CN114062539B - Method for quantitatively detecting glucan in biological sample - Google Patents

Method for quantitatively detecting glucan in biological sample Download PDF

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CN114062539B
CN114062539B CN202111304973.0A CN202111304973A CN114062539B CN 114062539 B CN114062539 B CN 114062539B CN 202111304973 A CN202111304973 A CN 202111304973A CN 114062539 B CN114062539 B CN 114062539B
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dextran
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顾景凯
任天明
张瑀峣
徐新
宋诗文
孙冬
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Zhongrong Kaite Beijing Biotechnology Co ltd
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Abstract

The invention discloses a method for quantitatively detecting glucan in a biological sample, which is characterized in that after the biological sample is pretreated, a liquid chromatogram-triple quadrupole tandem mass spectrometer is used for realizing the separation and quantitative analysis of glucan components of a substance to be detected; the mass spectrum acquisition mode is a mode combining pyrolysis in an ion source and multi-reaction monitoring; quantitative analysis of dextran in biological samples was performed using the specific fragment ion m/z 649.3 from the lysis in the dextran source and the characteristic oligosaccharide ion fragment ion m/z325.1 from the collision induced lysis of this fragment ion. The method provided by the invention is suitable for quantitative analysis of glucan in a complex biological matrix.

Description

Method for quantitatively detecting glucan in biological sample
Technical Field
The invention belongs to the technical field of drug analysis research, and relates to a biological mass spectrometry method for analyzing glucan.
Background
Dextran is macromolecular polymer composed of glucose as monosaccharide, has wide bioactivity, and has molecular formula of HO [ C ] 6 H 10 O 5 ] n H. In recent years, researches show that the beta-glucan has certain effects of resisting tumor growth and inhibiting tumor metastasis. The current research mainly focuses on the aspects of separation and purification, characteristic identification, content determination, structural analysis, pharmacological activity and the like, and no effective method is available for quantitative analysis of glucan in a biological sample. Whether a colorimetric method, a fluorescence labeling method, an isotope labeling method, an enzymolysis and LC-MS/MS combined method and the like exist, the endogenous interference in a complex biological matrix is strong, and the lower limit of the quantification is highHigh and the limitation of not being able to achieve absolute quantitative analysis of the complete profile for dextran. Therefore, there is a need to establish an absolute quantitative analysis method of polydisperse glucan in a biological sample with good selectivity, high sensitivity and good accuracy.
In recent years, rapidly developing LC-MS/MS techniques have provided the possibility of analysis of dextran in biological samples. Compared with the traditional immunology, HPLC, colorimetric method, fluorescent labeling and the like, the LC-MS/MS method has great advantages in the aspects of accuracy, precision, selectivity, sensitivity, quantitative dynamic range and the like. However, the current LC-MS/MS analysis techniques for dextran are still relatively rare, mainly due to the following, in addition to the common problems mentioned above: dextran is a polymer, the molecular weight of dextran is polydisperse, precursor ions without complex multiple charges can be generated in an ion source, and a traditional LC-MS/MS quantitative analysis scanning mode based on a Multiple Reaction Monitoring (MRM) scanning mode is limited by a scanning speed of a mass spectrum quadrupole rod, and only limited and molecular weight-determined target compounds can be quantitatively analyzed, so that the complete profile absolute quantitative analysis of the polydisperse dextran has great challenges at present.
