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ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR
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ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR

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ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR

ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR

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  • 1. prof. RavisankarVignan Pharmacy collegeValdlamudiGuntur Dist.Andhra PradeshIndia.banuman35@gmail.com00919059994000
  • 2.  Validation is a very necessary element ofany firm that falls under the scrutiny ofthe governing regulatory agencies TheFDA, under the authority of existingcGMP regulations guidelines anddirectives, considers validation isnecessary and because it makes goodsense of science / engineering.
  • 3.  Drug substances and drug productmanufacturers must performvalidations, it is very important thatthis understanding be sharedthroughout the organization The term validation generally tocover the entire spectrum of cGMP
  • 4. DEFINTIONThe process to Confirm that the analyticalThe process to Confirm that the analyticalprocedure employed for a specific test isprocedure employed for a specific test issuitable for intended use and that theysuitable for intended use and that theysupport the identity, quality, purity andsupport the identity, quality, purity andpotency of the drug substances and drugpotency of the drug substances and drugproducts.products.I. INTRODUCTIONI. INTRODUCTION
  • 5. NEED The process of method development andThe process of method development andvalidation has a direct impact on thevalidation has a direct impact on thequality of these dataquality of these data To trust the methodTo trust the method Regulatory requirementRegulatory requirementI. INTRODUCTION (Contd.,)
  • 6. Currently available guidelines from variousCurrently available guidelines from variousregulatory bodiesregulatory bodiesICHICHUSFDAUSFDACDERCDEREPEPUSP XXXIUSP XXXII. INTRODUCTION (Contd.,)
  • 7. Analytical procedures to be validatedAnalytical procedures to be validatedI. INTRODUCTION (Contd.,) Identification TestsIdentification Tests Quantitative Tests for ImpuritiesQuantitative Tests for ImpuritiesQuantitative tests for active moiety in samples ofQuantitative tests for active moiety in samples ofdrug substance & drug products and other selecteddrug substance & drug products and other selectedcomponents in drug productcomponents in drug product
  • 8. I. INTRODUCTION (Contd.,)I. INTRODUCTION (Contd.,)Recommended validation characteristics of the various types ofRecommended validation characteristics of the various types ofproceduresprocedures
  • 9. CONCEPT OF REVALIDATIONWhen we make any changes inWhen we make any changes in Analytical procedureAnalytical procedure Drug substance (e.g. synthetic route)Drug substance (e.g. synthetic route) Drug product (e.g. composition)Drug product (e.g. composition) The changes may necessitate revalidation ofThe changes may necessitate revalidation ofthe analytical proceduresthe analytical proceduresI. INTRODUCTION (Contd.,)I. INTRODUCTION (Contd.,)
  • 10. A few typical cases are presented below whereA few typical cases are presented below whererevalidation is necessaryrevalidation is necessaryChangeChange Parameters to be considered forParameters to be considered forrevalidationrevalidationSynthetic routeSynthetic route All parametersAll parametersAnalytical procedureAnalytical procedure All parametersAll parametersAddition of new impurityAddition of new impurity Specificity, stability, linearity, accuracy,Specificity, stability, linearity, accuracy,range, LOD & LOD (for new impurityrange, LOD & LOD (for new impurityonly)only)Composition of drug productComposition of drug product Specificity, stability, Precision andSpecificity, stability, Precision andaccuracyaccuracyChange in specificationChange in specification Linearity, accuracy and rangeLinearity, accuracy and rangeI. INTRODUCTION (Contd.,)*If solubility differs each other
