Method for detecting 146 pesticide residues in animal food by dispersive solid-phase extraction-gas chromatography-mass spectrometry

Abstract

The invention discloses a method for detecting 146 pesticide residues in animal food by using a dispersive solid-phase extraction-gas chromatography-mass spectrometry technology, and relates to the technical field of food safety detection, and the technical key points are as follows: a QuEChERS extraction method and a dSPEEMR-Lipid purification method are adopted; the 146 pesticide residues in the animal food sample are detected by combining a gas chromatography-triple quadrupole mass spectrometer GC-MS / MS (Gas Chromatography-Mass Spectrometer / Mass Spectrometer). The dSPEEMR-Lipid can selectively remove lipids in a sample extract without causing loss of an analyte, acetonitrile is used as an extraction solvent, the dSPEEMR-Lipid is subjected to purification, concentration and replacement solvent redissolution and then is subjected to GC-MS / MS quantitative analysis, and an analysis method for simply, conveniently, rapidly and accurately determining the pesticide multi-residues in the animal food is established. And a technical guarantee is provided for quality safety of animal food.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and in particular to a method for detecting 146 pesticide residues in animal food by using dispersed solid phase extraction-gas chromatography-mass spectrometry technology. Background Art

[0002] While promoting the increase of agricultural production, chemical pesticides not only affect the ecological environment, but also are introduced into animal food through the environment, feed, drinking water, breathing, food chain and other channels, resulting in pesticide residues in animal food. As people's demand for animal food increases, residual pesticides may accumulate in the human body through the food chain, causing potential harm to human health.

[0003] Pesticide residues are an indicator of global concern. Many countries have established strict maximum residue limits (MRLs) for pesticides in animal foods. With the increase in the coverage of pesticide types and the strict requirements for limit values, higher requirements are placed on the simplicity, rapidity, sensitivity and throughput of detection methods. At present, the standards and literature reports on animal food inspection methods mainly include gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), gas chromatography-tandem mass spectrometry (GC-MS / MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), high-resolution mass spectrometry, etc. At present, GC-MS and LC-MS / MS are the most commonly used methods in national standards and literature reports, and there are relatively few national standards for GC-MS / MS detection methods. Most of the reports on high-throughput detection of multiple pesticide residues using GC-MS / MS are for plant foods, and there are relatively few high-throughput methods for detecting multiple pesticide residues in animal foods. Therefore, it is very necessary to establish accurate and sensitive high-throughput detection methods for animal foods.

[0004] Animal foods contain a lot of fat and protein, which not only cause instrument contamination, but also may interfere with the detection of target pesticides. Removing fat is the key to the pretreatment of animal samples. Currently, most of the pretreatment methods reported for the analysis of pesticide multi-residues in animal foods use gel permeation chromatography, solid phase extraction, freeze filtration technology, QuEChERS pretreatment, etc., and most of them are combined with LC-MS / MS for detection. Gel permeation chromatography equipment is expensive and cumbersome to operate, which is not conducive to batch detection; solid phase extraction is time-consuming and the pretreatment operation is complicated; QuEChERS has the characteristics of fast, convenient, low cost, and high extraction efficiency. Although it has been widely used in recent years, it has disadvantages such as limitations in the selection of purification materials in the detection of multiple pesticide residues.

[0005] To this end, the present invention aims to provide a method for detecting 146 pesticide residues in animal food using dispersed solid phase extraction-gas chromatography-mass spectrometry technology to solve the above problems. Summary of the invention

[0006] The purpose of the present invention is to solve the above problems and provide a method for detecting 146 pesticide residues in animal food by using dispersed solid phase extraction-gas chromatography-mass spectrometry technology. The 146 pesticide residues in animal food samples are detected by using QuEChERS extraction method and dSPE EMR-Lipid cleanup method in combination with gas chromatography-triple quadrupole mass spectrometry GC-MS / MS.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] The present invention provides a method for detecting 146 pesticide residues in animal food by using dispersed solid phase extraction-gas chromatography-mass spectrometry technology. The method is to detect 146 pesticide residues in animal food samples by using QuEChERS extraction method and dSPE EMR-Lipid purification method in combination with gas chromatography-triple quadrupole mass spectrometry GC-MS / MS;

