5. LIST OF PARAMETERS FOR VALIDATIONS:^5,6,7

1) Specificity

2) Linearity

3) Range

4) Accuracy

5) Precision

6) Detection limit

7) Quantitation limit

8) Robustness

9) Ruggedness

6. COMPARISON OF VALIDATION PARAMETERS:

[A] Specificity:^6-12

ICH :

Definition: It may be defined as ability to assess distinctly the analyte in the presence of components (impurity).

Method:

· When the impurities are present in the sample, Spike of pure substance in appropriate level of impurities and determine that the result is unchanged.

· When the impurities are not available in the sample than correlate the test results of sample containing impurities or degradation product to predermined procedure. This correlation should include sample under compatible stress condition.

· In chromatographic method: Peak purity test to be done by diode array and mass spectrophotometry.

Expression:

· Evidance of intolerance of analyte in the presence of impurities

· For Peak purity test advice in demonstrating that the peak is not traceable to more than one component.

· For assay method two results should be correlated and for impurity tests two profiles should be compared.

Acceptance criteria: Not specified.

USFDA :

Definition: It may be defined as ability of an analytical method to characterized and evaluate the analyte in the existence of other components in the sample.

Method:

· Analysis of blank samples of relevant biological matrix obtained from at least six sample and test for interference.

· Selectivity should be ensured at lower limit of quantitation.

Expression:

Proof that the substance being quantified.

Acceptance criteria: Not specified.

AOAC:

Definition: It may be defined as ability of a method to measure only what it is proposed to measure.

Method:

· Analysis of blank samples should be run to ensure no interference of foreign compound are present.

· To justify that method for the analyte(s) of interest, results should be tested under different experimental conditions, e.g., two different analytical principles or two different detection techniques.

Expression: Not specified

Acceptance criteria: Not specified.

USP:

Definition: It is defined as ability to assess distinctly the analyte in the presence of othrer components like impurity which may be expected to be present.

Method:

· When the impurities are present in the sample, Spike of pure substance in appropriate level of impurities and determine that the result is unchanged.

Expression:

· Proof of discrimination of analyte in the presence of impurities e.g. for chromatography chromatogram should be submitted.

· Peak purity test helps in demonstrating that the peak is not attributable to more than one component.

· For assay two results should be compared and for impurity tests two profiles should be compared.

Acceptance criteria: Not specified.

JP:

Definition: It is defined as ability of analytical procedure to measure accurately an analyte in presence of components that may be expected to present in the sample matrix.

Method:

· The analytical procedure can identify an analyte or concentration of analyte in sample.

· It is analyzed by correlating analytical results obtained from a sample containing the analyte only with results obtained from sample containing excipients, related substance and degraded products.

· If reference standard is not available sample that are expected to contain impurities or sample after stress or accelerated study may be used.

Expression: Not specified

Acceptance criteria: Not specified.

APVMA:

Definition: It is defined as selectivity of a method attribute to the extent to which it can determine particular analyte in a sample without confliction from other components in the sample.

Method:

· The selectivity of the analytical method must be demonstrated by providing data to that provide information about presence of interference peaks with concern to degradation products, impurities and the matrix .

· Chromatographic methods may be applied for assessment of peak purity test.

Expression: Not specified

Acceptance criteria: Not specified.

IUPAC:

Definition: It is defined as degree to which a method can appraise the analyte accurately in the presence of impurities.

Method: Selectivity index should be calculated.

Expression: Selectivity Index

Acceptance criteria: Not specified.

[B] Linearity and range:^6-12

ICH :

Definition:

· Linearity: It may be defined as ability to obtain test results, which are directly proportional to the concentration.

· Range: It may be defined as interval between the upper and lower concentration of analyte in the sample.

Method:

· Drug and/or separately weighed synthetic mixture.

· Measurement of response and plot response vs. concentration of analyte and demonstration of linearity by Visual inspection of plot appropriate statistical methods.

For Linearity: Minimum of 5 concentrations are recommended for linearity

For Range: Assay of drug/finished product: 80–120% of test concentration.

