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.
D006916:5-Hydroxytryptophan / Q000032:analysis*; D000818:Animals; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004563:Electrochemistry; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains
D006916:5-Hydroxytryptophan