Determination of 5-hydroxytryptophol in urine by high-performance liquid chromatography: application of a new...

The aim of the present study was to develop a high-performance liquid chromatographic (HPLC) method for determination of the serotonin metabolite 5-hydroxytryptophol (5HTOL) in human urine. 5HTOL was liberated from its conjugated form by enzymatic hydrolysis and isolated by a sample clean-up procedure on a small Sephadex G-10 column. The eluate was injected onto an isocratically eluted C18 reversed-phase column and 5HTOL was converted into a fluorescent oxazole derivative by on-line post-column reaction with benzylamine in the presence of potassium hexacyanoferrate(III). The limit of detection was about 10 nM and the intra-assay coefficients of variation were below 4% with urine samples and standard solutions. The results indicate that the method can be used as a screening method to discriminate between normal and elevated levels of total (free + conjugated) 5HTOL in urine.