(2-Phthalimidyl)benzoyl Azides as Fluorescence Labeling Reagents for Alcohols in High Performance Liquid Chromatography

Abstract

Two fluorescence labeling reagents, 3- and 4-(2-phthalimidyl)benzoyl azide, were designed for primary and secondary alcohols for high performance liquid chromatography. These reagents, prepared from orthophthalaldehyde and aminobenzoic acid in several steps, react with alcohols at 125°C for 30 min to produce the fluorescent derivatives, which can be separated on a reversed-phase column ERC-ODS-1161 with acetonitrile/water (70:30) or acetonitrile/ethanol (70:30) as eluent. The extent of conversion of cholesterol into fluorescent derivative was 100%. The detection limit for the alcohols labeled with 3-(2-phthalimidyl)benzoyl azide was 0.1-0.4 pmol per injection (10 μl). 3-(2-Phthalimidyl)-benzoyl azide was applied for the determination of cholesterol in human serum by high performance liquid chromatography with fluorescence detection. The values of cholesterol in normal human sera were 4.8-9.4 μmol/ml.