6. AOACSPIFANMethods-2018Awards

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S CHIMPF ET AL . : J OURNAL OF AOAC I NTERNATIONAL V OL . 101, N O . 1, 2018 265

Figure 1. Chromatographic separation of lutein, zeaxanthin, a -carotene, and b -carotene.

tertiary butyl ether (MtBE). After extraction, a portion of the organic layer is evaporated to dryness, and the residue is redissolved in 75 + 25 10% butylated hydroxytoluene (BHT) in methanol – MtBE. Prepared samples are injected into a C30 HPLC column where cis and trans isomers of lutein, b -carotene, and lycopene are separated with a methanol – MtBE gradient and detected with UV-Vis spectroscopy at 445 nm. With this method, all carotenoid concentrations are calculated from their respective trans -carotenoid curves because cis isomer standards are not commercially available; however, when calculating concentrations of the major lutein cis isomers (13- cis and 13 0 - cis ) from the trans -lutein curve, appropriate relative response factors are used to correct for differences in absorptivity

adult nutritionals; however, this method has not been validated for the determination of lycopene. In response to the SPIFAN carotenoid SMPR, a new carotenoid method for the determination of trans- and cis - lutein isomers, b -carotene trans and cis isomers, and cis - and trans -lycopene isomers was developed and validated. This reversed-phase HPLC method uses C 30 chromatography and UV-Vis spectroscopy to determine cis and trans isomers of lutein, b -carotene, and lycopene in infant, pediatric, and adult nutritionals. With this new method, samples are saponified at 60°C with a mixture of potassium hydroxide, tetrahydrofuran (THF), and methanol; and carotenoids are extracted from saponified samples with a solution of 75 + 25 hexane – methyl

Figure 2. (a) Chromatographic separation of b-carotene isomers of a heated trans-b-carotene standard: entire chromatogram. (b) Chromatographic separation of b-carotene isomers of a heated trans-b-carotene standard: enlargement of the b-carotene region.