AN IMPROVED HPLC METHOD FOR THE SEPARATION OF 14 CAROTENOIDS, INCLUDING 15-13-CIS-BETA-CAROTENE AND 9-CIS-BETA-CAROTENE ISOMERS, PHYTOENE AND PHYTOFLUENE

The isocratic separation of 14 carotenoids, as well as retinol, retinyl acetate, retinyl palmitate, alpha-tocopherol and tocopherol acetate, is accomplished in 12 minutes, using a Spheri-5-ODS column and acetonitrile:dichloromethane:methanol (70:20:10) as mobile phase, with two-channel, programmable multiwavelength detection. The carotenoids separated are as follows: lutein/zeaxanthin, canthaxanthin, beta-apo-8' carotenal, beta-cryptoxanthin, echinenone, lycopene, gamma-carotene, alpha-carotene, beta-carotene, 9-cis-beta-carotene, 15-cis-/13-cis-beta-carotene, phytoene and phytofluene. The separation of lutein and zeaxanthin is obtained simply by changing the mobile phase to acetonitrile:methanol (85:15). The separation of the cis-isomers of beta-carotene is maintained, in spite of changes in the proportions and even after 250 hours of analysis. This chromatographic separation is not dependent on temperature (within a range of 15-37-degrees-C), on the injection solvent (THF, EtOH or the mobile phase) or flow rate (in a range of 1.3-2. 5 ml/min). the absorptivity value was unknown. The concentrations of the stock solutions were recalibrated monthly.