Soil pesticide residue grading extraction and triple quadrupole liquid chromatography-mass spectrometry detection method

Abstract

The invention relates to the technical field of pesticide residue analysis, and particularly discloses a soil pesticide residue graded extraction and triple quadrupole liquid chromatography-mass spectrometry detection method. Comprising the following steps: S1, removing foreign matters from a soil sample, freeze-drying, grinding and sieving; s2, mixing the soil powder with an inert adsorbent to obtain a dispersion matrix; s3, loading the dispersed matrix into an extraction tank, and performing first-stage extraction by adopting a mixed solvent; s4, performing second-stage extraction on the residues in S3 by adopting a halogenated hydrocarbon-alcohol mixed solvent; s5, extracting the residues in S4 by using a nitrile-acidic aqueous solution mixed solvent; and S6, mixing the extract liquids obtained in S3, S4 and S5, and purifying through a solid-phase extraction column to obtain a pesticide residue to-be-detected liquid subjected to graded extraction. The graded extraction method can be used for systematic screening and quantitative analysis of pesticide residues in a complex soil matrix in environmental monitoring, and has the advantage of improving the accuracy, reproducibility and flux of analysis for measuring multiple types of pesticide residues in a complex sample.

Description

Technical Field

[0001] This application relates to the field of pesticide residue analysis technology, and more specifically, to a method for graded extraction of pesticide residues in soil and detection using triple quadrupole liquid chromatography-mass spectrometry. Background Technology

[0002] Soil pesticide residue extraction, as a pretreatment step in pesticide residue analysis, is applied across fields such as environmental monitoring, agricultural product safety traceability, contaminated site assessment, and ecological risk research. By efficiently separating and enriching pesticide residues in soil, it lays the foundation for subsequent accurate detection. Its advantages lie in significantly improving the sensitivity and accuracy of analytical methods. With the help of modern extraction technologies such as solid-phase extraction and QuEChERS, matrix interference can be effectively reduced and recovery rates can be improved. At the same time, process optimization enables simultaneous analysis of multiple residues, greatly improving detection efficiency and providing technical support for comprehensively assessing soil pollution status, formulating remediation strategies, and ensuring agricultural sustainability.

[0003] Related soil pesticide residue extraction uses solid-phase extraction to achieve simultaneous analysis of multiple residues. However, this method is difficult to overcome the differences in extraction efficiency of pesticides with different polarities. It is not effective for extracting highly polar or ionic pesticide residues and is easily affected by soil matrix co-extractants. As a result, the overall recovery rate of pesticide residues with a wide range of polarities fluctuates greatly and the accuracy is limited. It is difficult to meet the high reliability analysis requirements of trace multi-category pesticide residues in complex soil environments. Summary of the Invention

[0004] To address the issue of large fluctuations in the overall recovery rate and limited accuracy of pesticide residues with a wide polarity range due to the simultaneous analysis of multiple residues through solid-phase extraction in soil pesticide residue extraction, this application provides a method for graded extraction of soil pesticide residues and triple quadrupole liquid chromatography-mass spectrometry (LC-MS) detection.

[0005] Firstly, this application provides a method for the graded extraction of pesticide residues in soil, employing the following technical solution: The method for graded extraction of pesticide residues in soil includes the following steps: S1. Remove foreign matter from the soil sample, freeze-dry until the moisture content is <5%, grind and sieve to obtain dry soil powder; S2. The soil powder obtained in S1 is mixed with an inert adsorbent at a mass ratio of (2:1) to (4:1) to obtain a dispersion matrix; S3. The dispersion matrix obtained in S2 is loaded into the extraction tank, and a first-stage extraction is performed using a non-polar-weakly polar mixed solvent. The non-polar-weakly polar pesticide residue extract is collected. S4. Then, the residue of S3 is subjected to a second stage of pressurized fluid extraction using a mixed solvent of halogenated hydrocarbons and alcohols to collect the extract of medium-polarity pesticide residues. S5. The residue from S4 is subjected to a third-stage vortex-assisted extraction using a nitrile-acidic aqueous solution mixture to collect the extract of strongly polar or ionic pesticide residues. The extract obtained by mixing S6, S3, S4 and S5 is purified by a composite adsorbent solid phase extraction column, and then concentrated by purging with inert gas to obtain the graded pesticide residue test solution.

[0006] By adopting the above technical solution, a staged extraction strategy is used to sequentially extract pesticide residues of different polarities from the soil using non-polar to weakly polar, moderately polar, and strongly polar solvents. This step utilizes the solvent polarity gradient to match the physicochemical properties of pesticide residues, thereby avoiding the limitations of single-solvent extraction. The first stage of extraction prioritizes the extraction of non-polar pesticide residues, reducing interference from subsequent steps. The second stage of pressurized fluid extraction improves the recovery rate of moderately polar pesticide residues under controllable conditions. The third stage of vortex-assisted extraction enhances the mass transfer efficiency of strongly polar pesticide residues through mechanical stirring. The final mixing and purification step removes matrix interference through a composite adsorbent and prevents oxidation loss through inert gas purging. This method can comprehensively cover pesticide residues of different polarities, improving extraction efficiency and accuracy.

