MICRO-HPLC & LC/MS METHODS FOR NEURAL SYSTEMS

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The objectives of this research program are to develop and utilize highly sensitive methods for the identification and quantitative analysis of phospholipids, glycosphingolipids, and oligosaccharides to enhance research efforts directed toward understanding the nervous system. Mass spectrometry can provide specific structural information and highly specific and sensitive detection. The proposed research is an effort to exploit the powerful analytical capabilities of high mass range Mass Spectrometry in combination with the high resolving power of micro HPLC. The specific aims of the proposal are: 1) to establish conditions for the liquid chromatographic/mass spectrometric (LC/MS) analysis of underivatized phospholipid classes and molecular species. We will utilize the in-source moving belt and instrumentation capabilities, such as fast atom bombardment (FAB), high resolution and linked scans, to develop methods for phospholipid molecular species analysis. 2) To develop methods for the LC/MS analysis of both underivatized and derivatized glycosphingolipids. We will explore the potential of FAB analysis of gangliosides with the LC/MS belt interface and negative ion detection. The permethylated derivatives have provided excellent results with direct chemical ionization and the extension of this approach to LC/MS will be undertaken. 3) To develop procedures for the LC/MS analysis of underivatized and derivatized oligosaccharides. 4) To test alternative methods of ionization and LC/MS interfacing which have potential for highly sensitive quantitation and structural analysis for the compounds of interest. These include thermospray ionization, plasmaspray, and dynamic (continuous flow) FAB for off-line and on-line LC/MS analysis. The overall significance of the proposed work lies in bringing the analysis of glycoconjugates and phospholipids to a level of specificity and sensitivity that will permit chemical- morphological correlations at the cellular level. Such capability should lead to a deeper understanding of the roles of glycoconjugates and phospholipids in normal and pathological processes.