Determination of diamide compounds in feed

Abstract

The invention discloses a method for determining diamide compounds in feed, comprising the following steps: S1 obtains supernatant: prepares 85% acetonitrile solution and inorganic salt components and adds them to feed, extracts cyanamide, chlorantraniliprole and tetrachlorantraniliprole in feed by water bath ultrasound and centrifugation to obtain supernatant; S2 obtains test liquid: uses purifier and CaptivaEMR-Lipid purification column to purify the obtained supernatant, collects the effluent of the purification column and filters it to obtain the test liquid; S3 test liquid detection and analysis: uses liquid chromatography tandem mass spectrometry to detect and analyze the obtained test liquid, and quantitatively adopts external standard method. The determination method of the present invention can simultaneously determine compounds such as cyanamide, chlorantraniliprole and tetrachlorantraniliprole in feed, has high detection accuracy and sensitivity, and is simple to operate and has good stability, which meets the current lack of determination methods of diamide compounds in feed and the need for supervision of pesticide residues in livestock products.

Description

Technical Field

[0001] The invention relates to a method for determining diamide compounds, in particular to a method for determining compounds such as chlorantraniliprole, cyantraniliprole and tetrachlorantraniliprole in feed. Background Art

[0002] As the world's largest category of pesticides, diamide compounds are widely used in agricultural production to protect crops from pests and diseases. The diamide compounds currently on the market mainly include chlorantraniliprole, cyantraniliprole, tetrachlorantraniliprole, etc. Their mechanism of action is to activate insect nicotinic acid (muscle) receptors. They have a rapid, long-lasting, and broad-spectrum control effect on most lepidopteran and coleopteran pests and are commonly used pesticides for cereals (Duan Jinsheng et al., 2016; Yan Ming et al., 2019). Diamide compounds are difficult to degrade in soil and water and tend to remain in the environment for a long time. Therefore, with the widespread use of diamide compounds in agricultural production, pesticide residues may enter poultry and livestock directly through feed on the one hand, and enter aquatic animals through water and soil on the other hand, and eventually transfer to humans through the food chain, causing potential safety and health risks (He Xufeng et al., 2021). GB2763-2021 "National Food Safety Standard Maximum Residue Limits of Pesticides in Food" has strict regulations on the maximum residue limits (MRLs) of diamide compounds in food. Some literature reports on the determination of diamide compounds in food and water by liquid chromatography-mass spectrometry, which has achieved a lower detection limit, good precision and reproducibility. However, there is no literature reporting a method for the simultaneous detection of three diamide compounds, cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole, in feed. Summary of the invention

[0003] The purpose of the present invention is to provide a method for determining diamide compounds in feed for the first time in response to the lack of methods for determining diamide compounds in feed and the need for supervision of pesticide residues in livestock products. The method can effectively and simultaneously determine compounds such as cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole in feed, has high detection accuracy and sensitivity, is easy to operate, has good stability, and is suitable for the determination of diamide compounds in feed.

[0004] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0005] The invention discloses a method for determining diamide compounds in feed, wherein the diamide compounds are cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole, and the method comprises the following steps: S1 obtaining a supernatant: firstly preparing an 85% acetonitrile solution and an inorganic salt component, wherein the 85% acetonitrile solution is prepared by adding water to 85 mL of acetonitrile to make the volume 100 mL, adding the prepared 85% acetonitrile solution and the inorganic salt component into feed, extracting the cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole compounds in the feed by water bath ultrasound and centrifugation to obtain a supernatant; S2 obtaining a test liquid: pre-cleaning the obtained supernatant by using a purifier, and purifying the pre-cleaned supernatant again by passing a CaptivaEMR-Lipid purification column; collecting the effluent of the CaptivaEMR-Lipid purification column for filtration to obtain a test liquid; S3 detecting and analyzing the test liquid: detecting and qualitatively analyzing the obtained test liquid by using a liquid chromatography tandem mass spectrometry method, and quantitatively analyzing by using an external standard method.

[0006] Through the above-mentioned disclosed steps, the present invention realizes the simultaneous determination of cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole in feed. The determination method is not only highly sensitive and accurate, but also simple and stable to operate, which meets the current lack of determination methods for diamide compounds in feed and the need for supervision of pesticide residues in livestock products.

