Method for separating and enriching abamectin drugs in soil
Through soil freeze-drying and acetonitrile water extraction combined with solid-phase extraction column activation technology, the detection problem of avermectin drugs in the soil is solved, efficient separation and enrichment and accurate detection are achieved, and the sensitivity and accuracy of the detection are improved.
Patent Information
- Application Number
- CN202411432853.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-14
- Publication Date
- 2025-01-10
AI Technical Summary
The prior art is difficult to effectively detect and separate the residues of avermectin drugs in soil, especially in the soil environment, where their detection methods are fewer and their sensitivity is insufficient, resulting in the neglect of potential hazards.
The soil was freeze-dried and ground, enriched using acetonitrile water extraction and PEP-2 solid phase extraction column, combined with ultrapure water and buffer, followed by ultrahigh performance liquid chromatography-triple quadratic rod tandem mass spectrometer (UPLC -MS/MS) carried out quantitative analysis to achieve the isolation and enrichment of avermectin drugs.
This method improves the enrichment efficiency and recovery rate of avermectin drugs in the soil. It is simple to operate, environmentally friendly, has high detection accuracy, and has a wide range of applications. It can detect multiple avermectin drugs and similar drugs at the same time. .
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Figure CN119291093A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of pre-treatment of trace organic pollutants in soil, and particularly relates to a method for separating and enriching avermectins in soil. Background Art
[0002] Avermectins (AVMs) are a class of structurally similar 16-membered macrolide compounds fermented by Streptomyces avermitilis. They are widely used in agriculture, medicine and veterinary medicine due to their very effective anthelmintic and insecticidal properties. In subsequent studies, many highly effective and low-toxic products were obtained by modifying and transforming ABM as the parent, such as Emamectin EAM, Eprinomectin EPR, Ivermectin IVM, Doramectin DOR and Moxidectin MOX.
[0003] However, the inadequate use of pesticides has led to the abuse of AVMs and the emergence of pesticide resistance, which has led to the continuous increase in the use of AVMs and the intensification of their harm. At the same time, many places have not included AVMs in the detection range, so the potential harm of AVMs has been ignored. In addition, during the use of pesticides, the dose directly acting on pests and diseases is less than 1% of the amount of pesticides applied, and more than 99% of pesticides flow into farmland soil, and even migrate to fields where pesticides are not used. Soil becomes the first receptor for pesticides. As a weakly leaching pesticide, AVMs are severely adsorbed on the soil surface, resulting in low pesticide utilization, thereby aggravating the pollution of AVMs pesticides. AVMs that enter the soil can be firmly adsorbed on various types of soil and are difficult to leach. Studies on the binding force between AVMs and soil have shown that AVMs are tightly bound to the soil and are non-flowing compounds.
[0004] At present, the main methods for residual detection of AVMs include liquid chromatography ultraviolet / fluorescence detection (HPLC-UV / FLD), enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Each detection method has its advantages and disadvantages. Due to the large molecular weight of AVMs, it is extremely difficult to vaporize and difficult to detect using gas chromatography. AVMs have a variety of interfering substances in the strong absorption spectrum range, so the liquid chromatography ultraviolet detection method is not sensitive enough when the amount to be detected is extremely low; the liquid chromatography fluorescence detection method has high sensitivity, high accuracy, and the detection limit meets the maximum residue limit requirements, but the sample needs to be derivatized; the enzyme-linked immunosorbent assay can meet the drug residue detection, but the detection has a certain degree of blindness. Most of the kits on the market in my country are imported from abroad, which limits the application of the enzyme-linked immunosorbent assay; the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) is characterized by high sensitivity, separation, strong qualitative and quantitative capabilities, and combines the advantages of chromatographic separation and mass spectrometry structure confirmation. Residue analysis methods in agricultural and livestock products are often reported in China, but there are fewer reports on the existence and detection of AVMs in soil environments, and most of them focus on avermectins in AVMs. There are even fewer reports on detection methods of other drugs in AVMs in soil. Summary of the invention
[0005] The purpose of the invention is to provide a method for separating and enriching avermectins in soil.
