Pharmaceutical Reference Standards

1. Pharmaceutical Reference Standards Part 1 Dr John H McB Miller Head of DLab EDQM Council of Europe Strasbourg France

2. Pharmaceutical Reference Standards Part 1 Definitions Guidelines Need Procurement Uses Establishment Adoption/Certification Part 2 Production/Filling Packaging Re-test Programme (Monitoring) Difference between reference material & reference standard Secondary reference standard (in-house/working standard)

3. PRIMARY CHEMICAL REFERENCE SUBSTANCE • A designated primary chemical reference substance is widely acknowledged as having appropriate qualities within a specified context, and whose value is accepted without reliance on comparison to another chemical substance • WHO/PHARM/96.590

4. European Pharmacopoeia Reference Standard A reference standard established under the aegis of and approved by the European Pharmacopoeia Commission European Pharmacopoeia Chemical Reference Substance A substance or mixture of substances intended for use as stated in a monograph or general chapter of the European Pharmacopoeia. EPCRS are primary standards, except for those, notably antibiotics, which are calculated in International Units. The latter are secondary standards traceable to the international standard.

5. DEFINITIONSFDA Guideline for Submitting Documentation in Drug Applications for the Manufacturing of Drug Substances Reference Standard A particular lot or batch of drug substance specifically prepared, either by independent synthesis or by additional purification of production material, and shown, by an extensive set of analytical tests, to be authentic material of the highest purity reasonably attainable. It is usually used for structural elucidation, and is the benchmark for working standards.

6. Reference Standard (ICH) Q6A A reference standard, or reference material, is a substance prepared for use as the standard in an assay, identification or purity test. It has a quality appropriate to its use. For new drug substances reference standards intended for use in assays, the impurities should be adequately identified and/or controlled and purity should be measured by a quantitative procedure.

7. Reference Material 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 a measurement process

8. Certified Reference Material An RM 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

9. BIOLOGICAL INTERNATIONAL STANDARD “An International Biological Standard is a biological or synthetic preparation, the mean value of which, based on a collaborative study, is used to define an international unit. The assignment of potency in international units of the first international standard is generally done arbitrarily. On the other hand, potency in international units are assigned to replacement international standards by calibration against the previous standard on the basis of an international collaborative trial. In all cases, these studies must demonstrate that the preparations are suitable to be used as international standards”. WHO Expert Committee on Biological Standardisation 40th Report World Health Organisation, Geneva (1990) (WHO Technical Report Series 800)

10. Biological Reference Preparations (Ph.Eur) Biological reference preparations are either primary or secondary standards. Secondary standards are usually calibrated in International Units. Primary standards may have an assigned potency in European Pharmacopoeia Units. Other assigned values may also be used for primary standards, for example virus titre, number of bacteria.

11. Guidelines 5.12 Reference Standards European Pharmacopoeia 6th Edition (2008) ISO Guide 34 General Requirements for the Competence of Reference Material Producers ISO Guide 35 Certification of reference materials. General and statistical principles.

12. GUIDELINES “WHO General Guideline for the Establishment, Maintenance & Distribution of Chemical Reference Substances” WHO Expert Committee for Pharmaceutical Preparations, Annex 3, 41st Report, Technical Report to Series 943

13. WHO Guideline Part A • Assessment of need • Obtaining source material • Evaluation of chemical reference substances • Chemical & physical methods used in evaluating chemical reference substances • Assignment of content • Handling & distribution

14. Need and Use Analytical procedures currently used in specifications for pharmaceutical substances & products that may require a chemical reference substance are: • Infrared (IR) spectrophotometry, whether for identification or quantitative purposes; • Quantitative methods based on ultraviolet (UV) absorption spectrophotometry; • Quantitative methods based on the development of a colour & the measurement of its intensity, whether by instrumental or visual comparison. • Methods based on chromatographic separation for identification or quantitative purposes;

15. Need and Use • Quantitative methods (including automated methods) based on other separation techniques that depend on partition of the substance to be determined between solvent phases, where the precise efficiency of the extraction procedure might depend upon ambient conditions that vary from time to time & from lab to lab; • Quantitative methods, often titrimetric but sometimes gravimetric, that are based on non-stoichiometric relationships; • Assay methods based on measurement of optical rotation; and • Methods that might require a chemical reference substance consisting of a fixed ratio of known components (eg cis/trans isomers, spiked samples)

16. Procurement Normal production batch: usually no further purification of active substance (> 99.0 per cent purity) Impurities: isolated or synthesised by the manufacturer (> 95.0 per cent purity)

17. Supplied with: • a certificate of analysis including test methods, test results with complete identification by appropriate physico-chemical methods, e.g. nuclear magnetic resonance spectroscopy, infrared spectrophotometry, mass spectroscopy etc… • Stability data of the substance with an indication of the storage conditions to be employed, • Information as to its hygroscopicity and its solid-state properties, e.g. amorphous, crystalline, polymorphic form etc… • A material safety data sheet, • A list of potential impurities (if an active substance) with response factors

18. The Uses of the Reference Substances/Preparations • Are clearly described in the individual monographs of the Pharmacopoeias; • Are applicable to active pharmaceutical substances, excipients and products

19. Reference substances required for: • identification (of substance for pharmaceutical use), • system suitability (to ensure adequate performance of the applied technique), • purity (identification and estimation of specified impurities in a substance for pharmaceutical use manufactured by a particular route of synthesis), • assay (of content of the substance for pharmaceutical use or its products).

