A pretreatment method for detecting bentazon in soil and broad beans using liquid chromatography-mass spectrometry

Abstract

This invention provides a pretreatment method for detecting metribuzin in soil and broad beans using liquid chromatography-mass spectrometry (LC-MS / MS). The method includes: weighing 10.0 g of soil into a 50.0 ml centrifuge tube, adding 10.0 ml of acetonitrile, vortexing for 3.0 min, adding 3.0 g of NaCl, vortexing for 1.0 min, centrifuging at 5000 rpm for 5.0 min, taking 1.5 ml of the supernatant and adding it to a 2.5 ml centrifuge tube containing 25.0 mg PSA, 25.0 mg C18, and 100 mg anhydrous MgSO4, vortexing for 1.0 min, centrifuging at 8000 rpm for 3.0 min, aspirating the supernatant with a 5.0 ml syringe, filtering through a membrane, and transferring to a 2.5 ml brown sample bottle. This invention is applicable to the detection of metribuzin residues in broad beans, a specialty crop grown in high-altitude regions.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, and in particular relates to a pretreatment method for detecting metribuzin in soil and broad beans using liquid chromatography-mass spectrometry. Background Technology

[0002] The existing technology establishes a method for determining the residues of bentazon and its metabolites in soil using a dispersion solid-phase extraction purification-liquid chromatography-tandem mass spectrometry (LC-MS / MS) approach. Soil samples are extracted with acetonitrile containing 0.5% acetic acid using ultrasound-assisted extraction, purified with a mixture of 7.5 mg GCB, 25 mg PSA, and 150 mg anhydrous magnesium sulfate, separated using a C18 column, and eluted with a gradient of formic acid aqueous solution (0.1% formic acid)-methanol as the mobile phase in multiple reaction monitoring (MRM) negative ion mode (ESI). - Ionization scanning and matrix standard curve external standard method were used for quantification. The correlation coefficients of bentazon and its metabolites were greater than 0.999 in the range of 0.1–200 μg / L. The limits of detection (S / N=3) were 0.3–2.0 μg / kg, the limits of quantitation (S / N=3) were 0.3–2.0 μg / kg, and the limits of quantitation (S / N=10) were 1.0–7.0 μg / kg. The recoveries were 90.1%–104.4%, and the relative standard deviations were 1.94%–6.56%.

[0003] The shortcomings of existing technologies are as follows: 1. There is a lack of detection methods for broad bean plants and fruits.

[0004] 2. Despite the superior performance of LC-MS / MS, its pretreatment steps still need to be optimized for different substrates (such as soil vs. broad beans), and the process remains complex.

[0005] 3. Existing standard methods (such as GC-MS) must rely on cumbersome derivation steps.

[0006] 4. Although methods such as ELISA are rapid, they may have risks of cross-reactivity, false positives / false negatives, and it is difficult to accurately quantify the parent compound and multiple metabolites at the same time. Summary of the Invention

[0007] This invention addresses the lack of a method for detecting metribuzin in broad bean plants, a specialty crop of the Qinghai Plateau. It provides a pretreatment method for detecting metribuzin in soil and broad beans using liquid chromatography-mass spectrometry, which is rapid and accurate.

[0008] The present invention adopts the following technical solution: A pretreatment method for detecting bentazon in soil using liquid chromatography-mass spectrometry includes: Weigh 10.0g of soil and place it in a 50.0ml centrifuge tube. Add 10.0ml of acetonitrile and vortex for 3.0min. Add 3.0g of NaCl and vortex for 1.0min. Centrifuge at 5000rpm for 5.0min. Take 1.5ml of the supernatant and add it to a 2.5ml centrifuge tube containing 25.0mg PSA, 25.0mg C18, and 100mg anhydrous MgSO4. Vortex for 1.0min and centrifuge at 8000rpm for 3.0min. Use a 5.0ml syringe to aspirate the supernatant, filter it through a membrane, and transfer it to a 2.5ml brown sample bottle.

[0009] A pretreatment method for detecting bentazon in broad beans using liquid chromatography-mass spectrometry includes: Weigh 5.0g of the aboveground parts of broad bean plants and place them in a 5.0ml centrifuge tube. Add 15.0ml of acetonitrile and vortex for 3.0min. Add 3.0g of NaCl and vortex for 1.0min. Centrifuge at 5000rpm for 5.0min. Take 1.5ml of the supernatant and add it to a 2.5ml centrifuge tube containing 25.0mg PSA, 25.0mg C18, and 100.0mg anhydrous MgSO4. Vortex for 1.0min and centrifuge at 8000rpm for 3.0min. Use a 5.0ml syringe to aspirate the supernatant, filter it through a membrane, and transfer it to a 2.5ml brown sample bottle.

