High-performance liquid chromatographic determination of 5-hydroxyindoles by post-column fluorescence derivatization.

A selective and sensitive high-performance liquid chromatographic method with post-column fluorescence detection has been developed for the determination of 5-hydroxyindoles. Five biogenic 5-hydroxyindoles (5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxyindol-3-ylacetic acid, 5-hydroxytryptophol and N-acetyl-5-hydroxytryptamine) were separated by isocratic elution on a reversed-phase column, TSKgel ODS-80Tm, and then converted into fluorescent derivatives by post-column reaction with benzylamine and potassium hexacyanoferrate(III) reagents. The detection limits (signal-to-noise ratio = 3) for the indoles were in the range 140-470 fmol per injection volume (100 microliters). The method was applied to the determination of 5-hydroxyindol-3-ylacetic acid in human urine, with direct sample injection.