AOAC SPIFAN Review Team Meeting Book-July 22, 2015

B-VIT-01 FOR ERP/WORKING GROUP 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

acid-5mM Ammonium formate

Remark: USP standard expiry information can be obtained using the following website: http://www.usp.org/reference-standards?gclid=CNaTqtu4wMUCFRcOjgodZXcAog

Preparation No. Procedure 1

75 mM Formic acid-20% MeOH mixture solution : 3.11 mL Formic acid and 200 mL MeOH in a 1000mL volumetric flask. Adjust and make up to volume with water. 1 N HCl : 83.33 mL of 37%HCl diluted to 1000mL with water. 50 mM HCl : 25 mL of 1 N HCl diluted to 500 mL with water. 1 % Formic acid : 5 mL Formic acid diluted to 500 mL with water. Weight 0.001x g IS Thiamin Cl, dissolved and diluted to 50 mL with water. Weight 0.001x g IS Riboflavin, dissolved and diluted to 50 mL with water. Weight 0.01xx g IS Nicotinamide, dissolved and diluted to 50 mL with water. Weight 0.001x g IS Nicotinic acid, dissolved and diluted to 50 mL with water. Weight 0.0015 g IS Pantothenic acid, Calcium salt monohydrate, dissolved and diluted to 50 mL with water. Weight 0.0015 g IS Pyridoxine HCl, dissolved and diluted to 50 mL with water. Weight 0.001x g IS Pyridoxal HCl, dissolved and diluted to 50 mL with water. Weight 0.001x g IS Pyridoxamine.2HCl, dissolved and diluted to 50 mL with water. Weight 0.001x g IS Biotin, dissolved and diluted to 50 mL with water. Pipet each stock internal standard solution into 100 mL volumetric flask as follows: 4 ml stock IS Thiamine HCl 8 ml stock IS Riboflavin 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. 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. Dry Nicotinamide in dessicator approx. 4 hours. Weight 0.05xx g Nicotinamide, dissolved and diluted with 50 mM HCl in 50 mL volumetric flask. Weight 0.005x g Nicotinic acid, dissolved and diluted with 50 mM HCl in 50 mL volumetric flask. 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. Weight 0.01xx g Pyridoxine HCl, dissolved and diluted with 50 mM HCl in 50 mL volumetric flask. Weight 0.005x g Pyridoxal HCl, dissolved and diluted with 50 mM HCl in 50 mL volumetric flask. 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. 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.

2 3 4 5 6 7 8 9

10 11 12 13 14

15

16

17

18

19

20

21

22

23

24

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

Page 5

PAGE 5 OF 13