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

Typical LC Time Program

Step

Total Time (min)

Flow rate (µL/min)

A (%)

B(%)

0 1 2 3 4 5 6 7

0.00 0.05 1.50 5.00 6.50 8.50 9.00

400 400 400 400 400 400 400 400

100 100

0 0

85 80 10 10

15 20 90 90

100 100

0 0

12.00

Stop Time

12 minutes

Retention Time

Nicotinic acid approximately 2.0-2.5 minutes Nicotinamide approximately 2.0-2.5 minutes Pyridoxal approximately 2.8-3.3 minutes Pantothenic acid approximately 2.8-3.3 minutes Pyridoxamine approximately 2.8-3.3 minutes Pyridoxine approximately 3.1-3.6 minutes Thiamin hydrochloride approximately 4.0-4.5 minutes Biotin approximately 6.0-6.5 minutes Riboflavin approximately 6.3-6.8 minutes

D. System Suitability Requirements a. Plot calibration between Concentration (ng/ml) and Area ratio ≥ 6 points . The correlation coefficient of each calibration should be not less than 0.995. % Error of concentration should be ≤ 10% . b. When stable baseline is obtained, inject a middle level of working standard three times in succession and determine the %RSD, The %RSD should be not more than 3%. c. Analyte a middle level of working standard between 10 to 12 samples. % Error od centration should be ≤ 10% d. For the confirmation, ion ratio should be with in 20%

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