Disclosure of Invention
The invention aims to solve the technical problem of establishing an absolute quantitative analysis method of the polydisperse glucan in the biological sample with good selectivity, high sensitivity and good accuracy, and solving the technical problem that parent ions and daughter ions of the glucan for quantification are difficult to determine. The aim of the invention is realized by the following technical scheme:
a method for quantitatively detecting glucan in a biological sample uses a triple quadrupole tandem mass spectrometer for quantitative analysis and detection, and adopts a method of in-source cleavage combined with multi-reaction monitoring to realize the quantitative analysis and detection of the glucan in the biological sample. The invention effectively separates endogenous substances in a glucan biological sample by using chromatography, and then carries out mass spectrum online quantitative analysis on the endogenous substances. The specific mass spectrometry flow is as follows: 1) The mass spectrum source internal cracking converts the precursor ions with multiple types and wide mass distribution and without counting into characteristic fragment ions with a finite unit number of refractive index, namely realizes dimensionality reduction treatment on the precursor ions which are complicated in numerous ways; 2) The fragment ions with common characteristics generated by the inner cracking of the glucan source with high and stable signal response are selected for MRM quantitative analysis, and the ion pairs used for quantitative reaction are as follows: m/z 649.3 (four anhydroglucose units) → m/z325.1 (two anhydroglucose units). The invention first utilizes mass spectrometry in-source fragmentation (giving high declustering voltage, i.e. DP) to perform data-independent fragmentation of all adducted ions of dextran in biological samples, which was found to produce the characteristic tetrasaccharide fragment ion m/z 649.3. Then, MRM quantitative analysis is carried out on the glucan, namely Q1 selects m/z 649.3, Q2 carries out collision induced fragmentation dissociation (CID) on the glucan, Q3 selects the characteristic fragment ion m/z325.1 (disaccharide) of m/z 649.3, and m/z 649.3 and 325.1 are taken as quantitative ion pairs, so that absolute quantitative analysis of the glucan with polydisperse molecular weight is realized for the first time. A schematic of in-source cleavage in combination with multiple reaction monitoring is shown in FIG. 1.
Furthermore, a ScextTRIPLEQUAD 6500plus type tandem mass spectrometer is selected for the mass spectrum, an ESI ionization source and a positive ion detection mode are adopted, and the MRM mass spectrum can effectively eliminate the interference of endogenous matrixes.
The optimized de-clustering voltage is 300V, so that the polydisperse glucan in the ion source can be effectively fragmented, the number of parent ions is reduced, the complex is simplified, and the characteristic ions m/z 649.3 with strong specificity and high abundance are generated. The optimized post-impact energy is 20eV.
Preferred mass spectrometry conditions are: the ion jet voltage is 4500 +/-500V; the temperature is 400 +/-50 ℃; source gas 1: nitrogen pressure 30 + -5 psi; gas 2: nitrogen pressure 20 + -5 psi; gas curtain gas: nitrogen pressure 20 + -5 psi; cluster-splitting voltage: 300 +/-50V; the collision energy is 20 + -5 eV.
Further, liquid chromatography conditions: an ultra-high performance liquid chromatography system; a normal phase chromatographic column; acidic acetonitrile/water mobile phase; gradient elution; the ultra-high performance liquid chromatography system and the acidic mobile phase can effectively retain glucan.
Preferred chromatographic conditions are: ACCHROM XAmide column (
Figure BDA0003339775200000021
4.6X 150mm, I.D5 μm); the flow rate is 1mL/min; the column temperature is 40 +/-10 ℃; the sample injection amount is 2-10 mu L; the running time per needle was 15. + -.5 min.
The method has good linear relation in the concentration range of 0.5-100 mug/mL, and the LLOQ is 0.5 mug/mL.
The pretreatment mainly comprises the step of extracting glucan from a biological sample by adopting a protein precipitation method, and the pretreatment methods of different types of complex biological samples are slightly different, wherein the biological samples comprise but are not limited to plasma, urine, bile, feces and tissues. When the biological sample is plasma, the pretreatment is as follows: precipitating protein from blood plasma obtained by anticoagulant treatment, centrifuging, and taking supernatant for dilution; when the biological sample is feces, the pretreatment is as follows: freeze drying, pulverizing, re-dissolving, vortex centrifuging, collecting supernatant, precipitating protein, centrifuging, and diluting supernatant; when the biological sample is urine or bile, the pretreatment is as follows: precipitating protein, centrifuging, and taking supernatant for dilution; when the biological sample is a tissue, the pretreatment is as follows: after the tissue is subjected to enzymolysis, protein precipitation and centrifugal dilution steps are carried out.
