Synchronous determination method for triazole pesticides in feed

Abstract

The invention relates to a synchronous determination method for triazole pesticides in a feed. The method comprises the following steps: S1, preparing a matrix matching series of standard working solutions; s2, extracting a to-be-detected feed sample with acetonitrile to obtain a to-be-purified solution; s3, enabling the to-be-purified solution to completely pass through a Captiva EMR-Lipid solid-phase extraction column, collecting all effluent, fixing the volume by using acetonitrile, uniformly mixing, and enabling the mixture to pass through a microfiltration membrane, so as to obtain a to-be-detected sample solution; and S4, carrying out high performance liquid chromatography-tandem mass spectrometry determination on the matrix matching series standard working solution and the to-be-detected sample solution, and quantifying each to-be-detected medicine by adopting an external standard method. The method provided by the invention is good in extraction and purification effect and high in sensitivity, can realize accurate qualitative and quantitative determination of chlorazophos, tebuconazole, tebuconazole, diniconazole, iminazole, flusilazole and tetraconazole residues in a feed sample with relatively high extraction efficiency and relatively low time cost and economic cost, and can provide technical support for feed quality safety detection.

Description

Technical Field

[0001] The invention relates to a method for determining pesticide residues in feed, and in particular to a method for synchronously determining triazole pesticides in feed. Background Art

[0002] Feed is the most important input in livestock and poultry farming, and feed quality and safety are the premise and fundamental guarantee for animal food safety. The main sources of feed in livestock and poultry farming are vegetables, fruits, crops, etc., and triazole pesticides, as a class of highly effective and broad-spectrum fungicides, are widely used in the prevention and control of pests and diseases in the cultivation of vegetables, fruits, rice and other crops. This makes it easy for triazole pesticides to remain in vegetables, fruits, crops, soil and water bodies, which not only brings serious environmental pollution problems, but also can be transmitted through the food chain, environmental accumulation and contaminated feed, etc., enriched in animal tissues, causing food quality and safety hazards, and posing a threat to consumer health. Moreover, GB 2763-2019 "National Food Safety Standard Maximum Limits of Pesticide Residues in Food" clearly stipulates the residue limits of some triazole pesticides. In order to control the residues of this type of pesticides, it is necessary to monitor the entire chain from drinking water, soil, feed, livestock products and other links.

[0003] However, in the prior art, the determination of triazole pesticide residues is mainly focused on water, soil, and plant samples such as vegetables and fruits. In the process, the sample purification is mostly carried out by solid phase extraction column purification, and the operation steps are cumbersome, time-consuming and laborious. In the prior art, there is a lack of methods for determining triazole pesticide residues in feed matrix samples, and there are few literatures discussing this.

[0004] Therefore, establishing a simple, rapid and accurate method for determining various triazole pesticides in feed is of great significance for controlling the risk of pesticide residues in animal food from the breeding stage and ensuring the safety of animal food. Summary of the invention

[0005] In view of this, the present invention provides a simultaneous determination method of triazole pesticides in feed, which uses a CaptivaEMR-Lipid solid phase extraction column to purify the extract of the feed sample, and establishes an analysis method for 7 triazole pesticides in feed, which can provide technical support for feed quality and safety testing, and has important research significance and practical application value.

[0006] The invention discloses a synchronous determination method of triazole pesticides in feed, wherein the triazole pesticides are chlorfenapyr, tebuconazole, fenthion, diniconazole, imipenem, flusilazole and flufenac. The method comprises the following steps:

[0007] S1, prepare a matrix-matched series of standard working solutions based on the corresponding standards of each drug to be tested;

[0008] S2, extracting the feed sample to be tested with acetonitrile to obtain a solution to be purified;

[0009] S3, passing the solution to be purified through a Captiva EMR-Lipid solid phase extraction column, collecting all the effluent, making up the volume with acetonitrile, mixing and passing through a microporous filter membrane to obtain a sample solution to be tested;

[0010] S4, subjecting the matrix-matching series standard working solutions and the test sample solutions to high performance liquid chromatography-tandem mass spectrometry, plotting a working curve with the concentration of the matrix-matching series standard working solutions as the abscissa and the measured chromatographic peak area of ​​each test drug as the ordinate, and quantifying each test drug using the external standard method.