[0007] Secondly, the present invention overcomes the existing single purification method and uses a secondary purification method to greatly improve the recovery rate of the target compound. The first purification using a purifier effectively reduces the solubility of impurity components, facilitates the removal of most of the excess impurity components in the supernatant, and obtains a supernatant containing the target compound with higher purity. The CaptivaEMR-Lipid purification column is used for the second time. Due to its high selectivity, it can further remove the impurity components remaining in the supernatant after the first purification, thereby obtaining a high-purity supernatant containing the target compound. In addition, its efficient lipid removal can also ensure that the ion suppression of the target compound is minimized, thereby significantly improving the reliability and durability of the determination method.

[0008] The purpose of adding inorganic salt in the present invention is to increase the ionic strength of acetonitrile, accelerate the ionization reaction of the substance, promote the interaction between the molecules of acetonitrile and the target compound, increase the reaction rate, so that the target compound can be completely and quickly dissolved in acetonitrile, and at the same time, the acetonitrile phase and the water phase are separated, which is convenient for subsequent separation.

[0009] In the determination method of diamide compounds in feed disclosed in the present invention, in step S1, the inorganic salt component is prepared by mixing anhydrous magnesium sulfate and sodium chloride in a mass ratio of 1:3. Since the matrix of the present invention is feed, its water content is relatively low, and the amount of anhydrous magnesium sulfate can be appropriately reduced to prevent excessive use from causing a reduction in the volume of acetonitrile. Therefore, the present invention can select a mass ratio of anhydrous magnesium sulfate and sodium chloride of 1:3 to obtain a better stratification effect.

[0010] In the method for determining diamide compounds in feed disclosed in the present invention, in step S1, after obtaining the supernatant by water bath ultrasound and centrifugation, anhydrous magnesium sulfate needs to be added and filtered. Since there is still a small amount or trace amount of water in the supernatant after separation, the purpose of adding anhydrous magnesium sulfate at this time is to completely absorb the water in the acetonitrile phase in the supernatant, thereby completely removing the water in the acetonitrile phase.

[0011] In the method for determining diamide compounds in feed disclosed in the present invention, in step S2, the purifier includes C18, PSA, GCB, multi-walled carbon nanotubes, and nano-zirconium oxide.

[0012] In the method for determining diamide compounds in feed disclosed in the present invention, in step S2, the purifiers selected are C18, PSA and nano zirconium oxide for combined purification.

[0013] In the determination method of diamide compounds in feed disclosed in the present invention, in step S1, the prepared acetonitrile solution and inorganic salt components are added to the feed and the supernatant is obtained by water bath ultrasound and centrifugation. The specific operation is as follows: the feed sample is placed in a centrifuge tube, firstly the prepared acetonitrile solution is added and vortex mixed for 2 minutes, then the prepared inorganic salt component is added and vortex mixed for 1 minute, extracted by water bath ultrasound for 20 minutes, and then centrifuged at 8000r / min for 5 minutes, and finally the supernatant is separated.

[0014] In the determination method of diamide compounds in feed disclosed in the present invention, in step S2, the specific operation of obtaining the test solution is as follows: remove the supernatant obtained in step S1, use a purifier to pre-purify the supernatant and then filter it, then purify the filtered supernatant again through a CaptivaEMR-Lipid purification column, and collect the effluent of the CaptivaEMR-Lipid purification column, finally make up the volume with water, vortex for 30 seconds, and obtain the test solution after filtering through a 0.22μm organic microporous filter membrane.

[0015] In the determination method of diamide compounds in feed disclosed in the present invention, in step S3, the liquid chromatography detection conditions include: the chromatographic column is a C18 column, the column oven temperature is 40°C, the injection volume is 5 μL, the mobile phase system includes mobile phase A and mobile phase B, mobile phase A is 5 mmol / L ammonium acetate solution, mobile phase B is methanol, and the flow rate of the mobile phase system is 0.3 mL / min;

[0016] The gradient elution program used was as follows:

[0017] 0.0~1.0min, the volume percentage of mobile phase A is 95%;

[0018] From 1.0 to 5.0 min, the volume percentage of mobile phase A decreases at a constant rate from 95% to 5%;

[0019] 5.0-7.0min, the volume percentage of mobile phase A is 5%;

[0020] From 7.0 to 7.1 min, the volume percentage of mobile phase A increased uniformly from 5% to 95%;

[0021] 7.1~10.0min, the volume percentage of mobile phase A is 95%.