[0007] Preferably, in step S1, the freeze-drying process further includes a pretreatment step: freezing the soil sample at -18°C to -25°C for 1.5 to 3.5 hours, followed by mechanical vibration to disperse the agglomerated particles, with a vibration frequency of 150 to 250 rpm and a time of 8 to 12 minutes.

[0008] By adopting the above technical solution, the low-temperature freeze-drying treatment causes the moisture and organic matter in the soil sample to form an ice crystal structure, which enhances the brittleness of the soil particles and facilitates subsequent mechanical vibration crushing. The vibration crushing step uniformly disperses the aggregated particles at a specific frequency and time, increasing the specific surface area and thus improving the uniformity and efficiency of freeze-drying. This pretreatment prevents uneven extraction caused by soil agglomeration during the drying process, providing a homogeneous matrix basis for subsequent fractional extraction and ensuring the representativeness and reproducibility of pesticide residue extraction.

[0009] Preferably, in step S2, the inert adsorbent is one or more of diatomaceous earth, Florisil, or quartz sand.

[0010] By adopting the above technical solution, diatomaceous earth, Florisil, or quartz sand are selected as inert adsorbents. These adsorbents have high specific surface area, porous structure, and chemical inertness, which can disperse soil powder, prevent agglomeration, and increase the contact interface. At the same time, the adsorbents themselves do not interfere with the target pesticide residues and can also remove interfering substances such as oils and pigments in the soil through physical adsorption, providing a uniform and pure matrix for subsequent extraction, thereby improving the reproducibility of the overall extraction process and the pesticide residue recovery rate.

[0011] Preferably, in step S3, the parameters of the first-stage extraction are as follows: pressure of 6~9 MPa, extraction temperature of 35~45℃, static extraction time of 8~12 min, and 1~2 cycles; and the non-polar-weakly polar mixed solvent is composed of the following components: the volume ratio of n-hexane: ethyl acetate: acetone is (4~5): (1~2): (0.5~1).

[0012] By adopting the above technical solution, the use of pressurized fluid extraction technology ensures solvent permeability by setting a pressure range, prevents degradation of heat-sensitive pesticide residues by temperature control, and ensures sufficient equilibrium by static time. In the solvent combination, n-hexane provides a non-polar environment, while ethyl acetate and acetone introduce weakly polar components. The polarity index is adjusted by volume ratio to match the dissolution requirements of non-polar pesticide residues. The number of cycles optimizes the extraction efficiency. This parameter combination enables the first-stage extraction to efficiently extract lipid-soluble pesticide residues, while reducing the matrix burden for the subsequent second-stage extraction and improving the overall selectivity of the method.

[0013] Preferably, in step S4, 0.05% to 0.3% of organic acid is added as a protonation aid in the second-stage extraction, the pH of the extract is controlled at 3.5 to 4.5, and dehydrating salt is added after extraction to dehydrate the water content to <0.2%; the halogenated hydrocarbon-alcohol mixed solvent is composed of the following components: the volume ratio of dichloromethane to methanol is (3 to 4): (1 to 2).

[0014] By adopting the above technical solution, the addition of organic acids such as formic acid or acetic acid as protonating agents lowers the pH of the extract, promoting the protonation of basic groups in moderately polar pesticide residues and enhancing their solubility in halogenated hydrocarbon-alcohol solvents. pH control prevents pesticide residue hydrolysis or ionization loss, and the dehydration step removes residual water using dehydrating salts such as anhydrous sodium sulfate, avoiding interference from the aqueous phase in subsequent analysis. This optimization ensures high recovery rates for moderately polar pesticide residues such as certain pesticides or metabolites in the second-stage extraction, while providing dry residue conditions for the third-stage extraction. The use of a mixed solvent of dichloromethane and methanol provides moderate polarity and high permeability, while methanol enhances the solubility of polar pesticide residues. The volume ratio optimization of solvent polarity matches the logP value of moderately polar pesticide residues, improving extraction selectivity. This solvent system works synergistically in pressurized fluid extraction to effectively extract pesticide residues such as organophosphorus or carbamates, while being compatible with the first-stage solvent, reducing cross-contamination.

[0015] Preferably, in step S6, the temperature of the inert gas purging and concentration is controlled at 25~35℃, and after concentration to near dryness, it is redissolved and brought to volume using a solvent compatible with the instrument, with a volume error of <1.5%.

