Method for determining enfenprop, flutolanil and / or triazifentyl in feedstuffs and use thereof

Abstract

The application discloses a kind of feed in enoxamium, pyrimethanil and / or triazolopyrimidines novel herbicide determination method and application.Belongs to pesticide residue detection technical field.The application provides a kind of feed in enoxamium, pyrimethanil and triazolopyrimidines novel herbicide determination method, based on dispersive solid phase extraction pretreatment technology, without solid phase extraction column, and the reagent cost used is low, realizes the effective purification and enrichment of three kinds of novel herbicides, combined with ultra-high performance liquid chromatography tandem mass spectrometry UPLC-MS / MS can be quickly, accurately determine whether the animal feed contains pyrimethanil, enoxamium and triazolopyrimidines herbicide and corresponding residual amount, the method is high in sensitivity, good in special property, detection result is accurate and reliable, for enoxamium, pyrimethanil and triazolopyrimidines this three kinds of novel herbicide simultaneous detection provides technical support.

Description

Technical Field

[0001] This invention relates to the field of pesticide residue detection technology, and more specifically to a method and application for determining clethodim, pyriflusulfonamide and / or triazole chlorpyrifos in feed. Background Technology

[0002] Clethodim is a novel acetylchloroaniline herbicide primarily used to control grassy and broadleaf weeds in corn and soybean fields. Pyriflusulfonamide is a second-generation sulfonamide herbicide for rice paddies, exhibiting extremely high efficacy against annual weeds such as barnyard grass, as well as perennial, difficult-to-control weeds, and showing excellent effectiveness against resistant weeds. Triazolam is a triazoline ketone herbicide for rice paddies, highly effective against grassy weeds, annual weeds, and some annual broadleaf weeds, especially barnyard grass, with a long residual effect and high safety even when applied during rice transplanting. Currently, these three new agricultural herbicides are widely used abroad, particularly in the cultivation of grain crops such as rice, wheat, corn, soybeans, and sorghum. These grain crops are the main raw materials for animal feed, leading to a high likelihood of significant residues of these three herbicides in the feed. These herbicide residues enter and accumulate in animal bodies through feed, harming animal health and subsequently being ingested by humans through animal-derived food products, thus endangering human health.

[0003] Currently, the EU, the US, Japan, and other countries have set residue limits for these three herbicides in plant-based foods. For example, Japan sets limits of ≤0.05 ppm for pyriflusulfone and ≤0.05 ppm for triazine in rice; the EU sets limits of ≤0.01 ppm for clethodim in fresh or frozen fruits, root and tuber vegetables, bulb vegetables, sunflower seeds, rapeseed, soybeans, pumpkin seeds, and corn cereals, and ≤0.05 ppm for clethodim in tea. It is clear that residues of these three herbicides are harmful to human health. Therefore, measuring and controlling the residue levels of these three herbicides in feed is of great significance for the health of livestock and for ensuring the safety of animal-derived foods for humans.

[0004] Currently, there are few standards for detecting herbicides in feed. The DB34 / T 3102-2018 standard, "Determination of Sulfonylurea Herbicides in Feed - Liquid Chromatography-Tandem Mass Spectrometry," utilizes a purification technique based on gel permeation chromatography. This technique is complex, costly, requires a gel permeation chromatograph, and demands a high level of operator skill. Furthermore, its detection range does not include clethodim, pyriflusulfonamide, and triazine. The GB / T 23744-2009 standard, "Determination of Multiple Residues of 36 Pesticides in Feed - Gas Chromatography-Mass Spectrometry," uses gas chromatography-mass spectrometry to determine four low- to medium-boiling-point herbicides: metribuzin, chlorpyrifos, chlorpyrifos, and chlorpyrifos. However, its gas chromatography-mass spectrometry method is not applicable to the three high-boiling-point (425.7℃~550.8℃) herbicides detected in this invention. Currently, there are no corresponding detection methods for the above three new herbicides, especially for the detection of residues of these three herbicides in feed.

