Determination of retinol and retinyl esters in human plasma by high-performance liquid chromatography with automated column switching and ultravioletdetection

A HPLC method with automated column switching and UV detection is described for the simultaneous determination of retinol and major retinyl esters (re tinyl palmitate, retinyl stearate, retinyl oleate and retinyl linoleate) in human plasma. Plasma (0.2 mi) was deproteinized by adding ethanol (1.5 mi) containing the internal standard retinyl propionate. Following centrifugat ion the supernatant was directly injected onto the pre-column packed with L iChrospher 100 RP-18 using 1.2% ammonium acetate-acetic acid-ethanol (80:1: 20, v/v) as mobile phase. The elution strength of the ethanol containing sa mple solution was reduced by on-line supply of 1% ammonium acetate-acetic a cid-ethanol (100:2:4, v/v). The retained retinol and retinyl esters were th en transferred to the analytical column (Superspher 100 RP-18, endcapped) i n the backflush mode and chromatographed under isocratic conditions using a cetonitrile-methanol-ethanol-2-propanol (1:1:1:1, v/v) as mobile phase. Com pounds of interest were detected at 325 nm. The method was linear in the ra nge 2.5-2000 ng/ml with a limit of quantification for retinol and retinyl e sters of 2.5 ng/ml. Mean recoveries from plasma were 93.4-96.5% for retinol (range 100-1000 ng/ml) and 92.7-96.0% for retinyl palmitate (range 5-1000 ng/ml). Inter-assay precision was less than or equal to5.1% and less than o r equal to6.3% for retinol and retinyl palmitate, respectively. The method was successfully applied to more than 2000 human plasma samples from clinic al studies. Endogenous levels of retinol and retinyl esters determined in f emale volunteers were in good accordance with published data. (C) 2001 Else vier Science B.V. All rights reserved.