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S

alvati

et al

.:

J

ournal of

AOAC I

nternational

V

ol

.

99, N

o

.

3, 2016 

9

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

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.

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

4.7

4.5

4.0

S02: Infant formula powder partially hydrolyzed, milk-based

3.7

3.9

4.6

3.6

S03: Infant formula powder partially hydrolyzed, soy-based

2.5

4.5

2.3

3.3

S04: Toddler formula powder, milk-based

3.5

5.6

2.4

6.2

S05: Infant formula powder, milk-based

2.8

5.4

2.0

3.0

S06: Adult nutritional powder, low-fat

3.4

9.0

2.8

5.1

S07: Child formula powder

3.6

5.3

2.4

3.6

S08: Infant elemental powder

3.0

5.7

1.4

4.3

S09: Infant formula powder, FOS/GOS-based

a

4.0

4.1

2.3

4.1

S10: Infant formula powder, milk-based

4.1

4.5

1.6

4.5

S11: Infant formula powder, soy-based

3.1

3.7

2.2

3.5

S12: Infant formula RTF, milk-based

4.0

5.8

3.3

5.7

S13: Adult nutritional RTF, high-protein

4.1

5.6

3.6

6.0

S14: Adult nutritional RTF, high-fat

3.2

4.2

3.2

5.0

a

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

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.

113