Binary eutectic solvent, application of binary eutectic solvent in pesticide residue detection and detection method

Abstract

The invention discloses a binary eutectic solvent, application thereof in pesticide residue detection and a detection method, and belongs to the technical field of pesticide testing and analysis. The binary eutecticevaporate solvent is composed of isopropyl methylphenol and phenyl alcohol substances, boscalid and difenoconazole in agricultural products can be efficiently extracted, the accuracy of pesticide residue detection in the agricultural products is improved, the UPLC-MS / MS method is matched, the use amount of an extracting agent is effectively reduced, and pesticide residue detection is more environmentally friendly and efficient.

Description

Technical Field

[0001] The present application relates to a binary low eutectic solvent and its application and detection method in pesticide residue detection, belonging to the technical field of pesticide testing and analysis. Background Art

[0002] Pesticide residues mainly come from the use of pesticides in agricultural production. Some pesticides will adhere to crops, and some will be scattered in the environment. Pesticides in the environment may be absorbed by plants. Agricultural products need to be tested for pesticide residues during the production process to ensure that the pesticide residues in agricultural products are within a safe range and protect consumer health.

[0003] Boscalid and difenoconazole have a broad bactericidal spectrum and are low-cost, and are widely used in the cultivation of agricultural products. However, residual pesticides enter the human body through consumption, and accumulation may endanger people's health. Therefore, it is necessary to remove boscalid and difenoconazole from agricultural products and detect pesticide residues.

[0004] Traditional organic solvents are used extensively in pesticide residue analysis and testing, and the detection methods are complex, potentially leading to further contamination and affecting detection efficiency. Therefore, a rapid and effective method is urgently needed to determine the residues of the two pesticides in fruits, vegetables, cereals, and water bodies in crop-growing environments. Summary of the Invention

[0005] In order to solve the above problems, a binary deep eutectic solvent and its application and detection method in pesticide residue detection are provided. The solvent can extract pesticide residues in agricultural products in an environmentally friendly and efficient manner, reduce the use of organic solvents, and lower the detection cost.

[0006] According to one aspect of the present application, a binary deep eutectic solvent is provided, wherein the binary deep eutectic solvent is composed of isopropyl methylphenol and phenyl alcohol substances.

[0007] Phenyl alcohols can form hydrogen bonds with isopropyl methylphenol, increasing the polarity of the solvent and thereby enhancing the extraction ability of polar pesticide components.

[0008] Optionally, the ratio of isopropyl methylphenol to phenyl alcohol is 1:(1-1.5).

[0009] By adjusting the ratio of the two solvents, the overall polarity of the solvent can be optimized, enabling it to effectively capture specific polar pesticide molecules during the extraction process, especially in samples where multiple pesticide components exist simultaneously, ensuring high-precision extraction of the target pesticide.

[0010] Optionally, the isopropyl methylphenol is one of thymol and carvacrol; the phenyl alcohol substance is one of benzyl alcohol, phenylethyl alcohol and phenylpropanol.

[0011] Preferably, the isopropyl methylphenol is thymol; and the phenyl alcohol substance is phenylpropanol.

[0012] Optionally, the preparation method of the binary deep eutectic solvent is to mix isopropyl methylphenol and phenyl alcohol, heat to 50-80° C., stir for 90-150 minutes, and cool to room temperature to obtain the binary deep eutectic solvent.

[0013] According to another aspect of the present application, there is provided a use of a binary deep eutectic solvent in pesticide residue detection, for detecting the residual amount of boscalid and / or difenoconazole.

