Method for quantitatively detecting AHLs in soil and bacterial suspension based on LC-MSMS

Abstract

The invention relates to the technical field of analysis, and discloses a method for quantitatively detecting AHLs in soil and bacterial suspension based on LC-MSMS. Aiming at the defects in the prior art, the invention aims to provide the AHLs detection method which is higher in sensitivity and lower in detection limit compared with the existing method. Verified by the method, the method is suitable for two matrixes of soil and bacterial suspension. According to the method, dispersive liquid phase extraction-solid phase supported liquid-liquid extraction is adopted, so that the extraction time, the matrix effect and the use amount of organic reagents are greatly reduced.

Description

Technical Field

[0001] The present invention relates to the field of analytical technology, and in particular to a method for quantitatively detecting AHLs in soil and bacterial suspension based on LC-MSMS. Background Art

[0002] Quorum sensing is a communication mechanism by which bacteria exchange information and coordinate behavior within a population or community through a special class of chemical signaling molecules. Through this mechanism, bacteria can respond to changes in the environment, adjust their metabolic activities, control downstream changes at the genetic level, adapt to various conditions together, and regulate many biological functions, such as the secretion of toxic factors, the synthesis of antibiotics, and the synthesis of biofilms. According to the form and sensing mode of quorum sensing molecules, signal molecules can be divided into three types: oligopeptides, N-acylated homoserine lactones (AHLs), and AI-2 autoinducers. AHLs are signal molecules for information exchange within Gram-negative bacteria. They are also the earliest and most fully characterized signal molecules discovered and currently the most comprehensively studied. In addition to regulating bacteria, AHLs can also be sensed by plants, thereby regulating plant physiological behaviors, including plant morphology, natural immunity, growth and development, and stress tolerance. Studies have shown that applying AHLs-secreting bacteria or directly applying AHLs in the soil can regulate plant defense responses. In agricultural production, applying AHLs to the rhizosphere of tomatoes can improve the systemic resistance of tomatoes to the pathogenic fungus Alternaria alternata; applying 3-oxo-C6-HSL and 3-oxo-C8-HSL can significantly increase the root length and survival rate of Arabidopsis under salt stress conditions, and can also significantly improve the salt and drought tolerance of wheat in the early growth stage. As an emerging immobilized microbial remediation technology, biofilm has attracted more and more attention in the remediation of POPs-contaminated soil, and low concentrations of exogenous AHLs can promote the degradation of phenanthrene and pyrene by biofilm.

[0003] In order to study, screen and prepare AHLs, AHLs-secreting bacteria are inoculated in a specified culture medium. The culture medium containing AHLs produced by the proliferation and metabolism of bacteria in the culture medium is called AHLs bacterial suspension. Detecting the content of AHLs in the bacterial suspension can not only screen AHLs-secreting strains, but can also be directly used in actual production to explore the effects of exogenous AHLs and AHLs-secreting strains on soil flora regulation and plant growth.

[0004] In summary, AHLs can affect the environment, gene expression and function of plants through quorum sensing effects, resulting in beneficial production attributes. Preparing bacterial suspensions is an important method for studying AHLs, so rapid and accurate detection of the content and change patterns of AHLs in soil and bacterial suspensions is an important basis for studying plant quorum sensing behaviors and mechanisms.

