Volume 46, Number 1 (January - February 2001)

Key words:  5-cholesten-3b-ol-7-one, sum of 5-cholestene-3b,7b-diol and 5-cholestene-3b,7a-diol, sum of 5a,6a-epoxycholestan-3b-ol and 5b,6b-epoxycholestan-3b-ol, human blood plasma, thin layer chromatography

Selected oxycholesterols: 5-cholesten-3b-ol-7-one, sum of 5-cholestene-3b,7b-diol and 5-cholestene-3b,7a-diol, sum of 5a,6a-epoxycholestan-3b-ol and 5b,6b-epoxycholestan-3b-ol were determined in human blood plasma samples by the use of TLC with densitometry. The plasma was hydrolysed with ethanol solution of  KOH and then lipid extraction with n-hexane was performed. After cleanup of organic phase on 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-cholesten-3b-ol-7-one and sum of  5-cholestene-3b,7b-diol and -3b,7a-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 5b,6b- and 5a,6a-epoxycholestan-3b-ol determination. Quantitation was carried out after Liebermann-Burchard reaction for all oxycholesterols, except 5-cholesten-3b-ol-7-one, for which visualization and determination was performed under UV light (l = 239 nm). Recoveries from plasma spiked with a known amount of 5-cholesten-3b-ol-7-one as well as sensitivity and repeatability of this method were sufficient.