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, 5-hydroxytryptophol) through post-column electrochemical derivatization fluorescence detection. These 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 isocratic elution; then they were subjected to oxidation with benzylamine produce their corresponding fluorescent derivatives. We detected these derivatives spectrofluorometrically at 480 nm upon excitation 345 nm. The detection limits (S/N = 3) in range 3.2–180 fmol per 20-µL injection.