[0005] There are few reports on the detection of N-acyl homoserine lactones (AHLs) in soil and bacterial suspensions. Currently, liquid chromatography, gas mass spectrometry, liquid phase mass spectrometry, etc. are mainly used for detection. For example, CN 103983729B, a method for detecting acyl homoserine lactones in soil solution by gas chromatography-mass spectrometry, determined 7 AHLs in soil solution, with a detection limit of 1.5-2.5 μg / L and a recovery rate of 54.29-96.67%; CN106290668A, a method for extracting, purifying and determining acyl homoserine lactones in soil and its application, used GC-MS to determine 7 AHLs in soil, with a detection limit of 0.75-1.25 μg / kg. The two articles on the use of GC-MS to determine AHLs have high detection limits because AHLs have high boiling points, all exceeding 300°C (C10-HSL boiling point is 466.1°C, C12-HSL boiling point is 485.8°C, 3-OH-C4-HSL boiling point is 504.6°C, etc.), which are not suitable for GC-MS detection. The high detection limit makes it difficult to fully evaluate the types and contents of low AHLs in the sample. Ma Chenchen detected AHLs in LB culture medium. The culture medium was centrifuged at 27000r / min for 20min, the supernatant was extracted with ethyl acetate and dried, and reconstituted with methanol for LC-MS analysis. This method also has serious limitations. Although high-speed centrifugation purifies the matrix, it causes a large amount of extracellular polymers containing AHLs to sink to the bottom without being extracted and purified. The quantitative results of AHLs are low, and the detection limit of this method is high (0.1-1.0μg / kg). Summary of the invention

[0006] In view of this, the present invention provides a method for quantitatively detecting AHLs in soil and bacterial suspensions based on LC-MSMS to solve the following problems: Currently, AHLs are mainly extracted by liquid-liquid extraction with ethyl acetate, which has a long extraction time, a large matrix effect of the extract, and uses a large amount of organic reagents, which is not conducive to the detection of large-scale samples; the method for purifying AHLs is to directly ultracentrifuge the sample, so that the extracellular polymers containing AHLs are precipitated to the bottom with impurities, resulting in low detection results; the existing method has a high detection limit of AHLs and cannot accurately and quantitatively analyze low concentrations of AHLs in soil and bacterial suspensions.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A method for quantitatively detecting AHLs in soil and bacterial suspension based on LC-MSMS comprises the following steps:

[0009] (1) Sample processing: The sample to be tested is extracted and purified twice in sequence to obtain a test solution;

[0010] (a) Extraction: The analyte and acetonitrile are ultrasonically vortexed to obtain an extract;

[0011] In the step (a), the test object is a bacterial suspension or soil; when the test object is a bacterial suspension, the sample weight is 2 to 15 g; when the test object is soil, the soil and water are first mixed and then mixed with acetonitrile, and the ratio of soil to water is 4 to 10 g: 9 to 12 mL;

[0012] (b) Purification: The extract is frozen and mixed with anhydrous magnesium sulfate, sodium chloride and sodium acetate, and then centrifuged to obtain the supernatant to complete the first purification; 4 to 10 mL of the supernatant is removed and sequentially dried, re-dissolved, solid phase supported liquid-liquid extraction, secondary purification, dried, re-dissolved and filtered to obtain the test solution;

[0013] In the step (b), the ratio of the extract, anhydrous magnesium sulfate, sodium chloride and sodium acetate is 12-35 g: 4-6 g: 1-4 g: 1-2 g;

[0014] The re-dissolution includes a first re-dissolution and a second re-dissolution; the reagent used in the first re-dissolution is a formic acid aqueous solution, and the mass fraction of the formic acid aqueous solution is 0.1-0.2%; the reagent used in the second re-dissolution is a methanol solution, and the mass fraction of the methanol solution is 20-40%;

[0015] The reagent used for the solid phase supported liquid extraction elution is a diethylamine dichloromethane solution, and the mass fraction of diethylamine in the dichloromethane solution is 0.1-0.2%;

[0016] (2) Preparation of standard substances: 3-OH-C4-HSL, 3-oxo-C6-HSL, 3-OH-C8-HSL, 3-oxo-C8-HSL, 3-oxo-C10-HSL, 3-OH-C12-HSL, 3-oxo-C12-HSL, and 3-OH-C14-HSL were prepared into eight kinds of AHLs mixed standard stock solutions; the mixed standard concentration for establishing the mass spectrometry method was 50 μg / L, and the standard curve concentrations were 0.001 to 300 μg / L;

[0017] The solvent used in the preparation of the AHLs mixed standard stock solution is a blank matrix solution obtained by pre-treatment of soil or bacterial suspension; the soil blank matrix is ​​a mixture of soil and quartz sand, with a ratio of 1:3 to quartz sand, which has been verified to contain no analyte through multiple verifications and is a blank matrix of soil;

