ASEAN AnalValdn 26Sep07 Effect 7May08

8/3/2019 ASEAN AnalValdn 26Sep07 Effect 7May08

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ASEAN GUIDELINES FOR

VALIDATION OF

ANALYTICALPROCEDURES

SupaneeDuangteraprecha, Ph.D.Bureau of Drug and Narcotic

Department of Medical Sciences


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Objective

The objective of validation of an analytical

procedure is to demonstrate that it is

suitable for its intended purpose.


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Scope

provide guidance and recommendation ofvalidation of the analytical procedures forsubmission as part of registration applicationswithin ASEAN.

it mainly adopts two ICH guidelines

Q2A:Validation of Analytical Methods: Definitions andTerminology, 27 October 1994 and ICH Q2B:Validation of Analytical Procedure: Methodology, 6November 1996.

the methodology applied for biological andbiotechnological products may be approacheddifferently than chemical entities.


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Types of Analytical Procedures

to be Validated

Identification tests.

Quantitative tests for impurities' content.

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

samples of drug substance or drug product

or other selected component(s) in the drugproduct.


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Criteria For Analytical Test Validation

PRECISION

LIMITS

ACCURACY

LINEARITY RANGE

SPECIFICITY

REPEATABILITY

QUANTIFICATION

INTERMEDIATEPRECISION

DETECTION


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Symbols

- signifies that this characteristic is not normallyevaluated

+ signifies that this characteristic is normallyevaluated

(1) in cases where reproducibility (see glossary)has been performed, intermediate precision is notneeded

(2) lack of specificity of one analytical procedure

could be compensated by other supportinganalytical procedure(s)

(3) may be needed in some cases


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Specificity

Is this analyticalprocedure specific

for the drug under test?

Suppose we altertest conditions

slightly?


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Specificity (1)

Specificity is the ability to assessunequivocally the analyte in the presence ofcomponents which may be expected to be

present. Typically these might include impurities,

degradants, matrix, etc.

Lack of specificity of an individualanalytical procedure may be compensatedby other supporting analytical procedure(s).


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Specificity (2)

An investigation of specificity should be conductedduring the validation of identification tests, thedetermination of impurities and the assay. The procedures used to demonstrate specificity will

depend on the intended objective of the analytical

procedure.

It is not always possible to demonstrate that ananalytical procedure is specific for a particularanalyte (complete discrimination).

In this case a combination of two or more analyticalprocedures is recommended to achieve the necessarylevel of discrimination.


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Identification

Suitable identification tests should be able todiscriminate between compounds of closely related

structures which are likely to be present.

The discrimination of a procedure may be confirmed by

obtaining positive results (perhaps by comparison with

a known reference material) from samples containing

the analyte, coupled with negative results from samples

which do not contain the analyte.

In addition, the identification test may be applied to

materials structurally similar to or closely related to the

analyte to confirm that a positive response is not

obtained.


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Assay and Impurity Test(s)

For chromatographic procedures, representativechromatograms should be used to demonstrate specificityand individual components should be appropriatelylabelled.

Critical separations in chromatography should be

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

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

In cases where a non-specific assay is used, other

supporting analytical procedures should be used todemonstrate overall specificity.

For example, where a titration is adopted to assay the drugsubstance for release, the combination of the assay and a suitabletest for impurities can be used.


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Impurities are available (1)

For the assay , this should involve demonstrationof the discrimination of the analyte in the presenceof impurities and/or excipients;

practically, this can be done by spiking puresubstances (drug substance or drug product) withappropriate levels of impurities and/or excipientsand demonstrating that the assay result is

unaffected by the presence of these materials (bycomparison with the assay result obtained onunspiked samples).


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Impurities are available (2)

For the impurity test, the discrimination

may be established by:

spiking drug substance or drug product with

appropriate levels of impurities and

demonstrating the separation of these

impurities individually and/or from other

components in the sample matrix.


