Method for detecting triazole fungicide pesticide residues in water extracted by hydrophobic deep-eutectic solvent

Abstract

The invention relates to a detection method for extracting triazole fungicide pesticide residues in water by using a hydrophobic deep-eutectic solvent. The detection method comprises the following steps: firstly, preparing the hydrophobic deep-eutectic solvent by using a heating method; determining the types of the deep eutectic solvents (DESs) and the proportion of hydrogen bond donors / acceptors (HBD / HBA) through a single factor experiment so as to screen out the DESs with the optimal extraction efficiency; the extraction process conditions of the QuEChERS method are optimized through an L9 (33) orthogonal experiment; inspecting the sensitivity, the detection limit, the accuracy and the precision of the method through a method verification experiment; and finally, carrying out actual sample detection. According to the method, detection can be completed in a short time with relatively high extraction efficiency and sensitivity, pretreatment operation steps are simplified, the detection cost is reduced, and potential risks of a traditional organic solvent on human health and environment are reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticide detection, and in particular to a method for detecting triazole fungicide residues in water by extraction with a hydrophobic low eutectic solvent. Background Art

[0002] Water resources and crops such as fruits and vegetables are essential for human survival. Fungal diseases often occur during the planting of crops. Triazole fungicides are effective in preventing and controlling fungal diseases. They can be used to treat leaf spot, black spot, black blight, etc. They are widely used in agricultural production and are one of the most popular fungicides in the world. This type of fungicide has stable chemical properties and is not easily decomposed or transformed, so it remains in different media for a long time. Pesticides remaining in crops are not only harmful to human health, but also to the natural environment and other organisms. Therefore, it is necessary to develop a more environmentally friendly, accurate and efficient pesticide residue detection technology; and to screen out a pretreatment method with high stability, high sensitivity and good reproducibility, so as to provide a simpler, accurate and reliable detection method for the risk assessment of primary agricultural product quality and safety. In the traditional QuEChERS method, organic solvents such as acetonitrile, toluene and acetone are often used as extractants. Since such solvents have certain toxicity, they are not only unfriendly to the environment but also harmful to the health of the experimenters. A green and environmentally friendly hydrophobic low eutectic solvent is now prepared to replace traditional toxic organic solvents such as acetonitrile. The extraction capacity of DESs synthesized with the same hydrogen bond acceptor (HBA) and different types and proportions of hydrogen bond donors (HBD) is explored, and the optimal extractant is screened. Summary of the invention

[0003] Purpose of the invention: The purpose of the present invention is to provide a method for detecting triazole fungicide residues in water by extraction with a hydrophobic low eutectic solvent, so as to solve the limitations of current traditional methods for detecting pesticide residues in crops, including reliance on harmful organic solvents, high-cost consumables, complex and time-consuming pretreatment processes, and unsuitability for large-volume sample processing.

[0004] Technical solution: The method for detecting non-polar triazole fungicide pesticide residues in water by extracting a hydrophobic low eutectic solvent described in the present invention comprises the following steps: first preparing a hydrophobic low eutectic solvent; then pre-treating the sample by the QuEChERS method: taking an appropriate amount of water sample containing a certain concentration of target analyte in a centrifuge tube; adding a certain amount of the prepared hydrophobic low eutectic solvent, centrifuging after vortex oscillation and ultrasound-assisted extraction; taking the upper clear liquid and putting it into another purification tube filled with an adsorbent, vortex oscillation and standing; taking the supernatant and diluting it with methanol, filtering it through an organic filter membrane, and injecting it into a sample injection bottle for HPLC-MS / MS detection; finally, verifying the accuracy, precision and sensitivity of the method through method verification and actual sample detection.

[0005] Furthermore, the preparation method of the low eutectic solvent is as follows: the hydrogen bond donor HBD and the hydrogen bond acceptor HBA are mixed in a certain molar ratio by a direct heating melting method, and heated and stirred until a transparent and uniform liquid is formed.

