Highly Sensitive and High-Throughput Identification Method for Carbohydrate Compounds Based on Chlorine-Enhanced Ionization

Abstract

The present invention discloses a new method for highly sensitive and high-throughput identification of saccharide compounds based on chlorine-enhanced ionization. The method includes: extracting saccharide compounds from a sample to be tested to obtain a saccharide compound extract; optimizing and selecting chloromethyltriphenylphosphonium chloride from 23 kinds of quaternary phosphonium salts, quaternary ammonium salts and inorganic ammonium salts as a chlorine affinity reagent added to the mobile phase, and performing targeted and non-targeted scanning analysis on the saccharide compound extract by liquid chromatography tandem high-resolution mass spectrometry (UPLC-ESI-HRMS); using the high-resolution mass spectrometry scanning results to identify unknown saccharide compounds and analyze the contents of known saccharides. The present invention uses the addition of chloromethyltriphenylphosphonium chloride as a chlorine affinity reagent in the mobile phase to significantly improve the detection sensitivity and separation effect of saccharide compounds, realizing the analysis of saccharide compounds with small sample amounts, low interference and high sensitivity; at the same time, the chlorine isotope-labeled saccharide compounds have isotope distribution characteristics, and the polysaccharides can produce double-charged molecular ion peaks, which significantly increase the mass spectrometry detection mass range, providing a new technology for the high-quality coverage screening of saccharide compounds in food, biological and environmental samples, and having originality.

Description

Technical Field

[0001] The invention belongs to the fields of biochemistry and analytical chemistry, and specifically relates to a method for highly sensitive and high-throughput identification of sugar compounds based on chlorine-enhanced ionization. Background Art

[0002] Carbohydrates are the main source of energy required to maintain life activities and play an important role in life activities, including monosaccharides (glucose, fructose, mannose and galactose, etc.), oligosaccharides (maltose, sucrose, lactose and other disaccharides and polymers formed by 3-10 monosaccharides), polysaccharides (polymers formed by more than 10 monosaccharides) and derivatives (sugar alcohols, cyclodextrins, etc.). Monosaccharides and disaccharides are a class of carbohydrate compounds that have important value at the nutritional level and even at the cellular level, while oligosaccharides with prebiotic properties and polysaccharide foods with recognized health properties have also received great attention from researchers and the food industry. In view of the wide range of sources, uses and diverse biological activities of carbohydrate compounds, accurately evaluating the occurrence types and content composition characteristics of carbohydrate compounds in food is of great significance for in-depth understanding and evaluation of the relationship between the structure and function of carbohydrate compounds, as well as their health effects.

[0003] At present, the main methods for determining sugar content are chemical methods and instrumental analysis methods. The chemical method can determine the total amount of sugar, but it cannot distinguish the types of sugar. Among the instrumental analysis methods, high performance liquid chromatography generally uses a differential refractometer, which has low sensitivity and is difficult to detect oligosaccharides above trisaccharides; high performance ion chromatography combined with a pulsed amperometric detector has high sensitivity, but cannot provide structural information, which is not conducive to the specific analysis of sugars in complex samples. In comparison, mass spectrometry has good selectivity and can display the structural characteristics of sugar compounds. It is considered to be an important technology for sugar analysis, but there are currently two main problems. First, the high hydrophilicity and low surface activity of sugars, especially trisaccharides and above, make them difficult to ionize by mass spectrometry and the detection sensitivity is not high. Studies have reported that adding dichloromethane, chloroform, etc. after the liquid column in a liquid chromatography-mass spectrometry system (LC-MS) can make monosaccharides and disaccharides form [M+Cl] - Adducts enhance their mass spectrometry ionization, but the post-column addition of the flow injection pump makes it difficult for the instrument to achieve continuous automatic sampling, severely limiting the analysis and detection of large quantities of samples; secondly, it is limited by the mass-to-charge ratio scanning range (usually set to m / z less than 1500). Electrospray ionization mass spectrometry polysaccharide analysis is mainly based on the spectral regularity of the target polysaccharide macromolecules being broken down into small molecule fragments, but actual samples are often mixtures of polysaccharides, and prior separation and purification are required to effectively apply the fingerprint spectrum of small molecule sugar product fragments to identify polysaccharide molecules. Moreover, polysaccharide standards must be purchased in advance or a comparable fingerprint library must be available, which severely limits the identification and screening of sugar compounds without standards and unknown sugar compounds in actual samples. Summary of the invention

