A method for detecting the enantiomers of indoxamide and its main metabolites in fruits and vegetables and cereals

Abstract

The present application belongs to the technical field of pesticide residue detection, and particularly relates to a method for detecting diacloden and its main metabolite enantiomers in fruits, vegetables and grains. The method comprises the following steps: mixing and oscillating the sample to be tested with acetonitrile, mixing and oscillating the mixture, sodium chloride and magnesium sulfate, separating the solid and liquid to obtain an extraction solution; mixing and purifying the extraction solution with multi-walled carbon nanotubes, separating the solid and liquid to obtain a test solution; detecting the test solution by liquid chromatography-mass spectrometry, and obtaining the content of diacloden and its two main chiral metabolite enantiomers in the sample to be tested according to the peak area and the standard curve. The method can simultaneously baseline separate and detect diacloden and its main metabolite enantiomers, and has high sensitivity. The method has good repeatability and reproducibility, and can simultaneously detect diacloden and its main metabolite M550I002 and M550I006 enantiomers in grapes, citrus, cabbage, cucumber, soybean and dry corn.

Description

Technical Field

[0001] This invention belongs to the field of pesticide residue detection technology, specifically relating to a method for detecting enantiomeric acetamiprid and its main metabolites in fruits, vegetables and grains. Background Technology

[0002] Grapes and citrus fruits are important economic crops, processed into many kinds of food, such as wine, raisins, and canned citrus fruits, which are very popular with consumers. Cabbage and cucumbers are common vegetables, frequently appearing on dining tables. Soybeans and corn are important food crops, especially soybeans, which are an important source of protein for the human body and play a vital role in ensuring public health and food security. The cultivation of grapes, citrus fruits, cabbage, cucumbers, soybeans, and corn is often accompanied by various pests and diseases, and chemical control, mainly using pesticides, is currently one of the fastest and most effective methods for controlling plant pests and diseases. Dimpropyridazine is a pyrazole amide insecticide with a novel mechanism of action, showing good control efficacy against aphids, silver leaf whiteflies, and greenhouse whiteflies in brassica vegetables, fruit vegetables, leafy vegetables, and cotton. Although dimpropyridazine is a highly effective and low-toxicity insecticide, improper application still poses a risk of residue to plants and the environment. These residues can enter the human body through food ingestion and ultimately harm human health. According to reports, when fenpropathrin is applied to field crops, it will produce five major metabolites, among which M550I002 and M550I006 are two major metabolites in plant samples.

[0003] Chiral pesticides account for nearly 40% of all pesticides, and this proportion is gradually increasing with the continuous market launch of new structural pesticides. Currently, most chiral pesticides are produced, sold, and used in racemic form, and their enantiomers often exhibit significant differences in biological activity, toxicity, and environmental impact. For these reasons, conducting safety evaluations of chiral pesticides at the enantiomer level is of great importance. Therefore, developing enantiomer separation methodologies for target chiral pesticides and establishing methods for residual analysis of chiral pesticide enantiomers in different samples and matrices is crucial for conducting the aforementioned safety evaluation studies. Structurally, chlorfenapyr and its two metabolites (M550I002 and M550I006) both contain a chiral carbon atom, meaning they each contain a pair of enantiomers. Currently, most reports on chlorfenapyr focus on its toxicity effects and efficacy evaluation; no reports have been found on the separation of chiral enantiomers from its parent compound and major metabolites, or on residue detection methods in plant-derived foods. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for detecting enantiomeric acetamiprid and its main metabolites in fruits, vegetables, and grains. This invention enables chiral separation of enantiomeric acetamiprid and its main metabolites, and simultaneous detection of residues in fruit, vegetable, and grain samples such as grapes, citrus fruits, cabbage, cucumbers, soybeans, and dried corn, with high sensitivity.

[0005] This invention provides a method for detecting enantiomeric acetamiprid and its main metabolites in fruits, vegetables, and grains, wherein the main metabolites include M550I002 and M550I006, comprising the following steps: The fruit, vegetable or grain sample to be tested is mixed with acetonitrile and shaken for the first time. The resulting mixture is then mixed with sodium chloride and magnesium sulfate and shaken for the second time. Solid-liquid separation is performed to obtain the extract. The extract was purified by mixing it with multi-walled carbon nanotubes with an outer diameter of 10-30 nm, followed by solid-liquid separation to obtain the test solution. The test solution is subjected to liquid chromatography-mass spectrometry detection. Based on the obtained peak area and the predetermined standard curve, the content of acetamiprid and its main metabolite enantiomerics in the test fruit, vegetable or grain sample is obtained. The liquid chromatography conditions for the liquid chromatography-mass spectrometry detection include: Chromatographic column: chiral column; mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.05-0.2% (v / v) aqueous solution of formic acid and mobile phase B is acetonitrile; flow rate: 0.3-0.5 mL / min, isocratic elution: 0-15 min, and the volume fraction of mobile phase B is maintained at 20-40%; The mass spectrometry conditions for the liquid chromatography-mass spectrometry detection include: Multiple reaction monitoring mode; Ionization source mode: ESI + Atomizing gas: nitrogen; Spray voltage: 5000~6000V; Ion source temperature: 500~600℃; Pressure of atomizing gas and auxiliary gas: 40~60psi and 40~60psi respectively.

