Ultra-high performance liquid chromatography-tandem mass spectrometry analysis method for contents of flufenamid and afidopyropen in bees

Abstract

The invention discloses an ultra-high performance liquid chromatography tandem mass spectrometry detection method for flufenamid and afidopyropen in bees, and the method comprises the following steps: (1) extraction: homogenizing an insect sample, mixing the homogenized insect sample with an extraction solvent and an extraction salt, carrying out oscillation extraction to obtain an extract, and taking a supernatant I; (2) purification: mixing the supernate I obtained in the step (1) with a purifying agent, purifying, collecting supernate II, and filtering to obtain a sample solution to be detected; and (3) determining by using an ultra-high performance liquid chromatograph-mass spectrometer: separating the residues of the flufenamid and the afidopyropen in the sample solution to be detected by using a liquid chromatographic separation column, and detecting by using a mass spectrum to obtain the content of the residues of the flufenamid and the afidopyropen in the bees. The pretreatment operation of the method is simple and convenient, the purification effect is good, the method has the advantages of high recovery rate, good reproducibility, small organic solvent dosage and the like, the minimum detection concentrations of the two pesticides are both low, and the method is suitable for analysis and detection of the flufenoxamid and the afidopyropen in the bees.

Description

Technical Field

[0001] The present invention relates to pesticide detection, and in particular to a method for simultaneously determining clothianidin and buprofezin in bees by ultra-high performance liquid chromatography tandem mass spectrometry. Background Art

[0002] Clothianidin (English name: dimpropyridaz; trade name: Axalion TM ) is a pyrazolecarboxamide insecticide with a novel mode of action developed by BASF. According to the latest classification of the Insecticide Resistance Action Committee (IRAC), clothianidin is a chordotonal organ modulator (site of action unknown), which inhibits the action of chordotonal organs by blocking the signal transduction upstream of the transient receptor potential vanilloid (TRPV) channel, interfering with the hearing, balance, sense of direction, gravity perception, locomotor ability, etc. of pests, making the poisoned pests lose coordination, unable to feed, and eventually die. Clothianidin has good systemic conductivity and is mainly used for fruit trees, vegetables, soybeans, cotton, cereals, potatoes and other field crops, as well as flowers, ornamental plants, etc. to control pests such as Lepidoptera, Coleoptera, Diptera, Hemiptera, Thysanoptera, etc., especially effective against piercing-sucking mouthpart pests such as aphids, whiteflies, and psyllids. Buprofezin is an acrylate compound with the same mode of action as clothianidin. Due to its strong systemicity, it is widely used on grapes, soybeans, potatoes, etc. to control piercing-sucking and sucking mouthpart pests such as aphids, whiteflies, psyllids, scale insects, mealybugs, and leafhoppers. The unique modes of action of these two insecticides make them have no cross-resistance with other insecticides on the market and have good control effects.

[0003] Bees are important pollinating insects for crops in China. As the world's largest beekeeping country, the economic value of bee pollination to agriculture accounts for about 12% of the total output value. Despite the important ecological and economic significance of bees, in recent years, the activity range of bees has shrunk significantly, and the population has also been decreasing continuously. Research around the world shows that exposure to pesticides, especially insecticides, is one of the reasons for the decline in the number of bees and wild bees. Bees often come into direct contact with insecticides when visiting flowers, and contaminated pollen and nectar are brought back to the beehive, causing the bees in the hive to be exposed as well, thus affecting the bee population. Therefore, carrying out the residue detection of insecticides in bees is of great significance for controlling the dosage of insecticides and determining the appropriate application period in production. Before instrumental analysis of pesticides, extraction and purification steps in the pretreatment process are required. They are important factors affecting the accuracy of quantitative results. Under the condition of meeting the sensitivity requirements, impurities need to be removed to the greatest extent, which can effectively ensure the residue amount of trace pesticides and avoid instrument contamination, ensuring the accuracy and repeatability of the results. Due to the extremely large differences in the physicochemical properties of flufenerim and cyantraniliprole, it is difficult to detect both simultaneously. Developing a method for simultaneously detecting flufenerim and cyantraniliprole is of great significance for ensuring agricultural production safety, protecting the health of pollinating insects, and maintaining ecological balance. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for simultaneously detecting flufenerim and cyantraniliprole, and meeting the requirements for accuracy, precision, and sensitivity in production.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] An ultra-high performance liquid chromatography tandem mass spectrometry analysis method for flufenerim and cyantraniliprole in bees, comprising the following steps:

[0007] (1) Extraction: Homogenize the bee sample with water, then mix it with an extraction solvent and an extraction salt, and obtain an extract after oscillating extraction. Take the supernatant I.

