What is Analytical Method Validation

1. What is Analytical Method Validation ?

2. Analytical Method validation?? USP 13(1996?) - ??? ??? ???? ?? ???? ?? ???? ??? ??? ???? ????? ????? ???? ?? ??? ?? ?? ???? ???? ???? ???? ??, ???? ??? ??? ??. JP(1996?) - ??? ???? ??? ??? ???? ???? ???? ???? ??? ????? ?. - ?, ???? ??? ??? ?? ??? ????? ??? ??? ???? ??(??)?? ?? ????? ???? ???.

3. ?? ? ???? ??? ? ???? ??? ? ???? ???? ?? ? ???? ??? ??? ??? ? ???? ???? ? ??? ???? ??? ??? ??? ?

4.

5. Method Validation Process

6. ??? ???? ????? ???? ????? ??? ??? ??? ??? ? ??? ?????? ??? ?? tailing? ???? ?? ? ?? ?? ???????? ????? ???? ??? ???? ??? HPLC? 80~90%? ?? ???????? ???? ????, ??? ???? ????? ???? ????? ??? ??? ??? ??? ? ??? ?????? ??? ?? tailing? ???? ?? ? ?? ?? ???????? ????? ???? ??? ???? ??? HPLC? 80~90%? ?? ???????? ???? ????,

7. ????? ?? ??? ??? ???? ???? ???? ???? ?? ??? ??? ????, ?????? ???? ??? ?? ?? ? ?????. ????? ?? ??? ??? ???? ???? ???? ???? ?? ??? ??? ????, ?????? ???? ??? ?? ?? ? ?????.

8. ????? ?? ??? ??? ???? ???? ???? ???? ?? ??? ??? ????, ?????? ???? ??? ?? ?? ? ???? ?? ?? ????? ?? ??? ??? ???? ???? ???? ???? ?? ??? ??? ????, ?????? ???? ??? ?? ?? ? ???? ?? ??

9. ?? ?? ?? ?? ?? ??? AcCN? ?? ??? ???? ??? ??? ?? ??? ??? ?? ??? ?? ???? UV???? ?? ?? ? ? ????., ? ???? ?? ????? MeOH, THF ??? ? ??? ??? ?? ?? ?? ???? ???? ?? ?? ????? ??? ?? ?????.?? ?? ?? ?? ?? ??? AcCN? ?? ??? ???? ??? ??? ?? ??? ??? ?? ??? ?? ???? UV???? ?? ?? ? ? ????., ? ???? ?? ????? MeOH, THF ??? ? ??? ??? ?? ?? ?? ???? ???? ?? ?? ????? ??? ?? ?????.

10. Particle size? 3~20micron?? ???? ??? Particle size? ??? ???? ??? ??? ??? ??? ?? ??? ?? ??? ??? ??? ?? ?? ?? ???? 5um? ???? ???? ??? ????,.Particle size? 3~20micron?? ???? ??? Particle size? ??? ???? ??? ??? ??? ??? ?? ??? ?? ??? ??? ??? ?? ?? ?? ???? 5um? ???? ???? ??? ????,.

12. ???? HPLC condition System : Dionex ChromelonTM Chromatography Data System with P680 pump and UVD 340U detector Column : Shiseido CAPCELL PAK C18 MG 5 ?m (4.6 x 150 mm), Mobile Phase : Acetonitrile � MeOH- 0.1% acetic acid (10:5:85) Flow rate : 0.5 ml/min Column Heater : 20? Wavelength : 220, 250, 260, 280 nm Chromatographic Run Time: 25.0 min HPLC ??? ? ??? ???? ? ???? ??? ??? ?? ??? ????? ?? ??? ??? ??(Isocratic elution)? ?? ? ???? ????? ?? ????? ?? ?? ?? ??? ???? ??? ???? ?? ?? ??? ??? ??? ??? ?????? ?? ?? ??, ?? ??? ??? ??? ??? ???? ???? ?? ???? ??? ??(Gradient elution)? ????. HPLC ??? ? ??? ???? ? ???? ??? ??? ?? ??? ????? ?? ??? ??? ??(Isocratic elution)? ?? ? ???? ????? ?? ????? ?? ?? ?? ??? ???? ??? ???? ?? ?? ??? ??? ??? ??? ?????? ?? ?? ??, ?? ??? ??? ??? ??? ???? ???? ?? ???? ??? ??(Gradient elution)? ????.

