Method for determining 10 kinds of pesticide residues in tea leaves

Abstract

The present application relates to a kind of method for determining 10 kinds of pesticide residues in tea, belong to detection technical field.The 10 kinds of pesticides are indanofan, hydroprene, methoprene, methoprene, clofentezine, fluorodifen, chlorobenzilate, dinocap, molinate, chloroneb;The method comprises: a. tea sample is mixed with water and then extracted by ultrasonic, to obtain solution A;B. solution A is mixed with 1% acetic acid-acetonitrile solution, sodium acetate, anhydrous magnesium sulfate and homogenous proton, shake uniformly, then centrifuged at 8500r / min for 3min at 4 DEG C to obtain supernatant A.The method of the present application first establishes the gas chromatography-tandem mass spectrometry determination method of 10 kinds of pesticide residues in tea, such as indanofan, hydroprene, methoprene, methoprene, clofentezine, fluorodifen, chlorobenzilate, dinocap, molinate, chloroneb.The method of the present application has the characteristics of simple operation, high sensitivity, good accuracy, low detection cost and the like.

Description

Technical Field

[0001] This invention relates to a method for determining the residues of 10 pesticides in tea, belonging to the field of detection technology. Background Technology

[0002] Tea is one of the world's three major beverages, and countries around the world have strict limits on pesticide residues in tea. Risk control regarding pesticide residues has become a crucial task for the tea industry and a green technology barrier that urgently needs to be overcome. GB 2763-2021, "National Food Safety Standard - Maximum Residue Limits for Pesticides in Food," and GB 2763.1-2022, "National Food Safety Standard - Maximum Residue Limits for 112 Pesticides including Sodium 2,4-D Butyrate in Food," set limits on 110 pesticide residues in tea. However, for more than ten pesticides with limited limits, such as indomethacin, cyclopyr, flufenoxuron, propargite, and tebufenozide, there are still missing supporting testing standards, and some reference standards are not applicable to tea testing. This creates a "blind spot" in the supervision of tea quality and safety, and also results in significant quality and safety loopholes in tea production.

[0003] T / FQIA 003—2022, "Determination of Residues of Herbicides, Flufenoxuron, Glyphosate, Cyclopyralid, Entampyr and Entampyr in Tea by Gas Chromatography-Tandem Mass Spectrometry," discloses a method for determining the residues of herbicides, flufenoxuron, glyphosate, cyclopyralid, entampyr and entampyr in tea. However, it can only detect 6 pesticide residues and cannot simultaneously detect 10 pesticide residues in tea, including indomethacin, entampyr, entampyr, entampyr, cyclopyralid, flufenoxuron, propyl ester, dicofol, fenpropathrin, and herbicides.

[0004] T / CSTEA 00051—2022, "Determination of acetamiprid, acetamiprid, cyprodinil, flufenoxuron and glyphosate in tea by gas chromatography-mass spectrometry," discloses a method for determining acetamiprid, acetamiprid, cyprodinil, flufenoxuron and glyphosate in tea. However, it can only detect 5 pesticide residues and cannot simultaneously detect 10 pesticide residues in tea, including indoxacarb, acetamiprid, acetamiprid, cyclopyr, flufenoxuron, propyl chlorpyrifos, chlorpyrifos, fenpropathrin, and glyphosate.

[0005] Pesticides vary in their chemical properties and structures, and the tea matrix is ​​complex, containing numerous interfering substances such as tea polyphenols, pigments, amino acids, and alkaloids, which significantly hinder detection. Overcoming matrix interference and achieving qualitative, quantitative, rapid, accurate, and sensitive detection of 10 pesticide residues with different structures and chemical properties in tea—including indomethacin, acetamiprid, acetamiprid, cyclopyrid, flufenoxuron, propylthiophanate-methyl, dicofol, fenpropathrin, and glyphosate—is of great significance for tea production and regulation. Summary of the Invention

[0006] The purpose of this invention is to provide a method for determining the residues of 10 pesticides in tea, including indomethacin, acetamiprid, acetamiprid, cyclopyrid, flufenoxuron, propyl chlorpyrifos, chlorpyrifos, fenpropathrin, and glyphosate.