[0008] (2) Purification: Mix the supernatant I obtained in step (1) with a purifying agent for purification, collect the supernatant II, and obtain the sample solution to be measured after filtration.

[0009] (3) Determination by ultra-high performance liquid chromatography-tandem mass spectrometry: Separate the residues of flufenerim and cyantraniliprole in the sample solution to be measured using a liquid chromatography separation column, and detect them by mass spectrometry, thereby obtaining the residue contents of flufenerim and cyantraniliprole in bees.

[0010] The present invention is applicable to bees and also includes other hymenopteran insects.

[0011] In the above method, in step (1), the extraction solvent may be acetonitrile, acetonitrile containing 0.1% formic acid, acetonitrile containing 0.1% acetic acid, acetonitrile containing 0.5% formic acid, or acetonitrile containing 0.5% acetic acid; preferably, it is acetonitrile containing 0.5% acetic acid.

[0012] In step (1), the mass ratio of the bee sample to the volume of the extraction solvent is 1 g:(5 - 10) mL, and specifically, it may be 1 g:5 mL.

[0013] Preferably, the aforementioned extraction salts may be a mixture of anhydrous magnesium sulfate and sodium chloride, and the mass ratio may be 1:1. For example, when analyzing a 1 ± 0.05 g bee sample, a mixture of 1 g anhydrous magnesium sulfate and 1 g sodium chloride can be added for water removal.

[0014] Preferably, water removal is carried out by adding the salts for 1 - 5 min, preferably 1 min.

[0015] Preferably, the extract is centrifuged at a rotation speed of 5000 - 10000 r / min, for a time of 5 - 10 min, and at a temperature of 0 - 5°C.

[0016] Preferably, in the above method, in step (2), the purifying agent may be one or a combination of several purification methods such as octadecylsilane bonded silica adsorbent (C 18 ), graphitized carbon black adsorbent (GCB), N-propylethylenediamine adsorbent (PSA), etc., and preferably, purification is carried out using PSA dispersive adsorbent. For example, when analyzing a 1 ± 0.05 g bee sample, 150 mg PSA can be used as the purifying agent.

[0017] Preferably, in the above method, in step (2), after the supernatant I is mixed with the purifying agent, vortexing is carried out to achieve purification, and then centrifugation is carried out at a rotation speed of 5000 - 10000 r / min, for a time of 5 - 10 min, and at a temperature of 0 - 5°C to obtain the supernatant II, which is filtered using a 0.22 μm filter membrane.

[0018] Preferably, in the above method, in step (3), the chromatographic column used in the liquid chromatography method is Agilent SB-C18.

[0019] Preferably, the specific specifications of the liquid chromatography column may be 2.1 mm × 75 mm, 2.7 - μm.

[0020] Preferably, in the above method, in step (3), the chromatographic conditions are as follows: the mobile phase A is a 2 mmol / L ammonium acetate aqueous solution containing 0.1% formic acid, and the mobile phase B is acetonitrile. An isocratic elution mode is adopted; the flow rate is set to 0.3 mL / min; the column temperature is 35°C, and the injection volume is 2 μL.

[0021] Preferably, in the above method, the mass spectrometry conditions in step (3) are as follows: electrospray positive ion mode (ESI+); capillary voltage: 4 KV; nebulizing gas flow rate 2.0 L / min, drying gas and heating gas flow rates are both 10.0 L / min, interface temperature 300 °C, desolvation temperature 526 °C, heating block temperature 400 °C; detection method: multiple reaction monitoring mode (MRM), the quantitative ion pair of cyantraniliprole: 302>109, the qualitative ion pair 302>179; the quantitative ion pair of cyclaniliprole: 594>202, the qualitative ion pair 594>148.

[0022] The present invention has the following advantages and effects compared with the prior art

[0023] The present invention establishes an analytical method for cyantraniliprole and cyclaniliprole in bees. Acetonitrile containing 0.5% acetic acid is used as the extractant, a mixture of anhydrous magnesium sulfate and sodium chloride is used as the extraction salt, and PSA dispersive adsorbent purification is used to further remove impurities in bees. External standard method is used for quantification. Compared with the prior art, the pretreatment of this method is simple and convenient, the purification effect is good, and it has the advantages of high recovery rate, good reproducibility, and less consumption of organic solvents. The minimum detection concentrations of the two pesticides are relatively low, which is suitable for the simultaneous analysis and detection of cyantraniliprole and cyclaniliprole in bees. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The beneficial effects of the present invention will be described in detail below in conjunction with the drawings and specific embodiments.

