ASEAN GUIDELINES FOR ASEAN GUIDELINES FOR VALIDATION OF VALIDATION OF

ANALYTICAL ANALYTICAL PROCEDURESPROCEDURES

Supanee Duangteraprecha, Ph.D.

Bureau of Drug and Narcotic

Department of Medical Sciences

ObjectiveObjective

The objective of validation of an analytical The objective of validation of an analytical procedure is to demonstrate that it is procedure is to demonstrate that it is suitable for its intended purpose. suitable for its intended purpose.

ScopeScope provide guidance and recommendation of provide guidance and recommendation of

validation of the analytical procedures for validation of the analytical procedures for submission as part of registration applications submission as part of registration applications within ASEAN. within ASEAN.

it mainly adopts two ICH guidelines it mainly adopts two ICH guidelines ““Q2A: Q2A: Validation of Analytical Methods: Definitions and Validation of Analytical Methods: Definitions and Terminology, 27 October 1994Terminology, 27 October 1994”” and and ““ICH Q2B: ICH Q2B: Validation of Analytical Procedure: Methodology, 6 Validation of Analytical Procedure: Methodology, 6 November 1996. November 1996.

the methodology applied for biological and the methodology applied for biological and biotechnological products may be approached biotechnological products may be approached differently than chemical entities.differently than chemical entities.

Types of Analytical Procedures Types of Analytical Procedures to be Validatedto be Validated

Identification tests.Identification tests. Quantitative tests for impurities' content.Quantitative tests for impurities' content. Limit tests for the control of impurities.Limit tests for the control of impurities. Quantitative tests of the active moiety in Quantitative tests of the active moiety in

samples of drug substance or drug product samples of drug substance or drug product or other selected component(s) in the drug or other selected component(s) in the drug product.product.

Criteria For Analytical Test ValidationCriteria For Analytical Test Validation

PRECISION

LIMITS

ACCURACY

LINEARITY RANGE

SPECIFICITY

REPEATABILITY

QUANTIFICATION

INTERMEDIATE PRECISION

DETECTION

TableTable

SymbolsSymbols

-- signifies that this characteristic is not normally signifies that this characteristic is not normally evaluatedevaluated

+ signifies that this characteristic is normally + signifies that this characteristic is normally evaluatedevaluated(1) in cases where reproducibility (see glossary) (1) in cases where reproducibility (see glossary) has been performed, intermediate precision is not has been performed, intermediate precision is not neededneeded(2) lack of specificity of one analytical procedure (2) lack of specificity of one analytical procedure could be compensated by other supporting could be compensated by other supporting analytical procedure(s)analytical procedure(s)(3) may be needed in some cases(3) may be needed in some cases

SpecificitySpecificity

Is this analytical procedure specific

for the drug under test?

Suppose we altertest conditions

slightly?

Specificity (1)Specificity (1)

Specificity is the ability to assess Specificity is the ability to assess unequivocally the analyte in the presence of unequivocally the analyte in the presence of components which may be expected to be components which may be expected to be present. present.

Typically these might include impurities, Typically these might include impurities, degradants, matrix, etc.degradants, matrix, etc.

Lack of specificity of an individual Lack of specificity of an individual analytical procedure may be compensated analytical procedure may be compensated by other supporting analytical procedure(s). by other supporting analytical procedure(s).

Specificity (2)Specificity (2) An investigation of specificity should be conducted An investigation of specificity should be conducted

during the validation of identification tests, the during the validation of identification tests, the determination of impurities and the assay. determination of impurities and the assay. The procedures used to demonstrate specificity will The procedures used to demonstrate specificity will

depend on the intended objective of the analytical depend on the intended objective of the analytical procedure. procedure.

It is not always possible to demonstrate that an It is not always possible to demonstrate that an analytical procedure is specific for a particular analytical procedure is specific for a particular analyte (complete discrimination). analyte (complete discrimination). In this case a combination of two or more analytical In this case a combination of two or more analytical

procedures is recommended to achieve the necessary procedures is recommended to achieve the necessary level of discrimination.level of discrimination.

IdentificationIdentification

Suitable identification tests should be able to Suitable identification tests should be able to discriminate between compounds of closely related discriminate between compounds of closely related structures which are likely to be present. structures which are likely to be present. The discrimination of a procedure may be confirmed by The discrimination of a procedure may be confirmed by

obtaining positive results (perhaps by comparison with obtaining positive results (perhaps by comparison with a known reference material) from samples containing a known reference material) from samples containing the analyte, coupled with negative results from samples the analyte, coupled with negative results from samples which do not contain the analyte. which do not contain the analyte.

In addition, the identification test may be applied to In addition, the identification test may be applied to materials structurally similar to or closely related to the materials structurally similar to or closely related to the analyte to confirm that a positive response is not analyte to confirm that a positive response is not obtained. obtained.

Assay and Impurity Test(s)Assay and Impurity Test(s) For chromatographic procedures, representative For chromatographic procedures, representative

chromatograms should be used to demonstrate specificity chromatograms should be used to demonstrate specificity and individual components should be appropriately and individual components should be appropriately labelled. labelled.

