Method for separating and enriching abamectin drugs in water environment
By using PEP-2 solid-phase extraction column and UPLC-MS/MS detection technology, the problem of separating and enriching avermectin drugs in aquatic environments has been solved, achieving efficient, low-cost, and low-toxicity separation and enrichment effects, and is applicable to the detection of a variety of avermectin drugs.
Patent Information
- Application Number
- CN202411432855.1
- 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
Existing technologies are difficult to efficiently and cost-effectively separate and enrich avermectin drugs from the aquatic environment, and conventional methods suffer from problems such as complex operation, high cost, and low sensitivity.
The PEP-2 solid-phase extraction column was used for activation and elution with ultrapure water, methanol and acetone solutions, and the samples were detected by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS/MS). The operation steps were simplified and the enrichment efficiency was improved by adjusting the pH of the water sample and pretreating it with a low-toxicity buffer.
It achieves efficient, low-toxicity, and low-cost separation and enrichment of avermectin drugs, with recoveries ranging from 64.99% to 110.02%, and relative standard deviations of less than 13.50% for the same batch of samples. It has a wide range of applications, high detection accuracy, and low detection limits.
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Figure CN119291094A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of pre-detection treatment of trace organic pollutants in water environments, and particularly relates to a method for separating and enriching avermectins in water environments. Background Art
[0002] Avermectins (AVMs) are a class of structurally similar 16-membered macrolide compounds fermented by Streptomyces avermitilis. They are the most widely used and largest-volume broad-spectrum insecticides, mainly used as agricultural or veterinary insecticides and acaricides. Although avermectin is considered a biological pesticide, it is considered a highly toxic compound according to the World Health Organization's classification standards. Avermectin is toxic to the nervous system and growth system, and is easily residual in animals. Long-term consumption can cause disorders of the human nervous system and reproductive system.
[0003] Abamectins have been shown to pose a threat to the aquatic environment and can affect the reproduction, biological function and survival of non-target aquatic and terrestrial organisms. When AVMs are used as pesticides, they may be washed into the water by rainwater; after use as veterinary drugs, they are rarely metabolized in animals, and most of them are excreted in their original form through feces, which are then washed into the water by rainwater. When animal feces are used as aquatic animal feed or water fertilizer, AVMs in the feces will directly enter the water body. AVMs entering the water can cause serious toxicity to some aquatic organisms. Almost every country has regulations on the maximum residue limit (MRL) of avermectins in food. The European Union (EU), the United States (US), the United Nations Codex Alimentarius Commission (CAC) and China have all established maximum residue limits for avermectins in mammals, of which the EU is 15 μg / kg, the United States is 20 μg / kg, and the United Nations Codex Alimentarius Commission and many countries have a residue limit of 10 μg / kg for this compound.
[0004] Avermectins have a large relative molecular weight and are difficult to vaporize, so there is no feasible gas chromatography analysis method. At present, the main detection methods include enzyme-linked immunosorbent assay (ELISA), liquid chromatography (HPLC), etc. ELISA has the lowest detection limit and can meet the requirements of AVMs residue detection. However, the blindness of immunoassay detection and the dependence of antibodies on foreign imports limit the widespread application of ELISA. HPLC technology is a commonly used drug analysis technology for AVMs, among which the HPLC-DAD detection method has low sensitivity and cannot accurately detect residues. HPLC-FLD optical detection is currently the most widely used AVMs residue detection method in China, but the sample pretreatment is complicated, derivatization is required, and the cost is high. UPLC-MS / MS method has been reported more at home and abroad. This method combines the high speed and high separation of ultra-high performance liquid chromatography with the high selectivity and high sensitivity of mass spectrometry. It does not require sample derivatization, can simultaneously measure multiple targets, and has the advantages of high selectivity, high sensitivity, and high throughput. Summary of the invention
[0005] The purpose of the present invention is to provide a method for separating avermectins in an enriched water environment.
[0006] The specific steps are:
[0007] (1) Use a buffer to adjust the pH value of the water sample containing avermectins to 7.0±0.05, filter it, and add 4% methanol extract to obtain the pretreated water sample.
[0008] (2) The PEP-2 solid phase extraction column is activated with n-heptane solution, acetone solution, methanol solution and ultrapure water with a pH value of 7 in sequence, so that the resin in the PEP-2 solid phase extraction column is in an activated state. The pretreated water sample obtained in step (1) is taken to pass through the PEP-2 solid phase extraction column at a flow rate of 3-5 mL / min for enrichment. After the enrichment is completed, 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. The extraction column is then freeze-dried in vacuum and then eluted with methanol solution and acetone solution in sequence. The eluent flow rate is less than or equal to 1.0 mL / min. The obtained eluent is slowly blown to 200 µL under a nitrogen flow. The obtained residue is added to an acetonitrile solution for re-dissolution, thereby completing the separation and enrichment of avermectins in an aqueous environment.
