Method for determination of pesticide residues in food samples by high-throughput semi-automated emulsion liquid microextraction

Abstract

The application provides a method for determining pesticide residues in food samples by high-flux semi-automatic emulsion liquid microextraction, and belongs to the technical field of food detection. A hydrophobic extractant and water are prepared into a primary oil-in-water emulsion, which is injected into a sample. The primary oil-in-water emulsion has the characteristic of being diluted by water, so that a secondary oil-in-water emulsion is quickly formed, thereby realizing rapid extraction of pesticides in the sample. Then, a saturated NaCl solution is injected into the secondary oil-in-water emulsion as a demulsifier to quickly separate the extractant, thereby omitting the centrifugation step. The application has been applied to the analysis of pesticide residues in food samples, and the reproducibility, accuracy, precision, detection limit and recovery rate of the method can meet the analysis requirements.

Description

Technical Field

[0001] This invention belongs to the technical field of food testing, and specifically discloses a high-throughput semi-automated method for determining pesticide residues in food samples using emulsion liquid microextraction. Background Technology

[0002] Pesticides are widely used in agricultural production to achieve high quality and high yields, and the issue of pesticide residues in food is receiving increasing attention worldwide. Pesticide residues can enter food through air, water, and soil, posing health problems to humans. Therefore, it is necessary to employ rapid, efficient, and accurate technologies to detect pesticide residues in food samples.

[0003] Currently, rapid detection methods for pesticide residues are typically evaluated based on factors such as operation time, precision, and sensitivity. Automation, as the latest trend in technological development, has broad application prospects in liquid-liquid microextraction (LLME). Automated processing can not only greatly improve sample processing efficiency but also significantly reduce human intervention and errors caused by manual operation, meeting the requirements of green sample preparation principles. However, currently reported automated or semi-automated LLME methods often use expensive peristaltic pumps, custom-made containers, and selector valves. Furthermore, these methods face difficulties in separating the extractant, still requiring centrifugation, which prolongs operation time and increases energy consumption. In addition, another way to reduce sample pretreatment time is to increase sample throughput, but high-throughput sample pretreatment methods are currently limited. Therefore, there is an urgent need to develop a high-throughput pesticide residue analysis method based on automation or semi-automation to improve detection speed and precision.

[0004] Liquid-liquid microextraction (LLME) is a technology that has gradually developed to address the problems of high extractant consumption and low enrichment coefficients in traditional LLME. It mainly includes single-droplet microextraction, hollow fiber membrane liquid-phase microextraction, dispersion LLME, and homogeneous LLME. However, these methods also have their own drawbacks. Single-droplet microextraction and hollow fiber membrane liquid-phase microextraction have long extraction times, and the poor mechanical stability of single droplets or bubbling on the fiber membrane surface leads to poor experimental repeatability. Furthermore, hollow fiber membrane liquid-phase microextraction requires specific pH levels, which limits the choice of extractant. Dispersion LLME requires the use of toxic or harmful dispersants to assist extraction. Homogeneous LLME can only use special solvents with switchable properties, resulting in limited extractant selection. Additionally, it requires control of pH and temperature during extractant separation, making it unsuitable for detecting environmentally sensitive pesticides. Therefore, shortening operation time, improving experimental repeatability, maximizing pesticide stability, and achieving high-throughput semi-automation are currently pressing technical challenges.

[0005] Emulsion-liquid microextraction (ELM) differs from traditional liquid-liquid microextraction (LLM) in that the extractant and sample achieve high mass transfer in the oil-in-water emulsion, facilitating extraction. However, ELM methods utilize ultrasound-assisted or other mechanically assisted emulsification, leading to time-consuming extraction and extractant separation processes. Furthermore, the need for individual sample handling limits processing efficiency and continuity, and operational errors and time differences between groups significantly impact parallelism. No reports have yet described the development of a simple, semi-automated ELM method using small-scale equipment capable of simultaneously processing multiple samples. Summary of the Invention

[0006] To address the aforementioned problems in the prior art, this invention provides a high-throughput semi-automated method for determining pesticide residues in food samples using emulsion-liquid microextraction.

[0007] The above-mentioned high-throughput semi-automated emulsion-liquid microextraction method for determining pesticide residues in food samples includes the following steps:

[0008] S1, n parts of extractant are added to n parts of water at the same time, and mixed according to the preset number of mixing times and preset mixing speed, so that water and extractant are emulsified to form n parts of primary oil-in-water emulsion, where n is the number of samples to be tested;

[0009] S2, add n portions of the primary oil-in-water emulsion from step S1 to n portions of the test sample to form n portions of the secondary oil-in-water emulsion, and simultaneously complete the extraction of n portions of the test sample.

