High-sensitivity and high-flux carbohydrate compound identification method based on chlorine enhanced ionization

Abstract

The invention discloses a novel high-sensitivity and high-flux carbohydrate compound recognition method based on chlorine enhanced ionization. The method comprises the following steps: extracting carbohydrates in a to-be-detected sample to obtain a carbohydrate extracting solution; chloromethyl triphenylphosphonium chloride is optimally selected from 23 quaternary phosphonium salts, quaternary ammonium salts and inorganic ammonium salts as a chlorine affinity reagent added to a mobile phase, and a carbohydrate extracting solution is subjected to targeted and non-targeted scanning analysis by liquid chromatography-tandem high-resolution mass spectrometry (UPLC-ESI-HRMS); and identifying unknown carbohydrate compounds and analyzing the content of known carbohydrates by utilizing a high-resolution mass spectrum scanning result. According to the method, the chloromethyl triphenylphosphonium chloride chloroaffinity reagent is added into the mobile phase, so that the detection sensitivity and the separation effect of the carbohydrates are remarkably improved, and the carbohydrates are analyzed with small sample amount, low interference and high sensitivity; meanwhile, the chlorine isotope labeled carbohydrates have the isotope distribution characteristic, and polysaccharide can generate double-charge molecular ion peaks, so that the mass spectrum detection mass range is remarkably increased, a new technology is provided for screening the high-quality coverage range of the carbohydrates in food, biological and environmental samples, and the method has 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] In order to solve at least one of the above technical problems, the purpose of the present invention is to propose a new method for high-sensitivity and high-throughput analysis of carbohydrate compounds based on chlorine-enhanced ionization. A quaternary phosphonium chloride or quaternary ammonium salt that can be dissolved in both the aqueous phase and the organic phase is added to the mobile phase, and a specific chlorine-enhanced ionization signal ([M+Cl]-) of the carbohydrate is formed in the electrospray ion source, which greatly improves the analytical sensitivity, and the mass spectrometry response signal is increased by 700 times compared with the LC-MS method reported to add chloroform or guanidine hydrochloride after the mobile phase column; further based on the characteristic isotope peak distribution characteristics of carbohydrate chlorine affinity products, and the characteristics that polysaccharide compounds with increased ionizable sites can obtain double-charged ions, thereby significantly increasing the mass range of mass spectrometry detection, a high-sensitivity and high-throughput scanning method based on the organic phosphonium chloride-enhanced ionization of intact carbohydrate molecules and liquid chromatography high-resolution mass spectrometry (UPLC-ESI-HRMS) combined analysis is established, which provides a new technology for targeted detection and non-targeted screening of carbohydrate compounds in food, biological and environmental samples.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A method for highly sensitive and high throughput identification of carbohydrate compounds based on chlorine enhanced ionization comprises the following steps:

[0007] Extracting sugar compounds in the sample to be tested to obtain an extract;

[0008] Select mobile phase additives and chromatographic columns, optimize mobile phase gradient and loading solution composition, and perform UPLC-ESI-HRMS scanning on the sugar compound extracts;

[0009] UPLC-ESI-HRMS scanning results were used for targeted detection of sugar compounds with standards and non-targeted identification of unknown sugars.

[0010] As a preferred embodiment, the extracted samples include but are not limited to food samples, biological samples or environmental samples.

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

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

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

[0014] Preferably, as a preferred embodiment, when the sample to be tested is a liquid sample, the amount of the sample to be tested is 10-2000 μL; when the sample to be tested is a solid sample, the amount of the sample to be tested is 0.1-100 mg.

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

[0016] As a preferred embodiment, the selection of the chlorine affinity reagent added to acetonitrile includes: selecting a carbohydrate chlorine affinity ([M+Cl] - ) The chlorine affinity reagent with the highest mass spectrometry sensitivity and its added concentration. The halogen-containing affinity reagent includes 8 quaternary phosphonium salts (tetraphenylphosphonium chloride, tetrabutylphosphonium chloride, methyltriphenylphosphonium chloride, ethyltriphenylphosphonium chloride, tetraphenylphosphonium bromide, chloromethyltriphenylphosphonium chloride, tetrabutylphosphonium bromide, methyltriphenylphosphonium bromide), 13 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, acryloyloxyethyldimethylbenzylammonium chloride, dimethylbenzyl-2-methylacrylic acid ethylamine chloride, benzethonium chloride and tetradecyldimethylbenzylammonium chloride hydrate) and 2 inorganic ammonium salts (ammonium chloride and ammonium bromide); the added concentration of the halogen-containing affinity reagent is selected to be 1 μM.

