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  • Title: Simple determination of retinol, alpha-tocopherol and carotenoids (lutein, all-trans-lycopene, alpha- and beta-carotenes) in human plasma by isocratic liquid chromatography.
    Author: Bui MH.
    Journal: J Chromatogr B Biomed Appl; 1994 Mar 18; 654(1):129-33. PubMed ID: 8004234.
    Abstract:
    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 microliters) is deproteinized with 200 microliters 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 microliters of a mixture of acetonitrile-tetrahydrofuran-methanol (68:22:7, v/v/v). Aliquots (15 microliters) 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 microns, 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.
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