Determination method of pyraclostrobin, flufenoxuron, quimacryd thiocyclam in tea leaf

Abstract

The application discloses a method for determining the residual amount of pydiflumetofen, flufenoxuron, quimacryd thiocyclam in tea leaves, and the method is gas chromatography-tandem mass spectrometry, and the method comprises the following steps: (1) preparation of standard stock solution and standard working solution; (2) preparation of the sample to be measured; (3) gas chromatography-tandem mass spectrometry analysis conditions; (4) drawing of a standard curve; and (5) result calculation. The method establishes a method for determining the residual amount of pydiflumetofen, flufenoxuron, quimacryd and thiocyclam in tea leaves by using gas chromatography-tandem mass spectrometry. The application fills the blank of the prior art that there is no detection method for simultaneously detecting pydiflumetofen, flufenoxuron, quimacryd and thiocyclam in tea leaves, and the method has the characteristics of good selectivity, high sensitivity and strong anti-background interference capacity and is suitable for daily detection of the residual amount of pydiflumetofen, flufenoxuron, quimacryd and thiocyclam in tea leaves.

Description

Technical Field

[0001] This invention relates to a method for determining the residues of azoxystrobin, flufenoxuron, quinfenoxuron, and thiamethoxam in tea. Background Technology

[0002] Flufenoxam, quinfenoxam, thiamethoxam, and azoxystrobin are all common agricultural pesticides in my country, widely used to control crop diseases and pests. my country's national standard for maximum residue limits of pesticides in food (GB 2763-2021) specifies the maximum residue limits for azoxystrobin, flufenoxam, quinfenoxam, and thiamethoxam in tea (limits of 20 mg / kg for azoxystrobin, 20 mg / kg for flufenoxam, 15 mg / kg for quinfenoxam, and 10 mg / kg for thiamethoxam, respectively). However, currently there is no method to simultaneously detect azoxystrobin, flufenoxam, quinfenoxam, and thiamethoxam in tea. Detection of these substances requires multiple methods, which is time-consuming and consumes a large amount of reagents and consumables. Furthermore, existing gas chromatography-tandem mass spectrometry standards for these substances all use selected ion detection (SIM) mode, which has limitations in selectivity, sensitivity, and resistance to background interference. Therefore, the invention of a detection method that can simultaneously detect the residues of azoxystrobin, flufenoxuron, quinalfen, and thiamethoxam in tea, and that has the characteristics of good selectivity, high sensitivity, and strong resistance to background interference, is of great significance for the detection of pesticide residues in tea on the market. Summary of the Invention

[0003] The purpose of this invention is to provide a method for determining the residues of azoxystrobin, flufenoxuron, quinfenoxuron, and thiamethoxam in tea, filling the current gap in the lack of detection methods that can simultaneously detect azoxystrobin, flufenoxuron, quinfenoxuron, and thiamethoxam in tea. By using a multiple reaction monitoring mode, this method has the characteristics of good selectivity, high sensitivity, and strong resistance to background interference.

[0004] To achieve the above objectives, the technical solution of the present invention is as follows: The method for determining the residues of azoxystrobin, flufenoxuron, quinfenoxam, and thiamethoxam in tea according to the present invention comprises the following steps: (1) Preparation of standard stock solutions and standard working solutions: The standard stock solutions of azoxystrobin, flufenoxuron, quinfenoxuron and thiamethoxam are all 100 μg / mL standard stock solutions. The standard stock solutions are prepared with acetonitrile to form a mixed standard working solution with a concentration of 1 μg / mL. The mixed standard working solution of azoxystrobin, flufenoxuron, quinfenoxuron and thiamethoxam is prepared with tea blank matrix solution by stepwise dilution to form a series of mixed standard working solutions with concentrations of 0.0020 μg / mL, 0.0050 μg / mL, 0.0100 μg / mL, 0.0500 μg / mL, 0.100 μg / mL, 0.200 μg / mL and 0.500 μg / mL. (2) Preparation of the test sample: Weigh 2 g of the sample (accurate to 0.01 g) into a 50 mL plastic centrifuge tube, add 10 mL of water, vortex and mix well, and let stand for 30 min. Add 15 mL of acetonitrile + acetic acid (99+1) solution, 6 g of anhydrous magnesium sulfate, and 1 ceramic homogenate, cap the centrifuge tube, shake vigorously for 1 min, and centrifuge at 4200 r / min for 5 min. Take 8 mL of the supernatant and add it to a 15 mL plastic centrifuge tube containing 1200 mg of anhydrous magnesium sulfate, 400 mg of PSA, 400 mg of C18, and 50 mg of GCB. Vortex and mix well for 1 min, and centrifuge at 4200 r / min for 5 min. Accurately take 4 mL of the supernatant into a 10 mL test tube and blow it to near dryness with nitrogen in a 40℃ water bath. Add 1 mL of ethyl acetate to reconstitute, filter through a microporous membrane, and use for determination.

