
organisms. These reactions can be linked to agglutination detection of specified micro-organisms.
phenomena, colorimetric or fluorimetric end-points offering
2-3-1-5. Biochemical assays based on physiological reactions
both quantitative and qualitative detection. Enzyme-linked
immunosorbent assays (ELISA) offer simple solid phase Principles of measurement. These assays are usually preceded
methodologies. by a Gram stain or other early differentiation test to decide on
the appropriate testing protocol. Microbial cell suspensions are
Critical aspects. Immunological detection methods depend
tested using biochemical test kits. Micro-organisms are known
upon the unique expression of specific identifiers but
to have particular reactions to these biochemical substances,
do not necessarily demonstrate the presence of viable
e.g. utilisation of specific carbon sources. The identification
micro-organisms.
of the culture is done by comparing the biochemical reaction
Potential uses. Detection and identification of specified profile with a database. These methods can be performed
micro-organisms. manually or by automated instruments.
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Critical aspects. A pure colony is needed which must not be Critical aspects of RNA amplification techniques. These
older than 3 days. The handling of the system is easy but the methods have proven to be very valuable for specific
interpretation of the results can be subjective. Depending on (quantitative) RNA detection. However, they may be more
the system used and the micro-organism under investigation, difficult to implement routinely.
the results can be available quickly. Critical aspects of (semi-) quantitative detection (real-time
Potential uses. Identification of environmental and product PCR). Classical PCR techniques are based on end-point
flora for contaminant tracing and detection of specified detection. In general fragment analysis is carried out using
micro-organisms. agarose gels and specific size markers. However, there is no
correlation between the amount of PCR product at the end
2-3-2. Genotypic of the reaction and the original amount of target molecule.
2-3-2-1. Nucleic acid amplification techniques (NAAT) In contrast the amount of PCR product detected at the
beginning of the exponential phase of the reaction correlates
General principles of measurement. NAAT rely on the very well with the initial starting amount of nucleic acid.
reiteration of the process of DNA polymerisation, leading to Modern real-time PCR techniques are developed to measure
an exponential increase of a specific fragment of the nucleic this exponential phase of the reaction. These techniques
acid, i.e. the use of the polymerase chain reaction (PCR). In generate amplification data from which the original amount
this thermophilic cyclic process a specific DNA fragment is of target molecule can be deduced. A specific labelled probe
amplified using oligonucleotide primers (see also general detects in real time the PCR product formed, allowing direct
method 2.6.21). RNA can also be amplified by PCR after visualisation of the exponential part of the PCR reaction.
transcription into cDNA using a reverse transcriptase. This By comparison with amplification plots of a standard
technique is known as reverse transcriptase PCR (RT-PCR). dilution series, a quantification of the target molecule can be
Alternatively, specific RNA-based amplification techniques, obtained. Automated real-time PCR systems are available
for example nucleic acid sequence-based amplification on the market. An additional advantage is that the chance
(NASBA) or transcription-mediated amplification (TMA) of cross-contamination is minimised, as PCR products are
are available to amplify multiple antisense copies of the RNA scanned with a laser while the tubes remain closed. However,
target. Amplified nucleic acid fragments can be analysed by generation of standards will be difficult to accomplish.
several methods : fragment size analysis ; specific sequence
analysis ; reamplification with a second primer pair ; or Critical aspects of amplification of genes coding for 16S or 23S
specific detection by hybridisation with a fluorescent labelled rRNA. A powerful application of PCR is the amplification
probe. Depending on the choice of analysis the amplification and subsequent sequence analysis of specific parts of the
technique can be qualitative, semi-quantitative or quantitative. genes coding for 16S or 23S rRNA. Analysis of these specific
For identification/characterisation purposes sequence analysis DNA sequences allows in most cases the identification of
of specific parts of the genome can be used (i.e. 16S or 23S a micro-organism at species level. Selection of appropriate
rRNA targets). universal primers, or even species-specific primer pairs, from
international databases allows a high specificity in fragment
General critical aspects. NAAT have many advantages over amplification. Modern systematic classification is based on
classical methods for the detection of micro-organisms : comparative sequence analysis.
– the methods are highly specific, provided that the primers Potential uses. Owing to the high specificity of the
chosen are specific for a particular micro-organism or amplification techniques, they are very suitable for
group of micro-organisms ; identification purposes. NAAT are suitable for the detection
– the procedures are rapid, overcoming the problem of of specified micro-organisms or certain groups such as
prolonged incubation times ; mycoplasmas. Real-time quantitative PCR is needed for
enumeration.
– the methods are highly sensitive allowing ideally the 2-3-2-2. Genetic fingerprinting
detection and amplification of one single nucleic acid
fragment in the reaction mix. Principles of measurement. This technique characterises
and identifies micro-organisms using restriction fragments
However, there are numerous practical restrictions to its use : of nucleic acids from bacterial and fungal genomes. DNA
– the sensitivity of the methods is highly dependent on how is extracted from a pure microbial cell lysate and cut into
successfully the target fragments can be concentrated in fragments by restriction enzymes. DNA fragments are
the sample ; size-separated by electrophoresis, visualised, and the pattern is
compared with other known patterns of microbial isolates. The
– the presence of inhibitors of the enzymatic process result genetic fingerprint is a stable marker that provides definitive
in false negative reactions ; species discrimination or even characterisation below species
– the starting volume of the sample tested is small ; level. Ribotyping is a typical example of this technique. There
are also fingerprinting methods based on PCR with primers
– the procedures are prone to cross-contamination from that bind to several sites in the microbial genome, creating
previously amplified fragments resulting in false positive amplicons with a characteristic size distribution.
results.
Critical aspects. There is a need for a pure colony, but
Depending on the aim, a choice must be made for amplification no preliminary cultivation step is necessary. The growth
of an RNA or DNA target. The target choice affects the conditions (temperature, type of media,) do not affect the
correlation with viability. The use of DNA as a marker has the outcome of the analysis. For the identification of bacteria
disadvantage that dead micro-organisms also contain DNA, semi-automated systems are on the market.
whereas mRNA is rapidly degraded in dead bacteria and is Potential uses. Genetic fingerprinting is more valuable for
considered a better marker for viability. strain discrimination (characterisation below species level)
Critical aspects of RT-PCR. Reverse transcriptase-PCR is than for identification of species.
characterised by the synthesis of cDNA using RNA as a
template. Reverse transcriptase is used for this step. A 3. GENERAL VALIDATION REQUIREMENTS
specific part of the cDNA is subsequently amplified by The purpose of this section is to provide guidance on the
PCR. Depending on the quality of the RNA isolation, the validation of methods for use as alternatives to microbiological
cDNA synthesis efficiency can vary. RT-PCR can be used to methods of the Pharmacopoeia. For microbial recovery and
specifically detect RNA if the DNA contamination of the RNA identification, microbiological testing laboratories sometimes
sample is minimal. use alternative test methods to those described in the
general chapters for a variety of reasons, such as economics, of this could be the sterility test where this would translate
throughput, and convenience. Validation of these methods into a comparison of the rate of positive and negative
is required. Some guidance on validation is provided in the results produced by the alternative method versus the
General Notices section 1.1 on the use of alternative methods. pharmacopoeial method for identical samples. However, in
Validation of alternative microbiological methods must take a case such as the sterility test, the low number of failures
into account the large degree of variability associated with would required thousands of comparison tests to establish
conventional methods. When conducting microbiological equivalency and thus would be problematic.
testing by conventional plate count, for example, one A more feasible method for evaluating the precision
frequently encounters a range of results that is broader than of an alternative qualitative method compared with a
ranges in commonly used chemical tests. pharmacopoeial method might be to observe the degree
Where specific equipment is critical for the application of of agreement between the two when the procedures are
the alternative method, the equipment, including computer performed repeatedly on different lots of the same product.
hardware and software, must be fully qualified as follows : The accuracy and precision of the alternative method may be
– design qualification (DQ) to provide documented evidence expressed as the relative rates of false positive and false negative
that the design of the equipment is suitable for correct results between the new method and the pharmacopoeial
performance of the method ; to be provided by the supplier ; method using a standardised, low-level inoculum.
– installation qualification (IQ) to provide documented The rate of occurrence of false negative results in the presence
evidence that the equipment has been provided and of the sample for the 2 methods can be estimated using low
installed in accordance with its specification ; levels of test micro-organisms. This design is similar to the
– operational qualification (OQ) to provide documented standard bacteriostasis/fungistasis test ; however, the level of
evidence that the installed equipment operates within micro-organisms inoculated must be very low, for example
pre-determined limits when used in accordance with its about 5 CFU per unit. The level of inoculum should ensure a
operational procedures ; frequency of failure rates high enough to provide a means to
compare the 2 methods. The alternative method must provide
– performance qualification (PQ) to provide documented at least as high a frequency of recovery as the pharmacopoeial
evidence that the equipment, as installed and operated method.
in accordance with operational procedures, consistently
performs in accordance with predetermined criteria 3-2-2. Specificity
and thereby yields correct results for the method. The specificity of an alternative qualitative method is its
This is typically done with a ‘model’ system (with test ability to detect the required range of micro-organisms that
micro-organisms) to make sure that the conditions used by may be present in the sample under test. This concern is
the user laboratory make it possible to satisfy the criteria adequately addressed by growth promotion of the media for
described by the supplier of the method in the laboratory. qualitative methods that rely upon growth to demonstrate
Some alternative methods depend on the use of databases. presence or absence of micro-organisms. For those methods
The extent of coverage of the database with respect to the that do not require growth as an indicator of microbial
range of micro-organisms of interest must be taken into presence, the specificity assures that extraneous matter in the
account for validation purposes. test system does not interfere with the test. Where relevant
The value of a new or modified method must be demonstrated for the purpose of the test, mixtures of micro-organisms are
in a comparative study between the official method and the used during validation.
alternative method. The characteristics defined in this chapter 3-2-3. Limit of detection
must be used to establish this comparison. The limit of detection of an alternative qualitative method
3-1. TYPES OF MICROBIOLOGICAL TESTS is the lowest number of micro-organisms in a sample that
It is critical to the validation effort to identify the portion of can be detected under the stated experimental conditions. A
the test addressed by the alternative method. For example, microbiological limit test determines the presence or absence
there are a variety of methods available to detect the presence of micro-organisms. Due to the nature of microbiology, the
of viable cells. These methods may have applications in limit of detection refers to the number of micro-organisms
a variety of tests (e.g. bioburden, sterility tests,) but may present in the original sample before any dilution or incubation
not, in fact, replace the critical aspects of the test entirely. steps ; it does not refer to the number of micro-organisms
For example, a sterility test by membrane filtration may be present at the time of testing.
performed according to the pharmacopoeial procedure up to The 2 methods (alternative and pharmacopoeial) must be
the point of combining the processed filter with the recovery assessed by using an inoculum containing a low number of test
media, and after that the presence of viable cells might then micro-organisms, for example about 5 CFU per unit, followed
be demonstrated by use of some of the available methods. by a measurement of recovery. The level of inoculation
Validation of this application would, therefore, require must be adjusted until at least 50 per cent of the samples
validation of the recovery system employed rather than the show growth in the pharmacopoeial method. It is necessary
entire test. to repeat this determination several times, as the limit of
General concerns. Validation of a microbiological method detection of a test is determined from an appropriate number
is the process by which it is experimentally established of replicates (for example not less than 5). The ability of the
that the performance characteristics of the method meet 2 methods to detect the presence of single organisms can be
the requirements for the intended application. Since demonstrated using the χ2 test.
microbiological tests have 3 basic applications, 3 separate
sets of validation criteria are required. These concerns are 3-2-4. Robustness
described below. The robustness of an alternative qualitative method is a
3-2. VALIDATION OF ALTERNATIVE QUALITATIVE measure of its capacity to remain unaffected by small but
TESTS FOR THE PRESENCE OR ABSENCE OF deliberate variations in method parameters, and provides an
MICRO-ORGANISMS indication of the method’s reliability under a variety of normal
test conditions, such as different analysts, instruments, batches
3-2-1. Accuracy and precision of reagents and laboratories. Robustness can be defined as the
A direct method to show the equivalence of 2 qualitative intrinsic resistance to the influences exerted by operational and
methods would be to run them side by side and determine environmental variables on the results of the microbiological
the degree to which the method under evaluation shows method. Robustness is a validation parameter best suited to
equivalence to the pharmacopoeial method. An example determination by the supplier of the method, but if critical
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parameters are modified by the user their effects on robustness sample containing a known number of micro-organisms, it is
have to be evaluated. Robustness of a qualitative method is essential that the quantification limit is determined from a
judged by its ability to detect the test micro-organisms under number of replicates, for example at least 5. The results of
the deliberate variations to the method parameters. the linearity and accuracy studies can also be used. Here,
3-3. VALIDATION OF ALTERNATIVE QUANTITATIVE the lowest concentration in the linear range is considered to
TESTS FOR ENUMERATION OF MICRO-ORGANISMS be the limit of quantification of the method. The limit of
quantification must not be a number greater than that of the
3-3-1. Accuracy pharmacopoeial method.
The accuracy of an alternative quantitative method is the
3-3-5. Linearity
closeness of the test results obtained by the alternative
method to the value obtained by the pharmacopoeial method. The linearity of an alternative quantitative method is its ability
Accuracy must be demonstrated across the practical range of to produce results that are proportional to the concentration
the test. Accuracy is usually expressed as the percentage of of micro-organisms present in the sample within a given
recovery of micro-organisms by the method. range. The linearity must be determined over the range
Accuracy may be shown by preparing a suspension of corresponding to the purpose of the alternative method.
micro-organisms at the upper end of the range of the test, A method to determine this would be to select different
serially diluted down to the lower end of the range of the test. concentrations of each test micro-organism and conduct
For example, if the alternative method is meant to replace several replicates of each concentration. The number of
the traditional plate count method for viable counts, then a replicates is chosen so that the entire test can be carried out
reasonable range might be 100-106 CFU per mL. If it is, instead, during the same working session. 2 more working sessions
a replacement for the MPN method, a much more narrow are then completed under conditions of maximum variability
range may be used. At least 5 suspensions across the range (different reagents, different operators, different days, etc.).
of the test must be analysed for each test micro-organism. If After checking the homogeneity of the variances of the
the alternative method is meant to replace the conventional results obtained for each concentration, the regression line is
method, it must provide an estimate of viable micro-organisms calculated. Linearity is demonstrated if the estimated slope
of not less than 70 per cent of the estimate provided by the is significant and if the test for deviation from linearity is
pharmacopoeial method. non-significant.
The protocol used to check the linearity (see 3-3-5.) of 3-3-6. Range
the method may also be used to check the accuracy : The range of an alternative quantitative method is the interval
the suspensions of micro-organisms prepared for the between the upper and lower levels of micro-organisms that
alternative method are counted at the same time using the have been determined with precision, accuracy, and linearity
pharmacopoeial method. Accuracy is demonstrated if the using the method as written. The range is determined from
suitability tests show that the slope of the regression line does studies of precision, accuracy and linearity.
not differ significantly from 1 and if the y-intercept is not 3-3-7. Robustness
significantly different from 0.
The robustness of an alternative quantitative method is a
3-3-2. Precision measure of its capacity to remain unaffected by small but
The precision of an alternative quantitative method is the deliberate variations in method parameters and provides an
degree of agreement among individual test results when the indication of its reliability under a variety of normal test
procedure is applied repeatedly to multiple samplings of conditions, such as different analysts, instruments, batches of
homogeneous suspensions of micro-organisms under the reagents and laboratories. Robustness can be defined as the
prescribed conditions. The precision is usually expressed as intrinsic resistance to the influences exerted by operational and
the variance, standard deviation or coefficient of variation of a environmental variables on the results of the microbiological
series of measurements. method. Robustness is a validation parameter best suited to
At the very least, a suspension of micro-organisms with a determination by the supplier of the method, but if critical
concentration usually in the middle of the range is counted parameters are modified by the user their effects on robustness
several times. The number of replicates is chosen so that have to be evaluated. Robustness of a quantitative method is
the entire test can be carried out during the same working judged by its ability to enumerate with statistical relevance
session, i.e. under the same operating conditions and without the test micro-organisms under the deliberate variations to
any change in the suspension of micro-organisms. Other the method parameters.
working sessions are then carried out under conditions of 3-4. VALIDATION OF ALTERNATIVE IDENTIFICATION
maximum variability (different reagents, different operators, TESTS
different days, etc.). The variance of the results observed in There is a large body of evidence that different methods vary
each of the working sessions (‘groups’) is calculated. If the considerably in their ability to identify micro-organisms in
variances are homogeneous, the variance of the repeatability pharmacopoeial products. It must be accepted that a method
can be calculated. The inter-group variance of the results is of systematics needs to be internally consistent, but may
calculated. The variance of the intermediate precision is the differ from others in identification of isolates. In other words,
sum of the variance of the repeatability and the inter-group identification of an isolate based on biochemical activity may
variance. The coefficients of variation are then calculated. lead to one conclusion, identification by fatty acid analysis
Generally, a coefficient of variation in the 10-15 per cent range to another, identification by DNA analysis may lead to a
is acceptable. Irrespective of the specific results, the alternative third, and other methods may lead to alternative conclusions.
method must have a coefficient of variation that is not larger Microbiological identifications by a particular system flow
than that of the pharmacopoeial method. directly from previous experience with that system, and
3-3-3. Specificity therefore may well differ from identifications by another
The specificity of an alternative quantitative method is system. It is critical that each system provides a consistent
demonstrated using a range of appropriate micro-organisms. identification of isolates from pharmacopoeial products.
Where relevant for the purpose of the test, mixtures of 3-4-1. Accuracy
micro-organisms are used during validation.
The accuracy of an alternative identification method is
3-3-4. Limit of quantification its ability to identify the desired micro-organism to the
The limit of quantification of an alternative quantitative required taxonomic level and to differentiate it from
method is the lowest number of micro-organisms that can be other micro-organisms present in the sample. It must be
accurately counted. As it is not possible to obtain a reliable demonstrated with a series of test micro-organisms or
micro-organisms obtained from a typical sample previously construction of a correct phylogenetic correlation tree, another
identified by another method. answer may be more useful in the context of pathogenicity or
3-4-2. Precision other properties of the differentiated micro-organisms.
The precision of an alternative identification method is the 4-1-1. Primary validation
degree of agreement among individual test results when the In order to characterise a specific microbiological method,
procedure is applied repeatedly to multiple samplings of the principle of detection must be clearly described by the
suspensions of test micro-organisms across the range of the supplier. The method must be fully detailed with respect to
test. the conditions required for application, the materials and
equipment needed, and the expected signal. The application
3-4-3. Robustness
principle should be described in a peer-reviewed journal.
The robustness of an alternative identification method is a The principle of detection must be characterised in a model
measure of its capacity to remain unaffected by small but system and/or with a panel of test micro-organisms, by at least :
deliberate variations in method parameters, and provides
an indication of its reliability under a variety of normal – prerequisite treatment of sample or micro-organisms ;
test conditions such as different analysts, instruments, – type of response ;
batches of reagents and laboratories. Robustness can be – specificity of the response ;
defined as the intrinsic resistance to the influences exerted – limit of detection ;
by operational and environmental variables on the results
– range ;
of the microbiological method. Robustness is a validation
parameter best suited to determination by the supplier of the – linearity of the response ;
method, but if critical parameters are modified by the user – accuracy and precision of the response ;
their effects on robustness have to be evaluated. Robustness – robustness of the method in a model system ;
of an identification method is judged by its ability to identify – limits of suitability.
consistently the test micro-organisms under the deliberate
variations to the method parameters. Once the method has been characterised in this way by the
supplier, the principle of detection need not be verified by
4. SPECIFIC VALIDATION REQUIREMENTS each user.
4-1. BACKGROUND 4-1-2. Validation of alternative microbiological method
Validation is defined in various contexts with some differences, 4-1-2-1. Risk-benefit analysis
but a consensus definition is to establish documented evidence For validation of specific alternative microbiological methods
that a process will consistently achieve what it is intended it is critical that the purpose of the procedure is precisely
to do. Hence, in order to perform correct validation of a outlined. Based on the purpose, the type and depth of
new method it is critical to understand and define what the information needed must be defined. The information
procedure is intended to achieve. obtained by, and the limitations of, the conventional method
2 levels of validation must be envisaged for the application and the alternative method must be considered and compared
of conventional or alternative microbiological methods. in a risk-benefit analysis.
Primary validation of a method is typically performed by An alternative method can be justified as being applicable if
the supplier of the new method, whereas validation for the the information obtained gives a scientifically sound answer
actual intended use, which is a verification of the suitability to the questions asked in the procedure, and if the limitations
or applicability of the method in a given situation, must be of the method are not more severe than the limitations of the
seen as the responsibility of the user. Before validation for the conventional method.
actual intended use, performance qualification is carried out 4-1-2-2. Validation for the actual intended use
by the user as described in 3. General validation requirements. The alternative method must be applied in the procedure used
Typically, microbiological methods use specific characteristics and with the samples to be analysed under the responsibility
of micro-organisms as indicators or detection principles for of the user, and must be shown to give comparable results
more general questions. The information needed is presence, as characterised in a model system by the supplier. Specific
number, viability, resistance or identity of micro-organisms in questions to be asked where applicable are :
a given product or environment. – compatibility of the response with the sample preparation
A given method will usually give an indirect and conditional needed for product testing ;
answer to the questions. For example, the total number and – limit and range of detection of the method with regard to
viability of micro-organisms is indicated by the number of sample size and sample availability ;
micro-organisms able to reproduce under a certain set of
– specificity of the response with regard to the influence of
conditions for sample preparation, cultivation and incubation.
the ingredients of the product ;
Reproduction in classical microbiology is hence taken as the
general indicator for viability. There are other parameters, – linearity of the response with regard to all types of samples
however, that can be used as an indication of viability. The to be analysed ;
level of ATP or accumulation or metabolism of substrates – accuracy and precision of the response with regard to all
in living cells can also be taken as an indicator for viability. types of samples to be analysed ;
The results of different indication methods for viability may – robustness of the method with regard to all types of samples
not always be identical. Micro-organisms may not be able to to be analysed.
reproduce on a given medium, but may still accumulate and Acceptance criteria for the method in routine use will need
metabolise a substrate. Micro-organisms may be unable at a to be defined as a function of the application and of the
given state of damage to accumulate a substrate, but may still validation data.
be able to recover and to reproduce.
4-2. BIOLUMINESCENCE FOR ENUMERATION OF
Another example is the various methods used for identification MICRO-ORGANISMS
of micro-organisms. Characterisation of the metabolic
pattern of micro-organisms is frequently used for species 4-2-1. Risk-benefit analysis
identification, whereas another method consists of the Extensive scientific evidence and use for years supports the
comparison of DNA sequences. Again, the answer obtained capability of the ATP viability marker to detect the same
may not be fully coincident for the different identification range of micro-organisms as is encountered using standard
methods, and while one answer may be appropriate for the plating methods. Since this method is growth-dependent,
General Notices (1) apply to all monographs and other texts 567
5.1.6. Alternative methods for control of microbiological quality EUROPEAN PHARMACOPOEIA 8.0
the improvement comparing to the plating methods is the with those obtained using standard plate count. Multiple
rapidity to obtain a result (from 5 days with the plating replicates (at least 5) from overnight cultures diluted across a
methods to 24 h for bioluminescence). It is possible to identify concentration range (e.g. 100 per cent, 75 per cent, 50 per cent,
the bioluminescence-detected micro-organisms from the 25 per cent and 10 per cent) must be used to evaluate linearity,
incubation step medium, but it has to be remembered that accuracy, precision, range, specificity, limit of quantification
in a mixed culture some micro-organisms may out-compete (quantitative method) and limit of detection (flow cytometry
others during incubation. This method provides evaluation of with pre-incubation step). Since cytometry has high sensitivity
samples within 24 h for filterable and non-filterable products (solid phase cytometry can detect single cells, whereas flow
(water, in-process control, environmental samples, solid and cytometry is sensitive to a level of around 10-50 cells per
liquid raw materials, solid and liquid finished products, etc.) millilitre), and detection is not growth based, the linearity of
and for a large number of samples, when the detection step the instrumentation can be tested by comparison of the actual
is automated. results with the expected value.
4-2-2. Validation for the actual intended use Following this step, validation proceeds in 2 phases : validation
The method relies upon the detection of ATP from viable with respect to the product to be examined and comparative
micro-organisms. Performance qualification is carried out testing. Results of each phase must be evaluated against
with test micro-organisms to make sure that under the pre-determined acceptance criteria using positive and negative
conditions applied by the user laboratory it is possible to controls :
satisfy the criteria described by the supplier for precision, – phase 1 : individual materials to be evaluated by cytometry
accuracy and linearity (quantitative method), or limit of must be ‘spiked’ with a defined level of micro-organisms to
detection (qualitative and semi-quantitative method) over ensure that the sample preparation process and the samples
the range required for the intended use. Following this step, themselves do not have an impact upon the performance
validation proceeds in 3 phases : of the detection system ; specifically, the sample matrix
– phase 1 : fertility of the medium in the presence of the must not affect detection (i.e. contain endogenous
product (if an incubation step is performed) ; chromophores, auto-fluorescent particles), and in the case
of flow cytometry, sample size/dilution and flow rate must
– phase 2 : search for interferences that may increase or inhibit
be determined for optimal performance ;
the ATP production (by addition of an ATP standard
solution to the product to test) ; – phase 2 : testing must be performed in which the results
– phase 3 : comparative testing with the pharmacopoeial obtained by cytometry and the pharmacopoeial method are
method. compared ; the number of samples and the testing period
must be defined in a comparability protocol ; the number
A detailed example of validation of the bioluminescence of samples required will vary, but must be representative of
method is given at the end of this chapter. the material evaluation process (i.e. time/number), and
4-3. CYTOMETRY (SOLID AND FLOW) FOR must allow for statistical evaluation ; all samples must be
ENUMERATION OF MICRO-ORGANISMS prepared according to defined procedures and evaluated
4-3-1. Risk-benefit analysis against selected validation and acceptance criteria, similar
to those used for pure culture evaluation.
Extensive scientific evidence supports the capability of
this fluorescence viability marker to detect and/or count 4-4. FATTY ACID PROFILES FOR IDENTIFICATION
a wider range of micro-organisms than are encountered 4-4-1. Risk-benefit analysis
using standard plating methods. Cytometry will detect all Identification by fatty acid profiles may be more precise
viable micro-organisms including some that may not be than the identification methods based on metabolic profiles
discernable by growth-based methods. Whilst being rapid, the in conventional microbiological culture methods. The
recovery of micro-organisms post-analysis is limited. Thus database is broader than for conventional culture methods.
the further processing of analysed samples for identification Pre-incubation is needed, but extraction and identification is
would require alternative fluorescent stains or an alternative faster than in biochemical methods and hence, the result is
method. Currently it is not possible to use this method for obtained faster. Other modern methods, such as 16S rRNA
routine identification of micro-organisms, although basic sequence analysis or genetic fingerprinting, have a similar
morphology is readily discernable in solid phase cytometry broad differentiation range and give a result as fast as this
under fluorescent microscopes. This method provides rapid method.
evaluation of samples and hence allows for a proactive
approach to pharmaceutical manufacturing, facilitating Separation of closely related micro-organisms (e.g. E. coli
building quality into pharmaceutical operations. This method and Salmonella spp.) can be difficult by fatty acid profiles.
is not growth-dependent and hence all metabolically active Where the identification of closely related micro-organisms
micro-organisms will be detected. However, the limit of is especially important, other systems may give more precise
detection for flow cytometry is currently such that it cannot results. For a given application it is important to specify which
be used for enumeration by direct examination for most types of micro-organisms are most important to be identified.
pharmaceutical samples. If pre-incubation is necessary, the If it is most critical to characterise the correct phylogenetic
estimation becomes semi-quantitative (limit test). species of the isolate, DNA sequence-based identification
methods will give more reliable results.
4-3-2. Validation for the actual intended use
Limitations of identification by fatty acid profiles are also seen
The method relies upon the detection of a fluorescent signal in the necessity to grow micro-organisms on standardised
from labelled micro-organisms. media under standard temperature conditions and durations
Performance qualification is carried out to ensure that of incubation. Micro-organisms that cannot be cultivated on
the instruments perform within their defined operational such media cannot be identified.
parameters. This involves the use of fluorescent standards of
4-4-2. Validation for the actual intended use
prescribed intensity and cultures of known type and number
of micro-organisms. These tests challenge the quantitative Using a range of test micro-organisms and at least 3 replicate
detection system. Reagents and consumables (negative determinations in each case, it must be demonstrated that the
controls) must also be utilised to ensure that the routine test method yields consistent results.
protocol is applicable, and that the quality of the materials A significant number of isolates from typical samples to be
used in the test do not contribute to the final result. Pure analysed by the user must be identified, at least 3 times each.
culture experiments involving test micro-organisms are used The results in each case should be consistent and in accord
to challenge the detection system, and to compare test results with those obtained using alternative identification methods.
Where a different identification result is found in another determination in the quantity analysed. Using more than a
identification system, the reason for the difference must be single sample quantity, the system may offer semi-quantitative
investigated. Where a scientifically plausible explanation exists determination (limit test). For example, the classical tested
for the recognition of a different species, a difference between quantity for viable aerobic count on non-sterile products is
identification systems may be acceptable. In such a case it 0.1 g or 0.1 mL leading to absence in 0.1 g or 0.1 mL, i.e.
must be assured that the recognition of the identified species less than 10 micro-organisms in 1 g or 1 mL for a negative
is robust. It must also be assured that the system does not result and more than or equal to 10 micro-organisms in 1 g
group poorly recognised isolates under one ‘species’ thereby or 1 mL in case of a positive result. If 0.01 g or 0.01 mL is
simulating the repeated isolation of a single species. tested simultaneously, a negative result corresponds to a
4-5. NUCLEIC ACID AMPLIFICATION TECHNIQUES number of micro-organisms less than 100 in 1 g or 1 mL.
The combination between negative for 0.01 g or 0.01 mL
4-5-1. Risk-benefit analysis and positive for 0.1 g or 0.1 mL permits an estimate of the
NAAT are widely used in diagnostics for their precision and contamination level of the product to be less than 100 but more
rapidity at a relatively low cost (for the analysis, but not for the than or equal to 10 micro-organisms in 1 g or 1 mL.
instruments,) when compared with the traditional methods. As mentioned in section 2., bioluminescence can be used as
Provided that specific validations have been performed, when a quantitative method if micro-organisms are captured on a
NAAT are appropriately used, they may offer advantages filtration membrane and later incubated in culture medium
in some fields in comparison to classical methods ; on the (bioluminescence on membrane).
other hand classical methods are generally more easily
The protocol below describes validation aspects for qualitative,
standardisable, need a lower level of technical competence and
semi-quantitative and quantitative methods.
may have lower costs. Even when NAAT are not more difficult
to perform than traditional methods, the interpretation of the PERFORMANCE QUALIFICATION OF THE
results generally needs a high degree of scientific competence. ALTERNATIVE METHOD
When used for identification, DNA-based methods cannot Specificity
discriminate between dead and live micro-organisms. That
means that they cannot be directly used on the product Screen the method with test micro-organisms appropriate to
but only after passage on a traditional culture medium, the method. For example, for microbial aerobic viable count
thereby losing part of the advantage in rapidity. Moreover, on non-sterile products, use at least the micro-organisms
if used directly on the product at the end of the analysis, described in chapter 2.6.12 for the fertility of the media in the
these methods do not result in a strain to be used in presence of product. This determination is performed at least
further experiments and may not give advantages when the 3 times with each micro-organism. Acceptance criterion : all
micro-organisms to be detected are poorly cultivable or test micro-organisms are successfully detected.
stressed. RNA amplification techniques (e.g. RT-PCR) may Limit of detection (only for semi-quantitative or qualitative
identify living micro-organisms (but not spores) directly in methods)
the products, but in comparison to traditional methods are Prepare a low inoculum (about 5 CFU in the initial sample)
much more difficult to use routinely. On the other hand, of each test micro-organism. Perform the analysis in at
where specific primers are used, identification (or typing) by least 5 replicates with the pharmacopoeial method and
NAAT is more precise than the traditional methods and in with the bioluminescence method concerned. Acceptance
some cases may have other advantages : for instance for the criterion : the ability of the 2 methods to detect the presence
identification of some vaccines (e.g. cholera vaccine, whole of a single micro-organism can be demonstrated using the
cell pertussis vaccine,) their use may substitute for that of χ2 test. Alternative procedure : prepare a series of dilutions
specific sera and contribute to reducing the use of animals, or of micro-organisms to have a count in the next dilution
may give a very specific identification where this is presently of about 5 CFU per inoculum (e.g. : 10 CFU/inoculum,
lacking (e.g. BCG vaccine). 5 CFU/inoculum, 2.5 CFU/inoculum, 1.25 CFU/inoculum,
These methods are in general non-quantitative (PCR) or 0.75 CFU/inoculum). Perform the test on 5 independent series
semi-quantitative (real-time PCR), meaning that their results of dilutions with the pharmacopoeial method and with the
cannot be compared with those of a colony count where an bioluminescence method concerned. Determine the limit of
exact enumeration of the micro-organisms present in the detection for each method. It corresponds to the last dilution
sample is requested, but even if colony count has a valence where the result is positive for the 5 series. Acceptance
consolidated in time this dogma may not be verified for criterion : the limit of detection of the bioluminescence
bacteria which have a tendency to clump (mycobacteria) or are method is equal to or lower than that of the pharmacopoeial
organised in chains or in clusters (streptococci, staphylococci), method.
therefore an accurate standardisation of the semi-quantitative Limit of quantification (quantitative method)
methods may give results of comparable reliability.
This can be performed at the same time as the linearity
4-5-2. Validation for the actual intended use determination. It corresponds to the lowest concentration
The method is validated according to chapter 2.6.21. of the chosen range that satisfies the criteria for linearity,
Comparison of conventional and PCR-based methodologies, accuracy and precision. Acceptance criterion : the limit of
which differ in sensitivity and specificity, is particularly quantification of the bioluminescence method is equal to or
difficult and may lead to divergent conclusions. lower than that of the pharmacopoeial method.
The following example is published for information and not Precision
for general application. Quantitative evaluation. For each test micro-organism,
perform at least 5 replicates during the same series including
Example validation of an alternative method : at least the concentration of micro-organisms corresponding
to the middle of the range. Perform 3 independent tests.
detailed protocol followed by a laboratory Carry out a statistical analysis to compare the precision of
for the implementation of bioluminescence the 2 methods or calculate the coefficient of variation (CV).
Acceptance criterion : CV 15 per cent to 30 per cent or
BACKGROUND precision not different with the risk alpha equal to 5 per
Methods using a pre-incubation step in liquid medium cent between the 2 methods. If precision is different, the
(bioluminescence in tube or microtitre plate) do not bioluminescence method is better than the pharmacopoeial
offer quantitative information but a presence/absence method, indicated by a smaller standard deviation.
General Notices (1) apply to all monographs and other texts 569
5.1.6. Alternative methods for control of microbiological quality EUROPEAN PHARMACOPOEIA 8.0
Qualitative or semi-quantitative evaluation. Use the alternative – phase 2 : the absence of interference from the product that
procedure described for setting the limit of detection and may increase or inhibit ATP production ;
report the frequency of positive results in parallel with the – phase 3 : the testing of the product in parallel with the
pharmacopoeial method. Acceptance criterion : the frequency pharmacopoeial method.
of positive results at the detection limit is 100 per cent and
this frequency is better than or equal to the pharmacopoeial These 3 parts of validation are performed on 3 independent
method. tests using for example at least 2 different batches of product.
Linearity Phase 1 : fertility of the medium in the presence of the
For each test micro-organism, prepare 5 concentrations in product
the range of the bioluminescence method (range is normally If the product has a known high contamination level (more
indicated by the supplier). Perform the pharmacopoeial and than 500 micro-organisms per gram or millilitre) the
the bioluminescence methods in parallel. Repeat this test incubation step is unnecessary, the micro-organisms can
2 further times to have results on 3 independent tests. Test be detected directly. In this case testing the fertility of the
for linear regression, presence of a slope, and lack of fit with medium in the presence of the product is not necessary.
the F test at alpha equal to 5 per cent. If statistical analysis is However, pharmaceutical products are generally contaminated
not possible, calculate the correlation coefficient (r2) and the at a much lower level and growth of the micro-organism is
slope between the 2 methods. Acceptance criterion : statistical necessary to obtain detection with bioluminescence. It must
analysis may show linear regression, the presence of a slope therefore be proven that the product does not inhibit the
and no lack of fit with a risk of 5 per cent. Equation y = a + bx growth of micro-organisms under the conditions of the test.
is determined where b is the slope and a the intercept. If In order to do so, separately add inoculum at not more than
no statistical analysis is available, r2 is at least 0.9 and the 100 CFU for each test micro-organism into the portion of
slope does not diverge by more than 20 per cent from 1 (b medium containing the product. For bioluminescence in
between 0.8 and 1.2). If the linearity is not demonstrated in tube or microtitre plate, perform the bioluminescence test.
such a large range, the range can be decreased and linearity For bioluminescence on membrane, incubate at 30-35 °C or
demonstrated with only 3 concentrations in place of 5. 20-25 °C for 5 days and count the bioluminescent colonies
Accuracy on the membrane. Acceptance criterion : the test is positive
(bioluminescence in tube or microtitre plate) ; the quantitative
Quantitative evaluation. Accuracy can be determined with recovery of the micro-organism is at least 70 per cent
data obtained in linearity. For each micro-organism use (bioluminescence on membrane).
3 to 5 concentrations within the linear range of the method.
Perform statistical analysis (Student’s t test at risk 5 per cent) Phase 2 : search for interference of the product
to test the conformity of the estimated slope (value = 1) The objective is to show that the product does not add stray
versus the obtained slope and to test the conformity of light or non-microbial ATP (does not lead to false positive
the estimated intercept (value = 0) versus the obtained result : criterion A) or does not decrease the ATP detection
intercept. For example, if the estimated slope is b with a (does not lead to a false negative result : criterion B).
standard deviation s(b) of 0.090 with 5 concentrations of
micro-organisms, calculate t = (b – 1)/s(b). For intercept a, Bioluminescence in tube or microtitre plate
with standard deviation equal to s(a), t = (a – 0)/s(a). Compare A. Perform the bioluminescence test with the culture broth
these values to the Student’s t at 5 per cent, for 13 degrees of alone and with the culture broth in the presence of the
freedom (3 tests, 5 concentrations). Acceptance criterion : if product. Determine the RLU value for culture broth alone
the t values obtained are less than the Student’s t, the method and the RLU value for culture broth in the presence of
is exact in the applied range. In the case that there is no product.
conformity for the slope (slope different from 1) or for the B. Perform the bioluminescence test with the culture broth
intercept (intercept different from 0) the method is not exact alone and the culture broth in the presence of ATP.
over the applied range. Determine the response coefficient for ATP concentration
Qualitative or semi-quantitative evaluations. Use the in per cent.
alternative procedure described for setting the limit of
Acceptance criterion :
detection. Calculate the proportion of false negatives for
bioluminescence and for the pharmacopoeial method over – criterion A : the RLU value of culture broth in the presence
all tested dilutions. Compare the extent of false negatives of product is less than twice the RLU value of culture
for the 2 or 3 concentrations of micro-organisms just broth alone (if criterion A is not satisfied, it is necessary to
under the detection limit (for example 5 CFU/inoculum, determine a specific threshold for this product) ;
2.5 CFU/inoculum or 1.25 CFU/inoculum) giving a positive – criterion B : the RLU value of culture broth in the presence
result. By definition, the detection limit corresponds to 0 per of product and ATP is within the interval 25 per cent
cent of false negatives. Acceptance criterion : the percentage to 200 per cent of the RLU value of culture broth in the
of false negatives for the bioluminescence method at sample presence of ATP.
concentrations below the detection limit must be equal to or
lower than that of the pharmacopoeial method. Bioluminescence on membrane : perform the complete
bioluminescence test to search for interference. Acceptance
Range criterion : the recovery of micro-organisms is greater than or
This is the interval between the lowest and the highest equal to 70 per cent and not more than 200 per cent.
concentrations of micro-organisms where linearity, precision
Phase 3 : analysis of the product in parallel with the
and accuracy have been demonstrated.
pharmacopoeial method
Robustness
Perform the test according to the validated method for the
The information is given by the supplier. product concerned in parallel with the pharmacopoeial
method to show the relationship between the 2 methods for
VALIDATION FOR THE ACTUAL INTENDED USE
the product concerned, on 3 independent tests and using
In the example given, there was no need to determine the at least 2 different batches. Express the result as positive or
accuracy and detection limit in the presence of the product. negative in a certain quantity (bioluminescence in tube or
The validation consists of 3 parts, verifying : microtitre plate) or express the count per filtered quantity
– phase 1 : the fertility of the medium in the presence of the (bioluminescence on membrane). Acceptance criterion :
product ; results must be correlated with the pharmacopoeial method.
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or, in the case of herbal drugs, of pre-treatment, would and the efficiency of the adopted sampling plan. Hence for
not reduce the level of organisms sufficiently to reach the the purpose of this text a batch is defined as a homogeneous
criteria required under B collection of sealed containers prepared in such a manner that
the risk of contamination is the same for each of the units
TAMC (2.6.12) Acceptance criterion : 105 CFU/g or CFU/mL
contained therein.
Maximum acceptable count : 500 000 CFU/g
or CFU/mL In the case of terminally sterilised products, physical proofs,
TYMC (2.6.12) Acceptance criterion : 104 CFU/g or CFU/mL biologically based and automatically documented, showing
Maximum acceptable count : 50 000 CFU/g correct treatment throughout the batch during sterilisation are
or CFU/mL of greater assurance than the sterility test. The circumstances
Bile-tolerant in which parametric release may be considered appropriate
gram-negative Acceptance criterion : 104 CFU/g or CFU/mL are described under 5.1.1. Methods of preparation of sterile
bacteria (2.6.31) products. The method of media-fill runs may be used to
Escherichia coli evaluate the process of aseptic production. Apart from that,
Absence (1 g or 1 mL)
(2.6.31) the sterility test is the only analytical method available for
Salmonella (2.6.31) Absence (25 g or 25 mL) products prepared under aseptic conditions and furthermore
it is, in all cases, the only analytical method available to the
authorities who have to examine a specimen of a product for
EXTRACTS sterility.
Extracts should fulfill the acceptance criteria for category B The probability of detecting micro-organisms by the test for
herbal medicinal products. However, where it can be sterility increases with their number present in the sample
demonstrated that the method of processing would not tested and varies according to the readiness of growth of
reduce the level of micro-organisms sufficiently to reach the micro-organism present. The probability of detecting very
category B criteria, the extracts shall meet the requirements low levels of contamination even when it is homogenous
for category C herbal medicinal products. throughout the batch is very low. The interpretation of the
The recommended acceptance criteria apply to extracts that results of the test for sterility rests on the assumption that
are to be incorporated into herbal medicinal products for the contents of every container in the batch, had they been
oral use. More-stringent acceptance criteria may be required tested, would have given the same result. Since it is manifest
for extracts that are to be incorporated into pharmaceutical that every container cannot be tested, an appropriate sampling
preparations to be administered by other routes in order plan should be adopted. In the case of aseptic production, it is
to satisfy the acceptance criteria for the intended route of recommended to include samples filled at the beginning and
administration (5.1.4). at the end of the batch and after significant intervention.
It is recognised that for some herbal medicinal products and OBSERVATION AND INTERPRETATION OF RESULTS
extracts used in their preparation the criteria given above for
TAMC, TYMC and bile-tolerant gram-negative bacteria cannot Conventional microbiological/biochemical techniques are
be met because of the typical level of microbial contamination. generally satisfactory for identification of micro-organisms
Less-stringent acceptance criteria may be applied on the basis recovered from a sterility test. However, if a manufacturer
of a risk assessment that takes account of qualitative and wishes to use condition (d) as the sole criterion for invalidating
quantitative characterisation of the microbial contamination a sterility test, it may be necessary to employ sensitive typing
and the intended use of the herbal medicinal product or extract. techniques to demonstrate that a micro-organism isolated
from the product test is identical to a micro-organism isolated
If it has been shown that none of the prescribed tests for a herbal
medicinal product or extract will allow valid enumeration of from the test materials and/or the testing environment. While
micro-organisms at the level prescribed, a validated method routine microbiological/biochemical identification techniques
with a limit of detection as close as possible to the indicated can demonstrate that 2 isolates are not identical, these
acceptance criterion is used. methods may not be sufficiently sensitive or reliable enough to
provide unequivocal evidence that 2 isolates are from the same
source. More sensitive tests, for example molecular typing
01/2009:50109 with RNA/DNA homology, may be necessary to determine
that micro-organisms are clonally related and have a common
5.1.9. GUIDELINES FOR USING THE origin.
TEST FOR STERILITY
01/2010:50110
The purpose of the test for sterility (2.6.1), as that of all
pharmacopoeial tests, is to provide an independent control
analyst with the means of verifying that a particular material 5.1.10. GUIDELINES FOR USING THE
meets the requirements of the European Pharmacopoeia. A TEST FOR BACTERIAL ENDOTOXINS
manufacturer is neither obliged to carry out such tests nor
precluded from using modifications of, or alternatives to, the 1. INTRODUCTION
stated method, provided he is satisfied that, if tested by the Endotoxins from gram-negative bacteria are the most
official method, the material in question would comply with common cause of toxic reactions resulting from contamination
the requirements of the European Pharmacopoeia. of pharmaceutical products with pyrogens ; their pyrogenic
activity is much higher than that of most other pyrogenic
PRECAUTIONS AGAINST MICROBIAL substances. These endotoxins are lipo-polysaccharides.
CONTAMINATION Although there are a small number of pyrogens which possess
Aseptic conditions for performance of the test can be achieved a different structure, the conclusion is generally justified that
using, for example, a class A laminar-air-flow cabinet located the absence of bacterial endotoxins in a product implies the
within a class B clean room, or an isolator absence of pyrogenic components, provided the presence of
non-endotoxin pyrogenic substances can be ruled out.
GUIDANCE TO MANUFACTURERS The presence of endotoxins in a product may be masked
The level of assurance provided by a satisfactory result of a by factors interfering with the reaction between the
test for sterility (the absence of contaminated units in the endotoxins and the amoebocyte lysate. Hence, the analyst
sample) as applied to the quality of the batch is a function of who wishes to replace the rabbit pyrogen test required in a
the homogeneity of the batch, the conditions of manufacture pharmacopoeial monograph by a test for bacterial endotoxins
has to demonstrate that a valid test can be carried out on the In setting a threshold concentration of endotoxin for the
product concerned ; this may entail a procedure for removing product to be tested, due attention should be paid to the dose
interfering factors. of the product : the threshold should be set so as to ensure that
as long as the endotoxin concentration in the product remains
As indicated in the test for bacterial endotoxins (2.6.14),
below this threshold even the maximal dose administered
information must be available on the 2 following aspects
by the intended route per hour does not contain sufficient
before a test on a sample can be regarded as valid.
endotoxin to cause a toxic reaction.
– The suitability of the material to be used for the test has When the endotoxin concentration in the product exactly
to be established. The absence of endotoxins in the water equals the threshold value, gelation will occur, as is the case
for BET and in the other reagents must be assured and the when the endotoxin concentration is much higher, and the
sensitivity of the amoebocyte lysate must be checked to product will fail the test, because the all-or-none character
confirm the sensitivity declared by the manufacturer. of the test makes it impossible to differentiate between a
– As the product to be examined may interfere with the concentration exactly equal to the threshold concentration
test, the sensitivity of the amoebocyte lysate is determined and one that is higher. It is only when no gelation occurs that
in the presence and in the absence of the product under the analyst may conclude that the endotoxin concentration is
examination. There must be no significant difference below the threshold concentration.
between the 2 sensitivity values. For products in the solid state, this threshold concentration
The text 2.6.14. Bacterial endotoxins indicates methods for of endotoxin per mass unit or per International Unit (IU) of
removing interfering factors ; in the case of interference, product has to be translated into a concentration of endotoxin
another test must be carried out after such a method has been per millilitre of solution to be tested, as the test can only be
applied to check whether the interference has indeed been carried out on a solution. The case of products that already
neutralised or removed. exist in the liquid state (such as infusion fluids) is discussed
below.
This general chapter explains the reasons for the requirements
Endotoxin limit : the endotoxin limit for active substances
in the test for bacterial endotoxins, then deals with the reading
administered parenterally, defined on the basis of dose, is
and interpretation of the results.
equal to :
Substitution of the rabbit pyrogen test required in a
pharmacopoeial monograph by an amoebocyte lysate test
constitutes the use of an alternative method of analysis and
hence requires validation ; some guidance on how to proceed
is given in section 11. K = threshold pyrogenic dose of endotoxin per
kilogram of body mass ;
The reference method for bacterial endotoxins is stated in the
M = maximum recommended bolus dose of product
monograph on a given product ; where no method is stated,
method A is the reference method. If a method other than the per kilogram of body mass.
reference method is to be used, the analyst must demonstrate When the product is to be injected at frequent intervals
that the method is appropriate for this product and gives a or infused continuously, M is the maximum total dose
result consistent with that obtained with the reference method administered in a single hour period.
(see also Section 13). The endotoxin limit depends on the product and its route of
administration and is stated in the monograph. Values for K
2. METHOD are suggested in Table 5.1.10.-1.
The addition of endotoxins to amoebocyte lysate may result in For other routes, the acceptance criterion for bacterial
turbidity, precipitation or gelation (gel-clot) ; only the gel-clot endotoxins is generally determined on the basis of results
method was used in the Pharmacopoeia as an evaluation obtained during the development of the preparation.
criterion in the first type of test for bacterial endotoxins. The
Table 5.1.10.-1
advantage was the simplicity of basing the decision to pass or
fail the product under examination on the absence or presence Route of administration K (IU of endotoxin per kilogram
of a gel-clot, visible with the naked eye. The quantitative of body mass)
methods described as methods C, D, E and F were developed Intravenous 5.0
later : they require more instrumentation, but they are easier Intravenous, for radiopharmaceuticals 2.5
to automate for the regular testing of large numbers of samples
of the same product. Intrathecal 0.2
Endotoxins may be adsorbed onto the surface of tubes Which dilution of the product is to be used in the test to
or pipettes made from certain plastics or types of glass. obtain maximal assurance that a negative result means that
Interference may appear due to the release of substances the endotoxin concentration of the product is less than the
from plastic materials. Hence, the materials used should be endotoxin limit and that a positive result means that the
checked ; subsequent batches of tubes or pipettes may have lysate detected an endotoxin concentration equal to or greater
a slightly different composition, and therefore the analyst is than the endotoxin limit? This dilution depends on the
advised to repeat such tests on starting with new batches of endotoxin limit and on the sensitivity of the lysate : it is called
materials. the Maximum Valid Dilution (MVD) and its value may be
The decision to use the test for bacterial endotoxins as a limit calculated using the following expression :
test implies first that a threshold endotoxin concentration
must be defined for the product to be tested, and second that
the objective of the test is to know whether the endotoxin
concentration in the product under examination is below or Concentration of test solution :
above this threshold. The quantitative methods C, D, E and F – mg/mL if the endotoxin limit is specified by mass (IU/mg) ;
make it possible to determine the endotoxin concentration
in the sample under examination, but for compliance with – Units/mL if the endotoxin limit is specified by unit of
the Pharmacopoeia and in routine quality control the final biological activity (IU/Unit) ;
question is whether or not this concentration exceeds a – mL/mL if the endotoxin limit is specified by volume
defined limit. (IU/mL).
General Notices (1) apply to all monographs and other texts 573
5.1.10. Guidelines for using the test for bacterial endotoxins EUROPEAN PHARMACOPOEIA 8.0
λ = the labelled lysate sensitivity in the gel-clot technique analysts may prefer to distil the water more than 3 times.
(IU/mL) or the lowest concentration used in the Whatever method is used, the resultant product must be free
standard curve of the turbidimetric or chromogenic of detectable endotoxins.
techniques.
5. pH OF THE MIXTURE
When the value of the maximum valid dilution is not a whole
number, a convenient whole number smaller than the MVD In the test for bacterial endotoxins, optimum gel-clot occurs
may be used for routine purposes (which means preparing a for a mixture at pH 6.0-8.0. However, the addition of the lysate
solution of the product which is less diluted than the MVD to the sample may result in a lowering of the pH.
indicates). In this case, a negative result indicates that the
endotoxin concentration of the product lies below the limit 6. VALIDATION OF THE LYSATE
value. However, when the endotoxin concentration of the It is important to follow the manufacturer’s instructions for
product in such a test is less than the endotoxin limit but the preparation of the solutions of the lysate.
high enough to make the reaction with the lysate result in a
The positive end-point dilution factors in gel-clot methods A
clot, the test may be positive under these conditions. Hence,
and B are converted to logarithms. The reason is that if the
when a test with this ‘convenient’ dilution factor is positive,
frequency distribution of these logarithmic values is plotted, it
the product should be diluted to the MVD and the test should
usually approaches a normal distribution curve much more
be repeated. In any case of doubt or dispute the MVD must
closely than the frequency distribution of the dilution factors
be used.
themselves ; in fact it is so similar that it is acceptable to use
This stresses the importance of the confirmation of the the normal frequency distribution as a mathematical model
sensitivity of the lysate. and to calculate confidence limits with Student’s t-test.
Example
A 50 mg/mL solution of phenytoin sodium (intended for 7. PRELIMINARY TEST FOR INTERFERING FACTORS
intravenous injection) has to be tested. Determine the MVD, Some products cannot be tested directly for the presence of
given the following variables : endotoxins because they are not miscible with the reagents,
M = maximum human dose = 15 mg per kilogram of they cannot be adjusted to pH 6.0-8.0 or they inhibit or
body mass ; activate gel formation. Therefore a preliminary test is required
to check for the presence of interfering factors ; when these
c = 50 mg/mL ; are found the analyst must demonstrate that the procedure to
K = 5 IU of endotoxin per kilogram of body mass ; remove them has been effective.
