A new method for analyzing triazole fungicides in fruit peel based on switchable deep eutectic solvent pretreatment

Abstract

The present invention discloses a novel method for analyzing triazole fungicides in fruit peels based on switchable low eutectic solvent pretreatment. The method is a simple, efficient and green QuEChERS-LPME coupling method. By introducing a pH-responsive switchable low eutectic solvent (SDES) to determine 8 triazole fungicides in rotten fruit peel waste, satisfactory pretreatment and analysis effects of complex matrix biomass-derived samples are finally obtained. The method is combined with a high performance liquid chromatography diode array detector (HPLC-DAD) to obtain a satisfactory recovery rate and a high enrichment factor in the rotten fruit peel after the matrix is ​​purified, showing the possibility of further applying the method to other biological resource pretreatment processes.

Description

Technical Field

[0001] The present invention relates to a new method for pretreating and analyzing triazole fungicides in rotten peel waste. Specifically, a new system for analyzing triazole fungicides in rotten peel waste is established by combining a pretreatment method based on a QuEChERS purification procedure and a pH-responsive switchable low eutectic solvent microextraction with a high performance liquid chromatography. Background Art

[0002] The analysis and utilization of biomass-derived wastes have become a hot topic in the current global greening process. How to analyze and screen valuable biomass components is a necessary prerequisite for biomass conversion and utilization. Due to China's large population and large area of ​​arable land, it is crucial to handle the relationship between biological resource utilization and safety. In addition to crops that can be used directly, by-products from crop production and use are also an important available resource. The increasing importance of biomass resources has brought about the need to monitor and control their quality and safety. Researchers have begun to focus on the separation and detection of residual organic compounds in biomass wastes. Among them, the excessive amount of toxic pesticide residues in a large amount of peel-derived waste has attracted great attention.

[0003] Triazole fungicides (TFs) are a class of fungicides that show good efficacy in crop sterilization and protection. It can affect the biosynthesis of sterols by destroying the cell membrane function of bacteria, thereby protecting crops from pathogens. However, the negative effects of these pesticides on the environment and organisms also lead to some safety issues. TFs can affect the growth rate and chlorophyll content of plants (such as algae), thereby inhibiting the photosynthesis rate of plants. They also have teratogenic effects on mammals, affect the gene expression of certain enzymes, and have a certain negative impact on the biosynthesis of steroids in humans. Triazole fungicide residues in crop wastes are released into the ecosystem with the growth and metabolism of crops, and enter the biomass conversion process with crops and their by-products. Therefore, the detection and analysis of TFs residues in reusable peel-derived wastes is crucial to protect humans and other organisms.

[0004] Organic analytes in samples can be easily separated by chromatographic techniques and combined with appropriate detectors for accurate and precise determination. The application of high-resolution analytical instruments is limited by their high cost of use and maintenance. Relatively low-cost high-performance liquid chromatography with diode array detector (HPLC-DAD) combined with sample pretreatment methods can achieve high-sensitivity detection and universal applicability. QuEChERS (quick, easy, cheap, effective, robust and safe) is a mature cleanup method for analyzing complex matrix samples. The sample pretreatment method of QuEChERS combined with solvent liquid phase microextraction can make up for the shortcomings of QeEChERS in preconcentration of analytes in complex biomass samples, eliminate matrix effects, and make the analysis of biomass samples more universal and convenient when combined with HPLC-DAD. Researchers have used switchable solvent liquid phase microextraction (SS-LPME) to enhance the QuEChERS method for the determination of organochlorine pesticides and hormones in food and environmental samples, which can analyze complex matrix samples in a short time.

[0005] Considering the complexity of the matrix in actual biomass waste samples and the high cost and difficulty of their analysis, pre-concentration or sample preparation plays an important role in the analytical procedure. Homogeneous liquid-liquid microextraction (HLLME) technology usually requires the participation of extractants, dispersants and emulsifiers to switch the extraction solvent and water system between single phase and two phases. Traditional organic solvents, ionic liquids (ILs) and switchable solvents (SS) are now a thing of the past due to their high toxicity, large dosage and strong irritation. The benign demand of the concept of green analytical chemistry has promoted the birth of deep eutectic solvents (DES), which are formed by intermolecular hydrogen bonds between hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs). Due to the low production cost, non-toxicity, biodegradability, recyclability and durability / stability of DES, it has been widely used in biomass pretreatment and production. Researchers have found that it is possible to dynamically switch DES between hydrophobic and hydrophilic forms by introducing inorganic acids or bases to adjust the pH of the solution. The hydrophilic state allows the switchable deep eutectic solvent (SDES) to disperse into the aqueous phase, thereby achieving a nearly infinite contact area, while the recovery of the hydrophobic state allows SDES to achieve high extraction efficiency. In this way, SDES is endowed with a dual role (extractant and dispersant), achieving organic solvent-free, efficient and timely extraction and separation. Summary of the invention

