Method for determining etoxazole nitrile and three metabolites thereof in fresh tea leaves, tea leaves and tea soup based on ultra-high performance liquid chromatography-tandem mass spectrometry

Abstract

The invention relates to a method for determining SYP-9625 and three metabolites thereof in fresh tea leaves, tea leaves and tea soup based on ultra-high performance liquid chromatography-tandem mass spectrometry, which comprises the following steps: 1) sample extraction and purification: extracting a fresh tea leaf, tea leaf or tea soup sample with acetonitrile, performing dispersive solid-phase extraction and purification with PSA, multi-walled carbon nanotubes and magnesium sulfate, concentrating, and fixing the volume with acetonitrile; (2) separating by adopting a chromatographic column Lux3mu m Celluse-2, and detecting the etoxazole nitrile and the three metabolites thereof by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry; 3, standard curves and linear correlation coefficients of the SYP-9625 and the three metabolites of the SYP-9625 are calculated, and 4, the adding recovery rate, the relative standard deviation and the quantitation limit of the method.By means of the method, the residue analysis requirement is met, and an analysis method can be provided for research on the SYP-9625 and the three metabolites of the SYP-9625 in fresh tea leaves, tea leaves and tea soup.

Description

Technical Field

[0001] The present invention belongs to the technical field of tea pesticide residue detection, and particularly relates to a method for determining cyetpyrafen and its three metabolites in fresh tea leaves, tea leaves and tea soup based on ultra-high performance liquid chromatography tandem mass spectrometry. Background Technique

[0002] The tea garden ecosystem is complex and diverse. Pests and diseases are the biggest problems in tea garden management, seriously affecting the yield and quality of tea. There are more than 400 known tea tree pests, and more than 50 common ones, such as Empoasca vitis, Ectropis obliqua, Euproctis pseudoconspersa, etc. With the extensive use of a large number of chemical insecticides in recent decades, harmful mites have developed into dominant populations without the restriction of natural enemy populations, endangering tea trees. The main harmful mites in tea gardens include Phyllocoptruta oleivora, Calacarus carinatus, Oligonychus coffeae, Polyphagotarsonemus latus (broad mite) and Brevipalpus obovatus, etc. These pests mainly suck the sap of tea tree leaves with adult and nymph mites, causing the leaves to gradually lose their luster and seriously resulting in leaf drop, affecting the growth and yield of plants. At present, the registered acaricides in tea gardens are lime sulfur, mineral oil and quinothrin to control spider mites, leaf mites and Phyllocoptruta oleivora. The long-term use of one acaricide will cause harmful mites to develop resistance, and even cause harmful mites to develop cross-resistance to other acaricides, resulting in the rampant growth of harmful mites. In order to reduce problems such as mite resistance and cross-resistance, methods such as rotating the use of acaricides with different action mechanisms, restricting the use times, and developing some new acaricides with new action sites can be adopted.

[0003] Cyetpyrafen,

[0004] [(Z)-2-(4-tert-Butylphenyl)-2-cyano-1-(2-ethyl-5-methylpyrazol-3-yl)ethenyl]2,2-dimethylpropanoate, C24H31N3O2), with the development code SYP-9625 and CAS registry number 1253429-01-4, is a novel acrylonitrile acaricide developed by Shenyang Sinochem Agrochemical R & D Co., Ltd. It is of low toxicity to mammals and non-target organisms. It is a novel acaricide with a completely new structure discovered through structural modification using cyenopyrafen as a lead compound. It has no systemic activity and mainly kills mites through contact and stomach poisoning actions. Cyazofamid mainly metabolizes and transforms into hydroxyl compounds in mites, inhibits the action of succinate dehydrogenase, and then acts on mitochondrial complex II in the respiratory electron transport chain to disrupt energy synthesis, causing cell synthesis to stop its life functions due to lack of energy, thereby achieving the effect of controlling harmful mites. Cyazofamid has currently been registered and commercialized for use on various crops such as vegetables and fruits. It has a highly efficient selective activity spectrum against spider mites and red spider mites, and is also active against other phytophagous mites, while being safe for predatory mites and other non-target organisms. It can be mixed with fungicides and insecticides. There are currently various insecticidal and acaricidal compositions formulated by combining cyazofamid with other active ingredients (such as cyflumetofen, pyflubumide, and fluazinam, etc.). These compositions have advantages such as significant synergistic effects and delaying resistance, and can be applied to the control of pests and mites on various crops such as fruit trees, vegetables, cotton, tea, and grains. M-309 is its main metabolite. Currently, there is relatively little research on metabolite M-309. Therefore, in this invention, it is taken as one of the main metabolites for research, and at the same time, metabolites M-325-1 and M-409-3 generated through plant or environmental metabolism are also studied.

