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

Pyridoxal: MwPyridoxal

= =

Molecular weight of Pyridoxal = 167.162 g/mol Molecular weight of Pyridoxal HCl = 203.62 g/mol

MwPyridoxal HCl

Mw

162 . 167

Pyridoal

F





Mw

62. 203

Cl PyridoxalH

Pyridoxamine: MwPydoxamine

=

Molecular weight of Pyridoxamine = 168.19 g/mol Molecular weight of Pyridoxamine 2HCl = 241.11 g/mol 11. 241 19. 168 Mw Mw F HCl 2emin Pyridoxa emin Pyridoxa  

MwPyridoxamine2HCl =

B. Sample Preparation

Step

Task

1

For powder samples; transfer 25g (W1) accurately weighed to a 200mL plastic beaker. Dissolve using warm water (about 40-45°C), cool, make up to 200mL(V1) with water. Pipet 25mL (V2) of the reconstituted sample to a 50 mL centrifuge tube. Sample weight (powder equivalent) g = (W1 x V2 ) / V1 = (25x25)/200 = 3.125 g. The exactly sample weight is used for the calculation. For liquid products, transfer 15mL of thoroughly homogenized sample directly to a 50mL centrifuge tube. For SRM1849a; weight 1 g in 50 mL centrifuge tube. Dissolve using 25 ml warm water (about 40-45°C). To the above sample solutions, add 10mL 75 mM Formic acid-20% MeOH mixture solution, except SRM1849a add 3.2 ml 75 mM Formic acid-20% MeOH mixture solution. Tightly cap, mix the sample by inverting the tubes for 10 second and place the tubes in a Ultrasonic bath for 30 minutes. Remove the samples from the ultrasonic. Transfer extracted sample solutions to 100 mL Volumetric flasks. Rinse the tubes with water 3- 4 times and make up volume to a mark with water. Transfer extracted sample to 2 ml microcentrifuge tubes. Centrifuge at 15000 rpm at 4  C for 20 minutes Pipet 0.5 mL of clear extracted sample solutions to 15 ml test tubes that contained 0.1 mL Mixed ISTD Int I and 0.4 mL water. Vortex dilute sample and determine water-soluble vitamins by LCMSMS

2

3

4

5

6

7

8

C.Chromatography

Parameters

Column

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

Mobile Phase A Mobile Phase B Injection Volume Wash location Tray temperature Column Oven MSMS condition MRM detection Flow Rate Wash time Run Time

0.04% HFBA-0.05% Formic acid-5mM Ammonium formate in water

MeOH

0.4mL/min

20 µL

Flush port 5 seconds

4±2 °C 35±2 °C

12 minutes

ESI Positive mode

70 seconds

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