[0006] In view of the shortcomings of the previously published QuEChERS and SS-LPM combined method, this study aims to improve the combined method by replacing the switchable solvent (SS) with a new green solvent, switchable deep eutectic solvent (SDES). Compared with SS, SDES has better targeted adsorption and pre-enrichment effects, which is attributed to its stable hydrogen bond network structure.

[0007] In addition, SDES microextraction uses fewer solvents and other reagents and SDES itself is highly green, while the amine compounds as SS have relatively unstable physical and chemical properties, are highly toxic and irritating, and require a large amount of solvent but have average results. Considering the shortcomings of this method, this study introduced SDESs based on natural products menthol, thyme, and medium / long-chain fatty acids to improve the extraction performance and greenness of the method combined with QuEChERS and solvent liquid phase microextraction.

[0008] Single factor experiment and Box-Behnken design response surface methodology were used to optimize the factors affecting the extraction process. Under the optimal conditions, eight triazole fungicides (triadimenol, chloranil, epoxiconazole, cyproconazole, flusilazole, tebuconazole, hexaconazole and difenoconazole) were separated and analyzed in rotten grapes, watermelon peels and orange peels.

[0009] The basic concept of the present invention makes full use of the high selectivity of green solvents with specific chemical structures for the target and the characteristics of the solvent switching between hydrophilic / hydrophobic states. The method combining the QuEChERS purification procedure with switchable deep eutectic solvent microextraction has a short pretreatment time and enhanced impurity removal and pre-enrichment effects. The present invention is simple to operate, low in cost, good in effect, highly green, and has the advantage of pretreatment of large-volume complex matrix samples in batches.

[0010] The technical solution of the present invention is as follows:

[0011] A method for analyzing triazole fungicides in rotten peel waste based on a pretreatment method combined with a QuEChERS cleanup procedure and a pH-responsive switchable deep eutectic solvent microextraction combined with high performance liquid chromatography, comprising the following steps:

[0012] (1) Synthesis of Extractant

[0013] The hydrogen bond acceptor (HBA) and the hydrogen bond donor (HBD) are mixed, stirred at 50-60° C. for 15-20 min, and then cooled to room temperature to obtain an extractant (SDES);

[0014] The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:2 to 2:1;

[0015] The hydrogen bond acceptor is a natural product selected from menthol or thymol;

[0016] The hydrogen bond donor is a medium-chain fatty acid selected from n-hexanoic acid, n-octanoic acid, n-dodecanoic acid or oleic acid;

[0017] A particularly preferred SDES has thymol as a hydrogen bond acceptor, n-hexanoic acid as a hydrogen bond donor, and the molar ratio of thymol to n-hexanoic acid is 1:1, i.e., SDES-12 in Table 1;

[0018] Preferably, the heating temperature of menthol-based SDES is 50°C, and the heating temperature of thymol-based SDES is 60°C;

[0019] (2) Sample pretreatment

[0020] The QuEChERS cleanup was performed by adding acetonitrile containing 0.1 wt% acetic acid, anhydrous magnesium sulfate A, and sodium chloride to a uniform rotten peel sample, vortexing and centrifuging, and then adding anhydrous magnesium sulfate B, PSA, and C to the supernatant. 18 , centrifuge (4000rpm, 3min), take the supernatant and dilute it with distilled water, then add the extractant prepared in step (1), add NaOH solution dropwise, mix thoroughly, add citric acid solution to form an emulsion, then ultrasonicate (preferably 90s), centrifuge (5000rpm, 2min), collect the upper liquid phase with a microsyringe, dilute it with methanol, and filter it through an organic filter membrane to obtain a sample solution;

[0021] Preferably, the ratio of the sample to be tested to acetonitrile containing 0.1wt% acetic acid, anhydrous magnesium sulfate A, and sodium chloride is 10g:10mL:5g:1.5g;

[0022] The preferred ratio of the sample to be tested to anhydrous magnesium sulfate B, PSA, and C18 is 10 g: 1.5 g: 25 mg: 25 mg;

[0023] The preferred ratio of the sample to be tested to the extractant, NaOH solution, and citric acid solution is 10 g: 90 μL: 600 μL: 700 μL;