[0014] According to another aspect of the present application, a UPLC-MS / MS method for detecting pesticide residues is provided. The detection method is based on the above-mentioned binary deep eutectic solvent and comprises the following steps:

[0015] (1) Preparing a test solution: placing the treated test sample in a centrifuge tube; adding a binary deep eutectic solvent extractant to the centrifuge tube, dispersing the sample and then centrifuging to obtain an upper binary deep eutectic solvent extract phase enriched with pesticide residues, and filtering the extract through an organic filter membrane to obtain a test solution;

[0016] When the sample to be tested is in liquid form, the sample to be tested is filtered to obtain a processed sample to be tested; when the sample to be tested is in solid form, the sample to be tested is crushed to obtain a processed sample to be tested;

[0017] (2) preparing a mixed standard solution: placing a boscalid standard stock solution and a difenoconazole standard stock solution in acetonitrile to obtain a mixed standard solution containing both boscalid and difenoconazole;

[0018] (3) Establishing a standard curve: inject the mixed standard solution prepared in step (2) into the UPLC-MS / MS instrument, establish a standard curve based on the obtained data, obtain the linear equation and correlation coefficient of each component and test it, determine the peak area of the component and its qualitative and quantitative ion pairs, draw a standard curve with the peak area as the ordinate and the mass concentration as the abscissa, and obtain the linear equation and correlation coefficient of each component;

[0019] (4) Calculation of pesticide residues: The test solution obtained in step (1) is injected into a UPLC-MS / MS instrument, and quantitative analysis is performed based on the peak areas and the abundance ratios of the qualitative and quantitative ion pairs. The content of boscalid and / or difenoconazole in the test sample is calculated according to the linear equation obtained in step (3).

[0020] Specifically, in step (1), when the sample to be tested is a liquid, the volume ratio of the sample to be tested to the binary deep eutectic solvent extractant is 1:50 to 200 (mL / μL); when the sample to be tested is a liquid, the volume ratio of the sample to be tested to the binary deep eutectic solvent extractant is 1:20 to 200 (g / μL).

[0021] Optionally, in step (1), the preparation process of the test solution is as follows: pipetting the sample into a centrifuge tube, adding a mixed standard solution, adding a binary deep eutectic solvent extractant, vortexing and dispersing, and then centrifuging in a high-speed centrifuge to obtain an upper binary deep eutectic solvent-enriched phase enriched with pesticide residues, transferring the binary deep eutectic solvent-enriched phase to another centrifuge tube, diluting it with acetonitrile, and filtering it through a 0.22 μm organic filter membrane to obtain the sample to be tested.

[0022] Optionally, the preparation of the mixed standard solution in step (3) includes:

[0023] Weigh boscalid in acetonitrile to prepare a boscalid standard stock solution;

[0024] Weigh difenoconazole in acetonitrile to prepare a difenoconazole standard stock solution;

[0025] The boscalid standard stock solution and the difenoconazole standard stock solution are placed in acetonitrile to prepare at least five sets of mixed standard solutions with different concentrations, wherein the concentrations of boscalid and difenoconazole in the mixed standard solutions are 1 to 0.0025 mg / L.

[0026] Optionally, the concentrations of boscalid and difenoconazole in the mixed standard solution are the same, and the concentrations of boscalid and difenoconazole in the five groups of mixed standard solutions are 0.0025, 0.025, 0.05, 0.5, and 1 mg / L, respectively.

[0027] Optionally, during step (3) and step (4), the test conditions are:

[0028] UPLC: C18-WP column 2.1 mm × 50 mm, capillary 3 μm, column temperature 25°C; flow rate: 0.3 mL / min, constant flow; injection volume 2 μl;

[0029] Mobile phase: acetonitrile: 0.1% formic acid aqueous solution = 70:30 (v / v);

[0030] MS / MS: electrospray ionization source mode was used with a capillary voltage of 4 kV; the collision energy of the secondary mass spectrometer was 25 eV; the drying gas temperature was 350°C; the drying gas flow rate was 8 L / min; the nebulizing gas pressure was 35 psi; and the reaction gas was N2.

[0031] The beneficial effects of this application include but are not limited to:

[0032] 1. According to the binary deep eutectic solvent of the present application, isopropyl methylphenol and phenyl alcohol are used to form a binary deep eutectic solution, which has a high extraction rate for pesticide components, stable chemical properties, and a green and safe extraction process.

[0033] 2. According to the application of the binary deep eutectic solvent in the present application in the detection of pesticide residues, the residual amount of boscalid and / or difenoconazole in agricultural products can be detected. Only a small amount of solvent is needed to specifically and efficiently detect the content of the two pesticide residues, thereby reducing the cost of pesticide residue detection.