Furthermore, because the glucan is a substance with large polarity and can be tightly combined with the protein in the biological sample, the precipitation effect is greatly influenced by the type and pH of the precipitation reagent selected for precipitating the protein, and through optimization and investigation, the invention determines that methanol (containing 0.03% formic acid) is the optimal extraction reagent of the glucan.
Furthermore, in the process of collecting the plasma samples (rats and dogs), because the commonly used anticoagulant heparin is polysaccharide and is exogenous interference for the substance to be detected, the sodium citrate is selected as the anticoagulant to avoid interference.
Furthermore, the matrix of the tissue sample is very complex, the endogenous interference is very strong, the pretreatment method of glucan in the tissue is optimized, and the interference is reduced by adopting an enzymolysis method before protein precipitation.
Further, the double-enzyme combination of amylase and saccharifying enzyme can effectively cut off alpha glycosidic bonds, so that the interference of complex matrix is reduced. The method comprises the following specific steps: the tissue is firstly enzymolyzed by amylase of 100ug/mL, is shaken in water bath at 40 ℃ for 30min, is adjusted to pH 4.5 +/-1 by acetic acid, is enzymolyzed by carbohydrase, is shaken in water bath at 60 ℃ for 30min, and then is subjected to protein precipitation, centrifugal dilution and other steps.
And further, measuring glucan in the biological sample, comprising the steps of sample pretreatment and standard curve preparation before measurement, taking the supernatant subjected to pretreatment, performing source-internal lysis and multi-reaction combined monitoring analysis, and recording a chromatogram. Substituting the peak area of the glucan analyte into the standard curve to obtain the concentration of the glucan.
The invention principle is as follows:
the magnitude of the declustering voltage (DP) in the ion source can affect the species and distribution of small molecule target analyte ions, the higher the DP of the cone hole, the faster the ion velocity, the smaller the loss, and the higher the detection sensitivity, while too high DP can increase the collision between ions, causing in-source fragmentation, and further reducing the sensitivity of the target analyte. However, for glucans having a repeating structural unit composition, the idea of the invention is: if the characteristic of DP can be utilized, the countless precursor ions with various types and wide mass distribution are converted into characteristic fragment ions with finite unit number of exponential characteristic, that is, the complex precursor ions are subjected to dimensionality reduction treatment. Then, a multi-reaction monitoring scanning (MRM) mode of tandem mass spectrometry is combined, common characteristic fragment ions generated by the glucan source internal cracking with high signal response and stability are selected, then MRM analysis is carried out, and the technical bottleneck of quantitative glucan analysis can be broken through. Repeated exploratory innovation experiments show that by adopting the technical idea of the invention, the absolute quantitative analysis of the complete profile of the polydisperse glucan in the biological sample is successfully realized by using an in-source lysis-combined MRM (ion pair for quantitative reaction: m/z 649.3 → m/z 325.1) scanning mode.
The invention has the beneficial effects that:
the invention provides a glucan analysis method based on in-source fragmentation combined with collision induced fragmentation for the first time, which breaks polydisperse glucan into specific fragment ions with lower molecular weight and stable signal intensity, and quantitatively analyzes the glucan by taking m/z 649.3 and m/z325.1 as parent ions and child ions for the first time, thereby effectively breaking through the technical bottleneck that MRM parent ions and child ions are not easy to determine during polydisperse glucan mass spectrometry. The method provided by the invention is suitable for quantitative analysis of glucan in a complex biological matrix.
Drawings
FIG. 1 is a schematic illustration of in-source cleavage coupled with multiple reaction monitoring;
FIG. 2 shows the fragmentation of dextran under in-source fragmentation (300V declustering voltage);
FIG. 3 is a typical chromatogram of different ion channels of dextran;
FIG. 4 is a typical chromatogram of dextran and internal standard obtained in example 1 (dextran for I, internal standard for m/z 649.3 → 325.1, m/z 425.2 → 204.2;
FIG. 5 is a time curve of plasma concentration of dextran administered to the tail vein of rats obtained in example 1;
FIG. 6 is a time curve of plasma concentration of the beagle dog obtained in example 3 after intravenous administration of dextran to hind limb.
