Method for measuring pesticide residues in environment through full-automatic dispersive liquid-liquid microextraction based on bio-derived solvent

Abstract

The invention discloses a green, efficient and economical method for measuring pesticide residues in an environment through full-automatic dispersive liquid-liquid microextraction based on a bio-derived solvent, belongs to the technical field of environmental hazard monitoring, and adopts a liquid treatment platform only provided with a pipetting function module to realize full-automatic pretreatment of multiple samples and eliminate manual errors. Green bio-derived solvents gamma-valerolactone and eudesmol are used as renewable resources respectively as a dispersing agent and an extracting agent, and have very high environmental friendliness. The cineole is automatically separated from a sample within a preset time, and centrifugation and demulsification are not needed. The ultra-high performance liquid chromatography-tandem mass spectrometry has obvious advantages in the aspect of sensitivity. The method is already applied to analysis of pesticide residues in tap water, river water, lake water and spring water samples, reproducibility, accuracy, precision, detection limit and recovery rate of the method can meet analysis requirements, operation continuity is good, sample treatment efficiency is high, and reference can be provided for rapid pretreatment of a large number of samples of a detection mechanism.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental hazard monitoring, and specifically discloses a method for determining pesticide residues in the environment by fully automatic dispersed liquid-liquid microextraction based on a bio-derived solvent. Background Art

[0002] Diamide insecticides exhibit unique targeting effects on lepidopteran pests, disrupting their feeding behavior and causing death. Their market share continues to increase, making them one of the best-selling insecticides. However, frequent use leads to excessive residues in agricultural soils, which in turn pollute aquatic environments such as rivers, lakes, and springs. Diamide insecticides are persistent and mobile in aquatic environments, spreading rapidly in aquatic ecosystems, posing environmental risks. Therefore, establishing a method for rapid detection of diamide insecticide residues in aquatic environments is crucial for monitoring environmental risks.

[0003] Liquid-liquid microextraction is a miniaturized liquid-liquid extraction technique that only requires microliter-level reagents to achieve the extraction and enrichment of analytes. The degree of automation is a key criterion for evaluating the greenness of analytical methods. Automation greatly shortens sample pretreatment time and minimizes errors caused by manual operations. However, the currently reported automated liquid-liquid microextraction methods usually rely on customized functional modules such as peristaltic pumps, customized containers, and selection valves. The high cost and complexity of these devices hinder the wider development and application of fully automated liquid-liquid microextraction.

[0004] Dispersive liquid-liquid microextraction is the most widely used liquid-liquid microextraction technique. Traditional dispersive liquid-liquid microextraction methods rely on toxic dispersants to achieve rapid dispersion of the extractant. In order to address environmental issues and avoid the use of toxic dispersants, some studies have used extraction devices (vortexers, oscillators, and ultrasonic generators) to help disperse the extractant. However, introducing these extraction devices and separation devices (centrifuges) as functional modules into fully automated dispersive liquid-liquid microextraction systems also incurs high costs. Therefore, there is an urgent need to develop inexpensive fully automated dispersive liquid-liquid microextraction methods that eliminate the need for special functional modules. Summary of the invention

[0005] In view of the defects of the prior art, the present invention proposes a method for determining pesticide residues in the environment by fully automatic dispersed liquid-liquid microextraction based on bio-derived solvents, which is green, efficient and low-cost. The entire pretreatment process, from sample addition to extraction completion, is fully automated; the impact on the environment is minimized by using green and renewable bio-derived solvents; and because eucalyptol has the unique property of spontaneous separation from the sample, the separation operation does not require centrifugation or the use of demulsifiers. This method can successfully extract diamide pesticides from various environmental samples (including tap water, river water, lake water and spring water), and can perform fully automatic simultaneous processing of multiple samples by using a liquid handling platform equipped with only a simple pipetting function module, which conforms to the principles of green analytical chemistry, improves the efficiency of sample pretreatment, and eliminates the need for expensive dedicated function modules commonly found in fully automatic pretreatment systems.