Critical separations in chromatography should be Critical separations in chromatography should be investigated at an appropriate level. investigated at an appropriate level. For critical separations, specificity can be demonstrated by the For critical separations, specificity can be demonstrated by the

resolution of the two components which elute closest to each other. resolution of the two components which elute closest to each other.

In cases where a non-specific assay is used, other In cases where a non-specific assay is used, other supporting analytical procedures should be used to supporting analytical procedures should be used to demonstrate overall specificity. demonstrate overall specificity. For example, where a titration is adopted to assay the drug For example, where a titration is adopted to assay the drug

substance for release, the combination of the assay and a suitable substance for release, the combination of the assay and a suitable test for impurities can be used. test for impurities can be used.

Impurities are available (1) Impurities are available (1)

For the assayFor the assay , this should involve demonstration , this should involve demonstration of the discrimination of the analyte in the presence of the discrimination of the analyte in the presence of impurities and/or excipients; of impurities and/or excipients;

practically, this can be done by spiking pure practically, this can be done by spiking pure substances (drug substance or drug product) with substances (drug substance or drug product) with appropriate levels of impurities and/or excipients appropriate levels of impurities and/or excipients and demonstrating that the assay result is and demonstrating that the assay result is unaffected by the presence of these materials (by unaffected by the presence of these materials (by comparison with the assay result obtained on comparison with the assay result obtained on unspiked samples).unspiked samples).

Impurities are available (2)Impurities are available (2)

For the impurity testFor the impurity test, the discrimination , the discrimination may be established by: may be established by: spiking drug substance or drug product with spiking drug substance or drug product with

appropriate levels of impurities andappropriate levels of impurities and demonstrating the separation of these demonstrating the separation of these

impurities individually and/or from other impurities individually and/or from other components in the sample matrix.components in the sample matrix.

Impurities are not available Impurities are not available If impurity or degradation product standards are If impurity or degradation product standards are

unavailable, specificity may be demonstrated by unavailable, specificity may be demonstrated by comparing the test results of samples containing comparing the test results of samples containing impurities or degradation products to a second impurities or degradation products to a second well-characterized procedure e.g.: well-characterized procedure e.g.: pharmacopoeial method or other validated pharmacopoeial method or other validated analytical procedure (independent procedure). analytical procedure (independent procedure).

As appropriate, this should include samples stored As appropriate, this should include samples stored under relevant stress conditions:under relevant stress conditions: light, heat, humidity, acid/base hydrolysis and light, heat, humidity, acid/base hydrolysis and

oxidation.oxidation. for the assay, the two results should be compared.for the assay, the two results should be compared. for the impurity tests, the impurity profiles should be for the impurity tests, the impurity profiles should be

compared. compared.

Linearity and RangeLinearity and Range

‘‘Know that it’s a straight line’Know that it’s a straight line’

vsvs

‘‘For what concentrations is it For what concentrations is it a straight line’a straight line’

Linearity and RangeLinearity and Range

‘‘Know that it’s a straight line’ Know that it’s a straight line’ versusversus ‘For what ‘For what concentrations is it a straight line’concentrations is it a straight line’

Is it a straight line between 0.4 & 0.6 mg/mL?Is it a straight line between 0.4 & 0.6 mg/mL? Over what range is it a straight line? Over what range is it a straight line? Answer: approx 0.25-0.70 mg/mLAnswer: approx 0.25-0.70 mg/mL

Concentrationmg/mL

Response

LINEARITY (1)LINEARITY (1)

A linear relationship should be evaluated A linear relationship should be evaluated across the range of the analytical across the range of the analytical procedure. procedure.

It may be demonstrated directly on the It may be demonstrated directly on the drug substance by: drug substance by: dilution of a standard stock solution and/ordilution of a standard stock solution and/or separate weighings of synthetic mixtures of the separate weighings of synthetic mixtures of the

drug product components drug product components

using the proposed procedure. using the proposed procedure.

LINEARITY (2)LINEARITY (2) Linearity should be evaluated by visual inspection of a plot of Linearity should be evaluated by visual inspection of a plot of

signals as a function of analyte concentration or content. signals as a function of analyte concentration or content. If there is a linear relationship, test results should be If there is a linear relationship, test results should be

evaluated by appropriate statistical methods, for example, by evaluated by appropriate statistical methods, for example, by calculation of a regression line by the method of least squares. calculation of a regression line by the method of least squares.

In some cases, to obtain linearity between assays and sample In some cases, to obtain linearity between assays and sample concentrations, the test data may need to be subjected to a concentrations, the test data may need to be subjected to a mathematical transformation prior to the regression analysis. mathematical transformation prior to the regression analysis. Data from the regression line itself may be helpful to provide Data from the regression line itself may be helpful to provide mathematical estimates of the degree of linearity.mathematical estimates of the degree of linearity.

For the establishment of linearity, a minimum of 5 For the establishment of linearity, a minimum of 5 concentrations is recommended. concentrations is recommended.

