Construction method and application of polysaccharide mass spectrum fingerprint spectrum

Abstract

The invention discloses a construction method and application of a polysaccharide mass spectrum fingerprint spectrum. The method comprises the following steps: mixing a polysaccharide standard substance with a methylation reagent, directly introducing the mixture into an in-situ ionization source for direct ionization, and carrying out tandem mass spectrometry to obtain a mass spectrum fingerprint spectrum of the polysaccharide; a sample to be tested is subjected to the same testing process to obtain a mass spectrum fingerprint spectrum of the sample to be tested, the monosaccharide unit type of the polysaccharide is rapidly judged by analyzing the mass difference of characteristic continuous neutral loss in the spectrum, and accurate identification of the polysaccharide type is achieved by utilizing characteristic precursor ions and diagnostic fragment ions thereof. The method realizes direct and rapid analysis of complete polysaccharides, does not need tedious hydrolysis, derivatization and chromatographic separation steps, can effectively distinguish isomeric polysaccharides with different structures, and provides a novel high-throughput and high-discriminability analysis method for polysaccharide components in food, plant and biological samples.

Description

Technical Field

[0001] This invention relates to a method and application for constructing polysaccharide mass spectrometry fingerprints, particularly a method for constructing fingerprints of intact polysaccharides through direct structural analysis using in-situ ionization mass spectrometry, and its application in polysaccharide identification and quantitative analysis. This invention belongs to the fields of analytical chemistry and biotechnology. Background Technology

[0002] Polysaccharides are biological macromolecules composed of ten or more monosaccharides linked by glycosidic bonds. They are essential components of living organisms, possessing various biological activities such as energy storage, structural support, and immune regulation. Unlike proteins and nucleic acids, whose sequences are defined, polysaccharides exhibit high structural complexity and microscopic heterogeneity, including variations in monosaccharide composition, glycosidic bond configuration, branching degree, and stereoconfiguration. This complexity makes the direct determination of the complete polysaccharide structure an extremely challenging analytical task.

[0003] Traditional strategies for polysaccharide structure analysis typically rely on indirect, degradation-based methods. These methods first break down polysaccharides into monosaccharides or oligosaccharides through chemical or enzymatic digestion, then analyze them using gas chromatography-mass spectrometry (GC-MS), and finally infer the structure of the intact polysaccharide from the degradation products. However, these methods generally suffer from cumbersome sample pretreatment, long processing times, and the potential loss of stereochemical information. While chemical derivatization (such as methylation) can improve detection sensitivity and amplify structural differences, it is usually performed offline, further increasing the complexity of the analytical process.

[0004] In recent years, direct analytical ion sources (DART) have attracted much attention for their ability to achieve rapid, high-throughput analysis in open atmospheric pressure environments with little or no sample pretreatment. When DART is combined with mass spectrometry (MS), it forms in-situ ionization mass spectrometry. However, in direct DART-MS analysis of polysaccharides, spontaneous fragmentation during thermal desorption primarily produces low-mass, non-diagnostic fragment ions (typically m / z < 300), leading to similar spectra for polysaccharides with different structures (such as isomers), making them difficult to distinguish. Therefore, developing a method that can directly, rapidly, and accurately resolve intact polysaccharide structures has significant scientific and practical value. Summary of the Invention

[0005] Purpose of the Invention: The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for constructing polysaccharide mass spectrometry fingerprints based on in-situ ionization mass spectrometry. This method enables rapid, high-throughput structural characterization and fingerprint construction of intact polysaccharides without hydrolysis, chromatographic separation, or offline derivatization. It is particularly suitable for the identification and quantitative analysis of polysaccharides in complex samples. Therefore, another objective of this invention is to provide the application of this polysaccharide mass spectrometry fingerprint in the rapid screening, identification, or compositional analysis of polysaccharide components in food, plant samples, or traditional Chinese medicine.

[0006] Technical solution: The method for constructing a polysaccharide mass spectrometry fingerprint according to the present invention includes the following steps:

[0007] (A1) Mix the polysaccharide sample with the derivatization reagent to obtain a polysaccharide standard solution;

[0008] (A2) The polysaccharide standard solution was directly introduced into the in-situ ionization source for DART-MS analysis to obtain the mass spectrum of the polysaccharide sample;

[0009] (A3) Mass spectrum fragments of polysaccharide samples were selected for MS / MS (tandem mass spectrometry) analysis to finally establish the mass spectrometric fingerprint of polysaccharide samples.