[0006] Furthermore, the hydrogen bond donor HBD and the hydrogen bond acceptor HBA are mixed by direct heating and melting method, and stirred while heating until a transparent and uniform liquid is formed, and then stored away from light for later use.

[0007] Furthermore, the direct heating melting method is specifically as follows: HBD and HBA are poured into a round-bottom flask according to a certain molar ratio, a magnet is added, the round-bottom flask is placed in a constant temperature water bath magnetic stirrer, and heating and stirring are performed until a transparent and uniform liquid is formed, which is a hydrophobic low eutectic solvent.

[0008] Furthermore, the temperature of the constant temperature water bath magnetic stirrer is set to 70-90° C., and the stirring time is 40-80 min.

[0009] Furthermore, the hydrophobic L-menthol is HBA, and the caprylic acid, lactic acid, thymol, n-butanol and n-decanoic acid are HBD; the molar ratio of HBA to HBD is 1:1 to 1:4.

[0010] Furthermore, five hydrophobic DESs were prepared by direct heating and melting method. 1 Its structure and thermal stability were characterized by H-NMR, FT-IR and TGA.

[0011] Furthermore, through single-factor experiments, HBA and HBD were mixed at a fixed molar ratio of 1:1, and the recovery rates of the three triazole fungicides were used as indicators to determine the type of optimal DESs.

[0012] Furthermore, the orthogonal experimental method was used to optimize the extraction conditions. Using DES5 (L-menthol / decanoic acid 1:1) as the extractant, three factors and three levels of L-menthol were optimized for factor A: DESs / Sample volume ratio (0.05 / 0.075 / 0.1), factor B: vortex extraction time (3 / 4 / 5 min) and factor C: extraction centrifugation time (2 / 3 / 4 min). 9 (3 3 ) Orthogonal test, with the recovery rate of the target substance as the evaluation indicator.

[0013]

[0014] Table 1 Level table of influencing factors of orthogonal experiment

[0015] Furthermore, the sensitivity and linear range of the method were investigated by establishing a calibration curve. A standard working curve between the mass concentration of the pesticides and their peak areas was constructed under the same experimental conditions by dissolving the mixed standard solutions of the three triazole fungicides in a methanol solution containing hydrophobic DES5. The detection limit was calculated according to the signal-to-noise ratio of 3:1 (S / N=3), and the quantification limit LOQ was calculated according to the signal-to-noise ratio of 10:1 (S / N=10). The linear range, correlation coefficient, detection limit and quantification limit of the three triazole fungicides were investigated.

[0016] Further, the HPLC-MS / MS instrument conditions are:

[0017] (1) Chromatographic conditions

[0018] Chromatographic column: Waters ACQUITY UPLC BEH C18 Column (2.1mm×100mm, 1.7um); sample room temperature: 15°C; column temperature: 35°C; flow rate: 0.3mL / min; injection volume: 1.0uL; mobile phase: phase A is 0.1% formic acid aqueous solution, phase B is acetonitrile, and high performance liquid separation is performed according to the gradient elution conditions in Table 2.

[0019]

[0020]

[0021] Table 2 Liquid chromatography gradient elution conditions

[0022] (2) Mass spectrometry conditions

[0023] Electrospray ion source: negative ion mode; multiple reaction monitoring mode (MRM); capillary voltage: 3.5 kV; cone backflush gas (nitrogen): 150 L / h; desolvation gas (nitrogen) flow rate: 800 L / h; desolvation gas temperature: 550°C; collision gas (argon) flow rate: 0.15 mL / min; ion source temperature: 150°C.

[0024] Furthermore, under the best experimental conditions, the precision, accuracy and actual sample detection of the optimized QuEChERS method were investigated, the recovery rate ER was used to evaluate the accuracy of the method, and the precision RSD was used to measure the accuracy of the method.