  • 11. PRE-REQUISITESPRE-REQUISITES AA well-designed experimental matrix (Writtenwell-designed experimental matrix (Writtenprotocol)protocol) Step-by-step methodology (STP)Step-by-step methodology (STP) Test samples of good quality (should meet the qualityTest samples of good quality (should meet the qualityas per defined specifications)as per defined specifications) Peak purity (preferably >99% purity)Peak purity (preferably >99% purity) Equipments & analytical instruments should be inEquipments & analytical instruments should be incalibrated state.calibrated state.I. INTRODUCTION (Contd.,)
  • 12. PARAMETERS TO BE EVALUATEDPARAMETERS TO BE EVALUATEDSpecificity *Specificity *Forced degradation study*Forced degradation study*LOD & LOQ (if applicable)*LOD & LOQ (if applicable)*Linearity*Linearity*Precision*Precision*System Precision (System suitability)*System Precision (System suitability)*Method Repeatability*Method Repeatability*Intermediate Precision (or) Ruggedness*Intermediate Precision (or) Ruggedness*Method Reproducibility**Method Reproducibility**
  • 13. • Accuracy (or) Recovery*• Solution Stability*• Robustness*• System Suitability** Included in ICH Guidelines** Terminology included in ICH guidelines but are notpart of required parameters
  • 14.  No exact methodology given for each parameterNo exact methodology given for each parameter Only ICH – Q2B and CDER guidelines are provided butOnly ICH – Q2B and CDER guidelines are provided butnot to the extent of 100%not to the extent of 100% Good understanding of each performance characteristicsGood understanding of each performance characteristicsmost important. This understanding must be beyond themost important. This understanding must be beyond thebasic definition of each parameterbasic definition of each parameter Understanding must be anchored by sufficient years ofUnderstanding must be anchored by sufficient years ofpractical experience and knowledge. It will permit soundpractical experience and knowledge. It will permit soundand logical decisions, even under the most intenseand logical decisions, even under the most intensesituationssituationsII. METHODOLOGY
  • 15. DEFINITION SpecificitySpecificity of analytical method as its ability to measureof analytical method as its ability to measureaccurately an analyte in the presence of interference, suchaccurately an analyte in the presence of interference, suchas synthetic precursors, excipients, enantiomers andas synthetic precursors, excipients, enantiomers andknown (or likely) degradation product that may beknown (or likely) degradation product that may beexpected to be present in the sample matrix.expected to be present in the sample matrix.1. SPECIFICITY
  • 16. DISCUSSION SpecificSpecific generally refers to a method that produces agenerally refers to a method that produces aresponse for a single analyte onlyresponse for a single analyte only SelectiveSelective refers to a method which provides responses for arefers to a method which provides responses for anumber of chemical entities that may / may not benumber of chemical entities that may / may not bedistinguished from each other. If each response isdistinguished from each other. If each response isdistinguished from all other responses, then the method isdistinguished from all other responses, then the method issaid to be selective.said to be selective. Use of the termUse of the term SelectivitySelectivity is appropriate for the methodsis appropriate for the methodsbased on techniques such as HPLC, GC methodsbased on techniques such as HPLC, GC methods..1. SPECIFICITY
  • 17. 1.SPECIFICITYEVALUATIONIdentification tests: Response for compound of interest onlyAssay: Peak purity of analyte peak.Impurities: Resolution between within the impurity(s) and/ordegradants and form analytePeak purity of - analyte peak (for un-spiked)- analyte and impurity(s) peaks (for spiked samples)
  • 18. 1.SPECIFICITYACCEPTANCE CRITERIAIdentification Tests: Positive response for compound of interest onlyAssay : No peak should be found at the retention time of analyte peakand Peak purity of analyte peak should pass.Impurities : Should pass Peak purity of -main analyte and ImpuritypeaksNo peak should be found at the retention time of analyte/ImpurityDissolution: No peak should be found at the retention time of analyte.In case of UV methodology, % difference should be not more than 2.0