[0005] As a major economic crop in China, tea plants are treated with a large amount of insecticides, fungicides, and acaricides for a long time to control tea garden pests and diseases, which may lead to pest resistance and the problem of pesticide residues. Therefore, the low-toxic and highly active acaricide cyazofamid has good prospects for application in tea gardens. This invention has established a UPLC-MS / MS residue analysis method for cyazofamid and its metabolites (M-309, M-325-1, and M-409-3) in fresh tea leaves, tea, and tea soup. This provides a basis for subsequent research on the residue degradation behavior of cyazofamid and its metabolites in tea and the dietary risk assessment after drinking tea. It is of great significance for determining the maximum residue limit of cyazofamid in tea, guiding the rational application of cyazofamid in tea gardens, and ensuring the quality and safety of tea. Summary of the Invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for determining ethazofop-butyl and its three metabolites in fresh tea leaves, tea leaves and tea soup based on ultra-high performance liquid chromatography-tandem mass spectrometry. By comparing extraction solvents with different pH values, different purification fillers and different mobile phases, a residual analysis method for ethazofop-butyl and its three metabolites in fresh tea leaves, tea leaves and tea soup was established.

[0007] This is achieved specifically through the following technical solutions:

[0008] A method for determining ethazofenyl and its three metabolites in fresh tea leaves, tea leaves and tea soup by ultra-high performance liquid chromatography-tandem mass spectrometry comprises the following steps:

[0009] 1) Sample extraction and purification: For fresh tea leaves, tea leaves or tea soup samples, acetonitrile extraction is used, and PSA, multi-walled carbon nanotubes (MWCNT) and magnesium sulfate (MgSO4) dispersed solid phase extraction are used for purification, and acetonitrile is used to make the volume after concentration;

[0010] 2) Using chromatographic columns 3μm Cellulose-2 separation, ultra-performance liquid chromatography tandem mass spectrometry detection of ethazofenyl and its three metabolites;

[0011] 3) preparing standard stock solutions of ethazofon and its three metabolites, diluting them with acetonitrile to form a mixed standard solution of 20 mg / L, and then preparing the fresh leaves, tea leaves or tea soup matrix treated in step 1) into standard solutions of 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.002 and 0.001 mg / L, respectively, and performing UPLC-MS / MS injection analysis. Each concentration was measured 3 times, with the concentration as the abscissa x and the average peak area as the ordinate y, to obtain the standard curve and linear correlation coefficient of ethazofon and its three metabolites;

[0012] 4) Calculate the spike recovery, relative standard deviation, and quantitative limit of the method to meet the requirements for residue analysis.

[0013] Furthermore, in step 1), the specific extraction and purification of fresh tea leaves is as follows: weigh 5.0 g of ground fresh tea leaves into a centrifuge tube, add 2 mL of water, vortex and mix thoroughly, then let stand for 10 min, then add 20 mL of acetonitrile, vortex and mix, oscillate for 5 min, ultrasonicate for 10 min, add 1.0 g of NaCl, vortex and mix, oscillate for 5 min, centrifuge at 5000 rpm for 5 min, draw 6 mL of the upper organic phase solution and add it to a 10 mL centrifuge tube containing 60 mg MWCNT + 100 mg MgSO4 + 40 mg PSA, vortex and purify for 1 min, then centrifuge, take 4 mL of the supernatant into a 50 mL chicken heart bottle, concentrate to dryness, add 1 mL of acetonitrile to make up the volume, ultrasonicate to dissolve, filter through a 0.22 μm filter membrane into a sample injection bottle, and test with UPLC-MS / MS.

[0014] Further, the specific extraction and purification of tea leaves in step 1) is as follows: Weigh 2.0 g of ground black tea or green tea into a centrifuge tube, add 2 mL of water, vortex thoroughly and let stand for 10 min, then add 20 mL of acetonitrile, vortex and oscillate for 5 min, sonicate for 10 min, add 1.0 g of NaCl, vortex and oscillate for 5 min, centrifuge at 5000 rpm for 5 min, aspirate 9 mL of the upper organic phase solution and add it to a 10 mL centrifuge tube containing 90 mg of MWCNT + 150 mg of MgSO4 + 60 mg of PSA, vortex and purify for 1 min and then centrifuge, take 8 mL of the supernatant and transfer it to a 50 mL pear-shaped flask, concentrate to dryness, add 1 mL of acetonitrile to make up the volume, dissolve with ultrasonic assistance, then freeze in a -24°C refrigerator for 1 h, take out and immediately centrifuge at 10000 rpm for 2 min, filter through a 0.22 μm filter membrane into an injection vial for UPLC-MS / MS determination.