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Impurities are not available

If impurity or degradation product standards areunavailable, specificity may be demonstrated bycomparing the test results of samples containingimpurities or degradation products to a secondwell-characterized procedure e.g.:

pharmacopoeial method or other validatedanalytical procedure (independent procedure).

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

oxidation.

for the assay, the two results should be compared.

for the impurity tests, the impurity profiles should becompared.


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Linearity and Range

Know that its a straight line

vs

For what concentrations is it astraight line


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Linearity and Range

Know that its a straight lineversus

For whatconcentrations is it a straight line Is it a straight line between 0.4 & 0.6 mg/mL?

Over what range is it a straight line?

Answer: approx 0.25-0.70 mg/mL

Concentration

mg/mL

Response


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LINEARITY (1)

A linear relationship should be evaluatedacross the range of the analytical procedure.

It may be demonstrated directly on the drug

substance by: dilution of a standard stock solution and/or

separate weighings of synthetic mixtures of thedrug product components

using the proposed procedure.


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LINEARITY (2)

Linearity should be evaluated by visual inspection of a plot ofsignals as a function of analyte concentration or content.

If there is a linear relationship, test results should beevaluated by appropriate statistical methods, for example, bycalculation of a regression line by the method of least squares.

In some cases, to obtain linearity between assays and sampleconcentrations, the test data may need to be subjected to amathematical transformation prior to the regression analysis.Data from the regression line itself may be helpful to providemathematical estimates of the degree of linearity.

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


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Example

Taken from:

ASEAN Operational Manual for Implementationof GMP ed. 2000 p.403

Seven solutions containing differentconcentrations (0.2800.520) mg/ml of ketotifenfumarate in tablet batch no. 2506 VAMG were

assayed using HPLC

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


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Example (continued)

Concentration of ketotifen fumarate Area detected Acceptance

criteriamg/ml %

0.280

0.320

0.360

0.400

0.440

0.480

0.520

70

80

90

100

110

120

130

1473566

1677013

1904848

2091215

2293647

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2670144

Regression: y = ax + b

a = 5055766.964

b = 67608.786

r2 = 0.9984

0.9981.002


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RANGE

The specified range is normally derived from

linearity studies and depends on the intended

application of the procedure.

It is established by confirming that the analyticalprocedure provides an acceptable degree of

linearity, accuracy and precision when applied to

samples containing amounts of analyte within or

at the extremes of the specified range of theanalytical procedure.


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Minimum Specified Ranges (1)

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

concentration

for content uniformity, covering a minimum of70 - 130 % of the test concentration

for dissolution testing: +/-20 % over thespecified range; e.g., if the specifications for a

controlled released product cover a region from20%, after 1 hour, up to 90%, after 24 hours, the

validated range would be 0-110% of the label claim


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Minimum Specified Ranges (2)

for the determination of an impurity: from the reportinglevel of an impurity to 120% of the specification; forimpurities known to be unusually potent or to producetoxic or unexpected pharmacological effects, the

detection/quantitation limit should be commensurate withthe level at which the impurities must be controlled.

if assay and purity are performed together as one test andonly a 100% standard is used, linearity should cover the

range from the reporting level of the impurities to 120% ofthe assay specification


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Accuracy vs precision

What youwould like

to see!


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Poor accuracy Good precision


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Poor precision Good accuracy


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Totally hopeless!

Poor precision Poor accuracy

What would youcall this?


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So what definitions do these

concepts lead us to in thecontext of assay validation?


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ACCURACY (1)

The accuracy of an analytical procedure

expresses the closeness of agreement

between the value which is accepted either

as a conventional true value or an acceptedreference value and the value found. This is

sometimes termed trueness.


