Factors affecting the stability and separation of biogenic amines and their metabolites. Simultaneous measurement by HPLC with electrochemical detection

Abstract

We describe a simple and sensitive method for the rapid and simultaneous quantification of dopamine, 3-methoxytyramine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, serotonin, 5-hydroxyindoleacetic acid, and 5-hydroxytryptophan in the picogram range in small samples of brain tissue. After minimal sample preparation the amines were analyzed utilizing isocratic separation and reversed-phase high-performance liquid chromatography with amperometric detection. The effects of pH and methanol concentration in the solvent on the retention times of the amines on two different C-18 columns were investigated. Stabilities of the amines in solution were determined under various conditions. Light and air were found to be detrimental to the stability of indoles. In the absence of light, their stability was dependent on temperature and the presence of air; however, in the absence of air, light and/or temperature had little effect. The catechols were stable under most of these conditions. The assay has been applied to study the postmortem stability of dopamine, serotonin, and their metabolites in the striatum of rat brain. In the striatum 4 hr after death, the content of dopamine and 3,4-dihydroxyphenylacetic acid decreased by less than 20%, and 3-methoxytyramine increased by 158%, with no changes in serotonin, 5-hydroxyindoleacetic acid, and homovanillic acid.