[0005] Therefore, how to provide a rapid and accurate method for determining the residues of pyriflusulfonamide, clethodim, and triazole chlorpyrifos in animal feed is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a method and application for the determination of clethodim, pyriflusulfonamide and / or triazole chlorpyrifos in feed. Based on the dispersion solid phase extraction pretreatment technology, combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), it can rapidly and accurately determine whether animal feed contains herbicides such as pyriflusulfonamide, clethodim and triazole chlorpyrifos and their corresponding residues. The method has high sensitivity and specificity, can simultaneously determine three herbicides, and the detection results are accurate and reliable.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for determining clethodim, pyriflusulfonamide, and / or triazole chlorpyrifos in feed, comprising the following steps:

[0009] (1) Sample pretreatment: Take the sample to be tested, add water and mix well, let stand for 15-30 minutes, add extraction solvent and homogenants, shake and mix well to obtain primary extract;

[0010] (2) Adsorption of impurities and moisture: Add adsorbent to the primary extract, shake at high speed, centrifuge and take the supernatant to obtain the secondary extract;

[0011] (3) Purification of the extract: Add purification reagent to the secondary extract, vortex and shake, centrifuge, take the supernatant, filter, and obtain the test solution;

[0012] (4) High performance liquid chromatography-tandem mass spectrometry detection: The detection solution is injected into the high performance liquid chromatography-tandem mass spectrometer for qualitative and quantitative detection.

[0013] Preferably, the extraction solvent in step (1) is a 1% acetic acid-acetonitrile solution, and the amount added is 15 mL / 5 g of the sample to be tested; the shaking time is 5 min.

[0014] Preferably, the adsorbent in step (2) is anhydrous magnesium sulfate and sodium acetate, with addition amounts of 6.0 g / 5 g of the sample to be tested and 1.5 g / 5 g of the sample to be tested, respectively; the high-speed shaking speed is 250 times / min and the time is 2 min; the centrifugation speed is 5000 r / min and the centrifugation time is 5 min.

[0015] Preferably, the purification reagents in step (3) are anhydrous magnesium sulfate, C18 and PSA, with addition amounts of 150 mg / mL secondary extract, 50 mg / mL secondary extract and 50 mg / mL secondary extract, respectively; the vortexing speed is 250 times / min and the time is 2min; the centrifugation speed is 5000 r / min and the centrifugation time is 5min; the filtration is performed using a microporous membrane with a pore size of 0.22 μm.

[0016] Preferably, the chromatographic conditions for high performance liquid chromatography in step (4) are as follows:

[0017] Column: ACQUITY UPLC HSS T31.8μm; 2.1×100mm;

[0018] Mobile phase: Phase A is a 0.1% formic acid aqueous solution; Phase B is an acetonitrile solution;

[0019] Elution: Gradient elution, the elution procedure is as follows:

[0020] 0-0.5 min, mobile phase A is 50% and mobile phase B is 50%;

[0021] Over a period of 0.5–4 min, the amount of mobile phase A was decreased from 50% to 20% at a constant rate, while the amount of mobile phase B was increased from 50% to 80% at a constant rate.

[0022] 4-5 min, with mobile phase A at 20% and mobile phase B at 80%;

[0023] Over 5-5.5 minutes, the amount of mobile phase A was increased from 20% to 50% at a constant rate, while the amount of mobile phase B was decreased from 80% to 50% at a constant rate.

[0024] 5.5-7 min, with mobile phase A at 50% and mobile phase B at 50%.

[0025] Preferably, in step (4), the flow rate of the mobile phase during high-performance liquid chromatography elution is 0.3 mL / min, the column temperature is 40 °C, and the injection volume is 1.0 μL.

[0026] Preferably, the mass spectrometry conditions for the tandem mass spectrometry in step (4) are as follows:

[0027] Ion source type: Electrospray ionization source;

[0028] Scanning mode: positive ion mode;

[0029] Capillary voltage: 3.0kV;

[0030] Desolvation gas temperature: 500℃;

[0031] Desolvation gas flow rate: 600 L / Hr;

[0032] Atomizer pressure: 7.0 bar;

[0033] Multiple response monitoring (MRM).

[0034] Another object of the present invention is to provide the application of the above-described assay method in the detection of residues of clethodim, pyriflusulfonamide and / or triazole chlorpyrifos in feed.

