CN113533588A - Method for simultaneously detecting nine lignin monomer cross-linked structures in wheat straw - Google Patents
Method for simultaneously detecting nine lignin monomer cross-linked structures in wheat straw Download PDFInfo
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- 239000000178 monomer Substances 0.000 title claims abstract description 36
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- 235000021307 Triticum Nutrition 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 29
- 239000010902 straw Substances 0.000 title claims abstract description 25
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 39
- -1 S (8-O-4) S (8-5) G Chemical compound 0.000 claims abstract description 13
- LCXGTSCVCJANHX-AATRIKPKSA-N buddlenol B Chemical compound O1C=2C(OC)=CC(\C=C\CO)=CC=2C(CO)C1C(C=C1OC)=CC(OC)=C1OC(CO)C(O)C1=CC=C(O)C(OC)=C1 LCXGTSCVCJANHX-AATRIKPKSA-N 0.000 claims abstract description 12
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- 238000010828 elution Methods 0.000 claims abstract description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 41
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- 239000012071 phase Substances 0.000 description 7
- LZFOPEXOUVTGJS-ONEGZZNKSA-N trans-sinapyl alcohol Chemical compound COC1=CC(\C=C\CO)=CC(OC)=C1O LZFOPEXOUVTGJS-ONEGZZNKSA-N 0.000 description 7
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- 239000003643 water by type Substances 0.000 description 3
- CDICDSOGTRCHMG-ONEGZZNKSA-N (E)-sinapaldehyde Chemical compound COC1=CC(\C=C\C=O)=CC(OC)=C1O CDICDSOGTRCHMG-ONEGZZNKSA-N 0.000 description 2
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- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
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- JXAZAUKOWVKTLO-UHFFFAOYSA-L sodium pyrosulfate Chemical compound [Na+].[Na+].[O-]S(=O)(=O)OS([O-])(=O)=O JXAZAUKOWVKTLO-UHFFFAOYSA-L 0.000 description 1
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- KCDXJAYRVLXPFO-UHFFFAOYSA-N syringaldehyde Chemical compound COC1=CC(C=O)=CC(OC)=C1O KCDXJAYRVLXPFO-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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Abstract
The invention discloses a method for simultaneously detecting nine lignin monomer cross-linked structures in wheat straws, wherein the lignin monomer cross-linked structure is S (8-O-4) S (8-8) S, S (8-O-4) G (8-O-4) S, G (8-O-4) S (8-5) G, S (8-O-4) S (8-5) G, G (8-O-4) S (8-8) S, G (8-O-4) G (8-5) G, S (8-O-4) G, G (8-8) G, G (8-5) H, UPLC-MS/MS is adopted, and the chromatographic conditions are as follows: mobile phase A: ultrapure water, mobile phase B: acetonitrile, gradient elution. The method has the advantages of strong specificity, high sensitivity, high flux and objective and easy analysis of results, can determine the change of the lignin monomer cross-linking structure in the growth and development of the wheat straw, and can provide guidance for the decomposition of the lignin of the wheat straw.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a detection method for simultaneously detecting nine lignin monomer cross-linked structures in wheat straws.
Background
In recent years, with the emission of greenhouse gases of fossil fuels, plant straws have become more and more important as an ethanol source. Ethanol produced from lignocellulosic biomass is referred to as second generation ethanol. One of the major obstacles to second generation ethanol production is the recalcitrant lignocellulosic materials. For example, wheat straw is composed primarily of cellulose, hemicellulose and lignin, and to separate them, it is important to determine the composition and content of the monolignol crosslinked structure in the straw, since this material must first be pretreated to extract and remove the lignin, and then the cellulose is hydrolyzed to release glucose, which is then fermented and converted to ethanol.
Lignin is a complex phenolic biopolymer and is difficult to break down. The biosynthesis of lignin is mainly based on the radical coupling of three hydroxyphenylalane monomers derived from phenylpropanol (p-hydroxyphenyllignin (H), guaiacyl lignin (G) and syringyl lignin (S)), by a series of oxidation reactions mediated by extracellular enzymes (peroxidases and laccases).