For content uniformity: 70–130% of test concentration.

For dissolution testing: _20% over specified range.

For impurity: from reporting level to 120% of specification.

Expression: Correlation coefficient, slope of regression line, residual sum of squares.

Acceptance criteria: Not specified.

USFDA :

Definition: It is expressed as relationship between response and known concentration of analyte.

Method: Sufficient number of standards in the expected matrix is taken and response measured.

· Plot of concentration vs. Response is plotted and linearity is demonstrated.

· Nature of analyte/ response relationship Conc. of sample depends on expected concentration range

· One blank sample which include matrix sample without internal standard was used for study.

· One zero sample that include matrix sample and internal standard was used for study.

· 6–8 non-zero samples covering expected range including LLOQ were used for the study.

Expression: Not specified.

Acceptance criteria:

· At LLOQ: Not more than 20% deviation from nominal value

· Other than LLOQ: 15% deviation from nominal value

· At least 4 out of 6 nonzero samples should meet the above criteria including LLOQ and highest concentration.

AOAC:

Definition:

Linearity: It is defined as the capacity of the method to obtain test results proportional to the concentration.

Range: Not specified.

Method: 4 Concentration level should be selected 1/2, 1,3/2,2 for 3 days.

Expression: Not specified.

Acceptance criteria: Not specified.

USP:

Definition:

Linearity: It is defined as the capacity to obtain test results, which are directly proportional to the concentration of analyte.

Range: It is defined as interval between the upper and lower concentration (amounts) of analyte.

Method: Drug (different dilution) and/or separately weighed synthetic mixture. Measurement of response and plot response vs. concentration of analyte and demonstration of linearity by Visual inspection of plot and Appropriate statistical methods

For linearity: Minimum of 5 concentrations are recommended.

For Range: Assay of drug/finished product: 80– 120% of test concentration.

For content uniformity: 70–130% of test concentration

For dissolution testing: 20% over specified range

For impurity: from reporting level to 120% of specification.

Expression: Correlation coefficient, y-intercept, slope of regression line, residual sum of squares

Acceptance Criteria: Not specified.

JP:

Definition:

Linearity: It may be defined as ability of analytical procedure to bring out the responses linearly relevant to amount of analyte in samples.

Range: It may be defined as as interval between lower and upper limit of the amount in the sample.

Method:

For linearity: Sample with five different amount or concentration should be investigated.

For range: 80-120% of specified limit of testing should be considered.

Expression: Correlation coefficient, y-intercept, slope of regression line

Acceptance Criteria: Not specified.

APVMA:

Definition:

Linearity: It may be expressed as the ability of analytical procedure to produce test results which are proportional to the concentration of analyte in samples.

Range: It may be expressed as the interval between the upper and lower concentration of analyte in the sample .

Method:

Linearity: Linearity should be determined by using a minimum of six solution in at least 80 - 120% concentration of the sample.

Range: For the assay normally from 80 –120% of the test concentration is used for the study.

For the determination of an impurity: from the specification level of the impurity to 120% of the specification.

Expression: Correlation coefficient, y-intercept, slope of regression line.

Acceptance Criteria: Not specified.

IUPAC

Definition: Not specified.

Method:

For linearity: Six or more calibration standards evenly spaced over the range of interest.

For Range: 0–150% or 50–150% of target concentration depending on which of this is more suitable. Calibration standard should be run at least in duplicate, preferably in triplicate or more.

Expression: Not specified.

Acceptance Criteria: Not specified.

[C] Accuracy:^6-12

ICH:

Definition: It may be defined as the closeness of agreement between the true value.

Method:

· Application of procedure to analyze synthetic mixture of known purity.

· Comparison of result with already established procedure.

· Minimum of nine determinations

For low concentration of range 3 replicates must be used for the study.

For medium concentration of range 3 replicates must be used for the study.

For high concentration of range 3 replicates must be used for the study.

Expression: Percent recovery should be found.

Acceptance Criteria: Not specified.

