ANALYTICAL METHOD VALIDATION BY P.RAVISANKAR

1. prof. Ravisankar
Vignan Pharmacy college
Valdlamudi
Guntur Dist.
Andhra Pradesh
India.
banuman35@gmail.com
00919059994000

2.  Validation is a very necessary element of
any firm that falls under the scrutiny of
the governing regulatory agencies The
FDA, under the authority of existing
cGMP regulations guidelines and
directives, considers validation is
necessary and because it makes good
sense of science / engineering.

3.  Drug substances and drug product
manufacturers must perform
validations, it is very important that
this understanding be shared
throughout the organization
 The term validation generally to
cover the entire spectrum of cGMP

4. DEFINTION
The process to Confirm that the analyticalThe process to Confirm that the analytical
procedure employed for a specific test isprocedure employed for a specific test is
suitable for intended use and that theysuitable for intended use and that they
support the identity, quality, purity andsupport the identity, quality, purity and
potency of the drug substances and drugpotency of the drug substances and drug
products.products.
I. INTRODUCTIONI. INTRODUCTION

5. NEED
 The process of method development andThe process of method development and
validation has a direct impact on thevalidation has a direct impact on the
quality of these dataquality of these data
 To trust the methodTo trust the method
 Regulatory requirementRegulatory requirement
I. INTRODUCTION (Contd.,)

6. Currently available guidelines from variousCurrently available guidelines from various
regulatory bodiesregulatory bodies
ICHICH
USFDAUSFDA
CDERCDER
EPEP
USP XXXIUSP XXXI
I. INTRODUCTION (Contd.,)

7. Analytical procedures to be validatedAnalytical procedures to be validated
I. INTRODUCTION (Contd.,)
 Identification TestsIdentification Tests
 Quantitative Tests for ImpuritiesQuantitative Tests for Impurities
Quantitative tests for active moiety in samples ofQuantitative tests for active moiety in samples of
drug substance & drug products and other selecteddrug substance & drug products and other selected
components 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 of
proceduresprocedures

9. CONCEPT OF REVALIDATION
When 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 of
the analytical proceduresthe analytical procedures
I. INTRODUCTION (Contd.,)I. INTRODUCTION (Contd.,)

10. A few typical cases are presented below whereA few typical cases are presented below where
revalidation is necessaryrevalidation is necessary
ChangeChange Parameters to be considered forParameters to be considered for
revalidationrevalidation
Synthetic routeSynthetic route All parametersAll parameters
Analytical procedureAnalytical procedure All parametersAll parameters
Addition of new impurityAddition of new impurity Specificity, stability, linearity, accuracy,Specificity, stability, linearity, accuracy,
range, LOD & LOD (for new impurityrange, LOD & LOD (for new impurity
only)only)
Composition of drug productComposition of drug product Specificity, stability, Precision andSpecificity, stability, Precision and
accuracyaccuracy
Change in specificationChange in specification Linearity, accuracy and rangeLinearity, accuracy and range
I. INTRODUCTION (Contd.,)
*If solubility differs each other

11. PRE-REQUISITESPRE-REQUISITES
 AA well-designed experimental matrix (Writtenwell-designed experimental matrix (Written
protocol)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 quality
as 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 in
calibrated state.calibrated state.
I. INTRODUCTION (Contd.,)

12. PARAMETERS TO BE EVALUATEDPARAMETERS TO BE EVALUATED
Specificity *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 not
part 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 but
not to the extent of 100%not to the extent of 100%
 Good understanding of each performance characteristicsGood understanding of each performance characteristics
most important. This understanding must be beyond themost important. This understanding must be beyond the
basic definition of each parameterbasic definition of each parameter
 Understanding must be anchored by sufficient years ofUnderstanding must be anchored by sufficient years of
practical experience and knowledge. It will permit soundpractical experience and knowledge. It will permit sound
and logical decisions, even under the most intenseand logical decisions, even under the most intense
situationssituations
II. METHODOLOGY