[0035] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for determining novel herbicides such as clethodim, pyriflusulfonamide, and / or triazole chlorpyrifos in feed. The method uses dispersion solid-phase extraction pretreatment technology to pretreat the sample, eliminating the need for solid-phase extraction columns and using low-cost reagents. It achieves effective purification and enrichment of the three novel herbicides. Combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), it can rapidly and accurately determine whether animal feed contains pyriflusulfonamide, clethodim, and triazole chlorpyrifos, as well as the corresponding residues. This method has high sensitivity, good specificity, and accurate and reliable detection results, providing technical support for the simultaneous detection of these three novel herbicides. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0037] Figure 1 For: the change in the amount of mobile phase B used over time in the elution process;

[0038] Figure 2For example: MRM mass spectrum of clethodim;

[0039] Figure 3 Here is the MRM mass spectrum of pyriflusulfonamide;

[0040] Figure 4 For example: MRM mass spectrum of triazole oxychloride;

[0041] Figure 5 TIC plots for: Pyrimisulfan, Petoxamid, and Ipfencarbazone;

[0042] Figure 6 For example: the standard working curve of clethodim matrix matching;

[0043] Figure 7 For example: the standard working curve of pyriflusulfonamide matrix matching;

[0044] Figure 8 For example: Triazole oxychloride matrix matching standard working curve;

[0045] Figure 9 Here are the formulas for calculating the content of clethodim, pyriflusulfonamide, and triazole chlorpyrifos;

[0046] Figure 10 The mass chromatogram of clethodim is as follows: (1: 296.39>250.22; 2: 296.39>131.14; 3: TIC).

[0047] Figure 11 The mass chromatogram of pyriflusulfonamide is shown below (1: 420.37>370.19; 2: 420.37>255.22; 3: TIC).

[0048] Figure 12 The mass chromatogram of triazolyl chlorpyrifos is shown below (1: 427.297>198.18; 2: 427.29>198.18; 3: TIC). Detailed Implementation

[0049] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] The instruments and equipment used in the embodiments of this invention are as follows:

[0051] UPLC Iclass-XEVOTQ-XS liquid chromatography-tandem mass spectrometry system (Waters, USA); electronic balance (BSA223S, Sartorius, Germany); high-speed refrigerated centrifuge (Sigma, Germany); nitrogen blowing parallel concentrator (Biotage); ultrapure water purification system (Millipore, USA); XW-80A micro vortex mixer (Shanghai Huxi); high-speed sample shaker (TAITEC SR-1, Japan); 0.22μm organic phase microporous filter membrane.

[0052] The reagents, materials, and standard solutions used in the embodiments of this invention are prepared as follows:

[0053] 1) Acetonitrile (chromatographic grade, CAS No.: 75-05-8);

[0054] 2) Formic acid (chromatographic grade, CAS number: 64-18-6);

[0055] 3) Acetic acid (chromatographic grade, CAS No.: 64-19-7);

[0056] 4) Sodium acetate (analytical grade, CAS No.: 127-09-3);

[0057] 5) Anhydrous magnesium sulfate (analytical grade, CAS No.: 7487-88-9);

[0058] 6) Ethylenediamine-N-propylsilylated silica gel (PSA): Particle size 40μm-60μm;

[0059] 7) Octadecylsilane-bonded silica gel (C18): Particle size 40μm-60μm;

[0060] 8) Standard substances: Clethodim (purity 99.5%); Pyriflusulfuron (purity 99.6%); Triazole chlorpyrifos (92.4%);

[0061] 9) Preparation of standard stock solution: Weigh 1 mg of standard substance accurately using a 1 / 100,000 balance, dissolve it in acetonitrile, and dilute to 10 mL with a volumetric flask to prepare a standard stock solution with a concentration of 100 μg / mL. Store at -18℃ for 1 year.

[0062] 10) Preparation of mixed standard stock solution: Take 1 mL of each of the standard stock solutions of clethodim, pyriflusulfonamide, and triazole chlorpyrifos into a 10 mL volumetric flask, and dilute to 10 mL with acetonitrile to prepare a mixed standard stock solution with a concentration of 10 μg / mL.

[0063] Example 1

[0064] A method for determining clethodim, pyriflusulfonamide, and / or triazole chlorpyrifos in feed, comprising the following steps:

[0065] (1) Sample pretreatment: Take 5g of sample (accurate to 0.01g) and place it in a 50ml plastic centrifuge tube. Add 10ml of water, vortex to mix, let stand for 30min, add 15ml of 1% acetic acid-acetonitrile solution and 1 homogenate, shake vigorously on a shaker for 5min to obtain the primary extract.