[0016] By adopting the above technical solution, the purging temperature is controlled within a mild range to prevent the volatilization or decomposition of heat-sensitive pesticide residues, and inert gas such as nitrogen is used for purging to avoid oxidation. After being concentrated to near dryness, the solution is redissolved and brought to volume using instrument-compatible solvents such as acetonitrile or methanol to ensure compatibility with subsequent detection methods. The volume adjustment error is strictly controlled to improve quantitative accuracy. This step ensures the stability and concentration consistency of the pesticide residue test solution, providing a reliable sample for liquid chromatography-mass spectrometry (LC-MS) detection.

[0017] Secondly, this application provides a triple quadrupole liquid chromatography-mass spectrometry (LC-MS) method for the graded extraction of pesticide residues in soil, employing the following technical solution: A triple quadrupole chromatography-mass spectrometry (LC-MS) method for detecting pesticide residues in soil by graded extraction includes the following steps: P1. The pesticide residue test solution is separated by reversed-phase chromatography column, wherein mobile phase A is a deionized aqueous solution containing volatile buffer salt and acidifying agent, and mobile phase B is a polar organic solvent, using a gradient elution program; P2. Use an electrospray ionization source to ionize in positive ion mode, and set the ion source temperature and the desolvation gas temperature. P3. Data acquisition was performed using multiple reaction monitoring (MRM) and quantitative analysis was conducted using the internal standard method. A standard working curve was established using internal standards labeled with deuterated or carbon-13 stable isotopes.

[0018] By employing the above technical solution, reversed-phase chromatography is used for separation. Volatile buffer salts such as ammonium formate and acidifying agents such as formic acid in mobile phase A adjust the pH, enhancing the retention behavior of polar pesticide residues. Mobile phase B, such as acetonitrile or methanol, achieves the separation of compounds of different polarities through gradient elution. Electrospray ionization promotes the protonation of pesticide residues in positive ion mode, and temperature control optimizes the solvent removal efficiency. Multiple reaction monitoring mode improves selectivity, and internal standard method compensates for matrix effects and instrument fluctuations. Thus, this method can achieve highly sensitive and specific quantitative detection of pesticide residues.

[0019] Preferably, in step P1, the gradient elution procedure is as follows: initially, a high proportion of mobile phase A is maintained; within 3-4 minutes, the proportion of mobile phase B is increased to a medium level; then, within 2-3 minutes, it is increased to a high proportion and maintained for a short time; finally, it is quickly restored to the initial proportion equilibrium, with the column temperature controlled at 30-40°C and the flow rate at 0.25-0.35 mL / min.

[0020] By adopting the above technical solution, due to the use of gradient elution program, the initial high proportion of aqueous phase retains strongly polar pesticide residues, while the proportion of organic phase is gradually increased to elute moderate to non-polar compounds, and the holding time ensures the degree of separation; column temperature control affects column efficiency and selectivity, and flow rate optimization balances separation time and back pressure; this program can effectively separate multiple pesticide residues in complex matrices, reduce co-elution phenomenon, and provide clear chromatographic peak shapes for mass spectrometry detection.

[0021] Preferably, in step P2, the mass spectrometry interface voltage is 2.8~3.2kV, the nebulization auxiliary gas flow rate is 1.8~2.5L / min, and the collision gas is a high-purity inert gas.

[0022] By adopting the above technical solutions, the ionization efficiency is optimized by setting the interface voltage, the droplet formation and desolvation process are controlled by the atomization-assisted gas flow rate, and the collision gas, such as nitrogen or argon, provides controllable collision-induced dissociation in multi-reaction monitoring mode, ensuring the generation of characteristic ion fragments. These parameters work together to improve the stability and sensitivity of the mass spectrometry signal, reduce background noise, and thus enhance the reliability and accuracy of detection.

[0023] Preferably, in step P3, the internal standard is added at the initial stage of sample preparation, and the concentration is set according to the expected residual level.

[0024] By adopting the above technical solution, since an internal standard, such as a deuterated analogue, is added at the beginning of sample extraction, its chemical properties are similar to those of the target pesticide residue but its mass spectrometry behavior is different. This allows for the tracking of losses and variations during the entire sample processing process. The concentration of the added standard matches the expected residue level, ensuring that the linear range of the standard working curve covers the actual sample. This internal standard method effectively corrects systematic errors in the extraction, purification, and detection stages, improving the accuracy and reproducibility of quantitative results.

[0025] In summary, this application has the following beneficial effects: 1. Because this application adopts a process that combines graded extraction with coupled detection, through the continuous coordination of three-stage extraction processes of non-polar to weakly polar, medium polar and strong polarity, the solvent polarity gradient is matched step by step with the physicochemical properties of pesticide residues. At the same time, combined with the improvement of parameters of subsequent chromatographic separation and mass spectrometry detection, the coverage and accurate quantification of pesticide residues in a wide range of polarities are obtained, thereby improving the extraction recovery rate and detection sensitivity of pesticide residues.

