SC IV M. Reference Standards

(Ph. Eur. general texts 5.12)

This chapter is published for information.

1 INTRODUCTION

‘Reference standard’ is used in this chapter as a general term covering reference substances, reference preparations and reference spectra.

Reference standards are frequently necessary to achieve adequate quality control of medicinal products and their components.

Reference standards are established using suitable procedures and their continued suitability for use is monitored according to a predefined programme. Where a reference standard is needed, it is an integral part of the pharmacopoeial monograph or the manufacturer’s specification. Where a European Pharmacopoeia reference standard is referred to in a monograph or general chapter, it represents the official standard that is alone authoritative in case of doubt or dispute.

2 TERMINOLOGY

Primary standard A standard designated or widely acknowledged as having the highest metrological qualities and whose property value is accepted without reference to other standards of the same property or quantity, within a specified context. This definition does not cover international standards.

International standard A primary standard provided to enable the results of biological or immunological assay procedures to be expressed in the same way throughout the world. The value assignment is in International Units (IU) or another suitable unit. The unitage is normally assigned to a first international standard by the World Health Organization (WHO) in an arbitrary manner based on an international interlaboratory study. Activities in International Units are assigned to replacement international standards, where appropriate, by comparing them with a previous standard.

Secondary standard A standard whose property value is assigned by comparison with a primary standard of the same property or quantity.

European Pharmacopoeia reference standard A reference standard established under the aegis of and adopted by the European Pharmacopoeia Commission.

European Pharmacopoeia chemical reference substance (CRS) A substance or mixture of substances intended for use as stated in a monograph or general chapter of the European Pharmacopoeia. CRSs are in general primary standards, except for those (notably antibiotics) that are calibrated in International Units. The latter are secondary standards traceable to the international standard.

European Pharmacopoeia herbal reference standard (HRS) A herbal drug preparation (usually an extract) or a herbal drug intended for use as stated in a monograph or general chapter of the European Pharmacopoeia. Unless otherwise specified, HRSs are designated as primary reference standards for their intended use.

European Pharmacopoeia biological reference preparation (BRP) A substance or mixture of substances intended for use as stated in a monograph or general chapter of the European Pharmacopoeia. BRPs are either secondary standards calibrated in International Units or primary standards, which may be used to define a European Pharmacopoeia Unit (Ph. Eur. U.). Other assigned contents may also be used, for example, virus titre or number of bacteria.

The specificity of pharmacopoeial reference standards has been acknowledged in ISO Guide 34 –General requirements for the competence of reference material producers. In particular, it is acknowledged that the measurement uncertainty of assigned content values is not normally stated since it is negligible in relation to the defined limits of the method-specific assays of the pharmacopoeias for which they are used.

Reference material (RM) A material sufficiently homogeneous and stable with respect to one or more specified properties, which has been established to be fit for its intended use in the measurement process.

Certified reference material (CRM) A reference material characterised by a metrologically valid procedure for one or more specified properties, accompanied by a certificate that states the value of the specified property, its associated uncertainty, and a statement of metrological traceability.

3 USE OF EUROPEAN PHARMACOPOEIA REFERENCE STANDARDS

European Pharmacopoeia reference standards are employed in the identification, purity testing and assay of articles subject to a European Pharmacopoeia monograph or general chapter. European Pharmacopoeia reference standards are shown to be suitable for their intended purpose; they are not necessarily suitable for other purposes. If a European Pharmacopoeia reference standard is to be used for any purpose other than that for which it has been established, its suitability for the new use has to be fully demonstrated and when applicable, to be described in the marketing authorisation application. Any value assigned to a reference standard is valid for the intended use and not necessarily for other uses.

A European Pharmacopoeia reference standard with an assigned content/potency for use in the assay of a substance for pharmaceutical use (see general monograph Substances for pharmaceutical use (2034)) may be suitable to determine the content/potency of that substance in a pharmaceutical preparation provided all of the following conditions are fulfilled:

— the chromatographic assay method described in the active substance monograph is employed;

— the applicability of the method to the particular pharmaceutical preparation (absence of interference) is verified by the user;

— any pre-treatment of the sample (e.g. extraction, filtration) is validated for the particular pharmaceutical preparation.