[0009] The avermectin drugs are one or more of avermectin (ABM), ivermectin (IVM), epramectin (EPR), doramectin (DOR), emamectin benzoate (EAM), and moxidectin (MOX).
[0010] The buffer used in step (1) is ammonium acetate.
[0011] In the step (1), the filtration is preferably performed using a 0.45 µm glass fiber filter.
[0012] The pH value of the ultrapure water used for activation in step (2) is adjusted by using ammonium acetate.
[0013] The beneficial effects of the present invention are:
[0014] 1) The reagents used in the water sample treatment process of this method are low-toxic, used in small amounts, and are environmentally friendly.
[0015] 2) This method has a high enrichment efficiency for water samples, with a recovery rate between 64.99% and 110.02%.
[0016] 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 13.50%, which meets the requirements of pretreatment.
[0017] 4) This method can simultaneously enrich multiple drugs of the same class of antibiotics 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
[0018] Figure 1 This 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): 4.78 min; b is epromectin (EPR): 6.09 min; c is doramectin (DOR): 6.10 min; d is avermectin (ABM): 6.48 min; e is moxidectin (MOX): 6.80 min; f is ivermectin (IVM): 7.36 min.
[0019] Figure 2 This is the quantitative ion standard curve of emamectin benzoate (EAM) in Example 1 of the present invention.
[0020] Figure 3 This is the quantitative ion standard curve of epremectin (EPR) in Example 1 of the present invention.
[0021] Figure 4 This is the quantitative ion standard curve of doramectin (DOR) in Example 1 of the present invention.
[0022] Figure 5 This is the quantitative ion standard curve of Avermectin (ABM) in Example 1 of the present invention.
[0023] Figure 6 This is the quantitative ion standard curve of moxidectin (MOX) in Example 1 of the present invention.
[0024] Figure 7 This is the ivermectin (IVM) quantitative ion standard curve in Example 1 of the present invention. DETAILED DESCRIPTION
[0025] (1) The pH value of the water sample containing avermectins was adjusted to 7.0±0.5 with 5 mol / L ammonium acetate, filtered using a GF / F (Whatman, 0.45 µm) glass fiber filter, and 4% methanol extract was added to obtain the pretreated water sample.
[0026] (2) The PEP-2 (Agela, 60 mg, 3 mL) solid phase extraction column was activated with 3 mL of n-heptane solution, 2 mL of acetone solution, 3 mL of methanol solution and 3 mL of ultrapure water with a pH value of 7, so that the polymer material in the PEP-2 solid phase extraction column was in an activated state. 250 mL of the pretreated water sample prepared in step (1) was taken and enriched through the PEP-2 solid phase extraction column at a flow rate of 3 mL / min. After the enrichment was completed, the PEP-2 solid phase extraction column was rinsed with 10 mL of ultrapure water to remove the water-soluble interfering impurities enriched on the PEP-2 solid phase extraction column. The PEP-2 extraction column was then freeze-dried in vacuum and eluted with 4 mL of methanol solution and 4 mL of acetone solution in sequence at a flow rate of 1.0 mL / min. The eluate was collected in a 10 mL conical bottom glass centrifuge tube and the obtained eluate was slowly blown to 200 μL under a nitrogen flow. The obtained residue was added with 800 mL of ethanol. The solution was re-dissolved in 1 μL acetonitrile solution and vortex mixed for about 1 min to complete the separation and enrichment of avermectins in water environment.
[0027] The avermectin drug is one of avermectin (ABM), ivermectin (IVM), epramectin (EPR), doramectin (DOR), emamectin benzoate (EAM), and moxidectin (MOX).
[0028] In order to meet the concentration range of antibiotic residues in water environment, this embodiment uses ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS / MS) to quantify the above 6 avermectins, with a calibration range of 1~400μg / L.
[0029] 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.
[0030] After the avermectins in the water environment of this embodiment are separated and enriched, they are 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 are measured by calculation. The recovery rate is calculated using the following formula:
[0031]
[0032] R-recovery rate, %.
[0033] C2- Concentration of avermectins in water sample spiked with standard solution, µg / L.
[0034] C1- Concentration of avermectins in water sample without adding standard solution, µg / L.
[0035] C S -Concentration of added standard solution, µg / L.
[0036] V2- Volume of water sample added with standard solution, L.
[0037] V1-volume of water sample without adding standard solution, L;
[0038] Vs- volume of standard solution added, L.
[0039] In the experiment of this embodiment, brown glassware is used to hold water samples. Colorless glassware can be placed with aluminum foil on the surface of the vessel. The samples should be protected from direct sunlight during the experiment. The glassware needs to be washed several times with organic solvents, tap water, and ultrapure water before use.
[0040] The following is an explanation of the method for detecting the concentrations of six avermectins in a water environment according to the present invention in conjunction with the accompanying drawings and examples.
