2016 OMB Summer Meeting

Multi Laboratory Test – AOAC First Action Method 2012.22 Ascorbic Acid (Vitamin C) in Infant Formula and

Adult/Pediatric Nutritionals.

RDLS-RD150006

E. Campos Giménez (NRC, Lausanne)

Page 16

CONFIDENTIAL

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This document may not be reproduced or disclosed to third parties without prior authorisation.

Annex B: Method,

Continued

Preparation of standards

Warning: Vitamin C is sensitive to light and oxygen, conduct operations under subdued light,

or use amber glassware. Keep all solutions away from direct light.

Ascorbic acid stock solution (500 µg/mL).-

Into a 25 mL amber glass volumetric flask,

weigh 12.5 mg ascorbic acid. Dissolve and make up to volume with TCEP solution. This

solution can be kept for 3 months if stored at 4 °C away from light.

b. Ascorbic acid intermediate standard solution (50 µg/mL).-

Into a 10 mL amber glass

volumetric flask, pipet 1.0 mL of stock solution. Make up to volume with TCEP solution.

This solution can be used for 1 month if stored at 4 °C away from light.

c. Ascorbic acid calibration standard solutions (0.5, 1.0, 2.0, 3.0, 5.0, 7.5, 10.0 µg/mL).-

Into 10 mL amber glass volumetric flasks, pipet 0.1, 0.2, 0.4, 0.6, 1.0, 1.5 and 2.0 mL of

intermediate standard solution. Make up to volume with mobile phase.

Sample preparation

a. Reconstitute powder samples

i. Weigh 25.0 g ± 0.1 g (m1) of powder into a brown glass 250 mL beaker. Add 10 mg

TCEP

ii. Add 200.0 g ± 0.1 g (m2) warm water (40 °C ± 5 °C). Mix well until complete

dissolution making sure there are no lumps.

Note: the powder samples must be reconstituted freshly before the analysis. Discard any

unused portion.

b. Extraction

i. Weigh 2.0 g ± 0.1 g (m) of liquid or reconstituted sample into a 10 mL (V

) amber-

glass volumetric flask.

ii. Add 4 mL of TCEP solution and 2 mL of TCA 15%.

iii. Bring up to volume with water.

iv. Filter solution through folded paper filter.

v. Transfer 1.0 mL (V

) of filtrate into a 10 mL (V

) amber glass volumetric flask

containing 1 mL acetate solution (pH 5.4) and bring up to volume with mobile phase.

Filter about 2 mL through 0.22 µm membrane into a LC vial

Continued on next page

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