Determination of retinol and retinylesters inhuman plasma by high-performance liquid chromatographywith automated column switching and ultraviolet detection

摘要

A HPLC method with automated column switching and UV detection is described for the simultaneous determination of and major retinyl esters (retinyl palmitate, retinyl stearate, retinyl oleate and retinyl linoleate) in human plasma. (0.2 ml) was deproteinized by adding ethanol (1.5 ml) containing the internal standard retinyl propionate. Following ifugation the supernatant was directly injected onto the pre-column packed with LiChrospher 100 RP-18 using 1.2%nmomum acetate—acetic acid—ethanol (80:1:20, v/v) as mobile phase. The elution strength of the ethanol containing solution was reduced by on-line supply of 1% ammonium acetate—acetic acid—ethanol (100:2:4, v/v). The retained-and retinyt esters were then tmnsf erred to the analytical column (Superspher 100 RP-18, endcapped) in the backflush and chromatographed under isocratic conditions using acetonitrile—methanol—ethanob..2propanol (1:1:1:1, v/v) as phase. Compounds of interest were detected at 325 nm. The method was linear in the range 2.5—2000~ ng/ml with aof quantification for retinol and retinyl esters of 2.5 ng/ml. Mean recoveries from plasma were 93.4—96.5% for retinol 100—1000 ng/ml) and 92.7—96.0% for retinyl palmitate (range 5-1000 ng/ml). Inter-assay precision was s5.l% and‘3% for retinol and retinyl palmitate, respectively. The method was successfully applied to more than 2000 human plasma from clinical studies. Endogenous levels of retinol and retinyl esters determined in female volunteers were in good with published data.