Liquid Chromatography-Tandem Mass Spectrometry in Studies of Neurotransmitters and Their Metabolites in the Brain
Year: 2009
Type: dissertation
Abstract: Neurotransmitters transfer chemically the electrical impulse from one neuron to another in the brain. The concentration of neurotransmitters in many neurological disorders is altered. The measurement of neurotransmitters in the brain is needed to understand how these diseases develop and how they can be treated. Neurotransmitters can be extracted from the brains of freely moving, alert animals by microdialysis technique. The concentration of neurotransmitters and their metabolites in brain microdialysates is low, and therefore highly sensitive and specific analytical methods are needed. The aim of this study was to develop such liquid chromatography-electrospray-tandem mass spectrometric (LC-ESI-MS/MS) methods for the analysis of neurotransmitters in brain microdialysates. Neurotransmitters and their metabolites are known to form sulfate and glucuronide conjugates in the brain. However, only non-specific indirect analysis methods employing acid or enzyme hydrolysis have been used in the analysis of brain and cerebrospinal fluid (CSF) samples. In this work sulfate and glucuronide conjugates of dopamine and serotonin, and their phase I metabolites were synthesized for reference compounds. With LC-MS/MS method developed the regioisomers of conjugates could be detected, which has not been possible with indirect methods. In this study, the serotonin and dopamine glucuronides was discovered in rat and mouse brain microdialysates for the first time. It was observed that neurotransmitters were glucuronidated while their acidic phase I metabolites were sulfated. The analysis of gamma-aminobutyric acid (GABA) in rat brain microdialysates by LC-MS has been problematic due to high LODs. In this study amino acids were derivatized to enhance their retention on reversed phase chromatography and ESI. Three different commonly used derivatization reagents (propyl chloroformate, butanol and 9-fluorenylmethyl chloroformate) were compared in the analysis of amino acids by LC-MS/MS. The limits of detection (LOD) for derivatives were 2-60 times lower than for native underivatized amino acids. After propyl chloroformate derivatization, GABA and nine other amino acids could be easily quantified in rat brain microdialysates by LC-MS/MS. In the analysis of acetylcholine (Ach) and choline, hydrophilic interaction liquid chromatography (HILIC) was utilised. In HILIC polar stationary phases are used and the retention of polar compounds increases with increasing organic solvent content. The high concentration of ACN (80 %) in the eluent was favourable to ESI enabling sensitive analysis of Ach. The limit of quantification (LOQ) for Ach was 0.1 nM, which allowed the analysis of Ach in rat and mouse brain microdialysates without the use of acetylcholinesterase inhibitors.
Source: Työväentutkimus Vuosikirja
Author: Päivi Uutela
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funder: Helsingin Yliopisto
OpenAlex ID: W814995654