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ACCURACY (2)

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

analyte of known purity (e.g. reference material);

b) comparison of the results of the proposedanalytical procedure with those of a second well-characterized procedure, the accuracy of which isstated and/or defined (independent procedure)

c) accuracy may be inferred once precision, linearityand specificity have been established


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ACCURACY (3)

Assay of Drug Product:a) application of the analytical procedure to syntheticmixtures of the drug product components to which knownquantities of the drug substance to be analysed have beenadded;

b) in cases where it is impossible to obtain samples of all drugproduct components, it may be acceptable either to:

add known quantities of the analyte to the drug product or

to compare the results obtained from a second, well characterizedprocedure, the accuracy of which is stated and/or defined

(independent procedure)

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


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ACCURACY (4)

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

substance/drug product) spiked with known amounts ofimpurities.

In cases where it is impossible to obtain samples of certain

impurities and/or degradation products, it is consideredacceptable to compare results obtained by an independentprocedure. The response factor of the drug substance canbe used.

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


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Example:

Taken from:

ASEAN Operational Manual for

Implementation of GMP ed. 2000 p.405

Nine solutions containing different

concentrations of ketotifen fumarate

reference standard added to ketotifen tablet

batch no. 2506VAMG were assayed


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Example (continued):

Conc. of ketotifen fumarate Area

detected

Recovery

(%)

Acceptance

Criteriamg/ml %

0.280

0.320

0.360

0.3800.400

0.420

0.440

0.480

0.520

70

80

90

95100

105

110

120

130

1473566

1677013

1904848

19058622091215

2180374

2293647

2518976

2670144

99.32

99.48

100.94

100.51100.06

100.03

100.07

101.01

98.99

Mean (recovery) : 100.04

Standard deviation : 0.699

Relative standard deviation (RSD) : 0.699 %

98.0102.0 %

< 2 %


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PRECISION

The precision of an analytical procedure expresses the

closeness of agreement (degree of scatter) between a seriesof measurements obtained from multiple sampling of thesame homogeneous sample under the prescribedconditions.

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

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

The precision of an analytical procedure is usuallyexpressed as the variance, standard deviation orcoefficient of variation of a series of measurements.


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Repeatability (1)

Repeatability expresses the precision under

the same operating conditions over a short

interval of time.

Repeatability is also termed intra-assay

precision.


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Repeatability (2)

Repeatability should be assessed using:

a) a minimum of 9 determinations

covering the specified range for the

procedure (e.g. 3 concentrations/3

replicates each) or

b) a minimum of6 determinations at

100% of the test concentration.


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Intermediate precision

Intermediate precision expresses within-laboratoriesvariations: different days, different analysts,different equipment, etc.

The extent to which intermediate precision should beestablished depends on the circumstances under which the

procedure is intended to be used.

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

Typical variations to be studied include days, analysts,

equipment, etc. It is not considered necessary to studythese effects individually. The use of an experimentaldesign (matrix) is encouraged.


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Reproducibility

Reproducibility is assessed by means of an

inter-laboratory trial.

Reproducibility should be considered in

case of the standardization of an analytical

procedure, for instance, for inclusion of

procedures in pharmacopoeias.

These data are not part of the marketing

authorization dossier.


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Recommended Data

The standard deviation, relativestandard deviation (coefficient of

variation) and confidence interval should bereported for each type of precision

investigated.


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Example

Taken from:

ASEAN Operational Manual for

Implementation of GMP ed. 2000 p.403

The active ingredient, ketotifen fumarate,

in tablets (batch no. 2506VAMG) was

assayed seven times using HPLC and the

reference standard


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Example (continued)

Sample no. Concentration (mg/ml) Area detected1

2

3

4

5

6

7

0.4

0.4

0.4

0.4

0.4

0.4

0.4

1902803

1928083

1911457

1915897

1913312

1897702

1907019

Mean : 1910896

Standard deviation : 9841.78

Relative standard deviation (RSD) : 0.515 %

Acceptance criteria:

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


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Detection limit vs

Quantitation limit

Know that its therevsKnow how much is there


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Detection limit(means)

Is any of it present?

Is it there?


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Quantitation limit

How much of it is present???

How much of it is there?