[0018] (4) Chromatographic conditions and mass spectrometry conditions: The chromatographic conditions are as follows: chromatographic column: biphenyl column; mobile phase A: 0.05% acetic acid aqueous solution containing 0.1-0.3 mM ammonium formate; mobile phase B: 0.05% acetic acid methanol solution containing 0.1-0.3 mM ammonium formate; column temperature: 30-50°C; injection volume: 2-10 μL; mass spectrometry conditions: electrospray ion source, curtain gas: 30-50 psi, collision gas: Medium, ionization voltage: 4500-5000 V, temperature: 450-500°C, spray gas: 50-55 psi, auxiliary heating gas: 55-60 psi;

[0019] (4) Calculate the AHLs content.

[0020] Furthermore, in the step (a), the power of ultrasonic mixing is 150-250w, the time of ultrasonic mixing is 15-25min; the time of vortex mixing is 1-2min; the speed of centrifugation is 4000-6000r / min, and the time of centrifugation is 4-10min.

[0021] Furthermore, in step (b), the mixture is frozen at -20°C for 10 to 15 minutes.

[0022] Furthermore, in the step (b), drying is performed by passing nitrogen through a water bath at 30-40°C.

[0023] Furthermore, the blank matrix of the bacterial suspension in step (2) is the corresponding bacterial culture medium; the soil used in the soil blank matrix is ​​dry soil aged for more than 2 years, which is fully mixed with 40-80 mesh quartz sand.

[0024] Furthermore, the solvent used in the preparation of the AHLs mixed standard stock solution is a blank matrix obtained by pre-treatment of soil or bacterial suspension.

[0025] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0026] In view of the shortcomings of the prior art, the present invention has higher sensitivity and lower detection limit than other methods for determining AHLs related to soil and bacterial suspensions; the present invention greatly shortens the detection time and reduces the use of organic reagents, and can process samples in large quantities; the present invention greatly reduces the matrix effect and reduces the pollution to the instrument through two purifications; the present invention has good linearity, accuracy and repeatability, and improves the detection limit of AHLs in soil and bacterial suspensions to 0.517-2.670 ng / kg, which is more than a hundred times higher than the existing methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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.

[0028] Figure 1 It is the extracted ion current chromatogram of 8 AHLs in Example 1; Note: the numbers in the figure represent the XIC peaks of the following substances: 1, 3-OH-C4-HSL, 2, 3-oxo-C6-HSL, 3, 3-OH-C8-HSL, 4, 3-oxo-C8-HSL, 5, 3-oxo-C10-HSL, 6, 3-OH-C12-HSL, 7, 3-oxo-C12-HSL, 8, 3-OH-C14-HSL.

[0029] Figure 2 is the standard curve of the eight AHLs in Example 1;

[0030] Figure 3 is the secondary mass spectra of 8 AHLs in Example 1;

[0031] Figure 4 This is the extracted ion current chromatogram of the soil blank matrix solution in Example 3. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Various common instruments, equipment and chemical reagents used in the examples can be purchased through commercial channels.

[0035] The instruments and key reagents and consumables used in the following examples are: triple quadrupole mass spectrometer liquid chromatography (AB SCIEX QTRAP 6500, USA); methanol, acetic acid, ammonium formate (mass spectrometry grade, Thermo Fisher Scientific, USA), chromatographic column (biphenyl chromatographic column, 2.1×100 mm×1.7 μm, Fenome, USA); standard products: 3-OH-C4-HSL, 3-oxo-C6-HSL, 3-OH-C8-HSL, 3-oxo-C8-HSL, 3-oxo-C10-HSL, 3-OH-C12-HSL, 3-oxo-C12-HSL, 3-OH-C14-HSL (Merck, USA), other drugs and consumables: Novum TM SLE column (8B-S138-KDG, Fenome Corporation, USA), CleanertSLE pH=9 (HC0006Q-9, Aeger Corporation, China).

