QuEChERS-UPLC-MS / MS-based detection method for simultaneously detecting multiple pesticide residues in apples and application of QuEChERS-UPLC-MS / MS-based detection method

Abstract

The invention provides a QuEChERS-UPLC-MS / MS (QuEChERS-Ultra Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry)-based detection method for simultaneously detecting various pesticide residues in apples and application of the QuEChERS-UPLC-MS / MS. The QuEChERS-UPLC-MS / MS-based detection method is combined with the apple pesticide residue limit standard in China, specifically aims at the apples, selects 60 common pesticides and metabolites thereof in apple planting, adopts QuEChERS pretreatment and UPLC-MS / MS technology, and can be used for simultaneously detecting various pesticide residues in the apples. The method for simultaneously detecting multiple pesticide residues in apples is established, has high sensitivity and good repeatability and reliability, and can provide technical support for detection and supervision of the pesticide residues in the apples.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticide residue detection, and in particular to a detection method and application of simultaneously detecting multiple pesticide residues in apples based on QuEChERS-UPLC-MS / MS. Background Art

[0002] Apple is the largest deciduous fruit tree in my country, with a cultivated area and output accounting for more than 50% of the world's total. It plays an important role in residents' fruit consumption, increasing farmers' income in fruit-producing areas, increasing agricultural efficiency and rural economic development. Diseases and pests frequently occur in apple production in my country, and chemical control using pesticides is the main method of disease and pest control. Pesticides inevitably form residues in apples after use, resulting in extremely common pesticide residues and multiple residues in apples. Carrying out apple pesticide residue testing, supported by accurate, efficient, economical, environmentally friendly, advanced and practical pesticide residue detection methods, is the premise and basis for protecting consumer health, conducting apple pesticide residue risk monitoring and control, and guiding the safe use of pesticides in apple production.

[0003] The simultaneous detection of multiple pesticide residues using QuEChERS pretreatment combined with HPLC-MS / MS is the main focus of research and development of fruit pesticide residue detection methods. The technology of simultaneously detecting multiple pesticide residues in apples based on QuEChERS sample pretreatment technology combined with UPLC-MS / MS method is of great significance to apple pesticide residue detection, supervision and apple safe production. Summary of the invention

[0004] The present invention provides a detection method and application of multiple pesticide residues in apples based on QuEChERS-UPLC-MS / MS. A simultaneous detection method for multiple pesticide residues in apples is established by combining QuEChERS pretreatment with UPLC-MS / MS technology. The detection method has high sensitivity, stable recovery rate and good reproducibility, and can provide technical support for the detection and supervision of pesticide residues in apples.

[0005] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions:

[0006] The present invention provides a detection method for simultaneously detecting multiple pesticide residues in apples based on QuEChERS-UPLC-MS / MS, and the specific detection steps are as follows:

[0007] (1) Preparation of pesticide standards: 60 pesticides and their metabolites commonly used in apple cultivation were selected;

[0008] (2) Preparation of acetonitrile standard solution: 60 pesticide standards were diluted with chromatographic grade acetonitrile solution to prepare a mixed standard stock solution, which was then diluted with chromatographic grade acetonitrile solution to prepare standard solutions of different gradient concentrations, filtered with an organic filter membrane, and stored in a refrigerator;

[0009] (3) QuEChERS method for sample pretreatment;

[0010] (4) UPLC-MS / MS method for pesticide multi-residue detection.

[0011] Furthermore, the sample pretreatment in step (3) specifically includes:

[0012] S1. Accurately weigh the homogenized apple sample to be tested;

[0013] S2. adding the apple sample to be tested into acetonitrile solvent, shaking and separating and extracting; then adding the extractant, shaking again, and centrifuging;

[0014] S3. Take the supernatant separated by S2, add a purifying agent, vortex and centrifuge;

[0015] S4. Take an appropriate amount of the supernatant separated in S3 and filter it through an organic filter membrane.

[0016] Furthermore, the extractant in step S2 is sodium chloride, anhydrous magnesium sulfate, disodium hydrogen citrate, and sodium citrate, with a mass ratio of 1:4:0.5:1; the mass ratio of the extractant to the sample to be tested is 6.5:10; the purifier in step S3 is N-propylethylenediamine and magnesium sulfate, accounting for 1.50% and 9.00% of the weight of the sample to be tested, respectively.