[0024] The concentration of the NaOH solution is 5 mol / L; the concentration of the citric acid solution is 50 wt%;

[0025] The anhydrous magnesium sulfate A and anhydrous magnesium sulfate B have no special meanings, and are marked as "A" and "B" only to distinguish the anhydrous magnesium sulfate used in different operation steps;

[0026] (3) Sample testing

[0027] Analyzing the sample solution obtained in step (2) using a high performance liquid chromatography instrument with a diode array detector to obtain the content of the triazole fungicide in the sample;

[0028] The HPLC conditions were as follows: triazole fungicides (TFs) were detected using an Agilent 1260 Infinity II HPLC system (Agilent Technologies, USA) equipped with a diode array detector (Agilent Technologies, USA); chromatographic separation was performed on an Agilent Poroshekk 120EC-C18 column (150 mm×4.6 mm, 4 μm) at a column temperature of 30°C; the flow rate was 1.0 mL / min, the injection volume was 20 μL, and the detection wavelength was 220 nm; a combination of methanol and water was used as the mobile phase, and the elution program was as follows: 0-6 min, the methanol ratio changed from 20% to 50%; 6-26 min, the methanol ratio changed from 50% to 70%, and then maintained to 32 min; from 32-40 min, the methanol ratio changed to 100%.

[0029] Taking the best extractant SDES-12 as an example, the mechanism of the present invention is analyzed as follows:

[0030] (I) pH-induced phase transformation mechanism of SDES / aqueous system

[0031] For the transformation process of the SDES-12 / aqueous phase system from a biphasic state to a monophasic state, when sodium hydroxide is added to raise the pH value of the system to the pKa of hexanoic acid (4.88), sodium hexanoate is gradually produced in the system. Due to the acid-base interaction between hexanoic acid and sodium hydroxide, the hydrogen bonds in SDES-12 are destroyed due to competition, and the SDES / aqueous phase system begins to gradually transform from a biphasic state to a monophasic state. When the pH value reaches the pKa of thymol (10.62), most of the hexanoic acid exists in the form of sodium hexanoate. At this time, SDES switches to a hydrophilic state, and the solution system is completely transformed from a biphasic state to a monophasic state. For the system that transforms from a monophasic state to a biphasic state, citric acid (C6H8O7) with three acidity coefficients (pKa = 3.13, 4.77 and 6.39) is added to the system as a protonating agent. As the pH value of the solution system gradually decreases, sodium hexanoate is protonated to produce hexanoic acid, and re-bonds with thymol through hydrogen bonds to form SDES-12. In addition, as the pKa gradually decreased to 6.39, 4.77, and 3.13, the salting-out effect of the corresponding sodium citrate salts (NaC6H7O7, Na2C6H6O7, and Na3C6H5O7) prompted the separation of the SDES-12 phase and the target analytes from the aqueous solution to form a two-phase system.

[0032] (II) Hydrophobic-hydrophilic transition mechanism of pH-responsive SDES

[0033] SDES composed of menthol / thymol (HBA) and fatty acids (HBD) can show excellent pH-induced phase transition characteristics and can show strong hydrophobicity in aqueous systems, resulting in extensive ionization and dissolution phenomena in alkaline solutions. For example, thymol (pKa = 10.62) and fatty acids (pKa of caproic acid, caprylic acid, lauric acid and oleic acid are in the range of 4.88 to 5.30) can exist as hydrophilic anions under alkaline conditions (pH>10.62) and form a uniform mixture with aqueous solutions. As the pH value decreases, the hydrophilic form is protonated, and the precursor molecules with limited solubility in aqueous media dominate the sample solution and form tiny droplets of SDES in situ (producing emulsification). In this case, the contact surface area between the solvent phase and the aqueous phase is close to the maximum, resulting in rapid mass transfer of the target analyte to the extractant.

[0034] In addition, the target analytes in this study, triazole fungicides (TFs), all contain one or more benzene ring structures, and the SDES synthesized based on thymol can produce π-π interactions with the benzene ring, which can improve the affinity between thymol-based SDESs and TFs. In addition, the hydrogen bonding between the N atoms on the heterocyclic rings of TFs and the hydroxyl groups of SDESs, as well as the hydrophobic and electrostatic interactions between TFs and the long alkyl chains of fatty acids, can further improve the efficiency of extraction and adsorption.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. The present invention provides an effective method for determining triazole fungicides in rotten peel waste;

[0037] 2. The present invention combines the QuEChERS method with switchable deep eutectic solvent liquid phase microextraction, overcoming the problems of high toxicity, large dosage and general extraction and enrichment effects of traditional switchable solvents, and has the advantage of good treatment effect on complex matrix samples;