  • 19. 1.SPECIFICITYFORCED DEGRADATION STUDIESINTRODUCTIONINTRODUCTIONForced degradation or stress testing is undertaken toForced degradation or stress testing is undertaken todemonstratedemonstrate specificityspecificity when developing stability-indicatingwhen developing stability-indicatingmethodsmethodsA stability-indicating method is one that accurately quantitatesA stability-indicating method is one that accurately quantitatesthe active ingredients without interference from degradationthe active ingredients without interference from degradationproducts, process impurities, excipients or other potentialproducts, process impurities, excipients or other potentialimpuritiesimpurities
  • 20.  Address the stability of the compound Establish the degradation pathway Identify the degradation products Validate the stability indicating power of theanalytical procedures used
  • 21. 1.SPECIFICITY / SELECTIVITY (Contd.,)FORCED DEGRADATION STUDIESPROCEDUREPROCEDUREPerform analysis for each stressed (acid / base / peroxide / thermal /Perform analysis for each stressed (acid / base / peroxide / thermal /photolytic / humidity) sample as per methodologyphotolytic / humidity) sample as per methodologyNormal initial stressed conditions to be appliedNormal initial stressed conditions to be applied1M HCl1M HCl1M NaOH1M NaOH10% H10% H22OO22105°C/at least 72 Hours105°C/at least 72 Hours12000 Lux/at least 72 Hours12000 Lux/at least 72 Hours92% RH/25°C/at least 72 Hours92% RH/25°C/at least 72 Hours
  • 22.  Initiated at an early stage of development Repeated as methods, processes or formulationschange, so it is an ongoing effort. Evaluate the each unique formulation before formalstability begins
  • 23. EVALUATIONEVALUATIONAssay:Assay: % Difference of assay for Control (Un-stressed) and each Stressed% Difference of assay for Control (Un-stressed) and each StressedsamplessamplesPeak purity of analyte peak for Control and stressed samplePeak purity of analyte peak for Control and stressed sampleImpurities:Impurities: Peak purity of analyte peak for Control and each Stressed samplePeak purity of analyte peak for Control and each Stressed sampleACCEPTANCE CRITERIAACCEPTANCE CRITERIAAssay:Assay: Peak purity of analyte peak in Control and each Stressed samples shouldPeak purity of analyte peak in Control and each Stressed samples shouldpasspassImpurities:Impurities: Peak purity of analyte peak in Control and each Stressed samplesPeak purity of analyte peak in Control and each Stressed samplesshould passshould pass1.SPECIFICITY / SELECTIVITY (Contd.,)FORCED DEGRADATION STUDIES
  • 24. Points to rememberPoints to rememberIf the degradation media degrades the drug substance/drug product to too great extentIf the degradation media degrades the drug substance/drug product to too great extentor do not degrade the drug substance/drug product at all, then alternative action shouldor do not degrade the drug substance/drug product at all, then alternative action shouldbe taken (e.g., change the strength of the degradation medium or exposure time or applybe taken (e.g., change the strength of the degradation medium or exposure time or applyheat over a period of time to achieve minimum level of degradation) heat over a period of time to achieve minimum level of degradation) Different numerical values were proposed for the extent of degradation in recentDifferent numerical values were proposed for the extent of degradation in recentliteratureliterature* Minimum 5%* Minimum 5%Some compound may not necessarily degrade under a given stress condition. NoSome compound may not necessarily degrade under a given stress condition. Nofurther stressing is advised in these casesfurther stressing is advised in these casesOver stressing may lead to the formation of secondary degradants, causes disturbance toOver stressing may lead to the formation of secondary degradants, causes disturbance tothethe selectivityselectivity of the method, requires further developmentof the method, requires further development1.SPECIFICITY / SELECTIVITY (Contd.,)FORCED DEGRADATION STUDIES