[0036] Example 1

[0037] This example measures and verifies the linear relationship of the method:

[0038] (1) Dilute the standard stock solution to a 50 μg / L working standard solution, then dilute the working standard solution with 20% methanol to a concentration of 0.02 μg / L, 0.05 μg / L, 0.1 μg / L, 0.5 μg / L, 1 μg / L, and 5 μg / L, and detect using LC-MSMS.

[0039] (2) Mass spectrometry conditions: Electrospray ion source, curtain gas 35 psi, collision gas Medium, ionization voltage 5000 V, temperature 500°C, spray gas 50 psi, auxiliary heating gas 55 psi. Chromatographic conditions: Mobile phase A: 0.05% acetic acid-water (containing 0.25 mM ammonium formate); Mobile phase B: 0.05% acetic acid-methanol (containing 0.25 mM ammonium formate), elution gradient: initial B = 20%, 0-3 min, B = 90%, 3-8 min, B = 90%, 8.1-10 min, A = 80%. Column temperature: 40°C; injection volume: 5 μL.

[0040] (3) The eight AHLs were completely separated with good peak shapes, no tailing peaks, front peaks, inverted peaks, or broad peaks, and good separation without baseline noise or drift.

[0041] The extracted ion chromatograms of 8 AHLs are shown in Figure 1 The standard curves of 8 AHLs are shown in Figure 2 The secondary mass spectra of 8 AHLs are shown in Figure 3The mass spectrometry parameters and ion parameters of the eight HSLs are shown in Table 1. The linear relationships of the eight HSLs are shown in Table 2. The results show that the standard curve concentration has a good linear relationship in the range of 0.02 to 5 μg / L, R 2 The above data show that the mass spectrometry and chromatography methods of this example are accurate, linear, and highly sensitive, and the detection time is short, the peak is completed within 8 minutes, and the detection efficiency is extremely high.

[0042] Table 1 Mass spectrometry parameters and ion parameters of 8 AHLS

[0043]

[0044]

[0045] Table 2 Linear relationships of 8 AHLs

[0046] Compound Name Chemical formula Linear range Linear equations R 3-OH-C4-HSL <![CDATA[C8H 13 NO4]]> 0.02~5μg / L y=6.6887e6x+4500.9102 0.9998 3-OH-C8-HSL <![CDATA[C 12 H 21 NO4]]> 0.02~5μg / L y=2.0779e7x+47.6692 0.9999 3-OH-C12-HSL <![CDATA[C 16 H 29 NO4]]> 0.02~5μg / L y=1.1213e7x+1499.2393 0.9999 3-OH-C14-HSL <![CDATA[C 18 H 33 NO4]]> 0.02~5μg / L y=1.0884e7x-96.8154 0.9999 3-oxo-C6-HSL <![CDATA[C 10 H 15 NO4]]> 0.02~5μg / L y=4.4522e6x-730.4636 0.9998 3-oxo-C8-HSL <![CDATA[C 12 H 19 NO4]]> 0.02~5μg / L y=2.5817e6x-181.5649 0.9998 3-oxo-C10-HSL <![CDATA[C 14 H 23 NO4]]> 0.02~5μg / L y=1.0697e7x-281.5666 0.9999 3-oxo-C12-HSL <![CDATA[C 16 H 27 NO4]]> 0.02~5μg / L y=1.5432e7x+509.3234 0.9999

[0047] Example 2

[0048] This embodiment explores and optimizes the pre-processing of the invention