15. DEFINITION
 SpecificitySpecificity of analytical method as its ability to measureof analytical method as its ability to measure
accurately an analyte in the presence of interference, suchaccurately an analyte in the presence of interference, such
as synthetic precursors, excipients, enantiomers andas synthetic precursors, excipients, enantiomers and
known (or likely) degradation product that may beknown (or likely) degradation product that may be
expected 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 a
response 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 a
number of chemical entities that may / may not benumber of chemical entities that may / may not be
distinguished from each other. If each response isdistinguished from each other. If each response is
distinguished from all other responses, then the method isdistinguished from all other responses, then the method is
said to be selective.said to be selective.
 Use of the termUse of the term SelectivitySelectivity is appropriate for the methodsis appropriate for the methods
based on techniques such as HPLC, GC methodsbased on techniques such as HPLC, GC methods..
1. SPECIFICITY

17. 1.SPECIFICITY
EVALUATION
Identification tests: Response for compound of interest only
Assay: Peak purity of analyte peak.
Impurities: Resolution between within the impurity(s) and/or
degradants and form analyte
Peak purity of - analyte peak (for un-spiked)
- analyte and impurity(s) peaks (for spiked samples)

18. 1.SPECIFICITY
ACCEPTANCE CRITERIA
Identification Tests: Positive response for compound of interest only
Assay : No peak should be found at the retention time of analyte peak
and Peak purity of analyte peak should pass.
Impurities : Should pass Peak purity of -main analyte and Impurity
peaks
No 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.SPECIFICITY
FORCED DEGRADATION STUDIES
INTRODUCTIONINTRODUCTION
Forced degradation or stress testing is undertaken toForced degradation or stress testing is undertaken to
demonstratedemonstrate specificityspecificity when developing stability-indicatingwhen developing stability-indicating
methodsmethods
A stability-indicating method is one that accurately quantitatesA stability-indicating method is one that accurately quantitates
the active ingredients without interference from degradationthe active ingredients without interference from degradation
products, process impurities, excipients or other potentialproducts, process impurities, excipients or other potential
impuritiesimpurities

20.  Address the stability of the compound
 Establish the degradation pathway
 Identify the degradation products
 Validate the stability indicating power of the
analytical procedures used

21. 1.SPECIFICITY / SELECTIVITY (Contd.,)
FORCED DEGRADATION STUDIES
PROCEDUREPROCEDURE
Perform 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 methodology
Normal initial stressed conditions to be appliedNormal initial stressed conditions to be applied
1M HCl1M HCl
1M NaOH1M NaOH
10% H10% H22OO22
105°C/at least 72 Hours105°C/at least 72 Hours
12000 Lux/at least 72 Hours12000 Lux/at least 72 Hours
92% 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 formulations
change, so it is an ongoing effort.
 Evaluate the each unique formulation before formal
stability begins

23. EVALUATIONEVALUATION
Assay:Assay: % Difference of assay for Control (Un-stressed) and each Stressed% Difference of assay for Control (Un-stressed) and each Stressed
samplessamples
Peak purity of analyte peak for Control and stressed samplePeak purity of analyte peak for Control and stressed sample
Impurities:Impurities: Peak purity of analyte peak for Control and each Stressed samplePeak purity of analyte peak for Control and each Stressed sample
ACCEPTANCE CRITERIAACCEPTANCE CRITERIA
Assay:Assay: Peak purity of analyte peak in Control and each Stressed samples shouldPeak purity of analyte peak in Control and each Stressed samples should
passpass
Impurities:Impurities: Peak purity of analyte peak in Control and each Stressed samplesPeak purity of analyte peak in Control and each Stressed samples
should passshould pass
1.SPECIFICITY / SELECTIVITY (Contd.,)
FORCED DEGRADATION STUDIES