[0010] Further, in step (A1), the polysaccharide is one or more of hexose polysaccharides, pentose polysaccharides, or nitrogen-containing polysaccharides, and the derivatizing reagent is tetramethylammonium hydroxide solution, 1-phenyl-3-methyl-5-pyrazolone, or 1-(4-carboxyphenyl)-3-methyl-5-pyrazolone, or a combination of iodomethane and sodium hydroxide. Specifically, the hexose polysaccharides are one or more of cellulose, amylose, dextran, mannan, or galactomannan; the pentose polysaccharides are one or more of arabinogalactan, arabinogalactan, or xylan; and the nitrogen-containing polysaccharides are one or two of chitin or hyaluronic acid.

[0011] Further, in step (A1), the polysaccharide concentration in the mixed solution of the polysaccharide sample and the derivatization reagent is 0.1-4 mg / mL. -1The mass concentration of the derivatization reagent solution is 0.1-20%. In step (A2), the in-situ ionization source is an excited-state molecular beam ion source capable of generating active species (excited-state helium atoms, protonated water clusters, and solvent radical cations). The operating parameters of the in-situ ionization source include: pump pressure of -60 to -95 kPa, working gas of helium or nitrogen, and working gas heating temperature of 200-500 ℃. In DART-MS analysis, the data acquisition mode is positive electrode, and the mass spectrometry detection range is m / z 50-2000; the tandem mass spectrometry uses collision-induced dissociation (CID), and the collision energy (CE) is 10-40 eV. DART-MS analysis of the characteristic mass number differences in the polysaccharide mass spectral fingerprint spectrum caused by continuous loss of neutral atoms includes: Δm = 204 Da, Δm = 160 Da, Δm = 245 Da, and / or Δm = 218 Da. In step (A3), stable polysaccharide fragment ions with a relative intensity >10% are selected from the mass spectrum fragments of the polysaccharide sample as the parent ion. The MS / MS analysis operation conditions include: collision-induced dissociation of the selected parent ion at a collision energy of 10-40 eV, and screening out ions with structure specificity from the generated secondary fragments as characteristic ions.

[0012] This invention employs high-resolution orbital trap mass spectrometry, with positive electrode acquisition mode. The m / z range is set within a reasonable range of 50-2000, based on the size of different carbohydrates. The ion transmission tube temperature is set at 350 °C. The automatic gain control (AGC) target is 2 × e-1. 5 The maximum ion implantation time is 100 ms; the tandem mass spectrometry uses collision-induced dissociation (CID) with collision energies between 10 and 40 eV.

[0013] It should be noted that the in-situ ionization source in this invention is DART, and the mass spectrometer can be any mass analyzer that can be connected to DART and can perform full scan and tandem mass spectrometry analysis.

[0014] Furthermore, in the ion source, the polysaccharide undergoes an online derivatization reaction with the derivatizing reagent, and the derivatized polysaccharide is simultaneously ionized and detected by mass spectrometry. This derivatization reaction occurs online and instantaneously in the in-situ ionization source. The in-situ ionization source generates heated, metastable, gaseous excited-state particles, providing both ionization conditions for the ionization source and reaction conditions for the derivatization reaction.

[0015] In full-scan mass spectrometry, the ionized form of the parent ion is protonated trimethylamine ([N(CH3)3H)2). + ]) adducts; in tandem mass spectrometry, the ionization form of fragment ions is mainly protonated hydrogen ions [H +] and partially protonated trimethylamine ([N(CH3)3H + ]) combination.

[0016] The derivatized polysaccharide mass spectrum fragments were selected as the parent ions and tandem mass spectrometry analysis was performed to obtain the corresponding fragment ions. Using the same parent ion, different polysaccharides can produce different fragment ions, among which the unique fragment ions serve as diagnostic ions for recognizing isomer molecules.

[0017] The mass spectrometry fingerprint of polysaccharides obtained by the construction method described in this invention can be used for rapid screening, identification, or compositional analysis of polysaccharide components in food, plant samples, or traditional Chinese medicine.

[0018] Furthermore, the application includes the following steps:

[0019] (B1) Mix the sample solution to be tested with the derivatization reagent to obtain the analytical solution;

[0020] (B2) The analytical solution is directly introduced into the in-situ ionization source for ionization and tandem mass spectrometry analysis to obtain the mass spectrum of the polysaccharide sample in the test sample;

[0021] (B3) Compare the mass spectrum of the sample to be tested obtained in step (2) with the mass spectrum of the polysaccharide to determine the type of polysaccharide;

[0022] or,

[0023] The precursor ions were selected for MS / MS analysis, and the analysis results were compared with the polysaccharide mass spectrometry fingerprint. The polysaccharide species were determined by matching characteristic precursor ions and their diagnostic fragment ions.

[0024] In addition, an internal standard is added to the analytical solution in step B1 for quantitative analysis of polysaccharides.