[0011] In the method disclosed in the present invention, since a Captiva EMR-Lipid solid phase extraction column is used for purification, not only lipid impurities can be removed with high selectivity and efficiency, but also the loss of analytes can be reduced to the greatest extent, thereby significantly improving the reliability and durability of the method of the present invention. At the same time, it does not require operations such as activation and elution, that is, a high recovery rate can be obtained without the steps of activation and balancing, thereby simplifying the sample pretreatment process.

[0012] The present invention adopts a flow-through solid phase extraction column Captiva EMR-Lipid combined with high performance liquid chromatography-tandem mass spectrometry to establish a rapid detection method that can accurately characterize and quantify the residues of chlorfenapyr, tebuconazole, chlorfenapyr, diniconazole, imipenem, flusilazole and fluconazole in feed, providing a new idea for controlling the risk of pesticide residues in animal food and ensuring the safety of animal food in the breeding process.

[0013] In the method disclosed in the present invention, in step S4, the chromatographic column of the high performance liquid chromatography-tandem mass spectrometry is C 18 The column has a mobile phase system of mobile phase A and mobile phase B, wherein mobile phase A is water, mobile phase B is methanol, and gradient elution is adopted. In the present invention, since water and methanol are used as mobile phases, the response values ​​of seven triazole pesticides in feed, including chlorfenapyr, tebuconazole, chlorfenapyr, diniconazole, imipenem, flusilazole and flufenazone, are all high, and good separation can be achieved between the chromatographic peaks, and the peak shape has good symmetry, which satisfies the detection requirements very well.

[0014] In the method disclosed in the present invention, in the high performance liquid chromatography, the flow rate of the mobile phase system is 0.1-0.5 mL / min, and the gradient elution program of the mobile phase system is:

[0015] 0.0~1.0min, the volume percentage of the mobile phase A is 30%;

[0016] 1.0-5.0min, the volume percentage of the mobile phase A is uniformly reduced from 30% to 5%;

[0017] 5.0~9.0min, the volume percentage of the mobile phase A is 5%;

[0018] 9.0-9.1min, the volume percentage of the mobile phase A is increased uniformly from 5% to 30%;

[0019] 9.1-12.0 min, the volume percentage of the mobile phase A is 30%.

[0020] The initial methanol ratio is set to 70%, which appropriately increases the organic phase ratio in the mobile phase to shorten the sample detection time and solve the problem of late peak time due to the small polarity of the seven triazole drugs; 1-5min, the methanol ratio gradually increases, which can make each test object flow out from the chromatographic column in turn; when 5-9min, the methanol ratio is 95%, and the non-polar impurities in the sample can flow out as much as possible with the help of a high proportion of organic phase, avoiding accumulation in the chromatographic column and contaminating the chromatographic column; and when 9-12min, it returns to the initial ratio, and after 3min of equilibrium, the next injection of the sample can be made. Based on the entire gradient elution program set by the present invention, it is possible to ensure that the seven drugs of chlorfenapyr, tebuconazole, chlorfenapyr, diniconazole, imipenem, flusilazole and fluconazole peak at the right time and obtain a good peak shape, while avoiding the accumulation of other impurities in the chromatographic column, ensuring that good repeatability can still be obtained when repeatedly injecting samples.

[0021] In the method disclosed in the present invention, the measurement conditions of mass spectrometry in high performance liquid chromatography-tandem mass spectrometry are as follows: the ion source is an electrospray ion source, the monitoring mode is multiple reaction monitoring, the scanning mode is positive ion scanning, the electrospray voltage is 5.5 Kv, the desolvation temperature is 550°C, the curtain gas is 35 psi, the spray gas is 50 psi, and the collision gas is 9 psi.