[0010] S3, inject n parts of demulsifier into n parts of secondary oil-in-water emulsion simultaneously to accelerate the separation of extractant and water;

[0011] S4. Take the extractant from step S3, dilute and filter it, transfer it to a sample vial along with the mobile phase, and use ultra-high performance liquid chromatography-tandem mass spectrometry to detect it and obtain the chromatographic peak area of ​​the target pesticide in the sample.

[0012] S5. Prepare standard solutions of the target pesticide with different mass concentrations, transfer them to injection bottles, and detect them using ultra-high performance liquid chromatography-tandem mass spectrometry. Plot 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 to obtain the linear equation.

[0013] S6. Substitute the chromatographic peak area of ​​the target pesticide in the sample to be tested obtained in step S4 into the linear equation in step S5 to calculate the concentration of the target pesticide in the sample to be tested.

[0014] In the above method, the target pesticide in the sample to be tested is any one, any two, any three, any four, or any five of the following: fluopyram, fluopyram, boscalid, bifenpyroxime, or fluopyram hydroxylamine.

[0015] A multichannel electronic micropipette was used to measure and transfer extractant, primary oil-in-water emulsion, and demulsifier. The multichannel electronic micropipette had 2n channels.

[0016] The extractant is octanoic acid, octanol, or a eutectic solvent; the eutectic solvent is synthesized from octanoic acid and menthol in a 1:1 molar ratio under boiling water conditions to obtain a homogeneous and transparent liquid; the demulsifier is a saturated NaCl solution.

[0017] In the above method, the volume ratio of extractant to water is 2:1 to 1:2; the preset number of mixing times is 0 to 8; the preset mixing speed is 100 to 360 μL / s; the volume ratio of primary oil-in-water emulsion to sample is 3:100 to 1:20; and the volume ratio of demulsifier to sample is 0 to 1:6.

[0018] Preferably, the volume ratio of extractant to water is 8:7 to 7:8; the preset number of mixing times is 4 to 8; the preset mixing speed is 360 μL / s; the volume ratio of primary oil-in-water emulsion to the sample to be tested is 1:25 to 1:20; and the volume ratio of demulsifier to the sample to be tested is 1:30 to 2:15.

[0019] Preferably, the extractant is octanoic acid, the volume ratio of extractant to water is 8:7, the preset number of mixing times is 4, the preset mixing speed is 360 μL / s, the volume ratio of primary oil-in-water emulsion to test sample is 1:20, the volume ratio of demulsifier to test sample is 1:30; the diluent is methanol-water solution, the volume ratio of methanol to water is 90:10, the volume ratio of extractant to diluent is 1:4, and a 0.22 μm filter membrane is used.

[0020] In the above method, the multichannel electronic micropipette has a volume range of 15–300 μL, the volume of the sample to be tested is 6 mL, the volume of the primary oil-in-water emulsion is 300 μL, the volume of the demulsifier is 200 μL, and the volume of the diluent is 160 μL.

[0021] In the above method, a 12-channel electronic micropipette is used to process six samples simultaneously.

[0022] In step S1, six parts of water are added at intervals in a 12×8×1.2mL deep well plate using a 12-channel electronic micropipette, and six parts of extraction solvent are added to six parts of water simultaneously using the 12-channel electronic micropipette to form six primary oil-in-water emulsions.

[0023] In step S2, six primary oil-in-water emulsions are simultaneously added to a 6×4×10mL deep-well plate containing six test samples using a 12-channel electronic micropipette to form six secondary oil-in-water emulsions, thereby completing the extraction of the six test samples.

[0024] In step S3, six portions of demulsifier are simultaneously injected into six portions of secondary oil-in-water emulsion using a 12-channel electronic micropipette.

[0025] In the above method, the chromatographic conditions for ultra-high performance liquid chromatography-tandem mass spectrometry detection are as follows: the mobile phase consists of phase A and phase B. Phase A is an aqueous phase, using a mixed aqueous solution of 0.01% formic acid and 2 mmol / L ammonium formate. Phase B is an organic phase, using methanol. The mobile phase gradient conditions are set according to the table below.