[0006] Preferably, the outer diameter of the multi-walled carbon nanotube is 10~20 nm.

[0007] Preferably, the volume ratio of the extract to the mass of the multi-walled carbon nanotubes is 1.5 mL: 4~6 mg.

[0008] Preferably, the chiral column is a Chiral MZ(2)-RH column with dimensions of 2.1×150mm and 3μm.

[0009] Preferably, the flow rate is 0.4 mL / min.

[0010] Preferably, the volume fraction of the mobile phase B is maintained at 30%.

[0011] Preferably, the liquid chromatography conditions for liquid chromatography-mass spectrometry detection further include a column temperature of 30~40℃.

[0012] Preferably, the predetermined standard curve is a matrix-matched standard curve with a linear range of 0.0005~0.1 mg / L.

[0013] Preferably, when the sample to be tested is a grain sample, the method further includes mixing the sample to be tested with water before mixing with acetonitrile; the mass ratio of the sample to the volume of water is 1g:1~3mL.

[0014] Preferably, the ratio of the mass of the fruit, vegetable or grain sample to the volume of acetonitrile is 4~6g:20~30mL.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for detecting acetamiprid and its main metabolite enantiomers in fruits, vegetables, and grains, wherein the main metabolites include M550I002 and M550I006. The method includes the following steps: mixing the fruit / vegetable or grain sample to be tested with acetonitrile and performing a first shaking; mixing the resulting mixture with sodium chloride and magnesium sulfate and performing a second shaking; performing solid-liquid separation to obtain an extract; purifying the extract by mixing it with multi-walled carbon nanotubes with an outer diameter of 10-30 nm; performing solid-liquid separation to obtain a test solution; performing liquid chromatography-mass spectrometry on the test solution; and quantitatively calculating the content of acetamiprid and its two main metabolite enantiomers in the fruit / vegetable or grain sample based on the obtained peak area and matrix matching standard curve.

[0016] The detection method provided by this invention can achieve chiral separation and simultaneous detection of enantiomers of fenproxil fumarate and its main metabolites, and has high sensitivity.

[0017] Furthermore, by optimizing the QuEChERS method, the present invention develops a stable, practical, and sensitive analytical method that can simultaneously detect the residues of acetonitrile enantiomers, M550I002 enantiomers, and M550I006 enantiomers in grapes, citrus fruits, cabbage, cucumbers, soybeans, and dried corn.

[0018] Test data show that the average recoveries of the six enantiomers in grapes, citrus fruits, cabbage, cucumbers, soybeans, and dried corn ranged from 74.4% to 95.3%, with relative standard deviations (RSDs) of 1.2% to 6.4%, indicating good repeatability and reproducibility of the detection method. The method of this invention features a rapid and simple pretreatment process, good instrumental sensitivity, and high accuracy. It can be used for the detection and analysis of imidazole enantiomers, M550I002 enantiomer, and M550I006 enantiomer residues in grapes, citrus fruits, cabbage, cucumbers, soybeans, and dried corn. This invention establishes a UHPLC-MS / MS enantiomeric detection method for the chiral pesticide acetamiprid and its two main chiral metabolites in grapes, citrus, cabbage, cucumber, soybeans, and dried corn. This provides an analytical method for future research on the residual degradation of acetamiprid enantiomeric, M550I002 enantiomeric, and M550I006 enantiomeric in grapes, citrus, cabbage, cucumber, soybeans, and dried corn during their growth process, as well as for residue analysis in other agricultural products, thus ensuring the quality and safety of agricultural products. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The diagram shows the structural formulas of the enantiomers of the three chiral compounds of this invention. Figure 2 The solvent standard chromatograms of the three chiral compounds (0.5 mg / L) in Example 1 are shown. Figure 3 The images show the effects of purifying the three blank sample matrices (grape, cucumber, and dried corn) in Example 2 using different purifying agents. Figure 4 This is a graph showing the effect of different purifying agents on the recovery rate in Example 2; Figure 5 This is a graph showing the effect of different amounts of purifying agent on the recovery rate in Example 2; Figure 6 and Figure 7 The graph shows the separation performance of six different stationary phase chiral columns in Example 2. Figure 6 For Chiral MY(2)-RH, MX(2)-RH and MZ(2)-RH columns, Figure 7 For MS(2)-RH, NQ(2)-RH and NT(2)-RH columns; Figure 8 and Figure 9This is a diagram showing the separation effect of different mobile phase compositions in Example 2. Figure 8 For different volume ratios of water / acetonitrile, Figure 9 Different volume ratios of water / methanol; Figure 10 This is a graph showing the separation effect of different proportions of mobile phases in Example 2, using 0.1% formic acid water as the aqueous phase and acetonitrile as the organic phase. Figure 11 This is a diagram showing the separation effect at different flow rates in Example 2; Figure 12 This is a diagram showing the separation effect at different column temperatures in Example 2. Detailed Implementation

