Sensitive and simultaneous quantification of 16 neurotransmitters and metabolites in murine microdialysate by fast liquid chromatography-tandem mass spectrometry

The sensitive and simultaneous measurement of multiple neurotransmitters in microdialysate (MD) of freely moving mice is a prerequisite to study neurochemical imbalances in specific brain regions. The quantitative analysis of 16 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), homovanillinic acid (HVA), acetylcholine (ACh), deoxy carnitine (iso-ACh), choline (Ch), and ɣ-aminobutyric acid (GABA), adenosine (ADE), glutamine (Gln), and glutamic acid (Glu) was achieved within a chromatographic separation time of 6.5 min by the application of a biphenyl column coupled to an API-QTrap 5500 (AB SCIEX) mass spectrometer. Optimized chromatographic separation as well as high sensitivity allow the simultaneous analysis and precise quantification of 16 neurotransmitters and metabolites in artificial cerebrospinal fluid (CSF). Sample preparation procedure consisted of simply adding isotopically labeled internal standard solution to the microdialysis sample. The limits of detection in aCSF ranged from 0.025 pg (Ch) to 9.75 pg (Gln) and 85.5 pg (HVA) on column. Recoveries were between 83 and 111% for neurotransmitter concentrations from 0.6 to 45 ng/ml or 200 ng/ml with a mean intra-day and inter-day coefficient of variation of 7.6% and 11.2%, respectively. Basal extracellular concentrations of the following analytes: 5-HT, 5-HIAA, ME, DA, 3-MT, HVA, ACh, iso-ACh, Ch, GABA, ADE, Gln, and Glu were determined in the striatum of mice with a MD flow rate of 0.5 μl/min. This LC-MS/MS method leads to an accurate quantification of ACh and its isobaric structure iso-ACh, which were detected in the MD samples at ratios of 1:8.6. The main advantage of the high sensitivity is the miniaturization of the MD protocol with short sample collection times and volumes down to 5 μl, which makes this method suitable for pharmacological intervention and optogenetic studies to detect neurochemical changes in vivo. © 2022 The Authors