Detailed Description
Example 1
Accurately weighing about 300mg of glucan standard, dissolving the glucan standard by using sodium chloride solution for injection after the purity is converted, diluting the glucan standard into 400 mu g/mL, and administrating the glucan standard into the tail vein of a rat, wherein the blood sampling time points are as follows: 1min, 3min, 5min, 10min, 15min, 20min, 30min, 40min, 50min, 60min, 80min. Plasma glucan content was measured after intravenous administration to rat tail and the time curve of plasma drug concentration is shown in figure 5.
The method mainly comprises the following steps:
A. pretreatment of a plasma sample:
1) 25 μ L of the biological sample was added to a polyethylene tube,
2) Adding 15 μ L of internal standard, mixing by vortex,
3) Protein precipitation: adding 75 μ L methanol containing 0.03% formic acid, mixing by vortex,
4) Centrifugation is carried out at 13000rpm for 5 minutes,
5) Transferring 50 mu L of centrifuged supernatant into a 2mL EP tube containing 50 mu L of acetonitrile-water solution (1/1,v/v), and mixing uniformly by vortex;
B. preparation of a standard curve:
1) Diluting the dextran stock solution to 0.5, 1.0, 3.0, 10.0, 30.0, 50.0 and 100.0 mu g/mL respectively by using acetonitrile-water (1/1,v/v) solution;
2) Preparing an internal standard working solution: the internal standard N-acetylated chitobiose was diluted to 1.00. Mu.g/mL using acetonitrile-water solution (1/1,v/v).
3) Taking 10 μ l for liquid chromatography-tandem mass spectrometry, recording chromatogram, taking dextran concentration as abscissa and dextran and internal standard peak area ratio as ordinate, and using weight W =1/x 2 The least square method is used for regression operation, the linear regression equation is obtained, the linear range of the working curve for determining the dextran concentration in the rat plasma is 0.5-100 mug/mL, and the LLOQ is 0.5 mug/mL.
C. Preparation of quality control samples:
the quality control samples with low, medium and high concentrations (1.4, 40.0, 80.0 μ g/mL) were prepared by operating under the "preparation of standard curve", and the concentration was obtained from the standard curve for at least three samples per concentration. The accuracy of the quality control samples was calculated, see table 1 for details, and the method accuracy was investigated.
TABLE 1 dextran BioMass Spectrometry Absolute quantitation method quality control sample accuracy (rat plasma)
Figure BDA0003339775200000051
The conditions involved in the absolute quantitation method of dextran biological mass spectrometry in the above steps are as follows:
the chromatographic conditions are as follows: and (3) chromatographic column: ACCHRROM XAmide (
Figure BDA0003339775200000052
4.6X 150mm, I.D5 μm) and a flow rate of 1mL/min. Mobile phase A:0.1% aqueous formic acid, mobile phase B: acetonitrile, needle wash: methanol-water solution (1/1,v/v). The column temperature is 40 deg.C, the sample injection amount is 10 μ L, and the total running time is 15min
The gradient elution in the chromatographic conditions described, the procedure is shown in Table 2,
TABLE 2 gradient elution procedure
Figure BDA0003339775200000053
Figure BDA0003339775200000061
The mass spectrum conditions are as follows: the SciextripleEQUAD 6500plus type triple quadrupole tandem mass spectrometer is provided with an ESI ionization source and analysis data processing software; ion source parameters: detecting in a positive ion mode; ion spray voltage 5000V; the temperature is 400 ℃; atomizing: nitrogen pressure 35psi; heating gas: nitrogen pressure 20psi; gas curtain gas: nitrogen pressure 20psi; cluster-splitting voltage: 300V; fragmentation energy: 20eV.
