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Analytical Method Validation

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Analytical Method Validation

  1. 1. What is Validation? • Validation is defined as establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its pre-determined specifications and quality attributes.  Identification  Determination of impurities  Assay 8 January 2016 Analytical Method Validation 2
  2. 2. Why Validation? • The objective of any analytical measurement is to obtain consistent, reliable and accurate data. Validated analytical methods play a major role in achieving this goal. • Validation of analytical methods is also required by most regulations.l. 8 January 2016 Analytical Method Validation 3
  3. 3. Typical Analytical Procedures To Be Validated • Four most common 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 8 January 2016 Analytical Method Validation 4
  4. 4. Revalidation • Revalidation may be necessary in the following circumstances: • Changes in the synthesis of the drug substance • Changes in the composition of the finished product • Changes in the analytical procedure 8 January 2016 Analytical Method Validation 5
  5. 5. Parameters 1. Linearity and Range 2. Specificity 3. Precision 4. Accuracy 5. Limit of Detection 6. Limit of Quantitation 7. Robustness 8. System Suitability 8 January 2016 Analytical Method Validation 6
  6. 6. Characteristic Identification Impurities Testing Assay Quantitative Limit Accuracy _ + _ + Precision a. Repeatability _ + _ + b. Intermediate precision _ + _ + Specificity + + + + LOD _ _ + _ LOQ _ + _ _ Linearity _ + _ + Range _ + _ + 8 January 2016 Analytical Method Validation 7 -signifies that this characteristic is not normally evaluated + signifies that this characteristic is normally evaluated
  7. 7. 1. Linearity and Range LINEARITY • Ability to obtain test results that are directly (or by a well-defined mathematical transformation) proportional to the concentration of analyte in samples within a given range. (y = mx + c) • The following parameters should be determined:  correlation coefficient  y-intercept(c)  slope of the regression line(m) 8 January 2016 Analytical Method Validation 8
  8. 8. Determination of Linearity • For establishment of linearity, minimum 5 concentrations are recommended. • Linearity results should be established by appropriate statistical methods. 8 January 2016 Analytical Method Validation 9 y = 0.0868x + 0.019 R² = 0.9988 0 0.2 0.4 0.6 0.8 1 0 2 4 6 8 10 12
  9. 9. • Transformations are also acceptable and may include log, square root, or reciprocal (other transformations are acceptable) 8 January 2016 Analytical Method Validation 10 Conc. (µg/ml) Response 1 0.0625 2 0.25 3 0.562 4 0.922 5 1.562 y = 0.3671x - 0.4296 R² = 0.9515 -0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0 2 4 6
  10. 10. • If linearity is not attainable, a nonlinear model may be used. The goal is to have a model (whether linear or nonlinear) that describes closely the concentration-response relationship. • Acceptance criteria: Linear regression r2 > 0.95 8 January 2016 Analytical Method Validation 11 Conc. (µg/ml) √Response 1 0.25 2 0.5 3 0.75 4 0.96 5 1.25 y = 0.246x + 0.004 R² = 0.9982 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 2 4 6
  11. 11. RANGE 8 January 2016 Analytical Method Validation 12 • The range of an analytical procedure is the interval between the upper and lower levels of analyte(including these levels) that have been demonstrated with a suitable level of precision, accuracy, and linearity. • For assay tests, ICH requires the minimum specified range to be 80 to 120 percent of the test concentration. It also requires the range for the determination of an impurity to extend from the limit of quantitation or from 50 percent of the specification of each impurity, whichever is greater, to 120 percent of the specification
  12. 12. 8 January 2016 Analytical Method Validation 13 • Acceptable range having linearity, accuracy, precision. • For Drug Substance & Drug product Assay – 80 to 120% of test Concentration • For Content Uniformity Assay – 70 to 130% of test Concentration • For Dissolution Test Method – +/- 20% over entire Specification Range • For Impurity Assays – From Reporting Level to 120% of Impurity Specification for Impurity Assays – From Reporting Level to 120% of Assay Specification for Impurity/Assay Methods
  13. 13. 2. Precision • The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. • Sample prepared in six replicates on the same day and analysed as per the method. • The precision is reported in terms of %RSD 8 January 2016 Analytical Method Validation 14
  14. 14. Concentration Absorbance 10 µg/ml Mean 0.22 0.35 0.39 0.53 0.36 Less Variation More Variation High Precision Low Precision 8 January 2016 Analytical Method Validation 15 Concentration Absorbance 10 µg/ml Mean 0.28 0.31 0.29 0.30 0.29
  15. 15. • Precision may be considered at three levels: 8 January 2016 Analytical Method Validation 16 Precision Repeatability Intermediate Precision Reproducibility
  16. 16. 1. Repeatability • Repeatability expresses the precision under the same operating conditions over a short interval of time. • Repeatability should be assessed using a minimum of 9 determinations covering the specified range. 2. Intermediate Precision • Intermediate precision expresses variations within laboratories, such as different days, different analysts, different equipment, and so forth 3. Reproducibility • Reproducibility expresses the precision between laboratories. It is assessed by means of an inter- laboratory trial. (Defined as ruggedness in USP, ISO 17025) 8 January 2016 Analytical Method Validation 17
  17. 17. • Following parameters should be reported: a. Standard deviation b. Relative standard deviation (coefficient of variation) 8 January 2016 Analytical Method Validation 18 Concentration µg/ml Absorbance SD & % RSD 8 0.337 0.00041, 1.223% 0.348 0.341 12 0.575 0.0106, 1.815% 0.583 0.596 20 0.967 0.0091, 0.933% 0.985 0.978 0 0.2 0.4 0.6 0.8 1 1.2 4 8 12 16 20 24 8 8 8 12 12 12 20 20 20