[0006] The specific steps are:
[0007] 1. A method for separating and enriching avermectins in soil, characterized in that the specific steps are:
[0008] (1) Place the soil in a freeze dryer for freeze drying, grind it through a 100-mesh sieve, weigh the sieved sample and place it in a centrifuge tube, extract it with an acetonitrile aqueous solution with a volume percentage concentration of 75%, repeat the above operation and combine the two extracts; the obtained extract is diluted to 500 mL with ultrapure water; the pH of the extract is adjusted to 7.00±0.05 with a buffer solution to obtain a pretreated soil sample.
[0009] (2) Activate the PEP-2 solid phase extraction column with n-heptane solution, acetone solution, acetonitrile solution and ultrapure water with a pH value of 7 in turn, so that the resin in the PEP-2 solid phase extraction column is in an activated state. After passing through the membrane, the sample is enriched by passing through the column at a flow rate of 3 mL / min; after the sample is loaded, the PEP-2 solid phase extraction column is rinsed with ultrapure water to remove the water-soluble interfering impurities enriched on the PEP-2 solid phase extraction column, dried in a vacuum freeze dryer, and then eluted with acetonitrile solution and acetone solution in turn. Finally, the collected eluate is slowly blown to 200 µL under a nitrogen flow, and the resulting residue is re-dissolved in acetonitrile solution, filtered through the membrane and placed in a brown injection bottle, thus completing the separation and enrichment of avermectins in soil;
[0010] The avermectin drugs are one or more of avermectin (ABM), ivermectin (IVM), epramectin (EPR), doramectin (DOR), emamectin benzoate (EAM), and moxidectin (MOX).
[0011] The buffer used in step (1) is preferably ammonium acetate solution.
[0012] In the step (2), the filtration preferably uses a 0.22 µm glass fiber filter membrane.
[0013] The pH value of the ultrapure water used for activation in step (2) is preferably adjusted with ammonium acetate solution.
[0014] The beneficial effects of the present invention are:
[0015] 1) The reagents used in this method for soil treatment are low-toxic, small in amount, and environmentally friendly.
[0016] 2) This method has a high soil enrichment efficiency, with a recovery rate between 50.93% and 103.45%.
[0017] 3) This method is simple to operate and has strong repeatability. The relative standard deviation (RSD) of the same batch of samples is less than 15.72%, which meets the requirements of pretreatment.
[0018] 4) This method can simultaneously enrich multiple types of drugs in the same class and other drugs with physical and chemical properties similar to avermectins. It has a wide range of applications, takes less time to use instrument detection, has a low detection limit, high accuracy, and high sample detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1This is the ion flow diagram of the 200 μg / L mixed standard solution of 6 avermectins in Example 1 of the present invention. Among them, a is emamectin benzoate (EAM): 4.810 min; b is epromectin (EPR): 6.32 min; c is doramectin (DOR): 6.33 min; d is avermectin (ABM): 6.74 min; e is moxidectin (MOX): 6.77 min; f is ivermectin (IVM): 7.82 min.
[0020] Figure 2 This is the quantitative ion standard curve of moxidectin (MOX) in Example 1 of the present invention.
[0021] Figure 3 This is the quantitative ion standard curve of emamectin benzoate (EAM) in Example 1 of the present invention.
[0022] Figure 4 This is the quantitative ion standard curve of Avermectin (ABM) in Example 1 of the present invention.
[0023] Figure 5 This is the ivermectin (IVM) quantitative ion standard curve in Example 1 of the present invention.
[0024] Figure 6 This is the quantitative ion standard curve of epremectin (EPR) in Example 1 of the present invention.
[0025] Figure 7 This is the quantitative ion standard curve of doramectin (DOR) in Example 1 of the present invention. DETAILED DESCRIPTION
[0026] (1) Freeze-dry the soil in a freeze dryer, grind it through a 100-mesh sieve, weigh the sieved sample and place it in an extraction cell, add 75% acetonitrile aqueous solution for extraction, vortex for 1 min, centrifuge at 4000 r / min for 10 min, transfer the supernatant to a 500-mL volumetric flask, and repeat the operation twice; add ultrapure water to the 500-mL mark and adjust the pH of the extract to 7.00±0.05 with ammonium acetate buffer to obtain a pretreated soil sample.
