Simultaneous determination of cholesterol and cholestanol in human serum by high-performance liquid chromatography using 3-(5,6-methylenedioxy-2-phthalimidyl)benzoyl azide as precolumn fluorescent labelling reagent

A fluorescent labelling reagent, 3-(5,6-methylenedioxy-2-phthalimidyl)benzoyl azide, designed for the determination of alcohols by precolumn high-performance liquid chromatography, has been applied to the simultaneous determination of cholesterol and cholestanol in human serum. The reagent reacts with cholesterol and cholestanol at 140°C for 10 min to produce the fluorescent derivatives, which can be separated on a reversed-phase column with acetonitrile—ethanol—water (60:35:7.5, v/v) as eluent. The detection limits for cholesterol and cholestanol were 45 and 50 fmol per injection (20 μl), respectively. The values of cholesterol and cholestanol in normal human sera were 135–212 mg/dl and 137–928 μg/dl, respectively.