[0025] Figure 1 shows the influence of adsorbent type and dosage on the recovery rates of the two pesticides

[0026] Figure 2 is 18 the high performance liquid chromatography diagrams of the impurity removal effects of three different adsorbents, C18, GCB, and PSA. In the figure, black is C18, gold is PSA, blue is GCB, and yellow is the unpurified sample.

[0027] Figure 3 is the high performance liquid chromatography diagram of the impurity removal effect of different contents of PSA. In the figure, the dosages of PSA are 50 mg (black), 100 mg (gold), 150 mg (blue), 200 mg (gold), 250 mg (yellow), and 400 mg (dark blue) according to the line colors.

[0028] Figure 4 is the standard curve diagrams of the two pesticides, A is cyantraniliprole, and B is cyclaniliprole.

[0029] Figure 5are the separation effects and response values of two pesticides, where A is clothianidin in a 50-mm chromatographic column, B is cyantraniliprole in a 50-mm chromatographic column, C is clothianidin in a 75-mm chromatographic column, D is cyantraniliprole in a 75-mm chromatographic column, E is clothianidin in a 100-mm chromatographic column, and F is cyantraniliprole in a 100-mm chromatographic column. Detailed implementation manners

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all obtained from conventional biochemical reagent stores unless otherwise specified.

[0032] The present invention discloses a method for detecting clothianidin and cyantraniliprole in bees. The main instruments and reagents used in this method are as follows:

[0033] 1. Instruments:

[0034] Ultra-high performance liquid chromatography tandem mass spectrometer (Shimadzu 8045), multi-tube vortex mixer (LICHEN Vorter-2500MT), centrifuge (GTR22-1).

[0035] 2. Reagents: Acetonitrile (chromatographic grade, Fisher Company, USA), anhydrous magnesium sulfate (analytical grade, Sinopharm Group), sodium chloride (analytical grade, Sinopharm Group), ethylenediamine-N-propylsilane adsorbent (PSA, Shanghai Anpu Company), graphitized carbon black adsorbent (GCB, Shanghai Anpu Company), octadecylsilyl silica gel adsorbent (C 18 , Shanghai Anpu Company), clothianidin (97.1%, Dr Ehrenstorfer), cyantraniliprole (99.5%, Dr Ehrenstorfer).

[0036] 3. Preparation of standard working solution: Weigh 10 mg of clothianidin and cyantraniliprole standard products accurately respectively, dissolve them with 10 mL of chromatographic-grade acetonitrile, and prepare a stock solution of 1000 mg / L, which is stored sealed in a refrigerator at 0-4°C, and the storage period is 6 months.

[0037] Preparation of working solution: Accurately pipette an appropriate amount of the stock solution and serially dilute it with chromatographically pure acetonitrile to working solutions with different concentrations of 1, 5, 10, 20, 100, and 500 μg / L. Prepare the solutions immediately before use.

[0038] Optimization of detection conditions in Example 1

[0039] 1. Instrument operating conditions:

[0040] Chromatographic conditions:

[0041] The chromatographic conditions of the liquid chromatography method are as follows: Mobile phase A: 2 mmol / L ammonium acetate aqueous solution containing 0.1% formic acid, mobile phase B: acetonitrile. Isocratic elution is used; the flow rate is set at 0.3 mL / min; column temperature: 35 °C, injection volume 2 μL.

[0042] Mass spectrometry conditions:

[0043] Electrospray positive ion mode (ESI+); capillary voltage: 4 kV; nebulizing gas flow rate 2.0 L / min, drying gas and heating gas flow rates are both 10.0 L / min, interface temperature 300 °C, desolvation temperature 526 °C, heating block temperature 400 °C; detection mode: multiple reaction monitoring mode (MRM), the quantitative ion pair of cyantraniliprole: 302>109, the qualitative ion pair 302>179; the quantitative ion pair of cyproconazole: 594>202, the qualitative ion pair 594>148.

[0044] 2. Sample pretreatment

[0045] (1) Extraction

[0046] Weigh 1.0 g (accurate to 0.01 g) of the ground bee sample, place it in a 10 mL centrifuge tube, add 1 mL of pure water, then add 5 mL of acetonitrile containing 0.5% acetic acid, vortex (LICHEN Vorter-2500MT) for 5 min, add 1 g of anhydrous magnesium sulfate and 1 g of sodium chloride, vortex (LICHEN Vorter-2500MT) and then centrifuge (GTR22-1 type tabletop centrifuge) at 5000 rpm and 2 °C for 10 min;

