Determination of 7-Dehydrocholesterol in Human Skin Surface Lipids by High Performance Liquid Chromatography with Fluorescence Detection

Abstract

A simple, highly sensitive, high performance liquid chromatographic method with fluorescence detection for the determination of 7-dehydrocholesterol in human skin surface is described. 7-Dehydrocholesterol, extracted with hexane-ethanol (1:1, v/v) from skin surface using a handy extraction device, is converted into a fluorescent derivative by reaction with 3,4-dihydro-6,7-dimethoxy-4-methyl-3-oxo-quinoxaline-2-carbonyl azide. The derivative is separated from biogenic interfering substances on a reversed-phase column (TSK gel ODS-120T) with aqueous 90% (v/v) acetonitrile and detected fluorometrically. The detection limit (S/N=3) is 1.6 pmol (0.61 ng) cm-2 of human skin surface [corresponding to 18fmol (6.8 pg) in a 10 μ injection volume]. The correlation between age and the concentration of 7-dehydrocholesterol in skin surface is also described.