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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

v)

α – Amylase from Aspergillus Oryzae, Sigma (A9857) or equivalent

w)

Acid Phosphotase From Potato grade II, Roche Dianostics (10 108 227 001)

x)

Drierite (desiccant), Anhydrous Calcium Sulfate, 8 mesh: (Stock# 23005), or equivalent.

y)

Glacial acetic acid: ACS reagent grade, or equivalent.

z)

Hydrochloric Acid: ACS reagent grade, or equivalent

NOTE: Drierite must be changed when the original blue color fades and

begins to turn pink.

2.

Solutions

.

a)

Mobile Phase A: 20mM Ammonium Formate in Water

Fill a 1-L volumetric flask with approximately 500 mL laboratory water. Weigh 1.3 grams of

Ammonium Formate, and add to the flask. Stir until dissolved, fill the flask to the mark with

laboratory water, and mix well. Transfer to a mobile phase solvent reservoir

.

Expiration: 3 days

b)

Mobile Phase B: Methanol

c)

50mM Ammonium Formate

Fill a 1-L volumetric flask with approximately 500 mL laboratory water. Weigh 3.2 grams of

Ammonium Formate, and add to the flask. Stir until dissolved, fill the flask to the mark with

laboratory water, and mix well. Transfer to a mobile phase solvent reservoir

.

Solution may be

scaled proportionately.

EXPIRATION: Make fresh daily

d)

Enzyme solution

This solution is scaled to the number of samples that are being analyzed (5 mL per sample). For

every 5 mLs of solution needed add, 5mg of Acid Phosphatase, 10mg Papain, and 2.0mg of α –

Amylase. Using an appropriate graduated cylinder measure out the amount of 50mM

Ammonium Formate needed for the preparation, using concentrated Formic Acid adjust the pH to

between 4.0 and 4.5, and transfer to an appropriate glass container. Add the necessary amount of

enzyme using the ratios above, and stir until dissolved.

EXPIRATION: Make fresh daily

32

BVit-02

FOR ERP USE ONLY

DO NOT DISTRIBUTE