AOAC Final Action Methods in 2019

( 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. G. Chromatography ( a )  Chromatographic conditions.— Set up the UHPLC system according to the specifications in Table 2016.13H . Follow the manufacturer’s instructions for column installation, cleaning, and storage. Although the method does not involve high system backpressure normally associated with UHPLC, the low system volume is recommended for resolution with a 2.0 mm id column. To minimize system dwell volume and extra-column volume, 0.12 mm id connecting tubing and a low volume flow cell designed for UHPLC systems are recommended. On some LC systems, it is helpful to convert the pump to low delay volume mode. ( 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, all-trans zeaxanthin, and apocarotenal .—From the chromatogram of the lutein system suitability solution, E(h) , determine the resolution between all- trans lutein, all -trans zeaxanthin, and apocarotenal. Resolution should be ≥3.7 between all- trans lutein and all -trans zeaxanthin and ≥2.5 between all -trans zeaxanthin and apocarotenal. See Figure 2016.13A . ( 3 )  Resolution between β-carotene cis and trans isomers and α-carotene .—Inject the β-carotene system suitability solution, E(g) , and determine the resolution between the two major cis isomers of β-carotene, all- trans β-carotene, and α-carotene. Resolution should be ≥1.4 between 13- cis β-carotene and cis/trans α-carotene and ≥2.6 between all- trans β-carotene and 9- cis β-carotene. See Figure  2016.13B .

Table 2016.13H. 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

3 8

15

25

100

25.5

3 3

32

Flow rate

0.25 mL/min ca 185 bar

Backpressure Injection volume UV-Vis detection

5 μL

450 nm, ref = off

( f ) Add 8 mL extraction solution, C(r) , 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 n -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. An adjustable pipet may be used. ( 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 solution and vortex to dissolve, shaking if necessary to include any residue on the sides of the vial.

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

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