[0049] For sample extraction and purification, this embodiment selected methanol, acetonitrile, ethyl acetate, and 1% acetic acid-acetonitrile as extraction solvents. The results showed that acetonitrile extraction had the best extraction effect; dispersed liquid extraction selected anhydrous magnesium sulfate, sodium chloride, sodium citrate, disodium citrate, sodium acetate, C18, PSA, and GCB as purifiers. The results showed that anhydrous magnesium sulfate, sodium chloride, and sodium acetate had good effects and low costs, and their usage was optimized. The optimal usage was: anhydrous magnesium sulfate (4-6 g), sodium chloride (1-4 g), and sodium acetate (1-2 g); solid phase support In the liquid-liquid extraction step, during the purification operation, five solutions, including ultrapure water, 20% methanol aqueous solution, sodium acetate buffer, 0.1% formic acid aqueous solution, and 5% ammonia aqueous solution, were selected to compare the sample adsorption effect. The results showed that the sample loading effect of 0.1% formic acid aqueous solution was the best; five different solutions, including ethyl acetate, dichloromethane, 1% acetic acid-ethyl acetate, 0.1% diethylamine-dichloromethane, and 0.1% diethylamine-ethyl acetate, were selected to compare the elution effect. The results showed that the elution recovery rate of 0.1% diethylamine-dichloromethane was the highest and the matrix effect was the lowest. Subsequently, 0.05%, 0.1%, 0.2%, 0.3%, and 0.5% formic acid aqueous solution and 0.05%, 0.1%, 0.2%, 0.3%, and 0.5% diethylamine-dichloromethane were used for sample loading and elution. The results showed that 0.2% had the highest recovery rate, but the difference with 0.1% was not significant. Other concentrations significantly reduced the recovery rate.

[0050] Example 3

[0051] This example measured the detection limit and quantification limit of 8 AHLs in soil and LB medium:

[0052] (1) Preparation of blank matrix solution: For the blank matrix of soil, dry soil aged for more than 2 years (verified to contain no target substance) and quartz sand were mixed in a ratio of 1:3. For the blank matrix of bacterial suspension, uninoculated LB medium was selected. Weigh 5g of soil blank matrix or 10g of LB medium into a 50mL centrifuge tube, homogenize with 2 glass balls, add 10mL of water, and vortex for 1min using a vortex shaker. Then add 10mL of acetonitrile, ultrasonicate at 170W for 15min, vortex for 1min, freeze at -20℃ for 10min, take out and add 4g of anhydrous magnesium sulfate, 2g of sodium chloride, and 1.5g of sodium acetate, vortex for 1min, and centrifuge at 4000r / min for 5min. Accurately transfer 8mL of supernatant and blow to nearly dryness with nitrogen at 30℃, and reconstitute with 2mL of 0.1% formic acid aqueous solution and vortex for 1min. Add 2mL of sample solution to Novum TM In the SLE column, apply vacuum pressure of 5 Hg column for 2 seconds, wait for 5-10 minutes, add 5mL×2 0.1% diethylamine-dichloromethane solution for elution, evacuate for 5 seconds, and dry with nitrogen at 30°C. Redissolve with 1ml 20% methanol. Filter with 0.2μm polytetrafluoroethylene microporous filter membrane, and use the above treated sample solution as blank matrix solution for standby use. The extracted ion current chromatogram of soil blank matrix solution is shown in Figure 4 .

[0053] (2) Preparation of matrix standards: Take an appropriate amount of AHLs mixed standard and dilute it step by step to 0.2, 0.5, 1, 2, 5, and 20 ng / L using the blank matrix treated above, and then analyze it by LC-MSMS.

[0054] (3) The matrix standard curve has a good linear relationship in the range of 0.5 to 20 ng / L. 2 The value was above 0.997, and the concentrations of 8 AHLs matrix standards (0.5-20 ng / L) were analyzed at the lowest possible concentration, and the signal-to-noise ratio (S / N) was calculated by the software. The 3-fold signal-to-noise ratio was used as the method detection limit, and the 10-fold signal-to-noise ratio was used as the method quantification limit. The detection limits and quantification limits of 8 AHLs in soil and LB culture medium are shown in Table 3. The detection limit of soil is between 0.517 and 2.114 ng / kg, and the quantification limit is between 1.723 and 7.047 ng / kg; the detection limit of bacterial suspension is between 0.855 and 2.670 ng / kg, and the quantification limit is between 2.850 and 8.90 ng / kg. This method has high sensitivity and low detection limit, and can analyze AHLs with extremely low content in samples.