[0004] To solve at least one of the above technical problems, the object of the present invention is to propose a new method for highly sensitive and high-throughput analysis of saccharide compounds based on chlorine-enhanced ionization. By adding quaternary phosphonium chloride or quaternary ammonium salt that can be simultaneously dissolved in the aqueous phase and the organic phase to the mobile phase, a specific chlorine-enhanced ionization signal ([M+Cl]−) of saccharides is formed in the electrospray ion source, greatly improving the analysis sensitivity. The mass spectrometry response signal is increased by up to 700 times compared with the reported LC-MS method of adding chloroform or guanidine hydrochloride post-column in the mobile phase; further based on the characteristic isotope peak distribution characteristics of the chlorine affinity product of saccharides, and the fact that polysaccharide compounds with increased ionizable sites can obtain double-charged ions, thus significantly increasing the mass range of mass spectrometry detection, a highly sensitive and high-throughput scanning method for liquid chromatography high-resolution mass spectrometry (UPLC-ESI-HRMS) combined analysis based on the chlorine-enhanced ionization of organic phosphonium salts of intact saccharide molecules is established, providing a new technology for targeted detection and non-targeted screening of saccharide compounds in food, biological and environmental samples.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for highly sensitive and high-throughput identification of saccharide compounds based on chlorine-enhanced ionization, comprising the following steps:

[0007] Extract the saccharide compounds in the sample to be tested to obtain an extract;

[0008] Select a mobile phase additive and a chromatographic column, optimize the mobile phase gradient and the composition of the sample loading solution, and perform UPLC-ESI-HRMS scanning on the extract of saccharide compounds;

[0009] Utilize the UPLC-ESI-HRMS scanning results to target the detection of saccharide compounds with standards and non-targetedly identify unknown saccharides.

[0010] As a preferred embodiment, the sample extraction includes but is not limited to food samples, biological samples or environmental samples.

[0011] As a preferred embodiment, the specific method for extracting saccharide compounds in the sample to be tested is:

[0012] Add 5 μg of glucose to the sample to be tested - 13For the C6 internal standard, first add 200 - 2000 μL of frozen methanol, vortex to mix evenly, and then centrifuge at 10000 rpm for 5 min at 4°C until complete phase separation to obtain the supernatant; then add 200 - 2000 μL of ice water, vortex to mix evenly, and centrifuge at 10000 rpm for 5 min at 4°C until complete phase separation to obtain the supernatant; then add 200 - 2000 μL of frozen acetonitrile, vortex to mix evenly, and centrifuge at 10000 rpm for 5 min at 4°C until complete phase separation to obtain the supernatant; collect all the supernatants, which is the extract of carbohydrate compounds.

[0013] As a preferred embodiment, determine the dilution factor of the extract according to the sample properties and the concentration level of the target analyte, and filter through a 0.22 μm filter membrane and then inject for UPLC-ESI-HRMS analysis.

[0014] Preferably, as a better implementation manner, when the sample to be tested is a liquid sample, the dosage of the sample to be tested is 10 - 2000 μL; when the sample to be tested is a solid sample, the dosage of the sample to be tested is 0.1 - 100 mg.

[0015] As a preferred embodiment, the UPLC-ESI-HRMS scanning method includes the following steps: using an HRMS equipped with an electrospray ion source, separating the carbohydrate compounds by normal-phase chromatography, using acetonitrile and water as the chromatographic mobile phase, optimizing by adding a chlorine affinity reagent to acetonitrile, and scanning in the negative ion - Full-MS mode.