[0009] The method comprises the following steps:

[0010] S1. Selection of instruments and materials: The instruments used were gas chromatography-triple quadrupole mass spectrometry, high-speed low-temperature centrifuge, ultrapure water machine and fully automatic parallel concentrator; the materials used were QuEChERS EN extraction kit, ethyl acetate and acetonitrile;

[0011] S2. Prepare reagents: Take 146 pesticide standards in a 10 mL volumetric flask and use acetone to make a standard stock solution; accurately pipette the standard stock solution and use ethyl acetate to make a mixed standard working solution; before use, use ethyl acetate pure solvent or blank matrix sample extract to prepare a series of standard solutions from the mixed standard working solution. All standard solutions are stored at -18°C away from light;

[0012] S3. Sample pretreatment: Weigh 5 g of sample and add ultrapure water and acetonitrile, perform ultrasonic extraction and freezing, then add QuEChERS EN-extraction salt package, centrifuge to obtain supernatant, take the supernatant and purify it using dSPE EMR-Lipid purification tube, then concentrate it by nitrogen blowing and redissolve it in ethyl acetate, and perform GC-MS / MS analysis;

[0013] S4. Data analysis: Qualitative and quantitative analysis was performed using Agilent MassHunter software, and data statistics and visualization were performed using Excel.

[0014] The samples are commercially available pork, beef, mutton, chicken, aquatic products, and eggs;

[0015] The chromatographic column is a HP-5MSUI capillary chromatographic column with a length of 30 m, an inner diameter of 0.25 mm, and a film thickness of 0.25 μm. The temperature of the chromatographic injection port is set to 280° C. The chromatographic program temperature rising conditions are: the initial temperature is 60° C., maintained for 1 minute, then heated to 170° C. at a rate of 40° C. / min, then heated to 310° C. at a rate of 10° C. / min, and maintained at 310° C. for 3 minutes; the carrier gas flow rate is 1.0 mL / min, the injection mode is splitless injection, and the injection volume is 1 μL;

[0016] The mass spectrometry conditions adopted electron impact EI ionization mode, and the ionization energy was set to 70 eV; the ion source temperature was 230° C., the transfer line temperature was 280° C., and the solvent delay time was 2.5 minutes; the instrument was operated in dynamic multiple reaction monitoring DMRM mode; nitrogen was used as collision gas, and helium was used as quenching gas.

[0017] Compared with the prior art, this solution has the following beneficial effects:

[0018] The present invention adopts QuEChERS extraction method and dSPEEMR-Lipid purification method, combined with gas chromatography-triple quadrupole mass spectrometry GC-MS / MS, to detect 146 pesticide residues in animal food samples. The pretreatment method is simple to operate, has good purification effect, can effectively reduce the interference of impurities in the matrix, has high selectivity and accuracy, is suitable for the simultaneous determination and analysis of multiple pesticide residues in animal food, can provide technical support for the daily monitoring and supervision of multiple pesticide residues in animal food, and can also provide a reference for the formulation and revision of subsequent standards. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a flow chart of the method for detecting 146 pesticide residues in animal food by dispersed solid phase extraction-gas chromatography-mass spectrometry in an embodiment of the present invention

[0020] Figure 2 is the total ion current diagram of 146 pesticides (240 μg / L) in pork matrix in the embodiment of the present invention;

[0021] Figure 3 Schematic diagram showing the effect of the amount of water added on the recovery rate of pesticide compounds in an embodiment of the present invention;

[0022] Figure 4 Schematic diagram of the effect of the purification scheme on the recovery rate of pesticide compounds in an embodiment of the present invention;

[0023] Figure 5Schematic diagram of matrix effect and intensity level ratio of target compound in blank matrix in an embodiment of the present invention;

[0024] Figure 6 It is a chromatogram of the standard solution and the positive sample in the embodiment of the present invention, wherein A is the standard solution, B is fish, C is egg, and D is chicken. DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0026] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0027] Example:

[0028] 1. Materials and Methods

[0029] 1.1 Reagents, instruments and materials

[0030] 146 pesticide standards: purity ≥95%.