[0006] The invention provides a method for determining pesticide residues in an environment by fully automatic dispersed liquid-liquid microextraction based on a bio-derived solvent. Eucalyptol, methyl linoleate, methyl laurate or guaiacol are used as extractants, and gamma-valerolactone is used as a dispersant. A mixed solution of the extractant and the dispersant is added to a sample containing a target pesticide and mixed thoroughly. After standing for a preset time, the extractant is spontaneously separated from the sample. The extractant and the mobile phase are transferred into a sample injection bottle, and ultra-high performance liquid chromatography-tandem mass spectrometry is used for detection to obtain the chromatographic peak area of ​​the target pesticide in the sample. The chromatographic peak area of ​​the target pesticide is brought into a linear equation of the target pesticide to calculate the mass concentration of the target pesticide in the sample. The target pesticide contained in the sample is either flubendiamide or chlorfenapyr or a mixture of the two.

[0007] The above-mentioned method for determining pesticide residues in the environment by fully automatic dispersive liquid-liquid microextraction based on bio-derived solvents specifically comprises the following steps:

[0008] S1, placing the pipette tip box, liquid reservoir, deep well plate I, deep well plate II and waste box into the five card slots of the liquid handling platform respectively, adding the sample, extractant and dispersant into different grids of the liquid reservoir for standby use, and multiple pipettes are installed on the pipetting arm of the liquid handling platform;

[0009] S2, the pipetting arm of the liquid handling platform moves to the position of the pipette tip box and installs the pipette tip of the pipette;

[0010] S3, the pipetting arm of the liquid handling platform moves to the well position corresponding to the sample in the liquid storage tank, the pipette quantitatively absorbs the sample, and then moves to the position of deep well plate I, adds the sample to the sample well of deep well plate I, and repeats the operation until the sample amount reaches the preset value;

[0011] S4, the pipetting arm of the liquid handling platform moves to the waste box position, unloads the pipetting tip, and then repeats step S2;

[0012] S5, the pipetting arm of the liquid handling platform moves to the well position corresponding to the extractant in the liquid storage tank, the pipette quantitatively absorbs the extractant, and then moves to the position of the deep well plate II, adds the extractant to the sample well of the deep well plate II, and then repeats step S4;

[0013] S6, the pipetting arm of the liquid handling platform moves to the well corresponding to the dispersant in the liquid storage tank, the pipette quantitatively absorbs the dispersant, then moves to the deep well plate II position, adds the dispersant to the extractant, and then quantitatively absorbs the mixed solution of the extractant and the dispersant;

[0014] S7, the pipetting arm of the liquid handling platform moves to the deep well plate I position, the pipette adds the mixed solution of the extractant and the dispersant to the sample, and performs several suction operations to accelerate the mixing, so that the dispersant is dissolved in the sample and the extractant is fully dispersed;

[0015] S8, after standing for a preset time, taking the extractant and the mobile phase and transferring them into a sample injection bottle, and using ultra-high performance liquid chromatography-tandem mass spectrometry for detection to obtain the chromatographic peak area of ​​the target pesticide in the sample;

[0016] S9, preparing target pesticide standard solutions with different mass concentrations, transferring them into injection bottles, and detecting them by ultra-high performance liquid chromatography-tandem mass spectrometry, plotting a standard working curve with the mass concentration of the target pesticide standard solution as the abscissa and the corresponding chromatographic peak area as the ordinate, and obtaining a linear equation;

[0017] S10, the chromatographic peak area of ​​the target pesticide in the sample after step S8 is substituted into the linear equation in step S9 to calculate the mass concentration of the target pesticide in the sample.

[0018] In the above-mentioned method for determining pesticide residues in the environment by fully automatic dispersive liquid-liquid microextraction based on bio-derived solvents, the volume ratio of the extractant to the dispersant is 1:0 to 1:8; the ratio between the total volume of the extractant and the dispersant and the sample volume is: 0.6 to 1.2: 4 to 7; the mixed solution of the extractant and the dispersant is mixed with the sample through a pipette, the mixing times are 0 to 8, and the mixing speed is 140 to 430 μL / s.