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

[0018] As a preferred embodiment, the targeted detection of sugar compounds in the sample to be tested includes the following steps: preparing a standard curve solution of the target sugar compound and measuring it by UPLC-ESI-HRMS. 13 C6), and the corresponding concentration linear regression analysis was performed on the peak area corrected by the internal standard to 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: the ion source is an ESI source, and the negative ion mode-full-MS mode is used for full scan, and the full scan mass range is 50-1500Da; the cone voltage is 30V; the desolvation temperature is 500°C; the capillary voltage is 2.0kV; the desolvation flow rate is 800L / Hr; and the source temperature is 100°C.

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

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

[0022] First, the mass spectrometry data was peak extracted to obtain the mass-to-charge ratio, retention time, and peak area of ​​the chemical signal. Then, non-targeted screening was performed based on the characteristic isotope peaks of the sugar chlorine affinity products, and the mass spectrometry signals of the sugar chlorine affinity product mass spectrometry cluster peaks were screened and paired based on the accurate mass number and peak height ratio of the chlorine isotope peaks. Finally, the elemental composition of the screened sugar chlorine affinity product cluster peaks was analyzed and compared 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: low energy intensity threshold 200.0, mass-to-charge ratio tolerance 3mDa; lock mass number 554.2620; maximum considered charge in 3D isotope cluster is 2, maximum number of isotopes in each cluster is 1, and minimum allowed single isotope / maximum isotope intensity ratio is 0.1. The parameters for mass spectrometry signal screening and pairing are as follows: the absolute value of the retention time difference of the ion pair is 0.02min; the absolute value of the mass-to-charge ratio difference of the ion pair is between 1.9921 and 2.0021Da, or between 0.996 and 1.001. The parameters for elemental composition analysis are as follows: 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 10ppm.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention can significantly enhance the mass spectrometry ionization signal intensity of carbohydrate compounds by simply adding quaternary phosphonium chloride and quaternary ammonium chloride affinity reagents before the liquid chromatography mobile phase column, with the detection sensitivity as low as 3.4 pg and the enhancement factor as high as 700 times, which can greatly reduce the amount of sample used.

[0026] (2) The present invention adds quaternary phosphonium chloride and quaternary ammonium salt to the mobile phase to form a chlorine affinity, which enhances the chromatographic retention behavior of the sugar compound in the amido normal phase chromatography column and can significantly improve the ability to separate the isomers and optical isomers of the sugar compound.

[0027] (3) The present invention significantly increases the mass spectrometry detection mass range by scanning polysaccharide compounds to generate double-charged molecular ion peaks, thereby improving the mass coverage of non-targeted screening from monosaccharides to polysaccharide compounds. In the implementation case based on this method, targeted detection of [M+Cl] - 10 monosaccharides, oligosaccharides and sugar alcohols with molecular ion peaks; non-targeted screening and identification of [M+2Cl] 2- The molecular ion peaks were polysaccharide compounds (nona to tridecanes [molecular weight over 2200 Da]), and unsaturated sugars (tri to hexasaccharides, with an unsaturation degree of 1) were also found. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0029] Figure 1The effect 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 spectrometric response intensity of sugar compounds (taking monosaccharide D-glucose and disaccharide maltose as examples);

[0030] Figure 2 The molecular ion mass spectra of some target sugar compounds analyzed by UPLC-ESI-HRMS after adding chlorine affinity reagent to the mobile phase are shown;

[0031] Figure 3 The chromatogram of UPLC-ESI-HRMS simultaneous analysis of 4 monosaccharides, 10 oligosaccharides and 7 derivatives (4 sugar alcohols and 3 cyclodextrins) standard solutions after adding chlorine affinity reagent to the mobile phase is shown, among which the reducing sugar compounds show double peaks due to the α-type and β-type terminal group diastereomers;

[0032] Figure 4 Shown are the chromatograms and mass spectra of newly identified sugar compounds in beer samples by non-targeted UPLC-ESI-HRMS scanning after adding chlorine affinity reagent to the mobile phase. DETAILED DESCRIPTION

[0033] In the following description, many specific details are set forth 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 to 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 sugar compounds in beer samples as an example, but this example does not constitute any limitation to the present invention.

[0035] 1. Instruments and reagents:

[0036] 1. Instrument

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

[0038] 2. Reagents

[0039] Chloromethyltriphenylphosphine 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) with a purity greater than 97%.

