Liquid Chromatography Mass Spectrometry Analysis And Fast Fingerprinting Study Of Plant Polysaccharides

Plant polysaccharides,especially extracted from traditional Chinese herbs,have medicinal functions including regulating blood pressure,modulating immune system,and anti-tumor and anti-fungal activities.But the quality of plant polysaccharides can be different based on the types of plants,regions of productions and even conditions of cultivation.Plant polysaccharides are polymeric mixtures with unequal molecular chain lengths.Their structures are extremely complex from sugar chain composition,sequence of monosaccharide residues,conformation of polysaccharide skeleton chain and the existence of numerous isomers.Until now,the majority of studies have focused on the determination of monosaccharide composition of large saccharides through complete hydrolysis and identification using liquid chromatography(LC),where derivatization is required prior to analysis since carbohydrates do not have chromophoric groups.However,due to the limited resolution of LC and sample complexity,different mono-or di-or tri-saccharide hydrolysates and other impurities can co-elute in LC,thus by simply matching the limited or available monomer standards can be inaccurate or even misleading.Mass spectrometry(MS)is a powerful tool for compound identification and structure analysis in many fields.It can give m/z values for every single compound eluted from LC or all the compounds in single LC peak,and tandem MS provides structural information with better sensitivity without the need for derivatization.In this thesis,the large polysaccharide was acid hydrolyzed to oligosaccharides and the products were efficiently separated and identified using liquid chromatography coupled to a high resolution tandem mass spectrometry(LC-MS2).Direct analysis in real time mass spectrometry(DART-MS)was used to generate polysaccharide fingerprints.In addition,a novel mechanochemical extraction(MCE)method was used to successfully extract saccharide components,it exhibits many advantages over traditional methods.Certain theoretical basis for in-depth analysis of chemical composition and chemical structures analysis was provided in this work.The concrete research content is as follows:1.A novel and highly efficient environment-friendly mechanochemical extraction method was successfully applied for the extraction of polysaccharide components from traditional Chinese herbs for the first time.Using water as solvent,the process is simple,fast(40 s),high yield and no radiation.The molecular weights of the intact saccharides from calibration curve and light scattering measurement were determined and compared after separation with size exclusion chromatography(SEC).The yields of polysaccharides from DO with mechanochemical extraction,hot Water extraction and ultrasonic extraction were 33.21%,13.91%and 18.81%respectively;the MWs of intact polysaccharide from both calibration and light scattering measurements after separation on size exclusion chromatography were determined and compared.The calibrated MW values are closest to the Mw values and the mass differences between these two sets of values ranged from 4.93%to 12.3%.2.An analytical methodology employing ultra-performance liquid chromatography(UPLC)coupled to a high resolution tandem MS was developed to characterize the oligosaccharide acid hydrolysis products of large polysaccharides.In this work,Dendrobium offcinale(DO)from different regions in China were selected as objectives of study,which is listed individually in the Pharmacopoeia of the People’s Republic of China from other Dendrobium genus.Hydrolysis is performed for sample preparation,since the large molecular weights of polysaccharides make direct analysis very challenging.Oligosaccharide digests were efficiently resolved in 14 min using the developed UPLC methods,obvious differences were detected among the liquid chromatograms of DO from different regions.The chemical structures for the main hydrolysis products were analyzed based on collision induced dissociation(CID)patterns using a high resolution orbitrap MS.The chemical formula and putative ionic forms of the oligosaccharide products were proposed based on accurate mass with less than 10 ppm mass errors.Moreover,isomeric digested products were detected and tandem fragmentation spectra for resolved isomeric LC peaks were obtained as well.Using chromatographic and advanced mass spectrometric techniques,certain structural information for polysaccharide was explored through the oligosaccharide products.3.Large saccharides can break down into characteristic small fragments(<500)in the DART source via pyrolysis,and the products are then detected by high resolution MS.In this study,six traditional Chinese herbal medicine,Dendrobium officinale(DO),Dendrobium nobile(DN),Codonopsis pilosula(CP),Astragalus membranaceus(AM),Poria cocos(PC)and Lentinus edodes(LE)were used to test the potential of using DART-MS based method for the molecular level characterization of polysaccharides.DART-MS was used to generate polysaccharide fingerprints in a few seconds,moreover,the investigations for the polysaccharides from the same herb but different production regions were performed as well,and the mixtures of different plant saccharides were analyzed.Temperature was shown to be the crucial parameter for the decomposition of large polysaccharide so the DART helium gas temperatures changed from 200 ℃ to 450℃.The general behavior of carbohydrates in DART-MS was also studied through the investigation of a number of mono-and oligosaccharide standards.The chemical formula and ion forms of the fragments were proposed based on accurate mass with less than 10 ppm mass errors.Multivariate data analysis shows the clear differentiation of different plant species.Intensities of marker ions compared among samples also showed obvious differences.DART-MS demonstrates a simple,fast,high throughput analytical protocol for the efficient evaluation of plant polysaccharide components.In all,a mechanochemical extraction method was applied to extract polysaccharides from plants.A variety of chromatography and high resolution mass spectrometry(SEC,UPLC-MS2,DART-MS)was used to comprehensively analyze plant polysaccharides.The research contains the determination of the absolute molecular weight of polysaccharide,the separation and structure analysis of the acid hydrolysate(oligosaccharide),and the establishment of the fast fingerprint of polysaccharide.The research results have some significance to promote the scientific research of polysaccharide.