[0022] Based on the weak polarity of diamide compounds, a 95% aqueous phase with a larger polarity is used in the initial stage, which enables the rapid elution of interfering impurities with large polarity; 1-5min, the proportion of methanol in the organic phase is gradually increased from 5% to 95% at a uniform rate, which gradually reduces the polarity of the mobile phase, thereby enhancing the elution ability of diamide compounds, and achieving the purpose of orderly separation and rapid elution of target compounds; and at 5-7min, the mobile phase maintains a high proportion of organic phase, which can further remove non-polar impurities remaining in the chromatographic column; finally, at 7-10min, the fluidity returns to the initial proportion, balances the system, and ensures the repeatability of sample detection. In the present invention, based on the setting of the gradient elution program as described above, the polarity of the mobile phase can be adjusted by changing the solvent ratio, thereby shortening the analysis time and improving the separation effect. And, more importantly, by adding an appropriate amount of ammonium acetate to the aqueous phase, this makes it possible to adjust the pH value of the mobile phase, thereby improving the peak shape of the analyte and increasing the accuracy of quantification.

[0023] In the determination method of diamide compounds in feed disclosed in the present invention, in step S3, the mass spectrometry detection conditions include: the ion source is an electrospray ion source, the monitoring mode is multiple reaction monitoring, and the scanning mode is a positive ion mode and a negative ion mode. The electrospray voltage in the positive ion mode is 4.5Kv, and the desolvation temperature is 550°C; the electrospray voltage in the negative ion mode is 3.5Kv, and the desolvation temperature is 550°C.

[0024] In the determination method of diamide compounds in feed disclosed in the present invention, in step S3, the specific operation of external standard method quantification is: accurately pipette an appropriate amount of mixed standard solution of diamide compounds and use acetonitrile to prepare a series of standard working solutions with concentrations of 1.0 μg / L, 2.0 μg / L, 5.0 μg / L, 10.0 μg / L, 20.0 μg / L and 50.0 μg / L, respectively, and sequentially determine the diamide compound standard solution and the test solution, draw a working curve with the concentration of the standard solution as the horizontal coordinate and the peak area of ​​the quantitative ion of the test object as the vertical coordinate, and quantify the sample.

[0025] The specific operation steps of the scheme of the present invention are as follows:

[0026] 5.0 g of the feed sample was weighed and placed in a 50 mL centrifuge tube, 25 mL of 85% acetonitrile solution was accurately added, mixed for 2 min, then 0.4 g of anhydrous magnesium sulfate and 1.2 g of sodium chloride were added and vortexed for 1 min, and after water bath ultrasonic extraction for 20 min, centrifuged at 8000 r / min for 5 min; 0.1 g of anhydrous magnesium sulfate was added to the supernatant after extraction and centrifugation and mixed for 1 min, 5 mL of the supernatant was accurately transferred after filtration, 95 mg of PSA, 95 mg of C18 and 10 mg of nano zirconium oxide purifier were added for joint purification, filtered through a 0.22 μm organic microporous filter membrane, and purified again through a Captiva EMR-Lipid purification column, the effluent was collected, the volume was made up to 5.0 mL with water, vortexed for 30 s, filtered through a 0.22 μm organic microporous filter membrane, and analyzed by HPLC-MS / MS, and quantified by the external standard method.

[0027] The specific steps of external standard method quantification are as follows:

[0028] Accurately pipette an appropriate amount of the mixed standard solution of bisamide compounds and use acetonitrile to prepare a series of standard working solutions with concentrations of 1.0μg / L, 2.0μg / L, 5.0μg / L, 10.0μg / L, 20.0μg / L and 50.0μg / L, respectively. Determine the standard solution and sample solution of bisamide compounds in turn, draw a working curve with the concentration of the standard solution as the horizontal coordinate and the peak area of ​​the quantitative ion of the analyte as the vertical coordinate, and quantify the sample.

[0029] The beneficial effects of the present invention are as follows:

[0030] 1) The determination method of the present invention can simultaneously determine compounds such as cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole in feed, with high detection accuracy and sensitivity, simple operation and good stability, which meets the current lack of determination methods for diamide compounds in feed and the need for supervision of pesticide residues in livestock products.

[0031] 2) The present invention overcomes the existing single purification method and uses a secondary purification method to greatly improve the recovery rate of the target compound. The first purification using a purifier effectively reduces the solubility of impurity components, facilitates the removal of most of the excess impurity components in the supernatant, and obtains a supernatant containing the target compound with higher purity. The CaptivaEMR-Lipid purification column is used for the second time. Due to its high selectivity, it can further remove the impurity components remaining in the supernatant after the first purification, thereby obtaining a high-purity supernatant containing the target compound. In addition, its efficient lipid removal can also ensure that the ion suppression of the target compound is minimized, thereby significantly improving the reliability and durability of the determination method.

