A method for determining four metabolites of tolfenpyrad in tea leaves and tea soup based on ultra-high performance liquid chromatography-tandem mass spectrometry

Abstract

A method for determining 4 metabolites of tolfenpyrad in tea leaves and tea infusions by ultra-high performance liquid chromatography-tandem mass spectrometry belongs to the technical field of detection of pesticide residues in tea. The present invention includes: 1) Sample extraction and purification: For fresh tea leaves or tea samples, extract with 1% formic acid acetonitrile, and purify by dispersive solid-phase extraction with C 18 , MgSO4, GCB and C NT -OH, and make the volume constant with acetonitrile after concentration; or 1) Extract the tea infusion sample with 1% formic acid acetonitrile, and make the volume constant with acetonitrile after concentration; 2) Separate with an ACQUITY UPLC HSS T3 chromatographic column, and detect 4 metabolites of tolfenpyrad by ultra-high performance liquid chromatography-tandem mass spectrometry; 3) Calculate the standard curve, linear correlation coefficient and matrix effect of the tolfenpyrad metabolites; 5) Calculate the spiked recovery rate, relative standard deviation, detection limit and quantification limit of the method. The method of the present invention meets the requirements of residue analysis and can provide an analytical method for the research and detection of 4 metabolites of tolfenpyrad in fresh tea leaves, tea leaves and tea infusions.

Description

Technical Field

[0001] The invention belongs to the technical field of tea pesticide residue detection, and specifically relates to a method for determining four metabolites of tolfenpyrad in tea leaves and tea soup based on ultra-high performance liquid chromatography tandem mass spectrometry. Background Art

[0002] Tea is an important economic crop in my country. According to statistics, in 2020, the tea plantation area in my country reached 3.261 million hectares, and the tea output was 3.18 million tons, an increase of 7.1% over 2019. Tea trees mostly grow in warm and humid environments and are susceptible to pests and diseases. At present, chemical control is still the main means of controlling tea tree pests and diseases. As a popular beverage, people are accustomed to drinking tea soup and discarding tea residues. Therefore, tea soup is an important carrier for evaluating safety. The selection of high-efficiency and low-water-soluble pesticides is of great significance for ensuring the quality and safety of tea and the safety of consumers drinking tea. Tolfenpyrad is a new type of pyrazole heterocyclic insecticide and acaricide developed by the former Mitsubishi Chemical Corporation of Japan. It mainly acts on the energy metabolism process of the insect respiratory system, inhibits the electron transfer of mitochondrial complex I, and thus prevents the oxidative phosphorylation process in energy metabolism. The drug is mainly contact-killing, has a broad insecticide spectrum, kills insects quickly, and does not have systemic conductivity. In addition, Tolfenpyrad has the advantages of low water solubility (0.087mg / L) and good control effect. It is registered and promoted in my country as an excellent agent for controlling tea green leafhoppers. From 2011 to 2013 alone, it has been demonstrated and promoted in 15 provinces in my country's four major tea-growing areas, with an application area of ​​about 350,000 mu. At present, Tolfenpyrad has been widely used in my country's tea gardens and has broad prospects. my country's national food safety standard GB2763-2021 stipulates that the maximum residue limit of Tolfenpyrad in tea is 50mg / kg; the United States and Canada are 30mg / kg, Japan is 20mg / kg, and the European Union is a uniform standard of 0.01mg / kg.

[0003] In recent years, in addition to the parent pesticides, the metabolic degradation products of pesticides in agricultural products and foods have also received extensive attention. The JMPR report of the International Joint Meeting on Pesticide Residues reported the metabolites of tolfenpyrad in some crops such as eggplant, cabbage, and peach, such as PT-OH, PT-CA, OH-PT, OH-T-CA, CA-T-CA, OH-T-OH, CA-T-AM, etc. Among them, the acute oral toxicity of PT-CA and OH-PT to rats is greater than that of the parent pesticides, which may bring greater safety risks. At present, the research on tolfenpyrad mainly focuses on the parent pesticides, including the prevention effect, detection methods, and the digestion and residue rules in different crops. The research on metabolites is still scarce, and there is no detection method for the metabolites of tolfenpyrad in agricultural products.