[0017] Furthermore, the chromatographic conditions of the UPLC-MS / MS method in step (4) are: chromatographic column: InfinityLabProshell 120EC-C18 chromatographic column 3.0 mm×100 mm, 2.7 μm; column temperature: 40°C; mobile phase A is 5 mmol / L ammonium formate + 0.1% formic acid aqueous solution, and B is methanol; injection volume: 2 μL; flow rate: 0.4 mL / min; gradient elution program.

[0018] Furthermore, the mass spectrometry conditions of the UPLC-MS / MS method in step (4) are as follows: ion source: electrospray ion source (ESI); scanning mode: electrospray ion source positive and negative simultaneous scanning; detection mode: multiple reaction monitoring (MRM); dryer temperature: 285°C; dryer flow rate: 10 L / min; nebulizer: 35 psi; ion source temperature: 150°C; sheath gas temperature: 375°C; sheath gas flow rate: 12 L / min.

[0019] Further, the gradient elution procedure is:

[0020] Time 0-1min, mobile phase A volume fraction is 90%, mobile phase B volume fraction is 10%;

[0021] From 1 to 5 min, the volume fraction of mobile phase A decreased from 90% to 50%, and the volume fraction of mobile phase B increased from 10% to 50%;

[0022] During the period of 5-10 min, the volume fraction of mobile phase A decreased from 50% to 0%, and the volume fraction of mobile phase B increased from 50% to 100%;

[0023] Time 10-12min, volume fraction of mobile phase A is 0, volume fraction of mobile phase B is 100%;

[0024] At 12-12.5 min, the volume fraction of mobile phase A increased from 0 to 90%, and the volume fraction of mobile phase B decreased from 100% to 10%;

[0025] The time was 12.5-14 min, the volume fraction of mobile phase A was 90%, and the volume fraction of mobile phase B was 10%.

[0026] Furthermore, the scanning mode is to perform parent ion scanning in Scan mode in an automatic tuning mode; and the sheath gas is nitrogen.

[0027] Further, the pesticide is specifically avermectin, difenoconazole, imidacloprid, pyraclostrobin, propiconazole, fenbumid, diflubenzuron, pyraclostrobin, dichlorvos, syringostyrin, acetamiprid, boscalid, chlorpyrifos, carbendazim, paclobutrazol, famectin, dinotefuran, fluopyram, sulfoxaflor, flonicamid, flusilazole, epoxiconazole, hexaflumuron, hexaconazole, emamectin benzoate, methylthiophanate-methyl myclobutanil, bifenazate, spirotetramat, spiro One or more of tetramethamine-monohydroxy, spirotetramethamine-keto-hydroxy, spirotetramethamine-enol, spirotetramethamine-enol-glycoside, spirodiclofen, chlorantraniliprole, malathion, prochloraz, kresoxim-methyl, myclobutanil, chlorfenapyr, chloranil ...

[0028] Furthermore, the linear correlation coefficient R 2 >0.99, the detection limit is 0.02μg / kg-8.87μg / kg, the quantification limit is 0.05μg / kg-29.55μg / kg, the added recovery rate is 75.9%-119.9%, and the relative standard deviation is 0.1%-17.7%.

[0029] The present invention also provides application of the detection method to quickly detect multiple pesticide residues in apple planting.

[0030] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0031] 1. The detection method provided by the present invention, combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) technology, can achieve the detection effect of 60 pesticide residues at the same time. It has the advantages of high efficiency, sensitivity, economy and environmental protection. It can effectively drive and promote the improvement of the technical capabilities of my country's food safety monitoring and testing institutions, and has good social benefits.

[0032] 2. The detection method provided by the present invention has a wide linear range and good linearity (R 2 The high precision of the detection method further improved the accuracy and sensitivity of the detection method.

[0033] 3. The detection method provided by the present invention has low detection limit and quantification limit, high method sensitivity, stable recovery rate, good reproducibility, and meets the requirements of multi-residue analysis. DETAILED DESCRIPTION

[0034] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0035] Embodiment 1:

[0036] 1. Materials and Methods

[0037] (1) Drugs and reagents

[0038] Standards: The concentrations of thiophanate-methyl, lufenuron, clofenazine, trifloxystrobin, penconazole, succinimid, etoxazole, and imazalil are all 100 mg / L; the concentration of carbendazim is 500 mg / L; the concentration of avermectin, difenoconazole, imidacloprid, pyraclostrobin, propiconazole, fenbuconazole, diflubenzuron, pyraclostrobin, dichlorvos, syringystrobin, acetamiprid, boscalid, chlorpyrifos, paclobutrazol, famoxadone, dinotefuran, fluopyram, sulfoxaflor, flonicamid, flusilazole, epoxiconazole, hexaflumuron, and hexachlor Alcohol, emamectin benzoate, methyl thiophanate, myclobutanil, bifenazate, spirotetramat, spirotetramat-monohydroxy, spirotetramat-keto-hydroxy, spirotetramat-enol, spirotetramat-enol-glycoside, spirodiclofen, chlorantraniliprole, malathion, prochloraz, kresoxim-butyl, myclobutanil, diflubenzuron, chloranil, malathion, prochloraz, kresoxim-butyl, myclobutanil, clothianidin, thiamethoxam, hexythiazox, buprofezin, triadimenol, triadimefon, tebuconazole, diniconazole, phoxim, ethopropanol, iprodione, the concentration of each of them is 1000 mg / L.

[0039] Reagents: acetonitrile (chromatographic grade, Sigma-Aldrich, Germany); formic acid and ammonium formate (mass spectrometry grade, ThermoFisher Scientific, USA); methanol (chromatographic grade, Thermo Fisher Scientific, USA); sodium citrate, sodium chloride, disodium hydrogen citrate, anhydrous magnesium sulfate and N-propylethylenediamine (PSA) were all purchased from Agilent Technologies, USA.

[0040] (2) Instruments and equipment

[0041] 1290Infinity-6460 ultra-high performance liquid chromatography triple quadrupole mass spectrometer (Agilent, USA); InfinityLab Proshell 120EC-C18 (3.0mm×100mm, 2.7μm); homogenizer (SMT-Y22B, Shenzhen Smart Intelligent Electric Development Co., Ltd.); balance (PL602-L, METTLER TOLEDO, Switzerland); vertical large-capacity high-speed refrigerated centrifuge (CF16RXⅡ, Hitachi, Japan); multi-tube oscillator (MTV-100, Hangzhou Aosheng Instrument Co., Ltd.); fully automatic ultrapure water machine (Milli-Q Gradient, Millipor, USA).

[0042] (3) Standard solution preparation

[0043] Pesticides include: avermectin, fenpropimorph, imidacloprid, pyraclostrobin, propiconazole, fenbumid, diflubenzuron, pyraclostrobin, dichlorvos, syringostyrin, acetamiprid, boscalid, chlorpyrifos, carbendazim, paclobutrazol, oxathiapiprolin, dimethoate, fluopyram, flupyrad, flupyrad, flupyrad, fluphenazine, flusilazole, fluepoxazole, hexaflumuron, hexaconazole, emamectin benzoate, methylthiophanate-methyl myclobutanil, bifenazate, spirotetramat, spirotetramat-monohydroxy, spirotetramat-ketone -hydroxy, spirotetramat-enol, spirotetramat-enol-glycoside, spirodiclofen, chlorantraniliprole, malathion, prochloraz, kresoxim-methyl, myclobutanil, diflubenzuron, chlorfenapyr, clothianidin, thiamethoxam, hexathiazolin, thiamethoxam, thiamethoxam, thiamethoxam, thiamethoxam, triadimenol, triadimenol, lufenuron, clofentezin, trifloxystrobin, penconazole, tebuconazole, diniconazole, cinclofos, phoxim, etoxazole, etoxazolidinone, isoprodinil, and imazalil, a total of 60 pesticides were detected by ultra performance liquid chromatography tandem mass spectrometry.

[0044] Preparation of pesticide standard solution: 60 kinds of pesticide standards were diluted with chromatographic grade acetonitrile solution to prepare a 5 mg / L mixed standard stock solution, and then diluted with chromatographic grade acetonitrile solution to prepare a series of concentration standard solutions such as 0.5 mg / L, 0.1 mg / L, 0.05 mg / L, 0.01 mg / L, 0.005 mg / L, and 0.001 mg / L. After passing through a 0.22 μm organic filter membrane, the solutions were frozen in a -20°C refrigerator.

