Determination of urinary 5-hydroxytryptophol by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method for the routine determination of elevated urinary levels of the serotonin metabolite 5-hydroxytryptophol (5-HTOL) is described. Urine samples were treated with beta-glucuronidase, and 5-HTOL was isolated by solid-phase extraction on a small Sephadex G-10 column prior to injection onto an isocratically eluted C18 reversed-phase column. Detection of 5-HTOL was performed electrochemically at +0.60 V vs. Ag/AgCl. The limit of detection was ca. 0.05 microM, and the intra-assay coefficients of variation were below 6% with urine samples containing 0.2 and 2.1 microM 5-HTOL and a standard solution of 2.0 microM (n = 5). The recovery of 5-HTOL after the sample clean-up procedure was close to 100%. A good correlation (r2 = 0.97; n = 12) was obtained between the present method and a sensitive and specific gas chromatographic-mass spectrometric method. The total (free plus conjugated) 5-HTOL levels in urine were normally below 0.2 microM, but after an acute dose of alcohol they increased to 0.5-15 microM.