An analytical method for detecting brassinolide in microbial inoculants based on nanomaterial QuEChERS-UPLC-MSMS

Abstract

This invention provides a method for detecting brassinolide in microbial inoculants based on nanomaterials QuEChERS-UPLC-MSMS, using specific amounts of nano-ZrO2, PSA, and C. 18 Using MWCNTs as a purification agent combination for sample pretreatment, and with 0.1% formic acid aqueous solution and methanol as the mobile phase, under specific gradient elution conditions, the content of brassinolide in microbial inoculant samples can be accurately analyzed. The method of this invention has advantages such as high sensitivity, strong versatility, high accuracy, good stability, and simple operation, providing technical support for the detection of plant-derived pesticides from microbial inoculants.

Description

Technical Field

[0001] This invention relates to the field of plant-derived pesticide detection technology, and in particular to a method for detecting plant-derived pesticide residues in microbial agents based on the QuEChERS technology using nanomaterials. Background Technology

[0002] With the development of agricultural planting, problems such as continuous cropping and excessive use of chemical fertilizers leading to soil-borne diseases and soil compaction have not only caused a decline in the quality and yield of agricultural products, but also resulted in environmental pollution, seriously affecting the development of the agricultural industry. Therefore, changing this situation has become an urgent problem to be solved in agricultural development.

[0003] Microbial inoculants are low-carbon, all-natural, non-toxic, harmless, and pollution-free organic microbial agents. Due to their functions such as regulating nutrient balance within plants, improving fertilizer utilization, and improving soil, their application in agricultural production has become increasingly widespread in recent years.

[0004] Natural brassinolide is a novel sterol substance with extremely high activity. It is an internationally recognized, highly efficient, and low-toxicity plant growth regulator, and one of the most dynamic and promising new substances for developing high-yield, high-quality, and high-efficiency agriculture and ecological agriculture in China. Its main functions include promoting cell division, enhancing crop resistance and photosynthesis, regulating the distribution of nutrients within the plant, increasing crop yield, and improving crop quality. It not only does not antagonize biological agents but also has a synergistic effect, allowing for their mixed use.

[0005] Currently, there are numerous types of commercially available microbial inoculants, with varying quality. Unscrupulous manufacturers, seeking to reduce costs and maximize profits, inappropriately add the types and amounts of the active ingredient brassinolide, a plant-derived pesticide, resulting in reduced crop yields and lower quality, causing significant economic losses to farmers. This also severely damages the legitimate interests of legitimate manufacturers. Furthermore, with the increasing use and expanding application of brassinolide plant growth regulators, their safety is receiving growing attention. Studies have shown that various plant growth regulators exhibit reproductive toxicity, and some can even increase the risk of tumors and affect myocardial function. Therefore, developing a rapid, simple, sensitive, and accurate analytical method for detecting the types and amounts of brassinolide active ingredients in microbial inoculants is urgently needed, and is of great significance for ensuring the quality of microbial inoculant products and promoting agricultural development.

[0006] QuEChERS technology is a novel pretreatment method developed based on dispersion solid-phase extraction technology. It offers advantages such as speed, simplicity, cost-effectiveness, high efficiency, reliability, and safety. For different types of samples, the optimal purification effect can be achieved by changing the type and amount of adsorbent. QuEChERS technology is currently widely used in the detection of fruit and vegetable samples, but its application in pesticide residue detection in microbial inoculants is limited. Ultra-high performance chromatography-tandem mass spectrometry (UPLC-MSMS) not only possesses the advantages of liquid chromatography—wide analytical range and high selectivity—but also enables accurate qualitative and quantitative analysis of complex samples by acquiring primary and secondary ion fragments of the analyte using a triple quadrupole mass spectrometer. It is currently widely adopted by relevant standards (National Drug Inspection Supplementary Inspection Method Approval Documents 2006004, 2012005, and BJS 201701 "Determination of Sibutramine and Other Compounds in Food," etc.).

[0007] Zhang Junjie et al. (Pesticide Science and Management, 2021, 42(6)) disclosed a method for detecting the residues of acaricides and their metabolites on leeks and ginger using gas chromatography-tandem mass spectrometry. The purification agent combination used was 40 mg PSA and 10 mg C. 18 5 mg MWCNT. Wu Xuejin et al. (Southern Agricultural Journal, 2020, 51(10): 2532-2539) disclosed a method for simultaneous determination of 10 plant growth regulator residues in litchi using QuEChERS purification-ultra-high performance liquid chromatography-tandem mass spectrometry. The method uses methanol-5 mmol / L ammonium acetate aqueous solution (containing 0.1% formic acid, v / v) buffer solution as the mobile phase, and 150 mg MgSO4, 50 mg PSA, and 50 mg C 18 As a purification composition.