[0026] Table 1. Gradient conditions of the mobile phase

[0027]

[0028] The column oven temperature was 40℃, the injection volume was 2μL, and the flow rate was 0.3mL / min;

[0029] The mass spectrometry conditions were as follows: ionization mode was electrospray positive ionization source, detection mode was multiple reaction monitoring, capillary voltage was 5.5 kV, ion source temperature was 550 °C, curtain gas was 40 psi, and nebulizing gases Gas1 and Gas2 were both 55 psi. The optimized mass spectrometry conditions for each pesticide are shown in the table below.

[0030] Table 2. Mass spectrometry optimization parameters for succinate dehydrogenase inhibitor bactericides.

[0031]

[0032] In step S5, accurately weigh the standards of fluopyram, fluopyram, boscalid, bifenthiophanate-methyl, and fluopyram hydroxylamine, prepare a mixed standard solution with a mass concentration of 1000 μg / mL for each, and dilute with methanol using a serial dilution method to obtain mixed standard solutions with concentrations of 200, 100, 20, 10, 2, 1, 0.1, and 0.02 μg / L.

[0033] Compared with the prior art, the present invention has the following beneficial effects.

[0034] 1. This method enables simultaneous extraction and separation of multiple samples. Compared with traditional emulsion-liquid microextraction methods, it significantly improves sample processing speed. The use of a multi-channel electronic micropipette enables high-throughput and semi-automated emulsification, extraction, and separation operations. Taking the simultaneous processing of six samples using a 12-channel electronic micropipette as an example, the 12-channel electronic micropipette can be used for the semi-automated simultaneous pretreatment of six 6mL samples by utilizing the correspondence between commercially available 12×8 96-well plates and 6×4 24-well plates. Extraction of the six samples can be completed within 1 second, and separation of the six samples can be completed within 3 minutes, which is more than six times shorter than the total time required for traditional emulsion-liquid microextraction.

[0035] 2. This method does not require the use of special containers and equipment. It uses deep-well plates instead of centrifuge tubes, achieving semi-automated operation in the deep-well plate, simplifying experimental operations, reducing costs, and using a multi-channel electronic micropipette to reduce errors caused by manual operation and time differences between processing groups. It has good operational continuity, high sample processing efficiency, and improves the precision of results, providing a certain reference for the rapid preprocessing of large numbers of samples by testing institutions.

[0036] 3. Compared with existing liquid-liquid microextraction methods, this method based on the emulsion liquid-liquid microextraction of sample dilution water-in-oil emulsion has the advantages of simple operation, short operation time, good repeatability, no need to adjust pH value and add dispersing solvent, less analyte loss, and no need for professional technicians.

[0037] 4. Under optimal extraction and detection conditions, this method has significant advantages in sensitivity, with a detection limit of 0.001 μg / L and a quantification limit of 0.003 μg / L. Furthermore, it can produce detection results within 8.5 minutes, which is superior to traditional liquid chromatography detection methods for succinate dehydrogenase inhibitors.

[0038] 5. This method has been applied to the analysis of pesticide residues in food samples. The reproducibility, accuracy, precision, limit of detection and recovery rate of the method all meet the analytical requirements. Attached Figure Description

[0039] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0040] Figure 1 A flowchart for a high-throughput, semi-automated method for determining pesticide residues in food samples using emulsion-liquid microextraction.

[0041] Figure 2 The graph shows the effect of extractant type on extraction efficiency.

[0042] Figure 3 The figure shows the effect of the organic phase / aqueous phase volume ratio in the primary oil-in-water emulsion on the extraction efficiency.

[0043] Figure 4 The graph shows the effect of the volume of the primary oil-in-water emulsion on the extraction efficiency.

[0044] Figure 5 The graph shows the effect of the number of mixing cycles on the extraction efficiency.

[0045] Figure 6 The graph shows the effect of mixing speed on extraction efficiency.

[0046] Figure 7 This is a graph showing the effect of demulsifier volume on extraction efficiency.

[0047] Figure 8 The graph shows the effect of sample volume on extraction efficiency. Detailed Implementation

[0048] This invention studies and optimizes the main factors affecting extraction efficiency, such as the type of extractant, the volume ratio of organic phase to aqueous phase in the primary oil-in-water emulsion, the volume of the primary oil-in-water emulsion, the number of mixing cycles, the mixing speed, the volume of the demulsifier, and the sample volume. It establishes a high-throughput semi-automated method for determining pesticide residues in food samples by emulsion microextraction, which is fast, has high experimental repeatability, and enables the simultaneous processing of multiple samples.