[0032] 3) The present invention uses 85% acetonitrile solution and inorganic salts to extract three diamide compounds in feed for the first time; then uses a purifier and CaptivaEMR-Lipid purification column to purify the supernatant; uses HPLC-MS / MS analysis and external standard method to quantify the three diamide compounds in the sample simultaneously. The results show that at the addition level of 10 to 100 μg / kg, the average recovery rate of the three diamide compounds in feed is between 83.5% and 105.0%, the relative standard deviation is less than 3%, the detection limit (LOD) is between 2.9 and 4.3 μg / kg, and the quantification limit (LOQ) is between 9.1 and 13.5 μg / kg, which further illustrates that this method is suitable for the simultaneous determination of three diamide compounds of cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole in feed, and has the advantages of high sensitivity, high accuracy, simple and stable operation, etc.

[0033] The method for determining diamide compounds 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

[0034] Figure 1 It is a flow chart of the method for determining diamide compounds in feed of the present invention;

[0035] Figure 2 The chemical structures of cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole in the feed of the present invention are shown in FIG.

[0036] Figure 3 This is a chromatogram of cyantraniliprole and chlorantraniliprole in the feed of the present invention in the positive ion scanning mode

[0037] (Mobile phase: 5mmol / L ammonium acetate solution-methanol);

[0038] Figure 4 This is a chromatogram of cyantraniliprole and chlorantraniliprole in the feed of the present invention in the positive ion scanning mode

[0039] (Mobile phase: 0.1% formic acid solution-methanol);

[0040] Figure 5 The chromatogram of tetrachlorantraniliprole in the feed of the present invention in the negative ion scanning mode (mobile phase: 5mmol / L

[0041] Ammonium acetate solution-methanol);

[0042] Figure 6 The chromatogram of tetrachlorantraniliprole in the feed of the present invention in the negative ion scanning mode (mobile phase: water-methyl

[0043] alcohol);

[0044] Figure 7 It is a blank feed matrix chromatogram detected simultaneously in positive and negative ion modes of the present invention;

[0045] Figure 8 It is a chromatogram of feed additive samples detected simultaneously in positive and negative ion modes according to the present invention. DETAILED DESCRIPTION

[0046] The technical solutions in the embodiments of the present invention will be described clearly and completely below 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 the field without creative work are within the scope of protection of the present invention. It should be noted that the embodiments in this application and the features in the embodiments can be combined with each other without conflict.

[0047] The instruments and reagents used in the embodiments of the present invention are as follows:

[0048] Instruments and equipment: ultra-high performance liquid chromatography-mass spectrometry (TripleQuad5500+, SCIEX, USA), ultrasonic cleaning machine (2127QTD, Beijing Kexi Century Technology Co., Ltd.), desktop pure water analyzer (STARA212, Thermo Fisher Scientific (China) Co., Ltd.), electronic balance (XS205DU, Mettler-Toledo Instrument Co., Ltd.), high-speed refrigerated centrifuge (3-30K, SIGMA, Germany) and nitrogen blowdown instrument (TTL-DCII, Beijing Tongtai Lian Technology Development Co., Ltd.).

[0049] Reagent materials: Cyantraniliprole (purity 99.2%), chlorantraniliprole (purity 99.2%) and tetrachlorantraniliprole (purity 99.6%) were purchased from Tianjin Alta Technology Co., Ltd.; chromatographic grade methanol, acetonitrile and formic acid were purchased from ACS Chemical Company, USA; Captiva EMR-Lipid solid phase extraction cartridge (200 mg / 6 mL, Agilent).

[0050] Example 1

[0051] like Figure 1 As shown, the present invention discloses a method for determining diamide compounds in feed, wherein the diamide compounds are cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole, comprising the following steps: S1 obtaining a supernatant: first preparing an 85% acetonitrile solution and an inorganic salt component, wherein the 85% acetonitrile solution is prepared by adding water to 85 mL of acetonitrile to make a fixed volume of 100 mL, adding the prepared 85% acetonitrile solution and the inorganic salt component to the feed, extracting the cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole compounds in the feed by water bath ultrasound and centrifugation to obtain a supernatant; S2 obtaining a test liquid: pre-purifying the obtained supernatant with a purifier, and purifying the pre-purified supernatant again with a CaptivaEMR-Lipid purification column; collecting the effluent of the CaptivaEMR-Lipid purification column and filtering to obtain a test liquid; S3 testing and analyzing the test liquid: using liquid chromatography tandem mass spectrometry to detect and qualitatively analyze the obtained test liquid, and simultaneously using an external standard method for quantification.