The 8-O-4 linkage is most common in monolignol cross-linked structures and is also more prone to cleavage than other chemical linkages. The relative proportion of monolignol determines the relative abundance of the linkages between the units. For example, G unit-rich lignin contains more recalcitrant 8-5, 5-5 and 5-O-4 bonds, while S unit-rich lignin has a lower degree of cross-linking and is less difficult to extract. Thus, the composition of lignin is generally described in terms of relative abundance and ratio of H, G and S units. Since the extractability and digestibility of ethanol produced from wheat straw are affected not only by the amount of lignin but also by the monomer composition and arrangement of monomers in plant cell walls, it is important to determine such information when studying how to produce ethanol from wheat straw.
Lignin content is usually measured by conventional gravimetric methods, most commonly by acid decomposition (based on separation of cell wall material) and spectrophotometric methods, such as bromoacetyl and mercaptoacetic acid, which degrade cell walls and lignin and determine soluble degradation products. However, the method of determining lignin in the same sample results in different estimates of lignin concentration. These methods also involve the content of insoluble lignin in the cell wall and may also involve the structure of lignin multimers, with very inaccurate results.
Disclosure of Invention
Based on the problems in the prior art, the invention establishes a simple method for polymerizing lignin in vitro by using three monomers and peroxidase in different proportions to simulate the natural polymerization process. The resulting compounds can be analyzed by Ultra Performance Liquid Chromatography (UPLC) in combination with tandem mass spectrometry (MS/MS). Soluble lignin found in plant tissues was then gently extracted without degradation and its structure and content determined by Ultra Performance Liquid Chromatography (UPLC) in combination with tandem mass spectrometry (MS/MS). The invention analyzes the soluble lignin oligomer of the wheat straw, more accurately determines the composition and the content of the lignin monomer cross-linked structure of the wheat straw, and provides scientific basis for preparing ethanol by the wheat straw.
One of the purposes of the invention is to provide a method for simultaneously detecting nine lignin monomer cross-linked structures in wheat straws, wherein the lignin monomer cross-linked structure is S (8-O-4) S (8-8) S, S (8-O-4) G (8-O-4) S, G (8-O-4) S (8-5) G, S (8-O-4) S (8-5) G, G (8-O-4) S (8-8) S, G (8-O-4) G (8-5) G, S (8-O-4) G, G (8-8) G, G (8-5) H, UPLC-MS/MS is adopted, and the chromatographic conditions are as follows: mobile phase A: ultrapure water, mobile phase B: acetonitrile, gradient elution (preferably: 0-0.5min, 95% A, 0.5-3min, 95% A-75% A linear decrease, 3-3.5min, 75% A-10% A linear decrease, 3-4.0min, 10% A, 4.0-4.1min, 10% A-95% A linear increase, 4.1-6.0min, 95% A).
Preferably: the chromatographic column is C18, 1.7 μm,2.1 × 100mm, and the column temperature is 40 deg.C.
Preferably: the mass spectrum conditions are as follows: an MS system: ACQUITY XEVO TQ-D, ionization mode: electrospray ion source negative ion mode (ESI-); source temperature: 150 ℃, desolventizing gas temperature: at 450 ℃; desolventizing air flow rate: 800L/h, taper hole air flow rate: 30L/h, collision airflow rate: 0.20mL/min, capillary voltage: the negative ion is 2.5 KV; desolventizing gas temperature: at 450 ℃; scanning mode: multiple reaction monitoring, MRM; atomizing: nitrogen gas.
A method for simultaneously detecting nine lignin monomer cross-linked structures in wheat straws comprises the following specific steps:
(1) preparation of a standard substance: 3 monomers (p-coumaryl alcohol (H), coniferyl alcohol (G), sinapyl alcohol (S)) were mixed (3 mg of each monomer) and dissolved in 2mL of a solution containing 27mmol L of L-1Of (CTA)2SO4pH 6.5 sodium phosphate buffer (10mmol mL)-1) In (1). 67 μ L of 3% H was then added2O2And 0.3mg of horseradish peroxidase type II. Bathing in water at 30 deg.C for 1h, then adding 1mL of 5% Na2S2O7The aqueous solution interrupts the reaction. Extracting lignin monomer and oligomer with 0.6ml ethyl acetate, washing with saturated NaCl water solution, vortex oscillating for 2min, centrifuging for 5min at 5000g, collecting supernatant, and evaporating to dryness in vacuum concentrator. Finally, the dried sample was dissolved in 2ml of an aqueous solution containing 35% acetonitrile and was ready to be loaded.
