The development of TLC method for the compounds determination

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A multitude of studies have revealed specific biological mechanisms that contribute to D-amino acid action and regulation in the mammalian central nervous system. The remarkable increase in our understanding of D-amino acid function and distribution in mammals is in many ways a result of the development of sensitive enantioselective separation strategies that allow for quantification in real biological samples. In capillary electrokinetic chromatography (cEKC) the most powerful chiral resolving agents are anionic cyclodextrins (CDs), yet these have not previously been investigated for chiral bioanalysis study of amino acids. The focus of this dissertation research was to investigate for the first time the feasibility of and application of anionic cyclodextrins as resolving agents in bioanalytical chiral separations of amino acids. This dissertation encompasses the development of a new bioanalytical separation utilizing capillary electrophoresis laser induced fluorescence (CE-LIF) with sulfated-β-cyclodextrin for analysis of D-serine (D-ser) and L-glutamate (L-glu) in mammalian brain, the first synthesis and characterization of 6 members of a new family of single isomer sulfoalkyl cyclodextrins, initial studies on chiral analysis of amino acids using single isomer sulfoalkyl CDs, and development and application of a novel microperfusion sampling approach for acute brain slices and coupling of this method to the developed chiral CE-LIF for studying magnitude and timing of D-ser and L-glu efflux from acute hippocampus in response to modeled cerebral ischemia. The results of these studies demonstrate that anionic CDs are powerful chiral selectors for amino acids and can be applied for sensitive bioanalysis study of D-amino acids including D-ser, D-glu, and D-asp in brain samples; single isomer sulfoalkyl CDs can be synthesized by regioselective reaction chemistry; single isomer sulfoalkyl CDs are excellent resolving agents for amino acid analysis and may be valuable for bioanalytical chiral applications; and microperfusion sampling coupled to CE-LIF can be used to analyze dynamic changes in the magnitude and timing of neurochemical efflux from single acute hippocampus slices exposed to modeled ischemia. Results of these latter studies suggest that D-ser and L-glu efflux occurs simultaneously in acute hippocampus with similar timing but differing magnitudes.