[0019] Preferably, the volume ratio of the extractant and the dispersant is 1:4 to 1:7; the ratio between the total volume of the extractant and the dispersant and the sample volume is 0.4 to 0.6:2 to 3; the mixed liquid of the extractant and the dispersant and the sample are mixed 3 to 7 times, and the mixing speed is 315 to 430 μL / s.

[0020] Preferably, the extractant is eucalyptol; the volume ratio of the extractant to the dispersant is 1:6; the ratio between the total volume of the extractant and the dispersant and the sample volume is 1:5; the number of mixing times of the mixed solution of the extractant and the dispersant and the sample is 5, and the mixing speed is 430 μL / s.

[0021] In the above-mentioned method for determining pesticide residues in the environment by fully automated dispersive liquid-liquid microextraction based on bio-derived solvents, in the ultra-high performance liquid chromatography-tandem mass spectrometry detection, the chromatographic conditions are as follows: the mobile phase is divided into phase A and phase B, phase A is an aqueous phase, using a mixed aqueous solution of 0.01% formic acid and 2mmoL / L ammonium formate, phase B is an organic phase, using methanol, and the mobile phase gradient conditions are set according to the following table

[0022]

[0023] The column oven temperature was 40°C, the injection volume was 2 μL, and the flow rate was 0.3 mL / min;

[0024] The mass spectrometry conditions are as follows: capillary voltage 5.5 kV, ion source temperature 550 °C, curtain gas 40 psi, nebulizer gas Gas1 and Gas2 both 55 psi, detection mode multiple reaction monitoring, target ionization mode, parent-daughter ion pair, declustering voltage and collision voltage are set according to the following table

[0025]

[0026] In step S9, chlorantraniliprole and flubendiamide standards are accurately weighed to prepare mixed standard solutions with a mass concentration of 1000 μg / mL each, and standard solutions of different mass concentrations are obtained by diluting with methanol using a multiple gradient dilution method.

[0027] In the above-mentioned method for determining pesticide residues in the environment by fully automatic dispersive liquid-liquid microextraction based on bio-derived solvents, the specifications of deep well plate I are 4×6×10mL, and the specifications of deep well plate II are 8×12×2mL. The sample is one of tap water, river water, lake water and spring water containing target pesticides.

[0028] Compared with the prior art, the present invention has the following beneficial effects.

[0029] 1. The present invention is based on a liquid processing platform, which can simultaneously extract multiple samples within 10 seconds and separate the extractants within 5 minutes. Compared with the traditional dispersed liquid-liquid microextraction method, it improves the degree of automation and sample processing speed, avoids the errors caused by manual operation, improves the precision of the results, and liberates labor to a great extent, reducing labor costs.

[0030] 2. The present invention does not require the use of special containers and equipment, and realizes inexpensive fully automated operation in a universal deep-well plate and liquid handling platform, thereby reducing instrument costs.

[0031] 3. Compared with the existing liquid-liquid microextraction method, the fully automatic dispersed liquid-liquid microextraction method based on bio-derived solvents provided by the present invention does not use toxic extractants and toxic dispersants, and has the significant advantage of being environmentally friendly.

[0032] 4. Based on the properties of eucalyptol and the assistance of γ-valerolactone, the novel extractant eucalyptol used in the present invention only needs to be left to stand after the extraction is completed to achieve spontaneous separation from the sample, thereby avoiding time-consuming centrifugation operations and eliminating the need to add expensive centrifugation function modules to the liquid processing platform, thereby achieving full automation from extraction to separation.

