Use of a Novel Type of Rotating Disc Electrode and a Flow Cell with Laminar Flow Pattern for the Electrochemical Detection of Biogenic Monoamines and Their Metabolites After Sephadex Gel Chromatographic Purification and High Performance Liquid Chromatographic Isolation from Rat Brain

Abstract

A novel type of rotating disc electrode and a flow cell with laminar flow pattern were developed and applied to the electrochemical detection of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, 3-methoxytyramine (3-MT), noradrenaline, 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid after HPLC of these compounds. The active surface of the rotating disc working electrode was made from solid paraffin (40% wt/wt) and graphite powder (60% wt/wt). The sensitivity of the detector was proportional to the square root of the angular velocity and was practically independent of the flow rate of the mobile phase. The surface of the working electrode was very large (radius = 12 mm), and so the percentage of oxidation was 24-67% (flow rate = 1.0 ml/min), depending on the compound. Electrical noise between 20 and 40 pA and background current of 20-60 nA were observed. In practice, the sensitivity for the detection of the compounds examined here was 8-16 nA/ng, and so a detection limit of 5 pg/injection could be achieved, when the detector was combined with reversed-phase HPLC. Supernatants obtained from the extracts of the tissue samples (nine brain parts of rat brain were studied) were purified by using Sephadex G-10 gel chromatography. Before this procedure, the proteins of the tissue extracts were precipitated by 0.2 M HClO4, and the excess of HClO4 was precipitated by KOH/HCOOH buffer. Simultaneously, the pH of the extracts was set to 2.4 by the above buffer.(ABSTRACT TRUNCATED AT 250 WORDS)