6. AOACSPIFANMethods-2018Awards

290

S CHIMPF ET AL . : J OURNAL OF AOAC I NTERNATIONAL V OL . 101, N O . 1, 2018 273

Table 2017.04B. Gradient time

Table 2017.04C. Peak resolution requirements

Time, min

A, %

B, %

Peaks

Minimum resolution

13- cis -Lutein and 13 0 - cis -lutein 13 0 - cis -Lutein and trans -lutein

0.0

100 100

0 0

1.3 1.3 7.0 1.3

10.0 10.1 18.0 18.1 27.0 27.1 30.0

13- cis - b -Carotene and trans - b -carotene

65 65 25 25

35 35 75 75

trans -Lycopene and 5- cis -lycopene

( 10 ) After 15 min, immediately place tubes in an ice bath to cool. (b) Extraction and concentration. — ( 1 ) Add 10.0 mL extraction solution (75 + 25 hexane – MtBE) to each cooled tube and immediately cap each tube after adding the extraction solution to prevent evaporation. ( 2 ) Stir, shake, or mix on a vortex mixer each tube for 5 min (if stirring, add stir bar to tube before adding the extraction solution). ( 3 ) After 5 min, add 15 mL water and stir, shake, or mix on a vortex mixer each tube for an additional 5 min. ( 4 ) Centrifuge until a complete separation of the aqueous/ methanol and organic layers is achieved. ( 5 ) Quantitatively pipet 4.0 mL organic layer into an appropriate container and evaporate the organic solvent with a gentle stream of nitrogen in a 37 + 2°C water bath. After evaporating the solvent, there should be a thin slightly cloudy layer of residue on the bottom of the container and no droplets of oil. If there are any oil droplets in the bottom or on the sides of the tube, the preparation should be repeated. ( 6 ) Remove one container at a time from the water bath and quantitatively add 0.25 mL dilution solution (75 + 25 10% BHT in methanol – MtBE), cover the container, mix on the vortex mixer for 30 s, and immediately transfer contents of container to an autosampler vial fitted with a 300 µL insert and cap vial. (a) Instrument operating conditions. — ( 1 ) Mobile phase A. — 85 + 15 methanol – MtBE. (2) Mobile phase B. — MtBE. ( 3 ) Flow rate. — 1 mL/min. (4) Injection volume. — 50 µL. (5) Column temperature. — 25°C. ( 6 ) Run time. — 30 min. (7) Detection wavelength. — 445 ± 4 nm. ( 8 ) Gradient ( see Table 2017.04B ). F. HPLC Analysis

100 100

0 0

E. Sample Preparation

(a) Saponification. — ( 1 ) Liquid samples. — Thoroughly mix or stir products prior to sampling. Homogenize if necessary. Accurately weigh 1.0 g ± 10% into a 50 mL centrifuge tube and record the weight to the nearest 0.0001 g. Proceed with step E(a) (3) . ( 2 ) Powder samples. — If powder sample homogeneity is unknown, assume it is nonhomogeneous and proceed as for dry-blended/nonhomogeneous powder. — ( i ) Dry-blended/ nonhomogeneous powder. — Accurately weigh approximately 25 g powder and add approximately 200 g water. Mix until homogeneous suspension is obtained. A homogenizer can be used when necessary. Accurately weigh 1 g ± 10% homogeneous suspension into a 50 mL centrifuge tube and record the weight to the nearest 0.0001 g. Proceed with step E(a) (3) . ( ii ) Wet blended powder. — Accurately weigh approximately 0.2 g powder into a 50 mL centrifuge tube and record the weight to the nearest 0.0001 g. Proceed with step E(a) (3) . ( 3 ) Add 3 mL laboratory water to liquid samples and 4 mL laboratory water to powder samples. Swirl to mix or suspend powder. ( 4 ) Add approximately 0.5 g or one-eighth of a teaspoon sodium ascorbate and swirl each tube gently to mix. ( 5 ) Add 10 mL methanol to each tube. ( 6 ) Add 1.0 mL 45% potassium hydroxide to each tube and swirl to mix. ( 7 ) Add 1.0 mL THF to each tube. ( 8 ) Cap tubes and mix for 30 s. If any of the tubes leak, reweigh the sample. ( 9 ) Heat tubes in a 60 ± 2°C water bath for 15 ± 2 min. shaking tubes for ~ 10 s every 5 min.

Figure 6. Chromatographic separation of carotenoids in an infant soy powder (E10NWZC).