ExampleExample

Taken from:Taken from:ASEAN Operational Manual for Implementation ASEAN Operational Manual for Implementation of GMP ed. 2000 p.403of GMP ed. 2000 p.403

Seven solutions containing different Seven solutions containing different concentrations (0.280 – 0.520) mg/ml of ketotifen concentrations (0.280 – 0.520) mg/ml of ketotifen fumarate in tablet “batch no. 2506 VAMG were fumarate in tablet “batch no. 2506 VAMG were assayed using HPLCassayed using HPLC

The results were evaluated statistically and the The results were evaluated statistically and the results shown on the following slideresults shown on the following slide

Example (continued)Example (continued)Concentration of ketotifen fumarateConcentration of ketotifen fumarate Area detectedArea detected Acceptance Acceptance

criteriacriteriamg/mlmg/ml %%

0.2800.280

0.3200.320

0.3600.360

0.4000.400

0.4400.440

0.4800.480

0.5200.520

7070

8080

9090

100100

110110

120120

130130

14735661473566

16770131677013

19048481904848

20912152091215

22936472293647

25189762518976

26701442670144

Regression: y = ax + bRegression: y = ax + b

a = 5055766.964a = 5055766.964

b = 67608.786b = 67608.786

rr2 2 = 0.9984= 0.9984

0.998 – 1.0020.998 – 1.002

RANGE RANGE

The specified range is normally derived from The specified range is normally derived from linearity studies and depends on the intended linearity studies and depends on the intended application of the procedure. application of the procedure.

It is established by confirming that the analytical It is established by confirming that the analytical procedure provides an acceptable degree of procedure provides an acceptable degree of linearity, accuracy and precision when applied to linearity, accuracy and precision when applied to samples containing amounts of analyte within or samples containing amounts of analyte within or at the extremes of the specified range of the at the extremes of the specified range of the analytical procedure. analytical procedure.

Minimum Specified Ranges (1) Minimum Specified Ranges (1)

for the for the assayassay of a drug substance or a finished of a drug substance or a finished (drug) product: normally from (drug) product: normally from 80 - 120 %80 - 120 % of the test of the test concentrationconcentration

for for content uniformitycontent uniformity, covering a minimum of , covering a minimum of 70 - 130 %70 - 130 % of the test concentration of the test concentration

for for dissolutiondissolution testing: testing: +/-20 % over the +/-20 % over the specified rangespecified range; e.g., if the specifications for a ; e.g., if the specifications for a controlled released product cover a region from controlled released product cover a region from 20%, after 1 hour, up to 90%, after 24 hours, the 20%, after 1 hour, up to 90%, after 24 hours, the validated range would be 0-110% of the label claimvalidated range would be 0-110% of the label claim

Minimum Specified Ranges (2)Minimum Specified Ranges (2)

for the determination of an impurity: from the reporting for the determination of an impurity: from the reporting level of an impurity to 120% of the specification; for level of an impurity to 120% of the specification; for impurities known to be unusually potent or to produce impurities known to be unusually potent or to produce toxic or unexpected pharmacological effects, the toxic or unexpected pharmacological effects, the detection/quantitation limit should be commensurate with detection/quantitation limit should be commensurate with the level at which the impurities must be controlled.the level at which the impurities must be controlled.

if assay and purity are performed together as one test and if assay and purity are performed together as one test and only a 100% standard is used, linearity should cover the only a 100% standard is used, linearity should cover the range from the reporting level of the impurities to 120% of range from the reporting level of the impurities to 120% of the assay specificationthe assay specification

Accuracy vs Accuracy vs precisionprecision

What you What you would would likelike

to seeto see!!

Accuracy vs Accuracy vs precisionprecision

Poor accuracyPoor accuracy Good precisionGood precision

Accuracy vs Accuracy vs precisionprecision

Poor precisionPoor precision Good accuracyGood accuracy

Accuracy vs Accuracy vs precisionprecision

Totally hopeless!Totally hopeless! Poor precisionPoor precision Poor accuracyPoor accuracy

What would What would you call you call this?this?

So what definitions do these So what definitions do these concepts lead us to in the concepts lead us to in the context of assay validation?context of assay validation?

ACCURACY (1)ACCURACY (1)

The accuracy of an analytical procedure The accuracy of an analytical procedure expresses the closeness of agreement expresses the closeness of agreement between the value which is accepted either between the value which is accepted either as a conventional true value or an accepted as a conventional true value or an accepted reference value and the value found. This is reference value and the value found. This is sometimes termed trueness. sometimes termed trueness.