13. ??? ?? ????? ??? ???? ??? ?? ???? ??? ???? ?? ???? ?? ???? ??? ?????. ?? ??? ???? ?? ???? ????? ?? ???? ODS-column? ?/AcCN ???? ???? ?? ??????. ?? ??? ????? ?? ?? ???? ????? ??? phosphoric acid? acetic acid, formic acid? ???? ?????? ?? ???? ????? ?? ????? ???? ???? UV???? ?? ?? ???? ???? ????? ?? ? ????. ??? ?? ????? ??? ???? ??? ?? ???? ??? ???? ?? ???? ?? ???? ??? ?????. ?? ??? ???? ?? ???? ????? ?? ???? ODS-column? ?/AcCN ???? ???? ?? ??????. ?? ??? ????? ?? ?? ???? ????? ??? phosphoric acid? acetic acid, formic acid? ???? ?????? ?? ???? ????? ?? ????? ???? ???? UV???? ?? ?? ???? ???? ????? ?? ? ????.

14. ????? ??? coptisine,palmatine,berberine? ????(??,???)? ????? peak? ??? ???? hesperidin? ??? ??? ???? ??? ??? ??? ??? ??? ????? ?? ??? ??? ?? ??? ???? ??? ? ?????.????? ??? coptisine,palmatine,berberine? ????(??,???)? ????? peak? ??? ???? hesperidin? ??? ??? ???? ??? ??? ??? ??? ??? ????? ?? ??? ??? ?? ??? ???? ??? ? ?????.

16. Analytical Method Validation ?? Identification of Sources and Quantitation of Potential errors Determination if Method is Acceptable for Intended Use Establish Proof that a Method Can be Used for Decision Making Satisfy FDA Requirements

17. What are the Benefits of Analytical Method Validation? Regulatory Compliance Assurance that Test data from Methods are Reliable Establishment that Test Data are Reproducible, Accurate, Specificity�. Analytical Method Validation? ??? ??? ???? ??? ?? ???? ??? ???? ???? ???,???,????? ??? ?? ???? ???? ??? ???? ??? ? ? ????.Analytical Method Validation? ??? ??? ???? ??? ?? ???? ??? ???? ???? ???,???,????? ??? ?? ???? ???? ??? ???? ??? ? ? ????.

18. GMP Validation Parameters Accuracy Specificity Sensitivity Reproducibility 21 CFR 211.165 (e) GMP-good manufacturing practice ? ??? ??? ?? ?? ?? ??? ????.GMP-good manufacturing practice ? ??? ??? ?? ?? ?? ??? ????.

19. FDA Validation Parameters Accuracy Precision Linearity (& Range) Specificity (& Determination Limit) Recovery Ruggedness 1987 FDA Guidelines

20. ICH/USP Validation Requirements & Parameters Specificity Linearity Range Accuracy Precision Repeatability Intermediate Precision Reproducibility Limit of Detection Limit of Quantitation

21. Guideline for Method Validation USP Chapter <1225> Validation of Compendial Methods ICH Guidelines Q2A, Text on Validation of Analytical procedures (March 1995) Q2B, Validation of Analytical Procedures: Methodology (May 1997) ICH Guidelines(International Conference on Harmornization)?? ??? ??? guideline ??? International Conference on Harmornization ??? ??? guideline ???International Conference on Harmornization ??? ??? guideline ???

22. Validation Parameter

23. Linearity ???? ???? ??? ??? ?? ?? ?????? ?????(????)? ? ?? ??? ??? ???? ????? ???? ? � A minimum of 5 concentrations nomally be used . -ICH � R2 > 0.99 for chromatographic method ? R2 (coefficient of determination) ? ????? ?? ?? ???? ?????? ????? ?? ?? ??? ????? ??? ??? ??? ?? ?, R2? 0.99?? ??? ?? ??? ???? ????? ?? ???? ???? (???)? 99% ???.

