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Abbott Nutrition Division
Simultaneous Determination of Total Vitamin B
6,
B
2,
B
3
and B
1
in Infant
Formula Products by LC-MS/MS Using Enzymatic Digestion
Table 6. Example Pyridoxine Standard Curve.
Standard
[PN] ng/mL
Response Ratio
WS1
3.195
0.312
WS2
7.984
0.779
WS3
15.976
1.592
WS4
31.952
2.931
WS5
79.84
7.48
WS6
159.76
18.113
Slope = 0.1123
y-intercept = -0.3894
Coefficient of Determination (r
2
) = 0.9961
7.
Liquid and Powder Product Calculation
MassLynx is used to calculate both the vitamin concentration in the analytical sample and in the
original sample. The following equation is used for this calculation
PW
FSW ASW RW Vit
Vit
AS
sample
× × ×
=
] [
] [
where:
[Vit]
sample
: Vitamin concentration in Product Sample,
m
g/kg
[Vit]
AS
: Vitamin concentration in the Analytical Sample; ng/g
RW: Reconstitution Weight (total), g. For direct weight (liquid) samples RW=1
ASW: Analytical Sample Weight, g
-1
. Due to limitations in MassLynx, the sample
weight must be entered as the inverse, i.e. for a 0.5 sample, SW=2.0
FSW: Final Sample Weight, g. As each compound has an internal standard added to
the sample this value is set to 25.0g for every sample
PW: Powder Weight (for reconstituted samples), g. For direct weight samples, this
value is 1
Example:
10.04 g of Similac Pwd. is reconstituted into laboratory water (PW) resulting in a final
recon weight (RW) of 102.386 g. 1.0298 g of slurry (ASW) is weighed into a tarred
centrifuge tube and prepared per method with a final sample weight (FSW) of 25.0. The
Pyridoxine concentration in the analytical sample ([Vit]
sample
) is determined to be 8.6318
ng/g. The Pyridoxine concentration in the original Similac pwd. sample is calculated as
follows:
kg g
g ng
g
g
g
g
g ng
PN
sample
/ 4.
2143
/ 4.
2143
04.10
0.25
9711 .0
386 . 102 /
6318 .8
] [
1
m
=
=
×
×
×
=
−
41
BVIT-02
FOR ERP/WORKING GROUP USE ONLY
DO NOT DISTRIBUTE