λ = 0.4 IU of endotoxin per millilitre. The object of the preliminary test is to test the null hypothesis
that the sensitivity of the lysate in the presence of the product
under examination does not differ significantly from the
sensitivity of the lysate in the absence of the product. A simple
criterion is used in methods A and B : the null hypothesis is
For routine tests on this product, it may be expedient to dilute accepted when the sensitivity of the lysate in the presence of
1 mL of the solution to be tested to 20 mL (MVD/2 rounded the product is at least 0.5 times and not more than twice the
to the next lower whole number). However, if this test result is sensitivity of the lysate by itself.
positive the analyst will have to dilute 1 mL to 41.67 mL and
A classical approach would have been to calculate the means
repeat the test. A dilution to 41.67 mL is also necessary when
of the log dilution factor for the lysate sensitivity with and
the test is performed to settle a dispute.
without the product and to test the difference between the
3. REFERENCE MATERIAL 2 means with Student’s t-test.
Endotoxin standard BRP is intended for use as the reference The test for interfering factors in gel-clot methods A and B
preparation. It has been assayed against the WHO requires the use of a sample of the product in which no
International Standard for Endotoxin and its potency is endotoxins are detectable. This presents a theoretical problem
expressed in International Units of endotoxin per ampoule. when an entirely new product has to be tested. Hence, a
The International Unit of endotoxin is defined as the specific different approach was designed for quantitative methods C,
activity of a defined mass of the International Standard. D, E and F.
For routine purposes, another preparation of endotoxin may
be used, provided it has been assayed against the International 8. REMOVAL OF INTERFERING FACTORS
Standard for Endotoxin or the BRP and its potency is The procedures to remove interfering factors must not
expressed in International Units of endotoxin. increase or decrease (for example, by adsorption) the amount
NOTE : 1 International Unit (IU) of endotoxin is equal to of endotoxin in the product under examination. The correct
1 Endotoxin Unit (E.U.). way of checking this is to apply the procedures to a spiked
sample of the product, that is, a sample to which a known
4. WATER FOR BET amount of endotoxin has been added, and then to measure
Testing the absence of endotoxin in this reagent by a technique the recovery of the endotoxin.
derived from the rabbit pyrogen test was rejected for practical Methods C and D. If the nature of the product to be analysed
and theoretical reasons : shows interference which cannot be removed by classical
– the rabbit test is not sensitive enough to detect endotoxin methods, it may be possible to determine the standard curve in
in water for BET intended for tests on products with a very the same type of product freed from endotoxins by appropriate
low endotoxin limit ; treatment or by dilution of the product. The endotoxins test is
– the relatively low precision of the rising temperature then carried out by comparison with this standard curve.
response in rabbits would call for many replications in Ultrafiltration with cellulose triacetate asymmetric membrane
rabbits ; filters has been found to be suitable in most cases. The
– the terms ‘pyrogens’ and ‘endotoxins’ denote groups of filters should be properly validated, because under some
entities that do not coincide completely. circumstances cellulose derivatives (β-D-glucans) can cause
The text 2.6.14. Bacterial endotoxins indicates that methods false positive results.
other than triple distillation may be used to prepare water for Polysulfone filters have been found to be unsuitable because
BET. Reverse osmosis has been used with good results ; some false positive results had been obtained by some users.
9. THE PURPOSE OF THE CONTROLS interest ; such data includes details of sample preparation
The purpose of the control made up with water for BET and of any procedures necessary to eliminate interfering
and the reference preparation of endotoxin at twice the factors ; in addition, any available parallel data for rabbit
concentration of the labelled lysate sensitivity is to verify the pyrogen testing that would contribute to an assurance that
activity of the lysate at the time and under the conditions of the replacement of a rabbit pyrogen test by the test for
the test. The purpose of the negative control is to verify the bacterial endotoxin is appropriate, must be provided.
absence of a detectable concentration of endotoxin in water Additional requirements are defined in the following sections.
for BET.
12. USE OF A DIFFERENT BACTERIAL ENDOTOXIN
The positive control, which contains the product to be TEST FROM THAT PRESCRIBED IN THE MONOGRAPH
examined at the concentration used in the test, is intended to
show the absence of inhibiting factors at the time and under When a test for bacterial endotoxins is prescribed in a
the conditions of the test. monograph and none of the 6 methods (A to F) described in
chapter 2.6.14 is specified, then method A, the gel-clot method
10. READING AND INTERPRETATION OF THE RESULTS limit test, has been validated for this product. If one of the
Minute amounts of endotoxin in the water for BET, or in other methods (B to F) is specified, this is the one which has
any other reagent or material to which the lysate is exposed been validated for this product.
during the test, may escape detection as long as they do not 13. VALIDATION OF ALTERNATIVE METHODS
reach the sensitivity limit of the lysate. However, they may
raise the amount of endotoxin in the solution containing the Replacement of a rabbit pyrogen test by a bacterial endotoxin
product under examination to just above the sensitivity limit test, or replacement of a stated or implied method for bacterial
and cause a positive reaction. endotoxins by another method, is to be regarded as the use of
an alternative method in the replacement of a pharmacopoeial
The risk of this happening may be reduced by testing the test, as described in the General Notices :
water for BET and the other reagents and materials with the
“The test and assays described are the official methods
most sensitive lysate available, or at least one that is more
upon which the standards of the Pharmacopoeia are based.
sensitive than the one used in the test on the product. Even
With the agreement of the competent authority, alternative
then, the risk of such a ‘false positive result’ cannot be ruled
methods of analysis may be used for control purposes,
out completely. It should be realised, however, that in this
provided that the methods used enable an unequivocal
respect the test design is ‘fail-safe’ in contrast to a test design
decision to be made as to whether compliance with the
permitting a false negative result, which could lead to the
standards of the monographs would be achieved if the
release of an unsatisfactory product, thus endangering the
official methods were used. In the event of doubt or
patient’s health.
dispute, the methods of analysis of the Pharmacopoeia are
11. REPLACEMENT OF THE RABBIT PYROGEN TEST BY alone authoritative.”
A TEST FOR BACTERIAL ENDOTOXINS The following procedures are suggested for validating a
Monographs on pharmaceutical products intended for method for bacterial endotoxins other than the one implied or
parenteral administration that may contain toxic amounts indicated in the monograph.
of bacterial endotoxins require either a test for bacterial 13-1. The procedure and the materials and reagents used
endotoxins or a rabbit pyrogen test. As a general policy : in the method should be validated as described for the test
– in any individual monograph, when a test is required, only concerned.
one test is included, either that for pyrogens or that for 13-2. The presence of interfering factors (and, if needed, the
bacterial endotoxins ; procedure for removing them) should be tested on samples
– in the absence of evidence to the contrary, the test for of at least 3 production batches. It should be borne in mind
bacterial endotoxins is preferred over the test for pyrogens, that methods D and E, using a chromogenic peptide, require
since it is usually considered to provide equal or better reagents that are absent in methods A, B, C and F, and hence
protection to the patient ; compliance of methods A, B, C or F with the requirements
for interfering factors cannot be extrapolated to method D or
– before including a test for bacterial endotoxins in a method E without further testing.
monograph, evidence is required that one of the tests
described in chapter 2.6.14 can be applied satisfactorily to 14. VALIDATION OF THE TEST FOR NEW PRODUCTS
the product in question ; The procedures described under 13-1 and 13-2 should
– the necessary information is sought from manufacturers ; be applied to all new products intended for parenteral
companies are invited to provide any validation data administration that have to be tested for the presence of
that they have concerning the applicability of the test for bacterial endotoxins according to the requirements of the
bacterial endotoxins to the substances and formulations of Pharmacopoeia.
General Notices (1) apply to all monographs and other texts 575
EUROPEAN PHARMACOPOEIA 8.0 5.2.2. SPF chicken flocks for vaccines
5.2. GENERAL TEXTS ON Single harvest. Material derived on one or more occasions
BIOLOGICAL PRODUCTS
from a single production cell culture inoculated with the same
working seed lot or a suspension derived from the working
seed lot, incubated, and harvested in a single production run.
01/2008:50201 Monovalent pooled harvest. Pooled material containing a
corrected 6.0 single strain or type of micro-organism or antigen and derived
from a number of eggs, cell culture containers etc. that are
5.2.1. TERMINOLOGY USED IN processed at the same time.
MONOGRAPHS ON BIOLOGICAL Final bulk vaccine. Material that has undergone all the steps
PRODUCTS of production except for the final filling. It consists of one or
more monovalent pooled harvests, from cultures of one or
For some items, alternative terms commonly used in more species or types of micro-organism, after clarification,
connection with veterinary vaccines are shown in parenthesis. dilution or addition of any adjuvant or other auxiliary
Seed-lot system. A seed-lot system is a system according to substance. It is treated to ensure its homogeneity and is used
which successive batches of a product are derived from the for filling the containers of one or more final lots (batches).
same master seed lot. For routine production, a working seed Final lot (Batch). A collection of closed, final containers or
lot may be prepared from the master seed lot. The origin and other final dosage units that are expected to be homogeneous
the passage history of the master seed lot and the working and equivalent with respect to risk of contamination during
seed lot are recorded. filling or preparation of the final product. The dosage units
Master seed lot. A culture of a micro-organism distributed are filled, or otherwise prepared, from the same final bulk
from a single bulk into containers and processed together in vaccine, freeze-dried together (if applicable) and closed in
a single operation in such a manner as to ensure uniformity one continuous working session. They bear a distinctive
and stability and to prevent contamination. A master seed number or code identifying the final lot (batch). Where a final
lot in liquid form is usually stored at or below − 70 °C. A bulk vaccine is filled and/or freeze-dried on several separate
freeze-dried master seed lot is stored at a temperature known sessions, there results a related set of final lots (batches) that
to ensure stability. are usually identified by the use of a common part in the
distinctive number or code ; these related final lots (batches)
Working seed lot. A culture of a micro-organism derived are sometimes referred to as sub-batches, sub-lots or filling
from the master seed lot and intended for use in production. lots.
Working seed lots are distributed into containers and stored
as described above for master seed lots. Combined vaccine. A multicomponent preparation
formulated so that different antigens are administered
Cell-bank system (Cell-seed system). A system whereby simultaneously. The different antigenic components are
successive final lots (batches) of a product are manufactured intended to protect against different strains or types of the
by culture in cells derived from the same master cell bank same organism and/or different organisms. A combined
(master cell seed). A number of containers from the master vaccine may be supplied by the manufacturer either as a single
cell bank (master cell seed) are used to prepare a working liquid or freeze-dried preparation or as several constituents
cell bank (working cell seed). The cell-bank system (cell-seed with directions for admixture before use.
system) is validated for the highest passage level achieved
during routine production.
Master cell bank (Master cell seed). A culture of cells 07/2010:50202
distributed into containers in a single operation, processed
together and stored in such a manner as to ensure uniformity 5.2.2. CHICKEN FLOCKS FREE
and stability and to prevent contamination. A master cell bank
(master cell seed) is usually stored at − 70 °C or lower. FROM SPECIFIED PATHOGENS FOR
Working cell bank (Working cell seed). A culture of cells THE PRODUCTION AND QUALITY
derived from the master cell bank (master cell seed) and CONTROL OF VACCINES
intended for use in the preparation of production cell cultures.
The working cell bank (working cell seed) is distributed into Where specified, chickens, embryos or cell cultures used for
containers, processed and stored as described for the master the production or quality control of vaccines are derived from
cell bank (master cell seed). eggs produced by chicken flocks free from specified pathogens
(SPF). The SPF status of a flock is ensured by means of the
Primary cell cultures. Cultures of cells obtained by system described below. The list of micro-organisms given is
trypsination of a suitable tissue or organ. The cells are based on current knowledge and will be updated as necessary.
essentially identical to those of the tissue of origin and are
no more than 5 in vitro passages from the initial preparation A flock is defined as a group of birds sharing a common
from the animal tissue. environment and having their own caretakers who have no
contact with non-SPF flocks. Once a flock is defined, no
Cell lines. Cultures of cells that have a high capacity for non-SPF birds are added to it.
multiplication in vitro. In diploid cell lines, the cells have Each flock is housed so as to minimise the risk of
essentially the same characteristics as those of the tissue of contamination. The facility in which the flock is housed must
origin. In continuous cell lines, the cells are able to multiply not be sited near to any non-SPF flocks of birds with the
indefinitely in culture and may be obtained from healthy or exception of flocks that are in the process of being established
tumoral tissue. Some continuous cell lines have oncogenic as SPF flocks and that are housed in facilities and conditions
potential under certain conditions. appropriate to SPF flocks. The SPF flock is housed within
Production cell culture. A culture of cells intended for use in an isolator or in a building with filtered air under positive
production ; it may be derived from one or more containers pressure. Appropriate measures are taken to prevent entry of
of the working cell bank (working cell seed) or it may be a rodents, wild birds, insects and unauthorised personnel.
primary cell culture. Personnel authorised to enter the facility must have no contact
Control cells. A quantity of cells set aside, at the time of virus with other birds or with agents potentially capable of infecting
inoculation, as uninfected cell cultures. The uninfected cells the flock. It is advisable for personnel to shower and change
are incubated under similar conditions to those used for the clothing or to wear protective clothing before entering the
production cell cultures. controlled facility.
General Notices (1) apply to all monographs and other texts 579
5.2.2. SPF chicken flocks for vaccines EUROPEAN PHARMACOPOEIA 8.0
Wherever possible, items taken into the facility are sterilised. required. Tests are performed on two 5 per cent samples of
In particular it is recommended that the feed is suitably the flock (minimum 10, maximum 200 birds) taken with an
treated to avoid introduction of undesirable micro-organisms interval of at least 4 weeks between the ages of 12-16 weeks
and that water is at least of potable quality, for example from and 16-20 weeks.
a chlorinated supply. No medication is administered to birds
within the flock that might interfere with detection of any All samples are collected and tested individually. Blood
disease. samples for antibody tests and suitable samples for testing for
leucosis antigen are collected. The test methods to be used are
A permanent record is kept of the general health of the flock as described under Routine testing of designated SPF flocks.
and any abnormality is investigated. Factors to be monitored Only when all tests have confirmed the absence of infection
include morbidity, mortality, general physical condition, feed may the new generation be designated as SPF.
consumption, daily egg production and egg quality, fertility
and hatchability. Records are maintained for a period of at
least 5 years. Details of any deviation from normal in these ROUTINE TESTING OF DESIGNATED SPF FLOCKS
performance parameters or detection of any infection are General examination and necropsy. Clinical examination
notified to the users of the eggs as soon as practicable. is carried out at least once per week throughout the life
The tests or combination of tests described below must have of the flock in order to verify that the birds are free from
suitable specificity and sensitivity with respect to relevant fowl-pox virus and signs of any other infection. In the event
serotypes of the viruses. Samples for testing are taken at of mortality exceeding 0.2 per cent per week, necropsy is
random. performed on all available carcasses to verify that there is
no sign of infection. Where appropriate, histopathological
A positive result for chicken anaemia virus (CAV) does and/or microbiological/virological studies are performed to
not necessarily exclude use of material derived from the confirm diagnosis. Specific examination for tuberculosis
flock, but live vaccines for use in birds less than 7 days old lesions is carried out and histological samples from any
shall be produced using material from CAV-negative flocks. suspected lesions are specifically stained to verify freedom
Inactivated vaccines for use in birds less than 7 days old may from Mycobacterium avium. Caecal contents of all available
be produced using material from flocks that have not been carcasses are examined microbiologically for the presence of
shown to be free from CAV, provided it has been demonstrated Salmonella spp. using the techniques described below. Where
that the inactivation process inactivates CAV. appropriate, caecal samples from up to 5 birds may be pooled.
ESTABLISHMENT OF AN SPF FLOCK Cultural testing for Salmonella spp. Cultural testing for
Salmonella spp. is performed either by testing samples of
A designated SPF flock is derived from chickens shown to be droppings or cloacal swabs or by testing of drag swabs. Where
free from vertically-transmissible agents listed in Table 5.2.2-1. droppings or cloacal swabs are tested, a total of 60 samples
This is achieved by testing of 2 generations prior to the within each 4-week period is tested throughout the entire
designated SPF flock. A general scheme for the procedure life of the flock. Tests may be performed on pools of up to
to be followed in establishing and maintaining an SPF flock 10 samples. Where drag swabs are tested, a minimum of
is shown diagrammatically in Table 5.2.2.-2. In order to 2 drag swabs are tested during each 4-week period throughout
establish a new SPF flock, a series of tests must be conducted the entire life of the flock. Detection of Salmonella spp. in
on 3 generations of birds. All birds in the 1st generation must these samples is performed by pre-enrichment of the samples
be tested at least once before the age of 20 weeks for freedom followed by culture using Salmonella-selective media.
from avian leucosis group-antigen and tested by an enzyme
immunoassay (EIA) or by virus neutralisation (VN) for Tests for avian leucosis antigen. Prior to the commencement
freedom of antibodies to avian leucosis virus subtypes A, B and of laying, cloacal swabs or blood samples (using buffy coat
J. All birds must also be tested for freedom from antibodies cultivation) are tested for the presence of group-specific
to the vertically-transmissible agents listed in Table 5.2.2-1. leucosis antigen. A total of 5 per cent (minimum 10, maximum
From the age of 8 weeks the flock is tested for freedom from 200) of the flock is sampled during each 4-week period.
Salmonella. Clinical examination is carried out on the flock During lay, albumen samples from 5 per cent (minimum 10,
from 8 weeks of age and the birds must not exhibit any signs maximum 200) of the eggs are tested in each 4-week period.
of infectious disease. The test methods to be used for these Tests are performed by EIA for group-specific antigen using
tests are given in the table and further guidance is also given methods that are capable of detecting antigen from subgroups
in the section below on routine testing of designated SPF A, B and J.
flocks. From 20 weeks of age, the flock is tested as described Test for antibodies to other agents. Tests for antibodies to all
under Routine testing of designated SPF flocks. All stages agents listed in Table 5.2.2.-1 are performed throughout the
of this testing regime are also applied to the subsequent laying period of the flock. In each 4-week period, samples are
2 generations, except the testing of every bird before lay for taken from 5 per cent (minimum 10, maximum 200) of the
vertically-transmissible agents. All test results must indicate flock. It is recommended that 1.25 per cent of the flock is
freedom from pathogens in all 3 generations for the flock sampled each week since some test methods for some agents
consisting of the 3rd generation to be designated as SPF. must be conducted on a weekly basis. Table 5.2.2.-1 classifies
SPF embryos derived from another designated SPF flock the agents into those that spread rapidly through the flock and
contained within a separate facility on the same site may be those that spread slowly or may not infect the entire flock. For
introduced. From 8 weeks of age, these replacement birds those agents listed as slowly spreading, each sample is tested
are regarded as a flock and are tested in accordance with test individually. For those agents listed as rapidly spreading,
procedures described above. at least 20 per cent of the samples collected in each 4-week
period are tested individually or, where serum neutralisation
INITIAL TESTING REQUIREMENTS FOR SUBSEQUENT or ELISA tests are employed, all of the samples may be tested
GENERATIONS DERIVED FROM A DESIGNATED SPF individually or by preparing pools of 5 samples, collected at
FLOCK the same time.
Where a replacement flock is derived exclusively from a fully Suitable methods to be used for detection of the agents
established SPF flock the new generation is tested prior to are shown in Table 5.2.2.-1. Subject to agreement by the
being designated as SPF. In addition to the tests for Salmonella competent authority, other test methods may be used provided
and monitoring of the general health and performance of they are shown to be at least as sensitive as those indicated
the flock, further specific testing from the age of 8 weeks is and of appropriate specificity.
Table 5.2.2.-1
Agent Test Vertical Rapid/slow
to be used** transmission spread
Avian adenoviruses, group 1 AGP, EIA yes slow
Table 5.2.2-2. – Schematic description of the establishment and maintenance of SPF flocks
NEW STOCK Establish freedom from vertically-transmissible agents
Test all birds for avian leucosis antigen and antibodies prior to 20 weeks of age
Test for Salmonella spp. and perform general clinical observation from 8 weeks of age
Carry out routine testing for specified agents from 20 weeks of age
2nd GENERATION Test all birds for avian leucosis antigen and antibodies prior to 20 weeks of age
Test for Salmonella spp. and perform general clinical observation from 8 weeks of age
Carry out routine testing for specified agents from 20 weeks of age
rd
3 GENERATION Test all birds for avian leucosis antigen and antibodies prior to 20 weeks of age
Test for Salmonella spp. and perform general clinical observation from 8 weeks of age
DESIGNATE FLOCK AS SPF IF ALL TESTS ARE SATISFACTORY
rd
3 GENERATION Carry out routine testing for specified agents from 20 weeks of age
Carry out routine testing for specified agents from 20 weeks of age
TESTS TO BE CONDUCTED AT THE END OF THE minimum of 5 per cent of the flock (minimum 10, maximum
LAYING PERIOD 200) is retained for at least 4 weeks. Blood samples are
Following the last egg collection, final testing to confirm collected from every bird in the group during the 4-week
the absence of vertically-transmissible agents indicated in period with at least 1.25 per cent of the birds (25 per cent
Table 5.2.2.-1 is performed. After the last egg collection, a of the sample) being bled not earlier than 4 weeks after
General Notices (1) apply to all monographs and other texts 581
5.2.3. Cell substrates for the production of vaccines for human use EUROPEAN PHARMACOPOEIA 8.0
the final egg collection. Serum samples are tested for working cell bank is prepared from one or more containers of
vertically-transmissible agents (as defined by Table 5.2.2.-1) the master cell bank. The use, identity and inventory control
using the methods indicated. Where sampling is performed of the containers is carefully documented.
on a weekly basis, at least 1.25 per cent of the birds (25 per Media and substances of human or animal origin. The
cent of the sample) are tested each week during this period. composition of media used for isolation and all subsequent
Alternatively, within 4 weeks of the final egg collection blood culture is recorded in detail, and if substances of human or
and/or other suitable sample materials are collected from at animal origin are used they must be free from extraneous
least 5 per cent of the flock and tested for the presence of agents (2.6.16) and must comply with the general chapter on
vertically-transmissible agents using validated nucleic acid 5.1.7. Viral safety.
amplification techniques (2.6.21).
If human albumin is used, it complies with the monograph
ACTION TO BE TAKEN IN THE EVENT OF DETECTION Human albumin solution (0255).
OF A SPECIFIED AGENT If bovine serum is used, it complies with the monograph
Bovine serum (2262).
If evidence is found of contamination of the flock by an
agent listed as slowly spreading in Table 5.2.2.-1, all materials Trypsin used for the preparation of cell cultures is examined
derived from the flock during the 4-week period immediately by suitable methods and shown to be sterile and free from
preceding the date on which the positive sample was collected mycoplasmas and viruses, notably pestiviruses, circoviruses
are considered unsatisfactory. Similarly, if evidence is found and parvoviruses.
of contamination of the flock by an agent listed as rapidly Cell seed. The data used to assess the suitability of the cell
spreading in Table 5.2.2.-1, all materials derived from the flock seed comprises information, where available, on source,
during the 2-week period immediately preceding the date history and characterisation.
on which the positive sample was collected are considered Source of the cell seed. For human cell lines, the following
unsatisfactory. Any product manufactured with such information concerning the donor is recorded : ethnic and
materials, and for which the use of SPF materials is required, is geographical origin, age, sex, general physiological condition,
considered unsatisfactory and must be discarded ; any quality tissue or organ used, results of any tests for pathogens.
control tests conducted using the materials are invalid.
For animal cell lines, the following information is recorded
Producers must notify users of all eggs of the evidence of concerning the source of the cells : species, strain, breeding
contamination as soon as possible following the outbreak. conditions, geographical origin, age, sex, general physiological
Any flock in which an outbreak of any specified agent is condition, tissue or organ used, results of any tests for
confirmed may not be redesignated as an SPF flock. Any pathogens.
progeny derived from that flock during or after the 4-week Cells of neural origin, such as neuroblastoma and P12 cell
period prior to the last negative sample being collected may lines, may contain substances that concentrate agents of
not be designated as SPF. spongiform encephalopathies and such cells are not used for
vaccine production.
History of the cell seed. The following information is recorded :
the method used to isolate the cell seed, culture methods,
01/2011:50203 any other procedures used to establish the master cell bank,
notably any that might expose the cells to extraneous agents.
5.2.3. CELL SUBSTRATES FOR THE Full information may not be available on the ingredients of
media used in the past for cultivation of cells, for example on
PRODUCTION OF VACCINES FOR the source of substances of animal origin ; where justified and
HUMAN USE authorised, cell banks already established using such media
may be used for vaccine production.
This general chapter deals with diploid cell lines and Characterisation of the cell seed. The following properties are
continuous cell lines used as cell substrates for the production investigated :
of vaccines for human use ; specific issues relating to vaccines
prepared by recombinant DNA technology are covered by the (1) the identity of the cells (for example, isoenzymes, serology,
monograph Products of recombinant DNA technology (0784). nucleic acid fingerprinting) ;
Testing to be carried out at various stages (cell seed, master (2) the growth characteristics of the cells and their
cell bank, working cell bank, cells at or beyond the maximum morphological properties (optical and electron microscopes) ;
population doubling level used for production) is indicated in (3) for diploid cell lines, karyotype ;
Table 5.2.3.-1. General provisions for the use of cell lines and (4) for diploid cell lines, the in vitro life span in terms of
test methods are given below. Where primary cells or cells population doubling level.
that have undergone a few passages without constitution of a
cell bank are used for vaccine production, requirements are Cell substrate stability. Suitable viability of the cell line in
given in the individual monograph for the vaccine concerned. the intended storage conditions must be demonstrated. For
a given product to be prepared in the cell line, it is necessary
Diploid cell lines. A diploid cell line has a high but finite to demonstrate that consistent production can be obtained
capacity for multiplication in vitro. with cells at passage levels at the beginning and end of the
Continuous cell lines. A continuous cell line has the capacity intended span of use.
to multiply indefinitely in vitro ; the cells often have differences Infectious extraneous agents. Cell lines for vaccine
in karyotype compared to the original cells ; they may be production shall be free from infectious extraneous agents.
obtained from healthy or tumoral tissue either from mammals Tests for extraneous agents are carried out as shown in
or from insects. Table 5.2.3.-1 using the methods described below.
For injectable vaccines produced in continuous cell lines, For cell lines of insect origin, tests for specific viruses relevant
the purification process is validated to demonstrate removal to the species of origin of the insect cells and for arboviruses
of substrate-cell DNA to a level equivalent to not more than (arthropod - borne viruses) are applied. The panel of viruses
10 ng per single human dose, unless otherwise prescribed. tested is chosen according to the current state of scientific
Cell-bank system. Production of vaccines in diploid or knowledge.
continuous cell lines is based on a cell-bank system. The in Cell lines that show the presence of retroviruses capable of
vitro age of the cells is counted from the master cell bank. Each replication are not acceptable for production of vaccines.
Morphology + + + +
2. EXTRANEOUS AGENTS
Mycoplasmas − + + −
Co-cultivation − − + (2)
+(2)
3. TUMORIGENICITY
(1) The diploid character is established for each working cell bank but using cells at or beyond the maximum population doubling level used
for production.
(2) Testing is carried out for each working cell bank, but using cells at or beyond the maximum population doubling level used for production.
(3) Testing is carried out for the master cell bank, but using cells at or beyond the maximum population doubling level used for production.
(4) The MRC-5, WI-38 and FRhL-2 cell lines are recognised as being non-tumorigenic and they need not be tested. Tests are not carried out on cell
lines that are known or assumed to be tumorigenic, for example CHO and BHK-21.
(5) Testing is carried out on the cell seed, but using cells at or beyond the maximum population doubling level used for production.
General Notices (1) apply to all monographs and other texts 583
5.2.3. Cell substrates for the production of vaccines for human use EUROPEAN PHARMACOPOEIA 8.0
Mycoplasmas (2.6.7). The master cell bank and each working Tests in eggs. Using an inoculum of 106 viable cells per
cell bank comply with the test for mycoplasmas. Use one or egg, inoculate the cells into the allantoic cavity of ten 9- to
more containers for the test. 11-day-old SPF embryonated hens’ eggs (5.2.2) and into the
Spiroplasmas (insect cell lines). The master cell bank and yolk sac of ten 5- to 6-day-old SPF embryonated hens’ eggs.
each working cell bank of insect cells are demonstrated to be Incubate for not less than 5 days. Test the allantoic fluids for
free of spiroplasmas by a validated method approved by the the presence of haemagglutinins using mammalian and avian
competent authority. Use one or more containers for the test. red blood cells ; carry out the test at 5 ± 3 °C and 20-25 °C
and read the results after 30-60 min. The cells comply with
Electron microscopy (insect cell lines). The master cell the test if no evidence of any extraneous agent is found. The
bank is examined by electron microscopy for the presence test is invalid if fewer than 80 per cent of the embryos remain
of adventitious agents. Cell lines are maintained at the healthy and survive to the end of the observation period.
temperature routinely used for production and taken at or
beyond the maximum population doubling level. In addition, Specific tests for possible contaminants depending on the
cell lines are maintained at temperatures above and below origin of the cells. Tests for specific pathogens are carried out
that routinely used for production and may also be subjected using nucleic acid amplification techniques (NAT) (2.6.21)
to other treatments such as exposure to chemical stressors. with or without prior amplification in cells. Alternatively,
The maintenance temperatures and treatments used are suitable serological techniques such as enzyme-linked
agreed with the competent authority along with the number immunosorbent assay, serum neutralisation and anti-body
of sectioned cells to be examined. production tests in suitable permissive animals may be used.
For cell lines of rodent origin, use either antibody production
Test for extraneous agents in cell cultures. The cells comply tests in mice, rats or hamsters or nucleic acid amplification
with the test for haemadsorbing viruses and with the tests in techniques (2.6.21) to detect species-specific viruses. Testing
cell cultures for other extraneous agents given in chapter 2.6.16 must take account of the origin and culture history of the cell
under Production cell culture : control cells. If the cells are of line. The tests are designed to detect potential contaminants,
simian origin, they are also inoculated into rabbit kidney cell particularly those that are known to infect latently the species
cultures to test for herpesvirus B (cercopithecid herpesvirus 1). of origin, for example simian virus 40 in rhesus monkeys or
Co-cultivation. For mammalian and avian cell lines, Flock house virus in insect cells.
co-cultivate intact and/or disrupted cells separately with other Tests for tumorigenicity in vivo. The test consists in
cell systems including human cells and simian cells. For insect establishing a comparison between the continuous cell line
cell lines, extracts of disrupted cells are incubated with other and a suitable positive control (for example, HeLa or Hep2
cell systems, including human, simian, and at least 1 cell line cells).
that is different from that used in production, is permissible to Animal systems that have been shown to be suitable for this
insect viruses and allows detection of human arboviruses (for test include :
example BHK-21). Carry out examinations to detect possible (1) athymic mice (Nu/Nu genotype) ;
morphological changes. Carry out tests on the cell culture
fluids to detect haemagglutinating viruses, or on cells to (2) newborn mice, rats or hamsters that have been treated
detect haemadsorbing viruses. The test for haemagglutinating with antithymocyte serum or globulin ;
viruses does not apply for arboviruses to be detected in insect (3) thymectomised and irradiated mice that have been
cells. The cells comply with the test if no evidence of any reconstituted (T–, B+) with bone marrow from healthy mice.
extraneous agent is found. Whichever animal system is selected, the cell line and the
Retroviruses. Examine for the presence of retroviruses using : reference cells are injected into separate groups of 10 animals
each. In both cases, the inoculum for each animal is 107 cells
(1) product-enhanced reverse transcriptase (PERT) assay suspended in a volume of 0.2 mL, and the injection may be by
(2.6.21) carried out for cell bank supernatants using cells at either the intramuscular or the subcutaneous route. Newborn
or beyond the maximum population doubling level that will animals are treated with 0.1 mL of antithymocyte serum or
be used for production ; globulin on days 0, 2, 7 and 14 after birth. A potent serum or
(2) transmission electron microscopy. globulin is one that suppresses the immune mechanisms of
growing animals to the extent that the subsequent inoculum
If test (1) and/or test (2) gives a positive result, test (3) is of 107 positive reference cells regularly produces tumours
carried out : and metastases. Severely affected animals showing evident,
(3) infectivity assays carried out on human cells with an progressively growing tumours are euthanised before the end
endpoint PERT assay on the supernatant. of the test to avoid unnecessary suffering.
Since the sensitivity of PERT assays is very high, interpretation At the end of the observation period all animals, including the
of a positive signal may be equivocal and a decision on the reference group(s), are euthanised and examined for gross and
acceptability of a cell substrate is based on all available data. microscopic evidence of the proliferation of inoculated cells at
the site of injection and in other organs (for example, lymph
Tests in animals. Inject intramuscularly (or, for suckling nodes, lungs, kidneys and liver).
mice, subcutaneously) into each of the following groups of
animals 107 viable cells divided equally between the animals In all test systems, the animals are observed and palpated
in each group : at regular intervals for the formation of nodules at the
sites of injection. Any nodules formed are measured in
(1) 2 litters of suckling mice less than 24 h old, comprising not 2 perpendicular directions, the measurements being recorded
fewer than 10 animals ; regularly to determine whether there is progressive growth of
(2) 10 adult mice. the nodule. Animals showing nodules that begin to regress
during the period of observation are euthanised before the
Inject intracerebrally into each of 10 adult mice 106 nodules are no longer palpable, and processed for histological
viable cells to detect the possible presence of lymphocytic examination. Animals with progressively growing nodules
choriomeningitis virus. are observed for 1-2 weeks. Among those without nodule
Observe the animals for at least 4 weeks. Investigate animals formation, half are observed for 3 weeks and half for 12 weeks
that become sick or show any abnormality to establish the before they are euthanised and processed for histological
cause of illness. The cells comply with the test if no evidence examination. A necropsy is performed on each animal and
of any extraneous agent is found. The test is invalid if fewer includes examination for gross evidence of tumour formation
than 80 per cent of the animals in each group remain healthy at the site of injection and in other organs such as lymph
and survive to the end of the observation period. nodes, lungs, brain, spleen, kidneys and liver.
All tumour-like lesions and the site of injection are examined Table 5.2.4.-1. – Cell culture stage at which tests
histologically. In addition, since some cell lines may give rise are carried out
to metastases without evidence of local tumour growth, any Master cell Working Cell from working
detectable regional lymph nodes and the lungs of all animals seed cell seed cell seed at highest
are examined histologically. passage level
General microscopy + + +
The test is invalid if fewer than 9 of the 10 animals injected
with the positive reference cells show progressively growing Bacteria and fungi + + −
tumours. + + −
Mycoplasmas
Tests for tumorigenicity in vitro. The following test systems + + −
Viruses
may be used :
Identification of species + − +
(1) colony formation in soft agar gels ;
Karyotype + − +
(2) production of invasive cell growth following inoculation
into organ cultures ; Tumorigenicity + − −
General Notices (1) apply to all monographs and other texts 585
5.2.4. Cell cultures for the production of veterinary vaccines EUROPEAN PHARMACOPOEIA 8.0
Detection of cytopathic viruses. Two monolayers of at least Wherever possible, particularly for mammalian cells, a
6 cm2 each are stained with an appropriate cytological stain. seed-lot system is used with, for example, a master cell seed
The entire area of each stained monolayer is examined for formed after less than five passages, the working cell seed
any inclusion bodies, abnormal numbers of giant cells or any being no more than five passages from the initial preparation
other lesion indicative of a cellular abnormality which might of the cell suspension from the animal tissues.
be attributable to a contaminant. Each master cell seed, working cell seed and cells of the
Detection of haemadsorbent viruses. Monolayers totalling highest passage of primary cells are checked in accordance
at least 70 cm2 are washed several times with an appropriate with Table 5.2.4.-2 and the procedure described below. The
buffer and a sufficient volume of a suspension of suitable red sample tested shall cover all the sources of cells used for the
blood cells added to cover the surface of the monolayer evenly. manufacture of the batch. No batches of vaccine manufactured
After different incubation times cells are examined for the using the cells may be released if any one of the checks
presence of haemadsorption. performed produces unsatisfactory results.
Detection of specified viruses. Tests are carried out for Table 5.2.4.-2. – Cell culture stage at which tests
freedom from contaminants specific for the species of origin are carried out
of the cell line and for the species for which the product is
intended. Sufficient cells on suitable supports are prepared Master Working Highest passage
to carry out tests for the agents specified. Suitable positive cell seed cell seed level
General microscopy + + +
controls are included in each test. The cells are subjected
to suitable tests, for example using fluorescein-conjugated Bacteria and fungi + + −
antibodies or similar reagents.
Mycoplasmas + + −
Tests in other cell cultures. Monolayers totalling at least
+ + −
140 cm2 are required. The cells are frozen and thawed at least Viruses
three times and then centrifuged to remove cellular debris. Identification of species + − −
Inoculate aliquots onto the following cells at any time up to
70 per cent confluency : Characteristics of cultures. The appearance of cell
– primary cells of the source species ; monolayers, before and after histological staining, is described.
Information, if possible numerical data, is recorded, especially
– cells sensitive to viruses pathogenic for the species for on the speed and rate of growth. Similarly, the presence or
which the vaccine is intended ; absence of contact inhibition, polynucleated cells and any
– cells sensitive to pestiviruses. other cellular abnormalities are specified.
The inoculated cells are maintained in culture for at least Identification of species. It shall be demonstrated by one
7 days, after which freeze-thawed extracts are prepared as validated test that the master cell seed comes from the
above and inoculated onto sufficient fresh cultures of the same specified species of origin.
cell types to allow for the testing as described below. The cells When a fluorescence test is carried out and the corresponding
are incubated for at least a further 7 days. The cultures are serum to the species of origin of cells is used and shows that
examined regularly for the presence of any cytopathic changes all the tested cells are fluorescent, it is not necessary to carry
indicative of living organisms. out other tests with reagents able to detect contamination by
At the end of this period of 14 days, the inoculated cells are cells of other species.
subjected to the following checks : Bacterial and fungal sterility. The cells comply with the
– freedom from cytopathic and haemadsorbent organisms, test for sterility (2.6.1). The sample of cells to be examined
using the methods specified in the relevant paragraphs consists of not less than the number of cells in a monolayer
above, with an area of 70 cm2 or for cells grown in suspension an
approximately equivalent number of cells. The cells are
– absence of pestiviruses and other specific contaminants maintained in culture for at least 15 days without antibiotics
by immunofluorescence or other validated methods as before carrying out the test.
indicated in the paragraph above on Detection of Specified
Mycoplasmas (2.6.7). The cells comply with the test for
Viruses.
mycoplasmas. The cells are maintained in culture for at least
Tumorigenicity. The risk of a cell line for the target species 15 days without antibiotics before carrying out the test.
must be evaluated and, if necessary, tests are carried out.
Absence of contaminating viruses. The cells must not be
contaminated by viruses ; suitably sensitive tests, including
PRIMARY CELLS those prescribed below are carried out.
For most mammalian vaccines, the use of primary cells is not The monolayers tested shall be at least 70 cm2, and shall be
acceptable for the manufacture of vaccines since cell lines can prepared and maintained in culture using the same medium
be used. If there is no alternative to the use of primary cells, and additives, and under similar conditions to those used for
the cells are obtained from a herd or flock free from specified the preparation of the vaccine.
pathogens, with complete protection from introduction of
diseases (for example, disease barriers, filters on air inlets, The monolayers are maintained in culture for a total of at
suitable quarantine before introduction of animals). Chicken least 28 days or for the longest period possible if culture for
flocks comply with the requirements prescribed in general 28 days is impossible. Subcultures are made at 7-day intervals,
chapter 5.2.2. Chicken Flocks Free from Specified Pathogens for unless the cells do not survive for this length of time when the
the Production and Quality Control of Vaccines. For all other subcultures are made on the latest day possible. Sufficient cells,
species, the herd or flock is shown to be free from relevant in suitable containers are produced for the final subculture to
specified pathogens. All the breeding stock in the herd or carry out the tests specified below.
flock intended to be used to produce primary cells for vaccine The monolayers are examined regularly throughout the
manufacture is subject to a suitable monitoring procedure incubation period for the possible presence of cytopathic
including regular serological checks carried out at least twice effects and at the end of the observation period for cytopathic
a year and two supplementary serological examinations effects, haemadsorbent viruses and specific viruses by
performed in 15 per cent of the breeding stock in the herd immunofluorescence and other suitable tests as indicated
between the two checks mentioned above. below.
Detection of cytopathic viruses. Two monolayers of at least 2. GENERAL PRINCIPLES AND REQUIREMENTS
6 cm2 each are stained with an appropriate cytological stain. Substances of animal origin comply with the requirements of the
Examine the entire area of each stained monolayer for any European Pharmacopoeia (where a relevant monograph exists).
inclusion bodies, abnormal numbers of giant cells or any
other lesion indicative of a cellular abnormality that might be Restrictions are placed on the use of substances of animal
attributable to a contaminant. origin because of safety concerns associated with pathogens
that may be present in them and epidemiological and/or
Detection of haemadsorbent viruses. Monolayers totalling regulatory concerns associated with the presence of particular
at least 70 cm2 are washed several times with a suitable buffer antigens (either live or inactivated).
solution and a sufficient volume of a suspension of suitable General principles :
red blood cells added to cover the surface of the monolayer
evenly. After different incubation times, examine cells for the – it is recommended to minimise, wherever practicable, the
presence of haemadsorption. use of substances of animal origin ;
Detection of specified viruses. Tests are be carried out for – unless otherwise justified, the use of substances of animal
freedom of contaminants specific for the species of origin of origin as constituents in the formulation of medicinal
the cells and for the species for which the product is intended. products is not acceptable except where such substances
are subject to a treatment validated for the inactivation of
Sufficient cells on suitable supports are prepared to carry out live extraneous agents.
tests for the agents specified. Suitable positive controls are General requirements :
included in each test. The cells are subjected to suitable tests
using fluorescein-conjugated antibodies or similar reagents. – any batch of substance (after inactivation and/or processing,
if relevant) found to contain or suspected of containing any
Tests in other cell cultures. Monolayers totalling at least living extraneous agent shall be discarded or used only in
140 cm2 are required. The cells are frozen and thawed at least exceptional and justified circumstances ; to be accepted for
three times and then centrifuged to remove cellular debris. use, further processing must be applied that will ensure
Aliquots are inoculated onto the following cells at any time elimination and/or inactivation of the extraneous agent,
up to 70 per cent confluency : and it shall then be demonstrated that the elimination
– primary cells of the source species ; and/or inactivation has been satisfactory ;
– cells sensitive to viruses pathogenic for the species for – any batch of substance that, as concluded from the risk
which the vaccine is intended ; assessment, may induce an unacceptable detectable
– cells sensitive to pestiviruses. immune response in the target species as a consequence
of contamination with inactivated extraneous agents,
The inoculated cells are maintained in culture for at least must not be used for the manufacture of that particular
7 days, after which freeze-thawed extracts are prepared as immunological veterinary medicinal product.
above, and inoculated onto sufficient fresh cultures of the
same cell types to allow for the testing as described below. The 3. RISK MANAGEMENT
cells are incubated for at least a further 7 days. All cultures are No single measure or combination of measures can guarantee
regularly examined for the presence of any cytopathic changes the safety of the use of substances of animal origin, but they
indicative of living organisms. can reduce the risk from such use. It is therefore necessary
At the end of this period of 14 days, the inoculated cells are for the manufacturer of immunological veterinary medicinal
subjected to the following checks : products to take account of this when choosing a substance
– freedom from cytopathic and haemadsorbent organisms is of animal origin to use in manufacture, and to conduct a risk
demonstrated using the methods specified in the relevant assessment, taking into account the origin of the substance
paragraphs above ; and the manufacturing steps applied to it.
– relevant substrates are tested for the absence of pestiviruses In addition, risk management procedures must be applied.
and other specific contaminants by immunofluorescence Any residual risk must be evaluated in relation to the
or other validated methods as indicated in the paragraph potential benefits derived from the use of the substance for
above on Detection of Specified Viruses. the manufacture of the immunological veterinary medicinal
product.
3-1. RISK ASSESSMENT
The risk assessment must take account of the animal diseases
07/2009:50205 occurring in the country of origin of the animals used as
a source of the substance, the potential infectious diseases
5.2.5. SUBSTANCES OF ANIMAL occurring in the source species and the likely infectivity in
the source organ or tissue. From this information, as part of
ORIGIN FOR THE PRODUCTION OF the risk assessment, a list can be prepared of the extraneous
IMMUNOLOGICAL VETERINARY agents that may be present in the substance.
The risk of contamination of the substance and the
MEDICINAL PRODUCTS resultant immunological veterinary medicinal product
with living extraneous agents needs to be assessed. The
1. SCOPE risk of contamination of the substance and the resultant
Substances of animal origin (for example serum, trypsin and immunological veterinary medicinal product with inactivated
serum albumin) may be used during the manufacture of extraneous agents may also need to be taken into account.
immunological veterinary medicinal products. This would be the case if, for example, the contaminant was
The requirements set out in this chapter apply to substances of one from which a European country is officially free and/or is
animal origin produced on a batch basis, for use at all stages the subject of a specific disease control program in a European
of manufacture, for example in culture media or as added country and where the presence of the inactivated agent could
constituents of products during blending. These requirements lead to the stimulation of a detectable immune response in
are not intended for the control of seed materials or substrates recipient animals.
of animal origin that are covered by requirements in other As part of the risk assessment, the presence in the substance
pharmacopoeial texts such as the monograph Vaccines for of antibodies that can interfere with the detection and/or
veterinary use (0062) and chapter 5.2.4. Cell cultures for the inactivation of living extraneous agents must also be taken
production of veterinary vaccines. into account.
General Notices (1) apply to all monographs and other texts 587
5.2.6. Evaluation of safety of veterinary vaccines and immunosera EUROPEAN PHARMACOPOEIA 8.0
The risk assessment may need to be repeated and the risk For inactivated immunological veterinary medicinal products,
management steps described below re-evaluated and revised the method used for inactivation of the active ingredient may
in order to take account of changes : also be validated for inactivation of possible contaminants
from substances of animal origin used in the manufacture of
– in the incidence of diseases occurring in the country or
this active ingredient.
countries of origin of animals used as the source for the
substance, including emerging diseases (new pathogens) ; 4-4. TESTS
Depending on the outcome of the risk assessment and the
– in the incidence of diseases and of disease control measures
validation data available for any procedure applied, tests for
applied in the European countries in which immunological extraneous agents may be conducted on each batch before
veterinary medicinal products manufactured with the and/or after the application of an inactivation/processing
substance are used.
step. For examination of the substance for freedom from
3-2. RISK CONTROL extraneous agents, any solids are dissolved or suspended in a
For each of the potential extraneous agents identified by the suitable medium to provide a suitable preparation for testing.
risk assessment, and taking into account the proposed use of A sufficient quantity of the preparation is tested to give a
the substance, the risk must be controlled by the use of one or suitably sensitive test, as established in the validation studies.
a combination of the followings measures : As well as tests for living extraneous agents, tests may need
to be conducted for the presence of inactivated extraneous
– placing restrictions on the source of the material and agents, depending on the risks identified.
auditing this ;
Freedom from living extraneous viruses. A sample from
– using validated inactivation procedures ; each batch of the substance is tested for extraneous viruses by
– demonstrating the ability of a production step to remove or general and specific tests. These tests are validated with respect
inactivate extraneous agents ; to sensitivity and specificity for detection of a suitable range
of potential extraneous viruses. Suitably sensitive cell cultures
– testing for extraneous agents. are used for the tests for extraneous viruses, including primary
cells from the same species as the substance to be examined.
4. CONTROL MEASURES General test. The inoculated cell cultures are observed
4-1. SOURCE regularly for 21 days for cytopathic effects. At the end of each
All substances of animal origin used in the manufacture 7-day period, a proportion of the original cultures is fixed,
(including blending) of immunological veterinary medicinal stained and examined for cytopathic effects, and a proportion
products must be from a known and documented source is tested for haemadsorbing agents.
(including species of origin and country of origin of source Specific tests. A proportion of the cells available at the end
animals and tissues). of the general test is tested for specific viruses. The specific
viruses to be tested for are potential extraneous viruses that
4-2. PREPARATION
are identified through the risk assessment and that would
Substances of animal origin are prepared from a homogeneous not be detected by the general test. A test for pestiviruses is
bulk designated with a batch number. A batch may contain conducted if the source species is susceptible to these.
substances derived from as many animals as desired but once
defined and given a batch number, the batch is not added to Bacteria and fungi. Before use, substances are tested for
or contaminated in any way. sterility (2.6.1), or sterilised to inactivate any bacterial or
The production method used to prepare the substance of fungal contaminants.
animal origin from the raw material may contribute to Mycoplasma. Before use, substances are tested for freedom
the removal and/or inactivation of extraneous agents (see from mycoplasma (2.6.7), or sterilised to inactivate any
section 4-3). mycoplasmal contaminants.
4-3. INACTIVATION AND/OR OTHER PROCESSING STEPS
FOR REMOVAL OF EXTRANEOUS AGENTS 04/2013:50206
The inactivation procedure and/or other processing steps
chosen shall have been validated and shown to be capable of 5.2.6. EVALUATION OF SAFETY
reducing the titre of potential extraneous agents described OF VETERINARY VACCINES AND
below in the substance concerned by a factor of at least 106.
If this reduction in titre cannot be shown experimentally, a IMMUNOSERA
maximum pre-treatment titre of the extraneous agent must be The term ‘product’ means either a vaccine or an immunoserum
set, taking into account the reduction in titre afforded by the throughout the text.
inactivation/processing step and including a safety margin During development, safety tests are carried out in the target
factor of 100 ; each batch of substance must be tested to species to show the risks from use of the product.
determine the pre-treatment starting titre and confirm it is no
greater than the specified limit, unless proper risk assessment, Immune status for tests on vaccines. The immune status of
based on valid and suitable data, shows that titres will always animals to be used for the safety test is specified in the specific
be at least 100-fold below the titre that can effectively be monograph. For most monographs, 1 of the 3 following
inactivated. categories is specified :
1) the animals must be free from antibodies against the
The validation of the procedure(s) is conducted with a suitable virus/bacterium/toxin etc. contained in the vaccine ;
representative range of viruses covering different types
and sizes (enveloped and non-enveloped, DNA and RNA, 2) the animals are preferably free from antibodies against
single- and double-stranded, temperature- and pH-resistant), the virus/bacterium/toxin etc. contained in the vaccine, but
including test viruses with different degrees of resistance, animals with a low level of antibody may be used as long as
taking into account the type of procedure(s) to be applied the animals have not been vaccinated and the administration
and the viruses that may be present in the material. The of the vaccine does not cause an anamnestic response ;
evidence for the efficacy of the procedure may take the form 3) the animals must not have been vaccinated against the
of references to published literature and/or experimental data disease that the vaccine is intended to prevent.
generated by the manufacturer, but must be relevant to the As a general rule, category 1 is specified for live vaccines.
conditions that will be present during the production and For other vaccines, category 2 is usually specified, but where
inactivation/processing of the substance. most animals available for use in tests would comply with
category 1, this may be specified for inactivated vaccines also. Unless otherwise prescribed in a specific monograph or, in the
Category 3 is specified for some inactivated vaccines where absence of a specific monograph, unless otherwise justified
determination of antibodies prior to testing is unnecessary or and authorised, the vaccine complies with the test if no animal
impractical. For poultry vaccines, as a general rule the use of shows abnormal local or systemic reactions or signs of disease,
specified-pathogen-free (SPF) birds is specified. or dies from causes attributable to the vaccine.
For avian vaccines, the safety test is generally carried out 1-2. SAFETY OF 1 ADMINISTRATION OF AN OVERDOSE
using SPF chickens (5.2.2), except that for vaccines not Overdose testing is required only for live vaccines. An
recommended for use in chickens it is carried out using birds overdose of the product is administered by each recommended
of one of the species for which the vaccine is recommended, route of administration to animals of the categories of the
the birds being free from antibodies against the disease agent target species that are expected to be the most sensitive, such
for which the vaccine is intended to provide protection. as animals of the youngest age. If multiple routes and methods
Vaccines. In laboratory tests, ‘dose’ means that quantity of of administration are specified for the product concerned,
the product to be recommended for use and containing the administration by all routes is recommended. If 1 route of
maximum titre or potency likely to be contained in production administration has been shown to cause the most severe
batches. Live vaccines are prepared only from strains of effects, this single route may be selected as the only one for use
organisms that have been shown to be safe. For live vaccines, in the study. The overdose normally consists of 10 doses of a
use a batch or batches of vaccine containing virus/bacteria live vaccine. For freeze-dried live vaccines, the 10 doses shall
at the least attenuated passage level that will be present in a be reconstituted in a suitable volume of diluent for the test. For
batch of vaccine. vaccines intended for use in mammals, in general 8 animals
For combined vaccines, the safety shall be demonstrated ; for per group are used unless otherwise justified or specified in a
live components of combined vaccines, compliance with the specific monograph. For vaccines intended for use in fish, in
special requirements for live vaccines stated below shall be general 50 fish per group are used unless otherwise justified or
demonstrated separately for each vaccine strain. specified in a specific monograph. For vaccines intended for
For inactivated vaccines, safety tests carried out on the use in birds older than 3 weeks, in general 8 birds per group
combined vaccine may be regarded as sufficient to demonstrate are used unless otherwise justified or specified in a specific
the safety of the individual components. monograph. For vaccines intended for use in birds younger
than 3 weeks, in general 10 birds per group are used unless
Immunosera. In the tests, ‘dose’ means the maximum quantity
otherwise justified or specified in a specific monograph. The
of the product to be recommended for use and containing
animals are observed and examined at least daily for signs
the maximum potency and maximum total protein likely
of local and systemic reactions. Other objective criteria are
to be contained in production batches. In addition, if
recorded, such as body temperature (for mammals) and
appropriate, the dose tested also contains maximum quantities
performance measurements. The animals are observed and
of immunoglobulin or gammaglobulin.
examined for at least 14 days after administration.
