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6

S

alvati

et al

.:

J

ournal of

AOAC I

nternational

V

ol

.

99, N

o

.

3, 2016

where [Vit]

WSx

= vitamin concentration in the working standard

in ng/mL; [Vit]

MWS

= concentration of vitamin in the MWS

in ng/mL; Vol

MWS

= volume of the MWS fortified in working

standard in μL; and 500 = dilution factor.

(d)

 Vitamin concentration calculated in product from

analytical result:

Vit

Vit

RW

SW PW

sample

AS

[ ]

[ ]

500

=

× ×

×

where [Vit]

sample

= vitamin concentration in product, μg/kg;

[Vit]

AS

= vitamin mass in the analytical sample as calculated

from calibration curve, ng/mL; RW = reconstitution weight

(total), g, for direct weight (liquid) samples RW = 1;

SW = analytical sample weight, g; PW = powder weight (for

reconstituted samples), g, for liquid samples, this value is 1; and

500 = dilution factor.

(e)

 For vitamins B

3

and B

6

, the reported concentration of

the individual forms is summed to report total. For example,

concentration of nicotinamide and nicotinic acid are summed to

report “Total B

3

” and concentration of pyridoxal, pyridoxamine,

and pyridoxine are summed to report “Total B

6

.” Thiamine and

riboflavin do not require this step.

Validation

Method performance was demonstrated against predefined

suitability criteria for these vitamins published in SMPRs

(1–4). Although each SMPR is slightly different, methods for

B

1

, B

2

, B

3

, and B

6

are required to achieve repeatability of ≤5%

RSD, reproducibility of ≤10% RSD, and over-spike recovery of

90–110%. This method met each of these requirements except

reproducibility, which was not evaluated. Instead, intermediate

precision is given and suggests the reproducibility requirement

will be met upon multilaboratory evaluation. Additional

measures of method performance are also discussed, including:

linearity, specificity, and robustness.

Table 1. Conditions for MS transitions on a Waters TQ-S are given along with retention time windows

Compound

Function No.

Start, min

End, min Molecular ion Fragment ion Cone voltage

Collision

energy (V)

Dwell time, s

Nicotinamide

a

1

2.71

3.20

122.9

80.1

20.0

16.0

0.025

Nicotinamide

1

2.71

3.20

122.9

96.0

20.0

16.0

0.025

2

H

4

-nicotinamide

a

1

2.71

3.20

127.0

84.0

20.0

16.0

0.025

2

H

4

-nicotinamide

1

2.71

3.20

127.0

100.0

20.0

16.0

0.025

Nicotinic acid

a

2

0.50

1.70

124.0

80.0

20.0

16.0

0.025

Nicotinic acid

2

0.50

1.70

124.0

106.0

20.0

16.0

0.025

2

H

4

-nicotinic acid

a

2

0.50

1.70

128.0

84.1

20.0

16.0

0.025

2

H

4

-nicotinic acid

2

0.50

1.70

128.0

109.0

20.0

16.0

0.025

Pyridoxal

3

1.76

2.70

168.0

94.0

20.0

22.0

0.025

Pyridoxal

a

3

1.76

2.70

168.0

150.0

20.0

12.0

0.025

2

H

3

-pyridoxal

3

1.76

2.70

171.0

97.0

20.0

22.0

0.025

2

H

3

-pyridoxal

a

3

1.76

2.70

171.0

153.0

20.0

12.0

0.025

Pyridoxamine

4

0.50

1.70

169.0

134.0

20.0

20.0

0.025

Pyridoxamine

a

4

0.50

1.70

169.0

152.0

20.0

12.0

0.025

2

H

3

-pyridoxamine

4

0.50

1.70

172.0

136.0

20.0

20.0

0.025

2

H

3

-pyridoxamine

a

4

0.50

1.70

172.0

155.0

20.0

12.0

0.025

Pyridoxine

a

5

2.41

3.00

170.0

134.0

20.0

18.0

0.025

Pyridoxine

5

2.41

3.00

170.0

152.0

20.0

12.0

0.025

13

C

4

-pyridoxine

a

5

2.41

3.00

174.0

138.0

20.0

18.0

0.025

13

C

4

-pyridoxine

5

2.41

3.00

174.0

156.0

20.0

12.0

0.025

Thiamine

6

3.01

3.60

265.1

81.0

20.0

30.0

0.025

Thiamine

a

6

3.01

3.60

265.1

122.0

20.0

12.0

0.025

13

C

4

-thiamine

6

3.01

3.60

269.0

81.0

20.0

30.0

0.025

13

C

4

-thiamine

a

6

3.01

3.60

269.0

122.0

20.0

12.0

0.025

Riboflavin

7

4.21

5.00

377.0

172.0

20.0

35.0

0.025

Riboflavin

a

7

4.21

5.00

377.0

243.0

20.0

20.0

0.025

13

C

4

,

15

N

2

-riboflavin

7

4.21

5.00

383.0

175.0

20.0

35.0

0.025

13

C

4

,

15

N

2

-riboflavin

a

7

4.21

5.00

383.0

249.0

20.0

20.0

0.025

Although the mass transitions are expected to remain the same across instrument platforms, the other parameters may need to be adjusted to maximize

sensitivity.

a

 Indicates primary transition used in quantitation.

110