1 5 AnalyticalMethodDevelopment Validation

8/20/2019 1 5 AnalyticalMethodDevelopment Validation

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Analytical Method Developmentand Validation

Bob Seevers


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Importance of Analytical Methods inDevelopment Stability Studies

• Without analytical methods it is notpossible to know what has happenedduring stability – Assay

– Impurities/Degradation roducts

– Dissolution

– !hiral urity – reservative !ontent


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Interactive "ole of Analytical

Methods and Stability Data

• #his is a chicken and egg problem – $eed analytical methods to generate stability data

– $eed stability information to develop methods

• Start with preliminary method based on what is

known of drug substance – !an determine content of drug substance

– %nown or e&pected degradants are separated

• 'se preliminary method to analy(e stress stability

studies – Stress deliberately creates signi)cant degradation

– !an evaluate ability of method to separate degradants

• 'se this information to modify/improve method


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#ypes of Stress Studies

• hotostability

• *igh #emperature

•

+ow #emperature• ,&idation

• p* e&tremes


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Selection of !hromatographicMode

• *+!

• "eversed-phase *+!

•

!hiral *+!• .!

• #+!

•

lectorphoresis


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Identifying Degradants

• redicting routes of degradation

– Acid/base hydrolysis of esters and amides

– ,&idation of thiols0 alcohols and amines

– +oss of methyl groups

• Synthesi(ing possible degradants – repare possible degradant

–

%nown Structure – #est it in potential analytical method

• Detectability

• Separation from parent peak


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Assay "un #ime vs1

Detection/"esolution

• +ong run times for an assay meanthat stability testing will take moretime and cost more

• Shorter run times mean that testingwill go faster and be less e&pensivebut – +ower resolution

– Some peaks may not be separated


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2alidation rotocol

• A validation protocol lays out ahead oftime the e&perimental design that will beused to establish the validity the

analytical methods – "eagent0 solvents

– Sample0 standard0 solution preparation

– Identify e3uipment to be used

– !hromatographic conditions

– System suitability

– !alculations


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I!* 45 2alidation of Analyticalrocedures

• #ypes of Analytical rocedures to be2alidated – Identi)cation tests6

–

4uantitative tests for impurities7 content6 – +imit tests for the control of impurities6

– 4uantitative tests of the active moiety in samplesof drug substance or drug product or otherselected component8s9 in the drug product1

• See also 'S .eneral !hapter :;55


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With "egard to Stability

• #ypes of Analytical rocedures to be2alidated – Identi)cation tests6

– 4uantitative tests for impurities7content6

– +imit tests for the control of impurities6

– 4uantitative tests of the active moietyin samples of drug substance or drugproduct or other selected component8s9in the drug product1• Drug product assay


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#ypical 2alidation !haracteristics

• Accuracy

• recision

– "epeatability

–

Intermediate recision• Speci)city

• Detection +imit

• 4uantitation +imit

• +inearity

• "ange

• "obustness


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Accuracy

• !loseness of agreement between thevalue found and either – the value accepted as a conventional

true value

– or an accepted reference value


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Accuracy for DrugSubstance

• Several methods of determining accuracyare available> – application of an analytical procedure to an

analyte of known purity 8e1g1 referencematerial96

– comparison of the results of the proposedanalytical procedure with those of a secondwell-characteri(ed procedure0 the accuracy ofwhich is stated and/or de)ned6

– accuracy may be inferred once precision0linearity and speci)city have been established1


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Accuracy for Drug roduct

• Several methods for determining accuracy areavailable> – application of the analytical procedure to synthetic

mi&tures of the drug product components to which known

3uantities of the drug substance to be analysed have beenadded6

– in cases where it is impossible to obtain samples of all drugproduct components 0 it may be acceptable either to addknown 3uantities of the analyte to the drug product or tocompare the results obtained from a second0 wellcharacteri(ed procedure0 the accuracy of which is statedand/or de)ned6

– accuracy may be inferred once precision0 linearity andspeci)city have been established1


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recision

• recision of an analytical procedure e&presses thecloseness of agreement 8degree of scatter9between a series of measurements obtained frommultiple sampling of the same homogeneous

sample under the prescribed conditions1 – "epeatability

– intermediate precision

– reproducibility1

•

#he standard deviation0 relative standard deviation8coe?cient of variation9 and con)dence intervalshould be reported for each type of precisioninvestigated1


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"epeatability

• "epeatability should be assessedusing> – a minimum of @ determinations covering

the speci)ed range for the procedure8e1g10 concentrations/ replicateseach96

–

or – a minimum of determinations at ;CC

of the test concentration1


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Intermediate recision

• Depends on the circumstances under whichthe procedure is intended to be used1

• +ook at eEects of random events on the

precision of the analytical procedure1 – Days

– Analysts

– e3uipment0 etc1

• It is not considered necessary to study theseeEects individually1 #he use of ane&perimental design 8matri&9 is encouraged1


