Determination of five new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry

Abstract

The invention relates to the technical field of food safety detection, discloses a method for determining 5 new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry, optimizes a beef sample pretreatment method based on QuEChERS by orthogonal experimental design, adopts a detection method of gas chromatography-tandem mass spectrometry, and combines external standard method quantification to establish a detection method for detecting 5 pesticide residues in animal-derived foods, which provides certain data support for future animal-derived food supervision and import and export risk monitoring. The method comprises the following steps: 1) preparation of standard solution: respectively preparing a standard reserve solution, a mixed standard working solution and a blank matrix mixed standard working solution; 2) sample pretreatment: placing the sample in a centrifuge tube, vortexing and centrifuging with an extraction solvent and an extraction dispersant, obtaining an upper organic phase, further vortexing and centrifuging in a centrifuge tube containing a dSPE mixture to obtain a supernatant, and filtering for standby use; 3) performing GC-MS / MS analysis on the filtered supernatant.

Description

Technical Field

[0001] The invention relates to the technical field of food safety detection, in particular to a method for determining five new pesticide residues in animal-derived foods by using gas chromatography-tandem mass spectrometry. Background Art

[0002] 2,6-Diisopropylnaphthalene is a hydrocarbon and a derivative of naphthalene. In 2021, South Korea developed an analytical method for its residues in plant-based agricultural products, while domestic research is all about its synthesis. Flumethrin belongs to the second-generation pyrethroid insecticide and acaricide. It is a common agent for the control of Varroa mites. Therefore, reports on this pesticide are basically residue detection in bee products. Benthiophene is a new type of insecticide with an aromatic alkoxy pyrimidine structure, and Flupyrimin is a new type of insecticide that acts on nicotinic acetylcholine receptors. There are only reports on their synthesis and biological activity at home and abroad. Flupyrimid is a new type of pyrazoleamide fungicide. The matrices involved in the residue analysis methods reported at home and abroad are mainly plant-based agricultural products.

[0003] In recent years, food safety accidents and international trade disputes caused by pesticide residues still occur from time to time. In response to the problems caused by the new pesticides included in the new changes in the food safety regulations of major trading countries, my country uses advanced analytical techniques to detect pesticide residues in food and monitors its content according to the prescribed limits to ensure food safety and prevent trade disputes. The "positive list system" implemented by Japan in 2006 has already stipulated the residue limit standards of the above-mentioned pesticides in animal-derived foods, and Japan has not established maximum permissible residue limits (MRLs) or agricultural chemicals that are not exempted substances. In food, they must comply with the "uniform standard" regulations, that is, they must not exceed 0.01 mg / kg. my country's GB 2763-2021 "National Food Safety Standard Maximum Residue Limits of Pesticides in Food" does not make any requirements for the MRLs values ​​of these five pesticides in animal foods, and no corresponding detection methods have been developed in China. The detection methods published by Japan have different pre-treatment methods according to different types of pesticides. For example, benzylpyridinol is extracted with acetonitrile and water and then passed through C 18 Solid phase extraction: Flumethrin was filtered through acetone, extracted by shaking with hexane and acetonitrile, and then evaporated and concentrated through C 18 The above process is complicated and cumbersome, and is not suitable for the simultaneous detection of multiple pesticide residues. Therefore, it is necessary to develop a detection method for the above five new pesticide residues in animal-derived foods that is suitable for my country's national conditions to ensure food safety.

[0004] The QuEChERS method was introduced by Anastassiades et al. in 2003. It is a technique that can remove water and unwanted matrix components from the sample matrix. It has the advantages of being fast, simple, cheap, effective, robust and safe. Recently, a large number of studies have applied this method to the analysis of pesticide residues in animal foods (such as meat, eggs, milk and aquatic products). Therefore, it is believed that the QuEChERS method can be used to analyze pesticides in animal foods. In order to achieve the best extraction efficiency, it is necessary to optimize parameters including extraction solvent, material-liquid ratio, salting-out agent and dispersive solid-phase extraction (dSPE) adsorbent, which makes the pretreatment optimization steps complicated and cumbersome. The orthogonal design method of multi-factor multi-level analysis can avoid the tediousness and time-consuming of comprehensive experiments and improve the efficiency of experimental design. Mao Xuejin et al. established a new analytical method for the simultaneous determination of trimethyltin, triphenyltin and tributyltin in laver by liquid chromatography-tandem mass spectrometry (LC-MS / MS), and used the orthogonal design method to find the best extraction conditions. Tian Tian et al. established a method for determining the residues of five pesticides in vegetables by dispersed solid phase extraction-gas chromatography-mass spectrometry and optimized the sample extraction conditions using the orthogonal design method. Summary of the invention