[0034] 3. According to the UPLC-MS / MS method for detecting pesticide residues of the present application, UPLC-MS / MS was used for detection, and the liquid chromatography and mass spectrometry detection conditions for detecting two pesticide components were explored, including the parameters of mobile phase composition, ratio, column temperature in liquid chromatography detection, and precursor ion, product ion, and collision energy in mass spectrometry detection. The results obtained showed that the method has good linearity, sensitivity, accuracy and repeatability. DETAILED DESCRIPTION

[0035] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0036] Unless otherwise specified, the raw materials in the examples and comparative examples of the present application were purchased through commercial channels.

[0037] Unless otherwise specified, the methods used in the examples and comparative examples of the present application are conventional methods in the prior art.

[0038] The instruments and reagents used in this application are:

[0039] Reagents

[0040] Acetonitrile (CH3CN, CAS No.: 75-05-8): analytical grade;

[0041] Formic acid (HCOOH, CAS No.: 64-18-6): chromatographically pure;

[0042] Liquid chromatography-mass spectrometry: Agilent 1290II-6460, Agilent Technologies, USA;

[0043] Multi-tube vortex mixing: MTV-100, Hangzhou Aosheng Instrument Co., Ltd.

[0044] Benchtop ventilated centrifuge: Sorvall ST16, ThermoFisher; FC5706, Ohaus, USA;

[0045] Boscalid standard: content is 99.5%;

[0046] Difenoconazole: content is 99.6%.

[0047] The HPLC-MS / MS detection parameters of the two pesticides are shown in Table 1

[0048] Table 1

[0049]

[0050] In the following examples, the binary deep eutectic solvent used is thymol and phenylpropanol in a molar ratio of 1:1; isopropylmethylphenol and phenyl alcohol substances in a molar ratio of 1: (1 to 1.5) can achieve efficient extraction of boscalid and difenoconazole. In this scheme, thymol and phenylpropanol in a molar ratio of 1:1 were selected for verification.

[0051] Recovery calculation:

[0052]

[0053] Where: F a is the peak area of the sample; F s is the peak area of the standard sample; Q s (μL) is the standard sample injection volume; Q x (μL) is the sample injection volume; V ex (mL) is the total volume of the extract; V ri (mL) is the volume of the extracted solution; V x (mL) is the final volume; C s (mg / L) is the standard concentration; C a (mg / kg) is the added concentration; M (g) is the sample weight.

[0054] Standard curve and matrix effect calculation:

[0055] The standard curve was fitted with a linear equation using the concentration of the standard solution as the abscissa and the peak area of the monitored ion pair as the ordinate to obtain the correlation coefficient (r).

[0056] Matrix effect calculation:

[0057]

[0058] Where: ME is the matrix effect, expressed in %; ɑ 基质 is the slope of the matrix standard sample; ɑ 溶剂 is the slope of the solvent standard.

[0059] Example 1

[0060] The embodiment relates to a UPLC-MS / MS method for detecting pesticide residues using a binary deep eutectic solvent, comprising the following steps:

[0061] (1) Preparation of the test solution: 2 mL of tap water sample was transferred to a 5 mL plastic centrifuge tube, 0.005 mg / kg, 0.05 mg / kg and 0.1 mg / kg of boscalid and difenoconazole were added to the tap water, respectively, and then 0.2 mL of binary deep eutectic solvent extractant was added. The binary deep eutectic solvent was fully dispersed into the sample by vortex-assisted method to achieve a good extraction effect. The vortex time was 1 min, and then centrifuged at 10,000 rpm for 2 min to obtain the upper binary deep eutectic solvent-enriched phase enriched with pesticide residues. The binary deep eutectic solvent-enriched phase was transferred to another 2 mL centrifuge tube, diluted 5 times with acetonitrile, filtered through a 0.22 μm organic filter membrane, and transferred to a sample injection vial for testing.

[0062] (2) Prepare mixed standard solution:

[0063] S1. Weigh 0.0103 g of boscalid standard and dilute to 10 mL with acetonitrile to prepare a 1025 mg / L boscalid standard stock solution.

