Detection method for determining benfuracarb, carbosulfan and metabolite of benfuracarb and carbosulfan

Abstract

The invention relates to the technical field of pesticide residue detection, in particular to a detection method for determining benfuracarb, carbosulfan and metabolites thereof, which comprises the steps of sample preparation and preservation, sample extraction, purification, standard working curve drawing and result calculation. An optimized QuEChERS pretreatment method is adopted, a citric acid buffer salt system is combined to adjust the pH value, a liquid chromatography-mass spectrometry / mass spectrometer is used for detection, and an external standard method is used for quantitative analysis. The method can effectively solve the problem that the target object is unstable under the acidic condition, simplifies the operation process, reduces the reagent consumption, is environment-friendly, enhances the detection applicability of the method in various matrixes, is suitable for rapid detection of large-batch samples, and has the characteristics of high efficiency, accuracy and environment friendliness.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticide residue detection, in particular to a method for determining benfuracarb, carbosulfan and their metabolites. Background Art

[0002] As common pesticide residues, the detection methods for benfuracarb, carbofuran and their metabolites still have room for improvement in terms of sensitivity, specificity and ease of operation. In practical applications, the demand for the determination of four target substances in multiple matrices through a single pretreatment has not yet been fully met. Among the currently valid standards, such as GB 23200.121-2021, carbofuran and 3-hydroxycarbofuran can be detected, but carbofuran and benfuracarb cannot be determined because the composition of the extraction salt (AOAC and EN) of the conventional QuEChERS method is acidic and not applicable. GB 23200.13-2016 is the recommended method for the determination of carbofuran in vegetables, fruits and edible fungi according to GB2763-2021, and there is no recommended method for benfuracarb. However, in GB23200.13-2016, the amount of acetonitrile extraction in the pretreatment is large, the purification method is complicated, and the use of organic reagents such as toluene, which is highly toxic, is also required.

[0003] Patent publication number CN105492907B provides a method for detecting cytokeratins 8, 18, and 19, and their soluble fragments, in samples through antibody fixation and labeled antibody binding. This technology demonstrates high sensitivity and specificity in biomarker detection, but is primarily applicable to protein-based assays and has limited applicability to small molecules such as benfuracarb, carbofuran, and their metabolites. Furthermore, the method involves complex antibody fixation and labeling steps, resulting in a lengthy and costly process and difficulty meeting the demands of rapid, high-throughput testing.

[0004] Patent publication number CN110291197B describes a method for isolating eukaryotic nucleic acids from human or non-human animal fecal samples and analyzing eukaryotic biomarkers. While this technology is highly efficient in nucleic acid extraction and analysis, it primarily targets nucleic acid-based substances and is difficult to directly apply to the detection of small molecule compounds such as pesticide residues. Furthermore, the method relies on specialized nucleic acid isolation materials and equipment, resulting in high testing costs and complexity, limiting its widespread application in resource-limited settings.

[0005] These issues demonstrate that existing detection methods for small-molecule compounds such as benfuracarb, carbosulfan, and their metabolites have limitations in terms of sensitivity, specificity, and ease of use. Therefore, the present invention aims to provide a novel assay method to improve detection performance, optimize operational procedures, and reduce testing costs, thereby better meeting practical needs. Summary of the Invention

[0006] The object of the present invention is to provide a method for determining benfuracarb, carbosulfan and their metabolites to solve the problems raised by the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solution: a method for determining benfuracarb, carbosulfan and their metabolites comprises the following steps:

[0008] S1: Preparation and preservation of samples; take the sample to be tested and pre-treat it according to the sample type; Preparation and preservation of samples; take 500g of the sample to be tested and pre-treat it according to the sample type;

[0009] For random sampling, the weight should not be less than 3kg. After the sample is divided into quarters, it should be directly put into a tissue crusher and crushed into a homogenous slurry. It should be stored in a polyethylene bottle. If it is stored for a long time, it should be stored at -20℃.

[0010] S2: Sample extraction: Weigh 10 g of sample accurately to 0.01 g into a 50 mL centrifuge tube, and add 10 mL of acetonitrile, 5 g of sodium chloride, 1 g of sodium citrate, 0.1 g of disodium hydrogen citrate, and a ceramic homogenizer in that order. Cover the centrifuge tube with a cap and place the tube in an oscillator at 300 rpm for 20 min.