  • 25. Hence, the forced degradation studies should mimic the conditions to which theHence, the forced degradation studies should mimic the conditions to which thedrug substance / drug product is actually exposed during its shelf lifedrug substance / drug product is actually exposed during its shelf lifeA simple logic behind these studies is, ability to separate the analyte ofA simple logic behind these studies is, ability to separate the analyte ofinterest from its degradation impurities formed, if any, caused byinterest from its degradation impurities formed, if any, caused byquality of chemicals used for its manufacturing, processing,quality of chemicals used for its manufacturing, processing,packaging, storage, shipment, and/or any unexpected exposurepackaging, storage, shipment, and/or any unexpected exposureduring shelf life of the drug substance/drug productduring shelf life of the drug substance/drug product1.SPECIFICITY / SELECTIVITY (Contd.,)FORCED DEGRADATION STUDIES
  • 26. 2. LIMIT OF DETECTION AND LIMIT OF QUANTIFICATION(LOD & LOQ)DEFINITIONDEFINITIONLOD:LOD: Lowest amount of analyte in a sample which can be detected but notLowest amount of analyte in a sample which can be detected but notnecessarily quantitated, under the stated experimental conditions (LOD)necessarily quantitated, under the stated experimental conditions (LOD)LOQ:LOQ: Lowest amount of analyte in a sample which can be quantitativelyLowest amount of analyte in a sample which can be quantitativelydetermined with suitable precision and accuracy (LOQ)determined with suitable precision and accuracy (LOQ)
  • 27. Different approaches suggested by ICH, USP & EPSeveral approaches are given in the ICH guidelines for estimating LOD and LOQSeveral approaches are given in the ICH guidelines for estimating LOD and LOQDepending on the procedure : instrumental / non-instrumentalDepending on the procedure : instrumental / non-instrumentalApproachApproach LODLOD LOQLOQVisual inspectionVisual inspection Minimum level detectableMinimum level detectable Minimum level quantifiableMinimum level quantifiableSignal-to-Noise ratioSignal-to-Noise ratio 2:1 or 3:12:1 or 3:1 10:110:1SD of response (SD of response (σσ) &) &Slope (S)Slope (S)[3:3 x[3:3 x σσ] / S] / S [10.0 x[10.0 x σσ] / S] / SRSD CriteriaRSD Criteria Concentration at whichConcentration at whichRSD10 to 33.0%RSD10 to 33.0%Concentration at which RSDConcentration at which RSD≤≤10.0%10.0%As per ICH and USP, other approaches suggested above are alsoacceptable2. LOD & LOQ (Contd.,)
  • 28. PROCEDUREPROCEDURESD of response (SD of response () & Slope (S)) & Slope (S): Prepare linearity curve with a series of related: Prepare linearity curve with a series of relatedsubstance(s) solutions at different concentrations (3 concentrations below 50 % ofsubstance(s) solutions at different concentrations (3 concentrations below 50 % ofspecification level and 3 more concentrations above 50 % specification level)specification level and 3 more concentrations above 50 % specification level)RSD criteria:RSD criteria: Prepare a series of related substance(s) solutions of differentPrepare a series of related substance(s) solutions of differentconcentrations below to specification level (generally about 10%, 20%, 30%, 40% andconcentrations below to specification level (generally about 10%, 20%, 30%, 40% and50% of specification concentration) and inject six replicate injections into HPLC.50% of specification concentration) and inject six replicate injections into HPLC.Precision should be established (if predicted from other than RSD criteria) at LOQ andPrecision should be established (if predicted from other than RSD criteria) at LOQ andLOD level as per ICH, USP & EP guidelinesLOD level as per ICH, USP & EP guidelines** Prepare the solution at predicted concentration (for LOQ/LOD) and inject in to sixPrepare the solution at predicted concentration (for LOQ/LOD) and inject in to sixreplicates as per methodologyreplicates as per methodology2. LOD & LOQ (Contd.,)
