SPIFAN Nutrients ERP Book_9-29-15

BVit-01 FOR ERP 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

23

Weight 0.005x g Pyridoxamine.2HCl, dissolved and diluted with 50 mM HCl in 50 mL volumetric flask. Weight 0.005x g Biotin, dissolved with water and 1 ml NH4OH, diluted with water in 50 mL volumetric flask.

24

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. Pipet 0.5 ml of Mixed water-soluble vitamin intermediate standard solution I into 100 mL volumetric flask. Make up to volume with water. 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. 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.

27

28

29

III. PROCEDURE A.

Calculation of Standard Concentrations A.1 Stock water-soluble vitamins solution (WSV)

x V W )mL / mcg ( WSV .  100 P x

TS

Conc

1000000 xFx

100

1

2

Where: Conc. WSV

= Concentration of stock water-soluble vitamin standard solution (mcg/mL)

W

= Standard weight (g)

V P

= Pipetted volume of stock water-soluble vitamin standard solution (mL)

= % Purity of each standard or % Assay = Convert factor of % Purity to mass fraction (g/g)

100 1

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

PAGE 6 OF 13