In view of the linearity of the typical standard curve of dextran and the accuracy of the quality control sample (table 1), the analytical method for quantitatively detecting dextran in biological samples has good linear relation (0.5-100.0 mug/mL), high accuracy, good reproducibility, sensitivity and reliability, and can be used for absolute quantification of dextran in rat plasma.
Example 2
After the rat is administrated with 3mg/kg by single intravenous injection, rat kidney samples are collected at 5min, 30min and 80min respectively, and the distribution concentration of the seaweed-beta-glucan in the kidney is determined.
The method mainly comprises the following steps:
A. pretreatment of kidney tissue samples:
1) Tissue enzymolysis: subjecting kidney tissue to enzymolysis with 100ug/mL amylase, shaking in water bath at 40 deg.C for 30min, adjusting pH to 4.5 + -1 with acetic acid, performing enzymolysis with saccharifying enzyme, shaking in water bath at 60 deg.C for 30min,
2) Centrifuging to obtain supernatant, adding 25 μ L into polyethylene tube,
3) Adding 15 μ L of internal standard, mixing by vortex,
4) Protein precipitation: adding 75 μ L of methanol containing 0.03% formic acid, mixing by vortex
5) Is centrifuged at 13000rpm for 5 minutes,
6) Transferring 50 mu L of centrifuged supernatant into a 2mL EP tube containing 50 mu L of acetonitrile-water solution (1/1,v/v), and mixing uniformly by vortex;
B. preparation of a standard curve:
1) Diluting the dextran stock solution to 1.0, 3.0, 5.0, 10.0, 30.0, 50.0 and 100.0 mu g/mL respectively by using acetonitrile-water (1/1,v/v) solution;
2) Preparing an internal standard working solution: the internal standard xylotetraose was diluted to 10.00. Mu.g/mL of internal standard working solution using acetonitrile-water solution (1/1,v/v).
3) Taking 10 μ l for liquid chromatography-tandem mass spectrometry, recording chromatogram, taking dextran concentration as abscissa and dextran and internal standard peak area ratio as ordinate, and using weight W =1/x 2 The least square method is used for carrying out regression operation to obtain a linear regression equation, the linear range of a working curve for determining the glucan concentration in the beagle plasma is 1.0-100.0 mu g/mL, and the LLOQ is 1.0 mu g/mL.
C. Quality control sample preparation
The quality control samples with low, medium and high concentrations (1.4, 40.0, 80.0 μ g/mL) were prepared by operating under the "preparation of standard curve", and the concentration was obtained from the standard curve for at least three samples per concentration. The accuracy of the quality control samples was calculated, see table 3 for details, and the method accuracy was investigated.
TABLE 3 Absolute quantitation of dextran Biometrics Mass Spectrometry quality control sample accuracy (rat Kidney)
Figure BDA0003339775200000071
The conditions involved in the absolute quantitation method of dextran biological mass spectrometry in the above steps are as follows:
the chromatographic conditions are as follows: a chromatographic column: ACCHRROM XAmide (
Figure BDA0003339775200000072
4.6X 150mm, I.D5 μm) and a flow rate of 1mL/min. Mobile phase A:0.1% aqueous formic acid, mobile phase B: acetonitrile, needle wash: methanol-water solution (1/1,v/v). The column temperature was 40 ℃ and the sample size was 10. Mu.LThe total run time was 15min and the gradient elution in the chromatographic conditions described, the procedure is as in table 2.
The mass spectrum conditions are as follows: the SciextripleEQUAD 6500plus type triple quadrupole tandem mass spectrometer is provided with an ESI ionization source and analysis data processing software; ion source parameters: detecting in a positive ion mode; ion spray voltage 5000V; the temperature is 400 ℃; atomizing: nitrogen pressure 35psi; heating gas: nitrogen pressure 20psi; gas curtain gas: nitrogen pressure 20psi; DP:300V; CE:20eV;
in view of the linearity of the typical standard curve of dextran and the accuracy of the quality control sample (table 3), the analytical method for quantitatively detecting dextran in biological samples has good linear relation (1.0-100.0 mug/mL), high accuracy, good reproducibility and high sensitivity, and can be used for absolute quantification of dextran in rat kidney tissues.