24. Points to rememberPoints to remember
If 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 extent
or 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 should
be 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 apply
heat 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 recent
literatureliterature
* 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. No
further stressing is advised in these casesfurther stressing is advised in these cases
Over stressing may lead to the formation of secondary degradants, causes disturbance toOver stressing may lead to the formation of secondary degradants, causes disturbance to
thethe selectivityselectivity of the method, requires further developmentof the method, requires further development
1.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 the
drug substance / drug product is actually exposed during its shelf lifedrug substance / drug product is actually exposed during its shelf life
A simple logic behind these studies is, ability to separate the analyte ofA simple logic behind these studies is, ability to separate the analyte of
interest from its degradation impurities formed, if any, caused byinterest from its degradation impurities formed, if any, caused by
quality 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 exposure
during shelf life of the drug substance/drug productduring shelf life of the drug substance/drug product
1.SPECIFICITY / SELECTIVITY (Contd.,)
FORCED DEGRADATION STUDIES

26. 2. LIMIT OF DETECTION AND LIMIT OF QUANTIFICATION
(LOD & LOQ)
DEFINITIONDEFINITION
LOD: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 not
necessarily 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 quantitatively
determined with suitable precision and accuracy (LOQ)determined with suitable precision and accuracy (LOQ)

27. Different approaches suggested by ICH, USP & EP
Several approaches are given in the ICH guidelines for estimating LOD and LOQSeveral approaches are given in the ICH guidelines for estimating LOD and LOQ
Depending on the procedure : instrumental / non-instrumentalDepending on the procedure : instrumental / non-instrumental
ApproachApproach LODLOD LOQLOQ
Visual inspectionVisual inspection Minimum level detectableMinimum level detectable Minimum level quantifiableMinimum level quantifiable
Signal-to-Noise ratioSignal-to-Noise ratio 2:1 or 3:12:1 or 3:1 10:110:1
SD 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] / S
RSD CriteriaRSD Criteria Concentration at whichConcentration at which
RSD10 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 also
acceptable
2. LOD & LOQ (Contd.,)

28. PROCEDUREPROCEDURE
SD of response (SD of response () & Slope (S)) & Slope (S): Prepare linearity curve with a series of related: Prepare linearity curve with a series of related
substance(s) solutions at different concentrations (3 concentrations below 50 % ofsubstance(s) solutions at different concentrations (3 concentrations below 50 % of
specification 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 different
concentrations below to specification level (generally about 10%, 20%, 30%, 40% andconcentrations below to specification level (generally about 10%, 20%, 30%, 40% and
50% 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 and
LOD 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 six
replicates as per methodologyreplicates as per methodology
2. LOD & LOQ (Contd.,)

29. 2. LOD & LOQ (Contd.,)
EVALUATIONEVALUATION
SD 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 mean
square 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 data
LOD = 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 declare
the 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 is
betweenbetween >> 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. LINEARITY
DEFINITIONDEFINITION
TheThe LinearityLinearity of an analytical procedure is its ability (within a given range) toof an analytical procedure is its ability (within a given range) to
elicit (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 sample
Range: 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 and
linearity using this method as writtenlinearity using this method as written
DISCUSSIONDISCUSSION
In 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 and
response is essentialresponse is essential
ANALYTE ------ PROCEDURE ------ANALYTE ------ PROCEDURE ------ RESPONSERESPONSE
ICH and USP encouraging Linear and non-Linear relation shipsICH and USP encouraging Linear and non-Linear relation ships

31. PROCEDUREPROCEDURE
Prepare 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 per
methodmethod
Assay:-Assay:- 80% to 120% of test concentration80% to 120% of test concentration
CU :-CU :- 70% to 130% of70% to 130% of test concentrationtest concentration
Dissolution:-Dissolution:- ± 20% of expected release (Q) for immediate release± 20% of expected release (Q) for immediate release
0 to 120% (for extended release)0 to 120% (for extended release)
Impurities:-Impurities:- LOQ to 200% of specificationLOQ to 200% of specification
3. LINEARITY (Contd.,)