[0025] Further, in step B2, the presence of characteristic mass number differences caused by consecutive neutral losses in the chromatogram is analyzed. These characteristic mass number differences indicate the type of monosaccharide units in the polysaccharide: if consecutive neutral losses Δm = 204 Da occur in the chromatogram, the polysaccharide is a hexose; if consecutive neutral losses Δm = 160 Da occur, the polysaccharide is a pentose; if consecutive neutral losses Δm = 245 Da occur, the polysaccharide is N-acetylated hexosamine; and if consecutive neutral losses Δm = 245 Da and 218 Da occur, the polysaccharide is composed of N-acetylated hexosamine and glucuronic acid.

[0026] Further, in step B3, the mass spectrum and MS / MS analysis results of the sample to be tested are compared with the polysaccharide mass spectrum fingerprint of the polysaccharide standard. By matching characteristic precursor ions and their diagnostic fragment ions, the type of polysaccharide to be tested is determined.

[0027] The derivatization mechanism of polysaccharides and derivatizing agents (TMAH) in an in-situ ion source can be considered as the result of complex molecular ionic and free radical reactions. The hydroxide ion of TMAH first undergoes an acid-base reaction with the hydroxyl group in the sugar molecule. Next, the oxygen anion of the sugar molecule attacks the NC bond of TMAH, undergoing a nucleophilic reaction, and the hydroxyl group is methylated. Simultaneously, TMAH transforms into trimethylamine, which has a strong proton affinity and combines with hydrogen ions in the ion source to become protonated trimethylamine. Under the influence of excited-state gas particles in the ion source, trimethylamine can generate methyl radicals, which further generate ethyl radicals. Both of these radicals can attack the hydroxyl groups of the sugar molecule, producing methylated products.

[0028] Theoretically, it is believed that in the in-situ ion source, carbohydrate molecules mainly undergo gas-phase reactions with TMAH, and in the gas phase, carbohydrate molecules can better exhibit their differences in stereostructure; combined with tandem mass spectrometry analysis, isomers can produce characteristic and distinct fragment ions.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0030] (1) Direct and fast: This invention enables direct analysis of intact polysaccharides without the time-consuming hydrolysis, pre-column / post-column derivatization and chromatographic separation steps of traditional methods. The analysis time of a single sample can be shortened to tens of seconds, which greatly improves the analytical throughput.

[0031] (2) Rich in information and strong identification ability: This invention utilizes a combination of online in-situ derivatization and intrasource fragmentation to generate fingerprint spectra rich in structural information. The monosaccharide composition type can be quickly determined through the continuous neutral loss pattern, and the "precursor ion-diagnostic fragment ion" data can effectively distinguish isomers of polysaccharides with different glycosidic bond linkages and branched structures, with identification ability far exceeding that of conventional DART-MS methods.

[0032] (3) Simple operation and environmentally friendly: The method of the present invention is simple to prepare samples, does not require complicated separation and purification processes, and hardly uses organic solvents, which is in line with the concept of green chemistry.

[0033] (4) High sensitivity and good reproducibility: The method of this invention combines high-resolution mass spectrometry. This method exhibits good linearity, sensitivity and a relative standard deviation of less than 12%, which meets the requirements of complex sample analysis.

[0034] (5) Wide range of applications: This invention can be successfully applied to the direct identification of polysaccharide components in a variety of real and complex samples such as fruits, vegetables, and Chinese medicinal materials, providing a powerful analytical tool for food science, phytochemistry and biomedical research. Attached Figure Description

[0035] Figure 1 shows the mass spectrum of the intact polysaccharide directly analyzed by DART-MS in situ methylation in Example 1, where (A) is cellulose, (B) is amylose, (C) is 20 kDa dextran, (D) is mannan, (E) is galactomannan, (F) is arabinogalactan, (G) is arabinogalactan, (H) is xylan, (I) is chitin, and (J) is hyaluronic acid.

[0036] Figure 2 shows the MS / MS spectra of polysaccharides analyzed online by DART-MS / MS using TMAH reagent in Example 1. (A) is the MS / MS spectrum of the precursor ion of cellulose at m / z 514.32; (B) is the MS / MS spectrum of the precursor ion of amylose at m / z 514.32; (C) is the MS / MS spectrum of the precursor ion of dextran (20 kDa) at m / z 514.32; (D) is the MS / MS spectrum of the precursor ion of mannan at m / z 514.32; (E) is the MS / MS spectrum of the precursor ion of galactomannan at m / z 514.32; and (F) is the MS / MS spectrum of the precursor ion of dextran (20 kDa) at m / z 760.43. (G) is the MS / MS spectrum of the precursor ion at m / z 760.43 for mannan; (H) is the MS / MS spectrum of the precursor ion of galactomannan at m / z 760.43.