[0010] The filter membrane has a pore size of 0.22 μm.

[0011] The beneficial effects of this invention are: 1. A detection technology for metribuzin residues in broad beans, a specialty crop of the plateau region, has been developed. Previous methods for detecting metribuzin were only applicable to other crops and were not specifically developed for broad beans, resulting in problems such as poor recovery rate and low detection accuracy.

[0012] 2. By improving the methods, detection can be made more efficient, faster, and more economical. Most of the detection methods developed previously were liquid chromatography, while mass spectrometry detection is faster and more sensitive. Attached Figure Description

[0013] Figure 1 Chromatogram when the mobile phase is acetonitrile.

[0014] Figure 2 Chromatogram when the mobile phase is methanol.

[0015] Figure 3 Chromatogram of a mobile phase of 0.1% formic acid aqueous solution.

[0016] Figure 4 Chromatogram of a mobile phase of 0.5 mmol / L aqueous solution (pH=7).

[0017] Figure 5Chromatogram of a mobile phase of 0.5 mmol / L aqueous solution (pH=7.5).

[0018] Figure 6 Chromatogram of a mobile phase of 0.5 mmol / L aqueous solution (pH=8).

[0019] Figure 7 Chromatogram of a mobile phase of 0.5 mmol / L aqueous solution (pH=8.5).

[0020] Figure 8 Chromatogram of a mobile phase of 0.5 mmol / L aqueous solution (pH=9).

[0021] Figure 9 Chromatogram at a flow rate of 0.2 ml / min.

[0022] Figure 10 Chromatogram at a flow rate of 0.25 ml / min.

[0023] Figure 11 Chromatogram at a flow rate of 0.3 ml / min.

[0024] Figure 12 Standard curve of metribuzin.

[0025] Figure 13 The effect of extractant dosage on bentazon recovery rate.

[0026] Figure 14 Standard curve of metribuzin substrate.

[0027] Figure 15 The effect of different amounts of extractant on the recovery rate of broad bean aerial parts.

[0028] Figure 16 The effect of different amounts of extractant on the recovery rate of broad bean underground parts.

[0029] Figure 17 Operation flowchart. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0031] This invention aims to solve three technical problems: Problem 1: Pretreatment methods suffer from low efficiency and poor versatility. Traditional methods (such as liquid-liquid extraction and conventional SPE) involve lengthy processes for soil and plant substrates, which are completely different, resulting in cumbersome operations and large amounts of organic solvents. This invention, employing the QuEChERS pretreatment method, can quickly, efficiently, and economically solve these problems.

[0032] Problem 2. Strong interference from mechanisms, resulting in insufficient accuracy. Humic acid in the soil and endogenous substances such as pigments and sugars in broad beans severely interfere with the determination, leading to a decrease in detection sensitivity and accuracy. This invention, through optimized pretreatment technology and the selection of multiple purification agent combinations, can effectively solve the interference from pigments, sugars, and lipids in the soil and broad bean plants.

[0033] Problem 3. Limited Detection Target. Most methods only detect the parent compound bentazon, failing to simultaneously monitor its key environmental metabolites (such as 6-hydroxy and 8-hydroxybenzidine), thus hindering a comprehensive assessment of residue risks. The pretreatment technology of this invention not only boasts high recovery rates for the parent compound but is also largely adaptable to its degradation products.

[0034] Risk 4: Limitations in instrument sensitivity and throughput. Traditional GC or HPLC methods often lack sufficient sensitivity or require complex derivatization steps, making them unsuitable for current trace detection and high-throughput screening needs. This invention utilizes high-performance liquid chromatography-mass spectrometry (HPLC-MS) to rapidly and efficiently detect target analytes in complex matrices.

[0035] Example 1 0. Determination and optimization of mass spectrometry conditions Table 1 Mass spectrometry information for the detection of metribuzin A Shimadzu LCMS-8040 triple quadrupole mass spectrometer was used in conjunction with an ultra-high performance liquid chromatograph (UHPLC) system. Mass spectrometry detection employed an electrospray ionization source in negative ion mode with multiple reaction monitoring (MRM). The nebulizer gas flow rate was 3 L / min, the drying gas flow rate was 15 L / min, the DL tube temperature was 250℃, the heating module temperature was 400℃, and the collision gas was argon (230 kPa). The MRM parameters of the bentazon compounds are shown in Table 1.