[0011] S3: Purification: Centrifuge the shaken centrifuge tube at 4500 rpm for 5 minutes. Transfer 6 mL of the supernatant to a 15 mL centrifuge tube containing 900 mg of magnesium sulfate and 150 mg of PSA for purification. Vortex the centrifuge tube containing the extract for 1 minute, then centrifuge at 4500 rpm for 5 minutes. Pass the supernatant through a 0.22 μm organic phase microporous filter membrane for liquid chromatography-mass spectrometry / mass spectrometry analysis. For samples with a strong acidic matrix, add 5 mol / L sodium hydroxide solution during the extraction process to maintain the pH value of the extract at 4.8-6.0.

[0012] S4: drawing a standard working curve; taking a blank sample and treating it according to the above steps, using the obtained sample solution to dilute the mixed standard solution of benfuracarb, carbofuran, carbofuran and 3-hydroxycarbofuran to obtain a series of standard working solutions with a concentration gradient; injecting and testing in order from low to high concentration, and drawing a standard working curve with the chromatographic peak area as the ordinate and the standard working solution concentration as the abscissa;

[0013] S5: Calculation of results; Inject equal volumes of standard working solution and sample solution for determination. If the response value of the target substance in the sample solution to be tested exceeds the linear range of the standard curve, the sample solution must be appropriately diluted before injection analysis; Calculate the content of benfuracarb, butfuracarb and their metabolites in the test sample based on the standard curve and the peak area of ​​the test sample.

[0014] Preferably, in step S3, the dehydrating agent and purification material in the plastic centrifuge tube are anhydrous magnesium sulfate and PSA; 900 mg of anhydrous magnesium sulfate and 150 mg of PSA are required for purification for every 6 mL of extract.

[0015] Preferably, in step S4, the concentration range of the standard working solution is 0.005 mg / L to 1.0 mg / L.

[0016] Preferably, in step S5, the calculation formula is Where W is the content of the target substance in the sample, A is the peak area of ​​the sample, b is the intercept of the standard curve, a is the slope of the standard curve, V is the volume of the sample extract, and m is the sample mass.

[0017] Preferably, in step S5, the measurement is repeated twice and the average value is taken as the final result.

[0018] Preferably, in step S4, the chromatographic column used in the liquid chromatography is Agilent Prosheld EC C18, with a specification of 2.1 mm×100 mm×2.7 μm, and the column temperature is set to 40°C.

[0019] Preferably, in step S4, the mobile phase consists of phase A and phase B, phase A is 0.1% formic acid + 2 mmol / L ammonium formate aqueous solution, phase B is methanol, the flow rate is 0.3 mL / min, and the injection volume is 1 μL.

[0020] Preferably, in step S4, the gradient elution program is: 0-2 min, the proportion of mobile phase A decreases from 90% to 80%; 2-3 min, the proportion of mobile phase A decreases from 80% to 50%; 3-6 min, the proportion of mobile phase A decreases from 50% to 1%; 6-8 min, the proportion of mobile phase A remains at 1%; 8-9 min, the proportion of mobile phase A recovers from 1% to 90%.

[0021] Preferably, in step S4, the ionization mode during mass spectrometry is electrospray ionization in positive ion mode; the capillary voltage is 3500 V, the sheath gas temperature is 300°C, the sheath gas flow rate is 10 L / min, the drying gas temperature is 120°C, the drying gas flow rate is 11 L / min, and the nebulizing gas pressure is 30 psi.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention primarily utilizes an optimized QuEChERS pretreatment method. By introducing a citric acid buffer system to adjust the pH of the extract, this method effectively addresses the problem of target instability caused by the acidic extract salt component in the conventional QuEChERS method, and enhances the method's applicability for detection in a variety of matrices. Furthermore, the pretreatment process is simplified, the use of toxic reagents is reduced, and the risk of environmental contamination is mitigated. While also improving detection efficiency, it can meet the needs of rapid detection of large quantities of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Flow chart of the steps of the present invention;

[0025] Figure 2 This is a bar graph of the stability of four pesticides in different sample matrices under the same conditions of the present invention;

[0026] Figure 3 The figure is a comparison of the separation and response of four pesticides in different mobile phases of the present invention;

[0027] Figure 4 The recovery rates of the four pesticides in different sample matrices under the same conditions of the present invention are as follows: DETAILED DESCRIPTION

[0028] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0029] The present invention provides a method for determining the residues of benfuracarb, carbosulfan and their metabolites. Figure 1-3 During the sample preparation and storage phase, the sample to be tested must first be pre-processed. For samples such as vegetables, fruits, and edible fungi, according to relevant standards such as NY / T 3304-2018 and NY / T 789-2004, a random sample of no less than 3 kg should be taken. After being quartered, the sample should be directly crushed into a homogenate using a tissue crusher and stored in polyethylene bottles. For longer-term storage, the sample should be stored at -20°C.

