Determination of selected oxycholesterols in human blood plasma by means of thin-layer chromatography with densitometry

Selected oxycholesterols: 5-cholesten-3 beta -ol-7-one, sum of 5-cholestene-3 beta ,7 beta -diol and 5-cholestene-3 beta ,7 alpha -diol, sum of 5 alpha ,6 alpha -epoxycholestan-3 beta -ol and 5 beta ,6 beta -epoxycholestan-3 beta -ol were determined in human blood plasma samples by the use of TLC with densitometry. The plasma was hydro lysed with ethanol solution of KOH and then extraction of lipids with n-hexane was performed. After cleanup of organic phase bn silica gel SPE cartridges, oxycholesterols fraction was eluted with 2-propanol in n-hexane. Next, the fraction, after separation by TLC, was quantificated by densitometry. For the determination of 5-choleslen-3 beta -ol-7-one and sum of 5-cholestene-3 beta ,7 beta -diol and -3 beta ,7 alpha -diol the plates coated with silica gel and acetone with chloroform (1:9,v/v) as mobile phase were used. RP-18 stationary phase and 3% 2-propanol in dichloromethane solution was used for the sum of 5 beta ,6 beta- and 5 alpha ,6 alpha -epoxycholestan-3 beta -ol determination. Quantitation was carried out after Liebermann-Burchard reaction for all oxycholesterols, except 5-cholesten-3 beta -ol-7-one, for which visualization and determination was performed under UV light (lambda = 239 nm). Recoveries from plasma spiked with known amount of 5-cholesten-3 beta -ol-7-one as well as sensitivity and repeatability of this method were sufficient.