[0008] Current research on the detection of brassinolide residues is relatively limited, and the types of active ingredients detected are also few. Furthermore, there are no reports on the detection of multiple brassinolide active ingredients in microbial inoculants. Therefore, establishing methods for the detection of three brassinolide active ingredients in microbial inoculants is urgently needed and of profound significance. Summary of the Invention

[0009] This invention employs QuEChERS-UPLC-MSMS to develop a method with advantages such as simple operation, low cost, rapid analysis, and high recovery rate, enabling the detection of trace amounts of three brassinolide active ingredients in microbial agents to meet market demands.

[0010] Therefore, this invention provides a method for detecting brassinolide in microbial inoculants based on QuEChERS-UPLC-MSMS using nanomaterials, the method comprising the following steps:

[0011] (1) Sample collection

[0012] Collect the microbial agent product to be tested, label it, and then seal and store it in a -18℃ refrigerator for later use.

[0013] (2) QuEChERS sample pretreatment

[0014] The frozen microbial agent sample was thoroughly ground in a clean mortar and pestle. 2-4g of the ground sample was accurately weighed into a 50mL centrifuge tube, 8-15mL of acetonitrile was added and shaken quickly. Then 0.5-2g of sodium chloride and 3-6g of anhydrous magnesium sulfate were added. The sample was extracted by vortexing at 2500rpm for 7-14min, and then centrifuged at 5000rpm for 3-6min. The precipitate was discarded and the supernatant was obtained.

[0015] Take 1.5 mL of the supernatant into a 2 mL centrifuge tube, which is pre-filled with a purification agent combination and anhydrous magnesium sulfate. Vortex at 2500 rpm for 3-8 min, then centrifuge at 10000 rpm for 2-4 min. Filter the supernatant through a 0.22 μm organic filter membrane for analysis.

[0016] (3) UPLC-MSMS analysis and detection

[0017] The processed samples were analyzed using UPLC-MSMS (ultra-high performance liquid chromatography-tandem mass spectrometry) to detect brassinolide and its content.

[0018] In one embodiment of the present invention, the brassinolide includes one or more of 28-epihomoprassinolide, 28-homoprassinolide, and 22,23,24-epihomoprassinolide.

[0019] In one embodiment of the present invention, step "(2) sample pretreatment" is preferably:

[0020] The frozen microbial agent sample was thoroughly ground in a clean mortar and pestle. 2.5g of the ground sample was accurately weighed into a 50mL centrifuge tube, 10mL of acetonitrile was added and shaken quickly, then 1g of sodium chloride and 4g of anhydrous magnesium sulfate were added. The sample was extracted by vortexing at 2500rpm for 10min, and then centrifuged at 5000rpm for 5min. The precipitate was discarded and the supernatant was obtained.

[0021] Take 1.5 mL of the above supernatant into a 2 mL centrifuge tube, which is pre-filled with a purification agent combination and anhydrous magnesium sulfate. Vortex at 2500 rpm for 5 min, then centrifuge at 10000 rpm for 3 min. Filter the supernatant through a 0.22 μm organic filter membrane for analysis.

[0022] In one embodiment of the present invention, in step "(2) sample pretreatment", the purifying agent combination is: 30 mg nano-ZrO2, 20 mg PSA, 30 mg C 18 3-8 mg MWCNTs (i.e., nano-ZrO2 / PSA / C 18 / MWCNTs=30 / 20 / 30 / 3-8mg). Preferably, the purifying agent combination is: 30mg nano-ZrO2, 20mg PSA, 30mg C 18 3mg MWCNTs (i.e., nano-ZrO2 / PSA / C) 18 / MWCNTs=30 / 20 / 30 / 3mg). The amount of anhydrous magnesium sulfate added is 200mg.

[0023] Wherein, nano-ZrO2 refers to nano-zirconium oxide; PSA refers to ethylenediamine-N-propylsilylated silica gel adsorbent; C 18 "Octadecylsilane adsorbent (50μm)" refers to 50μm adsorbent; "MWCNTs" refers to multi-walled carbon nanotubes.