[0055] Table 3 Detection limits of 8 AHLs in soil and bacterial suspension

[0056]

[0057] Example 4

[0058] This example analyzes the sample spike recovery and precision of 8 AHLs in soil and LB bacterial suspension:

[0059] (1) Weigh 5g of soil or 5g of MSB bacterial suspension into a 50mL centrifuge tube, add 0.1mL of 0.1μg / L and 1μg / L mixed standard solutions of 8 kinds of AHLs, and prepare the corresponding samples without adding standard. Add 2 glass balls to the centrifuge tube for protonation. Add 10mL of water to the soil and vortex for 1min using a vortex shaker. Then add 10mL of acetonitrile, ultrasonicate at 150W for 20min, vortex for 1min, freeze at -20℃ for 10min, take out and add 4g of anhydrous magnesium sulfate, 2g of sodium chloride, 1.5g of sodium acetate, vortex for 1min, and centrifuge at 4000r / min for 5min. Accurately transfer 8mL of supernatant and blow dry with nitrogen at 30℃, and reconstitute with 2mL of 0.1% formic acid aqueous solution and vortex for 1min. Add 2mL of sample solution to Novum TM In the SLE column, apply vacuum pressure of 5 Hg column for 2s, wait for 5-10min, add 5mL×2 0.2% diethylamine-dichloromethane solution for elution, evacuate for 5s, and dry with nitrogen at 40℃. Redissolve with 1mL 20% methanol. Filter with 0.2μm polytetrafluoroethylene microporous filter membrane and wait for loading.

[0060] (2) Preparation of standard curve: Use a pipette to draw an appropriate amount of AHLs mixed standard, dilute it step by step to 1, 5, 20, 100, and 200 ng / L using blank matrix solution, and use LC-MSMS for sample injection and analysis.

[0061] The recovery and precision are shown in Table 4.

[0062] Table 4 Recovery and precision of 8 AHLs in soil and bacterial suspension

[0063]

[0064]

[0065] In this example, sample spike recovery was performed on soil and LB bacterial suspension, and the recovery rates of the eight AHLs in soil were 79.21-107.9%, and the recovery rates in LB bacterial suspension were 84.59-112.32%.

[0066] Example 5

[0067] This example is to determine 8 AHLs in different types of soil and bacterial suspensions:

[0068] (1) Sample information: soil A (loam, pH = 4.76, Luzhou, Sichuan, walnut plantation forest, sampling depth 15 cm), soil B (loam, pH = 8.56, Chengdu, Sichuan, bamboo shoot plantation forest, sampling depth 25 cm), soil C (clay, pH = 5.10, Mianyang, Sichuan, garlic planting farmland, sampling depth 10 cm); bacterial suspension A (LB medium, inoculated with Shinellayambaruensis ZZ12), bacterial suspension B (MSB medium, inoculated with Sphingomonas sp. WK22), bacterial suspension C (AT medium, inoculated with Acinetobacter sp. ZY1), wherein the culture medium was purchased from Beijing Solebow Technology Co., Ltd. or self-prepared, and the inoculated bacteria were three AHLs-producing bacteria screened and purified from the soil.

[0069] (2) Sample pretreatment: Weigh 5g of soil or 2g of bacterial suspension into a 50mL centrifuge tube, and perform a blank matrix test at the same time. Add 2 glass balls to the centrifuge tube to homogenize, add 10mL of water to the soil sample, and vortex for 1min using a vortex shaker. Then add 10mL of acetonitrile, ultrasonicate at 150W for 25min, vortex for 1min, and freeze at -20℃ for 10min (the freezing step is critical. If it is not frozen or the freezing time is short, the addition of subsequent substances will generate a lot of heat and cause the test substance to be destroyed; if the freezing time is too long, it will affect the further mixing of subsequent substances). Take out and add 4g of anhydrous magnesium sulfate, 1g of sodium chloride, and 1g of sodium acetate. Vortex at 2500r / min for 1min, and centrifuge at 4000r / min for 10min. Accurately transfer 8mL of supernatant to dryness with nitrogen at 30℃, vortex for 1min with 1mL of 0.1% formic acid aqueous solution to dissolve, and transfer to a 2mL centrifuge tube for later use. Then clean the tube wall with 1mL 0.1% formic acid aqueous solution, and pipette the mixed solution into a 2mL centrifuge tube. Add 2mL of sample solution into Cleanert SLE column, apply positive pressure of 5 Hg vacuum for 1 second, wait for 5min, add 5mL×2 0.1% diethylamine-dichloromethane solution for elution, vacuum for 5s, and dry with nitrogen at 30℃. Redissolve with 1mL 20% methanol. Filter with 0.2μm polytetrafluoroethylene microporous filter membrane and wait for the machine.

