International Federation for Consulting GmbH IFC

1. Validation by Dr. Michael Scheutwinkel International Federation forConsulting GmbH IFC

2. Governmental • regulations • Books …. • Recommendations • of associations

3. References EURACHEM Guide The Fitness for Purpose of Analytical Methods - A Laboratory Guide to Method Validation and Related Topics (12/1998) www.eurachem.com CITAC / EURACHEM Guide Guide to Quality in Analytical Chemistry An Aid to Accreditation (2002) www.eurachem.com

5. Which chapter of ISO/IEC 17025 is dedicated to validation?

6. Do not ask me, I am only a simple guy from Alemania!

7. Definition in clause 5.4.5.1 of ISO/IEC 17025 Validation (ISO/IEC 17025) Validation is the confirmation by examination and the provision of objective evidence that the particular requirements for a specific intended use are fulfilled. ….fit for purpose

8. Validation of an analytical test method • Validation is the process of establishing the • Performance characteristics • Limitations of a method • Identification of the influences which may • change these characteristics and to what extent. Which analytes can be determined in which matrix in the presence of which interferences? Within these conditions what levels of precision and accuracy can be achieved?

9. Equipment and method valdiation Equipment validation DQ OQ Soft and hardware PQ IQ Method validation … different items

10. Validation in order to analytical method Standardized-methods Verification Modified standardized andin-house methods Validation

11. Verification of an analytical test method Verification is the process to demonstrate the competence in test performance of an already validated standardized test method. The tools are the same as for validation, the laboratory needs only to verify that the documented performance characteristics can be met (e.g. accuracy).

12. Changes in established methods New method developed for a particular problem Established method used in different laboratories, different equipment or different staff Out-of-control situations within internal quality assurance Non-successful participation in PTs Demonstration of equivalence between two methods (e.g. a rapid new test against a standard method) When should be validated?

13. A Validation Strategy Specific test requirements defined by customer or market B C D Characterization of the test method Comparison of method charcacteristics with requirements Verification to give proof that the requirements are fulfilled

14. Description of test method (clause 5.4.4) Identification / Scope Parameters in which matrix Reagents and materials, CRMs Equipment and environmental conditions Calibration Sampling and sample preparation Test performance including safety instructions Quality assurance Documentation , reporting evaluation criteria Uncertainty or procedure for its estimation

15. Please show us the way…

16. Tools Determination of method characteristics • Characteristics • of the range • Characteristics • of calibration • Characteristics • of accuracy • Robustness / • Ruggedness • Selectivity or • Specifity

17. Tools Determination of method characteristics • Perform more • point calibration Correlation coefficient: r² > 0.99 ?? • Characteristics • of calibration • Linearity • Sensitivity Linearity is defined by the correlation coefficient Sensitivity is defined by the slope of the calibration graph

18. Commission Decision 2002/657/EC

19. Validation of testing methods Calibration • Calibration values should be applied within the working range. • Lowest point should be the limit of quantification. • For calibration certified standard solutions shall be used. • In addition, also standard solutions have to be added to blank • materials, analysed over all process steps. • For linear calibration functions at least five concentrations are • necessary. • Test calibration functions for linearity and highlight failing • linearity.

20. Validation of testing methods Sensitivity (resolution) What is sensitivity?  It is the difference in an analytical concentration that corresponds to the smallest difference of a signal in a method which is still detectable. Sensitivity can be extracted from the calibration curve or defined by using samples with different concentrations.

21. Validation of testing methods Linearity Definition based on samples with varying concentrations and the calculation of the regression of results. Signal and concentration do not have to be fully related. Five standards suffice to produce a calibration curve if linearity is o.k. More standards are necessary once the linearity is unsatisfactory. Examine repeat samples and standards over a particular working range to experience whether a reliable line can be drawn between proof and detection limit.

22. Homogeneity of variances • Characteristic values for the comparison of standard variations for differing concentration. In case of great differences between standard deviations (in-homogeneity of variances), the working range needs to be split.

23. Tools Determination of method characteristics Characteristics of the range Limit of detection Limit of quantification • Characteristics • of calibration • Characteristics • of accuracy • Robustness / • Ruggedness • Selectivity or • Specifity

24. Validation of testing methods Working range • Finding out the appropriate working range using • different matrices • varying concentrations. • Concentration range within the achievement of acceptable • accuracy and • precision is possible. In general the working range is broader than the linear range.

25. Validation of testing methods Prerequisites for calibration • Standards must be faultless. • Precision has to be similar throughout the entire working range. • The model function is applicable: either linear or curved. • Errors only may occur randomly within signals. • Errors have to follow the normal distribution.