[0004] The applicant previously used time-of-flight mass spectrometry to analyze and identify four metabolites of tolfenpyrad in tea trees: PT-OH, PT-CA, OH-T-CA and CA-T-CA, and obtained standard products. The present invention establishes a residual analysis method for the four metabolites of tolfenpyrad in fresh tea leaves, green tea, black tea, green tea soup and black tea soup matrices. This provides a methodological basis for understanding the generation, transfer and safety assessment of metabolites of tolfenpyrad after application to tea trees and during subsequent processing and brewing, which is of great significance for ensuring the quality and safety of tea. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for determining four metabolites of tolfenpyrad in tea leaves and tea soup based on ultra-high performance liquid chromatography tandem mass spectrometry. This method solves the technical problems in the prior art that the residual amount of tolfenpyrad metabolites in tea leaves cannot be detected, and the residual behavior of tolfenpyrad metabolites cannot be monitored after application to tea trees and during subsequent tea processing and brewing.

[0006] The present invention is specifically implemented through the following technical solutions:

[0007] The method for determining four metabolites of tolfenpyrad in tea leaves and tea soup based on ultra-high performance liquid chromatography tandem mass spectrometry comprises the following steps:

[0008] 1) Sample extraction and purification: Take the crushed fresh tea leaves or tea samples, extract with 1% formic acid and acetonitrile, C 18 MgSO 4 , GCB and C NT -OH was purified by dispersed solid phase extraction, concentrated and fixed to volume with acetonitrile, and passed through a 0.22 μm organic membrane for testing;

[0009] Or 1) Sample extraction: extract the tea sample with 1% formic acid and acetonitrile, concentrate it, make it constant with acetonitrile, pass it through a 0.22 μm organic membrane, and then test it;

[0010] 2) Using ACQUITY UPLC HSS T3 column for separation and ultra-high performance liquid chromatography-tandem mass spectrometry UPLC-MS / MS to detect the four metabolites of tolfenpyrad;

[0011] 3) Prepare standard stock solutions of four metabolites of tolfenpyrad, dilute them with acetonitrile to a mixed standard solution of 100 mg / L, and then dilute them step by step with fresh tea leaves, tea leaves and tea soup matrix treated in step 1) and acetonitrile to standard solutions of 5.0, 1.0, 0.50, 0.10, 0.050 and 0.010 mg / L, respectively; measure each concentration three times by UPLC-MS / MS injection, with concentration as the abscissa x and the average peak area as the ordinate y, to obtain a standard curve of tolfenpyrad metabolites within a certain concentration range, and calculate the linear correlation coefficient, matrix effect and method detection limit LOD;

[0012] 4) Calculate the recovery rate, relative standard deviation and limit of quantification LOQ of the validation method through the addition recovery test to meet the requirements of residue analysis;

[0013] The names, chemical structures, molecular formulas and relative molecular masses of the above four metabolites of tolfenpyrad are as follows:

[0014]

[0015] Further, the extraction and purification of fresh tea leaves or tea samples in step 1) specifically includes: weighing 2.0 g of ground fresh tea leaves or 1.0 g of dry tea samples into a 50 mL polytetrafluoroethylene centrifuge tube, adding 5 mL of purified water and vortexing, standing for 30 min, adding 10 mL of 1% formic acid acetonitrile solution, and vortexing; after standing for 2 h, adding 5.0 g of NaCl, oscillating for 5 min, vortexing, ultrasonicating for 30 min, and centrifuging at 10000 r / min for 5 min; transferring all the supernatant, repeating the extraction once, combining the supernatant and vortexing; taking 7.5 mL of the supernatant into a heart-shaped bottle, concentrating to dryness by rotary evaporation in a water bath at 40° C., and adjusting the volume to 1.5 mL of acetonitrile; transferring to a 50 mg C 18 、50mgMgSO 4 , 20mgGCB and 20mgC NT -OH in a 2 mL centrifuge tube, vortex, centrifuge at 12000 r / min for 5 min, pass the supernatant through a 0.22 μm organic filter membrane, and then be tested by UPLC-MS / MS.