The tests described below, modified or supplemented by tests Unless otherwise prescribed in a specific monograph or, in the
described in the Production section of a monograph, may be absence of a specific monograph, unless otherwise justified
carried out as part of the tests necessary during development and authorised, the vaccine complies with the test if no animal
to demonstrate the safety of the product. shows abnormal local or systemic reactions or signs of disease,
1. LABORATORY TESTS or dies from causes attributable to the vaccine.
1-1. SAFETY OF THE ADMINISTRATION OF 1 DOSE 1-3. SAFETY OF THE REPEATED ADMINISTRATION OF
For each of the recommended routes of administration, 1 DOSE
administer 1 dose of product to animals of each species and Repeated administration of 1 dose may be required to
category for which use of the product is to be recommended. reveal any adverse effects induced by such administration.
This must include animals of the youngest recommended age These tests are particularly important where the product,
and pregnant animals, if appropriate. notably an immunoserum, may be administered on several
For vaccines intended for use in mammals, in general occasions over a relatively short period of time. These tests
8 animals per group are used unless otherwise justified or are carried out on the most sensitive categories of the target
specified in a specific monograph. species, using each recommended route of administration. If
For fish vaccines administered by immersion, bathe the fish multiple routes and methods of administration are specified
for twice the recommended time using a bath at twice the for the product concerned, administration by all routes is
recommended concentration. recommended. If 1 route of administration has been shown
to cause the most severe effects, this single route may be
For vaccines intended for use in fish, in general 50 fish per selected as the only one for use in the study. The number of
group are used unless otherwise justified or specified in a administrations must be not less than the maximum number
specific monograph. recommended ; for vaccines, this shall take account of the
For vaccines intended for use in birds older than 3 weeks, in number of administrations for primary vaccination and the
general 8 birds per group are used unless otherwise justified 1st re-vaccination ; for immunosera, it shall take account of
or specified in a specific monograph. For vaccines intended the number of administrations required for treatment. The
for use in birds younger than 3 weeks, in general 10 birds interval between administrations shall be suitable (e.g. period
per group are used unless otherwise justified or specified in of risk or required for treatment) and appropriate to the
a specific monograph. recommendations of use. Although, for convenience, as far
The animals are observed and examined at least daily for signs as vaccines are concerned, a shorter interval may be used in
of abnormal local and systemic reactions. Where appropriate, the study than that recommended in the field, an interval
these studies shall include detailed post-mortem macroscopic of at least 14 days must be allowed between administrations
and microscopic examinations of the injection site. Other for the development of any hypersensitivity reaction. For
objective criteria are recorded, such as body temperature immunosera, however, administration shall follow the
(for mammals) and performance measurements. The body recommended schedule. For vaccines intended for use in
temperatures are recorded on at least the day before and at the mammals, in general 8 animals per group are used unless
time of administration of the product, 4 h later and on the otherwise justified or specified in a specific monograph. For
following 4 days. The animals are observed and examined at vaccines intended for use in fish, in general 50 fish per group
least daily until reactions may no longer be expected but, in are used unless otherwise justified or specified in a specific
all cases, the observation and examination period extends at monograph. For vaccines intended for use in birds older than
least until 14 days after administration. 3 weeks, in general 8 birds per group are used unless otherwise
General Notices (1) apply to all monographs and other texts 589
5.2.6. Evaluation of safety of veterinary vaccines and immunosera EUROPEAN PHARMACOPOEIA 8.0
justified or specified in a specific monograph. For vaccines 1-7. ADVERSE EFFECTS FROM INTERACTIONS
intended for use in birds younger than 3 weeks, in general Studies are undertaken to show a lack of adverse effect on
10 birds per group are used unless otherwise justified or the safety of the product when simultaneous administration
specified in a specific monograph. The animals are observed is recommended or where administration of the product
and examined at least daily for at least 14 days after the last is recommended as part of a schedule of administration of
administration for signs of systemic and local reactions. Other products within a short period of time.
objective criteria are recorded, such as body temperature and 1-8. SPECIAL REQUIREMENTS FOR LIVE VACCINES
performance measurements.
The following laboratory tests must also be carried out with
Unless otherwise prescribed in a specific monograph or, in the
live vaccines.
absence of a specific monograph, unless otherwise justified
For the following tests except for the test for increase in
and authorised, the product complies with the test if no
virulence (section 1-8-3), use the vaccine strain at the least
animal shows abnormal local or systemic reactions or signs of
attenuated passage level that will be present between the
disease, or dies from causes attributable to the product.
master seed lot and a batch of vaccine.
1-4. EXAMINATION OF REPRODUCTIVE PERFORMANCE 1-8-1. Spread of the vaccine strain. Spread of the vaccine
When the vaccine is recommended for use or may be used strain from vaccinated to unvaccinated target animals is
in pregnant animals or laying birds, carry out a test for investigated using the recommended route of administration
safety in this category of animals. If the reproductive safety most likely to result in spread. Moreover, it may be necessary to
studies are not performed, an exclusion statement appears investigate the safety of spread to non-target species that could
on the label, unless a scientific justification for absence of be highly susceptible to a live vaccine strain. An assessment
risk is provided. Examination of reproductive performance must be made of how many animal-to-animal passages are
must also be considered when data suggest that the starting likely to be sustainable under normal circumstances together
material from which the product is derived may be a risk with an assessment of the likely consequences.
factor. Where appropriate, reproductive performance of males
and females and harmful effects on the progeny, including 1-8-2. Dissemination in vaccinated animal. Faeces, urine,
teratogenic or abortifacient effects, are investigated by each of milk, eggs, and oral, nasal and other secretions shall be tested
the recommended routes of administration. If multiple routes for the presence of the organism as appropriate. Moreover,
and methods of administration are specified for the product studies may be required of the dissemination of the vaccine
concerned, administration by all routes is recommended. If strain in the body, with particular attention being paid to
1 route of administration has been shown to cause the most the predilection sites for replication of the organism. In the
severe effects, this single route may be selected as the only case of live vaccines for well-established zoonotic diseases for
one for use in the study. food-producing animals, these studies are obligatory and shall
For vaccines intended for use in mammals, in general 8 particularly take into account the persistence of the strain at
animals per group are used unless otherwise justified or the injection site.
specified in a specific monograph. Vaccines recommended 1-8-3. Increase in virulence. Unless otherwise prescribed in a
for use or that may be used in pregnant animals, are tested in specific monograph or, in the absence of a specific monograph,
each of the specific periods of gestation recommended for use unless otherwise justified and authorised, the following
on the label. An exclusion statement will be required for those applies. This test is carried out using the master seed lot. If
gestation periods not tested. the quantity of the master seed lot sufficient for performing
the test is not available, the lowest passage material used for
The observation period is extended to parturition, to examine
the production that is available in sufficient quantity may be
any harmful effects during gestation or on progeny, unless
used. At the time of inoculation, the animals in all groups are
otherwise justified or specified in a specific monograph.
of an age suitable for recovery of the strain. Serial passages are
The following protocol is given as an example of an appropriate carried out in target animals using 5 groups of animals, unless
test for vaccines. there is justification to carry out more passages or unless
Safety in pregnant animals. Use not fewer than 8 animals the strain disappears from the test animal sooner. In vitro
per group, at the recommended stage of gestation or at a propagation may not be used to expand the passage inoculum.
range of stages of gestation according to the recommended The passages are carried out using animals most appropriate
schedule. Not fewer than 8 animals are used for each stage of to the potential risk being assessed.
pregnancy (i.e. 24 animals for 3 trimesters of pregnancy in The initial administration is carried out using the
cattle). Administer to each animal a recommended dose of recommended route of administration most likely to lead to
the vaccine. If the recommended schedule requires a 2nd dose, reversion to virulence, using an initial inoculum containing
administer another dose after an interval of at least 14 days. the maximum release titre. After this, not fewer than 4 further
Unless otherwise prescribed in a specific monograph, observe serial passages through animals of the target species are
the animals at least daily until 1 day after parturition. Unless undertaken. The passages are undertaken by the route of
otherwise prescribed in a specific monograph, or, in the administration most likely to lead to reversion to virulence.
absence of a specific monograph, unless otherwise justified If the properties of the strain allow sequential passage via
and authorised, the vaccine complies with the test if no animal natural spreading, this method may be used, otherwise
shows abnormal local or systemic reactions or signs of disease, passage as described in each specific monograph is carried
or dies from causes attributable to the vaccine, and if no out and the micro-organisms that have been recovered at
adverse effects on the pregnancy or the offspring are noted. the final passage are tested for increase in virulence. For
1-5. RESIDUES the first 4 groups, a minimum of 2 animals is used for
mammalian vaccines, and a minimum of 5 birds is used
In the case of live vaccines for well-established zoonotic
for avian vaccines. The last group consist of a minimum
diseases, the determination of residual vaccine organisms at
the injection site may be required, in addition to the studies of of 8 mammals or 10 birds. At each passage, the presence
of living vaccine-derived micro-organisms in the material
dissemination described below.
used for passage is demonstrated. Care must be taken to
1-6. ADVERSE EFFECTS ON IMMUNOLOGICAL avoid contamination by micro-organisms from previous
FUNCTIONS passages. When the micro-organism is not recovered from any
Where the product might adversely affect the immune intermediate in vivo passage, repeat the passage in 10 animals
response of the animal to which the product is administered or using in vivo passaged material from the last passage in which
of its progeny, suitable tests on the immunological functions the micro-organism was recovered. The micro-organism
are carried out. recovered is used as the inoculum for the next passage. If the
target micro-organism is not recovered, the experiment is significant exposure of the environment to the product, the
considered to be completed with the conclusion that the target potential ecotoxicity is evaluated, taking into account the
micro-organism does not show an increase in virulence. properties of the product.
General clinical observations are made during the study.
Animals in the last group are observed for 21 days unless 04/2008:50207
otherwise justified. These observations include all relevant
parameters typical for the disease that could indicate increase
in virulence. Compare the clinical signs and other relevant 5.2.7. EVALUATION OF EFFICACY
parameters with those observed in the animals used in the OF VETERINARY VACCINES AND
test for safety of the administration of 1 dose (section 1-1). If
the last group of animals shows no evidence of an increase in
IMMUNOSERA
virulence, further testing is not required. Otherwise, material The term ‘product’ means either a vaccine or an immunoserum
used for the 1st passage and the microorganisms recovered at throughout the text.
the final passage level are used in a separate experiment using During development of the product, tests are carried out to
at least 8 animals per group for mammal vaccines and at least demonstrate that the product is efficacious when administered
10 birds per group for avian vaccines, to compare directly the by each of the recommended routes and methods of
clinical signs and other relevant parameters. This study is administration and using the recommended schedule to
carried out using the route of administration that was used animals of each species and category for which use of the
for previous passages. An alternative route of administration product is to be recommended. The type of efficacy testing to
may be used if justified. be carried out varies considerably depending on the particular
Unless otherwise justified and authorised, the product type of product.
complies with the test if no animal dies or shows signs As part of tests carried out during development to establish
attributable to the vaccine strain and no indication of increased efficacy, the tests described in the Production section of a
virulence is observed in the animals of the last group. monograph may be carried out ; the following must be taken
1-8-4. Biological properties of the vaccine strain. Other into account.
tests may be necessary to determine as precisely as possible The dose to be used is that quantity of the product to be
the intrinsic biological properties of the vaccine strain (for recommended for use and containing the minimum titre or
example, neurotropism). For vector vaccines, evaluation is potency expected at the end of the period of validity.
made of the risk of changing the tropism or virulence of the For live vaccines, use vaccine containing virus/bacteria at the
strain and where necessary specific tests are carried out. Such most attenuated passage level that will be present in a batch
tests are systematically carried out where the product of a of vaccine.
foreign gene is incorporated into the strain as a structural
For immunosera, if appropriate, the dose tested also contains
protein.
minimum quantities of immunoglobulin or gammaglobulin
1-8-5. Recombination or genomic reassortment of strain. and/or total protein.
The probability of recombination or genomic reassortment The efficacy evidence must support all the claims being made.
with field or other strains shall be considered. For example, claims for protection against respiratory disease
must be supported at least by evidence of protection from
2. FIELD STUDIES clinical signs of respiratory disease. Where it is claimed that
Results from laboratory studies shall normally be there is protection from infection this must be demonstrated
supplemented with supportive data from field studies. using re-isolation techniques. If more than one claim is made,
Provided that laboratory tests have adequately assessed the supporting evidence for each claim is required.
safety and efficacy of a product under experimental conditions Vaccines. The influence of passively acquired and maternally
using vaccines of maximum and minimum titre or potency derived antibodies on the efficacy of a vaccine is adequately
respectively, a single batch of product may be used to assess evaluated. Any claims, stated or implied, regarding onset and
both safety and efficacy under field conditions. In these cases, duration of protection shall be supported by data from trials.
a typical routine batch of intermediate titre or potency may Claims related to duration of immunity are supported by
be used. evidence of protection. The test model described under
For food-producing mammals, the studies include Immunogenicity and/or Potency is not necessarily used to
measurement of the body temperatures of a sufficient number support claims regarding the duration of immunity afforded
of animals, before and after administration of the product ; by a vaccine.
for other mammals, such measurements are carried out if the The efficacy of each of the components of multivalent and
laboratory studies indicate that there might be a problem. The combined vaccines shall be demonstrated using the combined
size and persistence of any local reaction and the proportion vaccine.
of animals showing local or systemic reactions are recorded.
Immunosera. Particular attention must be paid to providing
Performance measurements are made, where appropriate.
supporting data for the efficacy of the regime that is to be
Performance measures for broilers include weekly mortality, recommended. For example, if it is recommended that
feed conversion ratios, age at slaughter and weight, down the immunoserum needs only to be administered once to
grading and rejects at the processing plant. For vaccines achieve a prophylactic or therapeutic effect then this must
for use in laying birds or in birds that may be maintained be demonstrated. Any claims, stated or implied, regarding
to lay, the effect of the vaccine on laying performance and onset and duration of protection or therapeutic effect must be
hatchability is investigated, as appropriate. supported by data from trials. For example, the duration of
the protection afforded by a prophylactic dose of an antiserum
3. ECOTOXICITY must be studied so that appropriate guidance for the user can
An assessment is made of the potential harmful effects of the be given on the label.
product for the environment and any necessary precautionary Studies of immunological compatibility are undertaken when
measures to reduce such risks are identified. The likely simultaneous administration is recommended or where it is a
degree of exposure of the environment to the product is part of a usual administration schedule. Wherever a product
assessed, taking into account : the target species and mode is recommended as part of an administration scheme, the
of administration ; excretion of the product ; and disposal of priming or booster effect or the contribution of the product to
unused product. If these factors indicate that there will be the efficacy of the scheme as a whole is demonstrated.
General Notices (1) apply to all monographs and other texts 591
5.2.8. Minimising the risk of transmitting TSE via medicinal products EUROPEAN PHARMACOPOEIA 8.0
continues to be warranted if biological materials from species must demonstrate that medicinal products are manufactured
naturally affected by TSE diseases, especially bovine species, in accordance with the latest version of this note for guidance
are used for the manufacture of medicinal products. published in the Official Journal of the European Union. This
In the course of active surveillance programs, two previously is a continuing obligation after the marketing authorisation
unrecognized forms of atypical BSE (BSE-L, also named has been granted.
BASE, and BSE-H) have been identified in rare sporadic By definition, the principle of Specified Risk Materials as
cases from Europe, North America, and Japan. The ‘L’ and defined in Regulation (EC) No 999/2001 of the European
‘H’ identify the higher and lower electrophoretic positions Parliament and of the Council(5) does not apply to medicinal
of their protease-resistant PrPTSE isoforms. It is noteworthy products. However, Regulation (EC) No 1774/2002 of the
that atypical cases have been found in countries that did not European Parliament and of the Council(6), which applies
experience classical BSE so far, like Sweden, or in which only since 1st May 2003, lays down health rules concerning
few classical BSE cases have been found like Canada or USA. animal by-products not intended for human consumption.
The atypical BSE agent has been experimentally transmitted As a general rule, and unless properly justified, all animal
to transgenic mice expressing the human prion protein and to by-products used as starting materials in the manufacture of
a cynomolgus monkey. medicinal products should be ‘Category 3 (i.e. safe) materials
Scrapie occurs worldwide and has been reported in most or equivalent’, as defined in Regulation (EC) No 1774/2002.
European countries. It has the highest incidence in Cyprus. Justification for the use of substances derived from other, high
While humans have been exposed to naturally occurring infectivity materials must follow an appropriate benefit/risk
scrapie for over 250 years, there is no epidemiological evidence evaluation (see further below).
directly linking scrapie to spongiform encephalopathies in The note for guidance should be read in conjunction with the
humans(1). However, there remains a theoretical and currently various EU legal instruments including Commission decisions
unquantifiable risk that some BSE-contaminated protein progressively implemented since 1991. Where appropriate,
supplement may have been fed to sheep. Further, it should references to these decisions are given in the text. Position
also be assumed that any BSE agent introduced into the small statements and explanatory notes made by the Committee for
ruminant population via contaminated feed is likely to be Medicinal Products for Human Use (CHMP) and Committee
recycled and amplified(2). for Medicinal Products for Veterinary Use (CVMP) are still
There is interest in infecting cells with TSE agents to develop applicable for the purpose of regulatory compliance unless
assays and for basic scientific reasons. Some success has been otherwise superseded by the note for guidance.
reported, usually but not always with neural cell lines. The The general monograph Products with risk of transmitting
conditions needed to infect a cell are not well understood and agents of animal spongiform encephalopathies of the European
the process is difficult requiring particular combinations of Pharmacopoeia refers to this chapter, which is identical
agent and cell. It is not considered appropriate to make specific with the note for guidance. The monograph forms the basis
recommendations in terms of cell substrates to be used for for issuing Certificates of Suitability as a procedure for
production of biological/biotechnology-derived substances. demonstrating TSE compliance for substances and materials
Nevertheless, the possibility of infection of cell lines with TSE used in the manufacture of human and veterinary medicinal
agents should be taken into account in risk assessments. products.
1-2. REGULATORY COMPLIANCE Clarification of note for guidance. As the scientific
understanding of TSEs, especially the pathogenesis of the
Risk assessment. Since the use of animal-derived materials diseases, is evolving, from time to time CHMP and its
is unavoidable for the production of some medicinal Biologics Working Party in collaboration with CVMP and
products and that complete elimination of risk at source is its Immunologicals Working Party may be required in the
rarely possible, the measures taken to manage the risk of future to develop supplementary guidance in the form of
transmitting animal TSEs via medicinal products represent position statements or explanatory notes for the purpose
risk minimisation rather than risk elimination. Consequently, of clarifying the note for guidance. The supplementary
the basis for regulatory compliance should be based on a risk guidance shall be published by the Commission and on the
assessment, taking into consideration all pertinent factors as website of the European Medicines Agency and taken into
identified in this chapter (see below). consideration accordingly in the scope of the certification
Legal basis. The note for guidance is published by the of the European Directorate for the Quality of Medicines &
European Commission following HealthCare (EDQM).
– Annex I, part I, module 3, section 3.2 : Content : 2. SCOPE
basic principles and requirements, point (9) of
Directive 2001/83/EC of the European Parliament and TSE-RELEVANT ANIMAL SPECIES
of the Council of 6 November 2001 on the Community Cattle, sheep, goats and animals that are naturally susceptible
code relating to medicinal products for human use(3), as to infection with transmissible spongiform encephalopathy
amended, and agents or susceptible to infection through the oral route other
– Annex I, Title I, part 2, section C Production and control of than humans(7) and non-human primates are defined as
starting material of Directive 2001/82/EC of the European “TSE-relevant animal species”(8).
Parliament and of the Council of 6 November 2001 on MATERIALS
the Community code relating to veterinary medicinal This chapter is concerned with materials derived from
products(4), as amended. “TSE-relevant animal species” that are used for the preparation
These directives require that applicants for marketing of :
authorisation for human and veterinary medicinal products – active substances,
(1) This is currently being assessed by EFSA and ECDC. For updated information, please refer to the following link : http://registerofquestions.efsa.europa.eu/roqFrontend/
questionsListLoader?mandate=M-2009-0221
(2) In January 2005, after confirmation of BSE in a goat in France, additional legislative measures were taken related to monitoring and an increased testing of small ruminants. The
increased surveillance did not identify additional cases of BSE in sheep and goats in the EU.
(3) OJ L 311, 28.11.2001, p. 67.
(4) OJ L 311, 28.11.2001, p. 1.
(5) OJ L 147, 31.5.2001, p. 1.
(6) OJ L 273, 10.10.2002, p. 1. Regulation (EC) 1774/2002 has been repealed by Regulation (EC) 1069/2009 that will apply from 4 March 2011 (OJ L 300, 14.11.2009, p. 1).
(7) Regulatory guidance and position papers have been issued by the Committee for Medicinal Products for Human Use and its Biologics Working Party on human tissue derived
medicinal products in relation to CJD and vCJD. Such guidance can be found on http://www.ema.europa.eu
(8) Pigs and birds, which are animal species of particular interest for the production of medicinal products, are not naturally susceptible to infection via the oral route. Therefore they are
not TSE-relevant animal species within the meaning of this chapter. Also dogs, rabbits and fish are non TSE-relevant animal species within the meaning of this chapter.
General Notices (1) apply to all monographs and other texts 593
5.2.8. Minimising the risk of transmitting TSE via medicinal products EUROPEAN PHARMACOPOEIA 8.0
– excipients and adjuvants, and However, where materials derived from the “TSE-relevant
– raw and starting materials and reagents used in production animal species” are used in fermentation/routine production
(e.g. bovine serum albumin, enzymes, culture media processes or in the establishment of working seeds and
including those used to prepare working cell banks, or new working cell banks, the applicant must demonstrate that they
master cell banks for medicinal products which are subject fulfil the requirements of the note for guidance.
to a new marketing authorisation). 3. GENERAL CONSIDERATIONS
This chapter is also applicable to materials that come into
3-1. SCIENTIFIC PRINCIPLES FOR MINIMISING RISK
direct contact with the equipment used in manufacture of the
medicinal product or that come in contact with the medicinal When manufacturers have a choice, the use of materials from
product and therefore have the potential for contamination. “non TSE-relevant animal species” or non-animal origin is
preferred. The rationale for using materials derived from
Materials used in the qualification of plant and equipment, “TSE-relevant animal species” instead of materials from
such as culture media used in media fill experiments to validate “non-TSE-relevant species” or of non-animal origin should be
the aseptic filling process, shall be considered in compliance given. If materials from “TSE-relevant animal species” have
with this chapter provided that the constituent or constituents to be used, consideration should be given to all the necessary
are derived from tissues with no detectable infectivity measures to minimise the risk of transmission of TSE.
(category IC tissues), where the risk of cross-contamination
with potentially infective tissues has been considered (see Readily applicable diagnostic tests for TSE infectivity in vivo
section 3-3) and where the materials are sourced from are not yet available. Diagnosis is based on post-mortem
countries with negligible BSE risk or controlled BSE risk confirmation of characteristic brain lesions by histopathology
(Categories A and B, respectively – see section 3-2). Such and/or detection of PrPTSE by Western blot or immunoassay.
information shall be provided in the dossier for a marketing The demonstration of infectivity by the inoculation of suspect
authorisation and verified during routine inspection for tissue into target species or laboratory animals is also used for
compliance with Good Manufacturing Practice (GMP). confirmation. However, due to the long incubation periods of
all TSEs, results of in vivo tests are available only after months
Other materials such as cleaning agents, softeners and or years.
lubricants that come into contact with the medicinal product
during its routine manufacture or in the finishing stage or in Several immunochemical tests have been developed for the
the primary packaging are considered in compliance with detection of PrPTSE in post-mortem samples and some are
this chapter if they are tallow derivatives prepared using the now considered to be extremely sensitive. However, their
rigorous physicochemical processes as described in section 6. ability to detect an infected animal depends on the timing
of sample collection in relation to timing of exposure, the
SEED LOTS, CELL BANKS AND ROUTINE type of tissue collected and infectious dose acquired, together
FERMENTATION/PRODUCTION(9) with consequential timing of onset of clinical disease. There
For the purpose of regulatory compliance, master seeds or is currently insufficient information on how this might be
master cell banks in marketing authorisation applications affected by strain variations.
lodged after 1 July 2000 (for human medicinal products) or Although screening of source animals by in vitro tests
1 October 2000 (for veterinary medicinal products) shall be may prevent the use of animals at late stages of incubation
covered by the note for guidance. of the disease and may provide information about the
Master seeds and master cell banks, epidemiological status of a given country or region, none of
– for vaccine antigens, the tests are considered suitable to unambiguously confirm
– for a biotechnology-derived medicinal product as described the negative status of an animal.
in the Annex to Regulation (EC) No 726/2004 of the Minimising the risks of transmission of TSE is based upon
European Parliament and of the Council(10), and three complementary parameters :
– for other medicinal products using seed lots or cell banking – the source animals and their geographical origin,
systems in their manufacture, – nature of animal material used in manufacture and any
that have already been approved for the manufacture of procedures in place to avoid cross-contamination with
a constituent of an authorised medicinal product shall be higher risk materials,
considered in compliance with the note for guidance even if – production process(es) including the quality assurance
they are incorporated in marketing authorisation applications system in place to ensure product consistency and
lodged after 1 July 2000 (for human medicinal products) or traceability.
1 October 2000 (for veterinary medicinal products). 3-2. ANIMAL SOURCE
Master cell banks and master seeds established before The source materials used for the production of materials
1 July 2000 (for human medicinal products) or 1 October 2000 for the manufacture of medicinal products shall be derived
(for veterinary medicinal products), but not yet approved from animals fit for human consumption following ante- and
as a constituent of an authorised medicinal product shall post-mortem inspection in accordance with EU or equivalent
demonstrate that they fulfil the requirements of the note for (third country) conditions, except for materials derived from
guidance. If, for some raw or starting materials or reagents live animals, which should be found healthy after clinical
used for the establishment of these cell banks or seeds, full examination.
documentary evidence is no longer available, the applicant 3-2-1. Geographical sourcing
should present a risk assessment as described in Section 4 of
the note for guidance. 3-2-1-1. Bovine materials
Established working seeds or cell banks used in the The World Organisation for Animal Health (OIE)(11) lays
manufacture of medicinal products authorised before down the criteria for the assessment of the status of countries
1 July 2000 (human medicines) or 1 October 2000 (veterinary in the chapter of the International Animal Health Code on
medicines), which have been subjected to a properly bovine spongiform encephalopathy. Countries or regions are
conducted risk assessment by a Competent Authority of classified as follows :
the Member States or the European Medicines Agency and A. countries or regions with a negligible BSE risk ;
declared to be acceptable, shall also be considered compliant. B. countries or regions with a controlled BSE risk ;
(9) See also : Position paper on the assessment of the risk of transmission of animal spongiform encephalopathy agents by master seed materials used in the production of veterinary
vaccines (EMEA/CVMP/019/01-February 2001 adopted by the Committee for Medicinal Products for Veterinary Use (CVMP) in July 2001, (OJ C 286, 12.10.2001, p. 12)).
(10) OJ L 136, 30.4.2004, p. 1.
(11) http://www.oie.int/eng/Status/BSE/en_BSE_free.htm
C. countries or regions with an undetermined BSE risk. in sheep, the use of a genotype(s) showing resistance to
BSE/scrapie infection could be considered in establishing TSE
As stipulated in Commission Regulation (EC) No 999/2001, free flocks(17). However, the possibility that genotypes resistant
as amended(12), the classification of countries or regions to scrapie could be susceptible to BSE (experimental oral
thereof according to their BSE risk, based on the rules laid exposure) or atypical scrapie (natural cases) should also be
down by OIE, is legally binding in the EU since 1 July 2007. taken into account. Goats have not been studied sufficiently
Commission Decision 2007/453/EC(13) as amended, provides with regard to a genotype specific sensitivity.
the classification of countries or regions according to their
BSE risk. Material of small ruminant origin should preferably be
sourced from countries with a long history of absence of
Previously, the European Commission Scientific Steering
scrapie. Justification shall be required if the material is sourced
Committee (SSC)(14) had established a temporary system for
from some other origin.
classifying the countries according to their geographical BSE
risk (GBR)(15). 3-2-2. BSE negligible risk (closed) bovine herds. The safest
sourcing is from countries or regions with a negligible risk
For the purposes of this chapter the BSE classification based (Category A countries). Other countries may have or have
on the OIE rules should be used. If a country, which was had cases of BSE at some point in time and the practical
previously classified in accordance to the SSC GBR criteria, concept of “Negligible risk (closed) bovine herds” has been
has not been classified yet according to the OIE rules, the GBR developed by the SSC and endorsed by the CHMP and CVMP.
classification can be used until OIE classification has taken Criteria for establishing and maintaining a “BSE negligible
place, provided that there is no evidence of significant change risk (closed) bovine herd” can be found in the SSC opinion
in its BSE risk(16). of 22-23 July 1999(18).
Where there is a choice, animals should be sourced from
countries with the lowest possible BSE risk (negligible BSE For the time being it is not possible to quantify the reduction
risk countries (Category A)) unless the use of material from of the geographical BSE risk for cattle from BSE ‘negligible
countries with a higher BSE risk is justified. Some of the risk (closed) bovine herds’. However, it is expected that this
materials identified in Section 6, “Specific Conditions” can be risk reduction is substantial. Therefore, sourcing from such
sourced from countries with controlled BSE risk (Category B) closed bovine herds shall be considered in the risk assessment
and, in some cases, from countries with undetermined BSE in conjunction with the OIE classification of the country.
risk (Category C), provided that the controls and requirements 3-3. ANIMAL PARTS, BODY FLUIDS AND SECRETIONS
as specified in the relevant sections below are applied. Apart AS STARTING MATERIAL
from these exceptions, animals must not be sourced from In a TSE infected animal, different organs and secretions have
countries with undetermined BSE risk (Category C), and different levels of infectivity. If materials from ‘TSE-relevant
justifications for the use of animals from countries with animal species’ have to be used, consideration should be given
undetermined BSE risk (Category C) must always be provided. to use materials of the lowest category of risk. The tables given
in the Annex of this chapter(19) summarise current data about
3-2-1-2. Sheep and goats (small ruminants)
the distribution of infectivity and PrPTSE in cattle with BSE,
Naturally occurring clinical scrapie cases have been reported and in sheep and goats with scrapie(20).
in a number of countries worldwide. As BSE in sheep and
goats could possibly be mistaken for scrapie, as a precautionary The information in the tables is based exclusively upon
measure, sourcing of materials derived from small ruminants observations of naturally occurring disease or primary
shall take into account the prevalence of both BSE and scrapie experimental infection by the oral route (in cattle) but
in the country and the tissues from which the materials are does not include data on models using strains of TSE
derived. that have been adapted to experimental animals, because
passaged strain phenotypes can differ significantly and
The principles related to “BSE negligible risk (closed) bovine unpredictably from those of naturally occurring disease.
herds” (see section 3-2-2) could equally be applied in the Because immunohistochemical and/or Western blot detection
context of small ruminants in order to develop a framework of misfolded host protein (PrPTSE) have proven to be a
to define the TSE status of a flock of small ruminants. For surrogate marker of infectivity, PrPTSE testing results have been
sheep, because of the concern over the possibility of BSE presented in parallel with bioassay data. Tissues are grouped
GBR level Presence of one or more cattle clinically or pre-clinically infected with BSE in a geographical region/country
I Highly unlikely
IV Confirmed at a higher level (≥ 100 cases/1 Million adult cattle per year)
Reports of the GBR assessment of the countries are available on the SSC website (http://ec.europa.eu/food/fs/sc/ssc/outcome_en.html)
(16) Experts consider that the GBR classification system is stable enough, so that it can continue to be used, during the interim period, for the demonstration of compliance with this chapter.
(17) Opinion of the Scientific Panel on Biological Hazards on ‘the breeding programme for TSE resistance in sheep’ : http://www.efsa.europa.eu/EFSA/efsa_locale-
1178620753812_1178620775678.htm
(18) SSC Scientific Opinion on the conditions related to “BSE Negligible Risk (Closed) Bovine Herds” adopted at the meeting of 22-23 July 1999. http://ec.europa.eu/
food/fs/sc/ssc/out56_en.html
(19) The tissue classification tables are based upon the most recent WHO Guidelines on Tissue Infectivity Distribution in Transmissible Spongiform Encephalopathies (2010)
http://www.who.int/bloddproducts/tablestissueinfectivity.pdf
(20) A Scientific opinion on BSE/TSE infectivity in small ruminant tissues is currently being reviewed by EFSA (Question No EFSA-Q-2010-052). For updated information please follow
this link : http://registerofquestions.efsa.europa.eu/roqFrontend/questionsListLoader?mandate=M-2010-0041
General Notices (1) apply to all monographs and other texts 595
5.2.8. Minimising the risk of transmitting TSE via medicinal products EUROPEAN PHARMACOPOEIA 8.0
into three major infectivity categories, irrespective of the stage – the extent of brain damage,
of disease : – the dissemination of brain particles in the animal body.
Category IA High-infectivity tissues These factors must be considered in conjunction with the
central nervous system (CNS) tissues that attain a high
OIE/GBR classification of the source animals, the age of the
titre of infectivity in the later stages of all TSEs, and animals in the case of cattle and the post-mortem testing of
certain tissues that are anatomically associated with the the cattle using a validated method.
CNS
Category IB Lower-infectivity tissues
The underlying principles indicated above would be equally
applicable to sheep and goats.
peripheral tissues that have tested positive for infectivity
and/or PrPTSE in at least one form of TSE
The risk posed by cross-contamination will be dependent on
Category IC Tissues with no detectable infectivity several complementary factors including :
– measures adopted to avoid contamination during collection
tissues that have been examined for infectivity, without
any infectivity detected, and/or PrPTSE, with negative of tissues (see above),
results – level of contamination (amount of the contaminating
Category IA tissues and substances derived from them shall tissue),
not be used in the manufacture of medicinal products, unless – amount and type of materials collected at the same time.
justified (see Section 5). Manufacturers or the marketing authorisation
Although the category of lower risk tissues (category IB holders/applicants should take into account the risk
tissues) almost certainly includes some (e.g. blood) with with respect to cross-contamination.
a lower risk than others (e.g. lymphoreticular tissues), the 3-4. AGE OF ANIMALS
data about infectivity levels in these tissues are too limited to As the TSE infectivity accumulates in bovine animals over
subdivide the category into different levels of risk. It is also an incubation period of several years, it is prudent to source
evident that the placement of a given tissue in one or another from young animals.
category can be disease and species specific, and subject to
revision as new data emerge. Presence of infectious material has essentially been reported
in the central nervous system and related tissues, as well as
For the risk assessment (see section 4), manufacturers and/or in the lymphoreticular system, depending on the TSE agent
marketing authorisation holders/applicants shall take into (BSE in cattle or scrapie in sheep and goat). The exact time
account the tissue classification tables in the Annex to this course of infectivity in the respective body parts and tissues,
chapter. from the date of infection, is not known in both species and,
The categories in the tables are only indicative and it is as such, it is difficult to give clear guidance on the age above
important to note the following points. which the various tissues may be infected and should not be
collected. The initial recommendation to collect tissues in the
– In certain situations there could be cross-contamination youngest age is still valid. In addition, it is noteworthy that the
of tissues of different categories of infectivity. The potential age criteria depend also on the geographical origin. Age is a
risk will be influenced by the circumstances in which more important parameter for materials from countries where
tissues were removed, especially by contact of tissues the risk is higher (Category B and C countries), than from
with lower-infectivity tissues or no detectable infectivity countries with a negligible BSE risk (Category A countries).
(categories IB and IC tissues) with high-infectivity tissues
(category IA tissues). Thus, cross-contamination of some 3-5. MANUFACTURING PROCESS
tissues may be increased if infected animals are slaughtered The assessment of the overall TSE risk reduction of a medicinal
by brain stunning (penetrative or non penetrative) or product shall take into account the control measures instituted
if the brain and/or spinal cord is sawed. The risk of with respect to :
cross-contamination will be decreased if body fluids are – sourcing of the raw/starting materials, and
collected with minimal damage to tissue and cellular – the manufacturing process.
components are removed, and if foetal blood is collected
without contamination from other maternal or foetal Controlled sourcing is a very important criterion in achieving
tissues including placenta, amniotic and allantoic fluids. acceptable safety of the product, due to the documented
For certain tissues, it is very difficult or impossible to resistance of TSE agents to most inactivation procedures.
prevent cross-contamination with category IA tissues (e.g. A quality assurance system, such as ISO 9000 certification,
skull). This has to be considered in the risk assessment. HACCP(22) or GMP, must be put in place for monitoring the
– For certain classes of substances the stunning/slaughtering production process and for batch delineation (i.e. definition
techniques used may be important in determining the of batch, separation of batches, cleaning between batches).
potential risk(21) because of the likelihood of disseminating Procedures shall be put in place to ensure traceability as well
the brain particles into the peripheral organs, particularly as self-auditing and to auditing suppliers of raw/starting
to the lungs. Stunning/slaughtering techniques should materials.
be described as well as the procedures to remove high Certain production procedures may contribute considerably
infectivity tissues. The procedures to collect the animal to the reduction of the risk of TSE contamination, e.g.
tissues/organs to be used and the measures in place to procedures used in the manufacture of tallow derivatives (see
avoid cross-contamination with a higher risk material must section 6). As such rigorous processing cannot be applied to
also be described in detail. many products, processes involving physical removal, such as
precipitation and filtration to remove prion-rich material, are
– The risk of contamination of tissues and organs with likely to be more appropriate than chemical treatments. A
BSE-infectivity potentially harboured in central nervous description of the manufacturing process, including in-process
material as a consequence of the stunning method used for controls applied, shall be presented and the steps that might
cattle slaughtering depends on the following factors : contribute to reduction or elimination of TSE contamination
– the amount of BSE-infectivity in the brain of the should be discussed. Whenever different manufacturing
slaughtered animal, sites are involved, the steps performed at each site shall be
(21) SSC opinion on stunning methods and BSE risk (The risk of dissemination of brain particles into the blood and carcass when applying certain stunning methods), adopted at the
meeting of 10-11 January 2002. http://ec.europa.eu/food/fs/sc/ssc/out245_en.pdf. Report of the EFSA Working group on BSE risk from dissemination of brain particles in blood and
carcass. Question No EFSA-Q-2003-122, adopted on 21 October 2004, http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_1178620777397.htm
(22) Hazard Analysis Critical Control Point.
clearly identified. The measures in place in order to ensure As indicated in the introduction to this chapter, regulatory
traceability of every production batch to the source material compliance is based on a favourable outcome from a
should be described. risk assessment. The risk assessments, conducted by the
Cleaning process. Cleaning of process equipment may be manufacturers and/or the marketing authorisation holders
difficult to validate for the elimination of TSE agents. It is or applicants for the different materials or substances from
reported that after exposure to high titre preparations of TSE “TSE-relevant animal species” used in the manufacture of a
agent, detectable infectivity can remain bound to the surface of medicinal product shall show that all TSE risk factors have
stainless steel. The removal of all adsorbed protein by the use been taken into account and, where possible, risk has been
of 1 M sodium hydroxide or chlorine releasing disinfectants minimised by application of the principles described in this
(e.g. 20 000 ppm chlorine for 1 h) have been considered chapter. TSE Certificates of suitability issued by the EDQM
acceptable approaches where equipment that cannot be may be used by the marketing authorisation holders or
replaced has been exposed to potentially contaminated applicants as the basis of the risk assessments.
material. Milder treatments with limited concentrations of An overall risk assessment for the medicinal product,
alkali or stabilized bleach, when properly formulated with conducted by the marketing authorisation holders or
detergents and used at specified temperatures, have been applicants, shall take into account the risk assessments for all
shown to exhibit similar efficiency for removing prions as did the different materials from “TSE-relevant animal species”
classical NaOH or chlorine treatments. A system based on and, where appropriate, TSE reduction or inactivation by the
vaporised hydrogen peroxide also appeared to be efficient manufacturing steps of the active substance and/or finished
for inactivating TSE agents. These new treatments are more product.
compatible with delicate materials and may be suitable for The final determination of regulatory compliance rests with
practical use(23). the competent authority.
If risk materials are used in the manufacture of a product,
cleaning procedures, including control measures, shall be put It is incumbent upon the manufacturers and/or the marketing
in place in order to minimise the risk of cross-contamination authorisation holders or applicants for both human and
between production batches. This is especially important veterinary medicinal products to select and justify the control
if materials from different risk categories are handled in measures for a given “TSE-relevant animal species” derivative,
the same plant with the same equipment. In the case of taking into account the latest scientific and technical progress.
using category IA materials in the manufacture of a product,
dedicated equipment shall be used, unless otherwise justified. 5. BENEFIT/RISK EVALUATION
Further research is needed to develop and validate In addition to the parameters as mentioned in sections 3 (that
new decontamination procedures to lower the risk of may be covered by a TSE Certificate of Suitability issued by
cross-contamination for material and devices which are not the EDQM) and 4, the acceptability of a particular medicinal
compatible with WHO-recommended procedures. product containing materials derived from a “TSE-relevant
animal species”, or which as a result of manufacture could
Removal/Inactivation validation. Validation studies of
contain these materials, shall take into account the following
removal/inactivation procedures for TSEs can be difficult
factors :
to interpret. It is necessary to take into consideration the
nature of the spiked material and its relevance to the natural – route of administration of the medicinal product,
situation, the design of the study (including scaling-down of – quantity of animal material used in the medicinal product,
processes) and the method of detection of the agent (in vitro
or in vivo assay). Further research is needed to develop an – maximum therapeutic dosage (daily dose and duration of
understanding of the most appropriate “spike preparation” for treatment),
validation studies. Therefore, validation studies are currently – intended use of the medicinal product and its clinical
not generally required. However, if claims are made for benefit,
the safety of the product with respect to TSEs based on the
ability of manufacturing processes to remove or inactivate – presence of a species barrier.
TSE agents, they must be substantiated by appropriate High-infectivity tissues (category IA tissues) and substances
investigational studies(24). derived thereof shall not be used in manufacture of medicinal
In addition to appropriate sourcing, manufacturers are products, their starting materials and intermediate products
encouraged to continue their investigations into removal and (including active substances, excipients and reagents), unless
inactivation methods to identify steps/processes that would justified. A justification why no other materials can be
have benefit in assuring the removal or inactivation of TSE used shall be provided. In these exceptional and justified
agents. In any event, a production process wherever possible circumstances, the use of high-infectivity tissues could be
shall be designed taking account of available information envisaged for the manufacture of active substances, when,
on methods which are thought to inactivate or remove TSE after performing the risk assessment as described in Section 4
agents. of this chapter, and taking into account the intended clinical
use, a positive benefit/risk assessment can be presented by
For certain types of products (see section 6-3 Bovine blood
the marketing authorisation applicant. Substances from
and blood derivatives), where validated removal/inactivation category IA materials, if their use is justified, must be
is not readily applicable, process evaluation might be required.
produced from animals of countries with negligible BSE risk
This should be based on the starting material and any
(Category A).
published data on TSE risk.
4. RISK ASSESSMENT OF MATERIALS OR SUBSTANCES 6. SPECIFIC CONSIDERATIONS
USED IN THE MANUFACTURE AND PREPARATION The following materials prepared from “TSE-relevant animal
OF A MEDICINAL PRODUCT IN THE CONTEXT OF species” are considered in compliance with this chapter
REGULATORY COMPLIANCE provided that they meet at least the conditions specified
The assessment of the risk associated with TSE needs careful below. The relevant information or a certificate of suitability
consideration of all of the parameters as outlined in section granted by the EDQM shall be provided by the marketing
3-1 (Scientific Principles for Minimising Risk). authorisation applicant/holder.
(23) WHO Guidelines on Tissue Infectivity Distribution in Transmissible Spongiform Encephalopathies (2006) http://www.who.int/bloodproducts/tse/WHO%20TSE%20Guide-
lines%20FINAL-22%20JuneupdatedNL.pdf
(24) Guideline on the investigation of manufacturing process for plasma-derived medicinal products with regard to vCJD risk CPMP/BWP/5136/03
General Notices (1) apply to all monographs and other texts 597
5.2.8. Minimising the risk of transmitting TSE via medicinal products EUROPEAN PHARMACOPOEIA 8.0
(25) Based on the Opinion of the Scientific Panel on Biological Hazards of the European Food Safety Authority on the ‘Quantitative assessment of the human BSE risk posed by gelatine
with respect to residual BSE risk’. The EFSA Journal, 312, (1-28). http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_1178620776107.htm
The requirements for source material selection and manufacture are appropriate for oral or parenteral gelatin for use in human and veterinary medicinal products.
the womb should be completely removed and the foetal blood Whenever a risk of cross-contamination of blood with brain
harvested in dedicated space or area by cardiac puncture into cannot be avoided at routine slaughtering in countries with
a closed collection system using aseptic technique. a controlled BSE risk (Category B), safety measures such
Newborn calf serum is obtained from calves under 20 days old as restriction of the age of the cattle and/or reduction of
and calf serum from animals under the age of 12 months. In infectious agents during manufacture have to be applied.
the case of donor bovine serum, given that it may be derived Age
from animals less than 36 months old, the TSE negative status
of the donor herd shall be well defined and documented. For countries with a controlled BSE risk (Category B), a
In all cases, serum shall be collected according to specified precautionary age limit of 21 months shall apply for bovine
protocols by personnel trained in these procedures to avoid blood or blood derivatives where no significant reduction of
cross-contamination with higher risk tissues. TSE agents can be assumed from manufacture. An age limit of
30 months is considered sufficient for blood derivatives where
For bovine blood and blood derivatives, documentation significant reduction of TSE agents can be demonstrated as
to demonstrate compliance with this chapter needs to be described below.
provided taking into account the provisions listed in sections 3
to 5. In addition, consideration should be given to the Reduction of TSE agents during manufacture
following. For blood derivatives, the capacity of the manufacturing
Traceability process to reduce/eliminate TSE agents should be estimated
from investigational studies. The estimation may be based on
Traceability to the slaughterhouse must be assured for each published data or in house data whenever it can be shown that
batch of serum or plasma. Slaughterhouses must have available such data is relevant to the specific manufacturing process.
lists of farms from which the animals are originated. If serum If it cannot be concluded that the reduction capacity is
is produced from living animals, records must be available for comparable, it is recommended that manufacturers undertake
each serum batch which assures the traceability to the farms. product-specific investigational studies. Investigations using
Geographical origin biochemical assay may be sufficient if there is scientific
Whilst tissue infectivity of BSE in cattle is more restricted evidence that this assay correlates with infectivity data.
than scrapie, as a precautionary measure bovine blood should General guidance for investigational studies on reduction
be sourced from Category A countries. Bovine blood from of TSE agents has been outlined(29). Brain-derived spike
Category B countries is also acceptable provided that there is preparations are appropriate for studies investigating the risk
no risk for cross-contamination of blood with brain material from brain-contaminated blood.
from the slaughter of animals over 21 months(26) of age. 6-4. TALLOW DERIVATIVES
Stunning methods Tallow is fat obtained from tissues including subcutaneous,
If it is sampled from slaughtered animals, the method of abdominal and inter-muscular areas and bones. Tallow
slaughter is of importance to assure the safety of the material. used as the starting material for the manufacture of tallow
It has been demonstrated that stunning by captive bolt stunner derivatives shall be ‘Category 3 material or equivalent’, as
with or without pithing as well as by pneumatic stunner, defined in Regulation (EC) No 1774/2002 of the European
especially if it injects air, can destroy the brain and disseminate Parliament and of the Council of 3 October 2002 laying down
brain material into the blood stream. Non-penetrative health rules concerning animal by-products not intended for
stunning is no more considered as an alternative to penetrative human consumption.
stunning because contamination of blood with brain material Tallow derivatives, such as glycerol and fatty acids,
has been demonstrated(27). Negligible risk can be expected manufactured from tallow by rigorous processes are thought
from electro-narcosis(28), but this even does not provide strict unlikely to be infectious and they have been the subject of
safety because, when unsuccessful, animals may have to be specific consideration by CHMP and CVMP. For this reason,
additionally stunned. The stunning methods must therefore such materials manufactured under the conditions at least as
be described for the bovine blood collection process. rigorous as those given below shall be considered in
(26) Opinion of the Scientific Panel on Biological Hazards on the assessment of the age limit in cattle for the removal of certain Specified Risk Materials (SRM). Question No
EFSA-Q-2004-146, adopted on 28 April 2005
(27) The tissue classification tables are based upon the most recent WHO Guidelines on Tissue Infectivity Distribution in Transmissible Spongiform Encephalopathies (2010)
http://www.who.int/bloodproducts/tablestissueinfectivity.pdf
(28) Report of the EFSA Working Group on BSE risk from dissemination of brain particles in blood and carcass. Question No EFSA-Q-2003-112, adopted on 21 October 2004,
http://www.efsa.europa.eu/en/sciencebiohaz/biohaz_opinions/opinion_annexes/733.html
(29) Guideline on the investigation of manufacturing process for plasma-derived medicinal products with regard to vCJD risk CPMP/BWP/5136/03.
General Notices (1) apply to all monographs and other texts 599
5.2.8. Minimising the risk of transmitting TSE via medicinal products EUROPEAN PHARMACOPOEIA 8.0
compliance for this chapter, irrespective of the geographical 6-7. WOOL DERIVATIVES
origin and the nature of the tissues from which tallow Derivatives of wool and hair of ruminants, such as lanolin
derivatives are derived. Examples of rigorous processes are : and wool alcohols derived from hair shall be considered in
– trans-esterification or hydrolysis at not less than 200 °C for compliance with this chapter, provided the wool and hair are
not less than 20 min under pressure (glycerol, fatty acids sourced from live animals.
and fatty acid esters production), Wool derivatives produced from wool which is sourced from
– saponification with 12 M NaOH (glycerol and soap slaughtered animals declared “fit for human consumption”
production) : and the manufacturing process in relation to pH, temperature
– batch process : at not less than 95 °C for not less than 3 h, and duration of treatment meets at least one of the stipulated
processing conditions listed below are unlikely to present any
– continuous process : at not less than 140 °C, under TSE risk and shall therefore be considered compliant with this
pressure for not less than 8 min, or equivalent, chapter.
– distillation at 200 °C. – Treatment at pH ≥ 13 (initial ; corresponding to a NaOH
Tallow derivatives manufactured according to these conditions concentration of at least 0.1 M NaOH) at 60 °C for at least
are unlikely to present any TSE risk and shall therefore be 1 h. This occurs normally during the reflux stage of the
considered compliant with this chapter. organic-alkaline treatment.
Tallow derivatives produced using other conditions must – Molecular distillation at ≥ 220 °C under reduced pressure.
demonstrate compliance with this chapter.
Wool derivatives produced using other conditions must
6-5. ANIMAL CHARCOAL demonstrate compliance with this chapter.
Animal charcoal is prepared by carbonisation of animal 6-8. AMINO ACIDS
tissues, such as bones, using temperatures higher than 800 °C.
Amino acids can be obtained by hydrolysis of materials from
Unless otherwise justified, the starting material for the
various sources.
manufacture of animal charcoal shall be Category 3 material
or equivalent, as defined in Regulation (EC) No 1774/2002 of Unless otherwise justified, the starting material for the
the European Parliament and of the Council of 3 October 2002 manufacture of amino acids shall be ‘Category 3 material or
laying down health rules concerning animal by-products equivalent’, as defined in Regulation (EC) No 1774/2002 of the
not intended for human consumption. Irrespective of the European Parliament and of the Council of 3 October 2002
geographical origin and the nature of the tissue, for the laying down health rules concerning animal by-products not
purpose of regulatory compliance, animal charcoal shall be intended for human consumption.
considered in compliance with this chapter. Amino acids prepared using the following processing
Charcoal manufactured according to these conditions is conditions are unlikely to present any TSE risk and shall be
unlikely to present any TSE risk and shall therefore be considered compliant with this chapter :
considered compliant with this chapter. Charcoal produced – amino acids produced from hides and skins by a process
using other conditions must demonstrate compliance with which involves exposure of the material to a pH of 1 to 2,
this chapter. followed by a pH of > 11, followed by heat treatment at
6-6. MILK AND MILK DERIVATIVES 140 °C for 30 min at 3 bar,
In the light of the current scientific knowledge and irrespective – the resulting amino acids or peptides must be filtered after
of the geographical origin, bovine milk is unlikely to present production, and
any risk of TSE contamination(30).
– analysis is performed using a validated and sensitive
Certain materials, including lactose, are extracted from whey, method to control any residual intact macromolecules,
the spent liquid from cheese production following coagulation. with an appropriate limit set.