[0036] 2. Determination and optimization of chromatographic conditions To achieve rapid baseline separation of the compound bentazon and improve peak tailing, the chromatographic method was systematically optimized. The optimization target was an analysis time of <10 min and an asymmetry factor (As) of all peaks between 0.8 and 1.2.

[0037] Based on the physicochemical properties of the target compound and relevant literature, a reversed-phase chromatography system was initially selected. The initial chromatographic conditions were: column: Shim-pack GIST-HPC18 (100 mm × 2.1 mm × 3 μm); mobile phase: methanol-water (0.5 mmol / L ammonium acetate, pH=8) (5:95, v / v) Figure 1 and Figure 2 , Figures 3 to 8 ); Flow rate: 0.25 mL / min Figures 9 to 11 ); Column temperature: 40℃. Time program: 0-1min B 95%, 1-4min B 5%, 4-5min B 5%, 5-7min B 95%, 7-8min B 95%.

[0038] 3. Determination and optimization of preprocessing conditions (1) Optimization of the extractant In UPLC-MS / MS analysis, the core optimization goal is not only to improve the extraction efficiency of the target analyte, but also to minimize co-extractants (matrix effect) and ensure the compatibility of the extractant to guarantee instrument sensitivity, stability, and data accuracy. Acetonitrile extraction has the following advantages: strong protein precipitation ability: effectively precipitates proteins in the sample, reducing matrix interference. Moderate polarity: good extraction efficiency for a wide range of pesticides (from polar to moderate polarity). Relatively low matrix effect: compared with methanol, acetonitrile co-extracts fewer non-polar interfering substances such as lipids and pigments, which is crucial in MS / MS. Good compatibility with subsequent steps: good compatibility with QuEChERS salt packs (MgSO4, NaCl), forming clear two-phase separation. Methanol extraction has the following characteristics: severe co-extraction: extracts more sugars, pigments, lipids, etc., leading to strong matrix inhibition or enhancement effects, seriously affecting the quantitative accuracy of MS / MS. Weaker protein precipitation ability than acetonitrile. Poor compatibility with salting-out agents: It is not easy to separate into layers with water, or the separation effect is not ideal. Since the samples in this experiment involved soil and plants in the later stages, both of which had high water content, considering that methanol and water are not easy to separate into layers, acetonitrile was chosen as the extraction agent.

[0039] This invention aims to determine the optimal acetonitrile dosage for subsequent UPLC-MS / MS analysis by systematically evaluating the effects of different volumes of acetonitrile extractant on the recovery of target analytes, matrix effects, and method precision. A single-factor variable method was used, fixing all pretreatment conditions except for the acetonitrile volume. For a 10.0 g test sample, the following four acetonitrile dosage levels were set:

[0040] Level A: 5.0 mL acetonitrile (sample:extractant = 1:0.5, w / v) Level B: 10.0 mL acetonitrile (sample:extractant = 1:1, w / v) Level C: 15.0 mL acetonitrile (sample:extractant = 1:1.5, w / v) Level D: 20.0 mL acetonitrile (sample:extractant = 1:2, w / v) At least five parallel samples are required for each level: three spiked samples (for recovery and precision calculations), one matrix blank sample (for background monitoring), and one post-extraction spiked sample (for matrix effect assessment). Accurately weigh 10.0 ± 0.1 g of homogenized blank sample into a 50 mL centrifuge tube. Add an appropriate amount (0.1 mg / L) of standard working solution and let stand for 15-30 min to allow the analyte to bind to the matrix. Matrix blank: No standard added. Post-extraction spiked: The sample is not pre-spikened and is processed after extraction. According to the above experimental design, add the corresponding volume of acetonitrile (5.0, 10.0, 15.0, or 20.0 mL) to each centrifuge tube. Vigorously shake manually to ensure the sample and extractant are fully wetted. Add 3.0 g of NaCl and vortex for 1 min to ensure thorough mixing of the solvent and salt and prevent clumping. Centrifuge at 5000 rpm for 35 min at room temperature to ensure complete separation of the two phases. Accurately transfer 1.5 mL of supernatant from each centrifuge tube into a 2 mL dSPE tube containing 100 mg MgSO4 + 25 mg PSA. Vortex for 1 minute, then centrifuge at 8000 rpm for 3 minutes at room temperature. Pit the supernatant, filter through a 2.0 μm organic filter membrane, and transfer to a 2.0 mL amber sample vial for analysis. Figure 12 As shown

[0041] Evaluation criteria: Recovery rate: Prioritize dosages that maintain stable recoveries of most target analytes between 70% and 120%. Precision: RSD < 20% at the same dosage level. Signal-to-noise ratio: Prioritize dosages with the highest signal-to-noise ratio at the same concentration. Economy and convenience: When performance is similar, prioritize dosages with smaller volume.

