AOAC SPIFAN Nutrients ERP Review (March 14, 2019)

( 2 )  Liquid RTF with individual carotenoid concentrations ≤200 μg/100 g. —Shake bottle or can on a reciprocating shaker 10 min before opening. Transfer approximately 5 g sample into a 50 mL centrifuge tube. ( 3 )  RTF sample with individual carotenoid concentrations >200 μg/100 g .—Shake bottle or can on a reciprocating shaker 10 min before opening. Transfer approximately 2 g sample into a 50 mL centrifuge tube. Add 3 mL water, cap, and vortex-mix 10 s. Let sit for up to 15 min at room temperature. ( 4 )  Infant formula concentrate .—Shake bottle or can on a reciprocating shaker 10min before opening. Transfer approximately 2.5 g sample into a 50 mL centrifuge tube. Add 2.5 mL water, cap, and vortex-mix 10 s. Let sit for up to 15 min at room temperature. ( c ) Pipet 5.0 mL of the appropriate ISTD solution from E(i) to each tube. ( d ) Add 1.5 mL KOH solution, C(l) , to each tube with a repeater pipet. ( e ) Shake on reciprocating shaker for 5 min. ( f ) Add 8 mL extraction solution, C(o) , to each tube with a repeater pipet. ( g ) Shake for 10 min. ( h ) With a repeater pipet or dispenser, add 10 mL water and 10 mL hexane to each tube. ( i ) Shake for 1 min. ( j ) Centrifuge at 1000 rpm (or equivalent to 200 × g ) for 5 min. ( k ) Transfer a portion of the supernatant to a 12 mL scintillation vial.—( 1 ) For samples with individual carotenoid concentrations ≤50 μg/100 g .—Use 10 mL supernatant. ( 2 )  For samples with individual carotenoid concentrations >50 μg/100 g .—Use 3 mL supernatant. ( l ) Dry under nitrogen at ≤40°C. ( m ) Reconstitute dried extract in sample solvent and vortex to mix.—( 1 ) For samples with individual carotenoid concentrations ≤100 μg/100 g .—Add 0.5 mL. ( 2 )  For samples with individual carotenoid concentrations of 100–500 μg/100 g .—Add 1 mL. ( 3 )  For samples with individual carotenoid concentrations of 500–1000 μg/100 g .—Add 2 mL. ( 4 )  For samples with individual carotenoid concentrations of 1000–1500 μg/100 g .—Add 3 mL. ( n ) Filter through 0.2 μm PTFE syringe filter before injection.

Table 2016.13C. Chromatographic conditions Parameter Condition Analytical column

YMC C30 3 μm, 250 × 2.0 mm YMC C30 3 μm, 10 × 2.0 mm

Guard column

Column temperature

30°C

Mobile phases

A: 20 mM ammonium acetate in MeOH–water 98 + 2; B: MTBE

Gradient

Time, min

Mobile phase B, %

0 1 8

5 8

15

25

100

25.5

5 5

32

Flow rate

0.25 mL/min ca 185 bar

Backpressure Injection volume UV/Vis detection

5 μL

450 nm, ref = off

F. Sample Preparation Note on range: Although the method can quantify carotenoids in the range of 1–1300 μg/100 g, it is recommended to quantify only a 100-fold difference with a single preparation. For example, the range of 1–100 μg/100 g works well for infant formula, but the range of 15–1500 μg/100 g would work best for samples with the highest carotenoid concentrations. ( a ) Prepare up to nine samples at a time. ( b )  Weights .—Weigh all samples (powders and liquids) to 0.1 mg.—( 1 ) Powders .—Weigh approximately 625 mg powder sample into a 50 mL centrifuge tube. Add 5 mL water, cap, and vortex-mix until dissolved. Let sit for up to 15 min at room temperature. Note : For nonhomogeneous powder samples, first dissolve 25 g powder sample with 200 mL water (record weights of both powder sample and water) and then transfer approximately 5 g reconstituted sample into a 50 mL centrifuge tube.

Figure 2016.13A. Chromatogram of lutein system suitability solution, E(h). Lut = Lutein, Zea = zeaxanthin, and Apo = apocarotenal.

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