[0037] Figure 3 A flowchart illustrating the workflow for online methylation analysis of polysaccharides using DART-MSn combined with TMAH;

[0038] Figure 4 The following is a representative MS / MS spectrum of the actual sample (goji berry) used for polysaccharide identification in Example 2, showing precursor ions with mass-to-charge ratios of m / z 514.32, m / z 556.33, m / z 584.33, m / z 614.34, m / z 394.24, m / z 526.29 and m / z 672.38. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0040] Obviously, the embodiments described above are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. It should be noted that the present invention will be further described in detail below with reference to specific examples. These examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0041] Example 1: Analysis of polysaccharide standards and construction of a diagnostic ion database

[0042] 1. Materials and Reagents

[0043] Polysaccharide standards: cellulose, amylose, dextran (20 kDa), mannan, galactomannan, arabinogalactan, arabinogalactan, xylan, chitin, hyaluronic acid, and 25% TMAH solution were all purchased from commercial companies.

[0044] 2. Sample Preparation

[0045] Each of the above polysaccharide standards was dissolved in a 10% tetramethylammonium hydroxide (TMAH) solution to prepare a final concentration of 1.0 mg / mL. -1 The standard solution. For water-insoluble polysaccharides, the strong basicity of TMAH can dissolve them by breaking hydrogen bonds.

[0046] 3. DART-Orbitrap MS / MS Analysis

[0047] An Orbitrap Fusion Lumos high-resolution mass spectrometer equipped with a DART ion source was used. DART source operating conditions: positive ion mode, helium as the ionizing gas, gas temperature 400℃. Sample introduction: 1 μL of standard solution was dipped into a dip-it glass tube and placed at the DART source outlet for analysis for 30 seconds. Mass spectrometry conditions: Orbitrap resolution 60000, ion transfer tube temperature 350℃. MS / MS analysis employed collision-induced dissociation, with collision energies optimized between 20-30 eV.

[0048] 4. Data Analysis and Database Construction

[0049] (1) Full-scan mass spectrometry fingerprint analysis: The DART-MS spectra of each polysaccharide standard were analyzed under TMAH assistance. The results are as follows: Figure 1 As shown, the results indicate that all 10 polysaccharides produced abundant ions distributed in the m / z range of 300-1500, rather than low-mass fragments as in the case of no derivatization.

[0050] Hexosaccharide polysaccharides—cellulose, amylose, and dextran (20 kDa)—are all composed of glucose units, but their different linkages and branching structures produce distinct derivatized DART-MS spectra. Similarly, mannan and galactomannan, both containing mannose basic units, also exhibit unique spectral characteristics due to the presence of galactose side chains in galactomannan. Despite the presence of some common ions, each polymer displays a unique fragmentation pattern. Notably, all hexosaccharide polysaccharide mass spectra show a continuous 204 Da (C6H) group. 10 The neutral loss of O5+3CH2) corresponds to the methylated hexose unit, indicating a breakage of its glycosidic bond. As an example, a series of key ions resulting from consecutive neutral losses of 204 Da were identified. For cellulose, these include m / z 1486.77, 1282.67, 1078.57, 874.47, and 670.37. A homologous series of ions with m / z 1222.65, 1018.55, 814.45, 610.34, and 406.24 was observed in amylose. Consecutive neutral losses of 204 Da (1168.63, 1022.53, 964.54, 818.44, 760.43, 614.14, and 556.33) were observed in dextran (20 kDa). Similarly, mannans show a series at m / z 1372.73, 1168.63, 922.53, 788.43, 584.43, and 380.23, while galactomannans show a series at m / z 862.50, 814.45, 658.40, 610.34, and 482.30. The main ionic product after derivatization is protonated trimethylamine {[N(CH3)3H]⁺}, derived from TMAH, due to its high proton affinity.

[0051] Pentosaccharide polysaccharides: Arabingalactan consists of a galactose backbone and extensive branching, with its side chains primarily composed of arabinose and galactose residues. In contrast, arabinan is mainly composed of α-(1→5)-linked arabinose residues, while xylan is composed of β-(1→4)-linked xylose residues. Their methylated derivatives were detected in the m / z range of 300–900. All three polymers exhibited intrasource cleavage ions resulting from the neutral loss of methylated pentose units (Δm = 160, corresponding to C5H8O4+2CH2), indicating glycosidic bond breakage. For arabingalactan, a characteristic 160 Da neutral loss precursor ion was observed at m / z 774.42, 730.39, and 686.40; for arabinan, it was observed at m / z 874.47 and 846.47. These ions primarily originate from pentose-based methylated tetrasaccharides and pentose oligosaccharides. Xylan shows a significant signal at m / z 332.21, belonging to {[C 10 H 18 [O9–2H2O–CH2O+4CH2+N(CH3)3H]⁺}, is generated by the successive loss of a precursor ion with m / z 682.36 by 160 Da. Furthermore, [O9–2H2O–CH2O+4CH2+N(CH3)3H]⁺} was also detected in arabinogalactan and arabinogalactan from 204 Da (corresponding to C6H). 10 (O5+3CH2) Neutral loss of fragment ions. For arabinogalactan, this observation may be attributed to modifications on its side chain residues.