[0042] Table 2. Recovery rate and RSD of bensulfuron-methyl in soil Because previous methods had low recovery rates and high RSDs, this invention investigated the extraction of bentazon at different extraction ratios. The results in the table show that ( Figure 13 The highest recoveries were observed at extractant volumes of 10 mL and 15 mL, at 89.30% and 90.72%, respectively, with relative standard deviations of 1.06% and 1.96%. The recoveries remained stable between 70% and 120%, with RSDs < 20%. However, there was no significant difference in recovery rates when extracting bentazon from soil using 10 mL and 15 mL extractants. Therefore, considering both economy and convenience, 10 mL of acetonitrile was chosen as the extractant volume. Furthermore, this invention is more economical and has a higher recovery rate compared to other methods.

[0043] (2) Optimization of purifying agent Soil contains abundant organic matter such as humic acid and fulvic acid, as well as interfering substances such as oils and pigments, which places higher demands on remediation. By systematically evaluating the combined effects of three remediation agents—PSA, C18, and GCB—and their different dosage combinations on the recovery rate, remediation effect, and matrix effect of target pesticides in the soil matrix, the optimal dSPE remediation scheme was determined.

[0044] Factor A: PSA dosage (mg / mL extract): Level 1 (A1): 25 mg; Level 2 (A2): 50 mg; Level 3 (A3): 75 mg Factor B: C18 dosage (mg / mL extract): Level 1 (B1): 25 mg; Level 2 (B2): 50 mg; Level 3 (B3): 75 mg Factor C: GCB dosage (mg / mL extract): Level 1 (C1): 0 mg; Level 2 (C2): 2.5 mg; Level 3 (C3): 5 mg L9 (3) 4 An orthogonal array is used, requiring a total of 9 experimental groups. For each experimental group, the following data for each target pesticide are recorded: Recovery rate (Y1): Calculate the average value.

[0045] Matrix effect (Y2): expressed as the absolute value of matrix inhibition rate. This indicates that (the smaller the value, the better).

[0046] (a) Data Analysis Methods (Taking recovery rate Y1 as an example) – Intuitive Analysis (range analysis), this method is used to determine the order of importance of each factor and the optimal combination of levels. Calculate the sum of the indices (Ki) and the mean (ki) of each factor at the same level.

[0047] For factor A (PSA): K_A1 = Y1_1 + Y1_2 + Y1_3 (the sum of the recovery rates of all experiments at level 1 with A as the base) K_A2 = Y1_4 + Y1_5 + Y1_6 (where A is the sum of the recovery rates of all experiments at level 2) K_A3 = Y1_7 + Y1_8 + Y1_9 (The sum of the recovery rates of all A's in level 3 experiments) k_A1 = K_A1 / 3, k_A2 = K_A2 / 3, k_A3 = K_A3 / 3 (Calculate the average value) Calculate the range (R) of each factor. R_A=max(k_A1,k_A2,k_A3)-min(k_A1,k_A2,k_A3) Similarly, calculate R_B(C18) and R_C(GCB).

[0048] (b) Interpretation of Results Factor importance: Based on the size of the range R, the larger the R value, the greater the impact of the factor on the recovery rate.

[0049] For example: if R_C > R_A > R_B, then the order of importance of factors is GCB usage > PSA usage > C18 usage.

[0050] Maximum level combination: Compare k_A1, k_A2, k_A3 for each factor and select the level that maximizes the recovery rate.

[0051] For example: if k_A2 is the largest, k_B2 is the largest, and k_C1 is the largest, then the theoretically optimal combination is A2B2C1 (i.e., 50mg PSA, 50mg C18, 0GCB).

[0052] (c) Analysis of variance (ANOVA) This method is used to quantify the significance of the effects of various factors and determine whether the difference in recovery rate is caused by changes in the amount of purifying agent, rather than experimental error.

[0053] Perform the analysis using professional statistical software (such as SPSS, Design-Expert, Minitab). Input the experimental data (recovery rates for 9 groups) into the software, specifying the factors and levels. Key outputs: F-value and P-value.

