A Simple High-Performance Liquid Chromatographic Method for the Simultaneous Determination of Monoamine Neurotransmitters and Relative Metabolites with Application in Mouse Brain Tissue

摘要

As a means to evaluate the role of organic solute carriers in CNS homeostasis, a simple and rapid high-performance liquid chromatographic method utilizing ultraviolet and electrochemical detectors was developed and validated for the simultaneous determination of the neurotransmitters dopamine, norepinephrine, and serotonin and their metabolites in mouse brain homogenate. For analyte separation, the method utilized a C-18 column with a mobile phase of 75mM sodium dihydrogen phosphate (monohydrate), 1.7mM 1-octanesulfonic acid sodium salt, 25 mu M EDTA, 10% acetonitrile, and 1% triethylamine with a final pH of 3.0 adjusted using phosphoric acid. The method was linear for dopamine, norepinephrine, serotonin, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid from 20-1000ng/mL (r >= 0.993) with detection limits of 5ng/mL for dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid, and of 20ng/mL for serotonin and 5-hydroxyindoleacetic acid. The method was linear over the range of 10-200 mu g/ml for quinolinic acid, xanthurenic acid, and nicotinic acid (r >= 0.993) with detection limits of 1 mu g/mL for all components. At a flow rate of 1mL/min, the analytical run time was less than 10min. The method was successfully applied to the quantitation of these compounds in mouse whole brain homogenates.