[0025] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) A green and environmentally friendly hydrophobic low eutectic solvent is prepared, which has the advantages of biodegradability, low cost and easy preparation. (2) The pretreatment method prepared by the present invention is simple and easy to operate, and phase separation can be achieved without adding salt, eliminating the salting-out step, high extraction efficiency, and effectively reducing the harm of toxic reagents to human health and the environment. (3) At the same time, the invention also determines the optimal molar ratio conditions for synthesizing DESs and further optimizes the extraction process. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the preparation process of the hydrophobic deep eutectic solvent of the present invention;

[0027] Figure 2 It is a schematic diagram of the pre-treatment process of the present invention;

[0028] Figure 3 The results are as follows: the effect of the type of the hydrophobic deep eutectic solvent on the extraction efficiency of the triazole fungicide;

[0029] Figure 4 The results are the effects of the ratio of L-menthol to n-capric acid on the extraction efficiency of triazole fungicides. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0031] Unless otherwise specified, the experimental methods used in the present invention are all conventional methods.

[0032] In the present invention, L-menthol, n-octanoic acid, lactic acid, thymol, n-butanol, n-decanoic acid, difenoconazole, hexaconazole, and myclobutanil were all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0033] like Figure 1 As shown, an embodiment of the present invention provides a method for detecting triazole fungicide residues in water by extraction with a hydrophobic deep eutectic solvent, comprising the following steps:

[0034] S1: Effect of hydrophobic deep eutectic solvent type on the extraction efficiency of triazole fungicides

[0035] (1) Preparation of hydrophobic deep eutectic solvent

[0036] The hydrophobic L-menthol was selected as HBA, and caprylic acid, lactic acid, thymol, n-butanol and n-decanoic acid were selected as HBD. The HBA and HBD were prepared by a direct heating and melting method. A certain amount of HBA and HBD were accurately weighed and mixed according to a certain molar ratio.

[0037] Heat in a water bath at 85°C, stirring while heating, until the liquid is transparent and clear. Store away from light for later use. Figure 1 shown.

[0038] (2) Sample pretreatment

[0039] Usually, when using the QuEChERS method for sample pretreatment, salt needs to be added to help achieve phase separation and remove water from the sample matrix. However, the extractant used in the present invention is a hydrophobic low eutectic solvent, which has a large density difference with the matrix water, so phase separation can be achieved without adding salt. The steps for using hydrophobic DESs for QuEChERS method to pretreat samples are as follows: Figure 2 First, take 10 mL of water sample into a 50 mL centrifuge tube with a cap; add an appropriate amount of prepared hydrophobic low eutectic solvent, vortex oscillation for 1 min, and ultrasonic assisted extraction for 10 min; centrifuge at 4500 r / min; take the supernatant and put it into another centrifuge tube that has been loaded with solid adsorbents (PSA, GCB and anhydrous MgSO 4 ) in a 15 ml purification tube, vortex for 1 min and let stand for 5 min; take 2 ml of the supernatant and pass it through an organic phase filter membrane with a pore size of 0.22 μm and inject it into the sampling bottle for testing on the machine.

[0040] Depend on Figure 3 The results of the effect of the type of low eutectic solvent on the extraction efficiency of triazole fungicides show that the extraction efficiency of the three triazole fungicides is greatly affected by the types of the two components of the low eutectic solvent. Compared with the other four DESs, for the same HBA (L-menthol), DES5 with n-capric acid as HBD has a higher extraction efficiency for the three triazole fungicides. This indicates that longer alkyl chains are more conducive to the extraction of non-(weakly) polar triazole fungicides through hydrophobic effects, and the -COOH group contained in n-capric acid has a strong adsorption affinity for the target fungicide. Therefore, DES5 was selected for the next study.

