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prof. Ravisankar
Vignan Pharmacy college
Valdlamudi
Guntur Dist.
Andhra Pradesh
India.
banuman35@gmail.com
00919059994000
 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.
 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
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
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.,)
Currently available guidelines from variousCurrently available guidelines from various
regulatory bodiesregulatory bodies
ICHICH
USFDAUSFDA
CDERCDER
EPEP
USP XXXIUSP XXXI
I. INTRODUCTION (Contd.,)
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
I. INTRODUCTION (Contd.,)I. INTRODUCTION (Contd.,)
Recommended validation characteristics of the various types ofRecommended validation characteristics of the various types of
proceduresprocedures
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.,)
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
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.,)
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**
• Accuracy (or) Recovery*
• Solution Stability*
• Robustness*
• System Suitability*
* Included in ICH Guidelines
** Terminology included in ICH guidelines but are not
part of required parameters
 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
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
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
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)
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
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
 Address the stability of the compound
 Establish the degradation pathway
 Identify the degradation products
 Validate the stability indicating power of the
analytical procedures used
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
 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
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
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
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
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)
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.,)
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.,)
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%
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
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.,)
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
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
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
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)
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
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
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)
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
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.
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
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..
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.
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)
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)
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
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
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)
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
1. Pharmaceutical Process Validation by
Robert.A.Nash, Alfred.H.Wachter
2. ICH guidelines
THANK YOU

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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