[0031] Reagents and kits: N-propylacetamide (PSA); octadecylsilane bonded silica gel (C18); QuEChERS EN extraction package: containing 4 g anhydrous magnesium sulfate, 1 g sodium chloride, 1 g sodium citrate, 0.5 g disodium hydrogen citrate; dSPE EMR-Lipid purification tube.

[0032] Analytical reagents: anhydrous magnesium sulfate (analytical grade); ethyl acetate, acetonitrile (chromatographic grade).

[0033] Equipment: Gas chromatography-triple quadrupole mass spectrometer: model 7890B-7000D, a product of Agilent Technologies, Inc., USA; high-speed low-temperature centrifuge: model 5810R, a product of Eppendor, Germany; ultrapure water machine: model Milli-Q, a product of Millipore, USA; fully automatic parallel concentrator: model AUTO-EVA-60, a product of Ruike Group (Xiamen) Co., Ltd.

[0034] The pork, beef, mutton, chicken, aquatic products and egg samples used were all commercially available.

[0035] 1.2 Preparation of standard solution

[0036] Weigh 10 mg (accurate to 0.01 mg) of each pesticide standard into a 10 mL volumetric flask and dilute to volume with acetone to prepare a standard stock solution. Accurately pipette an appropriate amount of the standard stock solution and dilute to volume with ethyl acetate to prepare a mixed standard working solution. Before use, use pure ethyl acetate solvent or blank matrix sample extract to prepare a series of standard solutions from the mixed standard working solution. All standard solutions are stored at -18°C in the dark.

[0037] 1.3 Sample pretreatment

[0038] Weigh 5g (accurate to 0.001g) of sample into a 50mL polypropylene centrifuge tube, add 2mL ultrapure water, shake well, add 10mL acetonitrile, ultrasonically extract for 10min, freeze at -20℃ for 10min, add QuEChERSEN-extraction salt package, shake immediately, vortex mix for 1min, centrifuge at 4000r / min for 5min at 4℃, and the supernatant is to be purified. Accurately transfer 3mL of supernatant to the dSPEEMR-Lipid purification tube pre-loaded with purification materials, vortex mix for 1min, centrifuge at 4000r / min for 5min at 4℃, accurately transfer 2mL of purification solution to a 40℃ water bath with nitrogen blown to near dryness, accurately add 1mL of ethyl acetate to dissolve, pass through a 0.22μm organic filter membrane, and wait for GC-MS / MS analysis.

[0039] 1.4 Instrument conditions

[0040] 1.4.1 Chromatographic conditions

[0041] The chromatogram was a HP-5MSUI capillary column (30m×0.25mm, 0.25μm). The chromatographic injection port temperature was set to 280°C. The chromatographic program temperature conditions were: initial temperature 60°C, maintained for 1 minute, then heated to 170°C at a rate of 40°C / min, then heated to 310°C at a rate of 10°C / min, and maintained at 310°C for 3 minutes. The carrier gas flow rate was 1.0mL / min, the injection mode was splitless injection, and the injection volume was 1μL.

[0042] 1.4.2 Mass spectrometry conditions

[0043] Electron impact (EI) ionization was used, and the ionization energy was set to 70 eV. The ion source temperature was 230°C, the transfer line temperature was 280°C, and the solvent delay time was 2.5 minutes. The instrument was operated in dynamic multiple reaction monitoring (DMRM) mode. Nitrogen (purity>99.999%) was used as collision gas, and helium (purity>99.999%) was used as quenching gas.

