A method for quantitative detection of AHLs in soil and bacterial suspensions based on LC-MSMS

Abstract

The application relates to the technical field of analysis, and discloses a method for quantitatively detecting AHLs in soil and bacterial suspension based on LC-MSMS. In view of the shortcomings of the prior art, the purpose of the application is to provide an AHLs detection method which has higher sensitivity and lower detection limit compared with the prior art. Through method verification, the method is suitable for two substrates of soil and bacterial suspension. The method adopts dispersed liquid phase extraction-solid phase support liquid-liquid extraction, greatly reduces extraction time, matrix effect and the amount of organic reagent.

Description

Technical Field

[0001] This invention relates to the field of analytical technology, and in particular to a method for quantitative detection of AHLs in soil and bacterial suspensions based on LC-MSMS. Background Technology

[0002] Quorum sensing is a communication mechanism in bacteria that uses specialized chemical signaling molecules to exchange information and coordinate behavior within a population or community. Through this mechanism, bacteria can respond to environmental changes, adjust their metabolic activities, control downstream changes at the genetic level, collectively adapt to various conditions, and regulate many biological functions, such as the secretion of toxic factors, antibiotic synthesis, and biofilm synthesis. Based on the form and sensing mode of quorum sensing molecules, these signaling molecules can be classified into three types: oligopeptides, N-acylated homoserine lactones (AHLs), and AI-2 type autoinducers. AHLs are signaling molecules for intraspecies communication in Gram-negative bacteria, and are currently the earliest discovered, most fully characterized, and most comprehensively studied class of signaling molecules. Besides regulating bacteria, AHLs can also be sensed by plants, thereby regulating plant physiological behaviors, including morphogenesis, innate immunity, growth and development, and stress tolerance. Studies have shown that applying AHL-secreting bacteria to the soil or directly applying AHLs 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. Applying 3-oxo-C6-HSL and 3-oxo-C8-HSL can significantly increase the root length and survival rate of Arabidopsis thaliana under salt stress, and also significantly improve the salt and drought tolerance of wheat in the early growth stages. Biofilms, as an emerging immobilized microbial remediation technology, have attracted increasing attention in the remediation of POPs-contaminated soils, and low concentrations of exogenous AHLs can promote the degradation of phenanthrene and pyrene by biofilms.

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

[0004] In summary, AHLs can influence the environment, plant gene expression, and function through quorum sensing effects, resulting in beneficial production attributes. Preparing bacterial suspensions is an important method for studying AHLs; therefore, rapidly and accurately detecting the content and variation patterns of AHLs in soil and bacterial suspensions is a crucial foundation for studying plant quorum sensing behavior and mechanisms.

[0005] Reports on the detection of N-acylhomoserine lactones (AHLs) in soil and bacterial suspensions are limited. Currently, liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry are the main methods used for detection. For example, CN 103983729B describes a gas chromatography-mass spectrometry method for detecting acylhomoserine lactones in soil solution, which determined seven AHLs in soil solution with a detection limit range of 1.5–2.5 μg / L and a recovery rate of 54.29–96.67%. CN106290668A describes an extraction, purification, and determination method for acylhomoserine lactones in soil and its application, which used GC-MS to determine seven AHLs in soil with a detection limit of 0.75–1.25 μg / kg. Two studies on the use of GC-MS for AHLs determination showed high detection limits because AHLs have high boiling points, both exceeding 300℃ (C10-HSL boiling point is 466.1℃, C12-HSL boiling point is 485.8℃, 3-OH-C4-HSL boiling point is 504.6℃, etc.), making them unsuitable for GC-MS detection. The high detection limits make it difficult to comprehensively evaluate the types and contents of low-level AHLs in a sample. Ma Chenchen detected AHLs in LB medium by centrifuging the medium at 27000 rpm for 20 min, extracting the supernatant with ethyl acetate, evaporating to dryness, and reconstituted with methanol for LC-MS analysis. This method also has serious limitations. While high-speed centrifugation purified the matrix, it caused a large amount of AHL-containing extracellular polymeric substances to settle to the bottom without being extracted and purified, resulting in low AHLs quantification results. Furthermore, this method has a high detection limit (0.1–1.0 μg / kg). Summary of the Invention