[0047] (2) Purification

[0048] Pipette 2 mL of the extract from the centrifuge tube, transfer it to a centrifuge tube containing 150 mg of PSA dispersive adsorbent for purification. After vigorous shaking, vortex for 10 s, then centrifuge at 10000 r / min for 2 min, take the supernatant, and filter it through a 0.22 μm organic syringe filter for determination;

[0049] 3. Experimental results

[0050] (1) Optimization of the extraction step

[0051] 1) Optimization of extraction solvent

[0052] When selecting the extraction solvent, acetonitrile, acetonitrile containing 0.1% formic acid, acetonitrile containing 0.1% acetic acid, acetonitrile containing 0.5% formic acid, and acetonitrile containing 0.5% acetic acid were compared. The extraction efficiencies of the 5 extraction solutions varied greatly for the two drugs. Only the recovery rate of the acetonitrile extraction solution containing 0.5% acetic acid was between 90% and 110% (see Table 1 for details). At the same time, considering that PSA was needed to remove some impurities subsequently, the acetonitrile extraction solution containing 0.5% acetic acid had a good recovery rate at this time. Therefore, the acetonitrile homogenization extraction containing 0.5% acetic acid was selected as the extraction solvent for thiazolezolamide and bipropyl dicarboxylate in bees.

[0053] Table 1 Effects of different extraction solvents on the recovery rates of 2 pesticides

[0054]

[0055]

[0056] 2) Selection of extraction salts

[0057] Anhydrous magnesium sulfate is often used to remove the moisture in the matrix. The purpose of adding sodium chloride is to separate the organic phase and the aqueous phase. 4 g of anhydrous magnesium sulfate and 1 g of sodium chloride are the commonly used masses in the extraction process of most fruits and vegetables; the effects of using 4 g of anhydrous magnesium sulfate and 1 g of sodium chloride in the extraction process of the present invention were average. Experiments with mass ratios of anhydrous magnesium sulfate to sodium chloride of (4:1), (3:1), (2:1), and (1:1) were carried out. The results showed that the extraction effect was the best when the ratio was 1:1 (see Table 2), and better dehydration and layering effects could be obtained.

[0058] Table 2 Effects of the masses of anhydrous magnesium sulfate and sodium chloride on the extraction recovery rates of 2 pesticides

[0059]

[0060] (2) Optimization of purification conditions

[0061] When selecting the purification material, the effects of 3 different purification materials, namely C 18 , PSA, and GCB, and different contents on the purification effects of thiazolezolamide and bipropyl dicarboxylate in bees were compared. The results showed that ( Figure 1 ), after being purified by the above 3 purification materials alone, the recovery rate after GCB purification was about 70%, indicating that GCB would cause serious adsorption of thiazolezolamide and bipropyl dicarboxylate, resulting in a low recovery rate; the recovery rates of the 2 purification materials C 18 and PSA were both between 83.6% and 89.5%, and Figure 2The impurities in the display matrix were correspondingly lower after PSA purification; meanwhile, the effect of different PSA contents on impurity removal was investigated, and it was found that the purification effect was the best with 100 mg of PSA, the effects of 150 - 400 mg were comparable, and the effect was the worst with 50 mg, as shown in Figure 3 (Data 1 - Data 6 are the amounts of PSA used as 50, 100, 150, 200, 250, and 400 mg respectively). Therefore, considering the purification effect, recovery rate, and cost, 100 mg - 150 mg of PSA can be selected. In this study, 150 mg of PSA was selected as the purification adsorbent, and the recovery rates of both pesticides were above 80%, meeting the requirements for pesticide residue detection.

[0062] (3) Optimization of detection conditions

[0063] The present invention investigated the effects of positive and negative ion scanning modes on the responses of cyantraniliprole and cyproconazole - ethyl and the formation of specific ion pairs. The results showed that in the negative ion mode, the responses of the two pesticides were lower and the formed specific ion pairs were not obvious. Therefore, the positive ion scanning mode was selected for this experiment.

[0064] The present invention investigated the separation, response, and retention effects of three commonly used liquid chromatography columns, namely 50 mm, 75 mm, and 100 mm, on cyantraniliprole and cyproconazole - ethyl. When using the Agilent SB - C18 column (2.1 mm × 75 mm, 2.7 - μm), the separation effect and response values of the two pesticides were better (see Table 3), and baseline separation could be better achieved ( Figure 5 ), obtaining a good symmetric peak shape and ensuring the accuracy of the quantitative results.