It is the policy of the European Pharmacopoeia to supply reference standards in adequate quantities for immediate use (i.e. the quantity needed to perform the test(s) described in the monograph or general chapter) after opening of the container. Use in other conditions is the responsibility of the analyst. If an unopened container is stored in the recommended conditions, it remains suitable for use as long as it is of the current batch. Information on current batch numbers is provided in the European Pharmacopoeia reference standards database (http://go.edqm.eu/RS). Storage of reconstituted or diluted solutions of reference standards is not recommended unless suitability has been demonstrated by the user.

Secondary standards A secondary standard is usually established to reduce the use of the primary standard and may be used for routine quality control purposes. A secondary standard shall exhibit the same property or properties as the primary standard to which it is traceable. It shall therefore be used for the same purpose as the primary standard.

International standards are usually available in relatively limited quantities and are intended to be used for the characterisation and calibration of secondary standards; these secondary standards may then be used as working standards.

4 ESTABLISHMENT OF REFERENCE STANDARDS

4-1 PRIMARY STANDARDS

A substance or preparation to be established as a primary standard is characterised by a variety of analytical techniques chosen to demonstrate its suitability for use.

For reference standards used for the control of medicinal products and their components, relevant parts of the following test programme are usually applied.

Test programme:

— Characterisation of the substance (structural elucidation) by appropriate chemical attributes such as structural formula, empirical formula, molecular mass or composition. A number of techniques may be used including:

— nuclear magnetic resonance spectrometry;

— mass spectrometry;

— infrared spectrophotometry;

— elemental analysis.

— Determination of purity:

— determination of the content of related substances by an appropriate separation technique and/or spectrometric method, where applicable;

— quantitative determination of water;

— determination of the content of residual solvents;

— determination of loss on drying, which may in certain circumstances replace the determinations of water and residual solvents;

— determination of inorganic impurities (e.g. sulfated ash, atomic absorption spectrometry, inductively coupled plasma spectrometry, X-ray fluorescence spectrometry); the results are usually not used to determine an assigned content, except where they would have an appreciable impact upon it;

— determination of purity by an independent method (e.g. quantitative nuclear magnetic resonance spectrometry, differential scanning calorimetry or titration where appropriate; the results of these determinations are usually used to support and confirm the results obtained from separation techniques; they are not used in the calculation of the assigned content).

For biologicals, guidance is given in the WHO recommendations for the preparation, characterisation and establishment of international and other biological reference standards (WHO Technical Report Series).

4-2 EUROPEAN PHARMACOPOEIA CHEMICAL REFERENCE substances

The extent of testing and the number of laboratories involved in the establishment of a CRS depend on the use of the CRS and are tailored to ensure fitness for purpose.

Where an interlaboratory study is carried out during establishment, a protocol is provided for each participant and only valid results derived according to the protocol are used to determine an assigned content or otherwise confirm suitability.

Relevant parts of the following programme are typically applied.

4-2-1 Identification

In general, the candidate batch is shown to comply with the relevant requirements of the monograph; full structural elucidation is carried out for the first batch.

4-2-2 Related substances test

A CRS corresponding to an impurity is characterised for identity and purity. Where a CRS is used to determine the content of a given impurity, the preferred minimum content is 95.0 per cent; where this is achieved the assigned content of the CRS is not given and it is considered to be 100.0 per cent; this approximation is acceptable since there will be no appreciable effect on the determination of impurities. When this minimum content cannot be obtained, an assigned content is given to the CRS.

CRSs used to determine the content of a given impurity are normally in the same acid, base or salt form as the substance that is the subject of the corresponding monograph. Where this is not the case, unless otherwise justified, a corresponding stoichiometric conversion factor is applied.

If an impurity is not available in a sufficient quantity to establish a CRS, a number of other options exist:

— preparation of a CRS that contains a mixture of the compound(s) and the impurity or impurities;

— preparation of a CRS containing a mixture of specified impurities.

Where such a mixture is also used to determine the content of a given impurity, the content of the impurity in the CRS is determined by appropriate separation methods and a content is assigned to the reference standard.

4-2-3 Assay

4-2-3-1 Chemical assay When a CRS is to be used for quantitative determination of a substance for pharmaceutical use (assay standard), the extent of testing is greater than when a CRS is used for other purposes. Unless the substance is of high purity, several collaborating laboratories are usually involved in testing. The results obtained are used to assign a content. It is particularly important to quantify the impurities if a selective assay is employed. In such a case, it is best to characterise the candidate substance by additional analytical procedures that are scientifically justified, including, where possible, independent methods and methods based on different principles.