[0026] 2. In this application, the pretreatment and extraction parameters are preferably controlled in a coordinated manner. The low-temperature freeze solidification and mechanical vibration pretreatment provide a uniform matrix basis for subsequent extraction. The solvent ratio, pressure, temperature and pH value are controlled in each extraction stage, forming a continuous process from pretreatment to separation and purification, so as to achieve high reproducibility of pesticide residue extraction and low matrix interference.

[0027] 3. The method of this application combines the extraction process with the detection process. In the staged extraction, inert gas purging and concentration ensures the stability of the sample, while the internal standard method in the detection stage intervenes to correct from the beginning of extraction. Gradient elution and multiple reaction monitoring mode are specifically matched to the chromatographic behavior of each stage of the extract. Therefore, the analysis results are achieved with controllable error throughout the process and strong data accuracy. Attached Figure Description

[0028] Figure 1 This is a flowchart of the soil pesticide residue grading and extraction method proposed in this application; Figure 2 This is a flowchart of the triple quadrupole liquid chromatography-mass spectrometry (LC-MS) method for the graded extraction of pesticide residues in soil proposed in this application. Detailed Implementation

[0029] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0030] Example 1: This example provides a method for graded extraction of pesticide residues in soil, comprising the following steps: S1. Remove foreign matter from the soil sample, freeze-dry until the moisture content is <5%, grind and sieve to obtain dry soil powder; In step S1, a pretreatment step is included before freeze drying: the soil sample is frozen at -18℃ for 1.5h, and then mechanically shaken to disperse the aggregated particles at a frequency of 150rpm for 8min.

[0031] S2. Mix the soil powder obtained in S1 with an inert adsorbent at a mass ratio of 2:1 to obtain a dispersion matrix; The inert adsorbent is diatomaceous earth.

[0032] S3. The dispersion matrix obtained in S2 is loaded into the extraction tank, and a first-stage extraction is performed using a non-polar-weakly polar mixed solvent. The non-polar-weakly polar pesticide residue extract is collected. The parameters for the first-stage extraction were: pressure 6 MPa, extraction temperature 35℃, static extraction time 8 min, and one cycle; the non-polar to weakly polar mixed solvent consisted of n-hexane, ethyl acetate, and acetone in a volume ratio of 4:1:0.5. S4. Then, the residue of S3 is subjected to a second stage of pressurized fluid extraction using a mixed solvent of halogenated hydrocarbons and alcohols to collect the extract of medium-polarity pesticide residues. In the second stage of extraction, 0.05% organic acid was added as a protonation aid to control the pH of the extract to 3.5. After extraction, dehydrating salt was added to dehydrate the extract so that the residual water content was <0.2%. The halogenated hydrocarbon-alcohol mixed solvent consisted of dichloromethane and methanol in a volume ratio of 3:1. S5. The residue from S4 is subjected to a third-stage vortex-assisted extraction using a nitrile-acidic aqueous solution mixture to collect the extract of strongly polar or ionic pesticide residues. S6, the extract obtained by mixing S3, S4 and S5, is purified by a composite adsorbent solid phase extraction column, and concentrated by inert gas purging to obtain the graded extract of pesticide residue test solution. The inert gas purging and concentration temperature is controlled at 25℃. After being concentrated to near dryness, it is redissolved and brought to volume using a solvent compatible with the instrument, with a volume error of <1.5%.

[0033] The above-mentioned method for detecting pesticide residues in soil using a triple quadrupole chromatography-mass spectrometry (LC-MS) technique involves the following steps: P1. The pesticide residue test solution is separated by reversed-phase chromatography column, wherein mobile phase A is a deionized aqueous solution containing volatile buffer salt and acidifying agent, and mobile phase B is a polar organic solvent, using a gradient elution program; The gradient elution procedure is as follows: initially, maintain a high proportion of mobile phase A, increase the proportion of mobile phase B to a medium level within 3 minutes, then increase it to a high proportion within 2 minutes and maintain it for a short time, and finally quickly restore it to the initial proportion equilibrium. The column temperature is controlled at 30℃ and the flow rate is 0.25 mL / min.

[0034] P2. Use an electrospray ionization source to ionize in positive ion mode, and set the ion source temperature and the desolvation gas temperature. The mass spectrometer interface voltage is 2.8 kV, the nebulization auxiliary gas flow rate is 1.8 L / min, and the collision gas is a high-purity inert gas.

[0035] P3. Data acquisition was performed using multiple reaction monitoring (MRM) and quantitative analysis was conducted using the internal standard method. A standard working curve was established using internal standards labeled with deuterated or carbon-13 stable isotopes.

