Review Team (February 2016)

BVit-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Determination of B-Vitamins in Infant Formula and Adult/Pediatric Formulas ESI LC-MSMS Applicable for the determination of B-Vitamins (Thiamine, Riboflavin, Nicotinic acid, Niacinamide, Pantothenic acid, Pyridoxine, Biotin and Folic acid) in infant, adult, and/or pediatric formula (powders, ready to feed liquids and liquid concentrates). The LOQs of Biotin and Folic acid are 0.05 mg/kg and for remaining vitamins, 0.5 mg/kg. Caution: Refer to Material Safety Data Sheet (MSDS) of chemicals prior to use, and follow the suggested personal protective equipment. A. Principle: Samples are extracted with water in presence of ascorbic acid and adjusted to pH 4.8 with ammonia solution to precipitate proteins. Extract is filtered and subjected to LC-MSMS. Results are quantified against certified reference materials by external standard method. B. Apparatus: a) Liquid Chromatograph: Agilent UHPLC Infinity series with Binary Solvent Manager capable of 1200 bar pressure. b) Column: Zorbax RRHD, C18, 3.0 x 100 mm, 1.8 μm.

c) Detector: Agilent 6460A triple quadrupole mass spectrometer. d) Injector: Agilent Infinity Series Auto sampler with column oven. e) Data System: Mass Hunter, Agilent. f) pH meter. g) Syringe filters: 0.22 μm, PTFE. C. Reagents: a) Ascorbic acid , AR grade. b) Ammonium Formate , MS grade. c) Formic acid , MS grade. d) Ammonia , Mass fraction of about 30%. e) Acetic acid , Glacial, AR grade. f) Potassium Hydrogen Carbonate , AR grade. g) Methanol, absolute, ULC/MS Grade h) De-ionized laboratory water - ≥ 18 Mohm/cm i) Volumetric flasks (Low actinic), 10ml, 25ml, 50ml, 100ml D. Standards: a) Thiamin Hydrochloride ,from Supelco, Cat. No. 47858 b) Riboflavin , from Supelco, Cat. No. 4-7861 c) Niacinamide or Nicotinamide , from Sigma, Cat. No. 72340 d) Nicotinic acid , from Supelco, Cat. No. 47864

e) D-Pantothenic acid hemicalcium salt , from Sigma, Cat. No. 21210 f) Pyridoxine Hydrochloride , from Sigma-Aldrich, Cat. No. P9755

g) Biotin , Sigma-Aldrich, Cat. No. B4501 h) Folic acid , from Sigma, Cat. No. F7876 E. Solutions Preparation: a) Mobile Phase A: Dissolve 0.63 g of ammonium formate in water, add 0.5ml formic acid and dilute to 500ml with water. Mix and degas for 10 min in ultrasonic bath. b) Mobile Phase B: Dissolve 0.63 g of ammonium formate in methanol, add 0.5ml formic acid and dilute to 500ml with methanol. Mix and degas for 10 min in ultrasonic bath.

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