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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 5 OF 13
53
Mixed water-soluble
vitamin working standard
solution 1-16
28
Freshly
prepared
54
Mobile Phase A: 0.04%
HFBA-0.05% Formic
acid-5mM Ammonium
formate
29
2 weeks
Room
temperature
Remark: USP standard expiry information can be obtained using the following website:
http://www.usp.org/reference-standards?gclid=CNaTqtu4wMUCFRcOjgodZXcAogPreparation No. Procedure
1
75 mM Formic acid-20% MeOH mixture solution : 3.1 mL Formic acid and 200 mL
MeOH in a 1000mL volumetric flask. Adjust and make up to volume with water.
2
300 mM Formic acid-20% MeOH mixture solution : 12.5 mL Formic acid and 200 mL
MeOH in a 1000mL volumetric flask. Adjust and make up to volume with water.
3
1 N HCl : 83.33 mL of 37%HCl diluted to 1000mL with water.
4
50 mM HCl : 25 mL of 1 N HCl diluted to 500 mL with water.
5
1 % Formic acid : 5 mL Formic acid diluted to 500 mL with water.
6
Weight 0.001x g IS Thiamin Cl, dissolved and diluted to 50 mL with water.
7
Weight
0.001x g IS Riboflavin, dissolved and diluted to 50 mL with water.
8
Weight
0.01xx g IS Nicotinamide, dissolved and diluted to 50 mL with water.
9
Weight
0.001x g IS Nicotinic acid, dissolved and diluted to 50 mL with water.
10
Weight
0.0015 g IS Pantothenic acid, Calcium salt monohydrate, dissolved and diluted
to 50 mL with water.
11
Weight
0.0015 g IS Pyridoxine HCl, dissolved and diluted to 50 mL with water.
12
Weight
0.001x g IS Pyridoxal HCl, dissolved and diluted to 50 mL with water.
13
Weight
0.001x g IS Pyridoxamine.2HCl, dissolved and diluted to 50 mL with water.
14
Weight
0.001x g IS Biotin, dissolved and diluted to 50 mL with water.
15
Pipet each stock internal standard solution into
100 mL volumetric flask as follows:
4 ml stock IS Thiamine HCl
8 ml stock IS Riboflavin
2 ml stock IS Nicotinamide
15 ml stock IS Nicotinic acid
10 ml stock IS Pantothenic acid
0.5 ml stock IS Pyridoxine
0.5 ml stock IS Pyridoxal
0.6 ml stock IS Pyridoxamine
0.8 ml stock IS Biotin
dissolved and diluted to 100 mL with water.
16
DryThiamine HCl at 105 ± 5
◦
C 2 hours. Weight
0.01xx g Thiamine HCl, dissolved and
diluted with 50 mM HCl in
50 mL volumetric flask.
17
Dry Riboflavin at 105 ± 5
◦
C 2 hours. Weight
0.005x g Riboflavin, dissolved and diluted
with 1 % Formic acid in
100 mL volumetric flask.
18
Dry Nicotinamide in
dessicator approx.
4 hours. Weight
0.05xx g Nicotinamide,
dissolved and diluted with 50 mM HCl in
50 mL volumetric flask.
19
Weight 0.005x g Nicotinic acid, dissolved and diluted with 50 mM HCl in
50 mL
volumetric flask.
20
Dry Calcium pantothenate at 105 ± 5
◦
C 3 hours. Weight
0.025x g Calcium
pantothenate, dissolved and diluted with water in
50 mL volumetric flask.
21
Weight
0.01xx g Pyridoxine HCl, dissolved and diluted with 50 mM HCl in
50 mL
volumetric flask.
22
Weight
0.005x g Pyridoxal HCl, dissolved and diluted with 50 mM HCl in
50 mL
volumetric flask.
BVit-01
FOR ERP USE ONLY
DO NOT DISTRIBUTE