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Single Laboratory Validation – AOAC First Action Method 2012.22 Vitamin C in Infant Formula and Adult/Pediatric
Nutritional Formula by UPLC-UV
Date of Issue: February 14
th
, 2013
RDLS-RD130018
E.Campos Giménez (NRC/QS, Lausanne)
Page 3
Methodology and Trials
Materials and Methods
Samples
A SPIFAN Single Laboratory Validation Material Test Kit was used (Attachment
2). The kit is aimed to represent the whole range of products in the scope of the
project (Infant Formulas and Adult Nutritionals made from any combination of
milk, soy, rice, whey, hydrolyzed protein, starch and amino acids, with and
without intact protein). It is composed of 6 non fortified (placebo) products and
12 fortified products. Furthermore, it includes a Certified Reference Material
(SRM 1849a) with certified value for vitamin C.
Analytical
method:
AOAC 2012.22
Sample reconstitution
Powder samples were reconstituted by dissolving 25 g of powder sample in
200 g warm water (40 °C). The SRM was reconstituted by dissolving 10 g of
powder in 90 g warm water (40 °C). Ten (10) mg of TCEP (Tris(2-carboxyethyl)-
phosphine hydrochloride) were added to the samples before reconstitution to
prevent oxidation.
Extraction
Two (2) grams of reconstituted or RTF sample were weighed into a 10 mL
amber-glass volumetric flask. 4 mL of TCEP solution (250 µg/mL) and 2 mL
trichloroacetic acid (TCA) solution (15 %) added before making up to volume
with water. After filtration through paper filter, 1 mL of filtrate was transferred to
a 10 mL amber-glass volumetric flask containing 1 mL acetate solution (pH 5.4)
and made up to volume with water.
Standard solutions
Stock standard solution (500 µg/mL) was diluted in mobile phase to obtain a
50 µg/mL intermediate solution. Into 10 mL amber-glass volumetric flasks,
aliquots of the intermediate solution were pipette and made up to volume with
mobile phase to yield working standard solutions. (0.5, 1.0, 2.0, 3.0, 5.0, 7.5 and
10.0 µg/mL).
Liquid chromatography
Five (5) µL of final extract were injected onto a UPLC system (Waters Acquity©)
equipped with a Waters Acquity UPLC BEH C18, 1.75 µm; 2.1 x 100 mm
column. Mobile phase was: sodium acetate, 25 mmol/L, pH 5.4, 1.6 g
decylamine, 1 % acetonitrile, 50 mg/L TCEP. Flow rate was 0.35 mL/min, with a
run time of 4.0 minutes.
The column was rinsed after each analytical series with 50 % acetonitrile in
water for 10 min at 0.4 mL/min.
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VitC-03/2012.22
For ERP Use Only
DO NOT DISTRIBUTE
AOAC OMB Meeting Book
313