[0049] To better explain and facilitate understanding of the present invention, a detailed description of the invention is provided below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, all methods employed are based on existing techniques in the art. The reagents used in the following examples were sourced as follows: fluopyram, fluopyram, boscalid, bifenthiophanate-methyl, and fluopyram (purity > 95%) were purchased from reagent companies; analytical grade menthol, octanoic acid, octanol, ammonium formate, and formic acid were purchased from Maclean Biotechnology Co., Ltd. (Shanghai, China); chromatographic grade methanol and acetonitrile were purchased from Merck Group (Darmstadt, Germany).

[0050] Example 1

[0051] This invention involves numerous factors affecting the pretreatment of food samples, necessitating a series of optimization experiments. Therefore, single-factor optimizations were performed on the type of extractant, the organic / aqueous phase volume ratio in the primary oil-in-water emulsion, the volume of the primary oil-in-water emulsion, the number and speed of mixing, the volume of the demulsifier, and the sample volume. Samples were purchased from the market in the applicant's location to verify the reliability of the detection results in practical applications of this invention. The following experiments were conducted:

[0052] 1. Optimization of extractant types

[0053] Different extractants have varying effects on extraction recovery rates. Traditional pesticide extraction techniques rely on toxic extractants (such as acetonitrile). Considering the physicochemical properties of extractants, such as water solubility, melting point, and extraction efficiency, three extractants were selected from a green chemistry perspective: fatty acids, fatty alcohols, and a eutectic solvent (fatty acid-menthol). Longer carbon chains in aliphatic compounds result in higher viscosity, which is detrimental to extractant emulsification; conversely, shorter carbon chains lead to higher solvent density, making it difficult to separate the extractant from the sample. Therefore, octanoic acid, octanol, and a eutectic solvent (octanoic acid-menthol) were chosen as green extractants. Using octanoic acid and menthol as raw materials, a homogeneous and transparent liquid, the eutectic solvent, was synthesized in a 1:1 molar ratio under boiling water conditions. The specific procedure involves adding six portions of water (140 μL) and six portions of octanoic acid (160 μL) to a deep-well plate (12 × 8 × 1.2 mL) at intervals, and continuously mixing four times at a rate of 360 μL / s using a 12-channel electronic micropipette to emulsify them. The resulting six portions of primary oil-in-water emulsion (300 μL) are then added to a deep-well plate (6 × 4 × 10 mL) containing six portions of spiked sample (6 mL), immediately yielding a secondary oil-in-water emulsion. Six portions of saturated NaCl solution (200 μL) are then injected into the secondary oil-in-water emulsion to accelerate phase separation. Finally, 40 μL of extractant is diluted with 160 μL of methanol-water solution (90:10, v / v), filtered through a 0.22 μm filter membrane, and finally detected by ultra-high performance liquid chromatography-tandem mass spectrometry. Figure 1 .

[0054] In the ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) detection, the chromatographic conditions were as follows: the mobile phase was methanol and water (a mixed aqueous solution of 0.01% formic acid and 2 mmol / L ammonium formate), and the mobile phase gradient conditions are shown in Table 1; the column oven temperature was 40℃; the injection volume was 2 μL; and the flow rate was 0.3 mL / min. The mass spectrometry conditions were as follows: ionization mode was electrospray positive ion source; detection mode was multiple reaction monitoring; capillary voltage was 5.5 kV; ion source temperature was 550℃; curtain gas was 40 psi; and nebulizer gases Gas1 and Gas2 were both 55 psi. The optimized mass spectrometry conditions for fluopyram, fluopyram, boscalid, bifenthiophanate-methyl, and fluopyram were shown in Table 2.

[0055] Table 1. Gradient conditions of the mobile phase

[0056]

[0057] Table 2. Mass spectrometry optimization parameters for succinate dehydrogenase inhibitor bactericides.

[0058]

[0059] Under the same conditions, only octanoic acid (as the extractant) was replaced with octanol and a eutectic solvent (octanoic acid-menthol), and the same treatment was performed. The results showed... Figure 2 The results show that octanoic acid, as the extractant, achieved a higher recovery rate. Therefore, octanoic acid is the optimal extractant for further experiments.

