AOACSPIFANMethods-2017Awards

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M c M ahon : J ournal of AOAC I nternational V ol . 99, N o . 1, 2016  225

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

7.0–382.6 b

Analytical range

2–450 0.099

2–450

≤2.0 b ≤7.0 b

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

0.85 2.83

0.33

7–300 b 7–300 b

Repeatability (RSD r

) (SLV)

≤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)

10–383 b

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) ≤16% 6.51–16.25%

( 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.

A. Principle This procedure utilizes the proteolytic enzyme papain to hydrolyze 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. 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.

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

Method performance requirements

Parameter

α-Tocopherol

α-Tocopherol acetate

0.2–8 b

Analytical range

0.03–8

0.02–9.4

≤0.1 b ≤0.2 b

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

0.01

0.023 0.075 ≤4.39

0.035

0.5–2.0 b

Repeatability (RSD r

) (SLV)

≤8% ≤6%

≤4.25% ≤3.78% ≤17.31% ≤9.24% 100.60%

4–8 b <2.0 b >2.0 b

≤3.53% ≤10.54% ≤8.25% 102.92%

Intermediate precision (RSD r

) (SLV)

— —

Recovery (SLV)

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

0.5–1.0 b 1.0–8.0 b

Reproducibility (RSD R

) (MLT)

≤22% ≤16% ≤22% ≤16%

3.84–43.56%

—

—

4.15–11.25%

) (MLT) total vitamin E 0.5–1.0 b

Reproducibility (RSD R

3.84–10.78%

1.0–8.0 b

≤12.47% 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  mg/100 g α-tocopherol and α-tocopheryl acetate in reconstituted final product.