Szabolcs Sofalvi, M.S., D-ABFT-FT Cleveland, Ohio

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1 Statistical Tools for SWGTOX Method Validation of 11 Benzodiazepines in Whole Blood by SPE and GC/MS Szabolcs Sofalvi, M.S., D-ABFT-FT Cleveland, Ohio

2 Disclaimer Neither I nor any member of my immediate family has a financial relationship with a company as defined in the AACC policy on disclosure of potential bias or conflict of interest. SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 2

3 Introduction Driving force for a new method? SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 3

4 Chromatographic Quality SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 4

5 Objective Validate Benzodiazepines (11) Assay 5 batches (different days/analysts) Customized Microsoft Excel Template ANOVA (8) Calibration Model (5) LOQ (1) Bias and Precision (2) [Low/High QC] LODs (Estimated) SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 5

6 Method Sample Size (2mL) Deuterated Internal Standards Solid Phase Extraction UCT Clean Screen (ZSDAU020) Derivatized with MTBSTFA Agilent 7890A/5975C GC/MS Phenomenex ZB-50 GUARDIAN Part No. (7CD-G GGT) SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 6

7 Results Analyte Lorazepam α-hydroxymidazolam Clonazepam Midazolam Alprazolam α-hydroxyalprazolam Temazepam Diazepam Oxazepam Nordiazepam Linearity [ng/ml] SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 7

8 Response Ratio Calibration Model Alprazolam Concentration (ng/ml) Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Average SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 8

9 Drug: Alprazolam Calibration Model Trial 1 SUMMARY OUTPUT Regression Statistics Multiple R R Square Adjusted R Square Standard Error Regression Statistics Observations 7 ANOVA df SS MS F Significance F Regression E-10 Residual Total Coefficients Standard Error t Stat P-value Lower 95% Upper 95% Intercept E X Variable E E ANOVA RESIDUAL OUTPUT Observation Predicted Y Residuals Standard Residuals Residual Output SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 9

10 Calibration Model 1. Regression Statistics Concentration Response Ratio Regression Statistics Multiple R =CORREL($B$7:$B$13,F7:F13) select ranges for x,y (use $ sign) R Square =CORREL($B$7:$B$13,F7:F13)^2 typed rsquare1 in upper left corner Adjusted R Square =1-((1-rsquare1)*(n-1)/(n-2)) Residuals Standard Error =SQRT(SUMSQ(D7:D13)/(n-2)) typed serror1 in variable name cell Observations 7 =COUNT($B$7:$B$13) entered below L7 (ULOL) cell & type n in upper left corner Look for this entry on the next slide! SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 10

11 Calibration Model 1. Regression Statistics SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 11

12 Residuals 101 y [Response Ratio] y-ŷ ŷ =m*x + b x [Concentration] SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 12

13 Calibration Model 2. ANOVA ANOVA df SS MS F Significance F Regression E-10 Residual Total Coefficients Standard Error t Stat P-value Lower 95% Upper 95% Intercept E X Variable E E SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 13

14 Calibration Model 3. Residual Output RESIDUAL OUTPUT ŷ N74 Observation Predicted Y Residuals Standard Residuals =(N74)/STDEV(D$7:D$13) SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 14

15 Standardized Residual Plot Alprazolam Standardized Residuals Concentration (ng/ml) Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Average SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 15

16 Limit of Quantitation (LOQ) L1 Concentration 10 ng/ml Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Calculated Mean Replicate Replicate Bias Replicate % SUMMARY Groups Count Sum Average Variance SS Trial Trial Trial SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 16

17 Limit of Quantitation (LOQ) ANOVA Source of Variation SS df MS F P-value F crit Between Groups Within Groups Total Within Run CV (%) 9.17% Between Run CV (%) 11.81% SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 17

18 LOQ = 10 ng/ml SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 18

19 Bias and Precision (QCs) Low QC Concentration 125 ng/ml Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Calculated Mean Replicate Replicate Bias Replicate % High QC Concentration 250 ng/ml Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Calculated Mean Replicate Replicate Bias Replicate % SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 19

20 SUMMARY Bias and Precision(ANOVA) Groups Count Sum Average Variance SS Trial Trial Trial Trial Trial ANOVA Source of Variation SS df MS F P-value F crit Between Groups Within Groups Total Within Run CV (%) 2.07% Between Run CV (%) 2.80% SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 20

21 Estimated LOD from Calibration Curves Limit of Detection (LOD) Estimated Slope y-intercept Trial Trial Trial Trial Trial Average Standard Deviation =SLOPE('Calibration Model'!G7:G13,'Calibration Model'!B7:B13) =INTERCEPT('Calibration Model'!G7:G13,'Calibration Model'!B7:B13) Estimated LOD 4.56 ng/ml =(3.3)*(G14)/D13 LLLLLL = SSSS yy iiiiiiiiiiiiiiiiii AAAAAA SSSSSSSSSS SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 21

22 Conclusions Improved chromatographic quality: Ion ratios Peak shapes Number of data points/peak Tailing Factor (< 2.00) SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 22

23 Conclusions Customized Microsoft Excel: Instant results Accurate No need to reformat every time Validation Summary Report Advantage: Other validations! (e.g. Speaker 42) SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 23

24 Harold E. Schueler Xin Xu (Speaker S42) Claire K. Kaspar Cassandra L. Clyde Eric S. Lavins Thomas P. Gilson Acknowledgments SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 24

Questions Email: ssofalvi@cuyahogacounty.

25 Questions SOFT 2016 Dallas, TX Development of Statistical Tools for Method Validation 25

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