[0060] 2. Optimization of the organic phase / aqueous phase volume ratio in primary oil-in-water emulsions

[0061] During the extraction process, a suitable organic phase / aqueous phase volume ratio in the primary oil-in-water emulsion is beneficial for the extractant to enter the aqueous phase and form droplets with smaller radii, thereby enhancing the emulsification effect and improving the recovery rate. Following the procedure described in section 1, the difference lies in using octanoic acid as the extractant, and adjusting the organic phase / aqueous phase volume ratios to 2:1, 3:2, 8:7, 1:1, 7:8, 2:3, and 1:2, respectively. The results show... Figure 3 As shown in the figure, the recovery rate initially increases and then decreases as the proportion of octanoic acid in the primary oil-in-water emulsion gradually increases, reaching its maximum when the organic phase / aqueous phase volume ratio of the primary oil-in-water emulsion is 8:7. This is because when the proportion of octanoic acid is too low, insufficient extractant cannot completely extract the target analyte from the sample, and it also makes subsequent collection operations difficult. Conversely, when the proportion of octanoic acid is too high, the water, as the continuous phase, is insufficient to accommodate the excessive octanoic acid, resulting in large octanoic acid droplet radii and rapid aggregation, leading to poor emulsification of the prepared primary oil-in-water emulsion and ultimately incomplete extraction. Therefore, the preferred organic phase / aqueous phase volume ratio of the primary oil-in-water emulsion is 8:7 to 7:8, with 8:7 being the most preferred.

[0062] 3. Optimization of the volume of the primary water-in-oil emulsion

[0063] Currently, commercially available 12-channel electronic micropipettes have a volume range of 15–300 μL. Considering the practicality of this method and the issue of extractant collection, the procedure described in section 1 above was followed, except that octanoic acid was used as the extractant, and the organic phase / aqueous phase volume ratio of the primary oil-in-water emulsion was 8:7. The volumes of the primary oil-in-water emulsion were processed as 180 μL, 210 μL, 240 μL, 270 μL, and 300 μL, respectively. The results are as follows: Figure 4 As shown, the recovery rate reaches its highest when the volume of the primary oil-in-water emulsion is 300 μL. This is likely because when the volume of the primary oil-in-water emulsion is too small, there are fewer extractant droplets per unit volume of sample, resulting in incomplete extraction and making it difficult to collect the extractant. Therefore, the preferred volume of the primary oil-in-water emulsion is 240 μL to 300 μL, with 300 μL being the most preferred.

[0064] 4. Optimization of mixing times

[0065] A single mixing step is defined as the entire process of using a 12-channel electronic micropipette to draw in the mixture of extractant and water into a deep-well plate (12×8×1.2mL) at a certain speed once, and then dispensing it once. Through continuous mixing, the contact between octanoic acid and water is increased, thereby forming a primary oil-in-water emulsion. The multiple mixing steps of the extractant and water using the 12-channel electronic micropipette are performed continuously. Compared to manual pipettes, there are no differences in pipetting pressure, intensity, or time intervals between each press, which are crucial factors affecting emulsification and, consequently, extraction. Following the above procedure, the difference lies in using octanoic acid as the extractant, and the organic phase / aqueous phase volume ratio and volume of the primary oil-in-water emulsion being 8:7 and 300μL, respectively, with mixing times of 0, 1, 2, 3, 4, 6, and 8. Figure 5 As shown, the recovery rate initially increases gradually with the number of mixing cycles, then stabilizes after four cycles. Therefore, the preferred number of mixing cycles is 4 to 8, with 4 cycles being the most suitable for further shortening the operation time.

[0066] 5. Optimization of mixing speed

[0067] The 12-channel electronic micropipette has five speed settings: 100 μL / s, 140 μL / s, 195 μL / s, 265 μL / s, and 360 μL / s. Due to the combined effects of liquid inertia and viscosity, the rapid acceleration generated by the 12-channel electronic micropipette breaks the extractant into fine droplets. As the mixing speed gradually increases, the emulsification degree of the primary oil-in-water emulsion in the deep-well plate (12×8×1.2 mL) increases. Compared with manual pipettes, the 12-channel electronic micropipette precisely controls the aspiration and dispensing speeds, further ensuring the parallelism of results and extraction efficiency. Following the above procedure, the difference lies in using octanoic acid as the extractant, with an organic phase / aqueous phase volume ratio of 8:7 and 300 μL for the primary oil-in-water emulsion, and performing four mixing cycles at the five speed settings mentioned above. The results show... Figure 6 The results showed that the recovery rate of the target substance increased significantly with increasing mixing rate, reaching its highest value at a mixing rate of 360 μL / s. Therefore, the optimal mixing rate is 360 μL / s.