[0052] In step S1, 5.0 g of the feed sample is weighed and placed in a 50 mL centrifuge tube. First, 25 mL of 85% acetonitrile solution is accurately added and mixed for 2 minutes. Then, 0.4 g of anhydrous magnesium sulfate and 1.2 g of sodium chloride are added and vortexed for 1 minute. After ultrasonic extraction in a water bath for 20 minutes, the supernatant is obtained by centrifugation at 8000 r / min for 5 minutes. 0.1 g of anhydrous magnesium sulfate is added to the supernatant after extraction and centrifugation, mixed for 1 minute, and then filtered.

[0053] In step S2, 5 mL of the filtered supernatant was accurately transferred, 95 mg PSA, 95 mg C18 and 10 mg nano zirconium oxide purifier were added for joint purification, and then filtered through a 0.22 μm organic microporous filter membrane, and then purified through a Captiva EMR-Lipid purification column, and the effluent of the Captiva EMR-Lipid purification column was collected, and finally the volume was adjusted to 5.0 mL with water, vortexed for 30 seconds, and filtered through a 0.22 μm organic microporous filter membrane to obtain the test solution.

[0054] In step S3, when liquid chromatography tandem mass spectrometry is used for detection and analysis, the liquid chromatography conditions are as follows: the chromatographic column is a C18 column, the column oven temperature is 40° C., the injection volume is 5 μL, the mobile phase system includes mobile phase A and mobile phase B, mobile phase A is a 5 mmol / L ammonium acetate solution, mobile phase B is methanol, the flow rate of the mobile phase system is 0.3 mL / min, and gradient elution is used. The gradient elution program is shown in Table 1 below:

[0055] Table 1 Gradient elution program

[0056] Time / min A volume fraction / % B volume fraction / % 0.00 95 5 1.00 95 5 5.00 5 95 7.00 5 95 7.10 95 5 10.00 95 5

[0057] The mass spectrometry condition parameters are as follows: Since cyantraniliprole and chlorantraniliprole have strong positive ion responses in the mass spectrometry, the positive ion scanning mode is selected; chlorantraniliprole has strong negative ion responses, so the negative ion scanning mode is selected, and multi-stage reaction monitoring (MRM) is used. The electrospray voltage in the positive ion mode is 5.5 kv, and the desolvation temperature is 550 ° C; the electrospray voltage in the negative ion mode is 4.5 kv, and the desolvation temperature is 550 ° C. The collision gas is high-purity nitrogen. Using A and B mobile phases (50:50, V / V) as solvents, standard solutions of cyantraniliprole, chlorantraniliprole and chlorantraniliprole with a concentration of 1000 μg / L were prepared respectively, and injected into the mass spectrometry analysis at a flow rate of 7 μL / min through a syringe pump. The mass spectrometry parameters such as cone voltage, collision energy and fragment ions are optimized as shown in Table 2 below to establish the mass spectrometry analysis conditions.

[0058] Table 2 MS / MS parameter settings for diamide compounds a. ESI+ monitoring mode

[0059]

[0060] b.ESI-monitoring mode

[0061]

[0062] The specific operation of external standard method quantification is as follows: accurately pipette an appropriate amount of mixed standard solution of diamide compounds and use acetonitrile to prepare a series of standard working solutions with concentrations of 1.0μg / L, 2.0μg / L, 5.0μg / L, 10.0μg / L, 20.0μg / L and 50.0μg / L, respectively; measure the standard solution of diamide compounds and the test solution in turn; draw a working curve with the concentration of the standard solution as the horizontal coordinate and the peak area of ​​the quantitative ion of the test object as the vertical coordinate, and quantify the sample.

[0063] The invention realizes the simultaneous determination of cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole in feed, has high detection accuracy and sensitivity, is simple to operate and has good stability, meets the current lack of a determination method for diamide compounds in feed and the need for supervision of pesticide residues in livestock products, and simultaneously uses 85% acetonitrile solution and inorganic salts to extract the three diamide compounds in the feed for the first time; then uses a purifier and a CaptivaEMR-Lipid purification column to purify the supernatant; and adopts HPLC-MS / MS analysis and an external standard method to simultaneously quantify the three diamide compounds in the sample. The results showed that at the addition level of 10 to 100 μg / kg, the average recovery rate of the three diamide compounds in feed was between 83.5% and 105.0%, the relative standard deviation was less than 3%, the detection limit (LOD) was between 2.9 and 4.3 μg / kg, and the quantification limit (LOQ) was between 9.1 and 13.5 μg / kg. This further demonstrated that this method is suitable for the simultaneous determination of three diamide compounds, cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole in feed, and has the advantages of high sensitivity, high accuracy, simple and stable operation.

