A New Method for Identifying Neurotransmitters and Their Metabolites

Neurotransmitters (NTs) are signaling molecules that can be released by a neuron at a chemical synapse. They act as chemical messengers from a presynaptic neuron to a postsynaptic cell, which may respond in either an excitatory or inhibitory manner. There are four classes of NTs, which are organized primarily according to structure: monoamines, amino acids, peptides and other structures. NTs and their metabolites are important in biological systems because their imbalance may lead to diseases such as schizophrenia, depression, Alzheimer’s disease and Parkinson’s disease. It is therefore important to be able to identify and quantify NTs. There are many challenges in detecting these molecules, however, including complex matrix interference, chemical instability and low concentrations.

Previous studies have used liquid chromatography–mass spectrometry (LC-MS) to identify NTs and their metabolites in samples. While these studies indicate a good platform for quantitative detection, many have poor resolution, which is not ideal for future analysis. Yang et al. show that pairing high-performance liquid chromatography with high-resolution mass spectrometry (HPLC-HRMS) could help alleviate this issue and provide results that have both quantitative detection and ideal resolution.¹ This study is the first to show simultaneous detection of NTs and their metabolites in the mouse hypothalamus using the Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific), which is a benchtop LC-MS/MS system that combines quadrupole precursor ion selection with high resolution as well as accurate mass orbitrap mass analyzer based detection.

Yang et al. wished to establish a stable, reliable, sensitive and convenient method to monitor NTs and their metabolites. To accomplish this, the group first identified the best mode to use with the mass spectrometer, from the three options of full scan, selected ion monitoring and targeted MS2. When they compared the modes based on reproducibility and sensitivity, the group identified targeted MS2 as the best mode for identifying NTs and their metabolites. Next, they evaluated the system using calibration standards and quality-control samples to observe the feasibility of applying targeted MS2 to regulated bioanalysis. Their evaluation showed that using this mode gives sensitive, accurate, precise and stable results. Finally, the group applied their system by looking at mouse hypothalamus samples. Results from a test of 10 male mice showed a high concentration of γ-aminobutyric acid (GABA) and L-tyrosine (L-AA) in hypothalamus tissue, while homovanillic acid (HVA) and epinephrine (E) were at lower concentrations. This method verified the results of other studies and showed greater resolution.

The methods described in this paper show that the Q Exactive mass spectrometer is reliable with high sensitivity in the detection of NTs and their metabolites in a biological system. Furthermore, using the MS2 mode gave good sensitivity, accuracy and precision, showing the feasibility of using HPLC-HRMS for quantitative analysis. This shows great promise for future clinical applications, including disease identification and therapeutic monitoring.

Reference
1. Yang, Z.-L., et al. (2016) “An optimized method for neurotransmitters and their metabolites analysis in mouse hypothalamus by high performance liquid chromatography—Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry,” Journal of Chromatography B, 1012–1013 (pp. 79–88).