[0024] In one embodiment of the present invention, in step "(3) UPLC-MSMS analysis and detection":

[0025] The ultra-high performance liquid chromatography conditions are as follows: the chromatographic column is C10 ... 18 (2.6μm*2.1mm*50mm); The target compound was separated by gradient elution using an aqueous solution containing 0.1% formic acid (phase A) and methanol (phase B) as the mobile phase. The gradient elution program was as follows: 0 min 90% A + 10% B, 4.0 min 5% A + 95% B, 5.5 min 5% A + 95% B, 5.6 min 90% A + 10% B, 7.0 min 90% A + 10% B; Flow rate: 0.40 mL / min; Injection volume: 2 μL; Column temperature: 35℃.

[0026] The mass spectrometry conditions were as follows: electrospray ionization (ESI); positive ion scanning mode, multiple reaction monitoring (MRM) mode, temperature 450℃, voltage 5.5kV; nebulizer gas GS1 pressure 40psi; nebulizer gas GS2 pressure 40psi.

[0027] Another aspect of the present invention relates to the use of a purification agent combination for purifying microbial agent samples when detecting brassinolide content in microbial agents using the QuEChERS-UPLC-MSMS method, wherein the purification agent combination comprises: 30 mg nano-ZrO2, 20 mg PSA, and 30 mg C. 183-8 mg MWCNTs. Preferably, the purifying agent combination is: 30 mg nano-ZrO2, 20 mg PSA, 30 mg C 18 3mg MWCNTs.

[0028] This invention uses nano-ZrO2, PSA, and C 18 Using MWCNTs as a purification agent, a method for the analysis of trace amounts of 28-epi-homosinolactone, 28-homosinolactone, and 22,23,24-mixed-epi-homosinolactone in microbial inoculants was established based on QuEChERS-UPLC-MSMS technology. This method has the advantages of high sensitivity, strong versatility, high accuracy, good stability, and simple operation, providing technical support for the detection of plant-derived pesticides in microbial inoculants and has guiding significance.

[0029] By adopting the above technical solution, the present invention has the following beneficial effects:

[0030] The method of the present invention has the advantages of simple operation, low cost, rapid analysis and high recovery rate, and can realize the detection of trace amounts of three brassinolide active ingredients in microbial agents.

[0031] In addition, the method of the present invention can simultaneously detect three brassinolide components in microbial agents—28-epihomopsamol, 28-homopsamol, and 22,23,24-epihomopsamol. Attached Figure Description

[0032] Figure 1 Total ion chromatogram of the target compound when the mobile phase is 0.1% formic acid aqueous solution-methanol(II);

[0033] Figure 2 The response intensity of three brassinolides in mobile phase systems of 0.05% formic acid aqueous solution-methanol (I), 0.1% formic acid aqueous solution-methanol (II) and 0.05% formic acid (containing 2 mmol / L ammonium acetate) aqueous solution-ethanol (III);

[0034] Figure 3 Different nano-ZrO2 / PSA / C 18 Me of three brassinolides under a combination of purifying agents;

[0035] Figure 4 Me of three brassinolides at different MWCNT dosages; Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] I. Methodological Validation

[0038] The 28-epibrassinolide standard used in the following embodiments of this application has a content of 90%, the 22,23,24-epibrassinolide standard has a content of 90%, and the 22,23,24-epibrassinolide standard has a content of 92%, all of which were purchased from Beijing Qincheng Yixin Technology Development Co., Ltd.; the types of microbial agents are biological pesticide agents and microbial fertilizer agents.

[0039] 1. Materials and Methods

[0040] 1.1 Preparation of standard solutions and matrix solutions

[0041] Accurately weigh a certain amount of brassinolide standards (three active ingredients: 28-epihomocysinolide, 28-homocysinolide, and 22,23,24-epihomocysinolide) and prepare standard stock solutions with acetonitrile to a concentration of 500 mg / L. Then, mix and dilute to obtain mixed standard solutions with a concentration of 10 mg / L for each of the three active ingredients. Then, dilute with acetonitrile and microbial agent matrix solution to 200, 100, 50, 25, 10, 5, and 2.5 μg / L (the microbial agent matrix solution is obtained through the sample pretreatment steps in 1.2 below).

[0042] 1.2 QuEChERS Sample Pretreatment

[0043] The frozen microbial agent sample was thoroughly ground using a clean mortar and pestle. 2.5g of the ground sample was accurately weighed into a 50mL centrifuge tube, 10mL of acetonitrile was added and shaken quickly, followed by 1g of sodium chloride and 4g of anhydrous magnesium sulfate. The sample was extracted by vortexing at 2500rpm for 10min, and then centrifuged at 5000rpm for 5min. The supernatant was then purified.