[0041] S2: Effect of the molar ratio of L-menthol / n-caprylic acid on the extraction efficiency of triazole fungicides

[0042] The experimental steps were the same as those in S1. The low eutectic solvents with different ratios of L-menthol: n-capric acid (1:1, 1:2, 1:3, 1:4) were prepared respectively. The extraction was performed according to the sample pretreatment steps in Example S1. The effect of the molar ratio of L-menthol to n-capric acid on the extraction efficiency of triazole fungicides was shown in FIG. Figure 4As shown in the figure, it can be seen that the extraction recovery rate of difenoconazole in the five hydrophobic DESs reached 91% to 66%, myclobutanil reached 72% to 56%, and hexaconazole reached 85% to 49%. Among them, hydrophobic DES5 (1:1) has the best extraction efficiency for the three fungicides, and with the increase of the proportion of n-decanoic acid, the extraction rate shows a downward trend, and hydrophobic DES6 to DES8 have poor stability at room temperature and are easy to solidify. Therefore, hydrophobic DES5 (1:1) was selected for further study.

[0043] S3: Effect of extraction conditions on the extraction efficiency of triazole fungicides

[0044] The experimental steps were the same as S1, and the comprehensive effects of the three factors A, B, and C on the extraction effect were investigated, namely, the volume ratio of DESs / Sample, the vortex extraction time, and the extraction centrifugation time during the extraction process. DES5 (L-menthol / decanoic acid 1:1) was used as the extraction solvent, and the orthogonal experimental method was used to conduct a 3-factor 3-level L 9 (3 3 ) orthogonal test, with the recovery rate of the target as the evaluation index. As shown in Table 3, the R values ​​of the three single factors A, B, and C show that the order of priority affecting the extraction effect is: A>B>C, that is, the DESs / Sample volume ratio has the greatest impact on the average recovery rate MR value, followed by the vortex extraction time, and finally the extraction centrifugation time. The optimal extraction conditions were determined to be: DESs / Sample=0.075, the vortex extraction time was 4min, and the extraction centrifugation time was 3min. Under the optimal experimental conditions, the water samples were pre-treated, and the average recovery rate of the three triazole fungicides (fenpropimorph, hexaconazole, and myclobutanil) could reach 87%.

[0045]

[0046] Table 3 Orthogonal experiment data table

[0047] S4: The three triazole fungicides were dissolved in a methanol solution containing hydrophobic DES5, and a standard working curve between the mass concentration of the pesticide and its peak area was constructed under the same experimental conditions. The detection limit was calculated according to the signal-to-noise ratio of 3:1 (S / N=3), and the quantification limit was calculated according to the signal-to-noise ratio of 10:1 (S / N=10). The sensitivity and linear range of the method were investigated by the calibration curve, the accuracy of the method was measured by the recovery rate ER, and the precision of the method was measured by the relative standard deviation RSD. The stream water near the farmland and the tap water soaked in fruits and vegetables for 30 minutes were added with mixed pesticide standards at low, medium and high levels, and each addition level was repeated 5 times to calculate the ER and RSD values.

Claims

1. A method for detecting triazole fungicide residues in water by extraction with a hydrophobic deep eutectic solvent, characterized in that: The following steps are involved: First, a hydrophobic low eutectic solvent is prepared; then the sample is pretreated using the QuEChERS method: an appropriate amount of water sample containing a certain concentration of target analyte is taken into a centrifuge tube; a certain amount of the prepared hydrophobic low eutectic solvent is added, and it is first vortexed and ultrasonic-assisted extracted before direct centrifugation; the upper clear liquid is taken and placed into another purification tube containing an adsorbent, and it is vortexed and allowed to stand after being allowed to stand; the supernatant is filtered through an organic filter membrane and injected into a sample injection bottle for HPLC-MS / MS detection; finally, the accuracy, precision and sensitivity of the method are verified through method verification and actual sample detection experiments.

2. The method for detecting triazole fungicide residues in fruits and vegetables by deep eutectic solvent extraction according to claim 1, characterized in that: The specific method for preparing the low eutectic solvent is as follows: the hydrogen bond donor HBD and the hydrogen bond acceptor HBA are mixed in a certain molar ratio by a direct heating melting method, and heated and stirred until a transparent and uniform liquid is formed.

