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 alph a ,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 wi th densitometry. The plasma was hydro lysed with ethanol solution of KOH an d then extraction of lipids with n-hexane was performed. After cleanup of o rganic phase bn silica gel SPE cartridges, oxycholesterols fraction was elu ted with 2-propanol in n-hexane. Next, the fraction, after separation by TL C, 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-propa nol in dichloromethane solution was used for the sum of 5 beta ,6 beta- and 5 alpha ,6 alpha -epoxycholestan-3 beta -ol determination. Quantitation wa s carried out after Liebermann-Burchard reaction for all oxycholesterols, e xcept 5-cholesten-3 beta -ol-7-one, for which visualization and determinati on was performed under UV light (lambda = 239 nm). Recoveries from plasma s piked with known amount of 5-cholesten-3 beta -ol-7-one as well as sensitiv ity and repeatability of this method were sufficient.