Determination of the biogenic amines and their major metabolites in single human brain tissue samples using a combined extraction procedure and high-performance liquid chromatography with electrochemical detection

A combined extraction system for the selective and quantitative isolation of the monoamines norepinephrine, epinephrine, dopamine, serotonin (5-hydroxytryptamine) and their metabolites 3-methoxy-4-hydroxyphenylethylene glycol, 3,4-dihydroxyphenylacetic acid, 5-hydroxyindoleacetic acid, homovanillic acid and 3-methoxytyramine from one single brain tissue sample is described. The extraction system is a combination of an ethyl acetate extraction for 3-methoxy-4-hydroxyphenylethylene glycol, 3,4-dihydroxyphenylacetic acid, 5-hydroxyindoleacetic acid and homovanillic acid, and two successive ion-pair extractions. In a first step, the catecholamines are quantitatively isolated by extracting with heptane—octanol (99:1) containing 0.25% tetraoctylammonium bromide as an ion-pairing agent in the presence of 0.2% diphenylborate. In a second step, 3-methoxytyramine and 5-hydroxytryptamine are isolated from the aqueous phase with di(2-ethylhexyl)phosphoric acid as counter-ion in chloroform. Dihydroxybenzylamine, isohomovanillic acid and 5-hydroxy-N-methyltryptamine are used as the internal standards.