Reversed-phase ion-pair high performance liquid chromatography with electrochemical detection for simultaneous quantification of neurotransmitters in mice brain tissues

Abstract

An analytical method for the simultaneous quantification of neurotransmitters has been developed using reversed-phase ion-pair high performance liquid chromatography (RPIP-HPLC) coupled with electrochemical detection (ECD). As a strategy to simultaneously analyze acetylcholine, glutamate, and γ-aminobutyric acid, which were previously difficult to analyze in ECD, along with serotonin and dopamine, the ion pair separation technique was optimized, and electroactivity was induced by eluting with strong alkaline solution. This method achieved improved sensitivity, showing limits of quantification of 0.02 to 0.1 μg/mL and linear regression coefficients of determination of 0.9938–0.9994 through the use of an 8% acetonitrile solution containing 15 mM H3PO4 and 0.4 mM sodium 1-octanesulfonate as the mobile phase and 200 mM NaOH as the post-column eluent, detected at the gold electrode. All inter- and intra-day precision were below 4.10%, and the average recoveries were 97.43–103.69%. In an application comparing cerebral cortical tissues and hippocampal tissues between a control group and a scopolamine-treated group, the levels of the five components showed a clear difference. As a result of evaluating the eco-friendliness of this method, the Analytical GREEnness metric was determined as 0.47. Our method is expected to be useful in screening for neurotransmitters in various biological and clinical samples.