Determination of retinyl esters and retinol in serum or plasma by normal-phase liquid chromatography: Method and applications

Abstract

Retinol and retinyl esters are measured in serum or plasma samples by gradient, normal-phase, adsorption 'high-performance' liquid chromatography, with ultraviolet detection at 325 nm. The four major circulating retinyl esters in humans (esters of palmitate, stearate, oleate, and linoleate) are co-eluted as a single peak. Retinyl acetate is included as an internal standard, to correct for variable recovery. Retinol values so measured correlated well (r = 0.88) with those by a widely used reversed-phase chromatographic technique (Clin Chem 1983;29:708-12). The mean retinol concentration was 570 (SEM 17) μg/L and the mean for retinyl esters was 33 (SEM 4) μg/L as determined in samples from 88 fasting young adults. Concentrations of retinol in plasma as low as 50 μg/L can be detected in 100-μL samples, as can 10 μg of retinyl esters per liter. Using this method, we measured absorption of low doses of vitamin A, which may provide a more physiological approach to assessment of fat malabsorption. Additionally, the procedure proved useful for quickly screening for vitamin A toxicity. Major advantages include small sample size, direct injection of the extracted sample without evaporation, rapid elution pattern, co-elution of major retinyl esters as a single peak, and low limit of detection.