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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 USE ONLY

DO NOT DISTRIBUTE