(2) Sample treatment: freeze-drying wheat stalks ground under the condition of liquid nitrogen, adding an extracting agent, and uniformly mixing, wherein the extracting agent is ethanol: pure water 80:20 (volume ratio); preferably: the ratio of the extracting agent to the wheat straw is 4:1, mL: g.
(3) ultrasonic water bath extraction (preferably: 1h), centrifuging (preferably: 5000g, centrifuging for 5min), collecting supernatant, and vacuum concentrating and drying;
(4) dissolving the dried sample in step (3) in ultrapure water;
(5) extracting the mixture treated in the step (4) with ethyl acetate, centrifuging (preferably: 5000g, centrifuging for 5min), and collecting the supernatant;
(6) concentrating the supernatant obtained in the step (5) in vacuum, drying, adding acetonitrile (preferably: acetonitrile: water ═ 35: 65) to dissolve, filtering, and measuring S (8-O-4) S (8-8) S, S (8-O-4) G (8-O-4) S, G (8-O-4) S (8-5) G, S (8-O-4) S (8-5) G, G (8-O-4) S (8-8) S, G (8-O-4) G (8-5) G, S (8-O-4) G, G (8-8) G, G (8-5) H by using ultra performance liquid chromatography tandem mass spectrometry;
the determination method of the step (6) is LC-MS, and the chromatographic conditions are as follows: mobile phase A: ultrapure water, mobile phase B: acetonitrile, the mass spectrometry conditions are: an MS system: ACQUITY XEVO TQ-D, ionization mode: electrospray ion source negative ion mode (ESI-); source temperature: 150 ℃, desolventizing gas temperature: at 450 ℃; desolventizing air flow rate: 1000L/h, taper hole air flow rate: 150L/h, collision airflow rate: 0.15mL/min, capillary voltage: 2.5 KV; desolventizing gas temperature: at 450 ℃; scanning mode: multiple reaction monitoring, MRM; atomizing: nitrogen gas.
Another object of the present invention is to provide a method for simultaneously detecting nine lignin monomer cross-linked structures of wheat straw, including any one of the above methods, wherein the nine lignin monomer cross-linked structures are: s (8-O-4) S (8-8) S, S (8-O-4) G (8-O-4) S, G (8-O-4) S (8-5) G, S (8-O-4) S (8-5) G, G (8-O-4) S (8-8) S, G (8-O-4) G (8-5) G, S (8-O-4) G, G (8-8) G, G (8-5) H.
The invention also aims to provide application of any one of the methods in detection of nine lignin monomer cross-linked structures in wheat straws, wherein the nine lignin monomer cross-linked structures are as follows: s (8-O-4) S (8-8) S, S (8-O-4) G (8-O-4) S, G (8-O-4) S (8-5) G, S (8-O-4) S (8-5) G, G (8-O-4) S (8-8) S, G (8-O-4) G (8-5) G, S (8-O-4) G, G (8-8) G, G (8-5) H.
Compared with the prior art, the invention has the following beneficial effects:
the invention establishes a lignin monomer cross-linking structure which has extremely low content and small difference in simultaneously detecting wheat stalks and other samples through the pretreatment of wheat samples and the optimization of an ultra-high performance liquid chromatography tandem mass spectrometry method, particularly simultaneously detects nine lignin monomer cross-linking structures of S (8-O-4) S (8-8) S, S (8-O-4) G (8-O-4) S, G (8-O-4) S (8-5) G, S (8-O-4) S (8-5) G, G (8-O-4) S (8-8) S, G (8-O-4) G (8-5) G, S (8-O-4) G, G (8-8) G, G (8-5) H, the method can be used for accurate qualitative and quantitative determination, and is a simple detection method with high flux and reliable result.
