AOAC SPIFAN Review Team Meeting Book-July 22, 2015

BVIT-02 FOR ERP/WORKING GROUP USE ONLY DO NOT DISTRIBUTE

Abbott Nutrition Division Simultaneous Determination of Total Vitamin B 6, B 2, B 3 in Infant Formula Products by LC-MS/MS Using Enzymatic Digestion and B 1

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

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