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 17

CONFIDENTIAL

The contents of this document constitute a trade secret and the intellectual property rights thereto belong to Nestlé S. A., Vevey, Switzerland.

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

Annex B: Method,

Continued

Analysis

a. Chromatographic conditions

i. Injection volume, 5 µL

ii. Autosampler temperature: 10 °C

iii. Column temperature: 25 °C

iv. Flow rate, 0.35 mL/min

v. Run time, 4.0 minutes

vi. Mobile phase for LC, see (D.d)

Note

At the end of each analytical series, rinse column with 50% acetonitrile:water for 10 minutes at

0.4 mL/min

System suitability test

.- Equilibrate the chromatographic system for ≥ 0.5 h. Inject a

working standard solution at least 6 times and check peak retention times and response

(peak height or area). Inject working standard solutions on a regular basis within a

series of analyses.

Calibration

.- Make single injections of each of the working standard solutions at least at

the beginning and the end of each analytical series. Establish the calibration curve (7-

point) by plotting peak response (height or area) vs ascorbic acid concentration.

Calculate the linear regression parameters. Calculate slope (S) and intercept (I).

Analysis

.- Make single injections of sample solutions.

Identification

.- Identify ascorbic acid peak in the chromatograms of the sample solutions

by comparison with the retention time and UV spectrum of the corresponding peak in the

standard solution (see example chromatograms).

Calculations

Calculate the concentration of vitamin C, in mg ascorbic acid/100 g expressed in product “as is” for

liquid products or as “reconstituted powder” for powder samples, as follows:

1000 x

x Vm x S

100 x

x V x V I)- (A



Where:

A = response (height or area) of the ascorbic acid peak obtained for the sample solution

I = intercept of the calibration curve

S = slope of the calibration curve

m = weight of the test portion, in g (2.0 g)

= volume of the test solution (volume used to dissolve the test portion) in mL (10 mL)

= volume used in the sample dilution (1.0 mL)

= volume of the final sample dilution (10 mL)

100 = conversion to 100 g basis

1000 = conversion from μg to mg.

Note: if results expressed in powder sample are needed, use the reconstitution rate to

calculate (c*225 g/25 g or c*100 g/10 g).

Continued on next page

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