[0038] 3. A green solvent with pH responsiveness was synthesized as an extractant, which does not require any dispersant or emulsifier, and the extraction process is simple;

[0039] 4. No organic solvent is used in the whole process, and the atomic utilization rate of the solvent synthesis process is 100%;

[0040] 5. The application of the present invention can be combined with practice to reduce the difficulty and cost of analyzing complex matrix samples, and provides a new method for the analysis of triazole fungicides in biomass waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1Schematic diagram of the process of analyzing triazole fungicides in rotten peel waste by a pretreatment method based on the QuEChERS purification procedure and pH-responsive switchable low eutectic solvent microextraction combined with high performance liquid chromatography established in the present invention.

[0042] Figure 2 This is the optimization result of the extractant type.

[0043] Figure 3 This is the optimization result of the amount of extractant.

[0044] Figure 4 This is the optimization result of ultrasound time.

[0045] Figure 5 The response surface optimization results for the concentration and dosage of the base and the dosage of the acid.

[0046] Figure 6 Infrared spectrum and H NMR spectrum of the best extractant obtained by optimization.

[0047] Figure 7 This is the chromatogram of the spiked recovery experiment and pretreatment effect of three kinds of fruit peel waste in actual sample analysis.

[0048] Figure 8 These are the contact angle measurement results of SDES-12, after switching by adding NaOH, and after switching by adding citric acid. DETAILED DESCRIPTION

[0049] The present invention is further described below by means of specific embodiments, but the protection scope of the present invention is not limited thereto.

[0050] The present invention uses menthol and thymol, two natural products, as HBA and different types of medium-chain fatty acids at molar ratios of 1:1, 1:2 and 2:1 to synthesize 22 different SDES (Table 1), all of which are in the upper layer of the aqueous phase.

[0051] Table 1 Basic information of the synthesized SDES

[0052]

[0053] Among them, the relevant characteristics of the best extractant SDES-12 are as follows:

[0054] (I) Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectroscopy ( 1 H NMR) characterization

[0055] In order to confirm that SDES-12 indeed formed a hydrogen bond grid structure between thymol (5-methyl-2-isopropylphenol) and hexanoic acid (HA), the stretching vibration wavelengths of thymol, hexanoic acid and SDES-12 were measured by FT-IR. Figure 6 As shown in (A), the OH stretching vibration peaks of HA and thymol appear at approximately 3200 cm -1 and 3300cm -1 SDES-12 3500cm -1 The appearance of vibration peaks around 1714 cm indicates that OH in HA participates in the formation of hydrogen bonds, resulting in the migration of electrons from oxygen atoms to hydrogen bonds, which also leads to a decrease in the bond force constant of carboxylate OH and a red shift. -1 The C=O stretching vibration of HA was observed at , and similar vibration peaks were also found in SDES-12, indicating that the C=O structure of HA was involved in the formation of SDES-12.

[0056] By investigating the 1 H NMR spectroscopy was used to study the formation of hydrogen bonds and structural changes during the synthesis of SDES. Figure 6 As shown in (B), the H signals at 6.70ppm, 6.84ppm and 7.13ppm in the NMR of thymol are all attributed to the hydrogen on the benzene ring, and the same result is found in the NMR of SDES-12. The H signal at 3.05ppm is generated by the CH at the isopropyl position adjacent to the benzene ring in thymol, while the H signals at 0.88ppm, 1.28ppm, 1.29ppm, 1.54ppm, and 2.21ppm are generated by the methylene in HA. All of these chemical shift peaks can be found in the NMR of SDES-12, indicating that SDES-12 is composed of thymol and HA.

[0057] (II) Contact angle measurement experiment

[0058] This part of the study intuitively reflects the hydrophilic and hydrophobic states of SDES-12 during the switching process. Figure 8 The contact angle states of SDES-12 in its synthesized form and two switched forms are shown. The contact angle of water on octadecylsilane (θ=129.64°) was used as a benchmark to evaluate the hydrophobicity of the initial SDES, the hydrophilicity of the SDES after base switching, and the hydrophobicity of the SDES after acid switching. All three states of SDES-12 were measured on the same hydrophobic surface (octadecylsilane-coated glass). Figure 8 As shown in (A), the contact angle of the initial SDES-12 is less than 13.05°, which indicates that the synthesized SDES-12 exhibits strong hydrophobicity. Figure 8As shown in (B), after the NaOH switch, the contact angle of SDES-12 is greater than 124.95°, which indicates that SDES is switched to a hydrophilic state under the action of acid and base, which is beneficial to the subsequent dispersion and regeneration of SDES. Figure 8 As shown in (C), after switching the polarity by citric acid, the contact angle of SDES-12 recovered to 15.65°, indicating that the hydrophobicity recovery effect of SDES was good. Due to the sharp change in contact angle, SDES-12 exhibited a high hydrophobic-hydrophilic conversion efficiency driven by NaOH and citric acid, which is meaningful for the separation and pre-concentration of TF analytes in biomass waste samples in a single step.