[0033] 5. Under the optimal extraction and detection conditions, this method has obvious advantages in sensitivity, with a detection limit of 0.002μg / L and a quantification limit of 0.006μg / L. The test results can be obtained within 4.5 minutes, which is superior to traditional diamide pesticide detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 is a flow chart of a method for the determination of pesticide residues in the environment using fully automated dispersive liquid-liquid microextraction based on bio-derived solvents;

[0036] Figure 2 is the effect of the type of extractant on the extraction efficiency;

[0037] Figure 3 is the effect of the volume ratio of the extractant and dispersant on the extraction efficiency;

[0038] Figure 4 is the effect of the total volume of the extractant and dispersant on the extraction efficiency;

[0039] Figure 5 It is the effect of the number of times the mixture of extractant and dispersant is mixed with the sample on the extraction efficiency;

[0040] Figure 6 It is the effect of the mixing speed of the mixture of extractant and dispersant with the sample on the extraction efficiency;

[0041] Figure 7 is the effect of sample volume on extraction efficiency. DETAILED DESCRIPTION

[0042] Eucalyptol is extracted from plant essential oils and has suitable hydrophobicity and density. It can be used as a new type of extractant and can achieve spontaneous separation from the sample after the extraction is completed. γ-Valerolactone is a green renewable solvent, commonly used as a food additive, and can be dissolved in both aqueous and organic phases. In addition, compared with water, γ-valerolactone has a higher density, which helps to spontaneously separate low-density eucalyptol from the sample by further increasing the density difference between the aqueous phase and the extractant. Based on this, the present invention provides a method for determining pesticide residues in the environment by fully automatic dispersed liquid-liquid microextraction. After the automated extraction is completed, the extractant and the sample undergo spontaneous phase separation, and eucalyptol is the upper liquid. Due to the influence of the liquid surface tension, eucalyptol is concentrated in the corners of each square deep well in the deep well plate I (4×6×10mL) for easy collection.

[0043] The present invention studies and optimizes the main factors affecting the extraction efficiency, such as the type of extractant, the volume ratio of the extractant to the dispersant, the total volume of the extractant and the dispersant, the number of mixing times, the mixing speed and the sample volume, and establishes a method for determining pesticide residues in the environment by fully automatic dispersive liquid-liquid microextraction based on bio-derived solvents combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). The method has fast operation, high experimental repeatability, and realizes simultaneous processing of multiple samples.

[0044] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below in conjunction with the accompanying drawings through specific embodiments. All that is not particularly specified is carried out using the prior art in the art. The sources of the reagents used in the following examples are as follows: flubendiamide and chlorfenapyr (purity> 95%) were purchased from a reagent company; chromatographically pure methyl laurate, methyl linoleate, guaiacol, eucalyptol, γ-valerolactone, ammonium formate and formic acid were purchased from MacLean Biochemical Technology Co., Ltd. (Shanghai, China); chromatographically pure methanol and acetonitrile were purchased from Merck Group (Darmstadt, Germany).

[0045] Example 1 Optimization experiment

[0046] The present invention has many influencing factors on the pretreatment of environmental samples, and a series of optimization experiments are required. Therefore, single-factor optimization is carried out on the type of extractant, the volume ratio of the extractant and the dispersant, the total volume of the extractant and the dispersant, the number of mixing of the mixed solution of the extractant and the dispersant with the sample, the mixing speed, and the sample volume. Sampling is carried out in different water environments at the applicant's location to verify the reliability of the detection results of the method of the present invention in practical applications, and the following experiments are carried out.

[0047] 1. Optimization of extraction agent types

[0048] Due to the different physical and chemical properties of the extractants, their ability to extract analytes can vary greatly. To optimize the selection of the extractant, four bioderived solvents were evaluated: eucalyptol, guaiacol, methyl laurate, and methyl linoleate.

[0049] like Figure 2 As shown, the extraction recovery rate corresponding to eucalyptol is the highest. This may be due to the different oil-water partition coefficients of the four extractants. The oil-water partition coefficient of eucalyptol is 3.4, which is closer to the target chlorantraniliprole (2.9) and flubendiamide (4.1). The closer the oil-water partition coefficient values ​​of the extractant and the target, the higher the affinity between the two, and the easier it is to extract. In the separation process, if the density of the extractant is too small, the separation speed will be too fast and the extraction efficiency will be reduced; the closer the density of the extractant is to water, the easier it will be to cause self-separation difficulties and it will not be completely separated within a certain period of time. Since the densities of eucalyptol and γ-valerolactone are slightly smaller than water and greater than water, respectively, when the sample is miscible with the dispersant, the density of the mixed system is slightly greater than water, so that the separation speed of eucalyptol and it is moderate, and rapid separation can be achieved under the premise of efficient extraction. Therefore, eucalyptol was selected as the best type of extractant.

