Development of Electrophoretic Methods for Monitoring Small Molecule Neuroactive Compounds in Brain Microdialysis Samples

Abstract

The burden of neurological disorders has been increasing across the United States over the last thirty years with stroke, neurodegenerative diseases such as Alzheimer’s and migraine contributing the most to the number of disability adjusted life years. As the population continues to age, the need for targeted therapies for these conditions grows. However, even today, the available treatments are aimed at symptom management and not the root cause of the disease. Development of new pharmacological interventions requires a detailed understanding of the underlying biological processes, and in the case of the disorders affecting the nervous system, these are notoriously complex. Therefore, there is a high demand for analytical tools capable of providing insight into in vivo neurochemical dynamics.Microdialysis (MD) is a powerful technique that makes it possible to perform continuous sampling from living tissues for long periods of time. The composition of the obtained sample is reflective of all small molecules present in the MD probe surroundings (e.g. tissues), which means that with an appropriate analytical method it is possible to quantify multiple compounds simultaneously. To this end, separation-based techniques such as capillary and microchip electrophoresis (known as CE and ME respectively) can be used in tandem with MD to monitor neurochemical concentration changes in biological systems. By direct coupling of MD with ME (MD-ME) we are able to produce a separation-based biosensor for near real-time on-line in vivo and in vitro monitoring of the species of interest in continuous sample flow.This thesis focuses on the development of ME and CE methods for the monitoring of neurotransmitters, ascorbic acid (AA), and adenosine energy metabolites. First, the development of a ME separation and electrochemical detection (EC) of four monoamine neurotransmitters, two dopamine metabolites DOPAC and HVA, and AA is described and evaluated by the analysis of a rat brain homogenate sample. Next, the composition of the background electrolyte (BGE) used for the ME separation was further reoptimized to ensure compatibility with MD perfusates containing physiological concentrations of Ca2+ and Mg2+. This required a switch of the buffering system from phosphate to an organic buffer, MES, and the addition of EDTA to prevent precipitation of the metal cations with the surfactant present in the BGE.The effect of MD perfusate NaCl content on the dialysate ionic composition and in vivo recovery of monoamine neurotransmitter metabolites was also studied using CE with capacitively coupled contactless conductivity detection and liquid chromatography with EC detection. It was shown that to reliably evaluate the effect of sodium content on the recovery of analytes, the post-surgery rest time must be longer than 1 hour. Evaluation of Na+ recovery from tissues showed that at 40% of physiological concentration of NaCl there was no significant flux of sodium into the perfusate. It was also determined that, given sufficient post-surgery rest time, the recoveries of both the metabolites (DOPAC, HVA, and 5-HIAA) and sodium did not vary following initial equilibration of the probe during 90–100 min of sampling.A small-scale animal study was carried out to investigate the effect of glutamate content in perfusate on the signal of AA in dialysates obtained from rat brain using in vivo on-line MD-ME-EC analysis. A non-physiological effect of decreased ascorbate recovery during MD sampling with glutamate-containing solutions was observed, although ultimately it appeared to be an artifact of the MD probe batch used in the experiments.Finally, a robust reproducible CE-UV separation was developed for adenosine triphosphate, diphosphate, and monophosphate. It was shown to be compatible with a previously developed transient isotachoforesis method for on-line preconcentration of these analytes from MD samples.