229 / 620
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
Pyridoxine:
MwPyridoxine
=
Molecular weight of Pyridoxine = 169.18 g/mol
MwPyridoxine HCl
=
Molecular weight of Pyridoxine HCl = 205.64 g/mol
64. 205
18. 169
M
M
F
HCl
Pyridoxine
Pydoxine
W
W
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; weight 25g (W1) sample into a 250mL plastic beaker. Add 200g water
and weight to 225g, (W2). Weight 25g (W3) of the reconstituted sample to a 50 mL centrifuge
tube.
Sample weight (powder equivalent) g = (W1 x W3 ) / W2 = (25x25)/225 = 2.78 g. The actual
sample weight is used for the calculation.
2
For liquid products, weight 15 g or pipet 15 ml of thoroughly homogenized sample directly to a
50mL centrifuge tube. Dilute to 25 ml with water. The sample weight or volume can be
decreased, depends on the concentration of analyte in sample. The example, take 1 or 2 g for
the liquid concentraes.
3
For SRM1849a; weight 1 g in 50 mL centrifuge tube. Dissolve using 25 ml water
.
4
To the above sample solutions, the extracting solution is added in the following:
- Milk based sample: add 10mL 75 mM Formic acid-20% MeOH mixture solution
- Soy based sample: add 10mL 300 mM Formic acid-20% MeOH mixture solution
- SRM1849a: add 3.2 ml 75 mM Formic acid-20% MeOH mixture solution
If the liquid sample is took lower than 15 ml, the extracting solution can be decrease as
proportion of sample, or add for adjust pH of sample solution to pH 3-4 for protein precipitation.
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. A few drops of MeOH can be used for
antifoaming.
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 filter pass though 0.2 µm Nylon
membrane. Determine water-soluble vitamins by LCMSMS
C.Chromatography
BVit-01
FOR ERP USE ONLY
DO NOT DISTRIBUTE