SIMULTANEOUS DETERMINATION OF RETINOL, ALPHA-TOCOPHEROL AND RETINYL PALMITATE IN PLASMA OF PREMATURE NEWBORNS BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A reversed-phase high-performance liquid chromatographic method is des cribed for the simultaneous determination of retinol, alpha-tocopherol and retinyl palmitate in plasma. Plasma containing an internal standa rd (tocol) was deproteinized with ethanol, then extracted with n-hexan e. The organic layer was removed and evaporated under a nitrogen strea m, and chromatographed on a reversed-phase RP-18 column using a water/ acetonitrile-ethyl acetate/2-propanol gradient solvent system over 15 min at 305 nm. The recovery exceeded 93%. The detection limit was 0.1 mug/ml for retinol, 1.3 mug/ml for alpha-tocopherol and 0.95 mug/ml fo r retinyl palmitate. The reproducibility, precision (expressed as coef ficients of variation) and accuracy were less than 8% for all analytes . The small sample requirement, the simplicity of extraction, the shor t run-time and the good reproducibility make this procedure particular ly useful for monitoring retinol and alpha-tocopherol supplementation in premature newborns.