Coagulation can involve the use of calf rennet, an extract from
abomasum, or rennet derived from other ruminants. The Amino acids prepared using other conditions must
CHMP/CVMP have performed a risk assessment for lactose demonstrate compliance with this chapter.
and other whey derivatives produced using calf rennet and 6-9 PEPTONES
concluded that the TSE risk is negligible if the calf rennet is Peptones are partial hydrolysates of protein, achieved by
produced in accordance with the process described in the risk enzymic or acid digestion. They are used in microbiological
assessment report(31). The conclusion was endorsed by the culture media to support the nutritional requirements of
SSC(32) which has also performed an assessment of the TSE micro-organisms, which might be used as seed stocks or in
risk of rennet in general(33). industrial scale fermentations for the production of human
Bovine milk derivatives manufactured according to the and veterinary medicinal products, including vaccines. There
conditions described below are unlikely to present any TSE is considerable interest in the use of vegetable protein as an
risk and shall therefore be considered compliant with this alternative to animal sourced protein. However :
chapter. – where gelatin is used as the protein source material,
– The milk is sourced from healthy animals in the same reference is made to section 6-2 Gelatin, of this chapter,
conditions as milk collected for human consumption, and – where casein is used as the protein source material,
– no other ruminant materials, with the exception of calf reference is made to section 6-6 Milk and milk derivatives,
rennet, are used in the preparation of such derivatives (e.g. of this chapter,
pancreatic enzyme digests of casein). – where tissue of TSE-relevant animal species is the protein
Milk derivatives produced using other processes or rennet source material, the tissue must be sourced from animals
derived from other ruminant species must demonstrate fit for consumption (see section 3-2 Source animals, of this
compliance with this chapter. chapter) with a maximum age of 30 months old for cattle
(30) For milk and milk derivatives from small ruminants, please see EFSA opinion on Question No EFSA-Q-2008-310, adopted on 22 October 2008,
http://www.efsa.europa.eu/en/scdocs/scdoc/849.htm
(31) Committee for Medicinal Products for Human Use and its Biologics Working Party conducted a risk and regulatory assessment of lactose prepared using calf rennet. The risk
assessment included the source of the animals, the excision of the abomasums and the availability of well-defined quality assurance procedures. The quality of any milk replacers used as
feed for the animals from which abomasums are obtained is particularly important. The report can be found on http://www.ema.europa.eu/pdfs/human/press/pus/057102.pdf
(32) Provisional statement on the safety of calf-derived rennet for the manufacture of lactose, adopted by the SSC at its meeting of 4-5 April 2002 (http://ec.europa.eu/
food/fs/sc/ssc/out255_en.pdf)
(33) The SSC issued an opinion on the safety of animal rennet in regard to risks from animal TSE and BSE in particular, adopted at its meeting of 16 May 2002
(http://ec.europa.eu/food/fs/sc/ssc/out265_en.pdf)
from countries with a controlled BSE risk (Category B). Data entries are shown as follows :
The age of animals is of minimal concern for animals from + = Presence of infectivity or PrPTSE
countries with a negligible BSE risk (Category A).
− = Absence of detectable infectivity or PrPTSE
Annex : major categories of infectivity NT = Not tested
The tables below are taken from the WHO Guidelines on NA = Not applicable
Tissue Infectivity Distribution in Transmissible Spongiform ? = Uncertain interpretation
Encephalopathies (2010).
() = Limited or preliminary data
[] = Infectivity or PrPTSE data based exclusively on
bioassays in transgenic (Tg) mice over-expressing
the PrP-encoding gene or PRPTSE amplification
methods
Category IA : High-infectivity tissues
Tissue Cattle Sheep and goats Elk and deer
BSE Scrapie CWD
Infectivity1 PrPTSE Infectivity1 PrPTSE Infectivity1 PrPTSE
Brain + + + + + +
Spinal cord + + + + NT +
Retina + NT NT + NT +
Optic nerve 2 + NT NT + NT +
Spinal ganglia + + + + NT +
Trigeminal ganglia + + NT + NT -
Pituitary gland 3 − NT + + NT +
3
Dura mater NT NT NT NT NT NT
Autonomic ganglia 4
NT + NT + NT +
Lymphoreticular tissues
Spleen − − + + NT +
Lymph nodes − − + + NT +
Tonsil + − + + NT +
Thymus − NT + + NT −
Alimentary tract 5
Oesophagus − NT [+] + NT +
Duodenum − − [+] + NT +
Jejunum 7 − + [+] + NT NT
Ileum 7 + + + + NT +
Appendix NA NA NA NA NA NA
Colon/caecum7 − − + + NT +
Rectum NT NT NT + NT +
Reproductive tissues
Placenta8 − NT + + NT −
Ovary3 − NT − − NT −
Uterus 3 − NT − − NT −
General Notices (1) apply to all monographs and other texts 601
5.2.8. Minimising the risk of transmitting TSE via medicinal products EUROPEAN PHARMACOPOEIA 8.0
Heart/pericardium − NT − NT NT +
Lung − NT − − NT +
Liver 3 − NT + − NT −
Kidney 3, 11 − − [+] + NT +
Adrenal [+] + + − NT +
Pancreas3 − NT + NT NT +
Bone marrow 12
[+] NT + NT NT −
Skeletal muscle 13
[+] NT [+] + [+] −
Tongue14 − NT [+] + NT −
Blood vessels − NT NT + NT −
Nasal mucosa15 − NT + + NT +
Salivary gland − NT + NT − −
Cornea16 NT NT NT NT NT NT
Blood 17 − ? + ? + ?
Saliva NT NT − NT + [–]
Milk 18 − − + [+] NT NT
Urine19 − NT − − –[+] [+]
Feces 19 − NT − NT –[+] NT
Prostate/Epididymis/Seminal − NT − − NT −
vesicle
Semen − NT − − NT NT
Placenta fluids − NT NT NT NT NT
Foetus 20 − NT − − NT (–)
Embryos 20 − NT ? NT NT NT
Musculo-skeletal tissues
Bone − NT NT NT NT NT
Tendon − NT NT NT NT NT
Other tissues
Gingival tissues NT NT NT NT NT NT
Dental pulp NT NT NT NT NT NT
Trachea − NT NT NT NT −
Thyroid gland NT NT − NT NT −
Cord blood21 − NT NT NT NT NT
Sweat NT NT NT NT NT NT
Tears NT NT NT NT NT NT
Nasal mucus NT NT NT NT NT NT
Bile NT NT NT NT NT NT
1. Infectivity bioassays of human tissues have been conducted in either primates or mice (or both), bioassays of cattle tissues have been conducted in
either cattle or mice (or both), and most bioassays of sheep and/or goat tissues have been conducted only in mice. In regard to sheep and goats not all
results are consistent for both species, for example, two goats (but no sheep) have contracted BSE naturally [Eurosurveillance, 2005, Jeffrey et al.,
2006]. Similarly, most of the results described for CWD were derived from studies in deer, and findings may not be identical in elk or other cervids.
2. In experimental models of TSE, the optic nerve has been shown to be a route of neuroinvasion, and contains high titres of infectivity.
3. No experimental data about infectivity in pituitary gland or dura mater in humans with all forms of human TSE have been reported, but cadaveric
dura mater patches, and growth hormone derived from cadaveric pituitaries have transmitted disease to hundreds of people and therefore must
be included in the category of high-risk tissues. PrPTSE was detected by immunoblot in the dura mater of a vCJD patient who died in the US
after an unusually long incubation period (see also Table IB for other positive tissues : skin, kidney, liver, pancreas, ovary and uterus) [Notari et
al., 2010]. It must be mentioned that earlier studies of numerous cases examined in the UK reported all of these tissues to be negative [Ironside
et al., 2002, Head et al., 2004].
4. In cattle, PrPTSE is reported to be inconsistently present in the enteric plexus in the distal ileum, but immunohistochemical examination of tissues
from a single ‘fallen stock’ case of BSE in Japan suggested (albeit equivocally) involvement of myenteric plexuses throughout the small and large
intestine [Kimura and Haritani, 2008].
5. In vCJD, PrPTSE is limited to gut-associated lymphoid and nervous tissue (mucosa, muscle, and serosa are negative).
6. Ruminant fore stomachs (reticulum, rumen, and omasum) are widely consumed, as is the true stomach (abomasum). The abomasum of cattle
(and sometimes sheep) is also a source of rennet.
7. When a large BSE oral dose was used to infect cattle experimentally, infectivity was detected in the jejunum and the ileo-caecum junction in Tg
mice overexpressing PrP [courtesy of Dr M Groschup]. PrPTSE was detected at low incidence in lymphoid tissue of ileum [Terry et al., 2003] and has
been detected at an even lower frequency in jejunal lymphoid tissue of cattle similarly infected by the oral route [EFSA, 2009].
8. A single report of transmission of sporadic CJD infectivity from human placenta has never been confirmed and is considered improbable.
9. PrPTSE has been detected in scrapie-infected sheep with chronic mastitis, but not from infected sheep without mastitis [Ligios et al., 2005].
10. Studies in hamsters orally infected with scrapie revealed that PrPTSE deposition in skin was primarily located within small nerve fibres. Also,
apical skin ‘velvet’ from the antlers of CWD-infected deer is reported to contain PrPTSE and infectivity [Angers et al., 2009].
11. PrPTSE detected by immunocytochemistry in the renal pelvis of scrapie-infected sheep [Siso et al., 2006], and in lymphoid follicles within
connective tissue adjacent to the renal pelvis in CWD-infected mule deer [Fox et al., 2006].
12. A single positive marrow in multiple transmission attempts from cattle orally dosed with BSE-infected brain [Wells et al., 1999, Wells et al.,
2005, Sohn et al., 2009].
13. Muscle homogenates have not transmitted disease to primates from humans with sporadic CJD, or to cattle from cattle with BSE. However,
intra-cerebral inoculation of a semitendinosus muscle homogenate (including nervous and lymphatic elements) from a single cow with clinical BSE
has transmitted disease to transgenic mice that overexpress PrP at a rate indicative of trace levels of infectivity [Buschmann and Groschup, 2005].
Also, recent published and unpublished studies have reported the presence of PrPTSE in skeletal muscle in experimental rodent models of scrapie and
vCJD [Beekes et al., 2005], in experimental and natural scrapie infections of sheep and goats [Andreoletti et al., 2004], in sheep orally dosed with BSE
[Andreoletti, unpublished data], and in humans with sporadic, iatrogenic, and variant forms of CJD [Glatzel et al., 2003, Kovacs et al., 2004, Peden et
al., 2006]. Bioassays of muscle in transgenic mice expressing cervid PrP have documented infectivity in CWD-infected mule deer [Angers et al.,
2006], and experiments are underway to determine whether detectable PrPTSE in other forms of TSE is also associated with infectivity.
14. In cattle, bioassay of infectivity in the tongue was negative, but the presence of infectivity in palatine tonsil has raised concern about possible
infectivity in lingual tonsillar tissue at the base of the tongue that may not be removed at slaughter [Wells et al., 2005, EFSA, 2008]. In sheep naturally
infected with scrapie, 7 of 10 animals had detectable PrPTSE in the tongue [Casalone et al., 2005, Corona et al., 2006].
15. Limited chiefly to regions involved in olfactory sensory reception.
16. Because only one case of iatrogenic CJD has been certainly attributed to a corneal transplant among hundreds of thousands of recipients (one
additional case is considered probable, and another case only possible), cornea has been categorized as a lower-risk tissue, other anterior chamber
tissues (lens, aqueous humour, iris, conjunctiva) have been tested with a negative result both in vCJD and other human TSEs, and there is no
epidemiological evidence that they have been associated with iatrogenic disease transmission.
17. A wealth of data from studies of blood infectivity in experimental rodent models of TSE have been extended by recent studies documenting
infectivity in the blood of sheep with naturally occurring scrapie and in sheep transfused with blood from BSE-infected cattle [Houston et al., 2008],
of deer with naturally occurring CWD [Mathiason et al., 2006], and (from epidemiological observations) in the red cell fraction (which includes
significant amounts of both plasma and leukocytes) of four blood donors in the pre-clinical phase of vCJD infections [reviewed in Brown, 2006,
Hewitt et al., 2006]. Plasma Factor VIII administration has also been potentially implicated in a subclinical case of vCJD in a haemophilia patient
[Peden et al., 2010]. Blood has not been shown to transmit disease from humans with any form of ‘classical’ TSE [Dorsey et al., 2009], or from
cattle with BSE (including fetal calf blood). A number of laboratories using new, highly sensitive methods to detect PrPTSE are reporting success in a
variety of animal and human TSEs. However, several have experienced difficulty obtaining reproducible results in plasma, and it is not yet clear that
positive results imply a potential for disease transmissibility, either because of false positives, or of ‘true’ positives that are due to sub-transmissible
concentrations of PrPTSE. Because of these considerations (and the fact that no data are yet available on blinded testing of specimens from naturally
infected humans or animals) the expert group felt that it was still too early to evaluate the validity of these tests with sufficient confidence to permit
either a negative or positive conclusion.
18. Evidence that infectivity is not present in milk from BSE-infected bovines includes temporo-spatial epidemiologic observations failing to detect
maternal transmission to calves suckled for long periods, clinical observations of over a hundred calves suckled by infected cows that have not
developed BSE, and experimental observations that milk from infected cows reared to an age exceeding the minimum incubation period has not
transmitted disease when administered intra-cerebrally or orally to mice [Middleton and Barlow, 1993, Taylor et al., 1995]. Also, PrPTSE has not been
detected in milk from cattle incubating BSE following experimental oral challenge [SEAC, 2005]. However, low levels (μg to ng/L) of normal PrP have
been detected in milk from both animals and humans [Franscini et al., 2006]. PrPTSE has been detected in the mammary glands of scrapie-infected
sheep with chronic mastitis [Ligios et al., 2005], and very recently it has been reported that milk (which in some cases also contained colostrum) from
scrapie-infected sheep transmitted disease to healthy animals [Konold et al., 2008, Lacroux et al., 2008].
General Notices (1) apply to all monographs and other texts 603
5.2.9. Safety of batches of immunosera for veterinary use EUROPEAN PHARMACOPOEIA 8.0
19. A mixed inoculum of urine and faeces from naturally infected CWD deer did not transmit disease during an 18-month observation period
after inoculation of healthy deer with a heterozygous (96 G/S) PRNP genotype [Mathiason et al., 2006]. However, recent bioassays in Tg mice have
transmitted disease from both urine [Haley et al., 2009] and faeces [Tamgüney et al., 2009]. In addition, mice with lymphocytic nephritis that were
experimentally infected with scrapie shed both PrPTSE and infectivity in urine, when bioassayed in Tg mice [Seegeret al., 2005]. Very low levels
of infectivity have also been detected in the urine (and histologically normal kidneys) of hamsters experimentally infected with scrapie [Gregori
and Rohwer, 2007, Gonzalez-Romero et al., 2008]. Finally, in an experimental scrapie-hamster model, oral dosing resulted in infectious faeces
when bioassayed in Tg mice over-expressing PrP [Safar et al., 2008].
20. Embryos from BSE-affected cattle have not transmitted disease to mice, but no infectivity measurements have been made on fetal calf tissues
other than blood (negative mouse bioassay) [Fraser and Foster, 1994]. Calves born of dams that received embryos from BSE- affected cattle have
survived for observations periods of up to seven years, and examination of the brains of both the unaffected dams and their offspring revealed no
spongiform encephalopathy or PrPTSE [Wrathall et al., 2002].
21. Early reports of transmission of sporadic CJD infectivity from human cord blood and colostrum have never been confirmed and are considered
improbable. A bioassay from a cow with BSE in transgenic mice over-expressing bovine PrP gave a negative result [Buschmann and Groschup, 2005],
and PrPTSE has not been detected in colostrum from cattle incubating BSE following experimental oral challenge [SEAC, 2005].
01/2008:50300 design and the sample size. The 95 per cent confidence
interval is usually chosen in biological assays. Mathematical
statistical methods are used to calculate these limits so as to
5.3. STATISTICAL ANALYSIS warrant the statement that there is a 95 per cent probability
that these limits include the true potency. Whether this
OF RESULTS OF BIOLOGICAL precision is acceptable to the European Pharmacopoeia
depends on the requirements set in the monograph for the
ASSAYS AND TESTS preparation concerned.
The terms “mean” and “standard deviation” are used here as
defined in most current textbooks of biometry.
The terms “stated potency” or “labelled potency”, “assigned
potency”, “assumed potency”, “potency ratio” and “estimated
1. INTRODUCTION potency” are used in this section to indicate the following
concepts :
This chapter provides guidance for the design of bioassays – “stated potency” or “labelled potency” : in the case of
prescribed in the European Pharmacopoeia (Ph. Eur.) and for a formulated product a nominal value assigned from
analysis of their results. It is intended for use by those whose knowledge of the potency of the bulk material ; in the case of
primary training and responsibilities are not in statistics, but bulk material the potency estimated by the manufacturer ;
who have responsibility for analysis or interpretation of the
results of these assays, often without the help and advice of – “assigned potency” : the potency of the standard
a statistician. The methods of calculation described in this preparation ;
annex are not mandatory for the bioassays which themselves – “assumed potency” : the provisionally assigned potency
constitute a mandatory part of the Ph. Eur. Alternative of a preparation to be examined which forms the basis of
methods can be used and may be accepted by the competent calculating the doses that would be equipotent with the
authorities, provided that they are supported by relevant data doses to be used of the standard preparation ;
and justified during the assay validation process. A wide range
of computer software is available and may be useful depending – “potency ratio” of an unknown preparation ; the ratio of
on the facilities available to, and the expertise of, the analyst. equipotent doses of the standard preparation and the
unknown preparation under the conditions of the assay ;
Professional advice should be obtained in situations where : a
comprehensive treatment of design and analysis suitable for – “estimated potency” : the potency calculated from assay
research or development of new products is required ; the data.
restrictions imposed on the assay design by this chapter are Section 9 (Glossary of symbols) is a tabulation of the more
not satisfied, for example particular laboratory constraints important uses of symbols throughout this annex. Where
may require customized assay designs, or equal numbers of the text refers to a symbol not shown in this section or uses
equally spaced doses may not be suitable ; analysis is required a symbol to denote a different concept, this is defined in that
for extended non-linear dose-response curves, for example as part of the text.
may be encountered in immunoassays. An outline of extended
dose-response curve analysis for one widely used model is
nevertheless included in Section 3.4 and a simple example is
given in Section 5.4.
General Notices (1) apply to all monographs and other texts 607
5.3. Statistical analysis EUROPEAN PHARMACOPOEIA 8.0
2) an under-estimation of the true confidence limits for the – The square transformation of y to y2 can be useful if, for
test, which, as shown in Section 3.2.5, are calculated from the example, the dose is more likely to be proportional to
estimate of s2, the residual error mean square. the area of an inhibition zone rather than the measured
diameter of that zone.
For some assays depending on quantitative responses, such as
3. ASSAYS DEPENDING UPON immunoassays or cell-based in vitro assays, a large number
of doses is used. These doses give responses that completely
QUANTITATIVE RESPONSES span the possible response range and produce an extended
non-linear dose-response curve. Such curves are typical for
3.1. STATISTICAL MODELS
all bioassays, but for many assays the use of a large number of
3.1.1. GENERAL PRINCIPLES doses is not ethical (for example, in vivo assays) or practical,
The bioassays included in the Ph. Eur. have been conceived as and the aims of the assay may be achieved with a limited
“dilution assays”, which means that the unknown preparation number of doses. It is therefore customary to restrict doses
to be assayed is supposed to contain the same active to that part of the dose-response range which is linear under
principle as the standard preparation, but in a different suitable transformation, so that the methods of Sections 3.2
ratio of active and inert components. In such a case the or 3.3 apply. However, in some cases analysis of extended
unknown preparation may in theory be derived from the dose-response curves may be desirable. An outline of one
standard preparation by dilution with inert components. To model which may be used for such analysis is given in
check whether any particular assay may be regarded as a Section 3.4 and a simple example is shown in Section 5.4.
dilution assay, it is necessary to compare the dose-response There is another category of assays in which the response
relationships of the standard and unknown preparations. If cannot be measured in each experimental unit, but in which
these dose-response relationships differ significantly, then only the fraction of units responding to each treatment can be
the theoretical dilution assay model is not valid. Significant counted. This category is dealt with in Section 4.
differences in the dose-response relationships for the standard
3.1.2. ROUTINE ASSAYS
and unknown preparations may suggest that one of the
preparations contains, in addition to the active principle, When an assay is in routine use, it is seldom possible to
other components which are not inert but which influence check systematically for conditions 1 to 3, because the limited
the measured responses. number of observations per assay is likely to influence the
sensitivity of the statistical tests. Fortunately, statisticians have
To make the effect of dilution in the theoretical model shown that, in symmetrical balanced assays, small deviations
apparent, it is useful to transform the dose-response from homogeneity of variance and normality do not seriously
relationship to a linear function on the widest possible range affect the assay results. The applicability of the statistical
of doses. 2 statistical models are of interest as models for model needs to be questioned only if a series of assays shows
the bioassays prescribed : the parallel-line model and the doubtful validity. It may then be necessary to perform a
slope-ratio model. new series of preliminary investigations as discussed in
The application of either is dependent on the fulfilment of the Section 3.1.1.
following conditions :
2 other necessary conditions depend on the statistical model
1) the different treatments have been randomly assigned to to be used :
the experimental units,
– for the parallel-line model :
2) the responses to each treatment are normally distributed,
4A) the relationship between the logarithm of the dose and
3) the standard deviations of the responses within each the response can be represented by a straight line over the
treatment group of both standard and unknown preparations
range of doses used,
do not differ significantly from one another.
When an assay is being developed for use, the analyst has to 5A) for any unknown preparation in the assay the straight
determine that the data collected from many assays meet these line is parallel to that for the standard.
theoretical conditions. – for the slope-ratio model :
– Condition 1 can be fulfilled by an efficient use of Section 2. 4B) the relationship between the dose and the response can
– Condition 2 is an assumption which in practice is almost be represented by a straight line for each preparation in the
always fulfilled. Minor deviations from this assumption assay over the range of doses used,
will in general not introduce serious flaws in the analysis 5B) for any unknown preparation in the assay the straight
as long as several replicates per treatment are included. In line intersects the y-axis (at zero dose) at the same point
case of doubt, a test for deviations from normality (e.g. the as the straight line of the standard preparation (i.e. the
Shapiro-Wilk(1) test) may be performed. response functions of all preparations in the assay must
– Condition 3 can be checked with a test for homogeneity of have the same intercept as the response function of the
variances (e.g. Bartlett’s(2) test, Cochran’s(3) test). Inspection standard).
of graphical representations of the data can also be very Conditions 4A and 4B can be verified only in assays in which
instructive for this purpose (see examples in Section 5). at least 3 dilutions of each preparation have been tested. The
When conditions 2 and/or 3 are not met, a transformation of use of an assay with only 1 or 2 dilutions may be justified when
the responses may bring a better fulfilment of these conditions. experience has shown that linearity and parallelism or equal
Examples are ln y, , y 2. intercept are regularly fulfilled.
– Logarithmic transformation of the responses y to ln y After having collected the results of an assay, and before
can be useful when the homogeneity of variances is not calculating the relative potency of each test sample, an
satisfactory. It can also improve the normality if the analysis of variance is performed, in order to check whether
distribution is skewed to the right. conditions 4A and 5A (or 4B and 5B) are fulfilled. For this,
– The transformation of y to is useful when the the total sum of squares is subdivided into a certain number
observations follow a Poisson distribution i.e. when they of sum of squares corresponding to each condition which has
are obtained by counting. to be fulfilled. The remaining sum of squares represents the
(1) Wilk, M.B. and Shapiro, S.S. (1968). The joint assessment of normality of several independent samples, Technometrics 10, 825-839.
(2) Bartlett, M.S. (1937). Properties of sufficiency and statistical tests, Proc. Roy. Soc. London, Series A 160, 280-282.
(3) Cochran, W.G. (1951). Testing a linear relation among variances, Biometrics 7, 17-32.
residual experimental error to which the absence or existence b) In the parallel-line model, the ratio of adjacent doses must
of the relevant sources of variation can be compared by a be constant for all treatments in the assay ; in the slope-ratio
series of F-ratios. model, the interval between adjacent doses must be constant
When validity is established, the potency of each unknown for all treatments in the assay.
relative to the standard may be calculated and expressed as c) There must be an equal number of experimental units to
a potency ratio or converted to some unit relevant to the each treatment.
preparation under test e.g. an International Unit. Confidence If a design is used which meets these restrictions, the
limits may also be estimated from each set of assay data. calculations are simple. The formulae are given in Sections 3.2
Assays based on the parallel-line model are discussed in and 3.3. It is recommended to use software which has been
Section 3.2 and those based on the slope-ratio model in developed for this special purpose. There are several programs
Section 3.3. in existence which can easily deal with all assay-designs
If any of the 5 conditions (1, 2, 3, 4A, 5A or 1, 2, 3, 4B, 5B) described in the monographs. Not all programs may use the
are not fulfilled, the methods of calculation described here same formulae and algorithms, but they should all lead to the
are invalid and an investigation of the assay technique should same results.
be made. Assay designs not meeting the above mentioned restrictions
The analyst should not adopt another transformation unless may be both possible and correct, but the necessary formulae
it is shown that non-fulfilment of the requirements is not are too complicated to describe in this text. A brief description
incidental but is due to a systematic change in the experimental of methods for calculation is given in Section 7.1. These
conditions. In this case, testing as described in Section 3.1.1 methods can also be used for the restricted designs, in which
should be repeated before a new transformation is adopted case they are equivalent with the simple formulae.
for the routine assays. The formulae for the restricted designs given in this text
Excess numbers of invalid assays due to non-parallelism may be used, for example, to create ad hoc programs in a
or non-linearity, in a routine assay carried out to compare spreadsheet. The examples in Section 5 can be used to clarify
similar materials, are likely to reflect assay designs with the statistics and to check whether such a program gives
inadequate replication. This inadequacy commonly results correct results.
from incomplete recognition of all sources of variability
affecting the assay, which can result in underestimation of the 3.2. THE PARALLEL-LINE MODEL
residual error leading to large F-ratios. 3.2.1. INTRODUCTION
It is not always feasible to take account of all possible sources The parallel-line model is illustrated in Figure 3.2.1.-I. The
of variation within one single assay (e.g. day-to-day variation). logarithm of the doses are represented on the horizontal axis
In such a case, the confidence intervals from repeated assays with the lowest concentration on the left and the highest
on the same sample may not satisfactorily overlap, and care concentration on the right. The responses are indicated on
should be exercised in the interpretation of the individual the vertical axis. The individual responses to each treatment
confidence intervals. In order to obtain a more reliable are indicated with black dots. The 2 lines are the calculated
estimate of the confidence interval it may be necessary to ln(dose)-response relationship for the standard and the
perform several independent assays and to combine these unknown.
into one single potency estimate and confidence interval (see
Section 6).
For the purpose of quality control of routine assays it is
recommended to keep record of the estimates of the slope of
regression and of the estimate of the residual error in control
charts.
– An exceptionally high residual error may indicate some
technical problem. This should be investigated and,
if it can be made evident that something went wrong
during the assay procedure, the assay should be repeated.
An unusually high residual error may also indicate the
presence of an occasional outlying or aberrant observation.
A response that is questionable because of failure to
comply with the procedure during the course of an assay
is rejected. If an aberrant value is discovered after the
responses have been recorded, but can then be traced
to assay irregularities, omission may be justified. The
arbitrary rejection or retention of an apparently aberrant
response can be a serious source of bias. In general, the
rejection of observations solely because a test for outliers is
significant, is discouraged.
– An exceptionally low residual error may once in a while
occur and cause the F-ratios to exceed the critical values.
In such a case it may be justified to replace the residual Figure 3.2.1.-I. – The parallel-line model for a 3 + 3 assay
error estimated from the individual assay, by an average Note : the natural logarithm (ln or loge) is used throughout
residual error based on historical data recorded in the this text. Wherever the term “antilogarithm” is used, the
control charts. quantity ex is meant. However, the Briggs or “common”
3.1.3. CALCULATIONS AND RESTRICTIONS logarithm (log or log10) can equally well be used. In this case
According to general principles of good design the following the corresponding antilogarithm is 10x.
3 restrictions are normally imposed on the assay design. For a satisfactory assay the assumed potency of the test sample
They have advantages both for ease of computation and for must be close to the true potency. On the basis of this assumed
precision. potency and the assigned potency of the standard, equipotent
a) Each preparation in the assay must be tested with the same dilutions (if feasible) are prepared, i.e. corresponding doses
number of dilutions. of standard and unknown are expected to give the same
General Notices (1) apply to all monographs and other texts 609
5.3. Statistical analysis EUROPEAN PHARMACOPOEIA 8.0
Table 3.2.3.-I. – Formulae for parallel-line assays with d doses of each preparation
Standard 1st Test sample 2nd Test sample
(S) (T) (U, etc.)
Total preparation
Linear contrast
Table 3.2.3.-II. – Additional formulae for the construction of the analysis of variance
Table 3.2.3.-III. – Formulae to calculate the sum of squares and degrees of freedom
Source of variation Degrees of freedom (f) Sum of squares
Preparations
Linear regression
Non-parallelism
Non-linearity*
Treatments
Blocks (rows)*
Columns**
Completely randomised
Latin square
Total
residual error (s2) and the significance of these values (known 3) The term for non-linearity is not significant, i.e. the
as F-ratios) are assessed by use of Table 8.1 or a suitable calculated probability is not less than 0.05. This indicates that
sub-routine of a computer program. condition 4A, Section 3.1, is satisfied.
3.2.4. TESTS OF VALIDITY A significant deviation from parallelism in a multiple assay
Assay results are said to be “statistically valid” if the outcome may be due to the inclusion in the assay-design of a preparation
of the analysis of variance is as follows. to be examined that gives an ln(dose)-response line with a
slope different from those for the other preparations. Instead
1) The linear regression term is significant, i.e. the calculated of declaring the whole assay invalid, it may then be decided
probability is less than 0.05. If this criterion is not met, it is to eliminate all data relating to that preparation and to restart
not possible to calculate 95 per cent confidence limits. the analysis from the beginning.
2) The term for non-parallelism is not significant, i.e. the When statistical validity is established, potencies and
calculated probability is not less than 0.05. This indicates that confidence limits may be estimated by the methods described
condition 5A, Section 3.1, is satisfied ; in the next section.
General Notices (1) apply to all monographs and other texts 611
5.3. Statistical analysis EUROPEAN PHARMACOPOEIA 8.0
(3.2.6.-1)
and the logarithm of the potency ratio of a test preparation,
for example T, is : where B′ is the sum of the responses in the block containing
the missing value, T′ the corresponding treatment total and G′
(3.2.5.-2)
is the sum of all responses recorded in the assay.
Latin square design
The calculated potency is an estimate of the “true potency” of The missing value y′ is obtained from :
each unknown. Confidence limits may be calculated as the
antilogarithms of : (3.2.6.-2)
(3.2.5.-3)
where B′ and C′ are the sums of the responses in the row and
column containing the missing value. In this case k = n.
where and
Cross-over design
If an accident leading to loss of values occurs in a cross-over
The value of t may be obtained from Table 8.2 for p = 0.05 design, a book on statistics should be consulted (e.g. D.J.
and degrees of freedom equal to the number of the degrees of Finney, see Section 10), because the appropriate formulae
freedom of the residual error. The estimated potency (RT) and depend upon the particular treatment combinations.
associated confidence limits are obtained by multiplying the 3.3. THE SLOPE-RATIO MODEL
values obtained by AT after antilogarithms have been taken.
If the stock solutions are not exactly equipotent on the basis 3.3.1. INTRODUCTION
of assigned and assumed potencies, a correction factor is This model is suitable, for example, for some microbiological
necessary (See Examples 5.1.2 and 5.1.3). assays when the independent variable is the concentration of
an essential growth factor below the optimal concentration
3.2.6. MISSING VALUES of the medium. The slope-ratio model is illustrated in
In a balanced assay, an accident totally unconnected with Figure 3.3.1.-I.
the applied treatments may lead to the loss of one or more
responses, for example because an animal dies. If it is
considered that the accident is in no way connected with
the composition of the preparation administered, the exact
calculations can still be performed but the formulae are
necessarily more complicated and can only be given within
the framework of general linear models (see Section 7.1).
However, there exists an approximate method which keeps
the simplicity of the balanced design by replacing the missing
response by a calculated value. The loss of information is
taken into account by diminishing the degrees of freedom
for the total sum of squares and for the residual error by the
number of missing values and using one of the formulae below
for the missing values. It should be borne in mind that this is
only an approximate method, and that the exact method is to
be preferred.
If more than one observation is missing, the same formulae
can be used. The procedure is to make a rough guess at all the
missing values except one, and to use the proper formula for
this one, using all the remaining values including the rough
guesses. Fill in the calculated value. Continue by similarly
calculating a value for the first rough guess. After calculating
all the missing values in this way the whole cycle is repeated Figure 3.3.1.-I. – The slope-ratio model for a 2 × 3 + 1 assay
from the beginning, each calculation using the most recent The doses are represented on the horizontal axis with zero
guessed or calculated value for every response to which the concentration on the left and the highest concentration on the
formula is being applied. This continues until 2 consecutive right. The responses are indicated on the vertical axis. The
cycles give the same values ; convergence is usually rapid. individual responses to each treatment are indicated with black
Provided that the number of values replaced is small relative dots. The 2 lines are the calculated dose-response relationship
to the total number of observations in the full experiment for the standard and the unknown under the assumption that
(say less than 5 per cent), the approximation implied in they intersect each other at zero-dose. Unlike the parallel-line
this replacement and reduction of degrees of freedom by model, the doses are not transformed to logarithms.
the number of missing values so replaced is usually fairly Just as in the case of an assay based on the parallel-line
satisfactory. The analysis should be interpreted with great care model, it is important that the assumed potency is close to
however, especially if there is a preponderance of missing the true potency, and to prepare equipotent dilutions of the
values in one treatment or block, and a biometrician should be test preparations and the standard (if feasible). The more
consulted if any unusual features are encountered. Replacing nearly correct the assumed potency, the closer the 2 lines
missing values in a test without replication is a particularly will be together. The ratio of the slopes represents the “true”
delicate operation. potency of the unknown, relative to its assumed potency. If
the slope of the unknown preparation is steeper than that A completely randomised, a randomised block or a Latin
of the standard, the potency was underestimated and the square design may be used, such as described in Section 3.2.2.
calculations will indicate an estimated potency higher than the The use of any of these designs necessitates an adjustment to
assumed potency. Similarly, if the slope of the unknown is less the error sum of squares as described for assays based on the
steep than that of the standard, the potency was overestimated parallel-line model. The analysis of an assay of one or more
and the calculations will result in an estimated potency lower test preparations against a standard is described below.
than the assumed potency. 3.3.3. ANALYSIS OF VARIANCE
In setting up an experiment, all responses should be
3.3.3.1. The (hd + 1)-design
examined for the fulfilment of the conditions 1, 2 and 3 in
Section 3.1. The analysis of variance to be performed in The responses are verified as described in Section 3.1 and,
routine is described in Section 3.3.3 so that compliance with if necessary, transformed. The responses are then averaged
conditions 4B and 5B of Section 3.1 may be examined. over each treatment and each preparation as shown in
Table 3.3.3.1.-I. Additionally, the mean response for blanks (B)
3.3.2. ASSAY DESIGN is calculated.
The use of the statistical analysis presented below imposes the
following restrictions on the assay : The sums of squares in the analysis of variance are calculated
as shown in Tables 3.3.3.1.-I to 3.3.3.1.-III. The sum of
a) the standard and the test preparations must be tested with squares due to non-linearity can only be calculated if at least
the same number of equally spaced dilutions, 3 doses of each preparation have been included in the assay.
b) an extra group of experimental units receiving no treatment The residual error is obtained by subtracting the variations
may be tested (the blanks), allowed for in the design from the total variation in response
c) there must be an equal number of experimental units to (Table 3.3.3.1.-IV).
each treatment. The analysis of variance is now completed as follows. Each
As already remarked in Section 3.1.3, assay designs not sum of squares is divided by the corresponding number of
meeting these restrictions may be both possible and correct, degrees of freedom to give mean squares. The mean square
but the simple statistical analyses presented here are no longer for each variable to be tested is now expressed as a ratio to the
applicable and either expert advice should be sought or residual error (s2) and the significance of these values (known
suitable software should be used. as F-ratios) are assessed by use of Table 8.1 or a suitable
sub-routine of a computer program.
A design with 2 doses per preparation and 1 blank, the
“common zero (2h + 1)-design”, is usually preferred, since 3.3.3.2. The (hd)-design
it gives the highest precision combined with the possibility The formulae are basically the same as those for the
to check validity within the constraints mentioned above. (hd + 1)-design, but there are some slight differences.
However, a linear relationship cannot always be assumed to
be valid down to zero-dose. With a slight loss of precision a – B is discarded from all formulae.
design without blanks may be adopted. In this case 3 doses per –
preparation, the “common zero (3h)-design”, are preferred to
2 doses per preparation. The doses are thus given as follows : – SSblank is removed from the analysis of variance.
1) the standard is given in a high dose, near to but not – The number of degrees of freedom for treatments becomes
exceeding the highest dose giving a mean response on the hd − 1.
straight portion of the dose-response line, – The number of degrees of freedom of the residual error
2) the other doses are uniformly spaced between the highest and the total variance is calculated as described for the
dose and zero dose, parallel-line model (see Table 3.2.3.-IV).
3) the test preparations are given in corresponding doses Validity of the assay, potency and confidence interval are
based on the assumed potency of the material. found as described in Sections 3.3.4 and 3.3.5.
Table 3.3.3.1.-I. – Formulae for slope-ratio assays with d doses of each preparation and a blank
Standard 1st Test sample 2nd Test sample
(S) (T) (U, etc.)
… … … …
Total preparation
Linear product
Intercept value
Slope value
Treatment value
Non-linearity*
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5.3. Statistical analysis EUROPEAN PHARMACOPOEIA 8.0
Table 3.3.3.1.-II. – Additional formulae for the construction of the analysis of variance
Table 3.3.3.1.-III. – Formulae to calculate the sum of squares and degrees of freedom
Regression
Blanks
Intersection
Non-linearity*
Treatments
Blocks (rows)*
Columns**
Completely
randomised
Residual error***
Randomised block
Latin square
Total
2) the variation due to intersection is not significant, i.e. the The slope of the standard, and similarly for each of the other
calculated probability is not less than 0.05. This indicates that preparations, is calculated from :
condition 5B, Section 3.1 is satisfied ;
3) in assays including at least 3 doses per preparation, the (3.3.5.1.-2)
variation due to non-linearity is not significant, i.e. the
calculated probability is not less than 0.05. This indicates that The potency ratio of each of the test preparations can now be
condition 4B, Section 3.1 is satisfied. calculated from :
The confidence interval for RT′ is calculated from : The general shape of the curves can usually be described by
a logistic function but other shapes are also possible. Each
curve can be characterised by 4 parameters : The upper
asymptote (α), the lower asymptote (δ), the slope-factor (β),
(3.3.5.1.-4) and the horizontal location (γ). This model is therefore
often referred to as a four-parameter model. A mathematical
representation of the ln(dose)-response curve is :
where and
General Notices (1) apply to all monographs and other texts 615
5.3. Statistical analysis EUROPEAN PHARMACOPOEIA 8.0
The most commonly used function is the cumulative normal the more extreme parts of the curve. This method, the analysis
distribution function. This function has some theoretical of variance, and the estimation of the potency and confidence
merit, and is perhaps the best choice if the response is a interval are described below.
reflection of the tolerance of the units. If the response is 4.2.1. TABULATION OF THE RESULTS
more likely to depend upon a process of growth, the logistic
Table 4.2.1.-I is used to enter the data into the columns
distribution model is preferred, although the difference in
indicated by numbers :
outcome between the 2 models is usually very small.
(1) the dose of the standard or the test preparation,
The maximum likelihood estimators of the slope and location
of the curves can be found only by applying an iterative (2) the number n of units submitted to that treatment,
procedure. There are many procedures which lead to the (3) the number of units r giving a positive response to the
same outcome, but they differ in efficiency due to the speed treatment,
of convergence. One of the most rapid methods is direct (4) the logarithm x of the dose,
optimisation of the maximum-likelihood function (see Section
7.1), which can easily be performed with computer programs (5) the fraction p = r/n of positive responses per group.
having a built-in procedure for this purpose. Unfortunately, The first cycle starts here.
most of these procedures do not yield an estimate of the (6) column Y is filled with zeros at the first iteration,
confidence interval, and the technique to obtain it is too
complicated to describe here. The technique described below (7) the corresponding value Φ = Φ(Y) of the cumulative
is not the most rapid, but has been chosen for its simplicity standard normal distribution function (see also Table 8.4).
compared to the alternatives. It can be used for assays in which The columns (8) to (10) are calculated with the following
one or more test preparations are compared to a standard. formulae :
Furthermore, the following conditions must be fulfilled :
1) the relationship between the logarithm of the dose and (8) (4.2.1.-1)
the response can be represented by a cumulative normal
distribution curve,
(9) (4.2.1.-2)
2) the curves for the standard and the test preparation are
parallel, i.e. they are identically shaped and may only differ
in their horizontal location, (10) (4.2.1.-3)
3) in theory, there is no natural response to extremely low
The columns (11) to (15) can easily be calculated from
doses and no natural non-response to extremely high doses.
columns (4), (9) and (10) as wx, wy, wx2, wy2 and wxy
4.2. THE PROBIT METHOD respectively, and the sum (Σ) of each of the columns (10) to
(15) is calculated separately for each of the preparations.
The sigmoid curve can be made linear by replacing each
response, i.e. the fraction of positive responses per group, by The sums calculated in Table 4.2.1.-I are transferred to
the corresponding value of the cumulative standard normal columns (1) to (6) of Table 4.2.1.-II and 6 additional columns
distribution. This value, often referred to as “normit”, ranges (7) to (12) are calculated as follows :
theoretically from − ∞ to + ∞. In the past it was proposed to
add 5 to each normit to obtain “probits”. This facilitated the
hand-performed calculations because negative values were (7) (4.2.1.-4)
avoided. With the arrival of computers the need to add 5 to
the normits has disappeared. The term “normit method”
would therefore be better for the method described below. (8) (4.2.1.-5)
However, since the term “probit analysis” is so widely spread,
the term will, for historical reasons, be maintained in this text.
(9) (4.2.1.-6)
Once the responses have been linearised, it should be possible
to apply the parallel-line analysis as described in Section 3.2.
Unfortunately, the validity condition of homogeneity of (10) (4.2.1.-7)
variance for each dose is not fulfilled. The variance is minimal
at normit = 0 and increases for positive and negative values of
the normit. It is therefore necessary to give more weight to (11) (4.2.1.-8)
responses in the middle part of the curve, and less weight to
dose n r x p Y Φ Z y w wx wy wx 2
wy 2
wxy
S . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
Σ= Σ= Σ= Σ= Σ= Σ=
T . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
Σ= Σ= Σ= Σ= Σ= Σ=
etc.
S . . . . . . . . . . . .
T . . . . . . . . . . . .
etc. . . . . . . . . . . . .
Σ= Σ=
The common slope b can now be obtained as : are slightly modified. t becomes the t-value (p = 0.05) with
the same number of degrees of freedom as used in the check
(4.2.1.-9) for linearity and s2 becomes the χ2 value divided by the same
number of degrees of freedom (and thus typically is greater
and the intercept a of the standard, and similarly for the test than 1).
preparations is obtained as : The test for parallelism is also slightly modified. The χ2 value
(12) (4.2.1.-10) for non-parallelism is divided by its number of degrees of
freedom. The resulting value is divided by s2 calculated above
Column (6) of the first working table can now be replaced to obtain an F-ratio with h - 1 and N - 2h degrees of freedom,
by Y = a + bx and the cycle is repeated until the difference which is evaluated in the usual way at the 0.05 significance
between 2 cycles has become small (e.g. the maximum level.
difference of Y between 2 consecutive cycles is smaller
than 10− 8). 4.3. THE LOGIT METHOD
4.2.2. TESTS OF VALIDITY As indicated in Section 4.1 the logit method may sometimes
Before calculating the potencies and confidence intervals, be more appropriate. The name of the method is derived
validity of the assay must be assessed. If at least 3 doses for from the logit function which is the inverse of the logistic
each preparation have been included, the deviations from distribution. The procedure is similar to that described for
linearity can be measured as follows : add a 13th column to the probit method with the following modifications in the
Table 4.2.1.-II and fill it with : formulae for Φ and Z.
(4.2.2.-1) (4.3.-1)
General Notices (1) apply to all monographs and other texts 617
5.3. Statistical analysis EUROPEAN PHARMACOPOEIA 8.0
(4.5.-1)
(4.5.-2)
5. EXAMPLES
330 267 360 280 273 283 Table 5.1.1.-III. – Analysis of variance without sample U
290 236 341 261 240 269 Source of Degrees of Sum of Mean Proba-
F-ratio
variation freedom squares square bility
364 250 321 241 307 251
390 229 303 223 317 223 Regression 1 66 830.6 66 830.6 90.5 0.000
360 269 334 254 312 250 Non-parallelism 1 34.2 34.2 0.05 0.831
306 259 315 235 265 265 Residual error 36 26 587.3 738.54
The analysis without preparation U results in compliance Table 5.1.2.-I. – Distribution of treatments over the plate
with the requirements with respect to both regression and
parallelism and so the potency can be calculated. The 1 2 3 4 5 6
formulae in Section 3.2.5 give :
1 S1 T1 T2 S3 S2 T3
3 T2 S3 S2 S1 T3 T1
4 S3 S2 T3 T1 S1 T2
5 S2 T2 S3 T3 T1 S1
– the ln(potency ratio) is :
6 T3 S1 T1 T2 S3 S2
1 2 3 4 5 6 Row mean
– and ln(confidence limits) are : 1 161 160 178 187 171 194 175.2 = R1
PT = 526.333 LT = 39.333
HP = =2 HL = =3
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5.3. Statistical analysis EUROPEAN PHARMACOPOEIA 8.0
Table 5.1.2.-IV. – Analysis of variance Table 5.1.3.-I. – Absorbances of the suspensions (× 1000)
Standard S Preparation T
Source of Degrees of Sum of Mean Proba-
F-ratio Block S1 S2 S3 S4 T1 T2 T3 T4
variation freedom squares square bility Mean
Preparations 1 11.1111 11.1111 1 252 207 168 113 242 206 146 115 181.1
Regression 1 8475.0417 8475.0417 408.1 0.000 2 249 201 187 107 236 197 153 102 179.0
Non-parallelism 1 18.3750 18.3750 0.885 0.358 3 247 193 162 111 246 197 148 104 176.0
Non-linearity 2 5.4722 2.7361 0.132 0.877 4 250 207 155 108 231 191 159 106 175.9
Rows 5 412 82.40 3.968 0.012 Mean 246.6 203.0 162.4 107.4 237.4 195.4 150.4 104.4
Total 35 9556
Figure 5.1.3.-I.
– and ln(confidence limits) are :
The formulae in Tables 3.2.3.-I and 3.2.3.-II lead to :
PS = 719.4 LS = − 229.1
PT = 687.6 LT = − 222
HP = = 1.25 HL = =1
The potency ratio is found by taking the antilogarithms,
resulting in 0.9763 with 95 per cent confidence limits from The analysis of variance is constructed with the formulae
0.9112-1.0456. in Tables 3.2.3.-III and 3.2.3.-IV. The result is shown in
Table 5.1.3.-II.
A correction factor of is Table 5.1.3.-II. – Analysis of variance
necessary because the dilutions were not exactly equipotent Source of Degrees of Sum of Mean square F-ratio Proba-
on the basis of the assumed potency. Multiplying by this variation freedom squares bility
correction factor and the assumed potency of 5600 IU/mg
Preparations 1 632.025 632.025
yields a potency of 5456 IU/mg with 95 per cent confidence
limits from 5092 to 5843 IU/mg. Regression 1 101 745.6 101 745.6 1887.1 0.000
5.1.3. FOUR-DOSE RANDOMISED BLOCK DESIGN Non-parallelism 1 25.205 25.205 0.467 0.500
Antibiotic turbidimetric assay Non-linearity 4 259.14 64.785 1.202 0.332
1:16 000 0.086 0.071 0.073 0.082 0.082 0.086 Residual error 40 0.267 0.0067
General Notices (1) apply to all monographs and other texts 621
5.3. Statistical analysis EUROPEAN PHARMACOPOEIA 8.0
PT = 162.25 LT = − 8.75
52 53 88 113 92 84 93 117 and with the formulae in Table 3.2.3.-III this leads to :
110 113 63 71 101 56 73 128 Day 1 Day 2 Pooled
116 91 50 65 66 55 39 87 SSprep = 18.000 SSprep = 13.781 SSprep = 0.141
Mean 95.6 82.8 69.6 93.3 89.9 66.6 72.4 106.8 SSpar = 144.5 SSpar = 1755.3 SSpar = 1453.5
The interaction terms are found as Day 1 + Day 2 - Pooled. – and ln(confidence limits) are :
63 53 423.2
Slope of standard :
Total
General Notices (1) apply to all monographs and other texts 623
5.3. Statistical analysis EUROPEAN PHARMACOPOEIA 8.0
Table 5.2.1.-I. – Absorbances external and the internal reactant is established, the zone of
the annulus precipitation area is measured. The results are
Blank Standard S Preparation T shown in Table 5.2.2.-I.
(in IU/mL) (in IU/mL)
0.022 0.138 0.219 0.299 0.121 0.191 0.255 A graphical presentation of the data shows no unusual features
(see Figure 5.2.2.-I). The formulae in Tables 3.3.3.1.-I and
0.023 0.137 0.218 0.302 0.121 0.190 0.254
3.3.3.1.-II yield
Mean 0.0235 0.1351 0.2176 0.2996 0.1200 0.1898 0.2554 PS = 108.2 PT = 103.85 PU = 85.8
and
HI = 0.0093 a′ = 11.04 K = 14 785.8
Source of
Degrees
Sum of Mean Proba- Slope of U is :
of F-ratio
variation squares square bility
freedom
Intersection 1 3 · 10 −9
3 · 10 −9
7 · 10 −4
0.978 This leads to a potency ratio of 6.056/6.356 = 0.953 for
vaccine T and 4.123/6.356 = 0.649 for vaccine U.
Non-linearity 2 2 · 10− 5 1 · 10− 5 2.984 0.061
Treatments 5 0.1917
Total 47 0.1919
And the confidence limits are found with formula 3.3.5.1.-4.
5.2.2. A COMPLETELY RANDOMISED (0,4,4,4)-DESIGN
For vaccine T :
An in-vitro assay of influenza vaccines
The haemagglutinin antigen (HA) content of 2 influenza
vaccines is determined by single radial immunodiffusion.
Both have a labelled potency of 15 μg HA per dose, which is For vaccine U :
equivalent with a content of 30 μg HA/mL. The standard has
an assigned content of 39 μg HA/mL.
Standard and test vaccines are applied in 4 duplicate The HA content in μg/dose can be found by multiplying the
concentrations which are prepared on the basis of the assigned potency ratios and confidence limits by the assumed content
and the labelled contents. When the equilibrium between the of 15 μg/dose. The results are given in Table 5.2.2.-III.
1.0 12 0 1.0 11 0
2.5 12 6 2.5 11 8
4.0 11 10 4.0 11 10
Table 5.2.2.-II. – Analysis of variance The observations are transferred to the first working table and
the subsequent columns are computed as described in Section
4.2.1. Table 5.3.1.-II shows the first cycle of this procedure.
Degrees Proba-
Source of Sum of Mean The sums of the last 6 columns are then calculated per
of F-ratio
variation squares square bility
freedom preparation and transferred to the second working table (see
Regression 3 1087.7 362.6 339.5 0.000
Table 5.3.1.-III). The results in the other columns are found
with formulae 4.2.1.-4 to 4.2.1.-10. This yields a common
Intersection 2 3.474 1.737 1.626 0.237 slope b of 1.655.
Non-linearity 6 5.066 0.844 0.791 0.594 The values for Y in the first working table are now replaced by
a + bx and a second cycle is carried out (see Table 5.3.1.-IV).
Treatments 11 1096.2
The cycle is repeated until the difference between 2 consecutive
Residual error 12 12.815 1.068 cycles has become small. The second working table should
then appear as shown in Table 5.3.1.-V.
Total 23 1109.0
Linearity is tested as described in Section 4.2.2. The χ2-value
with 4 degrees of freedom is 0.851 + 1.070 = 1.921 representing
a p-value of 0.750 which is not significant.