[0021] This invention provides a method for detecting enantiomeric acetamiprid and its main metabolites in fruits, vegetables, and grains, wherein the main metabolites include M550I002 and M550I006, comprising the following steps: The fruit, vegetable or grain sample to be tested is mixed with acetonitrile and shaken for the first time. The resulting mixture is then mixed with sodium chloride and magnesium sulfate and shaken for the second time. Solid-liquid separation is performed to obtain the extract. The extract was purified by mixing it with multi-walled carbon nanotubes with an outer diameter of 10-30 nm, followed by solid-liquid separation to obtain the test solution. The test solution is subjected to liquid chromatography-mass spectrometry detection. Based on the obtained peak area and the predetermined standard curve, the content of acetamiprid and its main metabolite enantiomerics in the test fruit, vegetable or grain sample is obtained. The liquid chromatography conditions for the liquid chromatography-mass spectrometry detection include: Chromatographic column: chiral column; mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.05-0.2% (v / v) aqueous solution of formic acid and mobile phase B is acetonitrile; flow rate: 0.3-0.5 mL / min, isocratic elution: 0-15 min, and the volume fraction of mobile phase B is maintained at 20-40%; The mass spectrometry conditions for the liquid chromatography-mass spectrometry detection include: Multiple reaction monitoring mode; Ionization source mode: ESI + Atomizing gas: nitrogen; Spray voltage: 5000~6000V; Ion source temperature: 500~600℃; Pressure of atomizing gas and auxiliary gas: 40~60psi and 40~60psi respectively.

[0022] Unless otherwise specified, all materials and equipment used in this invention are commercially available products in the field.

[0023] In this invention, the structural formula of the M550I002 enantiomer is shown in Formula I, and the structural formula of the M550I006 enantiomer is shown in Formula II: Formula I, Formula II.

[0024] In this invention, the fruit, vegetable or grain sample to be tested is mixed with acetonitrile and subjected to a first oscillation. The resulting mixture is then mixed with sodium chloride and magnesium sulfate and subjected to a second oscillation. Solid-liquid separation is then performed to obtain an extract.

[0025] In this invention, the fruits and vegetables preferably include grapes, citrus fruits, cabbage, or cucumbers, and the grains preferably include soybeans or corn, with the corn preferably being dried corn. The fruit / vegetable or grain samples are preferably homogenized samples.

[0026] In this invention, when the sample to be tested is grain, the method further includes mixing the sample to be tested with water before mixing with acetonitrile; the mass ratio of the sample to the volume of water is preferably 1g:1~3mL, specifically 1g:2mL.

[0027] In this invention, the preferred ratio of the mass of the fruit, vegetable or grain sample to the volume of acetonitrile is 4-6 g: 20-30 mL, specifically 5 g: 25 mL.

[0028] In this invention, the first oscillation time is preferably 5-15 minutes, specifically 10 minutes. This invention uses an oscillation method for extraction, resulting in high extraction efficiency.

[0029] In this invention, the magnesium sulfate is preferably anhydrous magnesium sulfate, and the mass ratio of the fruit / vegetable or grain sample to be tested, sodium chloride (NaCl), and anhydrous magnesium sulfate (MgSO4) is preferably 4~6:0.9~1.1:3~5, specifically 5:1:4. The second oscillation time is preferably 3~10 min, specifically 5 min. The solid-liquid separation is preferably centrifugation, and the centrifugation speed is preferably 4000 r / min, and the time is preferably 5 min. The addition of sodium chloride in this invention utilizes the salting-out effect to increase the ionic strength of the aqueous phase, reducing the solubility of the target pesticide in water, and promoting its easier partitioning into the acetonitrile phase, thereby improving the extraction efficiency of the method; the addition of anhydrous magnesium sulfate can effectively remove water from the extract, thereby reducing the interference of water in the sample on subsequent instrument analysis. Simultaneously, anhydrous magnesium sulfate and sodium chloride synergistically enhance the separation of the organic phase (acetonitrile) and the aqueous phase.