The wheat leaf pretreatment method used by the invention can effectively remove matrix interference substances such as pigments, fatty acids and the like in the leaves, reduce matrix effects such as mutual competition of sample matrix components and target compounds during ionization of an electrospray ion source during detection and the like, is simple and rapid to operate, and can sensitively detect the content of multiple lignin monomer cross-linked structures.
The lignin monomer cross-linked structure is an important lignin structure in the wheat stalks, and plays a key role in the quality of the wheat stalks.
The method has the advantages of strong specificity, high sensitivity, high flux and objective and easy analysis of results, determines the content of the lignin monomer cross-linked structure in the growth and development of the wheat straw, and can provide guidance for improving the lodging resistance of the wheat straw.
Drawings
FIGS. 1 to 9 show the H chromatograms of a mobile phase A, S (8-O-4) S (8-8) S, S (8-O-4) G (8-O-4) S, G (8-O-4) S (8-5) G, S (8-O-4) S (8-5) G, G (8-O-4) S (8-8) S, G (8-O-4) G (8-5) G, S (8-O-4) G, G (8-8) G, G (8-5), in this order;
FIGS. 10-18 are sequential H chromatograms of wheat stalks S (8-O-4) S (8-8) S, S (8-O-4) G (8-O-4) S, G (8-O-4) S (8-5) G, S (8-O-4) S (8-5) G, G (8-O-4) S (8-8) S, G (8-O-4) G (8-5) G, S (8-O-4) G, G (8-8) G, G (8-5) 14 days after jointing.
Detailed Description
In order to better understand the technical solution of the present invention, the following embodiments are provided to further explain the present invention.
The test instrument:
a mortar; vacuum centrifugal concentrator (eppendorf); a freeze dryer (Christ); 1mL pipette (Eppendolf); a temperature control shaking table; 10mL centrifuge tube; a ten-thousandth electronic balance; a multifunctional vortex oscillation mixer; an ultrasonic cleaner; ultra pure water instruments (Milli-Q A10); 0.22 μm organic filter (Millipore, Ireland, GNWP 04700); 0.22 μm water system filter membrane (Millipore, Ireland, JCWP 04700); sample vials (Waters,12 × 32 mm); ultra high performance liquid-triple quadrupole mass spectrometer (ACQUITY UPLC I-Class/Xevo TQ-D, Waters); c18 column (ACQUITY UPLC BEH C181.7 μm, 2.1X 100mm, Waters).
Reagents and standards:
ethanol; hydrogen peroxide (3%); sodium chloride; ultrapure water; ethyl acetate (chromatographic grade, CNW); acetonitrile (chromatographic grade, CNW);
sodium phosphate (Trisodium phosphate anbydrous, Macklin, CAS: 7601-54-9);
(CTA)2SO4(Hexadecyltrime thylammonium bisulfate,Macklin,CAS:68214-07-3)；
horseradish Peroxidase II (Peroxidase from horse radish type II, Sigma-Aldrich, CAS: 9003-99-0);
Na2S2O7(Sodium pyrosulfate,Macklin,CAS:13870-29-6)；
p-Coumaryl alcohol (p-Coumaryl alcohol, Yuanyeshengwu, CAS: 3690-05-9);
coniferyl alcohol (Coniferyl alcohol, Sigma-Aldrich, CAS: 458-35-5);
sinapyl alcohol (Sinapyl alcohol, Sigma-Aldrich, CAS: 537-33-7);
sinapinal (Sinapyl aldehyde, Sigma-Aldrich, CAS: 134-96-3).
Example 1
1. The lignin monomer cross-linked structure is unstable to heat, so a mortar for grinding a sample and a medicine spoon for weighing the sample need to be put into liquid nitrogen for precooling, an extractant is prepared in advance and put into a refrigerator for precooling at 4 ℃, and a test tube rack of a10 mL centrifuge tube is put into a heat-preservation foam box filled with crushed ice.