[0052] Nitrogenous polysaccharides: Chitin and hyaluronic acid are polysaccharides composed of different monosaccharide units: chitin is composed of N-acetylglucosamine, while hyaluronic acid is composed of disaccharide repeating units of N-acetylglucosamine and glucuronic acid. Their DART-MS spectra differ significantly from each other and from those of polysaccharides based on neutral hexoses and pentoses. Chitin produces characteristic ions with m / z values ​​of 318.15, 431.24, 491.26, 505.28, and 750.40, accompanied by Δm = 245 (C8H). 13 The neutral loss of (NO5+3CH2) indicates the breakage of the glycosidic bond in its N-acetylglucosamine unit. In contrast, hyaluronic acid exhibits a unique set of ion m / z values ​​of 446.20, 478.23, 635.34, and 723.35, accompanied by concurrent neutral losses of (C6H8O6+3CH2) at Δm = 245 and Δm = 218, corresponding to the breakage of the N-acetylglucosamine and glucuronic acid units, respectively. Therefore, chitin and hyaluronic acid can be directly distinguished using mass spectrometry.

[0053] The results show that methylation of hydroxyl groups on carbohydrate polymers using TMAH can occur successfully within seconds within a DART ion source. This methylation process is accompanied by extensive intrasource pyrolysis. These two processes, methylation and cleavage, occur simultaneously and may be interdependent: the detected methylated products may originate from methylation of pyrolytic fragments or from the subsequent decomposition of the initially methylated sugar chain.

[0054] The above steps establish a method for rapidly determining the monosaccharide composition type of polysaccharides through feature neutral loss.

[0055] (2) Tandem mass spectrometry (MS / MS) analysis and diagnostic ion screening:

[0056] While TMAH-assisted DART-MS produced rich and informative spectra for the various polysaccharides studied, direct identification of individual polysaccharide components in mixtures remains challenging due to signal overlap and the complexity of full-scan MS data. To address this issue, we performed in-situ ionization tandem mass spectrometry (DART-MS / MS) on methylated derivatives. We selected collision-induced dissociation of the major precursor ions (relative intensity >10%) at collision energies ranging from 10 eV to 40 eV to achieve more specific characterization of these saccharide polymers. For polysaccharides composed entirely of hexose units, including cellulose, amylose, dextran (20 kDa), mannan, and galactomannan, we selected different precursor ions. For example, MS / MS spectra at m / z 514.32 and m / z 760.43 are shown for hexose polysaccharides. Using hexose as the basic monomer unit, the symbols M1, M2, M3, and M4 represent the corresponding saccharide monomers (C6H4 ... 12 O6), disaccharide (C) 12 H 22 O 11 ), trisaccharides (C 18 H 32 O 16 ) and tetrasaccharides (C 24 H 42 O 21 The molecular formula of ). The mass spectrum peak m / z 514.32 corresponds to the fully methylated signal of the disaccharide unit {[M2+8CH2+N(CH3)3H] . + Common fragments from m / z 514.32 include m / z 455.25 {[M2+8CH2+H]}. +}, m / z 468.28{[M2+7CH2–CH3OH+N(CH3)3H] + ]} and m / z 482.30 {[M2+8CH2–CH3OH+N(CH3)3H] +It is noteworthy that although cellulose, amylose, and dextran (20 kDa) share the same glucose monomer unit, their characteristic MS / MS fragments differ significantly. Cellulose is characterized by the presence of a characteristic ion signal at m / z 374.23, identified as {[M2+8CH2–CH5O4+H]}. + In contrast, amylose exhibits a diagnostic ion at m / z 278.20, corresponding to {[M1–H2O+4CH2+N(CH3)3H]}. +}, while dextran (20 kDa) shows a unique ion at m / z 373.17, belonging to {[M2–H2O+8CH2–2CH3OH+H]}. + The relevant neutral loss is indicated in their respective MS / MS spectra. The precursor ion m / z 760.43 is believed to be a methylated fragment from a glycotetramer, with the ionic formula {[M4–C4H8O4+11CH2+N(CH3)3H]}. + The fragmentation at m / z 760.43 into dextran (20 kDa), mannan, and galactomannan produced distinct MS / MS spectra and unique fragment ions. MS / MS spectra of cellulose and amylose were not included because structurally information-rich fragment ions were not generated under the conditions employed. Specifically, the ion at m / z 556.33 {[M3–C4H8O4+8CH2+N(CH3)3H] + The presence of} is specific for dextran (20 kDa); while m / z 432.33 {[M2+8CH2-CH3OH+H] +}, 581.28{[M3-C2H8O2+10CH2+H] +}, 595.30 {[M3-C2H8O2+11CH2+H] +} and 644.35 {[M3–H2O+7CH2+N(CH3)3H] + The ion {} is a characteristic ion of mannan. Conversely, the ion m / z 672.38 {[M3–H2O+9CH2+N(CH3)3H] +} and m / z 701.35{[M4–C4H8O4 +11CH2+H] + These are unique to galactomannan. These diagnostic product ions provide strong identification evidence for differentiating different polysaccharide polymers.