[0005] The purpose of the present invention is to provide a method for determining five new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry. The QuEChERS-based beef sample pretreatment method is optimized by orthogonal experimental design. The detection method of gas chromatography-tandem mass spectrometry (GC-MS / MS) is adopted, and combined with the external standard method for quantification, a detection method for detecting five pesticide residues of 2,6-diisopropylnaphthalene, fluchlorfenvinphos, benzathine, flupyrimin and flupyrimid in animal-derived foods is established, which provides certain data support for the future supervision of animal-derived foods and import and export risk monitoring.

[0006] The present invention is achieved by the following technical scheme: A method for determining five new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry comprises the following steps:

[0007] 1) Preparation of standard solutions: prepare standard stock solution, mixed standard working solution and blank matrix mixed standard working solution respectively;

[0008] 2) Sample pretreatment: Place the sample in a centrifuge tube, vortex and centrifuge with the extraction solvent and extraction dispersant, and continue to vortex and centrifuge the upper organic phase in the centrifuge tube containing the dSPE mixture to obtain the supernatant, which is filtered and set aside;

[0009] 3) The filtered supernatant was subjected to GC-MS / MS analysis.

[0010] Among them, the standard substances of 5 new pesticide residues (i.e. pesticide standards) are 2,6-diisopropylnaphthalene (CAS: 24157-81-1), benzathine (CAS: 1449021-97-9), Flupyrimin (CAS: 1689566-03-7) (1000 mg / L, Tianjin Alta Technology Co., Ltd.); fluchlorfenapyr (CAS: 69770-45-2), fluazifop (CAS: 874967-67-6) (100 mg / L, Changzhou Manhage Testing Technology Co., Ltd.).

[0011] In order to better implement the method for determining the residues of five new pesticides in animal-derived foods by gas chromatography-tandem mass spectrometry of the present invention, the following setting is particularly adopted: the step 1) includes the following specific steps:

[0012] 1.1) Preparation of standard stock solutions: Accurately pipette 1 mL of each pesticide standard and prepare a standard stock solution with a mass concentration of 10 mg / L or 100 mg / L using methanol, acetonitrile and acetone. All standard stock solutions should be stored at -20°C for no more than 6 months.

[0013] 1.2) Preparation of mixed standard working solution: Accurately pipette a certain amount of standard stock solution and prepare a mixed standard working solution with a mass concentration of 1 mg / L using acetonitrile as solvent;

[0014] 1.3) Preparation of blank matrix mixed standard working solution: The sample was processed to obtain a blank matrix solution, and the mixed standard working solution was diluted stepwise with the blank matrix solution to a blank matrix mixed standard working solution of 0.0004, 0.0008, 0.0020, 0.0040, 0.0100, 0.0200, 0.0400, 0.0600, 0.0800, and 0.1000 μg / mL.

[0015] In order to better implement the method for determining the residues of five new pesticides in animal-derived foods by gas chromatography-tandem mass spectrometry of the present invention, the following setting is particularly adopted: the step 2) is specifically:

[0016] 2.1) Weigh 2.00±0.02g of sample into a 50mL centrifuge tube (preferably a polypropylene centrifuge tube), add 5mL of extraction solvent (preferably an acetonitrile / acetone solution (1:1, V:V) containing 1% acetic acid), vortex for 10min, and sonicate for 10min;

[0017] 2.2) Then add an appropriate amount of extraction dispersant (preferably 5 g NaCl) into the centrifuge tube, vortex again for 10 min, and refrigerate centrifuge at 8000 rpm for 5 min;

[0018] 2.3) Transfer 1.0 mL of the upper organic phase to a dSPE mixture (preferably 120 mg PSA, 30 mg C 18 , 180 mg MgSO4) in a 15 mL centrifuge tube (preferably a polypropylene centrifuge tube), vortex for 3 min, centrifuge at 4000 r / min for 3 min, and take the supernatant and filter it through a 0.22 μm filter membrane.

