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Pharmaceutical Reference

Standards

Part 1Dr John H McB Miller

Head of DLabEDQM

Council of EuropeStrasbourg

France


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QCL Training Seminar, Tanzania | 5-7 Dec 072 |

Pharmaceutical Reference Standards

Part 1 Definitions

Guidelines

Need

Procurement Uses

Establishment

Adoption/Certification

Part 2 Production/Filling

Packaging

Re-test Programme

(Monitoring)

Difference betweenreference material &reference standard

Secondary reference

standard (in-house/workingstandard)


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PRIMARYCHEMICAL REFERENCE SUBSTANCE

l A designated primary chemical referencesubstance is widely acknowledged as havingappropriate qualities within a specified context,and whose value is accepted without reliance on

comparison to another chemical substance

l WHO/PHARM/96.590


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European Pharmacopoeia Reference StandardA reference standard established under the aegis of and approvedby the European Pharmacopoeia CommissionEuropean Pharmacopoeia Chemical Reference SubstanceA substance or mixture of substances intended for use as stated ina 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.


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DEFINITIONSFDA Guideline for Submitting Documentation in Drug Applications for the

Manufacturing of Drug Substances

Reference Standard

A particular lot or batch of drug substance specificallyprepared, either by independent synthesis or by additional

purification of production material, and shown, by an extensiveset of analytical tests, to be authentic material of the highestpurity reasonably attainable. It is usually used for structuralelucidation, and is the benchmark for working standards.


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Reference Standard (ICH) Q6A

A reference standard, or reference material, is a substanceprepared for use as the standard in an assay, identificationor purity test. It has a quality appropriate to its use. For newdrug substances reference standards intended for use in

assays, the impurities should be adequately identifiedand/or controlled and purity should be measured by aquantitative procedure.


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Reference MaterialMaterial sufficiently homogeneous and stable with respect toone or more specified properties, which has beenestablished to be fit for its intended use in a measurementprocess


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Certified Reference Material

An RM characterised by a metrologically valid procedure forone or more specified properties, accompanied by acertificate that states the value of the specified property, itsassociated uncertainty and a statement of metrological

traceability


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BIOLOGICAL INTERNATIONAL STANDARD

An International Biological Standard is a biological orsynthetic preparation, the mean value of which, based on acollaborative study, is used to define an international unit.The assignment of potency in international units of the firstinternational standard is generally done arbitrarily. On theother hand, potency in international units are assigned toreplacement international standards by calibration againstthe previous standard on the basis of an internationalcollaborative trial. In all cases, these studies mustdemonstrate that the preparations are suitable to be used asinternational standards.

WHO Expert Committee on Biological Standardisation 40th Report World Health Organisation, Geneva (1990) (WHOTechnical Report Series 800)


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Biological Reference Preparations (Ph.Eur)

Biological reference preparations are either primary or secondarystandards. Secondary standards are usually calibrated inInternational Units. Primary standards may have an assigned potencyin European Pharmacopoeia Units. Other assigned values may also

be used for primary standards, for example virus titre, number ofbacteria.


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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.


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GUIDELINES

WHO General Guideline for the Establishment,

Maintenance & Distribution of Chemical ReferenceSubstances

WHO Expert Committee for Pharmaceutical Preparations,Annex 3, 41st Report, Technical Report to Series 943


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WHO Guideline Part A

Assessment of need

Obtaining source material

Evaluation of chemical reference substances

Chemical & physical methods used in evaluatingchemical reference substances

Assignment of content

Handling & distribution


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Need and Use

Analytical procedures currently used in specifications forpharmaceutical substances & products that may require a chemical

reference substance are:

a) Infrared (IR) spectrophotometry, whether for identification orquantitative purposes;

b) Quantitative methods based on ultraviolet (UV) absorptionspectrophotometry;

c) Quantitative methods based on the development of a colour &the measurement of its intensity, whether by instrumental orvisual comparison.

d) Methods based on chromatographic separation for

identification or quantitative purposes;


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Procurement

Normal production batch: usually no further purification ofactive substance (> 99.0 per cent purity)

Impurities: isolated or synthesised by the manufacturer(> 95.0 per cent purity)


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Supplied with:

a certificate of analysis including test methods, test results withcomplete identification by appropriate physico-chemical methods,e.g. nuclear magnetic resonance spectroscopy, infraredspectrophotometry, 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 responsefactors


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The Uses of the ReferenceSubstances/Preparations

Are clearly described in the individual monographs of thePharmacopoeias;

Are applicable to active pharmaceutical substances,excipients and products


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Reference substances required for:

identification (of substance for pharmaceutical use),

system suitability (to ensure adequate performance of theapplied technique),

purity (identification and estimation of specified impuritiesin a substance for pharmaceutical use manufactured by aparticular route of synthesis),

assay (of content of the substance for pharmaceutical useor its products).