[0044] 1.5 Data Analysis

[0045] Agilent MassHunter software was used for qualitative and quantitative analysis of pesticide compounds, and Excel software was used for statistical analysis, tabulation and drawing of data.

[0046] 2. Results and Discussion

[0047] 2.1 Selection of water addition amount

[0048] Pretreatment technology is an extremely important link in the analysis of trace components, mainly including extraction, purification, concentration and re-dissolution steps. Most of the pesticide residue detection standards and literature reports released in my country after 2018 use acetonitrile as the extraction solvent. Meat samples have a certain viscosity, and the solubility of fat in acetonitrile is low. Acetonitrile will cause rapid denaturation and precipitation of proteins, causing the target to be wrapped and difficult to release. After adding water, the sample can be evenly dispersed, and the permeability of acetonitrile to the sample is enhanced, which is beneficial to the extraction of the target. At the same time, salt needs to be added to remove excess water in the later stage. This study investigated the effect of adding 0mL, 2mL, and 5mL of ultrapure water to 5g of sample on the recovery rate of the target compound after pretreatment (see Figure 2 ).

[0049] 2.2 Selection of purification conditions

[0050] Animal food matrices are complex. Impurities in the extraction process will not only interfere with the ionization efficiency and response intensity of the target, but also easily cause blockage of the chromatographic column and instrument, resulting in decreased sensitivity. Effective purification of the extract can reduce detection interference and instrument contamination. Currently, commonly used purification materials include C18, PSA, carbon-based nanomaterials (MWCNTs, MWCNTs-OH), and degreasing materials dSPEEMR-Lipid. This study investigated experimental schemes such as C18, PSA, MWCNTs, MWCNTs-OH, dSPEEMR-Lipid, and ZIP-8 purification materials synthesized in the laboratory (such as scheme: ①MgSO 4 450mg+PSA 30mg, ②MgSO 4 450mg+C1830mg、③MgSO 4 450mg+PSA 15mg+C18 15mg, ④MgSO 4 450mg+MWCNTs 30mg, ⑤MgSO 4 450mg+MWCNTs-OH 30mg, ⑥dSPEEMR-Lipid, ⑦ZIP-830mg) on ​​the purification effect of pesticide compounds ( Figure 3 ).

[0051] 2.3 Matrix Effect

[0052] The matrix effect (ME) is the difference between the slope of the matrix-matched standard curve and the slope of the solvent standard curve, divided by the slope of the solvent standard curve. When ME is positive, it shows a matrix enhancement effect; when it is negative, it shows a matrix inhibition effect. When ||ME|| is less than 20%, the matrix effect is negligible; when ||ME|| is greater than 50%, it is a strong matrix effect; when ||ME|| is in the range of 20% to 50%, it is a moderate matrix effect ( Figure 4 ). The matrix effects of 146 pesticides in blank pork were investigated. The results showed that 92.5% of the pesticides showed matrix enhancement effects, and 7.5% of the pesticides showed matrix inhibition effects. Among them, 24.7% of the pesticides had negligible matrix effects, 37.0% of the pesticides had moderate matrix effects, and 38.3% of the pesticides had strong matrix effects. In order to compensate for the differences caused by matrix effects, the matrix matching standard curve method is required to reduce the influence of matrix effects.

[0053] 2.4 Methodological Review

[0054] A series of matrix mixed standard working solutions (5-320 μg / L) were injected and measured, with mass concentration as the horizontal axis and the corresponding peak area as the vertical axis. The standard curve was drawn and the regression equation and correlation coefficient were calculated. According to GB 5009.295-2023, blank pork matrix samples were spiked and diluted for determination. The estimated detection limit (LOD) and estimated quantification limit (LOQ) of the method were expressed as 3 and 10 when the signal-to-noise ratio was respectively. The spike recovery test was set at 3 levels (0.01, 0.02, 0.10 mg / kg), and each level was measured 6 times in parallel to investigate the accuracy and precision of the method. The correlation coefficients, LOD, LOQ, recovery and precision results of 146 pesticides are shown in Table 1. The results showed that the 146 pesticides had a good linear relationship, with correlation coefficients no less than 0.9939. The LODs of 141 compounds were 0.04-10.91 μg / kg, the LOQs were 0.11-32.72 μg / kg, the average recoveries of 146 compounds were 61.3%-119.3%, and the RSDs were 0.15%-16%.