[0064] S2. Weigh 0.0104 g of difenoconazole standard and dilute to 10 mL with acetonitrile to prepare a 1032 mg / L difenoconazole standard stock solution.

[0065] S3. Take the boscalid standard stock solution and the difenoconazole standard stock solution and place them in acetonitrile solution to obtain mixed standard solutions of different concentrations, with concentrations of 1, 0.5, 0.25, 0.025, and 0.0025 mg / L.

[0066] (3) Establishing a standard curve: Inject the mixed standard solution into the UPLC-MS / MS instrument, establish a standard curve based on the obtained data, obtain the linear equation and correlation coefficient of each component, determine the peak area of the component and its qualitative and quantitative ion pairs, and draw a standard curve with the peak area as the ordinate and the mass concentration as the abscissa to obtain the linear equation and correlation coefficient of each component;

[0067] (4) Calculation of pesticide residues:

[0068] The sample to be tested was injected into the UPLC-MS / MS instrument, and quantitative analysis was performed according to the peak area and the abundance ratio of the qualitative and quantitative ion pairs. The content of boscalid and / or difenoconazole in the sample to be tested was calculated according to the above linear equation.

[0069] During step (3) and step (4), the test conditions are:

[0070] UPLC: C18-WP column 2.1 mm × 50 mm, capillary 3 μm, column temperature 25°C; flow rate: 0.3 mL / min, constant flow; injection volume 2 μL;

[0071] Mobile phase: acetonitrile: 0.1% formic acid aqueous solution = 70:30 (v / v);

[0072] MS / MS: electrospray ionization source mode was used with a capillary voltage of 4 kV; the collision energy of the secondary mass spectrometer was 25 eV; the drying gas temperature was 350°C; the drying gas flow rate was 8 L / min; the nebulizing gas pressure was 35 psi; and the reaction gas was N2.

[0073] The standard curve in step (3) is shown in Table 2:

[0074] Table 2

[0075] substance Standard curve Correlation coefficient Boscalid y=2688126x+21602 r＝0.9997 Difenoconazole y=4196140x+20911 r＝0.9999

[0076] The recovery rates and RSDs of the two substances are shown in Table 3:

[0077] Table 3

[0078]

[0079] From Table 3, we can see that

[0080] Accuracy: When tap water was spiked with 0.005 mg / kg, 0.05 mg / kg, and 0.1 mg / kg of boscalid and difenoconazole, respectively, the recoveries of boscalid were 72%-87%, 75%-88%, and 79%-88%, respectively. The recoveries of difenoconazole were approximately 93%-98%, 83%-93%, and 101%-107%, respectively. It can be seen that the recoveries of both substances at different spike levels in tap water blank samples met the relevant requirements for pesticide residue testing.

[0081] Precision: When boscalid and difenoconazole were spiked in tap water at levels of 0.005 mg / kg, 0.05 mg / kg, and 0.1 mg / kg, respectively, the relative standard deviations for boscalid were 10%, 8%, and 6%, respectively; and for difenoconazole were 3%, 6%, and 3%, respectively. This indicates that the relative standard deviations for boscalid and difenoconazole at different spike levels in tap water blank samples meet the requirements for pesticide residue detection.

[0082] Example 2

[0083] The embodiment relates to a UPLC-MS / MS method for detecting pesticide residues using a binary deep eutectic solvent, comprising the following steps:

[0084] (1) Preparation of the test solution: 5.0 g ± 0.5 g of citrus blank sample (no pesticide residue) was crushed and accurately weighed into a 50 mL plastic centrifuge tube with a lid, and a mixed standard solution of boscalid and difenoconazole at different concentrations (0.01 mg / kg, 0.1 mg / kg, 1 mg / kg, with 4 replicates for each concentration) was added. The solution was allowed to stand for 10 min, 2.4 mL of water and 1 mL of binary deep eutectic solvent were added, and the solution was vortexed at 2500 r / min on an MTV-100 vortex machine for 1 min; the solution was centrifuged at 6000 r / min for 5 min, and 100 μL of the supernatant was diluted 5 times and filtered through a 0.22 μm organic filter membrane to obtain the test solution;

[0085] (2) Prepare mixed standard solution:

[0086] S1. Weigh 0.0103 g of boscalid standard and dilute to 10 mL with acetonitrile to prepare a 1025 mg / L boscalid standard stock solution.

