Method for detecting multiple pesticide residues in agricultural products based on improved QuEChERS-UPLC-MS / MS technology

Abstract

The invention provides a method for detecting pesticide residues in agricultural products based on nano-material improved QuEChERS-UPLC-MS / MS. PSA and MWCNTs with specific content are adopted as a cleaning agent combination to perform pretreatment on a sample, 0.05% formic acid (containing 2mmol / L ammonium acetate) aqueous solution and acetonitrile are adopted as mobile phases, and under a specific gradient elution condition, the content of the pesticide residues in the agricultural products is detected by using a high-performance liquid chromatography-mass spectrometry method, and the content of the pesticide residues in the agricultural products is detected by using the high-performance liquid chromatography-mass spectrometry method. The method can accurately analyze the residual content of abamectin, flonicamid, chlorfluazuron, isocarbophos, fenpropathrin, fludioxonil, chlorpyrifos, phorate, lufenuron, clothianidin, trichlorfon, imidacloprid, thiamethoxam, fluxapyroxad, thidiazuron, cyromazine, spirodiclofen, pyrimethanil, omethoate, acetamiprid, propiconazole and the like in agricultural products such as black vegetables, hyacinth beans, juicy peaches and the like. The method disclosed by the invention has the advantages of high sensitivity, strong universality, high accuracy, good stability, simplicity and convenience in operation and the like, and provides technical support for detection of pesticide residues in agricultural products.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural product pesticide residue detection, and particularly relates to a method for detecting pesticide residues in agricultural products based on the nano-material QuEChERS technology. Background Art

[0002] The long-term high-frequency and high-dose use of chemical pesticides is likely to cause excessive pesticide residues, threatening the health of residents. At the same time, the use of chemical pesticides will cause great harm to the ecological environment. Conducting research on the technology of multi-residue detection of pesticides in agricultural products is of great significance for promoting the healthy development of agriculture and ecological environment protection in China.

[0003] Currently, in the technology of multi-residue detection of pesticides in fruits and vegetables, the sample pretreatment method plays a crucial role, directly determining the accuracy and reliability of the analysis results. Due to its advantages such as rapidity, simplicity, low cost, effectiveness, robustness and safety, the QuEChERS technology has become the most widely used sample pretreatment method at present. However, the currently mainly used purifying agents have disadvantages such as large dosage, poor adsorption stability and high usage cost. Especially for samples with complex matrices, the purification effect cannot meet the detection requirements, and there is an urgent need to develop new purifying agents with large adsorption capacity, strong adsorption stability, good purification effect, economy and the ability to handle complex matrix samples. In recent years, a large number of scholars have made varying degrees of improvements to the extraction and purification parts of the QuEChERS method, mainly including the optimization of pH value, extraction solvent and purification. Among them, purification is the key step of the QuEChERS method, which can greatly affect the quantitative limit and detection limit of pesticide residue detection. The most common purifying agents are magnesium sulfate, graphitized carbon black (GCB), octadecylsilane (C18), etc. A single purifying agent can meet the purification of general fruits and vegetables (with low pigment content), while it is difficult to achieve a good purification effect for fruits and vegetables with high pigment content and high fat content.

[0004] Currently, nanomaterials have developed rapidly and are widely used in the field of adsorption separation due to their large specific surface area. It has been reported that MWCNTs are applied to the multi-residue detection of pesticides, but the reports are relatively few. Multi-walled carbon nanotubes (MWCNTs) have a very small inner diameter but extremely high strength, and have excellent adsorption ability for target organic compounds. A small amount of adsorbent can achieve effective purification and separation in a short time, and has been widely used in water samples, pesticide residues and veterinary detection.

[0005] Patent document CN117571855A discloses an analytical method for detecting brassinolide in microbial inoculants by nano-material QuEChERS-UPLC-MSMS, and discloses that the purifying agent combination is nano-ZrO2, PSA, C18, MWCNTs.

[0006] In this study, an improved QuEChERS method was adopted to simplify the pretreatment steps. Multi-walled carbon nanotubes were used as the purification adsorbent, and UPLC-MS / MS (ultra-high performance liquid chromatography-tandem mass spectrometry) was combined to carry out the research on the analytical method. It was applied to the detection of multi-pesticide residues in agricultural products in different ecological regions, with the expectation of finally formulating a number of pesticide residue detection technical standards and being promoted and used by relevant pesticide residue detection institutions. Relying on advanced detection technologies and scientific evaluation means to ensure the whole production, safety supervision, guiding consumption, and food safety of residents of agricultural products, and at the same time providing a strong guarantee for the sustainable development of the agricultural product economy. Summary of the Invention

[0007] The present invention uses QuEChERS-UPLC-MS / MS to develop a method with the advantages of simple operation, low cost, rapid analysis, high recovery rate, etc., to realize the detection of 38 pesticide residues in fruit and vegetable agricultural products to meet the market demand.