[0007] To achieve the above objectives, the 10 pesticides are indoxacarb, tebufenozide, tebufenozide, cyclopyr, flufenoxuron, propyl chlorpyrifos, chlorpyrifos, fenpropathrin, and glyphosate; the method includes:

[0008] a. After mixing the tea sample with water, extract it using ultrasound to obtain solution A;

[0009] b. Mix solution A with 1% acetic acid-acetonitrile solution, sodium acetate, anhydrous magnesium sulfate and homogenate, shake well, and then centrifuge at 8500 r / min for 3 min at 4℃ to obtain supernatant A; the mass-volume ratio of tea leaves, water, 1% acetic acid-acetonitrile, anhydrous magnesium sulfate and sodium acetate is 2 g:10 mL:10 mL:6 g:1.5 g;

[0010] c. Take the supernatant A and mix it with anhydrous magnesium sulfate, PSA, C18 and GCB, shake it evenly, and then centrifuge it at 8000 r / min for 3 min at 4℃ to obtain supernatant B. The volume-to-mass ratio of supernatant A, anhydrous magnesium sulfate, PSA, C18 and GCB in step c is 6 mL: 1.2 g: 50 mg: 50 mg: 50 mg.

[0011] d. Take the supernatant B and dry it with nitrogen gas; then add chromatographic grade ethyl acetate solution to redissolve it, and filter it through a 0.22 μm organic microporous membrane to obtain the sample to be tested. The volume ratio of the supernatant B to the chromatographic grade ethyl acetate solution is 2:1.

[0012] e. Gas chromatography-tandem mass spectrometry detection:

[0013] A series of mixed standard working solutions with different mass concentrations were sequentially injected into gas chromatography-tandem mass spectrometry for detection. A standard working curve was plotted with the mass concentration of each target compound as the x-axis and the peak area of ​​the target compound as the y-axis.

[0014] The sample to be tested was injected into gas chromatography-tandem mass spectrometry for detection. The peak area was substituted into the standard working curve to obtain the residues of 10 pesticides in the sample. The chromatographic column used was a DB-1701MS column.

[0015] Preparation of the series of mixed standard working solutions: First, prepare single standard stock solutions of 10 pesticides respectively. Then, mix the 10 single standard stock solutions and make up to a certain volume to prepare a mixed standard stock solution. Finally, dry the mixed standard stock solution with nitrogen gas, reconstitute it with blank matrix solution, and dilute it stepwise with blank matrix solution to prepare a series of mixed standard working solutions with different mass concentrations.

[0016] Preparation of blank matrix solution: Take a blank tea sample that does not contain the 10 pesticides to be tested and repeat steps a to d to obtain a blank matrix solution.

[0017] The homogenants can be ceramic homogenants, and the amount of homogenants added should be based on the appropriate amount for mixing the solution.

[0018] In one specific embodiment, the blank tea leaves are of the same type as the tea sample described in step a; the ultrasonic extraction time in step a is preferably 30 to 40 minutes.

[0019] In one specific implementation, the oscillation described in b is uniform oscillation for more than 1 minute.

[0020] In one specific embodiment, step c describes PSA with a mesh size of 40–60 μm, C18 with a mesh size of 100–120, and GCB with a mesh size of 40–60 μm.

[0021] In one specific embodiment, the chromatographic conditions are as follows: injection port temperature: 270°C; injection port pressure: 8.2 psi; injection volume: 1 μL; injection mode: splitless injection; constant flow mode, flow rate: 1.0 mL / min; temperature program: 80°C for 2 min, increase to 200°C at 15°C / min, and then increase to 280°C at 3°C / min for 5 min.

[0022] In one specific embodiment, the DB-1701MS column has dimensions of 30m × 0.25mm × 0.25μm.

[0023] In one specific embodiment, the mass spectrometry conditions are: electron energy 70 eV; electron bombardment ion source; ion source temperature 230°C; quadrupole temperature 150°C; transfer line temperature 280°C; scan mode: MRM mode.

[0024] In one specific embodiment, the mass concentration of the single-standard stock solution is 50.00 mg / L, and the solvent of the single-standard stock solution is chromatographically pure acetonitrile; the single-standard stock solution is preferably stored at -18°C.

[0025] In one specific embodiment, the mixed standard stock solution is prepared as follows: accurately transfer 0.10 mL of each of the 10 single standard stock solutions into a 10 mL volumetric flask, dilute to the mark with chromatographic grade acetonitrile, vortex mix, and prepare a mixed standard stock solution with a mass concentration of 0.50 mg / L. The mixed standard stock solution is preferably stored at -18°C.