[0057] Similarly, we performed tandem mass spectrometry analysis on multiple precursor ions of these polysaccharide samples and ultimately screened a series of precursor ions with diagnostic product ions for identifying different polysaccharides in the mixture. Relevant mass spectrometry information and characteristic product ions are shown in Table 1. For hexose-based polysaccharides, a total of 13 diagnostic precursor ions were identified. Among them, precursor ion m / z 514.32 could distinguish all five polysaccharides. Three ions (m / z 468.28, 556.33, and 614.34) could distinguish four polysaccharides; another three ions (m / z 718.42, 760.43, and 788.43) could distinguish three polysaccharides; and five ions (m / z 482.30, 584.33, 818.44, 874.47, and 992.53) could distinguish two polysaccharides. Furthermore, MS / MS fragment m / z 890.50 was specific for identifying amylose. For polysaccharides based on pentoses, a total of 10 diagnostic precursor ions were identified. Four precursor ions (m / z 406.24, 522.29, 526.29, and 554.32) could distinguish all three polysaccharides, while four ions (m / z 362.22, 392.24, 556.33, and 672.28) could distinguish two. Furthermore, fragment m / z 686.40 recognized arabinogalactan, while fragment m / z 890.46 recognized arabinogalactan.

[0058] Table 1. Characteristic precursor ions and diagnostic fragment ions of different polysaccharides

[0059]

[0060]

[0061] The names, characteristic precursor ions, and corresponding diagnostic fragment ion information of all the above-mentioned standards were compiled to construct a diagnostic ion database for polysaccharide identification. The results are shown in Table 1 and [Table data would be inserted here]. Figure 2 . Figure 2 Diagnostic ion fragments are marked (gray). Gray arrows indicate the loss of CH2; black arrows indicate the loss of CH3OH. Symbols M1, M2, M3, and M4 represent carbohydrate C6H, respectively. 12 O6, C 12 H 22 O 11 C 18 H 32 O 16 , and C 24 H 42 O 21 The collision energy of all compounds is 22 eV. This database serves as a basis for subsequent identification of unknown samples.

[0062] To evaluate the precision of this method, four structurally representative polysaccharides (cellulose, galactomannan, xylan, and hyaluronic acid) were selected, and their peak areas extracted from the precursor ions in the primary mass spectrometry spectroscopy were analyzed for intra-day and inter-day relative standard deviations. Intra-day precision was evaluated by performing at least three consecutive measurements on the same sample within one day; inter-day precision was evaluated by performing multiple measurements on the same sample over several consecutive days. The experimental procedure was the same as that for the four polysaccharides mentioned above, and the results are shown in Table 2.

[0063] Table 2 validates the reproducibility and repeatability of the online methylation method for polysaccharides based on the analysis of four different polysaccharide samples.

[0064]

[0065] Depend on Figure 2 As can be seen from the experimental results, the relative standard deviation of the peak area of ​​all target characteristic ions is less than 12%, indicating that the method has good reproducibility and further confirming the stability and reliability of the method in polysaccharide sample analysis.

[0066] Based on the above experiments, such as Figure 3 As shown, the polysaccharide mass spectrometry fingerprint constructed by the method of this invention can directly achieve rapid in-situ identification of polysaccharides (intact polymers) in actual samples. The core of this process lies in the direct introduction of the polysaccharide sample into the excited-state molecular beam ion source (DART) after mixing with TMAH. Utilizing the unique multi-process reaction chamber characteristics of the DART source, online methylation, in-situ ionization, and intrasource fragmentation of the sample are simultaneously achieved. The entire process takes no more than 10 seconds, greatly simplifying the sample pretreatment steps. After the final generated ions are analyzed by MS and MS / MS, they are compared with a pre-established diagnostic ion database to achieve direct identification of different polysaccharides.

