Development of a fast liquid chromatography-tandem mass spectrometry method for simultaneous quantification of neurotransmitters in murine microdialysate

Abstract

The continuous measurement of multiple neurotransmitters in microdialysate of freely moving mice to study neurochemical changes in specific brain regions requires a rapid and very sensitive quantitative analytical method. The quantitative analysis of 11 neurotransmitters and metabolites, including serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), melatonin (ME), dopamine (DA), levodopa (L-DOPA), 3-methoxytyramine (3-MT), norepinephrine (NE), epinephrine (EP), acetylcholine (ACh), choline (Ch), and γ-aminobutyric acid (GABA), was performed using a biphenyl column coupled to an API-QTrap 3200 (AB SCIEX) mass spectrometer in positive electrospray ionization mode. To the microdialysate samples, 0.5 ng of isotopically labeled standard was added for analyte quantification. A rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous analysis of monoamines, their precursor, and metabolites, as well as ACh, Ch, and GABA in murine microdialysate within 7.0 min. The limit of detection in artificial CSF ranged from 0.005 ng/mL (ME) to 0.75 ng/mL (NE and GABA). A comprehensive pre-analytical protocol was validated. Recovery was between 87 and 117% for neurotransmitter concentrations from 0.6 to 45 ng/mL with an inter-day accuracy of below 20%. Basal neurotransmitter values were determined in the striatum of mice over a time period of 3 h. This LC-MS/MS method, including a short and gentle sample preparation, is suitable for simultaneous measurements of neurotransmitters in murine cerebral microdialysate and enables the determination of basal neurotransmitter levels in specific brain regions to detect disease-related and drug-induced neurochemical changes.Graphical abstract.