[0044] Take 1.5 mL of the above supernatant into a 2 mL centrifuge tube (containing nano-ZrO2 / PSA / C). 18 / MWCNTs = 30 / 20 / 30 / 3mg (which is the optimal combination of purifiers; see the "QuEChERS Optimization" section for specific screening results) and 200mg anhydrous magnesium sulfate) were vortexed at 2500rpm for 5min, then centrifuged at 10000rpm for 3min, and the supernatant was filtered through a 0.22μm organic filter membrane.

[0045] 1.3 Instrumental Analysis Methods

[0046] The detection was performed using UPLC-MSMS (ultra-high performance liquid chromatography-tandem mass spectrometry).

[0047] The ultra-high performance liquid chromatography (UHPLC) conditions were as follows: the chromatographic column was C100000. 18 (2.6μm*2.1mm*50mm); The mobile phase consisted of an aqueous solution containing 0.1% formic acid (phase A) and methanol (phase B) (which was the optimal mobile phase; specific screening results are shown in the "Analytical Method Optimization" section). Gradient elution was used to separate the target compounds. The gradient elution program is shown in Table 1 (which was the optimal gradient elution program and was determined through screening in the "Analytical Method Optimization" section). The injection volume was 2μL, and the column temperature was 35℃.

[0048] Table 1. Gradient elution program for 28-epibrassinolide, 28-epibrassinolide, 22, 23, 24-epibrassinolide

[0049]

[0050] The mass spectrometry conditions were as follows: electrospray ionization (ESI); positive ion scanning mode, multiple reaction monitoring (MRM) mode, temperature 450℃, voltage 5.5kV; nebulizer gas GS1 pressure 40psi; nebulizer gas GS2 pressure 40psi.

[0051] 4. Matrix effect

[0052] Matrix effect (Me) refers to the phenomenon that the signal of a target compound is enhanced or weakened due to interference from co-eluting impurities. In the analysis of complex samples, the matrix effect can affect the degree of ionization of ions, thereby affecting the quantitative results of the target compound.

[0053] This study used the post-extraction addition experiment method to calculate the extraction addition matrix effect and used Me to evaluate the purification effect. The calculation formula is shown below.

[0054] Me = Se2 / Se1

[0055] In the formula, Se1 is the response intensity of the pure standard solution, and Se2 is the response intensity of the sample matrix with the same amount of analyte added; Se1 and Se2 must have the same amount of analyte added.

[0056] When Me is between 0.8 and 1.2, it is considered that there is no obvious matrix effect, that is, the purification effect is good; when Me < 0.8, it is a matrix inhibition effect, the smaller the value, the stronger the matrix inhibition effect, and the worse the purification effect; when Me > 1.2, it is a matrix enhancement effect, the larger the value, the stronger the matrix enhancement effect, and the worse the purification effect.

[0057] 2 Results

[0058] 2.1 Optimization of Analysis Methods

[0059] A 200 μg / L mixed standard solution of 28-epomobrassinolide, 28-homomobrassinolide, and 22,23,24-mixed epibrassinolide was injected into the mass spectrometer via a syringe pump for parameter tuning, and scanned in ESI positive and negative modes. The results showed that the parent ion of all three target compounds was [M+H]. + The mass spectrometry acquisition parameters for the three compounds in MRM mode were obtained by tuning and optimizing the parent and daughter ions of the target compounds, as well as the optimal cone voltage and collision energy (Table 2). Two sets of ion pairs with the best sensitivity were selected for detection, one for quantification and the other for qualitative analysis.

[0060] Table 2. Mass spectral information of 28-epibrassinolide, 28-epibrassinolide, and 22, 23, 24-epibrassinolide.

[0061]

[0062] Note: "*" indicates quantitative ion pairs.

[0063] Since formic acid and ammonium acetate are ESI + Enhancement of the target compound [M+H] under certain conditions + and [M+NH4] + Commonly used reagents for ionization degree, and both can effectively improve peak shape, making the peak shape sharper and more symmetrical. Therefore, the effects of 0.05% formic acid aqueous solution-methanol (I), 0.1% formic acid aqueous solution-methanol (II) and 0.05% formic acid (containing 2 mmol / L ammonium acetate) aqueous solution-ethanol (III) mobile phase systems on the sensitivity and peak shape of 28-epiobrassinolide, 28-homoisinolide, and 22,23,24-mixed epibrassinolide were investigated when performing gradient elution of target compounds.