[0059] Example 1: Enrichment and detection of triazole fungicides in rotten grape skin

[0060] (1) Extraction agent preparation

[0061] Weigh thymol and n-hexanoic acid in a molar ratio of 1:1 in a wide-mouth bottle, add a stirring magnetic bar, and stir continuously in a 60°C water bath for 15 minutes to obtain a hydrophobic, low-viscosity homogeneous liquid. After cooling, store the SDES extractant at room temperature.

[0062] (2) Sample pretreatment

[0063] The QuEChERS cleanup was performed by adding 10 mL of acetonitrile (containing 0.1% acetic acid), 5 g of anhydrous magnesium sulfate, and 1.5 g of sodium chloride to 10 g of uniform rotten grape skin sample (blank or spiked). After vortexing for 15 s and centrifuging, 1.5 g of anhydrous MgSO4, 25 mg of PSA, and 25 mg of C were added to the supernatant. 18 Centrifuge at 4000 rpm for 3 minutes. Then take the supernatant and dilute it to 10 mL with distilled water for subsequent extraction.

[0064] Take 90 μL of the prepared extractant and add it to the above solution. Add 600 μL of 5 mol / L NaOH solution under manual shaking. After the solution is allowed to stand for 30 seconds to fully mix, add 700 μL of 50% w / w citric acid solution to form an emulsion. Under the influence of a 90s ultrasonic water bath, the SDES droplets are evenly dispersed in various parts of the solution. After centrifugation at 5000 rpm for 3 minutes, collect the upper phase with a microsyringe and transfer it to a 1.5 mL centrifuge tube, dilute it 10 times with methanol and filter it into a sample injection bottle with a 0.45 μm organic filter membrane.

[0065] (3) Sample testing

[0066] The HPLC conditions were as follows: TFs were detected using an Agilent 1260 Infinity II HPLC system (Agilent Technologies, USA) equipped with a diode array detector (Agilent Technologies, USA). Chromatographic separation was performed on an Agilent Poroshekk 120EC-C18 column (150 mm × 4.6 mm, 4 μm) at a column temperature of 30 °C. The flow rate was 1.0 mL / min, the injection volume was 20 μL, and the detection wavelength was 220 nm. The mobile phase was a combination of methanol and water as follows: from 0 to 6 minutes, the methanol ratio was changed from 20% to 50%; from 6 to 26 minutes, the methanol ratio was changed from 50% to 70%, and then maintained until 32 minutes; from 32.0 minutes to 40 minutes, the methanol ratio was changed to 100%.

[0067] (4) Method evaluation

[0068] Under the optimal conditions, by considering the regression equation, linear range (LR), correlation coefficient (R 2 ), limit of detection (LOD), limit of quantification (LOQ), preconcentration factor (PF), and relative standard deviation (RSD) were used to evaluate the performance of the proposed SDES-HLLME method. Good linearity was obtained for all TFs in the range of 10-2000 ng / mL, and satisfactory R 2 The detection limit was in the range of 0.089-0.351 ng / mL, and the quantification limit was in the range of 0.297-1.172 ng / mL. The PFs of the eight target analytes obtained were in the range of 389-512 (after dilution with methanol). The precision of the method was verified by intra-day (n=6) and inter-day (n=6) analysis, with relative standard deviations ranging from 3.3%-7.9% and 2.1%-8.8%, respectively.

[0069] (5) Actual sample analysis

[0070] In this study, rotten grape skin samples were used as real samples to verify the applicability of the proposed method. TFs with known concentration levels (20, 50, 100 μg / kg) were added as matrix spiked samples to blank rotten grape skin samples to obtain the extraction recoveries (ERs) of eight TFs. Unspiked or spiked samples were pretreated under optimal conditions. The recoveries and actual concentrations of spiked samples before enrichment were calculated based on the calibration standards of target analytes prepared in methanol. Triadimenol, chlorfenapyr, flusilazole, tebuconazole, and difenoconazole were detected in the unspiked real samples, and their concentrations were much lower than the maximum residue limits of TFs in fruit pulp in my country. The spiked recoveries of TFs in rotten grape skin by this method were 91.0%-105.7%, and the relative standard deviations were less than 7.9%.