24. ??? ????? ??? ??? ???? ? ????? ??(???) ??. ????? ???: ????(SD) ?? ??? ????(STDEV)? ?? ? 3 concentrations ?? 3?? ??. ? RSD (Relative Standard Deviation) < 2% RSD= (????/??? ?)x100 ? Intermediate Precision: ????? ?? ?? ??, ?? ???, ?? ?? ?? ??? ??? ??? ??? ???? ?. (????? 2%???? ??) ? Reproducibility expresses the precision between laboratories (collaborative studies, usually applied to standardization of methodology)- USP? ??? Ruggedness? ??? ??. Precision

25. � ???(???)? ??? ??? ??? ?? ???? ???? ???? ?? ??. ? ?? ??? ????? ?? ? ? ???? ??? ?? ??? ?? ? ????. ? ????: ??? ??? ?? ???? ?? ? ? ??? ?? ???? ?????? ?????. Accuracy

26. Specificity ??? ???? ???, ???, ?????? ?? ??? ?? ?? ????? ??? ? ?? ??. Sensitivity ??? ????? ??? ? ?? ???? ??? ???? calivration curve? ??? ????. Range ??? ???? ??? ?? ???? ??? ????? ?? ??? ?? ???? ????? ???? ?? ?? ??(??). Specificity / Sensitivity/ Range

27. ?????? ??? ???? ?????? ??? ??? ??? ?? ????? ??? ??????? ??? ? ? ?? ??? ??. Based on Signal-to-Noise Acceptance criteria = 3:1 LOD = 3.3X(SD/Slope) S = slope of calibration curve SD : standard deviation of the respons LOD (Limit of detection)

28. ????? ? ? ?? ?? ?????? ?????? ??? ??? ??? ?? ???? ? ????? ??? ???? ?????? ??? ????? ???? ??? ???? ???? ??? ??? ??? ??? ? ?? ??? ? ?? ??? ????? ??. Based on Signal-to-Noise Acceptance criteria = 10:1 LOD = 10X(SD/Slope) S = slope of calibration curve SD : standard deviation of the respons LOQ (Limit of quantitation) ?? LOD? LOQ? signal-to-noise ratio? ?? ?????(?; LOD? S/N ? 2:1 ?? 3:1??, LOQ? 10:1 ??) ????? signal? baseline?? peak? ??? peak-to-peak noise? ????, noise? blank injection? ?? ?????.. ?? ????? baseline, noise? ??? ?? ??, ?? trace level?? ?? ?????. ??? International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use(ICH) Q2B Methology guideline??? analytical method validation? ?? ??? ??? ?????. ICH Q2B guideline??? LOD, LOQ ???? visual noninstrumental methods and limit calculation ??? ??? option? ?????, ??? ?? ???? ????. LOD = 3.3(s/S)LOQ = 10(s/S) s= standard deviation of the responseS=slope of the calibration curve ?? ????? ?? ???? ???? ??? s, S? ??? ??? ???? ???, LOD? LOQ? ?? ??, ??? ??? ???? ??? ???? ????. ?? LOD? LOQ? signal-to-noise ratio? ?? ?????(?; LOD? S/N ? 2:1 ?? 3:1??, LOQ? 10:1 ??) ????? signal? baseline?? peak? ??? peak-to-peak noise? ????, noise? blank injection? ?? ?????.. ?? ????? baseline, noise? ??? ?? ??, ?? trace level?? ?? ?????. ??? International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use(ICH) Q2B Methology guideline??? analytical method validation? ?? ??? ??? ?????. ICH Q2B guideline??? LOD, LOQ ???? visual noninstrumental methods and limit calculation ??? ??? option? ?????, ??? ?? ???? ????. LOD = 3.3(s/S)LOQ = 10(s/S) s= standard deviation of the responseS=slope of the calibration curve ?? ????? ?? ???? ???? ??? s, S? ??? ??? ???? ???, LOD? LOQ? ?? ??, ??? ??? ???? ??? ???? ????.

29. Robustness & Ruggedness Robustness - ????? ??? ??? ??? ??? ?? ??? ??? ???? ?? ???? ??. Examples of typical variations in LC 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 Ruggedness - ???? ????(???, ???, ????, ????, ?? ????, ????, ?????)? ?? ??? ??? ??? ???? ???? ????? ???. -???? ???? ? ????? ??? ?? ??.