[0040] 2. Embodiment 1

[0041] A method for targeted detection and non-targeted screening of sugar 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 into a 1.5 mL centrifuge tube, and 1 mL of ice-cold methanol and 5 μg of glucose were added. 13 C6 internal standard, vortex, centrifuge at 10000rpm for 5min at 4℃, and transfer the supernatant to a brown glass bottle. Add 1mL of ice methanol to the precipitate, vortex, centrifuge and transfer the supernatant; continue to add 1mL of ice water to the precipitate, vortex, centrifuge and transfer the supernatant; finally, add 1mL of frozen acetonitrile to the precipitate, vortex, centrifuge and transfer the supernatant, and repeat the above acetonitrile extraction step once. All supernatants were collected in the same brown glass bottle to obtain the carbohydrate compound extract, which was stored at 4℃ for UPLC-ESI-HRMS analysis.

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

[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 chloromethyltriphenylphosphine 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 effect 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 spectrometric response intensity of carbohydrate compounds (taking monosaccharide D-glucose and disaccharide maltose as examples) is shown in Figure 2. Figure 1 shown.

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

[0047] Standard mixed solutions of 21 oligosaccharides and their derivatives (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, and lactitol) were prepared, with concentration levels of 0.01 mg / L, 0.05 mg / L, 0.1 mg / L, 0.5 mg / L, 1 mg / L, and 5 mg / L. UPLC-ESI-HRMS analysis was performed with a 5 μL injection volume, and linear regression analysis was performed on the peak area and concentration of each substance to obtain a standard curve. The results showed that the peak area and concentration ratio of the standard substances within this concentration range showed a good linear relationship, and the correlation coefficients were all greater than 0.99.

[0048] The instrument detection limit is the concentration level of each sugar compound calculated when the signal-to-noise ratio (S / N) of the standard chromatographic peak is 3. The recovery rate is calculated by adding the target sugar standard mixed solution to the matrix sample, testing according to the sample pretreatment and instrumental analysis method, comparing the chromatographic peak area of ​​the spiked concentration level standard with the actual peak area measured in the spiked sample, and the specific results are listed in Table 1.

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

[0050]

[0051]

[0052] a Reducing sugar compounds have α-type and β-type end group diastereomers

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

[0054] The retention time and mass number of the carbohydrate organic matter cluster peak detected in the beer sample were compared with those of the target standard sample. As a result, three monosaccharides (D-glucose, D-(+)-galactose, and fructose), seven oligosaccharides (maltose, raffinose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, and maltooctaose) and two sugar alcohols (maltitol and lactitol) were detected in the beer sample.

[0055] Further, according to the internal standard method, the concentration levels of target sugar compounds in beer samples (n = 3) were calculated based on the peak areas corrected by the internal standard, which were: 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, and raffinose 2850.3 ± 22. 3mg / L, maltotetraose 3128.4±27.4mg / L, maltopentaose 2521.9±18.3mg / L, maltohexaose 4790.1±30.5mg / L, maltoheptaose 4473.8±40.4mg / L, maltooctaose 4155.0±6.6mg / L, maltitol 72.1±2.1mg / L and lactitol 69.6±0.4mg / L.

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

[0057] The mass spectrometry data of the beer samples were extracted using UNIFI software to obtain the mass-to-charge ratio, retention time, and intensity of the chemical signals. The elemental composition analysis was performed on the chemical signals of the molecular ion peaks of the unknown sugar compounds that did not match the standard substances. The results screened out 2 disaccharides, 3 trisaccharides, 2 tetrasaccharides, and 2 sugar alcohols that were different from the targeted sugars detected by the present invention; and [M+2Cl] 2- Five polysaccharide compounds (nona to tridecanes [molecular weight up to nearly 2125 Da]) were successfully identified for the molecular ion peaks, and the presence of four unsaturated sugars (tri to hexasaccharides, with an unsaturation degree of 1) were also found.

[0058] Table 2 Sugar compounds identified by high-sensitivity 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 detected accurately and targeted. Based on the chlorine isotope distribution characteristics and doubly charged molecular ion peaks, the mass spectrometry mass search range was increased to non-targetedly identify 18 sugar compounds, including 5 polysaccharides and 4 unsaturated oligosaccharides. This study provides an effective method for high-sensitivity and high-throughput scanning analysis of sugar compounds with a high coverage range.

[0063] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention.