[0015] Further, the specific extraction and purification of the tea soup in step 1) is as follows: Add boiling water to the tea powder according to the standard of a tea-to-water ratio of 1:50, filter twice with double-layer filter paper after 10 min to obtain the tea soup. Take 20 mL of the tea soup into a 50 mL centrifuge tube, add 20 mL of acetonitrile, vortex and oscillate for 5 min to mix evenly, add 7.0 g of NaCl, vortex and oscillate for 5 min, centrifuge at 5000 rpm for 5 min, take 15 mL of the supernatant, concentrate to dryness, add 1.5 mL of acetonitrile to make up the volume, dissolve with ultrasonic assistance, add it to a 2 mL centrifuge tube containing 30 mg of MWCNT + 50 mg of MgSO4 + 20 mg of PSA, vortex and purify for 1 min, centrifuge at 10000 rpm for 5 min, filter through a 0.22 μm filter membrane into an injection vial for UPLC-MS / MS determination.

[0016] Further, the chromatographic conditions in step 2) are as follows: 3 μm Cellulose-2 (150×2 mm); column temperature 45°C; injection volume 5 μL; flow rate 0.3 mL / min; mobile phase A is 0.1% formic acid acetonitrile, B is 0.05% formic acid aqueous solution; gradient elution program is: 0 - 1.0 min, 30% - 70% A; 1.0 - 1.5 min, 70 - 90% A; 1.5 - 2.0, 90 - 100% A, then hold 100% A for 3.5 min; 5.5 - 6.0 min, 100 - 30% A, and the whole analysis time is 8.5 min.

[0017] Further, the mass spectrometry conditions in step 2) are: electrospray positive ion multiple reaction monitoring mode; electrospray capillary voltage 3.5 KV; ion source temperature 130°C; desolvation gas N2 temperature 350°C, flow rate 700 L / Hr; cone hole counter-blowing gas N2 flow rate 60 L / Hr.

[0018] The linear correlation coefficients of the standard curves of all compounds in different matrices of the method of the present invention are all above 0.99, the average spiked recovery rate is between 73.4% and 106.2%, the relative standard deviation is less than or equal to 12.0%, and the method quantification limit is 0.005 mg / kg (0.2 μg / L) (except for cyetpyrafen in fresh tea leaves which is 0.001 mg / kg). This method meets the requirements of residue analysis and can provide an analytical method for the research and detection of cyetpyrafen and its three metabolites in fresh tea leaves, tea and tea soup. Description of the Drawings

[0019] Figure 1 Recovery rates of cyetpyrafen and its metabolites in fresh tea leaves and dry tea under different soaking solvents (A) and different volumes (B);

[0020] Figure 2 Recovery rates of cyetpyrafen and its metabolites in fresh tea leaves under different soaking solvents;

[0021] Figure 3 Effects of different amounts of biochar, GCB, multi-walled carbon nanotubes (A) and magnesium sulfate, PSA, ZrO2 (B) on the recovery rates of target compounds;

[0022] Figure 4 Response of cyetpyrafen and its metabolites to different organic phases (A) and aqueous phases (B) (A 、B、C、 D represent concentrations respectively, where formic acid water: 0.05%, 0.10%, 0.20%, 0.50%, aqueous solutions of ammonium formate and ammonium acetate: 1.0 mmol / L, 2.0 mmol / L, 5.0 mmol / L, 10 mmol / L). Detailed Embodiments

[0024] The following further describes the present invention with reference to the drawings of the specification to better understand the technical solution.

[0025] Examples

[0026] Experimental Part

[0027] Main Instruments and Equipment: UPLC / Quattra Premier XE ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, ESI source, Mass Lynx 4.1 mass spectrometry workstation software (Waters Corporation, USA); 3μm Cellulose-2 (Phenomenex, USA); R-210 Rotary Evaporator (BUCHI Labortechnik AG, Switzerland); 3K-5 Refrigerated High-Speed Centrifuge (Sigma, Germany); KQ-250DB CNC Ultrasonic Cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); Vortex Genie2 Vortex Shaker (Scientific, USA); DFT-200 Portable High-Speed Universal Crusher (Zhejiang Wenling Lind Machinery Co., Ltd.); Electronic Analytical Balance: 0.0001 g (Mettler-Toledo, Switzerland); FilterUnit Membrane: 0.22μm (Tianjin Bonna-Agela Technologies Co., Ltd.); 2 mL Sample Vial (Agilent, USA).

