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
Highlights
Neurochemistry provides insights into the pathophysiology of neurological diseases and mechanisms of drugs
For the final microdialysate setup, we evaluated the MD probe, the isoosmotic perfusate, the microdialysis flow rate, and the sampling time (Fig. 1)
A quantitative tandem mass spectrometric method was developed for the analysis of components of neurotransmitter pathways in murine microdialysate samples
Summary
Neurochemistry provides insights into the pathophysiology of neurological diseases and mechanisms of drugs. The specific analyte determines the selection of the MD probe (membrane cutoff and probe dimension) as well as the composition of the perfusate (i.e., ion composition, stabilizer, or uptake blockers) that modifies the neurotransmitter content which has to be considered to determine basal differences or dynamic changes [2]. All these aspects as well as the instrumental MD setup, the perfusion flow rate, fraction collection time, and stimulation methods influence the development of the following quantitative analysis (Fig. 1). Most demanding requirements for the subsequent analysis are as follows: (I) high selectivity of the chromatographic separation; (II) high sensitivity to quantify concentrations in the expected range from picograms to nanograms; (III) short and gentle sample preparation, including avoidance of derivatizations due to the low sample volume and simultaneous determination of instable monoamines
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