[0030] After obtaining the extract, the present invention mixes the extract with multi-walled carbon nanotubes with an outer diameter of 10~30nm for purification, and performs solid-liquid separation to obtain the test solution.

[0031] In this invention, the outer diameter of the multi-walled carbon nanotubes is preferably 10-20 nm. This invention uses 10-20 nm multi-walled carbon nanotubes for purification, resulting in good purification performance.

[0032] In this invention, the volume ratio of the extract to the mass of the multi-walled carbon nanotubes is preferably 1.5 mL: 4~6 mg, specifically 1.5 mL: 5 mg. The purification is preferably performed under vortex conditions, with the vortexing time preferably 1 min. The solid-liquid separation is preferably centrifugation, with the centrifugation speed preferably 4000 r / min and the centrifugation time preferably 5 min. The solid-liquid separation process preferably further includes: filtering the obtained clarified liquid through a membrane; the pore size of the filter membrane used for membrane filtering is preferably 0.22 μm.

[0033] After obtaining the test solution, the present invention performs liquid chromatography-mass spectrometry detection on the test solution, and obtains the content of acetamiprid and its main metabolite enantiomerics in the test fruit, vegetable or grain sample based on the obtained peak area and the predetermined standard curve. The liquid chromatography conditions for the liquid chromatography-mass spectrometry detection include: Chromatographic column: chiral column; mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.05-0.2% (v / v) aqueous solution of formic acid and mobile phase B is acetonitrile; flow rate: 0.3-0.5 mL / min, isocratic elution: 0-15 min, and the volume fraction of mobile phase B is maintained at 20-40%; The mass spectrometry conditions for the liquid chromatography-mass spectrometry detection include: Multiple reaction monitoring mode; Ionization source mode: ESI + Atomizing gas: nitrogen; Spray voltage: 5000~6000V; Ion source temperature: 500~600℃; Pressure of atomizing gas and auxiliary gas: 40~60psi and 40~60psi respectively.

[0034] In this invention, the chiral column is preferably a Chiral MZ(2)-RH column, with a preferred size of 2.1×150mm and 3μm.

[0035] In this invention, the mobile phase A is preferably an aqueous solution of formic acid with a volume fraction of 0.1%.

[0036] In this invention, the flow rate is preferably 0.4 mL / min; the volume fraction of the mobile phase B is preferably maintained at 30%.

[0037] In this invention, the liquid chromatography-mass spectrometry detection is preferably performed using an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) instrument.

[0038] In this invention, the liquid chromatography conditions for liquid chromatography-mass spectrometry detection preferably include: a column temperature of 30~40℃, specifically 30℃, 35℃ or 40℃; and an injection volume of 5μL.

[0039] In this invention, the spray voltage is preferably 5500V; the pressures of the atomizing airflow rate (GS1) and the auxiliary airflow rate (GS2) are preferably 50psi and 50psi, respectively.

[0040] In this invention, the mass spectrometry parameters of the enantiomeric forms of the imidazole and its main metabolites (see Table 1) preferably include: The parent ion m / z of the enantiocyanate is 302.4±0.2, the daughter ion m / z is 206.1±0.2 / 179.1±0.2, the declustering voltage is 95V, and the collision energy is 18V / 22V. The parent ion m / z of the enantiomer M550I002 is 274.3±0.2, the daughter ion m / z is 109.1±0.2 / 179.1±0.2, the declustering voltage is 92V, and the collision energy is 37V / 21V. The parent ion m / z of the enantiomer M550I006 is 197.4±0.2, the daughter ion m / z is 71.1±0.2 / 127.1±0.2, the declustering voltage is 47V, and the collision energy is 18V / 20V.

[0041] In this invention, the predetermined standard curve is preferably a matrix-matched standard curve, and the linear range of the six enantiomers of chlorfenapyr and its two major metabolites is preferably 0.0005~0.1 mg / L. This invention preferably uses the external standard method for quantification. This invention uses matrix-matched standard curves to quantify the six enantiomers, and the linear correlation coefficients of the standard curves for the enantiomers of chlorfenapyr and its major metabolites in different matrices are all greater than 0.99.