[0026] In one specific embodiment, the mixed standard working solution is prepared as follows: accurately pipette 2.00 mL of the mixed standard stock solution, blow dry with nitrogen, reconstitute with matrix blank solution to 2.00 mL, and then gradually dilute with matrix blank solution to form a series of mixed standard working solutions with mass concentrations of 0.008, 0.016, 0.031, 0.063, 0.125, 0.25, and 0.50 mg / L.

[0027] For accuracy, it is preferable to prepare 20 blank matrix solutions in parallel, collect them, and store them at 4°C.

[0028] Beneficial effects:

[0029] 1. The method of this invention establishes for the first time a gas chromatography-tandem mass spectrometry method for the determination of 10 pesticide residues in tea, including indomethacin, tebufenozide, tebufenozide, cyclopyridaben, flufenoxuron, propyl chlorpyrifos, chlorfenapyr, fenpropathrin, and glyphosate.

[0030] 2. The method of this invention is characterized by its simple operation, high sensitivity, and good accuracy. It is suitable for the simultaneous determination of residues of 10 pesticides in tea, including indomethacin, acetamiprid, acetamiprid, cyclopyrid, flufenoxuron, propargite, chlorpyrifos, fenpropathrin, and glyphosate. The correlation coefficient (R) of the 10 pesticides within the concentration range of 0.008–0.50 mg / L is [value missing]. 2 The values ​​were all greater than 0.999, indicating a good linear relationship. The method's limit of quantitation ranged from 0.005 to 0.010 mg / kg, and the recoveries at the three spiking levels (0.03, 0.05, and 0.10 mg / kg) were between 80.58% and 118.78%, with relative standard deviations between 0.40% and 6.80%.

[0031] The method's limit of quantitation fully meets the limits for relevant pesticides in tea as specified in GB 2763-2021 and the limits set by the EU and Japan. The method's accuracy and precision fully comply with the requirements of relevant standards such as GB 5009.295-2023 "General Rules for Validation of Chemical Analysis Methods in National Food Safety Standards" and GB / T 32465-2015 "Requirements for Validation and Internal Quality Control of Chemical Analysis Methods". Attached Figure Description

[0032] Figure 1 MRM chromatogram of Example 1;

[0033] Figure 2 MRM chromatogram of Comparative Example 10;

[0034] Figure 1 and Figure 2 In the formula, A is indoxacarb, B is tebufenozide, C is tebufenozide, D is tebufenozide, E is cyclopyridaben, F is flufenoxuron, G is chlorpyrifos, H is fenvalerate, I is propyl chlorpyrifos, and J is glyphosate.

[0035] Figure 3 This is a full scan image of Comparative Example 11. Detailed Implementation

[0036] To achieve the above objectives, the 10 pesticides are indoxacarb, tebufenozide, tebufenozide, cyclopyr, flufenoxuron, propyl chlorpyrifos, chlorpyrifos, fenpropathrin, and glyphosate; the method includes:

[0037] a. After mixing the tea sample with water, extract it using ultrasound to obtain solution A;

[0038] b. Mix solution A with 1% acetic acid-acetonitrile solution, sodium acetate, anhydrous magnesium sulfate and homogenate, shake well, and then centrifuge at 8500 r / min for 3 min at 4℃ to obtain supernatant A; the mass-volume ratio of tea leaves, water, 1% acetic acid-acetonitrile, anhydrous magnesium sulfate and sodium acetate is 2 g:10 mL:10 mL:6 g:1.5 g;

[0039] c. Take the supernatant A and mix it with anhydrous magnesium sulfate, PSA, C18 and GCB, shake it evenly, and then centrifuge it at 8000 r / min for 3 min at 4℃ to obtain supernatant B. The volume-to-mass ratio of supernatant A, anhydrous magnesium sulfate, PSA, C18 and GCB in step c is 6 mL: 1.2 g: 50 mg: 50 mg: 50 mg.

[0040] d. Take the supernatant B and dry it with nitrogen gas; then add chromatographic grade ethyl acetate solution to redissolve it, and filter it through a 0.22 μm organic microporous membrane to obtain the sample to be tested. The volume ratio of the supernatant B to the chromatographic grade ethyl acetate solution is 2:1.