USFDA:

Definition: It is expressed as a closeness of test results obtained by method to the true value.

Method:

It includes replicate analysis of samples containing known amounts of analyte.

Mainly three concentration levels are implemented for the study that includes

1. For low concentration of range 5 replicates must be used for the study.

2. For medium concentration of range 5 replicates must be used for the study.

3. For high concentration of range 5 replicates must be used for the study.

Expression: Comparison of Mean with true value

Acceptance Criteria:

· At LLOQ: Mean value should be within 20% of actual value.

· Other than LLOQ: Mean value should be within 15% of actual value.

AOAC:

Definition: It may be defined as closeness of the determined value to the true value.

Method:

Three concentration levels throughout the range

1. For low concentration of range 3 replicates must be used for the study.

2. For medium concentration of range 3 replicates must be used for the study.

3. For high concentration of range 3 replicates must be used for the study

Amount added for fortification should be more than the amount present in the sample.

Expression: % Recovery

Acceptance Criteria: Not specified.

USP:

Definition: It is expressed as a Closeness of test results obtained by that method to true value.

Method: Application of procedure to analyze synthetic mixture of known purity.

· Comparison of result with already established procedure.

· Accuracy may be inferred once precision, linearity and specificity have been established.

· Minimum of nine determinations

Low concentration of range 3 replicates

Medium concentration of range 3 replicates

High concentration of range 3 replicates

Expression:

· Percent recovery by the assay of known added amount of analyte.

· Comparison with mean with accepted true value with confidence interval.

Acceptance Criteria: Not specified.

JP:

Definition: It is expressed as a measurement of bias of observed values obtained by an analytical procedure.

Method:

· Difference between the total mean of observed values obtained during investigation of the reproducibility and the true value.

· Theoretical value is used as true value.

· When theoretical value is difficult to obtain then a certified value or consensus value may be used as a theoretical value.

Expression: It is expressed as the diversity between the average value obtained from series of observed values and the true value

Acceptance Criteria: Not specified.

APVMA:

Definition: It may be defined as degree at which the determined value of analyte in a sample correlate to the true value.

Method:It can be perform using any of the following method.

· At least 3 concentrations (80, 100 and 120%) must be used for study.

1. Comparing a sample of known concentration with sample by measured value to the ‘true’ value.

2. Spiked method: a known amount of pure active constituent is added to formulation blank and the resulting mixture is assayed, results obtained are compared with the expected result.

3. Standard addition method.: Sample is assayed, a known amount of pure active constituent is added, and the sample is again assayed. The difference between the results of the two assays is compared.

Expression: Percent recovery by the assay of known added amount of analyte.

Acceptance Criteria: The mean % recovery should be within following ranges.

%Active/impurity content	Acceptable mean recovery
0.1 – 1 mg	80 – 120%
< 0.1 mg	75 – 125%
≥ 1 mg	90 –110%
≥ 10 mg	98 –102%

IUPAC :

Definition: It may be defined as is the closeness of agreement between a test result and accepted reference value.

Method:Bias is determined by comparing response of method to a reference like,

Use of certified reference material.

Use of reference material

Use of reference methods

Use of spiking/recovery

Expression: Not specified.

Acceptance Criteria: Not specified.

[D] Precision:^6-12

ICH:

Definition: It may be defined as closeness of agreement among array of measurements obtained from multiple sampling of the same homogenous sample under the predetermined conditions.

Method:

Determination of % relative standard deviation (RSD) of response of multiple aliquots.

Repeatability study includes identical operating condition over short interval of time

Minimum of nine determinations should be analysed.

1. For low concentration of range 3 replicates must be used for the study.

2. For medium concentration of range 3 replicates must be used for the study.

3. For high concentration of range 3 replicates must be used for the study

(or)

6 determinations of identical concentration must be used for the study

Intermediate precision includes within laboratory variation like Different days

Different analysts, Different equipments etc.

Expression: Standard deviation, RSD and confidence interval

Acceptance Criteria: Not specified.