[0064] The results show that when mobile phase III is used, the sensitivity of the target compounds is low, while when mobile phase II is used, the peak shapes of the three compounds are optimal and the sensitivity is highest. Figure 2 ).

[0065] In summary, 0.1% formic acid-methanol was ultimately selected as the mobile phase system. Continuous optimization of the gradient elution program resulted in the effective separation of all compounds within 7 minutes. The total ion chromatogram of the target compounds is shown below. Figure 1 As shown in Table 1 above, the optimized elution procedure has been determined.

[0066] 2.2 QuEChERS Optimization

[0067] The nano-ZrO2 / PSA / C ratio was investigated at an addition level of 100 μg / kg and a fixed MWCNT dosage of 4 mg. 18 The purification effects of the purification agent combination on 28-epibrassinolide, 28-homibrassinolide, and 22,23,24-mixed epibrassinolide in the microbial agent matrix (Table 3).

[0068] The research results show that when nano-ZrO2 / PSA / C 18 When the dosage was 30 / 20 / 30 mg, the Me values ​​of the three compounds ranged from 0.81 to 0.96. Figure 3 (This method) has the best purification effect.

[0069] Therefore, this study ultimately selected nano-ZrO2 / PSA / C 18 =30 / 20 / 30mg for the next step of purification and optimization of the microbial agent matrix.

[0070] Table 3. Amount of purifying agent combination used in microbial agent matrix

[0071]

[0072]

[0073] MWCNTs are highly efficient adsorbents that effectively adsorb impurities such as pigments, heavy metals, inorganic non-metallic ions, aromatic hydrocarbons, and antibiotics. However, since MWCNTs adsorb both impurities and target compounds, their dosage needs to be optimized. Based on the above research results, a fixed nano-ZrO2 / PSA / C... 18 =30 / 20 / 30mg, the Me values ​​of 28-epibrassinolide, 28-homibrassinolide, and 22,23,24-mixed epibrassinolide in the microbial agent matrix were investigated when the MWCNTs addition amounts were 3, 5, 8 and 12 mg, respectively.

[0074] The results showed that when the dosage of MWCNTs was between 3 and 8 mg, the Me values ​​of the three compounds in the microbial agent matrix were between 0.77 and 1.11, indicating a good purification effect. Figure 4 To ensure good purification results while also considering cost savings, the final dosage of MWCNTs was determined to be 3 mg.

[0075] In summary, through optimization of the purification agent combination, the optimal combination of nano-ZrO2 / PSA / C for the analysis of trace amounts of 28-epomobrassinolide, 28-homomobrassinolide, and 22,23,24-mixed epibrassinolide in the microbial agent matrix was finally determined to be 1. 18 / MWCNTs=30 / 20 / 30 / 3mg.

[0076] 3. Method Validation

[0077] 3.1 Limit of detection, limit of quantitation, standard curve and R 2

[0078] The aforementioned screening method was used for analysis. Solvent standard curves and matrix standard curves of the microbial inoculant matrix were plotted with mass concentration as the X-axis and peak area as the Y-axis. The results showed that 28-epibrassinolide, 28-homibrassinolide, and 22,23,24-mixed epibrassinolide in the microbial inoculant matrix exhibited good linearity in the concentration range of 5–100 μg / L, with R0... 2 The LODs of the three compounds in the microbial agent were between 0.9995 and 0.9999. With a signal-to-noise ratio (S / N) of 3 times as LOD and a minimum addition level (LOQ) as LOQ, the LODs of the three compounds were 0.1 to 0.3 μg / kg and the LOQs were all 10 μg / kg (Table 4).

[0079] Table 4. Limits of detection, limits of quantitation, standard curves, and R values ​​for three brassinolides in solvents and microbial inoculant matrices. 2

[0080]

[0081] 3.2 Accuracy and Precision

[0082] my country has not yet established residue limits for brassinolide. Referring to the minimum values ​​in GB 2763-2021 MRLs, this study conducted five parallel experiments with four spiking levels (10, 100, 1000, and 5000 μg / kg) in a blank microbial inoculant. The results showed that at spiking levels of 10–5000 μg / kg, the average recoveries of the three compounds in the microbial inoculant matrix ranged from 79.8% to 103.2%, with relative standard deviations (RSDs) ranging from 2.6% to 9.4% (Table 5). In conclusion, all results meet the requirements of pesticide residue detection standards.