For a European Pharmacopoeia chemical reference substance established for assay purposes, the assigned content is usually calculated from the values obtained from the analyses performed for the determination of impurities (organic, inorganic, water and solvents) by applying the principle of mass balance; other suitable methods may also be used. When possible, the assigned content is confirmed by comparing with the result obtained by an independent method.

If a CRS is required for a non-chromatographic assay method (e.g. colorimetry or ultraviolet spectrophotometry), the relative reactivity or relative absorbance of the impurities present in a substance must be checked to ensure that they are not markedly different from those of the substance.

Unless otherwise stated, an assigned content is given for the substance or preparation as presented in the container (‘as is’), and the contents are not to be dried before use. For assay standards prepared by lyophilisation, the content of the pure substance is indicated in milligrams or International Units per vial.

4-2-3-2 Microbiological assay The potency is expressed in International Units or in European Pharmacopoeia Units if no international standard exists. The assigned potency together with the confidence limits are calculated from statistically valid results of an interlaboratory study, according to the usual statistical procedures (5.3).

4-2-3-3 Assay of components of herbal drugs and herbal drug preparations Reference standards used in monographs on herbal drugs vary in the extent of testing depending on the type of reference standard.

An active component or marker constituent used as a CRS is usually characterised and evaluated for identity and purity; a value for content is assigned irrespective of the purity.

4-2-4 Establishment report

A report containing the results of the establishment study as well as information concerning the use of the CRS is prepared by EDQM, approved by the relevant group of experts and adopted by the European Pharmacopoeia Commission. The report for an assay standard indicates the content assigned to the substance with the rationale for this assignment. The estimated uncertainty of the assigned content is calculated, and where it is less than a predefined value, which is considered to be negligible in relation to the acceptance criteria for the assay, then the study is accepted. Otherwise, the study may be repeated, in whole or in part, or the limits defined for the pharmaceutical substance may be widened. The uncertainty of the assigned content is usually not given as part of the information provided with the CRS, since the precision of the method and the uncertainty of the content assigned to the CRS are taken into account when setting the limit(s) in a monograph.

4-3 EUROPEAN PHARMACOPOEIA HERBAL REFERENCE STANDARDS

A HRS is used when the available quantity of pure constituent to be assayed is considered either insufficient or non-sustainable. HRSs may also be established for purposes other than assays, notably for use in tests for adulteration or for system suitability. A HRS is characterised by a variety of analytical techniques chosen to demonstrate its suitability for the intended use; relevant parts of the following test programme may be applied.

Test programme:

— macroscopy;

— microscopy;

— thin-layer chromatography;

— gas chromatography;

— liquid chromatography;

— quantitative determination of water;

— content of residual solvents;

— loss on drying;

— foreign matter;

— assay of constituents (e.g. constituents with known therapeutic activity, active markers, analytical markers) relevant to the intended use of the reference standard.

The extent of testing and the number of laboratories involved in the establishment of a HRS depend on its intended use.

For a European Pharmacopoeia herbal reference standard used for assay purposes, the assigned content is usually established by an interlaboratory study, using the assay method specified in the individual monograph in which the reference standard is intended to be used, comparing against a suitable pure sample of the constituent or constituents for which the content is to be assigned.

Establishment report

The establishment report for the HRS is prepared in the same manner as CRSs (see section 4-2-4).

4-4 EUROPEAN PHARMACOPOEIA BIOLOGICAL REFERENCE PREPARATIONS AND CHEMICAL REFERENCE SUBSTANCES FOR BIOLOGICALS

Most BRPs and some CRSs used for the testing of biological substances and preparations are established through the Biological Standardisation Programme, under the aegis of the Council of Europe and the European Commission. These reference standards are usually secondary standards calibrated against the corresponding WHO international standard. Where no international standard is available, they are primary standards with an assigned content/potency in European Pharmacopoeia Units or another suitable unit. They are established through interlaboratory studies where participating laboratories test the candidate material(s) and valid data are used to assign the official potency/content. Some of these studies are jointly organised with other organisations to establish a common material or batch of material as standard. In these cases, although the material constituting the European Pharmacopoeia reference standard may be identical to the international standard and its use may be validated through the same interlaboratory study, it is considered as a secondary standard for use as a working standard.