[0054] Significance assessment: P < 0.05 is typically used as the significance criterion. If the P value for a factor is < 0.05, it indicates that the factor has a significant impact on the recovery rate. Based on the theoretically optimal combination derived from intuitive analysis and analysis of variance, additional validation experiments (n ≥ 5) are conducted to confirm that its recovery rate, precision, and matrix effect are superior to or equal to other combinations, and that it meets the methodological requirements.

[0055] Table 3 Orthogonal Table of Purifying Agents Each experiment requires the preparation of n≥3 spiked parallel samples, including corresponding matrix blanks and extracted spiked samples for calculating matrix effects.

[0056] result Table 4 Recovery rates after treatment with different combinations of purifying agents Factor A (PSA) k_A1=(0.8142+0.7864+0.7507) / 3=0.7838 k_A2=(0.7332+0.7153+0.6988) / 3=0.7158 k_A3=(0.6902+0.6897+0.6758) / 3=0.6852 Factor B (C18) k_B1=(0.8142+0.7332+0.6902) / 3=0.7459 k_B2=(0.7864+0.7153+0.6897) / 3=0.7305 k_B3=(0.7507+0.6988+0.6758) / 3=0.7084 Factor C (GCB) k_C1=(0.8142+0.6988+0.6897) / 3=0.7342 k_C2=(0.7864+0.7332+0.6758) / 3=0.7318 k_C3=(0.7507+0.7153+0.6902) / 3=0.7187 Calculate the range (R) of each factor. R_A = 0.7838 - 0.6852 = 0.0986 R_B = 0.7459 - 0.7084 = 0.0375 R_C = 0.7342 - 0.7187 = 0.0155 Based on the range R (the larger the R value, the greater the impact): PSA dosage (A) >> C18 dosage (B) > GCB dosage (C). PSA dosage is the most decisive factor affecting recovery rate, C18 dosage has a moderate impact, and GCB dosage has the least impact on recovery rate within the dosage range of this experiment.

[0057] The levels with the highest k-values ​​for each factor were selected: Factor A (PSA): k_A1 (0.7838) was the highest, and the optimal level was A1 (25 mg); Factor B (C18): k_B1 (0.7459) was the highest, and the optimal level was B1 (25 mg); Factor C (GCB): k_C1 (0.7342) was the highest, and the optimal level was C1 (0 mg). The theoretically optimal combination was A1B1C1. The theoretically optimal combination A1B1C1 (25 mg PSA + 25 mg C18 + 0 mg GCB) corresponds exactly to Experiment 1, and its recovery rate (0.8142) is the highest among all 9 experiments, which strongly demonstrates the superiority of this combination.

[0058] Based on the experimental data obtained from the above experiments, this invention proposes a method for detecting metribuzin in soil using liquid chromatography, comprising: The pretreatment method for bentazon in soil is as follows: Weigh 10.0g of soil into a 50.0ml centrifuge tube, add 10.0ml of acetonitrile, vortex for 3.0min, add 3.0g of NaCl, vortex for 1.0min, centrifuge at 5000rpm for 5.0min, take 1.5ml of the supernatant and add it to a 2.5ml centrifuge tube containing 25.0mg PSA, 25.0mg C18, and 100.0mg anhydrous MgSO4, vortex for 1.0min, centrifuge at 8000rpm for 3.0min, use a 5.0ml syringe to aspirate the supernatant and filter it through a 0.22μm membrane, then transfer it to a 2.5ml brown sample bottle for analysis.

[0059] Methodological Validation The reliability, robustness, and applicability of the established QuEChERS-UPLC-MS / MS method will be evaluated to ensure the accuracy, reliability, and reproducibility of the analytical results. Method validation will be conducted through linear range, accuracy (recovery), precision, sensitivity, matrix effects, and stability.

[0060] Linear range and standard curve: Five standard working solutions with different concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 mg / L) were prepared using acetonitrile and the blank soil matrix extract after method treatment.

[0061] Accuracy (expressed as spike recovery rate) Spiked samples at three concentration levels (0.01, 0.05, and 0.1 mg / kg) were prepared by adding known amounts of a mixed standard solution to blank soil samples. Five parallel samples (n=5) were set up for each concentration level. The samples were processed according to the established full process (extraction, purification, and sample loading), the determination concentration of each target analyte was calculated, and the recovery rate was calculated by comparing it with the spiked concentration.

[0062] Precision Precision is divided into intra-day precision (repeatability) and inter-day precision (reproducibility). Intra-day precision: On the same day, the same operator uses the same set of instruments to perform a full analysis of spiked samples at three concentration levels (low, medium, and high) (n=5 for each concentration). Inter-day precision: Spiked samples at the above three concentration levels are analyzed by potentially different operators over consecutive days (at least 3 days). The relative standard deviation (RSD) of the results (recovery rate or concentration) for each group at each concentration level is calculated.