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DETECTION LIMIT

The detection limit of an individualanalytical procedure is the lowest amount ofanalyte in a sample which can be detected

but not necessarily quantitated as an exactvalue

Several approaches for determining thedetection limit are possible, depending on

whether the procedure is a non-instrumental or instrumental.


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Based on Visual Evaluation

Visual evaluation may be used for non-

instrumental methods but may also be used

with instrumental methods.

The detection limit is determined by the

analysis of samples with known

concentrations of analyte and by

establishing the minimum level at which theanalyte can be reliably detected .


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Based on Signal-to-Noise

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

Determination of the signal-to-noise ratio is

performed by comparing measured signals fromsamples with known low concentrations of analytewith those of blank samples and establishing theminimum concentration at which the analyte canbe reliably detected.

A signal-to-noise ratio between 3:1 or 2:1 isgenerally considered acceptable for estimating thedetection limit.

B d th St d d D i ti f


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Based on the Standard Deviation of

the Response and the Slope

The detection limit (DL) may be expressed

as:

DL = 3.3 s/Swhere s = the standard deviation of theresponse

S = the slope of the calibration curve

The slope S may be estimated from the

calibration curve of the analyte.


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Estimate ofs

Based on the Standard Deviation of the Blank

Measurement of the magnitude of analyticalbackground response is performed by analyzing anappropriate number of blank samples and calculating

the standard deviation of these responses Based on the Calibration Curve

A specific calibration curve should be studied usingsamples containing an analyte in the range of DL.

The residual standard deviation of a regression line orthe standard deviation of y-intercepts of regression linesmay be used as the standard deviation.


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Recommended Data

The detection limit and the method used fordetermining the detection limit should bepresented.

If DL is determined based on visual evaluation orbased on signal to noise ratio, the presentation ofthe relevant chromatograms is consideredacceptable for justification.

In cases where an estimated value for the detectionlimit is obtained by calculation or extrapolation,

this estimate may subsequently be validated by theindependent analysis of a suitable number ofsamples known to be near or prepared at thedetection limit


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QUANTITATION LIMIT

The quantitation limit of an individual analyticalprocedure is the lowest amount of analyte in asample which can be quantitatively determinedwith suitable precision and accuracy.

The quantitation limit is a parameter of

quantitative assays for low levels of compounds insample matrices, and is used particularly for thedetermination of impurities and/or degradationproducts.

Several approaches for determining thequantitation limit are possible, depending onwhether the procedure is a non-instrumental orinstrumental.


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Based on Visual Evaluation

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

The quantitation limit is generallydetermined by the analysis of samples withknown concentrations of analyte and byestablishing the minimum level at which the

analyte can be quantified with acceptableaccuracy and precision.

B d


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Based on

Signal-to-Noise Approach

This approach can only be applied to analytical

procedures that exhibit baseline noise.

Determination of the signal-to-noise ratio is

performed by comparing measured signals fromsamples with known low concentrations of analyte

with those of blank samples and by establishing

the minimum concentration at which the analyte

can be reliably quantified.

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

B d th St d d D i ti f


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Based on the Standard Deviation of

the Response and the Slope

The quantitation limit (QL) may be

expressed as:

QL = 10 s/Swhere s = the standard deviation of theresponse

S = the slope of the calibration curve

The slope S may be estimated from the

calibration curve of the analyte.


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Estimate ofs

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

background response is performed by analyzing anappropriate number of blank samples and calculating

the standard deviation of these responses. Based on the Calibration Curve

A specific calibration curve should be studied usingsamples, containing an analyte in the range of QL.

The residual standard deviation of a regression line orthe standard deviation of y-intercepts of regression linesmay be used as the standard deviation.


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Recommended Data

The quantitation limit and the method used

for determining the quantitation limit

should be presented.

The limit should be subsequently validated

by the analysis of a suitable number of

samples known to be near or prepared at

the quantitation limit.


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Robustness

Small changes do not affectthe parameters of the

assay


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ROBUSTNESS

The robustness of an analytical procedure is a measure of

its capacity to remain unaffected by small, but deliberatevariations in method parameters and provides anindication of its reliability during normal usage.

