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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 6 OF 13
23
Weight 0.005x g Pyridoxamine.2HCl, dissolved and diluted with 50 mM HCl in
50 mL
volumetric flask.
24
Weight
0.005x g Biotin, dissolved with water and 1 ml NH4OH, diluted with water in
50
mL volumetric flask.
25
Pipet
stock standard solution into
50 ml volumetric flask as follows :
2.5 ml 100 mcg/ml Thiamine HCl stock standard solution
15 ml 100 mcg/ml Riboflavin stock standard solution
4.3 ml 300 mcg/mL Nicotinamide stock standard solution
1 ml 100 mcg/ml Nicotinic acid stock standard solution
5 ml 150 mcg/ml Pantothenic acid stock standard
3 ml 100 mcg/ml Pyridoxine stock standard solution
0.7 ml 100 mcg/ml Pyridoxal stock standard solution
0.3 ml 100 mcg/ml Pyridoxamine stock standard
0.15 ml 100 mcg/ml Biotin stock standard solution
Diluted and make to volume with water.
26
Pipet
3 ml of Mixed water-soluble vitamin intermediate standard solution I into 100 mL
volumetric flask. Make up to volume with water.
27
Pipet
0.5 ml of Mixed water-soluble vitamin intermediate standard solution I into 100 mL
volumetric flask. Make up to volume with water.
28
Pipet 0.1 ml Mixed water-soluble vitamin intermediate internal standard solution I into
1
mL volumetric flask, Pipet 0.025, 0.05, 0.1, 0.15, 0.20 and 0.25 Mixed water-soluble
vitamin intermediate standard solution III and 0.05,
0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8
and 0.9 mL Mixed water-soluble vitamin intermediate standard solution II into each
volumetric flask. Make up to volume with water.
29
Mobile Phase A: 0.04% HFBA-0.05% Formic acid-5mM Ammonium formate : 0.4 mL
Heptafluorobutylric acid, 0.5 mL Formic acid and 3.15 g Ammonium formate in water.
Adjust and make up to 1000mL with water.
III. PROCEDURE
A.
Calculation of Standard Concentrations
A.1 Stock water-soluble vitamins solution (WSV)
1000000
xFx
100
TS
x
100
P
x
V
W
)mL / mcg ( WSV .
Conc
2
1
Where:
Conc. WSV
= Concentration of
stock water-soluble vitamin standard solution (mcg/mL)
W
= Standard weight
(g)
V
= Pipetted volume of stock water-soluble vitamin standard solution (mL)
P
= % Purity of each
standard or % Assay
100
1
= Convert factor of % Purity
to mass fraction (g/g)
TS
= % Total solid = 100-%moisture = Factor for dry weight basis or anhydrous form
100
2
= Convert factor of % Total solid to mass fraction (g/g)
1000000
= Convert factor from g to mcg
F
= Convert factor for standard
Pantothenic acid:
1 mg Calcium pantothenate = 0.92 g Pantothenic acid, Then F = 0.92
BVit-01
FOR ERP USE ONLY
DO NOT DISTRIBUTE