In 1964, the catecholamine assay described by Anton and Sayre (1) was modified by using a solution of Tris buffer, NaOH, and EDTA (2) to bring the tissue extract to the proper pH for adsorption of catecholamines onto alumina. This method was used to assay norepinephrine (NE) and dopamine (DA) in the same tissue extract from brain (2,3). It was necessary to perform duplicate experiments if one wished to measure 5-hydroxytryptamine
(5-HT) and with the increasing cost of research this constituted a very inefficient use of animals. As a result a start was made toward development of a simultaneous assay for NE, DA, and 5-HT from the same sample, utilizing the basic technique of Anton and Sayre for NE and DA extraction with isolation upon alumina. Since this effort began, others have published methods which also permit the simultaneous assay of these amines; however, to date, all of these utilize solvent extraction of homogenates (4-7).
This paper will describe a method which has the advantages of: (a) simple, quantitative extraction of amines in perchloric acid solution; (b) rapid adjustment of pH for alumina adsorption of catecholamines; (c) recoveries of amines in the range of 7&950/o ; (d) a single solvent step; (e) a combined oxidation procedure which permits rapid and accurate determination of NE and DA in a single sample; (f) solvent extraction of 5-HT at basic pH and the development of an accurate tissue blank permitting the utilization of the ninhydrin reaction for the determination of 5-HT.
This method provides for sensitive, rapid, and accurate estimation of NE, DA, and 5-HT and may be used with tissue samples ranging from 100 mg t'o 1.0 gm in weight from which quantities of amines ranging from 10 ng to 2 pg can be assayed.