[0066] (2) Adsorption of impurity components: Add 6g of anhydrous magnesium sulfate and 1.5g of sodium acetate to the primary extract, place it on a high-speed sample shaker and shake vigorously for 2min, then place it in a centrifuge and centrifuge at 5000r / min for 5min to collect the supernatant, which is the secondary extract;

[0067] (3) Purification of the extract: Accurately pipette 5 ml of the secondary extract, add 750 mg of anhydrous magnesium sulfate and 250 mg of C 18 250 mg PSA, vortex for 2 min, centrifuge at 5000 r / min for 5 min, take the supernatant, filter through a 0.22 μm organic phase microporous membrane to obtain the test solution;

[0068] (4) High performance liquid chromatography-tandem mass spectrometry detection: The detection solution is injected into a high performance liquid chromatography-tandem mass spectrometer for qualitative and quantitative detection;

[0069] The chromatographic conditions are as follows:

[0070] Column: ACQUITY UPLC HSS T31.8μm; 2.1×100mm;

[0071] Mobile phase: Phase A is a 0.1% formic acid aqueous solution; Phase B is an acetonitrile solution;

[0072] Elution: Gradient elution, the elution procedure is as follows:

[0073] 0-0.5 min, mobile phase A is 50% and mobile phase B is 50%;

[0074] Over a period of 0.5–4 min, the amount of mobile phase A was decreased from 50% to 20% at a constant rate, while the amount of mobile phase B was increased from 50% to 80% at a constant rate.

[0075] 4-5 min, with mobile phase A at 20% and mobile phase B at 80%;

[0076] Over 5-5.5 minutes, the amount of mobile phase A was increased from 20% to 50% at a constant rate, while the amount of mobile phase B was decreased from 80% to 50% at a constant rate.

[0077] 5.5-7 min, mobile phase A is 50% and mobile phase B is 50%;

[0078] The amount of mobile phase B used over time varies as follows: Figure 1As shown.

[0079] The mobile phase flow rate was 0.3 mL / min, the column temperature was 40 °C, and the injection volume was 1.0 μL.

[0080] The mass spectrometry conditions are as follows:

[0081] Ion source type: Electrospray ionization source;

[0082] Scanning mode: positive ion mode;

[0083] Capillary voltage: 3.0kV;

[0084] Desolvation gas temperature: 500℃;

[0085] Desolvation gas flow rate: 600 L / Hr;

[0086] Atomizer pressure: 7.0 bar;

[0087] Multiple response monitoring (MRM).

[0088] The retention time, parent ion, daughter ion, and ion pair mass spectrometry parameters for each target analyte are shown in Table 1. The MRM mass spectrum for each target analyte is shown in Table 1. Figures 2-4 As shown; the overall ion flow spectrum (TIC) is shown below. Figure 5 As shown:

[0089] Table 1 Target ion pair parameters

[0090]

[0091]

[0092] Qualitative determination: When performing sample determination, if the relative error between the retention time of the detected chromatographic peak and the retention time of the chromatographic peak of the blank sample with added reference standard is less than 2%, and the selected ion pairs appear in the sample mass spectrum after background subtraction, and the relative abundance ratio of the selected ion pairs and the relative abundance ratio of the ion pairs with added reference standard does not exceed the range specified in Table 2, then it can be determined that this compound exists in the sample.

[0093] Table 2. Maximum permissible error (%) for relative ion abundance during qualitative confirmation.

[0094] Relative ion abundance ＞50 ＞20-50 ＞10-20 ≤10 Permissible relative error ±20 ±25 ±30 ±50

[0095] Quantitative determination: Select a blank sample with the same or similar properties as the sample to be tested and pretreat it according to the sample pretreatment method in step (1) to obtain a blank matrix solution. Accurately pipette a certain amount of mixed standard stock solution and dilute it stepwise with blank matrix solution to prepare matrix-matched standard working solutions with mass concentrations of 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL and 100 ng / mL. Select no less than 5 concentration points according to instrument performance and detection needs for instrument measurement. Plot a matrix-matched standard working curve with the peak area of ​​the mass chromatogram of the target analyte as the ordinate and the corresponding mass concentration of the matrix-matched standard working solution as the abscissa. Quantify using the external standard method. The standard curves of clethodim, pyriflusulfonamide and triazole chlorpyrifos are shown below. Figures 6-8 As shown in Table 3, the linear equations are as follows. According to the results, the standard curve has good linearity in the concentration range of 0.5 to 100 ng / mL, and the correlation coefficient r > 0.999.