[0028] Materials and Reagents: Methanol and acetonitrile are of chromatographic grade (Merck, Germany); Chromatographic grade formic acid and analytical grade ammonia water (Shanghai Macklin Biochemical Co., Ltd.); Chromatographic grade ammonium acetate (Shanghai Anpu Experimental Technology Co., Ltd.); Analytical grade sodium chloride (Shanghai Sihui Hervey Chemical Industry Co., Ltd.); Multi-walled carbon nanotubes (>95%, ID: 5 - 10 nm, OD: 10 - 20 nm, Length: 10 - 30 um, Shanghai Macklin Biochemical Technology Co., Ltd.); PSA (40 - 63μm, Shanghai Anpu Experimental Technology Co., Ltd.); Analytical grade magnesium sulfate (Shanghai Lingfeng Chemical Reagent Co., Ltd.); Pure products of cyazofamid and its metabolites M-309, M-325-1 and M-409-3: The purity is greater than 95%, and they are prepared by commissioned synthesis.

[0029] Extraction and Purification of Fresh Tea Leaves: Weigh 5.0 g of fresh tea leaves ground by a video crusher into a centrifuge tube, add 2 mL of water, vortex thoroughly and let stand for 10 min, then add 20 mL of acetonitrile, vortex and oscillate for 5 min, sonicate for 10 min, add 1.0 g of NaCl, vortex and oscillate for 5 min, centrifuge at 5000 rpm for 5 min, pipette 6 mL of the upper organic phase solution into a 10 mL centrifuge tube containing 60 mg of MWCNT + 100 mg of MgSO4 + 40 mg of PSA, vortex for purification for 1 min and then centrifuge, take 4 mL of the supernatant into a 50 mL pear-shaped flask, concentrate to dryness, add 1 mL of acetonitrile to make up the volume, dissolve with ultrasonic assistance, filter through a 0.22μm membrane into a sample vial, and wait for UPLC-MS / MS analysis.

[0030] Tea extraction and purification: Weigh 2 g of tea ground by a food grinder into a centrifuge tube, add 2 mL of water, vortex thoroughly and let stand for 10 min. Then add 20 mL of acetonitrile, vortex and shake for 5 min, sonicate for 10 min, add 1.0 g of NaCl, vortex and shake for 5 min, centrifuge at 5000 rpm for 5 min. Pipette 9 mL of the upper organic phase solution into a 10 mL centrifuge tube containing 90 mg of MWCNT + 150 mg of MgSO4 + 60 mg of PSA, vortex for purification for 1 min and then centrifuge. Take 8 mL of the supernatant and transfer it to a 50 mL pear-shaped flask, concentrate to dryness, add 1 mL of acetonitrile to make the volume constant, dissolve with ultrasonic assistance, then freeze in a -24 °C refrigerator for 1 h, take it out and immediately centrifuge at 10000 rpm for 2 min, filter through a 0.22 μm filter membrane into an injection vial for UPLC-MS / MS analysis.

[0031] Tea soup extraction and purification: Add tea powder to boiling water according to the tea-water ratio of 1:50, filter twice with double-layer filter paper after 10 min to obtain the tea soup. Take 20 mL of the tea soup into a 50 mL centrifuge tube, add 20 mL of acetonitrile, vortex and shake for 5 min to mix evenly, add 7.0 g of NaCl, vortex and shake for 5 min, centrifuge at 5000 rpm for 5 min. Take 15 mL of the supernatant, concentrate to dryness, add 1.5 mL of acetonitrile to make the volume constant, dissolve with ultrasonic assistance, add it to a 2 mL centrifuge tube containing 30 mg of MWCNT + 50 mg of MgSO4 + 20 mg of PSA, vortex for purification for 1 min, centrifuge at 10000 rpm for 5 min, filter through a 0.22 μm filter membrane into an injection vial for UPLC-MS / MS analysis.

[0032] Chromatographic conditions: 3 μm Cellulose-2 (150×2 mm); column temperature 45 °C; injection volume 5 μL; flow rate 0.3 mL / min; mobile phase A is 0.1% formic acid in acetonitrile, B is 0.05% formic acid in aqueous solution; gradient elution program: 0 - 1.0 min, 30% - 70% A; 1.0 - 1.5 min, 70 - 90% A; 1.5 - 2.0 min, 90 - 100% A, then hold 100% A for 3.5 min; 5.5 - 6.0 min, 100 - 30% A, and the whole analysis time is 8.5 min. The retention times of cyazofamid and its three metabolites are shown in Table 1.