[0027] (2) The PEP-2 solid phase extraction column was activated with n-heptane solution, acetone solution, acetonitrile solution and ultrapure water with a pH value of 7, so that the resin in the PEP-2 solid phase extraction column was in an activated state. After the sample was taken through the membrane, it was enriched at a flow rate of 3.0 mL / min. After the sample was loaded, the PEP-2 solid phase extraction column was removed and rinsed with ultrapure water to remove the water-soluble interfering impurities enriched on the PEP-2 solid phase extraction column. Then, it was eluted with acetonitrile solution and acetone solution in turn. Finally, the collected eluate was slowly blown to 200 µL under nitrogen flow, and the obtained residue was re-dissolved in acetonitrile solution, filtered through the membrane and placed in a brown injection bottle, thus completing the separation and enrichment of avermectins in soil.
[0028] The avermectin drugs are one of moxidectin (MOX), avermectin benzoate (EAM), abamectin (ABM), ivermectin (IVM), epromectin (EPR), and doramectin (DOR).
[0029] In order to meet the concentration range of antibiotic residues in soil, this example uses ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS / MS) to quantify the above six avermectins, with a calibration range of 1-400 µg / L.
[0030] Drawing of the standard curve of the external standard method: Six 100 mg / L single-standard stock solutions of avermectins (FirstStandard ® ) Dilute the stock solution with methanol to a series of standard mixed solutions, transfer them to injection bottles, and measure them using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS / MS) under the above conditions. Plot the concentration as the abscissa and the peak area as the ordinate to obtain a standard curve for determining the amount of the analyte in the sample.
[0031] After the avermectins in the soil of this embodiment were separated and enriched, they were detected by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS / MS), substituted into the standard curve equation, and finally the concentrations of the six avermectins in the water sample to be tested were measured by calculation. The recovery rate was calculated using the following formula:
[0032]
[0033] R-recovery rate, %.
[0034] C 1 - Concentration of avermectins in soil spiked with standard solution, µg / L.
[0035] C S -Concentration of standard substance added to blank soil, µg / L.
[0036] V 1 - Volume of the spiked soil solution after concentration, mL.
[0037] Vs - volume of standard solution added to blank soil, mL.
[0038] m 1 -Weigh the mass of spiked soil sample, g.
[0039] m s -Mass of blank soil sample, g.
[0040] During the experiment of this embodiment, polytetrafluoroethylene sealed bags were used to hold the soil, which was frozen and stored at low temperature. After the sample collection was completed, it was immediately transported back to the laboratory.
[0041] The following is an explanation of the method for detecting the concentrations of six avermectins in soil according to the present invention in conjunction with the accompanying drawings and examples.
[0042] Embodiment 1:
[0043] (1) The samples were collected from farmland soil. During the collection process, impurities such as leaves and stones were removed and placed in polytetrafluoroethylene sealed bags. They were frozen and stored at low temperatures and immediately transported back to the laboratory after the sample collection was completed. The samples were placed in a freeze dryer for freeze drying and ground through a 100-mesh sieve.
[0044] (2) Weigh 5.000±0.005 g of the sieved sample in two portions and place them into 50 mL centrifuge tubes. Select 15 mL of acetonitrile-water (V / V, 3:1) solution as the extractant. Vortex the centrifuge tube for 1 min, centrifuge the sample at high speed (4000 r / min) for 10 min, transfer the supernatant to a 500 mL volumetric flask, add 10 mL of extraction solution to the centrifuge tube, and repeat the above steps.
[0045] (3) Place the extract in a 500 mL volumetric flask, add ultrapure water to 500 mL, adjust the pH to 7.00±0.05 with ammonium acetate, and filter through a 0.45 µm filter before performing solid phase extraction.
[0046] The PEP-2 solid phase extraction column was selected as the extraction column. Before the sample was loaded onto the column, the extraction column was activated with 3.0 mL of n-heptane, 2.0 mL of acetone, 3.0 mL of acetonitrile and 3.0 mL of ultrapure water at pH=7.00. After the sample was passed through the membrane, the sample was passed through the column at a flow rate of 3.0 mL / min. After the sample was loaded, the PEP-2 column was rinsed with 10.0 mL of ultrapure water, and then the PEP-2 column was eluted with 4.0 mL of acetonitrile and 4.0 mL of acetone in sequence. Finally, the collected eluate was blown to 200 µL under gentle nitrogen, and the acetonitrile solution was added to make it 1 mL. After passing through a 0.22 µm filter membrane, it was placed in a brown injection bottle and waited for determination on the machine.
