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S

alvati

et al

.:

J

ournal of

AOAC I

nternational

V

ol

.

99, N

o

.

3, 2016 

7

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

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

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).

Precision

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.

Robustness

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

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

7.2

2.3

WS2

Recovery (%)

100.4

99.9

100.8

99.1

97.9

100.3

98.8

RSD (%)

3.2

4.9

5.1

4.7

10.2

3.5

1.6

WS3

Recovery (%)

100.3

100.3

101.2

100.0

95.3

97.5

100.0

RSD (%)

2.1

3.7

3.2

2.2

10.7

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

100.7

99.9

RSD (%)

2.4

1.9

2.5

1.8

10.9

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.

111