Preamble:
Analytical method validation (AMV) is a prominent part of GLP in Pharma industry. It is mandatory to validate/verify an analytical method in a laboratory before it's intended use. Every Regulation recommends a systematic and dynamic way to validate the analytical methods in a chronological order.Currently the ICH (tripartite) guideline Q2(R1) is preffered. However other Regulation like as PIC/S , WHO GMP,ISO,AOAC-I,IUPAC and Pharmacopoeia's like USP, BP,EP etc recommends more or less the same methodology.Verification or revalidation should be performed when relevant, for example, when there are changes in the process for synthesis of the drug substance;changes in the composition of the finished product; changes in the analytical procedure; when analytical methods are transferred from one laboratory to another; or when major pieces of equipment instruments change.Justification should be provided when non-pharmacopoeial methods are used if pharmacopoeial methods are available. Justification should include data such as comparisons with the pharmacopoeial or other methods.
"Validation of an analytical method is a process by which it is established by laboratory studies that the performance characteristics of the analytical method meets the intended analytical applications."
1. Why the Verification/Validation is required for Non Compendial/Compendial methods?.
A majority of analysts often confuses when to perform the Verification and when to Validate an analytical method. As mentioned in USP 37 NF 32 Appendix name < Validation of Compendial methods>
" Users of analytical methods mentioned in USP and NF are not required to validate the accuracy and reliability of these methods but merely Verify their Suitability under actual condition of use".
The aforementioned lines are self explanatory. In a more legal way as recommended in 21 cfr Part 211.194 a(2)
"The suitability of all testing methods used shall be verified under actual conditions of use."
Validation procedure:
As per USP 37 NF 32 analytical validation is categorized in four parts.
Category I: In this Quantification technics are validated eg. Assay.
Category II: In this Qualitative technics are validated eg. Impurities.
Category III: Performance characteristics are validated eg. Dissolution.
Category IV: This is for Identification validation .
Characteristics that should be considered during validation of analytical methods(HPLC) include
Specificity
Linearity
Range
Accuracy
Precision
Detection limit
Quantitation limit
Robustness.
Specificity: Specificity of an analytical methods is its ability to assess unequivocally the analyte in the prescence of components that may be expected to be present such as degradation product, matrix components, impurities etc.Some communities e.g. AOAC-I, IUPAC prefer the term selectivity (The methods that are completely selective) in place of Specificity however both have the almost same meaning. Preferrebly single dilution at 100% concentration level and 6 replicates are performed and % RSD of Retention time is NMT 0.5%.
Note: It is acceptable to use a “spiked” placebo where a known quantity or concentration of a Reference material is used.
Linearity : "Linearity of an analytical method is it's ability to elicit test results that are directly or by a well defined mathematical transformation proportional to the concentration of analyte within a given range".In the case of assay five dilutions and three replicate of each from 80% to 120% concentration level and for impurity LOQ to 120% in the same manner.
The correlation coefficient, y-intercept, slope of the regression line and residual sum of squares should be submitted. %RSD < 2.0 ,Correlation coefficient = close to 1, Confidence level for Residuals and calibration curve should be NLT 97.0.
Range:" Range of an analytical method is the interval between upper and lower level of analytes that have been demonstrated to be determined with acceptable level of Accuracy, Precision and Linearity".
For assay two dilutions at concentration level 80% and 120% and for Impurity LOQ and 120% with 6 replicates of each should be prepared/performed. %RSD NMT 2.0%.
Accuracy : Accuracy means, "The correctness of a measurement". The concordance between true and obtained value. Three dilutions at concentration level LOQ, 100% and 120% are prepared and 3 replicate of each is carried out. Recovery from 100% concentration level should be within 98.0% to 102.0%
Precision: " This is the degree of agreement among individual test results obtained when the procedure is applied repeatadily to the multiple samplings of a homogeneous sample". This is of three type.
(1) Repeatability: Single dilution at 100% concentration level and 6 replicate of the same, % RSD NMT 2.0 and confidence level NLT 97.5.
(2) Intermediate Precision: This is also known as Ruggedness. Analysis of a homogeneous sample on different days, different analysts, different equipments but within the same laboratory. Assay/Related substance is analysed by three different analysts. Variation in assay NMT 2.0% and in Related substance NMT 0.05%(individual) , NMT 0.10% (total) is recommended.
(3) Reproducibility:Analysis of a homogeneous sample on different days, different analysts, different equipments and in different laboratories. Assay/Related substance is analysed by three different analysts. Variation in assay NMT 2.0% and in Related substance NMT 0.05%(individual) , NMT 0.10% (total) is recommended.
Detection limit: " The lowest amount of analyte in a sample that can be detected but not necessarily quantitated under stated experimental conditions". It should be noted that Detection limit of an analyte varies slightly from one instrument to another. There are three ways to find the detection limit.
(1) Based on visual observation: This is done by checking the lowest possible level at which analyte can be detected without any abnormality. Three replicate and % RSD NMT 2.0.
(2) Based on signal to noise:
This approach can only be applied to analytical procedures which exhibit baseline noise.Determination of the signal-to-noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected. A signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for estimating the detection limit.
(3) Based on standard deviation of the response and slope: This is obtained as following
3.3 X Standard deviation of the Response
The slope of the calibration curve
Quantitation limit:
It should be noted that Quantitation limit of an analyte varies slightly from one instrument to another. There are three ways to find the Quantitation limit.
(1) Based on visual observation:This is done by checking the lowest possible level at which analyte can be Quantitated without any abnormality (generally two times the LOD). Three replicate and % RSD NMT 2.0.
(2) Based on signal to noise:
This approach can only be applied to analytical procedures which exhibit baseline noise.Quantitation of the signal-to-noise ratio is performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably quantified.Sigal to noise ratio between 10:1 is generally considered acceptable for estimating the Quantitation limit.
(3) Based on standard deviation of the response and slope:
This is obtained as following
10X Standard deviation of the Response
The slope of the calibration curve
Robustness: "Robustness of an analytical method is it's ability to remain unaffected by small but deliberate variations in method parameters."
Some deliberate variations are as following.
Stability of analytical solution.
Extraction time .
Influence of varitions of pH in a mobile phase.
Influence of variations in mobile phase composition.
Different columns (different lots and/or suppliers).
Temperature.
Flow rate.
In the case of gas-chromatography
Different columns (different lots and/or suppliers).
Temperature.
System suitability: SST is an integral part of the AMV. This is carried out as per the corresponding testing method under validation.