[0016] As a preferred embodiment, the selection of the chlorine affinity reagent added to acetonitrile includes: according to the solubility of the chlorine affinity reagent in the organic phase / water phase and its ability to undergo an affinity reaction with carbohydrate compounds, select the chlorine affinity reagent and the added concentration with the highest mass spectrometry sensitivity for carbohydrate chloroaffinity ([M+Cl] - )). The halogen-containing affinity reagents include 8 kinds of quaternary phosphonium salts (tetraphenylphosphonium chloride, tetrabutylphosphonium chloride, methyltriphenylphosphonium chloride, ethyltriphenylphosphonium chloride, tetraphenylphosphonium bromide, chloromethyltriphenylphosphonium chloride, tetrabutylphosphonium bromide, methyltriphenylphosphonium bromide), 13 kinds of quaternary ammonium salts (phenyltrimethylammonium chloride, phenyltrimethylammonium bromide, dimethylbenzylphenylammonium chloride, N-triethyl-(4-vinylbenzyl)ammonium chloride, vulcanization accelerator DBU-B, benzalkonium chloride, benzyltrimethylammonium chloride, benzyltripropylammonium chloride, benzyltributylammonium chloride, acryloyloxyethyl dimethylbenzylammonium chloride, dimethylbenzyl-2-methylacrylate ethylamine chloride, benzethonium chloride, and tetradecyldimethylbenzylammonium chloride hydrate), and 2 kinds of inorganic ammonium salts (ammonium chloride and ammonium bromide); the added concentration of the halogen-containing affinity reagent is selected as 1 μM.

[0017] As a preferred embodiment, the optimization of the composition of the sample loading solution includes the following steps: According to the effect of the sample loading solution on the chromatographic separation of carbohydrate isomers and optical isomers and the mass spectrometry response intensity caused by the solvent effect of the chromatographic peak, the volume ratio of acetonitrile, methanol and water in the sample loading solution is optimized to 2:2:1.

[0018] As a preferred embodiment, the targeted detection of carbohydrate compounds in the sample to be tested includes the following steps: Prepare a standard curve solution of the target carbohydrate compound and determine it by UPLC-ESI-HRMS. According to the internal standard method (isotope internal standard: glucose- 13 C6), perform a linear regression analysis of the corresponding concentration with the peak area corrected by the internal standard, and calculate the concentration of the target carbohydrate compound in the sample to be tested.

[0019] Preferably, as a better embodiment, the mass spectrometry detection conditions are as follows: The ion source is an ESI source, and the full scan is performed in the negative ion mode-full-MS mode. The full scan mass range is 50-1500 Da; the cone voltage is 30 V; the desolvation gas temperature is 500 °C; the capillary voltage is 2.0 kV; the desolvation gas flow rate is 800 L / Hr; the source temperature is 100 °C.

[0020] Preferably, as a better embodiment, the chromatographic separation conditions are as follows: The chromatographic column is a Waters ACQUITY UPLC BEH Amide Column (2.1 mm × 100 mm, 1.7 μm), and the column temperature is 40 °C; The mobile phase is acetonitrile (1 μM chloromethyltriphenylphosphonium chloride) and water, the flow rate is 0.3 mL / min, the injection volume is 5 μL, and the gradient conditions are as follows: 0 min, 95% A; 0-3 min, 95% A; 3-40 min, 95%-60% A; 40-40.1 min, 60%-50% A; 40.1-42 min, 50% A; 42-42.1 min, 50%-95% A; 42.1-43 min, 95% A.

[0021] As a preferred embodiment, the non-targeted screening and identification method for unknown carbohydrates in the sample to be tested includes the following steps:

[0022] First, perform peak extraction on the mass spectrometry data to obtain the mass-to-charge ratio, retention time, and peak area of the chemical signal. Then, perform non-targeted screening based on the characteristic isotope peaks of the carbohydrate chloroaffinity products, and screen and pair the mass spectrometry signals of the carbohydrate chloroaffinity product mass spectrometry cluster peaks according to the exact mass number and peak height ratio of the chlorine isotope peaks. Finally, perform elemental composition analysis on the selected carbohydrate chloroaffinity product cluster peaks and compare them with the actual mass spectrometry signals to verify the accuracy of the elemental composition analysis.