20. Pharmacopoeial Reference Standards used for identification of substances for pharmaceutical use 1. Identification: Pharmaceutical reference standards can be used for confirmation of identity of the substance by, e.g. • spectroscopy usually, infrared spectrophotometry where the spectrum of the substance to be examined is compared to the spectrum of the CRS, or to the reference spectrum, • separation techniques where the retention times (or migration distance or migration time) of both the substance to be examined and the CRS are compared. • identification by peptide mapping requires these of both a CRS it’s chromatogram.

21. 2. Related Substances (organic impurities) The system suitability criteria for selectivity may be: • resolution of two closely eluting impurities or an impurity and the main compound; • peak-to-valley ratio of an impurity which is incompletely separated to the main compound. A mixture of the compound with a small amount of the impurity is required; • mixtures of impurities or a mixture of impurities and the compound as reference standard (may be supplied with a chromatogram if prescribed in the monograph) as a system suitability test based on the unadjusted relative retentions of the impurity peaks to the substance peak and the resolution or peak-to-valley ratio of specified peaks.

22. System suitability 4 mg of abacavir sulfate for system suitability Control No 107244 monitored at 254 nm.

23. Chromatogram of clazuril for system suitability CRS

24. Chromatogram of loperamide hydrochloride for system suitability CRS

25. Location of impurity peaks Confirmation of identification may be achieved by : • reference standards of individual specified impurities; • mixtures (as described above) where their chromatograms serve also to locate the impurities in the chromatogram of the test sample.

26. Impurity Standards By-products & degradation products Synthesis of by-products & degradation products is often challenging & the preparation of samples with greater than 95.0% purity may be impossible. Since such comparison substances are used to determine relative response factors, errors in the purity of 5.0% are not critical for the quantitation of impurities in the drug substance at the 0.5% level or less. However, purity correction must be made in the calculation of the response factor.

27. Pharmacopoeial Reference Standards used for Assay • physico-chemical methods chemical reference substances are employed to assay for content of synthetic and semi-synthetic pharmaceutical substances, herbal substances and peptides: • microbiological assay of antibiotics to determine their potencies. These microbiological reference standards are referred to a chemical reference substances in the European Pharmacopoeia and are secondary reference standards since they have been established against the International Standard; • biological assays (in vitro or in vivo) to determine the activity/potency of biological substances. The biological reference standards are referred to as Biological Reference Preparations in the European Pharmacopoeia and may be primary or secondary standards. • The assigned content/potency of the reference standard is method-specific i.e. it is to be applied only with the method described in the specification or the monograph.

28. Establishment of Reference Substances • The extent of analyses required for the establishment of Chemical Reference Substances depends on the purpose(s) for which it is employed. • In general a batch of good quality, selected from the normal production of the substance, is satisfactory.

29. Establishment of Reference Substances 1) to characterise the substance (proof of molecular structure) by appropriate chemical attributes such as structured formula, empirical formula and molecular weight. A number of techniques may be used including: • Nuclear magnetic resonance (NMR) spectroscopy ; • Mass spectroscopy; • Infrared spectroscopy. The spectra are to be interpreted to support the structure. • Elemental analysis to confirm the percentage composition of the elements.

30. Establishment of Reference Substances 2) determination the purity • determination of the content of organic impurities by an appropriate separation technique (eg gas chromatography (GC), liquid chromatography (LC) or capillary electrophoresis (CE)); • quantitative determination of water (eg micro or semi-microdetermination); • determination of the content of residual solvents; • determination of loss on drying may in certain circumstances replace the determinations of water and residual solvents; • determination of the purity by an absolute method (eg differential scanning calorimetry or phase solubility analysis where appropriate. The results of these determinations are to support and confirm the results obtained from separation techniques. They are not normally included in the calculation of the assigned value); • determination of inorganic impurities (test for heavy metals, sulphated ash, atomic absorption spectrophotometry, ICP, X-ray fluorescence) often the values obtained will have no consequence on the assignment of the purity of the standard.

31. Establishment of Reference Substances 3) Assay (for the active ingredient only) by an absolute method (volumetric titration) is recommended. The assigned content of the primary chemical reference standard is calculated from the values obtained from the analysis performed for the determination of purity and are verified by a calculation of the mass balance.