[0064] Example 2 Methodological Study

[0065] In the present invention, linearity is analyzed by a standard solution curve, and a series of standard working solutions of cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole with concentrations of 1.0, 2.0, 5.0, 10.0, 20.0 and 50 μg / L are prepared with a feed blank matrix solution, and a series of standard working solutions of cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole with concentrations of 1.0, 2.0, 5.0, 10.0, 20.0 and 50 μg / L are prepared with acetonitrile.

[0066] 10.0, 20.0, and 50 μg / L) standard working solution, by evaluating the correlation coefficient (r) of the above two standard curves, it should not be less than 0.99. At the same time, the sample matrix effect (ME) is evaluated according to the ratio of the slope of the working curve of the matrix matching standard solution and the slope of the working curve of the pure solvent standard solution. The ratio of the slopes should be between 80% and 120%. When the slope ratio is greater than 120% or less than 80%, the matrix effect will significantly affect the analysis results.

[0067] The detection limit and quantification limit of the method were investigated by adding low concentration (10 μg / kg) of diamide compounds into blank feed matrix. 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.

[0068] Add the mixed standard solution of diamide compounds to 5.0g of blank matrix feed (6 parallels), and set up three different concentration levels (10μg / kg, 40μg / kg and 100μg / kg) of spiked samples. After processing according to the above extraction and purification steps, the average recovery rate of diamide compounds was calculated by LC-MS / MS quantitative analysis. When the content of the measured component is less than 0.1mg / kg, the recovery rate should be between 60% and 120%, and when the content of the measured component is between 0.1 and 1mg / kg, the recovery rate should be between 80% and 110%.

[0069] The precision of the method needs to be repeated at least 6 times and expressed as relative standard deviation (RSD). When the content of the measured component is 10μg / kg, the relative standard deviation should be less than 21%, and when the content of the measured component is 100μg / kg, the relative standard deviation should be less than 15%.

[0070] Example 3 Optimization of mobile phase system

[0071] The positive ion scanning mode was used to compare the 5mmol / L ammonium acetate solution-methanol ( Figure 3 ) and 0.1% formic acid solution-methanol ( Figure 4 ) two mobile phase systems; tetrachlorantraniliprole was used in negative ion scanning mode to compare 5mmol / L ammonium acetate solution-methanol ( Figure 5 ) and water-methanol ( Figure 6 ) two mobile phase systems. The results showed that the sensitivity and peak shape obtained by the two mobile phase systems used for cyantraniliprole and chlorantraniliprole in the positive ion scanning mode were comparable, and that in the negative ion scanning mode, chlorantraniliprole using a 5mmol / L ammonium acetate solution-methanol mobile phase system could obtain greater sensitivity and optimal peak shape. Taking into account the simultaneous positive and negative ion scanning modes, this method adopted an ammonium acetate solution (A: containing 5mmol / L ammonium acetate) and methanol (B) system as the mobile phase. Under the optimized chromatographic conditions, the retention times of cyantraniliprole, chlorantraniliprole and chlorantraniliprole were 4.46min, 4.75min and 5.12min, respectively, and the analysis time was 10min.

[0072] Example 4 Selection of Extractant

[0073] The common solvents for the extraction of agricultural residues include acetonitrile, ethyl acetate, acetone and normal hexane mixed solution, etc., because acetone has a low solubility, high toxicity, and is volatile, and the ethyl acetate extraction efficiency is not high, and acetonitrile has a strong solubility for three kinds of diamide compounds, and the solubility for grease, pigment and protein substances is weak, and the dispersibility of the sample can be increased by adding an appropriate amount of water at the same time, and the extraction efficiency is enhanced, so it is considered that 85% acetonitrile solution is used as the first choice of extraction solvent. At the same time, in order to improve the ionic strength of acetonitrile in acetonitrile solution, accelerate the ionization reaction of the substance, add anhydrous magnesium sulfate and an inorganic salt of sodium chloride in a certain mass ratio, promote the interaction between acetonitrile and target compound molecules, increase the reaction rate, so that the target compound can be quickly and completely dissolved in acetonitrile, and acetonitrile phase and water phase are layered, which is convenient for subsequent separation. And considering that the matrix of the present invention is feed, its water content is relatively low, the consumption of anhydrous magnesium sulfate can be appropriately reduced, and the excessive consumption is prevented from causing the volume reduction of acetonitrile, so the mass ratio of anhydrous magnesium sulfate and sodium chloride can be selected to be 1:3, and a good layering effect can be obtained.