[0050] 2. Optimization of the volume ratio of extractant and dispersant

[0051] The low volume ratio of eucalyptol relative to the dispersant will lead to significant dissolution of eucalyptol in the sample, which is not conducive to complete extraction. On the contrary, excessive eucalyptol is not easy to disperse and the droplet radius formed is larger. In order to optimize the volume ratio of the extractant to the dispersant, eight different ratios were optimized: 1:0, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 and 1:8.

[0052] like Figure 3 As shown in the figure, the extraction recovery rate showed a trend of first increasing and then decreasing, reaching a maximum value at a ratio of 1:6. This trend shows that the appropriate volume ratio is essential to promote the uniform dispersion of eucalyptol in the sample in the form of smaller droplets. Therefore, 1:6 was selected as the optimal volume ratio of extractant to dispersant.

[0053] 3. Optimization of the total volume of extractant and dispersant

[0054] Insufficient eucalyptol will lead to incomplete extraction, however, a larger eucalyptol volume will reduce the sensitivity and environmental friendliness of the method. In order to optimize the total volume of the extractant and dispersant, seven different total volumes of the extractant and dispersant were optimized: 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 and 1.2 mL.

[0055] like Figure 4As shown in Figure 1, as the total volume increased from 0.6 mL to 1.0 mL, the extraction recovery rate gradually increased, and the subsequent further increase in the total volume did not significantly affect the extraction recovery rate. Therefore, 1 mL was selected as the optimal total volume of the extractant and dispersant.

[0056] 4. Optimization of the number of times the mixture of extractant and dispersant is mixed with the sample

[0057] Adequate mixing helps eucalyptol to form small and uniform droplets, which are then effectively dispersed in the sample. The larger specific surface area enhances mass transfer and ultimately improves the extraction efficiency. In order to optimize the mixing times, optimization experiments were conducted for different mixing times: 0, 1, 2, 3, 4, 5, 6, 7, 8, and 10 times.

[0058] like Figure 5 As shown in the figure, the extraction recovery rate gradually increased with the increase of mixing times and reached the maximum at 5 times. The slight decrease in extraction recovery rate when the mixing times were too many may be due to the strong binding of eucalyptol with the sample, which slowed down its spontaneous separation. Therefore, 5 times was selected as the optimal mixing number.

[0059] 5. Optimization of the mixing speed of the extractant and dispersant mixture with the sample

[0060] The relative motion between the liquids helps to achieve effective mixing, and high-speed mixing helps to achieve a balanced distribution of the diamide insecticide between the eucalyptol droplets and the sample. In order to optimize the mixing speed, different mixing speeds were optimized: 140, 180, 225, 270, 315, 360 and 430 μL / s.

[0061] like Figure 6 As shown in the figure, the extraction recovery rate continued to increase with the increase of mixing speed. Therefore, 430 μL / s was selected as the optimal mixing speed.

[0062] 6. Optimization of sample volume

[0063] To improve the enrichment factor and sensitivity of the fully automated dispersive liquid-liquid microextraction method based on bio-derived solvents, optimization experiments were performed with different sample volumes: 4, 5, 6, and 7 mL.

[0064] like Figure 7 As shown in the figure, the extraction recovery rate initially remained at a high level and then gradually decreased with the increase of sample volume. This may be due to the increased loss of eucalyptol and the fact that the quantitative extractant could not achieve complete extraction with the larger sample volume. Therefore, 5 mL was selected as the optimal sample volume.

[0065] Example 2 Analysis characteristics of diamide pesticides in samples

[0066] (1) Sample pretreatment

[0067] The tap water, river water, lake water and spring water samples obtained from different sampling sites were passed through a 0.45 μm filter for later use.

