2016 OMB Summer Meeting

AOAC OMB Meeting Book

390

AOAC 2012.22 (FINAL VERSION REVIEW) - JUNE 2016 FOR ERP USE ONLY DO NOT DISTRIBUTE

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Vitamin C in Infant Formula and Adult/Pediatric Nutritional Formula Ultra-

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Performance Liquid Chromatography with Ultraviolet Detection

(Applicable to the determination of vitamin C (L-ascorbic acid) in infant formula and 3

adult/pediatric nutritional formula by ultra-performance LC (UPLC ® )-UV. 4

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

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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

(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.

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(c). LC column.— Waters Acquity UPLC ® ethylene bridged hybrid (BEH) C 18

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column,

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1.75 µm, 2.1 x 100 mm, or equivalent.

(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

(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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