Since there are no significant deviations from linearity, the
test for parallelism can be carried out as described in the same
section. The χ2-value with 1 degree of freedom is
S 1.0 12 0 0.000 0.000 0 0.5 0.399 − 1.253 7.64 0.00 − 9.57 0.00 12.00 0.00
1.6 12 3 0.470 0.250 0 0.5 0.399 − 0.627 7.64 3.59 − 4.79 1.69 3.00 − 2.25
2.5 12 6 0.916 0.500 0 0.5 0.399 0.000 7.64 7.00 0.00 6.41 0.00 0.00
4.0 11 10 1.386 0.909 0 0.5 0.399 1.025 7.00 9.71 7.18 13.46 7.36 9.95
T 1.0 11 0 0.000 0.000 0 0.5 0.399 − 1.253 7.00 0.00 − 8.78 0.00 11.00 0.00
1.6 12 4 0.470 0.333 0 0.5 0.399 − 0.418 7.64 3.59 − 3.19 1.69 1.33 − 1.50
2.5 11 8 0.916 0.727 0 0.5 0.399 0.570 7.00 6.42 3.99 5.88 2.27 3.66
4.0 11 10 1.386 0.909 0 0.5 0.399 1.025 7.00 9.71 7.18 13.46 7.36 9.95
General Notices (1) apply to all monographs and other texts 625
5.3. Statistical analysis EUROPEAN PHARMACOPOEIA 8.0
S 29.92 20.30 − 7.18 21.56 22.36 7.70 7.79 12.58 20.64 0.68 − 0.24 − 1.36
T 28.65 19.72 − 0.80 21.03 21.97 12.11 7.46 12.66 21.95 0.69 − 0.03 − 1.17
S 1.0 12 0 0.000 0.000 − 1.36 0.086 0.158 − 1.911 3.77 0.00 − 7.21 0.00 13.79 0.00
1.6 12 3 0.470 0.250 − 0.58 0.279 0.336 − 0.672 6.74 3.17 − 4.53 1.49 3.04 − 2.13
2.5 12 6 0.916 0.500 0.15 0.561 0.394 − 0.001 7.57 6.94 − 0.01 6.36 0.00 − 0.01
4.0 11 10 1.386 0.909 0.93 0.824 0.258 1.260 5.07 7.03 6.39 9.75 8.05 8.86
T 1.0 11 0 0.000 0.000 − 1.17 0.122 0.202 − 1.769 4.20 0.00 − 7.43 0.00 13.14 0.00
1.6 12 4 0.470 0.333 − 0.39 0.349 0.370 − 0.430 7.23 3.40 − 3.11 1.60 1.34 − 1.46
2.5 11 8 0.916 0.727 0.35 0.637 0.375 0.591 6.70 6.14 3.96 5.62 2.34 3.63
4.0 11 10 1.386 0.909 1.13 0.870 0.211 1.311 4.35 6.03 5.70 8.36 7.48 7.90
S 18.37 14.80 − 2.14 14.85 17.81 5.28 2.93 7.00 17.56 0.81 − 0.12 − 2.05
T 17.96 12.64 − 0.55 11.86 18.35 6.76 2.96 7.15 18.34 0.70 − 0.03 − 1.72
Φ
The potency and confidence limits can now be found by taking
the antilogarithms and multiplying these by the assumed Z
potency of 140 IU/vial. This yields an estimate of 160.6 IU/vial
with 95 per cent confidence limits from 121.0-215.2 IU/vial.
slope b 4.101 2.590 1.717
Table 5.3.3.-I. – Dilutions (10x μL of the undiluted vaccine) So ln confidence limits are :
− 3.5 − 4.0 − 4.5 − 5.0 − 5.5 − 6.0 −6.5 −7.0 −7.5 −8.0
+ + + + − − − − − −
+ + + + − − − − − −
Further :
Figure 5.3.3.-I.
T 10 − 3.5
8 0 − 8.06 0.000 0.00 0.5 0.399 − 1.253 5.09 − 41.04 − 6.38 330.8 8.00 51.4
− 4.0
10 8 0 − 9.21 0.000 0.00 0.5 0.399 − 1.253 5.09 − 46.91 − 6.38 432.0 8.00 58.8
10− 4.5 8 1 − 10.36 0.125 0.00 0.5 0.399 − 0.940 5.09 − 52.77 − 4.79 546.8 4.50 49.6
10− 5.0 8 2 − 11.51 0.250 0.00 0.5 0.399 − 0.627 5.09 − 58.63 − 3.19 675.1 2.00 36.7
− 5.5
10 8 6 − 12.66 0.750 0.00 0.5 0.399 0.627 5.09 − 64.50 3.19 816.8 2.00 − 40.4
− 6.0
10 8 7 − 13.82 0.875 0.00 0.5 0.399 0.940 5.09 − 70.36 4.79 972.1 4.50 − 66.1
− 6.5
10 8 7 − 14.97 0.875 0.00 0.5 0.399 0.940 5.09 − 76.23 4.79 1140.8 4.50 − 71.7
− 7.0
10 8 8 − 16.12 1.000 0.00 0.5 0.399 1.253 5.09 − 82.09 6.38 1323.1 8.00 − 102.9
10− 7.5 8 8 − 17.27 1.000 0.00 0.5 0.399 1.253 5.09 − 87.95 6.38 1518.9 8.00 − 110.2
− 8.0
10 8 8 − 18.42 1.000 0.00 0.5 0.399 1.253 5.09 − 93.82 6.38 1728.2 8.00 − 117.6
T 50.93 − 674.3 11.17 9484.6 57.50 − 312.32 556.92 − 164.43 55.05 − 13.24 0.219 − 3.690
T 19.39 − 238.2 0.11 2981.1 26.05 − 37.45 55.88 − 36.11 26.05 − 12.28 0.006 − 7.931
General Notices (1) apply to all monographs and other texts 627
5.3. Statistical analysis EUROPEAN PHARMACOPOEIA 8.0
1/20 2.579 2.654 1/20 2.801 2.808 There are no significant deviations from parallelism and
1/40 2.130 2.212 1/40 2.401 2.450 linearity and thus the assay is satisfactory for potency
calculations. If the condition of equal upper and lower
1/80 1.651 1.638 1/80 1.918 1.963 asymptotes is not fulfilled, significant deviations from linearity
1/160 1.073 0.973 1/160 1.364 1.299 and/or parallelism are likely to occur because the tests for
linearity and parallelism reflect the goodness of fit of the
1/320 0.585 0.666 1/320 0.861 0.854 complete four-parameter model. The residual error in the
analysis of variance is always equal to 1 as a result of the
1/640 0.463 0.356 1/640 0.497 0.496
transformation. However, a heterogeneity factor (analogous
1/1280 0.266 0.234 1/1280 0.340 0.344 to that for the probit model) can be computed.
1/2560 0.228 0.197 1/2560 0.242 0.217 The relative potency of the test preparation can be
obtained as the antilogarithm of γS − γT. Multiplying by
1/5120 0.176 0.215 1/5120 0.178 0.125 the assigned potency of the standard yields an estimate of
1.459 × 0.4 = 0.584 IU/mL. Formula 4.2.3.-2 gives 95 per cent
For this example, it will be assumed that the laboratory has confidence limits from 0.557-0.612 IU/mL.
validated conditions 1 to 3 in Section 3.1.1 when the assay was
being developed for routine use. In addition, the laboratory
has validated that the upper limit and lower limit of the 6. COMBINATION OF ASSAY RESULTS
samples can be assumed to be equal. 6.1. INTRODUCTION
No unusual features are discovered in a graphical Replication of independent assays and combination of their
representation. A least squares method of a suitable computer results is often needed to fulfil the requirements of the
program is used to fit the parameters of the logistic function, European Pharmacopoeia. The question then arises as to
assuming that the residual error terms are independent and whether it is appropriate to combine the results of such assays
identically distributed normal random variables. In this case, and if so in what way.
3 parameters (α, β and δ) are needed to describe the common 2 assays may be regarded as mutually independent when
slope-factor and the common lower and upper asymptotes. the execution of either does not affect the probabilities of
2 additional parameters (γS and γT) are needed to describe the the possible outcomes of the other. This implies that the
horizontal location of the 2 curves. random errors in all essential factors influencing the result
(for example, dilutions of the standard and of the preparation
The following estimates of the parameters are returned by the to be examined, the sensitivity of the biological indicator) in
program : one assay must be independent of the corresponding random
errors in the other one. Assays on successive days using the
original and retained dilutions of the standard therefore are
not independent assays.
There are several methods for combining the results of
independent assays, the most theoretically acceptable being
In addition, the estimated residual variance (s2) is returned the most difficult to apply. 3 simple, approximate methods are
as 0.001429 with 20 degrees of freedom (within-treatments described below ; others may be used provided the necessary
variation). conditions are fulfilled.
Before potencies from assays based on the parallel-line
In order to obtain confidence limits, and also to check for or probit model are combined they must be expressed in
parallelism and linearity, the observed responses (u) are logarithms ; potencies derived from assays based on the
linearised and submitted to a weighted parallel-line analysis slope-ratio model are used as such. As the former models are
by the program. This procedure is very similar to that more common than those based on the slope-ratio model,
described in Section 4.2 for probit analysis with the following the symbol M denoting ln potency is used in the formulae
modifications : in this section ; by reading R (slope-ratio) for M, the analyst
may use the same formulae for potencies derived from assays where the number of degrees of freedom of t equals the sum of
based on the slope-ratio model. All estimates of potency must the number of degrees of freedom for the error mean squares
be corrected for the potency assigned to each preparation to in the individual assays.
be examined before they are combined. 6.2.4. WEIGHTED MEAN AND CONFIDENCE LIMITS
6.2. WEIGHTED COMBINATION OF ASSAY RESULTS BASED ON THE INTRA- AND INTER-ASSAY VARIATION
When results of several repeated assays are combined, the
This method can be used provided the following conditions (χ2-value may be significant. The observed variation is then
are fulfilled : considered to have two components :
1) the potency estimates are derived from independent assays ; – the intra-assay variation ,
2) for each assay C is close to 1 (say less than 1.1) ;
3) the number of degrees of freedom of the individual residual – the inter-assay variation
errors is not smaller than 6, but preferably larger than 15 ;
where is the unweighted mean. The former varies from
4) the individual potency estimates form a homogeneous set assay to assay whereas the latter is common to all M.
(see Section 6.2.2).
For each M a weighting coefficient is then calculated as :
When these conditions are not fulfilled this method cannot be
applied. The method described in Section 6.3 may then be
used to obtain the best estimate of the mean potency to be
adopted in further assays as an assumed potency.
which replaces W in Section 6.2.3. where t is taken to be
6.2.1. CALCULATION OF WEIGHTING COEFFICIENTS approximately 2.
It is assumed that the results of each of the n′ assays have been
analysed to give n′ values of M with associated confidence 6.3. UNWEIGHTED COMBINATION OF ASSAY RESULTS
limits. For each assay the logarithmic confidence interval L To combine the n′ estimates of M from n′ assays in the simplest
is obtained by subtracting the lower limit from the upper. A way, the mean is calculated and an estimate of its standard
weight W for each value of M is calculated from equation deviation is obtained by calculating :
6.2.1.-1, where t has the same value as that used in the
calculation of confidence limits.
(6.3.-1)
(6.2.1.-1)
and the limits are :
6.2.2. HOMOGENEITY OF POTENCY ESTIMATES
(6.3.-2)
By squaring the deviation of each value of M from the weighted
mean, multiplying by the appropriate weight and summing where t has (n′ − 1) degrees of freedom. The number n′ of
over all assays, a statistic is obtained which is approximately estimates of M is usually small, and hence the value of t is
2
distributed as χ (see Table 8.3) and which may be used to test quite large.
the homogeneity of a set of ln potency estimates :
6.4. EXAMPLE OF A WEIGHTED MEAN POTENCY WITH
where CONFIDENCE LIMITS
Table 6.4.-I lists 6 independent potency estimates of the same
(6.2.2.-1) preparation together with their 95 per cent confidence limits
and the number of degrees of freedom of their error variances.
If the calculated χ2 is smaller than the tabulated value Conditions 1, 2 and 3 in Section 6.2. are met. The ln potencies
corresponding to (n′ − 1) degrees of freedom the potencies are and the weights are calculated as described in Section 6.2.
homogeneous and the mean potency and limits obtained in
Section 6.2.3 will be meaningful. Table 6.4.-I. – Potency estimates and confidence intervals of
6 independent assays
If the calculated value of this statistic is greater than the
tabulated value, the potencies are heterogeneous. This means Potency Lower Upper Degrees ln Weight
that the variation between individual estimates of M is greater estimate limit limit of po- W
than would have been predicted from the estimates of the (IU/vial) (IU/vial) (IU/vial) free- tency
confidence limits, i.e. that there is a significant variability dom M
between the assays. Under these circumstances condition 4 is
18 367 17 755 19 002 20 9.8183 3777.7
not fulfilled and the equations in Section 6.2.3 are no longer
applicable. Instead, the formulae in Section 6.2.4 may be used. 18 003 17 415 18 610 20 9.7983 3951.5
6.2.3. CALCULATION OF THE WEIGHTED MEAN AND
18 064 17 319 18 838 20 9.8017 2462.5
CONFIDENCE LIMITS
The products WM are formed for each assay and their sum 17 832 17 253 18 429 20 9.7887 4003.0
divided by the total weight for all assays to give the logarithm
of the weighted mean potency. 18 635 17 959 19 339 20 9.8328 3175.6
General Notices (1) apply to all monographs and other texts 629
5.3. Statistical analysis EUROPEAN PHARMACOPOEIA 8.0
By taking the antilogarithms a potency of 18 187 IU/vial 7.3. OUTLIERS AND ROBUST METHODS
is found with 95 per cent confidence limits from The method of least squares described in this annex has the
17 946-18 431 IU/vial. disadvantage of being very sensitive to outliers. A clear outlier
may completely corrupt the calculations. This problem is often
remedied by discarding the outlying result from the dataset.
This policy can lead to arbitrary rejection of data and is not
7. BEYOND THIS ANNEX always without danger. It is not easy to give a general guideline
on how to decide whether or not a specific observation is an
It is impossible to give a comprehensive treatise of statistical outlier and it is for this reason that many robust methods
methods in a pharmacopoeial text. However, the methods have been developed. These methods are less sensitive to
described in this annex should suffice for most pharmacopoeial outliers because they give less weight to observations that are
purposes. This section tries to give a more abstract survey of far away from the predicted value. New problems usually arise
alternative or more general methods that have been developed. in computing confidence intervals or defining a satisfactory
The interested reader is encouraged to further explore the function to be minimised.
existing literature in this area. The use of more specialised
statistical methods should, in any case, be left to qualified 7.4. CORRELATED ERRORS
personnel. Absolute randomisation is not always feasible or very
undesirable from a practical point of view. Thus, subsequent
7.1. GENERAL LINEAR MODELS doses within a dilution series often exhibit correlated errors
The methods given in this annex can be described in terms leading to confidence limits that are far too narrow. Some
of general linear models (or generalised linear models to methods have been developed that take account of this
include the probit and logit methods). The principle is to autocorrelation effect.
define a linear structure matrix X (or design matrix) in which
each row represents an observation and each column a linear 7.5. EXTENDED NON-LINEAR DOSE-RESPONSE CURVES
effect (preparation, block, column, dose). For example : the Analysis of extended non-linear dose-response curves raises a
Latin square design in example 5.1.2 would involve a matrix number of statistical questions which require consideration,
with 36 rows and 13 columns. 1 column for each of the and for which professional advice is recommended. Some of
preparations, 1 column for the doses, 5 columns for each these are indicated below.
block except the first, and 5 columns for each row except the 1) An example using the four-parameter logistic function
first. All columns, except the one for doses, are filled with 0 or has been shown. However, models based on functions giving
1 depending on whether or not the observation relates to the other sigmoid curves may also be used. Models incorporating
effect. A vector Y is filled with the (transformed) observations. additional asymmetry parameters have been suggested.
The effects are estimated with the formula (XtX)− 1XtY from 2) Heterogeneity of variance is common when responses
which the potency estimate m can easily be derived as a ratio cover a wide range. If the analysis ignores the heterogeneity,
of relevant effects. Confidence intervals are calculated from interpretation of results may not be correct and estimates
Fieller’s theorem : may be biased. Use of the reciprocal of the error variances
as weights is unlikely to be reliable with limited numbers of
replicates. It may be appropriate to estimate a function which
m L , mU
relates variance to mean response.
3) The statistical curve-fitting procedures may give different
where g estimates depending on assumptions made about the
homogeneity of the variance and on the range of responses
and v11, v22, v12 represent the variance multipliers for the used.
numerator, the denominator and their covariance multiplier 4) In principle, equality of upper and lower response limits for
respectively. These are taken directly from (XtX)− 1 or the different preparations included in an assay can be directly
indirectly by noting that : tested in each assay. However, interpretation of the results of
these tests may not be straightforward. The tests for linearity
Var(a1 − a2) = Var(a1) + Var(a2)–2Cov(a1,a2) and parallelism given by the simplified method of analysis
(Example 5.4.1) indirectly incorporate tests for equality and
and Cov(a1 − a2 ,b) = Cov(a1 ,b) − Cov(a2 ,b) accuracy of upper and lower limits.
5) Many assays include “controls” which are intended to
A full analysis of variance in which all components are identify the upper and/or lower response limits. However,
partitioned is slightly more complicated as it involves a these values may not be consistent with the statistically fitted
renewed definition of X with more columns to relax the upper and lower response limits based on the extended
assumptions of parallelism and linearity, after which the dose-response curve.
linear hypotheses can be tested. For assays depending upon 6) The simplified method of analysis given in Example 5.4.1
quantal responses the linear effects (intercepts aS, aT etc. and provides approximate confidence intervals. Other methods
the common slope b are found by maximising the sum over may also be used, for example intervals based on lack-of-fit of
treatments of nln Φ(ai + bx) + (n − r)ln(1 − Φ(ai + bx)) where the completely specified model. For typical assay data, with
x is the ln(dose), Φ denotes the shape of the distribution and responses covering the complete range for each preparation
i ∈ {S, T, ...}. tested, all methods give similar results.
7.2. HETEROGENEITY OF VARIANCE 7.6. NON-PARALLELISM OF DOSE-RESPONSE CURVES
Heterogeneity of variance cannot always be solved by simply Similarity of dose-response relationships is a fundamental
transforming the responses. A possible way to cope with this criterion for assessing whether an assay may be regarded
problem is to perform a weighted linear regression. In order as a dilution assay and hence whether the estimation of
to obtain an unbiased estimate, the weight of the observations relative potency is valid (see Section 3.1.1). This criterion
is taken to be proportional to the reciprocal of the error is frequently met by showing that dose-response curves for
variances. Since the true error variance is not always known, standard and test samples do not deviate significantly from
an iterative reweighted linear procedure may be followed. parallelism. Underestimation of the residual error can lead
However, the calculation of the confidence interval involves to excess rejection of assays due to significant deviations
new problems. from parallelism and/or linearity. This is often an artefact of
inappropriate assay design or analysis. Minor modifications extensive tables. Many computer programs include statistical
to assay designs might in many cases substantially improve functions and their use is recommended instead of the tables
the estimation of the residual error. Analysis allowing for in this section. Alternatively, the generating procedures given
the actual level of replication may also improve the situation. below each table can be used to compute the probability
If estimation of the relevant residual error is not feasible corresponding to a given statistic and number of degrees of
for individual assays, for example because it is impractical freedom.
to create independent doses and/or replicates, it might be
possible to obtain a more correct estimate of the residual error
during the assay validation process. There may also be cases
8.1. THE F-DISTRIBUTION
where the assay system is sufficiently precise to detect slight
but genuine non-parallelism. If there is true non-parallelism If an observed value is higher than the value in Table 8.1.-I, it
this needs to be recognised and a suitable solution adopted. A is considered to be significant (upper lines, p = 0.05) or highly
solution might, for example, require a suitable standard that significant (lower lines, p = 0.01). df1 is the number of degrees
is similar in composition to, and therefore parallel to, the test of freedom of the numerator and df2 is the number of degrees
samples. If the assay system is responding in a non-specific of freedom of the denominator.
manner to extraneous components of the standard or test
samples, then a more specific assay system that does not Generating procedure. Let F be the F-ratio and df1 and df2
respond to the irrelevant components may be the solution. as described above. Let pi = = 3.14159265358979... The
No simple, generally applicable statistical solution exists to procedure in Table 8.1.-II will then generate the p-value.
overcome these fundamental problems. The appropriate
action has to be decided on a case-by-case basis with the help
of statistical expertise.
8.2. THE -DISTRIBUTION
8. TABLES AND GENERATING Generating procedures. The p-value for a given t with df
degrees of freedom can be found with the procedures in
PROCEDURES Section 8.1 where F = t2, df1 = 1 and df2 = df.
The tables in this section list the critical values for the most The t-value (p = 0.05) for a given number of degrees of
frequently occurring numbers of degrees of freedom. If a freedom df can be found with the procedure in Table 8.2.-II,
critical value is not listed, reference should be made to more which should be accurate up to 6 decimal places.
df2 ↓
10 4.965 4.103 3.708 3.478 3.326 3.217 3.072 2.978 2.913 2.845 2.774 2.538
10.044 7.559 6.552 5.994 5.636 5.386 5.057 4.849 4.706 4.558 4.405 3.909
12 4.747 3.885 3.490 3.259 3.106 2.996 2.849 2.753 2.687 2.617 2.544 2.296
9.330 6.927 5.953 5.412 5.064 4.821 4.499 4.296 4.155 4.010 3.858 3.361
15 4.543 3.682 3.287 3.056 2.901 2.790 2.641 2.544 2.475 2.403 2.328 2.066
8.683 6.359 5.417 4.893 4.556 4.318 4.004 3.805 3.666 3.522 3.372 2.868
20 4.351 3.493 3.098 2.866 2.711 2.599 2.447 2.348 2.278 2.203 2.124 1.843
8.096 5.849 4.938 4.431 4.103 3.871 3.564 3.368 3.231 3.088 2.938 2.421
25 4.242 3.385 2.991 2.759 2.603 2.490 2.337 2.236 2.165 2.089 2.007 1.711
7.770 5.568 4.675 4.177 3.855 3.627 3.324 3.129 2.993 2.850 2.699 2.169
30 4.171 3.316 2.922 2.690 2.534 2.421 2.266 2.165 2.092 2.015 1.932 1.622
7.562 5.390 4.510 4.018 3.699 3.473 3.173 2.979 2.843 2.700 2.549 2.006
50 4.034 3.183 2.790 2.557 2.400 2.286 2.130 2.026 1.952 1.871 1.784 1.438
7.171 5.057 4.199 3.720 3.408 3.186 2.890 2.698 2.563 2.419 2.265 1.683
∞ 3.841 2.996 2.605 2.372 2.214 2.099 1.938 1.831 1.752 1.666 1.571 1.000
6.635 4.605 3.782 3.319 3.017 2.802 2.511 2.321 2.185 2.039 1.878 1.000
General Notices (1) apply to all monographs and other texts 631
5.3. Statistical analysis EUROPEAN PHARMACOPOEIA 8.0
t=t*i/(i+1)*cs*cs
next i
for i=1 to (df1-2) step 2
v=v+w
w=w*(df2+i)/(i+2)*sn*sn
next i
p=1+(t*df2*v-x-s)/pi*4
Table 8.2.-I – Critical values of the t-distribution Table 8.2.-II – Generating procedure for the t-distribution
df p = 0.05 p = 0.01 df p = 0.05 p = 0.01
t = 1.959964+
1 12.706 63.656 22 2.074 2.819
2.37228/df+
2 4.303 9.925 24 2.064 2.797
2.82202/df^2+
3 3.182 5.841 26 2.056 2.779
2.56449/df^3+
4 2.776 4.604 28 2.048 2.763
1.51956/df^4+
5 2.571 4.032 30 2.042 2.750
1.02579/df^5+
6 2.447 3.707 35 2.030 2.724
0.44210/df^7
7 2.365 3.499 40 2.021 2.704
8.3. THE 2
-DISTRIBUTION Table 8.4.-I – Values of the Φ-distribution
x Φ x Φ x Φ
Table 8.3.-I – Critical values of the 2
-distribution
0.00 0.500 1.00 0.841 2.00 0.977
df p = 0.05 p = 0.01 df p = 0.05 p = 0.01
0.05 0.520 1.05 0.853 2.05 0.980
1 3.841 6.635 11 19.675 24.725
0.10 0.540 1.10 0.864 2.10 0.982
2 5.991 9.210 12 21.026 26.217
0.15 0.560 1.15 0.875 2.15 0.984
3 7.815 11.345 13 22.362 27.688
0.20 0.579 1.20 0.885 2.20 0.986
4 9.488 13.277 14 23.685 29.141
0.25 0.599 1.25 0.894 2.25 0.988
5 11.070 15.086 15 24.996 30.578
0.30 0.618 1.30 0.903 2.30 0.989
6 12.592 16.812 16 26.296 32.000
0.35 0.637 1.35 0.911 2.35 0.991
7 14.067 18.475 20 31.410 37.566
0.40 0.655 1.40 0.919 2.40 0.992
8 15.507 20.090 25 37.652 44.314
0.45 0.674 1.45 0.926 2.45 0.993
9 16.919 21.666 30 43.773 50.892
0.50 0.691 1.50 0.933 2.50 0.994
10 18.307 23.209 40 55.758 63.691
0.55 0.709 1.55 0.939 2.55 0.995
If an observed value is higher than the value in Table 8.3.-I, it 0.60 0.726 1.60 0.945 2.60 0.995
is considered to be significant (p = 0.05) or highly significant
(p = 0.01). 0.65 0.742 1.65 0.951 2.65 0.996
phi=0.5+s*exp(-x*x/2)/sqr(2*pi)
In this procedure phi is the cumulative standard normal
distribution function Φ (see Section 8.4).
General Notices (1) apply to all monographs and other texts 633
5.3. Statistical analysis EUROPEAN PHARMACOPOEIA 8.0
1. N=6 S1 S2 S3 T1 T2 T3 2 T3 S3 S1 T2 T1 S2
2. r=2 → ←
3 S2 T3 S3 S1 T2 T1
3. S1 T3 S3 T1 T2 S2
6 T1 S2 T3 S3 S1 T2
4. N=5
1 T2 T1 S2 T3 S3 S1
2. r=4 → ←
4 S1 T2 T1 S2 T3 S3
3. S1 T3 S3 T2 T1 S2
4. N=4 5 S3 S1 T2 T1 S2 T3
2. r=4 ↓ ↓
3. S1 T3 S3 T2 T1 S2
1 2 3 4 5 6
4. N=3
1 S1 T3 T2 T1 S3 S2
2. r=1 → ←
2 S2 T2 T3 S3 T1 S1
3. S3 T3 S1 T2 T1 S2
4. N=2 3 T1 S1 S2 T3 T2 S3
2. r=1 → ← 4 S3 S2 S1 T2 T3 T1
3. T3 S3 S1 T2 T1 S2 5 T3 T1 S3 S1 S2 T2
4. N=1
6 T2 S3 T1 S2 S1 T3
S2 T3 S3 S1 T2 T1
General Notices (1) apply to all monographs and other texts 635
EUROPEAN PHARMACOPOEIA 8.0 5.4. Residual solvents
General Notices (1) apply to all monographs and other texts 639
5.4. Residual solvents EUROPEAN PHARMACOPOEIA 8.0
The lists are not exhaustive and other solvents can be used Class 2 solvents : Solvents to be limited
and later added to the lists. Recommended limits of Class 1 Non-genotoxic animal carcinogens or possible causative
and 2 solvents or classification of solvents may change as new agents of other irreversible toxicity such as neurotoxicity
safety data becomes available. Supporting safety data in a or teratogenicity.
marketing application for a new medicinal product containing
Solvents suspected of other significant but reversible
a new solvent may be based on concepts in this guideline
toxicities.
or the concept of qualification of impurities as expressed in
the guideline for active substances (Q3A, Impurities in New Class 3 solvents : Solvents with low toxic potential
Active Substances) or medicinal products (Q3B, Impurities in Solvents with low toxic potential to man ; no health-based
New Medicinal Products), or all three guidelines. exposure limit is needed. Class 3 solvents have PDEs of
50 mg or more per day.
3.2. METHODS FOR ESTABLISHING EXPOSURE LIMITS
2. SCOPE OF THE GUIDELINE The method used to establish permitted daily exposures for
residual solvents is presented in Appendix 3. Summaries of the
Residual solvents in active substances, excipients, and in toxicity data that were used to establish limits are published in
medicinal products are within the scope of this guideline. Pharmeuropa, Vol. 9, No. 1, Supplement April 1997.
Therefore, testing should be performed for residual solvents 3.3. OPTIONS FOR DESCRIBING LIMITS OF CLASS 2
when production or purification processes are known to result SOLVENTS
in the presence of such solvents. It is only necessary to test Two options are available when setting limits for Class 2
for solvents that are used or produced in the manufacture solvents.
or purification of active substances, excipients, or medicinal
product. Although manufacturers may choose to test the Option 1 : The concentration limits in parts per million stated
medicinal product, a cumulative method may be used to in Table 2 can be used. They were calculated using equation (1)
calculate the residual solvent levels in the medicinal product below by assuming a product mass of 10 g administered daily.
from the levels in the ingredients used to produce the
medicinal product. If the calculation results in a level equal to (1)
or below that recommended in this guideline, no testing of the
medicinal product for residual solvents need be considered. If
however, the calculated level is above the recommended level, Here, PDE is given in terms of mg/day and dose is given
the medicinal product should be tested to ascertain whether in g/day.
the formulation process has reduced the relevant solvent level These limits are considered acceptable for all substances,
to within the acceptable amount. Medicinal product should excipients, or products. Therefore this option may be applied
also be tested if a solvent is used during its manufacture. if the daily dose is not known or fixed. If all excipients and
active substances in a formulation meet the limits given
This guideline does not apply to potential new active in Option 1, then these components may be used in any
substances, excipients, or medicinal products used during the proportion. No further calculation is necessary provided
clinical research stages of development, nor does it apply to the daily dose does not exceed 10 g. Products that are
existing marketed medicinal products. administered in doses greater than 10 g per day should be
considered under Option 2.
The guideline applies to all dosage forms and routes of Option 2 : It is not considered necessary for each component
administration. Higher levels of residual solvents may be of the medicinal product to comply with the limits given
acceptable in certain cases such as short term (30 days or less) in Option 1. The PDE in terms of mg/day as stated in
or topical application. Justification for these levels should be Table 2 can be used with the known maximum daily dose
made on a case by case basis. and equation (1) above to determine the concentration
of residual solvent allowed in a medicinal product. Such
See Appendix 2 for additional background information related limits are considered acceptable provided that is has been
to residual solvents. demonstrated that the residual solvent has been reduced to the
practical minimum. The limits should be realistic in relation
to analytical precision, manufacturing capability, reasonable
3. GENERAL PRINCIPLES variation in the manufacturing process, and the limits should
reflect contemporary manufacturing standards.
3.1. CLASSIFICATION OF RESIDUAL SOLVENTS BY RISK Option 2 may be applied by adding the amounts of a residual
ASSESSMENT solvent present in each of the components of the medicinal
The term “tolerable daily intake” (TDI) is used by the product. The sum of the amounts of solvent per day should be
International Program on Chemical Safety (IPCS) to describe less than that given by the PDE.
exposure limits of toxic chemicals and “acceptable daily intake”
Consider an example of the use of Option l and Option 2
(ADI) is used by the World Health Organization (WHO)
applied to acetonitrile in a medicinal product. The permitted
and other national and international health authorities and
daily exposure to acetonitrile is 4.1 mg per day ; thus, the
institutes. The new term “permitted daily exposure” (PDE)
Option 1 limit is 410 ppm. The maximum administered
is defined in the present guideline as a pharmaceutically
daily mass of a medicinal product is 5.0 g, and the medicinal
acceptable intake of residual solvents to avoid confusion of
product contains two excipients. The composition of the
differing values for ADI’s of the same substance.
medicinal product and the calculated maximum content of
residual acetonitrile are given in the following table.
Residual solvents assessed in this guideline are listed in
Appendix 1 by common names and structures. They were Component Amount in Acetonitrile Daily
evaluated for their possible risk to human health and placed formulation content exposure
into one of three classes as follows : Active substance 0.3 g 800 ppm 0.24 mg
Excipient 1 0.9 g 400 ppm 0.36 mg
Class 1 solvents : Solvents to be avoided
Excipient 2 3.8 g 800 ppm 3.04 mg
Known human carcinogens, strongly suspected human Medicinal product 5.0 g 728 ppm 3.64 mg
carcinogens, and environmental hazards.
Excipient l meets the Option l limit, but the drug substance, If solvents of Class 2 or Class 3 are present at greater than
excipient 2, and medicinal product do not meet the Option l their Option 1 limits or 0.5 per cent, respectively, they should
limit. Nevertheless, the product meets the Option 2 limit of be identified and quantified.
4.l mg per day and thus conforms to the recommendations
in this guideline. 4. LIMITS OF RESIDUAL SOLVENTS
Consider another example using acetonitrile as residual 4.1. SOLVENTS TO BE AVOIDED
solvent. The maximum administered daily mass of a medicinal Solvents in Class 1 should not be employed in the
product is 5.0 g, and the medicinal product contains two manufacture of active substances, excipients, and medicinal
excipients. The composition of the medicinal product and the products because of their unacceptable toxicity or their
calculated maximum content of residual acetonitrile is given deleterious environmental effect. However, if their use is
in the following table. unavoidable in order to produce a medicinal product with
a significant therapeutic advance, then their levels should
Component Amount in Acetonitrile Daily
formulation content exposure be restricted as shown in Table 1, unless otherwise justified.
0.3 g 800 ppm 0.24 mg
1,1,1-Trichloroethane is included in Table 1 because it is an
Active substance
environmental hazard. The stated limit of 1500 ppm is based
Excipient 1 0.9 g 2000 ppm 1.80 mg on a review of the safety data.
Excipient 2 3.8 g 800 ppm 3.04 mg Table 1. – Class 1 solvents in pharmaceutical products
Medicinal product 5.0 g 1016 ppm 5.08 mg (solvents that should be avoided)
Solvent Concentration limit Concern
In this example, the product meets neither the Option 1 (ppm)
Benzene 2 Carcinogen
nor the Option 2 limit according to this summation. The
manufacturer could test the medicinal product to determine Carbon tetrachloride 4 Toxic and environmental hazard
if the formulation process reduced the level of acetonitrile. If
1,2-Dichloroethane 5 Toxic
the level of acetonitrile was not reduced during formulation
to the allowed limit, then the manufacturer of the medicinal 1,1-Dichloroethene 8 Toxic
product should take other steps to reduce the amount of
acetonitrile in the medicinal product. If all of these steps fail 1,1,1-Trichloroethane 1500 Environmental hazard
to reduce the level of residual solvent, in exceptional cases
the manufacturer could provide a summary of efforts made 4.2. SOLVENTS TO BE LIMITED
to reduce the solvent level to meet the guideline value, and Solvents in Table 2 should be limited in pharmaceutical
provide a risk-benefit analysis to support allowing the product products because of their inherent toxicity. PDEs are given to
to be utilised containing residual solvent at a higher level. the nearest 0.1 mg/day, and concentrations are given to the
3.4. ANALYTICAL PROCEDURES nearest 10 ppm. The stated values do not reflect the necessary
analytical precision of determination. Precision should be
Residual solvents are typically determined using determined as part of the validation of the method.
chromatographic techniques such as gas chromatography.
Any harmonised procedures for determining levels of residual Table 2. – Class 2 solvents in pharmaceutical products
solvents as described in the pharmacopoeias should be used, Solvent PDE Concentration limit
if feasible. Otherwise, manufacturers would be free to select (mg/day) (ppm)
the most appropriate validated analytical procedure for a Acetonitrile 4.1 410
particular application. If only Class 3 solvents are present, a 3.6 360
Chlorobenzene
non-specific method such as loss on drying may be used.
Validation of methods for residual solvents should conform to Chloroform 0.6 60
ICH guidelines “Text on Validation of Analytical Procedures” Cyclohexane 38.8 3880
and “Extension of the ICH Text on Validation of Analytical
Procedures”. 1,2-Dichloroethene 18.7 1870
– Only Class 2 solvents X, Y, ... and Class 3 solvents are Methylbutylketone 0.5 50
likely to be present. Residual Class 2 solvents are below 11.8 1180
Methylcyclohexane
the Option 1 limit and residual Class 3 solvents are below
0.5 per cent. N-Methylpyrrolidone 5.3 530
If Class 1 solvents are likely to be present, they should be Nitromethane 0.5 50
identified and quantified. “Likely to be present” refers to the
solvent used in the final manufacturing step and to solvents Pyridine 2.0 200
that are used in earlier manufacturing steps and not removed Sulfolane 1.6 160
consistently by a validated process.
General Notices (1) apply to all monographs and other texts 641
5.4. Residual solvents EUROPEAN PHARMACOPOEIA 8.0
Solvent PDE Concentration limit which to base a PDE was found. Manufacturers should
(mg/day) (ppm)
Tetrahydrofuran 7.2 720
supply justification for residual levels of these solvents in
pharmaceutical products.
Tetralin 1.0 100
8.9 890
Table 4. – Solvents for which no adequate toxicological data
Toluene
was found
1,1,2-Trichloroethene 0.8 80
1,1-Diethoxypropane Methylisopropylketone
Xylene* 21.7 2170
1,1-Dimethoxymethane Methyltetrahydrofuran
*usually 60 per cent m-xylene, 14 per cent p-xylene, 9 per cent o-xylene
with 17 per cent ethyl benzene 2,2-Dimethoxypropane Petroleum ether
Isooctane Trichloroacetic acid
4.3. SOLVENTS WITH LOW TOXIC POTENTIAL
Solvents in Class 3 (shown in Table 3) may be regarded as less Isopropyl ether Trifluoroacetic acid
toxic and of lower risk to human health. Class 3 includes no
solvent known as a human health hazard at levels normally
accepted in pharmaceuticals. However, there are no long-term
GLOSSARY
toxicity or carcinogenicity studies for many of the solvents in
Class 3. Available data indicate that they are less toxic in acute Genotoxic carcinogens : Carcinogens which produce cancer by
or short-term studies and negative in genotoxicity studies. It affecting genes or chromosomes.
is considered that amounts of these residual solvents of 50 mg
per day or less (corresponding to 5000 ppm or 0.5 per cent LOEL : Abbreviation for lowest-observed effect level.
under Option l) would be acceptable without justification. Lowest-observed effect level : The lowest dose of substance in a
Higher amounts may also be acceptable provided they are study or group of studies that produces biologically significant
realistic in relation to manufacturing capability and good increases in frequency or severity of any effects in the exposed
manufacturing practice. humans or animals.
Table 3. – Class 3 solvents which should be limited by GMP or
other quality-based requirements Modifying factor : A factor determined by professional
Acetic acid Heptane judgement of a toxicologist and applied to bioassay data to
relate that data safely to humans.
Acetone Isobutyl acetate
Neurotoxicity : The ability of a substance to cause adverse
Anisole Isopropyl acetate effects on the nervous system.
1-Butanol Methyl acetate NOEL : Abbreviation for no-observed-effect level.
2-Butanol 3-Methyl-1-butanol
No-observed-effect level : The highest dose of substance
Butyl acetate Methylethylketone at which there are no biologically significant increases in
frequency or severity of any effects in the exposed humans
tert-Butylmethyl ether Methylisobutylketone
or animals.
Cumene 2-Methyl-l-propanol
PDE : Abbreviation for permitted daily exposure.
Dimethyl sulfoxide Pentane
Permitted daily exposure : The maximum acceptable intake per
Ethanol 1-Pentanol day of residual solvent in pharmaceutical products.
Ethyl acetate 1-Propanol
Reversible toxicity : The occurrence of harmful effects that are
Ethyl ether 2-Propanol caused by a substance and which disappear after exposure to
the substance ends.
Ethyl formate Propyl acetate
Formic acid
Strongly suspected human carcinogen : A substance for which
there is no epidemiological evidence of carcinogenesis but
there are positive genotoxicity data and clear evidence of
4.4. SOLVENTS FOR WHICH NO ADEQUATE carcinogenesis in rodents.
TOXICOLOGICAL DATA WAS FOUND
The following solvents (Table 4) may also be of interest to Teratogenicity : The occurrence of structural malformations in
manufacturers of excipients, active substances, or medicinal a developing foetus when a substance is administered during
products. However, no adequate toxicological data on pregnancy.
General Notices (1) apply to all monographs and other texts 643
5.4. Residual solvents EUROPEAN PHARMACOPOEIA 8.0
*usually 60 per cent m-xylene, 14 per cent p-xylene, 9 per cent o-xylene with 17 per cent ethyl benzene.
APPENDIX 2. ADDITIONAL BACKGROUND The method is outlined here to give a better understanding of
A2.1. ENVIRONMENTAL REGULATION OF ORGANIC the origin of the PDE values. It is not necessary to perform
VOLATILE SOLVENTS these calculations in order to use the PDE values tabulated in
Section 4 of this document.
Several of the residual solvents frequently used in the
production of pharmaceuticals are listed as toxic chemicals PDE is derived from the no-observed-effect level (NOEL), or
in Environmental Health Criteria (EHC) monographs and the lowest-observed effect level (LOEL), in the most relevant
the Integrated Risk Information System (IRIS). The objectives animal study as follows :
of such groups as the International Programme on Chemical
Safety (IPCS), the United States Environmental Protection
Agency (USEPA) and the United States Food and Drug
Administration (USFDA) include the determination of The PDE is derived preferably from a NOEL. If no NOEL
acceptable exposure levels. The goal is protection of human is obtained, the LOEL may be used. Modifying factors
health and maintenance of environmental integrity against proposed here, for relating the data to humans, are the same
the possible deleterious effects of chemicals resulting from kind of “uncertainty factors” used in Environmental Health
long-term environmental exposure. The methods involved in Criteria (Environmental Health Criteria 170, World Health
the estimation of maximum safe exposure limits are usually Organization, Geneva, 1994), and “modifying factors” or
based on long-term studies. When long-term study data “safety factors” in Pharmacopoeial Forum. The assumption
are unavailable, shorter term study data can be used with of 100 per cent systemic exposure is used in all calculations
modification of the approach such as use of larger safety regardless of route of administration.
factors. The approach described therein relates primarily to
long-term or life-time exposure of the general population in The modifying factors are as follows :
the ambient environment, i.e. ambient air, food, drinking F1 = A factor to account for extrapolation between
water and other media. species
A2.2. RESIDUAL SOLVENTS IN PHARMACEUTICALS
F1 = 2 for extrapolation from dogs to humans
Exposure limits in this guideline are established by referring
to methodologies and toxicity data described in EHC and F1 = 2.5 for extrapolation from rabbits to humans
IRIS monographs. However, some specific assumptions about
F1 = 3 for extrapolation from monkeys to humans
residual solvents to be used in the synthesis and formulation
of pharmaceutical products should be taken into account in F1 = 5 for extrapolation from rats to humans
establishing exposure limits. They are :
F1 = 10 for extrapolation from other animals to
1) Patients (not the general population) use pharmaceuticals humans
to treat their diseases or for prophylaxis to prevent infection F1 = 12 for extrapolation from mice to humans
or disease.
2) The assumption of life-time patient exposure is not F1 takes into account the comparative surface area : body
necessary for most pharmaceutical products but may be weight ratios for the species concerned and for man. Surface
appropriate as a working hypothesis to reduce risk to human area (S) is calculated as :
health.
3) Residual solvents are unavoidable components in
pharmaceutical production and will often be a part of in which m = body mass, and the constant k has been taken to
medicinal products. be 10. The body weight used in the equation are those shown
below in Table A3.-1.
4) Residual solvents should not exceed recommended levels
except in exceptional circumstances. Table A3.-1. – Values used in the calculations in this document
5) Data from toxicological studies that are used to determine Rat body weight 425 g
acceptable levels for residual solvents should have been
generated using appropriate protocols such as those described Pregnant rat body weight 330 g
for example, by OECD and the FDA Red Book. Mouse body weight 28 g
The Gaylor-Kodell method of risk assessment (Gaylor, D. W. Rhesus monkey body weight 2.5 kg
and Kodell, R. L. Linear Interpolation algorithm for low dose Rabbit body weight (pregnant or not) 4 kg
assessment of toxic substance. J. Environ. Pathology, 4, 305,
1980) is appropriate for Class 1 carcinogenic solvents. Only in Beagle dog body weight 11.5 kg
cases where reliable carcinogenicity data are available should Rat respiratory volume 290 L/day
extrapolation by the use of mathematical models be applied to
setting exposure limits. Exposure limits for Class 1 solvents Mouse respiratory volume 43 L/day
could be determined with the use of a large safety factor (i.e., Rabbit respiratory volume 1440 L/day
10 000 to 100 000) with respect to the no-observed-effect level
(NOEL). Detection and quantification of these solvents should Guinea-pig respiratory volume 430 L/day
be by state-of-the-art analytical techniques. Human respiratory volume 28800 L/day
Acceptable exposure levels in this guideline for Class 2 Dog respiratory volume 9000 L/day
solvents were established by calculation of PDE values
according to the procedures for setting exposure limits in Monkey respiratory volume 1150 L/day
pharmaceuticals (Pharmacopeial Forum, Nov-Dec 1989), Mouse water consumption 5 mL/day
and the method adopted by IPCS for Assessing Human
Health Risk of Chemicals (Environmental Health Criteria Rat water consumption 30 mL/day
170, WHO, 1994). These methods are similar to those used Rat food consumption 30 g/day
by the USEPA (IRIS) and the USFDA (Red Book) and others.
General Notices (1) apply to all monographs and other texts 645
5.4. Residual solvents EUROPEAN PHARMACOPOEIA 8.0
F2 = A factor of 10 to account for variability between weights of 60 kg or 70 kg that are often used in this type of
individuals. A factor of 10 is generally given for calculation. It is recognised that some adult patients weigh less
all organic solvents, and 10 is used consistently in than 50 kg ; these patients are considered to be accommodated
this guideline. by the built-in safety factors used to determine a PDE. If the
solvent was present in a formulation specifically intended for
F3 = A variable factor to account for toxicity studies of paediatric use, an adjustment for a lower body weight would
short-term exposure. be appropriate.
F3 = 1 for studies that last at least one half-lifetime (1 As an example of the application of this equation, consider the
year for rodents or rabbits ; 7 years for cats, dogs toxicity study of acetonitrile in mice that is summarised in
and monkeys). Pharmeuropa, Vol. 9. No. 1, Supplement, April 1997, page S24.
F3 = 1 for reproductive studies in which the whole The NOEL is calculated to be 50.7 mg kg–1 day–l. The PDE for
period of organogenesis is covered. acetonitrile in this study is calculated as follows :
F3 = 2 for a 6 month study in rodents, or a 3.5 year
study in non-rodents.
F3 = 5 for a 3 month study in rodents, or a 2 year
study in non-rodents.
F3 = 10 for studies of a shorter duration. In this example,
In all cases, the higher factor has been used for study durations F1 = 12 to account for the extrapolation from mice to
between the time points, e.g. a factor of 2 for a 9 month humans
rodent study.
F2 = 10 to account for differences between individual
F4 = A factor that may be applied in cases of severe humans
toxicity, e.g. non-genotoxic carcinogenicity, F3 = 5 because the duration of the study was only
neurotoxicity or teratogenicity. In studies of 13 weeks
reproductive toxicity, the following factors are
used : F4 = 1 because no severe toxicity was encountered
General Notices (1) apply to all monographs and other texts 649
5.5. Alcoholimetric tables EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 651
5.5. Alcoholimetric tables EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 653
5.5. Alcoholimetric tables EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 655
5.5. Alcoholimetric tables EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 657
5.5. Alcoholimetric tables EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 659
5.5. Alcoholimetric tables EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 663
5.6. Assay of interferons EUROPEAN PHARMACOPOEIA 8.0
Nitrogen-13 (13N) 9.965 (4) min β+ 0.492 (I) (max: 1.198) 99.8 γ 0.511 199.6 (II)
Oxygen-15 (15O) 122.24 (16) s β+ 0.735 (I) (max: 1.732) 99.9 γ 0.511 199.8 (II)
18
Fluorine-18 ( F) 109.77 (5) min β +
0.250 (I)
(max: 0.633) 96.7 γ 0.511 193.5 (II)
Phosphorus-32 (32P) 14.26 (4) days β− 0.695 (I) (max: 1.71) 100
33 − (I) 100
Phosphorus-33 ( P) 25.34 (12) days β 0.076 (max: 0.249)
Sulfur-35 (35S) 87.51 (12) days β− 0.049 (I) (max: 0.167) 100
β+ 0.179 (I)
0.9 γ 0.511 38.0 (II)
(I)
0.631 18.1 0.847 100.0
1.038 14.1
1.175 2.2
1.238 66.1
Cobalt-56 (56Co)
1.360 4.3
1.771 15.5
2.015 3.0
2.035 7.8
2.598 17.0
3.202 3.1
3.253 7.6
General Notices (1) apply to all monographs and other texts 667
5.7. Table of physical characteristics of radionuclides EUROPEAN PHARMACOPOEIA 8.0
0.692 0.15
β+ 0.201 (I)
14.9 γ 0.511 29.9 (II)
Cobalt-58 (58Co)
0.811 99.4
0.864 0.7
1.675 0.5
β+ 0.157 (I)
1 γ 0.511 112 (II)
(I)
0.331 0.7 0.834 5.9
(I)
0.397 3.8 1.039 37
(I)
0.782 0.3 1.333 1.2
(I)
1.90 50 1.919 2.1
2.190 5.6
Gallium-66 (66Ga)
2.423 1.9
2.752 23.4
3.229 1.5
3.381 1.5
3.792 1.1
4.086 1.3
4.295 4.1
4.807 1.8
0.300 16.8
0.394 4.7
0.888 0.15
Gallium-68 (68Ga)
β+ 0.353 (I)
1.2 γ 0.511 178.3
(I)
0.836 88.0 1.077 3.0
0.538 2.2
(81mKr: 13.10 (3) s)
50.53 (7) days β− 0.583 (I) (max: 1.492) 99.99 γ 0.909 0.01
Strontium-89 (89Sr)
in equilibrium with
Yttrium-89m (89mY)
(89mY: 16.06 (4) s)
28.74 (4) years β− 0.196 (I) (max: 0.546) 100
Strontium-90 (90Sr)
in equilibrium with
Yttrium-90 (90Y)
(90Y: 64.10 (8) hours)
64.10 (8) hours β− 0.934 (I) (max: 2.280) 100
Yttrium-90 (90Y)
(I)
65.94 (1) hours β− 0.133 16.4 X 0.018-0.021 3.6
(I)
0.290 1.1
0.443 (I)
82.4 γ 0.041 1.1
99
Molybdene-99 ( Mo) 0.141 4.5
in equilibrium with
Technetium-99m 0.181 6
(99mTc)
0.366 1.2
99m 0.740 12.1
( Tc: 6.01 (1) hours)
0.778 4.3
0.137-0.140 1.3
5 − (I)
2.11 × 10 years β 0.085 (max: 0.294) 100
Technetium-99 (99Tc)
General Notices (1) apply to all monographs and other texts 669
5.7. Table of physical characteristics of radionuclides EUROPEAN PHARMACOPOEIA 8.0
γ 0.642 25.9
0.885 92.9
0.938 68.4
0.997 10.5
0.658 97.8
2.129 2.1
0.023-0.026 82.3
ce 0.145 7.8
Indium-111 (111In)
0.167-0.171 1.3 γ 0.171 90.2
0.241-0.245 1.0
0.186-0.190 40
Indium-114m (114mIn)
in equilibrium with γ 0.190 15.6
Indium-114 (114In)
*β− 0.777 (I) (max: 1.985) 95 0.558 3.2
114 0.725 3.2
( In: 71.9 (1) s)
0.022-0.023 7.4
0.508 17.7
0.573 80.3
0.027-0.031 86.6
ce 0.127 13.6
0.440 0.4
0.505 0.3
0.529 1.4
0.538 0.4
γ 0.035 6.7
1.420 0.3
(I)
β+ 0.530 1
0.330 1.6
γ 0.080 2.6
(I)
Iodine-131 (131I) β− 0.069 2.1 0.284 6.1
(I)
0.097 7.3 0.365 81.7
(I)
0.192 89.9 0.637 7.2
0.723 1.8
0.030 44.0
0.159 28.5
General Notices (1) apply to all monographs and other texts 671
5.7. Table of physical characteristics of radionuclides EUROPEAN PHARMACOPOEIA 8.0
0.031 40.3
ce 0.045 55.1 0.035 9.4
Xenon-133 (133Xe)
0.075-0.080 9.9
γ 0.080 38.3
− (I)
β 0.101 99.0
0.030 45.9
Xenon-133m (133mXe)
(decays to radioactive ce 0.199 64.0 0.034 10.6
Xenon-133)
0.228 20.7
1.678 9.6
1.791 7.8
Xenon-135 (135Xe)
β− 0.171 3.1 γ 0.250 90.2
0.032-0.036 7
ce 0.624 8.0
Caesium-137 (137Cs)
in equilibrium with 0.656 1.4 γ 0.662 85.1
Barium-137m (137mBa)
(I)
β− 0.174 94.4
(I)
0.416 5.6
(137mBa: 2.552 (1) min)
26.1 (1) hours ce 0.285 3.4 X 0.010 32.0
0.08 17.5
+ (I)
β 0.495 0.3
γ 0.368 87.2
0.579 13.8
Thallium-200 (200Tl)
0.828 10.8
1.206 29.9
1.226 3.4
1.274 3.3
1.363 3.4
1.515 4.0
0.083 19
ce 0.246 8.5
0.276 2 γ 0.331 79
0.406 2.0
201
Lead-201 ( Pb) 0.585 3.6
(decays to radioactive
Thallium-201) 0.692 4.3
0.767 3.2
0.826 2.4
0.908 5.7
0.946 7.9
1.099 1.8
1.277 1.6
0,167 10.0
0.069-0.071 61.6
202
Thallium-202 ( Tl) ce 0.357 2.4 0.080 17.1
γ 0.440 91.4
0.071-0.073 69.6
ce 0.194 13.3 0.083 19.4
Lead-203 (203Pb)
γ 0.279 80.8
0.401 3.4
General Notices (1) apply to all monographs and other texts 673
EUROPEAN PHARMACOPOEIA 8.0 5.8. Pharmacopoeial harmonisation
between black diamonds (♦♦) in the corresponding Ph. Eur. the European Pharmacopoeia.
texts., while the local requirements are placed between white Methodologies of amino acid analysis : general principles
diamonds (◊◊). (USP). The USP has replaced ‘6-aminoquinolyl-N-
The 3 pharmacopoeias have undertaken not to make unilateral hydroxysuccinimidyl carbamate or o-phthalaldehyde’ with
changes to harmonised monographs and general chapters ‘6-aminoquinolyl-N-hydroxysuccinimidyl carbonate’.
but rather to apply the agreed revision procedure whereby all These reagents are different but compatible and the use of one
partners adopt a revision simultaneously. or the other does not affect harmonisation.
