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CN-122016991-A - Construction method and application of polysaccharide mass spectrum fingerprint

CN122016991ACN 122016991 ACN122016991 ACN 122016991ACN-122016991-A

Abstract

The invention discloses a method for constructing a polysaccharide mass spectrum fingerprint and application thereof. Mixing polysaccharide standard substance and methylation reagent, directly introducing into in-situ ionization source to make direct ionization, using tandem mass spectrometry analysis to obtain mass spectrum fingerprint of polysaccharide, using same test process to obtain mass spectrum fingerprint of sample to be tested, using analysis to make characteristic continuous neutral loss quality difference to quickly judge monosaccharide unit type of polysaccharide, and using characteristic precursor ion and its diagnostic fragment ion to implement accurate identification of polysaccharide type. The invention realizes the direct and rapid analysis of the complete polysaccharide, does not need complicated hydrolysis, derivatization and chromatographic separation steps, can effectively distinguish the isomerism polysaccharide with different structures, and provides a novel analysis method with high flux and high discrimination for polysaccharide components in food, plants and biological samples.

Inventors

  • LI HONGLI
  • Qiu Zhichen
  • XU LIPING
  • CHEN DAYONG

Assignees

  • 南京师范大学

Dates

Publication Date
20260512
Application Date
20260320

Claims (10)

  1. 1. The method for constructing the polysaccharide mass spectrum fingerprint is characterized by comprising the following steps of: (A1) Mixing a polysaccharide sample with a derivatization reagent to obtain a polysaccharide standard solution; (A2) Directly introducing a polysaccharide standard solution into an in-situ ionization source, and performing DART-MS analysis to obtain a mass spectrum of a polysaccharide sample; (A3) And selecting fragment ions in a mass spectrum of the polysaccharide sample for MS/MS analysis, and finally establishing a mass spectrum fingerprint of the polysaccharide sample.
  2. 2. The method for constructing a mass spectrum fingerprint of polysaccharide according to claim 1, wherein in the step (A1), the polysaccharide is one or more of hexose polysaccharide, pentose polysaccharide or nitrogenous polysaccharide, and the derivatizing agent is tetramethylammonium hydroxide solution, 1-phenyl-3-methyl-5-pyrazolone or 1- (4-carboxyphenyl) -3-methyl-5-pyrazolone, or a combination of methyl iodide and sodium hydroxide.
  3. 3. The method for constructing a mass spectrum fingerprint of polysaccharide according to claim 2, wherein the hexose polysaccharide is one or more of cellulose, amylose, glucan, mannan or galactomannan, the pentosan polysaccharide is one or more of arabinogalactan, arabinan or xylan, and the nitrogenous polysaccharide is one or two of chitin or hyaluronic acid.
  4. 4. The method for constructing a mass spectrum fingerprint of polysaccharide according to claim 1, wherein in the step (A1), the concentration of polysaccharide in the mixed solution of the polysaccharide sample and the derivatization reagent is 0.1-4 mg mL -1 , and the mass concentration of the derivatization reagent solution is 0.1-20%.
  5. 5. The method for constructing a polysaccharide mass spectrum fingerprint according to claim 1, wherein in the step (A2), the in-situ ionization source is an excited molecular beam ion source, and the operation parameters of the in-situ ionization source include pumping pressure of-60 to-95 kPa, working gas of helium or nitrogen, and heating temperature of the working gas of 200-500 ℃.
  6. 6. The method for constructing a polysaccharide mass spectrum fingerprint according to claim 1, wherein in the step (A2), in DART-MS analysis, a data acquisition mode is positive, a mass spectrum detection range is m/z 50-2000, tandem mass spectrum adopts collision induced dissociation, and collision energy is 10-40 eV.
  7. 7. The method for constructing a polysaccharide mass spectrum fingerprint according to claim 1, wherein in the step (A3), mass spectrum fragments of the polysaccharide sample are selected to select stable polysaccharide fragment ions with a relative intensity of >10% as parent ions, and the MS/MS analysis operation conditions include collision-induced dissociation of the selected parent ions at a collision energy of 10-40 eV, and screening out ions with structural specificity from the generated secondary fragments as characteristic ions.
  8. 8. The use of mass spectrogram polysaccharide mass spectrogram obtained by the construction method of any one of claims 1-7 in rapid screening, identification or composition analysis of polysaccharide components in food, plant samples or Chinese medicinal materials.
  9. 9. The use according to claim 8, characterized by the steps of: (B1) Mixing a sample solution to be detected with a derivatization reagent to obtain an analysis solution; (B2) Directly introducing an analysis solution into an in-situ ionization source, and carrying out ionization and tandem mass spectrometry analysis to obtain a mass spectrum of a polysaccharide sample in a sample to be detected; (B3) Comparing the mass spectrum of the sample to be measured obtained in the step (2) with the mass spectrum of the polysaccharide to determine the polysaccharide type; Or alternatively And selecting parent ions for MS/MS analysis, comparing the analysis result with a polysaccharide mass spectrum fingerprint, and determining the polysaccharide type by matching characteristic precursor ions and diagnostic fragment ions thereof.
  10. 10. Use according to claim 9, characterized in that an internal standard is added to the analysis solution of step (B1) for quantitative analysis of the polysaccharide.