[0030] During the sample extraction stage, 10 g of sample was weighed to the nearest 0.01 g and placed in a 50 mL plastic centrifuge tube. 10 mL of acetonitrile was added as the extraction solvent, along with 5 g of sodium chloride to facilitate phase separation, 1 g of sodium citrate, and 0.1 g of disodium hydrogen citrate to create a buffer system. A ceramic homogenizer was also added to enhance extraction efficiency. After capping the tube, the tube was shaken at 300 rpm for 20 minutes. During this process, the physical action of the ceramic homogenizer effectively disrupted the sample structure, allowing the target compound to be fully released into the extraction solvent. After shaking, the sample was centrifuged at 4500 rpm for 5 minutes to achieve solid-liquid separation. 6 mL of the supernatant was then aspirated and transferred to another 15 mL plastic centrifuge tube. This tube was pre-loaded with 900 mg of anhydrous magnesium sulfate and 150 mg of PSA as purification materials for subsequent cleanup steps. For highly acidic matrices such as lemon, the pH of the extract must be adjusted to a neutral range with the addition of 5 mol / L sodium hydroxide solution to ensure target stability. For most other samples, the currently optimized extraction salt ratio ensures stable extraction and detection of all four target compounds.

[0031] During the purification stage, the 15mL plastic centrifuge tube containing the extract is vortexed for 1 minute to allow the anhydrous magnesium sulfate and PSA to fully contact the extract and absorb impurities. The main function of anhydrous magnesium sulfate is to remove water from the extract, while PSA is used to adsorb interfering substances such as organic acids and sugars. After vortex mixing, centrifuge at 4500r / min for 5 minutes to achieve solid-liquid separation. The supernatant is aspirated and passed through a 0.22μm organic phase microporous filter membrane to further remove particulate matter for liquid chromatography-mass spectrometry / mass spectrometry analysis. For highly acidic matrix samples, an appropriate amount of 5mol / L sodium hydroxide solution should be added during the extraction process to maintain the pH value of the extract at around 4.8-6.0 to improve the stability of the target.

[0032] During the drawing stage of the standard working curve, a blank sample is taken and processed according to the above steps, and the obtained sample solution is used to dilute the mixed standard solution of benfuracarb, butyralcarb, carbofuran and 3-hydroxycarbofuran to obtain a series of standard working solutions with concentration gradients. The concentration range of the standard working solution must cover the concentration of the target substance that may exist in the sample to be tested, and is usually set to 0.005 mg / L, 0.01 mg / L, 0.05 mg / L, 0.1 mg / L, 0.2 mg / L, 0.5 mg / L, and 1.0 mg / L. The samples are injected and tested in order from low to high concentration, and the chromatographic peak area of ​​the target substance at each concentration is recorded. The standard working curve is drawn with the chromatographic peak area as the vertical axis and the concentration of the standard working solution as the horizontal axis to ensure the linear correlation coefficient R 2 ≥0.99.

[0033] For liquid chromatography-mass spectrometry / mass spectrometry analysis, an Agilent Prosheld EC C18 column (2.1 mm × 100 mm × 2.7 μm) was used, with the column temperature set at 40°C. The mobile phase consisted of phases A and B: 0.1% formic acid in a 2 mmol / L aqueous solution of ammonium formate; phase B was methanol. The flow rate was 0.3 mL / min, and the injection volume was 1 μL. The gradient elution program was as follows: 0-2 min, mobile phase A decreased from 90% to 80%; 2-3 min, mobile phase A decreased from 80% to 50%; 3-6 min, mobile phase A decreased from 50% to 1%; 68 min, mobile phase A remained at 1%; 8-9 min, mobile phase A was restored from 90% to 90%. The ionization mode for mass spectrometry was electrospray ionization in positive ion mode, with a capillary voltage of 3500 V, a sheath gas temperature of 300°C, a sheath gas flow rate of 10 L / min, a drying gas temperature of 120°C, a drying gas flow rate of 11 L / min, and a nebulizing gas pressure of 30 psi.