[0006] In view of this, the present invention provides a method for quantitative detection of AHLs in soil and bacterial suspensions based on LC-MSMS to solve the following problems: Currently, the extraction of AHLs mainly uses ethyl acetate for liquid-liquid extraction, which has a long extraction time, a large matrix effect in the extract, and uses a large amount of organic reagents, which is not conducive to the detection of large-scale samples; the method of AHLs purification is to directly ultracentrifuge the sample, causing the extracellular polymers containing AHLs to settle to the bottom with impurities, resulting in low detection results; existing methods have high detection limits for AHLs, which cannot accurately quantify low concentrations of AHLs in soil and bacterial suspensions.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for quantitative detection of AHLs in soil and bacterial suspensions based on LC-MSMS includes the following steps:

[0009] (1) Sample preparation: The analyte is extracted and purified twice in sequence to obtain the test solution;

[0010] (a) Extraction: The analyte and acetonitrile were mixed by ultrasonic vortex to obtain the extract;

[0011] In step (a), the analyte is a bacterial suspension or soil. When the analyte is a bacterial suspension, the sample weight is 2-15g. When the analyte is soil, the soil and water must be mixed first and then mixed with acetonitrile. The ratio of soil to water is 4-10g:9-12mL.

[0012] (b) Purification: After freezing the extract, mix it with anhydrous magnesium sulfate, sodium chloride and sodium acetate, centrifuge and collect the supernatant to complete the first purification; transfer 4 to 10 mL of the supernatant and sequentially perform the following purification steps: drying, resolution, solid-phase support liquid-liquid extraction, drying, resolution and filtration to obtain the test solution;

[0013] In 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 redissolution includes a first redissolution and a second redissolution; the reagent used for the first redissolution is an aqueous formic acid solution with a mass fraction of 0.1-0.2%; the reagent used for the second redissolution is a methanol solution with a mass fraction of 20-40%.

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

[0016] (2) Preparation of standards: 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 8 AHLs mixed standard stock solutions; the mixed standard concentration for 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 preparing the AHLs mixed standard stock solution is a blank matrix solution obtained by pretreatment of soil or bacterial suspension; the soil blank matrix is ​​a mixture of soil and quartz sand in a ratio of 1:3, which has been verified multiple times to be free of the analyte and is therefore a soil blank matrix.

[0018] (3) Chromatographic and mass spectrometry conditions: The chromatographic conditions are as follows: Column: diphenyl column; Mobile phase A: 0.05% aqueous acetic acid solution containing 0.1-0.3mM ammonium formate; Mobile phase B: 0.05% methanolic acetic acid solution containing 0.1-0.3mM ammonium formate; Column temperature: 30-50℃; Injection volume: 2-10μL; The mass spectrometry conditions are as follows: Electrospray ionization source, curtain gas 30-50psi, collision gas Medium, ionization voltage 4500-5000V, temperature 450-500℃, spray gas 50-55psi, auxiliary heating gas 55-60psi;

[0019] (4) Calculate the AHL content.

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

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

[0022] Furthermore, in step (b), the drying is carried out using a water bath at 30-40°C with nitrogen gas.

[0023] Furthermore, the blank substrate for the bacterial suspension in step (2) is the corresponding bacterial culture medium; the soil used for the blank substrate is dry soil that has been aged for more than 2 years and is thoroughly mixed with 40-80 mesh quartz sand.

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

[0025] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] To address the shortcomings of existing technologies, this invention offers higher sensitivity and lower detection limits compared to other methods for determining AHLs in soil and bacterial suspensions. It significantly reduces detection time and the use of organic reagents, enabling large-scale sample processing. Through two purification processes, it substantially reduces matrix effects and instrument contamination. Furthermore, it exhibits excellent linearity, accuracy, and repeatability, raising the detection limits for AHLs in soil and bacterial suspensions to 0.517–2.670 ng / kg, more than a hundredfold improvement over existing methods. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0028] Figure 1 The above are the extracted ion chromatograms of the eight 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 The standard curves for the eight AHLs in Example 1;

[0030] Figure 3 The following are the secondary mass spectra of the eight AHLs in Example 1;