[0036] The internal standard is added at the initial stage of sample preparation, and the concentration is set according to the expected residual level.

[0037] Example 2: This example provides a method for graded extraction of pesticide residues in soil, comprising the following steps: S1. Remove foreign matter from the soil sample, freeze-dry until the moisture content is <5%, grind and sieve to obtain dry soil powder; In step S1, a pretreatment step is included before freeze drying: the soil sample is frozen solidified at -21.5℃ for 2.5h, and then mechanically shaken to disperse the aggregated particles at a frequency of 200rpm for 10min.

[0038] S2. Mix the soil powder obtained in S1 with an inert adsorbent at a mass ratio of 3:1 to obtain a dispersion matrix; The inert adsorbent is Florisil.

[0039] S3. The dispersion matrix obtained in S2 is loaded into the extraction tank, and a first-stage extraction is performed using a non-polar-weakly polar mixed solvent. The non-polar-weakly polar pesticide residue extract is collected. The parameters for the first-stage extraction were: pressure 7.5 MPa, extraction temperature 40℃, static extraction time 10 min, and one cycle; the non-polar-weakly polar mixed solvent consisted of n-hexane, ethyl acetate, and acetone in a volume ratio of 4.5:1.5:0.75. S4. Then, the residue of S3 is subjected to a second stage of pressurized fluid extraction using a mixed solvent of halogenated hydrocarbons and alcohols to collect the extract of medium-polarity pesticide residues. In the second stage of extraction, 0.175% organic acid was added as a protonation aid to control the pH of the extract to 4.0. After extraction, dehydrating salt was added to dehydrate the extract so that the residual water content was <0.2%. The halogenated hydrocarbon-alcohol mixed solvent consisted of dichloromethane and methanol in a volume ratio of 3.5:1.5. S5. The residue from S4 is subjected to a third-stage vortex-assisted extraction using a nitrile-acidic aqueous solution mixture to collect the extract of strongly polar or ionic pesticide residues. S6, the extract obtained by mixing S3, S4 and S5, is purified by a composite adsorbent solid phase extraction column, and concentrated by inert gas purging to obtain the graded extract of pesticide residue test solution. The inert gas purging and concentration temperature is controlled at 30℃. After being concentrated to near dryness, it is redissolved and brought to volume using a solvent compatible with the instrument, with a volume error of <1.5%.

[0040] The above-mentioned method for detecting pesticide residues in soil using a triple quadrupole chromatography-mass spectrometry (LC-MS) technique involves the following steps: P1. The pesticide residue test solution is separated by reversed-phase chromatography column, wherein mobile phase A is a deionized aqueous solution containing volatile buffer salt and acidifying agent, and mobile phase B is a polar organic solvent, using a gradient elution program; The gradient elution procedure is as follows: initially, maintain a high proportion of mobile phase A, increase the proportion of mobile phase B to a medium level within 3.5 minutes, then increase it to a high proportion within 2.5 minutes and maintain it for a short time, and finally quickly restore it to the initial proportion equilibrium. The column temperature is controlled at 35℃ and the flow rate is 0.3 mL / min.

[0041] P2. Use an electrospray ionization source to ionize in positive ion mode, and set the ion source temperature and the desolvation gas temperature. The mass spectrometer interface voltage is 3.0 kV, the nebulization auxiliary gas flow rate is 2.15 L / min, and the collision gas is a high-purity inert gas.

[0042] P3. Data acquisition was performed using multiple reaction monitoring (MRM) and quantitative analysis was conducted using the internal standard method. A standard working curve was established using internal standards labeled with deuterated or carbon-13 stable isotopes.

[0043] The internal standard is added at the initial stage of sample preparation, and the concentration is set according to the expected residual level.

[0044] Example 3: This example provides a method for graded extraction of pesticide residues in soil, comprising the following steps: S1. Remove foreign matter from the soil sample, freeze-dry until the moisture content is <5%, grind and sieve to obtain dry soil powder; In step S1, a pretreatment step is included before freeze drying: the soil sample is frozen at -25°C for 3.5 hours, and then mechanically shaken to disperse the aggregated particles at a frequency of 250 rpm for 12 minutes.

[0045] S2. Mix the soil powder obtained in S1 with an inert adsorbent at a mass ratio of 4:1 to obtain a dispersion matrix; The inert adsorbent is quartz sand.