[0045] (4) Sample pretreatment

[0046] In a 50mL centrifuge tube, accurately weigh 10.00g of homogenized apple sample, add 10mL acetonitrile, vortex at 2500r / min for 5min; then add sodium chloride (1g), anhydrous magnesium sulfate (4g), disodium hydrogen citrate (0.5g), sodium citrate (1g), vortex again for 5min; centrifuge at 6000r / min for 5min; transfer 6mL of supernatant to a 15mL centrifuge tube, add 149.9mg PSA and 900.1mg magnesium sulfate, vortex at 2500r / min for 1min, and centrifuge at 6000r / min for 5min. Take an appropriate amount of supernatant, filter through a 0.22μm organic filter membrane and place in a sample injection bottle for UPLC-MS / MS detection.

[0047] (5) UPLC-MS / MS detection method

[0048] Chromatographic conditions: InfinityLab Proshell 120EC-C18 (3.0 mm×100 mm, 2.7 μm); mobile phase A was 5 mmol / L ammonium formate + 0.1% formic acid aqueous solution, and mobile phase B was methanol; gradient elution program was shown in Table 1; column temperature: 40°C; injection volume: 2 μL; flow rate: 0.4 mL / min.

[0049] Mass spectrometry conditions: ion source: electrospray ion source (ESI); scanning mode: ESI source positive and negative simultaneous scanning; detection mode: multiple reaction monitoring (MRM); dryer temperature: 285°C; dryer flow rate: 10 L / min; nebulizer: 35 psi; ion source temperature: 150°C; sheath gas (nitrogen) temperature: 375°C; sheath gas flow rate: 12 L / min.

[0050] Table 1 Gradient elution conditions for ultra-high performance liquid chromatography tandem mass spectrometry

[0051]

[0052] (6) Method validation

[0053] To ensure the accuracy, consistency and reliability of the analytical results, the linear range, limit of detection (LOD), limit of quantification (LOQ), matrix effect (ME), accuracy and precision of the method were verified according to EC SANTE / 11312 / 2021. Before the experiment, the instrument was calibrated by linear analysis. Solvent standard solutions and blank matrix-matched standard solutions with concentrations of 0.0001-1 mg / L were prepared respectively. The correlation coefficient (R 2 ) was used to estimate the goodness of fit. The matrix effect was determined by calculating the ratio of the slope of the matrix standard curve to the slope of the solvent standard curve. Accuracy and precision were evaluated by recovery and reproducibility tests, and the recovery test was performed at four spike levels (0.005 mg / kg, 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg) (n=3). Precision was evaluated using the relative standard deviation (RSD) of 60 pesticides and their metabolites. The detection limit and quantification limit were 3 times and 10 times the signal-to-noise ratio (S / N) of the quantitative ion pair, respectively, and the undetected value was set to LOQ / 2 for statistical analysis.

[0054] 2. Instrument Testing Conditions

[0055] (1) UPLC-MS / MS detection parameters

[0056] A single acetonitrile standard solution with a pesticide concentration of 100 mg / L was directly input into the mass spectrometer through a syringe pump. The positive ion mode (ESI+) and negative ion mode (ESI-) were used, and parent ion scanning was performed in Scan mode by automatic tuning. The mass spectrometry parameters of the pesticides are detailed in Table 2.

[0057] Table 2 UPLC-MS / MS detection parameters for 60 pesticides

[0058]

[0059]

[0060] Table 2 (continued)

[0061]

[0062]

[0063] Note: Emamectin Benzoate -1 It is emamectin benzoate; -1 is the quantitative ion.

[0064] (2) Standard equation and sensitivity

[0065] Based on the QuEChERS-UPLC-MS / MS method, it can be seen from Table 3 that the 60 detected compounds in the apple matrix have a good linear relationship in the concentration range of 0.0001 mg / kg-1 mg / kg, R 2 All of them were greater than 0.99. Among the 60 pesticides, the detection limits of bifenthrin, penconazole, methyl thiophanate, pyraclostrobin, carbendazim, tebuconazole, spirotetramat-monohydroxy, chlorpyrifos, myclobutanil, cinclostrobin, dichlorvos, trifloxystrobin, acetamiprid, hexaconazole, and spirotetramat-enol-glycoside were 0.02μg / kg-0.19μg / kg, and the quantification limits were 0.05μg / kg-0.63μg / kg, and the quantification limits were 0.05μg / kg-0.63μg / kg, and the quantification limits were 0.05μg / kg-0.63μg / kg, and those of clofentezine, avermectin, tebuconazole, spirotetramat-keto-hydroxy, etoxazole, spirotetramat-enol, myclobutanil, prochloraz, dimethoate, epoxiconazole, propiconazole, and triadimenol , boscalid, fenpropathrin, ethomethacin, imazalil, diflubenzuron, isoprodinil, flonicamid, and lufenuron have detection limits of 0.22μg / kg-0.94μg / kg, and quantification limits of 0.73μg / kg-3.13μg / kg, and sulfoxaflor, spirotetramat, fluopyram, buprofezin, pyridaben, hexaflumuron, imidacloprid, triadimefon, hexathiapyr, paclobutrazol, thiamethoxam, diniconazole, spirodiclofen, oxathiapiprolin, and clothianidin have detection limits of 1.01μg / kg-8.87μg / kg, and quantification limits of 3.35μg / kg-29.55μg / kg. The above results show that the QuEChERS-UPLC-MS / MS method established in the present invention meets the professional requirements for the detection of pesticide multi-residues.