[0067] Example 2: Identification of polysaccharides in actual samples

[0068] 1. Sample processing

[0069] Taking wolfberry as an example, mechanochemical extraction (MCE) was performed using a FastPrep®-24 device (MP Biomedicals LLC, USA) to extract polysaccharide components from the original sample. All samples were prepared with pure water, and the initial concentration was 0.1 g / mL. -1 (For powder) or 1:1 volume ratio (for oral liquid), then in 4 ms -1Extraction was performed at a running speed of 60 s. The crude solution after MCE extraction was subjected to five cycles of ethanol precipitation and protein removal (Sevage method). Finally, the purified polysaccharide powder was obtained by freeze drying (LGJ-18, Henan Brothers Instrument Co., Ltd.). All polysaccharide powders were prepared with water to a concentration of 2.0 mg / mL. -1 The polysaccharide solution was then purified by ultrafiltration (Nanjing Formass Biotechnology Co., Ltd.) to remove small molecule impurities with molecular weights below 3 kDa (MWCO), such as oligosaccharides, before being used for mass spectrometry analysis. The purified polysaccharide was dissolved in 10% TMAH solution to prepare a 1 mg / mL solution. -1 The analytical solution. This example uses commercially available wolfberry powder.

[0070] 2. Analysis and Identification

[0071] This method utilizes in-situ methods and MS / MS diagnostic ions within the DART ion source to directly identify different polysaccharides in real samples. Polysaccharide samples premixed with TMAH are directly introduced into the DART ion source. The DART ion source can be viewed as a reaction chamber where multiple processes occur, including online methylation, in-situ ionization, and autogenous cleavage. All these processes are completed within 10 seconds, after which the resulting ions are analyzed by MS and MS / MS. The interpretation of MS data is based on characteristic neutral losses: a consecutive neutral loss with a mass difference (Δm) of 204 Da indicates a hexose-based polysaccharide; Δm of 160 Da suggests a pentose-based polysaccharide; Δm of 245 Da points to an N-acetylglucosamine-based polysaccharide; and consecutive losses of 245 Da and 218 Da indicate a polysaccharide composed of N-acetylglucosamine and glucuronic acid.

[0072] Different polysaccharides exhibited differentiated recognition characteristics in mass spectrometry analysis. Based on the experimental results of Example 1, chitin and hyaluronic acid could be directly identified using their characteristic ion signals: chitin presented characteristic ion clusters at m / z 318.15, 431.24, 491.26, 505.28, and 750.40; hyaluronic acid showed unique MS signals at m / z 446.20, 478.23, 635.34, and 723.35. These signals can be directly used to identify chitin and hyaluronic acid. The remaining eight polysaccharides, while exhibiting different fragmentation patterns, lacked unique characteristic ion fragment signals. For these polysaccharides, it is necessary to select specific parent ions for collision-induced dissociation, obtain their secondary fragment spectra, and then screen for characteristic fragment ions with identification significance.

[0073] Based on the above analysis, the correspondence between precursor ions and diagnostic fragment ions for each polysaccharide was established in Table 1 of Example 1. By matching the MS / MS data of the actual sample in this example with the diagnostic ions in Table 1, the types of polysaccharides contained in the sample can be accurately identified based on the presence or absence of specific diagnostic ions. This workflow provides an efficient and robust strategy for identifying macromolecular polysaccharides without requiring complex sample pretreatment.

[0074] The Lycium barbarum polysaccharide extract, i.e., the analytical solution from step 1, was analyzed according to the DART-Orbitrap MS / MS analytical conditions in step 3 of Example 1. The results are as follows: Figure 4 As shown. First, observe the full-scan mass spectrometry. Figure 3 As can be seen from the first-order spectrum, there are indeed continuous Δm = 204 Da (C6H) values ​​of m / z 1018.55, 814.44, 760.43, 610.34, 556.33, and 406.24. 10The loss of neutral ions (O5+3CH2) and the consecutive neutral losses of 160 Da (C5H8O4+2CH2) at m / z 846.47, 714.39, 686.40, 554.32, etc., along with the absence of characteristic ion signals for chitin and hyaluronic acid, suggest the possible co-existence of hexose and pentose polysaccharides in the sample. Further, all the identifying precursor ions summarized in Table 1 were selected and sequentially analyzed by tandem mass spectrometry at collision energies of 10-40 eV, and then compared with the established database and strategy. The following observations were made in the actual sample: Secondary fragment ions at m / z 394.24 included xylan characteristic ions (m / z 379.16); secondary fragment ions at m / z 514.32 included amylose characteristic ions (m / z 278.20), mannan characteristic ions (m / z 377.18), and galactomannan characteristic ions (m / z 423.22); secondary fragment ions at m / z 526.29 included arabinomannan characteristic ions (m / z 290.20); secondary fragment ions at m / z 556.33 included cellulose characteristic ions (m / z 293.10, m / z 353.23), amylose characteristic ions (m / z 359.17, m / z 475.22); and secondary fragment ions at m / z 584.33 included galactomannan characteristic ions (m / z 359.17) and mannan characteristic ions (m / z 423.22). Secondary fragment ions at m / z 391.20 and m / z 614.34 contain characteristic ions of dextran (20 kDa) at m / z 255.09 and m / z 351.17, and characteristic ions of mannan at m / z 391.20; secondary fragment ions at m / z 672.28 contain characteristic ions of arabinogalactan at m / z 654.30. In summary, diagnostic fragment ions corresponding to arabinogalactan, arabinogalactan, xylan, cellulose, amylose, dextran (20 kDa), galactomannan, and mannan were found in the sample, thus successfully identifying these eight polysaccharide components in wolfberry. This is consistent with existing knowledge of wolfberry components.