[0063] Sensitivity (limit of detection and limit of quantitation) Signal-to-noise ratio (SNR) method: This method analyzes a low-concentration standard solution or spiked sample by measuring the ratio of the target analyte's response signal (S) to the baseline noise (N). Limit of detection (LOD): Typically the concentration corresponding to a SNR (S / N) ≥ 3. Limit of quantitation (LOQ): Typically the concentration corresponding to a SNR (S / N) > 10. The LOQ should also meet acceptable standards for recovery and precision.

[0064] Matrix effect Prepare three sets of standard curves: a pure solvent standard curve, a matrix-matched standard curve (spiked in the blank extract after method treatment), and calculate the matrix effect (ME) for each target analyte. ME (%) = [(Slope of matrix-matched standard curve / Slope of pure solvent standard curve) - 1] × 100% Table 5. Recovery and RSD of metribuzin at different spiked concentrations Limit of detection (LOD): 0.0001 mg / L Limit of Quantitation (LOQ): 0.05 mg / L ME (%) = [(Slope of matrix-matched standard curve / Slope of pure solvent standard curve) - 1] × 100% = (2966466.54 / 3304749.18-1) * 100% = 10.03%.

[0065] Example 2 This invention systematically evaluates the effects of different volumes of acetonitrile extractant on the recovery, mechanism effect, and method precision of the target analyte, determining the optimal acetonitrile dosage for subsequent UPLC-MS / MS analysis. A single-factor variable method was used, fixing all pretreatment conditions except for the acetonitrile volume. For a 5.0 g test sample, the following four acetonitrile dosage levels were set:

[0066] Level A: 5.0 mL acetonitrile (sample:extractant = 1:1, w / v) Level B: 10.0 mL acetonitrile (sample:extractant = 1:2, w / v) Level C: 15.0 mL acetonitrile (sample:extractant = 1:3, w / v) Level D: 20.0 mL acetonitrile (sample:extractor = 1:4, w / v) At least five parallel samples are required for each level: three spiked samples (for recovery and precision calculations), one matrix blank sample (for background monitoring), and one post-extraction spiked sample (for matrix effect assessment). Accurately weigh 5.0 ± 0.1 g of homogenized blank sample into a 50 mL centrifuge tube. Add an appropriate amount (0.1 mg / L) of standard working solution and let stand for 15-30 min to allow the analytes to bind to the matrix. Matrix blank: No standard added. Post-extraction spiked: Samples are not pre-spikened and are processed after extraction. According to the above experimental design, add the corresponding volume of acetonitrile (5.0, 10.0, 15.0, or 20.0 mL) to each centrifuge tube. Vigorously shake manually to ensure the sample and extractant are fully wetted. Add 3.0 g of NaCl and vortex for 1 min to ensure thorough mixing of the solvent and salt and prevent clumping. Centrifuge at 5000 rpm for 35 min at room temperature to ensure complete separation of the two phases. Accurately transfer 1.5 mL of supernatant from each centrifuge tube into a tube containing 100 mg MgSO4. Vortex for 1 minute, then centrifuge at 8000 rpm for 3 minutes at room temperature. Pivot the supernatant, filter through a 2.0 μm organic filter membrane, and transfer to a 2.0 mL amber sample vial for analysis.

[0067] Evaluation criteria: Recovery rate: Prioritize dosages that maintain stable recoveries of most target analytes between 70% and 120%. Precision: RSD < 20% at the same dosage level. Signal-to-noise ratio: Prioritize dosages with the highest signal-to-noise ratio at the same concentration. Economy and convenience: When performance is similar, prioritize dosages with smaller volume. (The results show...) Figure 14 , Figure 16 The optimal extraction solvent dosage is 1:1.