[0074] Example 5 Selection of Purifier and Purification Column

[0075] The present invention, when selecting a purifier, respectively compares the impact of 5 purifiers of C18, PSA, GCB, multi-walled carbon nanotubes and nano-zirconia on the purification effects of cyanantraniliprole, chlorantraniliprole and tetrachlorantraniliprole in feed. Wherein, it is found by purification alone that the purification effects of multi-walled carbon nanotubes and GCB are not good, and serious adsorption will be produced during purification, resulting in a low recovery rate. And C18, PSA, nano-zirconia purification effects perform better, and a higher recovery rate can be obtained, therefore investigating the impact of different combinations of C18, PSA and nano-zirconia (as shown in Table 1 below) on the recovery rate (as shown in Table 2 below).

[0076] Table 1 (different combinations)

[0077]

[0078]

[0079] Table 2 (Effect of different combinations on recovery rate)

[0080]

[0081] It can be seen from Table 2 that the recovery rates of the target compounds in Schemes 1, 2, 3 and 4 are all above 85%, which meets the requirements of the analysis. Finally, considering the factors such as impurity removal efficiency, interference near the peak and recovery rate, 95 mg PSA, 95 mg C18 and 10 mg nano zirconium oxide in Scheme 3 were selected as purifiers for combined purification application.

[0082] In this paper, pig feed was used as the matrix for optimizing purification conditions, and the HLB purification column, CaptivaEMR-Lipid purification column and C 18 The effect of purification column on the recovery rate of diamide compounds in feed. The results showed that the average recovery rate of the compounds after purification by CaptivaEMR-Lipid column was 83.5% to 105.0%, which was higher than that of HLB column and C 18 The average recovery rate of the compounds after column purification was 79.7% to 91.2%, which was relatively high. The CaptivaEMR-Lipid column has a better recovery rate after purification, firstly because it can remove lipids / matrix with high selectivity and efficiency without causing analyte loss; secondly, its efficient lipid removal can ensure that the ion suppression of the target compound is minimized, thereby significantly improving the reliability and durability of the method. In addition, the CaptivaEMR-Lipid purification column is easy to operate, can meet the easy flow under vacuum or positive pressure drive, is easy and convenient to use, and is very suitable for feeds with complex matrices.

[0083] Example 6 Methodology Verification

[0084] Matrix effect refers to the influence of other components in the sample other than the target compound on the response value of the target compound. This effect usually leads to ion suppression or ion enhancement of the target compound and affects the accuracy and precision of the established method. Common matrix effect compensation methods include isotope internal standard method, standard addition method and matrix matching standard solution method.

[0085] Table 3 shows the matrix effects of the three diamide compounds. From the results, the matrix effect (ME / %) is between 95% and 103%, which meets the range requirement of 80% to 120%. The matrix effects of cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole in feed are not obvious, so the pure solvent standard working curve within the linear range is used for quantification. Other methodological evaluation results are shown in Table 3.

[0086] Table 3 Methodological evaluation results a. Standard curve regression equation, detection limit and quantification limit

[0087]

[0088] b. Accuracy and precision

[0089]

[0090] The standard working solutions of cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole mixed standard samples with a series of concentrations (1.0, 2.0, 5.0, 10.0, 20.0 and 50 μg / L) were prepared with acetonitrile solvent, and the correlation coefficients (r) of the standard curves were all greater than 0.999. The detection limits of the three compounds in feed were between 2.9 and 4.3 μg / kg, and the quantification limits were between 9.1 and 13.5 μg / kg. The average recoveries of three different added concentration levels (10 μg / kg, 40 μg / kg and 100 μg / kg) were between 83.5% and 105.0%, and the relative standard deviation of precision was between 1.49% and 2.99%, with high sensitivity, accuracy and good reproducibility.

[0091] In summary, the present invention uses 85% acetonitrile solution and inorganic salts to extract three diamide compounds (cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole) in feed for the first time, and uses a purifier and CaptivaEMR-Lipid purification column to purify the extract, and realizes the simultaneous quantification of cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole by HPLC-MS / MS analysis method, and obtains satisfactory results in sensitivity, accuracy and precision. The method is suitable for the simultaneous detection of three diamide compounds in feed.

[0092] The above are only preferred embodiments of the present invention and are 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.