[0042] This invention provides a method for detecting enantiomers of acetamiprid, M550I002 enantiomers, and M550I006 enantiomers in grapes, citrus fruits, cabbage, cucumbers, soybeans, and dried corn using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). This invention improves the QuEChERS pretreatment technique and, combined with UHPLC-MS / MS, establishes a method for simultaneously detecting the residues of acetamiprid and its two main metabolites, M550I002 and M550I006 enantiomers. This method features a rapid and simple pretreatment process, good instrument sensitivity, a limit of quantitation of 10 μg / kg, and high accuracy. It can provide a basis for risk assessment and safe use studies of acetamiprid enantiomers and their two main metabolite enantiomers in grapes, citrus fruits, cabbage, cucumbers, soybeans, and dried corn.

[0043] To further illustrate the present invention, the detection method for imizoamide and its main metabolites in fruits, vegetables and grains provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1 Experiments and Methods 1. Materials and Reagents Standards: Racemic chlorfenapyr (purity > 99.5%), Dr. Ehrenstorfer GmbH, Germany, product code: DRE-C1277600, catalog number: 1270935; Racemic M550I002 (purity > 95.0%), Racemic M550I006 (purity > 95.0%), synthesized by Chang Le County Han's Agricultural Technology Service Co., Ltd.; Structural formulas of the enantiomers of the three chiral compounds are as follows: Figure 1 As shown; Formic acid [mass spectrometry grade, Thermo Fisher Scientific (China) Co., Ltd.]; Acetonitrile [chromatographic grade, Thermo Fisher Scientific (China) Co., Ltd.]; N -Propylethylenediamine (PSA); graphitized carbon (GCB); multi-walled carbon nanotubes (MWCNTs with outer diameters of <8 nm, 10–20 nm, and 20–30 nm); water is distilled water.

[0045] 2. Instruments and Equipment AB SCIEX 6500+ triple quadrupole liquid chromatography-mass spectrometry system (UHPLC-MS / MS, AB SCIEX, USA); Chiral MZ(2)-RH column (2.1 mm × 150 mm, 3 μm, Guangzhou Philomen Scientific Instruments Co., Ltd.); CK2000 high-throughput tissue homogenizer (Beijing Tomogen Biotechnology Co., Ltd.).

[0046] 3. Preparation of standard solutions and plotting of standard curves Accurately weigh the three compound standards into 10 mL volumetric flasks, dissolve and dilute with methanol to prepare a 200 mg / L standard stock solution, and store at -20 °C.

[0047] Fresh grapes, citrus fruits, cabbage, cucumbers, soybeans, and dried corn blank samples were taken and processed according to "4. Pretreatment Method" to obtain blank matrices. The above standard stock solutions were diluted with blank matrices or methanol solvent to 0.0005, 0.001, 0.0025, 0.005, 0.025, 0.05, and 0.1 mg / L, respectively. The concentrations were measured according to "5. Instrument Conditions". A standard curve was plotted with concentration as the abscissa (x) and peak area as the ordinate (y).

[0048] 4. Pretreatment methods Weigh 5.00 g of homogenized fresh grapes, citrus fruits, cabbage, cucumbers, soybeans, and dried corn samples into a 50 mL centrifuge tube. Add 10 mL of purified water (no water is needed for grape, citrus, cabbage, and cucumber samples), then add 25 mL of acetonitrile. Shake for 10 min, add 1.0 g of NaCl and 4 g of anhydrous magnesium sulfate, shake for 5 min, and centrifuge at 8000 r / min for 5 min. Pipette 1.5 mL of the upper organic phase solution into a 5 mL centrifuge tube containing 5 mg of multi-walled carbon nanotubes (MWCNTs, outer diameter 10–20 nm), vortex for 1 min, centrifuge at 4000 r / min for 5 min, and filter the supernatant through a 0.22 μm filter membrane into a sample vial to obtain the test solution. Perform UHPLC-MS / MS analysis.

[0049] 5. Instrument conditions Chromatographic conditions: Chiral MZ(2)-RH column (2.1×150 mm, 3 μm); column temperature 35℃; injection volume 5 μL; flow rate 0.40 mL / min; mobile phase A is 0.1% formic acid aqueous solution and B is acetonitrile; elution program is: hold 30% B for 15 min.

[0050] Mass spectrometry conditions: Ionization source mode: Electrospray ionization (ESI); Ion source polarity: ESI + Nebulizing gas: nitrogen; spray voltage: 5500 V; ion source temperature: 550 ℃; GS 1: 50 psi, GS 2: 50 psi; mass spectrometry parameters of acetamiprid and its metabolites are shown in Table 1.

[0051] Table 1 Mass spectrometric parameters of acetamiprid and its metabolites

[0052] Note: * indicates quantitative ions. The three chiral compounds were finally confirmed using the established instrumental method. The UHPLC-MS / MS spectra of the three chiral compounds (concentration 0.5 mg / L) are shown below. Figure 2 As shown.