[0008] Therefore, the present invention provides a method for detecting pesticide residues in agricultural products based on nano-material QuEChERS-UPLC-MSMS, and the method comprises the following steps:

[0009] (1) Sample collection

[0010] Collect agricultural product samples, mark them, and then store them sealed in a -20°C refrigerator for later use.

[0011] (2) QuEChERS sample pretreatment

[0012] Use a high-speed homogenizer to fully grind the frozen sample. Accurately weigh 10 g of the ground sample into a 50 mL centrifuge tube, add 10 - 20 mL of acetonitrile and shake well quickly, then add 0.5 - 2 g of sodium chloride and 3 - 6 g of anhydrous magnesium sulfate, vortex and oscillate for extraction at 2500 rpm for 7 - 14 min, and then centrifuge at 5000 rpm for 3 - 6 min, discard the precipitate to obtain the supernatant;

[0013] Take 1.5 mL of the above supernatant into a 2 mL centrifuge tube, which is pre-filled with a purification agent combination and anhydrous magnesium sulfate, vortex and oscillate at 2500 rpm for 3 - 8 min, and then centrifuge at 10000 rpm for 2 - 4 min. The supernatant passes through a 0.22 μm organic filter membrane for detection.

[0014] (3) UPLC-MS / MS analysis and detection

[0015] Use UPLC-MS / MS (ultra-high performance liquid chromatography-tandem mass spectrometry) method to detect the processed sample to be detected, and detect the pesticide residues and their contents in the sample.

[0016] In one embodiment of the present invention, the pesticide residues in agricultural products include one or more of the 38 pesticides in Table 1.

[0017] In one embodiment of the present invention, the step of "(2) sample pretreatment" is preferably as follows: Use a high-speed homogenizer to thoroughly grind the frozen sample, weigh 10 g of the homogenized sample into a 50 mL centrifuge tube respectively, then add 20 mL of acetonitrile, and vortex at 2500 rpm for 5 min; then add 2 g of NaCl and 3 g of anhydrous magnesium sulfate and vortex at 2500 rpm for 5 min; centrifuge at 4000 rpm for 10 min with a centrifuge, and the supernatant is to be purified;

[0018] Take 1.5 mL of the above supernatant into a 2 mL centrifuge tube, which is pre-filled with a purification agent combination and anhydrous magnesium sulfate, vortex at 2500 rpm for 5 min, then centrifuge at 10000 rpm for 5 min, and the supernatant passes through a 0.22 μm organic filter membrane for detection.

[0019] In one embodiment of the present invention, in the step of "(2) sample pretreatment", the purification agent combination is: 40 mg of PSA, 2 - 8 mg of MWCNTs (i.e., PSA / MWCNTs = 40 / 2 - 8 mg). Preferably, the purification agent combination is: 40 mg of PSA, 3 mg of MWCNTs (i.e., PSA / MWCNTs = 40 / 3 mg). The addition amount of the anhydrous magnesium sulfate is 15 mg.

[0020] Among them, PSA refers to ethylenediamine-N-propylsilylated silica gel adsorbent; MWCNTs refers to multi-walled carbon nanotubes.

[0021] In one embodiment of the present invention, in the step of "(3) UPLC-MS / MS analysis and detection": The ultra-high performance liquid chromatography conditions are as follows: The chromatographic column is C18 (2.6 μm * 2.1 mm * 100 mm); An aqueous solution containing 0.05% formic acid and 2 mmol ammonium acetate (phase A) and acetonitrile (phase B) are used as the mobile phase, and gradient elution is used for the separation of target compounds. The gradient elution program is: 0 min 90% A + 10% B, 8.5 min 5% A + 95% B, 10.5 min 5% A + 95% B, 10.51 min 90% A + 10% B, 12.5 min 90% A + 10% B; Flow rate: 0.3 mL / min; Injection volume 2 μL, column temperature 35 °C.

[0022] The mass spectrometry conditions are as follows: Electrospray ionization (ESI); Positive ion scan mode, the scan method is multiple reaction monitoring (MRM), temperature 450 °C, voltage 5.5 kV; Nebulizing gas GS1 pressure 40 psi; Nebulizing gas GS2 pressure 40 psi.