[0066] Table 3 UPLC-MS / MS detection limit and quantification limit

[0067]

[0068]

[0069] Table 3 (continued)

[0070]

[0071]

[0072] Note: Emamectin Benzoate -1 It is emamectin benzoate.

[0073] (3) Matrix effect

[0074] In the detection of pesticide residues in fruits, the matrix effect refers to the influence of the tissue structure, composition and characteristics of the fruit itself on the detection results of pesticide residues. Fruits themselves contain various chemical substances, such as sugars, acids, fats, etc. These components may inhibit or enhance the signal of the analyte, thereby causing quantitative errors. In actual detection, the matrix of the fruit needs to be processed to reduce the influence of the matrix effect and improve the accuracy of the detection. Common processing methods include sample extraction, purification and concentration to separate pesticide residues and eliminate the interference of matrix effects on the detection results. The matrix effect is determined by the slope ratio of the matrix standard curve to the pure solution standard curve. The matrix effect is divided into three types: weak matrix effect (ME range -20% to 20%), moderate matrix effect (ME range -50% to -20% or 20% to 50%) and strong matrix effect (less than -50% or more than 50%). As shown in Table 3, among the 60 pesticides detected by UPLC-MS / MS, 49 pesticides have weak matrix effects, including diflubenzuron, avermectin, imidacloprid, propiconazole, tebuconazole, acetamiprid, dinotefuran, sulfoxaflor, epoxiconazole, hexaconazole, bifenazate, spirotetramat, spirotetramat-monohydroxy, spirotetramat-keto-hydroxy, spirotetramat-enol, spirotetramat-enol-glycoside, malathion, azoxystrobin, clothianidin, and chloranil. , thiamethoxam, thiamethoxam, triadimenol, triadimefon, sphenanthamide and etoxazolidinone showed matrix enhancement effect; fenpropimorph, pyraclostrobin, pyraclostrobin, syringosterone, boscalid, carbendazim, paclobutrazol, flonicamid, flusilazole, fluopyram, methylthiophanate-methyl myclobutanil, spirodiclofen, chlorantraniliprole, prochloraz, kresoxim-methyl, propargyl cypermethrin, buprofezin, penconazole, tebuconazole, diniconazole, phoxim, iprodione and imazalil showed matrix inhibition effect. 11 pesticides showed medium matrix effect, among which lufenuron and hexaflumuron showed matrix enhancement effect; dichlorvos, chlorpyrifos, oxadone, avermectin benzoate, diflubenzuron, hexythiazox, clofentezin, trifloxystrobin and etoxazolidinone showed matrix inhibition effect. There was no pesticide with strong matrix effect among the 60 tested pesticides.

[0075] (4) Recovery and precision

[0076] The precision and accuracy of the test were evaluated by the spike recovery test. The spike recovery rates of the apple samples determined by QuEChERS-UPLC-MS / MS are shown in Table 4. The spike recovery test was carried out at four concentration levels (0.005 mg / kg, 0.01 mg / kg, 0.05 mg / kg, and 0.1 mg / kg), and each level was repeated 3 times in parallel. At the 0.005 mg / kg level, the added recovery rates of 60 pesticides in the apple matrix were 78.87%-119.87%, and the RSD was 0.45%-17.74%; at the 0.01 mg / kg level, the added recovery rates of 60 pesticides in the apple matrix were 77.26%-116.50%, and the RSD was 0.29%-11.33%; at the 0.05 mg / kg level, the added recovery rates of 60 pesticides in the apple matrix were 75.93%-116.10%, and the RSD was 0.08%-9.72%; at the 0.1 mg / kg level, the added recovery rates of 60 pesticides in the apple matrix were 79.90%-117.78%, and the RSD was in the range of 0.26%-14.73%. The precision and accuracy of this method meet the requirements of EC SANTE / 11312 / 2021 and meet the requirements for the detection of multiple pesticide residues in apple matrix in this test.