[0033] Mass spectrometry conditions: Electrospray positive ion multiple reaction monitoring mode; electrospray capillary voltage 3.5 KV; ion source temperature 130 °C; desolvation gas N2 temperature 350 °C, flow rate 700 L / Hr; cone hole counter gas N2 flow rate 60 L / Hr. The other secondary mass spectrometry parameters of cyazofamid and its three metabolites are shown in Table 1.

[0034] Preparation of Standard Solutions and Standard Curves: Weigh 10 mg of the standard samples of cyazofamid and its three metabolites into 50 mL volumetric flasks respectively, dissolve and make up the volume with acetonitrile to prepare standard stock solutions with a concentration of 200 mg / L, and store them at -18 °C. Dilute the standard stock solutions with acetonitrile to a mixed standard solution with a concentration of 20 mg / L, and then prepare a series of standard solutions (2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.002, and 0.001 mg / L) using fresh tea leaves, tea, tea soup matrix and acetonitrile solvent obtained by treating according to the method in Section 1.3. Inject and analyze by UPLC-MS / MS, measure each concentration 3 times, use the concentration as the abscissa (x) and the average peak area as the ordinate (y) to obtain the standard curves and linear correlation coefficients of cyazofamid and its three metabolites.

[0035] The matrix effect is calculated using the following formula:

[0036] ME = (A / B - 1) × 100%

[0037] In the formula, A is the slope of the matrix standard curve, and B is the slope of the solvent standard curve. If ME is greater than 0, it indicates the existence of matrix enhancement effect; conversely, if ME is less than 0, it indicates the existence of matrix weakening effect.

[0038] Spiked Recovery Rates, Precision and Method Quantification Limits: Weigh (measure) fresh tea leaves, tea and tea soup blank samples that have been determined to contain no cyazofamid and its three metabolites, add mixed standard solutions at different concentration levels respectively. The addition levels are shown in Table 2. After vortex mixing, let it stand for 2 h to be closer to the actual situation of pesticide residues in samples, and then perform extraction, purification and determination according to the method in Section 1.3. Repeat each concentration 5 times; at the same time, add standard solutions with corresponding concentrations to the blank fresh tea leaves, tea and tea soup sample solutions obtained after treatment and make up the volume to prepare corresponding matrix standard solutions for determination, and calculate the spiked recovery rates, relative standard deviations and method quantification limits.

[0039] Table 1 Information and Mass Spectrometry Parameters of Cyazofamid and Its Three Metabolites

[0040] Note: "*" indicates the quantitative ion

[0041] Table 2 Linear Equations, Correlation Coefficients, Matrix Effects, Average Recovery Rates, Standard Deviations, Relative Standard Deviations, Detection Limits and Quantification Limits of Cyazofamid and Its Metabolites in Different Tea Matrices

[0042]

[0043] Note: C1, C2, C3, and C4 represent the added concentrations respectively. Among them, for fresh tea leaves: 0.005, 0.01, 0.1, and 1 mg / kg (the lowest addition level of CYE is 0.001 mg / kg); for green tea and black tea: 0.005, 0.025, 0.25, 2.5 mg / kg; for green tea soup and black tea soup: 0.0002, 0.001, 0.01, 0.1 mg / L.

[0044] Results and Discussion:

[0045] Optimization of the soaking solvent: Before organic solvent extraction, adding a certain amount of deionized water can make the sample absorb water and expand, which is more conducive to extracting the target compound from the sample matrix. pH is very important for extracting certain pesticides, as it affects the dissociation and solvation of pesticides, and sometimes even affects their stability. In this experiment, the recovery rates of cyazofamid and its metabolites in different soaking solvents (water, 1% formic acid in water, 2% formic acid in water, 5% formic acid in water, 1% ammonium acetate in water, 2% sodium acetate in water, 5% sodium acetate in water) were first investigated using fresh tea leaf matrix. The results are as Figure 1 shown. Compared with without adding a soaking solvent, adding a soaking solvent can improve the recovery rates of cyazofamid and its metabolites. The extraction effects of metabolites M-309 and M-409-3 are poor in alkaline solvents, and there is no significant difference between the formic acid in water condition and pure water. Considering the situation, water was finally selected as the soaking solvent. On this basis, different volumes (2, 5, 8, 10 mL) of water were optimized. Increasing the volume of water had no significant effect on the recovery rates of cyazofamid and its metabolites. Therefore, 2 mL of water was finally selected as the soaking solvent for fresh tea leaves and tea.