Example 3
Accurately weighing about 300mg of glucan standard substance, dissolving the glucan standard substance by using sodium chloride solution for injection after the purity is converted, diluting the glucan standard substance to 12mg/mL, weighing the beagle dog, then administrating the glucan standard substance by hind limb intravenous injection according to the dosage of 1mg/kg, and freely eating and drinking water after the administration. The blood sampling time points were as follows: 1min, 3min, 5min, 10min, 15min, 20min, 30min, 40min, 50min, 60min, 80min. The plasma glucan content after beagle administration was determined and the plasma drug concentration time curve is shown in fig. 6.
The method mainly comprises the following steps:
A. pretreatment of a plasma sample:
1) 25 μ L of the biological sample was added to a polyethylene tube,
2) Adding 15 μ L of internal standard, mixing by vortex,
3) Precipitating protein: adding 75 μ L methanol containing 0.03% formic acid, mixing by vortex
4) Centrifugation is carried out at 13000rpm for 5 minutes,
5) Transferring 50 mu L of centrifuged supernatant into a 2mL EP tube containing 50 mu L of acetonitrile-water solution (1/1,v/v), and mixing uniformly by vortex;
B. preparation of a standard curve:
1) Diluting the dextran stock solution to 0.5, 1.0, 3.0, 5.0, 10.0, 30.0 and 50.0 mu g/mL by using acetonitrile-water (1/1,v/v) solution;
2) Preparing an internal standard working solution: the internal standard N-acetylated chitobiose was diluted to 1.00. Mu.g/mL of internal standard working solution using acetonitrile-water solution (1/1,v/v).
3) Taking 10 μ l for liquid chromatography-tandem mass spectrometry, recording chromatogram, taking dextran concentration as abscissa and dextran and internal standard peak area ratio as ordinate, and using weight W =1/x 2 The least square method is used for carrying out regression operation to obtain a linear regression equation, the linear range of a working curve for determining the glucan concentration in the beagle dog plasma is 0.5-50 mu g/mL, and the LLOQ is 0.5 mu g/mL.
C. Quality control sample preparation
The operation is carried out under the item of 'standard curve preparation', low, medium and high concentration (1.4, 15.0 and 40.0 mu g/mL) quality control samples are prepared, at least three samples are obtained for each concentration, and the concentration is obtained according to the standard curve. The accuracy of the quality control samples was calculated, see table 4 for details, and the method accuracy was investigated.
TABLE 4 dextran BioMass Spectrometry Absolute quantitation method quality control sample accuracy (beagle plasma)
Figure BDA0003339775200000091
The conditions related to the absolute quantitative method of glucan biological mass spectrum in the steps are as follows:
the chromatographic conditions are as follows: a chromatographic column: ACCHRROM XAmide (
Figure BDA0003339775200000092
4.6X 150mm, I.D5 μm) and a flow rate of 1mL/min. Mobile phase A:0.1% aqueous formic acid, mobile phase B: acetonitrile, needle wash: methanol-water solution (1/1,v/v). The column temperature was 40 ℃, the sample size was 10 μ L, the total run time was 15min, and the gradient elution in the chromatographic conditions was performed according to the procedure in table 2.
The mass spectrum conditions are as follows: the Sciex TRIPLEQUAD 6500plus type triple quadrupole tandem mass spectrometer is provided with an ESI ionization source and analysis data processing software; ion source parameters: detecting in a positive ion mode; ion spray voltage 5000V; the temperature is 400 ℃; atomizing: nitrogen pressure 35psi; heating gas: nitrogen pressure 20psi; gas curtain gas: nitrogen pressure 20psi; DP:300V; CE:20eV;
in view of the linearity of the typical standard curve of dextran and the accuracy of the quality control sample (Table 4), the analytical method for quantitatively detecting dextran in biological samples has good linear relation (0.5-50.0 μ g/mL), high accuracy, good reproducibility and high sensitivity, and can be used for absolute quantification of plasma dextran of beagle dogs.
