Development of an Analytical Method for the Detection and Quantitation of Nitrite as a Biomarker of Nitric Oxide and Reactive Nitrogen Species in Biological Samples By

The brain is a complex organ, consisting of blood vessels, neurons, neuronal support cells, and meninges. The extracellular space surrounding the neurons is a very complex microenvironment, containing proteins, growth factors, neurotransmitters, and other small molecules. Microdialysis has been extensively used for sampling analytes from extracellular spaces in tissues. The microdialysis membrane excludes large molecules, making it possible to obtain protein free samples from this space that do not require any cleanup steps. The use of microdialysis allows the sampling of multiple analytes in extracellular spaces from multiple regions, making it possible to obtain a temporal profile over the time course of an experiment. High performance liquid chromatography (LC) coupled with ultraviolet (UV) and electrochemical (EC) detection makes it possible to analyze samples collected via microdialysis with high throughput. Our lab has developed an LC UV-EC based separation and detection method that is capable of the high throughput needed to analyze small sample volumes directly from animal studies to study changes in nitric oxide synthase (NOS) activity and reactive nitrogen species formation in the brain and other tissues with high temporal resolution. In this thesis, an LC based ion-pair chromatography method is described in conjunction with EC and UV detection methods for the determination of primary and iv secondary products of nitric oxide metabolism (nitrite and nitrate) in the microdialysate. The development of an ion-pair based separation and combined UV-EC detection of nitrite and nitrate were the fundamental steps for the optimization of separation of nitrite and nitrate present in microdialysates. Detection optimization for nitrite and nitrate was carried out in the same matrix as that of perfusate used in animal experiments. Separation conditions consisted of 1 mM tetrabutylammonium hydroxide (TBAOH), 15 mM sulfate at pH 4.0, a flow rate of 0.1 mL/min, using a C-18 4 µM 1x150 mm column as the stationary phase. Microdialysis samples were then injected, separated, and detected using LC with UV-EC detection. The first application of this method involved monitoring changes in nitrite and nitrate concentrations in the rat hippocampus region as a result of chemically-induced excitotoxicity. 3-Mercaptopropionic acid (MPA) was perfused through a microdialysis probe into the hippocampus brain region of a rat, as had occurred in past studies of our research group. In contrast to the elevation of other biomarkers seen by the administration of this compound, this method was unable to detect changes in the concentration of nitrite, and the changes …