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"eproducibility

• "eproducibility is assessed by meansof an inter-laboratory trial1"eproducibility should be considered

in case of the standardi(ation of ananalytical procedure0 for instance0 forinclusion of procedures in

pharmacopoeias1 #hese data are notpart of the marketing authori(ationdossier1


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Speci)city

• Speci)city is the ability to assessune3uivocally the analyte in thepresence of components which may

be e&pected to be present1 #ypicallythese might include impurities0degradants0 matri&0 etc1

• +ack of speci)city of an individualanalytical procedure may becompensated by other supporting

analytical procedure8s91


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Identity

• Able to discriminate between compoundsof closely related structures which arelikely to be present1

• #he discrimination of a procedure may becon)rmed by obtaining positive results8perhaps by comparison with a knownreference material9 from samples

containing the analyte0 coupled withnegative results from samples which donot contain the analyte1


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Detection +imit

• #he detection limit of an individualanalytical procedure is the lowest amountof analyte in a sample which can be

detected but not necessarily 3uantitatedas an e&act value1

• !an be determined

– 2isually

– Signal to $oise

– Standard Deviation of the "esponse and theSlope


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Detection +imit> Signal to$oise

• #his approach can only be applied to analyticalprocedures which e&hibit baseline noise1

• !omparing measured signals from samples

with known low concentrations of analyte withthose of blank samples and establishing theminimum concentration at which the analytecan be reliably detected1

•

A signal-to-noise ratio between or 5>; isgenerally considered acceptable for estimatingthe detection limit1


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Detection +imit> Standard Deviationof the "esponse and the Slope

• #he detection limit 8D+9 may be e&pressedas> D+ F 1 G/S – G F the standard deviation of the response

–

S F the slope of the calibration curve• #he slope S may be estimated from the

calibration curve of the analyte1 #heestimate of G may be carried out in a

variety of ways0 for e&ample> – Based on the Standard Deviation of the Blank

– Based on the !alibration !urve


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4uantitation +imit

• #he 3uantitation limit of an individualanalytical procedure is the lowestamount of analyte in a sample which

can be 3uantitatively determinedwith suitable precision and accuracy1

#he 3uantitation limit is a parameter

of 3uantitative assays for low levelsof compounds in sample matrices0and is used particularly for the

determination of impurities and/or


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4uantitation +imit

• 2isual valuation

• Signal-to-$oise

• Standard Deviation of the "esponse and the Slope – 3uantitation limit 84+9 may be e&pressed as> 4+ F ;C

G/S

– G F the standard deviation of the response

• S F the slope of the calibration curve

• #he estimate of G may be carried out in a varietyof ways0 for e&ample> – Based on the Standard Deviation of the Blank

– Based on the !alibration !urve


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+inearity

• #he linearity of an analytical procedure is itsability 8within a given range9 to obtain test resultswhich are directly proportional to theconcentration 8amount9 of analyte in the sample1

• #est results should be evaluated by appropriatestatistical methods0 for e&ample0 by calculation ofa regression line by the method of least s3uares1 – correlation coe?cient0 y-intercept0 slope of the

regression line and residual sum of s3uares should besubmitted

– minimum of < concentrations is recommended


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"ange

• #he range of an analytical procedure is the intervalbetween the upper and lower concentration 8amounts9 ofanalyte in the sample 8including these concentrations9 forwhich it has been demonstrated that the analyticalprocedure has a suitable level of precision0 accuracy and

linearity1

• #he following minimum speci)ed ranges should beconsidered>

– Hor the assay of a drug substance or a )nished 8drug9product> normally from C to ;5C percent of the test

concentration6

– Hor content uniformity0 covering a minimum of JC to ;Cpercent of the test concentration0 unless a wider moreappropriate range0 based on the nature of the dosageform 8e1g10 metered dose inhalers90 is Kusti)ed6

– Hor dissolution testing> L/-5C over the speci)ed range


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"obustness

• #he robustness of an analytical procedure is ameasure of its capacity to remain unaEected bysmall0 but deliberate variations in methodparameters and provides an indication of its

reliability during normal usage1 – stability of analytical solutions6

– e&traction time1

– inuence of variations of p* in a mobile phase

–

inuence of variations in mobile phase composition – diEerent columns 8diEerent lots and/or suppliers9

– #emperature

– ow rate1


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Method 2alidation "eport

• rovide results of validation eEort

• &plain – !hoice of acceptance criteria

– *ow method was developed

• Method 2alidation "eport will besubmitted to regulators


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"evalidation May be$ecessary

• !hanges in the synthesis of the drugsubstance6

• !hanges in the composition of the)nished product6

• !hanges in the analytical procedure1

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1 5 AnalyticalMethodDevelopment Validation