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 F. Procedure a) Preparation of food samples: Add 25.0 g of powdered infant formula/nutrtionals to 200 g of water at 40 ˚C. Mix until a homogenous suspension is obtained . b) Extraction: 1) Weigh 2.5 g aliquot of homogenized sample suspension into a 50 mL polypropylene centrifuge tube. Add 25 mL of 1% Ascorbic acid solution in water and vortex for 10 minutes. 2) Adjust the pH to 4.8 ± 0.2 with aqueous ammonia. 3) Transfer quantitatively to a 50 mL volumetric flask and makeup to 50 mL with laboratory water and mix. 4) Filter an aliquot through 0.22 μm PTFE syringe filter and inject into LC-MSMS. c) Chromatographic Conditions: Table 3: Column Zorbax RRHD, 1.8 μm, 3 x 100 mm Mobile phase A 20 mM Ammomium Formate with 0.1% Formic acid in water Mobile phase B 20 mM Ammomium Formate with 0.1% Formic acid in Methanol Flow rate 0.5 mL/minute Injection Volume 5.0 μL Column Temperature 55°C Total Run Time 8.0 minutes Gradient Method Time (minutes) A % B% 0.00 99.0 1.0 0.50 99.0 1.0 0.60 90.0 10.0 5.00 15.0 85.0 5.10 99.0 1.0 8.00 99.0 1.0

Source Parameters

Parameter

ESI (+)

Gas Temp (°C) Gas Flow (l/min) Nebulizer (psi) SheathGasHeater

300

7

35

400

SheathGasFlow(l/min)

11

Capillary (V) VCharging

2500

0

3