[0023] Preferably, the parameters for peak extraction are set as follows: the low energy intensity threshold is 200.0, the mass-to-charge ratio tolerance is 3 mDa; the locked mass number is 554.2620; the maximum considered charge in the 3D isotope cluster is 2, the maximum number of isotopes per cluster is 1, and the allowed minimum single isotope / maximum isotope intensity ratio is 0.1. The parameters for mass spectrometry signal screening and pairing are: the absolute value of the retention time difference of the ion pair is 0.02 min; the absolute value of the mass-to-charge ratio difference of the ion pair is between 1.9921 and 2.0021 Da, or between 0.996 and 1.001. The parameters for elemental composition analysis are: the upper limit of the number of atoms of each element in the chemical formula is 90C, 160H, 80O, 5N, 3Cl; the mass tolerance is 10 ppm.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] (1) By simply adding quaternary phosphonium chloride and quaternary ammonium salt chlorine affinity reagents in front of the liquid chromatography mobile phase column, the present invention can significantly enhance the mass spectrometry ionization signal intensity of saccharide compounds, with the detection sensitivity as low as 3.4 pg and the improvement multiple as high as 700 times, which can greatly reduce the sample usage amount.

[0026] (2) By adding quaternary phosphonium chloride and quaternary ammonium salt in the mobile phase to form a chlorine affinity substance, the present invention enhances the chromatographic retention behavior of saccharide compounds in the acylamino normal phase chromatographic column, and can significantly improve the ability to separate saccharide compound isomers and optical isomers.

[0027] (3) By scanning polysaccharide compounds to generate double-charge molecular ion peaks, the present invention significantly increases the mass spectrometry detection mass range and improves the mass coverage range for non-target screening from monosaccharides to polysaccharide compounds. In the implementation cases based on this method, 10 kinds of monosaccharides, oligosaccharides and sugar alcohols with [M+Cl] - as the molecular ion peak were targeted detected in beer samples; polysaccharide compounds (nonasaccharides to tridecasaccharides [molecular weight exceeding 2200 Da]) with [M+2Cl] 2- as the molecular ion peak were non-target screened and identified, and the existence of unsaturated saccharides (trisaccharides to hexasaccharides, unsaturation degree of 1) was also found. Description of the Drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0029] Figure 1The effects of adding 23 chlorine affinity reagents (8 quaternary phosphonium salts, 13 quaternary ammonium salts, and 2 inorganic ammonium salts) to the mobile phase on the mass spectrometry response intensity of saccharide compounds are shown (taking the monosaccharide D-glucose and the disaccharide maltose as examples);

[0030] Figure 2 The molecular ion mass spectrometry diagrams of some target saccharide compounds in UPLC-ESI-HRMS analysis after adding chlorine affinity reagents to the mobile phase are shown;

[0031] Figure 3 The chromatograms of the standard solutions of 4 monosaccharides, 10 oligosaccharides, and 7 derivatives (4 sugar alcohols and 3 cyclodextrins) in UPLC-ESI-HRMS simultaneous analysis after adding chlorine affinity reagents to the mobile phase are shown, where reducing saccharide compounds show double peaks due to the presence of α- and β-anomers;

[0032] Figure 4 The chromatograms and mass spectrometry diagrams of newly identified saccharide compounds in beer samples in UPLC-ESI-HRMS non-targeted scanning after adding chlorine affinity reagents to the mobile phase are shown. Detailed implementation mode

[0033] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited by the specific embodiments disclosed below.

[0034] The technical solution of the present invention will be described in more detail below by taking the detection of saccharide compounds in beer samples as an example, but this example does not constitute any limitation to the present invention.

[0035] I. Instruments and reagents:

[0036] 1. Instruments

[0037] Ultra-high performance liquid chromatography high-resolution mass spectrometry combined system (Waters), including ACQUITY I CLASS PLUS ultra-high performance liquid system, Xevo G2-XS QTof mass spectrometry; high-speed refrigerated centrifuge; vortex oscillator.