[0070] (3) Preparation of standard curve: Pipette an appropriate amount of AHLs mixed standard and dilute it step by step to 1, 5, 20, 50, 100, and 200 ng / L using matrix blank solution. Then use LC-MSMS for sample injection and analysis.

[0071] The contents of AHLs in different soil and bacterial suspension samples are shown in Table 5.

[0072] Table 5 AHLs content in different soil and bacterial suspension samples

[0073]

[0074] Example 6

[0075] This example is to determine 8 AHLs in different types of soil and bacterial suspensions. The experimental parameter range is different from that of Example 5, and the test results are similar to those of Example 5:

[0076] (1) Sample information: Same as Example 5(1).

[0077] (2) Sample pretreatment: Weigh 10g of soil or 15g of bacterial suspension into a 50mL centrifuge tube, and perform a blank matrix test at the same time. Add 2 glass balls to the centrifuge tube to homogenize the protons. Add 15mL of water to the soil sample and vortex for 2min using a vortex shaker. Then add 12mL of acetonitrile, ultrasonicate at 250W for 15min, vortex for 2min, freeze at -20℃ for 15min, take out and add 6g of anhydrous magnesium sulfate, 4g of sodium chloride, and 2g of sodium acetate, vortex at 3000r / min for 2min, and centrifuge at 6000r / min for 5min. Accurately transfer 10mL of supernatant and blow dry with nitrogen at 30℃, re-dissolve with 1mL of 0.2% formic acid aqueous solution, vortex for 1min, and transfer to a 2mL centrifuge tube for later use. Then clean the test tube wall with 1mL of 0.2% formic acid aqueous solution, and transfer to a 2mL centrifuge tube to mix the solution. Add 2mL of sample solution to Novum TM In the SLE column, apply vacuum pressure of 5 Hg column for 2 seconds, wait for 10 minutes, add 5mL×3 0.2% diethylamine-dichloromethane solution for elution, evacuate for 5 seconds, and dry with nitrogen at 30℃. Redissolve with 1mL 40% methanol. Filter with 0.2μm polytetrafluoroethylene microporous filter membrane and wait for loading.

[0078] (3) Preparation of standard curve: Pipette an appropriate amount of AHLs mixed standard and dilute it step by step to 1, 5, 10, 20, 50, and 200 ng / L using matrix blank solution. Then use LC-MSMS for sample injection and analysis.

[0079] The contents of AHLs in different soil and bacterial suspension samples are shown in Table 6.

[0080] Table 6 AHLs content in different soil and bacterial suspension samples

[0081]

[0082] Examples 1 to 6 describe a method for quantifying AHLs in soil and bacterial suspensions based on LC-MSMS. The method includes verification of the calibration curve, determination of the detection limit, quantification limit, recovery rate and precision, screening of pretreatment extraction solvents, loading reagents and elution reagents, etc. A method for detecting AHLs with high sensitivity, high recovery rate and accuracy is obtained. At the same time, the method takes a short time to detect and uses less organic reagents, which is in line with the concept of green chemistry.