[0068] (2) Sample extraction

[0069] Samples, extractants, and dispersants were stored in three separate reservoirs within the liquid handling platform. First, four 5 mL samples were transferred into a 24-well cube deep-well plate (4×6×10 mL). Subsequently, four 0.143 mL portions of eucalyptol and four 0.857 mL portions of γ-valerolactone were transferred sequentially into a 96-well deep-well plate (8×12×1.2 mL) to form four 1 mL mixtures, in which the volume ratio of eucalyptol to γ-valerolactone was 1:6. Finally, the four mixtures were transferred into the samples in the corresponding wells and then mixed five times at a speed of 430 μL / s. After standing for 5 min, the extractants were collected from the corners of the 24-well cube deep-well plate and analyzed using UHPLC-MS / MS. Figure 1 A flowchart of the process is shown in Figure 1.

[0070] (3) Preparation of standard solution

[0071] Accurate flubendiamide and chlorantraniliprole standards were prepared by preparing a mixed standard solution with a mass concentration of 1000 μg / mL each. The mixed standard solution was diluted with methanol using a multiple gradient dilution method to obtain standard solutions with mass concentrations of 3, 0.6, 0.3, 0.06, 0.03, and 0.006 μg / L for flubendiamide and chlorantraniliprole, respectively. The standard solutions were then stored in a light-proof environment.

[0072] (4) UHPLC-MS / MS detection

[0073] Chromatographic conditions: mobile phase is methanol and water (mixed aqueous solution of 0.01% formic acid and 2mmoL / L ammonium formate), mobile phase gradient conditions are shown in Table 1; column oven temperature is 40°C; injection volume is 2μL; flow rate is 0.3mL / min. Mass spectrometry conditions are: capillary voltage is 5.5kV; ion source temperature is 550°C; curtain gas is 40psi; nebulizer gas Gas1 and Gas2 are both 55psi; detection mode is multiple reaction monitoring; ionization mode of target; parent-daughter ion pair, declustering voltage and collision voltage and other conditions are shown in Table 2.

[0074] Table 1 Mobile phase gradient conditions

[0075]

[0076] Table 2 Mass spectrometry optimization parameters for diamide insecticides

[0077]

[0078] Drawing of standard curve: Prepare the standard solution and spiked sample in step (3) and perform the test according to the operation of step (2) and step (4). Draw the standard curve with the mass concentration x of the mixed standard solution of the two pesticides as the horizontal axis and the corresponding chromatographic peak area y as the vertical axis. The linear equations of different sample matrices are shown in Table 3. The determination coefficient R of the linear equation is 2 ≥0.998. According to LOD being 3 times of the signal-to-noise ratio and LOQ being 10 times of the signal-to-noise ratio, the limits of detection (LOD) and quantification (LOQ) of the two pesticide residues were both 0.002 μg / L and 0.006 μg / L, which were much better than the sensitivity in the literature methods. The intra-day relative standard deviation (RSD) and inter-day RSD were 1.4-5.9% and 4.0-5.3%, respectively, meeting the analysis requirements.

[0079] Table 3 Analytical characteristics of diamide insecticides

[0080]

[0081] Example 3 Recovery of diamide pesticides in samples

[0082] First, flubendiamide and chlorantraniliprole in tap water, river water, lake water and spring water samples were detected, and the content was below the detection limit. Then, flubendiamide and chlorantraniliprole were added to tap water, river water, lake water and spring water at final mass concentrations of 0.006μg / L, 0.06μg / L and 0.6μg / L, respectively, and then detected according to the operations of steps (2) and (4), and the detection mass concentration was obtained through the standard curve. The results in Table 4 show that the recovery rate of flubendiamide in tap water, river water, lake water and spring water is 91.1-107.7%; the recovery rate of chlorantraniliprole in tap water, river water, lake water and spring water is 83.4-97.6%, indicating that this method has good accuracy and applicability. The relative standard deviations (RSDs) of the two pesticides are both between 1.7% and 5.4%, indicating that the method has good precision.

