Application Of LC-MS In Quantitative Analysis Of Plasma Glucose And Metabolomics Of C.elegans

Liquid chromatography-mass spectroscopy?LC-MS?,especially,ultra-high performance liquid chromatography?UPLC?coupled with high resolution mass spectrometry?HRMS?,is a powerful tool for qualitative and quantitative analysis of target components in complex biological samples.UPLC can greatly enhance the efficiency of chromatographic separation of various components in biological samples.Furthermore,HRMS provides more precise molecular weight and isotopic pattern,which considerably improve the reliability and accuracy of qualitative and quantitative analysis.In this thesis,we applied UPLC-HRMS in qualitative and quantitative analysis and metabolomics study of different biological samples?plasma and C.elegans extracts?.In the first part of this thesis,we developed a new approach to accurate quantification of endo-/exogenous glucose in plasma.Most of traditional quantitative methods cannot distinguish between endo-/exogenous glucose in plasma.Furthermore,low ionization efficiency of the neutral carbohydrates also limited the application of LC-MS.Combining selective derivatization with isotope dilution LC-MS and using D-[¹³C₆]glucose as a tracer of exogenous glucose can not only improve the sensitivity but also comprehensively evaluate the glucose metabolic status of the organism.Good linearity was obtained with a linear correlation coefficient?R?of 0.9969 in the range of D-glucose from 0.5 to 20.0 mM,which covers the healthy and diabetic physiologic scenarios,and D-[¹³C₆]glucose at the fixed concentration of 0.035 mM.Satisfactory reproducibility were obtained by evaluation of the intra-and inter-day precisions with relative standard deviations?RSDs?less than 5.16%,and relative recoveries ranging from 85.96%to 95.92%.The proposed strategy has proven to be less sample consumption,sensitive,accurate and robust.The second part of this thesis is the metabolomics study of the anti-aging effect of metformin by LC-MS using C.elegans as model organism.Metabolomics,the most downstream of systems biology,is most sensitive to the perturbations of biochemical networks.In this thesis,the C.elegans were cultivated and treated with metformin firstly.Then samples were collected and the methods of extracting metabolites were optimized.Unsupervised principal component analysis?PCA?and supervised partial least square discriminant analysis?PLS-DA?and independent-samples t test were performed to screen out the variables with significant differences.Preliminary identification of 13 differential variables showed that administration of metformin resulted in up-regulation of LysoPEs.In present thesis,based on the LC-MS analysis,we established new method and strategy to analyze complex biological samples?plasma and C.elegans extracts?.These methods expand the applicability and establish the foundation of LC-MS in qualitative and quantitative analysis and metabolism.These methods were successfully applied to:?1?simultaneous and accurate quantitation of endo-/exogenous glucose in plasma of non-diabetic and type II diabetic cynomolgus monkeys,which provides objective evidence for the diagnosis and prevention of the diseases related to glucose metabolism;?2?metabolomics study of the anti-aging effect of metformin on C.elegans,guiding the research of anti-aging mechanism of metformin in the organism.