2. Taking a wheat stalk or fresh stalk sample placed in a refrigerator at minus 80 ℃, placing the sample in a heat-preserving container filled with liquid nitrogen, shearing off the uppermost and lowermost stem nodes of the stalk with scissors, reserving the middle part of the stalk, shearing the middle part of the stalk into pieces, and uniformly mixing. Putting the uniformly mixed sample in a precooled mortar, adding liquid nitrogen, grinding the sample into powder, accurately weighing 0.5g of sample powder, adding the sample powder into a10 mL centrifuge tube, and putting the centrifuge tube on a test tube rack.
3. Accurately add 2mL of extractant (ethanol: ultrapure water: 80:20, volume ratio) with a pipette.
4. And oscillating and mixing the extractant and the sample in the centrifugal tube for 2min by using a multifunctional vortex oscillation mixer.
5. The centrifuge tube rack containing 10mL centrifuge tubes was placed in an ultrasonic cleaner and sonicated at room temperature for 30 min.
6.5000g was centrifuged for 5min at 4 ℃ and the supernatant carefully removed with a pipette and transferred to a new 10mL centrifuge tube.
7. The sample residue was added with 2mL of ethanol again, vortexed, mixed for 1min, centrifuged (5000g, 5min), and the supernatant carefully removed with a pipette and transferred to a corresponding new 10mL centrifuge tube.
8. Adding 1mL ethanol into the residual sample residue, vortex, shaking, mixing for 1min, centrifuging (5000g, 5min), carefully taking supernatant with pipette gun, and transferring to corresponding centrifuge tube.
9. Combining the three supernatants.
10. And (3) putting the supernatant obtained in the step (9) into a vacuum centrifugal concentrator, adding 2mL of ethyl acetate to dissolve and extract lignin after the supernatant is evaporated to dryness, and performing vortex oscillation and uniform mixing for 2 min.
5000g for 5min, the supernatant was carefully removed with a pipette and transferred to a new 10mL centrifuge tube.
12. 2mL of ethyl acetate was added again to the sample residue, vortexed and mixed for 1min, centrifuged (5000g, 5min), and the supernatant was carefully removed with a pipette and transferred to a corresponding new 10mL centrifuge tube.
13. The two supernatants were combined.
14. The supernatant obtained in step 13 was placed in a vacuum centrifugal concentrator, and after evaporation to dryness, 2mL of an acetonitrile aqueous solution (acetonitrile: ultrapure water 35: 65) was added and dissolved.
15. After passing through a 0.22 μm organic filter, the sample was measured by the ultra performance liquid mass spectrometry method, and the specific parameters are shown in tables 1 and 2.
TABLE 1 Mass Spectrometry parameter Table for Cross-linking Structure of Lignin monomers
Denotes quantitative ions
TABLE 2 liquid phase method Table
The mobile phase A is ultrapure water, the mobile phase B is pure acetonitrile, the sample introduction is 5 mu L, the column temperature is set to be 40 ℃, the sample temperature is set to be 4 ℃, and the standard substance prepared by the method is a mixture, so that the lignin monomer cross-linked structure is quantified by taking 50ppb sinapaldehyde as a reference. FIGS. 1-9 are chromatograms of standards prepared according to the present invention.
FIGS. 10 to 18 are chromatograms of detection of an actual sample, the retention time being the same as compared with the standard, and the retention time of the quantitative ions and the qualitative ions is the same as that of the standard, the cross-linking structures of the lignin monomers detected in the sample can be determined to be S (8-O-4) S (8-8) S, S (8-O-4) G (8-O-4) S, G (8-O-4) S (8-5) G, S (8-O-4) S (8-5) G, G (8-O-4) S (8-8) S, G (8-O-4) G (8-5) G, S (8-O-4) G, G (8-8) G, G (8-5) H respectively.
TABLE 3 content of cross-linked structure of 9 lignin monomers in wheat straw 14 days after node removal
The above description is only a preferred embodiment of the application and is illustrative of the principles of the technology employed. It will be appreciated by a person skilled in the art that the scope of the invention as referred to in the present application is not limited to the embodiments with a specific combination of the above-mentioned features, but also covers other embodiments with any combination of the above-mentioned features or their equivalents without departing from the inventive concept. For example, the above features may be replaced with (but not limited to) features having similar functions disclosed in the present application.