[0046] Optimization of the extraction solvent: Acetonitrile can dissolve and extract various polar and non-polar pesticides, and it can be miscible with water; moreover, acetonitrile has a large polarity and is not suitable for mixing with non-polar solvents. Some non-polar impurities such as chlorophyll will not be extracted together with pesticides, and the impurities in the extracted samples are relatively few, which is widely used in reversed-phase liquid chromatography. In this experiment, the recovery rates of cyazofamid and its metabolites in different acidified acetonitriles (pure acetonitrile, 1% formic acid in acetonitrile, 2% formic acid in acetonitrile, 1% ammonia in acetonitrile, 2% ammonia in acetonitrile) were first investigated using fresh tea leaf matrix. The results are as Figure 2 shown. In ammonia in acetonitrile, the recovery rates of the parent cyazofamid and metabolite M-409-3 cannot meet the requirements. Among them, pure acetonitrile has the best extraction effect, and the recovery rates of each compound can meet the requirements. The tea soup is slightly different from fresh tea leaves and dry tea. Without the need for a soaking solvent, it can be directly extracted using organic solvents. In this experiment, it was found that the recovery rates of the tea soup could meet the requirements after one extraction with 20 mL of acetonitrile. Therefore, 20 mL of acetonitrile was finally selected to extract fresh tea leaves, dry tea, and tea soup.

[0047] Screening of purification fillers: The matrix components of tea are complex, rich in various chemical components such as tea polyphenols, pigments, and lipids. Therefore, the interference in the analysis of pesticide residues in tea is greater than that in other plants, increasing the analysis difficulty. In this experiment, the adsorption of cyhexatin and three metabolites by the new purification filler biochar, multi-walled carbon nanotubes (MWCNT), and the traditional purification filler GCB was first investigated, and the recovery rates are as Figure 3 shown. Although biochar can better adsorb impurities such as pigments in the matrix and is even close to colorless and transparent after 50 mg purification, its adsorption of target compounds is also large, resulting in the recovery rate not meeting the analysis requirements. The adsorption of multi-walled carbon nanotubes and GCB on target compounds is small, and the recovery rates can meet the requirements. However, from the perspective of the color after purification, the effect of GCB is not as good as that of multi-walled carbon nanotubes. Considering the above factors, 30 mg of multi-walled carbon nanotubes was selected to adsorb impurities such as pigments in the sample. On this basis, nano-zirconia (ZrO2), which has the characteristics of high stability and strong inertness, can be used to adsorb impurities such as lipids and heavy metals. PSA can remove polar compounds such as fatty acids. During the extraction process, water immersion is added, and MgSO4 is needed to remove water. Therefore, PSA, MgSO4, and ZrO2 were added for comparison. Finally, a combination of 30 mg MWCNT + 50 mg MgSO4 + 20 mg PSA was selected to purify the sample.

[0048] Optimization of chromatographic conditions: To optimize the mobile phase in the experiment and obtain a higher response, referring to the literature reports, the responses of cyhexatin and its metabolites in the organic phases of methanol and acetonitrile, and the aqueous phases of formic acid-water, ammonium acetate aqueous solution, and ammonium formate aqueous solution were comprehensively compared. The results showed that the peak shape of metabolite M-325-1 was not good when the organic phase was methanol. Therefore, acetonitrile was selected as the mobile phase. Subsequently, the responses of cyhexatin and its metabolites after optimization with formic acid added at concentrations of 0.05%, 0.1%, 0.2%, and 0.5% were compared as Figure 4 shown. The compound response was the best under the condition of 0.05% formic acid addition, but the peak shape of metabolite M-325-1 was poor. Therefore, 0.1% formic acid-acetonitrile was selected for the subsequent experiment. For the aqueous phase, formic acid-water (0.05%, 0.10%, 0.20%, 0.50%), ammonium formate aqueous solution (1.0 mmol / L, 2.0 mmol / L, 5.0 mmol / L, 10 mmol / L), and ammonium acetate aqueous solution (1.0 mmol / L, 2.0 mmol / L, 5.0 mmol / L, 10 mmol / L) were compared. It was found that compared with formic acid aqueous solution, the responses of the parent compound and metabolite M-325-1 were lower in the ammonium solution; in formic acid-water, the compound response decreased with the increase in the concentration of formic acid-water. Subsequently, the responses in the tea matrix were compared, and 0.05% formic acid aqueous solution was comprehensively selected as the aqueous phase. The mobile phase was finally determined to be a combination of 0.1% formic acid-acetonitrile - 0.05% formic acid-water.