ACCURACY (2)ACCURACY (2)

Assay of Drug Substance:Assay of Drug Substance:a) application of an analytical procedure to an a) application of an analytical procedure to an

analyte of known purity (e.g. reference material);analyte of known purity (e.g. reference material);b) comparison of the results of the proposed b) comparison of the results of the proposed

analytical procedure with those of a second well-analytical procedure with those of a second well-characterized procedure, the accuracy of which is characterized procedure, the accuracy of which is stated and/or defined (independent procedure)stated and/or defined (independent procedure)

c) accuracy may be inferred once precision, linearity c) accuracy may be inferred once precision, linearity and specificity have been establishedand specificity have been established

ACCURACY (3)ACCURACY (3)

Assay of Drug Product:Assay of Drug Product:a) application of the analytical procedure to synthetic a) application of the analytical procedure to synthetic

mixtures of the drug product components to which known mixtures of the drug product components to which known quantities of the drug substance to be analysed have been quantities of the drug substance to be analysed have been added;added;

b) in cases where it is impossible to obtain samples of all drug b) in cases where it is impossible to obtain samples of all drug product components, it may be acceptable either to:product components, it may be acceptable either to: add known quantities of the analyte to the drug product or add known quantities of the analyte to the drug product or to compare the results obtained from a second, well characterized to compare the results obtained from a second, well characterized

procedure, the accuracy of which is stated and/or defined procedure, the accuracy of which is stated and/or defined (independent procedure)(independent procedure)

c) accuracy may be inferred once precision, linearity and c) accuracy may be inferred once precision, linearity and specificity have been established. specificity have been established.

ACCURACY (4)ACCURACY (4)Impurities (Quantitation):Impurities (Quantitation): Accuracy should be assessed on samples (drug Accuracy should be assessed on samples (drug

substance/drug product) spiked with known amounts of substance/drug product) spiked with known amounts of impurities. impurities.

In cases where it is impossible to obtain samples of certain In cases where it is impossible to obtain samples of certain impurities and/or degradation products, it is considered impurities and/or degradation products, it is considered acceptable to compare results obtained by an independent acceptable to compare results obtained by an independent procedure. The response factor of the drug substance can procedure. The response factor of the drug substance can be used.be used.

It should be clear how the individual or total impurities It should be clear how the individual or total impurities are to be determined e.g., weight/weight or area percent, in are to be determined e.g., weight/weight or area percent, in all cases with respect to the major analyte.all cases with respect to the major analyte.

Recommended DataRecommended Data

Accuracy should be assessed using a Accuracy should be assessed using a min. of min. of 9 determinations over a min. of 3 9 determinations over a min. of 3 concentration levelsconcentration levels covering the specified covering the specified range (e.g. 3 concentrations/3 replicates each of range (e.g. 3 concentrations/3 replicates each of the total analytical procedure). the total analytical procedure).

Accuracy should be reported as: Accuracy should be reported as: % recovery% recovery by the assay of known added amount of by the assay of known added amount of

analyte in the sample or as analyte in the sample or as the difference between the mean and the accepted true the difference between the mean and the accepted true

value together with the confidence intervalsvalue together with the confidence intervals

Example:Example:

Taken from:Taken from:

ASEAN Operational Manual for ASEAN Operational Manual for Implementation of GMP ed. 2000 p.405Implementation of GMP ed. 2000 p.405

Nine solutions containing different Nine solutions containing different concentrations of ketotifen fumarate concentrations of ketotifen fumarate reference standard added to ketotifen tablet reference standard added to ketotifen tablet batch no. 2506VAMG were assayedbatch no. 2506VAMG were assayed

Example (continued):Example (continued):Conc. of ketotifen fumarateConc. of ketotifen fumarate Area Area

detecteddetectedRecovery Recovery

(%)(%)Acceptance Acceptance

CriteriaCriteriamg/mlmg/ml %%

0.2800.280

0.3200.320

0.3600.360

0.3800.380

0.4000.400

0.4200.420

0.4400.440

0.4800.480

0.5200.520

7070

8080

9090

9595

100100

105105

110110

120120

130130

14735661473566

16770131677013

19048481904848

19058621905862

20912152091215

21803742180374

22936472293647

25189762518976

26701442670144

99.3299.32

99.4899.48

100.94100.94

100.51100.51

100.06100.06

100.03100.03

100.07100.07

101.01101.01

98.9998.99

Mean (recovery) : 100.04Mean (recovery) : 100.04

Standard deviation : 0.699Standard deviation : 0.699

Relative standard deviation (RSD) : 0.699 %Relative standard deviation (RSD) : 0.699 %

98.0–102.0 98.0–102.0 %%

<< 2 % 2 %

PRECISIONPRECISION The precision of an analytical procedure expresses the The precision of an analytical procedure expresses the

closeness of agreement (degree of scatter) between a series closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed same homogeneous sample under the prescribed conditions. conditions.

Precision may be considered at three levels:Precision may be considered at three levels: repeatability, repeatability, intermediate precision and intermediate precision and reproducibilityreproducibility..

Precision should be investigated using homogeneous, Precision should be investigated using homogeneous, authentic samples. However, if it is not possible to obtain a authentic samples. However, if it is not possible to obtain a homogeneous sample it may be investigated using homogeneous sample it may be investigated using artificially prepared samples or a sample solution. artificially prepared samples or a sample solution.

