Simultaneous determination of urinary catecholamines and 5-hydroxyindoleamines by high-performance liquid chromatography with fluorescence detection.

Abstract

A simple, selective and sensitive high-performance liquid chromatographic method with fluorescence detection is described for the simultaneous determination of catecholamines and 5-hydroxyindoleamines. The method is based on the derivatization of the amines with benzylamine in the presence of potassium hexacyanoferrate(III) under mild conditions. The resulting fluorescent derivatives are separated on a reversed-phase column, Cosmosil 5C18, using a mixture of acetonitrile and 10 mmol l-1 acetate buffer (pH 6.0) (35 + 65 v/v) as mobile phase, and are detected spectrofluorimetrically at 480 nm with excitation at 345 nm. The detection limits (signal-to-noise ratio = 3) for the amines are 2.8-236.0 fmol for an injection volume of 50 microliters. The method was successfully applied to the determination of 5-hydroxyindol-3-ylacetic acid, serotonin, norepinephrine and epinephrine in human urine. The method is simple and does not require clean-up of urine samples.