General Notices (1) apply to all monographs and other texts 677
5.8. Pharmacopoeial harmonisation EUROPEAN PHARMACOPOEIA 8.0
The USP has added a detailed example to describe each 2.6.12. MICROBIOLOGICAL EXAMINATION OF
method listed below : NON-STERILE PRODUCTS : MICROBIAL ENUMERATION
– Method 1 : post-column ninhydrin detection ; TESTS
– Method 2 : post-column OPA derivatisation ; As a result of an evaluation of the texts 4.05 Microbiological
Examination of Non-sterile Products : I. Microbiological
– Method 3 : pre-column PITC derivatisation ; Examination of Non-sterile Products – Microbial Enumeration
– Method 4 : pre-column AQC derivatisation ; Tests in the Japanese Pharmacopoeia XV 1st Supplement
– Method 5 : pre-column OPA derivatisation ; and <61> Microbiological Examination of Non-sterile
Products : Microbial Enumeration Tests in the United
– Method 6 : pre-column DABS-Cl derivatisation ; States Pharmacopeia USP30 NF25, and chapter 2.6.12.
– Method 7 : pre-column FMOC-Cl derivatisation ; Microbiological examination of non-sterile products : microbial
– Method 8 : pre-column NBD-F derivatisation. enumeration tests in the European Pharmacopoeia, the texts
of the 3 pharmacopoeias are considered harmonised.
The above examples are given for further information and
do not affect harmonisation. NOTE : ICH has declared this method interchangeable within
the ICH regions.
The texts of the 3 pharmacopoeias are therefore considered
harmonised. 2.6.13. MICROBIOLOGICAL EXAMINATION OF
NON-STERILE PRODUCTS : TEST FOR SPECIFIED
2.4.14. SULFATED ASH MICRO-ORGANISMS
The following comparative commentary refers to the texts 2.44 As a result of an evaluation of the texts 4.05 Microbiological
Residue on Ignition Test in the Japanese Pharmacopoeia XV Examination of Non-sterile Products : II. Microbiological
and <281> Residue on Ignition in the United States Examination of Non-sterile Products – Test for Specified
Pharmacopeia USP32 NF27 1st Supplement, and chapter Micro-organisms in the Japanese Pharmacopoeia XV 1st
2.4.14. Sulfated ash in the European Pharmacopoeia. Supplement and <62> Microbiological Examination of
The JP has added a non-harmonised introductory part, Non-sterile Products : Test for Specified Micro-organisms in the
included between black diamonds, at the beginning of this United States Pharmacopeia USP30 NF25, and chapter 2.6.13.
chapter. It is given for further information and therefore does Microbiological examination of non-sterile products : test for
not affect harmonisation. specified micro-organisms in the European Pharmacopoeia,
The USP text allows for the test to be performed at an the texts of the 3 pharmacopoeias are considered harmonised.
ignition temperature other than 600 ± 50 °C if prescribed in NOTE : ICH has declared this method interchangeable within
an individual monograph. In the same way, a sample mass the ICH regions.
different from the usual quantity of 1-2 g can be used if
prescribed in an individual monograph. 2.9.1. DISINTEGRATION OF TABLETS AND CAPSULES
The USP has added a section, included between black The following comparative commentary refers to the texts
diamonds, on the use of a muffle furnace and its calibration. 6.09 Disintegration Test in the Japanese Pharmacopoeia
XV and <701> Disintegration in the United States
The above differences in the USP text do not affect Pharmacopeia USP32 NF27 1st Supplement, and chapter 2.9.1.
harmonisation. Disintegration of tablets and capsules (Test A) in the European
The texts of the 3 pharmacopoeias are therefore considered Pharmacopoeia.
harmonised. In the Ph. Eur chapter, test A corresponds to the harmonised
NOTE : ICH has declared this method interchangeable within chapter while test B does not and is intended for tablets and
the ICH regions. capsules that are greater than 18 mm long. Test B is not within
the scope of pharmacopoeial harmonisation and has been
2.6.1. STERILITY placed between black diamonds (♦♦).
The following comparative commentary refers to the texts The JP and USP specify procedures and acceptance criteria for
4.06 Sterility Test in the partial revision of the Japanese different types of dosage forms. The equivalent statements are
Pharmacopoeia XV made official March 31, 2009, by included in the Ph. Eur. general monographs on dosage forms.
the Ministry of Health, Labour and Welfare Ministerial These statements are not within the scope of pharmacopoeial
Notification No. 190 and <71> Sterility Tests in the United harmonisation.
States Pharmacopeia as presented in Pharmacopeial Forum, In addition, the JP describes an auxiliary tube, and a metal
Volume 34(6), Interim Revision Announcement No. 6, plate to secure the glass tubes. This has been placed between
December 1, 2008, official on May 1, 2009, and chapter 2.6.1. black diamonds (♦♦). The use of this tube and this plate
Sterility in the European Pharmacopoeia.
may have an impact on hydrodynamics and thus may affect
The USP has added requirements that cover either pharmacy harmonisation.
bulk packages of antibiotics (which are not of concern in The texts of the 3 pharmacopoeias are therefore considered
Europe and Japan) or medical devices (which are outside the harmonised.
scope of the Ph. Eur. and JP). The corresponding parts, which
are not within the scope of pharmacopoeial harmonisation, NOTE : ICH has declared this method interchangeable within
have been placed between black diamonds ( ♦). ♦ the ICH regions subject to the conditions detailed below.
The JP has deleted the requirements for ‘Catgut and other For tablets and capsules larger than 18 mm long, for which
surgical sutures for veterinary use’ in Table 2, in the section a different apparatus is used, the disintegration test is not
‘Direct inoculation of the culture medium’ and in Table 3. considered to be interchangeable in the 3 regions.
Catgut and other surgical sutures are outside the scope of The test for disintegration is not considered to be
the JP. interchangeable in the 3 regions for dosage forms referred to
The above differences in the JP and USP texts do not affect in the pharmacopoeias as delayed-release, gastro-resistant or
harmonisation. enteric-coated.
The texts of the 3 pharmacopoeias are therefore considered 2.9.7. FRIABILITY OF UNCOATED TABLETS
harmonised. As a result of an evaluation of the texts 26. Tablet Friability
NOTE : ICH has declared this method interchangeable within Test in the Japanese Pharmacopoeia XV and <1216> Tablet
the ICH regions. Friability in the United States Pharmacopeia USP31 NF26
1st Supplement, and chapter 2.9.7. Friability of uncoated Multi-point measurement (JP). The JP does not state the
tablets in the European Pharmacopoeia, the texts of the meaning of the 22400 constant in the definition of the specific
3 pharmacopoeias are considered harmonised. surface area S and does not require a test to determine the
linearity of the method.
2.9.17. TEST FOR EXTRACTABLE VOLUME OF Single-point measurement (JP). The JP does not state
PARENTERAL PREPARATIONS the equivalent quantity of gas corresponding to the value
The following comparative commentary refers to the texts of P/P0, which is less precise (0.30) than in the other
6.05 Test for Extractable Volume of Parenteral Preparations in pharmacopoeias (0.300).
the Japanese Pharmacopoeia XV and <1> Injections in the The JP does not assume the material constant C to be invariant.
United States Pharmacopeia USP32 NF27 1st Supplement,
and chapter 2.9.17. Test for extractable volume of parenteral Measurements (JP). The JP does not specify the temperature
preparations in the European Pharmacopoeia. required to perform the test for either method.
The JP has added a non-harmonised introductory part, The JP limits its volumetric method to classical instruments
included between black diamonds, at the beginning of this and does not take alternative instruments into account.
chapter. It is given for further information and does not affect The above differences in the JP text might affect harmonisation.
harmonisation. Therefore only the texts of the Ph. Eur. and the USP are
The USP has included this test in general chapter <1> considered harmonised.
Injections, under a specific part entitled Determination of
Volume of Injection in Containers. This does not affect 2.9.36. POWDER FLOW
harmonisation. The following comparative commentary refers to the texts
18. Powder Flow in the Japanese Pharmacopoeia XV and
The texts of the 3 pharmacopoeias are therefore considered <1174> Powder Flow in the United States Pharmacopeia
harmonised. USP31 NF26 1st Supplement, and chapter 2.9.36. Powder flow
NOTE : ICH has declared this method interchangeable within in the European Pharmacopoeia.
the ICH regions. Flow through an orifice (JP). The JP limits the use of orifices
2.9.19. PARTICULATE CONTAMINATION : SUB-VISIBLE to classical ones and does not allow vibrators or moving
PARTICLES orifices. A test result using the JP method will be compatible
with the Ph. Eur and the USP. A Ph. Eur. or USP test result
The following comparative commentary refers to the texts 6.07 will not comply with the JP when a vibrator or moving orifice
Insoluble Particulate Matter Test for Injections in the Japanese is used.
Pharmacopoeia XV (corrected version dated September 2007)
and <788> Particulate Matter in Injections in the United States 2.9.37. OPTICAL MICROSCOPY
Pharmacopeia USP32 NF27 2nd Supplement, and chapter As a result of an evaluation of the texts 3.04 Particle
2.9.19. Particulate contamination : sub-visible particles in the Size Determination in the Japanese Pharmacopoeia XV
European Pharmacopoeia. and <776> Optical Microscopy in the United States
The USP specifies that system suitability can be verified using Pharmacopeia USP31 NF26 2nd Supplement, and chapter
USP Particle Count RS. This statement is not within the scope 2.9.37. Optical microscopy in the European Pharmacopoeia,
of pharmacopoeial harmonisation. It has been placed between the texts of the 3 pharmacopoeias are considered harmonised.
black diamonds (♦♦) and it does not affect harmonisation.
2.9.38. PARTICLE-SIZE DISTRIBUTION ESTIMATION BY
The JP includes a detailed section on calibration of the ANALYTICAL SIEVING
apparatus. In particular, requirements for the quality of
The following comparative commentary refers to the
particle-free water are given, which differ from those stated in
texts 3.04 Particle Size Determination in the Japanese
the USP (see section Reagents, Indicators and Solutions) and
Pharmacopoeia XV and <786> Particle-size Distribution
in the Ph. Eur (see chapter 4.1.1). The section on calibration
Determination by Analytical Sieving in the United States
is not within the scope of pharmacopoeial harmonisation. It
♦ Pharmacopeia USP31 NF26 1st Supplement, and chapter
has been placed between black diamonds ( ♦) and it does not
2.9.38. Particle-size distribution estimation by analytical
affect harmonisation.
sieving in the European Pharmacopoeia.
In addition, the JP describes more stringent acceptance
criteria for parenteral preparations having a nominal Sieving methods - Dry sieving method (JP). The JP permits
any powder on the down surface of the sieve to be brushed
volume of 100 mL. This was acknowledged by the PDG as
a non-harmonised item. It has been placed between black and combined with the fraction of the next sieve.
diamonds (♦♦). The acceptance criteria for parenteral The above difference in the JP text might affect harmonisation.
preparations having a nominal volume of 100 mL are therefore Therefore only the texts of the Ph. Eur. and the USP are
considered non-harmonised. considered harmonised.
The texts of the 3 pharmacopoeias are therefore considered 5.1.4. MICROBIOLOGICAL QUALITY OF NON-STERILE
harmonised except for the acceptance criteria for parenteral PHARMACEUTICAL PREPARATIONS AND SUBSTANCES
preparations having a nominal volume of 100 mL. FOR PHARMACEUTICAL USE
NOTE : ICH has declared this method interchangeable within The following comparative commentary refers to the texts 12.
the ICH regions except the acceptance criteria for parenteral Microbial Attributes of Non-sterile Pharmaceutical Products in
preparations having a nominal volume of 100 mL. the Japanese Pharmacopoeia XV 1st Supplement and <1111>
Microbiological Attributes of Non-sterile Pharmaceutical
2.9.26. SPECIFIC SURFACE AREA BY GAS ADSORPTION Products in the United States Pharmacopeia USP30 NF25,
The following comparative commentary refers to the texts and chapter 5.1.4. Microbiological quality of non-sterile
3.02 Specific Surface Area by Gas Adsorption in the Japanese pharmaceutical preparations and substances for pharmaceutical
Pharmacopoeia XV and <846> Specific Surface Area in the use in the European Pharmacopoeia.
United States Pharmacopeia USP31 NF26 1st Supplement, and A special Ph. Eur. provision for oral dosage forms containing
chapter 2.9.26. Specific surface area by gas adsorption in the raw materials of natural origin is included within table 5.1.4.-1.
European Pharmacopoeia. Also, a reference to chapter 5.1.8 giving recommended
The JP has chosen to express all the temperatures of this acceptance criteria for the microbiological quality of herbal
chapter in degrees Celsius. medicinal products for oral use and extracts used in their
General Notices (1) apply to all monographs and other texts 679
5.8. Pharmacopoeial harmonisation EUROPEAN PHARMACOPOEIA 8.0
Loss on drying + + + Each pharmacopoeia adapts the text to take account of local
reference materials and reagent specifications.
Microbial contamination + - +
Labelling + - +
POTATO STARCH (0355)
Legend As a result of the process of pharmacopoeial harmonisation,
+ : will adopt and implement the following reflects the agreement settled by the Ph. Eur.,
the JP and the USP.
– : will not stipulate
Non-harmonised attributes Harmonised attributes
Description/Characters, Heavy metals, Container/Packaging
and storage Attribute Ph. Eur. JP USP
Local requirements + + +
Definition
Second identification (specific optical rotation, melting point,
TLC) (Ph. Eur.), Absence of Salmonella (Ph. Eur.) Identification
+ + +
Reagents and reference materials – A
+ + +
– B
Each pharmacopoeia adapts the text to take account of local + + +
– C
reference materials and reagent specifications.
(sign-off date : 6 June 2012) pH + + +
Definition + + + + - +
Microbial contamination
Labelling + + +
Legend
Identification
– A + + + + : will adopt and implement
– B + + +
– C + + + – : will not stipulate
– D + + +
+ + + Non-harmonised attributes
– E
Viscosity Characters, Storage
– Method 1 + + +
+ + +
Local requirements
– Method 2
pH + + + Foreign matter (Ph. Eur.), Absence of Salmonella (Ph. Eur.)
Heavy metals + + + Reagents and reference materials
Loss on drying + + +
Each pharmacopoeia adapts the text to take account of local
Sulfated ash + + + reference materials and reagent specifications.
Assay + + + (sign-off date : 15 June 2011)
General Notices (1) apply to all monographs and other texts 681
5.8. Pharmacopoeial harmonisation EUROPEAN PHARMACOPOEIA 8.0
pH + + +
Iron + + +
Total protein + + +
Oxidising substances + + +
Sulfur dioxide + + +
Loss on drying + + +
Sulfated ash + + +
Microbial contamination + - +
Legend
+ : will adopt and implement
− : will not stipulate
General Notices (1) apply to all monographs and other texts 685
EUROPEAN PHARMACOPOEIA 8.0 5.10. Impurities in substances for pharmaceutical use
General Notices (1) apply to all monographs and other texts 689
5.10. Impurities in substances for pharmaceutical use EUROPEAN PHARMACOPOEIA 8.0
pharmaceutical use (2034)) is prescribed, this is valid only threshold. Pending editorial adaptation of already published
for specified impurities mentioned in the Impurities section. monographs using unequivocal terminology, the decision tree
The need for identification (wherever possible), reporting, (Figure 5.10.-1) may be used to determine the acceptance
specification and qualification of other impurities that occur criterion to be applied.
must be considered according to the requirements of the Recommendations to users of monographs of active
general monograph. It is the responsibility of the user of the substances
substance to determine the validity of the acceptance criteria
Monographs give a specification for suitable quality of
for impurities not mentioned in the Impurities section and for
substances with impurity profiles corresponding to those
those indicated as other detectable impurities.
taken into account during elaboration and/or revision of the
Acceptance criteria for the related substances test are presented monograph. It is the responsibility of the user of the substance
in different ways in existing monographs ; the decision tree to check that the monograph provides adequate control of
(Figure 5.10.-1) may be used as an aid in the interpretation impurities for a substance for pharmaceutical use from a given
of general acceptance criteria and their relation with the source, notably by using the procedure for certification of
Impurities section of the monograph. suitability of the monographs of the European Pharmacopoeia.
General acceptance criteria for “other” impurities are A monograph with a related substances test based on a
expressed in various ways in the monographs : “any other quantitative method (such as liquid chromatography, gas
impurity”, “other impurities”, “any impurity”, “any spot”, “any chromatography and capillary electrophoresis) provides
band”, etc. The general acceptance criteria may apply to adequate control of impurities for a substance from a given
certain specified impurities only or to unspecified impurities source if impurities present in amounts above the applicable
and certain specified impurities, depending on the nature identification threshold are specified impurities mentioned in
of the active substance and the applicable identification the Impurities section.
* The requirements of this section apply to active substances, with the exception of : biological and biotechnological products; oligonucleotides ;
radiopharmaceuticals; products of fermentation and semi-synthetic products derived therefrom ; crude products of animal or plant origin;
herbal products.
** To apply the Related substances section of the monograph Substances for pharmaceutical use (2034):
– an individual acceptance criterion must be defined for any impurity that may be present above the identification threshold ;
– any impurity with an acceptance criterion above the identification threshold must wherever possible be identified;
– any impurity with an acceptance criterion above the qualification threshold must be qualified.
Figure 5.10.-1. – Decision tree for interpretation of general acceptance criteria for ‘other’ impurities in monographs
If the substance contains impurities other than those Information is available via the EDQM website on commercial
mentioned in the Impurities section, it has to be verified names for columns and other reagents and equipment found
that these impurities are detectable by the method described suitable during monograph development, where this is
in the monograph, otherwise a new method must be considered useful.
developed and revision of the monograph must be requested.
Depending on the contents found and the limits proposed, GLOSSARY
the identification and/or the qualification of these impurities Disregard limit : in chromatographic tests, the nominal
must be considered. content at or below which peaks/signals are not taken into
account for calculating a sum of impurities. The numerical
Where a single related substances test covers different impurity values for the disregard limit and the reporting threshold are
profiles, only impurities for the known profile from a single usually the same.
source need to be reported in the certificate of analysis unless Identification threshold : a limit above which an impurity
the marketing authorisation holder uses active substances is to be identified.
with different impurity profiles.
Identified impurity : an impurity for which structural
Identification of impurities (peak assignment) characterisation has been achieved.
Where a monograph has an individual limit for an impurity, Impurity : any component of a substance for pharmaceutical
it is often necessary to define means of identification, use that is not the chemical entity defined as the substance.
for example using a reference substance, a representative Nominal concentration : concentration calculated on the
chromatogram or relative retention. The user of the substance basis of the concentration of the prescribed reference and
may find it necessary to identify impurities other than those taking account of the prescribed correction factor.
for which the monograph provides a means of identification, Other detectable impurities : potential impurities with a
for example to check the suitability of the specification for defined structure that are known to be detected by the tests in
a given impurity profile by comparison with the Impurities a monograph but not known to be normally present above
section. The European Pharmacopoeia does not provide the identification threshold in substances used in medicinal
reference substances, representative chromatograms or products that have been authorised by the competent
information on relative retentions for this purpose, unless authorities of Parties to the Convention. They are unspecified
prescribed in the monograph. Users will therefore have to impurities and are thus limited by a general acceptance
apply the available scientific techniques for identification. criterion.
New impurities/Specified impurities above the specified Potential impurity : an impurity that theoretically can arise
limit during manufacture or storage. It may or may not actually
appear in the substance. Where a potential impurity is known
Where a new manufacturing process or change in an to be detected by the tests in a monograph but not known to
established process leads to the occurrence of a new be normally present in substances used in medicinal products
impurity, it is necessary to apply the provisions of the general that have been authorised by the competent authorities of
monograph on Substances for pharmaceutical use (2034) Parties to the Convention, it will be included in the Impurities
regarding identification and qualification and to verify the section under Other detectable impurities for information.
suitability of the monograph for control of the impurity.
A certificate of suitability is a means for confirming for Qualification : the process of acquiring and evaluating data
a substance from a given source that the new impurity is that establishes the biological safety of an individual impurity
adequately controlled or the certificate contains a method for or a given impurity profile at the level(s) specified.
control with a defined acceptance criterion. In the latter case Qualification threshold : a limit above which an impurity
revision of the monograph will be initiated. is to be qualified.
Related substances : title used in monographs for general
Where a new manufacturing process or change in an tests for organic impurities.
established process leads to the occurrence of a specified
impurity above the specified limit, it is necessary to apply Reporting threshold : a limit above which an impurity is to
the provisions of the general monograph on Substances for be reported. Synonym : reporting level.
pharmaceutical use (2034) regarding qualification. Specified impurity : an impurity that is individually listed and
Expression of acceptance criteria limited with a specific acceptance criterion in a monograph. A
specified impurity can be either identified or unidentified.
The acceptance criteria for related substances are expressed Unidentified impurity : an impurity for which a structural
in monographs either in terms of comparison of peak areas characterisation has not been achieved and that is defined
(comparative tests) or as numerical values. solely by qualitative analytical properties (for example, relative
Chromatographic methods retention).
Unspecified impurity : an impurity that is limited by a general
General chapter 2.2.46. Chromatographic separation acceptance criterion and not individually listed with its own
techniques deals with various aspects of impurities control. specific acceptance criterion.
General Notices (1) apply to all monographs and other texts 691
EUROPEAN PHARMACOPOEIA 8.0 5.11. Characters section in monographs
01/2008:51100 CRYSTALLINITY
This method is employed to establish the crystalline or
amorphous nature of a substance.
5.11. CHARACTERS SECTION IN Mount a few particles of the substance to be examined in
MONOGRAPHS mineral oil on a clean glass slide. Examine under a polarising
microscope. Crystalline particles exhibit birefringence and
The General Notices indicate that the statements included in extinction positions when the microscope stage is revolved.
the Characters section are not to be interpreted in a strict SOLUBILITY
sense and are not requirements. For information of users, the
methods recommended to authors of monographs as the basis For this test a maximum of 111 mg of substance (for each
for statements concerning hygroscopicity, crystallinity and solvent) and a maximum of 30 mL of each solvent are
solubility are given below. necessary.
Dissolving procedure
Shake vigorously for 1 min and place in a constant temperature
HYGROSCOPICITY
device, maintained at a temperature of 25.0 ± 0.5 °C for
This method is to be carried out on a substance that complies 15 min. If the substance is not completely dissolved, repeat
with the test for loss on drying or water content of the the shaking for 1 min and place the tube in the constant
monograph. The method gives an indication of the degree of temperature device for 15 min.
hygroscopicity rather than a true determination. Method
Use a glass weighing vessel 50 mm in external diameter and Weigh 100 mg of finely powdered substance (90) (2.9.12) in
15 mm high. Weigh the vessel and stopper (m1). Place the a stoppered tube (16 mm in internal diameter and 160 mm
amount of substance prescribed for the test for loss on drying long), add 0.1 mL of the solvent and proceed as described
or water in the vessel and weigh (m2). Place the unstoppered under Dissolving Procedure. If the substance is completely
vessel in a desiccator at 25 °C containing a saturated solution dissolved, it is very soluble.
of ammonium chloride or ammonium sulfate or place it in a If the substance is not completely dissolved, add 0.9 mL of the
climatic cabinet set at 25 ± 1 °C and 80 ± 2 per cent relative solvent and proceed as described under Dissolving Procedure.
humidity. Allow to stand for 24 h. Stopper the weighing vessel If the substance is completely dissolved, it is freely soluble.
and weigh (m3). If the substance is not completely dissolved, add 2.0 mL of the
Calculate the percentage increase in mass using the expression : solvent and proceed as described under Dissolving Procedure.
If the substance is completely dissolved, it is soluble.
If the substance is not completely dissolved, add 7.0 mL of the
solvent and proceed as described under Dissolving Procedure.
If the substance is completely dissolved, it is sparingly soluble.
The result is interpreted as follows : If the substance is not completely dissolved, weigh 10 mg
– deliquescent : sufficient water is absorbed to form a liquid, of finely powdered substance (90) (2.9.12) in a stoppered
tube, add 10.0 mL of the solvent and proceed as described
– very hygroscopic : increase in mass is equal to or greater under Dissolving Procedure. If the substance is completely
than 15 per cent, dissolved, it is slightly soluble.
– hygroscopic : increase in mass is less than 15 per cent and If the substance is not completely dissolved, weigh 1 mg
equal to or greater than 2 per cent, of finely powdered substance (90) (2.9.12) in a stoppered
tube, add 10.0 mL of the solvent and proceed as described
– slightly hygroscopic : increase in mass is less than 2 per cent under Dissolving Procedure. If the substance is completely
and equal to or greater than 0.2 per cent. dissolved, it is very slightly soluble.
General Notices (1) apply to all monographs and other texts 695
EUROPEAN PHARMACOPOEIA 8.0 5.12. Reference standards
General Notices (1) apply to all monographs and other texts 699
5.12. Reference standards EUROPEAN PHARMACOPOEIA 8.0
Secondary standards. A secondary standard may be used for For chemical reference substances, relevant parts of the
routine quality control purposes for any of the uses described following programme are typically applied.
above for primary standards, provided that it is established 4-2-1. Identification. In general, a batch selected from the
with reference to the primary standard. A secondary standard normal production of the substance is satisfactory. It is shown
is established and employed to reduce the use of the primary to comply with the requirements of the monograph ; full
standard, which requires more extensive characterisation and structural elucidation is carried out for the first batch.
evaluation and may be available only in a limited quantity.
A secondary standard is used only for the same purpose as 4-2-2. Related substances test. A reference standard
the primary standard with reference to which it has been corresponding to an impurity is characterised for identity
established. and purity. Where a reference standard is used to determine
the content of a given impurity, the preferred minimum
4. ESTABLISHMENT OF REFERENCE STANDARDS content is 95.0 per cent ; where this is achieved no assigned
4-1. PRIMARY STANDARD value is given, the content being considered as 100.0 per
cent ; this approximation is acceptable since there will be no
A substance or preparation to be established as a primary appreciable effect on the determination of impurities. When
standard is characterised by a variety of analytical techniques this minimum content cannot be obtained, the standard has
chosen to demonstrate its suitability for use. an assigned content.
For substances for pharmaceutical use and their impurities, If an impurity is not available in a sufficient quantity to
relevant parts of the following test programme are usually establish a reference standard, a number of other options exist :
applied.
– preparation of a reference standard that contains a mixture
– Characterisation of the substance (structural elucidation) of the compound(s) and the impurity or impurities ;
by appropriate chemical attributes such as structural
formula, empirical formula and molecular weight. A – preparation of a reference standard containing a mixture of
number of techniques may be used including: specified impurities.
– nuclear magnetic resonance spectrometry ; Where such a mixture is also used to determine the content of
a given impurity, the content of the impurity in the reference
– mass spectrometry ; standard is determined by appropriate separation methods
– infrared spectrophotometry ; and a value assigned to the reference standard.
– elemental analysis. 4-2-3. Assay
– Determination of the purity : 4-2-3-1. Chemical assay. When a reference standard is to be
– determination of the content of organic impurities by used for quantitative determination of an active substance
an appropriate separation technique or spectrometric or an excipient (assay standard), the extent of testing is
method, where applicable ; greater. In general, several collaborating laboratories examine
– quantitative determination of water ; the proposed substance, following a detailed protocol that
describes the procedures to be followed. The results obtained
– determination of the content of residual solvents ; are used to assign a content. It is particularly important to
– determination of loss on drying, which may in certain quantify the impurities if a selective assay is employed. In
circumstances replace the determinations of water and such a case, it is best to examine the proposed substance by
residual solvents ; additional analytical procedures that are scientifically justified,
– determination of inorganic impurities (test for heavy including, where possible, absolute methods.
metals, sulfated ash, atomic spectrometry, inductively If a reference standard is required for a non-chromatographic
coupled plasma spectrometry, X-ray fluorescence) ; the assay method (e.g. colorimetry or ultraviolet
results are not used in determining an assigned content, spectrophotometry), the relative reactivity or relative
except where they would have an appreciable impact absorbance of the impurities present in a substance must be
upon it ; checked to ensure that they are not markedly different from
– determination of the purity by an absolute method those of the substance.
(e.g. differential scanning calorimetry or phase A protocol is prepared and must be strictly followed by the
solubility analysis where appropriate ; the results of these participants of the collaborative trial to assign the content.
determinations are used to support and confirm the The protocol usually requires :
results obtained from separation techniques ; they are – determination of water (or loss on drying) ;
not used in the calculation of the assigned value). – estimation of the organic impurities (including residual
For a primary chemical reference substance to be established solvents when appropriate) using the prescribed separation
for assay purposes, the assigned content is generally calculated techniques ;
from the values obtained from the analyses performed for the – and possibly, determination of the content of the substance
determination of impurities (organic, inorganic, water and by an absolute method ; this would be a confirmatory
solvents) by applying the principle of mass balance ; other determination not necessarily performed by all participants
suitable methods are also used. and the results would not be used in the calculation of the
An establishment report for the reference standard is prepared assigned value.
and approved by the qualified person. The protocol also indicates the system suitability tests and
4-2. EUROPEAN PHARMACOPOEIA REFERENCE acceptance criteria for each of the tests performed.
STANDARDS Unless otherwise stated, an assigned value is given for the
The candidate standards are tested against a wide variety of substance or preparation as presented in the container (‘as
analytical methods. The extent of testing and the number is’), and the contents are not to be dried before use. For assay
of laboratories involved depends on the use of the reference standards prepared by lyophilisation the content of the pure
standard. Compliance with the relevant monograph is usually substance is indicated in milligrams or International Units
required, unless otherwise justified. per vial.
Where a collaborative trial is carried out during establishment, 4-2-3-2. Microbiological assay. A reference standard for
a protocol is provided for each participant and only valid the microbiological assay is first shown to comply with the
results derived according to the protocol are used for monograph. If the results are satisfactory a collaborative
establishing an assigned value or otherwise confirming microbiological assay is carried out, using the international
suitability. standard. The potency is expressed in International Units.
If an international standard does not exist, European 5. PRODUCTION, LABELLING, STORAGE AND
Pharmacopoeia Units are used. The assigned potency is DISTRIBUTION
calculated from the results of a collaborative trial. Various 5-1. PRODUCTION
validity criteria are applied including parallelism, linearity, All operations are carried out according to the relevant
and quadratic fit, according to the usual statistical procedures norms of best practice to ensure the traceability and integrity
(5.3). The assigned potency with the confidence limits is of the reference standard. The production record includes
calculated from statistically valid results. information regarding filling, labelling and storage. Reference
4-2-3-3. Assay of components of herbal drugs and herbal drug standards are dispensed into containers under appropriate
preparations. Reference standards used in monographs of filling and closure conditions, to ensure the integrity of
herbal drugs vary in the extent of testing depending on the the reference standard. The containers employed may be
type of reference standard. multi-use or single use, but the latter is preferred to minimise
the risk of decomposition, contamination, or water uptake.
– An active component or marker constituent is characterised 5-2. LABELLING
and evaluated for identity and purity ; a value for content The labelling bears the name of the reference standard,
is assigned irrespective of the purity. the name of the supplier, the batch number, and any other
information necessary to the proper use of the reference
– An extract is used as a reference standard when insufficient standard. If used as an assay standard the following
active principle or marker constituent is available. The information is also given:
assigned content of the extract is established by means of a
– the assigned percentage content ;
collaborative trial using a well-characterised sample of the
active principle or marker component for which a value – or, the content in milligrams or millilitres of the chemical
is to be assigned. entity in the container ;
– or, the assigned potency (for biological assays or
4-2-4. Establishment report. A report containing the results
microbiological assays) in units either per milligram or per
of the establishment study as well as information concerning
vial.
the use of the reference standard is prepared. The report
for a chemical assay standard has a value assigned to the For a manufacturer’s reference standard, the label indicates a
substance with the rationale for attributing that value. The re-test or expiry date. For European Pharmacopoeia reference
estimated uncertainty of the assigned value is calculated, and standards, no re-test or expiry date is given since the re-test
where it is less than a predefined value, which is considered programme (see below) monitors continued fitness for use.
to be negligible in relation to the acceptance criteria for the Leaflets. An accompanying explanatory leaflet may also
assay, then the study is accepted. Otherwise, the trial may be be provided giving information needed for correct use of
repeated, in whole or in part, or the limits defined for the the reference standard. An explanatory leaflet is considered
pharmaceutical substance may be widened. The uncertainty as part of the labelling. Where stated in a monograph, a
of the assigned value is not given as part of the information chromatogram is included in the leaflet.
provided with the reference standard, since the precision of 5-3. STORAGE AND DISTRIBUTION
the method and the uncertainty of the value attributed to the
reference standard are taken into account when setting the Reference standards are to be stored and distributed in
limit(s) in a monograph. conditions suitable to ensure optimal stability.
European Pharmacopoeia reference standards. European
4-3. SECONDARY STANDARD Pharmacopoeia reference standards are mostly stored in
A secondary standard should exhibit the same property or temperature-controlled rooms at 5 ± 3 °C. However, a number
properties as the primary standard, relevant for the test(s) for of reference standards that are relatively unstable are stored
which it is established. The extent of testing is not so great at − 20 ± 5 °C or, in a few cases (e.g. live virus preparations),
as is required for the establishment of a primary standard. at − 80 ± 10 °C, and for cell cultures, under liquid nitrogen
The secondary standard is established by comparison with (− 180 °C).
the primary standard to which it is traceable. An official
Special packaging is employed to minimise the risk of damage
primary standard is used wherever possible for establishment
of secondary standards. during transport.
Reference standards that are normally stored at 5 ± 3 °C are
Identification dispatched by normal mail since short excursions from the
long-term storage temperature are not deleterious to the
– For use in infrared spectrophotometry : the absorbance reference standard. Reference standards stored at − 20 °C are
bands correspond in position and relative size to the packed on ice and dispatched by express courier. Reference
absorbance bands of the primary standard. standards stored at − 80 °C or stored under liquid nitrogen
are packed on solid carbon dioxide and dispatched by express
– For use in separation techniques : the migration distance,
courier.
migration time and retention time of the secondary
standard are the same as those of the primary standard for 6. RE-TEST PROGRAMME
thin-layer chromatography or electrophoresis, capillary
A system is established and implemented to ensure the
electrophoresis and gas or liquid chromatography
continued fitness-for-use of the reference standards. Normally,
respectively.
a re-test programme is applied, taking account of the
Purity test. For use in separation techniques : as for known physico-chemical properties and stability data for
identification but when used for quantification, a content the reference standard. Reference standards are periodically
relative to the signal from the primary standard is to be tested for stability during storage. A monitoring programme
established. is applied that is designed to detect at an early stage any sign
of decomposition using appropriate analytical techniques.
Assay. Secondary standards are assayed against a primary
The methods employed should be chosen from amongst those
standard with an assigned content or potency. The property
performed during establishment so that baseline data are
for which a value is to be assigned for the secondary standard
available.
is similar in magnitude to that of the primary reference
standard with which it is compared. Both the number of The periodicity and extent of re-testing reference standards
independent replicate determinations to be performed and the depends on a number of factors including:
acceptance criteria to be applied are predefined. – stability ;
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MCBs and WCBs are qualified by testing an aliquot of the ADENOVIRUS VECTORS
banked material or testing a subculture of the cell bank. FOR HUMAN USE
The following table indicates the tests required at each stage
of production.
DEFINITION
Assay Host MCB WCB EOPCs* Adenovirus vectors for human use are freeze-dried or liquid
strain
preparations of recombinant adenoviruses, genetically
Identity and purity
modified to transfer genetic material to human somatic cells
Viability + + + + in vivo or ex vivo.
Bacterial strain characterisation + + – +
+ + – +
PRODUCTION
Genotyping / phenotyping
VECTOR CONSTRUCTION
Presence of the plasmid
There are different approaches for the design and construction
– Sequence of the DNA plasmid – + – + of an adenovirus vector. The purpose of clinical use
– + + + determines which approach is optimal. A method is chosen
– Copy number
that minimises the risk of generating replication-competent
– Restriction map – + + + adenovirus vectors or that effectively eliminates helper viruses
– + + + that might be used during production.
– Percentage of cells retaining
the plasmid VECTOR PRODUCTION
Adventitious agents The production method shall have been shown to yield
Purity by plating + + + + a vector of consistent quality. Unless otherwise justified
and authorised, the vector in the final product shall have
Presence of bacteriophage + + – + undergone no more passages from the master seed lot than
were used to prepare the vector shown in clinical trials to be
* EOPCs are cells with a passage number at least equivalent to that used
for production. The analysis has to be done once to validate each new satisfactory with respect to safety and efficacy.
WCB, except for purity, which has to be tested for each fermentation.
The genetic and phenotypic stability of the vector at or beyond
the maximum passage level used for production is assessed by
IDENTITY AND PURITY TESTING suitable methods.
Viability. The number of viable cells is determined by plating SUBSTRATE FOR VECTOR PROPAGATION
a diluted aliquot of bacterial cells on an appropriate medium The vector is propagated in continuous cell lines
and counting individual colonies. (5.2.3) based on a cell bank system. The occurrence of
Biochemical and physiological bacterial strain replication-competent adenoviruses may be significant when
characterisation. Depending on the bacterial strain used large regions of homology exist between the viral genome and
for production, relevant biochemical and physiological the genome of the complementation cells. This occurrence
characterisation is performed to confirm cell identity at the may be minimised by minimising the homology between both
species level. genomes. The use of cells with no sequence homology with
the vector is recommended for production.
Genotyping / phenotyping. The genotype of bacterial cells is
verified by determination of the suitable specific phenotypic VECTOR SEED LOT
markers or by appropriate genetic analysis. Production of the vector is based on a seed-lot system.
Presence of the plasmid The strain of adenovirus used is identified by historical records
Sequencing. The whole nucleotide sequence of the plasmid that include information on its origin and its subsequent
is verified. manipulation, notably deleted or modified regions. A detailed
description of the genetic insert(s) and the flanking control
Copy number. The plasmid DNA is isolated and purified from regions is established, including the nucleotide sequence. The
a known number of bacteria and the copy number determined method by which the genetic insert is introduced into the
by a suitable method such as quantitative PCR (2.6.21). vector is documented.
Restriction map. Restriction endonuclease digestion is Only a seed lot that complies with the following requirements
performed with sufficient resolution to verify that the may be used for vector production.
structure of the plasmid is unaltered in bacterial cells.
Identification. The vector is identified in the master seed
Percentage of cells retaining the plasmid. Bacterial elements lot and each working seed lot by immunochemical methods
present in the plasmid, such as selectable genetic markers, are (2.7.1), NAT (2.6.21) or restriction enzyme analysis.
used to define the percentage of bacteria retaining the plasmid.
Genetic and phenotypic characterisation. The following
ADVENTITIOUS AGENTS AND ENDOGENOUS VIRUSES tests are carried out.
Purity by plating. Bacterial cells are streaked out onto – The entire genome of the vector is sequenced at a passage
suitable media and incubated in the required conditions in level comparable to a production batch and the analytically
order to detect potential bacterial contaminants. In order to determined sequence is compared to the theoretical
test for inhibition of the growth of contaminating organisms, sequence based on vector construction and available
additional tests in the presence of a definite amount of relevant databases.
positive control bacteria are carried out. A suitable number of – Restriction enzyme analysis is performed on the vector
colonies is examined ; no contamination is detected. DNA of the master seed lot, each working seed lot and
Presence of bacteriophage. Bacterial cells are plated a production batch. The viral DNA is extracted, purified
and incubated in a medium allowing proliferation of and digested with sufficient resolution. The digested
bacteriophages, to test for bacteriophage presence. The test is fragments are separated by gel electrophoresis or capillary
validated by the use of a reference bacteriophage strain and electrophoresis and the observed restriction pattern is
permissive cells as positive controls. A suitable number of compared to the theoretical restriction pattern based on
colonies is examined ; no contamination is detected. vector construction.
– A suitable number of isolated sub-clones are tested for Vector concentration. The titre of infectious vector and
expression of the genetic insert product(s) and biological the concentration of vector particles in purified harvests are
activity at a passage level comparable to a production batch. determined.
Sub-clones giving lower levels of expression or biological Residual host-cell protein. The concentration of residual
activity need further characterisation. host-cell protein is determined by a suitable immunochemical
Vector concentration. The titre of infectious vector or the method (2.7.1), unless the process has been validated to
concentration of vector particles in the master seed lot and demonstrate suitable clearance.
each working seed lot are determined. Residual host-cell DNA. The content of residual host-cell
Extraneous agents (2.6.16). The master seed lot and each DNA is determined using a suitable method, unless
working seed lot comply with the tests for extraneous agents. the process has been validated to demonstrate suitable
Replication-competent adenoviruses. Replication- clearance. Quantitative polymerase chain reaction (PCR) is
competent adenoviruses are generated by homologous recommended for its sensitivity and specificity, but other
recombination between the recombinant viral DNA and suitable techniques may also be used.
the adenovirus sequences integrated into the genome of the Residual reagents. Where reagents are used during the
complementation cells. production process, tests for these substances are carried out
Detection of replication-competent adenoviruses is performed on the purified harvest, unless the process has been validated
by a suitable method approved by the competent authority. to demonstrate suitable clearance.
It is generally performed by an infectivity assay on sensitive Residual antibiotics. Where antibiotics are used during
detector cell lines, which are not able to complement for the production process, their residual concentration is
the genes deleted from the vector. Other indicators of viral determined by a microbiological assay (adapted from general
replication may be used as appropriate. method 2.7.2) or by other suitable methods (for example,
When replication-competent adenoviruses are not supposed to liquid chromatography), unless the process has been validated
be present in the test sample, considering vector construction to demonstrate suitable clearance.
and cell lines used, at least 2, but preferably 3 or 4 successive FINAL BULK
passages are performed on the detector cell line, where Several purified harvests may be pooled during preparation of
applicable. Detection of a cytopathic effect at the end of the final bulk. A stabiliser and other excipients may be added.
the passages reveals the presence of replication-competent The formulated product is filtered through a bacteria-retentive
adenoviruses in the preparation. Positive controls are included filter.
in each assay to monitor its sensitivity. Only a final bulk that complies with the following requirement
When replication-competent adenoviruses are expected to may be used in the preparation of the final lot.
be present in the test sample, plaque-assays or limit dilution
assays on a detector cell line may be performed. Sterility (2.6.1). It complies with the test for sterility.
PROPAGATION AND HARVEST FINAL LOT
All processing of the cell bank and subsequent cell cultures Only a final lot that complies with each of the requirements
is done in an area with a suitable containment level where given below under Identification, Tests and Assay may be
no other cells or vectors are handled at the same time. Any released for use.
material of human or animal origin used in the preparation Provided that the tests for bovine serum albumin (when
of cell suspensions and culture media is qualified. The bovine serum is used to manufacture the vector) and
cell culture medium may contain a pH indicator such as replication-competent adenoviruses have been carried out
phenol red and suitable antibiotics at the lowest effective with satisfactory results on the final bulk, they may be omitted
concentration, but it is preferable to have a substrate free from on the final lot.
antibiotics during production. Unless otherwise justified and
authorised, at no stage during production is penicillin or IDENTIFICATION
streptomycin used. A portion of the production cell cultures
is set aside as uninfected cell cultures (control cells). The vector is identified by immunochemical methods (2.7.1),
NAT (2.6.21) or restriction enzyme analysis.
Each single harvest that complies with the following
requirements may be used in the preparation of the purified
TESTS
harvest.
Osmolality (2.2.35) : within the limits approved for the
Identification. The vector is identified by immunochemical
particular preparation.
methods (2.7.1), NAT (2.6.21) or restriction enzyme analysis.
pH (2.2.3) : within the limits approved for the particular
Vector concentration. The titre of infectious vector and
preparation.
the concentration of vector particles in single harvests are
determined. Extractable volume (2.9.17). It complies with the test for
Extraneous agents (2.6.16). The single harvest complies with extractable volume.
the tests for extraneous agents. Residual moisture (2.5.12) : within the limits approved for
Control cells. Control cells comply with a test for the particular freeze-dried preparation.
identification (5.2.3) and a test for extraneous agents (2.6.16). Bovine serum albumin : not more than the limit approved
PURIFIED HARVEST for the particular preparation, determined by a suitable
immunochemical method (2.7.1), where bovine serum has
Several single harvests may be pooled before the purification been used during production.
process. The purification process is validated to demonstrate
the satisfactory removal of impurities. Replication-competent adenovirus concentration : within
Purified harvests that comply with the following requirements the limits approved for the particular preparation.
may be used in the preparation of the final bulk. Vector aggregates. Vector aggregates are determined by
Identification. The vector is identified by immunochemical suitable methods (for example, light scattering).
methods (2.7.1), NAT (2.6.21) or restriction enzyme analysis. Sterility (2.6.1). It complies with the test for sterility.
Genomic integrity. Genomic integrity of the vector is verified Bacterial endotoxins (2.6.14) : less than the limit approved
by suitable methods such as restriction enzyme analysis. for the particular preparation.
General Notices (1) apply to all monographs and other texts 709
5.14. Gene transfer medicinal products for human use EUROPEAN PHARMACOPOEIA 8.0
Thermal stability. Maintain samples of the vector final lot cultured in vitro, which are simultaneously infected with the
at a temperature and for a length of time that are adapted parental poxvirus. DNA recombination occurs within the
and authorised for the particular preparation. Determine infected cells, between homologous sequences in the viral
the total infectious vector concentration after heating, as genome and viral sequences in the plasmid so as to transfer
described below under Assay. Determine in parallel the vector the genetic insert into the targeted site of the viral genome.
concentration of a non-heated sample. The estimation of the The correct targeting of the inserted DNA is checked by
difference between the total vector concentration without restriction-enzyme mapping, NAT (2.6.21) and sequencing.
heating and after heating is within the limits approved for the Successive plaque-cloning steps are performed to purify the
particular preparation. recombinant poxvirus from the mixture of parental and
recombinant poxviruses. A variety of methods (for example,
ASSAY foreign marker genes, DNA hybridisation, immunological
Vector particle concentration. Physical titration is performed detection, phenotypic changes in the virus) are employed to
by a suitable technique (for example, liquid chromatography, facilitate recognition and/or selection of the recombinant
absorbance measurement or NAT (2.6.21)). Use an appropriate poxvirus from the background of parental virus. Where
vector reference standard to validate each assay. foreign marker genes have been transiently employed, they are
The vector particle concentration of the preparation to be removed by appropriate methods from the final recombinant
examined is not less than the concentration stated on the label. poxvirus.
Infectious vector titre. Titrate the preparation to be examined An alternative strategy for creating poxvirus vectors begins
by inoculation into cell cultures. Titrate an appropriate vector with the in vitro construction of a full-length virus genome
reference standard to validate each assay. harbouring the expression cassette within a chosen target site.
This recombinant genome is then introduced into host cells
The assay is invalid if : simultaneously infected with a helper poxvirus that is unable
– the confidence interval (P = 0.95) of the logarithm of the to multiply. The helper virus may be a poxvirus of the same
vector concentration is greater than a value authorised by species whose ability to multiply has been inactivated, or
the competent authority ; another poxvirus species that does not multiply in the host
– the infectious vector titre of the reference standard is cells.
outside limit values defined by a control chart. The construction of non-replicative poxvirus vectors relies
Ratio of vector particle concentration to infectious vector on specific host cell lines or primary cells that are naturally
titre : within the limits approved for the particular preparation. permissive, or on host cell lines that have been modified to
express an essential poxvirus gene. These cells fulfill the
Expression of the genetic insert product. The expression of general requirements for the production of medicinal products
the genetic insert product(s) is determined wherever possible, (5.2.3) and do not allow the generation of replicative vectors.
following inoculation of cell cultures with the particular
preparation at a predetermined multiplicity of infection, by VECTOR PRODUCTION
suitable immunochemical (2.7.1) or biochemical assays or by The production method shall have been shown to yield
flow cytometry (2.7.24). a vector of consistent quality. Unless otherwise justified
and authorised, the vector in the final product shall have
Biological activity. Unless otherwise justified and authorised,
undergone no more passages from the master seed lot than
biological activity is determined by a suitable in vitro or in
were used to prepare the vector shown in clinical trials to be
vivo test.
satisfactory with respect to safety and efficacy.
LABELLING The genetic and phenotypic stability of the vector at or beyond
The label states : the maximum passage level used for production is assessed by
suitable methods.
– the content of active substance ;
SUBSTRATE FOR VECTOR PROPAGATION
– the recommended human dose, expressed in vector particle
concentration ; The vector is propagated under aseptic conditions in human
diploid cells (5.2.3), in continuous cell lines (5.2.3) or in
– for freeze-dried preparations : cultures of chick-embryo cells derived from a chicken flock
– the name or composition and the volume of the free from specified pathogens (5.2.2). When the vector is
reconstituting liquid to be added ; propagated in a continuous cell line or in human diploid cells,
– the time within which the product is to be used after a cell-bank system is established.
reconstitution. VECTOR SEED LOT
Production of the vector is based on a seed-lot system.
POXVIRUS VECTORS FOR HUMAN USE The strain of poxvirus used is identified by historical records
that include information on its origin and its subsequent
DEFINITION manipulation, notably deleted or modified regions. A detailed
Poxvirus vectors for human use are freeze-dried or liquid description of the genetic insert(s) and the flanking control
preparations of recombinant poxviruses, genetically modified regions is established, including the nucleotide sequence. The
to transfer genetic material to human somatic cells in vivo method by which the genetic insert is introduced into the
or ex vivo. vector is documented.
PRODUCTION Only a seed lot that complies with the following requirements
may be used for vector production.
VECTOR CONSTRUCTION
Identification. The vector is identified in the master seed
The general design of a poxvirus vector is currently as follows : lot and each working seed lot by immunochemical methods
the genetic insert is inserted downstream of a poxvirus (2.7.1) or NAT (2.6.21).
promoter. This expression cassette is inserted into the
poxvirus genome in such a manner that it interrupts a viral Genetic and phenotypic characterisation. The following
gene non-essential for replication or is positioned between tests are carried out.
2 virus open reading frames. – The entire genome of the vector is sequenced at a passage
In most strategies used so far for the construction of the level comparable to a production batch and the analytically
vector, the expression cassette is first inserted within the determined sequence is compared to the theoretical
target site of a virus DNA fragment cloned into a bacterial sequence based on vector construction and available
plasmid. The plasmid is then introduced into host cells, databases.
– Restriction enzyme analysis is performed on the vector Ratio of infectious vector titre to total protein
DNA of the master seed lot, each working seed lot and concentration. The total protein concentration is determined
a production batch. The viral DNA is extracted, purified by a suitable method (2.5.33). The ratio between infectious
and digested with sufficient resolution. The digested vector titre and total protein concentration is calculated.
fragments are separated by gel electrophoresis or capillary Residual host-cell protein. The concentration of residual
electrophoresis and the observed restriction pattern is host-cell protein is determined by a suitable immunochemical
compared to the theoretical restriction pattern based on method (2.7.1), unless the process has been validated to
vector construction. demonstrate suitable clearance.
– A suitable number of isolated sub-clones are tested for Residual host-cell DNA. The content of residual host-cell
expression of the genetic insert product(s) and biological DNA is determined using a suitable method, unless the
activity at a passage level comparable to a production batch. process has been validated to demonstrate suitable clearance.
Sub-clones giving lower levels of expression or biological Quantitative PCR is recommended for its sensitivity and
activity need further characterisation. specificity, but other suitable techniques may also be used.
– The host range is verified by determining the replication Residual reagents. Where reagents are used during the
properties of the vector and comparing them with that production process, tests for these substances are carried out
of the parental virus, at a passage level comparable to a on the purified harvest, unless the process has been validated
production batch. to demonstrate suitable clearance.
Infectious vector titre. The titre of infectious vector in the Residual antibiotics. Where antibiotics are used during
master seed lot and each working seed lot is determined. the production process, their residual concentration is
Extraneous agents (2.6.16). The master seed lot and each determined by a microbiological assay (adapted from general
working seed lot comply with the tests for extraneous agents, method 2.7.2) or by other suitable methods (for example,
except where cytopathic strains cannot be neutralised and the liquid chromatography), unless the process has been validated
vector causes interference. Where a test cannot be performed, to demonstrate suitable clearance.
carry out a suitable validated alternative. FINAL BULK
PROPAGATION AND HARVEST Several purified harvests may be pooled during preparation of
All processing of the cell bank and subsequent cell cultures the final bulk. A stabiliser and other excipients may be added.
is done under aseptic conditions in an area with a suitable Only a final bulk that complies with the following requirement
containment level where no other cells or vectors are handled may be used in the preparation of the final lot.
at the same time. Any material of human or animal origin Sterility (2.6.1). It complies with the test for sterility.
used in the preparation of cell suspensions and culture FINAL LOT
media is qualified. The cell culture medium may contain
a pH indicator such as phenol red and suitable antibiotics Only a final lot that complies with each of the requirements
at the lowest effective concentration, but it is preferable to given below under Identification, Tests and Assay may be
have a substrate free from antibiotics during production. released for use.
Unless otherwise justified and authorised, at no stage during Provided that the test for bovine serum albumin (when bovine
production is penicillin or streptomycin used. A portion serum is used to manufacture the vector) has been carried out
of the production cell culture is set aside as uninfected cell with satisfactory results on the final bulk, it may be omitted
cultures (control cells). on the final lot.
Each single harvest that complies with the following IDENTIFICATION
requirements may be used in the preparation of the purified
harvest. The vector is identified by immunochemical methods (2.7.1)
or NAT (2.6.21).
Identification. The vector is identified by immunochemical
methods (2.7.1) or NAT (2.6.21). TESTS
Infectious vector titre. The titre of infectious vector in single Osmolality (2.2.35) : within the limits approved for the
harvests is determined. particular preparation.