[0068] 6. Optimization of demulsifier volume

[0069] When a saturated NaCl solution is added as a demulsifier, the stability of the emulsion can be affected by lowering the zeta potential, thereby accelerating the separation of the extractant from the sample. It can also reduce the solubility of the target analyte in the aqueous phase through salting out, thus improving the extraction efficiency. Following the procedure described in section 1, the difference lies in using octanoic acid as the extractant. The volume ratio of the organic phase to the aqueous phase in the primary oil-in-water emulsion is 8:7 and 300 μL, respectively. The mixture is stirred four times at a speed of 360 μL / s. The volumes of the saturated NaCl solution as the demulsifier are 0 μL, 100 μL, 200 μL, 400 μL, 600 μL, 800 μL, and 1000 μL, respectively. The results show... Figure 7 In the process, as the volume of the saturated NaCl solution increases, the recovery of the target substance initially increases gradually, and then stabilizes within a certain range. If the volume of the saturated NaCl solution is too small, insufficient contact between the demulsifier and the secondary oil-in-water emulsion will lead to reduced separation efficiency of the extractant; therefore, the preferred volume of the saturated NaCl solution is 200–800 μL, and to reduce reagent usage, 200 μL is the most preferred.

[0070] 7. Sample volume optimization

[0071] The procedure described in section 1 above was followed, except that octanoic acid was used as the extractant, the volume ratio of the organic phase to the aqueous phase in the primary oil-in-water emulsion was 8:7 and the volume was 300 μL, the mixing was performed four times at a mixing speed of 360 μL / s, the volume of the demulsifier saturated NaCl solution was 200 μL, and the sample volumes were 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, and 8 mL, respectively. The results are as follows: Figure 8 As shown, the recovery of the target analyte initially remained constant with increasing sample volume, then decreased significantly. This may be because when the sample volume is too large, the extractant is insufficient to completely extract the target analyte. Therefore, the sample volume is preferably 3–6 mL, and most preferably 6 mL.

[0072] Example 2

[0073] Analytical characteristics of succinate dehydrogenase inhibitors in samples

[0074] (1) Sample pretreatment

[0075] Samples of purchased water, juice, and alcoholic beverages were filtered through a 0.45μm filter for later use.

[0076] (2) Sample extraction

[0077] First, six parts water (140 μL) and six parts octanoic acid (160 μL) were added sequentially and alternately to a deep-well plate (12 × 8 × 1.2 mL). The mixture was continuously mixed four times at a rate of 360 μL / s using a 12-channel electronic micropipette to achieve immediate emulsification. The resulting six parts of the primary oil-in-water emulsion (300 μL) were then added to a deep-well plate (6 × 4 × 10 mL) containing six parts of spiked sample (50 μL working solution + 6 mL sample), immediately generating a secondary oil-in-water emulsion. Six parts of saturated NaCl solution (200 μL) were then added to the secondary oil-in-water emulsion to accelerate phase separation. Finally, 40 μL of the extractant was diluted with 160 μL of methanol-water solution (90:10, v / v), filtered through a 0.22 μm filter membrane, and detected using ultra-high performance liquid chromatography-tandem mass spectrometry.

[0078] (3) Preparation of standard solutions

[0079] Accurately weigh the standards of fluopyram, fluopyram, boscalid, bifenthiophanate-methyl, and fluopyram hydroxylamine, and prepare mixed standard solutions with a mass concentration of 1000 μg / mL for each. Dilute with methanol using a serial dilution method to obtain mixed standard solutions with concentrations of 200, 100, 20, 10, 2, 1, 0.1, and 0.02 μg / L as working solutions. Then store the standard solutions in a light-protected environment.

[0080] (4) Detection by ultra-high performance liquid chromatography-tandem mass spectrometry

[0081] In the ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) detection, the chromatographic conditions were as follows: the mobile phase was methanol and water (a mixed aqueous solution of 0.01% formic acid and 2 mmol / L ammonium formate), and the mobile phase gradient conditions are shown in Table 1; the column oven temperature was 40℃; the injection volume was 2 μL; and the flow rate was 0.3 mL / min. The mass spectrometry conditions were as follows: ionization mode was electrospray positive ion source; detection mode was multiple reaction monitoring; capillary voltage was 5.5 kV; ion source temperature was 550℃; curtain gas was 40 psi; and nebulizer gases Gas1 and Gas2 were both 55 psi. The optimized mass spectrometry conditions for fluopyram, fluopyram, boscalid, bifenthiophanate-methyl, and fluopyram were shown in Table 2.