[0038] 2. Reagents

[0039] Chloromethyltriphenylphosphonium chloride, methanol (LC / MS grade), acetonitrile (LC / MS grade), Milli-Q water. Oligosaccharide and derivative standards include D-glucose, D-mannose, D-(+)-galactose, fructose, maltose, sucrose, lactose, raffinose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose, oligofructose, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, D-sorbitol, D-mannitol, maltitol, lactitol, glucose-13 C6 was purchased from Merck Sigma-Aldrich (USA), and the purity was greater than 97%.

[0040] II. Example 1

[0041] A method for targeted detection and untargeted screening of saccharide compounds in beer samples, comprising the following steps: (1) Pretreatment of the sample to be tested

[0042] The beer sample was ultrasonically degassed for 40 min. 10 μL was taken and placed in a 1.5 mL centrifuge tube. 1 mL of frozen methanol and 5 μg of glucose- 13 C6 internal standard were added, vortexed, centrifuged at 10000 rpm for 5 min at 4 °C, and the supernatant was transferred to a brown glass bottle. 1 mL of ice-cold methanol was added to the precipitate, vortexed, centrifuged, and the supernatant was transferred; 1 mL of ice-cold water was added to the precipitate, vortexed, centrifuged, and the supernatant was transferred; finally, 1 mL of frozen acetonitrile was added to the precipitate, vortexed, centrifuged, and the supernatant was transferred. Then the above acetonitrile extraction step was repeated once. All supernatants were collected in the same brown glass bottle to obtain a saccharide compound extract, which was stored at 4 °C for UPLC-ESI-HRMS analysis.

[0043] The mass spectrometry detection conditions for the ultra-high performance liquid chromatography high-resolution mass spectrometry system were as follows: The ion source was the ESI source, and the negative ion mode-full-MS mode was used for full scan. The full scan mass range was 50-1500 Da; the cone voltage was 30 V; the desolvation gas temperature was 500 °C; the capillary voltage was 2.0 kV; the desolvation gas flow rate was 800 L / Hr; the source temperature was 100 °C.

[0044] The chromatographic separation conditions were as follows: The chromatographic column was a Waters ACQUITY UPLC BEH Amide Column (2.1 mm × 100 mm, 1.7 μm), the column temperature was 40 °C; the mobile phase was acetonitrile (1 μM chloromethyltriphenylphosphonium chloride) and water, the flow rate was 0.3 mL / min, the injection volume was 5 μL, and the gradient conditions were: 0 min, 95% A; 0-3 min, 95% A; 3-40 min, 95%-60% A; 40-40.1 min, 60%-50% A; 40.1-42 min, 50% A; 42-42.1 min, 50%-95% A; 42.1-43 min, 95% A.

[0045] The influence of adding 23 kinds of chlorine affinity reagents (8 kinds of quaternary phosphonium salts, 13 kinds of quaternary ammonium salts and 2 kinds of inorganic ammonium salts) to the mobile phase on the mass spectrometry response intensity of saccharide compounds (taking monosaccharide D-glucose and disaccharide maltose as examples) was as Figure 1 shown.

[0046] The molecular ion mass spectra of some target saccharide compounds analyzed by UPLC-ESI-HRMS after adding a chlorine affinity reagent to the mobile phase are as follows Figure 2 as shown.

[0047] Prepare a standard mixed solution of 21 oligosaccharides and derivatives (D-glucose, D-mannose, D-(+)-galactose, fructose, maltose, sucrose, lactose, raffinose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose, fructooligosaccharide, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, D-sorbitol, D-mannitol, maltitol, lactitol), with concentration levels including 0.01 mg / L, 0.05 mg / L, 0.1 mg / L, 0.5 mg / L, 1 mg / L, and 5 mg / L. Perform UPLC-ESI-HRMS analysis with an injection volume of 5 μL, and conduct linear regression analysis on the peak area measured for each substance and its concentration to obtain a standard curve. The results show that the ratio of the peak area to the concentration of the standard substances presents a good linear relationship within this concentration range, and the correlation coefficients are all greater than 0.99.

