Quantitative Methodological Study On Endogenous Carbohydrate And Serotonin In Rat Plasma Based On Hydrophilic Liquid Chromatography - Tandem Mass Spectrometry

During the last two decades, metabolomics has become a new approach to study different diseases. Endogenous carbohydrates were frequently highlighted as the most differential metabolites in the metabolic profiling studies of biosamples such as plasma, serum and urine. Thus, a rapid and simple quantification method to analyze endogenous carbohydrates in the biosamples becomes necessary for expanded studies in the metabolomics approaches. Endogenous serotonin plays an essential role in neural communication in mammalians. Dysregulation of serotonin was frequently reported in the studies of monoamine neurotransmitter disorders and psychiatric disorders, including depression, anxiety, and schizophrenia. In this dissertation, we developed simple, high throughput quantitative methods on endogenous carbohydrates and endogenous serotonin in rat plasma respectively, using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry.Firstly, a simple, fast, simultaneous quantitative method for 16 endogenous carbohydrates in plasma has been developed using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry. In order to quantify 16 endogenous carbohydrates in plasma, various conditions, including columns, chromatographic conditions, MS conditions, and plasma preparation methods, were investigated. Different conditions in this quantified analysis were performed and optimized. The reproducibility, precision, recovery, and stability of the method were verified. The results indicated that methanol/acetonitrile(v/v, 50/50) mixture could effectively and reproducibly precipitate rat plasma proteins. Cold organic solvents coupled with vortex for 1 min and incubated at-20?C for 20 min were the most optimal conditions for protein precipitation and extraction. The results, according to the linearity, matrix, recovery, precision, and stability, showed that the method was satisfactory in the quantification of endogenous carbohydrates in rat plasma. The quantified analysis of endogenous carbohydrates in rat plasma performed excellently in the sensitivity, high throughput and simple sample preparation, which met the requirement of quantification in specific expanded metabolomic studies after the global metabolic profiling research.Secondly, a simple, fast and sensitive method was developed to simultaneously determine endogenous serotonin in rat plasma, using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry. Various conditions, including columns, chromatographic conditions, ion source, MS/MS conditions, and brain tissue preparation methods, were optimized and validated. The results indicated that methanol/acetonitrile(v/v, 50/50) mixture could effectively and reproducibly precipitate rat plasma proteins. Cold organic solvents coupled with vortex for 1 min and incubated at-20?C for 20 min were the most optimal conditions for protein precipitation and extraction. This method exhibited excellent linearity for all analytes(regression coefficients > 0.999). The results, according to the linearity, matrix, recovery, precision, and stability, showed that the method was satisfactory in the quantification of endogenous carbohydrates in rat plasma. The quantified analysis of endogenous serotonin in rat plasma performed excellently in the sensitivity, high throughput and simple sample preparation.These studies in this dissertation covered the specific quantitative metabonomics methodology on rat plasma. The results in these studies enriched the understanding of quantitative metabonomics, and powered the potential of methodology of other polar endogenous compounds.