  • 29. 2. LOD & LOQ (Contd.,)EVALUATIONEVALUATIONSD of response (SD of response () & Slope (S)) & Slope (S):: Calculate residual standard deviation on response ‘Calculate residual standard deviation on response ‘’’(also called residual standard deviation on Y- intercept or residual standard error or mean(also called residual standard deviation on Y- intercept or residual standard error or meansquare error or residual sum of squares) and slope (S) obtained from regression datasquare error or residual sum of squares) and slope (S) obtained from regression dataLOD = 3.3 xLOD = 3.3 x  / S LOQ = 10 x/ S LOQ = 10 x  / S/ S  RSD criteria:RSD criteria: Determine RSD for impurity response for each concentration and declareDetermine RSD for impurity response for each concentration and declarethe LOQ concentration in ‘µg/mL’ at which RSD of six replicate injections isthe LOQ concentration in ‘µg/mL’ at which RSD of six replicate injections is ≤≤ 10.0%10.0%and similarly, LOD concentration in ‘µg/mL’ at which RSD of six replicate injections isand similarly, LOD concentration in ‘µg/mL’ at which RSD of six replicate injections isbetweenbetween >> 10.0% and10.0% and ≤≤ 33.0%33.0%ACCEPTANCE CRITERIAACCEPTANCE CRITERIA(if predicted from other than RSD criteria)(if predicted from other than RSD criteria)RSD of six replicate injections isRSD of six replicate injections is ≤≤ 10.0% for LOQ and between10.0% for LOQ and between >> 10.0% and10.0% and ≤≤ 33.0%33.0%
  • 30. 3. LINEARITYDEFINITIONDEFINITIONTheThe LinearityLinearity of an analytical procedure is its ability (within a given range) toof an analytical procedure is its ability (within a given range) toelicit (obtain) test results that are directly proportional to the concentrationelicit (obtain) test results that are directly proportional to the concentration(amount) of analyte in the sample(amount) of analyte in the sampleRange: The interval between the upper and lower level( Including these level)Range: The interval between the upper and lower level( Including these level)that have been demonstrated to be determined with precision, accuracy andthat have been demonstrated to be determined with precision, accuracy andlinearity using this method as writtenlinearity using this method as writtenDISCUSSIONDISCUSSIONIn order to determine the quantity of any analyte present in unknown sample,In order to determine the quantity of any analyte present in unknown sample,some kind of relation ship (mathematical/empirical) between concentration andsome kind of relation ship (mathematical/empirical) between concentration andresponse is essentialresponse is essentialANALYTE ------ PROCEDURE ------ANALYTE ------ PROCEDURE ------ RESPONSERESPONSEICH and USP encouraging Linear and non-Linear relation shipsICH and USP encouraging Linear and non-Linear relation ships
  • 31. PROCEDUREPROCEDUREPrepare a series of solutions (not less than five is recommended) with standard /Prepare a series of solutions (not less than five is recommended) with standard /reference samples in the specified concentration range and analyze them as perreference samples in the specified concentration range and analyze them as permethodmethodAssay:-Assay:- 80% to 120% of test concentration80% to 120% of test concentrationCU :-CU :- 70% to 130% of70% to 130% of test concentrationtest concentrationDissolution:-Dissolution:- ± 20% of expected release (Q) for immediate release± 20% of expected release (Q) for immediate release0 to 120% (for extended release)0 to 120% (for extended release)Impurities:-Impurities:- LOQ to 200% of specificationLOQ to 200% of specification3. LINEARITY (Contd.,)
  • 32. 3. LINEARITY (Contd.,)EVALUATIONEVALUATIONSlopeSlope- indicates sensitivity of the method- indicates sensitivity of the methodInterceptIntercept- indicates response for no analyte (interference)- indicates response for no analyte (interference)Residual sum of squaresResidual sum of squares- indicates uncertainty of intercept(in blank response)- indicates uncertainty of intercept(in blank response)Correlation CoefficientCorrelation Coefficient- indicates the relation ship chosen is correct- indicates the relation ship chosen is correctACCEPTANCE CRITERIAACCEPTANCE CRITERIACorrelation Coefficient should be not less than 0.999 for assay, CU, dissolution testCorrelation Coefficient should be not less than 0.999 for assay, CU, dissolution testmethods and 0.99 for impurities test methodmethods and 0.99 for impurities test method