[0019] In order to better implement the method for determining the residues of five new pesticides in animal-derived foods by gas chromatography-tandem mass spectrometry described in the present invention, the following setting is particularly adopted: the sample in step 2.1) is a beef sample that is sealed and stored at -20°C after homogenization and then thawed before use; or it is a beef sample that is homogenized on site.

[0020] In order to better realize the method for determining 5 new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry described in the present invention, the following setting is particularly adopted: the extraction solvent is acetonitrile or 1% acetic acid acetonitrile, acetonitrile / acetone (1:1, V:V) or acetonitrile / acetone solution containing 1% acetic acid (1:1, V:V); the appropriate amount of extraction dispersant is 5g of NaCl or 4g MgSO4+1g NaCl or 6g MgSO4+1.5g NaOAc or 4g MgSO4+1g NaCl+1g Na3C6H5O7+0.5g C6H6O7Na2·1.5H2O or 6g MgSO4+1.5g CH3COONa or 4g MgSO4+1g NaCl+1g C6H5O7Na3+0.5gC6H6O7Na2·1.5H2O; the dSPE mixture uses 0, 30, 60, 90, 120 mg of PSA or 3, 5, 10, 15 mg of MWCNT, 30, 60, 90, 120 mg of C 18 and 30, 60, 90, 120, 180 mg of MgSO4; the centrifuge tube is a polypropylene centrifuge tube.

[0021] In order to better implement the method for determining five new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry of the present invention, the following configuration is particularly adopted: the extraction solvent is an acetonitrile / acetone solution (1:1, V:V) containing 1% acetic acid; the appropriate amount of extraction dispersant is 5g of NaCl; the dSPE mixture is 120mg of PSA, 30mg of C 18 and 180 mg of MgSO4.

[0022] In order to better implement the method for determining five new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry of the present invention, the following setting is particularly adopted: when performing GC-MS / MS analysis,

[0023] The chromatographic conditions are:

[0024] A SH-I-5Sil mass spectrometry capillary column (30m×0.25mm×0.25μm) was used; the injection volume was 1μL, non-split injection, and the carrier gas was helium; the column oven temperature program was as follows: the initial temperature was set at 70°C and maintained for 1.0min, then increased to 150°C at 30°C / min; increased to 280°C at a rate of 12°C / min; and finally increased to 290°C at a rate of 5°C / min and maintained for 1.0min; the flow rate was 1.6mL / min, the collision gas was argon, and the injection port temperature was 280°C;

[0025] Mass spectrometry conditions: ionization mode was electron impact (EI) source, detection voltage (relative to tuning result) was 0.3 Kv, ion source temperature was 230 °C, scanning mode was multiple reaction monitoring (MRM) mode, solvent delay time was 5.0 min, and total running time was 17.5 min.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] The present invention optimizes the QuEChERS-based beef sample pretreatment method by orthogonal experimental design, adopts gas chromatography-tandem mass spectrometry (GC-MS / MS) detection means, and combines external standard method quantification to establish a detection method for detecting five pesticide residues of 2,6-diisopropylnaphthalene, fluchlorfenvinphos, benzathine, flupyrimin and fluazifop-butyl in animal-derived food, which provides certain data support for the future supervision of animal-derived food and import and export risk monitoring.

[0028] The present invention uses a pass-through SPE pretreatment technology to develop an HPLC-MS / MS method for determining 17 new veterinary drug residues in animal-derived foods. The present invention optimizes influencing factors, including mass spectrometry parameters, mobile phase, extraction solvent, SPE column type and extraction solvent dosage. Evaluation indicators include matrix effect, linearity, recovery rate and precision. The method is simple, efficient and economical, and has been successfully applied to actual sample testing in local markets. It can provide valuable technical support for strengthening cross-border food safety and eliminating trade barriers.

[0029] The method of the present invention has simple pretreatment operation and high sensitivity. The recovery test also confirms the high accuracy, precision and repeatability of the method, which can meet Japan's limit requirements for these five new pesticides and fungicides. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the optimization result diagram of PSA addition amount.

[0031] Figure 2 C 18 Optimization result diagram of addition amount.

[0032] Figure 3 This is the optimization result diagram of MWCNT content.

