Simultaneous Determination of Serotonin and 5-Hydroxyindole-3-acetic Acid in Human Urine by Automated Precolumn Derivatization and Semi-microbore Column Liquid Chromatography with Fluorescence Detection

Abstract

An improved high-performance liquid-chromatographic (HPLC) assay for the simultaneous determination of serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA) in human urine is reported. Following the automated precolumn derivatization of urinary 5-HT and 5-HIAA with benzylamine, the derivatives are separated by isocratic elution on a reversed-phase C₁₈ semi-microbore column, and are fluorometrically detected at an excitation wavelength of 345 nm and an emission wavelength of 480 nm. The detection limits of 5-HT and 5-HIAA are 7 and 5 nmol/l in urine (0.7 and 0.5 fmol/20-µl injection). The proposed automatic method permits a highly selective and sensitive determination of 5-HT and 5-HIAA in human urine without any sample purification.