[0053] Example 2 In this embodiment, a spiking dose of 0.1 mg / kg was used for the experiment. Unless otherwise specified, the conditions were the same as in Example 1.

[0054] 1. The influence of purification conditions Grape, citrus, cabbage, cucumber, soybean, and dried corn matrices are relatively complex. Acetonitrile can extract or dissolve many substances within them, which may interfere with detection and affect residue testing. Therefore, the selection of a purification agent is particularly important. PSA, GCB, and C18 are commonly used as purification agents in the QuEChERS method. PSA (… N C18 (octadecylsilane-bonded silica gel) can remove sugars, organic acids and some polar pigments from the sample matrix; C18 (octadecylsilane-bonded silica gel) is used to adsorb non-polar impurities such as lipids and esters from polar samples; GCB (graphitized carbon black) can remove hydrophobic compounds such as pigments and sterols from the matrix.

[0055] The experiment selected grapes, cucumbers, and dried corn as representative complex sample matrices containing a large amount of pigments and sterols. The effects of PSA, GCB, PSA+GCB, and multi-walled carbon nanotubes with different outer diameters on the purification effect of sample matrices and the recovery rate of target compounds were systematically compared. Figure 3 The images show the effects of different adsorbents on three blank sample matrices: grape, cucumber, and dried corn. 1 represents the blank control without any adsorbent purification. 2-9 represent: 50 mg PSA (2), 100 mg PSA (3), 50 mg GCB (4), 100 mg GCB (5), 50 mg PSA + 50 mg GCB (6), 10 mg <8 nm MWCNTs (7), 10 mg 10-20 nm MWCNTs (8), and 10 mg 20-30 nm MWCNTs (9). Figure 3 It is clear that PSA's ability to remove pigments is significantly weaker than that of GCB, PSA+GCB, and multi-walled carbon nanotubes of different outer diameters.

[0056] This experiment also investigated and compared the effects of adding PSA, GCB, PSA+GCB, and multi-walled carbon nanotubes of different outer diameters on the recovery rates of chlorfenapyr enantiomers, M550I002 enantiomers, and M550I006 enantiomers. In this example, the recovery rate effects were all tested using a spiking dose of 0.1 mg / kg, see [link to details]. Figure 4Using PSA as a purifying agent significantly reduced the recovery rate of M550I006 (average recovery rate below 40%), possibly because the basic groups in PSA react with the carboxylic acid groups on metabolite M550I006. Furthermore, PSA's removal effect on pigments was also unsatisfactory. The use of GCB significantly reduced the recovery rate of metabolite M550I002, possibly due to the specific adsorption of planar compounds by GCB. Using three different sizes of multi-walled carbon nanotubes (10 mg dosage) as purifying agents, satisfactory recoveries were achieved for both enantiomers of chlorfenapyr and both enantiomers of metabolite M550I006 (average recovery rates between 70% and 120%). However, some adsorption was observed for the two enantiomers of metabolite M550I002, with an average recovery rate significantly lower than that of chlorfenapyr and M550I006.

[0057] Different amounts of adsorbent may affect the recovery rate of the target analyte; therefore, this experiment optimized the optimal amount of multi-walled carbon nanotubes with an outer diameter of 10–20 nm. The results are shown below. Figure 5 Using 5 mg of multi-walled carbon nanotubes with an outer diameter of 10-20 nm as a purifying agent, the enantiomers of chlorfenapyr, M550I002, and M550I006 all achieved the required recovery rates and RSDs. At the same time, they can effectively remove pigment impurities from the extract and provide better protection for the instrument.

[0058] Taking into account factors such as purification effect, recovery rate, and experimental cost, the three compounds were extracted with acetonitrile, and 5 mg of multi-walled carbon nanotubes with an outer diameter of 10-20 nm were used as the purification agent.

[0059] 2. Influence of liquid phase conditions The composition and ratio of the chiral stationary phase and mobile phase, flow rate, and column temperature are the four most important factors affecting enantiomer separation. In order to obtain better separation, we will explore the effects of the above four factors on the chiral enantiomer separation of fenproxil fumarate and its main metabolites M550I002 and M550I006.

[0060] First, six different chiral columns with different stationary phases were tested: Chiral MY(2)-RH, MX(2)-RH, MZ(2)-RH, MS(2)-RH, NQ(2)-RH, and NT(2)-RH, all with a diameter of 2.1 × 150 mm and a thickness of 3 μm. To reduce the influence of mobile phase pH on separation, pure water was selected as the aqueous phase for testing. The results are shown in [Figure number missing]. Figure 6 and Figure 7Among them, Chiral MX(2)-RH (2.1×150 mm, 3 μm) and Chiral MZ(2)-RH (2.1×150 mm, 3 μm) can achieve better separation results.

