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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

Weigh 14.0

±

0.1 mg into a tarred weighing vessel. Quantitatively transfer to a 25-mL

volumetric flask with laboratory water and dilute to volume with laboratory water. Mix well

and transfer to a 50-mL amber bottle and Store refrigerated (2˚-8˚C).

Expiration: Until exhausted or evidence of contamination.

f)

Stable-Isotope Labeled Compounds – Internal Standard Stock Mixture (ISSM)

i)

Combine the stable-isotope labeled internal standard stock solutions in a 5:1:1:1:1:1 volume ratio

of Lab Water:

2

H

3

-Pyridoxal:

13

C

4

-Pyridoxine:

2

H

3

-Pyridoxamine:

13

C

3

-Thiamine Chloride:

13

C

4

,

15

N

2

-Riboflavin Stock Solution

For example, a sample set containing 18 samples and two

sets of 6 working standards, a minimum ISSM volume of approx. 1.5 mL is required. A 2.0

mL (allowing for excess) volume of ISSM is prepared by mixing 1250

m

L Lab Water with 250

m

L of

2

H

3

-Pyridoxal, 250

m

L of

13

C

4

-Pyridoxine, and 250

m

L of

2

H

3

-Pyridoxamine Labeled

Internal Standard Stock Solutions. Mix solution well prior to use.

Expiration: Prepare fresh daily.

NOTE: Prepare a sufficient volume of this mixture on each day of analysis to allow

for all sample preparations and standard solutions to be inoculated with 50

m

L from one ISSM solution.

g)

Native Compounds – Stock Standard Solutions

i)

Pyridoxine Stock Solution; Approximate Concentration: 1.2 mg/mL

Weigh approximately 15 mg (

±

0.50mg) of Pyridoxine Hydrochloride into a weighing funnel

or other appropriate weighing vessel. Quantitatively transfer to a 100-mL volumetric flask

with laboratory water. After mixing thoroughly, QS to volume with laboratory water.

Store refrigerated (2˚-8˚C).

Expiration: 3 months.

ii)

Pyridoxal Stock Solution; Approximate Concentration: 1.2 mg/mL

Weigh approximately 15 mg (

±

0.50mg) of Pyridoxal Hydrochloride into a weighing funnel

or other appropriate weighing vessel. Quantitatively transfer to a 100-mL volumetric flask

with laboratory water. Mix thoroughly, then QS to volume with laboratory water.

Store refrigerated (2˚-8˚C).

Expiration: 3 months.

34

BVit-02

FOR ERP USE ONLY

DO NOT DISTRIBUTE