[0096] The contents of clethodim, pyriflusulfonamide, and triazole chlorpyrifos in the sample were determined according to... Figure 9 The formula shown calculates:

[0097] In the formula:

[0098] ω—The content of the analyte in the sample, in milligrams per kilogram (mg / kg);

[0099] ρ—The mass concentration of the analyte in the sample solution obtained from the matrix-matched standard working curve, in milligrams per liter (mg / L);

[0100] V—The numerical value of the volume of the extract, in milliliters (mL);

[0101] m—the numerical value of the sample mass, in grams (g);

[0102] The calculation result should be rounded to three significant figures.

[0103] Table 3. Linear equations for pyriflusulfonamide, clethodim, and triazine.

[0104] Compound Name Regression equation Concentration range (ng / mL) Correlation coefficient pyriflusulfonamide Y = 56373.6 * x - 120.977 0.5~100ng / mL 0.999812 Clethodim Y = 79066.9 * x + 9957.33 0.5~100ng / mL 0.999125 Triazolyl chlorpyrifos Y = 12776*x - 357.229 0.5~100ng / mL 0.999029

[0105] The mass chromatograms of standard samples of clethodim, pyriflusulfonamide, and triazole chlorpyrifos are shown below. Figures 10-12 As shown.

[0106] Based on the detection limits and quantitation limits calculated using the signal-to-noise ratio (S / N) ≥ 3 and S / N ≥ 10, the detection limits for clethodim and pyriflusulfonamide in this invention are 0.005 mg / kg and the quantitation limits are 0.01 mg / kg; the detection limit for triazole chlorpyrifos is 0.01 mg / kg and the quantitation limit is 0.02 mg / kg.

[0107] Precision determination

[0108] Spike recovery and relative standard deviation

[0109] Spiked recovery experiments were conducted on blank feed samples. Clethodim and pyrifluquinazon were added at concentrations of 0.01 mg / kg, 0.02 mg / kg, and 0.1 mg / kg, respectively; triazole chlorpyrifos was added at three concentration levels of standard at 0.02 mg / kg, 0.04 mg / kg, and 0.2 mg / kg, with six replicates for each spiking level. After vortexing, the samples were allowed to stand for 30 min to allow the standard and feed samples to mix thoroughly. Pretreatment and UPLC-MS / MS analysis were then performed according to the corresponding procedures. The recovery rate and precision were calculated, and the results are shown in Table 4.

[0110] Table 4. Recovery and precision of analytes when added to blank feed samples

[0111]

[0112]

[0113] Example 2

[0114] Determination of novel herbicides clethodim, pyriflusulfonamide, and triazole chlorpyrifos in different feeds:

[0115] 1. Feeds containing high amounts of starch: Weigh 5g of feed sample (feeds with wheat, rice, corn, and potatoes as the main components), add 0.1μg each of clethodim, pyriflusulfonamide, and triazole chlorpyrifos, and process and determine according to the operating steps of this invention. The results showed that clethodim was 0.0180mg / kg; pyriflusulfonamide was 0.0156mg / kg; and triazole chlorpyrifos was 0.0214mg / kg. The recoveries were 90%, 78%, and 107%, respectively, meeting the detection requirements.

[0116] 2. Oily feed: Weigh 5g of feed sample (feeds with high oil content such as soybeans, peanuts, and rapeseed as the main components), add 0.1μg of clethodim, pyriflusulfonamide, and triazole chlorpyrifos, and process and determine according to the operation steps of this invention. The results showed that clethodim was 0.0230mg / kg; pyriflusulfonamide was 0.0166mg / kg; and triazole chlorpyrifos was 0.0221mg / kg. The recoveries were 115%, 83%, and 110.5%, respectively, meeting the detection requirements.