  18. 18. 3. Accuracy • Closeness of agreement between the conventional true value / an accepted reference value and the value found. High Accuracy Less Accuracy (Less Precision) (High Precision) 8 January 2016 Analytical Method Validation 19
  19. 19. Determination of Accuracy Drug Substance a)application of analytical procedure to a reference material b) to compare the results c) accuracy may be inferred once precision, linearity and specificity have been established. Drug Product a)application of the analytical procedure to synthetic mixtures to which known quantities of the drug substance have been added b) to compare the results c) accuracy may be inferred once precision, linearity and specificity have been established. 8 January 2016 Analytical Method Validation 20 1. Assay
  20. 20. 2. Impurities (Quantitation)  Assessed on samples (drug substance/drug product) spiked with known amounts of impurities.  Accuracy should be assessed using a minimum of 9 determinations over a minimum of 3 concentration levels covering the specified range (e.g., 3 concentrations/3 replicates each of the total analytical procedure).  Accuracy should be reported as percent recovery by the assay of known added amount of analyte in the sample or as the difference between the mean and the accepted true value. 8 January 2016 Analytical Method Validation 21
  21. 21. 4. Limit of Detection & Limit of Quantitation LOD  Lowest amount of analyte in a sample that can be detected but not necessarily quantitated.  Estimated by Signal to Noise Ratio of 3:1. LOQ Lowest amount of analyte in a sample that can be quantified with suitable accuracy and precision. Estimated by Signal to Noise Ratio of 10:1. 8 January 2016 Analytical Method Validation 22
  22. 22. 4. Limit of Detection & Limit of Quantitation • Limit of Detection: • It is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated. • Limit of Quantitation: • It is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. 8 January 2016 Analytical Method Validation 23
  23. 23. Determination of LOD & LOQ Limit of Detection  Method  Based on visual evaluation  Based on standard deviation of response and slope LOD = 3.3 σ / Slope  Signal to noise ratio 2:1 or 3:1 Limit of Quantitation  Method  Based on visual evaluation  Based on standard deviation of response and slope LOD = 10 σ / Slope  Signal to noise ratio 10:1 8 January 2016 Analytical Method Validation 24
  24. 24. 8 January 2016 Analytical Method Validation 25
  25. 25. 6. Specificity • The ability to detect the analyte of interest in the presence of interfering substances (typically impurities, degradants, matrix). 1. Identification • Suitable identification tests should be able to discriminate between compounds of closely related structures which are likely to be present. • The discrimination of a procedure may be confirmed by obtaining positive results from samples containing the analyte, coupled with negative results from samples which do not contain the analyte. • 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. 8 January 2016 Analytical Method Validation 26
  26. 26. 2. Assay and impurity test: a. Impurities are available • For the assay , this should involve demonstration of the discrimination of the analyte in the presence of impurities and/or excipients. • This can be done by spiking pure substances with appropriate levels of impurities and/or excipients and demonstrating that the assay result is unaffected by the presence of these materials. • 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. 8 January 2016 Analytical Method Validation 27
  27. 27. b. Impurities are not available • If impurity or degradation product standards are unavailable, specificity may be demonstrated by comparing the test results of samples containing impurities or degradation products to a second well-characterized procedure e.g. pharmacopoeial method or other validated analytical procedure. • As appropriate, this should include samples stored under relevant stress conditions: light, heat, humidity, acid/base hydrolysis and oxidation. 8 January 2016 Analytical Method Validation 28
  28. 28. 7. Robustness • The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. • If measurements are susceptible to variations in analytical conditions, the analytical conditions should be suitably controlled or a precautionary statement should be included in the procedure, such as: • Use solution within 24 hours • Maintain temperature below 25 degrees 8 January 2016 Analytical Method Validation 29
  29. 29. • In the case of liquid chromatography, examples of typical variations are:  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 • In the case of gas-chromatography, examples of typical variations are:  different columns (different lots and/or suppliers)  temperature  flow rate 8 January 2016 Analytical Method Validation 30
  30. 30. 8. System Suitability • System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such. • Determination: repeatability, tailing factor (T), capacity factor (k’), resolution (R), and theoretical Plates (N) 8 January 2016 Analytical Method Validation 31
  31. 31. System Suitability Requirements Parameters Recommendations K’ In general k’ ≥ 2.0 R R >2, between the peak of interest and the closest potential interferent (degradant, internal STD, impurity, excipient, etc…) T T ≤ 2 N In general N > 2000 Repeatability RSD ≤ 2.0% (n ≥ 5) 8 January 2016 Analytical Method Validation 32
  32. 32. Conclusion • When the method is properly validated consistent, reliable and accurate results are obtained. Also, Validation of analytical methods is also required by regulations. Hence it is very important to validate any analytical method that has been developed. 8 January 2016 Analytical Method Validation 33
  33. 33. 8 January 2016 Analytical Method Validation 34

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