'Validation of system Protocol for Students'.pptx

Validation of System

1. Dr. Muhammad Shahid Associate Professor Department of Biochemistry, UAF. Validation of Bioanalytical Methods

2. METHOD VALIDATION It is set of parameters/process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Generation of data Well defined & fully validated Method does- intended to do Quantitative measurement Reliable and reproducible

3. Analytical method validation Reliable and reproducible for the intended use . Fundamental parameters Accuracy Precision Selectivity Sensitivity Linearity Stability

4. Why Validation? Documenting Performance characteristics Acceptability of data – criteria Methods are Modified Modifications should be validated Suitable performance

5. Validation Full Validation Partial Validation Cross-Validation Pre-study Validation In-study Validation

6. Full Validation Developing and implementing a bioanalytical method for the first time. A new compound/drug entity. Revised assay is important if metabolites are added to an existing assay for quantification

7. Partial Validation one intra-assay accuracy and precision determination to a nearly full validation. Typical bioanalytical method changes : Method transfers between laboratories or analysts Analytical methodology (detection systems) Anticoagulant in harvesting biological fluid Matrix within species (human plasma to urine )

8. Sample processing procedures Species within matrix (e.g. rat plasma to mouse ) Relevant concentration range Instruments and/or software platforms Limited sample volume (e.g. pediatric study) Rare matrices Selectivity demonstration of an analyte in the presence of concomitant medications Selectivity demonstration of an analyte in the presence of specific metabolites

9. Cross-Validation Comparison of validation parameters two or more analytical methods within the same study or across different studies. An original method ( reference) Revised analytical method ( comparator). Interlaboratory reliability within a single study more than one site or lab, spiked matrix standards and subject samples Different analytical techniques (LC-MS vs. ELISA) in different studies.

10. In Study Validation Application of validated method for routine analysis Accuracy & precision should be monitored Method works satisfactorily QC sample in duplicate at 3 concentration Low , Medium & High QCs Should be incorporated in each assay run

11. Pre-study Validation Analytical method development and documentation Each Biological Matrix and Chemical species Selectivity Calibration curve & Linearity Accuracy, Precision, Recovery Stability of analyte Acceptance criteria Documentation

12. Analytical laboratory conducting Good Laboratory Practices (GLPs) Sound principles of quality assurance Standard Operating Procedures (SOPs) QC & Assurance All aspects of analysis Time of sample collection, reaches, results report

13. SOPs Record keeping, security and chain of sample custody Sample preparation Analytical tools Methods Reagents Equipment Instrumentation Procedures for quality control Verification of results

14. Process of Analytical method Developed, validated and used divided into Reference standard preparation Method development - assay procedure Routine analysis and acceptance criteria for analytical run and/or batch ( In Study validation)

15. REFERENCE STANDARD Highly purified compound, well characterized To provide accurate data Quality and purity Types of reference standards Chemical Nuclidic , radiolabel purity & chemical purity

16. Chemical reference standards 1. Certified reference standards (USP compendial (standards; No need characterization) Commercial Other materials of documented purity (identity, strength, quality and purity)

17. REFERENCE STANDARD Source Lot number Expiration date Certificates of analyses when available Internally or externally generated evidence of identity and purity

18. For quantitation . External standards internal standards External standards Analyzed on a separate chromatogram from the sample comparison of the peak area/height (HPLC or GC) or spot intensity (TLC) of the sample to that of a reference standard of the analyte of interest.

19. Internal standard Known purity No interference in the analysis Added to the sample mixture. Response ratio of Compound of interest to IS vs reference standard (HPLC or GC). 1. Complex sample preparation procedures, (multiple extractions) 2. Low concentration sample (sensitivity) 3. Wide range of concentrations expected

20. Method development & establishment phase Selectivity Calibration curve & Linearity Accuracy, Precision & Recovery Stability of analyte Acceptance criteria Documentation

21. Specificity/ Selectivity Ability to assess analyte in the presence of endogenous compounds Ability to separate analyte from degradation products, metabolites and co-administerded drugs

22. Selectivity Differentiate and quantify the analyte in the presence of other components. Blank samples - 6 sources. No interference- LLOQ If more than one analyte , no interference Reject Blank with significant interference > 10 % blank show interference additional blanks, > 10 % still show interference Modify method to eliminate interference