[0031] Figure 4 This is the extractive ion chromatogram of the blank soil matrix solution in Example 3. Detailed Implementation

[0032] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

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

[0034] All commonly used 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-mass spectrometry system (AB SCIEX QTRAP 6500, USA); methanol, acetic acid, ammonium formate (mass spectrometry grade, Thermo Fisher Scientific, USA); chromatographic column (bisphenyl column, 2.1×100mm×1.7μm, Finova Corporation, USA); standards: 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 reagents and consumables: Novum. TM SLE column (8B-S138-KDG, Fenoxac, USA), CleanertSLE pH=9 (HC0006Q-9, Aijieer, China).

[0036] Example 1

[0037] This embodiment measures and verifies the linearity of the method:

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

[0039] (2) Mass spectrometry conditions: Electrospray ionization source was used, 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: Column was a diphenyl column; 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: initially mobile phase B was 20%, from 0 to 3 min, mobile phase B linearly increased from 20% to 90%, from 3 to 8 min, B remained at 90%, from 8.1 to 10 min, B recovered to 20%; column temperature: 40 °C; injection volume: 5 μL.

[0040] (3) The eight AHLs were completely separated by measurement. The peak shape was very good, with no tailing peaks, leading peaks, inverted peaks, or broad peaks. The separation was very good, with no baseline noise or drift.

[0041] Extraction ion chromatograms of the eight AHLs are shown below. Figure 1 The standard curves for the eight AHLs are shown below. Figure 2 Secondary mass spectra of the eight AHLs are shown below. 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 0.02–5 μg / L, R0 2 All values ​​are greater than 0.999. The above data indicate that the mass spectrometry and chromatographic methods in this example are accurate, linear, and highly sensitive, while also having a short detection time, with peak elution completed within 8 minutes, demonstrating extremely high detection efficiency.

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

[0043]

[0044] Table 2. Linear relationships among 8 types of AHLs

[0045]

[0046]

[0047] Example 2

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

[0049] For sample extraction and purification, this embodiment selected methanol, acetonitrile, ethyl acetate, and 1% acetic acid-acetonitrile as extraction solvents. Results showed that acetonitrile extraction yielded the best results. For dispersion-phase extraction, anhydrous magnesium sulfate, sodium chloride, sodium citrate, disodium citrate, sodium acetate, C18, PSA, and GCB were selected as purification agents. Results showed that anhydrous magnesium sulfate, sodium chloride, and sodium acetate were effective and cost-efficient. The optimal dosage was determined to be: 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 process, five solutions were selected for comparison of adsorption effects: ultrapure water, 20% methanol aqueous solution, sodium acetate buffer, 0.1% formic acid aqueous solution, and 5% ammonia aqueous solution. The results showed that 0.1% formic acid aqueous solution had the best loading effect. Five different elution solutions were selected for comparison of elution effects: ethyl acetate, dichloromethane, 1% acetic acid-ethyl acetate, 0.1% diethylamine-dichloromethane, and 0.1% diethylamine-ethyl acetate. The results showed that 0.1% diethylamine-dichloromethane eluted the highest recovery rate with the lowest matrix effect. Subsequently, experiments were conducted using 0.05%, 0.1%, 0.2%, 0.3%, and 0.5% formic acid aqueous solutions and 0.05%, 0.1%, 0.2%, 0.3%, and 0.5% diethylamine-dichloromethane for loading and elution. The results showed that 0.2% had the highest recovery rate, but the difference from 0.1% was not significant. Other concentrations significantly decreased the recovery rate.

[0050] Example 3

[0051] This example demonstrates the determination of the limits of detection and limits of quantitation for eight AHLs in soil and LB medium:

[0052] (1) Preparation of blank substrate solution: The blank substrate for soil was selected from dried soil aged for more than 2 years (verified to be free of target substances) and quartz sand mixed in a 1:3 ratio. The blank substrate for bacterial suspension was selected from uninoculated LB medium. Weigh 5g of soil blank substrate or 10g of LB medium into a 50mL centrifuge tube, homogenize two glass bulbs, add 10mL of water, and vortex for 1min. Then add 10mL of acetonitrile, sonicate at 170W for 15min, vortex for 1min, freeze at -20℃ for 10min, remove 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 it to near dryness with nitrogen at 30℃, then reconstitute it with 2mL of 0.1% formic acid aqueous solution by vortexing for 1min. Add 2mL of sample solution to Novum TM In an SLE column, apply a vacuum of 5 MJ column for 2 seconds, wait 5–10 min, add 5 mL × 2 of 0.1% diethylamine-dichloromethane solution for elution, apply vacuum for 5 s, and dry under nitrogen at 30°C. Redissolve in 1 mL of 20% methanol. Filter using a 0.2 μm polytetrafluoroethylene microporous membrane. Use the above-treated sample solution as a blank matrix solution. The extraction ion chromatogram of the soil blank matrix solution is shown below. Figure 4 .

[0053] (2) Preparation of matrix standards: Take an appropriate amount of AHLs mixed standard and use the above-prepared blank matrix to dilute stepwise to 0.2, 0.5, 1, 2, 5, 20 ng / L, and perform LC-MSMS analysis.

[0054] (3) The matrix standard curve showed a good linear relationship in the range of 0.5–20 ng / L, R0 2 For samples with a signal-to-noise ratio (SNR) above 0.997, the concentrations (0.5–20 ng / L) of eight AHL matrix standards were analyzed at the lowest detectable concentrations. The SNR was calculated using software. The limit of detection (LOD) was set at 3 times the SNR, and the limit of quantitation (LOQ) at 10 times the SNR. The LODs and LOQs for the eight AHLs in soil and LB broth are shown in Table 3. The LOD for soil ranged from 0.517 to 2.114 ng / kg, and the LOQ ranged from 1.723 to 7.047 ng / kg. The LOD for bacterial suspension ranged from 0.855 to 2.670 ng / kg, and the LOQ ranged from 2.850 to 8.90 ng / kg. This method exhibits high sensitivity and low LOD, enabling the analysis of AHLs at extremely low concentrations in samples.

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

[0056]

[0057]

[0058] Example 4

[0059] This example analyzes the recovery and precision of eight AHLs in soil and LB bacterial suspension samples:

[0060] (1) Weigh 5g of soil or 5g of MSB bacterial suspension into a 50mL centrifuge tube, add 0.1mL of a mixed standard solution of 0.1μg / L and 1μg / L of 8 AHLs, and prepare the corresponding unspiked sample at the same time. Add two glass bulbs to the centrifuge tube for homogenization, and add 10mL of water to the soil. Vortex for 1min. Add 10mL of acetonitrile, sonicate at 150W for 20min, vortex for 1min, freeze at -20℃ for 10min, remove 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 the supernatant and blow dry with nitrogen at 30℃. Reconstitute with 2mL of 0.1% formic acid aqueous solution by vortexing for 1min. Add 2mL of sample solution to Novum TM In the SLE column, apply a vacuum of 5 mercury column for 2 seconds, wait 5–10 minutes, add 5 mL × 2 of 0.2% diethylamine-dichloromethane solution for elution, apply vacuum for 5 seconds, and dry under nitrogen at 40°C. Redissolve in 1 mL of 20% methanol. Filter using a 0.2 μm polytetrafluoroethylene microporous membrane before use.

[0061] (2) Preparation of standard curve: Use a pipette to draw an appropriate amount of AHLs mixed standard, and use blank matrix solution to dilute stepwise to 1, 5, 20, 100 and 200 ng / L, and use LC-MSMS for analysis.

[0062] Recovery rate and precision are shown in Table 4.

[0063] Table 4. Recovery and precision of eight AHLs in soil and bacterial suspensions.

[0064]

[0065]

[0066] In this embodiment, spiked recovery of soil and LB bacterial suspensions was performed. The recovery rates of the eight AHLs in soil were 79.21%–107.9%, and the recovery rates in LB bacterial suspensions were 84.59%–112.32%.