[0083] Table 5. Average recovery and RSDs% of three brassinolides in microbial inoculant matrix at four spiking levels

[0084]

[0085]

[0086] In summary, this invention utilizes nano-ZrO2 and C 18Using PSA and MWCNTs as purification agents, a method for analyzing trace amounts of 28-epi-homosinolactone, 28-homosinolactone, and 22,23,24-mixed-epi-homosinolactone in microbial inoculants was established based on QuEChERS-UPLC-MSMS technology. This method has the advantages of high sensitivity, strong versatility, high accuracy, good stability and simple operation, providing technical support for the detection of plant-derived pesticides in microbial inoculants.

[0087] II. Implementation Examples

[0088] The optimal analytical method determined in this study was used to analyze the brassinolide content in commercially available microbial inoculants. The brassinolide content in commercially available microbial inoculants was calculated using a standard curve of the microbial inoculant matrix. The analytical results are shown in Table 6.

[0089] Table 6. Detection results of brassinolide content in different microbial inoculants on the market.

[0090]

[0091]

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting brassinolide in microbial inoculants based on nanomaterial QuEChERS-UPLC-MSMS, characterized in that, The brassinolides include one or more of 28-epi-homocysteine, 28-homocysteine, and 22,23,24-epi-homocysteine. The method includes the following steps: (1) Sample collection Collect the microbial agent product to be tested, label it, and then seal and store it in a -18℃ refrigerator for later use; (2) QuEChERS sample pretreatment The frozen microbial agent sample was thoroughly ground in a clean mortar and pestle. 2-4g of the ground sample was accurately weighed into a 50mL centrifuge tube, 8-15mL of acetonitrile was added and shaken quickly. Then 0.5-2g of sodium chloride and 3-6g of anhydrous magnesium sulfate were added. The sample was extracted by vortexing at 2500rpm for 7-14min, and then centrifuged at 5000rpm for 3-6min. The precipitate was discarded and the supernatant was obtained. Take 1.5 mL of the above supernatant into a 2 mL centrifuge tube, which is pre-filled with a purification agent combination and anhydrous magnesium sulfate. Vortex at 2500 rpm for 3-8 min, then centrifuge at 10000 rpm for 2-4 min. Filter the supernatant through a 0.22 μm organic filter membrane for analysis. The purification agent combination consists of: 30mg nano-ZrO2, 20mg PSA, and 30mg C. 18 and 3 mg MWCNTs; (3) UPLC-MSMS analysis and detection The processed samples were analyzed using UPLC-MSMS to detect brassinolide and its content. The ultra-high performance liquid chromatography (UHPLC) conditions were as follows: the chromatographic column was C100000. 18 The column diameter is 2.6 μm * 2.1 mm * 50 mm. The mobile phase consists of phase A containing 0.1% formic acid aqueous solution and phase B containing methanol. Gradient elution is used to separate the target compounds. The gradient elution program is as follows: 0 min 90% A + 10% B, 4.0 min 5% A + 95% B, 5.5 min 5% A + 95% B, 5.6 min 90% A + 10% B, 7.0 min 90% A + 10% B; flow rate: 0.40 mL / min; injection volume: 2 μL; column temperature: 35℃. Mass spectrometry conditions were as follows: electrospray ionization (ESI); positive ion scanning mode, multiple reaction monitoring (MRM) mode, temperature 450℃, voltage 5.5kV; nebulizer gas GS1 pressure 40psi; nebulizer gas GS2 pressure 40psi.

2. The method according to claim 1, characterized in that, In step (2): The frozen microbial agent sample was thoroughly ground in a clean mortar and pestle. 2.5g of the ground sample was accurately weighed into a 50mL centrifuge tube, 10mL of acetonitrile was added and shaken quickly, then 1g of sodium chloride and 4g of anhydrous magnesium sulfate were added. The sample was extracted by vortexing at 2500rpm for 10min, and then centrifuged at 5000rpm for 5min. The precipitate was discarded and the supernatant was obtained. Take 1.5 mL of the above supernatant into a 2 mL centrifuge tube, which is pre-filled with a purification agent combination and anhydrous magnesium sulfate. Vortex at 2500 rpm for 5 min, then centrifuge at 10000 rpm for 3 min. Filter the supernatant through a 0.22 μm organic filter membrane for analysis.

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