The above-mentioned embodiments are only part of the present invention, and do not cover the whole of the present invention, and on the basis of the above-mentioned embodiments and the attached drawings, those skilled in the art can obtain more embodiments without creative efforts, so that the embodiments obtained without creative efforts are all included in the protection scope of the present invention.

Claims (10)

1. A method for quantitatively detecting glucan in a biological sample, comprising: after the biological sample is pretreated, separating and quantitatively analyzing glucan components of the object to be detected by using a liquid chromatogram-triple quadrupole tandem mass spectrometer; liquid chromatography conditions: an ultra-high performance liquid chromatography system; a XAmide chromatography column; acidic acetonitrile/water mobile phase; gradient elution; the mass spectrum acquisition mode is a mode combining pyrolysis in an ion source and multi-reaction monitoring; utilizing specific fragment ions generated by in-vivo cleavage of a dextran sourcem/z649.3 and fragment ions of oligosaccharide ions characteristic of collision-induced fragmentation of said fragment ionsm/z325.1 performing a quantitative analysis of dextran in the biological sample; an ESI ionization source; positive ion detection mode.
2. The method of claim 1, wherein the dextran is quantitatively detected in the biological sample by: mass spectrum conditions: sciex TRIPLEQUAD 6500plus type tandem mass spectrometer.
3. The method of claim 2, wherein the dextran is quantitatively detected in the biological sample by: chromatographic conditions are as follows: an acchmrom XAmide chromatography column; the flow rate is 1mL/min; the column temperature is 40 +/-10 ℃; the sample injection amount is 2-10 mu L; the operation time of each needle is 15 +/-5 min; mass spectrum conditions: ion spray voltage 4500 ± 500V; the temperature is 400 +/-50 ℃; source gas 1: nitrogen pressure 30 + -5 psi; gas 2: nitrogen pressure 20 + -5 psi; gas curtain gas: nitrogen pressure 20 + -5 psi; cluster-splitting voltage: 300 +/-50V; impact energy 20 ± 5eV.
4. The method of claim 3, wherein the dextran is quantitatively detected in the biological sample by: the method has the detection concentration range of 0.5-100 mug/mL and the LLOQ of 0.5 mug/mL.
5. The method of claim 1, wherein the dextran is quantitatively detected in the biological sample by: the biological sample is any one of plasma, urine, bile, feces and tissue samples.
6. The method of claim 5, wherein the dextran is quantitatively detected in the biological sample by: when the biological sample is plasma, the pretreatment is as follows: precipitating protein from blood plasma obtained by anticoagulant treatment, centrifuging, and taking supernatant for dilution; when the biological sample is feces, the pretreatment is as follows: freeze drying, pulverizing, re-dissolving, vortex centrifuging, collecting supernatant, precipitating protein, centrifuging, and diluting supernatant; when the biological sample is urine or bile, the pretreatment is as follows: precipitating protein, centrifuging, and taking supernatant for dilution; when the biological sample is a tissue, the pretreatment is as follows: after the tissue is subjected to enzymolysis, protein precipitation and centrifugal dilution steps are carried out.
7. The method of claim 6, wherein the dextran is quantitatively detected in the biological sample by: when the biological sample is plasma, the anticoagulant used is sodium citrate.
8. The method of claim 6, wherein the dextran is quantitatively detected in the biological sample by: the precipitated protein reagent was a methanol solution containing 0.03% formic acid.
9. The method of claim 6, wherein the dextran is quantitatively detected in the biological sample by: when the biological sample is tissue, the tissue enzymolysis adopts a double-enzyme combination of amylase and saccharifying enzyme.
10. The method of claim 9, wherein the dextran is quantitatively detected in the biological sample by: the tissue is firstly enzymolyzed by 100ug/mL amylase, is shaken in water bath at 40 ℃ for 30min, is adjusted to pH 4.5 +/-1 by acetic acid, is enzymolyzed by carbohydrase, and is shaken in water bath at 60 ℃ for 30 min.
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