Analytical Method Validation basics by Dr. A. Amsavel
- 1. Analytical Method Validation- Basic Dr. A. Amsavel
- 2. Analytical methods are used to test and ensure the Quality of Drug substances / Drug products Analytical methods are followed from Official Pharmacopeia or developed by users.Pharmacopeia or developed by users. How can assure the analytical methods are suitable for it purpose. Hence Analytical Method has to be validated or confirm its suitability?
- 3. Method validation is the process of planned and systematic study to confirm that it is suitable for its intended use. Method is reliable, accurate & reproducible Pharmaceutical Industries governed by rules & regulations. Method validation is mandatory as per regulatory agencies.
- 4. Guidance for Method Validation CDER : “Analytical Procedures and Method Validation” Draft guidance August 2000 ICH: Validation Of Analytical Procedures: Text And Methodology Q2(R1) 2005.Text And Methodology Q2(R1) 2005. USP 41: General Chapter <1225> Validation of Compendial Methods
- 5. Four most common types of analytical procedures: ◦ Identification tests - Eg FTIR spectrum ◦ Quantitative tests for impurities: Eg. 10ppm to 2%◦ Quantitative tests for impurities: Eg. 10ppm to 2% ◦ Limit tests for the control of impurities: Eg < 100ppm ◦ Quantitative tests of the active substance - Assay / purity : Eg 99-101%
- 6. Validation Parameters/characteristics Accuracy Precision Repeatability Intermediate Precision ICH Q2R1 Intermediate Precision Specificity Linearity Range Detection Limit Quantitation Limit
- 7. Analytical procedure Characteristics Identification Tests for impurities Assay Quantitative Limit test Accuracy No Yes No Yes Precision- Repeatability No Yes No Yes Intermediate precision No Yes No YesIntermediate precision No No Specificity Yes Yes Yes Yes Limit of Detection No Yes Yes No Limit of Quantitation No Yes No No Linearity No Yes No Yes Range No Yes No Yes Robustness * No Yes No Yes
- 8. Specificity Ability of an analytical method to measure the analyte free from interference from components. Example: • HPLC/ GC: well separation of all peaks. Drug from Impurities Analytical Method Validation • HPLC/ GC: well separation of all peaks. Drug from Impurities • Peak purity- No overlapping of peaks / co elution
- 9. Analytical Method Validation Accuracy: 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 accepted reference value and the value found.accepted reference value and the value found. The accuracy is closeness between the experimental value and true value.
- 10. Assay: Test the assay at 80%, 100% & and 120% Acceptance criteria: Recovery : 99.0-101.0% % RSD should be less than 2.0. Analytical Method Validation Impurities -Related substances: Test impurities at 50% - 150% to the limit value. Acceptance criteria: Recovery 90.0% to 110.0% % RSD should be less than 10.0%.
- 11. Linearity The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. Linearity :should be proportional response to analyte concentration. Experiment: Detection level to 150% to limit Acceptance criteria: Plot Linear Regression (y = mx +c) Linear regression r2 > 0.98
- 12. Range: The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity.accuracy and linearity. ◦ For Drug Substance & Drug product Assay 80 to 120% of test Concentration ◦ For Impurities: LOQ Level to 120% of Limit
- 13. Limit of Detection Lowest amount of analyte in a sample that can be detected Limit of Quantitation: Lowest amount of analyte in a sample that can be quantified Analytical Method ValidationAnalytical Method Validation detected • Estimated by Signal to Noise Ratio of 3:1 or • 3.3 x Standard Deviation / Slope with suitable accuracy and precision. • Estimated by Signal to Noise Ratio of 10:1. or • 10 x Standard Deviation / Slope
- 14. Analytical Method ValidationAnalytical Method Validation LOD and LOQLOD and LOQ
- 15. Analytical Method ValidationAnalytical Method Validation LOD and LOQLOD and LOQ 3.3X Standard Deviation / Slope Ybl LOD LOQ Statistical estimate of LOD & LOQ LOD = 3.3 Sbl / b LOQ = 10 Sbl / b Y = b X + a
- 16. Precision: Closeness of agreement (degree of scatter) between a series of measurements obtained from multiple testing under the prescribed conditions. Precision to be established : Repeatability, Intermediate precision and Reproducibility. The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation
- 17. Repeatability Repeatability expresses the precision under the same operating conditions over a short interval of time. Intermediate precision Intermediate precision expresses within-laboratories variations: Intermediate precision expresses within-laboratories variations: different days, different analysts, different equipment, etc. Reproducibility Reproducibility expresses the precision between laboratories
- 18. Test Day 1 Day 2 Analyst-1 Analyst-2 1 100.3 99.8 100.4 99.6 2 100.5 99.9 100.2 99.9 3 100.1 98.9 100.3 98.9 4 100.3 99.4 99.8 100.14 100.3 99.4 99.8 100.1 5 100.5 99.7 99.9 99.7 6 100.7 99.6 99.6 99.8 Mean 100.40 99.55 100.03 99.67 % RSD 0.21 0.36 0.31 0.41 Sdv 0.21 0.36 0.31 0.41
- 19. Robustness: Analytical procedure is capable to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. Examples of typical variations in LC pH of mobile phase ( 5.0±0.2) Mobile phase composition (±10 % of solvent) Different columns (different lots and/or suppliers) Temperature ( 30±° 2C) Flow rate (1.5 ml ±0.1ml) Comparison results under differing conditions with under normal
- 20. www.fda.gov www.fda.gov/cder/ www.usp.org www.usp.org www.ich.org www.pharmweb.net