[0023] On the other hand, the present invention relates to the use of a purification agent combination for purifying fruit and vegetable samples when detecting the pesticide residue content in fruit and vegetable agricultural products by the QuEChERS-UPLC-MS / MS method. Among them, the purification agent combination is: 40 mg of PSA, 3-8 mg of MWCNTs. Preferably, the purification agent combination is: 40 mg of PSA, 3 mg of MWCNTs.

[0024] By adopting the above technical solutions, the present invention has the following beneficial effects:

[0025] The present invention uses PSA and MWCNTs as purification agents, and at the same time, based on the QuEChERS-UPLC-MS / MS technology, an analytical method for 38 pesticide residues in agricultural products is established. This method has the advantages of high sensitivity, strong versatility, simple operation, low cost, rapid analysis, high recovery rate, high accuracy, good stability and simple operation, and can realize the detection of 38 pesticide residue components in agricultural products. It provides technical support for the detection of pesticide residues in agricultural products and has guiding significance. Description of the Drawings

[0026] Figure 1 : Total ion chromatogram of target compounds when the mobile phase is 0.05% formic acid (containing 2 mmol / L ammonium acetate) aqueous solution - acetonitrile (II);

[0027] Figure 2 : Response intensities of 38 pesticides under the mobile phase systems of 0.05% formic acid aqueous solution - acetonitrile (1), 0.05% formic acid (containing 2 mmol / L ammonium acetate) aqueous solution - acetonitrile (2) and 0.1% formic acid aqueous solution - methanol (3);

[0028] Figure 3 : Me of 38 pesticides in Chinese cabbage matrix under different dosages of MWCNTs;

[0029] Figure 4 : Me of 38 pesticides in kidney bean matrix under different dosages of MWCNTs;

[0030] Figure 5 : Me of 38 pesticides in peach matrix under different dosages of MWCNTs. Detailed Embodiments

[0031] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art without making creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0032] I. Methodological Verification

[0033] The contents of 38 pesticide standards used in the following embodiments of this application are shown in Table 1, and all are purchased from Beijing Qincheng Yixin Technology Development Co., Ltd.; agricultural products are purchased from different shopping malls, farmers' markets and collected from different planting areas, including black leafy greens, lentils, and peaches.

[0034] Table 1 Contents and Sources of 38 Pesticide Ingredients

[0035]

[0036] 1 Materials and Methods

[0037] 1.1 Preparation of Standard Solution and Matrix Solution

[0038] Accurately weigh a certain amount of standard products (38 pesticide ingredients), and separately prepare standard stock solutions with a concentration of 1000 mg / L using acetonitrile. Then, mix and dilute to obtain a mixed standard solution with a concentration of 10 mg / L for all 38 active ingredients, and then dilute it with acetonitrile and matrix solution to 200, 100, 50, 10, 5, and 1 μg / L (the matrix solution is obtained through the following 1.2 sample pretreatment steps).

[0039] 1.2 QuEChERS Sample Pretreatment

[0040] Use a high-speed homogenizer to thoroughly grind the frozen samples. Weigh 10 g of the homogenized samples into 50 mL centrifuge tubes, then add 20 mL of acetonitrile, and vortex at 2500 rpm for 5 min; then add 2 g of NaCl and 3 g of anhydrous magnesium sulfate and vortex at 2500 rpm for 5 min; centrifuge at 4000 rpm for 10 min, and the supernatant is ready for purification.

[0041] Take 1.5 mL of the above supernatant into a 2 mL centrifuge tube (containing PSA / MWCNTs = 40 / 3 mg (which is the most preferred purification agent combination, and the specific screening results can be seen in the "QuEChERS Optimization" section) and 150 mg of anhydrous magnesium sulfate). The centrifuge tube is pre-filled with the purification agent combination and anhydrous magnesium sulfate, vortex at 2500 rpm for 5 min, then centrifuge at 10000 rpm for 5 min, and the supernatant is filtered through a 0.22 μm organic filter membrane for detection.

[0042] 1.3 Instrumental Analysis Method

[0043] Detection is carried out by UPLC-MS / MS (Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry) method.