23. Calibration/Standard Curve Relationship b/w instrument response & concentrations Each analyte - same biological matrix A blank sample (matrix sample processed without IS) A zero sample (matrix sample processed with IS) 5 minimum standards (including LLOQ ) Expected concentration range in the study

24. Concentration-Response Simplest model ,Concentration-response relationship Selection of weighting and use of a complex Regression equation should be justified. Conditions to be met 20% deviation of the LLOQ 15% deviation of standards other than LLOQ 4/ 6 non-zero standards meet the above criteria LLOQ and the highest calibration standard 0.95 or more correlation coefficient

25. Sensitivity/Lower limit of detection ( LOD) Smallest conc. distinguishable from noise level Detected only, not quantified Lower Limit of Quantification (LLOQ) Twice the response of LOD Lowest standard on the Calibration curve 5 times the response compared to blank response Identifiable, discrete, and reproducible with a precision of 20% and accuracy of 80-120%

26. Quality Control Samples Low QC ( < 3X LLOQ) 2. Medium QC ( midway) 3. High QC ( 75-90% highest standard) Intra- & inter-day precision, accuracy, recovery & stability studies

27. Accuracy, Precision Determines the error Primary criteria for Quality Accuracy ( Trueness) Closeness of test results to the true value 3 QCs concentrations in range of calibration curve 3 determinations per concentration of QCs Deviation within 15% of the actual value Should not deviate by > 20% at LLOQ

28. Precision Closeness of individual measures of an analyte procedure is applied repeatedly to multiple aliquots 3 QCs concentrations in calibration range. 3 determinations per QC concentration. Should not exceed 15% of CV LLOQ should not exceed 20% of CV.

29. Precision Within-run intra-batch precision or repeatability Between-run inter-batch precision or repeatability time, different analysts, equipment , reagents, and laboratories.

30. Recovery Detector response about analyte added to and extracted from the biological matrix Compared to the true concentration of standard. Extraction efficiency need not be 100% Extent of recovery of an analyte and IS Consistent, precise and reproducible Compare 3 conc. (low, medium, and high QCs) with unextracted standards

31. Stability Storage conditions, Chemical properties of the drug Matrix & Container system Freeze and Thaw Stability 2. Short-Term Temperature Stability 3. Long-Term Stability 4. Stock Solution Stability 5. Post-Preparative Stability

32. Freeze and Thaw Stability 3 freeze and thaw cycles 3 LQC and HQC stored at the intended storage temperature for 24 hours, thawed unassisted at room temperature. Again refrozen for 12 to 24 hours, 2 times more If unstable,frozen at -70 ° C during 3 freeze/thaw

33. 2. Short-Term Temperature Stability Three LQC and HQC 4 to 24 hours ( room temp) & analyzed 3. Long-Term Stability Should exceed the time between the date of first and last sample analysis. 3 Low and High QCs compared with 1 st day long term stability testing

34. 4. Stock Solution Stability Analyte , Room temperature - 6 hours. Refrigerated or frozen - relevant period, Stability should be documented. Desired storage time Instrument response - fresh solutions.

35. 5. Post-Preparative Stability Stability of processed samples Resident time in the auto sampler Analyte Anticipated run time for the batch size original calibration standards.

36. Ruggedness/Reproducibility Studying the eventual effect of different sets of conditions on the method ( cross validation). How? Multiple chemists in multiple labs run samples. Results should be reproducible and can be compared to method precision. Result – Samples were run in 3 labs by 3 chemists on 3 different instruments. IF the results were nearly 100% Then the method showed Raggedness

37. Robustness A measure of the analytical procedure’s capability to remain unaffected by small but deliberate variations Should be performed during development of the analytical procedure and the data discussed and / or submitted. Effect observed, representative instrument output should be submitted. Conditions are pH Buffer Concentration Temperature % of Organic part of Mobile phase Column lot/ type

38. Documentation Validity- established & verified Assay Validation Report SOP & Good record keeping (Essential part) Data should be documented ( Note book) Protocols & SOPs : signed & dated Regularly updated, available for Audit & inspection

39. Documentation Analytical method Stability studies & supporting data Selectivity Accuracy, Precision, Recovery Linearity & LOQ (equations and weighting functions) Relevant data

40. Summary information Method development & establishment Analytical reports of routine sample analysis Other information applicable

41. Validation though Tedious but solves most post analytical problems Quality of data Consequences of invalid methods Amount of time & resources exceeds Wend your way efficiently through the validation maze & eliminate many of the problems common to inadequately validated analytical methods

42. THANKS