The Determination of Catecholamines, Indoleamines, Metabolites, and Related Enzymatic Activities Using Three Micron Liquid Chromatography Columns

Abstract

Liquid chromatography with electrochemical detection has become an established technique for the determination of catechol-amines, indoleamines, precursors, metabolites, and related enzymatic activities in tissues and fluids. Previously available instrumentation, however, has limited the number of individual species readily and simultaneously accessible with reasonable throughput to only a few. Determinations of other species required either extended amounts of time per individual chromatogram or the use of an entirely separate chromatographic setup employing different columns and eluting solvents. Using reversed-phase columns packed with 3 micron particles, we have been able to produce the separation of 16 different catecholamine and in-doleamine related species and two different internal standard compounds in 5 or 7 minutes. Samples may be analyzed directly after only homogenization, centrifugation, and clarification by filtration. No further purification steps are required. The enzymatic activities of 6 separate enzymes may be determined using the same chromatographic apparatus and simply monitoring selected metabolites following appropriate incubation of pretreatment. The metabolites and transmitters currently accessible with this apparatus include norepinephrine, dopamine, epinephrine, serotonin, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenylalanine, normetanephrine, metanephrine, 3-methoxytyramine, 3,4-dihydroxy-phenylethyleneglycol, vanillylmandelic acid, homovanillic acid, 5-hydroxytryptophan, 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, and N-acetyl-5-hydroxytryptamine. The enzymatic activities include tyrosine hydroxylase, tryptophan hydroxylase, dopa decarboxylase, 5-hydroxytryptophan decarboxylase, monoamine oxidase, and catechol-O-methyltransferase.