The precision of an analytical procedure is usually The precision of an analytical procedure is usually expressed as the expressed as the variancevariance, , standard deviation or standard deviation or coefficient of variationcoefficient of variation of a series of measurements. of a series of measurements.

Repeatability (1)Repeatability (1)

Repeatability expresses the precision under Repeatability expresses the precision under the same operating conditions over a short the same operating conditions over a short interval of time. interval of time.

Repeatability is also termed intra-assay Repeatability is also termed intra-assay precision.precision.

Repeatability (2)Repeatability (2)

Repeatability should be assessed using:Repeatability should be assessed using:

a) a) a minimum of 9 determinationsa minimum of 9 determinations covering the specified rangecovering the specified range for the for the procedure (e.g. 3 concentrations/3 procedure (e.g. 3 concentrations/3 replicates each) orreplicates each) or

b) a minimum of b) a minimum of 6 determinations at6 determinations at 100%100% of the test concentration. of the test concentration.

Intermediate precisionIntermediate precision Intermediate precision expresses within-laboratories Intermediate precision expresses within-laboratories

variations: variations: different daysdifferent days, , different analystsdifferent analysts, , different equipmentdifferent equipment, etc. , etc.

The extent to which intermediate precision should be The extent to which intermediate precision should be established depends on the circumstances under which the established depends on the circumstances under which the procedure is intended to be used. procedure is intended to be used.

The applicant should establish the effects of random events The applicant should establish the effects of random events on the precision of the analytical procedure.on the precision of the analytical procedure.

Typical variations to be studied include days, analysts, Typical variations to be studied include days, analysts, equipment, etc. It is not considered necessary to study equipment, etc. It is not considered necessary to study these effects individually. The use of an experimental these effects individually. The use of an experimental design (matrix) is encouraged.design (matrix) is encouraged.

Reproducibility Reproducibility

Reproducibility is assessed by means of an Reproducibility is assessed by means of an inter-laboratory trial. inter-laboratory trial.

Reproducibility should be considered in Reproducibility should be considered in case of the standardization of an analytical case of the standardization of an analytical procedure, for instance, for inclusion of procedure, for instance, for inclusion of procedures in pharmacopoeias. procedures in pharmacopoeias.

These data are not part of the marketing These data are not part of the marketing authorization dossier.authorization dossier.

Recommended Data Recommended Data

The The standard deviationstandard deviation, , relative relative standard deviationstandard deviation (coefficient of (coefficient of variation) and confidence interval should be variation) and confidence interval should be reported for each type of precision reported for each type of precision investigated.investigated.

ExampleExample

Taken from:Taken from:

ASEAN Operational Manual for ASEAN Operational Manual for Implementation of GMP ed. 2000 p.403Implementation of GMP ed. 2000 p.403

The active ingredient, ketotifen fumarate,The active ingredient, ketotifen fumarate,

in tablets (batch no. 2506VAMG) was in tablets (batch no. 2506VAMG) was assayed seven times using HPLC and the assayed seven times using HPLC and the reference standardreference standard

Example (continued)Example (continued)Sample no.Sample no. Concentration (mg/ml)Concentration (mg/ml) Area detectedArea detected

11

22

33

44

55

66

77

0.40.4

0.40.4

0.40.4

0.40.4

0.40.4

0.40.4

0.40.4

19028031902803

19280831928083

19114571911457

19158971915897

19133121913312

18977021897702

19070191907019

Mean : 1910896Mean : 1910896

Standard deviation : 9841.78Standard deviation : 9841.78

Relative standard deviation (RSD) : 0.515 %Relative standard deviation (RSD) : 0.515 %

Acceptance criteria:

Relative standard deviation (RSD): not more than 2 %

Detection limit vs Detection limit vs Quantitation limitQuantitation limit

‘ ‘Know that it’s there’ Know that it’s there’ vs vs

‘ ‘Know how much is there’Know how much is there’

Detection limit Detection limit (means)(means)

Is any of it present?Is any of it present?

Is it there?Is it there?

Quantitation limitQuantitation limitHow much of it is present???How much of it is present???

How much of it is there?How much of it is there?

DETECTION LIMITDETECTION LIMIT

The detection limit of an individual The detection limit of an individual analytical procedure is analytical procedure is the lowest amount of the lowest amount of analyte in a sample which can be detectedanalyte in a sample which can be detected but not necessarily quantitated as an exact but not necessarily quantitated as an exact value value

Several approaches for determining the Several approaches for determining the detection limit are possible, depending on detection limit are possible, depending on whether the procedure is a non-whether the procedure is a non-instrumental or instrumental. instrumental or instrumental.

Based on Visual Evaluation Based on Visual Evaluation

Visual evaluation may be used for non-Visual evaluation may be used for non-instrumental methods but may also be used instrumental methods but may also be used with instrumental methods.with instrumental methods.

The detection limit is determined by the The detection limit is determined by the analysis of samples with known analysis of samples with known concentrations of analyte and by concentrations of analyte and by establishing the minimum level at which the establishing the minimum level at which the analyte can be reliably detected .analyte can be reliably detected .