[0034]

[0035] Note: *Quantitative ion

[0036] Table: Mass spectrometric parameters and retention times of benfuracarb, carbofuran, carbofuran and 3-hydroxycarbofuran.

[0037] During the result calculation phase, equal volumes of the standard working solution and sample solution are injected for determination. If the response value of the target substance in the sample solution exceeds the linear range of the standard curve, the sample solution must be appropriately diluted before injection analysis. Based on the standard curve and the peak area of ​​the test sample, the content of benfuracarb, butfuracarb and their metabolites in the test sample is calculated. The calculation formula is Where W is the content of the target substance in the sample, A is the peak area of ​​the sample, b is the intercept of the standard curve, a is the slope of the standard curve, V is the volume of the sample extract, and m is the sample mass.

[0038] In practical applications, this method can be used for rapid testing of large quantities of samples. For example, in food safety monitoring, vegetables, fruits and edible fungi on the market can be sampled and tested to assess whether they meet the pesticide residue limit standards. For different types of samples, the extraction and cleanup conditions can be adjusted according to their matrix characteristics. For example, for samples with high fat content, C18 filler can be added to the cleanup step to remove fat-like interfering substances. For samples with high pigment content, graphitized carbon black can be added to the cleanup step to adsorb pigments. In addition, this method can also be extended to the detection of other pesticide residues, simply by adjusting the extraction solvent and cleanup materials according to the chemical properties of the target.

[0039] Throughout the testing process, sample preparation and preservation laid the foundation for subsequent extraction. Sample extraction, using the optimized QuEChERS method, achieved efficient extraction of the target compound. Cleanup steps removed interfering substances to ensure accurate test results. Standard working curves were developed to provide a reliable basis for quantitative analysis, while liquid chromatography-mass spectrometry / mass spectrometry enabled highly sensitive detection of the target compound. The results calculation phase, through scientific data processing, yielded highly accurate results.

[0040] In order to better enable relevant personnel in this technical field to fully understand and implement the present invention, the specific implementation principle of the present invention is supplemented below with reference to a specific application scenario.

[0041] During the preparation and preservation of the sample, the representativeness of the sample to be tested must be ensured first. For solid samples such as apples, they need to be cut into small pieces of about 1-2 cm with a clean knife. After sampling by quartering, they are crushed into a homogenate in a tissue grinder and transferred to a clean container for sealed storage. After marking with relevant information, they are frozen below -20°C. The key to this step is to prevent the sample from deteriorating or contaminating during storage, thereby affecting the accuracy of subsequent test results. For samples with highly acidic matrices such as lemons, an appropriate amount of 5 mol / L sodium hydroxide solution needs to be added during the pretreatment process to adjust the pH value of the extract to the range of 4.8-6.0. The purpose of this adjustment is to maintain the stability of the target through the buffer system to prevent it from degradation or transformation during the extraction process.

[0042] During the sample extraction stage, after weighing 10g of sample to an accuracy of 0.01g, acetonitrile, sodium chloride, sodium citrate and disodium hydrogen citrate were added to the centrifuge tube in sequence. The selection and ratio of these reagents have been optimized. Acetonitrile as an extraction solvent can effectively dissolve the target, while sodium chloride is used to promote phase separation, and citrate constructs a buffer system to stabilize the pH value of the extract. The addition of ceramic homogenizers further improves the extraction efficiency. Its physical action can break up the sample tissue structure and fully release the target into the extraction solvent. During the oscillation extraction process, the oscillation speed of 300rpm and the extraction time of 20min have been experimentally verified to ensure the extraction efficiency while avoiding the loss of the target due to excessive operation. Subsequently, the centrifugal speed of 4500r / min ensures the thoroughness of solid-liquid separation, thereby obtaining a clear supernatant.