Claims (7)
1. A method for simultaneously detecting a lignin monomer cross-linking structure in wheat straws is characterized by comprising the following steps: the lignin monomer cross-linked structure is S (8-O-4) S (8-8) S, S (8-O-4) G (8-O-4) S, G (8-O-4) S (8-5) G, S (8-O-4) S (8-5) G, G (8-O-4) S (8-8) S, G (8-O-4) G (8-5) G, S (8-O-4) G, G (8-8) G, G (8-5) H, UPLC-MS/MS is adopted, and the chromatographic conditions are as follows: mobile phase A: pure water, mobile phase B: acetonitrile, gradient elution.
2. The method of claim 1, further comprising: the gradient elution is: 0-0.5min, 95% A, 0.5-3min, 95% A-75% A linear decrease, 3-3.5min, 75% A-10% A linear decrease, 3-4.0min, 10% A, 4.0-4.1min, 10% A-95% A linear increase, 4.1-6.0min, 95% A.
3. The method of claim 1, further comprising: the chromatographic column is C18, 1.7 μm,2.1 × 100mm, and the column temperature is 40 deg.C.
4. The method of claim 1, further comprising: the mass spectrum conditions are as follows: an MS system: ACQUITY XEVO TQ-D, ionization mode: an electrospray ion source negative ion mode; source temperature: 150 ℃, desolventizing gas temperature: at 450 ℃; desolventizing air flow rate: 1000L/h, taper hole air flow rate: 150L/h, collision airflow rate: 0.15mL/min, capillary voltage: 2.5 KV; desolventizing gas temperature: at 450 ℃; scanning mode: multiple reaction monitoring, MRM; atomizing: nitrogen gas.
5. The method of any of claims 1-4, wherein: the method comprises the following specific steps:
(1) preparation of a standard substance: 3 monomers p-coumarol (H), coniferyl (G), sinapyl (S)) were dissolved in 2mL of a solution containing 27mmol L of L-1Of (CTA)2SO4Sodium phosphate buffer pH 6.5, then 3% H2O2And 0.3mg of horseradish peroxidase type II in water at 30 ℃ for 1 hour, followed by 1mL of 5% Na2S2O7The aqueous solution is interruptedExtracting lignin monomer and oligomer with 0.6ml ethyl acetate, washing with saturated NaCl aqueous solution, performing vortex oscillation for 2min, centrifuging for 5min at 5000g, sucking supernatant, evaporating to dryness in a vacuum concentrator, dissolving dried sample in 2ml aqueous solution containing 35% acetonitrile, and waiting for loading;
(2) sample treatment: freeze-drying wheat stalks ground under the condition of liquid nitrogen, adding an extracting agent, and uniformly mixing, wherein the extracting agent is ethanol: water is 80:20, volume ratio;
(3) performing ultrasonic water bath extraction, centrifuging, collecting supernatant, and vacuum concentrating and drying;
(4) dissolving the dried sample in step (3) in ultrapure water;
(5) extracting the mixture treated in the step (4) with ethyl acetate, centrifuging and collecting supernatant;
(6) and (3) carrying out vacuum concentration and drying on the supernatant obtained in the step (5), adding acetonitrile for dissolving, filtering, and then measuring S (8-O-4) S (8-8) S, S (8-O-4) G (8-O-4) S, G (8-O-4) S (8-5) G, S (8-O-4) S (8-5) G, G (8-O-4) S (8-8) S, G (8-O-4) G (8-5) G, S (8-O-4) G, G (8-8) G, G (8-5) H by adopting ultra-performance liquid chromatography tandem mass spectrometry.
6. A method for simultaneously detecting nine lignin monomer cross-linked structures in wheat straws is characterized in that: the method of any of claims 1-8, wherein the nine monolignol crosslinked structures are: s (8-O-4) S (8-8) S, S (8-O-4) G (8-O-4) S, G (8-O-4) S (8-5) G, S (8-O-4) S (8-5) G, G (8-O-4) S (8-8) S, G (8-O-4) G (8-5) G, S (8-O-4) G, G (8-8) G, G (8-5) H.
7. Use of the method of any one of claims 1 to 6 for detecting monolignol crosslinked structures in wheat straw.
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