Based on Signal-to-Noise Based on Signal-to-Noise

This approach can only be applied to analytical This approach can only be applied to analytical procedures which exhibit baseline noise.procedures which exhibit baseline noise.

Determination of the signal-to-noise ratio is Determination of the signal-to-noise ratio is performed by comparing measured signals from performed by comparing measured signals from samples with known low concentrations of analyte samples with known low concentrations of analyte with those of blank samples and establishing the with those of blank samples and establishing the minimum concentration at which the analyte can minimum concentration at which the analyte can be reliably detected. be reliably detected.

A signal-to-noise ratio between A signal-to-noise ratio between 3:1 or 2:13:1 or 2:1 is is generally considered acceptable for estimating the generally considered acceptable for estimating the detection limit.detection limit.

Based on the Standard Deviation of Based on the Standard Deviation of the Response and the Slopethe Response and the Slope

The detection limit (DL) may be expressed The detection limit (DL) may be expressed as:as:

DL = 3.3 DL = 3.3 /S/S

where where = the standard deviation of the = the standard deviation of the responseresponse

S = the slope of the calibration curveS = the slope of the calibration curve

The slope S may be estimated from the The slope S may be estimated from the calibration curve of the analyte. calibration curve of the analyte.

Estimate of Estimate of

Based on the Standard Deviation of the BlankBased on the Standard Deviation of the Blank Measurement of the magnitude of analytical Measurement of the magnitude of analytical

background response is performed by analyzing an background response is performed by analyzing an appropriate number of blank samples and calculating appropriate number of blank samples and calculating the standard deviation of these responsesthe standard deviation of these responses

Based on the Calibration CurveBased on the Calibration Curve A specific calibration curve should be studied using A specific calibration curve should be studied using

samples containing an analyte in the range of DL.samples containing an analyte in the range of DL. The residual standard deviation of a regression line or The residual standard deviation of a regression line or

the standard deviation of y-intercepts of regression lines the standard deviation of y-intercepts of regression lines may be used as the standard deviation. may be used as the standard deviation.

Recommended DataRecommended Data The The detection limitdetection limit and the and the methodmethod used for used for

determining the detection limit should be determining the detection limit should be presented. presented.

If DL is determined based on visual evaluation or If DL is determined based on visual evaluation or based on signal to noise ratio, the presentation of based on signal to noise ratio, the presentation of the relevant chromatograms is considered the relevant chromatograms is considered acceptable for justification. acceptable for justification.

In cases where an estimated value for the detection In cases where an estimated value for the detection limit is obtained by calculation or extrapolation, limit is obtained by calculation or extrapolation, this estimate may subsequently be validated by the this estimate may subsequently be validated by the independent analysis of a suitable number of independent analysis of a suitable number of samples known to be near or prepared at the samples known to be near or prepared at the detection limit detection limit

QUANTITATION LIMIT QUANTITATION LIMIT The quantitation limit of an individual analytical The quantitation limit of an individual analytical

procedure is the lowest amount of analyte in a procedure is the lowest amount of analyte in a sample which can be quantitatively determined sample which can be quantitatively determined with suitable precision and accuracy. with suitable precision and accuracy.

The quantitation limit is a parameter of The quantitation limit is a parameter of quantitative assays for low levels of compounds in quantitative assays for low levels of compounds in sample matrices, and is used particularly for the sample matrices, and is used particularly for the determination of impurities and/or degradation determination of impurities and/or degradation products.products.

Several approaches for determining the Several approaches for determining the quantitation limit are possible, depending on quantitation limit are possible, depending on whether the procedure is a non-instrumental or whether the procedure is a non-instrumental or instrumental. instrumental.

Based on Visual Evaluation Based on Visual Evaluation

Visual evaluation may be used for non-Visual evaluation may be used for non-instrumental methods but may also be used instrumental methods but may also be used with instrumental methods. with instrumental methods.

The quantitation limit is generally The quantitation limit is generally determined by the analysis of samples with determined by the analysis of samples with known concentrations of analyte and by known concentrations of analyte and by establishing the minimum level at which the establishing the minimum level at which the analyte can be quantified with acceptable analyte can be quantified with acceptable accuracy and precision.accuracy and precision.

Based on Based on Signal-to-Noise Approach Signal-to-Noise Approach

This approach can only be applied to analytical This approach can only be applied to analytical procedures that exhibit baseline noise.procedures that exhibit baseline noise.

Determination of the signal-to-noise ratio is Determination of the signal-to-noise ratio is performed by comparing measured signals from performed by comparing measured signals from samples with known low concentrations of analyte samples with known low concentrations of analyte with those of blank samples and by establishing with those of blank samples and by establishing the minimum concentration at which the analyte the minimum concentration at which the analyte can be reliably quantified. can be reliably quantified.

A typical signal-to-noise ratio is A typical signal-to-noise ratio is 10:110:1..