[0043] During the purification stage, the centrifuge tube containing the extract is vortexed for 1 minute to allow the anhydrous magnesium sulfate and PSA to fully contact the extract. The main function of anhydrous magnesium sulfate is to remove water from the extract, while PSA purifies the extract by adsorbing interfering substances such as organic acids and sugars. After vortex mixing, centrifuge again to ensure that the impurities are completely removed. The step of aspirating the supernatant through a 0.22μm organic phase microporous filter membrane further removes particulate matter and ensures that the purity of the extract meets the injection requirements of the liquid chromatography-mass spectrometry / mass spectrometer. For highly acidic matrix samples such as lemons, an appropriate amount of 5mol / L sodium hydroxide solution must be added during the extraction process to maintain the pH value of the extract at around 4.8-6.0. This operation effectively improves the stability of the target by adjusting the acid-base environment of the extract and avoids decomposition or transformation caused by pH fluctuations.

[0044] During the drawing of the standard working curve, a blank sample was taken and processed according to the above steps, and then the mixed standard solutions of benfuracarb, butasulfuron, carbofuran and 3-hydroxycarbofuran were diluted into a series of standard working solutions with a concentration gradient. The concentration range of the standard working solution was set to 0.005 mg / L to 1.0 mg / L, covering the target concentrations that may exist in the sample to be tested. The samples were injected and tested in order from low to high concentration, and the chromatographic peak area of ​​the target at each concentration was recorded. The standard working curve was drawn with the chromatographic peak area as the vertical axis and the concentration of the standard working solution as the horizontal axis to ensure the linear correlation coefficient R 2 ≥0.99. This process achieves accurate quantitative analysis of the target compound through the external standard method, providing a reliable basis for subsequent sample testing.

[0045] In the liquid chromatography-mass spectrometry / mass spectrometry determination stage, an Agilent Prosheld EC C18 column was used for separation. The mobile phase consisted of phase A (0.1% formic acid + 2mmol / L ammonium formate aqueous solution) and phase B (methanol), the flow rate was set to 0.3mL / min, and the injection volume was 1μL. The gradient elution program was designed to achieve effective separation of the target, in which the proportion of mobile phase A was gradually reduced from 90% to 1% and then restored to 90%, ensuring the retention time and separation effect of the target in the chromatographic column. Electrospray ionization in positive ion mode was used for mass spectrometry determination, and the settings of parameters such as capillary voltage, sheath gas temperature, and drying gas temperature were optimized to achieve high-sensitivity detection of the target. The monitoring method was multiple reaction monitoring, and the selection of quantitative ion pairs and qualitative ion pairs was based on the molecular structure characteristics of the target, ensuring the specificity and accuracy of the detection.

[0046] In the result calculation stage, equal volumes of standard working solution and sample solution are injected for determination. If the response value of the target substance in the sample solution exceeds the linear range of the standard curve, the sample solution needs to be appropriately diluted before injection analysis. According to the standard curve and the peak area of ​​the test sample, the calculation formula is: Where W is the target compound content in the sample, A is the peak area of ​​the sample, b is the intercept of the standard curve, a is the slope of the standard curve, V is the volume of the sample extract, and m is the sample mass. Repeating the measurement twice and taking the average further enhances the reliability of the results. This process, through scientific data processing, yields accurate test results.

[0047] In practical applications, this method can be used for rapid testing of large quantities of samples. For example, in food safety monitoring, vegetables, fruits and edible fungi on the market can be sampled and tested to assess whether they meet the pesticide residue limit standards. For different types of samples, the extraction and cleanup conditions can be adjusted according to their matrix characteristics. For example, for samples with high fat content, C18 filler can be added to the cleanup step to remove fat-like interfering substances; for samples with high pigment content, graphitized carbon black can be added to the cleanup step to adsorb pigments. In addition, this method can also be extended to the detection of other pesticide residues, simply by adjusting the extraction solvent and cleanup materials according to the chemical properties of the target.