Claims

1. A method for highly sensitive and high throughput identification of carbohydrate compounds based on chlorine enhanced ionization, characterized in that: The steps include: Extracting sugar compounds in the sample to be tested to obtain an extract; Select mobile phase additives and chromatographic columns, optimize mobile phase gradient and loading solution composition, and subject the sugar compound extract to UPLC-ESI-HRMS detection; Utilize UPLC-ESI-HRMS scanning results for targeted detection of sugar compounds with standards and non-targeted identification of unknown sugar compounds; The extracted samples include but are not limited to food samples, biological samples or environmental samples; The UPLC-ESI-HRMS detection method includes the following steps: using HRMS equipped with an electrospray ion source to separate sugar compounds by normal phase chromatography, using acetonitrile and water as the chromatographic mobile phase, adding a chlorine affinity reagent to the acetonitrile, and scanning in negative ion-Full-MS mode.

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

3. The method for highly sensitive and high-throughput identification of carbohydrate compounds based on chlorine enhanced ionization according to claim 1, characterized in that: When the sample to be tested is a liquid sample, the amount of the sample to be tested is 10-2000 μL; when the sample to be tested is a solid sample, the amount of the sample to be tested is 0.1-100 mg.

4. The method for highly sensitive and high-throughput identification of carbohydrate compounds based on chlorine enhanced ionization according to claim 1, characterized in that: The mass spectrometry detection conditions are as follows: the ion source is an ESI source, and the full-MS mode is used for full scan, and the full scan mass range is 50-1500Da; the cone voltage is 30V; the desolvation temperature is 500°C; the capillary voltage is 2.0kV; the desolvation flow rate is 800L / Hr; and the source temperature is 100°C. The chromatographic separation conditions are as follows: the chromatographic column is Waters ACQUITY UPLC BEH Amide Column, 2.1 mm×100 mm, 1.7 μm; the column temperature is 40° C.; the mobile phase is acetonitrile added with 1 μM chloromethyltriphenylphosphine chloride and pure water without additives, 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; 40-40.1min, 60%-50%A; 40.1-42min, 50%A; 42-42.1min, 50%-95%A; 42.1-43min, 95%A.

5. The method for highly sensitive and high-throughput identification of carbohydrate compounds based on chlorine enhanced ionization according to claim 1, characterized in that: The selection of the chlorine affinity reagent added to acetonitrile includes: selecting the chlorine affinity reagent with the highest mass spectrometric sensitivity of the sugar chlorine affinity ([M+Cl]-) and the addition concentration according to the organic phase / aqueous phase solubility of the chlorine affinity reagent and its ability to react with the sugar compound by affinity. The halogen-containing affinity reagent comprises 8 quaternary phosphonium salts (tetraphenylphosphonium chloride, tetrabutylphosphonium chloride, methyltriphenylphosphonium chloride, ethyltriphenylphosphonium chloride, tetraphenylphosphonium bromide, chloromethyltriphenylphosphonium chloride, tetrabutylphosphonium bromide, methyltriphenylphosphonium bromide), 13 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, acryloyloxyethyldimethylbenzylammonium chloride, dimethylbenzyl-2-methylacrylic acid ethylamine chloride, benzethonium chloride and tetradecyldimethylbenzylammonium chloride hydrate) and 2 inorganic ammonium salts (ammonium chloride and ammonium bromide); the addition concentration of the halogen-containing affinity reagent is selected to be 1 μM.

6. The method for highly sensitive and high-throughput identification of carbohydrate compounds based on chlorine enhanced ionization according to claim 1, characterized in that: The optimization of the loading solution composition comprises the following steps: according to the chromatographic peak solvent effect caused by the loading solution affecting the chromatographic separation effect of sugar compound isomers and optical isomers and the mass spectrometry response intensity, the volume ratio of acetonitrile, methanol and water in the loading solution is optimized to 2:2:

1.

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

8. The method for highly sensitive and high-throughput identification of carbohydrate compounds based on chlorine enhanced ionization according to claim 1, characterized in that: The non-targeted identification of unknown carbohydrate compounds comprises: First, the mass spectrometry data is peak extracted to obtain the mass-to-charge ratio, retention time, and peak area of ​​the chemical signal. Then, non-targeted screening is performed based on the characteristic isotope peaks of chlorine-labeled sugar compounds, the extracted mass spectrometry signals are mass aligned, and the mass spectrometry signals of the chlorine-labeled organic material cluster peaks are screened and paired in combination with the accurate mass number and peak height ratio of the chlorine isotope peaks; finally, the elemental composition of the screened chlorine-labeled sugar compounds is analyzed, and the isotope peak theoretical simulation is performed on the determined chemical formula, and compared with the actual mass spectrometry signal to verify the accuracy of the elemental composition analysis.

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