[0016] Furthermore, the extraction and purification of the tea soup sample in the step 1) specifically includes: brewing dry tea at a tea / water ratio of 1 / 50 v / v and a water temperature of 100° C. for 5 minutes, filtering with double-layer filter paper to obtain a tea soup sample; taking 20 mL of tea soup and placing it in a 50 mL polytetrafluoroethylene centrifuge tube, adding 20 mL of 1% formic acid acetonitrile solution, vortexing, adding 8.0 g of NaCl, vortexing, oscillating for 5 minutes, and centrifuging at 10,000 r / min for 5 minutes; after stratification, transferring all the supernatant, repeating the extraction once, combining the supernatant and vortexing to mix, taking 20 mL of the supernatant and concentrating to dryness by rotary evaporation in a water bath at 40° C., making the volume of acetonitrile to 1.5 mL, passing through a 0.22 μm organic filter membrane, and determining by UPLC-MS / MS.

[0017] Further, the chromatographic conditions in the step 2) are: ACQUITY UPLC HSS T3 chromatographic column specifications 100 mm×2.1 mm, 1.8 μm, column temperature 40°C, flow rate 0.2 mL / min, injection volume 5 μL; mobile phase A is 0.1% formic acid methanol, and B is 0.1% formic acid aqueous solution; gradient elution program is: 10%-65% A, 0-1.5 min; 65%-85% A, 1.5-5.0 min; 85%-99% A, 5.0-7.5 min; 99%-100% A, 7.5-9 min; 100% A, 9-9.8 min, 100%-90% A, 9.8-10.3 min.

[0018] Furthermore, the mass spectrometry conditions in step 2) are: electrospray positive ionization / negative ionization multiple reaction monitoring mode; electrospray capillary voltage 3.5 kV; ion source temperature 150 ° C; desolvation gas N 2 Temperature 350℃, flow rate 650L / hr; cone backflush gas N 2 The flow rate was 50 L / hr; the collision gas Ar flow rate was 0.25 mL / min.

[0019] The linear correlation coefficients of the standard curves of the four metabolites in different matrices of the method of the present invention are all above 0.99, the average recovery rate of addition is 75.85%-114.97%, the relative standard deviation is less than 18.5%, and the method quantitative limit is less than or equal to 0.05 mg / kg. The method of the present invention meets the requirements of residue analysis and can provide an analytical method for the research and detection of the four metabolites of tolfenpyrad in fresh tea leaves, tea leaves and tea soup. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Comparison of the recovery rates of four metabolites of tolfenpyrad by different extraction solvents;

[0021] Figure 2 UPLC-MS / MS chromatograms of four metabolites of tolfenpyrad (2.5 mg / kg) in green tea matrix;

[0022] Figure 3 This is the matrix effect of the four metabolites of tolfenpyrad in different tea matrices. DETAILED DESCRIPTION

[0023] The present invention will be further described below with reference to the embodiments.

[0024] Example:

[0025] 1. Experimental part:

[0026] 1.1 Main instruments and equipment

[0027] UPLC-Xevo TQ-S Micro ultra-high performance liquid chromatography-triple quadrupole mass spectrometer: a product of Waters, USA, equipped with an electrospray ionization (ESI) source, MassLynx 4.1 workstation; chromatographic column: ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm); 3K-5 refrigerated high-speed centrifuge: a product of Sigma, Germany; R-210 rotary evaporator: a product of BUCHI Labortechnik AG, Switzerland; KQ-250DB CNC ultrasonic cleaner: a product of Kunshan Ultrasonic Instrument Co., Ltd.; Vortex Genie2 vortex oscillator: a product of Scientific, USA; DFT-200 portable high-speed universal pulverizer: a product of Linda Machinery Co., Ltd., Wenling, Zhejiang; electronic analytical balance: 0.0001 g, a product of Mettler-Toledo, Switzerland; Filter Unit filter membrane 0.22μm, product of Tianjin Bona Agilent Technology Co., Ltd.; 2mL injection bottle: product of Agilent Co., Ltd., USA.

[0028] 1.2 Materials and reagents

[0029] Methanol (MeOH) and acetonitrile (MeCN) were all chromatographically pure, from Merck, Germany; chromatographically pure formic acid (FA) and analytically pure ammonia (AHA) were from Shanghai Macklin Biochemical Co., Ltd.; chromatographically pure ammonium acetate (AA) was from Shanghai Anpu Experimental Technology Co., Ltd.; analytically pure acetic acid (HAC) was from Shanghai Lingfeng Chemical Reagent Co., Ltd.; anhydrous magnesium sulfate (MgSO 4 ), sodium chloride (NaCl) were analytical grade and were from Shanghai Shisi Hewei Chemical Co., Ltd.; graphitized carbon black (GCB, 120-400 mesh) and C 18 (40-60μm), Tianjin Bona Aiger Technology Co., Ltd.; Hydroxylated carbon nanotubes (C NT-OH, 10-20nm), Chengdu Timesnano Organic Chemicals Co., Ltd.; metabolite CA-T-CA standard, Shanghai Anqu Chemical Co., Ltd.; metabolite PT-OH, PT-CA and OH-T-CA standards were commissioned for synthesis (purity greater than 95%).