  • 33. 4. PRECISION (Contd.,)DEFINITIONDEFINITIONCloseness of agreement (degree of scatter) between a series of measurementsCloseness of agreement (degree of scatter) between a series of measurementsobtained from multiple sampling of the same homogenous sample under theobtained from multiple sampling of the same homogenous sample under theprescribed conditions.prescribed conditions.Precision may be considered at three levels.Precision may be considered at three levels.1.1. System precision (System suitability)System precision (System suitability)2.2. Method RepeatabilityMethod Repeatability3.3. Intermediate precisionIntermediate precision4.4. ReproducibilityReproducibility
  • 34. 4. PRECISION (Contd.,)DISCUSSIONDISCUSSION Repeatability:Repeatability: precision under same operating conditions (with-in a laboratoryprecision under same operating conditions (with-in a laboratoryover a short period of time using the sameover a short period of time using the same analyst with the same equipment)analyst with the same equipment)Measurement / Injection repeatability (System Precision)Measurement / Injection repeatability (System Precision)Method repeatability (Method Precision)Method repeatability (Method Precision)Intermediate precisionIntermediate precision: precision under different laboratory conditions (within-: precision under different laboratory conditions (within-laboratory variation, as on different days, or with different analysts, or equipmentslaboratory variation, as on different days, or with different analysts, or equipmentswithin the same laboratory)within the same laboratory)ReproducibilityReproducibility: precision between laboratories / intermediate precision can be: precision between laboratories / intermediate precision can beconsidered during the standardization of a procedure before it is submitted to theconsidered during the standardization of a procedure before it is submitted to thepharmacopoeiapharmacopoeiaAs per CDER guidelines, it is not normally expected if intermediate precision isAs per CDER guidelines, it is not normally expected if intermediate precision isaccomplishedaccomplished
  • 35. 4.PRECISION (Contd.,)PROCEDUREPROCEDURE Six replicate measurements/injections of standard preparation (System Precision)Six replicate measurements/injections of standard preparation (System Precision) Six replicate analysis of the samples through the complete analytical procedure fromSix replicate analysis of the samples through the complete analytical procedure fromsample preparation to final result (Method Precision)sample preparation to final result (Method Precision) Three different matrices at 80%, 100% and 120%(Triplicate preparation atThree different matrices at 80%, 100% and 120%(Triplicate preparation attriplicate concentration)triplicate concentration) Six replicate analysis of the samples through the complete analytical procedure fromSix replicate analysis of the samples through the complete analytical procedure fromsample preparation to final result by two different analysts, columns, instruments,sample preparation to final result by two different analysts, columns, instruments,different daysdifferent days (Ruggedness)(Ruggedness)
  • 36. 4.PRECISION (Contd.,)ACCEPTANCE CRITERIAProcedureProcedure%RSD%RSDSystem PrecisionSystem Precision Method PrecisionMethod Precision RuggednessRuggednessAPIAPI DPDP APIAPI DPDP APIAPI DPDPAssayAssay 1.0/2.01.0/2.0 1.0/2.0*1.0/2.0* 1.01.0 2.02.0 1.01.0 3.03.0DissolutionDissolution NANA 1.0 / 2.0*1.0 / 2.0* NANA 6.06.0 NANA 5.05.0ImpuritiesImpurities 5.0*5.0* 5.0*5.0* 10.010.0 10.010.0 10.010.0 15.015.0NA – Not applicableAPI – Active Pharmaceutical IngredientDP – Drug Product* : As given in STP
  • 37. 5. ACCURACYDEFINITIONDEFINITIONThe accuracy of an analytical procedure expresses theThe accuracy of an analytical procedure expresses thecloseness of agreement between the value, which is acceptedcloseness of agreement between the value, which is acceptedeither as a conventional true value or an accepted reference valueeither as a conventional true value or an accepted reference valueand the value foundand the value found