[0071] Example 2: Enrichment and detection of triazole fungicides in rotten watermelon rind

[0072] (1) Extraction agent preparation

[0073] Weigh thymol and n-hexanoic acid in a molar ratio of 1:1 in a wide-mouth bottle, add a stirring magnetic bar, and stir continuously in a 60°C water bath for 15 minutes to obtain a hydrophobic, low-viscosity homogeneous liquid. After cooling, store the SDES extractant at room temperature.

[0074] (2) Sample pretreatment

[0075] The QuEChERS cleanup was performed by adding 10 mL of acetonitrile (containing 0.1% acetic acid), 5 g of anhydrous magnesium sulfate, and 1.5 g of sodium chloride to 10 g of uniform rotten watermelon rind sample (blank or spiked). After vortexing for 15 s and centrifuging, 1.5 g of anhydrous MgSO4, 25 mg of PSA, and 25 mg of C were added to the supernatant. 18 Centrifuge at 4000 rpm for 3 minutes. Then take the supernatant and dilute it to 10 mL with distilled water for subsequent extraction.

[0076] Take 90 μL of the prepared extractant and add it to the above solution. Add 600 μL of 5 mol / L NaOH solution under manual shaking. After the solution is allowed to stand for 30 seconds to fully mix, add 700 μL of 50% w / w citric acid solution to form an emulsion. Under the influence of a 90s ultrasonic water bath, the SDES droplets are evenly dispersed in various parts of the solution. After centrifugation at 5000 rpm for 3 minutes, collect the upper phase with a microsyringe and transfer it to a 1.5 mL centrifuge tube, dilute it 10 times with methanol and filter it into a sample injection bottle with a 0.45 μm organic filter membrane.

[0077] (3) Sample testing

[0078] The HPLC conditions were as follows: TFs were detected using an Agilent 1260 Infinity II HPLC system (Agilent Technologies, USA) equipped with a diode array detector (Agilent Technologies, USA). Chromatographic separation was performed on an Agilent Poroshekk 120EC-C18 column (150 mm × 4.6 mm, 4 μm) at a column temperature of 30 °C. The flow rate was 1.0 mL / min, the injection volume was 20 μL, and the detection wavelength was 220 nm. The mobile phase was a combination of methanol and water as follows: from 0 to 6 minutes, the methanol ratio was changed from 20% to 50%; from 6 to 26 minutes, the methanol ratio was changed from 50% to 70%, and then maintained until 32 minutes; from 32.0 minutes to 40 minutes, the methanol ratio was changed to 100%.

[0079] (4) Method evaluation

[0080] Under the optimal conditions, by considering the regression equation, linear range (LR), correlation coefficient (R 2 ), limit of detection (LOD), limit of quantification (LOQ), preconcentration factor (PF), and relative standard deviation (RSD) were used to evaluate the performance of the proposed SDES-HLLME method. Good linearity was obtained for all TFs in the range of 10-2000 ng / mL, and satisfactory R 2 The detection limit was in the range of 0.089-0.351 ng / mL, and the quantification limit was in the range of 0.297-1.172 ng / mL. The PFs of the eight target analytes obtained were in the range of 389-512 (after dilution with methanol). The precision of the method was verified by intra-day (n=6) and inter-day (n=6) analysis, with relative standard deviations ranging from 3.3%-7.9% and 2.1%-8.8%, respectively.

[0081] (5) Volatile component analysis

[0082] In this study, rotten watermelon rind samples were used as real samples to verify the applicability of the proposed method. TFs with known concentration levels (20, 50, 100 μg / kg) were added as matrix spiked samples to blank rotten watermelon rind samples to obtain the extraction recoveries (ERs) of eight TFs. Unspiked or spiked samples were pretreated under optimal conditions. The recoveries and actual concentrations of spiked samples before enrichment were calculated based on the calibration standards of target analytes prepared in methanol. Methiconazole and difenoconazole were detected in the unspiked real samples at concentrations far below the maximum residue limits of TFs in fruit pulp in my country. The spiked recoveries of TFs in rotten watermelon rind by this method were 90.6%-108.3%, with relative standard deviations less than 8.8%.