[0030] 1.3 Sample extraction and purification

[0031] 1.3.1 Weigh 2.0g of ground fresh tea leaf sample (1.0g of dry tea sample) into a 50mL polytetrafluoroethylene centrifuge tube, add 5mL of pure water and vortex mix, let stand for 30min, add 10mL of 1% formic acid acetonitrile solution and vortex mix. After standing for 2h, add 5.0g of NaCl, oscillate for 5min, vortex mix, ultrasonicate for 30min, and centrifuge at 10000r / min for 5min. Transfer all the supernatant, repeat the extraction once, combine the supernatant and vortex mix. Take 7.5mL of supernatant in a chicken heart bottle, concentrate to dryness by rotary evaporation in a water bath at 40℃, and make up to 1.5mL of acetonitrile. Transfer to a container containing 50mgC 18 、50mgMgSO 4 , 20mgGCB and 20mgC NT -OH in a 2 mL centrifuge tube, vortex, centrifuge at 12000 r / min for 5 min, pass the supernatant through a 0.22 μm organic filter membrane, and then be tested by UPLC-MS / MS.

[0032] 1.3.2 Dry tea was brewed at a tea / water ratio of 1 / 50 (v:v) and a water temperature of 100°C for 5 minutes, and then filtered with double-layer filter paper to obtain tea soup (green tea soup, black tea soup) samples. Take 20mL of tea soup and place it in a 50mL polytetrafluoroethylene centrifuge tube, add 20mL of 1% formic acid acetonitrile solution, vortex, add 8.0gNaCl, vortex, oscillate for 5min, and centrifuge at 10000r / min for 5min. After stratification, transfer all the supernatant, repeat the extraction once, combine the supernatant and vortex to mix, take 20mL of supernatant and concentrate to dryness by rotary evaporation in a water bath at 40°C, dilute to 1.5mL with acetonitrile, pass through a 0.22μm organic filter membrane, and measure with UPLC-MS / MS.

[0033] 1.4 Chromatographic mass spectrometry conditions

[0034] 1.4.1 Chromatographic conditions: 100 mm × 2.1 mm, 1.8 μm ACQUITY UPLC HSS T3 column, 40 °C column temperature, 0.2 mL / min flow rate, injection volume 5 μL. Mobile phase A was 0.1% formic acid in methanol, and mobile phase B was 0.1% formic acid in water; gradient elution program was: 10%-65% A, 0-1.5 min; 65%-85% A, 1.5-5.0 min; 85%-99% A, 5.0-7.5 min; 99%-100% A, 7.5-9 min; 100% A, 9-9.8 min, 100%-90% A, 9.8-10.3 min.

[0035] 1.4.2 Mass spectrometry conditions: electrospray positive / negative ionization multiple reaction monitoring mode; electrospray capillary voltage 3.5 kV; ion source temperature 150 °C; desolvation gas N 2 Temperature 350℃, flow rate 650L / hr; cone backflush gas N 2 The flow rate was 50 L / hr, and the collision gas Ar flow rate was 0.25 mL / min. The MRM parameters of the four metabolites of tolfenpyrad are shown in Table 1.

[0036] Table 1 Mass spectrometry MRM parameters of four metabolites of tolfenpyrad

[0037]

[0038] 1.5 Standard solution, standard curve and matrix effect

[0039] Weigh a certain amount of tolfenpyrad metabolite standard into a 10 mL brown volumetric flask, dilute with acetonitrile to prepare a 1000 mg / L standard stock solution, and store in a -18 °C refrigerator. The standard stock solutions of the four metabolites were prepared into a 100 mg / L mixed standard solution with acetonitrile, and the fresh tea leaves, green tea, black tea, green tea soup and black tea soup matrices obtained after treatment with the method in Section 1.3 and acetonitrile were gradiently diluted to obtain a series of standard solutions of 5.0, 1.0, 0.50, 0.10, 0.050, and 0.010 mg / L. UPLC-MS / MS analysis was performed, with concentration as the abscissa (x) and peak area as the ordinate (y), to obtain the standard curves and linear correlation coefficients R of tolfenpyrad metabolites in the solvent and the above different matrices. 2 .