[0067] Example 5

[0068] This example describes the determination of eight AHLs in different types of soil and bacterial suspensions:

[0069] (1) Sample information: Soil A (loam, pH=4.76, walnut plantation in Luzhou, Sichuan, sampling depth 15cm), soil B (loam, pH=8.56, bamboo shoot plantation in Chengdu, Sichuan, sampling depth 25cm), soil C (clay, pH=5.10, garlic plantation in Mianyang, Sichuan, sampling depth 10cm); 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 Acinetobacters sp.ZY1), wherein the culture medium was purchased from Beijing Solarbio Science & Technology Co., Ltd. or prepared by ourselves, and the inoculated bacteria were three bacteria that produce AHLs that were screened and purified in the soil.

[0070] (2) Sample pretreatment: Weigh 5g of soil or 2g of bacterial suspension into a 50mL centrifuge tube. Simultaneously perform a blank matrix test. Add two glass bulbs to the centrifuge tube to homogenize the sample. Add 10mL of water to the soil sample and vortex for 1min. Add 10mL of acetonitrile, sonicate at 150W for 25min, vortex for 1min, and freeze at -20℃ for 10min (the freezing step is crucial; failure to freeze or short freezing time will generate excessive heat when adding subsequent substances, potentially damaging the analyte; excessive freezing time will affect further mixing). Remove the sample 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 the supernatant, dry it under nitrogen at 30℃, and reconstitute it with 1mL of 0.1% formic acid aqueous solution by vortexing for 1min. Transfer the reconstituted solution to a 2mL centrifuge tube for later use. Wash the test tube wall with 1 mL of 0.1% formic acid aqueous solution, and transfer the solution to a 2 mL centrifuge tube. Add 2 mL of sample solution to a Cleanert SLE column, apply a vacuum of 5 mercury column for 1 second, wait 5 min, add 5 mL × 2 of 0.1% diethylamine-dichloromethane solution for elution, apply vacuum for 5 s, and dry under nitrogen at 30°C. Redissolve in 1 mL of 20% methanol. Filter using a 0.2 μm polytetrafluoroethylene microporous membrane before processing.

[0071] (3) Preparation of standard curve: Use a pipette to draw an appropriate amount of AHLs mixed standard, and use matrix blank solution to dilute stepwise to 1, 5, 20, 50, 100 and 200 ng / L, and use LC-MSMS for sample analysis.

[0072] The contents of AHLs in different soil and bacterial suspension samples are shown in Table 5.

[0073] Table 5. Content of AHLs in different soil and bacterial suspension samples

[0074]

[0075]

[0076] Example 6

[0077] This example measures eight AHLs in different types of soil and bacterial suspensions. The experimental parameters differ from those in Example 5, but the detection results are similar to those in Example 5.

[0078] (1) Sample information: Same as Example 5(1).

[0079] (2) Sample pretreatment: Weigh 10g of soil or 15g of bacterial suspension into a 50mL centrifuge tube. Simultaneously perform a blank matrix test. Add two glass bulbs to the centrifuge tube to homogenize the sample. Add 15mL of water to the soil sample and vortex for 2min. Add 12mL of acetonitrile, sonicate at 250W for 15min, vortex for 2min, freeze at -20℃ for 15min, remove and add 6g of anhydrous magnesium sulfate, 4g of sodium chloride, and 2g of sodium acetate. Vortex at 3000r / min for 2min, centrifuge at 6000r / min for 5min. Accurately transfer 10mL of the supernatant and dry it under nitrogen at 30℃. Reconstitute with 1mL of 0.2% formic acid aqueous solution by vortexing for 1min, and transfer to a 2mL centrifuge tube for later use. Wash 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 a vacuum of 5 mercury column for 2 seconds, wait 10 minutes, add 5 mL × 3 of 0.2% diethylamine-dichloromethane solution for elution, apply vacuum for 5 seconds, and dry under nitrogen at 30°C. Redissolve in 1 mL of 40% methanol. Filter using a 0.2 μm polytetrafluoroethylene microporous membrane, ready for instrumentation.

[0080] (3) Preparation of standard curve: Use a pipette to draw an appropriate amount of AHLs mixed standard, and use matrix blank solution to dilute stepwise to 1, 5, 10, 20, 50, 200 ng / L, and use LC-MSMS for analysis.

[0081] The contents of AHLs in different soil and bacterial suspension samples are shown in Table 6.

