Quantitative determination of 5-hydroxytryptophan in dissected brain regions by on-line trace enrichment HPLC with electrochemical detection

A sensitive, specific and rapid quantitative HPLC assay for 5-hydroxytryptophan (5-HTP) in samples of brain regions of widely differing size is described. The method utilizes off-line prepurification of tissue supernatants on gravity-fed strong cation exchange columns, on-line enrichment of the entire cation exchange column eluate on short reverse phase enrichment precolumns, further separation by reverse phase chromatography on an analytical column and electrochemical detection. On-line trace enrichment permits the efficient incorporation of off-line column chromatography to maximize assay specificity without compromising assay sensitivity. A reliable, working limit of detection of 200 pg 5-HTP/sample permits the estimation of in vivo tryptophan hydroxylase activity by determining the rate of 5-HTP accumulation following L-aromatic amino acid decarboxylase inhibition in small discrete brain regions or larger tissue samples only poorly innervated by 5-HT terminals.