The study reports are endorsed by the study participants and approved by the relevant European Pharmacopoeia group of experts, where applicable, and by the Steering Committee of the Biological Standardisation Programme. The study results are then submitted to the European Pharmacopoeia Commission. Reference standards/materials established through the Biological Standardisation Programme are officially adopted by the European Pharmacopoeia Commission. The establishment reports are published in Pharmeuropa Bio & Scientific Notes (http://pharmeuropa.edqm.eu/PharmeuropaBioSN).

4-5 SECONDARY STANDARDS

A secondary standard should exhibit the same property or properties as the primary standard, relevant for the test(s) for which it is established. The extent of testing may not be as comprehensive as is required for the establishment of a primary standard. The secondary standard is established by comparison with the primary standard to which it is traceable. An official primary standard is used wherever possible for establishment of secondary standards.

5 manufacturing, LABELLING, STORAGE AND DISTRIBUTION of european pharmacopoeia reference standards

5-1 manufacturing

All operations are carried out according to the relevant best practices to ensure the traceability and integrity of the reference standard. The manufacturing record includes information regarding filling, labelling and storage. Reference standards are dispensed into containers under appropriate filling and closure conditions to ensure the integrity of the reference standard. The containers employed may be multi-use or single-use, but the latter is preferred to minimise the risk of decomposition, contamination or water uptake.

5-2 LABELLING

The label bears the name of the reference standard, the name and address of the supplier, the batch number and unit quantity (quantity per vial/ampoule).

An accompanying leaflet, considered as part of the labelling, is normally provided.

If used as an assay standard, the following information is also given:

— the assigned percentage content;

— or, the content in milligrams or millilitres of the chemical entity in the container;

— or, the assigned potency (for biological assays or microbiological assays) in units either per milligram, per millilitre or per vial/ampoule.

For European Pharmacopoeia reference standards, no re-test or expiry date is given since the re-test programme (see section 6) monitors continued fitness for use. A batch validity statement (BVS) for each European Pharmacopoeia reference standard is available from the European Pharmacopoeia reference standards database (http://go.edqm.eu/RS).

5-3 STORAGE AND DISTRIBUTION

Reference standards are to be stored and distributed in conditions suitable to ensure optimal stability.

Most European Pharmacopoeia reference standards are stored in temperature-controlled rooms at 5 ± 3 °C. However, a number of reference standards are stored at - 20 ± 5 °C and some (e.g. live virus preparations) are stored at - 80 ± 10 °C or at -196 °C to -170 °C under liquid nitrogen.

Appropriate packaging is used to minimise the risk of damage during transport, to keep the reference standard at the appropriate temperature when necessary and to comply with the current transport regulations.

Reference standards that are stored at 5 ± 3 °C are normally transported without cooling when short-term excursions from the long-term storage temperature are not deleterious to the reference standard. Some of them may nevertheless be sent at + 5 °C, packed with cold packs in cases where an increase in temperature is detrimental to their stability. Reference standards stored at - 20 °C are packed with cold packs or on dry ice (solid carbon dioxide) and dispatched by express courier or air freight. Reference standards stored at - 80 °C or stored under liquid nitrogen are packed on solid carbon dioxide.

Delivery conditions, dispatch and storage temperatures are available in the Reference Standards Catalogue, the Terms of Supply and the European Pharmacopoeia reference standards database (http://go.edqm.eu/RS).

6 RE-TEST PROGRAMME of european pharmacopoeia standards

A system is established and implemented to ensure the continued fitness-for-use of the European Pharmacopoeia reference standards. Normally, a re-test programme is applied, taking account of the known physico-chemical properties and stability data for the reference standard. Reference standards are periodically tested for stability during storage. A monitoring programme is applied that is designed to detect at an early stage any sign of degradation using appropriate analytical techniques. The methods employed are typically chosen from amongst those performed during establishment of the reference standard so that baseline data is available.

The periodicity and extent of re-testing reference standards depends on a number of factors including:

— stability;

— container and closure system;

— storage conditions;

— hygroscopicity;

— physical form;

— intended use;

— presentation (single use/multiple use).

The re-test period may be lengthened with the support of sufficient data. The maximum permitted variation from the assigned content should be pre-defined, and if exceeded, the batch should be re-established or replaced.