[0041] Embodiment 1:
[0042] (1) Take 250 mL of ultrapure water in a brown glass bottle, add 0.05 mL of 1 mg / L mixed standard working solution of six avermectins to each bottle of water sample, adjust the water sample to pH = 7.0 with a buffer, and filter it using a GF / F (Whatman, 0.45 µm) glass fiber filter to obtain the pretreated water sample.
[0043] (2) Activate the PEP-2 (Agela, 60 mg, 3 mL) solid phase extraction column with 3 mL of n-heptane solution, 2 mL of acetone solution, 3 mL of methanol solution and 3 mL of ultrapure water at pH 7 in sequence to activate the polymer material in the solid phase extraction column. Take 250 mL of water sample and pass it through the column at a flow rate of 3-5 mL / min. After the enrichment is completed, rinse the solid phase extraction column with 10 mL of ultrapure water to remove the water-soluble interfering impurities enriched on the PEP-2 solid phase extraction column. Then, freeze-dry the PEP-2 solid phase extraction column in vacuum. Finally, elute the solid phase extraction column with 4 mL of methanol solution and 4 mL of acetone solution in sequence at a flow rate of 1 mL / min. Collect the eluate in a 10 mL conical bottom glass centrifuge tube. Slowly blow the eluate to 200 µL under nitrogen flow. Add acetonitrile solution to dissolve the residue. Vortex mix for about 1 min and test.
[0044] (3) The concentrations of the six avermectins were determined using ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS / MS). The recovery rates were calculated and the results are shown in Table 1.
[0045] 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 as follows: avermectin benzoate (EAM): 4.78 min; epramectin (EPR): 6.09 min; doramectin (DOR): 6.10 min; avermectin (ABM): 6.48 min; moxidectin (MOX): 6.80 min; ivermectin (IVM): 7.36 min.
[0046] 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.
[0047] Figures 2 to 7 This is the standard curve for the quantitative ions of six avermectins.
[0048] Embodiment 2:
[0049] Take 250 mL of water sample from Yanshan District of Guilin City in a brown bottle and add the spiked water sample according to the method in Example 1. At the same time, take 250 mL of water sample without adding the spiked water sample as a blank control.
[0050] Substitute the spiked water sample results into the standard curve equation, and finally calculate the concentrations of the six avermectins in the water sample to be tested. Calculate their recovery rates, and the results are shown in Table 2.
[0051] Embodiment 3:
[0052] 250 mL of river and pond water samples from Yanshan District, Guilin were collected in brown glass bottles, 5 mol / L ammonium acetate was added to adjust the water sample to pH=7, and the water sample was stored below 4°C and transported back to the laboratory. The water 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), and the concentrations of the six avermectins in the actual water sample were finally obtained by substituting them into the standard curve equation and calculating. The recovery rate was calculated, and the results are shown in Table 3.
[0053] Table 1 Ultrapure water spike recovery test results
[0054]
[0055] Table 2 Recovery results of spiked pond water samples in Yanshan District, Guilin
[0056]
[0057] Table 3 Test results of water samples in Yanshan District, Guilin
[0058]
[0059] ND: means not detected.
Claims
1. A method for separating avermectins from an enriched water environment, characterized in that: The specific steps are: (1) The pH value of the water sample containing avermectins was adjusted to 7 ± 0.05 with a buffer, filtered, and a 4% (V:V) methanol extract was added to obtain a pretreated water sample; (2) Activate the PEP-2 solid phase extraction column with n-heptane, acetone, methanol and ultrapure water with a pH value of 7 in sequence, so that the polymer material in the PEP-2 solid phase extraction column is in an activated state, take the pretreated water sample obtained in step (1) and pass it through the PEP-2 solid phase extraction column at a flow rate of 3-5 mL / min for enrichment, after the enrichment is completed, rinse the PEP-2 solid phase extraction column with ultrapure water to remove the water-soluble interfering impurities enriched on the PEP-2 solid phase extraction column, then vacuum freeze-dry the PEP-2, and then elute with methanol solution and acetone solution in sequence, the eluent flow rate is less than or equal to 1.0 mL / min, and the obtained eluent is slowly blown to 200 μL under nitrogen flow, and the obtained residue is added to acetonitrile solution for re-dissolution, and the filter membrane is placed in a brown injection bottle, so as to complete the separation and enrichment of avermectins in the water environment; 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 avermectins in an enriched water environment according to claim 1, characterized in that The buffer used in step (1) is ammonium acetate.
3. The method for separating avermectins in an enriched water environment according to claim 1, characterized in that In step (1), a 0.45 µm glass fiber filter is used for filtration.
4. The method for separating avermectins in an enriched water environment according to claim 1, characterized in that The pH value of the ultrapure water used for activation in step (2) is adjusted with ammonium acetate.