MS based approaches for the analysis of small molecules such as sugar nucleotides and gangliosides
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The field of metabolomics has become a hot spot of analytical investigations since the discovery of the encompassing significance of the metabolome in complex eukaryotic systems. Most notably, emphasis in this field is currently being placed in the discovery of potential biomarkers through development of highly sensitive methods that are capable of identifying and quantifying small metabolites of interest in complex samples. In this thesis, a concise review is described in chapter 1 over the field of metabolomics as it pertains to the analytical developments of sugar nucleotides and ganglioside analysis. In addition, targeted mass spectrometric approaches are presented in which analytical methodologies are developed for sugar nucleotide and ganglioside profiling for the analysis of diseased biological samples (such as cancer) , with interest focused on the establishment of metabolic profiles of diseased states. Chapter 2 details the analytical method for targeted mass spectrometric analysis of sugar nucleotides. The method described permitted the establishment of a sugar nucleotide linear dynamic range of quantification expanding over almost three orders of magnitude with an extremely reproducible chromatographic separation in less than six minutes. Viability of this method was then tested by successfully quantifying the sugar nucleotide content of human cells from 3 different cancer cell lines. The developed short chromatographic method potentially permits for high throughputs analysis for its integration into population studies for the search for potential biomarkers. Chapter 3 describes the developed methodology for targeted ganglioside analysis. A chromatographic method was developed based on the use of a mixture of bovine brain gangliosides that allowed for complete separation in less than 7.5 minutes. Quantificationof gangliosides was also achieved by the construction of a calibration curve from ganglioside standards. The established method was then successfully employed for the ganglioside profiling of 3 diseased samples of human blood serum along with one disease free control. As a result, ganglioside profiles were quantitatively established at high sensitivity for the different ganglioside samples.