[0022] In the method disclosed by the present invention, in the step S1, an appropriate amount of seven pesticide standards, namely, chlorfenapyr, tebuconazole, chlorfenapyr, diniconazole, imipenem, flusilazole and fluconazole, are weighed, diluted to a fixed volume with acetonitrile to form a mixed standard solution of 1 μg / mL, and then diluted with a blank matrix solution to form a matrix-matched series standard working solutions with concentrations of 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL and 100 ng / mL, respectively.

[0023] In the method disclosed in the present invention, in step S2, it includes:

[0024] Step S21, taking 1-5 g of the feed sample to be tested, placing it in a 50 mL centrifuge tube, adding 5-15 mL of acetonitrile, ultrasonically extracting for 15 min, centrifuging at 4000 r / min for 3-5 min, and taking the supernatant into another centrifuge tube;

[0025] Step S22, the lower layer residue is extracted again with 5-15 mL of acetonitrile, and the supernatant is taken;

[0026] Step S23, combining the supernatants obtained in step S21 and step S22, vortexing, adding anhydrous magnesium sulfate and anhydrous sodium acetate, vortexing and shaking, to obtain a final supernatant;

[0027] Step S24, blowing the supernatant finally obtained to about 2-5 mL with nitrogen in a 40° C. water bath, adding water to make the volume to 5 mL, and mixing to obtain a solution to be purified.

[0028] Among them, preferably, in step S21, 5 g of the feed sample to be tested is taken, placed in a 50 mL centrifuge tube, 10 mL of acetonitrile is added, ultrasonic extraction is performed for 15 min, centrifuged at 4000 r / min for 5 min, and the supernatant is taken into another centrifuge tube; in step S22, the lower layer residue is repeatedly extracted with 10 mL of acetonitrile again, and the supernatant is taken; and then in step S24, the final supernatant is blown to about 4 mL in a 40°C water bath with nitrogen, water is added to make the volume to 5 mL, and mixed to obtain a solution to be purified.

[0029] Since the sample to be tested is pretreated with acetonitrile, anhydrous magnesium sulfate and anhydrous sodium acetate, the interfering substances such as fat, protein, pigment in the supernatant obtained in the end are greatly removed, so that the residues of seven drugs, including chlorfenapyr, tebuconazole, chlorfenapyr, diniconazole, imipenem, flusilazole and flufenac, are released to the greatest extent. Moreover, the Captiva EMR-Lipid solid phase extraction column is used immediately afterwards, so that the lipid impurities therein are further removed with high selectivity and efficiency. At the same time, as mentioned above, the analyte is not reduced in this process. That is, by pretreating the sample to be tested with acetonitrile, anhydrous magnesium sulfate and anhydrous sodium acetate in the present invention and by means of the Captiva EMR-Lipid solid phase extraction column, the loss of the analyte can be minimized while removing impurities to the greatest extent.

[0030] In the method disclosed in the present invention, in step S4, the conditions of liquid chromatography include: the column oven temperature of the chromatographic column is 40° C., and the injection volume is 5 μL.

[0031] Beneficial effects: In the method of the present invention, acetonitrile constant volume extraction and purification by means of a Captiva EMR-Lipid column are adopted, and liquid chromatography-tandem mass spectrometry detection means are used for on-machine determination, and an accurate, rapid and simple method for simultaneous determination of seven triazole pesticide residues in feed is established. The method provided by the present invention has good extraction and purification effects and high sensitivity, and can achieve accurate characterization and quantification of chlorfenapyr, tebuconazole, chlorfenapyr, diniconazole, imipenem, flusilazole and flufenacil residues in feed samples with high extraction efficiency, low time cost and economic cost. This method can provide technical support for feed quality and safety detection.

[0032] The method for synchronous determination of triazole pesticides in feed of the present invention is disclosed in detail below in conjunction with the embodiments shown in the accompanying drawings and the accompanying drawing numbers. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The present invention is a flowchart of the steps of the method for synchronously determining triazole pesticides in feed.

[0034] Figure 2 It is the MRM spectrum of the standard solution with water and methanol as the mobile phase system and acetonitrile as the sample solvent.

[0035] Figure 3 is the recovery rate of seven triazole pesticides under different purification conditions.