Based on the Standard Deviation of Based on the Standard Deviation of the Response and the Slope the Response and the Slope

The quantitation limit (QL) may be The quantitation limit (QL) may be expressed as:expressed as:

QL = 10 QL = 10 /S/S

where where = the standard deviation of the = the standard deviation of the responseresponse

S = the slope of the calibration curveS = the slope of the calibration curve The slope S may be estimated from the The slope S may be estimated from the

calibration curve of the analyte. calibration curve of the analyte.

Estimate of Estimate of

Based on Standard Deviation of the BlankBased on Standard Deviation of the Blank Measurement of the magnitude of analytical Measurement of the magnitude of analytical

background response is performed by analyzing an background response is performed by analyzing an appropriate number of blank samples and calculating appropriate number of blank samples and calculating the standard deviation of these responses.the standard deviation of these responses.

Based on the Calibration CurveBased on the Calibration Curve A specific calibration curve should be studied using A specific calibration curve should be studied using

samples, containing an analyte in the range of QL.samples, containing an analyte in the range of QL. The residual standard deviation of a regression line or The residual standard deviation of a regression line or

the standard deviation of y-intercepts of regression lines the standard deviation of y-intercepts of regression lines may be used as the standard deviation.may be used as the standard deviation.

Recommended Data Recommended Data

The The quantitation limitquantitation limit and the and the methodmethod used used for determining the quantitation limit for determining the quantitation limit should be presented. should be presented.

The limit should be subsequently validated The limit should be subsequently validated by the analysis of a suitable number of by the analysis of a suitable number of samples known to be near or prepared at samples known to be near or prepared at the quantitation limit.the quantitation limit.

RobustnessRobustnessSmall changes do not Small changes do not

affect the parameters of affect the parameters of the assaythe assay

ROBUSTNESS ROBUSTNESS The robustness of an analytical procedure is a measure of The robustness of an analytical procedure is a measure of

its capacity to remain unaffected by small, but deliberate its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an variations in method parameters and provides an indication of its reliability during normal usage.indication of its reliability during normal usage.

The evaluation of robustness should be considered during The evaluation of robustness should be considered during the development phase and depends on the type of the development phase and depends on the type of procedure under study. procedure under study.

If measurements are susceptible to variations in analytical If measurements are susceptible to variations in analytical conditions, the analytical conditions should be suitably conditions, the analytical conditions should be suitably controlled or a precautionary statement should be controlled or a precautionary statement should be included in the procedure. included in the procedure.

One consequence of the evaluation of robustness should be One consequence of the evaluation of robustness should be that a series of system suitability parameters (e.g., that a series of system suitability parameters (e.g., resolution test) is established to ensure that the validity of resolution test) is established to ensure that the validity of the analytical procedure is maintained whenever used. the analytical procedure is maintained whenever used.

Typical Variations Typical Variations stability of analytical solutions,stability of analytical solutions, extraction timeextraction timeLiquid chromatography:Liquid chromatography: influence of variations of pH in a mobile phase,influence of variations of pH in a mobile phase, influence of variations in mobile phase composition,influence of variations in mobile phase composition, different columns (different lots and/or suppliers),different columns (different lots and/or suppliers), temperature,temperature, flow rate.flow rate.Gas chromatography:Gas chromatography: different columns (different lots and/or suppliers),different columns (different lots and/or suppliers), temperature,temperature, flow rate.flow rate.

SYSTEM SUITABILITY TESTING SYSTEM SUITABILITY TESTING

System suitability testing is an integral part of System suitability testing is an integral part of many analytical procedures. many analytical procedures.

The tests are based on the concept that the The tests are based on the concept that the equipment, electronics, analytical operations and equipment, electronics, analytical operations and samples to be analyzed constitute an integral samples to be analyzed constitute an integral system that can be evaluated as such.system that can be evaluated as such.

System suitability test parameters to be System suitability test parameters to be established for a particular procedure depend on established for a particular procedure depend on the type of procedure being validated. They are the type of procedure being validated. They are especially important in the case of especially important in the case of chromatographic methods. chromatographic methods.

System Suitability in System Suitability in ChromatographyChromatography

To verify that the resolution and reproducibility of the To verify that the resolution and reproducibility of the chromatographic system are adequate for the analysis to chromatographic system are adequate for the analysis to be donebe done

The resolution, R, is specified to ensure that closely eluting The resolution, R, is specified to ensure that closely eluting compounds are resolved from each othercompounds are resolved from each other

Replicate injections of a standard preparation are Replicate injections of a standard preparation are compared to ascertain whether requirements for precision compared to ascertain whether requirements for precision are metare met

The tailing factor, T, has to meet a certain requirement, The tailing factor, T, has to meet a certain requirement, because as peak asymmetry increases, integration, and because as peak asymmetry increases, integration, and hence precision, becomes less reliablehence precision, becomes less reliable

Evaluating validation data Evaluating validation data for for an HPLCan HPLC procedure procedure

Here are some suggestions………Here are some suggestions……… But please note!But please note!- The slides that follow The slides that follow do not represent requirementsdo not represent requirements; they are ; they are

suggestions. suggestions. - There is more than one way to do this!There is more than one way to do this!- Use judgement.Use judgement.