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Pharmacopoeial Reference Standards used foridentification of substances for pharmaceutical use

1. Identification:

Pharmaceutical reference standards can be used forconfirmation of identity of the substance by, e.g.

spectroscopy usually, infrared spectrophotometry where the

spectrum of the substance to be examined is compared to thespectrum of the CRS, or to the reference spectrum,

separation techniques where the retention times (or migrationdistance or migration time) of both the substance to beexamined and the CRS are compared.

identification by peptide mapping requires these of both a CRS

its chromatogram.


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2. Related Substances (organic impurities)

The system suitability criteria for selectivity may be:

resolution of two closely eluting impurities or an impurity and the maincompound;

peak-to-valley ratio of an impurity which is incompletely separated to themain compound. A mixture of the compound with a small amount of theimpurity is required;

mixtures of impurities or a mixture of impurities and the compound asreference standard (may be supplied with a chromatogram if prescribedin the monograph) as a system suitability test based on the unadjustedrelative retentions of the impurity peaks to the substance peak and theresolution or peak-to-valley ratio of specified peaks.


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System suitability

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


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Chromatogram of clazuril for system suitability CRS


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Chromatogram ofloperamide hydrochloride for system suitability CRS


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Location of impurity peaks

Confirmation of identification may be achieved by :

reference standards of individual specified impurities;

mixtures (as described above) where their chromatogramsserve also to locate the impurities in the chromatogram ofthe test sample.


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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 determinerelative 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 orless. However, purity correction must be made in the calculation ofthe response factor.


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Pharmacopoeial Reference Standards used for Assay

physico-chemical methods chemical reference substances are employed toassay for content of synthetic and semi-synthetic pharmaceutical substances,herbal substances and peptides:

microbiological assay of antibiotics to determine their potencies. Thesemicrobiological reference standards are referred to a chemical referencesubstances in the European Pharmacopoeia and are secondary reference

standards since they have been established against the International Standard; biological assays (in vitroor in vivo) to determine the activity/potency of

biological substances. The biological reference standards are referred to asBiological Reference Preparations in the European Pharmacopoeia and may beprimary or secondary standards.

The assigned content/potency of the reference standard is method-specific i.e. itis to be applied only with the method described in the specification or themonograph.


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Establishment of Reference Substances

The extent of analyses required for the establishmentof Chemical Reference Substances depends on thepurpose(s) for which it is employed.

In general a batch of good quality, selected from thenormal production of the substance, is satisfactory.


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1) to characterise the substance (proof of molecularstructure) by appropriate chemical attributes such asstructured formula, empirical formula and molecularweight. 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 ofthe elements.


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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 ofwater and residual solvents;

determination of the purity by an absolute method (eg differential scanning calorimetry orphase solubility analysis where appropriate. The results of these determinations are tosupport and confirm the results obtained from separation techniques. They are notnormally included in the calculation of the assigned value);

determination of inorganic impurities (test for heavy metals, sulphated ash, atomicabsorption spectrophotometry, ICP, X-ray fluorescence) often the values obtained willhave no consequence on the assignment of the purity of the standard.


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3) Assay (for the active ingredient only) by an absolutemethod (volumetric titration) is recommended.

The assigned content of the primary chemical referencestandard is calculated from the values obtained from the

analysis performed for the determination of purity and areverified by a calculation of the mass balance.


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Purity of Impurities

at least 90.0 per cent when used only to locate a peak in thechromatogram 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 aspecified impurity (for the purpose of the estimation it is consideredto be 100 per cent). When the minimum purity cannot be obtained,then a purity value is assigned to the standard.


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Most chemical reference substances are fullycharacterised & are traceable to the molecular weight.They are considered to be Primary ReferenceSubstances - no comparison to existing standards.

Chemical Reference Substances (some antibiotics) usedfor the microbiological assay of potency & many of theBiological Reference Preparations are secondaryreference substances since they are calibrated against

the international standard established by WHO.