[0055] Table 1146 Correlation coefficients of pesticide compounds (r 2 ), detection limit, quantification limit, average recovery rate and relative standard deviation (n=6)

[0056]

[0057]

[0058]

[0059]

[0060] 2.5 Determination of actual samples

[0061] The method established in this paper was used to test 10 batches of pork, beef, mutton, chicken, fish and eggs. The results are shown in ( Figure 5 ), boscalid (1.80μg / kg) was detected in 1 batch of chicken, fipronil sulfone (1.74μg / kg and 1.90μg / kg) was detected in 2 batches of eggs, and 4,4-DDE (1.80μg / kg-2.18μg / kg) was detected in 5 batches of fish, all of which were far below the limit requirements of GB 2763-2021. No pesticide residues were detected in other samples.

[0062] 3 Conclusion

[0063] In this study, dSPEEMR-Lipid purification technology was used as a pretreatment degreasing method, and combined with GC-MS / MS, a qualitative and quantitative analysis method for rapid and accurate determination of 146 pesticide residues in animal foods was established. This pretreatment method is simple to operate, has good purification effect, can effectively reduce the interference of impurities in the matrix, has high selectivity and accuracy, and is suitable for the simultaneous determination and analysis of multiple pesticide residues in animal foods. It can provide technical support for the daily monitoring and supervision of multiple pesticide residues in animal foods, and can also provide reference for the formulation and revision of subsequent standards.

[0064] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for detecting 146 pesticide residues in animal food using dispersed solid phase extraction-gas chromatography-mass spectrometry technology, characterized in that: The method is to detect pesticide residues in animal food samples by using QuEChERS extraction method and dSPE EMR-Lipid cleanup method combined with gas chromatography-triple quadrupole mass spectrometry GC-MS / MS; The method comprises the following steps: S1. Selection of instruments and materials: The instruments used were gas chromatography-triple quadrupole mass spectrometry, high-speed low-temperature centrifuge, ultrapure water machine and fully automatic parallel concentrator; the materials used were QuEChERS EN extraction kit, ethyl acetate and acetonitrile; S2. Prepare reagents: Dissolve 146 pesticide standards and prepare standard stock solutions, and further prepare mixed standard working solutions and series standard solutions; S3. Sample pretreatment: Weigh 5 g of sample and add ultrapure water and acetonitrile, perform ultrasonic extraction and freezing, then add QuEChERS EN-extraction salt package and centrifuge to obtain supernatant. The supernatant is purified using dSPE EMR-Lipid purification tube, concentrated by nitrogen blowing and redissolved in ethyl acetate, and analyzed by GC-MS / MS. S4. Data analysis: Qualitative and quantitative analysis was performed using Agilent MassHunter software, and data statistics and visualization were performed using Excel.

2. The method for detecting 146 pesticide residues in animal food by using dispersed solid phase extraction-gas chromatography-mass spectrometry technology as claimed in claim 1, characterized in that: The samples are commercially available pork, beef, mutton, chicken, aquatic products, and eggs.

3. The method for detecting 146 pesticide residues in animal food by using dispersed solid phase extraction-gas chromatography-mass spectrometry technology as claimed in claim 1, characterized in that: The specific steps of preparing the reagents in step S2 are: taking the pesticide standard into a 10 mL volumetric flask, using acetone to make a standard stock solution; accurately pipetting the standard stock solution, and using ethyl acetate to make a mixed standard working solution; before use, using ethyl acetate pure solvent or blank matrix sample extract to make a series of standard solutions from the mixed standard working solution, and all standard solutions are stored at -18°C in the dark.

