6. AOACSPIFANMethods-2018Awards

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S alvati et al .: J ournal of AOAC I nternational V ol . 99, N o . 3, 2016  7

and pyridoxal-5′-phosphate were spiked at 4.9% and 4.3% and pyridoxamine and pyridoxamine-5′-phosphate were spiked at 5.8% and 5.0% of total spiked B 6 . On an RTF concentration basis, over-spikes were 2.60 and 21.0 μg/100 g of total pyridoxal; 3.00 and 24.0 μg/100 g of total pyridoxamine; 22.5 and 180 μg/100 g of total pyridoxine; 31.5 and 250 μg/100 g of total thiamine; 24.0 and 190 μg/100 g of total riboflavin; and 190 and 1500 μg/100 g of total B 3 . Good over-spike recovery was demonstrated (Table 3). Repeatability and intermediate precision were determined from six independent preparations of all 14 products over 6 days. The experiments were performed by two analysts and on one instrument. Repeatability and intermediate precision are reported as %RSD in Tables 4 and 5. SPIFAN SMPRs for repeatability and reproducibility are ≤5% and ≤10% RSD, respectively. Method robustness was evaluated during development by using three analysts and two instruments. The method was tested over 6 days as well with independent preparations for each data point, and accuracy was done over an additional three days for each matrix. Data were collected over the course of about 8 weeks. Given these variables, precision and accuracy were excellent suggesting good method robustness. Further, a review of sample weights collected during sample preparation show that the powder weight varied by up to 6%, the reconstitution weight varied by up to 8%, and the liquid sample weight varied by up to 9%. Given the demonstrated precision and accuracy, this method shows good robustness toward sample size variation. Within a run, there is notable signal suppression in some matrixes. Suppression is most easily observed by noting the absolute change in the internal standard intensity in samples compared with standards. The degree of suppression is matrix- and vitamin-dependent and ranged from negligible up to loss of 50% of the signal. Ion suppression is not uncommon with ESI, and necessitates the use of stable-isotope labeled internal Precision Robustness

Linearity

This method includes six working standards to bracket the distribution of vitamin concentrations in SPIFAN II products. Calibration curves were generated at the beginning and end of each analysis as required by the method. Each standard in the curve has its percent deviation calculated as the percent difference between the calculated concentration and the true concentration. Percent deviation of ±4% is typical for vitamins B 1 , B 2 , B 3 , pyridoxamine, and pyridoxine; and percent deviation of ±11% is typical for pyridoxal, which has lower response. Good performance was observed (Table 2). Accuracy was evaluated by over-spike recovery in the five SPIFAN II placebos and three of select SPIFAN II products (Table 3). The placebos were manufactured without fortification of vitamins and minerals, but do contain some inherent vitamins and minerals by contribution of the proteins, carbohydrates, and fats. An additional three fortified SPIFAN II samples were chosen for over-spike studies because they were different enough from the placebos to warrant additional inquiry: partially hydrolyzed, milk-based infant formula powder; partially hydrolyzed, soy-based infant formula powder; and soy-based infant formula powder. For over-spike recovery, each matrix was spiked at both low and high levels corresponding to approximately 50% and 200% of fortification, respectively. Each spike level was performed with independent sample preparation, and the experiment was repeated on three different days for a total of n = 6 data points at each level in each matrix. Recovery was calculated as the reported concentration divided by the inherent contribution plus the amount spiked. All vitamin forms required by the SMPRs were combined in the spiking solution except thiamine triphosphate, which was not available for purchase. Over-spike levels for each form were targeted to mimic ratios previously reported in infant formulas and milk: thiamine monophosphate and thiamine diphosphate were spiked at 12.3% and 8.6% of total B 2 ; riboflavin phosphate and flavin adenine dinucleotide were spiked at 18.1% and 8.8% of total B 2 ; nicotinic acid was spiked at 7.2% of total B 3 ; and pyridoxal Accuracy

Table 2. Calibration curve % deviation from true concentration is reported at each calibration level a Standard Overall ( n = 12) Thiamine Riboflavin Niacin Nicotinic acid Pyridoxal

Pyridoxamine Pyridoxine

WS1

Recovery (%)

99.2

99.6

98.3

100.5

104.4

101.3

100.9

RSD (%)

3.5

6.6

5.9

7.3

15.4 97.9 10.2 95.3 10.7

7.2

2.3

WS2

Recovery (%)

100.4

99.9

100.8

99.1

100.3

98.8

RSD (%)

3.2

4.9

5.1

4.7

3.5

1.6

WS3

Recovery (%)

100.3

100.3

101.2

100.0

97.5

100.0

RSD (%)

2.1

3.7

3.2

2.2

4.6

1.3

WS4

Recovery (%)

100.5

100.6

100.5

100.4

104.3

99.8

100.4

RSD (%)

2.2

3.4

3.4

2.6

9.9

3.6

2.0

WS5

Recovery (%)

99.6

99.6

99.3

99.8

98.9 10.9

100.7

99.9

RSD (%)

2.4

1.9

2.5

1.8

4.0

1.4

WS6

Recovery (%)

100.1

100.1

100.2

100.0

100.1

99.8

100.0

RSD (%)

2.2

1.1

3.0

1.8

8.7

3.2

1.1

a  The reported value is averaged across 6 days and reported along with %RSD.