[0005] (3) The instrument used was a gas chromatography-tandem mass spectrometer. The gas chromatography conditions were as follows: column: 5% cyanopropylphenyl-95% dimethyl polysiloxane quartz capillary column: 30 m × 0.25 mm × 0.25 μm; column temperature: 60 ℃ for 1 min, then increased to 170 ℃ at a programmed rate of 40 ℃ / min, then increased to 310 ℃ at a programmed rate of 10 ℃ / min, and held for 5 min; carrier gas: helium, purity ≥99.999%, flow rate 1.0 mL / min; injection port temperature: 280 ℃; injection volume: 1 μL; injection method: splitless injection; mass spectrometry conditions: ion source type: electron impact source (EI): 70 eV; ion source temperature: 280 ℃; transfer line temperature: 280 ℃; solvent delay: 3 min; multiple reaction monitoring; detection ion pair mass spectrometry parameters: flufenoxuron (retention time 8.81). (Retention time: 12.04 min, ion pair I: 126.0 / 98.0, collision energy 20 V, ion pair II: 126.0 / 80.0, collision energy 10 V); Thiamethoxam (retention time: 12.04 min, ion pair I: 104.0 / 51.0, collision energy 25 V, ion pair II: 104.0 / 77.0, collision energy 25 V); Azoxystrobin (retention time: 11.54 min, ion pair I: 145.0 / 102.1, collision energy 25 V, ion pair II: 145.0 / 115.1, collision energy 15 V); Quinalox (retention time: 11.53 min, ion pair I: 145.0 / 117.1, collision energy 10 V, ion pair II: 160.0 / 117.1, collision energy 20 V); (4) Construction of standard curve: A series of mixed standard working solutions of flufenoxuron, thiamethoxam, azoxystrobin, and quinclorac were loaded onto a gas chromatograph-tandem mass spectrometer and detected under the chromatographic and mass spectrometric conditions in step (3). The retention time and ion pairs were used for qualitative analysis. The standard curve was constructed by the relationship between the peak area of ​​the compound measured at each concentration and its mass concentration. The formula is Z = FW + G, where Z is the concentration of the compound to be tested, in mg / mL; W is the peak area of ​​the compound to be tested obtained on the instrument; F is the slope of the standard curve constructed by the instrument based on the relationship between the peak area of ​​the compound and its concentration; and G is the intercept of the standard curve constructed by the instrument based on the relationship between the peak area of ​​the compound and its concentration. (5) Calculation of results: Take the sample obtained in (2) and perform detection in a gas chromatograph-tandem mass spectrometer under the chromatographic and mass spectrometric conditions in step (3). Measure the peak areas of the quantitative ions of flufenoxuron, thiamethoxam, azoxystrobin, and quinacrine in the sample. Use retention time and ion pairs for qualitative analysis. Quantify according to the standard curve drawn in (4). Calculate the residual amounts of flufenoxuron, thiamethoxam, azoxystrobin, and quinacrine in the sample.

