Rapid and sensitive method for measuring norepinephrine, dopamine, 5-hydroxytryptamine and their major metabolites in rat brain by high-performance liquid chromatography. Differential effect of probenecid, haloperidol and yohimbine on the concentrations of biogenic amines and metabolites in various regions of rat brain.

Abstract

A rapid and sensitive method has been developed for the simultaneous determination of norepinephrine, dopamine, 5-hydroxytryptamine and their respectively metabolites 3-methoxy-4-hydroxyphenylglycol, homovanillic acid, 3,4-dihydroxyphenylacetic acid, and 5-hydroxyindoleacetic acid in discrete brain regions of rats. The supernatants of tissue homogenates were injected directly into a reversed-phase liquid chromatography system, coupled with electrochemical detection. Each of these compounds gave a linear response over the range 5.5-200 ng/ml cerebellar homogenate (0.11-4.0 ng on column). Analytical recoveries of these compounds, added to the homogenate, were essentially complete when compared with standards dissolved in perchloric acid. The average between-assay coefficients of variation for all these compounds were lower than 6.9% over the range 5.5-200 ng/ml. The within-assay coefficients of variation were lower than 9.7%, measured at 5.5 or 23.6 ng/ml. With the present test parameters and mobile phase conditions, all compounds were readily oxidized at 0.8 V vs. a Ag/AgCl reference electrode. The method was applied to an analysis of the differential activity of biogenic amines in the rat striatum, hypothalamus, and hippocampus, produced by probenecid, haloperidol and yohimbine.