32. 3. LINEARITY (Contd.,)
EVALUATIONEVALUATION
SlopeSlope
- indicates sensitivity of the method- indicates sensitivity of the method
InterceptIntercept
- 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 correct
ACCEPTANCE CRITERIAACCEPTANCE CRITERIA
Correlation 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 test
methods and 0.99 for impurities test methodmethods and 0.99 for impurities test method

33. 4. PRECISION (Contd.,)
DEFINITIONDEFINITION
Closeness of agreement (degree of scatter) between a series of measurementsCloseness of agreement (degree of scatter) between a series of measurements
obtained from multiple sampling of the same homogenous sample under theobtained from multiple sampling of the same homogenous sample under the
prescribed 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 Repeatability
3.3. Intermediate precisionIntermediate precision
4.4. ReproducibilityReproducibility

34. 4. PRECISION (Contd.,)
DISCUSSIONDISCUSSION
 Repeatability:Repeatability: precision under same operating conditions (with-in a laboratoryprecision under same operating conditions (with-in a laboratory
over 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 equipments
within the same laboratory)within the same laboratory)
ReproducibilityReproducibility: precision between laboratories / intermediate precision can be: precision between laboratories / intermediate precision can be
considered during the standardization of a procedure before it is submitted to theconsidered during the standardization of a procedure before it is submitted to the
pharmacopoeiapharmacopoeia
As per CDER guidelines, it is not normally expected if intermediate precision isAs per CDER guidelines, it is not normally expected if intermediate precision is
accomplishedaccomplished

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 from
sample 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 at
triplicate 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 from
sample 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 CRITERIA
ProcedureProcedure
%RSD%RSD
System PrecisionSystem Precision Method PrecisionMethod Precision RuggednessRuggedness
APIAPI DPDP APIAPI DPDP APIAPI DPDP
AssayAssay 1.0/2.01.0/2.0 1.0/2.0*1.0/2.0* 1.01.0 2.02.0 1.01.0 3.03.0
DissolutionDissolution NANA 1.0 / 2.0*1.0 / 2.0* NANA 6.06.0 NANA 5.05.0
ImpuritiesImpurities 5.0*5.0* 5.0*5.0* 10.010.0 10.010.0 10.010.0 15.015.0
NA – Not applicable
API – Active Pharmaceutical Ingredient
DP – Drug Product
* : As given in STP

37. 5. ACCURACY
DEFINITIONDEFINITION
The accuracy of an analytical procedure expresses theThe accuracy of an analytical procedure expresses the
closeness of agreement between the value, which is acceptedcloseness of agreement between the value, which is accepted
either as a conventional true value or an accepted reference valueeither as a conventional true value or an accepted reference value
and the value foundand the value found

38. 5.ACCURACY (Contd.,)
PROCEDURE
Assay/Dissolution:- Known amount of drug substance spiked with synthetic
mixtures of drug product components (excipients) – minimum of three levels
80%, 100% & 120% of test concentration-assay;
± 20% of expected release-dissolution), each level is triplicate
Impurities:- 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 triplicate
EVALUATION
- Recovery from amount added and amount found
- Precision (%RSD) at each level (for three replicate preparations)

39. 5.ACCURACY (Contd.,)
ACCEPTANCE CRITERIA
Assay:- Recovery should be between 98% to 102%( Depends upon the your
Strength)
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 whether
the procedure is capable of estimating a true value or not

40. 7. STABILITY
DISCUSSIONDISCUSSION
 It is often essential that solutions (standards, test samples) be stable enoughIt is often essential that solutions (standards, test samples) be stable enough
to 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 for
chromatographic 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.,)
PROCEDUREPROCEDURE
Prepare 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 time
intervals by keeping the sample at room temperature ( 25°C) / refrigeratorintervals by keeping the sample at room temperature ( 25°C) / refrigerator
condition (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 impurity
ACCEPTANCE CRITERIAACCEPTANCE CRITERIA
% Difference is not more than% Difference is not more than
Assay, CU :Assay, CU : 2.02.0
Dissolution :Dissolution : 3.03.0
Impurities :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 period
of time, a solution can be held before analysis withoutof time, a solution can be held before analysis without
compromising accuracycompromising accuracy..