[0036] Figure 4 Matrix effects of seven triazole pesticides under different purification conditions.

[0037] Figure 5 This is the chromatogram of the blank sample after Captiva EMR-Lipid cartridge cleanup.

[0038] Figure 6 Add sample chromatogram for blank after Captiva EMR—Lipid cartridge cleanup. DETAILED DESCRIPTION

[0039] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application may be combined with each other.

[0041] The feed in the present invention is animal feed, which contains vegetable oil, starch and vegetable protein impurities. The detection objects of the present application are seven triazole pesticides in animal feed, namely, chlorfenapyr, tebuconazole, chlorfenapyr, diniconazole, imipenem, flusilazole and flufenazone.

[0042] In view of this, the present invention provides an accurate, rapid and simple synchronous determination method, that is, firstly, seven triazole pesticides in feed are extracted with acetonitrile, then the extract is purified by Captiva EMR-Lipid solid phase extraction column, and the seven triazole pesticides are qualitatively and quantitatively determined by liquid chromatography-tandem mass spectrometry. With the help of the method of the present invention, not only good extraction and purification effects are achieved, but also accurate qualitative and quantitative determination of chlorfenapyr, tebuconazole, chlorfenapyr, diniconazole, imipenem, flusilazole and flufenacil residues in feed samples can be achieved with high extraction efficiency, low time cost and economic cost.

[0043] When using a chromatographic column for separation and determination, the selection of the chromatographic column is the core, and it is necessary to set it in a targeted manner according to the result characteristics of the target object. In the present invention, the Captiva EMR-Lipid solid phase extraction column is used, which can well achieve the separation of the stereoisomers of chlorfenapyr, tebuconazole, chlorfenapyr, diniconazole, imipenem, flusilazole and flufenazone, compared with the solid phase adsorption dispersion extraction and other types of solid phase extraction columns in the prior art, thereby laying the foundation for the separation of the chromatographic peaks of each triazole pesticide to be tested in the chromatographic analysis.

[0044] The specific embodiments disclosed in the present invention are introduced below.

[0045] The following instruments and reagents were used in the specific embodiments of the present invention: liquid spectrometer-mass spectrometer (SCIEX 5500+, SCIEX, USA), electronic analytical balance (ME204E, Mettler Toledo, Switzerland), centrifuge (Hettich, Germany), nitrogen blowdown instrument (Beijing Tongtai Lian Technology Development Co., Ltd.), seven triazole pesticides (100 μg / mL, Tianjin Alta Company), methanol, acetonitrile (chromatographic grade, Sigma), Captiva EMR-Lipid solid phase extraction cartridge (200 mg / 6 mL, Agilent). Specific embodiment 1

[0047] In this embodiment, the detection method of seven triazole pesticides in feed includes the following steps: S1, preparing a matrix-matched series of standard working solutions based on the corresponding standard products of each drug to be tested; S2, extracting the feed sample to be tested with acetonitrile to obtain a solution to be purified; S3, passing the solution to be purified through a Captiva EMR-Lipid solid phase extraction column, collecting all the effluent, making up the volume with acetonitrile, mixing and passing through a microporous filter membrane to obtain a sample solution to be tested; S4, subjecting the matrix-matched series of standard working solutions and the sample solution to be tested to high performance liquid chromatography-tandem mass spectrometry, drawing a working curve with the concentration of the matrix-matched series of standard working solutions as the horizontal coordinate and the measured chromatographic peak area of ​​each drug to be tested as the vertical coordinate, and quantifying each drug to be tested by an external standard method.

[0048] Among them, in step S1, an appropriate amount of 7 pesticide standards are accurately pipetted respectively, diluted with acetonitrile to a mixed standard solution with a concentration of 1 μg / mL, and then diluted with a blank matrix solution to form a matrix-matched series standard working solution with concentrations of 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL, respectively.

