HPLC method for the simultaneous determination of beta-carotene, retinol and alpha-tocopherol in serum

A method is described for the simultaneous analysis of alpha-tocopherol, beta-carotene, and retinol in n-hexane extracts of serum. The technique uses normal-phase HPLC in a silica gel column with n-hexane-2-propanol (97:3, v/v) as the mobile phase. The compounds are eluted from the column in the order alpha-tocopherol, beta-carotene, tocol (internal standard) and retinol. The compounds are quantified by use of the following detectors connected in series: retinol and tocol by a spectrophotometer, beta-carotene by a filter photometer, and alpha-tocopherol and tocol by a spectrophotofluorimeter. The time taken for separation is less than 6 min but a further 14 min is allowed for elution of the more polar compounds before the next run. With an automatic injector, the method is suitable for the analysis of a large number of samples because of the relatively short time for analysis and the small volume (0.2 ml) of serum required. The method has been used in a study of the relationships between serum levels of the fat-soluble vitamins and subsequent cancer in 2332 subjects.