[0077] Table 4 Recovery and relative standard deviation of pesticides in apples based on QuEChERS-UPLC-MS / MS (n=3)

[0078]

[0079]

[0080] Table 4 (continued)

[0081]

[0082]

[0083] Note: The added concentration unit is mg / kg; Emamectin benzoate -1 It is emamectin benzoate.

[0084] Embodiment 2:

[0085] The test results of 60 pesticide residues in 448 apple samples:

[0086] 448 apple samples were collected from four major production areas in China, and the pesticide residue detection was performed using the present invention. It can be seen from Table 5 that a total of 43 pesticides were detected, but the residual levels were not high, and there was no residue exceeding the standard. The detected pesticides included 15 insecticides (35%), 20 fungicides (47%), 7 miticides (16%) and 1 plant growth regulator (2%). The detection rates and residual levels of these pesticides are shown in Appendix 5. Among them, the detection rate of carbendazim was the highest (90.0%), followed by tebuconazole (85.0%), methyl thiophanate (43.3%), acetamiprid (33.7%), difenoconazole (29.2%), pyraclostrobin (24.6%), diflubenzuron (22.1%), etc.

[0087] Table 5 Detection rates and residue levels of 60 pesticide residues in 448 apple samples

[0088]

[0089] Note: Pesticide residues not listed were not detected.

[0090] In summary, the present invention established a method for simultaneous detection of 60 pesticides in apples by combining QuEChERS with UPLC-MS / MS. Through methodological evaluation of the 60 pesticides detected, all of them had a wide linear range and good linearity (R 2 The linear range is 0.0001mg / L-0.5mg / L (methyl thiophanate and tebuconazole), 0.0005mg / L-0.5mg / L (8 pesticide residues such as bifenazate), 0.001mg / L-0.5mg / L (7 pesticide residues such as chlorpyrifos), 0.001mg / L-1.0mg / L (carbendazim), 0.002mg / L-0.5mg / L (7 pesticide residues such as tebuconazole) or 0.005mg / L-0.5mg / L (35 pesticide residues such as myclobutanil); the detection limit is 0.02μg / kg-8.87μg / kg, the quantification limit is 0.05μg / kg-29.55μg / kg, the added recovery rate is 75.9%-119.9%, and the relative standard deviation is 0.1%-17.7%.

[0091] The present invention combines QuEChERS pretreatment technology with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) to rapidly screen and qualitatively screen and quantitatively analyze 60 pesticide residues in apples, and establishes a mass spectrum database of 60 pesticides in apples for rapid screening and confirmation of multiple pesticide residues. The detection method provided by the present invention is simple, rapid, efficient, and accurate, and also meets the requirements for apple pesticide residue analysis, and can provide effective detection technology support for apple pesticide residue detection, supervision, and apple safety production.

[0092] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A method for simultaneously detecting multiple pesticide residues in apples based on QuEChERS-UPLC-MS / MS, characterized in that: The specific detection steps include: (1) Preparation of pesticide standards: 60 pesticides and their metabolites commonly used in apple cultivation were selected; (2) Preparation of acetonitrile standard solution: 60 pesticide standards were diluted with chromatographic grade acetonitrile solution to prepare a mixed standard stock solution, which was then diluted with chromatographic grade acetonitrile solution to prepare standard solutions of different gradient concentrations, filtered with an organic filter membrane, and stored in a refrigerator; (3) QuEChERS method for sample pretreatment; (4) UPLC-MS / MS method for pesticide multi-residue detection.

2. The method for simultaneously detecting multiple pesticide residues in apples based on QuEChERS-UPLC-MS / MS according to claim 1, characterized in that: The step (3) sample pretreatment specifically includes: S1. Accurately weigh the homogenized apple sample to be tested; S2. adding the apple sample to be tested into acetonitrile solvent, shaking and separating and extracting; then adding the extractant, shaking again, and centrifuging; S3. Take the supernatant separated by S2, add a purifying agent, vortex and centrifuge; S4. Take an appropriate amount of the supernatant separated in S3 and filter it through an organic filter membrane.