[0049] In step S2, acetonitrile is used to extract twice to obtain a supernatant. Specifically, 5 g of the feed sample is weighed, placed in a 50 mL centrifuge tube, 10 mL of acetonitrile is added, ultrasonic extraction is performed for 15 min, centrifuged at 4000 r / min for 5 min, and the supernatant is taken into another centrifuge tube. The lower layer residue is repeatedly extracted once with 10 mL of acetonitrile, the supernatants are combined, anhydrous magnesium sulfate and anhydrous sodium acetate are added after vortexing, and vortexing and shaking are performed to obtain a final supernatant. The final supernatant is blown to about 4 mL in a 40°C water bath with nitrogen, and water is added to make the volume 5 mL, and mixed to serve as the liquid to be purified.

[0050] The actual technical problem to be solved in the present application is to provide a method in which a liquid phase-tandem mass spectrometry scheme is provided to measure seven triazole drug residues of chlorfenthion, tebuconazole, chlorfenthion, diniconazole, imipenem, flusilazole and fluconazole in feed. The matrix effect is smaller. In the field of pesticide detection, the elimination method of the matrix effect of different detection matrices is different. The detection object of the present invention is animal feed, specifically the determination of seven triazole drug residues of chlorfenthion, tebuconazole, chlorfenthion, diniconazole, imipenem, flusilazole and fluconazole in animal feed, and animal feed is rich in vegetable protein, vegetable oil and starch, etc., which have adverse effects on the determination. Therefore, in step S2, a series of pre-treatment means are utilized to eliminate the influence of matrix effect on the detection of pesticides by feed matrix. Specifically, acetonitrile is used as an extractant in the present invention, and anhydrous magnesium sulfate and anhydrous sodium acetate are added for pretreatment, so that the obtained supernatant does not contain dry meat substances such as lipids, proteins, pigments, etc., thereby allowing seven triazole drugs, namely, chlorfenapyr, tebuconazole, chlorfenapyr, diniconazole, imipenem, flusilazole and fluconazole, to be released to the maximum extent.

[0051] Moreover, after extraction, in the next purification step, Captiva EMR-Lipid is used for purification, which can further remove most of the lipids and other impurities in the feed while further eliminating the matrix effect of pesticide detection in the feed matrix, and can minimize the loss of analytes while removing impurities to the greatest extent. Specifically, in step S3, the liquid to be purified is passed through the Captiva EMR-Lipid solid phase extraction column, all the effluent is collected, the volume is made up to 5 mL with acetonitrile, mixed, and filtered through a 0.22 μm microporous filter membrane to obtain a sample solution to be tested.

[0052] In step S4, high performance liquid chromatography-tandem mass spectrometry is used to measure the matrix matching series standard working solution and the test sample solution. In step S4, the liquid chromatography conditions include: the chromatographic column is C 18 The column was set at 40°C, the injection volume was 5 μL, the mobile phase system was mobile phase A and mobile phase B, the mobile phase A was water, the mobile phase B was methanol, the flow rate of the mobile phase system was 0.3 mL / min, and the gradient elution program of the mobile phase system was as shown in Table 1:

[0053] Table 1 Gradient elution program of mobile phase system

[0054]

[0055]

[0056] Additionally, in step S4, the mass spectrometry detection conditions include: the ion source is an electrospray ion source, the monitoring mode is multiple reaction monitoring, the scanning mode is a positive ion mode, the electrospray voltage is 5.5 Kv, the desolvation temperature is 550°C, the curtain gas is 35 psi, the spray gas is 50 psi, and the collision gas is 9 psi. The multiple reaction monitoring ion pairs, cone voltage and collision energy in the present invention are shown in Table 2.

[0057] Table 2 Multiple reaction monitoring ion pairs, cone voltages and collision energies of seven triazole pesticides

[0058]

[0059] In addition, step S4 also includes a step of performing quantitative analysis on the drug to be tested, which is specifically: performing liquid chromatography tandem mass spectrometry detection on the matrix matching standard series working solution and the test sample solution to obtain the peak area of ​​each analyte, using the concentration of the series matrix standard working solution as the abscissa and the chromatographic peak area of ​​the quantitative ion of each analyte as the ordinate to draw a standard curve, obtaining the standard curve regression equation shown in Table 3, and quantifying the sample content.