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Assignment of content/potency of

Reference Substance/Preparation


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Assay

Pharmacopoeial reference standards with an assignedcontent required for comparative assay techniques:

Infrared spectrophotometry

Ultraviolet/visible spectrophotometry

Chromatography

Characterising Reference Material


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Characterising Reference Material(ISO Guide 35)

Single definitive method by a single organisation

Two or more independent reference methods by oneorganisation

Number of methods of known and acceptable accuracyand precision by a network of qualified organisations

Method specific approach (inter-lab study) giving only amethod specific assessed property value


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Assignment of Content

Ph.Eur. Policy

The value assigned is method specific and isestimated from the results obtained by acollaborative trial (min. 6 participants). Othertechniques may be employed to determine thecontent but these results are not used forassigning the value but give complementarysupporting information

E Ph i


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European PharmacopoeiaCollaborative Trials

Participation by:

Experts of the European Pharmacopoeia

Academia Industry

OMCLs

Initiating Laboratory


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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


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Protocol

Loss on drying or

Semi-micro determination of water

AC for repeatability

AC for repeatability

Estimation of organic impurities by theassay method

AC for repeatability, selectivity,sensitivity, symmetry, retentiontime

Estimation of Residual Solvents (> 0.5%) AC for repeatability, selectivity,symmetry, retention time

Estimation of inorganic impurities (ifnecessary)

Representative chromatogram is supplied for info

AC = Acceptance Criteria


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Content of the Reference Standard

The content that is used to correct the purity of the referencestandard is generally calculated as follows:

No correction of % loss on drying or sum water and solvents isdone if the value is


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CHEMICAL REFERENCE SUBSTANCESUNCERTAINTY OF THE ASSIGNED VALUE

Estimated uncertainty =

i2w

2 s2

nt

n1

Wheren= number of participating laboratories

i2 = is the variance for the estimation of impurities

w2 = is the variance for the estimatin of water

s2 = is the variance for the estimation of residual solvents

ASSAY STANDARD FOR LIQUID


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ASSAY STANDARD FOR LIQUIDCHROMATOGRAPHY

Normal limit accepted for analytical error 2.0%

Uncertainty of 0.24% implies 0.1% probability of rejectinga good result (n=3)

If this is considered to be insignificant then there is nojustification for giving uncertainty values with the assignedvalue

Maximum permitted uncertainty varies proportionally to

the limits set e.g. maximum permitted uncertainty forlimits of 1.0% would 0.12


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Uncertainty

The estimated uncertainty of the result of a collaborativetrial is employed to assess the acceptability of the trial.

If the estimated uncertainty is greater than a pre-definedcriterion 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 thiseventuality is unlikely.


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DETERMINATION OF THE UNCERTAINTY OF THE ASSIGNED


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DETERMINATION OF THE UNCERTAINTY OF THE ASSIGNEDVALUE

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

Approx uncertainty =0.005

6 0.005

7 2.50.1


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Extended use of Ph. Eur Reference Standards

1. Assay reference standard used for determination of content in amonograph for a substance for pharmaceutical use can be usedfor the assay of that substance in a pharmaceutical product.

Provided that:

- the assay method employed for the product is that described inthe monograph of the substance for pharmaceutical use;

- any pre-treatment of the sample (eg extraction) is validated by theuser.

Misuse of European Pharmacopoeia Reference


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Misuse of European Pharmacopoeia ReferenceStandards

If used for another purpose other than those described in

the monograph it is the responsibility of the user tovalidate appropriately the reference standard

CERTIFICATEP d I t ti l Ch i l R f S b t


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Proposed International Chemical Reference SubstanceABACAVIR SULFATE

Control No 106238

Intended useThe International Chemical Reference Substance for abacavir sulfate isintended to be used in the infrared absorption spectrophotometric andthin-layer chromatographic tests for identity according to the monographfor Abacavir sulfate in The International Pharmacopoeia.Analytical data mg of1.3: A spectrum,nfrared absorption spectrophotometry (IR)Iabacavir sulfate in 300 mg of potassium bromide, is given in figureW106238T.


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4000,0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400,0

0,0

10

20

30

40

50

60

70

80

90

100,0

cm-1

%T

W106238T


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Ultraviolet spectrophotometric absorption data: = 416 at 297 nm,calculated with reference to the dried substance. Abacavir sulfate wasdissolved in 0.1 M hydrochloric acid.

High performance liquid chromatography (HPLC): The purity wasestimated by peak area normalization to 99.8% at 254 nm.

Thin-layer chromatography (TLC): The purity was estimated to100.0% at 254 nm, when 100 g were applied.

Thermogravimetric analysis (TG): When heated to 105 C a loss of

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