[0093] 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 determining diamide compounds in feed, characterized in that: The diamide compounds are cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole, comprising the following steps: S1 obtaining a supernatant: first preparing an 85% acetonitrile solution and an inorganic salt component, adding the prepared 85% acetonitrile solution and the inorganic salt component to the feed, extracting cyantraniliprole, chlorantraniliprole and tetrachlorantraniliprole compounds in the feed by water bath ultrasound and centrifugation to obtain a supernatant; S2: obtaining the liquid to be tested: pre-cleaning the obtained supernatant with a purifying agent, and purifying the pre-cleaned supernatant again through a CaptivaEMR-Lipid purification column; collecting the effluent of the CaptivaEMR-Lipid purification column and filtering it to obtain the liquid to be tested; S3 Detection and analysis of the test liquid: Use liquid chromatography tandem mass spectrometry to detect and qualitatively analyze the obtained test liquid, and use the external standard method for quantitative analysis; Among them, 85% acetonitrile solution is prepared by adding water to 85mL acetonitrile to make up to 100mL; In step S1, the inorganic salt component is prepared by mixing anhydrous magnesium sulfate and sodium chloride; In step S1, after obtaining the supernatant by water bath ultrasound and centrifugation, anhydrous magnesium sulfate is added, mixed and then filtered; In step S2, the purifier is selected from C18, PSA and nano zirconium oxide for combined purification; In step S3, when liquid chromatography tandem mass spectrometry is used for detection and analysis, the liquid chromatography conditions are: the chromatographic column is a C18 column, the mobile phase system includes mobile phase A and mobile phase B, mobile phase A is a 5 mmol / L ammonium acetate solution, mobile phase B is methanol, and gradient elution is used. The gradient elution procedure is as follows: 0.0~1.0min, the volume percentage of mobile phase A is 95%; From 1.0 to 5.0 min, the volume percentage of mobile phase A decreases at a constant rate from 95% to 5%; 5.0-7.0min, the volume percentage of mobile phase A is 5%; From 7.0 to 7.1 min, the volume percentage of mobile phase A increased uniformly from 5% to 95%; 7.1-10.0min, the volume percentage of mobile phase A is 95%; In step S3, the mass spectrometry condition parameters are: the ion source is an electrospray ion source, the monitoring mode is multiple reaction monitoring, the scanning mode is positive ion mode and negative ion mode, positive ion scanning mode is selected for cyantraniliprole and chlorantraniliprole, and negative ion scanning mode is selected for tetrachlorantraniliprole.

2. The method for determining diamide compounds in feed according to claim 1, characterized in that: In the step S1, the inorganic salt component is prepared by mixing anhydrous magnesium sulfate and sodium chloride in a mass ratio of 1:

3.

3. The method for determining diamide compounds in feed according to claim 1, characterized in that: In the step S1, the prepared acetonitrile solution and inorganic salt component are added to the feed and the supernatant is obtained by water bath ultrasound and centrifugation as follows: the feed sample is placed in a centrifuge tube, firstly the prepared acetonitrile solution is added and vortexed for 2 minutes, then the prepared inorganic salt component is added and vortexed for 1 minute, extracted by water bath ultrasound for 20 minutes, then centrifuged at 8000r / min for 5 minutes, and finally the supernatant is collected.

4. The method for determining diamide compounds in feed according to claim 1, characterized in that: In the step S2, the operation of obtaining the test liquid is as follows: the supernatant obtained in the step S1 is removed, the supernatant is pre-purified by using a purifier and then filtered, and the filtered supernatant is purified again by a CaptivaEMR-Lipid purification column, and the effluent of the CaptivaEMR-Lipid purification column is collected, and finally the volume is fixed with water, vortexed for 30 seconds, and filtered through a 0.22 μm organic microporous filter membrane to obtain the test liquid.

5. The method for determining diamide compounds in feed according to any one of claims 1 to 4, characterized in that: In step S3, the column oven temperature is 40° C., the injection volume is 5 μL, and the flow rate of the mobile phase system is 0.3 mL / min.

6. The method for determining diamide compounds in feed according to claim 5, characterized in that: In the step S3, the electrospray voltage in the positive ion mode is 4.5 Kv, and the desolvation temperature is 550°C; the electrospray voltage in the negative ion mode is 3.5 Kv, and the desolvation temperature is 550°C.

7. The method for determining diamide compounds in feed according to claim 1, characterized in that: In step S3, the operation of external standard method quantification is as follows: an appropriate amount of mixed standard solution of diamide compounds is pipetted and prepared with acetonitrile into a series of standard working solutions with concentrations of 1.0 μg / L, 2.0 μg / L, 5.0 μg / L, 10.0 μg / L, 20.0 μg / L and 50.0 μg / L, respectively; the standard solution of diamide compounds and the test solution are sequentially measured by liquid chromatography tandem mass spectrometry; a working curve is drawn with the concentration of the standard solution as the horizontal coordinate and the peak area of ​​the quantitative ion of the test object as the vertical coordinate, and the sample is quantified.

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