| Providing the material basis for the active ingredients in traditional Chinese medicine,the polysaccharides are natural macromolecules consisting of 10 or more monosaccharide groups and linked by the glycosidic linkages.The polysaccharides exhibit a wide range of biological activities with regard to being an antioxidant,besides possessing immunomodulatory,hypoglycemic,anti-inflammatory,and antitumor properties.The polysaccharide physiological activities,on the other hand,are closely related to their monosaccharide compositions,while the monosaccharides are the most basic units of the polysaccharides.In addition,monosaccharides are the most fundamental research object for investigating the structural characteristics of the polysaccharides,physicochemical properties,and the structure-activity relationships.It is of great importance to accurately assess the monosaccharide content in the traditional Chinese medicine polysaccharides to elucidate the nature and structure of the polysaccharides,given the many flaws of the existing methods for the analysis of sugar composition.As a result,a simple,accurate,comprehensive,and rapid analytical method was established,which used the pre-column derivatization method of aldononitrile acetate,equipped with Liquid Chromatography-Electrospray Mass Spectrometry（UPLC-ESI-MS/MS）system.Within 14 min,the method enables the simultaneous isolation and detection of eight neutral saccharides,two uronic acids,one ketose,and eight alditols.The UPLC-ESI-MS/MS method was successfully used for the quantitative analysis of free carbohydrates in water extracts of Crataegus pinnatifida as well as the determination of Polygonatum cyrtonema,and Ganoderma lucidum polysaccharides.The major research of the experiment was as follows:（1）In this study,an aldononitrile acetate derivatization method was developed,equipped with the UPLC-ESI-MS/MS system,enabling the simultaneous analysis of neutral monosaccharides,ketoses,uronic acids,and sugar alcohols in under 14 min.The derivatization method was optimized by a single factor experiment and the optimum reaction time and temperature of the sugar oxime reaction was 15 min and 60℃,respectively,while the highest derivatization efficiency was obtained with a 25:1 solvent ratio between the sample and AC2O in around 30 min.Besides,the range of the RSD values for precision,repeatability,and stability were within 0.36%-4.03%,0.69%-2.53%,and 1.61%-3.93%respectively,which indicated that the method developed by this study possessed high precision,and repeatability besides being capable of wide used for high-throughput analysis of the monosaccharide composition of traditional Chinese medicine polysaccharides.（2）The applications of the UPLC-ESI-MS/MS methods.To begin with,the single-factor experiments were applied to optimize the hydrolysis conditions of the polysaccharides of P.cyrtonema and G.lucidum to obtain the conditions of ideal polysaccharides hydrolysis.Moreover,the new method established in this experiment was applied to establish the sugar composition of P.cyrtonema polysaccharides,G.lucidum polysaccharides,and the C.pinnatifida free sugars.The polysaccharides of P.cyrtonema were found to be composed of galacturonic acid,fructose,galactose,mannose,glucose,xylose,arabinose,and rhamnose,with the monosaccharide recovery ranging between 97.26%and 101.55%.The G.lucidum polysaccharides were composed of xylose,arabinose,glucose,galactose,mannose,fucose,sorbitol,mannitol,xylitol,and glucuronic acid,while the recovery of each monosaccharide ranged between 97.95%and 101.51%.The C.pinnatifida free sugars consisted of xylose,fructose,sorbitol,glucose,mannitol,and fucose,with the recovery of each monosaccharide ranging between 98.06%and 102.58%.Consequently,these results further confirmed the significance of the method for the comprehensive analysis of monosaccharides in complex carbohydrate samples,besides its appropriateness in the analysis of complicated natural polysaccharides. |
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