[0048] The instrument detection limit is the concentration level of each saccharide compound calculated when the signal-to-noise ratio (S / N) of the standard chromatographic peak is 3. The recovery rate is obtained by adding the target saccharide standard mixed solution to the matrix sample, detecting it according to the sample pretreatment and instrument analysis methods, comparing the chromatographic peak area of the standard substance at the spiked concentration level with the peak area actually measured in the spiked sample, and calculating the recovery rate of this method. The specific results are listed in Table 1.

[0049] Table 1 Quantitative ion mass-to-charge ratio, retention time, instrument detection limit, and recovery rate results of standard substances

[0050]

[0051]

[0052] a-reducing saccharide compounds have α- and β-anomeric isomers

[0053] (2) Targeted detection of saccharide compounds in beer samples

[0054] Compare the retention time and mass number of the cluster peaks of saccharide organic compounds detected in the beer sample with those of the target standard sample. As a result, 3 monosaccharides (D-glucose, D-(+)-galactose, fructose), 7 oligosaccharides (maltose, raffinose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose), and 2 sugar alcohols (maltitol and lactitol) are detected in the beer sample.

[0055] Furthermore, according to the internal standard method, the concentration levels of target carbohydrate compounds in beer samples (n = 3) were calculated based on the peak areas corrected by the internal standard, and were respectively: D-glucose 192.7 ± 4.0 mg / L, D-(+)-galactose 116.0 ± 3.9 mg / L, fructose 451.6 ± 3.1 mg / L, maltose 1629.3 ± 13.1 mg / L, raffinose 2850.3 ± 22.3 mg / L, maltotetraose 3128.4 ± 27.4 mg / L, maltopentaose 2521.9 ± 18.3 mg / L, maltohexaose 4790.1 ± 30.5 mg / L, maltoheptaose 4473.8 ± 40.4 mg / L, maltooctaose 4155.0 ± 6.6 mg / L, maltitol 72.1 ± 2.1 mg / L, and lactitol 69.6 ± 0.4 mg / L.

[0056] (3) Non-targeted scanning identification of unknown carbohydrate compounds in beer samples

[0057] The UNIFI software was used to extract peaks from the mass spectrometry data of beer samples to obtain the mass-to-charge ratio, retention time, and intensity of chemical signals. Elemental composition analysis was performed on the chemical signals of the molecular ion peak clusters of unknown carbohydrate compounds that did not match the reference substances. As a result, 2 disaccharides, 3 trisaccharides, 2 tetrasaccharides, and 2 sugar alcohols different from the targeted detection of carbohydrates in the present invention were screened out; and 2- Using [M+2Cl] as the molecular ion peak, 5 polysaccharide compounds (nonasaccharide to tridecasaccharide [molecular weight reaching nearly 2125 Da]) were successfully identified, and the presence of 4 unsaturated carbohydrate compounds (trisaccharide to hexasaccharide, degree of unsaturation is 1) was also found.

[0058] Table 2 Carbohydrate compounds identified by high-sensitivity and high-throughput scanning of beer samples

[0059]

[0060]

[0061]

[0062] In summary, the present invention was successfully applied to beer samples, and 12 monosaccharides, oligosaccharides, and sugar alcohol compounds were targeted and accurately detected. Based on the chlorine isotope distribution characteristics and double-charged molecular ion peaks, 18 carbohydrate compounds were non-targetedly identified, including 5 polysaccharides and 4 unsaturated oligosaccharides. This study provides an effective method for high-sensitivity and high-throughput scanning analysis of carbohydrate compounds with a high coverage range.

[0063] The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention.