[0049] Optimization of mass spectrometry conditions: In this example, UPLC-ESI+-MS / MS was used to perform full-scan analysis on cyazofamid and its three metabolites, the cone voltage was optimized, and precursor ions were obtained respectively. The precursor ions were subjected to daughter ion scanning, the collision dissociation energy was optimized, the fragment ion information and the optimal collision energy were obtained. Finally, the optimized MS / MS conditions are shown in Table 1.

[0050] Standard curve, sensitivity and matrix effect: After the optimization of conditions, solvent standard solutions, fresh tea leaves, tea leaves and tea soup matrix standard solutions of cyazofamid and its metabolites in the concentration ranges of 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.002 and 0.001 mg / L were investigated. The relevant linear equations and correlation coefficients obtained by UPLC-MS / MS are shown in Table 2. The results show that in the above various matrices, the linear relationships of cyazofamid and its metabolites are good, and the correlation coefficients (R2) are all above 0.99, all meeting the requirements. After purification of samples such as tea leaves, the results show that there is still a certain matrix effect. Therefore, matrix standard external standard method needs to be used for quantitative analysis.

[0051] Spiked recovery rate, precision and limit of quantification: According to the above, 5 parallel spiked recovery rate tests of cyazofamid and its metabolites in fresh tea leaves, tea leaves and tea soup were carried out at low, medium and high concentration levels. The average spiked recovery rate (A.R.) and relative standard deviation (RSD) results are shown in Table 2. The limit of quantification LOQS of cyazofamid and its metabolites in fresh leaves and tea leaves is ≤0.005 mg / kg (0.001 mg / kg for cyazofamid in fresh leaf dry tea matrix), and the limit of quantification LOQS in tea soup is ≤0.5 μg / L. The method can meet the requirements for the residue analysis of cyazofamid and its three metabolites in fresh tea leaves, tea leaves and tea soup, etc.

[0052] Determination of actual samples: After spraying 30% cyazofamid suspension at a dose of 0.75 g·ai / 100 m2 on tea garden tea leaves, fresh tea leaf samples were collected at 2 h, 1, 2, 3, 5, 7, 10 and 14 d after spraying, and detected by this method. The results show that cyazofamid and metabolite M-309 were detected, the residue of M-409-3 was lower than the limit of quantification, and the residue of cyazofamid and M-309 were <LOQ~4.370 mg / kg and 0.011 - 0.136 mg / kg respectively, and M-325-1 was not detected. Therefore, this batch of samples has been stored in a -20°C refrigerator for one year, and there may be some errors in the residue amount. Next, a field experiment will be carried out again for verification.

[0053] Conclusion: In the present invention, by optimizing the extraction and purification conditions, acetonitrile extraction was used for fresh leaves, tea leaves and tea soup samples, and multi-walled carbon nanotubes, PSA and magnesium sulfate were used for dispersive solid-phase extraction and purification, and after concentration, it was made up to volume with acetonitrile. Chromatographic separation of cyetpyrafen and its three metabolites was carried out on a 3μm Cellulose-2 chromatographic column, and the matrix external standard method of UPLC-MS / MS was used for quantification. A residue analysis method for cyetpyrafen and its three metabolites in various matrices such as fresh tea leaves, tea leaves and tea soup was established. The linear correlation coefficients of the standard curves of all compounds in different matrices were above 0.99, the average spiked recovery rates were between 73.4% and 106.2%, the RSD was less than 12.0%, and the method quantification limit was 0.005 mg / kg (0.2 μg / L) (except for cyetpyrafen in fresh tea leaves which was 0.001 mg / kg). The results showed that the method met the requirements of residue analysis and could provide an analytical method for the research and detection of cyetpyrafen and its three metabolites in fresh tea leaves, tea leaves and tea soup.