3. The method for simultaneously detecting multiple pesticide residues in apples based on QuEChERS-UPLC-MS / MS according to claim 2, characterized in that: The extractant in step S2 is sodium chloride, anhydrous magnesium sulfate, disodium hydrogen citrate, and sodium citrate, and the mass ratio thereof is 1:4:0.5:1; the mass ratio of the extractant to the sample to be tested is 6.5:10; the purifier in step S3 is N-propylethylenediamine and magnesium sulfate, and the weight proportions of the sample to be tested are 1.50% and 9.00%, respectively.

4. The method for simultaneously detecting multiple pesticide residues in apples based on QuEChERS-UPLC-MS / MS according to claim 1, characterized in that: The chromatographic conditions of the UPLC-MS / MS method in step (4) are as follows: chromatographic column: InfinityLab Proshell 120EC-C18 chromatographic column 3.0 mm×100 mm, 2.7 μm; Column temperature: 40°C; mobile phase A is 5 mmol / L ammonium formate + 0.1% formic acid aqueous solution, and mobile phase B is methanol; injection volume: 2 μL; flow rate: 0.4 mL / min; gradient elution program.

5. The method for simultaneously detecting multiple pesticide residues in apples based on QuEChERS-UPLC-MS / MS according to claim 1, characterized in that: The mass spectrometry conditions of the UPLC-MS / MS method in step (4) are as follows: ion source: electrospray ion source ESI; scanning mode: electrospray ion source positive and negative simultaneous scanning; detection mode: multiple reaction monitoring MRM; dryer temperature: 285°C; dryer flow rate: 10 L / min; Nebulizer: 35psi; ion source temperature: 150°C; sheath gas temperature: 375°C; sheath gas flow rate: 12L / min.

6. The method for simultaneously detecting multiple pesticide residues in apples based on QuEChERS-UPLC-MS / MS according to claim 5, characterized in that: The gradient elution procedure is: Time 0-1min, mobile phase A volume fraction is 90%, mobile phase B volume fraction is 10%; From 1 to 5 min, the volume fraction of mobile phase A decreased from 90% to 50%, and the volume fraction of mobile phase B increased from 10% to 50%; During the period of 5-10 min, the volume fraction of mobile phase A decreased from 50% to 0%, and the volume fraction of mobile phase B increased from 50% to 100%; Time 10-12min, volume fraction of mobile phase A is 0, volume fraction of mobile phase B is 100%; At 12-12.5 min, the volume fraction of mobile phase A increased from 0 to 90%, and the volume fraction of mobile phase B decreased from 100% to 10%; The time was 12.5-14 min, the volume fraction of mobile phase A was 90%, and the volume fraction of mobile phase B was 10%.

7. The method for simultaneously detecting multiple pesticide residues in apples based on QuEChERS-UPLC-MS / MS according to claim 6, characterized in that: The scanning mode is an automatic tuning mode to perform parent ion scanning in Scan mode; the sheath gas is nitrogen.

8. The method for simultaneously detecting multiple pesticide residues in apples based on QuEChERS-UPLC-MS / MS according to any one of claims 1 to 7, characterized in that: The pesticide is specifically avermectin, difenoconazole, imidacloprid, pyraclostrobin, propiconazole, fenbumid, diflubenzuron, pyridabenzamide, dichlorvos, syringostyrin, acetamiprid, boscalid, chlorpyrifos, carbendazim, paclobutrazol, famectin, dinotefuran, fluopyram, sulfoxaflor, flonicamid, flusilazole, epoxiconazole, hexaflumuron, hexaconazole, emamectin benzoate, methylthiophanate-methyl myclobutanil, bifenazate, spirotetramat, spirotetramat -monohydroxy, spirotetramat-keto-hydroxy, spirotetramat-enol, spirotetramat-enol-glycoside, spirodiclofen, chlorantraniliprole, malathion, prochloraz, kresoxim-methyl, myclobutanil, chlorfenapyr, chloranil ...

9. The method for simultaneously detecting multiple pesticide residues in apples based on QuEChERS-UPLC-MS / MS according to claim 8, characterized in that: Linear correlation coefficient R 2 >0.99, the detection limit is 0.02μg / kg-8.87μg / kg, the quantification limit is 0.05μg / kg-29.55μg / kg, the added recovery rate is 75.9%-119.9%, and the relative standard deviation is 0.1%-17.7%.

10. Use of the detection method according to any one of claims 1 to 7 for rapid detection of multiple pesticide residues in apple cultivation.

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