[0006] Compared with the prior art, the present invention has the following outstanding advantages: This invention fills the gap in the existing technology for the simultaneous detection of azoxystrobin, flufenoxuron, quinfenoxuron, and thiamethoxam in tea. Only one pretreatment and instrument testing are required to complete the simultaneous determination of azoxystrobin, flufenoxuron, quinfenoxuron, and thiamethoxam in tea. This invention uses multiple reaction monitoring (MRM) mode, which, compared to the selected ion detection (SIM) mode used in existing standards, has the advantages of good selectivity, high sensitivity, and strong resistance to background interference. This invention employs the QuEChERS rapid sample pretreatment technique, which, compared with conventional pretreatment methods, is low-cost, easy to operate, has a short pretreatment time, high recovery rate, precision and accuracy, can analyze a wide range of pesticides, uses less solvent, and causes less environmental pollution. Attached Figure Description

[0007] Figure 1 This is a multiple reaction monitoring (MRM) ion chromatogram of pyridoxine.

[0008] Figure 2 This is a multiple reaction monitoring (MRM) ion chromatogram of flufenoxuron.

[0009] Figure 3 This is a multiple reaction monitoring (MRM) ion chromatogram of quinacrine.

[0010] Figure 4 This is a multiple reaction monitoring (MRM) ion chromatogram of thiamethoxam. Detailed Implementation

[0011] The present invention will be further illustrated below through specific embodiments. Example

[0012] 1. Reagents and Materials Unless otherwise specified, only analytical grade reagents were used in the analysis, and all test water was Grade I water as specified in GB / T 6682.

[0013] 1.1 Reagents 1.1.1 Acetonitrile (CH3CN).

[0014] 1.1.2 Ethyl acetate (CH3COOC2H5).

[0015] 1.1.3 Acetic acid (CH3COOH).

[0016] 1.1.4 Anhydrous magnesium sulfate (MgSO4).

[0017] 1.2 Solution Preparation 1.2.1 Acetonitrile-acetic acid solution (99+1): Measure 10 mL of acetic acid and add it to 990 mL of acetonitrile, then mix well.

[0018] 1.3 Standard Products Standard stock solutions of flufenoxuron (CAS No.: 101463-69-8), thiamethoxam (CAS No.: 69327-76-0), azoxystrobin (CAS No.: 117428-22-5), and quinclorac (CAS No.: 120928-09-8), all with a concentration of 100 μg / mL, were provided by Fujian Chemical Glass Educational Instrument Co., Ltd.

[0019] 1.4 Preparation of Standard Solutions Preparation of standard stock solutions and standard working solutions: The standard stock solution was prepared into a mixed standard working solution with a concentration of 1 μg / mL using acetonitrile. The mixed standard working solution of azoxystrobin, flufenoxuron, quinclorac, and thiamethoxam was prepared into a series of mixed standard working solutions with concentrations of 0.0020 μg / mL, 0.0050 μg / mL, 0.0100 μg / mL, 0.0500 μg / mL, 0.100 μg / mL, 0.200 μg / mL, and 0.500 μg / mL using tea blank matrix solution through a stepwise dilution method. All standard working solutions and standard working solutions were stored in a refrigerator at 0 ℃ to 4 ℃, protected from light.

[0020] 1.5 Materials 1.5.1 Ethylenediamine-N-propylsilylated silica gel (PSA): 40 μm~60 μm.

[0021] 1.5.2 Octadecylsilane bonded silica gel (C18): 40 μm~60 μm.

[0022] 1.5.3 Graphitized carbon black (GCB): 40–120 μm.

[0023] 1.5.4 Ceramic homogeneous proton: 2 cm (length) × 1 cm (outer diameter).

[0024] 1.5.5 Microporous filter membrane (organic phase): 13 mm × 0.22 μm.

[0025] 2. Instruments and Equipment 2.1 Gas Chromatography-Tandem Mass Spectrometry: Equipped with an electron impact source (EI).

[0026] 2.2 Analytical balance: sensitivity 0.1 mg and 0.01 g.

[0027] 2.3 Centrifuge: speed not less than 4200 r / min.

[0028] 2.4 Tissue homogenizer.

[0029] 2.5 Nitrogen Evaporator: Temperature controllable.

[0030] 2.6 Vortex mixer.

[0031] 3 Methods 3.1 Reference conditions for gas chromatography determination Chromatographic column: 5% acrylonitrile phenyl-95% dimethyl polysiloxane quartz capillary column: 30 m × 0.25 mm × 0.25 μm; Column temperature: 60 ℃ for 1 min, then increase to 170 ℃ at 40 ℃ / min, then increase to 310 ℃ at 10 ℃ / min and hold for 5 min; Carrier gas: Helium, purity ≥99.999%, flow rate 1.0 mL / min; Inlet temperature: 280 ℃; Injection volume: 1 μL; Injection method: splitless injection.