[0060] Table 3 Standard curve regression equations for seven triazole pesticides

[0061] Object to be tested Standard curve regression equation Correlation coefficient r Chlorpheniramine Y=4578.32*X+5846.07 0.9992 Tebuconazole Y=6932.11*X+4512.15 0.9993 Imidazol Y=6056.78*X-3056.55 0.9995 Diniconazole Y=4672.51*X+3858.47 0.9968 Imidazole Y=5326.24*X+2586.41 0.9987 Flusilazole Y=5146.59*X-7512.65 0.9992 Fluconazole Y=6632.17*X+8532.45 0.9996

[0062] Based on the above results, it can be seen that according to the determination method provided by the present invention, the seven triazole pesticides all showed a good linear relationship in the concentration range of 0.5 ng / mL to 100 ng / mL, and the correlation coefficient (r) was greater than 0.996.

[0063] Optimization of the mobile phase system in specific embodiment 2

[0064] In order to verify the advantages of using water and methanol as mobile phases for the determination of seven triazole pesticides in feed in the present invention, specific embodiment 2 was specially designed. In this embodiment, a mixed standard solution of seven triazole pesticides with a concentration of 50 ng / mL was detected on the liquid chromatography-tandem mass spectrometry according to the conditions of embodiment 1. Different from specific embodiment 1, a comparative analysis of the mobile phase system was carried out in this embodiment.

[0065] Specifically, in the present embodiment, the mobile phase system consisting of mobile phase A and mobile phase B is respectively constructed as water and methanol, water and acetonitrile, 0.1% formic acid water and methanol, 5mmol / L ammonium acetate solution and methanol. The results show that when water and acetonitrile are used as mobile phases, the analyte peaks are faster, and there is no complete separation between the chromatographic peaks. When a small amount of formic acid and ammonium acetate are added to water, the response values ​​of chlorfenapyr and flutriafol decrease significantly. When water and methanol system are used as mobile phases, the response values ​​of 7 triazole pesticides are all high, and the separation is good, the peak shape is symmetrical, and the detection requirements are met.

[0066] It can be proved that the use of water and methanol as mobile phases makes the response values ​​of seven triazole pesticides in feed, including chlorfenapyr, tebuconazole, thiamethoxam, diniconazole, imipenem, flusilazole and fluconazole, all high, and the chromatographic peaks can achieve good separation and the peak shape is symmetrical, which perfectly meets the detection requirements.

[0067] Specific Example 3 Selection of sample solvent

[0068] In order to verify the advantage of using acetonitrile as a solvent for the determination of seven triazole pesticides in feed in the present invention, a specific embodiment 3 is specially designed. In this embodiment, acetonitrile, 0.1% formic acid acetonitrile, 50% acetonitrile aqueous solution, and 50% acetonitrile aqueous solution (containing 0.1% formic acid) are used as solvents to prepare seven kinds of pesticide mixed standard solutions with a concentration of 50 ng / mL, respectively. The liquid chromatography-tandem mass spectrometry detection conditions of Example 1 are used for the determination. The results show that when acetonitrile and 0.1% formic acid acetonitrile are used as solvents to prepare the standard solution, the chromatographic peak response value of each analyte is higher and the peak shape is better. In order to simplify the operating steps, the present invention uses acetonitrile as the sample solvent.

[0069] The MRM spectrum of the standard solution with water and methanol as the mobile phase system and acetonitrile as the sample solvent is as follows: Figure 2 As shown. It can be seen that, since water and methanol are used as the mobile phase system and acetonitrile is used as the sample solvent in the present invention, a very good synergistic effect is achieved, which is specifically reflected in: a good separation degree can be achieved between the chromatographic peaks, the chromatographic peak response value of each analyte is high, and the peak shape is good, thereby achieving the purpose of determination extremely well.