Claims

1. A method for highly sensitive and high-throughput identification of saccharide compounds based on chlorine-enhanced ionization, characterized in that, It includes the following steps: Extract the saccharide compounds from the sample to be tested to obtain an extract; Select a mobile phase additive and a chromatographic column, optimize the mobile phase gradient and the composition of the sample loading solution, and perform UPLC-ESI-HRMS detection on the saccharide compound extract; Utilize the UPLC-ESI-HRMS scan results to target the detection of saccharide compounds with standards and non-targetedly identify unknown saccharide compounds; The sample to be tested includes a food sample, a biological sample or an environmental sample; when the sample to be tested is a liquid sample, the dosage of the sample to be tested is 10 - 2000 μL; when the sample to be tested is a solid sample, the dosage of the sample to be tested is 0.1 - 100 mg; The UPLC-ESI-HRMS detection method includes the following steps: Use an HRMS equipped with an electrospray ionization source to separate the saccharide compounds by normal-phase chromatography and perform scanning in the negative ion-Full-MS mode; Mass spectrometry detection conditions: The ion source is an ESI source, and full scanning is performed in the negative ion mode-full-MS mode. The full scanning mass range is 50 - 1500 Da; the cone voltage is 30 V; the desolvation gas temperature is 500 °C; the capillary voltage is 2.0 kV; the desolvation gas flow rate is 800 L / Hr; the source temperature is 100 °C; The chromatographic separation conditions: The chromatographic column is a Waters ACQUITY UPLC BEH Amide Column, 2.1 mm × 100 mm, 1.7 μm; the column temperature is 40 °C; the mobile phase is selected with pure water without addition as phase B and acetonitrile with 1 μM chloromethyltriphenylphosphonium chloride as phase A. The flow rate is 0.3 mL / min, the injection volume is 5 μL, and the gradient conditions are: 0 min, 95%A; 0 - 3 min, 95%A; 3 - 40 min, 95% - 60%A; 40 - 40.1 min, 60% - 50%A; 40.1 - 42 min, 50%A; 42 - 42.1 min, 50% - 95%A; 42.1 - 43 min, 95%A.

2. The method for highly sensitive and high-throughput identification of saccharide compounds based on chlorine-enhanced ionization according to claim 1, wherein The specific method for extracting saccharide compounds from the sample to be tested is: Add 5 μg of glucose- 13 C6 internal standard to the sample to be tested. First, add 200 - 2000 μL of ice-cold methanol, vortex to mix well, and then centrifuge at 10000 rpm for 5 min at 4 °C until complete phase separation to obtain the supernatant; then add 200 - 2000 μL of ice water, vortex to mix well, and centrifuge at 10000 rpm for 5 min at 4 °C until complete phase separation to obtain the supernatant; then add 200 - 2000 μL of ice-cold acetonitrile, vortex to mix well, and centrifuge at 10000 rpm for 5 min at 4 °C until complete phase separation to obtain the supernatant; collect all the supernatants, which is the extract of saccharide compounds.

3. A method for highly sensitive and high-throughput identification of saccharide compounds based on chlorine-enhanced ionization according to claim 1, wherein, The optimization of the composition of the sample loading solution includes the following steps: According to the effect of the sample loading solution causing solvent effect on the chromatographic separation of saccharide compound isomers and optical isomers and the mass spectrometry response intensity, optimize the volume ratio of acetonitrile, methanol and water in the sample loading solution to 2:2:

1.

4. A method for highly sensitive and high-throughput identification of saccharide compounds based on chlorine-enhanced ionization according to claim 1, characterized in that The content analysis of saccharide compounds includes: preparing a standard curve solution of the target saccharide compound, measuring it by UPLC-ESI-HRMS, and based on the internal standard method, the isotope internal standard: glucose- 13 C6, performing linear regression analysis on its corresponding concentration with the peak area corrected by the internal standard, and calculating the concentration of the target saccharide compound in the sample to be measured.

5. A method for highly sensitive and high-throughput identification of saccharide compounds based on chlorine-enhanced ionization according to claim 1, characterized in that, The non-targeted identification of unknown saccharide compounds includes: First, perform peak extraction on the mass spectrometry data to obtain the mass-to-charge ratio, retention time, and peak area of the chemical signals; then, conduct non-target screening based on the characteristic isotope peaks of chlorine-labeled carbohydrate compounds, align the extracted mass spectrometry signals in terms of mass, and screen and pair the mass spectrometry signals of the mass spectrometry clusters of chlorine-labeled organic substances in combination with the exact mass numbers and peak height ratios of the chlorine isotope peaks; finally, perform elemental composition analysis on the screened chlorine-labeled carbohydrate compounds, simultaneously conduct theoretical simulation of isotope peaks for the determined chemical formula, and compare with the actual mass spectrometry signals to verify the accuracy of the elemental composition analysis.

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