  • 38. 5.ACCURACY (Contd.,)PROCEDUREAssay/Dissolution:- Known amount of drug substance spiked with syntheticmixtures of drug product components (excipients) – minimum of three levels80%, 100% & 120% of test concentration-assay;± 20% of expected release-dissolution), each level is triplicateImpurities:- drug substance/drug product spiked with known amounts of impurities- minimum of three levelsLOQ level to 200% of specification andAt LOQ level, each level is triplicateEVALUATION- Recovery from amount added and amount found- Precision (%RSD) at each level (for three replicate preparations)
  • 39. 5.ACCURACY (Contd.,)ACCEPTANCE CRITERIAAssay:- Recovery should be between 98% to 102%( Depends upon the yourStrength)Dissolution:- 95% to 105%Impurities:- if, Specification is ≤ 0.2% : 85% to 115%if, Specification is > 0.2% : 90% to 110%At LOQ level : 80% to 120%A simple logic behind this performance characteristic is whetherthe procedure is capable of estimating a true value or not
  • 40. 7. STABILITYDISCUSSIONDISCUSSION It is often essential that solutions (standards, test samples) be stable enoughIt is often essential that solutions (standards, test samples) be stable enoughto allow for delays covering instrument break downs / overnight analysesto allow for delays covering instrument break downs / overnight analyses A minimum of 12 Hrs, 18 Hrs, or 24 Hrs is routinely recommended forA minimum of 12 Hrs, 18 Hrs, or 24 Hrs is routinely recommended forchromatographic methods for which vialed solutions may remain on an auto-chromatographic methods for which vialed solutions may remain on an auto-samplers at ambient temperatures due to various delays.samplers at ambient temperatures due to various delays.
  • 41. 7. STABILITY (Contd.,)PROCEDUREPROCEDUREPrepare test sample as per procedure and analyze at initial and at different timePrepare test sample as per procedure and analyze at initial and at different timeintervals by keeping the sample at room temperature ( 25°C) / refrigeratorintervals by keeping the sample at room temperature ( 25°C) / refrigeratorcondition (2-8°C)condition (2-8°C)EVALUATIONEVALUATION% Difference from initial response to specified interval for analyte / each impurity% Difference from initial response to specified interval for analyte / each impurityACCEPTANCE CRITERIAACCEPTANCE CRITERIA% Difference is not more than% Difference is not more thanAssay, CU :Assay, CU : 2.02.0Dissolution :Dissolution : 3.03.0Impurities :Impurities : 10.010.0
  • 42. 7. STABILITY (Contd.,)A simple logic behind this study is to determine the periodA simple logic behind this study is to determine the periodof time, a solution can be held before analysis withoutof time, a solution can be held before analysis withoutcompromising accuracycompromising accuracy..
  • 43. 8. ROBUSTNESSDEFINTIONDEFINTIONMeasure of its capacity to remain unaffected by small, but deliberate variationsMeasure of its capacity to remain unaffected by small, but deliberate variationsin method parameters and provides indication of its reliability during its normalin method parameters and provides indication of its reliability during its normalusageusageDISCUSSIONDISCUSSIONvarying method parameters within a realistic range and the quantitativevarying method parameters within a realistic range and the quantitativeinfluence of the variables is determined, and, if the influence of the parameter isinfluence of the variables is determined, and, if the influence of the parameter iswithin a previously specified tolerance, then, the parameter is said to be withinwithin a previously specified tolerance, then, the parameter is said to be withinthe method’s robustness rangethe method’s robustness rangeAccording to ICH guidelines, robustness should be considered early in theAccording to ICH guidelines, robustness should be considered early in thedevelopment stage of a method, but it is not required to be included as part of adevelopment stage of a method, but it is not required to be included as part of aregistration application.registration application.
