6. AOACSPIFANMethods-2018Awards

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

prepared and analyzed to confirm that it will not interfere with the chromatographic separation of b -carotene. Chromatograms from the analysis of individual lutein, zeaxanthin, a -carotene, and b -carotene standards are overlaid in Figure 1 and show that trans- a -carotene is separated from trans - b -carotene and a cis - b -carotene isomer. A review of Figures 4 – 10 also shows that most nutritional products do not contain a -carotene. Because cis isomers of b -carotene and lycopene are expected to form during the manufacture of most infant and adult nutritionals, experiments were completed to tentatively identify some of the cis - b -carotene and lycopene isomers separated with this method. Because cis isomers of b -carotene and lycopene are not commercially available, cis isomers were formed by heating trans- b -carotene and lycopene standards in closed containers flushed with nitrogen. The heated standards were analyzed chromatographically ( see Figures 2 and 3). The chromatographic separation of the heated b -carotene standard shows that the major isomer formed is 13- cis - b -carotene, and the chromatographic separation of the heated lycopene standard shows that the major isomers formed are 5- cis -lycopene, a group of peaks eluting between 22 and 23.5 min, and another group of peaks eluting between 24 and 24.5 min. Individual cis isomers were tentatively identified from information available in the literature (5, 10). It should be noted that trans- lutein was not heated to form isomers because the major isomers formed are 13- cis and 13 0 - cis , and these peaks are easily identified using information readily available in the literature (11).

of the cis and trans isomers. As noted previously in the literature, if total carotenoid concentrations are calculated using only the extinction coefficient of the trans isomer, serious errors in the total carotenoid concentrations will occur when cis isomers are present in samples (6). For lutein, the correction factor was determined experimentally to be 1.43. Briefly, portions of an all - trans -lutein standard of known concentration were heated in sealed vials, protected from light, at 65 and 95°C for 10 – 120 min. Immediately after the standards were removed from the heat, they were cooled and analyzed chromatographically and spectrophotometrically. Based on the results of these analyses (not shown), >90% recoveries of lutein under all experimental conditions were obtained when correction factors of 1.43 were applied to 13- cis- and 13 0 - cis- lutein, the major thermally induced isomers of lutein. No correction factors were applied to the major cis - b -carotene and lycopene isomers. all - trans - b -Carotene, 15- cis - b -carotene, 13- cis - b -carotene, and 9- cis - b -carotene have been reported to exhibit similar molar absorptivities between 410 and 436 nm (7, 8), and all - trans -lycopene and major cis -lycopene isomers exhibit similar molar absorptivities between 443 and 487 nm (9). During method development, total b -carotene results calculated from chromatograms collected simultaneously at 410 and 445 nm were not significantly different; so, to simplify the method, a wavelength of 445 nm was chosen for the detection of all cis - and trans - b -carotene, -lycopene, and -lutein isomers. Although this method has not been validated for the determination of a -carotene, an a -carotene standard was

Figure 3. (a) Chromatographic separation of lycopene isomers of a heated trans-lycopene standard: entire chromatogram. (b) Chromatographic separation of lycopene isomers of a heated trans-lycopene standard: enlargement of the lycopene region.