[0040] At the same time, the matrix effect (ME) was calculated using the formula ME = (A / B-1) × 100%

[0041] Where A is the slope of the matrix standard curve, and B is the slope of the solvent standard curve. ME>0 indicates the presence of a matrix enhancement effect; ME<0 indicates the presence of a matrix inhibition effect. The closer the ME is to 0, the smaller the matrix effect. In addition, the method detection limit (LOD) is calculated based on the response at the lowest concentration level on the matrix standard curve when the signal-to-noise ratio S / N=3.

[0042] 1.6 Add recovery, precision and limit of quantitation

[0043] Four different concentration levels of tolfenpyrad metabolite standard solutions were added to blank tea leaves, green tea, black tea (0.005, 0.5, 1 and 10 mg / L), green tea soup and black tea soup (0.0005, 0.005, 0.01 and 0.1 mg / L), vortexed and placed overnight to be closer to the actual pesticide residues in the samples. Extraction and purification were performed according to the steps in 1.3, and 5 parallels were set for each added concentration. The average recovery and relative standard deviation (RSD) of tolfenpyrad metabolites in different tea matrices at different added concentration levels were calculated by comparison with the matrix standards of the corresponding concentrations. The method quantification limit (LOQ) was defined by the minimum added concentration level that met the recovery and relative standard deviation requirements.

[0044] 2. Results and Discussion

[0045] 2.1 Optimization of pre-treatment conditions

[0046] 2.1.1 Optimization of soaking solvent: For compounds with high water solubility, soaking in water before the extraction step can effectively improve the recovery rate. This example investigates the recovery rate of tolfenpyrad metabolites in different soaking solvents (water, 1% formic acid aqueous solution, 5% formic acid aqueous solution, 1% acetic acid aqueous solution, and 5% acetic acid aqueous solution). The results show that under the above-mentioned soaking solutions, the recovery rate of the target substance is greater than 80%, and the relative standard deviation is less than 20%. In line with the purpose of environmental protection and conservation, water is selected as the soaking solvent.

[0047] 2.1.2 Optimization of extraction solvent: Acetonitrile is often used as the extraction solvent in the QuEChERS method. Different pH values ​​have a significant impact on the extraction effect. Considering the structural differences between the metabolites of tolfenpyrad, this example compares the recovery rates of acetonitrile, acidified acetonitrile (1% formic acid acetonitrile, 2% formic acid acetonitrile, 1% acetic acid acetonitrile, 2% acetic acid acetonitrile) and alkalinized acetonitrile (2% ammonia acetonitrile) as extraction solvents. The results are shown in Figure 1 As shown, it can be seen that the recovery rates of tolfenpyrad metabolites in acidified acetonitrile all meet the analytical requirements, among which 1% formic acid acetonitrile solution has the best effect as the extraction solvent.

[0048] 2.1.3 Screening of purification fillers: This example first investigated 6 commonly used fillers (PWAX, SCX, C 18 、PSA、C 18 -N, and MgSO 4 ) for the adsorption of four metabolites. The results showed that 50 mg C 18 +50mgMgSO 4 There was no obvious adsorption interference on the analytes. Considering that the tea matrix is ​​relatively complex and has a high pigment content, GCB and C with strong adsorption capacity were selected. NT -OH and optimized its dosage. The results showed that 50mgC 18 +50mgMgSO 4 +20mgGCB+20mgC NT -OH can achieve better purification effect under the premise of ensuring the recovery rate.

[0049] 2.2 Optimization of chromatography-mass spectrometry conditions

[0050] 2.2.1 Optimization of chromatographic conditions: In order to obtain a higher response of the target substance, the 0.1% FA-MeOH + 0.1% FA-H 2 O, 0.1% FA-MeOH+10mmol / L AA-H 2 O, 0.1% FA-MeCN+10mmol / L AA-H 2 O, 0.1% FA-MeCN + 0.1% FA-H 2 The response of the target compound when 0.1% FA-MeOH + 0.1% FA-H 2 When O was used as the mobile phase, all four metabolites could obtain high responses, so 0.1% formic acid methanol and 0.1% formic acid water were selected as the mobile phase.

