Validation: an Example

1. W. Wegscheider Validation: an Example Validation: an Example • Learning Objectives: Student • is able to inquire in sufficient depth on the purpose of analytical work • is able to translate this information into stepwise strategy keeping in mind suitable performance characteristics at each step • can devise experiments to test against these criteria • performs statistical analysis of obtained data with the goal to make a valid judgement regarding the suitability of the method

2. W. Wegscheider Validation: an Example Validation Must Be Performance Based • Definition of the need of the client translated to analytical quality criteria including cost and time • Compilation of candidate methods • Critical appraisal of candidate methods with respect to expected method performance • Design and execution of validation study • Evaluation of results, go-ahead or modifications or alternative method • Measure the unknown with due regard to previously established quality criteria

3. W. Wegscheider Validation: an Example Worked Example: Fe2O3 in MgO • For certain industrial applications of MgO the upper limit of Fe oxide needs to be controlled very tightly • Definition of the need of the customer translated to analytical quality criteria including cost and time • In this application an upper limit of 0.5 % was guaranteed by the supplier, the customer found 0.64 %, the supplier 0.41 %.

4. W. Wegscheider Validation: an Example Deriving Minimum Performance Characteristics from Requirements As the differences from one another was about 0.1 % Fe2O3 the proposed method needs to have the following characteristics: • overall uncertainty (including sampling): <0.03 % • uncertainty at required level of concentrationfrom analysis itself: ~0.01% • limit of quantitation (with matrix): <0.03 % • limit of detection (with matrix): <0.01 % • selectivity/interferences: CRM • linearity: at least 0.3-0.7 %

5. W. Wegscheider Validation: an Example Preliminary Examination of Candidate Methods (1)

6. W. Wegscheider Validation: an Example Preliminary Examination of Candidate Methods (2)

7. W. Wegscheider Validation: an Example Validation Plan • Produce calibration curves for Fe by flame AA under standard conditions with and without MgO • Compare calibration functions • Evaluate limit of detection and limit of quantitation • Estimate precision for meeting previously established requirements (+ margin): allowance for day to day variability, etc. • Produce recovery data by mixing various aliquots of CRM MgO with high purity MgO • Compare with alternative method ? • Estimate (laboratory) sampling error for sample as received

8. W. Wegscheider Validation: an Example Calibration without MgO

9. W. Wegscheider Validation: an Example Calibration with MgO

10. W. Wegscheider Validation: an Example without matrix with matrix mg/mL Graphical Comparison

11. W. Wegscheider Validation: an Example EXCEL Macro ValiData • Basis of the program is ISO 8466 and DIN 32645 • Homogeneity of variances test • Calibration functions • linear OR quadratic following Mandel´s test • weighted linear and weighted quadratic • standard additions • Performance characteristics • Decision limit and detection limit from calibration • Limit of quantitation • Linear range, sensitivity, confidence limits • Recovery studies • orthogonal regression • robust regression

12. W. Wegscheider Validation: an Example Numerical Comparison without matrix with matrix

13. W. Wegscheider Validation: an Example without matrix LOD 0.006 % with matrix LOD 0.005 % LOQ 0.018 % Limits of DetectionLimits of Quantitation

14. W. Wegscheider Validation: an Example Slope 1.015187226 found/(given) IC (slope) 0.994663053 1.035711399 found/(given) Ordinate intersect -0.008726667 found IC (Ordinate intersect) -0.016368629 -0.001084705 found Estimation of Recovery serial dilution of Burned Magnesite SRM 104 Fe content of 7.07+0.04 % (1 s) with pure MgO between 0.1 and 0.6 % - 0.009 is about - 1.8 % at 0.5 % level

15. W. Wegscheider Validation: an Example Alternative Method: Titrimetry 1. dissolve SRM in HCl, dilute to 100 ml 2. reduce with SnCl2 3. add 20 ml HgCl2 and 50 ml Zimmermann-Reinhart- Solution 4. titrate with 0.01 n KMnO4 Result: 7.01 % Fe2O3 Certified: 7.07 + 0.04 %

16. W. Wegscheider Validation: an Example Estimation of Uncertainty from Sampling • about 500 g laboratory sample delivered • sampled “according to ISO” by customer • max. concentration about 1 % Fe in dark pieces • min. concentration below LOD in light pieces • many larger grains: ca 1 cm3 corresponds to 3 g • total of 500 g/3 g ~ 160 pieces • 80 pieces give a relative uncertainty of 9 % • at the 0.5 % level this is 0.045 % (0.02 % requ. !!!) • CONCLUSION: • the laboratory sample must be about 2000 g

17. W. Wegscheider Validation: an Example Content of Validation Report Introduction Test principle Definitionof the type of the analyticaltest procedure Definition of validation parameters Performance of the test Test outline and acceptance criteria Equipment qualification System suitability Qualification of samples and standards Summary References Results