B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 W. WegscheiderValidation: an Example 1 Learning Objectives: Student n is able to inquire in sufficient depth on the purpose of analytical work n is able to translate this information into stepwise strategy keeping in mind suitable performance characteristics at each step n 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 performs statistical analysis of obtained data with the goal to make a valid judgement regarding the suitability of the method Validation: an Example

B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 W. WegscheiderValidation: an Example 2 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

B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 W. WegscheiderValidation: an Example 3 Worked Example: Fe 2 O 3 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 %.

B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 W. WegscheiderValidation: an Example 4 Deriving Minimum Performance Characteristics from Requirements  overall uncertainty (including sampling):<0.03 % uncertainty at required level of concentration from analysis itself:~0.01%uncertainty at required level of concentration from analysis itself:~0.01%  limit of quantitation (with matrix):<0.03 %  limit of detection (with matrix):<0.01 %  selectivity/interferences:CRM  linearity:at least % As the differences from one another was about 0.1 % Fe 2 O 3 the proposed method needs to have the following characteristics:

B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 W. WegscheiderValidation: an Example 5 Preliminary Examination of Candidate Methods (1)

B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 W. WegscheiderValidation: an Example 6 Preliminary Examination of Candidate Methods (2)

B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 W. WegscheiderValidation: an Example 7 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

B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 W. WegscheiderValidation: an Example 8 Calibration without MgO

B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 W. WegscheiderValidation: an Example 9 Calibration with MgO

B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 W. WegscheiderValidation: an Example 10 Graphical Comparison without matrix with matrix mg/mL

B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 W. WegscheiderValidation: an Example 11 EXCEL Macro ValiData  Basis of the program is ISO 8466 and DIN 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 robustregression

B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 W. WegscheiderValidation: an Example 12 Numerical Comparison without matrix with matrix

B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 W. WegscheiderValidation: an Example 13 Limits of Detection Limits of Quantitation without matrix LOD % with matrix LOD % LOQ %

B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 W. WegscheiderValidation: an Example 14 Estimation of Recovery serial dilution of Burned Magnesite SRM 104 Fe content of % (1 s) with pure MgO between 0.1 and 0.6 % found/(given) found/(given) found found Slope IC (slope) Ordinate intersect IC (Ordinate intersect) is about % at 0.5 % level

B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 W. WegscheiderValidation: an Example 15 Alternative Method: Titrimetry 1. dissolve SRM in HCl, dilute to 100 ml 2. reduce with SnCl 2 3. add 20 ml HgCl 2 and 50 ml Zimmermann-Reinhart- Solution 4. titrate with 0.01 n KMnO 4 Result: 7.01 % Fe 2 O 3 Certified: %

B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 W. WegscheiderValidation: an Example 16 Estimation of Uncertainty from Sampling P about 500 g laboratory sample delivered P sampled “according to ISO” by customer P max. concentration about 1 % Fe in dark pieces P min. concentration below LOD in light pieces P many larger grains: ca 1 cm 3 corresponds to 3 g P total of 500 g/3 g ~ 160 pieces P 80 pieces give a relative uncertainty of 9 % P at the 0.5 % level this is % (0.02 % requ. !!!) CONCLUSION: Pthe laboratory sample must be about 2000 g

B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 W. WegscheiderValidation: an Example 17 Content of Validation Report Introduction Test principle Definition of the type of the analytical test procedure Definition of validation parameters Performance of the test Test outline and acceptance criteria Equipment qualification System suitability Qualification of samples and standards SummaryReferencesResults