[0046] S3. The dispersion matrix obtained in S2 is loaded into the extraction tank, and a first-stage extraction is performed using a non-polar-weakly polar mixed solvent. The non-polar-weakly polar pesticide residue extract is collected. The parameters for the first-stage extraction were: pressure 9 MPa, extraction temperature 45 °C, static extraction time 12 min, and 2 cycles; the non-polar to weakly polar mixed solvent consisted of n-hexane, ethyl acetate and acetone in a volume ratio of 5:2:1. S4. Then, the residue of S3 is subjected to a second stage of pressurized fluid extraction using a mixed solvent of halogenated hydrocarbons and alcohols to collect the extract of medium-polarity pesticide residues. In the second stage of extraction, 0.3% organic acid was added as a protonation aid to control the pH of the extract to 4.5. After extraction, dehydrating salt was added to dehydrate the extract so that the residual water content was <0.2%. The halogenated hydrocarbon-alcohol mixed solvent consisted of dichloromethane and methanol in a volume ratio of 4:2. S5. The residue from S4 is subjected to a third-stage vortex-assisted extraction using a nitrile-acidic aqueous solution mixture to collect the extract of strongly polar or ionic pesticide residues. S6, the extract obtained by mixing S3, S4 and S5, is purified by a composite adsorbent solid phase extraction column, and concentrated by inert gas purging to obtain the graded extract of pesticide residue test solution. The inert gas purging and concentration temperature is controlled at 35℃. After being concentrated to near dryness, it is redissolved and brought to volume using a solvent compatible with the instrument, with a volume error of <1.5%.

[0047] The above-mentioned method for detecting pesticide residues in soil using a triple quadrupole chromatography-mass spectrometry (LC-MS) technique involves the following steps: P1. The pesticide residue test solution is separated by reversed-phase chromatography column, wherein mobile phase A is a deionized aqueous solution containing volatile buffer salt and acidifying agent, and mobile phase B is a polar organic solvent, using a gradient elution program; The gradient elution procedure is as follows: initially, maintain a high proportion of mobile phase A, increase the proportion of mobile phase B to a medium level within 4 minutes, then increase it to a high proportion within 3 minutes and maintain it for a short time, and finally quickly restore it to the initial proportion equilibrium. The column temperature is controlled at 40℃ and the flow rate is 0.35 mL / min.

[0048] P2. Use an electrospray ionization source to ionize in positive ion mode, and set the ion source temperature and the desolvation gas temperature. The mass spectrometer interface voltage is 3.2kV, the nebulization auxiliary gas flow rate is 2.5L / min, and the collision gas is a high-purity inert gas.

[0049] P3. Data acquisition was performed using multiple reaction monitoring (MRM) and quantitative analysis was conducted using the internal standard method. A standard working curve was established using internal standards labeled with deuterated or carbon-13 stable isotopes.

[0050] The internal standard is added at the initial stage of sample preparation, and the concentration is set according to the expected residual level.

[0051] Comparative Example 1: This comparative example refers to the content of Example 1, except that in step S2, the mass ratio of soil powder to inert adsorbent is changed to 1.5:1, and the rest is the same as Example 1.

[0052] Comparative Example 2: This comparative example refers to the content of Example 1, except that in step S3, the static extraction time of the first stage extraction is changed to 6 minutes, and the rest is the same as Example 1.

[0053] Comparative Example 3: This comparative example is based on the content of Example 1, except that in step S4, the concentration of the organic acid added in the second-stage extraction is changed to 0.04%, and the rest is the same as in Example 1.

[0054] Comparative Example 4: This comparative example refers to the content of Example 1, except that in step S4, the volume ratio of the haloalkane-alcohol mixed solvent is changed to 2.5:1, and the rest is the same as Example 1.

[0055] Comparative Example 5: This comparative example refers to the content of Example 1, except that in step S6, the temperature of inert gas purging and concentration is changed to 40°C, and the rest is the same as Example 1.

[0056] Comparative Example 6: This comparative example is based on the content of Example 1, except that in the detection method P1 step, the flow rate of chromatographic separation is changed to 0.2 mL / min, and the rest is the same as Example 1.

[0057] Performance testing Sample preparation: Blank soil matrix was selected for the experiment, and a mixture of pesticide standards of chlorfenapyr, chlorpyrifos and glyphosate was quantitatively added to it to simulate the actual residue level. The spiked sample was then uniformly packaged and processed in parallel using the methods described in Examples 1-3 and Comparative Examples 1-6 to prepare pesticide residue test solutions for subsequent performance testing.

[0058] Overall extraction recovery test: The test solutions prepared in each example and comparative example were analyzed by triple quadrupole liquid chromatography-mass spectrometry. By comparing the detection signal value of the target pesticide residue in the test solution with the signal value of the standard of the same concentration directly injected, the extraction recovery rate of each target pesticide residue was obtained, and the average recovery rate and relative standard deviation of all target pesticide residues were calculated. Test standard: Refer to "Laboratory Guidelines for Recovery in Pesticide Residue Analysis" (SANCO / 12571 / 2013).

