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AOAC FIRST ACTION 2012.22
MULTI LABORATORY TESTING
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AOAC First Action Official Method 2012.22 - Vitamin C (ascorbic acid) in Infant Formula
and Adult/Pediatric Nutritional Formula by UHPLC-UV
Caution: Refer to Material Safety Data Sheets prior to use of chemicals. Use appropriate personal
protective equipment when performing testing.
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 the 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.
UHPLC 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.
2012.22/VitC-03 (February 2016) - MLT
FOR ERP USE ONLY
DO NOT DISTRIBUTE