Determination of alpha-tocopherol, free cholesterol, esterified cholesterols and triacylglycerols in human lipoproteins by high-performance liquid chromatography.

The determination of alpha-tocopherol, free cholesterol, esterified cholesterols and triacylglycerols in human plasma and in fractions containing individual lipoproteins was achieved by reversed-phase high-performance liquid chromatography (HPLC). The lipoprotein fractions, such as chylomicron, VLDL, LDL, HDL2 and HDL3, were collected by ultracentrifugation of human plasma. The chromatographic separation was accomplished with a column packed with Hitachi Gel 3057, which is a spherical octadecylsilica of particle size 3 microns. The mobile phase was acetonitrile-2-propanol (75:25, v/v), and the eluate was monitored with ultraviolet absorption and fluorescence detectors connected in series. Qualitative analysis of the main chromatographic peaks collected during the HPLC of a plasma sample was done with the use of field-desorption mass spectrometry. The determination analysis of alpha-tocopherol, free cholesterol and esterified cholesterols was effected with a single chromatographic run with n-hexane extracts of plasma or lipoprotein fraction. The separation and determination of these fat-soluble components required as little as 5 microliters of plasma or lipoprotein fraction.