[0070] Specific Example 4 Selection of purification conditions

[0071] Similarly, in order to verify the advantages of using the Captiva EMR-Lipid solid phase extraction column in the present invention, a specific embodiment 4 is specially designed. In this embodiment, a mixed standard solution prepared according to Example 1 is added to 2g of blank feed sample to prepare blank added samples with 7 pesticide concentrations of 10μg / kg. The test solution is measured according to the extraction, purification steps and liquid chromatography-tandem mass spectrometry detection conditions of Example 1. The difference from Example 1 is that four solid phase extraction columns, namely C18 column, graphitized carbon black GCB column, HLB column and Captiva EMR-Lipid column, are used in the purification process. The recovery rates and matrix effects of 7 triazole pesticides under different purification conditions are shown in Figure 2. Figure 3 , Figure 4 shown.

[0072] Comprehensive consideration of the recovery rate and matrix effect shows that when using the Captiva EMR-Lipid solid phase extraction cartridge, the recovery rate is the highest, between 81.5% and 88.4%, and the matrix effect is closest to 100%, indicating that the Captiva EMR-Lipid solid phase extraction cartridge minimizes the ion suppression of the target analyte by the sample matrix. Compared with other solid phase extraction cartridges, the Captiva EMR-Lipid cartridge omits steps such as activation and equilibration during operation, and is the simplest to operate and takes the shortest time. Therefore, the present invention uses the Captiva EMR-Lipid cartridge for sample purification. The blank sample chromatogram and the blank added sample chromatogram after purification by the Captiva EMR-Lipid cartridge are shown in the figure. Figure 5 , Figure 6 shown.

[0073] Specific Example 5 Method Sensitivity Determination

[0074] The detection limit and quantification limit of 7 pesticides were determined by adding spikes to a blank feed matrix. The specific steps are as follows: the mixed standard solution prepared in Example 1 was added to a blank feed matrix (not containing triazole pesticides), and extraction, purification and machine determination were performed according to Example 1. The detection limit was determined by a signal-to-noise ratio S / N≥3, and the quantification limit was determined by a signal-to-noise ratio S / N≥10. The detection limits of the 7 triazole pesticides were all 0.5 μg / kg, and the quantification limits were all 2.0 μg / kg.

[0075] Specific Example 6 Accuracy and Precision Determination

[0076] Spiked samples with concentrations of 2.0 μg / kg, 10.0 μg / kg, and 100.0 μg / kg were prepared respectively, and 6 parallel samples were set for each concentration point. The extraction, purification, and machine determination were carried out according to Example 1. The recovery and precision results are shown in Table 4.

[0077] Table 4 Accuracy and precision test results

[0078]

[0079] As can be seen from the table, the average recovery rate of spiked feed samples is between 77.5% and 91.9% (n=6), and the relative standard deviation of precision is less than 2.6%. The method provided by the present invention has high sensitivity, good accuracy and reproducibility, and is suitable for the simultaneous detection of 7 triazole pesticides in feed samples.

[0080] As shown in the test results of the above examples, the detection limit of the detection method provided by the present invention is 0.5 μg / kg, the quantitative limit is 2.0 μg / kg, the average recovery rate is between 77.5% and 91.9% (n=6), and the precision relative standard deviation is less than 2.6%. From these results, it can be seen that the extraction and purification effect of the method provided by the present invention is good, the sensitivity is high, and the accurate determination and quantification of the residues of chlorfenapyr, tebuconazole, chlorfenapyr, diniconazole, imipenem, flusilazole, and fluconazole in feed samples can be achieved with higher extraction efficiency, lower time cost and economic cost, and the method can provide technical support for feed quality and safety detection. That is, compared with the prior art, the detection method of the present invention can realize the quantitative detection of seven drugs, namely, chlorfenapyr, tebuconazole, chlorfenapyr, diniconazole, imipenem, flusilazole and fluconazole, and has a lower detection limit and quantification limit, which is more conducive to the detection and accurate quantification of low content of seven drugs, namely, chlorfenapyr, tebuconazole, chlorfenapyr, diniconazole, imipenem, flusilazole and fluconazole in animal feed.