USFDA:

Definition: It may be defined as the closeness of individual measures of an analyte when the procedure is repeated for single identical sample matrix.

Method:

Mainly three concentration levels are implemented for the study that includes

· For low concentration of range 5 replicates must be used for the study.

· For medium concentration of range 5 replicates must be used for the study.

· For high concentration of range 5 replicates must be used for the study.

Intra batch precision(repeatability): Measures precision during single run.

Inter batch precision (reproducibility): Measures precision with time.

Expression: Coefficient of variation

Acceptance Criteria:

At LLOQ: CV 20%

Other than LLOQ: 15%

AOAC:

Definition: may be defined as degree of agreement of measurements under distinct conditions.

Method:

Method include determination of % relative standard deviation of collective aliquots.

4 conc. levels are to be selected.

1/2×

1×

3/2×

2×

Repeatability:

It should be carried out within laboratory like Different days, Different analysts, Different calibration curves, Different batches of reagents etc

Reproducibility:

It should be carried out in different labs.

· Expression: RSD

· Acceptance Criteria: Not specified.

USP:

Definition: It may be defined as degree of agreement between individual test results for multiple samplings of a homogenous sample.

Method:

Determination of % relative standard deviation (RSD) of multiple aliquots.

Repeatability (Same operating condition over short interval of time):

Minimum of nine determinations over the entire range

Minimum of nine determinations should be analyzed.

1. For low concentration of range 3 replicates must be used for the study.

2. For medium concentration of range 3 replicates must be used for the study.

3. For high concentration of range 3 replicates must be used for the study

(or)

At target concentration 6 determinations of identical sample.

Intermediate precision includes within laboratory variation like:

Different days, Different analysts, Different equipment etc.

Expression:

Standard deviation, RSD and confidence interval

Acceptance Criteria: Not specified.

JP:

Definition: It is measure of closeness of agreement between observed values obtained from multiple sampling of a homogeneous sample.

Method:

Three levels with different repetition conditions like repeatability, intermediate precision and reproducibility.

Repeatability ( Intra-assay precision)

Multiple sampling of a homogeneous sample for short period of time interval within laboratory by using Same analyst Same equipments and reagents, etc.

Intermediate Precision:

It is Precision of observed values obtained from multiple sapling of a homogeneous sample by changing a part of or all operating conditions within laboratory including:

Analyst, Experimental dates, Apparatus and instruments, Reagents, etc

Reproducibility:

It is the precision of observed values obtained from multiple sapling of a homogeneous sample in different laboratories.

Expression:

Standard deviation, RSD and confidence interval

Acceptance Criteria: Not specified.

APVMA:

Definition: It may be defined as closeness of agreement between a series of measurements obtained from multiple sampling of the identical sample under the predetermined condition.

Method:

Three types

1) Repeatability

2) Intermediate precision

3) Reproducibility.

A minimum of 5 replicate samples should be analyzed.

The following levels of precision are recommended.

≥ 10.0%	≤ 2%
1.0 up to 10.0%	≤ 5%
0.1 up to 1.0%	≤ 10%
< 0.1 %	≤ 20%

Expression: Standard deviation, RSD and confidence interval

Acceptance Criteria: Not specified.

IUPAC:

Definition: It may be defined as a Closeness of agreement between test results obtained under identical conditions.

Method:

Determination of % relative standard deviation (RSD) and F-test are used for the study.

Minimum of two concentrations are indicated

· At or near highest concentration in range

· At or near lowest concentration in range.

Expression: SD or RSD

Acceptance Criteria: Not specified.

[E] LOD^6-12

ICH:

Definition: It may be defined as lowest amount of analyte in the sample, which can be detected but not necessarily quantitated under stated experimental conditions.

Method:

1. visual evaluation

2. Use of signal to noise ratio

Actual lowest concentration of analyte detected in compared with blank response

3. Use of standard deviation and slope foe calculation

LOD = 3.3 δ /s

Where

δ= Slope of calibration curve

s = S.D. of response;

Standard deviation can be obtained by

· Standard deviation of blank response

· Residual standard deviation of the regression line

· Standard deviation of the y-intercept of the regression line

Expression:

· If based on visual examination or S/N ratio.

