An isocratic high-performance liquid chromatography method for the simultaneous analysis of plasma retinol, alpha-tocopherol, and various carotenoids

Abstract

An isocratic high-performance liquid chromatography method for the simultaneous determination of various fat-soluble vitamins and carotenoids is reported. The method utilizes a Radial-Pak C-18, 5-microns column and an elution solvent composed of methanol:acetonitrile:chloroform (25:60:15). Only 100 microliters of plasma sample is required for one determination. Retinol, alpha-tocopherol, alpha-carotene, beta-carotene, lycopene, zeaxanthin, and two other unidentified carotenoids can be clearly separated and quantified in one HPLC run using alpha-tocopheryl acetate or tocol as the internal standard. The eluted peaks are quantified by either a photodiode-array detector at preprogrammed wavelengths at the absorption maxima of the compounds or a dual-wavelength detector at 280 and 436 nm. The total run time is 16 min. With an automatic injector and a programmable detector, the system allows unattended operation. The within-run and day-to-day coefficients of variation range from 1 to 8%. The lower limits of determination are 2, 40, and 2 ng for retinol, alpha-tocopherol, and carotenes, respectively. In addition, the system monitors the absorption spectra of the eluant during the HPLC run; this allows the spectral identification of various compounds separated in the same run.