Ultra-performance liquid chromatographic determination of tocopherols and retinol in human plasma.

A rapid, selective and sensitive ultra-performance liquid chromatography method has been developed for the detection and quantification of tocopherols and retinol in human plasma. Alpha-tocopherol, gamma-tocopherol and retinol are assayed using fluorescence detection. Excitation/emission wavelengths are 295/330 nm and 325/470 nm for the analysis of both tocopherols and retinol, respectively. Retinol acetate is employed as the internal standard. The reversed-phase method incorporates gradient elution with a mobile phase consisting of methanol and acetonitrile. Separation of vitamin compounds is achieved using a bridged ethyl hybrid C18 column. The retention times for retinol, retinol acetate, gamma-tocopherol and alpha-tocopherol are 1.6, 1.8, 3.9 and 4.3 min, respectively. The limits of quantification for retinol, gamma-tocopherol and alpha-tocopherol were 0.02, 0.02 and 0.1 µg/mL, respectively. The assay method is suitable for the analysis of tocopherols and retinol in human plasma. The method may be applied following the ingestion of foods fortified with these fat-soluble vitamins.