[0081] It should be noted that the terms "first", "second", etc. in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable where appropriate, so that the embodiments of the present application described herein can be implemented in an order other than those illustrated or described herein, for example. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for synchronous determination of triazole pesticides in feed, characterized in that: The triazole pesticides are chlorfenapyr, tebuconazole, fenthion, diniconazole, imipenem, flusilazole and flufenazone; the method comprises the following steps: S1, prepare a matrix-matched series of standard working solutions based on the corresponding standards of each drug to be tested; S2, extracting the feed sample to be tested with acetonitrile to obtain a solution to be purified; S3, passing the solution to be purified through a Captiva EMR-Lipid solid phase extraction column, collecting all the effluent, making up the volume with acetonitrile, mixing and passing through a microporous filter membrane to obtain a sample solution to be tested; S4, subjecting the matrix-matching series standard working solutions and the test sample solutions to high performance liquid chromatography-tandem mass spectrometry, plotting a working curve with the concentration of the matrix-matching series standard working solutions as the abscissa and the measured chromatographic peak area of ​​each test drug as the ordinate, and quantifying each test drug using the external standard method.

2. The method for synchronous determination of triazole pesticides in feed according to claim 1, characterized in that: In step S4, the chromatographic column of the high performance liquid chromatography-tandem mass spectrometry is C 18 The column has a mobile phase system of mobile phase A and mobile phase B, wherein mobile phase A is water and mobile phase B is methanol, and gradient elution is adopted.

3. The method for synchronous determination of triazole pesticides in feed according to claim 2, characterized in that: In the high performance liquid chromatography, the flow rate of the mobile phase system is 0.1-0.5 mL / min, and the gradient elution program of the mobile phase system is: 0.0~1.0min, the volume percentage of the mobile phase A is 30%; 1.0-5.0min, the volume percentage of the mobile phase A is uniformly reduced from 30% to 5%; 5.0~9.0min, the volume percentage of the mobile phase A is 5%; 9.0-9.1 min, the volume percentage of the mobile phase A is increased uniformly from 5% to 30%; 9.1-12.0 min, the volume percentage of the mobile phase A is 30%.

4. The method for synchronous determination of triazole pesticides in feed according to claim 3, characterized in that: The mass spectrometry conditions in the HPLC-MS / MS are as follows: the ion source is an electrospray ion source, the monitoring mode is multiple reaction monitoring, the scanning mode is positive ion scanning, the electrospray voltage is 5.5 Kv, the desolvation temperature is 550° C., the curtain gas is 35 psi, the spray gas is 50 psi, and the collision gas is 9 psi.

5. The method for synchronous determination of triazole pesticides in feed according to any one of claims 1 to 4, characterized in that: In the step S1, an appropriate amount of seven pesticide standards, namely, chlorfenapyr, tebuconazole, chlorfenapyr, diniconazole, imipenem, flusilazole and fluconazole, are weighed, diluted to a fixed volume with acetonitrile to form a mixed standard solution of 1 μg / mL, and then diluted with a blank matrix solution to form a matrix-matched series standard working solutions with concentrations of 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL and 100 ng / mL, respectively.

6. The method for synchronous determination of triazole pesticides in feed according to any one of claims 1 to 4, characterized in that: In the step S2, it includes: Step S21, taking 1-5 g of the feed sample to be tested, placing it in a 50 mL centrifuge tube, adding 5-15 mL of acetonitrile, ultrasonically extracting for 15 min, centrifuging at 4000 r / min for 3-5 min, and taking the supernatant into another centrifuge tube; Step S22, the lower layer residue is extracted again with 5-15 mL of acetonitrile, and the supernatant is taken; Step S23, combining the supernatants obtained in step S21 and step S22, vortexing, adding anhydrous magnesium sulfate and anhydrous sodium acetate, vortexing and shaking, to obtain a final supernatant; Step S24, blowing the supernatant finally obtained to about 2-5 mL with nitrogen in a 40° C. water bath, adding water to make the volume to 5 mL, and mixing to obtain a solution to be purified.

7. The method for synchronous determination of triazole pesticides in feed according to any one of claims 1 to 4, characterized in that: In step S4, the liquid chromatography conditions include: the column oven temperature of the chromatographic column is 40° C., and the injection volume is 5 μL.

Images