Development Of Multi-label Chemical Isotope Labeling Reagents And Application Of UHPLC-MS/MS Analysis

Nowadays,ultra-high performance liquid chromatography tandem mass spectrometry(UHPLC-MS/MS)technology is widely used in various fields such as biochemistry,environmental science,and clinical medicine due to its strong separation ability,wide analysis range,and accurate analysis results.In order to improve detection sensitivity and correct for changes in MS response,it is necessary to develop effective sample pre-treatment technology.Multiplex tags chemical isotope labeling(MTCIL)technology further innovates on the basis of stable isotope labeling derivatization(SILD).MTCIL integrates derivatization technology,internal standard method and high-throughput labeling technology.Derivatization can increase the ionization efficiency,thereby increasing the sensitivity of MS,changing the chromatographic performance of the analytes and improving chromatographic separation.The internal standard method can correct the response of MS to achieve accurate quantification.High-throughput labeling technology can meet the needs of large sample detection and large-scale experiments,multiple samples can be detected simultaneously in a single LC-MS/MS run.In order to avoid damage to the instrument with complex matrices,effective sample enrichment and purification are necessary before detection.Dummy magnetic molecularly imprinted technology(DMMIT)uses derivatives as dummy template.This not only solves the problem of inaccurate quantification due to template molecule leakage,but also achieves high selective enrichment of multiple structural analogs.Dummy magnetic molecularly imprinted polymers(DMMIPs)can enrich targets and their structural analogs with high selectivity by virtue of its specific recognition function.The magnetic responsive function and dummy template technology can achieve both rapid separation and accurate quantification.This thesis mainly introduces two new label-extraction techniques,combined with UHPLC-MS/MS to detect the disease biomarker lyso-Gb3 of Fabry disease and the hexanal and heptanal in the serum of lung cancer patients.The details are as follows:Chapter One: Briefly describes the development and application of ultraperformance liquid chromatography-mass spectrometry technology,molecular imprinting technology,surface molecular imprinting technology,dummy template molecular imprinting technology,chemical derivatization technology,stable isotope labeling derivatization technology and multiplex tags chemical isotope labeling technology.Chapter Two: For the first time,a series of levofloxacin carbonyl chloride-based mass tags(LMTs)were designed and synthesized to label the disease biomarker lysoGb3 of Fabry disease.The dummy magnetic molecular imprinting technology(DMMIT)uses derivatives as dummy templates,combined with the multiplex tags chemical isotope labeling(MTCIL)technology for effective sample pretreatment.UHPLC-MS/MS detection successfully achieved the simultaneous quantification of lyso-Gb3 in 8 plasma samples.The LOD of this method is as low as 0.01 n M,which has great potential in the analysis of trace concentrations of lyso-Gb3.Chapter Three: The levofloxacin-hydrazide-based mass tags(LHMTs)were designed and synthesized for high-throughput labeling of potential biomarkers of lung cancer: hexanal and heptanal.Dummy magnetic molecularly imprinted polymers(DMMIPs)were synthesized based on derivatization-dummy template technique.DMMIPs were used to extract and enrich multiple derivatives,combined with UHPLC-MS/MS detection,a single LC-MS/MS run could simultaneously quantify hexanal and heptanal in 12 serum samples.This method has a good application prospect in the screening of patients with suspected lung cancer.