3. The method for detecting triazole fungicide residues in water by extraction with a hydrophobic deep eutectic solvent according to claim 2, characterized in that: The hydrogen bond donor HBD and the hydrogen bond acceptor HBA are mixed by heating and melting method, and stirred while heating until a transparent and uniform liquid is formed, and then stored away from light for later use.

4. The method for detecting triazole fungicide residues in water by extraction with a hydrophobic deep eutectic solvent according to claim 3, characterized in that: The specific steps of the direct heating melting method are as follows: HBD and HBA are poured into a round-bottom flask according to a certain molar ratio, a magnetic bar is added, and the round-bottom flask is placed in a constant temperature water bath magnetic stirrer, and heated and stirred until a transparent and uniform liquid is formed, which is a hydrophobic low eutectic solvent.

5. The method for detecting triazole fungicide residues in water by extraction with a hydrophobic deep eutectic solvent according to claim 4, characterized in that: The temperature of the constant temperature water bath magnetic stirrer was set to 70-90°C, and the stirring time was 40-80 min.

6. The method for detecting triazole fungicide residues in fruits and vegetables by deep eutectic solvent extraction according to claim 3, characterized in that: The hydrophobic L-menthol is HBA, and caprylic acid, lactic acid, thymol, n-butanol and n-decanoic acid are HBD; the molar ratio of HBA to HBD is 1:1~1:

4.

7. The method for detecting triazole fungicide residues in water by extraction with a hydrophobic deep eutectic solvent according to claim 3, characterized in that: Through single factor experiments, HBA and HBD were mixed at a fixed molar ratio, and the recovery rates of the three target substances were used as indicators to determine the type of optimal DESs.

8. The method for detecting triazole fungicide residues in water by extraction with a hydrophobic deep eutectic solvent according to claim 6, characterized in that: Five hydrophobic DESs were prepared by direct heating and melting method. 1 Its structure and thermal stability were characterized by H-NMR, FT-IR and TGA.

9. The method for detecting triazole fungicide residues in water by extraction with a hydrophobic deep eutectic solvent according to claim 1, characterized in that: The orthogonal experimental method was used to optimize the extraction conditions. DES5 (L-menthol / decanoic acid 1:1) was used as the extraction solvent. The three influencing factors, factor A: DESs / Sample volume ratio (0.05 / 0.075 / 0.1), factor B: vortex extraction time (3 / 4 / 5min) and factor C: extraction centrifugation time (2 / 3 / 4min), were analyzed by 3-factor 3-level L9 (3 3 ) Orthogonal test was conducted, with the recoveries of three fungicides: Difenoconazole, Myclobutanil and Hexaconazole as evaluation indicators.

10. The method for detecting triazole fungicide residues in water by extraction with a hydrophobic deep eutectic solvent according to claim 1, characterized in that: The sensitivity and linear range of the method were investigated by establishing a calibration curve. A standard working curve between the mass concentration of the pesticides and their peak areas was constructed by dissolving the mixed standard solutions of the three triazole fungicides in a methanol solution containing hydrophobic DES5 under the same experimental conditions. The limit of detection (LOD) was calculated according to the signal-to-noise ratio of 3:1 (S / N=3), and the limit of quantification (LOQ) was calculated according to the signal-to-noise ratio of 10:1 (S / N=10). The linear range, correlation coefficient, limit of detection and limit of quantification of the three triazole fungicides were investigated.

11. The method for detecting triazole fungicide residues in water by extraction with a hydrophobic deep eutectic solvent according to claim 1, characterized in that: Under the best experimental conditions, the precision, accuracy and actual sample detection of the optimized QuEChERS method were investigated. The recovery rate ER was used to evaluate the accuracy of the method, and the precision RSD was used to measure the accuracy of the method.

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