[0041] e. Gas chromatography-tandem mass spectrometry detection:

[0042] A series of mixed standard working solutions with different mass concentrations were sequentially injected into gas chromatography-tandem mass spectrometry for detection. A standard working curve was plotted with the mass concentration of each target compound as the x-axis and the peak area of ​​the target compound as the y-axis.

[0043] The sample to be tested was injected into gas chromatography-tandem mass spectrometry for detection. The peak area was substituted into the standard working curve to obtain the residues of 10 pesticides in the sample. The chromatographic column used was a DB-1701MS column.

[0044] Preparation of the series of mixed standard working solutions: First, prepare single standard stock solutions of 10 pesticides respectively. Then, mix the 10 single standard stock solutions and make up to a certain volume to prepare a mixed standard stock solution. Finally, dry the mixed standard stock solution with nitrogen gas, reconstitute it with blank matrix solution, and dilute it stepwise with blank matrix solution to prepare a series of mixed standard working solutions with different mass concentrations.

[0045] Preparation of blank matrix solution: Take a blank tea sample that does not contain the 10 pesticides to be tested and repeat steps a to d to obtain a blank matrix solution.

[0046] The homogenants can be ceramic homogenants, and the amount of homogenants added should be based on the appropriate amount for mixing the solution.

[0047] In one specific embodiment, the blank tea leaves are of the same type as the tea sample described in step a; the ultrasonic extraction time in step a is preferably 30 to 40 minutes.

[0048] In one specific implementation, the oscillation described in b is uniform oscillation for more than 1 minute.

[0049] In one specific embodiment, step c describes PSA with a mesh size of 40–60 μm, C18 with a mesh size of 100–120, and GCB with a mesh size of 40–60 μm.

[0050] In one specific embodiment, the chromatographic conditions are as follows: injection port temperature: 270°C; injection port pressure: 8.2 psi; injection volume: 1 μL; injection mode: splitless injection; constant flow mode, flow rate: 1.0 mL / min; temperature program: 80°C for 2 min, increase to 200°C at 15°C / min, and then increase to 280°C at 3°C / min for 5 min.

[0051] In one specific embodiment, the DB-1701MS column has dimensions of 30m × 0.25mm × 0.25μm.

[0052] In one specific embodiment, the mass spectrometry conditions are: electron energy 70 eV; electron bombardment ion source; ion source temperature 230°C; quadrupole temperature 150°C; transfer line temperature 280°C; scan mode: MRM mode.

[0053] In one specific embodiment, the mass concentration of the single-standard stock solution is 50.00 mg / L, and the solvent of the single-standard stock solution is chromatographically pure acetonitrile; the single-standard stock solution is preferably stored at -18°C.

[0054] In one specific embodiment, the mixed standard stock solution is prepared as follows: accurately transfer 0.10 mL of each of the 10 single standard stock solutions into a 10 mL volumetric flask, dilute to the mark with chromatographic grade acetonitrile, vortex mix, and prepare a mixed standard stock solution with a mass concentration of 0.50 mg / L. The mixed standard stock solution is preferably stored at -18°C.

[0055] In one specific embodiment, the mixed standard working solution is prepared as follows: accurately pipette 2.00 mL of the mixed standard stock solution, blow dry with nitrogen, reconstitute with matrix blank solution to 2.00 mL, and then gradually dilute with matrix blank solution to form a series of mixed standard working solutions with mass concentrations of 0.008, 0.016, 0.031, 0.063, 0.125, 0.25, and 0.50 mg / L.

[0056] For accuracy, it is preferable to prepare 20 blank matrix solutions in parallel, collect them, and store them at 4°C.

[0057] The specific embodiments of the present invention will be further described below with reference to examples, but the present invention is not limited to the scope of the embodiments described herein.

[0058] Instruments, Equipment and Materials

[0059] Gas chromatography-tandem mass spectrometry (Agilent 7890-7000C, Agilent Technologies, USA); Agilent DB-1701 capillary column (30m × 0.25mm × 0.25μm, Agilent Technologies, USA); ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.); electronic balance (METTLER TOLEDOAL 204, Mettler Toledo Instruments (Shanghai) Co., Ltd.); parallel concentrator (RELABOR); high-speed refrigerated centrifuge (SIGMA 3K15, Sigma-Aldrich, USA).

