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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 15
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Annex B: Method
AOAC First Action Official Method 2012.22 - Vitamin C (ascorbic acid) in Infant Formula and
Adult/Pediatric Nutritional Formula by UHPLC-UV
A.
Principle
Ascorbic acid is extracted using trichloroacetic acid in the presence of TCEP (
Tris (2-
carboxyethyl)-phosphine hydrochloride)
as a reducing agent to ensure dehydroascorbic acid
is reduced to ascorbic acid before extraction. Ascorbic acid is then determined by liquid
chromatography with UV detection at 265 nm. Separation takes place on a C
18
column with
sodium acetate (pH = 5.4) as eluent, combined with TCEP, and decylamine as pairing agent.
B.
Apparatus
a.
Balances.-
With readability of 0.1 mg and 0.01 g
b.
pH meter.-
Metrohm 691 (Herisau, Switzerland) or equivalent
c.
LC column.-
Waters Acquity UPLC
®
BEH C
18
column, 1.75 µm, 2.1 x 100 mm or
equivalent
d.
LC system.-
UHPLC system equipped with a UV or photodiode array detector (e.g.
Waters Acquity UPLC
®
or equivalent).
C.
Reagents and standards
a. Acetonitrile.-
HPLC grade, Merck (Geneva, Switzerland) or equivalent.
b. Ascorbic acid.- > 99 %
(Sigma-Aldrich, Switzerland) or equivalent
c. Decylamine.- Aldrich
(Switzerland) or equivalent
d. Phosphoric acid.- 85 %,
Merck (Geneva, Switzerland) or equivalent
e. Purified water.-
Millipore (Switzerland), or equivalent
f. Sodium acetate trihydrate.-
Merck (Geneva, Switzerland) or equivalent.
g. Trichloroacetic acid (TCA).-
Merck (
G
eneva, Switzerland) or equivalent
h. Tris [2-carboxyethyl] phosphine hydrochloride (TCEP).-
(
Sigma,
S
witzerland)
or
equivalent.
D.
Preparation of solutions
a. Sodium acetate solution pH 5.4.-
Into a 500 mL volumetric flask, weigh 34.0 g sodium
acetate trihydrate, add 400 mL water and dissolve. Adjust pH to 5.4 with phosphoric acid
85 %. Make up to volume with water.
b. Trichloroacetic acid (TCA) 15 %.-
Into a 500 mL volumetric flask, weigh 75.0 g of TCA,
dissolve and make up to volume with water.
c. Tris (2-carboxyethyl)-phosphine hydrochloride (TCEP) 250 µg/mL.-
Into a 500 mL
volumetric flask, weigh 125 mg of TCEP, dissolve and make up to volume with water.
d. Mobile phase for LC.-
Into a 250 mL flask, mix 0.4 g decylamine, 2.5 mL acetonitrile, 25
mL sodium acetate solution pH 5.4 (a.) and 205 mL of water. Adjust pH to 5.4 with
phosphoric acid 85 %. Add 10 mg TCEP and mix well.
Precipitation may occur; a clear
solution is obtained once the pH is adjusted.
Continued on next page
AOAC OMB Meeting Book
349