[0047] (4) The concentrations of six avermectins were determined using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS / MS). A total of five avermectins were detected in the surface soil of 26 sampling points in farmland in Yanshan District, Guilin City to varying degrees. The results are shown in Table 1.
[0048] Figure 1 This is the ion flow diagram of the 200 µg / L mixed standard solution of six avermectins in this example. The peak order and retention time are avermectin benzoate (EAM): 4.810 min; epramine (EPR): 6.32 min; doramectin (DOR): 6.33 min; avermectin (ABM): 6.74 min; moxidectin (MOX): 6.77 min; ivermectin (IVM): 7.82 min.
[0049] Drawing of standard curve by external standard method: dilute the stock solution with methanol solution into 6 kinds of avermectin drug standard mixed solutions with concentrations of 1 µg / L, 2 µg / L, 5 µg / L, 10 µg / L, 20 µg / L, 50 µg / L, 100 µg / L, 200 µg / L, and 400 µg / L, transfer them into injection bottles and measure them with ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS / MS). Plot the concentration as the abscissa and the peak area as the ordinate to obtain the standard curve.
[0050] Figures 2 to 7 This is the standard curve for the quantitative ions of six avermectins.
[0051] Embodiment 2:
[0052] (1) Blank soil samples were taken from soil far away from densely populated areas and were pretreated in the same way as the samples. Six AVMs were spiked into the blank soil samples at 20, 50, and 100 ng / g, respectively. Three replicates were set for each spike level (n=3). The spike recovery and relative standard deviation (RSD) are shown in Table III.
[0053] (2) The blank soil sample was pretreated according to the method in Example 1, and then detected by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS / MS). The determination was repeated 8 times (n=8), and the standard deviation (SD) of the determination results was calculated. The method detection limit (LOD) was 3SD, and the quantification limit (LOQ) was 4 times the LOD. The linear equation, linear range, correlation coefficient, detection limit and quantification limit of the six avermectin pesticides are shown in Table 2.
[0054] Table 1 Detection concentrations of avermectins in farmland soil in Yanshan District, Guilin
[0055]
[0056] ND: means not detected.
[0057] Table 2 Linear equation, linear range, correlation coefficient, detection limit and quantification limit of 6 avermectins
[0058]
[0059] Table 3 Blank soil spike recovery test results
[0060]
Claims
1. A method for separating and enriching avermectins antibiotics in soil, characterized in that: The specific steps are: (1) Place the soil in a freeze dryer for freeze drying, grind it through a 100-mesh sieve, weigh the sieved sample and place it in a centrifuge tube, extract it with a 75% by volume acetonitrile aqueous solution, vortex for 1 min, and centrifuge it at 4000 r / min for 10 min to obtain an extract. Repeat the above operation and combine the two extracts; add ultrapure water to the obtained extract to 500 mL; adjust the pH of the extract to 7.00±0.05 with a buffer solution to obtain a pretreated soil sample; (2) The PEP-2 solid phase extraction column was activated with n-heptane solution, acetone solution, acetonitrile solution and ultrapure water with a pH value of 7 in sequence, so that the PEP-2 solid phase extraction column was in an activated state. After the sample was taken through the membrane, it was enriched by passing through the column at a flow rate of 3 mL / min. After the sample was loaded, the PEP-2 solid phase extraction column was rinsed with ultrapure water to remove the water-soluble interfering impurities enriched on the PEP-2 solid phase extraction column, dried under vacuum freezing, and then eluted with acetonitrile solution and acetone solution in sequence. Finally, the collected eluate was slowly blown to 200 μL under a nitrogen flow, and the obtained residue was re-dissolved in acetonitrile solution, filtered through the membrane and placed in a brown injection bottle, thus completing the separation and enrichment of avermectins in soil. The avermectin drugs are one or more of avermectin (ABM), ivermectin (IVM), epramectin (EPR), doramectin (DOR), emamectin benzoate (EAM), and moxidectin (MOX).
2. The method for separating and enriching avermectins in soil as claimed in claim 1, characterized in that The buffer used in step (1) is ammonium acetate.
3. The method for separating and enriching avermectins in soil as claimed in claim 1, characterized in that In step (2), a 0.45 µm glass fiber filter is used for filtration.
4. The method for separating and enriching avermectins in soil as claimed in claim 1, characterized in that The pH value of the ultrapure water used for activation in step (2) is adjusted with 5 mol / L ammonium acetate.