Description

Construction method and application of polysaccharide mass spectrum fingerprint Technical Field The invention relates to a method for constructing a polysaccharide mass spectrum fingerprint and application thereof, in particular to a method for constructing a fingerprint by directly carrying out structural analysis on complete polysaccharide by utilizing an in-situ ionization mass spectrum technology and application of the method in polysaccharide identification and quantitative analysis, belonging to the technical fields of analytical chemistry and biology, Background Polysaccharides are biological macromolecules formed by connecting more than ten monosaccharides through glycosidic bonds, are important components of life bodies, and have various biological activities such as energy storage, structural support, immune regulation and the like. Unlike proteins and nucleic acids of which the sequence is defined, the structure of polysaccharides has a high degree of complexity and microscopic heterogeneity, including a variety of changes in monosaccharide composition, glycosidic linkage, branching degree, and steric configuration. This complexity makes direct resolution of the intact polysaccharide structure a very challenging analytical task. Traditional polysaccharide structural analysis strategies typically rely on indirect, degradation-based methods. The method comprises the steps of firstly decomposing polysaccharide into monosaccharide or oligosaccharide by a chemical or enzymolysis mode, then analyzing by using a gas phase or liquid chromatography-mass spectrometry technology, and finally reversely pushing the structure of the complete polysaccharide by degradation products. However, these methods generally have problems of complicated sample pretreatment, long time consumption, easy loss of stereochemical information, and the like. While chemical derivatization (e.g., methylation) can increase detection sensitivity and amplify structural differences, it is typically performed as an off-line step, further increasing the complexity of the analytical procedure. In recent years, direct analysis of ion sources (DART) in real time has received attention to the ability to achieve rapid, high throughput analysis in open atmosphere environments with little or no sample pretreatment. When DART and Mass Spectrometry (MS), in situ ionization mass spectrometry techniques are formed. DART-MS directly analyzes polysaccharide, and mainly generates non-diagnostic fragment ions with low mass number (m/z is generally < 300) due to spontaneous cleavage in the thermal desorption process, so that spectrograms of polysaccharides with different structures (such as isomers) are similar and are difficult to distinguish. Therefore, the development of a method capable of directly, rapidly and accurately analyzing the whole polysaccharide structure has important scientific significance and application value. Disclosure of Invention The invention aims to overcome the defects of the prior art and provide a method for directly analyzing and constructing polysaccharide mass spectrum fingerprint based on polysaccharide by using an in-situ ionization mass spectrum technology. The method can realize rapid and high-flux structural characterization and fingerprint construction of the complete polysaccharide on the premise of not carrying out hydrolysis, chromatographic separation or off-line derivatization, and is particularly suitable for identifying and quantitatively analyzing polysaccharide in complex samples, so that the invention further aims to provide application of the polysaccharide mass spectrum fingerprint in rapid screening, identification or composition analysis of polysaccharide components in foods, plant samples or Chinese medicinal materials. The technical scheme is that the method for constructing the polysaccharide mass spectrum fingerprint comprises the following steps of: (A1) Mixing a polysaccharide sample with a derivatization reagent to obtain a polysaccharide standard solution; (A2) Directly introducing a polysaccharide standard solution into an in-situ ionization source, and performing DART-MS analysis to obtain a mass spectrum of a polysaccharide sample; (A3) And (3) selecting mass spectrum fragments of the polysaccharide sample for MS/MS (tandem mass spectrometry) analysis, and finally establishing a mass spectrum fingerprint of the polysaccharide sample. Further, in the step (A1), the polysaccharide is one or more of hexose polysaccharide, pentose polysaccharide or nitrogenous polysaccharide, and the derivatizing agent is tetramethylammonium hydroxide solution, 1-phenyl-3-methyl-5-pyrazolone or 1- (4-carboxyphenyl) -3-methyl-5-pyrazolone, or a combination of methyl iodide and sodium hydroxide. Wherein the hexose polysaccharide is one or more of cellulose, amylose, glucan, mannan or galactomannan, the pentosan polysaccharide is one or more of arabinogalactan, arabinan or xylan, and the nitrogenous polysaccharide