[0083] Example 3: Enrichment and detection of triazole fungicides in rotten orange peel

[0084] (1) Extraction agent preparation

[0085] Weigh thymol and n-hexanoic acid in a molar ratio of 1:1 in a wide-mouth bottle, add a stirring magnetic bar, and stir continuously in a 60°C water bath for 15 minutes to obtain a hydrophobic, low-viscosity homogeneous liquid. After cooling, store the SDES extractant at room temperature.

[0086] (2) Sample pretreatment

[0087] The QuEChERS cleanup was performed by adding 10 mL of acetonitrile (containing 0.1% acetic acid), 5 g of anhydrous magnesium sulfate, and 1.5 g of sodium chloride to 10 g of uniform rotten orange peel sample (blank or spiked). After vortexing for 15 s and centrifuging, 1.5 g of anhydrous MgSO4, 25 mg of PSA, and 25 mg of C were added to the supernatant. 18 Centrifuge at 4000 rpm for 3 minutes. Then take the supernatant and dilute it to 10 mL with distilled water for subsequent extraction.

[0088] Take 90 μL of the prepared extractant and add it to the above solution. Add 600 μL of 5 mol / L NaOH solution under manual shaking. After the solution is allowed to stand for 30 seconds to fully mix, add 700 μL of 50% w / w citric acid solution to form an emulsion. Under the influence of a 90s ultrasonic water bath, the SDES droplets are evenly dispersed in various parts of the solution. After centrifugation at 5000 rpm for 3 minutes, collect the upper phase with a microsyringe and transfer it to a 1.5 mL centrifuge tube, dilute it 10 times with methanol and filter it into a sample injection bottle with a 0.45 μm organic filter membrane.

[0089] (3) Sample testing

[0090] The HPLC conditions were as follows: TFs were detected using an Agilent 1260 Infinity II HPLC system (Agilent Technologies, USA) equipped with a diode array detector (Agilent Technologies, USA). Chromatographic separation was performed on an Agilent Poroshekk 120EC-C18 column (150 mm × 4.6 mm, 4 μm) at a column temperature of 30 °C. The flow rate was 1.0 mL / min, the injection volume was 20 μL, and the detection wavelength was 220 nm. The mobile phase was a combination of methanol and water as follows: from 0 to 6 minutes, the methanol ratio was changed from 20% to 50%; from 6 to 26 minutes, the methanol ratio was changed from 50% to 70%, and then maintained until 32 minutes; from 32.0 minutes to 40 minutes, the methanol ratio was changed to 100%.

[0091] (4) Method evaluation

[0092] Under the optimal conditions, by considering the regression equation, linear range (LR), correlation coefficient (R 2 ), limit of detection (LOD), limit of quantification (LOQ), preconcentration factor (PF), and relative standard deviation (RSD) were used to evaluate the performance of the proposed SDES-HLLME method. Good linearity was obtained for all TFs in the range of 10-2000 ng / mL, and satisfactory R 2 The detection limit was in the range of 0.089-0.351 ng / mL, and the quantification limit was in the range of 0.297-1.172 ng / mL. The PFs of the eight target analytes obtained were in the range of 389-512 (after dilution with methanol). The precision of the method was verified by intra-day (n=6) and inter-day (n=6) analysis, with relative standard deviations ranging from 3.3%-7.9% and 2.1%-8.8%, respectively.

[0093] (5) Volatile component analysis

[0094] In this study, rotten orange peel samples were used as real samples to verify the applicability of the proposed method. TFs with known concentration levels (20, 50, 100 μg / kg) were added as matrix spiked samples to blank rotten orange peel samples to obtain the extraction recoveries (ERs) of eight TFs. Unspiked or spiked samples were pretreated under optimal conditions. The recoveries and actual concentrations before enrichment of spiked samples were calculated based on the calibration standards of target analytes prepared in methanol. Flusilazole and difenoconazole were detected in the unspiked real samples, and their concentrations were much lower than the maximum residue limits of TFs in fruit pulp in my country. The spiked recoveries of TFs in rotten orange peels by this method were 91.7%-110.9%, and the relative standard deviations were less than 8.5%.