4. The method for detecting 146 pesticide residues in animal food by using dispersed solid phase extraction-gas chromatography-mass spectrometry technology as claimed in claim 1, characterized in that: The chromatographic column is a HP-5MSUI capillary chromatographic column with a length of 30 m, an inner diameter of 0.25 mm, and a film thickness of 0.25 μm. The temperature of the chromatographic injection port is set to 280° C. The chromatographic program temperature rising conditions are: the initial temperature is 60° C., maintained for 1 minute, then heated to 170° C. at a rate of 40° C. / min, then heated to 310° C. at a rate of 10° C. / min, and maintained at 310° C. for 3 minutes; the carrier gas flow rate is 1.0 mL / min, the injection mode is splitless injection, and the injection volume is 1 μL; The mass spectrometry conditions adopted electron impact EI ionization mode, the ionization energy was set to 70 eV; the ion source temperature was 230° C., the transmission line temperature was 280° C., and the solvent delay time was 2.5 minutes; The instrument was operated in dynamic multiple reaction monitoring (DMRM) mode; nitrogen was used as collision gas and helium as quenching gas.

5. The method for detecting 146 pesticide residues in animal food by using dispersed solid phase extraction-gas chromatography-mass spectrometry technology as claimed in claim 1, characterized in that: The 146 pesticides include dichlorvos, methamidophos, captan, graminetobac, tetrachloronitrobenzene, hexaflumuron, ethoprophos, cadusin, diphenylamine, trifluralin, chlorfenapyr, chlorpyrifos, phorate, α-BHC, hexachlorobenzene, dimethoate, atrazine, chlornitrile, thiamethoxam, terbuthion, terbuthion, β-BHC, propyzamide, pentachloronitrobenzene, difenothion, diazinon, γ-BHC, pyrimethanil, trichlorfon, chlorpyrifos, δ-BHC, monocrotophos, vinylpyrifos, methyl chlorpyrifos, methyl parathion, methyl tolclofos , amethoxam, fluazifop, heptachlor, metalaxyl, methyl pirimiphos, fenitrothion, pentamethylenediamine, malathion, ethoprocarb, phorate sulfoxide, aldrin, chlorpyrifos, parathion, triadimefon, fenthion, tetrafluthrin, isocarb, bisbencarb, methyl isoflavone, cyprodinil, pendimethalin, phorate sulfone, penconazole, fipronil, triadimenol, terbuthion sulfone, quinalphos, methyl thiocyanate, thiocyanate, α-endosulfan, benzylpyridin, fipronil sulfoxide, triflumizole, procymidone, flupyralid, methidathion, chlordane, 2,4'- DDI, flumetralin, fenoxaprop-butyl, picoxystrobin, myclobutanil, fenamiphos, dieldrin, β-endosulfan, blastifungin, profenofos, pretilachlor, hexaconazole, 4,4'-DDI, myclobutanil, 2,4'-DDI, flusilazole, methoxypropyl, thiofuran, endrin, fipronil sulfone, herbicide, cypermethrin, 2,4'-DDT, oxadone, 4,4'-DDT, clethodim, triazophos, bensulfuron, trifloxystrobin, propiconazole, dichlorvos, 4,4'-DDT, endosulfan sulfate, propargyl, tebuconazole, increase ether, bio-resmethrin, triphenyl phosphate, isoprodinil, xinyanling, bifenthrin, bromocriptine, azinphos-methyl, phosmet, bifenazate, cypermethrin, acetamiprid, tebufenpyrad, quinazofen, phosalone, pyriproxyfen, anthelmintic, chlorfenapyr, cyhalothrin, permethrin, cyfluthrin, spirocyclobutanil, coumaphos, prochloraz, paraoxon, pyraclostrobin, cyfenapyr, boscalid, flucythrin, pyraclostrobin, quinoline, cypermethrin, ethomethrin, cypermethrin, fluvalinate, fenpropimorph, deltamethrin, myclobutanil and oxazolidinone.

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