Advances in capillary liquid chromatography: Multi-stage mass spectrometry for online monitoring of a neuropeptides and neurotransmitters in dialysate.
Shackman, Holly Merideth
2005
Abstract
Neurotransmitters are secreted from neurons to facilitate chemical communication with receptors on neighboring cells so that information necessary for daily function may be transmitted. Because defects present in the nervous system are responsible for many diseases and addictions, it is imperative to understand the multiple roles of both small molecule neurotransmitters as well as neuropeptides, as they are present in varying concentrations and locations within the body. However, studying neurochemistry has proven difficult as small volumes and concentrations provided by sampling methods challenge instrumental limits of detection, requiring development and refinement of analytical methods. Capillary liquid chromatography (cLC) was coupled to triple-stage mass spectrometry (MS) in order to increase the sensitivity, selectivity, specificity, and reproducibility of monitoring the enkephalin neuropeptides present in microdialysate. By utilizing high-pressure sample loading and washing along with a rapid gradient, temporal analysis of a single injection onto the system was also decreased. These improvements allowed for the measurement of basal and K<super>+</super> stimulated enkephalin levels taken from the striatum of anesthetized and freely-moving rats. The system demonstrated versatility by measuring changes in enkephalin dialysate concentrations upon administration of pharmacological compounds. Intravenous (i.v.) and intraperitoneal (i.p.) injections of vehicle or the enkephalinase inhibitor RB101 were given to anesthetized and freely-moving rats. Unexpected decreases in both enkephalin levels were seen after i.v. RB101 and vehicle i.p. injections. An increase in [Met]enkephalin dialysate concentrations was detected after i.p. RB101 administration, although [Leu]enkephalin did not exhibit any deviations. Further analytical advancements are necessary to routinely monitor changes induced by RB101. To extend the application of cLC-MS technology to other transmitters, a method was developed for determining acetylcholine (ACh). Strong cation exchange capillary liquid chromatography was coupled to single reaction monitoring tandem mass spectrometry to improve the sensitivity and temporal resolution of current methodology to routinely monitor the small molecule neurotransmitter ACh in microdialysate without acetylcholinesterase inhibition commonly used to augment endogenous concentrations. Basal and K<super>+</super> stimulated levels of ACh sampled from the striatum were monitored in rats anesthetized with chloral hydrate or ketamine. Absolute and percent basal levels of ACh from rats receiving different anesthetics were also compared.Subjects
Advances Capillary Liquid Chromatography Dialysate Mass Spectrometry Monitoring Multi Neuropeptides Neurotransmitters Online Stage
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