[0032] 3.2 Mass Spectrometry Reference Conditions Ion source type: Electron impact source (EI): 70 eV; Ion source temperature: 280 ℃; Transmission line temperature: 280 ℃; Solvent delay: 3 min; Multiple reaction monitoring (MRM): For each pesticide, one pair of quantitative ions and one pair of qualitative ions were selected. All ion pairs to be detected in each group were detected separately at different time points according to the elution order. The retention time, quantitative ion pairs, qualitative ion pairs, and collision voltage for each pesticide are shown in Table 1.

[0033] Table 1. Detection Ion Pair Mass Spectrometry Parameters

[0034] 3.3 Plotting the matrix standard working curve A series of mixed standard working solutions of flufenoxuron, thiamethoxam, azoxystrobin, and quinclorac were loaded onto a gas chromatograph-tandem mass spectrometer and analyzed under the chromatographic and mass spectrometric conditions described in 3.1-3.2. Qualitative analysis was performed using retention time and ion pairs. A standard curve was plotted based on the relationship between the peak area and mass concentration of each compound at different concentrations, using the formula Z = FW + G, where Z is the concentration of the analyte (mg / mL); W is the peak area of ​​the analyte obtained on the instrument; F is the slope of the standard curve plotted by the instrument based on the relationship between the peak area and concentration; and G is the intercept of the standard curve plotted by the instrument based on the relationship between the peak area and concentration.

[0035] 4. Sample Testing Procedures 4.1 Sample preparation and storage After grinding the tea leaves at room temperature and mixing them thoroughly, place them in a polyethylene bottle or bag and store the sample at -18 ℃ for later use.

[0036] 4.2 Sample Pretreatment Weigh 2 g of the sample (accurate to 0.01 g) into a 50 mL plastic centrifuge tube, add 10 mL of water, vortex to mix, and let stand for 30 min. Add 15 mL of acetonitrile + acetic acid (99+1) solution, 6 g of anhydrous magnesium sulfate, and 1 ceramic homogenate. Cap the centrifuge tube, shake vigorously for 1 min, and centrifuge at 4200 r / min for 5 min. Pipette 8 mL of the supernatant into a 15 mL plastic centrifuge tube containing 1200 mg of anhydrous magnesium sulfate, 400 mg of PSA, 400 mg of C18, and 50 mg of GCB. Vortex to mix for 1 min, centrifuge at 4200 r / min for 5 min, and accurately pipette 4 mL of the supernatant into a 10 mL test tube. Purge the test tube with nitrogen at 40 °C until nearly dry. Redissolve the sample in 1 mL of ethyl acetate, filter through a microporous membrane, and use for determination.

[0037] 4.3 Sample Testing The sample solution is injected sequentially into the gas chromatograph-tandem mass spectrometer. Qualitative analysis is performed by retention time and ion pairing. The quantitative ion peak area is measured. The response value of the compound in the sample solution should be within the linear range of the instrument's quantitative determination. If it exceeds the linear range, it should be diluted appropriately according to the concentration to be measured before analysis.

[0038] 4.4 Qualitative and Quantitative 4.4.1 Retention Time The relative error between the retention time of the target pesticide chromatographic peak in the test sample and the retention time of the corresponding standard chromatographic peak should be within ± 0.1 min.

[0039] 4.4.2 Ion abundance ratio When samples are measured under the same experimental conditions, if the retention time of the detected chromatographic peak is consistent with that of the standard sample, and both the quantitative and qualitative ions of the target compound appear in the mass spectrum of the sample after background subtraction, and for the same batch of samples, the relative abundance ratio of the qualitative and quantitative ions of the target compound in the sample does not exceed the range specified in Table 2 compared with the matrix standard solution of equivalent mass concentration, then the target pesticide can be determined to be present in the sample.