[0061] The composition of the mobile phase was optimized based on Chiral MX(2)-RH and Chiral MZ(2)-RH as stationary phases. The results are shown in [Figure 1]. Figure 8 (Different volume ratios of water / acetonitrile) and Figure 9 (Different volume ratios of water / methanol) were used to find that MZ(2)-RH had the best chiral separation effect for the three compounds. At the same time, acetonitrile, as an organic phase, had a better separation effect than methanol and caused less damage to the column.

[0062] Using MZ(2)-RH as the stationary phase, and to improve peak shape and increase the response value of the target compound, a 0.1% (v / v) formic acid solution was used as the aqueous phase, and acetonitrile as the organic phase. The optimal mobile phase ratio was investigated to obtain the best chiral separation effect. The results are shown below. Figure 10 It was found that baseline separation (Rs>1.5) could be achieved for all three pesticides and their metabolites when the ratio of 0.1% formic acid aqueous solution to acetonitrile was 70 / 30 (v / v). The flow rate was subsequently optimized, and the results are shown in [Figure missing]. Figure 11 It was found that a flow rate of 0.4 mL / min balanced good separation with a short analysis time (high detection efficiency); finally, the column temperature was optimized, and the results are shown in [Figure number missing]. Figure 12 It was found that 35℃ as the column temperature can ensure the resolution and sensitivity of chiral separation.

[0063] In summary, the optimal liquid phase conditions for this method were selected as follows: stationary phase Chiral MZ(2)-RH (2.1×150 mm, 3 μm), mobile phase 0.1% formic acid aqueous solution / acetonitrile = 70 / 30 (v / v), flow rate 0.4 mL / min, column temperature 35℃, and injection volume 5 μL.

[0064] Example 3 Method Verification 1. Addition, recovery, and precision testing In accordance with the requirements of the agricultural industry standard "Guidelines for Pesticide Residue Testing in Crops" (NY / T 788-2018), this experiment was designed with three spiking levels. Three compounds were added to blank samples of fresh grapes, citrus fruits, cabbage, cucumbers, soybeans, and dried corn at concentrations of 0.01 mg / kg, 0.1 mg / kg, and 1.0 mg / kg for recovery. Each concentration was repeated five times. Analysis was performed according to the detection procedure in Example 1, and the recovery rate and relative standard deviation (RSD) were calculated.

[0065] 2. Matrix effect In UHPLC-MS / MS detection, matrix effects caused by sample matrix components are common and difficult to avoid. The presence of matrix components may enhance or inhibit the analyte response during ionization, thus potentially interfering with quantification and producing erroneous results. In this invention, the matrix effect (Me) is calculated according to the following formula: Me = km / ks-1 In the formula, km is the slope of the matrix matching standard curve, ks is the slope of the solvent standard curve, Me>0.2 is the matrix enhancement effect, -0.2≤Me≤0.2 is the weak matrix effect, which can be ignored, and Me<-0.2 is the matrix inhibition effect.

[0066] The method was verified according to the conditions in Example 1, and the results are shown in Tables 2, 3 and 4.

[0067] Table 2. Linear equations, correlation coefficients, and matrix effects for the determination of six enantiomers of acetamiprid and its main metabolites in different matrix solutions by UHPLC-MS / MS.

[0068] Table 2 shows that after optimizing the pretreatment conditions, the peak areas of the six enantiomers in methanol, grape, citrus, cabbage, cucumber, soybean, and dry corn matrices exhibited good linearity with their mass concentrations within the linear range of 0.0005–0.1 mg / L, with a correlation coefficient (R0). 2 All values ​​were greater than 0.99. The matrix effects of the six enantiomers in grapes, citrus, cabbage, cucumber, soybean, and dry corn showed certain differences, indicating the presence of both matrix-enhancing and matrix-inhibiting effects. Therefore, this invention employs matrix-matched standard curve quantification to eliminate the influence of matrix effects and ensure the accuracy of the analytical results.

[0069] Table 3. Spiking recovery results of acetamiprid and its six major enantiomers in grape, citrus, and cucumber matrices.

[0070] Table 4. Spiking recovery results of acetamiprid and its six major enantiomers in cabbage, corn, and soybean matrices.