[0087] S2. Weigh 0.0104 g of difenoconazole standard and dilute to 10 mL with acetonitrile to prepare a 1032 mg / L difenoconazole standard stock solution.

[0088] S3. The boscalid standard stock solution and the difenoconazole standard stock solution were placed in acetonitrile to obtain mixed standard solutions of different concentrations, with concentrations of 1, 0.5, 0.25, 0.025, and 0.0025 mg / L.

[0089] (3) Establishing a standard curve: Inject the mixed standard solution into the UPLC-MS / MS instrument, establish a standard curve based on the obtained data, obtain the linear equation and correlation coefficient of each component, determine the peak area of the component and its qualitative and quantitative ion pairs, and draw a standard curve with the peak area as the ordinate and the mass concentration as the abscissa to obtain the linear equation and correlation coefficient of each component;

[0090] (4) Calculation of pesticide residues: The sample to be tested was injected into the UPLC-MS / MS instrument, and quantitative analysis was performed based on the peak area and the abundance ratio of the qualitative and quantitative ion pairs. The content of boscalid and / or difenoconazole in the sample to be tested was calculated according to the above linear equation.

[0091] During step (3) and step (4), the test conditions are:

[0092] UPLC: C18-WP column 2.1 mm × 50 mm, capillary 3 μm, column temperature 25°C; flow rate: 0.3 mL / min, constant flow; injection volume 2 μL;

[0093] Mobile phase: acetonitrile: 0.1% formic acid aqueous solution = 70:30 (v / v);

[0094] MS / MS: electrospray ionization source mode was used with a capillary voltage of 4 kV; the collision energy of the secondary mass spectrometer was 25 eV; the drying gas temperature was 350°C; the drying gas flow rate was 8 L / min; the nebulizing gas pressure was 35 psi; and the reaction gas was N2.

[0095] The recoveries and RSDs of the two substances are shown in Table 4:

[0096] Table 4

[0097]

[0098] From Table 4, we can see that

[0099] Accuracy: When citrus blank samples were spiked with boscalid and difenoconazole at levels of 0.01 mg / kg, 0.1 mg / kg, and 1 mg / kg, the recoveries of boscalid were 87%-92%, 87%-90%, and 70%-74%, respectively; and the recoveries of difenoconazole were 72%-85%, 81%-84%, and 73%-76%, respectively. This indicates that the recoveries of difenoconazole and difenoconazole in citrus blank samples at different spike levels met the requirements for pesticide residue testing.

[0100] Precision: When citrus blank samples were spiked with boscalid and difenoconazole at levels of 0.01 mg / kg, 0.1 mg / kg, and 1 mg / kg, the relative standard deviations for boscalid were 3%, 2%, and 3%, respectively; and for difenoconazole, 8%, 2%, and 1%, respectively. This indicates that the relative standard deviations of boscalid and difenoconazole at different spike levels in citrus blank samples meet the requirements for pesticide residue detection.

[0101] The foregoing is merely an embodiment of the present application, and the scope of protection of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application may have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc. made within the technical ideas and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A binary deep eutectic solvent, characterized in that: The binary deep eutectic solvent consists of isopropyl methylphenol and phenyl alcohol substances.

2. The binary deep eutectic solvent according to claim 1, characterized in that The molar ratio of the isopropyl methylphenol to the phenyl alcohol substance is 1:(1-1.5).

3. The binary deep eutectic solvent according to claim 1, characterized in that The isopropyl methylphenol is one of thymol and carvacrol; the phenyl alcohol substance is one of benzyl alcohol, phenylethyl alcohol and phenylpropanol.

