1. Industrial Technology Institute Test Method Validation & Verification H.P.P.S.Somasiri Principal Research Scientist / SDD-QAD /QM Industrial Technology Institute SLAB 2015/11/10, BMICH VER 6

2. Industrial Technology Institute 2 Introduction An analytical method is the series of procedures from receipt of a sample to the production of the final report. Validation is the process of verifying that a method is fit for purpose.

3. Industrial Technology Institute 3 Basic Concepts in Method Validation Specificity Analyte Stability Analytical Range Calibration Range Recovery (Accuracy) Precision Limit of Detection (LOD) Limit of Quantification (LOQ) Measurement uncertainty

4. Industrial Technology Institute 4 Specificity Shows that the detected signal is due to the analyte, not another compounds/ s. Use matrix blanks, reagent blanks for conformation of specificity

5. 5 Example: Confirmation of Identity

6. Industrial Technology Institute 6 Analyte Stability Analyte stability during sample storage, analyte stability in sample processing, analyte stability in sample extract should be studied.

7. Industrial Technology Institute 7 Analytical Range Range of the analyte that can be analysed with the proposed selected method.

8. Industrial Technology Institute 8 Calibration Range If a linear calibration is used, the range in which the response for the anayte shows a linear correlation with the analyte’s concentration should be determined. This is known as linear dynamic range.

9. Industrial Technology Institute 9 Linear Dynamic Range

10. Industrial Technology Institute 10 Recovery % Recover of the anlalyte on extraction, clean-up, derivatization and measurement. Can be done with -Certified Reference Material (CRM,s) -Standard Reference materials (SRM,s) -Interlaboratory comparisons -Spiking (@ 20 %, 50 % and 80 %)

11. Industrial Technology Institute 11 Accuracy (Trueness) How close the result to the expected (assigned) value

12. 12 Example: Accuracy SampleBlank value (mg/L) RM value (mg/L) 10.0011.46 20.0181.80 30.0011.17 40.0181.20 50.0021.22 60.0020.74 70.0021.16 80.0141.23 90.0011.20 100.0021.29 Mean value0.0041.25 RM assigned value: 1.22 ± 0.08 mg/L; Laboratory value for RM: 1.246 mg/L Mean laboratory Value from RM value certificate 1.246 mg/L 1.22 mg/L Bias value

13. 13 Accuracy versus Precision Accuracy (Trueness) Precision Accuracy & Precision

14. Industrial Technology Institute 14 Precision Is measured as “repeatability “ or reproducibility Repeatability Measurement is repeated with a minimum of variations (same person, same laboratory, same equipment, over a short period of time. Reproducibility Measurement is repeated with as much variation as possible. (Different analyst, different laboratory, different equipment, over a long period of time).

15. Industrial Technology Institute 15 Limit of Detection (LOD) Point where the variability of the measurement is such that it is impossible to distinguish between random fluctuation in the value measured and actual signal. –LOD = Y B + 3XS B Y B = Mean value measured from the blank S B = Standard Deviation of the value measured from the blank

16. 16 Replicate (n)Instrument response Calculated Concentration (ppb) 1295500.100 2286530.097 3294010.099 4285320.097 5297120.101 6299830.101 7281670.095 Mean 0.099 StdDev 0.002 3 Times StdDev 0.007 Detection Limit 0.106 TBT in Biota Limit of Detection

17. Industrial Technology Institute 17 Limit of Quantification (LOQ) Point where the measured value could be quantified reliably LOQ = Y B + 10XS B Y B = Mean value measured from the blank S B = Standard Deviation of the value measured from the blank

18. 18 Example: TBT in Biota Replicate (n)Instrument response Calculated Concentration (ppb) 1295500.100 2286530.097 3294010.099 4285320.097 5297120.101 6299830.101 7281670.095 Mean 0.099 StdDev 0.002 3 Times StdDev 0.007 Detection Limit 0.106 Limit of Quantitation Blk + 5s 0.110 Blk + 6s 0.113 Blk + 10s 0.122 Limit of Quantification (LOQ)

19. 19 Sensitivity- Cadmium Analysis by AAS 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.00 0.20 0.400.60 0.80 1.00 1.20 Cd Concentration (mg/L) Absorbance slope = 0.241 ± 0.011 intercept = 0.009 ± 0.003 r-square = 0.99442 X X x x x x Sensitivity

20. 20 Measurement Uncertainty Uncertainty estimates should take account of: Overall long-term precision Bias and its uncertainty, including the statistical uncertainty involved in the bias measurements, and the reference material or method uncertainty Calibration uncertainties Any significant effects operating in addition to those listed above

21. 21 Test Method Verification Any standard method shall be verified before being used in the laboratory Verify the Recovery Accuracy Precision ( Repeatability and Reproducibility) LOD and LOQ

22. Industrial Technology Institute 22