6. AOACSPIFANMethods-2018Awards

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G ill et al .: J ournal of AOAC I nternational V ol . 99, N o . 5, 2016  1329

Bias was evaluated by replicate analyses of the National Institute of Standards and Technology (NIST) 1849a Standard Reference Material (SRM). Differences between the measured value and the certified value were determined with the mean and SD of the differences, and the test statistic was calculated. A p (α = 0.05) of 0.25 indicates that there was no bias between the measured results and the certified value (Table 5). As part of initial method validation, the LC-MS/MS was evaluated for bias against an HPLC–UV method based on AOAC 2002.05 (8, 9). A p (α = 0.05) of 0.09 indicates that there was no bias between the methods (Table 6). Bias against a certified reference material or a reference method is not a defined parameter within the SMPR. Table 5. Results for the bias experiment against NIST 1849a SRM a Parameter Value Certified value, μg/hg 11.1 Uncertainty, μg/hg 1.7 Certified range, μg/hg 9.4–12.8 Coverage factor, k 2 Degrees of freedom, DF CRV 60 Mean, x 10.1 SD 0.53 Number of replicates, n 13 95% Confidence interval, μg/hg 9.8–10.4 T stat 1.165 Degrees of freedom 63.92 p (α = 0.05) 0.25 a  SRM=Standard Reference Material.

Table 6. Results for the bias experiment against AOAC 2002.05 Parameter

Reference method LC-MS/MS method

Mean, μg/hg

10.5 3.18

10.8 3.66

SD, μg/hg

Number of replicates, n

40

40

95% Confidence interval, μg/hg 10.0–11.0

10.2–11.4

Mean of paired differences SD of paired differences

–0.3 1.27 1.73

T stat

Degrees of freedom

38

0.09

p (α = 0.05)

SMPR, which specifies total vitamin D 2 , including their previtamin isomers. It was assumed in this analysis, as with all analytical methods for vitamin D that use calciferol internal standards, that the previtamin D:vitamin D ratio was equivalent for the sample analyte and the internal standard. For deuterated internal standards, the labeled site must be remote from the triene center because of the difference in interconversion behavior between the analyte and the internal standard (10). To confirm this assumption, the effect of temperature on the final results was evaluated. Experiments were performed with saponification assessed in three different ways: ( 1 ) at 70°C for 1 h, according to the described method protocol; ( 2 ) at 20°C for 7.5 h; and ( 3 ) at 70°C for 7.5 h. A 7.5 h saponification was chosen because this is the time needed, as previously reported, for a pure solution of vitamin D to reach equilibrium with previtamin D at 70°C (11). Samples 1–6 and 13–18, which were saponified at 70°C, showed significantly lower absolute peak areas for the vitamin D–PTAD quantifier ion than samples 7–12, which were saponified at 20°C. This was as expected because a higher proportion of vitamin D is converted to previtamin D at the elevated temperature. This effect was seen for both the analyte vitamin D in the sample and the SIL d6 -vitamin D internal standard, illustrating the appropriateness of the internal standard to account for any temperature-induced interconversion between previtamin D and vitamin D (Figure 5). The final results obtained showed that, within sample error, there or vitamin D 3

Vitamin D–Previtamin D Interconversion

Although the described method specifically detects vitamin D and not the previtamin D isomer, the method quantifies an aggregate result for both previtamin D and vitamin D. This satisfies the requirement of the applicability statement of the

Figure 5. Effect of saponification time/temperature on vitamin D and d6 -vitamin D.