Concurrent liquid chromatographic separation and photodiode array detection of retinol, tocopherols, all-trans-alpha-carotene, all-trans-beta-carotene and the mono-cis isomers of beta-carotene in extracts of human plasma.

In this report we describe the development of a method for the concurrent reversed-phase high-performance liquid chromatographic separation and photodiode array detection of human plasma retinol, tocopherols and carotenes. For a single sample injection, retinol, retinyl acetate, alpha-tocopherol, gamma-tocopherol, alpha-tocopheryl acetate, all-trans-alpha-carotene and all-trans-beta-carotene, as well as the mono-cis geometrical isomers of beta-carotene were separated and detected. Analytical separations were performed at a subambient temperature (0 degree C) over a Suplex pKb-100 reversed-phase analytical column with an isocratic mobile phase of methanol-methyl tert.-butyl ether-water (80:20:5, v/v/v) at a flow-rate of 0.8 ml/min for 60 min. Standards and samples were reconstituted in ethanol, and typically, 50 microliters was injected for analysis. By HPLC, compounds of interest were clearly resolved and detectable at the picomole level.