[0044] The conditions for ultra performance liquid chromatography are as follows: the chromatographic column is C 18(2.6μm * 2.1mm * 100mm); An aqueous solution containing 0.05% formic acid (containing 2 mmol / L ammonium acetate) (phase A) and acetonitrile (phase B) were used as the mobile phase (which is the most preferred mobile phase, and the specific screening results can be seen in the "Analysis Method Optimization" section). The separation of the target compound was carried out by gradient elution, and the gradient elution program is shown in Table 1 (which is the most preferred gradient elution program, determined by screening through the "Analysis Method Optimization" section). The injection volume was 2 μL, and the column temperature was 35 °C.

[0045] Table 2 Gradient Elution Program

[0046]

[0047] The described mass spectrometry conditions were as follows: electrospray ionization (ESI); positive ion scan mode, the scan method was multiple reaction monitoring (MRM), the temperature was 450 °C, the voltage was 5.5 kV; the pressure of nebulizing gas GS1 was 40 psi; the pressure of nebulizing gas GS2 was 40 psi.

[0048] 1.4. Matrix Effect

[0049] Matrix effect (Me) refers to the phenomenon that the signal of the target compound is enhanced or weakened due to the interference of co-eluting impurities. During the analysis of complex samples, the matrix effect will affect the ionization degree of ions, and thus affect the quantitative results of the target compound.

[0050] In this study, the ratio of the slope (K2) of the matrix standard curve to the slope (K1) of the solvent standard curve (k2 / k1) was used for evaluation, and Me was used to evaluate the purification effect. The calculation formula is shown below.

[0051] Me = K2 / K1

[0052] In the formula, K1 is the slope of the solvent standard curve, and K2 is the slope of the matrix standard curve; when Me is between 0.8 and 1.2, it is considered that there is no obvious matrix effect, that is, the purification effect is good; when Me < 0.8 or > 1.2, it indicates that there are more residual interfering substances in the matrix, and the greater the difference from the numerical values in this range, the worse the purification effect.

[0053] 2 Results

[0054] 2.1 Analysis Method Optimization

[0055] Inject a 200 μg / L mixed standard solution of 38 pesticides into the mass spectrometer through an injection pump for parameter tuning, and scan in both positive and negative ESI modes. The results show that only the parent ion of fludioxonil is in the [M-H]− mode, while the parent ions of the remaining 37 target compounds are all in the [M+H]+ mode. By tuning and optimizing the parent ions, daughter ions, and the best cone voltage and collision energy of the target compounds, the mass spectrometry acquisition parameters of 38 compounds in the MRM mode are finally obtained (Table 3). Select two sets of ion pairs with the best sensitivity for detection, one for quantification and the other for auxiliary qualitative analysis.

[0056] Table 3 Mass spectrometry information of 38 pesticides

[0057]

[0058]

[0059] Note: "*" represents the quantitative ion pair. Since formic acid and ammonium acetate are common reagents for enhancing the ionization degree of [M+H]+ and [M+NH4]+ of target compounds in the ESI+ mode, and both can effectively improve the peak shape, making the peak shape sharper and more symmetrical at the same time. Therefore, when performing gradient elution of target compounds, the effects of mobile phase systems of 0.05% formic acid aqueous solution - acetonitrile (1), 0.05% formic acid (containing 2 mmol / L ammonium acetate) aqueous solution - acetonitrile (2), and 0.1% formic acid aqueous solution - acetonitrile (3) on the sensitivity and peak shape of 38 pesticides were investigated respectively.

[0060] Sorted in ascending order according to the response values are: abamectin, flonicamid, chlorfluazuron, isocarbophos, fenpropathrin, fludioxonil, chlorpyrifos, phorate, lufenuron, clothianidin, trichlorfon, imidacloprid, thiamethoxam, fluxapyroxad, thidiazuron, cyromazine, spirodiclofen, pyrimethanil, omethoate, acetamiprid, propiconazole, napropamide, thiacloprid, thiram, pyraclostrobin, acephate, prochloraz, hexaconazole, flusilazole, azoxystrobin, propamocarb, carbofuran, difenoconazole, isofenphos-methyl, carbendazim, triazophos, pyridaben, picoxystrobin. The research results show that when the mobile phase is 2, the peak shapes of 38 compounds are the best and the sensitivity is the highest ( Figure 2 ).

[0061] To sum up, it is finally determined that 0.05% formic acid (containing 2 mmol ammonium acetate) - acetonitrile is the mobile phase system. By continuously optimizing the gradient elution program, all compounds can be effectively separated within 12.5 min. The total ion chromatogram of the target compounds is as shown in Figure 1 shown. The optimized elution program is as shown in Table 1 above.