30. Today�s Validation Requirements

31. ???,???,???,????,????? USP,ICH guideline? ???? ???? ????? ? ??(?? ?? ??? ??)? ???? ???? ??? ???? ?????. ??? ?? ?? ??? ?? ???? ?? ?? ??? ??? ???? ???? ?? ??? ? ????, ????? ??? -1=R=1? ???. ??? R2 (Coefficient of determination) ? ????? ?? ?? ????? ?? ?? ?????? ????? ?? ????? ??? ??? ??? ??? ?????., ?, R2 ? 0.99?? ??? ?? ??? ???? ????? ?? ???? ???? 99% ???? ???. ???? ??? ???? ??? ?? ???? ???? ?? ??? 3??? ??? ???? ? ??? ?3?? ?? ???? ??????? 3??? ?? ??? ???? ??? ?? ???? ???? ??? ?? ???? ??? ???? ???? ? ???? ??? ? ?? ?????. ???? ?? ????? ?? ? ?? ???? ????? ??? ??? ??? ???? ? ???? ?? ?????. LOD?? ICH Q2B guideline ? ???? ??? s? standard deviation of response? y??? ?????, S? slope of the calibration curve ? ???? ????? ?????. ???,???,???,????,????? USP,ICH guideline? ???? ???? ????? ? ??(?? ?? ??? ??)? ???? ???? ??? ???? ?????. ??? ?? ?? ??? ?? ???? ?? ?? ??? ??? ???? ???? ?? ??? ? ????, ????? ??? -1=R=1? ???. ??? R2 (Coefficient of determination) ? ????? ?? ?? ????? ?? ?? ?????? ????? ?? ????? ??? ??? ??? ??? ?????., ?, R2 ? 0.99?? ??? ?? ??? ???? ????? ?? ???? ???? 99% ???? ???. ???? ??? ???? ??? ?? ???? ???? ?? ??? 3??? ??? ???? ? ??? ?3?? ?? ???? ??????? 3??? ?? ??? ???? ??? ?? ???? ???? ??? ?? ???? ??? ???? ???? ? ???? ??? ? ?? ?????. ???? ?? ????? ?? ? ?? ???? ????? ??? ??? ??? ???? ? ???? ?? ?????. LOD?? ICH Q2B guideline ? ???? ??? s? standard deviation of response? y??? ?????, S? slope of the calibration curve ? ???? ????? ?????.

32. HPLC? ??? ?????? ?? ????? validation - ??? : ????? 6? ??? ??? ?? ??? ?? - ??? (Precision) intra-day variability : ??? ?? 3?? ??? ??? 3? ?? ?? inter-day variability : ????? ???? 3?? ??? ??? 3? ?? ?? - ??? (Accuracy) ??? ????? ?? ? ????? ??? ?? ?? ? ????? ?? ?? : ??? 3?? ??? ???? ?? ?? ??????? ?? : ??? ?????? ??? ??????? ?? ????? ??? ????

33. ? ? ?

34.  HPLC conditions ??? ????? ??? ??? ????? ????? ??? ???? C18??? ??? ????, ?? ?? ??? ????? ??? ?? ??? (? ?????? ??? ) ????? ???????.??? ????? ??? ??? ????? ????? ??? ???? C18??? ??? ????, ?? ?? ??? ????? ??? ?? ??? (? ?????? ??? ) ????? ???????.

35. ??? ???? ???? ??? ??? ?? ?????? ?? ??? ??? ?? ??? ??? ??? ?????? ??? ???? ??? ? ????.??? ???? ???? ??? ??? ?? ?????? ?? ??? ??? ?? ??? ??? ??? ?????? ??? ???? ??? ? ????.

36. ??? ? ????? ??? ?????? ?? validation? ???????. ???? ?? ??? ?? ???? ??? ?????. ??? ??? ??? R2?? ?? 0.99 ???? ?? ???? ???? ? ? ??? ?? ???? LOD? ???? LOQ? ??? ?? ? ?? 1 �g??? ?? ??? ???? ?? ???? ? ? ???? ??? ? ????? ??? ?????? ?? validation? ???????. ???? ?? ??? ?? ???? ??? ?????. ??? ??? ??? R2?? ?? 0.99 ???? ?? ???? ???? ? ? ??? ?? ???? LOD? ???? LOQ? ??? ?? ? ?? 1 �g??? ?? ??? ???? ?? ???? ? ? ????

37. ????? ??: ??? ?? inter-day, intra-day variability ??? ??? ???? ??? ?? ????? ??????? RSD? ?? 3%???? ????????? inter-day, intra-day variability ??? ??? ???? ??? ?? ????? ??????? RSD? ?? 3%???? ???????

38. ?????? ??? ?? ??