[0068] Table 6 Orthogonal Table of Different Combinations of Purifying Agents Table 7 Recovery rates after treatment with different combinations of purifying agents Range analysis calculation Calculate the average recovery rate of each factor at different levels: Factor A (GCB): k1 (20mg): (85.78+82.07+80.35) / 3=82.73% k2 (35mg): (85.50+87.92+80.56) / 3=84.66% k3 (50mg): (88.46+84.18+81.31) / 3=84.65% Factor B (PSA): k1 (50mg): (85.78+85.50+88.46) / 3=86.58% k2 (75mg): (82.07+87.92+84.18) / 3=84.72% k3 (100mg): (80.35+80.56+81.31) / 3=80.74 Factor C (C18): k1 (20mg): (85.78+80.56+84.18) / 3=83.51% k2 (25mg): (82.07+85.50+81.31) / 3=82.96% k3 (30mg): (80.35+87.92+88.46) / 3=85.58% Table 8 Summary of Range Analysis Results Analysis of primary and secondary factors: 0. PSA dosage (Factor B) is the most significant factor affecting recovery rate (maximum range: 5.84%). The dosage of C18 (factor C) was a minor factor (range: 2.62%). 0. The amount of GCB used (Factor A) has the least impact (range: 1.93%) Optimal level combination: Analysis based on the k-value (the larger the k-value, the lower the recovery rate). PSA·k1 (50mg) = 86.58% → Optimal level: 50mg C18:k3 (30mg) = 85.58% → Optimal level: 30mg GCB: k2 (35mg) = 84.66% → Optimal level: 35mg Optimal combination: A2B1C3 = GCB 35mg + PSA 50mg + C1 830mg Trend Analysis Significant impact of PSA dosage: When the concentration was increased from 50 mg to 100 mg, the recovery rate decreased significantly from 86.58% to 80.74%, indicating that PSA has a strong adsorption effect on the target pesticide and the dosage should not be too high.

[0069] The effect of C18 dosage: As the amount of C18 increased, the recovery rate showed a trend of first decreasing and then increasing, with the highest recovery rate obtained at 30 mg. This indicates that an appropriate amount of C18 helps to remove interfering substances without significantly affecting the adsorption of the target analyte, GCB dosage. The impact is relatively small; the effects of 35mg and 50mg are comparable, and it is recommended to choose 35mg to save costs.

[0070] This invention also proposes a method for detecting metribuzin in broad bean plants using liquid chromatography-mass spectrometry, comprising: The pretreatment method for bentazon in the aerial parts of broad beans is as follows: Weigh 5.0g of aerial parts into a 50.0ml centrifuge tube, add 15.0ml of acetonitrile, vortex for 3.0min, add 3.0g of NaCl, vortex for 1.0min, centrifuge at 5000rpm for 5.0min, take 1.5ml of the supernatant and add it to a 2.5ml centrifuge tube containing 50.0mg PSA, 30mg C18, 35.0mg GCB, and 100.0mg anhydrous MgSO4, vortex for 1.0min, centrifuge at 8000rpm for 3.0min, use a 5.0ml syringe to aspirate the supernatant and filter it through a 0.22μm membrane, then transfer it to a 2.5ml brown sample bottle for analysis.

[0071] Table 9. Recovery rates and RSDs at different spiking concentrations Therefore, the limit of detection (LOD) is 0.0001 mg / L, the limit of quantitation (LOQ) is 0.0005 mg / L, and the linearity is good. Figure 15 ).

[0072] The table below is an orthogonal array of different combinations and dosages of purifying agents, used for pretreatment optimization with different combinations.

[0073] Table 10 Orthogonal Table of Purifying Agent Treatment Combinations Table 11 Recovery rates after treatment with different combinations of purifying agents The mean recovery rate (k) of each factor at the same level was calculated from different combinations in the table above: Factor A (PSA) k_A1=(86.59+88.77+95.23) / 3=90.20 k_A2=(92.79+94.83+94.00) / 3=93.87 k_A3=(92.14+91.55+92.27) / 3=91.99 Factor B (GCB) k_B1=(86.59+92.79+92.14) / 3=90.51 k_B2=(88.77+94.83+91.55) / 3=91.72 k_B3=(95.23+94.00+92.27) / 3=93.83 Factor C (C18) k_C1=(86.59+94.00+91.55) / 3=90.71 k_C1=(88.77+92.79+92.27) / 3=91.28 k_C3=(95.23+94.83+92.14) / 3=94.07 Calculate the range (R) of each factor: R_A=max(90.20,93.87,91.99)-min(90.20,93.87,91.99)=93.87-90.20=3.67 R_B=max(90.51,91.72,93.83)-min(90.51,91.72,93.83)=93.83-90.51=3.32 R_C=max(90.71,91.28,94.07)-min(90.71,91.28,94.07)=94.07-90.71=3.36 in conclusion: Order of importance of factors Based on the magnitude of the range R (the larger the R value, the greater the impact on the recovery rate): PSA dosage (A) > C18 dosage (C) > GCB dosage (B) The amount of PSA used is the most decisive factor affecting the recovery rate.

[0074] The effect of C18 dosage is secondary.