[0059] Recovery rate test of pesticide residues of different polarities: For three representative pesticides of non-polarity, medium polarity and strong polarity, the individual recovery rates of each pesticide were statistically analyzed under the methods of the examples and comparative examples, and the cross-contamination or incomplete extraction of pesticide residues of different polarities in the incorrect extraction fraction was investigated; Test standard: Refer to the "Validation Procedure for the Detection Method of Maximum Residue Limits of Pesticides in Food" (GB / T27404-2008).

[0060] Method precision testing: Intra-day precision is measured by the same operator repeatedly processing the same spiked sample at least five times within one day according to the same embodiment method; inter-day precision is measured by different operators processing the same spiked sample once a day within five consecutive days according to the same embodiment method. The relative standard deviation of the recovery rate of each target pesticide residue is used as the evaluation index. Test standard: Refer to the "General Rules for Validation of Chromatographic Analysis Methods" (JJF1317-2011). Generally, the relative standard deviation of intra-day precision is required to be no greater than 15%, and the relative standard deviation of inter-day precision is required to be no greater than 20%.

[0061] Matrix effect evaluation: The matrix effect factor was determined by comparing the slope of the standard curve of the target pesticide residue in the extracted blank matrix solution with the slope of the standard curve in the pure solvent. Simultaneously, the deviation between the quantitative results corrected using the internal standard method and the actual spiked values ​​under the comparative examples and comparative methods was evaluated to assess the compensation effect of the internal standard method on the matrix effect. Test standards: A matrix effect factor between 85% and 115% is considered a weak matrix effect; after correction using the internal standard method, the absolute value of the relative error of the quantitative results should not exceed 15%.

[0062] Table 1: Comparison of extraction recovery results between the examples and comparative examples

[0063] Table 2: Comparison of performance parameters between the examples and comparative examples

[0064] Example Conclusion: Based on Examples 1-3 and Comparative Example 1, and in conjunction with Tables 1 and 2, it can be seen that controlling the mass ratio of soil powder to inert adsorbent within a certain range can form a loose dispersion matrix, thereby ensuring the contact and penetration of subsequent extraction solvents with soil particles. This is the basis for achieving a high and stable extraction recovery rate. In contrast, the excessively high adsorbent ratio in Comparative Example 1 leads to excessive matrix dilution and increased mass transfer resistance, reducing the overall extraction efficiency and method stability.

[0065] Based on Examples 1-3 and Comparative Example 2, and in conjunction with Table 1, it can be seen that ensuring sufficient static extraction time in the first stage of extraction guarantees that non-polar pesticide residues are fully desorbed and dissolved from the soil matrix; sufficient equilibrium time allows the solvent to penetrate and extract the target substance, while insufficient time leads to incomplete extraction of such pesticide residues, affecting the effectiveness of the first stage of the graded extraction process.

[0066] Based on Examples 1-3 and Comparative Example 3, and in conjunction with Table 1, it can be seen that maintaining an appropriate amount of organic acid in the second-stage extraction to control the pH of the extract promotes the protonation of moderately polar pesticide residues and enhances their solubility in such solvents. A suitable acidic environment is a condition for achieving efficient and selective extraction of moderately polar pesticide residues; deviation from this condition will lead to a decrease in the extraction effect of this stage.

[0067] Based on Examples 1-3 and Comparative Example 4, and in conjunction with Table 1, it can be seen that the volume ratio of halogenated hydrocarbons to alcohol solvents in the second-stage extraction affects the polarity and elution capacity of the mixed solvent; the improved ratio can match the physicochemical properties of moderately polar pesticide residues, achieving efficient extraction; deviations in the ratio will change the selectivity of the solvent system, resulting in insufficient extraction capacity for the target pesticide residues and undermining the targeted nature of the fractional extraction.

[0068] Based on Examples 1-3 and Comparative Example 5, and in conjunction with Tables 1 and 2, it can be seen that using a mild inert gas purging temperature in the concentration step can avoid the loss of heat-sensitive pesticide residues due to volatilization or decomposition, while ensuring the accuracy of the final test solution concentration. Excessively high temperatures will not only cause loss of the target substance and reduce the recovery rate, but also introduce additional errors due to uneven volatilization, thereby affecting precision and accuracy.

[0069] Based on Examples 1-3 and Comparative Example 6, and in conjunction with Table 2, it can be seen that setting an appropriate chromatographic flow rate in the detection method is an influencing factor for achieving efficient separation and rapid analysis. The improved flow rate can maintain analytical efficiency while ensuring the degree of separation, while a flow rate that is too slow will prolong the analysis cycle, increase system fluctuations and diffusion effects, thereby reducing the reproducibility and precision of the analytical process.