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for quantitatively detecting AHLs in soil and bacterial suspension based on LC-MSMS, characterized in that: The steps include: (1) Sample processing: The sample to be tested is extracted and purified twice in sequence to obtain a test solution; (a) Extraction: The analyte and acetonitrile are ultrasonically vortexed to obtain an extract; In the step (a), the test object is a bacterial suspension or soil; when the test object is a bacterial suspension, the sample weight is 2 to 15 g; when the test object is soil, the soil and water are first mixed and then mixed with acetonitrile, and the ratio of soil to water is 4 to 10 g: 9 to 12 mL; (b) Purification: The extract is frozen and mixed with anhydrous magnesium sulfate, sodium chloride and sodium acetate, and then centrifuged to obtain the supernatant to complete the first purification; 4 to 10 mL of the supernatant is removed and sequentially dried, re-dissolved, solid phase supported liquid-liquid extraction, secondary purification, dried, re-dissolved and filtered to obtain the test solution; In the step (b), the ratio of the extract, anhydrous magnesium sulfate, sodium chloride and sodium acetate is 12-35 g: 4-6 g: 1-4 g: 1-2 g; The re-dissolution includes a first re-dissolution and a second re-dissolution; the reagent used in the first re-dissolution is a formic acid aqueous solution, and the mass fraction of the formic acid aqueous solution is 0.1-0.2%; the reagent used in the second re-dissolution is a methanol solution, and the mass fraction of the methanol solution is 20-40%; The reagent used for the solid phase supported liquid extraction elution is a diethylamine dichloromethane solution, and the mass fraction of diethylamine in the dichloromethane solution is 0.1-0.2%; (2) Preparation of standard substances: 3-OH-C4-HSL, 3-oxo-C6-HSL, 3-OH-C8-HSL, 3-oxo-C8-HSL, 3-oxo-C10-HSL, 3-OH-C12-HSL, 3-oxo-C12-HSL, and 3-OH-C14-HSL were prepared into eight kinds of AHLs mixed standard stock solutions; the mixed standard concentration for establishing the mass spectrometry method was 50 μg / L, and the standard curve concentrations were 0.001 to 300 μg / L; The solvent used in the preparation of the AHLs mixed standard stock solution is a blank matrix solution obtained by pre-treatment of soil or bacterial suspension; the soil blank matrix is ​​a mixture of soil and quartz sand, with a ratio of 1:3 to quartz sand, which has been verified to contain no analyte through multiple verifications and is a blank matrix of soil; (3) Chromatographic conditions and mass spectrometry conditions: The chromatographic conditions are as follows: chromatographic column: biphenyl column; mobile phase A: 0.05% acetic acid aqueous solution containing 0.1-0.3 mM ammonium formate; mobile phase B: 0.05% acetic acid methanol solution containing 0.1-0.3 mM ammonium formate; column temperature: 30-50°C; injection volume: 2-10 μL; mass spectrometry conditions: electrospray ion source, curtain gas: 30-50 psi, collision gas: Medium, ionization voltage: 4500-5000 V, temperature: 450-500°C, spray gas: 50-55 psi, auxiliary heating gas: 55-60 psi; (4) Calculate the AHLs content.

2. The method for quantitatively detecting AHLs in soil and bacterial suspension based on LC-MSMS according to claim 1, characterized in that: In the step (a), the power of ultrasonic mixing is 150-250W, and the time of ultrasonic mixing is 15-25min; the time of vortex mixing is 1-2min; the speed of centrifugation is 4000-6000r / min, and the time of centrifugation is 4-10min.

3. A method for quantitatively detecting AHLs in soil and bacterial suspension based on LC-MSMS according to claim 1 or 2, characterized in that: In the step (b), the mixture is frozen at -20°C for 10 to 15 minutes.

4. The method for quantitatively detecting AHLs in soil and bacterial suspension based on LC-MSMS according to claim 1 or 2, characterized in that: In the step (b), drying is performed by passing nitrogen through a water bath at 30-40°C.

5. The method for quantitatively detecting AHLs in soil and bacterial suspension based on LC-MSMS according to claim 1 or 2, characterized in that: The blank matrix of the bacterial suspension in step (2) is the corresponding bacterial culture medium; the soil used in the soil blank matrix is ​​dry soil aged for more than 2 years, which is fully mixed with 40-80 mesh quartz sand.

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