Claims

1. A method for the determination of cyetpyrafen and its three metabolites in fresh tea leaves, tea leaves and tea infusions by ultra-high performance liquid chromatography-tandem mass spectrometry, characterized in that, Including the following steps: 1)) Sample extraction and purification: For fresh tea leaves, tea or tea soup samples, extract with acetonitrile, and purify by dispersive solid-phase extraction with PSA, multi-walled carbon nanotubes (MWCNT) and magnesium sulfate (MgSO4). After concentration, make up the volume with acetonitrile. 2) Use a chromatographic column Separate with 3μm Cellulose-2 and detect cyazofamid and its three metabolites by ultra-high performance liquid chromatography-tandem mass spectrometry; 3) Prepare a standard stock solution of cyenopyrafen and its three metabolites, dilute it with acetonitrile to a mixed standard solution of 20 mg / L. Then, prepare standard solutions of 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.002 and 0.001 mg / L from the fresh tea leaves, tea or tea soup matrix treated in step 1) respectively. Inject and analyze by UPLC-MS / MS, measure each concentration 3 times. Take the concentration as the abscissa x and the average peak area as the ordinate y to obtain the standard curves and linear correlation coefficients of cyenopyrafen and its three metabolites. 4) Calculate the spiked recovery rate, relative standard deviation and the limit of quantitation of the method to meet the requirements of residue analysis.

2. The method for determining cyazofamid and its three metabolites in fresh tea leaves, tea leaves and tea soup based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that In step 1), the extraction and purification of fresh tea leaves are specifically as follows: Weigh the ground fresh tea leaves into a centrifuge tube, add water, vortex thoroughly and mix evenly, then let it stand. Then add acetonitrile, vortex and mix evenly, shake, ultrasonicate, add NaCl, vortex and mix evenly and shake, centrifuge at 5000 rpm, aspirate the upper organic phase solution and add it to a centrifuge tube containing MWCNT + MgSO4 + PSA, vortex and purify, then centrifuge. Take the supernatant into a pear-shaped flask, concentrate to dryness, add acetonitrile to make up the volume, ultrasonically assist in dissolution, filter through a 0.22 μm filter membrane into an injection vial for UPLC-MS / MS determination.

3. The method for determining cyazofamid and its three metabolites in fresh tea leaves, tea leaves and tea soup based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, wherein In step 1), the extraction and purification of tea are specifically as follows: Weigh the ground black tea or green tea into a centrifuge tube, add water, vortex thoroughly and mix evenly, then let it stand. Then add acetonitrile, vortex and mix evenly, shake, ultrasonicate, add NaCl, vortex and mix evenly and shake, centrifuge at 5000 rpm, aspirate the upper organic phase solution and add it to a centrifuge tube containing MWCNT + MgSO4 + PSA, vortex and purify, then centrifuge. Take the supernatant into a pear-shaped flask, concentrate to dryness, add acetonitrile to make up the volume, ultrasonically assist in dissolution, then freeze in a -24 °C refrigerator for 1 h, take it out and immediately centrifuge at 10000 rpm for 2 min, filter through a 0.22 μm filter membrane into an injection vial for UPLC-MS / MS determination.

4. The method for determining cyazofamid and its three metabolites in fresh tea leaves, tea leaves and tea soup based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that In step 1), the extraction and purification of tea soup are specifically as follows: Add tea powder to boiling water according to the standard of 1:50 of tea to water ratio, filter twice with double-layer filter paper after 10 min to obtain tea soup; Take the tea soup into a centrifuge tube, add acetonitrile, vortex and mix evenly, add NaCl, vortex and mix evenly and shake, centrifuge at 5000 rpm, take the supernatant, concentrate to dryness, add acetonitrile to make up the volume, ultrasonically assist in dissolution, add it to a centrifuge tube containing MWCNT + MgSO4 + PSA, vortex and purify, then centrifuge at 10000 rpm for 5 min, filter through a 0.22 μm filter membrane into an injection vial for UPLC-MS / MS determination.

5. A method for determining cyazofamid and its three metabolites in fresh tea leaves, tea leaves and tea soup based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that In step 2), the chromatographic conditions are as follows: 3μm Cellulose-2 (150×2mm); column temperature 45°C; injection volume 5μL; flow rate 0.3mL / min; mobile phase A is 0.1% formic acid acetonitrile, B is 0.05% formic acid aqueous solution; gradient elution program: 0 - 1.0 min, 30% - 70% A; 1.0 - 1.5 min, 70 - 90% A; 1.5 - 2.0, 90 - 100% A, then hold 100% A for 3.5 min; 5.5 - 6.0 min, 100 - 30% A, and the whole analysis time is 8.5 min.

6. The method for determining cyazofamid and its three metabolites in fresh tea leaves, tea leaves and tea soup based on ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, wherein In step 2), the mass spectrometry conditions are as follows: electrospray positive ion multiple reaction monitoring mode; electrospray capillary voltage 3.5 KV; ion source temperature 130 °C; desolvation gas N2 temperature 350 °C, flow rate 700 L / Hr; cone orifice counter gas N2 flow rate 60 L / Hr.

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