A double column procedure for the simultaneous estimation of norepinephrine, normetanephrine, dopamine, 3-methoxytyramine and 5-hydroxytryptamine in brain tissue.

A double column procedure was devised for simultaneous estimation of norepinephrine, normetanephrine, dopamine, 3-methoxytyramine, and 5-hydroxytryptamine in brain tissue. Columns of aluminum oxide were placed on columns of Amberlite CG-50 by use of a specially devised column holder so that the effluent from the upper columns could flow directly into the lower ones. Perchloric acid extracts of brain samples were passed through the doubled columns after the pH was adjusted with K2CO3. Norepinephrine and dopamine in the extracts were adsorbed on aluminum oxide in the upper columns while normetanephrine, 3-methoxytyramine, and 5-hydroxytryptamine were retained by Amberlite CG-50 placed under the aluminum oxide columns. The amines adsorbed on both columns were eluted in a small volume of dilute HC1 and each of the amines was determined fluorometrically. Some modifications were incorporated into the existing methods for developing fluorescence from the amines to increase the sensitivity. The recovery rate throughout the entire procedure for the authentic amines added to brain homogenates was in the neighborhood of 90% or higher with little column to column variation. The procedure was tested in the estimation of the five amines on various parts of single brains of normal or pargyline-treated rats.