[0082] Table 6. Content of AHLs in different soil and bacterial suspension samples

[0083]

[0084]

[0085] Examples 1-6 describe a method for the quantitative detection of AHLs in soil and bacterial suspensions based on LC-MSMS. This includes validation of the calibration curve, determination of the limit of detection, limit of quantitation, recovery rate, and precision, and screening of pretreatment extraction solvents, loading reagents, and elution reagents. A highly sensitive, highly recoverable, and accurate method for AHLs detection was obtained. Furthermore, this method has a short detection time and requires minimal organic reagents, aligning with the principles of green chemistry.

[0086] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for quantitative detection of AHLs in soil and bacterial suspensions based on LC-MSMS, characterized in that, Includes the following steps: (1) Sample preparation: The analyte is extracted and purified twice in sequence to obtain the test solution; (a) Extraction: The analyte and acetonitrile were mixed by ultrasonic vortex to obtain the extract; In step (a), the analyte is a bacterial suspension or soil. When the analyte is a bacterial suspension, the sample weight is 2-15g. When the analyte is soil, the soil and water must be mixed first and then mixed with acetonitrile. The ratio of soil to water is 4-10g:9-12mL. (b) Purification: After freezing the extract, it is mixed with anhydrous magnesium sulfate, sodium chloride and sodium acetate, and centrifuged to collect the supernatant to complete the first purification; 4-10 mL of the supernatant is transferred and sequentially subjected to drying, first redissolution, solid-phase support liquid-liquid extraction for secondary purification, drying, second redissolution and filtration to obtain the test solution; In step (b), the ratio of the extract, anhydrous magnesium sulfate, sodium chloride, and sodium acetate is 12-35g: 4-6g: 1-4g: 1-2g. The reagent used for the first resolution is an aqueous formic acid solution with a mass fraction of 0.1-0.2%; the reagent used for the second resolution is a methanol solution with a mass fraction of 20-40%. The reagent used for solid-phase supported liquid-liquid extraction elution is a diethylamine solution in dichloromethane, wherein the mass fraction of diethylamine in the dichloromethane solution is 0.1-0.2%. (2) Preparation of standards: 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 8 AHLs mixed standard stock solutions; the mixed standard concentration for the mass spectrometry method was 50 μg / L, and the standard curve concentrations were 0.001~300 μg / L; The solvent used in preparing the AHLs mixed standard stock solution is a blank matrix solution obtained by pretreatment of soil or bacterial suspension; the soil blank matrix is ​​a mixture of soil and quartz sand in a ratio of 1:3, which has been verified multiple times to be free of the analyte and is therefore a soil blank matrix. (3) Chromatographic and mass spectrometric conditions: The chromatographic conditions are as follows: Column: diphenyl column; Mobile phase A: 0.05% aqueous acetic acid solution containing 0.1-0.3 mM ammonium formate; Mobile phase B: 0.05% methanolic acetic acid solution containing 0.1-0.3 mM ammonium formate; Column temperature: 30-50℃; Injection volume: 2-10 μL; Gradient elution conditions: Initially, mobile phase B is 20%, and within 0-3 min, mobile phase B linearly increases from 20% to... 90%, 3-8 min, B remains at 90%, 8.1-10 min, B recovers to 20%; the mass spectrometry conditions are: electrospray ionization 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. The parent ion, daughter ion, declustering voltage and collision energy parameters used for detecting 8 AHLs are shown in the table below. ； (4) Calculate the AHL content.

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

3. A method for quantitative detection of AHLs in soil and bacterial suspensions based on LC-MSMS according to claim 1 or 2, characterized in that, In step (b), the extract is frozen at -20°C for 10-15 minutes.

4. A method for quantitative detection of AHLs in soil and bacterial suspensions based on LC-MSMS according to claim 1 or 2, characterized in that, In step (b), the drying is carried out using a 30-40℃ water bath with nitrogen gas.

5. A method for quantitative detection of AHLs in soil and bacterial suspensions based on LC-MSMS according to claim 1 or 2, characterized in that, The blank substrate for the bacterial suspension in step (2) is the corresponding bacterial culture medium; the soil used for the blank substrate is dry soil that has been aged for more than 2 years and is thoroughly mixed with 40-80 mesh quartz sand.

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