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&le> – 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&le> – 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&le0 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