Establishment And Application Discriminate Method Of Monosaccharide And Disaccharide Isomers Based On LC-Q/TOF-MS Technology

Monosaccharide isomers and disaccharide isomers widely exist in nature,playing a key role in a number of important biological processes.However,due to the existence of many isomers of monomers and disaccharides,these isomers have the same molecular formula and high polarity,but there are differences in their biological activities.Moreover,these sugars lack UV absorption and are non-volatile.Therefore,compared with other small-molecule organic compounds,it is difficult to identify and differentiate these sugars,especially since the identification of trace sugars has always been a challenge.The distinction and identification methods that have been reported are complicated operations and suitable for sugars with high concentrations to a certain extent.Therefore,the establishment of a highly sensitive and convenient method for distinguishing monosaccharide and disaccharide isomers is helpful for carbohydrate-related drugs quality control and the search for carbohydrate potential markers of related diseases.Based on this problem,this study developed two methods for the differentiation of oligosaccharide isomers and validated and applied these two methods.The main content of this research includes the following two parts:1.Establishment and Application discriminate method of monosaccharide and disaccharide isomers based on HILIC-Q/TOF-MS technology.In this experiment,we propose a sensitive hydrophilic interaction liquid chromatography coupled to quadrupole/time-of-flight mass spectrometry(HILIC-Q/TOF-MS)method to detect monosaccharide isomers and disaccharide isomers by optimizing the chromatographic and mass spectrometry parameters.Based on summarizing the retention time,the relative abundance of characteristic fragment ions,and the ratio of the relative abundance of characteristic fragment ions,the rules for distinguishing monosaccharide and disaccharide isomers were established and applied.The method was validated by disaccharide(sucrose,lactose,maltose),tamarind gum,and pullulan,and was applied to the analysis of monosaccharide composition in the Moringa.oleifera seeds aqueous extract,hydrolysate of Moringa.oleifera seeds aqueous extract,Stephania tetrandra S.Moore.polysaccharides,and plasma.The results showed that the monosaccharide species in Moringa.oleifera seeds aqueous extract are glucose,fructose,galactose and rhamnose;hydrolysate of Moringa.oleifera seeds aqueous extract are arabinose,xylose,glucose,fructose,galactose and rhamnose.In addition,it was determined that Stephania tetrandra S.Moore.polysaccharide 1was composed of glucose,Stephania tetrandra S.Moore.polysaccharide 2 includes arabinose,xylose,glucose,and galactose,and plasma samples from different diseases were glucose,galactose,and xylose.The experimental results revealed that the HILIC-Q/TOF-MS is an effective and convenient strategy for rapid differentiation of monosaccharide isomers and disaccharide isomers,which may serve as a general platform for the analysis of neutral polysaccharides,food,medicinal plants,and herbs.2.Establishment and Application discriminate method for trace aldose enantiomers based on UPLC-DAD-Q-TOF-MS technology.The research content of this part is to establish an ultra-performance liquid chromatography with diode array detector coupled to quadrupole/time-of-flight mass spectrometry(UPLC-DAD-Q-TOF/MS)combined with one-pot monosaccharide derivatized method for the identification and differentiation of absolute trace aldoses by optimizing the chromatographic conditions and mass spectrometry parameters,considering sensitivity and resolution.Based on the inductive analysis of the retention time(t_R)of D/L aldose extracted ion chromatogram(EIC)and the relative abundance ratio(RAR)of characteristic fragment ions,the absolute configuration distinction of eight pairs of monosaccharides was achieved.More importantly,one-pot derivatization reaction,with all reactions performed in an Agilent screw capped sample vial,contained the completion of hydrolysis of the glycosidic bond,followed by the reaction of monosaccharide with L-cysteine methyl ester hydrochloride and o-toluene isothiocyanate in anhydrous pyridine solution.In addition,this study also focused on examining the sensitivity and resolution of method.This strategy was verified by commercially available trace D-lactose and narirutin standards and finally applied to the monosaccharide structure analysis of moringa seed glycosides and plasma biological samples.The results show that this study established an efficient and convenient derivatized UPLC-DAD-Q-TOF-MS method for the identification and differentiation of the absolute configuration of trace aldoses.This method was applied to the identification of the absolute configuration of 0.01μg trace monosaccharides and 0.1μg glycosides.Therefore,this study provides an effective strategy for the identification of the absolute configuration of trace aldoses in food,natural medicines,and biological samples.