Claims

1. A method for analyzing triazole fungicides in rotten peel waste by a pretreatment method based on a QuEChERS purification procedure and a pH-responsive switchable deep eutectic solvent microextraction combined with high performance liquid chromatography, characterized in that: The steps include: (1) Synthesis of Extractant The hydrogen bond acceptor and the hydrogen bond donor are mixed, stirred at 50-60° C. for 15-20 minutes, and then cooled to room temperature to obtain an extractant; The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:2 to 2:1; The hydrogen bond acceptor is menthol or thymol; The hydrogen bond donor is n-hexanoic acid, n-octanoic acid, n-dodecanoic acid or oleic acid; (2) Sample pretreatment The QuEChERS cleanup was performed by adding acetonitrile containing 0.1 wt% acetic acid, anhydrous magnesium sulfate A, and sodium chloride to a uniform rotten peel sample, vortexing and centrifuging, and then adding anhydrous magnesium sulfate B, PSA, and C to the supernatant. 18 , centrifuge, take the supernatant and dilute it with distilled water, then add the extractant prepared in step (1), add NaOH solution dropwise, mix thoroughly, add citric acid solution to form an emulsion, then ultrasonicate, centrifuge, collect the upper liquid phase with a microsyringe, dilute it with methanol, and filter it through an organic filter membrane to obtain a sample solution; (3) Sample testing The sample solution obtained in step (2) is analyzed using a high performance liquid chromatography instrument with a diode array detector to obtain the content of the triazole fungicide in the sample.

2. The method for analyzing triazole fungicides in rotten peel waste by a pretreatment method based on a QuEChERS purification procedure and a pH-responsive switchable deep eutectic solvent microextraction combined with a high performance liquid chromatography as claimed in claim 1, characterized in that: In step (1), the extractant is synthesized with thymol as a hydrogen bond acceptor and n-hexanoic acid as a hydrogen bond donor, and the molar ratio of thymol to n-hexanoic acid is 1:

1.

3. The method for analyzing triazole fungicides in rotten peel waste by a pretreatment method based on a QuEChERS purification procedure and a pH-responsive switchable deep eutectic solvent microextraction combined with a high performance liquid chromatography as claimed in claim 1, characterized in that: In step (2), the ratio of the sample to be tested to acetonitrile containing 0.1 wt % acetic acid, anhydrous magnesium sulfate A, and sodium chloride is 10 g: 10 mL: 5 g: 1.5 g.

4. The method for analyzing triazole fungicides in rotten peel waste by a pretreatment method based on a QuEChERS purification procedure and a pH-responsive switchable deep eutectic solvent microextraction combined with a high performance liquid chromatography as claimed in claim 1, characterized in that: In step (2), the ratio of the sample to be tested to anhydrous magnesium sulfate B, PSA, and C18 is 10 g: 1.5 g: 25 mg: 25 mg.

5. The method for analyzing triazole fungicides in rotten peel waste by a pretreatment method based on a QuEChERS purification procedure and a pH-responsive switchable deep eutectic solvent microextraction combined with a high performance liquid chromatography as claimed in claim 1, characterized in that: In step (2), the ratio of the sample to be tested to the extractant, the NaOH solution, and the citric acid solution is 10 g: 90 μL: 600 μL: 700 μL.

6. The method for analyzing triazole fungicides in rotten peel waste by a pretreatment method based on a QuEChERS purification procedure and a pH-responsive switchable deep eutectic solvent microextraction combined with a high performance liquid chromatography as claimed in claim 1, characterized in that: In step (2), the concentration of the NaOH solution is 5 mol / L.

7. The method for analyzing triazole fungicides in rotten peel waste by a pretreatment method based on a QuEChERS purification procedure and a pH-responsive switchable deep eutectic solvent microextraction combined with a high performance liquid chromatography as claimed in claim 1, characterized in that: In step (2), the concentration of the citric acid solution is 50 wt %.

8. The method for analyzing triazole fungicides in rotten peel waste by a pretreatment method based on a QuEChERS purification procedure and a pH-responsive switchable deep eutectic solvent microextraction combined with a high performance liquid chromatography as claimed in claim 1, characterized in that: In step (2), the ultrasonic time is 90s.

9. The method for analyzing triazole fungicides in rotten peel waste by a pretreatment method based on a QuEChERS purification procedure and a pH-responsive switchable deep eutectic solvent microextraction combined with a high performance liquid chromatography as claimed in claim 1, characterized in that: In step (3), the HPLC conditions are as follows: triazole fungicides are detected using an Agilent 1260 Infinity II HPLC system equipped with a diode array detector; chromatographic separation is performed on an Agilent Poroshekk 120EC-C18 column, and the column temperature is 30°C; the flow rate is 1.0 mL / min, the injection volume is 20 μL, and the detection wavelength is 220 nm; a combination of methanol and water is selected as the mobile phase, and the elution procedure is as follows: from 0 to 6 min, the methanol ratio changes from 20% to 50%; from 6 to 26 min, the methanol ratio changes from 50% to 70%, and then maintained to 32 min; from 32 to 40 min, the methanol ratio changes to 100%.

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