Mass Spectrometry Based Techniques for In Vivo Neurochemical Monitoring.

Abstract

Neurotransmitter concentration in vivo reflects various behavioral and pharmacological events. Therefore, the measurement of neurochemical dynamics in live behaving animals’ brain is of crucial importance for the fundamental understanding of the central nervous system and the development of therapies for neurological disorders. Previously published neurochemical monitoring methods, although being capable of measuring one or one class of neurotransmitters, fall short of truly multiplexed neurochemical monitoring. We developed a liquid chromatography-tandem mass spectrometry based assay of all commonly studied small molecule neurotransmitters, detecting 17 neurotransmitters and metabolites sampled in vivo at basal level. Prior to LC-MS analysis, amine and phenol containing analytes were benzoylated using benzoyl chloride, significantly improving chromatographic separation and mass spectrometric sensitivity. Additionally, stable isotope labeled internal standard was produced for each analyte, by reacting with commercially available C13 benzoyl chloride. The quantitative performance was enhanced. It demonstrated the ability to work with high spatial (1 mm) and high temporal resolution (1 min) monitoring. A benzoylation UHPLC-MS method was further developed into a faster and scaled down version. A two-fold improvement in throughput and four-fold reduction in sample consumption was achieved using a capillary column packed with high efficiency particles. It was suitable for high temporal resolution monitoring with conventional microdialysis sampling. For the neurotransmitter, acetylcholine, in vivo monitoring, high temporal resolution and high sensitivity are strongly desired due to its low basal levels and rapid changes during neuroexcitation such as behavioral activation. Current technologies do not meet these specific characteristics well. Electrochemical sensors have high temporal resolution but lack sensitivity while the opposite holds true for HPLC based methods. We developed an online acetylcholine monitoring method using segmented flow microdialysis - mass spectrometry. This novel acetylcholine “sensor” combines 5 s temporal resolution with 5 nM detection limit capable of detecting basal levels. Additionally, other analytes such as choline and the acetylcholine esterase inhibitor, neostigmine can both be detected simultaneously, demonstrating the versatility of mass spectrometry for detection. Tested in vivo, this setup captured both acetylcholine increase in response to acetylcholine esterase inhibition and decrease due to neuronal inhibition by tetradotoxin.