· If by calculation than suitable no. of samples known to be near or prepared at detection limit.

Acceptance Criteria: S/N ratio > 2–3; Not specified in other cases

USFDA:

LOD is not explained

AOAC:

Definition: It may be defined as lowest amount of content that can be measured.

Method: Based on more than 20 blank readings.

Expression: It may be expressed as considering mean value of the matrix blank readings where n ≥ 20 and standard deviations of the mean.

Acceptance Criteria: Not specified.

USP:

Definition: It may be defined as lowest amount of analyte in the sample, which can be detected but not necessarily quantitated.

Method:

Mainly two types

Non-instrumental method: Analysis of sample with known concentration of analyte

Instrumental Method : Process for non-instrumental can be adopted. Detection limit should be sufficiently low for analysis of samples with known concentration of analyte above and below the predetermined detection limit.

Expression: Not specified.

Acceptance Criteria: Not specified.

JP:

Definition: It may be defined as lowest amount or concentration of the analyte in the sample which can be detected, but not necessarily quantified.

Method:

It can be calculated using standard deviation and slope of response.

DL = 3.3 δ /s

Where

DL= Detection limit

δ= Slope of calibration curve

s = S.D. of response;

Expression: Not specified

Acceptance Criteria: Not specified.

APVMA:

Definition: It can be defined as lowest amount of an analyte in a sample that can be detected, but not necessarily quantified.

Method:

The LOD may be determined by the analysis of samples with known concentrations of analyte and by building the minimum level at which the analyte can be accurately identified.

LOD = X + (3 x SD).

Where:

X= average response

SD= Standard deviation

Expression: Not specified

Acceptance Criteria: Not specified.

IUPAC:

Definition: It may be defined as smallest amount of concentration of analyte in the sample that can be accurately diffentiate from zero.

Method: Not described

Expression: Not specified

Acceptance Criteria: Not specified.

[F] LOQ:^6-12

ICH:

Definition: It may be defined as lowest amount of analyte in a sample, which can be quantitatively determined with appropriate precision and accuracy.

Method:

1 visual evaluation

2. Use of signal to noise ratio Actual lowest concentration of analyte detected in compared with blank response

3. Use of standard deviation and slope foe calculation

Based on S.D. of response and slope

LOD = 10 δ /s

Where

δ= Slope of calibration curve

s = S.D. of response;

Standard deviation can be obtained by

· Standard deviation of blank response

· Residual standard deviation of the regression line

· Standard deviation of the y-intercept of the regression line

Sy/x i.e. standard error of estimate

Expression: Limits of quantitation and method used for determining should be presented.

Acceptance Criteria: Not specified

USFDA:

Definition: It may be defined as lowest amount of analyte that can be quantitatively determined with suitable precision and accuracy also called Lower limit of quantification.

Method: Preparation of standard curve and lowest concentration on the calibration curve should be accepted as lower limit of quantitation (LLOQ) if it satisfies following condition.

Response at LLOQ ¼ = 5× Response by blank Analyte peak should be identifiable discrete and reproducible with precision of 20% and accuracy of 80–120%

Expression: Not specified

Acceptance Criteria: Not specified

AOAC:

Definition: It may be defined as lowest concentration of analyte in sample, which can be quantitatively determined with precision and accuracy. Method: Not specified.

Expression: Not specified.

Acceptance Criteria: Not specified.

USP:

Definition: It may be defined as lowest amount of analyte in a sample, which can be determined quantitatively with relavant precision and accuracy.

Method:

1. By visual evaluation

2. Based on S/N ratio

Applicable to procedure, which exhibit baseline noise.

Actual lowest concentration of analyte detected in compared with blank response

3. Based on S.D. of response and slope

LOD = 10 δ /s

Where δ= Slope of calibration curve

s = S.D. of response;

Which can be obtained by

· Standard deviation of blank response

· Residual standard deviation of the regression line

· Standard deviation of the y-intercept of the regression line

Sy/x i.e. standard error of estimate

Expression: Expressed as analyte concentration (% or ppb)

Acceptance Criteria: Not specified.