[0082] (5) Plotting the standard curve

[0083] Prepare the standard solution and spiked sample in step (3), and perform the detection according to the procedures in steps (2) and (4). Plot a standard curve with the mass concentration x of the mixed standard solution of the five pesticides as the abscissa and the corresponding peak area y as the ordinate. The linear equations for different sample matrices are shown in Table 3. The coefficient of determination R of the linear equation is... 2With a sensitivity ≥0.998, and based on a limit of detection (LOD) of 3 times the signal-to-noise ratio and a limit of quantitation (LOQ) of 10 times the signal-to-noise ratio, the LOD for all five pesticide residues was 0.001 μg / L, and the LOQ was 0.003 μg / L, significantly better than the sensitivity of methods in the literature. The intra-day relative standard deviation (RSD) and inter-day RSD were 0.9–4.0% and 1.1–4.7%, respectively, meeting the analytical requirements. The matrix effect was between 80% and 120%, within an acceptable range.

[0084] Table 3 Analytical characteristics of succinate dehydrogenase inhibitor bactericides

[0085]

[0086] Example 3

[0087] Recovery rate of succinate dehydrogenase inhibitors in samples

[0088] First, the levels of fluopyram, fluopyram, boscalid, bifenthiophanate-methyl, and fluopyram were detected in water, fruit juice, and alcoholic beverage samples, respectively, and the levels were found to be below the detection limit. Then, fluopyram, fluopyram, boscalid, bifenthiophanate-methyl, and fluopyram were added to water, fruit juice, and alcoholic beverage samples at final concentrations of 0.003 μg / L, 0.03 μg / L, and 0.3 μg / L, respectively. The samples were then tested according to steps (2) and (4), and the concentrations were obtained using a standard curve. Table 4 shows that the recoveries of fluopyram, fluopyram, and fluopyram in water, fruit juice, and alcoholic beverages were 85.7%–106.9%; the recoveries of fluopyram, fluopyram, and fluopyram were 88.5%–96.6%; the recoveries of bifenthiophanate-methyl, and fluopyram were 83.1%–94.3%; and the recoveries of fluopyram were 82.6%–101.8%. This indicates that the method has good accuracy and applicability. The relative standard deviations (RSDs) of the five pesticides were all between 0.8% and 5.8%, indicating that the method has good precision.

[0089] Table 4. Recovery rates and relative standard deviations of the target analytes in water, fruit juice, and alcoholic beverages.

[0090]

[0091] Example 4

[0092] Comparison of this method with previously reported detection methods for succinate dehydrogenase inhibitors.

[0093] The detection method of this invention can simultaneously detect five pesticide residues. Compared with various gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry detection methods for succinate dehydrogenase inhibitor fungicides (Table 5), this method achieves semi-automatic simultaneous pretreatment of six samples. The entire extraction and separation process uses only 500 μL of octanoic acid, water, and NaCl solution, reducing the use of various toxic and harmful solvents or materials, and the required extraction time is less than 1 second. Furthermore, a 12-channel electronic micropipette is used instead of other auxiliary equipment (vortex mixer, ultrasonic water bath, shaker, or centrifuge). Compared with other methods, it has a wider linear range and lower LOD and LOQ, good recovery rate and RSD, and high detection sensitivity, indicating that the method of this invention is reliable, accurate, and has good applicability.

[0094] Table 5. Comparison of sample pretreatment and detection methods of this method with other succinate dehydrogenase inhibitor bactericides.

[0095]

[0096] In Table 5:

[0097] DSPE: Dispersive Solid Phase Extraction;

[0098] DLLME: Dispersion Liquid-Liquid Microextraction;

[0099] MSPE: Magnetic Solid Phase Extraction;

[0100] TMGO: Tetraenepentamine Magnetic Graphene Oxide;

[0101] Eutectic solvent: L-menthol and butylated hydroxytoluene, in a molar ratio of 3:1.

[0102] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art can make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A high-throughput semi-automated method for determining pesticide residues in food samples using emulsion-liquid microextraction, characterized in that, The food samples are water, fruit juice, or alcoholic beverages, and the process includes the following steps: S1, using a multichannel electronic micropipette with 2n channels, add n parts of water at intervals in the deep well plate. Simultaneously add n parts of extractant to n parts of water using the multichannel electronic micropipette. The extractant is octanoic acid, and the volume ratio of extractant to water is 8:

7. Mix according to the preset number of mixing times and preset mixing speed. The preset number of mixing times is 4 times, and the preset mixing speed is 360 μL / s, so that water and extractant emulsify to form n parts of primary oil-in-water emulsion, where n is the number of samples to be tested. The target pesticide in the sample to be tested is any one, two, three, four, or five of the following: fluopyram, fluopyram, cyproconazole, bifenpyroxime, or fluopyram hydroxylamine. S2, using a multi-channel electronic micropipette, n portions of the primary oil-in-water emulsion from step S1 are simultaneously added to a deep-well plate containing n portions of the sample to be tested. The volume ratio of the primary oil-in-water emulsion to the sample to be tested is 1:20, forming n portions of the secondary oil-in-water emulsion, and simultaneously completing the extraction of n portions of the sample to be tested. S3, using a multi-channel electronic micropipette, n portions of demulsifier are simultaneously injected into n portions of secondary oil-in-water emulsion to accelerate the separation of extractant and water. The demulsifier is a saturated NaCl solution, and the volume ratio of demulsifier to sample is 1:

30. S4. Take the extractant from step S3, dilute and filter it, transfer it to a sample vial along with the mobile phase, and use ultra-high performance liquid chromatography-tandem mass spectrometry to detect it and obtain the chromatographic peak area of ​​the target pesticide in the sample. S5. Prepare standard solutions of the target pesticide with different mass concentrations, transfer them to injection bottles, and detect them using ultra-high performance liquid chromatography-tandem mass spectrometry. Plot 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 to obtain the linear equation. S6. The chromatographic peak area of ​​the target pesticide in the sample to be tested obtained in step S4 is substituted into the linear equation in step S5 to calculate the concentration of the target pesticide in the sample to be tested.

2. The method for determining pesticide residues in food samples using high-throughput semi-automatic emulsion-liquid microextraction according to claim 1, characterized in that, The diluent was a methanol-water solution with a methanol-water volume ratio of 90:10, and the extractant-diluent volume ratio was 1:

4. A 0.22 μm filter membrane was used.

3. The method for determining pesticide residues in food samples using high-throughput semi-automated emulsion-liquid microextraction according to claim 2, characterized in that, The multichannel electronic micropipette has a volume range of 15~300μL. The volume of the sample to be tested is 6 mL, the volume of the primary oil-in-water emulsion is 300μL, the volume of the demulsifier is 200 μL, and the volume of the diluent is 160 μL.

4. The method for determining pesticide residues in food samples using high-throughput semi-automated emulsion-liquid microextraction according to claim 3, characterized in that, Six samples were processed simultaneously using a 12-channel electronic micropipette. In step S1, six parts of water are added at intervals in a 12×8×1.2 mL deep well plate using a 12-channel electronic micropipette, and six parts of extraction solvent are added to six parts of water simultaneously using the 12-channel electronic micropipette to form six primary oil-in-water emulsions. In step S2, six primary oil-in-water emulsions are simultaneously added to a 6×4×10 mL deep-well plate containing six test samples using a 12-channel electronic micropipette to form six secondary oil-in-water emulsions, thereby completing the extraction of the six test samples. In step S3, six portions of demulsifier are simultaneously injected into six portions of secondary oil-in-water emulsion using a 12-channel electronic micropipette.

5. The method for high-throughput semi-automated emulsion-liquid microextraction determination of pesticide residues in food samples according to any one of claims 1-4, characterized in that, In ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), the chromatographic conditions are as follows: the mobile phase consists of two phases, A and B. Phase A is an aqueous phase, using a mixed aqueous solution of 0.01% formic acid and 2 mmol / L ammonium formate. Phase B is an organic phase, using methanol. The mobile phase gradient conditions are set according to the table below. The column oven temperature was 40℃, the injection volume was 2 μL, and the flow rate was 0.3 mL / min; The mass spectrometry conditions were as follows: ionization mode was electrospray positive ionization source, detection mode was multiple reaction monitoring, capillary voltage was 5.5 kV, ion source temperature was 550 °C, curtain gas was 40 psi, and nebulizing gases Gas1 and Gas2 were both 55 psi. The mass spectrometry conditions for each pesticide are shown in the table below. 。 6. The method for determining pesticide residues in food samples using high-throughput semi-automated emulsion-liquid microextraction according to claim 1, characterized in that, In step S5, accurately weigh the standards of fluopyram, fluopyram, boscalid, bifenthiophanate-methyl, and fluopyram hydroxylamine, prepare a mixed standard solution with a mass concentration of 1000 μg / mL for each, and dilute with methanol using a serial dilution method to obtain mixed standard solutions with concentrations of 200, 100, 20, 10, 2, 1, 0.1, and 0.02 μg / L, respectively.

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