[0070] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for graded extraction of pesticide residues in soil, characterized in that, Includes the following steps: S1. Remove foreign matter from the soil sample, freeze-dry until the moisture content is <5%, grind and sieve to obtain dry soil powder; S2. The soil powder obtained in S1 is mixed with an inert adsorbent at a mass ratio of (2:1) to (4:1) to obtain a dispersion matrix; S3. The dispersion matrix obtained in S2 is loaded into the extraction tank, and a first-stage extraction is performed using a non-polar-weakly polar mixed solvent. The non-polar-weakly polar pesticide residue extract is collected. S4. Then, the residue of S3 is subjected to a second stage of pressurized fluid extraction using a mixed solvent of halogenated hydrocarbons and alcohols to collect the extract of medium-polarity pesticide residues. S5. The residue from S4 is subjected to a third-stage vortex-assisted extraction using a nitrile-acidic aqueous solution mixture to collect the extract of strongly polar or ionic pesticide residues. The extract obtained by mixing S6, S3, S4 and S5 is purified by a composite adsorbent solid phase extraction column, and then concentrated by purging with inert gas to obtain the graded pesticide residue test solution.

2. The method for graded extraction of pesticide residues in soil according to claim 1, characterized in that, In step S1, the freeze-drying process includes a pretreatment step: freezing the soil sample at -18°C to -25°C for 1.5 to 3.5 hours, followed by mechanical vibration to disperse the aggregated particles, with a vibration frequency of 150 to 250 rpm and a time of 8 to 12 minutes.

3. The method for graded extraction of pesticide residues in soil according to claim 1, characterized in that, In step S2, the inert adsorbent is one or more of diatomaceous earth, Florisil, or quartz sand.

4. The method for graded extraction of pesticide residues in soil according to claim 1, characterized in that, In step S3, the parameters for the first-stage extraction are as follows: pressure 6~9MPa, extraction temperature 35~45℃, static extraction time 8~12min, and 1~2 cycles; the non-polar-weakly polar mixed solvent is composed of the following components: the volume ratio of n-hexane: ethyl acetate: acetone is (4~5): (1~2): (0.5~1).

5. The method for graded extraction of pesticide residues in soil according to claim 1, characterized in that, In step S4, 0.05% to 0.3% of organic acid is added as a protonation aid in the second-stage extraction to control the pH of the extract to 3.5 to 4.

5. After extraction, dehydrating salt is added to dehydrate the extract so that the residual water content is <0.2%. The halogenated hydrocarbon-alcohol mixed solvent is composed of the following components: the volume ratio of dichloromethane to methanol is (3 to 4): (1 to 2).

6. The method for graded extraction of pesticide residues in soil according to claim 1, characterized in that, In step S6, the temperature of the inert gas purging and concentration is controlled at 25~35℃. After being concentrated to near dryness, it is redissolved and brought to volume using a solvent compatible with the instrument, with a volume error of <1.5%.

7. A triple quadrupole chromatography-mass spectrometry (LC-MS) method for the graded extraction of pesticide residues in soil, characterized in that, The method for graded extraction of pesticide residues in soil according to any one of claims 1-6 includes the following steps: P1. The pesticide residue test solution is separated by reversed-phase chromatography column, wherein mobile phase A is a deionized aqueous solution containing volatile buffer salt and acidifying agent, and mobile phase B is a polar organic solvent, using a gradient elution program; P2. Use an electrospray ionization source to ionize in positive ion mode, and set the ion source temperature and the desolvation gas temperature. P3. Data acquisition was performed using multiple reaction monitoring (MRM) and quantitative analysis was conducted using the internal standard method. A standard working curve was established using internal standards labeled with deuterated or carbon-13 stable isotopes.

8. The method for detecting pesticide residues in soil by graded extraction using triple quadrupole chromatography-mass spectrometry (LC-MS) according to claim 7, characterized in that, In step P1, the gradient elution procedure is as follows: initially, maintain a high proportion of mobile phase A, increase the proportion of mobile phase B to a medium level within 3-4 minutes, then increase it to a high proportion within 2-3 minutes and maintain it for a short time, and finally quickly restore it to the initial proportion equilibrium. The column temperature is controlled at 30-40℃ and the flow rate is 0.25-0.35 mL / min.

9. The method for detecting pesticide residues in soil by graded extraction using a triple quadrupole chromatography-mass spectrometry (LC-MS) according to claim 7, characterized in that, In step P2, the mass spectrometry interface voltage is 2.8~3.2kV, the nebulization auxiliary gas flow rate is 1.8~2.5L / min, and the collision gas is a high-purity inert gas.

10. The method for detecting pesticide residues in soil by graded extraction using a triple quadrupole chromatography-mass spectrometry (LC-MS) according to claim 7, characterized in that, In step P3, the internal standard is added at the initial stage of sample preparation, and the concentration is set according to the expected residual level.

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