[0048] The above contents are only specific embodiments of the present invention and do not limit the scope of protection of the present invention. Any modification, equivalent replacement or improvement within the spirit and principle of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for determining benfuracarb, carbosulfan and their metabolites, characterized in that: The following steps are involved: S1: Preparation and preservation of samples; take the sample to be tested and pre-treat it according to the sample type; For random sampling, the sample should be no less than 3kg. After being divided into quarters, it should be directly put into a tissue crusher and crushed into a homogenate. It should be stored in a polyethylene bottle. If it is stored for a long time, it should be stored at -20℃. S2: Sample extraction: Weigh 10 g of sample accurately to 0.01 g into a 50 mL centrifuge tube, add 10 mL of acetonitrile, 5 g of sodium chloride, 1 g of sodium citrate, 0.1 g of disodium hydrogen citrate, and a ceramic homogenizer in that order; cover the centrifuge tube with a cap and place the tube in an oscillator at 300 rpm for 20 min; S3: Purification; Centrifuge the shaken centrifuge tube at 4500r / min for 5 minutes. Pipette 6mL of supernatant and transfer it to a 15mL centrifuge tube containing 900mg of magnesium sulfate and 150mg of PSA for purification; Vortex the centrifuge tube containing the extract for 1 minute, then centrifuge at 4500r / min for 5 minutes; Pipette the supernatant through a 0.22μm organic phase microporous filter membrane for liquid chromatography-mass spectrometry / mass spectrometry analysis; For samples with strong acidic matrices, add an appropriate amount of 5mol / L sodium hydroxide solution during the extraction process to maintain the pH value of the extract at 4.8-6.0; S4: drawing a standard working curve; taking a blank sample and treating it according to the above steps, using the obtained sample solution to dilute the mixed standard solution of benfuracarb, carbofuran, carbofuran and 3-hydroxycarbofuran to obtain a series of standard working solutions with a concentration gradient; injecting and testing in order from low to high concentration, and drawing a standard working curve with the chromatographic peak area as the ordinate and the standard working solution concentration as the abscissa; S5: Calculation of results; Inject equal volumes of standard working solution and sample solution for determination. If the response value of the target substance in the sample solution to be tested exceeds the linear range of the standard curve, the sample solution must be appropriately diluted before injection analysis; Calculate the content of benfuracarb, butfuracarb and their metabolites in the test sample based on the standard curve and the peak area of ​​the test sample.

2. The method for determining benfuracarb, carbosulfan and their metabolites according to claim 1, wherein: In step S3, the dehydrating agent and purification material in the plastic centrifuge tube are anhydrous magnesium sulfate and PSA; 900 mg of anhydrous magnesium sulfate and 150 mg of PSA are required for purification for every 6 mL of extract.

3. The method for determining benfuracarb, carbosulfan and their metabolites according to claim 1, wherein: In step S4, the concentration of the standard working solution ranges from 0.005 mg / L to 1.0 mg / L.

4. The method for determining benfuracarb, carbosulfan and their metabolites according to claim 1, wherein: In step S5, the calculation formula is: Where W is the content of the target substance in the sample, A is the peak area of ​​the sample, b is the intercept of the standard curve, a is the slope of the standard curve, V is the volume of the sample extract, and m is the sample mass.

5. The method for determining benfuracarb, carbosulfan and their metabolites according to claim 1, wherein: In step S5, the measurement is repeated twice and the average value is taken as the final result.

6. The method for determining benfuracarb, carbosulfan and their metabolites according to claim 1, wherein: In step S4, the chromatographic column used in the liquid chromatography is Agilent Prosheld EC C18, with a specification of 2.1 mm×100 mm×2.7 μm, and the column temperature is set at 40°C.

7. The method for determining benfuracarb, carbosulfan and their metabolites according to claim 1, wherein: In step S4, the mobile phase consists of phase A and phase B, phase A is 0.1% formic acid + 2 mmol / L ammonium formate aqueous solution, phase B is methanol, the flow rate is 0.3 mL / min, and the injection volume is 1 μL.

8. The method for determining benfuracarb, carbosulfan and their metabolites according to claim 1, wherein: In step S4, the gradient elution program is: 0-2 min, the proportion of mobile phase A decreases from 90% to 80%; 2-3 min, the proportion of mobile phase A decreases from 80% to 50%; 3-6 min, the proportion of mobile phase A decreases from 50% to 1%; 6-8 min, the proportion of mobile phase A remains at 1%; 8-9 min, the proportion of mobile phase A recovers from 1% to 90%.

9. The method for determining benfuracarb, carbosulfan and their metabolites according to claim 1, wherein: In step S4, the ionization mode during mass spectrometry is electrospray ionization in positive ion mode; the capillary voltage is 3500 V, the sheath gas temperature is 300° C., the sheath gas flow rate is 10 L / min, the drying gas temperature is 120° C., the drying gas flow rate is 11 L / min, and the nebulizing gas pressure is 30 psi.

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