Sensitive and Selective LC Determination of 5-Hydroxyindoles Through Online Electrochemical Fluorescence Derivatization Using Benzylamine

In this paper we describe a highly sensitive and selective LC method for the determination of 5-hydroxyindoles (serotonin, N -acetylserotonin, 5-hydroxyindole-3-acetamide, 5-hydroxytryptophan, 5-hydroxyindole-3-acetic acid, and 5-hydroxytryptophol) through post-column electrochemical derivatization and fluorescence detection. These 5-hydroxyindoles can be separated within 40 min by reversed-phase liquid chromatography using 250 mM acetate buffer/acetonitrile [pH 6.5; 95:5 (v/v)] under conditions of isocratic elution; then they were subjected to electrochemical oxidation with benzylamine to produce their corresponding fluorescent derivatives. We detected these derivatives spectrofluorometrically at 480 nm upon excitation at 345 nm. The detection limits ( S / N = 3) of these 5-hydroxyindoles were in the range 3.2–180 fmol per 20-µL injection.