Extraneous agents (2.6.16). The single harvest complies with pH (2.2.3) : within the limits approved for the particular
the tests for extraneous agents, except where cytopathic strains preparation.
cannot be neutralised and the vector causes interference. Extractable volume (2.9.17). It complies with the test for
Where a test cannot be performed, carry out a suitable extractable volume.
validated alternative.
Residual moisture (2.5.12) : within the limits approved for
Control cells. If human diploid cells or a continuous cell the particular freeze-dried preparation.
line are used for production, the control cells comply with a
test for identification (5.2.3). They comply with the tests for Bovine serum albumin : not more than the limit approved
extraneous agents (2.6.16). for the particular preparation, determined by a suitable
immunochemical method (2.7.1), where bovine serum has
PURIFIED HARVEST been used during production.
Processing is carried out under aseptic conditions. Several
single harvests may be pooled before the purification process. Sterility (2.6.1). It complies with the test for sterility.
The harvest is first clarified to remove cells and then, where Bacterial endotoxins (2.6.14) : less than the limit approved
applicable, purified by validated methods. for the particular preparation.
Purified harvests that comply with the following requirements Thermal stability. Maintain samples of the vector final lot
may be used in the preparation of the final bulk. at a temperature and for a length of time that are adapted
Identification. The vector is identified by immunochemical and authorised for the particular preparation. Determine
methods (2.7.1) or NAT (2.6.21). the total infectious vector concentration after heating, as
described below under Assay. Determine in parallel the vector
Genomic integrity. Genomic integrity of the vector is verified concentration of a non-heated sample. The estimation of the
by suitable methods such as restriction enzyme analysis. difference between the total vector concentration without
Infectious vector titre. The titre of infectious vector in heating and after heating is within the limits approved for the
purified harvests is determined. particular preparation.
General Notices (1) apply to all monographs and other texts 711
5.14. Gene transfer medicinal products for human use EUROPEAN PHARMACOPOEIA 8.0
Residual host-cell DNA. The content of residual host-cell Replication-competent viruses. Detection of
DNA is determined using a suitable method, unless the replication-competent viruses is performed by suitable
production process has been validated to demonstrate suitable methods. It is generally performed by amplification on
clearance. Quantitative PCR is recommended for its sensitivity permissive cells followed by NAT (2.6.21), by detection of a
and specificity, but other suitable techniques may also be used. viral antigen (for example, p24 by ELISA) or by marker-rescue
Bacterial endotoxins (2.6.14) : less than the limit approved assay. Positive controls are included in each assay to monitor
for the particular preparation. its sensitivity.
Detection of replication-competent viruses is performed on the
Sterility (2.6.1). It complies with the test for sterility. purified harvest or on the final lot. No replication-competent
Producer cells used in a stable production system viruses are found.
Copy number. The copy number of the integrated viral genes Residual host-cell protein. The concentration of residual
and expression cassette is determined by a suitable method. host-cell protein is determined by a suitable immunochemical
method (2.7.1), unless the process has been validated to
Genetic stability. Genetic stability of the producer cells at demonstrate suitable clearance.
or beyond the maximum number of cell doublings used for
production is confirmed. Residual host-cell DNA. The content of residual host-cell
DNA is determined using a suitable method, unless the
Sequence integrity of the viral genes and expression process has been validated to demonstrate suitable clearance.
cassette. Complete nucleotide sequencing of the inserted viral Quantitative PCR is recommended for its sensitivity and
genes, the expression cassette and their respective regulation specificity, but other suitable techniques may also be used.
elements (for example, LTRs, promoters, psi sequence,
polyadenylation signal) is performed. Residual reagents. Where reagents are used during
production, tests for these substances are carried out on the
Replication-competent viruses. The detection of purified harvest, unless the process has been validated to
replication-competent viruses is performed by suitable demonstrate suitable clearance.
methods. Detection may be based on a co-cultivation for Residual antibiotics. Where antibiotics are used during
several cell doublings of the producer cells with a permissive
the production process, their residual concentration is
cell line, followed by detection (either by observation of a
determined by a microbiological assay (adapted from general
cytopathic or haemadsorbing effect on indicator cells like PG4 method 2.7.2) or by other suitable methods (for example,
S+L-, by detection using indicator cell lines by NAT (2.6.21)
liquid chromatography), unless the process has been validated
or by marker-rescue assay). Positive controls are included in
to demonstrate suitable clearance.
each assay to monitor its sensitivity. No replication competent
viruses are found. Residual plasmids. Where a transient production process is
used, the concentration of residual contaminating plasmids
PRODUCTION AND HARVEST must be quantified.
All processing of the cell bank and subsequent cell cultures
is done in an area with a suitable containment level where FINAL BULK
no other cells or vectors are handled at the same time. Any Several purified harvests may be pooled during preparation of
material of human or animal origin used in the preparation the final bulk. A stabiliser and other excipients may be added.
of cell suspensions and culture media must be qualified. It The formulated product is filtered through a bacteria-retentive
is preferable to have a substrate free from antibiotics during filter.
production. Unless otherwise justified and authorised, at no Only a final bulk that complies with the following requirement
stage during production is penicillin or streptomycin used. may be used in the preparation of the final lot.
Sterility (2.6.1). It complies with the test for sterility.
Each single harvest that complies with the following
requirements may be used in the preparation of the purified FINAL LOT
harvest. Only a final lot that complies with each of the requirements
Identification. The vector is identified by immunochemical given below under Identification, Tests and Assay may be
methods (2.7.1), NAT (2.6.21) or restriction enzyme analysis. released for use.
Provided that the tests for bovine serum albumin (when
Vector concentration. The titre of infectious vector and/or bovine serum is used to manufacture the vector) and
the concentration of vector particles in single harvests is replication-competent viruses have been carried out with
determined. satisfactory results on the purified harvest, they may be
Extraneous agents. Each single harvest complies with the omitted on the final lot.
tests for extraneous agents (2.6.16). IDENTIFICATION
Control cells. Where a transient production process is used, Retroviridae-derived vectors are identified by NAT (2.6.21),
control cells comply with a test for identification (5.2.3) and a immunochemical methods (2.7.1) or restriction enzyme
test for extraneous agents (2.6.16). analysis.
PURIFIED HARVEST
TESTS
Several single harvests may be pooled before purification.
Purified harvests that comply with the following requirements Osmolality (2.2.35) : within the limits approved for the
may be used in the preparation of the final bulk. particular preparation.
Identification. The vector is identified by immunochemical pH (2.2.3) : within the limits approved for the particular
methods (2.7.1), NAT (2.6.21) or restriction enzyme analysis. preparation.
Genomic integrity. Genomic integrity of the vector is verified Extractable volume (2.9.17). It complies with the test for
by a suitable method. extractable volume.
Vector concentration. The infectious particle titre is Residual moisture (2.5.12) : within the limits approved for
determined by a suitable method, for example infection of the particular freeze-dried preparation.
permissive cells followed by quantitative NAT (for example, Bovine serum albumin : where bovine serum has been
quantitative PCR), Southern blot or protein expression. For used during production, not more than the limit approved
lentivirus vectors, the physical titre is measured, for example for the particular preparation, determined by a suitable
by ELISA (p24). immunochemical method (2.7.1).
General Notices (1) apply to all monographs and other texts 713
5.14. Gene transfer medicinal products for human use EUROPEAN PHARMACOPOEIA 8.0
Replication-competent viruses. Detection of function as the origin of replication. The rep and cap genes are
replication-competent viruses is performed by suitable required in trans and function for replication and packaging
methods. It is generally performed by amplification on respectively. In summary, the rAAV vector contains the ITRs
permissive cells followed by NAT (2.6.21), detection of a viral and the genetic insert.
antigen (for example, p24 by ELISA) or marker-rescue assay. Wild-type AAV normally replicate only in the presence of
Positive controls are included in each assay to monitor its helper functions, provided by a coinfecting adenovirus or
sensitivity. herpes virus. Therefore, there are different approaches to the
Detection of replication-competent viruses is performed on the manufacture of an AAV vector. The manufacturing strategy
purified harvest or on the final lot. No replication-competent chosen is designed to minimise the risk for the generation of
viruses are found. replication-competent AAV vectors and effectively eliminate
helper viruses that might be used during production.
Sterility (2.6.1). It complies with the test for sterility.
VECTOR PRODUCTION
Bacterial endotoxins (2.6.14) : less than the limit approved
for the particular preparation. The production method shall have been shown to yield a
vector of consistent quality and stability.
ASSAY To produce AAV vectors, several strategies are currently used,
Vector-particle concentration. Physical titration for example :
is performed by a suitable technique (for example, – transient co-transfection of a cell line with plasmids
immunochemical methods (2.7.1) or NAT (2.6.21)). Use an containing the ITRs and the genetic insert, rep and cap
appropriate vector reference standard to validate each assay. genes and helper functions ;
Infectious vector titre. Titrate the preparation to be examined – infection with a replication-deficient helper virus of a
by inoculation into cell cultures. Titrate an appropriate vector producer cell line harbouring rep and cap genes, the ITRs
reference standard to validate each assay. and the genetic insert ;
The infectious vector titre of the preparation to be examined – infection of a permissive cell line with 1 or several
is not less than the minimum titre stated on the label. production viruses encoding rep and/or cap and/or the
The assay is invalid if : genetic insert and the ITRs, and that may or may not
provide helper functions (helper viruses and baculoviruses,
– the confidence interval (P = 0.95) of the logarithm of the respectively).
vector concentration is greater than a value authorised by
the competent authority ; Depending on the strategy used to produce AAV vectors,
different production intermediates are required (plasmids,
– the infectious vector titre of the reference standard is viruses used for production, packaging cells).
outside limit values defined by a control chart.
The occurrence of replication-competent AAV may be
Ratio of vector-particle concentration to infectious vector significant when regions of homology exist between the
titre : within the limits approved for the particular product, genomes of the production intermediates and the rAAV
where applicable. vector. This occurrence may be minimised by reducing the
Expression of the genetic insert product. The expression of homology between these genomes to a minimum. The use
the genetic insert product(s) is determined wherever possible, of production intermediates with no sequence homology is
following inoculation of cell cultures with the product recommended for production.
at a predetermined multiplicity of infection, by suitable The genetic and phenotypic stability of the vector at or beyond
immunochemical (2.7.1) or biochemical assays or by flow the maximum number of passage levels used for production is
cytometry (2.7.24). assessed by suitable methods.
Biological activity. Unless otherwise justified and authorised, PRODUCTION INTERMEDIATES
biological activity is determined by a suitable in vitro or in
vivo test. Viruses used for production and the rAAV vector are
produced in continuous cell lines (5.2.3) using a seed lot and a
LABELLING cell-bank system.
The label states : Packaging and producer cells
– the minimum vector titre per human dose ; Copy number. The genomic DNA is isolated and purified
– the recommended human dose ; from a known number of cells and the copy number of
– for freeze-dried preparations : the inserted viral genes and of the expression cassette is
determined by a suitable method such as quantitative PCR
– the name or composition and the volume of the (2.6.21).
reconstituting liquid to be added ;
Sequence integrity of the viral genes and expression
– the time within which the product is to be used after cassette. Complete nucleotide sequencing of the inserted viral
reconstitution. genes, of their regulatory elements and where applicable, of
the expression cassette is performed.
ADENO-ASSOCIATED-VIRUS VECTORS Genetic stability. Genetic stability of the cells is verified at
FOR HUMAN USE or beyond the maximum number of cell doublings used for
production.
DEFINITION
Adeno-associated-virus (AAV) vectors for human use are Wild-type AAV. The absence of wild-type AAV is verified
freeze-dried or liquid preparations of recombinant AAV using NAT (2.6.21).
(rAAV), genetically modified to transfer genetic material to Plasmids
human somatic cells in vivo or ex vivo. Production of the AAV vector by transient co-transfection
requires the use of plasmid intermediates. For each plasmid
PRODUCTION DNA used during production, a complete description
VECTOR CONSTRUCTION is established, including identification, source, means of
rAAV vectors are developed by replacement of the rep and cap isolation and nucleotide sequence. The source and function
genes with the genetic insert of interest. The inverted terminal of component parts of these plasmids, such as the origin
repeat (ITR) sequences are retained in the rAAV vector since of replication, viral and eukaryotic promoters and genes
these are the only AAV sequences absolutely required in cis to encoding selection markers, are documented.
General Notices (1) apply to all monographs and other texts 715
5.14. Gene transfer medicinal products for human use EUROPEAN PHARMACOPOEIA 8.0
method 2.7.2) or by other suitable methods (for example, Vector aggregates. Vector aggregates are determined by
liquid chromatography), unless the process has been validated suitable methods (for example, light scattering).
to demonstrate suitable clearance. Sterility (2.6.1). It complies with the test for sterility.
FINAL BULK Bacterial endotoxins (2.6.14) : less than the limit approved
Several purified harvests may be pooled during preparation of for the particular preparation.
the final bulk. A stabiliser and other excipients may be added.
The formulated product is filtered through a bacteria-retentive ASSAY
filter. Vector-particle concentration. Vector-particle concentration
Only a final bulk that complies with the following requirement is determined using a suitable method such as quantitative
may be used in the preparation of the final lot. PCR by comparison with a standard curve obtained using the
Sterility (2.6.1). It complies with the test for sterility. recombinant AAV plasmid or an AAV reference standard.
This concentration is within the limits approved for the
FINAL LOT particular product.
Only a final lot that complies with each of the requirements
given below under Identification, Tests and Assay may be Infectious vector titre. Titrate the preparation to be examined
released for use. by inoculation into cell cultures. Titrate an appropriate vector
Provided that the tests for bovine serum albumin reference standard to validate each assay.
(when bovine serum is used to manufacture the vector), The infectious vector titre of the preparation to be examined
replication-competent AAV and residual viruses used for is not less than the minimum amount stated on the label.
production have been carried out with satisfactory results on The assay is invalid if :
the final bulk, they may be omitted on the final lot. – the confidence interval (P = 0.95) of the logarithm of the
vector concentration is greater than a value authorised by
IDENTIFICATION the competent authority ;
The vector is identified by immunochemical methods (2.7.1), – the infectious vector titre of the reference standard is
NAT (2.6.21) or restriction enzyme analysis. outside limit values defined by a control chart.
TESTS Ratio of vector-particle concentration to infectious vector
titre : within the limits approved for the particular product.
Osmolality (2.2.35) : within the limits approved for the
particular preparation. Expression of the genetic insert product. The expression of
the genetic insert product is determined wherever possible,
pH (2.2.3) : within the limits approved for the particular following inoculation of cell cultures with the product
preparation. at a predetermined multiplicity of infection, by suitable
Extractable volume (2.9.17). It complies with the test for immunochemical (2.7.1) or biochemical assays or by flow
extractable volume. cytometry (2.7.24).
Residual moisture (2.5.12) : within the limits approved for the Biological activity. Unless otherwise justified and authorised,
particular freeze-dried product. biological activity is determined by a suitable in vitro or in
Bovine serum albumin : where bovine serum has been vivo test.
used during production, not more than the limit approved LABELLING
for the particular preparation, determined by a suitable
immunochemical method (2.7.1). The label states :
– the content of active substance ;
Replication-competent AAV concentration : within the
limits approved by the competent authority. – the recommended human dose ;
Detection of replication-competent AAV is performed by a – for freeze-dried preparations :
replication assay on a permissive cell line previously infected – the name or composition and the volume of the
with a helper virus and analysis of the replicative forms by reconstituting liquid to be added ;
Southern blot on low-molecular-weight DNA, or by detection – the time within which the product is to be used after
of the rep gene by quantitative PCR. reconstitution.
General Notices (1) apply to all monographs and other texts 719
5.15. Functionality-related characteristics of excipients EUROPEAN PHARMACOPOEIA 8.0
Examples of solid-state properties to be considered in the A given characteristic may be the subject of a mandatory
development of solid dosage forms include polymorphism, requirement in the monograph. If it is relevant for certain
pseudopolymorphism, crystallinity and density. Techniques to uses, it is also referred to in the FRCs section as a relevant
study them are given in general chapters 5.9. Polymorphism, characteristic that the manufacturer of the medicinal product
5.16. Crystallinity and 2.2.42. Density of solids. may choose to specify for the grade used of a particular
pharmaceutical preparation.
CHEMICAL GRADES The section on FRCs is intended to reflect current knowledge
related to the major uses of an excipient. In view of the
Excipients that are available in different chemical grades multiple uses of some excipients and the continuous
are of natural, semi-synthetic or synthetic origin. Specific development of new uses, the section may not be complete.
monographs usually control the chemical composition In addition, the methods cited for the determination of a
of excipients that are composed of a mixture of related particular characteristic are given as recommendations for
compounds, for example the composition of fatty acids in methods that are known to be satisfactory for the purpose,
vegetable oils or surfactants. There are, however, specific and the use of other methods is not excluded.
monographs in the Pharmacopoeia each describing a class
of polymeric materials that may vary in their composition PHARMACOPOEIAL HARMONISATION
with regard to the structure of homopolymers, block polymers A number of excipient monographs are subject to
and copolymers, the degree of polymerisation, and thus pharmacopoeial harmonisation among the European,
the molecular mass and mass distribution, the degree of Japanese and United States pharmacopoeias (see 5.8.
substitution and in some cases even different substituents on Pharmacopoeial harmonisation). Introduction of the FRCs
the polymer backbone. This variation may, however, have section in the monographs of the European Pharmacopoeia
a profound effect on the functionality of the excipient and means that the presentation of harmonised monographs
should be subject to investigations during the pharmaceutical differs. Tests for physical and chemical characteristics regarded
development, preferably to establish the acceptable range of as both quality-related and functionality-related in the
each characteristic being critical to the manufacturing process European Pharmacopoeia are, in the 2 other pharmacopoeias,
and performance of the end-product. included only in the body of the monograph. The different
format has no implications on the specification of excipient
FUNCTIONALITY-RELATED CHARACTERISTICS characteristics for the manufacturer of the medicinal product.
SECTION IN MONOGRAPHS Current regulatory guidance recommends the identification
and specification of only such critical properties that
Monographs on excipients may have a section entitled impact the manufacturing process and the performance
‘Functionality-related characteristics’. This section is included of the end-product. The different legal environments
for information for the user and is not a mandatory part of the of the 3 pharmacopoeias allow for different formats of
monograph. The section gives a statement of characteristics the monographs without affecting the pharmacopoeial
that are known to be relevant for certain uses of the excipient. harmonisation status.
The use for which the characteristic is relevant is stated.
For other uses, the characteristic may be irrelevant. For this GLOSSARY
reason, the section should not be seen simply as a supplement Critical characteristic : any physical or chemical material
to the monograph. It is the responsibility of the manufacturer characteristic that has been demonstrated to impact
of the medicinal product to decide how the information on significantly on the manufacturability and/or performance of
FRCs will be applied in the manufacturing process in light the medicinal product.
of the use of the excipient and data from pharmaceutical
development. Design space : the multidimensional combination and
interaction of input variables (e.g. material attributes) and
The information on the functionality-related characteristics process parameters that have been demonstrated to provide
may be given in different ways : assurance of quality.
– name of the FRC ; Functionality-related characteristic : a controllable physical
or chemical characteristic of an excipient that is shown to
– name of the FRC and a recommended method for its impact on its functionality.
determination, referring wherever possible to a general
chapter of the Pharmacopoeia ; Process analytical technology (PAT) : a system for designing,
analysing and controlling manufacturing through timely
– name of the FRC with a recommended method for its measurements (i.e. during processing) of critical quality and
determination and typical values, which may be in the form performance attributes of raw and in-process materials and
of tolerances from the nominal value. processes with the goal of ensuring final product quality.
General Notices (1) apply to all monographs and other texts 723
5.16. Crystallinity EUROPEAN PHARMACOPOEIA 8.0
amorphous content covered by this method depends on the Infrared absorption spectrophotometry and Raman
individual substance to be tested ; in favourable cases limits of spectrometry. Infrared absorption spectrophotometry
detection below 1 per cent can be reached. (2.2.24) and Raman spectrometry (2.2.48) are other techniques
Solution calorimetry (2.2.61). Solution calorimetry provides used to measure the degree of crystallinity, and have also
a means of determining enthalpy of solution for a solid been proven to be useful in studies of polymorphism. The
substance. The crystallinity of the solid sample to be examined IR spectrum and Raman spectrum of a sample contain both
is given by the enthalpy of solution of the solid sample ( ) physical and chemical information.
minus the enthalpy of solution of the chosen reference Solid-state NMR. Solid-state nuclear magnetic resonance
standard of the same substance ( ) when determined spectrometry (ss NMR) (2.2.33) can be used to provide
under the same conditions. Because the reference standard is information about polymorphism and related relative
usually chosen for its perceived high crystallinity, its enthalpy molecular conformations. However, some caution has to
of solution is usually algebraically greater (more endothermic be exercised in the interpretation of results, since it is not
or less exothermic) than that of the solid sample to be always simple to distinguish between samples that comprise a
examined in the same solvent. Consequently, the crystallinity mixture of different physical forms (2-state model) and those
determined is a negative quantity with the SI units kJ/mol or that comprise crystals having disorder with exchange that is
J/g (J/kg is avoided because of its unwieldiness and potential slow on the NMR timescale. Similarly, samples that contain
for error). The preference for a negative value with respect to defects arising from different molecular conformations or
a highly crystalline reference standard recognises the fact that slightly different packing arrangements (1-state model) may
most samples have a lower crystallinity than this reference show additional signals in the spectra. Solid-state NMR may
standard. be quite sensitive to this, even if lattice parameters are hardly
Near-infrared (NIR) spectroscopy. Near-infrared (NIR) affected and, consequently, little or no change is observed
spectroscopy (2.2.40) is another technique used to measure by XRPD. It is evident that the crystallinity of substances
the degree of crystallinity, and has also been proven to be for pharmaceutical use can be complex, and both crystalline
useful in studies of polymorphism. The NIR spectrum of a defects and amorphous material may co-exist.
sample contains both physical and chemical information. Optical microscopy. A method to detect whether or not
Being non-invasive, non-destructive and operable at room particles are crystalline is to use a polarising microscope
temperature, the method is a valuable tool to assess changes (2.9.37), where particles show birefringence and extinction
in the amorphous and crystalline state. positions when the microscope stage is revolved.
General Notices (1) apply to all monographs and other texts 727
5.17.1. Recommendations on dissolution testing EUROPEAN PHARMACOPOEIA 8.0
20 per cent to 30 per cent. The 2nd specification point defines Gastro-resistant dosage forms require at least 2 specification
the dissolution pattern and so is set at around 50 per cent points in a sequential test and 2 different specifications in a
release. The final specification point is intended to ensure parallel test. In a sequential test, the 1st specification point
almost complete release, which is generally understood as represents an upper limit and is set after 1 h or 2 h in acidic
more than 80 per cent release. medium, and the 2nd after a pre-set time period of testing in
Delayed-release dosage forms an adequate buffer solution (preferably pH 6.8).
In most cases the acceptance criteria at level B1 are that at
A delayed-release dosage form may release the active least 80 per cent of the active substance is released. This
substance(s) fractionally or totally according to the corresponds to a Q value of 75 per cent, since, as referred to
formulation design when tested in different dissolution media, in Table 2.9.3.-4, for level B1 the individual value of each of
e.g. in increasing pH conditions. Dissolution specifications the 6 units tested is not less than Q + 5 per cent, i.e. not less
therefore have to be decided on a case-by-case basis. than 80 per cent.
General Notices (1) apply to all monographs and other texts 729
EUROPEAN PHARMACOPOEIA 8.0 5.20. Metal catalyst or metal reagent residues
General Notices (1) apply to all monographs and other texts 733
5.20. Metal catalyst or metal reagent residues EUROPEAN PHARMACOPOEIA 8.0
The classification and concentration limits of the currently – Note for Guidance on Impurities : Residual
included metals may also change when new safety data Solvents (CPMP/ICH/283/95 in conjunction with
becomes available. CPMP/ICH/1507/02, CPMP/ICH/1940/00 corr,
The following assumptions and/or default values have been CPMP/QWP/450/03 and CPMP/QWP/8567/99)
used during establishment of the concentration limits: – Guideline on the Limits of Genotoxic
– body weight (bw) of an adult : 50 kg ; Impurities (EMEA/CHMP/QWP/251344/2006 and
– breathing volume of an adult : 20 m3 per day (24 h) ; CPMP/SWP/5199/02)
– occupational (workplace) inhalation exposure : 8 h per day – Validation of Analytical Procedures : Text and Methodology
(24 h) ; (CHMP/ICH/381/95, ICHQ2(R1))
– exposure limits were established using uncertainty factors 4. MAIN GUIDELINE TEXT
as described in appendix 3 of the ICH Q3C guidance ;
– for pragmatic reasons a number of uncertainty factors 4.1. CLASSIFICATION
were adapted to arrive at a final safe and practical The term ‘tolerable daily intake’ (TDI) is used by the
PDE setting - Q3C method for uncertainty factor (UF) International Program on Chemical Safety (IPCS) to
calculation plus additional pragmatic factor for PDE describe exposure limits of toxic chemicals, whereas the term
calculation ; ‘acceptable daily intake’ (ADI) is used by the World Health
Organization (WHO) and other national and international
– acceptable additional lifetime cancer risk : an increased health authorities and institutes. Following the ICH Q3C
cancer risk of 1 in 100,000 was identified as acceptable for guideline on residual solvents, a ‘new’ term was chosen to
genotoxic impurities in pharmaceuticals by the CHMP. avoid confusion of terms and their meaning. As for the ICH
2. DEFINITION AND SCOPE Q3C guideline, the new term is called the ‘permitted daily
Metal catalysts and metal reagents are defined here as exposure’ (PDE). For the purpose of this guideline, the PDE
chemical substances that are used to change the rate of is defined as the pharmaceutically maximum acceptable
chemical reactions or which act on other chemical substances exposure to a metal on a chronic basis that is unlikely to
in chemical reactions. For the purpose of this guideline, produce any adverse health effect.
metal catalysts and metal reagents refer to metals used in the Metal residues should be evaluated for their potential risk to
synthesis of the active pharmaceutical ingredient, the synthesis human health and placed into one of the following 3 classes:
of any of the pharmaceutical excipients, or the synthesis of any Class 1 metals : metals of significant safety concern. This
of the pharmaceutical excipients used during the manufacture group includes metals that are known or suspect human
of the drug product but no longer present in the drug product carcinogens, or possible causative agents of other significant
itself. Metal residues can either be present in the original form toxicity.
of the metal or as a form of the metallic element altered by
downstream chemical processing. Class 2 metals : metals of low safety concern. This group
includes metals with lower toxic potential to man. They are
This guideline applies to new and existing marketed drug generally well tolerated up to exposures that are typically
products. However, for existing marketed drug products encountered with administration of medicinal products. They
a time limit of 5 years is set for the implementation of the may be trace metals required for nutritional purposes or they
guideline in case an earlier implementation is not feasible. are often present in food stuffs or readily available nutritional
Following this 5 years implementation, transitional period supplements.
only drug products which have been manufactured using
pharmaceutical substances which comply with the guideline Class 3 metals : metals of minimal safety concern. This
can be released to the market. This guideline does not apply group includes metals with no significant toxicity. Their safety
to potential new drug substances or to excipients used during profile is well established. They are generally well tolerated
the clinical research stages of development of a medicinal up to doses that are well beyond doses typically encountered
product. During the clinical research stages of development, with the administration of medicinal products. Typically they
higher limits of metal residues might be acceptable. are ubiquitous in the environment or the plant and animal
The guideline does also not apply to metals that are kingdoms.
deliberate components of the pharmaceutical substance 4.2. EXPOSURE LIMITS
(such as a counter-ion of a salt) or metals that are used as A general set of safety based limits is defined for residues of
a pharmaceutical excipient in the drug product (e.g. an each particular class of metals, taking into account the route
iron oxide pigment). As described in the introduction, of administration.
the guideline does normally not apply to extraneous metal Table 1 provides information on acceptable PDEs and
contaminants that are more appropriately addressed by GMP, concentration limits for residues of the currently included
GDP or any other relevant quality provision. 14 metals following oral, parenteral and/or inhalation
The route of administration may influence the actual exposure exposure. The metals that are currently included in Class 1 are
of the human body to the metal. Due to the limited oral further subdivided into 3 subclasses called class 1A, 1B and 1C.
bioavailability of many metals, this guideline applies different The exposure limits in class 1A (platinoids) and 1C relate to
limits to oral and parenteral routes of administration. As the individual metals, whereas the exposure limits in class 1B
other routes of exposure may have different toxicological (also platinoids) relate to the total amount of the listed metals.
implications, specific limits have also been set for the For the platinoid metals in class 1B, a conservative approach
inhalation exposure to some metals. When the exposure is was adopted because the currently available toxicity data is
short, the PDEs mentioned in this guideline may be adapted rather limited. Therefore the indicated limit for Class 1B is the
as indicated in section 4.3. limit for the total amount of these platinoid metals that, based
3. LEGAL BASIS on the used synthesis procedures, is anticipated to be present.
This guideline should be read in conjunction with Directive 4.3. SETTING CONCENTRATION LIMITS METAL
2001/83/EC as amended and in conjunction with all relevant RESIDUES
CHMP guidance documents, with special emphasis on: 4.3.1. General
– Note for Guidance on Impurities in New Drug Products If synthetic processes of pharmaceutical substances are known
(CPMP/ICH/2738/99, ICHQ3B (R)) or suspected to lead to the presence of metal residues due
– Note for Guidance on Impurities Testing : Impurities in to the use of a specific metal catalyst or metal reagent, a
New Drug Substances (CPMP/ICH/2737/99, ICHQ3A) concentration limit and validated test for residues of each
Table 1. – Class exposure and concentration limits for individual metal catalysts and metal reagents
Oral Parenteral Inhalation
exposure exposure exposure*
Classification
PDE Concentration PDE Concentration PDE
(μg/day) (ppm) (μg/day) (ppm) (ng/day)
Class 1A:
100 10 10 1 Pt : 70*
Pt, Pd
Class 1B:
100** 10** 10** 1**
Ir, Rh, Ru, Os
Class 1C:
Mo, Ni, Cr, V 250 25 25 2.5
Ni : 100
Metals of significant safety Cr (VI) : 10
concern
Class 2:
Cu, Mn 2500 250 250 25
Metals with low safety concern
Class 3:
Fe, Zn 13 000 1300 1300 130
Metals with minimal safety
concern
specific metal should be set. All concentration limits should Option 2b : alternatively, it is not considered necessary for each
be realistic in relation to analytical precision, manufacturing pharmaceutical substance to comply with the limits given in
capability, and reasonable variation in the manufacturing Option 1 or the calculated limits using Option 2a.
process. Since the use of metal catalysts or metal reagents The PDE in terms of μg/day as stated in Table 1 can also be
during synthesis is restricted to defined chemical reactions, used with the known maximum daily dose of the drug product
limitation of their residues in pharmaceutical substances itself to determine the concentration of a metal residue originating
will normally be sufficient. A limit for a metal residue in from any of the pharmaceutical substances in the drug product
the pharmaceutical substance may however be replaced by a (not the substance). This approach is considered acceptable
limit for that metal residue in the final medicinal product, as provided that it has been demonstrated that the metal
described below. residue has been reduced to the practical minimum in every
4.3.2. Pharmaceutical products applied via the oral, substance. This approach implies that the maximum levels of
parenteral or inhalation route of administration a metal in certain substances may be higher than the Option 1
or Option 2a limit, but that this should then be compensated
Two options are available when setting a concentration limit by lower maximum levels in the other substances.
for a metal residue.
4.3.3. Pharmaceutical products applied via other routes
Option 1 : for each metal, the concentration limit in parts of administration
per million (ppm) as stated in Table 1 can be used. The
concentration limits (in ppm) in Table 1 have been calculated The concentration limits should be set in consideration of the
using expression (1) below by assuming a daily dose of route of administration.
10 grams of the drug product. Without proper justification, parenteral limits/PDEs should
be used for pharmaceutical substances that are administered
by other routes of administration, including inhalation.
Oral limits/PDEs may be applied if the absorption by other
routes of administration is not likely to exceed the absorption
If all pharmaceutical substances in a drug product meet the following oral administration. For example, for cutaneous
option 1 concentration limit for all metals potentially present, administration, oral concentration limits/PDEs are considered
then all these substances may be used in any proportion in the acceptable.
drug product as long as the daily dose of the drug product Platinum salts have been shown to be allergenic, with
does not exceed 10 g per day. When the daily dose of the hexachloroplatinic acid being clearly the most allergenic
drug product is greater than 10 g per day, Option 2 should (Malo, J-L, 2005). Consequently a specific limit for inhalation
be applied. exposure for this molecule has been set at 70 ng/day(1).
Option 2a : the PDE in terms of μg/day as stated in Table 1 can Chromium VI and Nickel, when inhaled, have been associated
be used together with the actual daily dose of a pharmaceutical with carcinogenicity. Therefore specific limits for inhalation
substance in the drug product to calculate the concentration exposure have been set for Chromium VI at 10 ng/day and for
of residual metal allowed in that pharmaceutical substance. Nickel at 100 ng/day(1).
(1) See Appendix 2 ‘Monographs on elements’ of the guideline, in its integral version.
General Notices (1) apply to all monographs and other texts 735
5.20. Metal catalyst or metal reagent residues EUROPEAN PHARMACOPOEIA 8.0
4.3.4. Pharmaceutical products used for short-term and quantity of all metal residues present in their compounds to
for life-saving indications the drug product manufacturers. The following statements are
given as acceptable examples of such information.
As the PDEs and concentration limits mentioned in this
guideline are based on chronic use, higher PDEs and – Only Class 3 metals are likely to be present. All are below
concentration limits may be acceptable in cases of short-term the Option 1 limit for <oral> or <parenteral> exposure.
use (30 days or less). For instance, this may be applicable to (Here the supplier would define the applicability, either
contrasting agents, antidotes, or products for diagnostic use. oral or parenteral product.)
This may however only be applied if neither an Option 1 nor an – Only Class 2 metals X, Y, ... are likely to be present. All
Option 2 limit is feasible. Specific risk-benefit considerations, are below the Option 1 limit for <oral> or <parenteral>
such as for compounds used for life-saving indications, may exposure.
also warrant the use of higher limits. Justifications should be (Here the supplier would name the Class 2 metals
made on a case-by-case basis. represented by X, Y, ... and define the applicability, oral or
parenteral of the product.)
4.4. ANALYTICAL PROCEDURES
For the determination of each metal residue an appropriate – Class 1 metal Z is likely to be present. The metal is
and validated method should be used. Attention should present in a concentration of zzz ppm which is below <the
be paid to the fact that metal residues may be present in a acceptance criterion>.
different form than the form of the element in the original (Here the supplier would state the identity of the metal,
catalyst or reagent. Unless otherwise justified, the test should the actual concentration found and the applied acceptance
be specific for each element. Where sufficient justification can criterion. If the metal is found below the LOD or LOQ of
be derived, a more general analytical method encompassing the applied analytical method, then the LOD and LOQ of
one or more metal residues with a general concentration limit this method are given.)
can be appropriate if it can be shown that the exposure limit ‘Likely to be present’ refers to the metal used in the final
for none of the specified metals would be exceeded. manufacturing step and to metals that are used in earlier
manufacturing steps and not removed consistently by the
Any harmonized procedures for determining levels of metallic manufacturing process.
residues as described in the pharmacopoeias should be used, if
feasible. Otherwise, manufacturers are free to select the most 5. GLOSSARY
appropriate validated analytical procedure for a particular ACGIH : American Conference of Governmental Industrial
application. If only residuals of Class 2 or Class 3 metals are Hygienists
present, a non-specific method may be used. Specifically with ADI : Acceptable daily intake
respect to platinoid Class 1B, where a group limits applies, it
is accepted that due to technical limitations, the lower limit of ATSDR : Agency for Toxic Substances and Disease Registry
detection may not be below 0.5 ppm for individual platinoids. Body weight of an adult : the weight adjustment assumes an
arbitrary adult human body weight for either sex of 50 kg.
General semi-quantitative metal limit tests based on the This relatively low weight provides an additional safety factor
precipitation at pH 3.5 of coloured metal sulfides are against the standard weights of 60 kg or 70 kg that are often
described in several publications (e.g. Ph. Eur.). Such tests used as well
are not suitable to quantitatively determine the actual levels
of a specific metal residue in a pharmaceutical substance. bw : body weight of an adult
If adjusted (e.g. by using standard addition methods) and Daily dose : maximum daily dose related to the product mass
properly validated (including cross-validation with an of a pharmaceutical substance or a drug product
element-specific test), a test based on the principle of sulfide ESADDI : estimated safe and adequate daily intake
precipitation, may be suitable for routine testing in some cases. FSA : Food Standard Agency
4.5. BATCH RESULTS, TESTING FREQUENCY AND GDP : Good Distribution Practice (for medicinal products for
DELETING OF A TEST FROM THE SPECIFICATION human use)
If synthetic processes are known or suspected to lead to the GMP : Good Manufacturing Practice
presence of metal residues due to the use of a specific metal IPCS : International Program on Chemical Safety
catalyst or metal reagent, element specific assays should be
LOEL : Lowest-observed effect level
undertaken to determine the actual amount of these metal
residues, particularly during the development of the synthetic LOD : Limit of Detection
process. LOQ : Limit of Quantification
If the synthetic or manufacturing processes have shown to MDD : Maximum daily dose
result in the removal of a potential metal residue, routine NOEL : No-observed effect level
testing of that metal residue may be replaced by non-routine PDE : Permitted daily exposure
(skip) testing. A metal residue can be considered adequately Pharmaceutical substance : a substance in the drug product
removed if, in 6 consecutive pilot scale batches or 3 consecutive (normally an active pharmaceutical ingredient or an excipient)
industrial scale batches less than 30 per cent of the appropriate
PMTDI : Provisional maximum tolerable daily intake
concentration limit was found. A change from routine to
non-routine testing does not mean that the test may also be ppm : Parts per million
deleted from the specification. RfD : Reference Dose
Only for Class 3 metals, the test may be deleted from the TDI : Tolerable daily intake
relevant specification if the drug product manufacturer TTC : Threshold of toxicological concern
sufficiently demonstrates that the adequate removal of the UF : Uncertainty factor
metal residue from the pharmaceutical substance or the drug US EPA : United States Environmental Protection Agency
product is guaranteed. WHO : World Health Organization
4.6. REPORTING LEVELS OF METALLIC RESIDUES
Manufacturers of medicinal products need information 6. REFERENCES (SCIENTIFIC AND/OR LEGAL)
about the content of metallic residues in pharmaceutical – Directive 2001/83/EC (as amended by Directive
substances in order to meet the criteria of this guideline. 2004/24/EC)
Thus, it is necessary that the manufacturers of pharmaceutical – Guideline on the Limits of Genotoxic Impurities
substances provide a clear statement on the identity and (CPMP/SWP/5199/02)
– Impurities Testing Guideline : Impurities in New Drug – Note for Guidance on Validation of Analytical Methods :
Substances (CPMP/ICH/2737/99, ICHQ3A) Definitions and Terminology (CPMP/ICH/381/95,
– Maintenance Note for Guidance on Impurities : Residual ICHQ2A)
Solvents (CPMP/ICH/1507/02, ICHQ3C (M))
– Malo, J-L. Occupational rhinitis and asthma due to metal – Note for Guidance on Validation of Analytical Procedures :
salts. Allergy 60 : 138-139, 2005 Methodology (CPMP/ICH/281/95, ICHQ2B)
– Note for Guidance on Impurities in New Drug Products – Uter et al., Contact Dermatitis, 1995, 32, 135-142
(CPMP/ICH/2738/99, ICHQ3B (R))
– Note for Guidance on Impurities : Residual Solvents – Validation of Analytical Procedures : Text and Methodology
(CPMP/ICH/283/95, ICHQ3C) (CHMP/ICH/381/95, ICHQ2(R1))
General Notices (1) apply to all monographs and other texts 737
EUROPEAN PHARMACOPOEIA 8.0 Allergen products
General Notices (1) apply to all monographs and other texts 741
Allergen products EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 743
Extracts EUROPEAN PHARMACOPOEIA 8.0
Figure 2098.-1. – Chromatogram for the test for chromatographic profile of essential oils
preliminary treatment, for example, inactivation of enzymes, Liquid extracts – extracta fluida
grinding or defatting. In addition, unwanted matter may be
removed after extraction. DEFINITION
Liquid extracts are liquid preparations of which, in general,
Herbal drugs, animal matter and organic solvents used
1 part by mass or volume is equivalent to 1 part by mass of the
for the preparation of extracts comply with any relevant
dried herbal drug or animal matter. These preparations are
monograph of the Pharmacopoeia. For soft and dry extracts
adjusted, if necessary, so that they satisfy the requirements for
where the organic solvent is removed by evaporation,
content of solvent, and, where applicable, for constituents.
recovered or recycled solvent may be used, provided that the
recovery procedures are controlled and monitored to ensure PRODUCTION
that solvents meet appropriate standards before re-use or Liquid extracts are prepared by using ethanol of a suitable
admixture with other approved materials. Water used for the concentration or water to extract the herbal drug or animal
preparation of extracts is of a suitable quality. Except for the matter, or by dissolving a soft or dry extract (which has been
test for bacterial endotoxins, water complying with the section produced using the same strength of extraction solvent as is
on Purified water in bulk in the monograph on Purified used in preparing the liquid extract by direct extraction) of
water (0008) is suitable. Potable water may be suitable if it the herbal drug or animal matter in either ethanol of a suitable
complies with a defined specification that allows the consistent concentration or water. Liquid extracts may be filtered, if
production of a suitable extract. necessary.
Where applicable, concentration to the intended consistency A slight sediment may form on standing, which is acceptable
is carried out using suitable methods, usually under reduced as long as the composition of the liquid extract is not changed
pressure and at a temperature at which deterioration of the significantly.
constituents is reduced to a minimum. Essential oils that
have been separated during processing may be restored to the TESTS
extracts at an appropriate stage in the manufacturing process. Relative density (2.2.5). Where applicable, the liquid extract
Suitable excipients may be added at various stages of the complies with the limits prescribed in the monograph.
manufacturing process, for example to improve technological
qualities such as homogeneity or consistency. Suitable Ethanol (2.9.10). For alcoholic liquid extracts, carry out
stabilisers and antimicrobial preservatives may also be added. the determination of ethanol content. The ethanol content
complies with that prescribed.
Extraction with a given solvent leads to typical proportions
of characterised constituents in the extractable matter ; Methanol and 2-propanol (2.9.11) : maximum 0.05 per
during production of standardised and quantified extracts, cent V/V of methanol and maximum 0.05 per cent V/V of
purification procedures may be applied that increase these 2-propanol for alcoholic liquid extracts, unless otherwise
proportions with respect to the expected values ; such extracts prescribed.
are referred to as ‘refined’. Dry residue (2.8.16). Where applicable, the liquid extract
complies with the limits prescribed in the monograph,
IDENTIFICATION corrected if necessary, taking into account any excipient used.
Extracts are identified using a suitable method. STORAGE
Protected from light.
TESTS
LABELLING
Where applicable, as a result of analysis of the herbal drug
or animal matter used for production and in view of the The label states in addition to the requirements listed above :
production process, tests for microbiological quality (5.1.4), – where applicable, the ethanol content in per cent V/V in
heavy metals, aflatoxins and pesticide residues (2.8.13) in the the final extract.
extracts may be necessary.
Tinctures – tincturae
ASSAY
DEFINITION
Wherever possible, extracts are assayed by a suitable method. Tinctures are liquid preparations that are usually obtained
using either 1 part of herbal drug or animal matter and
LABELLING 10 parts of extraction solvent, or 1 part of herbal drug or
The label states : animal matter and 5 parts of extraction solvent.
– the herbal drug or animal matter used ; PRODUCTION
– whether the extract is liquid, soft or dry, or whether it is a Tinctures are prepared by maceration or percolation (outline
tincture ; methodology is given below) using only ethanol of a suitable
concentration for extraction of the herbal drug or animal
– for standardised extracts, the content of constituents with
matter, or by dissolving a soft or dry extract (which has been
known therapeutic activity ;
produced using the same strength of extraction solvent as
– for quantified extracts, the content of constituents is used in preparing the tincture by direct extraction) of
(markers) used for quantification ; the herbal drug or animal matter in ethanol of a suitable
– the ratio of the starting material to the genuine extract concentration. Tinctures are filtered, if necessary.
(extract without excipients) (DER) ; Tinctures are usually clear. A slight sediment may form on
standing, which is acceptable as long as the composition of
– the solvent or solvents used for extraction ; the tincture is not changed significantly.
– where applicable, that a fresh herbal drug or fresh animal Production by maceration. Unless otherwise prescribed,
matter has been used ; reduce the herbal drug or animal matter to be extracted to
– where applicable, that the extract is ‘refined’ ; pieces of suitable size, mix thoroughly with the prescribed
extraction solvent and allow to stand in a closed container
– the name and amount of any excipient used including for an appropriate time. The residue is separated from the
stabilisers and antimicrobial preservatives ; extraction solvent and, if necessary, pressed out. In the latter
– where applicable, the percentage of dry residue. case, the 2 liquids obtained are combined.
General Notices (1) apply to all monographs and other texts 745
Herbal drug preparations EUROPEAN PHARMACOPOEIA 8.0
Production by percolation. If necessary, reduce the herbal Dry extracts – extracta sicca
drug or animal matter to be extracted to pieces of suitable size.
Mix thoroughly with a portion of the prescribed extraction DEFINITION
solvent and allow to stand for an appropriate time. Transfer Dry extracts are solid preparations obtained by evaporation of
to a percolator and allow the percolate to flow at room the solvent used for their production. Dry extracts have a loss
temperature slowly making sure that the herbal drug or animal on drying of not greater than 5 per cent m/m, unless a loss on
matter to be extracted is always covered with the remaining drying with a different limit or a test on water is prescribed in
extraction solvent. The residue may be pressed out and the the monograph.
expressed liquid combined with the percolate.
TESTS
TESTS Water (2.2.13). Where applicable, the dry extract complies
Relative density (2.2.5). Where applicable, the tincture with the limits prescribed in the monograph.
complies with the limits prescribed in the monograph. Loss on drying (2.8.17). Where applicable, the dry extract
Ethanol (2.9.10). The ethanol content complies with that complies with the limits prescribed in the monograph.
prescribed. Solvents. Residual solvents are controlled as described in
Methanol and 2-propanol (2.9.11): maximum 0.05 per chapter 5.4, unless otherwise prescribed or justified and
cent V/V of methanol and maximum 0.05 per cent V/V of authorised.
2-propanol, unless otherwise prescribed.
STORAGE
Dry residue (2.8.16). Where applicable, the tincture complies In an airtight container, protected from light.
with the limits prescribed in the monograph, corrected if
necessary, taking into account any excipient used.
STORAGE 07/2010:1434
Protected from light.
HERBAL DRUG PREPARATIONS
LABELLING
The label states in addition to the requirements listed above : Plantae medicinales praeparatae
– for tinctures other than standardised and quantified DEFINITION
tinctures, the ratio of starting material to extraction liquid
Herbal drug preparations are homogeneous products
or of starting material to final tincture ;
obtained by subjecting herbal drugs to treatments such as
– the ethanol content in per cent V/V in the final tincture. extraction, distillation, expression, fractionation, purification,
concentration or fermentation.
Soft extracts – extracta spissa Herbal drug preparations include, for example, extracts,
essential oils, expressed juices, processed exudates, and herbal
DEFINITION drugs that have been subjected to size reduction for specific
Soft extracts are semi-solid preparations obtained by applications, for example herbal drugs cut for herbal teas or
evaporation or partial evaporation of the solvent used for powdered for encapsulation.
extraction. Herbal teas comply with the monograph Herbal teas (1435).
NOTE : the term comminuted used in European Community
TESTS legislation on herbal medicinal products describes a herbal
Dry residue (2.8.16). The soft extract complies with the limits drug that has been either cut or powdered.
prescribed in the monograph. The term herbal drug preparation is synonymous with the
Solvents. Residual solvents are controlled as described in term herbal preparation used in European Community
chapter 5.4, unless otherwise prescribed or justified and legislation on herbal medicinal products.
authorised.
STORAGE 01/2012:1433
Protected from light.
HERBAL DRUGS
Oleoresins – oleoresina
Plantae medicinales
DEFINITION
DEFINITION
Oleoresins are semi-solid extracts composed of a resin in
solution in an essential and/or fatty oil and are obtained by Herbal drugs are mainly whole, fragmented, or broken plants,
evaporation of the solvent(s) used for their production. parts of plants, algae, fungi or lichen, in an unprocessed state,
usually in dried form but sometimes fresh. Certain exudates
This monograph applies to oleoresins produced by extraction that have not been subjected to a specific treatment are also
and not to natural oleoresins. considered to be herbal drugs. Herbal drugs are precisely
defined by the botanical scientific name according to the
TESTS
binominal system (genus, species, variety and author).
Water (2.2.13). The oleoresin complies with the limits Whole describes a herbal drug that has not been reduced
prescribed in the monograph. in size and is presented, dried or undried, as harvested ; for
Solvents. Residual solvents are controlled as described in example : dog rose, bitter fennel or sweet fennel, Roman
chapter 5.4, unless otherwise prescribed or justified and chamomile flower.
authorised. Fragmented describes a herbal drug that has been reduced in
size after harvesting to permit ease of handling, drying and/or
STORAGE packaging ; for example : cinchona bark, rhubarb, passion
In an airtight container, protected from light. flower.
Broken describes a herbal drug in which the more-fragile Where necessary herbal drugs comply with other tests, such
parts of the plant have broken during drying, packaging as the following, for example.
or transportation ; for example : belladonna leaf, matricaria Total ash (2.4.16).
flower, hop strobile.
Ash insoluble in hydrochloric acid (2.8.1).
Cut describes a herbal drug that has been reduced in size,
other than by powdering, to the extent that the macroscopic Extractable matter.
description in the monograph of the herbal drug can no longer Swelling index (2.8.4).
be applied. When a herbal drug is cut for a specific purpose
that results in the cut herbal drug being homogeneous, Bitterness value (2.8.15).
for example when cut for herbal teas, it is a herbal drug Aflatoxin B1 (2.8.18). Where necessary, limits for aflatoxins
preparation. Certain cut herbal drugs processed in this way may be required.
may be the subject of an individual monograph. Ochratoxin A (2.8.22). Where necessary, a limit for
A herbal drug that complies with its monograph and is ochratoxin A may be required.
subsequently cut for extraction shall comply in its cut form,
Radioactive contamination. In some specific circumstances,
except for its macroscopic description, with the monograph
the risk of radioactive contamination is to be considered.
for that herbal drug, unless otherwise justified.
The term herbal drug is synonymous with the term herbal Microbial contamination. Where a herbal drug is used whole,
substance used in European Community legislation on herbal cut or powdered as an ingredient in a medicinal product, the
medicinal products. microbial contamination is controlled (5.1.8. Microbiological
quality of herbal medicinal products for oral use and extracts
PRODUCTION used in their preparation or 5.1.4. Microbiological quality of
Herbal drugs are obtained from cultivated or wild plants. non-sterile pharmaceutical preparations and substances for
Suitable collection, cultivation, harvesting, drying, pharmaceutical use.
fragmentation and storage conditions are essential to ASSAY
guarantee the quality of herbal drugs.
Unless otherwise prescribed or justified and authorised, herbal
Herbal drugs are, as far as possible, free from impurities such drugs are assayed by an appropriate method.
as soil, dust, dirt and other contaminants such as fungal, insect
and other animal contaminations. They are not rotten. STORAGE
If a decontaminating treatment has been used, it is necessary to Protected from light.
demonstrate that the constituents of the plant are not affected
and that no harmful residues remain. The use of ethylene
oxide is prohibited for the decontamination of herbal drugs.
01/2013:1435
IDENTIFICATION
Herbal drugs are identified using their macroscopic and
microscopic descriptions and any further tests that may be
HERBAL TEAS
required (for example, thin-layer chromatography).
Plantae ad ptisanam
TESTS
Foreign matter (2.8.2). Carry out a test for foreign matter, DEFINITION
unless otherwise prescribed or justified and authorised. The Herbal teas consist exclusively of one or more herbal drugs
content of foreign matter is not more than 2 per cent m/m, intended for oral aqueous preparations by means of decoction,
unless otherwise prescribed or justified and authorised. An infusion or maceration. The preparation is prepared
appropriate specific test may apply to herbal drugs liable to immediately before use.
be adulterated. It may not be possible to perform the test for Herbal teas are usually supplied in bulk form or in bags for
foreign matter on a herbal drug that is cut, as described under single use.
Definition, for either a specific purpose or for extraction.
The herbal drugs used comply with the appropriate individual
Under these circumstances the cut material is presumed to
European Pharmacopoeia monographs or in their absence
comply with the test for foreign matter providing that the
with the general monograph Herbal drugs (1433).
herbal drug prior to cutting was compliant with this test.
Loss on drying (2.2.32). Carry out a test for loss on drying, IDENTIFICATION
unless otherwise prescribed or justified and authorised. The identity of herbal drugs present in herbal teas is checked
Water (2.2.13). A determination of water may be carried out by suitable methods such as botanical examinations and/or
instead of a test for loss on drying for herbal drugs with a high chromatographic profiles.
essential-oil content.
TESTS
Pesticides (2.8.13). Herbal drugs comply with the
Recommendations on the microbiological quality of herbal
requirements for pesticide residues. The requirements take
teas (5.1.8) take into account the prescribed preparation
into account the nature of the plant, where necessary the
method (use of boiling or non-boiling water).
preparation in which the plant might be used, and where
available the knowledge of the complete record of treatment The proportion of herbal drugs present in herbal teas is
of the batch of the plant. checked by appropriate methods.
