6. AOACSPIFANMethods-2018Awards

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S alvati et al .: J ournal of AOAC I nternational V ol . 99, N o . 3, 2016  9

Table 5. Intermediate precision for six independent preparations is expressed as %RSD Matrix Total B 1 Total B 2

Total B 3

Total B 6

S01: 1849a

3.0 3.7 2.5 3.5 2.8 3.4 3.6 3.0 4.0 4.1 3.1 4.0 4.1 3.2

4.7 3.9 4.5 5.6 5.4 9.0 5.3 5.7 4.1 4.5 3.7 5.8 5.6 4.2

4.5 4.6 2.3 2.4 2.0 2.8 2.4 1.4 2.3 1.6 2.2 3.3 3.6 3.2

4.0 3.6 3.3 6.2 3.0 5.1 3.6 4.3 4.1 4.5 3.5 5.7 6.0 5.0

S02: Infant formula powder partially hydrolyzed, milk-based S03: Infant formula powder partially hydrolyzed, soy-based

S04: Toddler formula powder, milk-based S05: Infant formula powder, milk-based S06: Adult nutritional powder, low-fat

S07: Child formula powder S08: Infant elemental powder

S09: Infant formula powder, FOS/GOS-based a S10: Infant formula powder, milk-based S11: Infant formula powder, soy-based S12: Infant formula RTF, milk-based S13: Adult nutritional RTF, high-protein S14: Adult nutritional RTF, high-fat

a  FOS/GOS = Fructo-oligosaccharides/galacto-oligosaccharides.

the signal intensity was higher, the variation in the ion ratio was reduced and approached the ±3% level of the more abundant vitamin forms. Finally, the choice of enzyme is important for method performance. During method development, two different acid phosphatases were investigated, one from Roche Diagnostics and one from Sigma-Aldrich. The acid phosphatase from Roche did not fully hydrolyze pyridoxamine-5′-phosphate and generally recovered about 50% of the over-spiked level. Further, it generated significant amounts of nicotinic acid during digestion on the order of up to 10% of the total vitamin B 3 . Although the source of the nicotinic acid is not entirely clear, it appears to result from conversion of nicotinamide to nicotinic acid because the total B 3 concentration (sum of nicotinamide and nicotinic acid) did not increase significantly in the three matrixes studied in detail. The method was validated using the acid phosphatase from Sigma-Aldrich. This acid phosphatase contains higher levels of pyridoxamine and pyridoxal, riboflavin, and nicotinic acid; but was chosen because it eliminates problems with nicotinic acid conversion and pyridoxamine-5′-phosphate recovery. The background vitamin levels in the Sigma-Aldrich acid phosphatase as a percent of their concentrations in SRM 1849a are 0.1% thiamine, 2.8% riboflavin, 0.2% nicotinamide, 18% nicotinic acid, 6.2% pyridoxal, 0.5% pyridoxamine, and 0.2% pyridoxine. However, these data need additional context. Nicotinic acid, pyridoxal, and pyridoxamine in SRM 1849a are virtually absent. From a total vitamin perspective, the overall contribution of vitamins from the enzyme in SRM 1849a is 0.1% total B 1 , 2.8% total B 2 , 0.3% total B 3 , and 0.5% total B 6 . Despite the small contribution from the enzyme, the standards are prepared as samples to mitigate any impact on method accuracy. The development work presented serves as caution: substitution of enzymes for other than those specified by this method may be deleterious to method performance. The use of an alternative enzyme would require significant investigation to the efficacy, digestion, and background contribution of vitamins to ensure adequate method performance.

Thiamine was a notable exception because of chromatographic interference in the first transition for the 13 C 4 -thiamine internal standard (Table 1). This chromatographic interference does not impact method accuracy because the first transition is not used for quantitation. However, it does mean that ion ratio suitability criteria cannot be specified for the thiamine internal standard. Ion ratios for the lower intensity vitamins (pyridoxal, pyridoxamine, and nicotinic acid) had a larger degree of variation because of the lower signal intensity. They averaged 102 ± 12% of the ion ratio in the standards. During the over-spike studies in which

Figure 1. A chromatogram for the seven vitamin forms in the child formula powder. The data are unsmoothed, and the intensity of each peak is normalized to aid visualization. There is more than two orders of magnitude difference in signal intensity, which makes the small features such as pyridoxamine and pyridoxal difficult to see when all are plotted on the same scale.