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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

Thiamin

250

Riboflavin

800

QS to the mark with laboratory water.

Expiration: Prepare fresh daily.

i)

Working Standard Solutions

NOTE: As a general rule, use the Gilson pipette with the smallest maximum volume to

dispense the specified volumes of IWSS and ISSM solutions.

Combine the IWSS and ISSM solution volumes shown in Table 8 into a 50-mL self-standing

centrifuge tube. Pipette in 5 mL of enzyme solution, and then add 50mM Ammonium Formate up

to a final volume of 30mL.

Expiration: Prepare fresh daily.

Table 5. Working Standard solution preparation.

Solution

IWSS

Volume

(µL)

Gilson Pipette

(recommended)

ISSM

Volume

(µL)

Gilson Pipette

(recommended)

Final

Volume

(mL)

WS 1

25

M25

50

M100

30

WS 2

50

M100

50

M100

30

WS 3

100

M100

50

M100

30

WS 4

250

M250

50

M100

30

WS 5

500

M1000

50

M100

30

WS 6

1000

M1000

50

M100

30

.

NOTE: The accurate and precise addition of the IWSS and the ISSM solutions are critical

to the accuracy and precision of the method. Extreme care must be taken in

aliquoting the IWSS into the standards and the ISSM into the standards and the

samples.

NOTE: Due to the addition of internal standards for each compound the final volume of

the working standards is not a critical volume. Small errors (on the order of

approximately 1-2 mL) in the final volume will not adversely affect the calibration

accuracy.

36

BVit-02

FOR ERP USE ONLY

DO NOT DISTRIBUTE