[0071] As shown in Tables 3 and 4 (RSD in the table) r The intraday relative standard deviation, RSD R(Inter-day relative standard deviation) In six matrices (grape, citrus, cabbage, cucumber, soybean, and dried corn), the average recoveries of the six enantiomers at a spiking level of 0.01 mg / kg were 74.4%–95.3%, with RSDs of 1.3%–6.4%; at a spiking level of 0.1 mg / kg, the average recoveries were 75.7%–94.7%, with RSDs of 1.4%–6.3%; and at a spiking level of 1.0 mg / kg, the average recoveries were 76.2%–93.2%, with RSDs of 1.2%–5.6%, all meeting the requirements for pesticide residue analysis. According to EU regulations, the limit of quantitation (LOQ) is defined as the lowest spiking concentration required to meet the recovery and relative standard deviation requirements. The LOQ for all six enantiomers in the six matrices (grape, citrus, cabbage, cucumber, soybean, and dried corn) was 0.01 mg / kg.

[0072] This invention optimizes sample pretreatment conditions, employing acetonitrile extraction and purification with 10-20 nm outer diameter multi-walled carbon nanotubes in six matrices (grape, citrus, cabbage, cucumber, soybean, and dried corn). UHPLC-MS / MS matrix external standard method is used for the quantification of enantiomers of acetamiprid, M550I002, and M550I006 in these six matrices. A method for the simultaneous detection of these enantiomers in grape, citrus, cabbage, cucumber, soybean, and dried corn is established. The linear correlation coefficients of the standard curves for all compounds in different matrices are above 0.99, the average recoveries range from 74.4% to 95.3%, the relative standard deviation is less than 6.4%, and the limit of quantitation is 0.01 mg / kg. The results show that the method of the present invention meets the requirements for residue analysis and can simultaneously detect the enantiomeric acetamiprid, the enantiomeric methyl methacrylate (EMM) 550I002, and the enantiomeric methyl methacrylate (EMM 550I006) in grapes, citrus fruits, cabbage, cucumbers, soybeans, and dried corn.

[0073] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detecting enantiomeric acetamiprid and its main metabolites in fruits, vegetables, and grains, wherein the main metabolites include M550I002 and M550I006, characterized in that, Includes the following steps: The fruit, vegetable or grain sample to be tested is mixed with acetonitrile and shaken for the first time. The resulting mixture is then mixed with sodium chloride and magnesium sulfate and shaken for the second time. Solid-liquid separation is performed to obtain the extract. The extract was purified by mixing it with multi-walled carbon nanotubes with an outer diameter of 10-30 nm, followed by solid-liquid separation to obtain the test solution. The test solution is subjected to liquid chromatography-mass spectrometry detection. Based on the obtained peak area and the predetermined standard curve, the content of acetamiprid and its main metabolite enantiomerics in the test fruit, vegetable or grain sample is obtained. The liquid chromatography conditions for the liquid chromatography-mass spectrometry detection include: Chromatographic column: chiral column; mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is a 0.05-0.2% (v / v) aqueous solution of formic acid and mobile phase B is acetonitrile; flow rate: 0.3-0.5 mL / min, isocratic elution: 0-15 min, and the volume fraction of mobile phase B is maintained at 20-40%; The mass spectrometry conditions for the liquid chromatography-mass spectrometry detection include: Multiple reaction monitoring mode; Ionization source mode: ESI + Atomizing gas: nitrogen; Spray voltage: 5000~6000V; Ion source temperature: 500~600℃; Pressure of atomizing gas and auxiliary gas: 40~60psi and 40~60psi respectively.

2. The detection method according to claim 1, characterized in that, The outer diameter of the multi-walled carbon nanotubes is 10~20 nm.

3. The detection method according to claim 1 or 2, characterized in that, The volume ratio of the extract to the mass of the multi-walled carbon nanotubes is 1.5 mL: 4~6 mg.

4. The detection method according to claim 1, characterized in that, The chiral column is a Chiral MZ(2)-RH column with dimensions of 2.1×150mm and 3μm.

5. The detection method according to claim 1 or 4, characterized in that, The flow rate is 0.4 mL / min.

6. The detection method according to claim 5, characterized in that, The volume fraction of the mobile phase B is maintained at 30%.

7. The detection method according to claim 1, characterized in that, The liquid chromatography conditions for liquid chromatography-mass spectrometry detection also include: column temperature of 30~40℃.

8. The detection method according to claim 1, characterized in that, The predetermined standard curve is a matrix-matched standard curve with a linear range of 0.0005~0.1 mg / L.

9. The detection method according to claim 1, characterized in that, When the sample to be tested is a grain sample, the process before mixing with acetonitrile also includes: mixing the sample to be tested with water; the mass ratio of the sample to the volume of water is 1g:1~3mL.

10. The detection method according to claim 1 or 9, characterized in that, The mass ratio of the fruit, vegetable, or grain sample to the volume of acetonitrile is 4-6 g: 20-30 mL.

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