43. 8. ROBUSTNESS
DEFINTIONDEFINTION
Measure of its capacity to remain unaffected by small, but deliberate variationsMeasure of its capacity to remain unaffected by small, but deliberate variations
in method parameters and provides indication of its reliability during its normalin method parameters and provides indication of its reliability during its normal
usageusage
DISCUSSIONDISCUSSION
varying method parameters within a realistic range and the quantitativevarying method parameters within a realistic range and the quantitative
influence 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 is
within a previously specified tolerance, then, the parameter is said to be withinwithin a previously specified tolerance, then, the parameter is said to be within
the method’s robustness rangethe method’s robustness range
According to ICH guidelines, robustness should be considered early in theAccording to ICH guidelines, robustness should be considered early in the
development 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 a
registration 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.,)
PROCEDUREPROCEDURE
Assay:Assay:Analysis of Resolution (if applicable), Standard & Test samples (2Analysis of Resolution (if applicable), Standard & Test samples (2
replicates) by proposed analytical methodology and the method operated atreplicates) by proposed analytical methodology and the method operated at
variable conditions.variable conditions.
Impurities:Impurities:Analysis of Resolution & Test sample by proposed analyticalAnalysis of Resolution & Test sample by proposed analytical
methodology and the method operated at variable conditions.methodology and the method operated at variable conditions.
EVALUATIONEVALUATION
Assay: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 conditions
Impurities:Impurities: System Suitability parameters at all variable conditionsSystem Suitability parameters at all variable conditions
RRTs 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 CRITERIA
Assay:Assay:System Suitability criteria should meet at each variable conditionSystem Suitability criteria should meet at each variable condition
Overall RSD for % assay results obtained at STP condition and variable conditionOverall RSD for % assay results obtained at STP condition and variable condition
for each variabilityfor each variability
Impurities:Impurities:System Suitability criteria should meet at each variable conditionSystem Suitability criteria should meet at each variable condition

47. III Time management in validation
There are no official guidelines on the sequence of validation
experiments and the optimal sequence can be depending on the
method itself. Based on experience, for HPLC method the
following sequence has been proven to be useful for time
management
If the method is proved as stable andIf the method is proved as stable and
robust under method developmentrobust under method development
(Pre validation) programme(Pre validation) programme
In case of stability and robustnessIn case of stability and robustness
data is not available with methoddata is not available with method
development datadevelopment data
SpecificitySpecificity StabilityStability
LinearityLinearity RobustnessRobustness
LOD & LOQ (if applicable)LOD & LOQ (if applicable) SpecificitySpecificity
PrecisionPrecision LinearityLinearity
AccuracyAccuracy LOD & LOQ (if applicable)LOD & LOQ (if applicable)
RangeRange PrecisionPrecision
StabilityStability AccuracyAccuracy
RobustnessRobustness RangeRange

48. IV VALIDATION REPORT
Every DMF / ANDA / COS data package submitting for US FDA and
European community etc., should consist method validation data. For
easy 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 critical
issues, recommendations etc., for the application of method)

49. IV. VALIDATION REPORT (Contd.,)
Instrument out puts, which should represent critical method
parameters
Specificity and LOD – for Identification Test (Generally,
photographs)
Selectivity / Specificity data (discriminating chromatogram, peak
purity data, blank and placebo chromatograms and stressed samples
chromatograms)
Linearity graphs
Resolution and related system suitability chromatograms
any out puts which are significant

50. 1. Pharmaceutical Process Validation by
Robert.A.Nash, Alfred.H.Wachter
2. ICH guidelines

51. THANK YOU