The determination of dopamine by a modification of the dihydroxyindole fluorimetric assay

Abstract

A simplified sensitive fluorimetric assay for dopamine based on the hydroxyindole principle is described. Oxidation of dopamine by ferricyanide and subsequent tautomerization both occur in the same, strongly alkaline, ethanolic solution in the presence of metabisulphite. The reaction is self regulating, and since times of additions of reagents are relatively unimportant and the final fluorescence is very stable, a large number of samples can be assayed together. The method was developed for assaying dopamine in N ethanolic (50%)-HCl eluates, after purification on a strongly acidic cation exchange column as previously described (Attack & Magnusson, 1970). Portions, up to 1 ml volume, of the 3.5 ml eluate can be taken. No preliminary, time consuming, neutralization step is required before oxidation. Fluorescence intensity shows a linear relation with concentration for 1 to 5,000 ng of dopamine per 1.6 ml of final solution, both in pure solutions and in column eluates. Fluorescence readings from biological material are given, with evidence for their reproducibility. These, together with other data, demonstrate the relative accuracy of the 'tissue blank' and indicate that amounts of dopamine greater than 3 ng per column can confidently be detected, and amounts greater than 10 ng can be measured quantitatively. Fluorescence spectra are presented for the fluorophore derived from authentic dopamine, and from suspected dopamine extracted from spinal cord of normal rats and rats treated with reserpine plus an inhibitor of synthesis. Values for the concentration of dopamine of 20 and <3 ng/g respectively, were obtained.