AOAC SPIFAN ERP & Working Group Method Reviews (July 10, 2019)

AOAC Official Method 2012.10 Simultaneous Determination of Vitamins E and A in Infant Formula and Adult Nutritionals Normal-Phase High-Performance Liquid Chromatography

B. Apparatus Common laboratory glassware and equipment and, in particular, the following: ( a )  HPLC system.— Consisting of pump, autosampler, programmable UV detector operating at 325 nm for vitamin A, and a fluorescence detector (FLD) at an excitation wavelength of 280 nm and an emission wavelength of 310 nm for vitamin E. ( b )  HPLC column .—Analytical normal-phase column, e.g., Zorbax NH2, 5 µm, 150 × 4.6 mm, or equivalent. ( c )  Water bath .—Set at 37 ± 2°C. ( d )  Centrifuge .—With adapters for 50 mL centrifuge tubes, capable of 4000 min –1 . ( e )  UV-Vis spectrophotometer .—With 1 cm quartz cells. ( f )  Analytical balance .—Weighing to four decimal places. ( g )  Amber HPLC vials .—2 mL, with plastic caps and polytetrafluoroethylene (PTFE) seals. ( h )  Disposable centrifuge tubes .—50 mL, e.g., Falcon (Fisher, Pittsburgh, PA), or equivalent. ( i )  Laboratory mechanical test tube shaker . ( j )  Sonic bath . ( k )  One-mark volumetric flasks .—50 and 100 mL. ( l )  Vacuum filtration apparatus .—With 0.45 μm nylon membrane. ( m )  Laboratory glass bottles .—250 mL and 1 and 2 L, e.g., Duran (Wertheim/Main, Germany), or equivalent. ( n )  Pipettors and tips.— Gilson P10002, or equivalent. C. Standards ( a )  Vitamin A palmitate reference standard.— Primary standard, U.S. Pharmacopeial Convention (USP; Rockville, MD, USA), or equivalent. The standard shall contain antioxidant. ( b )  Vitamin A acetate reference standard.— Primary standard, USP, or equivalent. ( c )  α-Tocopherol acetate reference standard.— Primary standard, USP, or equivalent. ( d )  α-Tocopherol reference standard.— Primary standard, USP, or equivalent. D. Chemicals and Reagents During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and distilled or demineralized water or water of equivalent purity.

First Action 2012 Final Action 2014

ISO–AOAC Method* [Applicable to the concurrent quantitative analysis of vitamin E (α-tocopherol and α-tocopherol acetate), vitamin A palmitate, and vitamin A acetate ( cis - and trans -isomers) present in milk- and soy- based infant formula and adult nutritionals and formulas containing hydrolyzed protein. Vitamin A is defined as 13- cis and all-trans retinol (CAS No. 68-26-8), retinyl esters (retinyl palmitate; CAS No. 79-81-2), and retinyl acetate (CAS No. 127-47-9). The determination of vitamin E focuses on α-tocopherol (CAS No. 59- 02-9), all -racemic α-tocopherol (CAS No. 1406-18-4), and their esters. α-Tocopherol and esters can be reported separately.] Caution: Correct personal and environmental safety standards shall be used while performing this analytical method. Laboratory personnel handling solvents, acids, and reagents should be knowledgeable of their potential hazards. Consult the Material Safety Data Sheets (MSDS) for information on the hazards and take proper precautions. Transfer solvents and acids inside efficient fume hoods and extractors. Ensure all glassware is free from chipping and hairline cracks. See Tables 2012.10A and B for results of the method performance studies supporting acceptance of the method. A. Principle This procedureutilizes theproteolytic enzymepapain tohydrolyze the hydrophilic protein coating of fat micelles in milk- or soy-based infant formulations in an aqueous solution. The hydrophobic contents of the micelles are then extracted quantitatively into iso- octane in a single extraction and chromatographed by normal-phase HPLC using a Zorbax ® NH2 analytical column. The analytes are eluted with a gradient and α-tocopherol and α-tocopherol acetate quantified using fluorescence detection, excitation/emission, 280/310 nm. Vitamin A palmitate ( cis and trans ) and vitamin A acetate ( cis and trans ) are quantified using UV detection at 325 nm.

Table 2012.10A. Method performance requirements: Single-laboratory validation (SLV) and multilaboratory testing (MLT) results summary—Vitamin A a

Method performance requirements

Parameter

Retinyl palmitate

Retinyl acetate

Analytical range

7.0–382.6 b

2–450 0.099

2–450

Limit of detection (LOD) Limit of quantitation (LOQ)

≤2.0 b ≤7.0 b

0.85 2.83

0.33

Repeatability (RSD r

) (SLV)

7–300 b 7–300 b

≤8%

≤4.03% ≤6.23% 99.13%

≤6.56% ≤10.63% 96.53%

Intermediate precision (RSD r

) (SLV)

—

Recovery (SLV)

90–110% (mean spiked recovery over the range of the assay)

Reproducibility (RSD R 11.73–22.61% a  Concentrations apply to ( 1 ) ‘ready-to-feed’ liquids; ( 2 ) reconstituted powders (25 g into 200 g water); and ( 3 ) liquid concentrate diluted 1:1 by weight. b  µg/100 g reported separately as cis -13 retinol and all- trans retinol in reconstituted final product. ) (MLT) 10–383 b ≤16% 6.51–16.25%

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