[0051] 2.2.2 Optimization of mass spectrometry conditions: In electrospray positive ionization mode (ESI + ) and electrospray negative ionization mode (ESI - ) performed a full scan analysis on the metabolites of tolfenpyrad to determine the parent ion and ionization mode, and optimized the cone voltage, and then applied collision energy to obtain characteristic product ions and optimal collision energy. The final optimized MRM parameters are shown in Table 1.

[0052] 2.3 Standard curve, correlation coefficient, matrix effect and detection limit

[0053] After the optimization conditions were established, the linear equations and correlation coefficients of the solvent standard solutions of the four metabolites of tolfenpyrad and the matrix standard solutions of fresh tea leaves, green tea, black tea, green tea soup and black tea soup with gradient concentrations (5.0, 1.0, 0.50, 0.10, 0.050, 0.010 mg / L) were determined by UPLC-MS / MS. The results are shown in Table 2. The results show that in the above matrices, the metabolites of tolfenpyrad have a good linear relationship within a certain concentration range, and the correlation coefficient (R 2 ) were all above 0.99, and the method detection limit (LOD) was less than 0.01 mg / kg, meeting the requirements for residue analysis. The typical UPLC-MS / MS chromatograms of the four metabolites of tolfenpyrad (2.5 mg / kg) in green tea matrix are shown in Figure 2 .

[0054] This example compares the matrix effect (ME) of tolfenpyrad metabolites in different tea matrices. Figure 3 As shown in the figure, it shows that the target compound has matrix inhibition effect in most matrices, so the matrix matching standard curve is used for quantitative analysis.

[0055] Table 2 Linear equations and correlation coefficients (R) of tolfenpyrad metabolites in different tea matrices 2 ), matrix effect (ME) and limit of detection (LOD)

[0056]

[0057] 2.4 Addition recovery, precision and limit of quantification

[0058] According to the above steps, the addition recovery test (n=5) of the metabolites of tolfenpyrad in fresh tea leaves, green tea, black tea, green tea soup and black tea soup was carried out at four addition levels, and the average addition recovery rate (AR), relative standard deviation (RSD) and limit of quantification (LOQ) results are shown in Table 3. At the four concentration addition levels of 0.005, 0.5, 1 and 10 mg / L, the average recovery rates of the metabolites of tolfenpyrad PT-OH and PT-CA in fresh tea leaves, green tea and black tea were 77.46-107.69%, and the corresponding RSDs were 1.43-17.84%; at the four addition levels of 0.0005, 0.005, 0.01 and 0.1 mg / L, the average recovery rates and RSDs of the metabolites of tolfenpyrad PT-OH and PT-CA in green tea soup and black tea soup were 81.28-114.97% and 4.21-17.08%, respectively. The metabolites OH-T-CA and CA-T-CA were detected in negative ion mode. At the three concentration addition levels of 0.5, 1 and 10 mg / L, the average recoveries of OH-T-CA and CA-T-CA in fresh tea leaves, green tea and black tea were 75.85-109.90%, and the corresponding RSDs were 2.69-16.24%; at the three addition levels of 0.005, 0.01 and 0.1 mg / L, the average recoveries and RSDs of OH-T-CA and CA-T-CA in green tea soup and black tea soup were 81.78-108.98% and 3.07-18.50%, respectively. The LOQs of the metabolites of tolfenpyrad in fresh tea leaves, green tea and black tea were ≤0.05 mg / L, and the LOQs in green tea soup and black tea soup were ≤0.005 mg / L. This method can meet the requirements for the residual analysis of the four metabolites of tolfenpyrad in tea leaves, tea soup, etc.

[0059] Table 3 Average recoveries (AR), relative standard deviations (RSDs) and limits of quantification (LOQs) of the four metabolites of tolfenpyrad in different tea matrices

[0060]

[0061]

[0062]

[0063] 2.5 Actual sample measurement

[0064] 20 samples of green tea and black tea were randomly selected from the market and tested according to the established method. The results showed that PT-CA was detected 5 times in 40 samples, with a detection rate of 12.5% ​​and a residual amount at LOQ-0.222 mg / kg. PT-OH was detected once with a residual amount of 0.035 mg / kg. The metabolites OH-T-CA and CA-T-CA were not detected.