  • 44. 8. ROBUSTNESS (Contd.,)Typical variations include under validation programmeTypical variations include under validation programme Flow rate (Flow rate ( 10%)10%) Wavelength (Wavelength ( 2 nm)2 nm) Mobile phase composition, generally, Organic composition (Mobile phase composition, generally, Organic composition ( 2 (or) 5%)2 (or) 5%) Temperature (Temperature ( 5° C)5° C) pH of the mobile phase (pH of the mobile phase ( 0.2 units)0.2 units)
  • 45. 8. ROBUSTNESS (Contd.,)PROCEDUREPROCEDUREAssay:Assay:Analysis of Resolution (if applicable), Standard & Test samples (2Analysis of Resolution (if applicable), Standard & Test samples (2replicates) by proposed analytical methodology and the method operated atreplicates) by proposed analytical methodology and the method operated atvariable conditions.variable conditions.Impurities:Impurities:Analysis of Resolution & Test sample by proposed analyticalAnalysis of Resolution & Test sample by proposed analyticalmethodology and the method operated at variable conditions.methodology and the method operated at variable conditions.EVALUATIONEVALUATIONAssay:Assay:System Suitability parameters at all variable conditionsSystem Suitability parameters at all variable conditions% Assay of samples at all variable conditions% Assay of samples at all variable conditionsImpurities:Impurities: System Suitability parameters at all variable conditionsSystem Suitability parameters at all variable conditionsRRTs at all variable conditionsRRTs at all variable conditions( monitor the separation at each variable condition)( monitor the separation at each variable condition)
  • 46. 8. ROBUSTNESS (Contd.,)ACCEPTANCE CRITERIAACCEPTANCE CRITERIAAssay:Assay:System Suitability criteria should meet at each variable conditionSystem Suitability criteria should meet at each variable conditionOverall RSD for % assay results obtained at STP condition and variable conditionOverall RSD for % assay results obtained at STP condition and variable conditionfor each variabilityfor each variabilityImpurities:Impurities:System Suitability criteria should meet at each variable conditionSystem Suitability criteria should meet at each variable condition
  • 47. III Time management in validationThere are no official guidelines on the sequence of validationexperiments and the optimal sequence can be depending on themethod itself. Based on experience, for HPLC method thefollowing sequence has been proven to be useful for timemanagementIf the method is proved as stable andIf the method is proved as stable androbust under method developmentrobust under method development(Pre validation) programme(Pre validation) programmeIn case of stability and robustnessIn case of stability and robustnessdata is not available with methoddata is not available with methoddevelopment datadevelopment dataSpecificitySpecificity StabilityStabilityLinearityLinearity RobustnessRobustnessLOD & LOQ (if applicable)LOD & LOQ (if applicable) SpecificitySpecificityPrecisionPrecision LinearityLinearityAccuracyAccuracy LOD & LOQ (if applicable)LOD & LOQ (if applicable)RangeRange PrecisionPrecisionStabilityStability AccuracyAccuracyRobustnessRobustness RangeRange
  • 48. IV VALIDATION REPORTEvery DMF / ANDA / COS data package submitting for US FDA andEuropean community etc., should consist method validation data. Foreasy to review, method validation report is usually attached to package. Generally method validation report should haveObjective and scope of the methodMolecule details (IUPAC name, CAS No. Molecular Formulae,Molecular weight and its Molecular Structure etc.,)Detailed list of chemicals, reagents, reference standardsListing of equipment and its functional and performance requirementsMethodology followedValidation data (parameter wise – procedure, results, conclusion etc.,)Summary of data (results in brief – parameter wise)Summary of report (overall view of validation exercise, any criticalissues, recommendations etc., for the application of method)
  • 49. IV. VALIDATION REPORT (Contd.,)Instrument out puts, which should represent critical methodparametersSpecificity and LOD – for Identification Test (Generally,photographs)Selectivity / Specificity data (discriminating chromatogram, peakpurity data, blank and placebo chromatograms and stressed sampleschromatograms)Linearity graphsResolution and related system suitability chromatogramsany out puts which are significant
  • 50. 1. Pharmaceutical Process Validation byRobert.A.Nash, Alfred.H.Wachter2. ICH guidelines
  • 51. THANK YOU

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