SIMPLE DETERMINATION OF RETINOL, ALPHA-TOCOPHEROL AND CAROTENOIDS (LUTEIN, ALL-TRANS-LYCOPENE, ALPHA-CAROTENE AND BETA-CAROTENE) IN HUMAN PLASMA BY ISOCRATIC LIQUID-CHROMATOGRAPHY

Retinol, alpha-tocopherol, lutein, all-trans-lycopene, and alpha- and beta-carotenes can be simultaneously determined in human plasma by reversed-phase liquid chromatography with a programmable and variable UV wavelength detector. Plasma (200 mul) is deproteinized with 200 mul of ethanol containing retinyl acetate, as internal standard, then extracted with 1.0 ml of n-hexane-2,6-di-tert.-butyl-p-cresol (BHT). The organic layer is removed and evaporated and the residue is dissolved in 100 mul of a mixture of acetonitrile-tetrahydrofuran-methanol (68:22:7, v/v/v). Aliquots (15 mul) are injected onto a 250 x 4.6 mm I.D. column of Nucleosil 100-5 C18 with a pre-column of Lichrosorb RP-18, 7 mum, 15 x 3.2 mm I.D. A mobile phase of acetonitrile-tetrahydrofuran-methanol (68:22:7, v/v/v), adjusted to 100 (v/v) with 1% ammonium acetate, at a flow-rate of 1.5 ml/min is used. Usual run time is 15 min. Retinol and retinyl acetate are monitored at 325 nm, tocopherol at 290 nm, lycopene at 470 nm, lutein and alpha- and beta-carotenes at 450 nm. The intra-batch coefficients of variation (C.V.) were 2.5, 2.2, 2.4, 5.8, 5.1 and 4.7% for retinol, alpha-tocopherol, lutein, trans-lycopene, alpha- and beta-carotenes, respectively. The inter-batch C.V.s of experiments performed on 30 different days over 12 weeks were 5.7, 3.9, 4.5, 10.9, 11.3 and 10.5%, respectively.