Claims

1. A method for constructing a polysaccharide mass spectrometry fingerprint, characterized in that, Includes the following steps: (A1) Mix the polysaccharide sample with the derivatization reagent to obtain a polysaccharide standard solution; (A2) The polysaccharide standard solution was directly introduced into the in-situ ionization source for DART-MS analysis to obtain the mass spectrum of the polysaccharide sample; (A3) Select fragment ions from the mass spectrum of the polysaccharide sample for MS / MS analysis, and finally establish the mass spectrometric fingerprint of the polysaccharide sample.

2. The method for constructing a polysaccharide mass spectrometry fingerprint according to claim 1, characterized in that, In step (A1), the polysaccharide is one or more of hexose polysaccharides, pentose polysaccharides, or nitrogen-containing polysaccharides, and the derivatizing reagent is tetramethylammonium hydroxide solution, 1-phenyl-3-methyl-5-pyrazolone or 1-(4-carboxyphenyl)-3-methyl-5-pyrazolone, or a combination of iodomethane and sodium hydroxide.

3. The method for constructing a polysaccharide mass spectrometry fingerprint according to claim 2, characterized in that, Hexose polysaccharides are one or more of cellulose, amylose, glucan, mannan or galactomannan; pentosaccharide polysaccharides are one or more of arabinogalactan, arabinogalactan or xylan; and nitrogen-containing polysaccharides are one or two of chitin or hyaluronic acid.

4. The method for constructing a polysaccharide mass spectrometry fingerprint according to claim 1, characterized in that, In step (A1), the polysaccharide concentration in the mixed solution of the polysaccharide sample and derivatization reagent is 0.1-4 mg / mL. -1 The mass concentration of the derivatization reagent solution is 0.1-20%.

5. The method for constructing a polysaccharide mass spectrometry fingerprint according to claim 1, characterized in that, In step (A2), the in-situ ionization source is an excited-state molecular beam ion source. The operating parameters of the in-situ ionization source include: pump pressure of -60 to -95 kPa, working gas of helium or nitrogen, and working gas heating temperature of 200-500 ℃.

6. The method for constructing a polysaccharide mass spectrometry fingerprint according to claim 1, characterized in that, In step (A2), during DART-MS analysis, the data acquisition mode is positive, and the mass spectrometry detection range is m / z 50-2000; the tandem mass spectrometry uses collision-induced dissociation with a collision energy of 10-40 eV.

7. The method for constructing a polysaccharide mass spectrometry fingerprint according to claim 1, characterized in that, In step (A3), stable polysaccharide fragment ions with a relative intensity >10% are selected from the mass spectrum fragments of the polysaccharide sample as the parent ion. The MS / MS analysis conditions include: collision-induced dissociation of the selected parent ion at a collision energy of 10-40 eV, and screening out ions with structure specificity from the generated secondary fragments as characteristic ions.

8. The application of the mass spectrometry fingerprint of polysaccharides obtained by the construction method according to any one of claims 1-7 in the rapid screening, identification or composition analysis of polysaccharide components in food, plant samples or traditional Chinese medicine.

9. The application according to claim 8, characterized in that, Includes the following steps: (B1) Mix the sample solution to be tested with the derivatization reagent to obtain the analytical solution; (B2) The analytical solution is directly introduced into the in-situ ionization source for ionization and tandem mass spectrometry analysis to obtain the mass spectrum of the polysaccharide sample in the test sample; (B3) Compare the mass spectrum of the sample to be tested obtained in step (2) with the mass spectrum of the polysaccharide to determine the type of polysaccharide; or, The precursor ions were selected for MS / MS analysis, and the analysis results were compared with the polysaccharide mass spectrometry fingerprint. The polysaccharide species were determined by matching characteristic precursor ions and their diagnostic fragment ions.

10. The application according to claim 9, characterized in that, An internal standard is added to the analytical solution in step (B1) for quantitative analysis of polysaccharides.

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