[0065] The above description is a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining four metabolites of tolfenpyrad in tea leaves and tea soup based on ultra-high performance liquid chromatography tandem mass spectrometry, characterized in that The following steps are involved: 1) Extraction and purification of fresh tea leaves or tea samples specifically include: weighing 2.0g of ground fresh tea leaves or 1.0g of dry tea samples into a 50mL polytetrafluoroethylene centrifuge tube, adding 5mL of purified water and vortexing, letting stand for 30 min, adding 10mL of 1% formic acid acetonitrile solution and vortexing; after standing for 2h, adding 5.0g of NaCl, shaking for 5min, vortexing, ultrasonicating for 30min, and centrifuging at 10000r / min for 5min; transferring all the supernatant, repeating the extraction once, combining the supernatant and vortexing; taking 7.5mL of the supernatant into a chicken heart bottle, concentrating to dryness by rotary evaporation in a water bath at 40℃, and making up the volume to 1.5mL with acetonitrile; transferring to a 50mgC 18 , 50mgMgSO4, 20mgGCB and 20mgC NT -OH in a 2 mL centrifuge tube, vortex, centrifuge at 12000 r / min for 5 min, and filter the supernatant through a 0.22 μm organic filter for UPLC-MS / MS analysis; The extraction and purification of tea soup samples specifically include: dry tea is brewed at a tea / water ratio of 1 / 50 v / v and a water temperature of 100°C for 5 minutes, and then filtered with double-layer filter paper to obtain a tea soup sample; 20 mL of tea soup is placed in a 50 mL polytetrafluoroethylene centrifuge tube, 20 mL of 1% formic acid acetonitrile solution is added, vortexed, 8.0 g of NaCl is added, vortexed, shaken for 5 minutes, and centrifuged at 10000 r / min for 5 minutes; after stratification, all supernatants are transferred, the extraction is repeated once, the supernatants are combined and vortexed to mix, 20 mL of supernatant is concentrated to dryness by rotary evaporation in a water bath at 40°C, the volume of acetonitrile is adjusted to 1.5 mL, and the solution is filtered through a 0.22 μm organic filter membrane and determined by UPLC-MS / MS; 2) ACQUITY UPLC HSS T3 column was used for separation, and ultra-high performance liquid chromatography-tandem mass spectrometry UPLC-MS / MS was used to detect the four metabolites of tolfenpyrad. The chromatographic conditions were as follows: ACQUITY UPLC HSS T3 column size: 100 mm × 2.1 mm, 1.8 μm, column temperature: 40 °C, flow rate: 0.2 mL / min, injection volume: 5 μL; mobile phase A: 0.1% formic acid in methanol, mobile phase B: 0.1% formic acid in water; gradient elution program: 10%-65% A, 0-1.5 min; 65%-85% A, 1.5-5.0 min; 85%-99% A, 5.0-7.5 min; 99%-100% A, 7.5-9min; 100%A, 9-9.8min, 100%-90%A, 9.8-10.3min; mass spectrometry conditions: electrospray positive ionization / negative ionization multiple reaction monitoring mode; electrospray capillary voltage 3.5kV; ion source temperature 150℃; desolvation gas N2 temperature 350℃, flow rate 650L / hr; cone hole backwash gas N2 flow rate 50L / hr; collision gas Ar flow rate 0.25mL / min; 3) Prepare standard stock solutions of four metabolites of tolfenpyrad and dilute them with acetonitrile to a mixed standard solution of 100 mg / L. Then, dilute them step by step with fresh tea leaves, tea leaves and tea soup matrix treated in step 1) and acetonitrile to standard solutions of 5.0, 1.0, 0.50, 0.10, 0.050 and 0.010 mg / L respectively; measure each concentration three times by UPLC-MS / MS injection, with concentration as the abscissa x and the average peak area as the ordinate y, to obtain a standard curve of tolfenpyrad metabolites within a certain concentration range, and calculate the linear correlation coefficient, matrix effect and method detection limit LOD; 4) Calculate the recovery rate, relative standard deviation and limit of quantification LOQ of the validation method through the addition recovery test to meet the requirements of residue analysis; The chemical structural formulas of the four metabolites of tolfenpyrad are: 。

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