[0060] Indomethacin, tebufenozide, tebufenozide, cyclopyr, flufenoxuron, propargite, chlorpyrifos, fenpyroximate, glyphosate (1000.0 mg / L, Tanmo Quality Inspection - Standard Material Center); chromatographic grade glacial acetic acid, chromatographic grade n-hexane, chromatographic grade acetonitrile, chromatographic grade ethyl acetate (Sigma-Aldrich, USA); anhydrous magnesium sulfate analytical grade (Chengdu Kelong Chemical Reagent Factory); PSA: 40-60 μm, C18: 100-120 mesh, GCB: 40-60 μm (Yantai Qingyun Instrument Equipment Co., Ltd.); tea sample: commercially available. The above pesticide residue parameters were tested before the experiment, and the test results were all undetectable.

[0061] Example 1

[0062] 1.1 Preparation of standard solutions

[0063] 1.1.1 Preparation of single-standard stock solutions

[0064] Accurately transfer 0.50 mL of each of the following standard solutions (indoxacarb, acetamiprid, acetamiprid, cyclopyrid, flufenoxuron, propyl chlorpyrifos, chlorpyrifos, fenpropathrin, and glyphosate) into a 10 mL volumetric flask, dilute to the mark with chromatographic grade acetonitrile, vortex mix, and prepare a single-standard stock solution with a mass concentration of 50.00 mg / L. Store at -18℃.

[0065] 1.1.2 Preparation of mixed standard stock solutions

[0066] Accurately transfer 0.10 mL of each of the 10 single-standard stock solutions prepared in 1.2.1 into a 10 mL volumetric flask, dilute to the mark with chromatographic grade acetonitrile, vortex to mix, and prepare a mixed standard stock solution with a mass concentration of 0.50 mg / L. Store at -18℃.

[0067] 1.1.3 Preparation of matrix blank solution

[0068] Weigh 2g of blank tea sample containing the pesticide to be tested (accurate to 0.001g) into a 50mL centrifuge tube and proceed according to step 1.2. Perform 20 parallel treatments, collect the processed solutions into 50mL centrifuge tubes, and store at 4℃.

[0069] 1.1.4 Preparation of mixed standard working solutions

[0070] Accurately pipette 2.00 mL of the mixed standard stock solution, dry it with nitrogen, reconstitute it with matrix blank solution to 2.00 mL, and then serially dilute it with matrix blank solution to prepare a series of mixed standard working solutions with mass concentrations of 0.008, 0.016, 0.031, 0.063, 0.125, 0.25, and 0.50 mg / L.

[0071] 1.2 Pretreatment Method

[0072] Sample pretreatment: Weigh 2g of sample (accurate to 0.0001g) into a 50mL centrifuge tube, add 10.00mL of water, mix well, and extract by sonication for 40min. Add 10.00mL of 1% acetic acid-acetonitrile solution, 1.5g of sodium acetate, 6g of anhydrous magnesium sulfate, and one homogeneous proton. Vigorously shake for 1min, then centrifuge (8500r / min, 4℃) for 3min. Transfer 6.00mL of the supernatant to a 15mL centrifuge tube containing 1.2g of anhydrous magnesium sulfate, 50mg of PSA, 50mg of GCB, and 50mg of C18. Shake and extract for 1min, then centrifuge (8000r / min, 4℃) for 3min. Transfer 2.00mL of the supernatant to a 15mL centrifuge tube and incubate under nitrogen at 40℃ until nearly dry. Reconstitute with chromatographically pure ethyl acetate to 1.00mL, filter through a 0.22μm organic phase microporous membrane, and proceed with GC-MS / MS analysis.

[0073] 1.3 Chromatographic and Mass Spectrometric Conditions

[0074] 1.3.1 Chromatographic conditions

[0075] Chromatographic column: DB-1701MS capillary column (30m×0.25mm×0.25μm); Injector temperature: 270℃; Injector pressure: 8.2psi; Injection volume: 1μL; Injection mode: splitless injection; constant flow mode, flow rate: 1.0mL / min; Temperature program: 80℃ for 2min, increase to 200℃ at 15℃ / min, then increase to 280℃ at 3℃ / min and hold for 5min.

[0076] 1.3.2 Mass Spectrometry Conditions

[0077] Electron energy: 70 eV; Electron impact (EI) ion source; Ion source temperature: 230℃; Quadrupole temperature: 150℃; Transmission line temperature: 280℃; Scan mode: MRM mode. The CAS numbers, retention times, and MRM parameters of the 10 pesticides are listed in Table 1.

