Method Validation

1. Method Validation

2. Validation Defination Establishing documentary evidence which provides a high degree of assurance that specification process will consistently produce a product meeting its predetermined specifications and quality attributes.

3. Methods need to be validated or revalidated • before their introduction into routine use • whenever the conditions change for which the method has been validated, e.g., instrument with different characteristics • whenever the method is changed, and the change is outside

4. Validation parameters evaluated (Q2R1) • Specificity • Linearity • Accuracy and range • Precision • Repeatability • Intermediate precision • Robustness

5. Specificity • Specificity Is ability to measure analyte Of interest accurately & specifically in presence of other componants that could be expected to be present in sample matrix.

6. Linearity • Minimum of 5 concentrations is recommended. • Range studied 0 to 150% of nominal (7 solutions, including a ‘blank’ solution) • Linearity experiments conducted in the presence and absence of excipients • Determine equation of the line, correlation coefficient, 95% confidence interval of the slope and the intercept. Verify that the 95% confidence interval of the intercept includes zero, or that the result is insignificant in the context of the experiment • Check that the response for both experiments is the same – calculate the slopes as a % of each other • Typically results of 98 to 102% would be considered acceptable

7. Range • The following minimum specified ranges should be considered: For the assay of a drug substance or a finished (drug) product: normally from 80 to 120 percent of the test concentration; • For content uniformity, covering a minimum of 70 to 130 percent of the test concentration, unless wider more appropriate range, based on the nature of the dosage form (e.g., metered dose inhalers), is justified;- • For dissolution testing: +/-20 % over the specified range;

8. Precision - Repeatability • One analyst conducting multiple replicates of the same sample, on the same day using the same equipment, and applying the method as written. • Determine the mean, the range, the standard deviation and the relative standard deviation. • 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)

9. Precision – Intermediate precision • A second operator conducts the ‘repeatability experiment’ on a different day using different equipment where possible. • Determine the individual data and overall mean, range, standard deviation and relative standard deviation. • Consider testing statistically that there is no significant difference between operators before combining the data set.

10. System Suitability • System suitability is checking of systemto ensure system performance before or durring the analysis of unknown • Plate count (N), Tailing Factor (T) , Resolution (R) & Reproducibilty (RSD) are measured for rplicate injections of sample • If RSD specification is less than 2%,Five replicate injections should be used. • If RSD specification is more than 2%,Six replicate injections should be used. • System suitability should be done Before,Durring,after the analysis.

11. Accuracy • Accuracy is a measure of Exactness of analytical method • It is measured as % analyte recovered by assay ,by spiking samples • Data from minimum Three conentrations & three replicate of each concentration to be reported.

12. Limit of Detection • The limit of Detection (LOD) is lowest conc of analyte in the sample that can be detected & Not quantified. • It can be measured by A signal-to-noise ratio . • A signal-to-noise ratio should be between 3 or 2:1 is generally considered acceptable for estimating the detection limit.

13. Limit of Quantitation • The limit of Quantitation (LOQ) is lowest conc of analyte in the sample that can be determined (quantitated). • It can be measured by A signal-to-noise ratio . • A signal-to-noise ratio should be between 10:1 is generally considered acceptable for estimating the detection limit.

14. Robustness • A number of attributes were evaluated prior to validation using an experimental design approach: • Injector temperature • Detector temperature • Initial temperature • Ramp rate • Hold time • Split flow • Column head pressure

15. A validation report should be prepared that includes: • objective and scope of the method (applicability, type) • type of compounds and matrix • detailed chemicals, reagents, reference standards and control sample • preparations • procedures for quality checks of standards and chemicals used • safety considerations • method parameters • critical parameters indicated from robustness testing • listing of equipment and its functional and performance requirements, e.g. cell

16. A validation report should be prepared that includes: • dimensions, baseline noise, column temperature range • detailed conditions on how the experiments were conducted, including sample preparation • statistical procedures and representative calculations • procedures for quality control in the routine (e.g., system suitability tests) • representative plots, e.g. chromatograms, spectra and calibration curves • method acceptance limit performance data • the expected uncertainty of measurement results • criteria for revalidation • person who developed and initially validated the method • summary and conclusions

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