1. Kyiv, 2005-10-061 TRAINING WORKSHOP ON PHARMACEUTICAL QUALITY, GOOD MANUFACTURING PRACTICE & BIOEQUIVALENCE Validation of Analytical Methods Used For Bioequivalence Studies Presented by John Gordon, Ph.D. Consultant to WHO e-mail: john_gordon@hc-sc.gc.ca

2. Kyiv, 2005-10-062 Bioanalytical Method Validation Quantitative determinations of drugs in biological samples, such as blood or plasma, play a significant role in evaluation and interpretation of bioequivalence data.

3. Kyiv, 2005-10-063 Bioanalytical Method Validation Essential to use a well-characterised and fully validated analytical method to yield reliable results.

4. Kyiv, 2005-10-064 Development / Use of a Bioanalytical Method Reference standard preparation Method procedure development Application of validated method to routine analysis

5. Kyiv, 2005-10-065 Reference Standards Calibration standards / Quality control samples Authenticated reference –Known identity –Known purity Reference standard should be identical to analyte, if possible

6. Kyiv, 2005-10-066 Method Development Chromatographic assays –e.g., gas chromatography, liquid chromatography Ligand-binding assays –e.g., radioimmunoassay (RIA)

7. Kyiv, 2005-10-067 Bioanalytical Method Validation Method Validation should include –Accuracy –Precision –Sensitivity –Specificity –Recovery –Stability

8. Kyiv, 2005-10-068 Bioanalytical Method Validation Accuracy Closeness of determined value to the true value. The acceptance criteria is mean value  15% deviation from true value. At LOQ, 20% deviation is acceptable.

9. Kyiv, 2005-10-069 Bioanalytical Method Validation Precision The closeness of replicate determinations of a sample by an assay. The acceptance criteria is  15% CV. At LOQ, 20% deviation is acceptable.

10. Kyiv, 2005-10-0610 Bioanalytical Method Validation Sensitivity The limit of quantitation (LOQ) is the lowest concentration which can be measured with acceptable accuracy and precision.

11. Kyiv, 2005-10-0611 Bioanalytical Method Validation Calibration Curve Sufficient number of calibration standards must be employed –Typically 6 – 8 non-zero standards –Blank sample –Zero sample –Lower Limit of Quantification (LLOQ) Goodness of fit statistics

12. Kyiv, 2005-10-0612 Bioanalytical Method Validation Specificity (selectivity) Ability of the method to measure only what it is intended to measure in the presence of other components in the sample. Blank samples of the biological matrix should be tested for interfering peaks.

13. Kyiv, 2005-10-0613 Bioanalytical Method Validation Recovery The extraction efficiency of an analytical process, reported as a percentage of the known amount of an analyte carried through the sample extraction and processing steps of the method. Recovery does not have to be 100%, but the extent of recovery of an analyte and of the internal standard should be consistent.

14. Kyiv, 2005-10-0614 Bioanalytical Method Validation Stability Stability of the analyte in the biological matrix –Sample collection –Sample storage –Sample analysis

15. Kyiv, 2005-10-0615 Bioanalytical Method Validation Stability Freeze-thaw stability Short-term stability Long-term stability Post-preparation stability Stock solution stability

16. Kyiv, 2005-10-0616 Bioanalytical Method Validation Dilution integrity Confirm ability to dilute samples above Upper Limit of Quantitation (ULOQ) Accuracy and precision

17. Kyiv, 2005-10-0617 Bioanalytical Method Validation Additional considerations for ligand- binding assays Cross-reactivity Matrix effects Concentration-response relationship

18. Kyiv, 2005-10-0618 Clinical Sample Analysis Single determination of calibration standards Calibration curve for each analytical run Extrapolation below or above calibration range not acceptable

19. Kyiv, 2005-10-0619 Clinical Sample Analysis Quality control (QC) At least three concentrations –Low concentration –Midrange concentration –High concentration Duplicate determinations 4 out of 6 QC samples must be  15% of nominal value; two outside cannot be at the same concentration

20. Kyiv, 2005-10-0620 Clinical Sample Analysis Repeat sample analysis Clear SOP and acceptance criteria Reasons for re-analysis –Analytical issues –Problematic pharmacokinetic fit Explanation for missing samples

21. Kyiv, 2005-10-0621 Standard Operating Procedures Complete written set of SOPs –Quality control and assurance –All aspects of analysis should be covered including Record keeping Security Chain of sample custody Sample preparation Analytical tools Procedures for quality control and verification of results

22. Kyiv, 2005-10-0622 Full / Partial Validation Full validation –During method development Partial validation –Transfer between laboratories –Change in instrument / software –Change in anticoagulant –Minor changes in sample processing procedures

23. Kyiv, 2005-10-0623 Bioanalytical Method Validation Useful references: FDA Guidance for Industry Bioanalytical Method Validation (May 2001) (http://www.fda.gov/cder/guidance/4252fnl.pdf) Published Workshop Reports Shah, V.P. et al, Pharmaceutical Research: 1992; 9:588-592 Shah, V.P. et al, Pharmaceutical Research: 2000; 17: 1551-1557