[0078] The CAS numbers, retention times, and MRM parameters of the 10 pesticides are detailed in Table 1:

[0079] Table 1. Target compound name, CAS number, retention time, and MRM parameters

[0080]

[0081] * indicates a quantitative ion.

[0082] 1.4 Data Processing

[0083] This experiment used OriginPro 2017C 64-bit software and Excel to plot and perform calculations on the data.

[0084] A series of matrix-mixed standard working solutions ranging from 0.008 to 0.50 mg / L were analyzed under the conditions described in section 1.3. Standard working curves were plotted with the target analyte concentration (x) on the x-axis and the corresponding analyte peak area (y) on the y-axis. Table 2 shows that the ten pesticides exhibited good linearity within the concentration range of 0.008–0.5 mg / L, with correlation coefficients (R0.05). 2 The signal-to-noise ratio of the ten pesticides was calculated when the signal-to-noise ratio of the blank sample was about 10 after adding a certain concentration. The method limit of quantitation was 0.005 to 0.010 mg / kg, which fully meets the limit requirements of GB 2763-2021 and the limits of relevant pesticides in tea set by the European Union, Japan and other countries.

[0085] Table 2 Linear range, linear equation, correlation coefficient (R), limit of quantitation, and matrix effect

[0086]

[0087]

[0088] Comparative Examples 1-3

[0089] The other steps were similar to Example 1, except that Comparative Examples 1-3 used acetonitrile, ethyl acetate, and acetone instead of the 1% acetic acid-acetonitrile solution, respectively. Specific results are shown in Table 3. As can be seen from Table 3, when Comparative Example 1 (acetonitrile), Comparative Example 2 (ethyl acetate), and Comparative Example 3 (acetone) were used as extraction solvents, the recovery rates ranged from 76.54% to 133.31%, 70.20% to 137.97%, and 93.15% to 196.55%, respectively. In all cases, 4 to 7 pesticides had recovery rates exceeding 120%. Only when Example 1 (1% acetic acid-acetonitrile) was used as the extraction solvent did the recovery rate range from 84.05% to 116.40%, meeting the requirements for additive recovery rates in relevant standards such as GB 5009.295-2023 "General Rules for Validation of Chemical Analysis Methods in National Food Safety Standards".

[0090] Table 3 Extraction recoveries of four different solvents

[0091]

[0092] Comparative Examples 4-9

[0093] The other steps were similar to Example 1, except that the dosages of C18 and PSA were 80 mg and 100 mg respectively in Comparative Example 4, 120 mg and 120 mg respectively in Comparative Example 6, 25 mg and 100 mg respectively in Comparative Example 7, and 150 mg and 100 mg respectively in Comparative Example 8 and Comparative Example 9. The tests were repeated six times with 10 pesticides at a concentration of 0.03 mg / kg added to blank tea leaves. The specific results are shown in Tables 4 and 5.

[0094] As shown in Table 4, the average recovery rate of Comparative Example 4 ranged from 60.68% to 97.22%; the average recovery rate of Comparative Example 5 ranged from 59.01% to 101.20%; the average recovery rate of Comparative Example 6 ranged from 60.68% to 102.39%; and only Example 1 had an average recovery rate range of 84.70% to 116.17%, which met the requirements of relevant national standards for method recovery rate, indicating that the purification effect was the best under this condition.

[0095] As shown in Table 5, the average recovery rate of Comparative Example 7 ranged from 98.09% to 129.07%; the average recovery rate of Comparative Example 8 ranged from 78.00% to 96.53%; the average recovery rate of Comparative Example 9 ranged from 61.68% to 90.44%; only Example 1 had an average recovery rate range of 92.40% to 117.94%, which met the requirements of relevant standards for method recovery rate. This indicates that when the amount of GCB exceeds 50 mg, it may also adsorb some target substances with different planar structures while adsorbing interfering impurities.

[0096] Table 4. Effects of different PSA and C18 dosages on the recovery of the target analyte.