If you are unsure, consult with experienced analysts!!If you are unsure, consult with experienced analysts!!

SpecificitySpecificity (selectivity)(selectivity)

Use some or all of these procedures:Use some or all of these procedures:- Add a synthetic mixture of excipients to the sample & Add a synthetic mixture of excipients to the sample &

check whether the assay result for the drug is the samecheck whether the assay result for the drug is the same- Add some known impurities to the test sample & check Add some known impurities to the test sample & check

whether they are resolved (separated from) the drugwhether they are resolved (separated from) the drug- Forcably degrade the active & test whether degradants are Forcably degrade the active & test whether degradants are

separated from the intact drugseparated from the intact drug- Assess peak purity by diode arrayAssess peak purity by diode array

LinearityLinearity- Minimum of 5 concentrationsMinimum of 5 concentrations- rr2 2 >0.99 if possible>0.99 if possible- Intercept NMT Intercept NMT ±±2% of response of 100% the 2% of response of 100% the

working concentrationworking concentration- Confirm accuracy & precision over the Confirm accuracy & precision over the

required rangerequired range

AccuracyAccuracy- Generally within Generally within ++2%2%

- Recoveries after spiking, orRecoveries after spiking, or- Comparison with ‘well-established’ methods & by Comparison with ‘well-established’ methods & by

inferenceinference

- Arguably can be up to +10% for Arguably can be up to +10% for related substancesrelated substances

- What is known about the reference What is known about the reference standard?standard?

PrecisionPrecision- - repeatabilityrepeatability

System repeatabilitySystem repeatability

Method repeatabilityMethod repeatability

%CV (of detector %CV (of detector response) response) <<2.0% for 6 2.0% for 6 injectionsinjections

%CV %CV <<2.0%2.0% andand accuracy accuracy should be within 2%should be within 2%

PrecisionPrecision - - intermediateintermediate[= ruggedness USP][= ruggedness USP]

- Use same Use same completecomplete analytical procedure for analytical procedure for comparisonscomparisons

- Compare results across different analysts, days, Compare results across different analysts, days, equipmentequipment

- Means preferably within 2% Means preferably within 2% - Compare %CV with that for method repeatabilityCompare %CV with that for method repeatability

PrecisionPrecision- - reproducibilityreproducibility

- This is not normally a component of a dossier for This is not normally a component of a dossier for an application to register, but if you do have to an application to register, but if you do have to evaluate these data then……evaluate these data then……

- For interlab comparisonsFor interlab comparisons- Means should preferably be within 2%Means should preferably be within 2%- Compare the %CV with that for method Compare the %CV with that for method

repeatabilityrepeatability- Can use an F test, normally with 95% Can use an F test, normally with 95%

confidenceconfidence

Limit of detectionLimit of detection- Use some or all of these procedures:Use some or all of these procedures:- -- Visual evaluation: A clear & symmetrical peak Visual evaluation: A clear & symmetrical peak

is visibleis visible- Signal to noise ratio of 3:1 or 2:1Signal to noise ratio of 3:1 or 2:1- Based on statistical information: Based on statistical information:

- Detection limit = Detection limit = - 3.3 x (std dev at that concentration)3.3 x (std dev at that concentration)- slopeslope

Limit of quantitationLimit of quantitation

- Use some or all of these procedures:Use some or all of these procedures:- ‘‘Visual’ evaluation: A clear & symmetrical peak Visual’ evaluation: A clear & symmetrical peak

is visibleis visible- Signal to noise ratio of 10:1Signal to noise ratio of 10:1- Based on statistical information: Based on statistical information:

- Detection limit = Detection limit = - 10 x (std dev at that concentration)10 x (std dev at that concentration)- slopeslope

RobustnessRobustness- Use some or all of these procedures:Use some or all of these procedures:- Compare results after altering HPLC parameters, Compare results after altering HPLC parameters,

eg mobile phase composition, buffer composition, eg mobile phase composition, buffer composition, pH, column type, flow rate:pH, column type, flow rate:

- NMT NMT ±± 2% difference in assay2% difference in assay

- Compare results after storage of test solution, eg Compare results after storage of test solution, eg for 24h at say 25for 24h at say 2500CC

- NMT NMT ±± 2% difference in assay2% difference in assay

Are the data concerning analytical validation satisfactory? YES/NOIf NO, recommended questions to the applicant appear in

……………………………………………………………………(eg page number below, or draft letter to the company on page……)

Evaluation of analytical validation dataThe objective of the analytical procedure

The analytical technique

Item Data provided by applicant (very briefly)

Acceptable or not? (add comments if necessary, & reasons if unacceptable)

Is a chromatogram, spectrum or similar provided?

Specificity

Linearity

Range

Accuracy

Precision: Repeatability

Precision: Intermediate

Precision: Reproducibility

Detection limit

Quantitation limit

Robustness

System suitability (if necessary)

Data on the reference standard

Other evaluator comments:

Thank youThank you