JP:

Definition: It may be defined as lowest amount or concentration of the analyte in the sample that can be quantitatively determined.

Method:

· Quantitation limit can be calculated using standard deviation response of blank samples or sample containing analyte close to quantitation limit and slope of calibration curve.

· It can be carried out using standard deviation of responses of blank samples or samples containing an analyte close to the detection limit and the slope of the calibration curve close to the detection limit.

QL= 10 δ /slope

Where

QL= Quantitation Limit

δ= S.D. of response

Slope= slope of calibration curve

Expression: Not specified

Acceptance Criteria: Not specified.

APVMA:

Definition: It may be defined as the lowest amount of the analyte in the sample that can be quantitatively determined with relevant precision.

Method:

· The LOQ may be estimated using preparing standard solutions at estimated LOQ concentration .

· The solution should be injected and analysed.

· The average response and the standard deviation (SD) of the results should be calculated and the SD should be less than 20%.

· If the SD exceeds 20%, a new standard solution of higher concentration should be prepared and the above procedure repeated.

LOQ = X + (10 x SD).

Where:

X= average response

SD= Standard deviation

Expression: Not specified

Acceptance Criteria: Not specified.

IUPAC:

Definition: Not defined.

Method: Not described.

Expression: Not specified.

Acceptance Criteria: Not specified.

Reference	Definition
ICH guideline⁵	“Validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose.” ICH Q2A: Includes documents on validation of analytical methods. ICH Q2B: Explain g proper guideline on validation of analytical scheme. ICHQ2R1: Q2A+Q2B
FDA⁶	“Process validation can be defined as establishing documented evidence that provides a high degree of assurance for specific process will constantly produce a product meeting its prearranged specifications and quality characteristics.”
USP⁷	“Validation is the process of contributing documented declaration that the method does what it is proposed to do”
WHO: GMP⁸	“It is of critical priority that attention is paid to validation of analytical methods, Automated system, cleaning procedure etc.”
ISO 16140-1⁹	“Endowment of the performance criteria for any method and outline the evidence that the predetermine performance criteria are successfully fulfilled.”
Health Canada¹⁰	“Verification inspection is required when a laboratory implements a properly validated, published or reference standard method. The laboratory should provide evidence, by organizing a secondary appraisal study, that must be able to of achieve the predetermined performance criteria for method.”
Japan pharmacopoeia¹¹	“The validation of an analytical method is the techniques of certifying that the analytical procedure selected for a test of pharmaceutical products is relevant for expected use.
APVMA (Australian pesticides and veterinary medicine and authority.)¹²	It is guidelines for the validation of analytical methods for active pharmaceutical ingredients, agricultural and veterinary products. It mainly includes the procedures involved in process is proper and it adhere with standards and compliance with the requirement specifications.”

Validation parameters	ICH(Q2A)	FDA	AOAC	JP	USP	IUPAC	APVMA	Ph. Eur.
Specificity	Yes	Yes	Yes	Yes	Yes	Yes	Yes	NO
Linearity	Yes	Yes	Yes	Yes	Yes	Yes	Yes	NO
Range	Yes	Yes	Yes	Yes	Yes	Yes	NO	NO
Accuracy	Yes	Yes	Yes	Yes*	No	Yes	NO	NO
Precision: Repeatability	Yes	Yes	Yes	Yes^#	No	Yes	Yes	NO
Precision: Intermediate Precision	Yes	Yes	Yes	No	No^$	Yes		NO
Precision: Reproducibility	Yes	Yes	Yes	Yes	No	Yes		NO
Detection Limit	Yes	Yes	Yes	No	Yes	NO	NO	NO
Quantitation limit	Yes	Yes	Yes	No	Yes	NO	NO	NO
Robustness	Yes	Yes	-	Yes	Yes	Yes	-	NO