[0097]

[0098] Table 5. Effect of different GCB dosages on analyte recoveries

[0099]

[0100] Comparative Example 10

[0101] Everything else is similar to Example 1, except that Comparative Example 10 uses a DB-17MS column. The MRM chromatograms of the mixed standard solutions of green tea matrix (all at a mass concentration of 0.5 mg / L) from Example 1 are detailed below. Figure 1 .Depend on Figure 1As can be seen, all ten target compounds in Example 1 were effectively separated, with indoxacarb, tebufenozide, and cyclopyridinium showing significantly better responses and sharp, symmetrical peaks. For details, see the MRM chromatogram of Comparative Example 10, which uses a mixed standard solution of green tea matrix at the same mass concentration. Figure 2 .Depend on Figure 2 It is evident that the target peaks of propyl ester and chlorpyrifos in Comparative Example 10 could not be effectively separated, and the responses of indomethacin, tebufenozide, and cyclopyrimethanil were significantly poor.

[0102] Example 2

[0103] Eighteen tea samples were weighed, and different amounts of mixed standard stock solutions of ten pesticides were added to each sample to prepare six positive additive samples with target pesticide concentrations of 0.03, 0.05, and 0.10 mg / kg. These samples were processed according to the method in section 1.2 of Example 1, and analyzed according to the method in section 1.3. Table 6 shows that the average recovery rate of the method ranged from 80.58% to 118.78%, and the RSD ranged from 0.40% to 6.80%, fully meeting the accuracy and precision requirements of relevant standards such as GB 5009.295-2023 "General Rules for Validation of Chemical Analysis Methods in National Food Safety Standards" and GB / T 32465-2015 "Requirements for Validation and Internal Quality Control of Chemical Analysis Methods". This indicates that the method of this invention can meet the needs of accurate determination of 10 pesticide residues, including indomethacin and propyl chlorpyrifos, in tea.

[0104] Table 6. Spike recoveries and relative standard deviations of ten pesticides at different levels.

[0105]

[0106] Example 3

[0107] Using the methods described in 1.2 and 1.3 of Example 1, ten samples of green tea, ten samples of black tea, ten samples of flower tea, ten samples of dark tea, and ten samples of oolong tea sold in the Chengdu market were analyzed. Three batches of dark tea were found to contain acetamiprid, with a content ranging from 0.037 mg / kg to 0.15 mg / kg. Two batches of flower tea were found to contain indoxacarb, with contents of 0.061 mg / kg and 0.056 mg / kg, respectively. The acetamiprid or indoxacarb content in these five batches of tea exceeded the relevant limits set by the European Union and Japan.

[0108] This invention establishes for the first time an accurate quantitative analytical method for the simultaneous determination of ten pesticide residue parameters in tea that are not supported by existing standards or for which reference standards are inapplicable: indoxacarb, acetamiprid, acetamiprid, cyclopyr, flufenoxuron, propyl chlorpyrifos, chlorpyrifos, fenpropathrin, and glyphosate. This method is characterized by its simplicity, high sensitivity, good accuracy, and low detection cost, and fully meets the requirements of current GB 2763-2021 and relevant regulations and standards of the EU and Japan.

[0109] Comparative Example 11

[0110] The following ten pesticides were detected in tea using gas chromatography-mass spectrometry (GC-MS / MS) according to T / CSTEA 00051—2022, "Determination of Indomethacin, Entamethrin, Chlorpyrifos, Flufenoxuron, and Glyphosate in Tea". A full scan of these pesticides at a concentration of 0.5 mg / L was performed using this group standard method. Figure 3 As shown, not all target substances showed peaks, and some pesticides had low responses.

Claims

1. A method for determining the residues of 10 pesticides in tea, characterized in that, The 10 pesticides are indoxacarb, tebufenozide, tebufenozide, cyclopyr, flufenoxuron, propyl chlorpyrifos, chlorpyrifos, fenpropathrin, and glyphosate; the method includes: a. After mixing the tea sample with water, extract it using ultrasound to obtain solution A; b. Mix solution A with 1% acetic acid-acetonitrile solution, sodium acetate, anhydrous magnesium sulfate and homogenate, shake well, and then centrifuge at 8500 r / min for 3 min at 4℃ to obtain supernatant A; the mass-volume ratio of tea leaves, water, 1% acetic acid-acetonitrile, anhydrous magnesium sulfate and sodium acetate is 2g:10mL:10mL:6g:1.5g; c. Take the supernatant A and mix it with anhydrous magnesium sulfate, PSA, C18 and GCB, shake it evenly, and then centrifuge it at 8000 r / min for 3 min at 4℃ to obtain supernatant B. The volume-to-mass ratio of supernatant A, anhydrous magnesium sulfate, PSA, C18 and GCB in step c is 6 mL : 1.2 g : 50 mg : 50 mg : 50 mg; d. Take the supernatant B and dry it with nitrogen gas; then add chromatographic grade ethyl acetate solution to redissolve it, and filter it through a 0.22 μm organic microporous membrane to obtain the sample to be tested. The volume ratio of the supernatant B to the chromatographic grade ethyl acetate solution is 2:

1. e. Gas chromatography-tandem mass spectrometry detection: A series of mixed standard working solutions with different mass concentrations were sequentially injected into gas chromatography-tandem mass spectrometry for detection. A standard working curve was plotted with the mass concentration of each target compound as the x-axis and the peak area of ​​the target compound as the y-axis. The sample to be tested was injected into gas chromatography-tandem mass spectrometry for detection. The peak area was substituted into the standard working curve to obtain the residues of 10 pesticides in the sample. The chromatographic column used was a DB-1701MS column. Preparation of the series of mixed standard working solutions: First, prepare single standard stock solutions of 10 pesticides respectively. Then, mix the 10 single standard stock solutions and make up to a certain volume to prepare a mixed standard stock solution. Finally, dry the mixed standard stock solution with nitrogen gas, reconstitute it with blank matrix solution, and dilute it stepwise with blank matrix solution to prepare a series of mixed standard working solutions with different mass concentrations. Preparation of the blank matrix solution: Take a blank tea sample that does not contain the 10 pesticides to be tested and repeat steps a to d to obtain the blank matrix solution; The chromatographic conditions were as follows: injection port temperature: 270 °C; injection port pressure: 8.2 psi; injection volume: 1 μL; injection mode: splitless injection; constant flow mode, flow rate: 1.0 mL / min; temperature program: 80 °C for 2 min, increase to 200 °C at 15 °C / min, then increase to 280 °C at 3 °C / min and hold for 5 min. The DB-1701MS column has dimensions of 30 m × 0.25 mm × 0.25 μm; The mass spectrometry conditions are as follows: electron energy 70 eV; electron bombardment ion source; ion source temperature 230℃; quadrupole temperature 150℃; transfer line temperature 280℃; scan mode: MRM mode.

2. The method for determining 10 pesticide residues in tea according to claim 1, characterized in that, The blank tea leaves are of the same type as the tea sample described in step a.

3. The method for determining 10 pesticide residues in tea according to claim 1, characterized in that, The ultrasonic extraction time in step a is 30-40 minutes.

4. The method for determining 10 pesticide residues in tea according to claim 1 or 2, characterized in that, The oscillation described in b is uniform oscillation for more than 1 minute.

5. The method for determining 10 pesticide residues in tea according to claim 1 or 2, characterized in that, Step c: PSA: 40-60 µm, C18: 100-120 mesh, GCB: 40-60 µm.

6. The method for determining 10 pesticide residues in tea according to claim 1 or 2, characterized in that, The mass concentration of each single-standard stock solution is 50.00 mg / L, and the solvent for the single-standard stock solutions is chromatographically pure acetonitrile.

7. The method for determining 10 pesticide residues in tea according to claim 6, characterized in that, The single-standard stock solution is stored at -18°C.

8. The method for determining 10 pesticide residues in tea according to claim 1 or 2, characterized in that, Preparation of the mixed standard stock solution: Accurately transfer 0.10 mL of each of the 10 single standard stock solutions into a 10 mL volumetric flask, dilute to the mark with chromatographic grade acetonitrile, vortex to mix, and prepare a mixed standard stock solution with a mass concentration of 0.50 mg / L.

9. The method for determining 10 pesticide residues in tea according to claim 8, characterized in that, The mixed standard stock solution is stored at -18°C.

10. The method for determining 10 pesticide residues in tea according to claim 1 or 2, characterized in that, Preparation of the series of mixed standard working solutions: Accurately pipette 2.00 mL of the mixed standard stock solution, blow dry with nitrogen, reconstitute with blank matrix solution to 2.00 mL, and then dilute stepwise with blank matrix solution to prepare a series of mixed standard working solutions with mass concentrations of 0.008, 0.016, 0.031, 0.063, 0.125, 0.25, and 0.50 mg / L.

11. The method for determining 10 pesticide residues in tea according to claim 10, characterized in that, The blank matrix solution was prepared in 20 parallel processes, and the samples were collected and stored at 4°C.

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