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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
PAGE 7 OF 13
Pyridoxal:
MwPyridoxal
=
Molecular weight of Pyridoxal = 167.162 g/mol
MwPyridoxal HCl
=
Molecular weight of Pyridoxal HCl = 203.62 g/mol
62. 203
162 . 167
Mw
Mw
F
Cl
PyridoxalH
Pyridoal
Pyridoxamine:
MwPydoxamine
=
Molecular weight of Pyridoxamine = 168.19 g/mol
MwPyridoxamine2HCl =
Molecular weight of Pyridoxamine 2HCl = 241.11 g/mol
11. 241
19. 168
Mw
Mw
F
HCl
2emin
Pyridoxa
emin
Pyridoxa
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.
2
For liquid products, transfer 15mL of thoroughly homogenized sample directly to a 50mL
centrifuge tube.
3
For SRM1849a; weight 1 g in 50 mL centrifuge tube. Dissolve using 25 ml warm water (about
40-45°C).
4
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.
5
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.
6
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.
7
Transfer extracted sample to 2 ml microcentrifuge tubes. Centrifuge at 15000 rpm at 4
C for
20 minutes
8
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
C.Chromatography
Parameters
Column
Shishedo, CAPCELL PAK C18 column, MGIII-
H, 100mm x 2.0mm x 3μm or equivalent.
Mobile Phase A
0.04% HFBA-0.05% Formic acid-5mM Ammonium formate in water
Mobile Phase B
MeOH
Flow Rate
0.4mL/min
Injection Volume
20 µL
Wash location
Flush port
Wash time
5 seconds
Tray temperature
4±2 °C
Column Oven
35±2 °C
Run Time
12 minutes
MSMS condition
ESI Positive mode
MRM detection
70 seconds
B-VIT-01
FOR ERP/WORKING GROUP USE ONLY
DO NOT DISTRIBUTE
Page 7