[0062] 2.2 QuEChERS optimization

[0063] When the addition level was 0.05 mg / kg and the amount of PSA was fixed at 40 mg, the purification effect of MWCNTs on 38 pesticides in fruit and vegetable agricultural product matrices was investigated.

[0064] MWCNTs can effectively adsorb impurities such as pigments, heavy metals, inorganic non-metallic ions, aromatic hydrocarbons, and antibiotics, and is an efficient adsorbent. However, since MWCNTs will also adsorb the target compounds while adsorbing impurities, it is necessary to optimize its dosage. Based on the above research results, with PSA = 40 mg fixed, the Me of 38 pesticides in 3 fruit and vegetable sample matrices was investigated when the addition amounts of MWCNTs were 3, 5, 8, and 12 mg, respectively.

[0065] The results showed that when the dosage of MWCNTs was between 3 and 5 mg, the Me of 3 compounds in the three matrices was between 0.7 and 1.2, and the purification effect was good ( Figures 3 - 5 ). On the basis of ensuring a good purification effect and taking into account the principle of cost saving, the dosage of MWCNTs was finally determined to be 3 mg.

[0066] In summary, through the optimization of the purifying agent combination, the optimal dosage of the purifying agent combination for the analysis of 38 pesticide residues in the dominant fruit and vegetable products in Jiangsu Province was finally determined as PSA / MWCNTs = 40 / 3 mg.

[0067] 3 Method validation

[0068] 3.1 Detection limit, quantification limit, standard curve and R 2

[0069] Analysis was carried out using the method screened above. With the mass concentration as the X-axis and its corresponding peak area as the Y-axis, the solvent standard curve and the matrix standard curve of the sample matrix were plotted. The results showed that the linear relationships of 38 pesticides in the three sample matrices were good in the concentration range of 1 - 200 μg / L, and R 2 was between 0.99 and 1; with 3 times the signal-to-noise ratio (S / N) as the LOD and the lowest addition level as the LOQ, the LODs and LOQs of 38 compounds in the three matrices were both 5 μg / kg.

[0070] Table 4 Detection limit, quantification limit, standard curve and R of 38 pesticides in the matrix of black pak choi 2

[0071]

[0072] Table 5 Detection limit, quantification limit, standard curve and R of 38 pesticides in the matrix of kidney bean 2

[0073]

[0074] Table 6 Detection limits, quantification limits, standard curves and R of 38 pesticides in the honey peach matrix 2

[0075]

[0076] Referring to the lowest value of GB2763-2021 MRLs, in this study, three spiking levels of 5, 50, and 500 μg / kg were set in three fruit and vegetable matrices, and 5 parallel tests were carried out. The results showed that when the spiking levels were 5-500 μg / kg, the average recoveries of 38 compounds in the three matrices were between 75.8% and 105.3%, and the relative standard deviations (RSDs) were 1.1% - 6.3% (Tables 7-9). In summary, all results met the requirements of pesticide residue detection standards.

[0077] Table 7 Average spiking recoveries and RSDs% of 38 pesticides in the pakchoi matrix at 3 spiking levels

[0078]

[0079] Table 8 Average spiking recoveries and RSDs% of 38 pesticides in the kidney bean matrix at 3 spiking levels

[0080]

[0081] Table 9 Average spiking recoveries and RSDs% of 38 pesticides in the honey peach matrix at 3 spiking levels

[0082]

[0083] In summary, the present invention established an analytical method for the determination of 38 pesticide residues in fruits and vegetables based on QuEChERS-UPLC-MS / MS technology with PSA and MWCNTs as purifying agents. This method has the advantages of high sensitivity, strong generality, high accuracy, good stability and simple operation, providing technical support for the detection of pesticide residues in agricultural products.

[0084] II. Examples

[0085] Using the optimized analytical method determined in this study, the contents of 38 pesticides in the collected fruit and vegetable samples were analyzed, and the contents of 38 pesticide residues in the collected samples were calculated through the matrix standard curves of each sample. The analysis results are shown in Tables 10-12.

