A validated liquid chromatography method for the simultaneous determination of vitamins A and E in human plasma

A fast (15 min) and simple HPLC method for determination of all-trans-retinol and alpha-, gamma- and delta-tocopherols in human plasma has been developed. The assay utilized 200 microl of plasma to which 20 microl of internal standard solution (retinol acetate) was added followed by 200 microl of water, 400 microl of ethanol and 800 microl of hexane. The hexane layer was collected, evaporated, the residue dissolved in 200 microl of methanol and analyzed on a Zorbax Eclipse XDB-C18 column using a step gradient with a polar organic mobile phase composed of acetonitrile and methanol and variable wavelength fluorescence detection. The quantification limits for all-trans-retinol and gamma-tocopherol were 20 ng/ml and for alpha- and delta-tocopherols 500 ng/ml and 10 ng/ml, respectively. The procedure was validated and applied to the analysis of plasma samples from the Baltimore Longitudinal Study of Aging.