[Microbore liquid chromatography with dual electrode electrochemical detection of monoamine transmitters in brain...

In the present work, the method of microbore liquid chromatography(MLC) with dual electrode electrochemical detection (DEED) was used to determine the monoamine transmitters and their metabolites in the striatal microdialysates from the anaesthetized rats. The potentials applied on the upstream (anodic) electrode and downstream (cathodic) electrode were set at +0.72V and +0.05V versus an Ag/AgCl reference electrode respectively. The interference of the large early-eluting peaks from brain dialysate was well eliminated on cathodic detection on the basis of electrochemical reversibility of catechol compounds. The collection efficiency of catecholamine (NA, A and DA) and DOPAC, that is the ratio of their responses in downstream electrode to those in upstream, was found to be in the range of 0.690 to 0.768, thereby providing reliable identification and measurement of early elutes. MLC-DEED is one of the best methods for the determination of monoamine transmitters at trace level. To optimize separation and detection, these adaptations were made to the system with respect to the injection valve, flow rate of the pump, connections between injector, column and detector, and cell volume of the detector. The detection limit could reach 1pg or less per injection, and the amounts, at least in the range of 2-200pg, were linearly correlated with the peak areas both at anodic detection and at cathodic detection. Prior to the collection of samples the probe recovery was measured in vitro at room temperature, and the recovery (%) for NA, DA, DOPAC, 5-HIAA, 5-HT and HVA were 19.5, 18.9, 22.1, 14,6, 16.5 and 19.3 respectively. The reported concentration of each compound was corrected according to probe recovery. The basal concentration of DA and 5-HT in striatal microdialysates was 12.5 +/- 1.8 and 5.0 +/- 0.7nmol/L. The perfussion of high K+ (100 mmol/L) through the dialysis probe induced a large increase of the concentration of NA, DA and 5-HT in microdialysates, and further increase of the concentration was observed in rat postmortem. The critical points for MLC-DEED are briefly discussed.