Heavy metals (2.4.27). Unless otherwise stated in an Herbal teas in bags comply with the following test :
individual monograph or unless otherwise justified and Uniformity of mass. Determine the individual and the
authorised : average mass of the contents of 20 randomly chosen units as
– cadmium : maximum 1.0 ppm ; follows : weigh a single full bag of herbal tea, open it without
– lead : maximum 5.0 ppm ; losing any fragments. Empty it completely using a brush.
Weigh the empty bag and calculate the mass of the contents
– mercury : maximum 0.1 ppm. by subtraction. Repeat the operation on the 19 remaining
Where necessary, limits for other heavy metals may be bags and calculate the average mass of the contents of the
required. 20 units. Unless otherwise justified, not more than 2 of the
General Notices (1) apply to all monographs and other texts 747
Herbal teas, instant EUROPEAN PHARMACOPOEIA 8.0
DEFINITION PRODUCTION
Instant herbal teas consist of 1 or more herbal drug GENERAL PROVISIONS
preparations (primarily extracts with or without added The production method shall have been shown to yield
essential oils), and are intended for the preparation of an oral
consistently immunosera of acceptable safety, potency in man
solution immediately before use. and stability.
Instant herbal teas may also contain, in addition to herbal Any reagent of biological origin used in the production of
drug preparations, suitable excipients such as maltodextrin immunosera shall be free of contamination with bacteria,
and added flavourings. fungi and viruses. The general requirements of chapter 5.1.7.
Instant herbal teas are presented as a powder or granules and Viral safety apply to the manufacture of animal immunosera
are usually supplied in bulk form or in sachets. for human use, in conjunction with the more specific
The herbal drug preparations used comply with the requirements relating to viral safety in this monograph. The
appropriate individual European Pharmacopoeia monographs method of preparation includes a step or steps that have been
or, in the absence of such individual monographs, with the shown to remove or inactivate known agents of infection.
general monograph Herbal drug preparations (1434) and Methods used for production are validated, effective,
with other appropriate general monographs, for example reproducible and do not impair the biological activity of the
Extracts (0765) or Essential oils (2098). product.
IDENTIFICATION The production method is validated to demonstrate that the
product, if tested, would comply with the test for abnormal
The identity of herbal drug preparations present in instant toxicity for immunosera and vaccines for human use (2.6.9).
herbal teas is checked by suitable methods.
Reference preparation. A batch shown to be suitable in clinical
TESTS trials, or a batch representative thereof, is used as the reference
General chapter 5.1.8 contains recommendations on the preparation for the tests for high molecular mass proteins and
microbiological quality of extract-containing herbal medicinal purity.
products such as instant herbal teas. ANIMALS
The proportion of herbal drug preparations present in instant The animals used are of a species approved by the competent
herbal teas is checked by suitable methods. authority, are healthy and are exclusively reserved for
Instant herbal teas in sachets comply with the following test. production of immunoserum. They are tested and
shown to be free from a defined list of infectious agents.
Uniformity of mass. Determine the individual and the The introduction of animals into a closed herd follows
average mass of the contents of 20 randomly chosen units specified procedures, including definition of quarantine
as follows : weigh a single full sachet of instant herbal tea, measures. Where appropriate, additional specific agents are
open it without losing any fragments. Empty it completely considered depending on the geographical localisation of the
using a brush. Weigh the empty sachet and calculate the mass establishment used for the breeding and production of the
of the contents by subtraction. Repeat the operation on the animals. The feed originates from a controlled source and
19 remaining sachets and calculate the average mass of the no animal proteins are added. The suppliers of animals are
contents of the 20 units. Unless otherwise justified, not more certified by the competent authority.
than 2 of the individual masses deviate from the average mass If the animals are treated with antibiotics, a suitable withdrawal
by more than the percentage deviation shown in the table period is allowed before collection of blood or plasma. The
below and none deviates by more than twice that percentage. animals are not treated with penicillin antibiotics. If a live
Average mass Percentage deviation vaccine is administered, a suitable waiting period is imposed
between vaccination and collection of serum or plasma for
less than 1.5 g 15 per cent immunoserum production.
1.5 g to 2.0 g included 10 per cent IMMUNISATION
more than 2.0 g 7.5 per cent The antigens used are identified and characterised, where
appropriate ; where relevant, they are shown to be free from
extraneous infectious agents. They are identified by their
STORAGE names and a batch number ; information on the source and
Protected from light. preparation are recorded.
The selected animals are isolated for at least 1 week before After purification and treatment for removal and/or
being immunised according to a defined schedule, with inactivation of viruses, a stabiliser may be added to the
booster injections at suitable intervals. Adjuvants may be used. intermediate product, which may be stored for a period
Animals are kept under general health surveillance and defined in light of stability data.
specific antibody production is controlled at each cycle of Only an intermediate product that complies with the following
immunisation. requirements may be used in the preparation of the final bulk.
Animals are thoroughly examined before collection of blood Purity. Examine by non-reducing polyacrylamide gel
or plasma. If an animal shows any pathological lesion not electrophoresis (2.2.31), by comparison with the reference
related to the immunisation process, it is not used, nor are preparation. The bands are compared in intensity and no
any other of the animals in the group concerned, unless it is additional bands are found.
evident that their use will not impair the safety of the product.
FINAL BULK
COLLECTION OF BLOOD OR PLASMA
The final bulk is prepared from a single intermediate product
Collection of blood is made by venepuncture or or from a pool of intermediate products obtained from
plasmapheresis. The puncture area is shaved, cleaned animals of the same species. Intermediate products with
and disinfected. The animals may be anaesthetised under different specificities may be pooled.
conditions that do not influence the quality of the product.
Unless otherwise prescribed, an antimicrobial preservative An antimicrobial preservative and a stabiliser may be added.
may be added. The blood or plasma is collected in such a If an antimicrobial preservative has been added to the blood
manner as to maintain sterility of the product. The blood or or plasma, the same substance is used as the antimicrobial
plasma collection is conducted at a site separate from the area preservative in the final bulk.
where the animals are kept or bred and the area where the Only a final bulk that complies with the following requirements
immunoserum is purified. If the serum or plasma is stored may be used in the preparation of the final lot.
before further processing, precautions are taken to avoid
Antimicrobial preservative. Where applicable, determine
microbial contamination.
the amount of antimicrobial preservative by a suitable
Several single plasma or serum samples may be pooled before physico-chemical method. It contains not less than 85 per
purification. The single or pooled samples are tested before cent and not more than 115 per cent of the amount stated on
purification for the following tests. the label.
Tests for contaminating viruses. If an antimicrobial
Sterility (2.6.1). It complies with the test for sterility.
preservative is added, it must be neutralised before carrying
out the tests, or the tests are carried out on a sample taken FINAL LOT
before addition of the antimicrobial preservative. Each pool is The final bulk of immunoserum is distributed aseptically into
tested for contaminating viruses by suitable in vitro tests. sterile, tamper-proof containers. The containers are closed so
Each pool is tested for viruses by inoculation to cell cultures as to prevent contamination.
capable of detecting a wide range of viruses relevant for the Only a final lot that complies with the requirements prescribed
particular product. below under Identification, Tests and Assay may be released
for use. Provided that the tests for osmolality, protein content,
Potency. Carry out a biological assay as indicated in the
molecular-size distribution, antimicrobial preservative,
monograph and express the result in International Units per
stabiliser, purity, foreign proteins and albumin and the assay
millilitre, where applicable. A validated in vitro method may
have been carried out with satisfactory results on the final
also be used.
bulk, they may be omitted on the final lot.
Protein content. Dilute the product to be examined with
a 9 g/L solution of sodium chloride R to obtain a solution Reconstitute the preparation to be examined as stated on the
containing about 15 mg of protein in 2 mL. To 2 mL of this label immediately before carrying out the identification, tests
solution in a round-bottomed centrifuge tube add 2 mL (except those for solubility and water) and assay.
of a 75 g/L solution of sodium molybdate R and 2 mL of a
mixture of 1 volume of nitrogen-free sulfuric acid R and IDENTIFICATION
30 volumes of water R. Shake, centrifuge for 5 min, decant
the supernatant and allow the inverted tube to drain on filter The identity is established by immunological tests and, where
paper. Determine the nitrogen in the residue by the method necessary, by determination of biological activity. The assay
of sulfuric acid digestion (2.5.9) and calculate the content of may also serve for identification.
protein by multiplying by 6.25. The protein content is within
approved limits. CHARACTERS
PURIFICATION AND VIRAL INACTIVATION Immunosera are clear to opalescent and colourless to
The immunoglobulins are concentrated and purified by very faintly yellow liquids. They are free from turbidity.
fractional precipitation, chromatography, immunoadsorption Freeze-dried products are white or slightly yellow powders or
or by other chemical or physical methods. They may be solid friable masses. After reconstitution they show the same
processed further by enzyme treatment. The methods are characteristics as liquid preparations.
selected and validated to avoid contamination at all steps of
processing and to avoid formation of protein aggregates that TESTS
affect the immunobiological characteristics of the product. For
products intended to consist of immunoglobulin fragments, Solubility. To a container of the preparation to be examined,
the methods are validated to guarantee total fragmentation. add the volume of the liquid for reconstitution stated on the
The methods of purification used are such that they do not label. The preparation dissolves completely within the time
generate additional components that compromise the quality stated on the label.
and the safety of the product. Extractable volume (2.9.17). It complies with the requirement
Unless otherwise justified and authorised, validated for extractable volume.
procedures are applied for removal and/or inactivation of
viruses. The procedures are selected to avoid the formation of pH (2.2.3). The pH is within the limits approved for the
polymers or aggregates and, unless the product is intended to particular product.
consist of Fab′ fragments, to minimise the splitting of F(ab′)2 Osmolality (2.2.35) : minimum 240 mosmol/kg after dilution,
into Fab′ fragments. where applicable.
General Notices (1) apply to all monographs and other texts 749
Immunosera for veterinary use EUROPEAN PHARMACOPOEIA 8.0
Protein content: 90 per cent to 110 per cent of the amount – the route of administration ;
stated on the label, and, unless otherwise justified and – the storage conditions ;
authorised, not more than 100 g/L. – the expiry date, except for containers of less than 1 mL
Dilute the preparation to be examined with a 9 g/L solution which are individually packed ; the expiry date may be
of sodium chloride R to obtain a solution containing about omitted from the label on the container, provided it is
15 mg of protein in 2 mL. To 2 mL of this solution in a shown on the package and the label on the package states
round-bottomed centrifuge tube add 2 mL of a 75 g/L solution that the container must be kept in the package until
of sodium molybdate R and 2 mL of a mixture of 1 volume of required for use ;
nitrogen-free sulfuric acid R and 30 volumes of water R. Shake, – the animal species of origin ;
centrifuge for 5 min, decant the supernatant and allow the
inverted tube to drain on filter paper. Determine the nitrogen – the name and amount of any antimicrobial preservative,
in the residue by the method of sulfuric acid digestion (2.5.9) any stabiliser and any other excipient.
and calculate the content of protein by multiplying by 6.25.
Molecular-size distribution. Examine by liquid 01/2008:0030
chromatography (2.2.29 or 2.2.30). It complies with the
specification approved for the particular product. IMMUNOSERA FOR VETERINARY USE
Antimicrobial preservative. Where applicable, determine
the amount of antimicrobial preservative by a suitable Immunosera ad usum veterinarium
physicochemical method. The amount is not less than the
minimum amount shown to be effective and is not greater DEFINITION
than 115 per cent of that stated on the label. Immunosera for veterinary use are preparations containing
Phenol (2.5.15) : maximum 2.5 g/L for preparations containing immunoglobulins, purified immunoglobulins or
phenol. immunoglobulin fragments obtained from serum or plasma
of immunised animals. They may be preparations of crude
Stabiliser. Determine the amount of stabiliser by a suitable polyclonal antisera or purified preparations.
physico-chemical method. The preparation contains not
less than 80 per cent and not more than 120 per cent of the The immunoglobulins or immunoglobulin fragments have
quantity stated on the label. the power of specifically neutralising the antigen used for
immunisation. The antigens include microbial or other toxins,
Purity. Examine by non-reducing polyacrylamide gel bacterial and viral antigens, venoms of snakes and hormones.
electrophoresis (2.2.31), by comparison with the reference The preparation is intended for parenteral administration to
preparation. No additional bands are found for the preparation provide passive immunity.
to be examined.
Foreign proteins. When examined by precipitation tests PRODUCTION
with specific antisera, only protein from the declared animal GENERAL PROVISIONS
species is shown to be present, unless otherwise prescribed, Immunosera are obtained from the serum or plasma of
for example where material of human origin is used during healthy animals immunised by administration of one or more
production. suitable antigens.
Albumin. Unless otherwise prescribed in the monograph, The production method shall have been shown to yield
when examined electrophoretically, the content of albumin is consistently batches of immunosera of acceptable safety (5.2.6)
not greater than the limit approved for the particular product and efficacy (5.2.7).
and, in any case, is not greater than 3 per cent. DONOR ANIMALS
Water (2.5.12): maximum 3 per cent. The animals used are exclusively reserved for production
Sterility (2.6.1). It complies with the test for sterility. of immunoserum. They are maintained under conditions
protecting them from the introduction of disease, as far as
Pyrogens (2.6.8). Unless otherwise justified and authorised, possible. The donor animals, and any animals in contact with
it complies with the test for pyrogens. Unless otherwise them, are tested and shown to be free from a defined list of
prescribed, inject 1 mL per kilogram of the rabbit’s body mass. infectious agents and re-tested at suitable intervals. The list
of agents for testing includes not only those agents that are
ASSAY
relevant to the donor animal, but also those that are relevant to
Carry out a biological assay as indicated in the monograph and the recipient target species for the product. Where the donor
express the result in International Units per millilitre, where animals have not been demonstrated to be free from a relevant
appropriate. A validated in vitro method may also be used. pathogen, a justification must be provided and a validated
STORAGE inactivation or purification procedure must be included in
the manufacturing procedure. The feed originates from a
Protected from light, at the temperature stated on the label. controlled source. Where the donor animals are chickens, use
Do not allow liquid preparations to freeze. chickens from a flock free from specified pathogens (5.2.2).
Expiry date. The expiry date is calculated from the beginning Where applicable for the species used, measures are taken to
of the assay. avoid contamination with agents of transmissible spongiform
encephalopathies.
LABELLING As far as possible, animals being introduced into the herd are
The label states : from a known source and have a known breeding and rearing
– the number of International Units per millilitre, where history. The introduction of animals into the herd follows
applicable ; specified procedures, including defined quarantine measures.
– the amount of protein per container ; During the quarantine period the animals are observed and
tested to establish that they are free from the list of agents
– for freeze-dried preparations : relevant for the donor animals. It may be necessary to test the
– the name and volume of the reconstituting liquid to be animals in quarantine for freedom from additional agents,
added ; depending on their known breeding and rearing history or
– that the immunoserum is to be used immediately after any lack of information on their source.
reconstitution ; Any routine or therapeutic medicinal treatment administered
– the time required for complete dissolution ; to the animals in quarantine or thereafter must be recorded.
General Notices (1) apply to all monographs and other texts 751
Immunosera for veterinary use EUROPEAN PHARMACOPOEIA 8.0
It is recognised that, in accordance with General Notices Total protein. For products where claims are being made
(section 1.1. General statements), for an established antiserum which relate to the protein content, as well as demonstrating
the routine application of the safety test will be waived by the that the batch contains not more than the stated upper limit,
competent authority in the interests of animal welfare when a the batch shall be shown to contain not less than that in the
sufficient number of consecutive batches have been produced batch or batches shown to be effective in the efficacy studies.
and found to comply with this test, thus demonstrating Extraneous agents. In addition to the test described under
consistency of the manufacturing process. Significant changes Tests, specific tests may be required depending on the nature
to the manufacturing process may require resumption of of the preparation, its risk of contamination and the use of the
routine testing to re-establish consistency. The number product. In particular, specific tests for important potential
of consecutive batches to be tested depends on a number pathogens may be required when the donor and recipient
of factors such as the type of antiserum, the frequency of species are the same and when these agents would not be
production of batches, and experience with the immunoserum detected reliably by the general screening test described under
during developmental safety testing and during application of Tests.
the batch safety test. Without prejudice to the decision of the
competent authority in the light of information available for Water. Where applicable, the freeze-drying process is checked
a given antiserum, testing of 10 consecutive batches is likely by a determination of water and shown to be within the limits
to be sufficient for the majority of products. For products set for the product.
with an inherent safety risk, it may be necessary to continue IDENTIFICATION
to conduct the safety test on each batch.
The identity of the product is established by immunological
tests and, where necessary, by determination of biological
Animal tests. In accordance with the provisions of the activity. The potency test may also serve for identification.
European Convention for the Protection of Vertebrate
Animals Used for Experimental and Other Scientific Purposes, TESTS
tests must be carried out in such a way as to use the minimum The following requirements refer to liquid immunosera and
number of animals and to cause the least pain, suffering, reconstituted freeze-dried immunosera.
distress or lasting harm. The criteria for judging tests in
monographs must be applied in the light of this. For example, Foreign proteins. When examined by precipitation tests with
if it is indicated that an animal is considered to show positive,specific antisera against plasma proteins of a suitable range
infected etc. when typical clinical signs occur then as soon of species, only protein from the declared animal species is
as sufficient indication of a positive result is obtained the shown to be present.
animal in question shall be either euthanised or given suitable Albumin. Purified immunosera comply with a test for
treatment to prevent unnecessary suffering. In accordance albumin. Unless otherwise prescribed in the monograph,
with the General Notices, alternative test methods may be when examined electrophoretically, purified immunosera
used to demonstrate compliance with the monograph and the show not more than a trace of albumin, and the content
use of such tests is particularly encouraged when this leads of albumin is in any case not greater than 30 g/L of the
to replacement or reduction of animal use or reduction of reconstituted preparation, where applicable.
suffering. Total protein. Dilute the preparation to be examined with
pH (2.2.3). The pH of crude and purified immunosera is a 9 g/L solution of sodium chloride R to obtain a solution
shown to be within the limits set for the products. containing about 15 mg of protein in 2 mL. To 2 mL of this
solution in a round-bottomed centrifuge tube add 2 mL of a
Formaldehyde. If formaldehyde is used for production of 75 g/L solution of sodium molybdate R and 2 mL of a mixture
immunoserum, a test for free formaldehyde is carried out as of 1 volume of nitrogen-free sulfuric acid R and 30 volumes of
prescribed under Tests. water R. Shake, centrifuge for 5 min, discard the supernatant
Other inactivating agents. When other inactivation methods and allow the inverted tube to drain on filter paper. Determine
are used, appropriate tests are carried out to demonstrate that the nitrogen in the residue by the method of sulfuric acid
the inactivating agent has been removed or reduced to an digestion (2.5.9) and calculate the content of protein by
acceptable residual level. multiplying by 6.25. The results obtained are not greater than
the upper limit stated on the label.
Batch potency test. If a specific monograph exists for the
product, the test described under Potency is not necessarily Antimicrobial preservative. Determine the amount of
carried out for routine testing of batches of antiserum. The antimicrobial preservative by a suitable physicochemical
type of batch potency test to be carried out will depend on method. The amount is not less than the minimum amount
the claims being made for the product. Wherever possible, shown to be effective and is not greater than 115 per cent of
in vitro tests must be used. The type of test required may that stated on the label.
include measurement of antibodies against specific infectious Formaldehyde (2.4.18). Where formaldehyde has been used
organisms, determination of the type of antibody (e.g. in the preparation, the concentration of free formaldehyde
neutralising or opsonising). All tests must be validated. The is not greater than 0.5 g/L, unless a higher amount has been
criteria for acceptance must be set with reference to a batch shown to be safe.
that has been shown to comply with the requirements specified Sterility (2.6.1). Immunosera for veterinary use comply with
under Potency if a specific monograph exists for the product, the test for sterility. When the volume of liquid in a container
and which has been shown to have satisfactory efficacy, in is greater than 100 mL, the method of membrane filtration
accordance with the claims being made for the product. is used wherever possible. If this method is used, incubate
Total immunoglobulins. A test for the quantities of total the media for not less than 14 days. Where the method of
immunoglobulins and/or total gammaglobulins and/or membrane filtration cannot be employed, the method of direct
specific immunoglobulin classes is carried out. The results inoculation may be used. Where the volume of liquid in each
obtained must be within the limits set for the product and container is at least 20 mL, the minimum volume to be used
agreed with the competent authority. The batch contains not for each culture medium is 10 per cent of the contents of the
more than the level shown to be safe in the safety studies and, container or 5 mL, whichever is the least. The appropriate
unless the batch potency test specifically covers all appropriate number of items to be tested (2.6.1) is 1 per cent of the batch
immunoglobulins, the level in the batch is not less than that with a minimum of 4 and a maximum of 10.
in the batch or batches shown to be effective in the efficacy Mycoplasmas (2.6.7). Immunosera for veterinary use comply
studies. with the test for mycoplasmas.
General Notices (1) apply to all monographs and other texts 753
Monoclonal antibodies for human use EUROPEAN PHARMACOPOEIA 8.0
– adequate removal of product- and process-related – for recombinant DNA products, consistency of the coding
impurities (for example, host-cell protein and DNA, sequence of the expression construct in cells cultivated to
protein A, antibiotics, cell-culture components) ; the limit of in vitro cell age for production use or beyond,
– specificity and biological activity of the monoclonal by either nucleic acid testing or product analysis.
antibody ;
– absence of non-endotoxin pyrogens, where applicable ; CELL BANKS
– reusability of purification components (for example, The master cell bank is a homogeneous suspension of the cell
column material), limits or acceptance criteria being set as line producing the monoclonal antibody, distributed in equal
a function of the validation ; volumes in a single operation into individual containers for
– methods used for conjugation, where applicable. storage.
A working cell bank is a homogeneous suspension of the cell
Product characterisation. The product is characterised material derived from the master cell bank at a finite passage
to obtain adequate information including : structural level, distributed in equal volumes in a single operation into
integrity, isotype, amino-acid sequence, secondary structure, individual containers for storage.
carbohydrate moiety, disulfide bridges, conformation,
specificity, affinity, biological activity and heterogeneity Post-production cells are cells cultured up to or beyond the
(characterisation of isoforms). population doubling level or generation number used for
routine production.
A battery of suitable analytical techniques is used including
chemical, physical, immunochemical and biological The following tests are performed on the master cell bank :
tests (for example, peptide mapping, N- and C-terminal viability, identity, absence of bacterial, fungal and mycoplasmal
amino-acid sequencing, mass spectrometry, chromatographic, contamination, characterisation of the monoclonal antibody
electrophoretic and spectroscopic techniques). Additional produced. Adventitious viral contamination is tested with
tests are performed to obtain information on cross-reactivity a suitable range of in vivo and in vitro tests. Retrovirus
with human tissues. and other endogenous viral contamination is tested using a
suitable range of in vitro tests.
For those products that are modified by fragmentation
or conjugation, the influence of the methods used on the The following tests are performed on the working cell bank :
antibody is characterised. viability, identity, absence of bacterial, fungal and mycoplasmal
contamination. Adventitious viral contamination is tested
Process intermediates. Where process intermediates are with a suitable range of in vivo and in vitro tests. For
stored, an expiry date or a storage period justified by stability the first working cell bank, these tests are performed on
data is established for each. post-production cells, generated from that working cell bank ;
Biological assay. The biological assay is chosen in terms for working cell banks subsequent to the first working cell
of its correlation with the intended mode of action of the bank, a single in vitro and in vivo test can be done either
monoclonal antibody. directly on the working cell bank or on post-production cells.
Reference preparation. A batch shown to be stable and For the master cell bank and working cell bank, tests for
shown to be suitable in clinical trials, or a batch representative specific viruses are carried out when potentially contaminated
thereof, is used as a reference preparation for the identification, biological material has been used during preparation of the
tests and assay. The reference preparation is appropriately cell banks, taking into account the species of origin of this
characterised as defined under Product characterisation, material. This may not be necessary when this material is
except that it is not necessary to examine cross-reactivity for inactivated using validated procedures.
each batch of reference preparation. The following tests are performed on the post-production
Definition of a batch. Definition of a batch is required cells : absence of bacterial, fungal and mycoplasmal
throughout the process. contamination. Adventitious viral contamination is tested
with a suitable range of in vivo and in vitro tests. Retrovirus
and other endogenous viral contamination is tested using a
SOURCE CELLS suitable range of in vitro tests.
Source cells include fusion partners, lymphocytes, myeloma
cells, feeder cells and host cells for the expression of the
recombinant monoclonal antibody. CULTURE AND HARVEST
The origin and characteristics of the parental cell are Production at finite passage level (single harvest). Cells
documented, including information on the health of the are cultivated up to a defined maximum number of passages
donors, and on the fusion partner used (for example, myeloma or population doublings, or up to a fixed harvest time (in
cell line, human lymphoblastoid B-cell line). accordance with the stability of the cell line). Product is
Wherever possible, source cells undergo suitable screening harvested in a single operation.
for extraneous agents and endogenous agents. The choice of Continuous-culture production (multiple harvest). Cells
viruses for the tests is dependent on the species and tissue of are continuously cultivated for a defined period (in accordance
origin. with the stability of the system and production consistency).
Monitoring is necessary throughout the life of the culture ; the
required frequency and type of monitoring will depend on the
CELL LINE PRODUCING THE MONOCLONAL ANTIBODY
nature of the production system.
The suitability of the cell line producing the monoclonal
antibody is demonstrated by : Each harvest is tested for antibody content, bioburden,
endotoxin and mycoplasmas. General or specific tests
– documentation on the history of the cell line including for adventitious viruses are carried out at a suitable stage
description of the cell fusion, immortalisation or depending on the nature of the manufacturing process and
transfection and cloning procedure ; the materials used. For processes using production at finite
– characterisation of the cell line (for example, phenotype, passage level (single harvest), at least 3 harvests are tested for
isoenzyme analysis, immunochemical markers and adventitious viruses using a suitable range of in vitro methods.
cytogenetic markers) ; The acceptance criteria for harvests for further processing
– characterisation of relevant features of the antibody ; are clearly defined and linked to the schedule of monitoring
– consistency of critical quality attributes for the antibody up applied. If any adventitious viruses are detected, the process
to or beyond the population doubling level or generation is carefully investigated to determine the cause of the
number used for routine production ; contamination and the harvest is not further processed.
Harvests in which an endogenous virus has been detected are Solubility. Freeze-dried preparations dissolve completely
not used for purification unless an appropriate action plan has in the prescribed volume of reconstituting liquid, within a
been defined to prevent transmission of infectious agents. defined time, as approved for the particular product.
pH (2.2.3). It complies with the limits approved for the
PURIFICATION particular product.
Harvests or intermediate pools may be pooled before Osmolality (2.2.35) : minimum 240 mosmol/kg, unless
further processing. The purification process includes otherwise justified and authorised.
steps that remove and/or inactivate non-enveloped and
enveloped viruses. A validated purification process, for Extractable volume (2.9.17). It complies with the test for
which removal and/or inactivation of infectious agents and extractable volume.
removal of product- and process-related impurities has been Total protein (2.5.33). It complies with the limits approved
demonstrated, is used. Defined steps of the process lead to a for the particular product.
purified monoclonal antibody (active substance) of consistent Molecular-size distribution. Molecular-size distribution is
quality and biological activity. determined by a suitable method, for example size-exclusion
chromatography (2.2.30). It complies with the limits approved
ACTIVE SUBSTANCE for the particular product.
The test programme for the active substance depends on the Molecular identity and structural integrity. Depending on
validation of the process, on demonstration of consistency the nature of the monoclonal antibody, its microheterogeneity
and on the expected level of product- and process-related and isoforms, a number of different tests can be used to
impurities. The active substance is tested for appearance, demonstrate molecular identity and structural integrity.
identity, bioburden and bacterial endotoxins, product-related These tests may include peptide mapping, isoelectric focusing,
substances, product- and process-related impurities including ion-exchange chromatography, hydrophobic interaction
tests for host-cell-derived proteins and host-cell- and chromatography, oligosaccharide mapping, monosaccharide
vector-derived DNA, as well as structural integrity, protein content and mass spectrometry.
content and biological activity by suitable analytical methods,
comparing with the reference preparation where necessary. Purity. Tests for process- and product-related impurities are
When the active substance is a conjugated or transformed carried out by suitable validated methods. Provided that tests
antibody, appropriate tests must be performed before and for process-related impurities have been carried out on the
after the antibody conjugation/modification. active substance or on the final bulk with satisfactory results,
they may be omitted on the final lot.
If storage of intermediates is intended, adequate stability of
these preparations and its impact on quality or shelf-life of the Stabiliser. Where applicable, it complies with the limits
finished product are evaluated. approved for the particular product.
Water (2.5.12). Freeze-dried products comply with the limits
FINAL BULK approved for the particular product.
One or more batches of active substance may be combined to Sterility (2.6.1). It complies with the test for sterility.
produce the final bulk. Suitable stabilisers and other excipients
Bacterial endotoxins (2.6.14). It complies with the limits
may be added during preparation of the final bulk.
approved for the particular product.
The final bulk must be stored under validated conditions with
respect to bioburden and stability. Tests applied to modified antibodies. Suitable tests are
carried out depending on the type of modification.
FINAL LOT
The final bulk is sterile-filtered and distributed under aseptic ASSAY
conditions into sterile containers, which may subsequently be Carry out a suitable biological assay compared to the reference
freeze-dried. preparation. Design of the assay and calculation of the results
As part of the in-process control each container (vial, syringe are made according to the usual principles (for example, 5.3).
or ampoule) is inspected after filling to eliminate containers
that contain visible particles. During development of the
product it must be demonstrated that either the process will STORAGE
not generate visible proteinaceous particles in the final lot As stated on the label.
or such particles are reduced to a low level as justified and
Expiry date. The expiry date is calculated from the date of
authorised.
sterile filtration, the date of filling (for liquid preparations) or
CHARACTERS the date of freeze-drying (where applicable).
Liquid preparations are clear or slightly opalescent, colourless
LABELLING
or slightly coloured liquids. Freeze-dried products are white
or slightly coloured powders or solid friable masses. After The label states :
reconstitution they show the same characteristics as liquid – the number of units per millilitre, where applicable ;
preparations. – the quantity of protein per container ;
IDENTIFICATION – the quantity of monoclonal antibody in the container ;
The identity is established by suitable validated methods – for liquid preparations, the volume of the preparation in
comparing the product with the reference preparation, where the container ;
appropriate. The assay also contributes to identification. – for freeze-dried preparations :
TESTS – the name and the volume of the reconstitution liquid
to be added ;
Appearance. Liquid or reconstituted freeze-dried preparations
comply with the limits approved for the particular product – the period of time within which the monoclonal
with regard to degree of opalescence (2.2.1) and degree of antibody is to be used after reconstitution ;
coloration (2.2.2). They are without visible particles, unless – the dilution to be made before use of the product, where
otherwise justified and authorised. applicable.
General Notices (1) apply to all monographs and other texts 755
Pharmaceutical preparations EUROPEAN PHARMACOPOEIA 8.0
Microbiological quality. The formulation of the Appearance. The appearance (e.g. size, shape and colour) of
pharmaceutical preparation and its container must ensure that the pharmaceutical preparation is controlled.
the microbiological quality is suitable for the intended use. Identity and purity tests. Where applicable, the following
During development, it shall be demonstrated that the tests are carried out on the pharmaceutical preparation:
antimicrobial activity of the preparation as such or, if – identification of the active substance(s) ;
necessary, with the addition of a suitable preservative or
preservatives, or by the selection of an appropriate container, – identification of specific excipient(s), such as preservatives ;
provides adequate protection from adverse effects that may – purity tests (e.g. investigation of degradation products,
arise from microbial contamination or proliferation during residual solvents (2.4.24) or other related impurities,
the storage and use of the preparation. A suitable test method sterility (2.6.1)) ;
together with criteria for evaluating the preservative properties – safety tests (e.g. safety tests for biological products).
of the formulation are provided in general chapter 5.1.3.
Efficacy of antimicrobial preservation. Uniformity (2.9.40 or 2.9.5/2.9.6). Pharmaceutical
preparations presented in single-dose units comply with the
If preparations do not have adequate antimicrobial efficacy
test(s) as prescribed in the relevant specific dosage form
and do not contain antimicrobial preservatives they are
monograph. If justified and authorised, general chapter 2.9.40
supplied in single-dose containers, or in multidose containers
can be applicable only at the time of release.
that prevent microbial contamination of the contents after
opening. Special uniformity requirements apply in the following cases :
In the manufacture/preparation of non-sterile pharmaceutical – for herbal drugs and herbal drug preparations, compliance
preparations, suitable measures are taken to ensure their with general chapter 2.9.40 is not required ;
microbial quality ; recommendations on this aspect are – for homoeopathic preparations, the provisions of general
provided in general chapters 5.1.4. Microbiological quality chapters 2.9.6 and 2.9.40 are normally not appropriate,
of non-sterile pharmaceutical preparations and substances however in certain circumstances compliance with these
for pharmaceutical use and 5.1.8. Microbiological quality of chapters may be required by the competent authority ;
herbal medicinal products for oral use and extracts used in
– for single- and multivitamin and trace-element
their preparation.
preparations, compliance with general chapters 2.9.6 and
Sterile preparations are manufactured/prepared using 2.9.40 (content uniformity only) is not required ;
materials and methods designed to ensure sterility and to
– in justified and authorised circumstances, for other
avoid the introduction of contaminants and the growth
preparations, compliance with general chapters 2.9.6 and
of micro-organisms ; recommendations on this aspect are
2.9.40 may not be required by the competent authority.
provided in general chapter 5.1.1. Methods of preparation of
sterile products. Reference standards. Reference standards may be needed
at various stages for quality control of pharmaceutical
Containers. A suitable container is selected. Consideration is
preparations. They are established and monitored taking due
given to the intended use of the preparation, the properties of
account of general chapter 5.12. Reference standards.
the container, the required shelf-life, and product/container
incompatibilities. Where applicable, containers for ASSAY
pharmaceutical preparations comply with the requirements
for containers (3.2 and subsections) and materials used for the Unless otherwise justified and authorised, contents of active
manufacture of containers (3.1 and subsections). substances and specific excipients such as preservatives are
determined in pharmaceutical preparations. Limits must be
Stability. Stability requirements of pharmaceutical defined and justified.
preparations are dependent on their intended use and on the
desired storage time. Suitable and validated methods are used. If assay methods
prescribed in the respective active substance monographs are
Where applicable, the probability and criticality of possible used, it must be demonstrated that they are not affected by the
degradation products of the active substance(s) and/or presence of the excipients and/or by the formulation.
reaction products of the active substance(s) with an excipient
and/or the immediate container must be assessed. Depending Reference standards. See Tests.
on the result of this assessment, limits of degradation and/or
reaction products are set and monitored in the pharmaceutical LABELLING AND STORAGE
preparation. Licensed products require a stability exercise. The relevant labelling requirements given in the general
Methods used for the purpose of stability testing for all dosage form monographs apply. In addition, relevant
relevant characteristics of the preparation are validated as European Union or other applicable regulations apply.
stability indicating, i.e. the methods allow the quantification of
the relevant degradation products and physical characteristic GLOSSARY
changes. Formulation : the designing of an appropriate formula
(including materials, processes, etc.) that will ensure that the
TESTS patient receives the suitable pharmaceutical preparation in an
Relevant tests to apply in order to ensure the appropriate appropriate form that has the required quality and that will be
quality of a particular dosage form are described in the specific stable and effective for the required length of time.
dosage form monographs. Licensed pharmaceutical preparation : a medicinal product
Where it is not practical, for unlicensed pharmaceutical that has been granted a marketing authorisation by a
preparations, to carry out the tests (e.g. batch size, time competent authority. Synonym : authorised pharmaceutical
restraints), other suitable methods are implemented to ensure preparation.
that the appropriate quality is achieved in accordance with the Manufacture : all operations of purchase of materials and
risk assessment carried out and any local guidance or legal products, Production, Quality Control, release, storage,
requirements. distribution of medicinal products and the related controls.
Stock preparations are normally tested to a greater extent than Preparation (of an unlicensed pharmaceutical
extemporaneous preparations. preparation) : the ‘manufacture’ of unlicensed pharmaceutical
The following tests are applicable to many preparations and preparations by or at the request of pharmacies or other
are therefore listed here. healthcare establishments (the term ‘preparation’ is used
General Notices (1) apply to all monographs and other texts 757
Products of fermentation EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 759
Radiopharmaceutical preparations EUROPEAN PHARMACOPOEIA 8.0
In the International System (SI), radioactivity is expressed in Carrier-free preparation : a preparation free from stable
becquerel (Bq), which is 1 nuclear transformation per second. isotopes of the same element as the radionuclide concerned
Absolute radioactivity measurements require a specialised present in the preparation in the stated chemical form or at
laboratory but identification of radioactivity and quantitative the position of the radionuclide in the molecule concerned.
measurement of radioactivity can be carried out relatively No-carrier-added preparation : a preparation to which
by comparing the measured samples with standardised no stable isotopes of the same element as the radionuclide
preparations provided by laboratories recognised by the concerned are intentionally added in the stated chemical
competent authority or by using a calibrated instrument. form or at the position of the radionuclide in the molecule
Radioactive decay : any radionuclide decays at an exponential concerned.
rate with its characteristic decay constant. Specific radioactivity : the radioactivity of a radionuclide
The curve of exponential decay (decay curve) is described by per unit mass of the element or of the chemical form
the following expression : concerned, e.g. becquerel per gram or becquerel per mole.
Radioactivity concentration : the radioactivity of a
radionuclide per unit volume or unit mass of the preparation.
For radiopharmaceutical solutions, it is expressed as
At = the radioactivity at time t ;
radioactivity per unit volume of the preparation.
A0 = the radioactivity at time t = 0 ; Total radioactivity : the radioactivity of the radionuclide,
λ = the decay constant, characteristic of each expressed per unit (vial, capsule, ampoule, generator, etc).
radionuclide ; Chemical precursors for synthesis of radioactive
e = the base of natural logarithms. substances : if the active substance of a radiopharmaceutical
preparation is not isolated, the chemical precursor for its
The half-life (T1/2) is the time in which a given radioactivity synthesis is considered as a substance for pharmaceutical use.
(amount) of a radionuclide decays to half its initial value. It is recommended to test each batch of chemical precursor
It is related to the decay constant (λ) by the following equation : material in production runs before its use for the manufacture
of radiopharmaceutical preparations to ensure that, under
specified production conditions, the substance yields the
radiopharmaceutical preparation in the desired quantity and
The equation of exponential decay can thus be expressed of the quality specified.
also in the following way, useful for the fast estimation of the Period of validity : the time during which specifications
radioactivity left after elapsing time t : described in the monograph must be fulfilled.
PRODUCTION
A radiopharmaceutical preparation contains its radionuclide :
133
The penetrating power of each radiation varies considerably – as an element in atomic or molecular form, e.g. Xe,
according to its nature and its energy. Alpha particles are [15O]O2 ;
completely absorbed in a thickness of a few micrometres – as an ion, e.g. [131I]iodide, [99mTc]pertechnetate ;
to some tens of micrometres of matter. Beta particles are
completely absorbed in a thickness of several millimetres to – included in, adsorbed on or attached to molecules by
several centimetres of matter. Gamma rays are not completely chelation, e.g. [111In]indium oxine, or by covalent bonding,
absorbed but only attenuated and a tenfold reduction may e.g. 2-[18F]fluoro-2-deoxy-D-glucose.
require, for example, several centimetres of lead. The denser Radionuclides can be produced in the following ways :
the absorbent, the shorter the range of alpha and beta particles
and the greater the attenuation of gamma rays. – in reactions of neutrons (target irradiation in nuclear
reactors) ;
Each radionuclide is characterised by an invariable
half-life, expressed in units of time and by the nature – in reactions of charged particles (target irradiation using
and energy of its radiation or radiations. The energy is accelerators, in particular cyclotrons) ;
expressed in electronvolts (eV), kilo-electronvolts (keV) or – by its separation from radionuclide generators.
mega-electronvolts (MeV). The probability of nuclear reaction occurrence depends on
Radionuclidic purity : the ratio, expressed as a percentage, of the nature and energy of the incident particles (protons,
the radioactivity of the radionuclide concerned to the total neutrons, deuterons etc.) and on the nature of the nucleus
radioactivity of the radiopharmaceutical preparation. The that is irradiated by them. The rate of production (yield) of
relevant potential radionuclidic impurities are listed with their a given radionuclide resulting from the irradiation depends
limits in the individual monographs. in addition on the isotopic composition of the target material
Radiochemical purity : the ratio, expressed as a percentage, and its chemical purity, and in the case of neutrons on their
of the radioactivity of the radionuclide concerned which is flux, and in the case of charged particles on beam current.
present in the radiopharmaceutical preparation in the stated In addition to the desired nuclear reaction, simultaneous
chemical form, to the total radioactivity of that radionuclide transformations usually occur. Probability of their occurrence
present in the radiopharmaceutical preparation. The relevant is given by the same factors as mentioned in the previous
potential radiochemical impurities are listed with their limits paragraph. Such simultaneous transformations may give rise
in the individual monographs. to radionuclidic impurities.
Chemical purity : in monographs on radiopharmaceutical The nuclear reaction (transformation) can be written in the
preparations, chemical purity is controlled by specifying limits form : target nucleus (incident particle, emitted particle)
for chemical impurities. produced nucleus.
Isotopic carrier : a stable isotope of the element concerned 58
either present in or added to the radioactive preparation in Examples : Fe(n,γ)59Fe
the same chemical form as that in which the radionuclide is 18
O(p,n)18F
present.
General Notices (1) apply to all monographs and other texts 761
Radiopharmaceutical preparations EUROPEAN PHARMACOPOEIA 8.0
The individual monographs prescribe the radionuclidic The individual monograph prescribes the details concerning
purity required and may set limits for specific radionuclidic the conduct of the test and the physiological distribution
impurities (for example, molybdenum-99 in technetium-99m). requirements that must be met.
While these requirements are necessary, they are not in
themselves sufficient to ensure that the radionuclidic In general, the test is performed as follows.
purity of a preparation is sufficient for its clinical use. The Each of 3 animals is injected intravenously with the
manufacturer must examine the product in detail and preparation. In some cases, dilution immediately before
especially must examine preparations of radionuclides with injection may be necessary.
a short half-life for impurities with a long half-life after a
suitable period of decay. In this way, information on the Immediately after injection each animal is placed in a separate
suitability of the manufacturing processes and the adequacy of cage for collection of excreta and prevention of contamination
the testing procedures is obtained. In cases where 2 or more of the body surface of the animal. At the specified time
positron-emitting radionuclides need to be identified and/or after injection, the animals are euthanised by an appropriate
differentiated, for example the presence of 18F-impurities in method and dissected. Selected organs and tissues are assayed
13
N-preparations, half-life determinations need to be carried for their radioactivity. The physiological distribution is
out in addition to gamma-ray spectrometry. then calculated and expressed in terms of the percentage of
the administered radioactivity that is found in each of the
Due to differences in the half-lives of the different selected organs or tissues, taking into account corrections for
radionuclides present in a radiopharmaceutical preparation, radioactive decay. For some radiopharmaceutical preparations
the radionuclidic purity changes with time. it is necessary to determine the ratio of the radioactivity in
RADIOCHEMICAL PURITY weighed samples of selected tissues (radioactivity/mass).
Radiochemical impurities may originate from : A preparation meets the requirements of the test if the
– radionuclide production ; distribution of radioactivity in at least 2 of the 3 animals
complies with all the specified criteria.
– subsequent chemical procedures ;
Disregard the results from any animal showing evidence of
– incomplete preparative separation ; extravasation of the injection (observed at the time of injection
– chemical changes during storage. or revealed by subsequent assay of tissue radioactivity). In
The determination of radiochemical purity requires that case the test may be repeated.
separation of the different chemical substances containing Sterility
the radionuclide and determination of the percentage of
radioactivity of the radionuclide concerned associated with Radiopharmaceutical preparations for parenteral
the stated chemical form. The radiochemical purity section administration comply with the test for sterility. They must
of an individual monograph may include limits for specified be prepared using precautions designed to exclude microbial
radiochemical impurities, including isomers. contamination and to ensure sterility. The test for sterility is
carried out as described in the general method (2.6.1). Special
In principle, any method of analytical separation may difficulties arise with radiopharmaceutical preparations
be used in the determination of radiochemical purity. because of the short half-life of some radionuclides, the small
For example, the monographs for radiopharmaceutical size of batches and the radiation hazards. In the case that the
preparations may include paper chromatography (2.2.26), monograph states that the preparation can be released for
thin-layer chromatography (2.2.27), electrophoresis (2.2.31), use before completion of the test for sterility, the sterility test
size-exclusion chromatography (2.2.30), gas chromatography must be started as soon as practically possible in relation to
(2.2.28) and liquid chromatography (2.2.29). The technical the radiation. If not started immediately, samples are stored
description of these analytical methods is set out in the under conditions that are shown to be appropriate in order to
monographs. Moreover, certain precautions special to prevent false negative results. Parametric release (5.1.1) of
radiopharmaceuticals must also be considered, such as the product manufactured by a fully validated process is the
radiation protection, measurement geometry, detector method of choice in such cases. When aseptic manufacturing
linearity, use of carriers, dilution of the preparation. is used, the test for sterility has to be performed as a control
Specific radioactivity of the quality of production.
Specific radioactivity is usually calculated taking into account When the size of a batch of the radiopharmaceutical
the radioactivity concentration and the concentration of preparation is limited to 1 or a few samples, sampling the
the chemical substance being studied, after verification that batch for sterility testing according to the recommendations
the radioactivity is attributable only to the radionuclide of the general method (2.6.1) may not be applicable.
(radionuclidic purity) and the chemical species (radiochemical
purity) concerned. When the half-life of the radionuclide is less than 5 min, the
administration of the radiopharmaceutical preparation to the
Specific radioactivity changes with time. The statement of the patient is generally on-line with a validated production system.
specific radioactivity therefore includes reference to a date
and, if necessary, time. For safety reasons (high level of radioactivity) it is not possible
to use the quantity of radiopharmaceutical preparations
Physiological distribution as required in the test for sterility (2.6.1). The method
Tests involving animals should be avoided wherever possible. of membrane filtration is preferred to limit irradiation of
Where the tests for identity and for radiochemical purity personnel.
are not adequate to completely define and control the
Notwithstanding the requirements concerning the use of
radiochemical species in a radiopharmaceutical preparation, antimicrobial preservatives in the monograph Parenteral
a physiological distribution test may be required. The preparations (0520), their addition to radiopharmaceutical
distribution pattern of radioactivity observed in specified
preparations in multidose containers is not obligatory, unless
organs, tissues or other body compartments of an appropriate prescribed in the monograph.
animal species can be a reliable indication of the suitability for
the intended purpose. Bacterial endotoxins - pyrogens
Alternatively, a physiological distribution test can serve to Radiopharmaceuticals for parenteral administration comply
establish the biological equivalence of the preparation under with the test for bacterial endotoxins (2.6.14) or with the test
test with similar preparations known to be clinically effective. for pyrogens (2.6.8).
General Notices (1) apply to all monographs and other texts 763
Recombinant DNA technology, products of EUROPEAN PHARMACOPOEIA 8.0
General Notices (1) apply to all monographs and other texts 765
Substances for pharmaceutical use EUROPEAN PHARMACOPOEIA 8.0
Whether or not it is specifically stated in the individual Certain monographs give two or more sets of tests for the
monograph that the substance for pharmaceutical use : purpose of the first identification, which are equivalent
– is a recombinant protein or another substance obtained and may be used independently. One or more of these sets
as a direct gene product based on genetic modification, usually contain a cross-reference to a test prescribed in the
where applicable, the substance also complies with the Tests section of the monograph. It may be used to simplify
requirements of the general monograph Products of the work of the analyst carrying out the identification and
recombinant DNA technology (0784) ; the prescribed tests. For example, one identification set
cross-refers to a test for enantiomeric purity while the other
– is obtained from animals susceptible to transmissible set gives a test for specific optical rotation : the intended
spongiform encephalopathies other than by experimental purpose of the two is the same, that is, verification that the
challenge, where applicable, the substance also complies correct enantiomer is present.
with the requirements of the general monograph Products
with risk of transmitting agents of animal spongiform
TESTS
encephalopathies (1483) ;
– is a substance derived from a fermentation process, Polymorphism (5.9). If the nature of a crystalline or
whether or not the micro-organisms involved are modified amorphous form imposes restrictions on its use in
by traditional procedures or recombinant DNA (rDNA) preparations, the nature of the specific crystalline or
technology, where applicable, the substance also complies amorphous form is identified, its morphology is adequately
with the requirements of the general monograph Products controlled and its identity is stated on the label.
of fermentation (1468). Related substances. Unless otherwise prescribed or justified
If solvents are used during production, they are of suitable and authorised, organic impurities in active substances are
quality. In addition, their toxicity and their residual level to be reported, identified wherever possible, and qualified as
are taken into consideration (5.4). If water is used during indicated in Table 2034.-1 or in Table 2034.-2 for peptides
production, it is of suitable quality. obtained by chemical synthesis.
If substances are produced or processed to yield a certain Table 2034.-1. – Reporting, identification and qualification of
form or grade, that specific form or grade of the substance organic impurities in active substances
complies with the requirements of the monograph. Certain
Use Maximum Report- Identification Qualification
functionality-related tests may be described to control daily ing
properties that may influence the suitability of the substance threshold threshold
dose threshold
and subsequently the properties of dosage forms prepared Human ≤ 2 g/day > 0.05 per > 0.10 per > 0.15 per
from it. use or cent cent or a cent or a
Powdered substances may be processed to obtain a certain human daily intake daily intake
and of > 1.0 mg of > 1.0 mg
degree of fineness (2.9.35). veterinary (whichever is (whichever is
Compacted substances are processed to increase the particle use the lower) the lower)
size or to obtain particles of a specific form and/or to obtain Human > 2 g/day > 0.03 per > 0.05 per cent > 0.05 per
use or cent cent
a substance with a higher bulk density. human
Coated active substances consist of particles of the active and
substance coated with one or more suitable excipients. veterinary
use
Granulated active substances are particles of a specified Veterinary Not > 0.10 per > 0.20 per cent > 0.50 per
size and/or form produced from the active substance by use only applicable cent cent
granulation directly or with one or more suitable excipients.
If substances are processed with excipients, these excipients Table 2034.-2. – Reporting, identification and qualification of
comply with the requirements of the relevant monograph or, organic impurities in peptides obtained by chemical synthesis
where no such monograph exists, the approved specification. Reporting Identification Qualification
Where active substances have been processed with excipients threshold threshold threshold
to produce, for example, coated or granulated substances, > 0.1 per cent > 0.5 per cent > 1.0 per cent
the processing is carried out under conditions of good
manufacturing practice and the processed substances are Specific thresholds may be applied for impurities known
regarded as intermediates in the manufacture of a medicinal to be unusually potent or to produce toxic or unexpected
product. pharmacological effects.
If the individual monograph does not provide suitable
CHARACTERS
control for a new impurity, a suitable test for control must be
The statements under the heading Characters (e.g. statements developed and included in the specification for the substance.
about the solubility or a decomposition point) are not to be
interpreted in a strict sense and are not requirements. They The requirements above do not apply to biological
are given for information. and biotechnological products, oligonucleotides,
radiopharmaceuticals, products of fermentation and
Where a substance may show polymorphism, this may be semi-synthetic products derived therefrom, to crude products
stated under Characters in order to draw this to the attention of animal or plant origin or herbal products.
of the user who may have to take this characteristic into
consideration during formulation of a preparation. For active substances in a new application for a medicinal
product for human use, the requirements of the guideline
IDENTIFICATION on the limits of genotoxic impurities and the corresponding
Where under Identification an individual monograph questions and answers documents published on the website
contains subdivisions entitled ‘First identification’ and of the European Medicines Agency (or similar evaluation
‘Second identification’, the test or tests that constitute the principles for non-European Union member states) must be
‘First identification’ may be used in all circumstances. The followed.
test or tests that constitute the ‘Second identification’ may be Residual solvents are limited according to the principles
used in pharmacies provided it can be demonstrated that the defined in chapter 5.4, using general method 2.4.24 or another
substance or preparation is fully traceable to a batch certified suitable method. Where a quantitative determination of a
to comply with all the other requirements of the monograph. residual solvent is carried out and a test for loss on drying is
not carried out, the content of residual solvent is taken into – granulated ;
account for calculation of the assay content of the substance, – sterile ;
the specific optical rotation and the specific absorbance. – free from bacterial endotoxins ;
Microbiological quality. Individual monographs give – free from pyrogens ;
acceptance criteria for microbiological quality wherever such – containing gliding agents.
control is necessary. Table 5.1.4.-2. – Acceptance criteria
for microbiological quality of non-sterile substances for Where applicable, the label states :
pharmaceutical use in chapter 5.1.4. Microbiological quality – the degree of hydration ;
of non-sterile pharmaceutical preparations and substances for – the name and concentration of any excipient.
pharmaceutical use gives recommendations on microbiological
quality that are of general relevance for substances subject to 01/2013:0153
microbial contamination. Depending on the nature of the
substance and its intended use, different acceptance criteria VACCINES FOR HUMAN USE
may be justified.
Sterility (2.6.1). If intended for use in the manufacture of Vaccina ad usum humanum
sterile dosage forms without a further appropriate sterilisation
procedure, or if offered as sterile grade, the substance for DEFINITION
pharmaceutical use complies with the test for sterility. Vaccines for human use are preparations containing antigens
Bacterial endotoxins (2.6.14). If offered as bacterial capable of inducing a specific and active immunity in man
endotoxin-free grade, the substance for pharmaceutical against an infecting agent or the toxin or antigen elaborated
use complies with the test for bacterial endotoxins. The by it. Immune responses includ