AOAC SPIFAN Review Team Meeting Book-July 22, 2015

B-VIT-01 FOR ERP/WORKING GROUP USE ONLY DO NOT DISTRIBUTE

Simultaneous Determination of Thiamine HCl, Riboflavin, Niacin (Nicotinic Acid and Nicotinamide), Pantothenic acid, Vitamin B6 (Pyridoxine, Pyridoxal and Pyridoxamine) and Biotin in Infant Formula and Adult Nutritionals

CONFIDENTIAL METHOD FOR ERP ASSESSMENT ONLY

I. SUMMARY Water-soluble vitamins in infant formula or adult nutritional samples are extracted with a mixture of mild acid-Methanol. Quantitation of the extracted solution is carried out via LC-MS/MS using ESI in the positive mode. a. Intended use of method: The method is applicable for the concurrent quantitative analysis of Thiamine HCl, Riboflavin, Niacin (Nicotinic Acid and Nicotinamide), Pantothenic acid, Vitamin B6 (Pyridoxine, Pyridoxal and Pyridoxamine) and Biotin present in milk- and soy- based infant formula and adult nutritionals and formulas containing hydrolyzed protein. b. Critical components: Correct personal and environmental safety standards shall be used while performing this analytical method. Laboratory personnel handling solvents, acids and reagents should be knowledgeable of their potential hazards. Consult the Material Safty Data Sheets for information on the hazards and take proper precautions.Transfer solvents and acids inside efficient fume hoods and extractors. Ensure all glassware is free from chipping and hairline cracks. II. EQUIPMENT & APPARATUS Equivalents may be used unless otherwise specified.

Equipment LC-MS/MS

Description (Brand, make, model, etc.)

Agilent UHPLC 1290 series- ABSCIEX QTRAP 6500 ESI Positive mode or equivalent.

HPLC Column

Shishedo, CAPCELL PAK C18 column, MGIII-H, 100mm x 2.0mm x 3 μm or equivalent.

Vortex mixer Ultrasonic bath

Labnet VX100 or equivalent. Ultrasonik 208H or equivalent.

High speed centrifuge

Hettich Rotina 420R, with rotor for 2mL centrifuge tubes and capable of 15000 rpm at 4  C or equivalent. Mettler Teledo XP204, capable of weighing to four decimal places or equivalent. Agilent Technologies, amber, 2mL capacity, with crimp caps and PTFE seals. 50 ml PP, Amber Falcon tube with screw cap or equivalent. Memmert or equivalent. EYELA Multishaker MMS or equivalent Eppendorf, 10-100 µl, 100-1000 µl and 1-5 ml or equivalent. Witeg, class A: 2, 3, 4, 5, 10, 15, 25 ml or equivalent. Witeg, class A, amber: 1 (clear), 10, 100, 200, 1000 ml or equivalent. Filtrex, 0.2 µm, 13 mm or equivalent and MSF filter 0.2 µm, 47mm or equivalent. 2 ml PP, Amber, Axygen or equivalent.

Hot-Air oven (105  C) Analytical balance

Mechanical Shaker

HPLC vials

Centrifuge tube

Microcentrifuge tube Auto pipette with tip Volumetric pipet Volumetric flask Syringe Filter Nylon membrane

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