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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.
Candidates for 2016 Method of the Year
203