[0065] Table 3 Effects of different chromatographic columns on the response values of two pesticides

[0066]

[0067] (4) Methodology investigation

[0068] The external standard method was used for quantification. Taking cyantraniliprole and cyproconazole - ethyl as the abscissa and the peak area of the quantitative ion as the ordinate, the calibration solution curve was plotted, as shown in Figure 4 , and the regression equations were: for cyantraniliprole, y = 7.21471e+007x + 44395.9 (R 2 = 0.9996); for cyproconazole - ethyl, y = 1.01630e+006x - 2596 (R 2 = 0.9998). The detection limit of cyantraniliprole by ultra - high performance liquid chromatography - tandem mass spectrometry was 1 μg / kg, and the quantification limit was 5 μg / kg; the detection limit of cyproconazole - ethyl was 0.1 μg / kg, and the quantification limit was 1 μg / kg.

[0069] Take blank bee samples, and set four spiking concentrations of 20 μg / kg, 50 μg / kg, 100 μg / kg, and 200 μg / kg respectively for the spiking recovery test, with 5 replicates set for each spiking concentration. As can be seen from Table 4, when the spiking concentrations of cyazofamid and cyprodinil in bees are 20 μg / kg, 50 μg / kg, 100 μg / kg, and 200 μg / kg, the average recoveries are 84.6% - 105.1%, and the relative standard deviations are 1.33% - 4.11%.

[0070] Table 4 Recoveries and standard deviations corresponding to different spiking concentrations

[0071]

[0072] The present invention has established a detection method for cyazofamid and cyprodinil in bees. The cyazofamid and cyprodinil of the present invention show a good linear relationship in the concentration range of 1 - 500 μg / L. The average recoveries of the two pesticides spiked in bees are 83.6 - 117%, and the relative standard deviation (RSD) is 1.33% - 7.10%. The method quantification limit is 1 - 5 μg / kg. The method of the present invention has the characteristics of simple and rapid operation, good sample reproducibility, and strong applicability, and all technical indicators can meet the requirements of pesticide residue detection and analysis.

[0073] Determination results of actual samples in Example 2

[0074] Randomly capture 4 Italian honeybee samples, and use the above method to detect cyazofamid and cyprodinil. The results are as follows:

[0075] Table 5 Detection results of two pesticides in bees

[0076]

[0077] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed as above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to the equivalent embodiments by using the disclosed technical content within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any brief modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. An ultra-high performance liquid chromatography tandem mass spectrometry analysis method for methoxazole and dimethoate, comprising the following steps: (1) Extraction: The sample to be tested is homogenized and mixed with an extraction solvent and an extraction salt, and then subjected to oscillation extraction to obtain an extract, and the supernatant I is taken; (2) Purification: the supernatant I obtained in step (1) is mixed with a purifying agent for purification, the supernatant II is collected, and the sample solution to be tested is obtained by filtration; (3) Ultra-high performance liquid chromatography-mass spectrometry: The residues of imipenem and dichlorvos in the sample solution are separated by a liquid chromatography column, and then the residues are detected by mass spectrometry to obtain the residue contents of the two pesticides in the sample solution.

2. The method according to claim 1, characterized in that: The samples to be tested are insects.

3. The method according to claim 1, characterized in that The samples to be tested are Hymenoptera insects.

4. The method according to claim 1, characterized in that The extraction solvent in step (1) is acetonitrile containing 0.5% acetic acid.

5. The method according to claim 1, characterized in that In step (1), the volume ratio of the mass of the sample to be tested to the extraction solvent is 1 g:5 mL to 1 g:10 mL.

6. The method according to claim 1, characterized in that The extraction salt in step (1) can be a mixture of anhydrous magnesium sulfate and sodium chloride.

7. The method according to claim 1, characterized in that Step (1) comprises centrifuging the extract at a speed of 5000 to 10000 r / min, a time of 5 to 10 min, and a temperature of 0 to 5° C., and taking the supernatant I after centrifugation.

8. The method according to claim 1, characterized in that In step (2), the purifier may be an octadecyl bonded silica gel adsorbent (C 18 ), graphitized carbon black adsorbent (GCB), N-propylethylenediamine adsorbent (PSA) and other purification methods or a combination of several of them.

9. The method according to claim 1, characterized in that: In step (2), the supernatant I is mixed with the purifier and then vortexed for purification, and then centrifuged at a speed of 5000-10000 r / min, a time of 5-10 min, and a temperature of 0-5°C to obtain the supernatant II, which is filtered using a 0.22 μm filter membrane.

10. The method according to claim 1, characterized in that The chromatographic conditions in step (3) are as follows: mobile phase A: 2 mmol / L ammonium acetate aqueous solution containing 0.1% formic acid, mobile phase B: acetonitrile. Isocratic elution is used; flow rate is set to 0.3 mL / min; column temperature: 35°C, injection volume 2 μL.

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