[0075] The amount of GCB used had the least impact on the recovery rate within the range specified in this experiment.

[0076] Optimal level combination Select the level with the largest k value for each factor. Factor A (PSA): k_A2 (93.87) Maximum → Optimal Level: A2 (75mg) Factor B (GCB): k_B3 (93.83) Maximum → Optimal Level: B3 (30mg) Factor C (C18): k_C3 (94.07) Maximum → Optimal Level: C3 (40mg) Theoretically optimal combination: A2B3C3 Analysis and Discussion Experiment 3: A1B3C3 (Recovery rate 95.23%) Experiment 5: A2B2C3 (Recovery rate 94.83%) Experiment 6: A2B3C1 (Recovery rate 94.00%) Based on trend analysis, A2B3C3 is expected to achieve the highest recovery rate. PSA: Recovery rate initially increases with PSA dosage, then slightly decreases. 75mg is the optimal value; dosages that are too low (50mg) or too high (100mg) both lead to decreased recovery rates, indicating that PSA has an adsorption effect on certain pesticides. GCB: Recovery rate continuously increases with increasing GCB dosage. In the 20-30mg range, no strong adsorption of the target pesticide was observed; increasing the dosage may actually improve the recovery rate by better removing interfering substances such as pigments. C18: Recovery rate continuously increases with increasing C18 dosage. In the 30-40mg range, no significant adsorption side effects were observed; increasing the dosage may have improved the recovery rate by better removing non-polar interfering substances.

[0077] in conclusion: The theoretically optimal combination A2B3C3 (75mg PSA, 30mg GCB, 40mg C18) is recommended as the standard method.

[0078] It is strongly recommended that a verification experiment (n≥3) be conducted according to this ratio to measure its recovery rate and confirm whether its performance is as superior as predicted.

[0079] The recovery rates of all experimental groups were above 86%, indicating that within the tested dosage range, the adsorption of the target analyte during the purification process was not severe, and the method as a whole had good robustness.

[0080] Summarize Range analysis using orthogonal experiments revealed the optimal combination of purifying agents: 75g PSA, 30mg GCB, and 40mg C18. The dosage of PSA is a parameter requiring precise control. A final verification experiment should be conducted to confirm the actual performance of this optimal combination.

[0081] Table 12 Recovery and RSD at different spike concentrations Detection Item (LOD): 0.0001 mg / L Limit of Quantification (LOQ): 0.0005 mg / L Comparative Example Compared to methods for determining metribuzin residues in plant-derived foods using liquid chromatography-mass spectrometry (LC-MS / MS), this invention offers a simpler and faster pretreatment process. While LC-MS / MS methods employ strong anion exchange solid-phase extraction columns, this invention utilizes the QuECHERS method, which is rapid, efficient, economical, and safe. Furthermore, compared to other methods, this invention has lower detection limits and quantitation limits, enabling the detection of even smaller concentrations. Figure 17 ).

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pretreatment method for detecting monuron in soil using liquid chromatography mass spectrometry, characterized by, include: Weigh 10.0g of soil and place it in a 50.0ml centrifuge tube. Add 10.0ml of acetonitrile and vortex for 3.0min. Add 3.0g of NaCl and vortex for 1.0min. Centrifuge at 5000rpm for 5.0min. Take 1.5ml of the supernatant and add it to a 2.5ml centrifuge tube containing 25.0mg PSA, 25.0mg C18, and 100mg anhydrous MgSO4. Vortex for 1.0min and centrifuge at 8000rpm for 3.0min. Use a 5.0ml syringe to aspirate the supernatant, filter it through a membrane, and transfer it to a 2.5ml brown sample bottle.

2. A pretreatment method for detecting bentazone in Vicia faba by liquid chromatography-mass spectrometry, characterized in that, include: Weigh 5.0g of the aboveground parts of broad bean plants and place them in a 5.0ml centrifuge tube. Add 15.0ml of acetonitrile and vortex for 3.0min. Add 3.0g of NaCl and vortex for 1.0min. Centrifuge at 5000rpm for 5.0min. Take 1.5ml of the supernatant and add it to a 2.5ml centrifuge tube containing 25.0mg PSA, 25.0mg C18, and 100.0mg anhydrous MgSO4. Vortex for 1.0min and centrifuge at 8000rpm for 3.0min. Use a 5.0ml syringe to aspirate the supernatant, filter it through a membrane, and transfer it to a 2.5ml brown sample bottle.

3. The method according to claim 1 or 2, characterized in that, The filter membrane has a pore size of 0.22 μm.

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