6. AOACSPIFANMethods-2018Awards

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4 H ostetler : J ournal of AOAC I nternational V ol . 100, N o . 3, 2017

(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)  Supernatant volume .—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)  Reconstitution volume .—Reconstitute dried extract in sample solvent and vortex-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 μmPTFE syringe filter before injection. (a)  Chromatographic conditions.— Set up the UHPLC system according to the specifications in Table 2016.13C . Follow the manufacturer’s instructions for column installation, cleaning, and storage. (b)  System suitability checks .—( 1 ) Resolution between lutein cis and trans isomers.— Inject the lutein system suitability solution, E(h) , and determine the resolution between the two major cis isomers and all- trans -lutein. Resolution should be ≥1.4 between 13- cis - and 13′- cis -lutein and ≥2.2 between 13′- cis - and all- trans -lutein using the half-width method. See Figure 2016.13A . ( 2 )  Resolution between all-trans-lutein, zeaxanthin, and apocarotenal .—From the chromatogram of the lutein system suitability solution, E(h) , determine the resolution between all- trans -lutein, zeaxanthin, and apocarotenal. Resolution should be G. Chromatography

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 Time, min Mobile phase B, % 0 5 1 8

Gradient

8

15

25

100

25.5

5 5

32

Flow rate

0.25 mL/min

∼ 185 bar

Backpressure Injection volume UV/Vis detection

5 μL

450 nm, ref = off

( 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 10 min 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 a 5.0 mL aliquot of the appropriate ISTD 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.

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