32. Purity of Impurities • at least 90.0 per cent when used only to locate a peak in the chromatogram of the substance for pharmaceutical use; • at least 90.0 per cent when used in the system suitability test for resolution; • at least 95.0 per cent when used to estimate the content of a specified impurity (for the purpose of the estimation it is considered to be 100 per cent). When the minimum purity cannot be obtained, then a purity value is assigned to the standard.

33. Most chemical reference substances are fully characterised & are traceable to the molecular weight. They are considered to be Primary Reference Substances - no comparison to existing standards. • Chemical Reference Substances (some antibiotics) used for the microbiological assay of potency & many of the Biological Reference Preparations are secondary reference substances since they are calibrated against the international standard established by WHO.

34. Assignment of content/potency of Reference Substance/Preparation

35. Assay Pharmacopoeial reference standards with an assigned content required for comparative assay techniques: • Infrared spectrophotometry • Ultraviolet/visible spectrophotometry • Chromatography

36. Characterising Reference Material(ISO Guide 35) • Single definitive method by a single organisation • Two or more independent reference methods by one organisation • Number of methods of known and acceptable accuracy and precision by a network of qualified organisations • Method specific approach (inter-lab study) giving only a method specific assessed property value

37. Assignment of Content • Ph.Eur. Policy • The value assigned is method specific and is estimated from the results obtained by a collaborative trial (min. 6 participants). Other techniques may be employed to determine the content but these results are not used for assigning the value but give complementary supporting information

38. European PharmacopoeiaCollaborative Trials • Participation by: • Experts of the European Pharmacopoeia • Academia • Industry • OMCLs

39. Initiating Laboratory(European Pharmacopoeia Laboratory) • Monograph requirements • Residual Solvents • Determination of Response Factors of Impurities • Absolute Analytical Method (Volumetric titration, DSC) Protocol prepared by the Ph.Eur Laboratory

40. Protocol • the determination of water (or loss on drying); • the estimation of the organic impurities (including residual solvents when appropriate) using the prescribed separation techniques; • the determination of the content of the substance by an absolute method (usually volumetric titration) may be included. These are confirmatory determinations and the results are not used in the calculation of the assigned value and are not necessarily performed by all participants.

41. Protocol Representative chromatogram is supplied for info AC = Acceptance Criteria

42. Content of the Reference Standard • The content that is used to correct the purity of the reference standard is generally calculated as follows: • No correction of % loss on drying or sum water and solvents is done if the value is <0.10%. • For a chiral chromatographic quantitative methods, the content of antipode is not taken in account in the % impurities. • Impurities are corrected for their response factors. • Impurities are determined at ≥ limit of quantification.

43. CHEMICAL REFERENCE SUBSTANCESUNCERTAINTY OF THE ASSIGNED VALUE Estimated uncertainty =

44. ASSAY STANDARD FOR LIQUID CHROMATOGRAPHY • Normal limit accepted for analytical error  2.0% • Uncertainty of 0.24% implies 0.1% probability of rejecting a good result (n=3) • If this is considered to be insignificant then there is no justification for giving uncertainty values with the assigned value • Maximum permitted uncertainty varies proportionally to the limits set e.g. maximum permitted uncertainty for limits of  1.0% would 0.12

45. Uncertainty • The estimated uncertainty of the result of a collaborative trial is employed to assess the acceptability of the trial. • If the estimated uncertainty is greater than a pre-defined criterion then the trial is considered to be unsatisfactory & after investigation & identification of the causes of failure the monograph may be revised & the trial repeated. • With in-built acceptance criteria in the protocol this eventuality is unlikely.

46. Assigned value of reference substances Assigned value = 100 – (water content % + organic impurities % + residual solvent %) or 100 – (loss on drying % + organic impurities %)

48. DETERMINATION OF THE UNCERTAINTY OF THE ASSIGNED VALUE TICARCILLIN SODIUM CRS Per cent Impurities Related Substances Water Mean 4.03 (n=6) 4.69 (n=7) 4.61(n=6) SD () 0.074 0.224 0.0758 Variance (2) 0.005 0.05 0.0057

49. Assay Ph.Eur. Active pharmaceutical ingredient CRS with assigned content only for assay method described in the corresponding monograph for the API & pharmaceutical product USP/BP Active pharmaceutical ingredient + pharmaceutical products If no content is given for RS assumed to be 100 per cent for assay.

50. Extended use of Ph. Eur Reference Standards • Assay reference standard used for determination of content in a monograph for a substance for pharmaceutical use can be used for the assay of that substance in a pharmaceutical product. Provided that: • the assay method employed for the product is that described in the monograph of the substance for pharmaceutical use; • any pre-treatment of the sample (eg extraction) is validated by the user.