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Vitamin C in Infant Formula and Adult/Pediatric Nutritional Formula Ultra-
1
Performance Liquid Chromatography with Ultraviolet Detection
2
(Applicable to the determination of vitamin C (L-ascorbic acid) in infant formula and
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adult/pediatric nutritional formula by ultra-performance LC (UPLC
®
)-UV.
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Caution:
Refer to Material Safety Data Sheets prior to use of chemicals. Use appropriate
5
personal protective equipment when performing testing.
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A.
Principle
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Ascorbic acid is extracted from the sample using trichloroacetic acid (TCA) in the
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presence of Tris[2-carboxyethyl]phosphine (TCEP) as a reducing agent. Ascorbic acid
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is then determined by UHPLC-UV at 265 nm. Separation takes place on a C
18
column
11
with sodium acetate (pH = 5.4) as eluent, combined with TCEP and decylamine as
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ion-pairing agent.
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B.
Apparatus
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(a).
Balances.—
With readability of 0.1 mg and 0.01 g.
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(b).
pH meter.—
Metrohm 691 (Herisau, Switzerland), or equivalent.
16
(c).
LC column.—
Waters Acquity UPLC
®
ethylene bridged hybrid (BEH) C
18
column,
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1.75 µm, 2.1 x 100 mm, or equivalent.
18
(d).
UHPLC system.—
Waters
Acquity UPLC
®
system equipped with a Waters
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Acquity UPLC
®
photodiode array detector, or equivalent.
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C.
Reagents and standards
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(a).
Acetonitrile.—
HPLC grade, Merck (Geneva, Switzerland), or equivalent.
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(b).
Ascorbic acid.-
Fluka (Buchs, Switzerland), or equivalent.
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(c).
Decylamine.-
Fluka, or equivalent.
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(d).
Phosphoric acid. —85 %,
Merck, or equivalent.
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(e).
Purified water.—
Millipore (Le Mont-sur-Lausanne, Swizerland), or equivalent.
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(f).
Sodium acetate trihydrate.—
Merck, or equivalent.
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AOAC 2012.22 (FINAL VERSION REVIEW) - JUNE 2016
FOR ERP USE ONLY
DO NOT DISTRIBUTE
AOAC OMB Meeting Book
390