[0083] Table 4 Recovery rate and relative standard deviation of target substances in tap water, river water, lake water and spring water

[0084]

[0085] Example 4 Comparison of this method with reported diamide pesticide detection methods

[0086] This method has obvious advantages over other pretreatment methods for the analysis of diamide pesticides in the environment (Table 5). It is worth noting that this method only uses 1mL of bio-derived solvents (eucalyptol and γ-valerolactone), which significantly reduces solvent consumption and improves its greenness compared to most methods. Although the dSPE method may use less solvent (200μL), the toxicity of solvents such as acetonitrile is significantly higher than that of eucalyptol and γ-valerolactone. In addition, compared with the long preparation time of more than 72 and 24 hours for IL-MMIP in MSPE and MOF in dSPE, respectively, the present invention requires almost no pre-preparation time. In addition, the present invention eliminates multiple steps such as vortexing, centrifugation, water bath, evaporation and redissolution, simplifying the sample pretreatment process. In addition, the present invention is particularly suitable for automation and multi-sample pretreatment, and has lower detection limit and quantification limit. In summary, the present invention has the advantages of environmental protection, high efficiency and high sensitivity.

[0087] Table 5 Comparison of sample pretreatment and detection methods of this method with other diamide pesticides

[0088]

[0089] MSPE: magnetic solid phase extraction;

[0090] dSPE: dispersive solid phase extraction;

[0091] SPE: solid phase extraction;

[0092] PSA: N-propylethylenediamine;

[0093] IL-MMIP: magnetic ionic liquid molecular imprinted polymer (synthesis time > 72 h);

[0094] MOF: Metal-organic framework (synthesis time > 24 h).

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for determining pesticide residues in the environment by fully automated dispersive liquid-liquid microextraction based on bio-derived solvents, characterized in that: Eucalyptol, methyl linoleate, methyl laurate or guaiacol are used as extractants, and gamma-valerolactone is used as dispersant. A mixed solution of the extractant and the dispersant is added to a sample containing a target pesticide and mixed thoroughly. After standing for a preset time, the extractant is spontaneously separated from the sample. The extractant and the mobile phase are transferred into an injection bottle, and ultra-high performance liquid chromatography-tandem mass spectrometry is used for detection to obtain the chromatographic peak area of ​​the target pesticide in the sample. The chromatographic peak area of ​​the target pesticide is substituted into the linear equation of the target pesticide to calculate the mass concentration of the target pesticide in the sample. The target pesticide contained in the sample is either flubendiamide or chlorfenapyr or a mixture of the two.

2. The method for determining pesticide residues in the environment by fully automated dispersive liquid-liquid microextraction based on bio-derived solvents according to claim 1, characterized in that: The steps include: S1, placing the pipette tip box, liquid reservoir, deep well plate I, deep well plate II and waste box into the five card slots of the liquid handling platform respectively, adding the sample, extractant and dispersant into different grids of the liquid reservoir for standby use, and multiple pipettes are installed on the pipetting arm of the liquid handling platform; S2, the pipetting arm of the liquid handling platform moves to the position of the pipette tip box and installs the pipette tip of the pipette; S3, the pipetting arm of the liquid handling platform moves to the well position corresponding to the sample in the liquid storage tank, the pipette quantitatively absorbs the sample, and then moves to the position of deep well plate I, adds the sample to the sample well of deep well plate I, and repeats the operation until the sample amount reaches the preset value; S4, the pipetting arm of the liquid handling platform moves to the waste box position, unloads the pipetting tip, and then repeats step S2; S5, the pipetting arm of the liquid handling platform moves to the well position corresponding to the extractant in the liquid storage tank, the pipette quantitatively absorbs the extractant, and then moves to the position of the deep well plate II, adds the extractant to the sample well of the deep well plate II, and then repeats step S4; S6, the pipetting arm of the liquid handling platform moves to the well corresponding to the dispersant in the liquid storage tank, the pipette quantitatively absorbs the dispersant, then moves to the deep well plate II position, adds the dispersant to the extractant, and then quantitatively absorbs the mixed solution of the extractant and the dispersant; S7, the pipetting arm of the liquid handling platform moves to the deep well plate I position, the pipette adds the mixed solution of the extractant and the dispersant to the sample, and performs several suction operations to accelerate the mixing, so that the dispersant is dissolved in the sample and the extractant is fully dispersed; S8, after standing for a preset time, taking the extractant and the mobile phase and transferring them into a sample injection bottle, and using ultra-high performance liquid chromatography-tandem mass spectrometry for detection to obtain the chromatographic peak area of ​​the target pesticide in the sample; S9, preparing target pesticide standard solutions with different mass concentrations, transferring them into injection bottles, and detecting them by ultra-high performance liquid chromatography-tandem mass spectrometry, plotting a standard working curve with the mass concentration of the target pesticide standard solution as the abscissa and the corresponding chromatographic peak area as the ordinate, and obtaining a linear equation; S10, the chromatographic peak area of ​​the target pesticide in the sample after step S8 is substituted into the linear equation in step S9 to calculate the mass concentration of the target pesticide in the sample.

