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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