[0040] Table 2 Maximum permissible deviation of relative ion abundance during qualitative analysis

[0041] 4.4.3 Quantitative analysis Quantitative analysis using the external standard method.

[0042] 5 Result Calculation The pesticide residues in the sample are represented by X, and the values ​​are expressed in milligrams per kilogram (mg / kg), calculated using the following formula:

[0043] In the formula: X: The content of the analyte in the sample, in milligrams per kilogram (mg / kg). C: Mass concentration of the analyte in the matrix-matched standard working solution, in micrograms per liter (μg / mL). A: Peak area of ​​the analyte in the sample solution; A S : Peak area of ​​the analyte in the matrix-matched standard working solution; V: The volume of the sample diluted during the test, in milliliters (mL). m: Sample weight, in grams (g); The calculation results are expressed as the arithmetic mean of two independent determinations obtained under repeatability conditions, and are retained to two significant figures. When the content exceeds 1 mg / kg, three significant figures are retained.

[0044] 6. Methodological evaluation: including linearity, limit of detection, limit of quantitation, recovery rate, and precision.

[0045] 6.1 Linearity, Limit of Detection, and Limit of Quantification: A series of mixed standard working solutions of flufenoxuron, thiamethoxam, azoxystrobin, and quinaloxone with concentrations ranging from 0.0020 μg / mL to 0.500 μg / mL were prepared. Linear regression was performed on peak area against concentration. The linear equation and correlation coefficient are shown in Table 3. The results showed that flufenoxuron, thiamethoxam, azoxystrobin, and quinaloxone exhibited good linearity within the range of 0.0020 μg / mL to 0.500 μg / mL, with correlation coefficients (R) all greater than 0.999. The method fully meets the detection requirements for flufenoxuron, thiamethoxam, azoxystrobin, and quinaloxone residues in tea. The limits of detection and limits of quantification of the method are shown in Table 4.

[0046] Table 3. Linear range, regression equation, and correlation coefficient of flufenoxuron, thiamethoxam, azoxystrobin, and quinclorac.

[0047] Y: Peak area; x: Mass concentration, μg / mL.

[0048] Table 4. Limits of Detection and Limits of Quantification for Flufenoxuron, Thiazide, Azoxystrobin, and Quinalox.

[0049] 6.2 Recovery and Precision: Under optimized detection conditions, blank matrix tea samples were used for spiked recovery testing. Spiking levels were 0.005 mg / kg, 0.01 mg / kg, and 0.05 mg / kg, with each level analyzed six times. The results are shown in Table 5. Table 5 shows that the spiked recovery rate of this method ranged from 79.2% to 91.4%, and the relative standard deviation (RSD) ranged from 2.9% to 4.3%. These results indicate that this method is suitable for routine analysis and detection of flufenoxuron, thiamethoxam, azoxystrobin, and quinclorac residues in tea.

[0050] Table 5. Results of spiked recovery of tea samples (n=6)