[0086] Table 10 Detection results of the contents of 38 pesticides in the collected pakchoi (10 portions) (mg / kg)

[0087]

[0088] Detection Results of 38 Pesticide Contents in the Collected Lentils (10 Samples) (mg / kg)

[0089]

[0090] Detection Results of 38 Pesticide Contents in the Collected Peaches (10 Samples) (mg / kg)

[0091]

[0092] "-" indicates not detected.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting pesticide residues in agricultural products based on nano-material QuEChERS-UPLC-MSMS, characterized in that, The method includes the following steps: (1) Sample collection Collect fruit and vegetable products, label them, and then store them sealed in a -20°C refrigerator for later use; (2) QuEChERS sample pretreatment Use a high-speed homogenizer to thoroughly grind the frozen samples. Weigh 10 g of the homogenized samples into 50 mL centrifuge tubes respectively, then add 10 - 20 mL of acetonitrile, and vortex at 2500 rpm for 3 - 5 min; then add 0.5 - 2 g of NaCl and 3 - 6 g of anhydrous magnesium sulfate and vortex at 2500 rpm for 3 - 5 min; centrifuge at 4000 rpm for 5 - 10 min, and the supernatant is to be purified; Take 1.5 mL of the above supernatant into a 2 mL centrifuge tube which is pre-filled with a purification agent combination and anhydrous magnesium sulfate, vortex at 2500 rpm for 3 - 8 min, then centrifuge at 10000 rpm for 2 - 5 min, and the supernatant passes through a 0.22 μm organic filter membrane for detection; (3) UPLC-MS / MS analysis and detection Use UPLC-MS / MS (Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry) method to detect the processed samples to be detected, and detect the pesticide residues and their contents in the samples.

2. The method according to claim 1, characterized in that, The pesticide residues in the agricultural products include one or more of abamectin, difenoconazole, imidacloprid, pyraclostrobin, propiconazole, pyridaben, trichlorfon, napropamide, acetamiprid, picoxystrobin, chlorpyrifos, carbendazim, flonicamid, chlorfluazuron, flusilazole, fluxapyroxad, thiram, fludioxonil, hexaconazole, phorate, isofenphos-methyl, fenpropathrin, carbofuran, spirodiclofen, prochloraz, azoxystrobin, pyrimethanil, cyromazine, thidiazuron, thiamethoxam, thiacloprid, thiamethoxam, triazophos, lufenuron, propamocarb, isocarbophos, omethoate, acephate.

3. The method according to claim 1, characterized in that The step "(2) Sample pretreatment" is as follows: Use a high-speed homogenizer to thoroughly grind the frozen samples. Weigh 10 g of the homogenized samples into 50 mL centrifuge tubes respectively, then add 20 mL of acetonitrile, and vortex at 2500 rpm for 5 min; then add 2 g of NaCl and 3 g of anhydrous magnesium sulfate and vortex at 2500 rpm for 5 min; centrifuge at 4000 rpm for 10 min, and the supernatant is to be purified; Take 1.5 mL of the above supernatant into a 2 mL centrifuge tube which is pre-filled with a purification agent combination and anhydrous magnesium sulfate, vortex at 2500 rpm for 5 min, then centrifuge at 10000 rpm for 5 min, and the supernatant passes through a 0.22 μm organic filter membrane for detection.

4. The method according to claim 3, characterized in that, The purification agent combination is: 20 - 50 mg PSA, 2 - 8 mg MWCNTs.

5. The method according to claim 4, wherein The purification agent combination is: 40 mg PSA, 3 mg MWCNTs.

6. The method according to claim 1, characterized in that The ultra-high performance liquid chromatography conditions in the step "(3) UPLC-MS / MS analysis and detection" are as follows: the chromatographic column is C18 (2.6 μm * 2.1 mm * 100 mm); an aqueous solution containing 0.05% formic acid (containing 2 mmol / L ammonium acetate) (phase A) and acetonitrile (phase B) are used as the mobile phase, and gradient elution is used for the separation of target compounds. The gradient elution program is: 90% A + 10% B at 0 min, 5% A + 95% B at 8.5 min, 5% A + 95% B at 10.5 min, 90% A + 10% B at 10.6 min, 90% A + 10% B at 12.5 min; flow rate: 0.3 mL / min; injection volume 1 μL, column temperature 35 °C; The mass spectrometry conditions are: electrospray ionization (ESI); positive ion scan mode, the scan method is multiple reaction monitoring (MRM), temperature 450 °C, voltage 5.5 kV; nebulizing gas GS1 pressure 40 psi; nebulizing gas GS2 pressure 40 psi.

7. A purifying agent combination, characterized in that, The use for purifying agricultural product samples.

8. The use according to claim 7, characterized in that, The purification agent combination is: 40 mg PSA, 2 - 8 mg MWCNTs.

9. The purifying agent combination according to claim 8, characterized in that, The combination is: 40 mg PSA, 3 mg MWCNTs.

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