[0117] 3. Sugar-containing feed: Weigh 5g of feed sample (feed with sugarcane stalks, sugar beets, or other crops with high sugar content as the main components), add 0.1μg of clethodim, pyriflusulfonamide, and triazole chlorpyrifos, and process and determine according to the operation steps of this invention. The results showed that clethodim was 0.0191mg / kg; pyriflusulfonamide was 0.0216mg / kg; and triazole chlorpyrifos was 0.0176mg / kg. The recoveries were 95.5%, 108%, and 88%, respectively, meeting the detection requirements.

[0118] 4. High-protein feed: Weigh 5g of feed sample (feed mainly composed of defatted soybeans, soybean meal, and other high-protein crops), add 0.1μg each of clethodim, pyriflusulfonamide, and triazole oxychloride, and process and determine according to the procedure of this invention. The results showed that clethodim was 0.0187mg / kg; pyriflusulfonamide was 0.0194mg / kg; and triazole oxychloride was 0.0196mg / kg. The recoveries were 95.5%, 108%, and 88%, respectively, meeting the detection requirements.

[0119] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0120] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for determining clethodim, pyriflusulfonamide, and / or triazole chlorpyrifos in feed, characterized in that, Includes the following steps: (1) Sample pretreatment: Take the sample to be tested, add water and mix well, let stand for 15-30 minutes, add extraction solvent and homogenizing proton, shake well to obtain primary extract; (2) Adsorption of impurities and moisture: Add adsorbent to the primary extract, shake at high speed, centrifuge and take the supernatant to obtain the secondary extract; (3) Purification of the extract: Add purification reagent to the secondary extract, vortex and shake, centrifuge, take the supernatant, filter, and obtain the test solution; (4) High performance liquid chromatography-tandem mass spectrometry detection: The detection solution is injected into a high performance liquid chromatography-tandem mass spectrometer for qualitative and quantitative detection; The extraction solvent in step (1) is a 1% acetic acid-acetonitrile solution, and the amount added is 15 mL / 5 g of the sample to be tested; The adsorbents in step (2) are anhydrous magnesium sulfate and sodium acetate, with addition amounts of 6.0 g / 5 g of the sample to be tested and 1.5 g / 5 g of the sample to be tested, respectively; The purification reagent in step (3) is anhydrous magnesium sulfate, C 18 And PSA, added at concentrations of 150 mg / mL secondary extract, 50 mg / mL secondary extract and 50 mg / mL secondary extract, respectively; The chromatographic conditions for high performance liquid chromatography in step (4) are as follows: Chromatographic column: ACQUITY UPLC HSS T3 1.8μm; 2.1×100mm; Mobile phase: Phase A is a 0.1% formic acid aqueous solution; Phase B is an acetonitrile solution; Elution: Gradient elution, the elution procedure is as follows: 0-0.5 min, mobile phase A is 50% and mobile phase B is 50%; Over a period of 0.5–4 min, the amount of mobile phase A was decreased uniformly from 50% to 20%, while the amount of mobile phase B was increased uniformly from 50% to 80%. 4-5 min, with mobile phase A at 20% and mobile phase B at 80%; Over 5-5.5 minutes, the amount of mobile phase A was increased uniformly from 20% to 50%, while the amount of mobile phase B was decreased uniformly from 80% to 50%. 5.5-7 min, mobile phase A is 50% and mobile phase B is 50%; The mass spectrometry conditions for the tandem mass spectrometry in step (4) are as follows: Ion source type: Electrospray ionization source; Scanning mode: positive ion mode; Capillary voltage: 3.0 kV; Desolvation gas temperature: 500℃; Desolvation gas flow rate: 600 L / Hr; Atomizer pressure: 7.0 bar; Multiple response monitoring (MRM).

2. The determination method according to claim 1, characterized in that, The shaking time in step (1) is 5 minutes.

3. The determination method according to claim 1, characterized in that, The high-speed oscillation in step (2) is 250 times / min for 2 minutes; the centrifugation speed is 5000 r / min for 5 minutes.

4. The determination method according to claim 1, characterized in that, The vortex oscillation speed in step (3) is 250 times / min, and the time is 2min; the centrifugation speed is 5000r / min, and the centrifugation time is 5min; The filtration process uses a microporous membrane with a pore size of 0.22 μm.

5. The determination method according to claim 1, characterized in that, In step (4), the flow rate of the mobile phase during high-performance liquid chromatography elution is 0.3 mL / min, the column temperature is 40 °C, and the injection volume is 1.0 μL.

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