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for detecting the residues of pydiflumetofen, flufenoxuron, quimacryd thiocyclam in tea leaves, characterized in that The detection method specifically comprises the following steps: (1) Preparation of standard stock solution and standard working solution: the standard stock solutions of the pyridalyl, flufenoxuron, quimacryd thiocyclam are each 100 μg / mL, the standard stock solution is prepared into a mixed standard use solution with a concentration of 1 μg / mL by using acetonitrile, and the mixed standard use solution of the pyridalyl, flufenoxuron, quimacryd thiocyclam is prepared into a series of mixed standard working solutions with concentrations of 0.0020 μg / mL, 0.0050 μg / mL, 0.0100 μg / mL, 0.0500 μg / mL, 0.100 μg / mL, 0.200 μg / mL and 0.500 μg / mL by using a tea blank matrix solution through a step-by-step dilution method; (2) Preparation of a sample to be detected: 2 g of a sample is weighed in a 50 mL plastic centrifugal tube, 10 mL of water is added and mixed uniformly by vortexing, is left to stand for 30 min, 15 mL of acetonitrile+acetic acid solution, 6 g of anhydrous magnesium sulfate and 1 ceramic homogenate are added, a centrifugal tube cover is covered, is violently shaken for 1 min and then centrifuged at 4200 r / min for 5 min, 8 mL of supernatant is taken and added to a 15 mL plastic centrifugal tube containing 1200 mg of anhydrous magnesium sulfate, 400 mg of PSA, 400 mg of C18 and 50 mg of GCB, is mixed uniformly by vortexing for 1 min and then centrifuged at 4200 r / min for 5 min, 4 mL of supernatant is accurately taken in a 10 mL test tube, is blown to near dryness under a nitrogen atmosphere in a 40°C water bath environment, 1 mL of ethyl acetate is added for redissolution, is filtered through a microporous filter membrane and is used for determination; (3) The instrument used is a gas chromatograph-tandem mass spectrometer, the gas chromatograph conditions are as follows: a chromatographic column: 5% cyanopropylphenyl-95% dimethylpolysiloxane quartz capillary column: 30 m x 0.25 mm x 0.25 μm; chromatographic column temperature: 60°C is kept for 1 min, then is programmed to increase the temperature to 170°C at a rate of 40°C / min, and then is increased to 310°C at a rate of 10°C / min, and is kept for 5 min; Carrier gas: helium, purity ≥ 99.999%, flow rate 1.0 mL / min; injection port temperature: 280 ℃; injection volume: 1 μL; injection mode: splitless injection; mass spectrometry conditions: ion source type: electron impact source: 70 eV; ion source temperature: 280 ℃; transfer line temperature: 280 ℃; solvent delay: 3 min; multiple reaction monitoring; detection ion pair mass spectrometry parameters: flufenoxuron: retention time 8.81 min, ion pair I: 126.0 / 98.0, collision energy 20 V, ion pair II: 126.0 / 80.0, collision energy 10 V; buprofezin: retention time 12.04 min, ion pair I: 104.0 / 51.0, collision energy 25 V, ion pair II: 104.0 / 77.0, collision energy 25 V; picoxystrobin: retention time 11.54 min, ion pair I: 145.0 / 102.1, collision energy 25 V, ion pair II: 145.0 / 115.1, collision energy 15 V; quimacryd thiourea: retention time 11.53 min, ion pair I: 145.0 / 117.1, collision energy 10 V, ion pair II: 160.0 / 117.1, collision energy 20 V; (4) Preparation of standard curve: the series of mixed standard working solutions of flufenoxuron, buprofezin, picoxystrobin and quimacryd thiourea are injected into a gas chromatography-tandem mass spectrometer, and detected according to the chromatography-mass spectrometry conditions of step (3). The standard curve is drawn according to the corresponding relationship between the peak area of each compound and its mass concentration, and the formula is Z=FW+G, wherein Z: the concentration of the compound to be detected, unit: μg / mL; W: the peak area of the compound to be detected obtained by the instrument; F: the slope of the standard curve drawn by the instrument according to the corresponding relationship between the peak area of the compound and its concentration; G: the intercept of the standard curve drawn by the instrument according to the corresponding relationship between the peak area of the compound and its concentration; (5) Result calculation: the sample obtained in step (2) is detected in a gas chromatography-tandem mass spectrometer under the chromatography-mass spectrometry conditions of step (3). The peak area of the flufenoxuron, buprofezin, picoxystrobin and quimacryd thiourea quantitative ion in the sample is measured, and the retention time and ion pair are used for qualitative analysis. The standard curve drawn in step (4) is used for quantitative analysis, and the residual amount of flufenoxuron, buprofezin, picoxystrobin and quimacryd thiourea in the sample is calculated. In step (3), the chromatographic column is a 5% cyanopropylphenyl-95% dimethylpolysiloxane quartz capillary column: 30 m x 0.25 mm x 0.25 μm, the carrier gas is helium with a purity of ≥ 99.999%, the ion source is an electron impact source, and a multiple reaction monitoring mode is used.

2. The method according to claim 1, wherein the tea is tea leaves. The 8 mL supernatant in step (2) is added to a 15 mL plastic centrifuge tube containing 1200 mg of anhydrous magnesium sulfate, 400 mg of PSA, 400 mg of C18 and 50 mg of GCB for purification.

3. The method according to claim 1, wherein the tea is tea leaves. The correlation coefficient of the standard curve in step (4) is not less than 0.99.

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