4. The binary deep eutectic solvent according to claim 1, characterized in that The preparation method of the binary deep eutectic solvent comprises the following steps: mixing isopropyl methylphenol and phenyl alcohol, heating the mixture to 50-80° C., stirring the mixture for 90-150 minutes, and cooling the mixture to room temperature to obtain the binary deep eutectic solvent.

5. Application of a binary deep eutectic solvent in pesticide residue detection, characterized in that: Used to detect the residues of boscalid and / or difenoconazole.

6. A UPLC-MS / MS method for detecting pesticide residues using the binary deep eutectic solvent according to claims 1 to 4, characterized in that: The following steps are involved: (1) Preparing a test solution: placing the treated test sample in a centrifuge tube; adding a binary deep eutectic solvent extractant to the centrifuge tube, dispersing the sample and then centrifuging to obtain an upper binary deep eutectic solvent extract phase enriched with pesticide residues, and filtering the extract through an organic filter membrane to obtain a test solution; When the sample to be tested is in liquid form, the sample to be tested is filtered to obtain a processed sample to be tested; when the sample to be tested is in solid form, the sample to be tested is crushed to obtain a processed sample to be tested; (2) preparing a mixed standard solution: placing a boscalid standard stock solution and a difenoconazole standard stock solution in acetonitrile to obtain a mixed standard solution containing both boscalid and difenoconazole; (3) Establishing a standard curve: inject the mixed standard solution prepared in step (2) into the UPLC-MS / MS instrument, establish a standard curve based on the obtained data, obtain the linear equation and correlation coefficient of each component and test it, determine the peak area of the component and its qualitative and quantitative ion pairs, draw a standard curve with the peak area as the ordinate and the mass concentration as the abscissa, and obtain the linear equation and correlation coefficient of each component; (4) Calculation of pesticide residues: The test solution obtained in step (1) is injected into a UPLC-MS / MS instrument, and quantitative analysis is performed based on the peak areas and the abundance ratios of the qualitative and quantitative ion pairs. The content of boscalid and / or difenoconazole in the test sample is calculated according to the linear equation obtained in step (3).

7. The method for detecting pesticide residues according to claim 6, wherein In step (1), the test solution is prepared as follows: the sample is transferred to a centrifuge tube, a binary deep eutectic solvent extractant is added, vortexed and dispersed, and then centrifuged in a high-speed centrifuge to obtain an upper binary deep eutectic solvent-enriched phase enriched with pesticide residues, the binary deep eutectic solvent-enriched phase is transferred to another centrifuge tube, diluted with acetonitrile, and filtered through a 0.22 μm organic filter membrane to obtain the test sample.

8. The method for detecting pesticide residues according to claim 6, wherein The preparation of the mixed standard solution in step (3) comprises: Weigh boscalid in acetonitrile to prepare a boscalid standard stock solution; Weigh difenoconazole in acetonitrile to prepare a difenoconazole standard stock solution; The boscalid standard stock solution and the difenoconazole standard stock solution are placed in acetonitrile to prepare at least five sets of mixed standard solutions with different concentrations, wherein the concentrations of boscalid and difenoconazole in the mixed standard solutions are 0.0025-1 mg / L.

9. The method for detecting pesticide residues according to claim 8, wherein The concentrations of boscalid and difenoconazole in the mixed standard solutions are the same, and the concentrations of boscalid and difenoconazole in the five groups of mixed standard solutions are 0.0025, 0.025, 0.05, 0.5, and 1 mg / L, respectively.

10. The method for detecting pesticide residues according to claim 6, wherein: During steps (3) and (4), the test conditions are: UPLC: C18-WP column 2.1 mm × 50 mm, capillary 3 μm, column temperature 25°C; flow rate: 0.3 mL / min, constant flow; injection volume 2 μl; Mobile phase: Acetonitrile: 0.1% formic acid aqueous solution = 70:30 (v / v); MS / MS: electrospray ionization source mode was used with a capillary voltage of 4 kV; the collision energy of the secondary mass spectrometer was 25 eV; the drying gas temperature was 350°C; the drying gas flow rate was 8 L / min; the nebulizing gas pressure was 35 psi; and the reaction gas was N2.

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