The evaluation of robustness should be considered duringthe development phase and depends on the type of

procedure under study.

If measurements are susceptible to variations in analyticalconditions, the analytical conditions should be suitablycontrolled or a precautionary statement should be includedin the procedure.

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

T i l V i ti


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Typical Variations

stability of analytical solutions,

extraction timeLiquid chromatography:

influence of variations of pH in a mobile phase,

influence of variations in mobile phase composition,

different columns (different lots and/or suppliers), temperature,

flow rate.

Gas chromatography:

different columns (different lots and/or suppliers), temperature,

flow rate.


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SYSTEM SUITABILITY TESTING

System suitability testing is an integral part ofmany analytical procedures.

The tests are based on the concept that theequipment, electronics, analytical operations andsamples to be analyzed constitute an integralsystem that can be evaluated as such.

System suitability test parameters to beestablished for a particular procedure depend onthe type of procedure being validated. They areespecially important in the case ofchromatographic methods.

System Suitability in


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System Suitability in

Chromatography

To verify that the resolution and reproducibility of thechromatographic system are adequate for the analysis tobe done

The resolution, R, is specified to ensure that closely eluting

compounds are resolved from each other

Replicate injections of a standard preparation arecompared to ascertain whether requirements for precisionare met

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

Evaluating validation data for an


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Evaluating validation data for anHPLC procedure

Here are some suggestions

But please note!

- The slides that follow do not represent requirements; they

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

- Use judgement.

If you are unsure, consult with experienced analysts!!

Specificity


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Specificity(selectivity)

Use some or all of these procedures:

- Add a synthetic mixture of excipients to the sample &

check whether the assay result for the drug is the same

- Add some known impurities to the test sample & check

whether they are resolved (separated from) the drug

- Forcably degrade the active & test whether degradants are

separated from the intact drug

- Assess peak purity by diode array


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Linearity

- Minimum of 5 concentrations

- r2 >0.99 if possible

- Intercept NMT 2% of response of 100% the

working concentration

- Confirm accuracy & precision over the

required range


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Accuracy

- Generally within +2%- Recoveries after spiking, or

- Comparison with well-established methods & byinference

- Arguably can be up to +10% forrelated substances

-

What is known about the referencestandard?

Precision


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Precision- repeatability

System repeatability

Method repeatability

%CV (of detectorresponse)


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Precision -intermediate[= ruggedness USP]

- Use same complete analytical procedure for

comparisons

-

Compare results across different analysts, days,equipment

- Means preferably within 2%

- Compare %CV with that for method repeatability

Precision


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Precision- reproducibility

- This is not normally a component of a dossier for

an application to register, but if you do have to

evaluate these data then

- For interlab comparisons

- Means should preferably be within 2%

- Compare the %CV with that for method

repeatability

- Can use an F test, normally with 95%

confidence

f d


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Limit of detection

- Use some or all of these procedures:

- - Visual evaluation: A clear & symmetrical peak isvisible

- Signal to noise ratio of 3:1 or 2:1

- Based on statistical information:- Detection limit =

- 3.3 x (std dev at that concentration)

- slope

L f


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Limit of quantitation

- Use some or all of these procedures:

- Visual evaluation: A clear & symmetrical peak

is visible

- Signal to noise ratio of 10:1

- Based on statistical information:- Detection limit =

- 10 x (std dev at that concentration)

- slope

R b tn


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Robustness

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

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

- NMT 2% difference in assay

- Compare results after storage of test solution, egfor 24h at say 250C

- NMT 2% difference in assay

Evaluation of analytical validation data

Th bj ti f th l ti l d


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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

)

The objective of the analytical procedure

The analytical technique

Item Data provided by applicant(very briefly)

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

Is a chromatogram, spectrum orsimilar 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:


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Thank you

Documents

ASEAN AnalValdn 26Sep07 Effect 7May08