Determination of retinol, alpha-tocopherol, alpha- and beta-carotene by direct extraction of human serum using high performance liquid chromatography.

In this report we describe a modified reverse phase HPLC method that avoids the solvent evaporation step and allows simple and rapid determination of retinol, alpha-tocopherol, alpha-carotene and beta-carotene and achieves complete separation of alpha- and beta-carotene. Retinyl acetate, alpha-tocopheryl acetate and retinyl palmitate in ethanol were added to serum as internal standards. Serum was then deproteinized with an equal volume of ethanol, and the lipid was extracted with ethyl acetate-butanol (1:1 v/v). A portion of this solution was injected into a C18 reverse phase chromatographic column and absorbencies of the vitamins and internal standards were measured at 292 nm for tocopherols, 325 nm for retinoids and 450 nm for carotenoids; peak-height ratios were used to quantify each vitamin. The analytical recoveries for retinol, alpha-tocopherol alpha- and beta-carotene at various concentrations tested were 95-103, 90-98, 92-99 and 94-96%, respectively. The intra- and interassay variations for low and high concentrations of retinol, alpha-tocopherol, alpha- and beta-carotene ranged from 2.4 to 6.7 for intraassay and from 4.3 to 8.5 for interassay replication. The detection limits were 1.25 (0.04), 19 (0.44), 0.35 (0.006) and 0.94 (0.017) micrograms/dL (delta mol/L) for retinol, alpha-tocopherol, alpha- and beta-carotene, respectively.