26. LOD Characteristics of the range Limit of detection by calculation LOD xLOD = 3 . sL / b (Blank value method) xLOD = 4 . sxo (Calibration function method) xLd = Limit of detection sL = Standard deviation of the blanks Sxo = Standard deviation of the calibration function b = Slope of calibration function Various conventions XLOD = Mean of blanks + 3 x SL

27. LOQ Characteristics of the range by calculation Limit of quantification LOQ xLOQ = 9 . sL/ b (Blank value method) xLOQ = 11 . sxo (Calibration function method) xLOQ = Limit of quantification sL = Standard deviation of the blanks Sxo = Standard deviation of the calibration function b = Slope of calibration function Various conventions XLOQ = 5 or 6 or 10 x SL

28. Commission Decision 2002/657/EC

29. LOD Characteristics of the range by signal to noise ratio Limit of detection From 2 : 1 to 5 : 1 Limit of quantification From 5 : 1 to 10 : 1

30. Effect of peak shape on LOD / LOQ

31. Tools Determination of method characteristics • Characteristics • of the range • Characteristics • of calibration • Characteristics • of accuracy • Robustness or • Ruggedness • Selectivity or • Specifity

32. Selectivity / Specifity Extent to which particular analytes can be determined in complex mixtures. A method which is selective for an analyte is said to be specific. • Selectivity • Specifity Selective detectors in instrumental analysis Selective media in microbiology Use of antibodies • Cross-reactions ?

33. The difference between selectivity and specificity Selectivity Specifity • A selective method gives correct results for all interesting analytes whereas a specific test method gives correct results for the interesting analyte whereas other analytes might interfere each other. • Outlines the analytical extent to which an analytical substance or substance group can be determined without interference from sample related components. No problem, usually both words are mixed …

34. Validation of testing methods Selectivity • Outlines the extent to which an analytical substance can be determined without interference from other components. • Method selective to one analytical substance specific. • Assess selectivity while application to pure solution up to complex matrices. • Document disturbances and restrictions of the method.

35. Tools Determination of method characteristics • Characteristics • of the range • Characteristics • of calibration • Characteristics • of accuracy • Robustness / • Ruggedness • Selectivity or • Specifity

36. Validation of testing methods Robustness  Degree of proneness of a method to conscious and unconscious alterations of a working instruction. • Part of the routine. • Learn from other analytical institutes, as • the method-establishing laboratory will have proved • robustness before publishing a new method.

37. Sensitivity degree of the test method against small deviations in experimental conditions. Examples: Times within process steps Environmental conditions (e.g. temperatures) Minor process changes (e.g. pH, flow rates in HPLC) Robustness

38. Ruggedness is the degree of reproducibility obtained under a variety of conditions, expressed as relative SD, e.g.: different analysts, different equipment, different trade marks of reagents, etc. Ruggedness, according to USP Internal reproducibility

39. Tools Determination of method characteristics • Characteristics • of the range Characteristics of accuracy: Correctness Uncertainty Precision (Repeatability/ Recovery) Reproducibility • Characteristics • of calibration • Robustness or • Ruggedness • Selectivity or • Specifity

40. Accuracy = Precision and correctness I Exactness of an analytical method Accuracy Degree of repeatability of an analytical method Precision Repeatability Uncertainty Reproducibility Correctness / Trueness Systematic error / Correct value Recovery / Bias

41. Random and systematic deviations

42. Accuracy = Precision and correctness II Characteristics of accuracy Correct- ness Measure of correctness, covering systematic and non-systematic mistakes (use of CRM, comparison to a well characterized method) Precision Measure of the degree of repeatability, covering systematic mistakes Repeatability: one laboratory; Better: internal reproducibility Reproducibility: more than one laboratory

43. Validation of testing methods Repeatability Use at least one of the following procedures for determination: • Measurement of reference materials. • Fortification of a blank sample with the analytical substance. • Comparison to a reference procedure. • Participation in a co-operative (interlaboratory) test in which the “true content” is known.

44. Validation of testing methods Precision  Information on accuracy of two coinciding and independent analytical results. Precision depends on the concentration of the analyte. Repeatability is part of the precision with respect to repeated measurements: • same material, • same method, • same analyst, • same laboratory and • short time-span in-between the analyses.

45. Validation of testing methods Correctness / Trueness Use the one of the following methods to assure correctness: • Analysis of certified reference material. • Participation in interlaboratory comparisons. • Comparison to a known procedure. If not available: • Document any data that prove correctness. • Make an approximation as a first approach to correctness. • Purchase reference material. • Use a similar method in parallel.

46. Validation of testing methods Recovery • Performed by addition of the analytical substance to the matrix. • A matrix shall be free of the analytical substance. • If not the sample will be enriched with a weak concentration of the analytical sample. • Or a simulated matrix will be used. • Recovery can be determined only when the analytical sample is available in pure form. • List the recovery rate (in %) and the standard deviation when recovery rates are constant. • Otherwise issue recovery rate as a function.

47. Validation of testing methods Frame conditions for the determination of the recovery rate The analytical substance needs to be added in that form in which it occurs in nature. The samples requires good homogenisation. The native content shall be below the determination limit.

48. Validation of testing methods Reproducibility internally Prove reproducibility externally • Statistically • Comparative standard deviation • Replicate standard deviation • Through participation in proficiency testing schemes • or participation in interlaboratory comparisons.

49. Tools Determination of method characteristics • Characteristics • of the range • Limit of detection • Limit of quantification • Characteristics • of calibration • Linearity • Sensitivity • Characteristics • of accuracy • Correctness • Uncertainty • Precision (Repeatability/ Recovery) • Reproducibility • Selectivity or • Specifity • Robustness or • Ruggedness

50. Validation of testing methods Classification of the test method Distinguish between • Analysis of contaminants (e.g. pesticides, heavy metals, • food additives) • Analysis of food constituents (salt and minerals) • Methods which are determining true values • Test methods which have to be calibrated with instruments • Physical measurement (pH value, conductivity) • Qualitative determinations • Microbiological methods