3. The method for determining pesticide residues in the environment by fully automatic dispersive liquid-liquid microextraction based on bio-derived solvents according to claim 1 or 2, characterized in that: The volume ratio of the extractant to the dispersant is 1:0 to 1:8; The ratio between the total volume of the extractant and dispersant and the sample volume is: 0.6-1.2:4-7; The mixed solution of the extractant and the dispersant is mixed with the sample through a pipette, the mixing times are 0 to 8, and the mixing speed is 140 to 430 μL / s.

4. The method for determining pesticide residues in the environment by fully automated dispersive liquid-liquid microextraction based on bio-derived solvents according to claim 3, characterized in that: The volume ratio of the extractant to the dispersant is 1:4 to 1:7; The ratio between the total volume of the extractant and dispersant and the sample volume is 0.4-0.6:2-3; The mixed solution of the extractant and the dispersant is mixed with the sample 3 to 7 times at a mixing speed of 315 to 430 μL / s.

5. The method for determining pesticide residues in the environment by fully automatic dispersive liquid-liquid microextraction based on bio-derived solvents according to claim 1 or 2, wherein the extractant is eucalyptol; The volume ratio of extractant to dispersant is 1:6; The ratio between the total volume of the extractant and dispersant and the sample volume was 1:5; The mixed solution of the extractant and the dispersant was mixed with the sample 5 times at a mixing speed of 430 μL / s.

6. The method for determining pesticide residues in the environment by fully automated dispersive liquid-liquid microextraction based on bio-derived solvents according to claim 1 or 2, characterized in that: In the ultra-high performance liquid chromatography-tandem mass spectrometry detection, the chromatographic conditions are as follows: the mobile phase is divided into phase A and phase B, phase A is an aqueous phase, using a mixed aqueous solution of 0.01% formic acid and 2mmoL / L ammonium formate, phase B is an organic phase, using methanol, and the mobile phase gradient conditions are set according to the following table The column oven temperature was 40°C, the injection volume was 2 μL, and the flow rate was 0.3 mL / min; The mass spectrometry conditions are as follows: capillary voltage 5.5 kV, ion source temperature 550 °C, curtain gas 40 psi, nebulizer gas Gas1 and Gas2 both 55 psi, detection mode multiple reaction monitoring, target ionization mode, parent-daughter ion pair, declustering voltage and collision voltage are set according to the following table 7. The method for determining pesticide residues in the environment by fully automated dispersive liquid-liquid microextraction based on bio-derived solvents according to claim 2, characterized in that: In step S9, chlorantraniliprole and flubendiamide standards are accurately weighed to prepare mixed standard solutions with a mass concentration of 1000 μg / mL each, and standard solutions of different mass concentrations are obtained by diluting with methanol using a multiple gradient dilution method.

8. The method for determining pesticide residues in the environment by fully automated dispersive liquid-liquid microextraction based on bio-derived solvents according to claim 1 or 2, characterized in that: The specifications of deep well plate I are 4×6×10mL, and the specifications of deep well plate II are 8×12×2mL; the sample is one of tap water, river water, lake water and spring water containing target pesticides.

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