[0033] Figure 4 This is the total ion chromatogram obtained from standard solutions of five pesticide compounds. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0035] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are 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 are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention.

[0036] In order to achieve better technical effects, the present invention involves the following instruments, materials and reagents:

[0037] GCMS-TQ8040 gas chromatograph-mass spectrometer (Shimadzu Corporation, Japan); SH-I-5Sil mass spectrometry capillary column (30m×0.25mm×0.25μm, Restek Corporation, USA); Mill-Q Integral 3 ultrapure water instrument (Millipore Corporation, USA); KQ-800DE CNC ultrasonic cleaning machine (Shanghai Kunshan Ultrasonic Instrument Co., Ltd.); MS3 vortex oscillator (IKA Company, Germany); XS205DU electronic analytical balance (precision 0.1mg, Mettler Toledo Company, Switzerland); 3H2RI desktop refrigerated centrifuge (Hexi Company, China).

[0038] All animal-origin food samples were obtained from local markets and supermarkets.

[0039] 2,6-Diisopropylnaphthalene (CAS: 24157-81-1), benzathine (CAS: 1449021-97-9), Flupyrimin (CAS: 1689566-03-7) (1000 mg / L, Tianjin Alta Technology Co., Ltd.); Flumethrin (CAS: 69770-45-2), Flupyrimid (CAS: 874967-67-6) (100 mg / L, Changzhou Manhage Testing Technology Co., Ltd.).

[0040] Acetone, acetonitrile, methanol (chromatographic grade, Fisher, USA); anhydrous magnesium sulfate (MgSO4), sodium acetate (NaAc), sodium chloride (NaCl) (analytical grade, Chengdu Kelun Reagent Co., Ltd.); trisodium citrate (C6H5O7Na3), disodium hydrogen citrate sesquihydrate (C6H6O7Na2·1.5H2O) (analytical grade, Shanghai MacLean Biochemical Co., Ltd.); octadecylsilane (C 18 ), ethylenediamine-N-propylsilane (primary secondary amine, PSA), filter head (0.22μm nylon) (Shanghai Anpu Experimental Technology Co., Ltd.); multi-walled carbon nanotube (Multi-walled carbon nanotube, MWCNT) [TCI (Shanghai) Chemical Industry Development Co., Ltd.].

[0041] Embodiment 1:

[0042] The method for determining five new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry comprises the following steps:

[0043] 1) Preparation of standard solutions: prepare standard stock solution, mixed standard working solution and blank matrix mixed standard working solution respectively;

[0044] 2) Sample pretreatment: Place the sample in a centrifuge tube, vortex and centrifuge with the extraction solvent and extraction dispersant, and continue to vortex and centrifuge the upper organic phase in the centrifuge tube containing the dSPE mixture to obtain the supernatant, which is filtered and set aside;

[0045] 3) The filtered supernatant was subjected to GC-MS / MS analysis.

[0046] Among them, the standard substances of 5 new pesticide residues (i.e. pesticide standards) are 2,6-diisopropylnaphthalene (CAS: 24157-81-1), benzathine (CAS: 1449021-97-9), Flupyrimin (CAS: 1689566-03-7) (1000 mg / L, Tianjin Alta Technology Co., Ltd.); fluchlorfenapyr (CAS: 69770-45-2), fluazifop (CAS: 874967-67-6) (100 mg / L, Changzhou Manhage Testing Technology Co., Ltd.).

[0047] Embodiment 2:

[0048] This embodiment is further optimized on the basis of the above embodiment, and the same points as the above technical solution are not repeated here. In order to better realize the method of determining 5 new pesticide residues in animal-derived food by gas chromatography-tandem mass spectrometry according to the present invention, the following setting is particularly adopted: the step 1) includes the following specific steps:

[0049] 1.1) Preparation of standard stock solutions: Accurately pipette 1 mL of each pesticide standard and prepare a standard stock solution with a mass concentration of 10 mg / L or 100 mg / L using methanol, acetonitrile and acetone. All standard stock solutions should be stored at -20°C for no more than 6 months.

[0050] 1.2) Preparation of mixed standard working solution: Accurately pipette a certain amount of standard stock solution and prepare a mixed standard working solution with a mass concentration of 1 mg / L using acetonitrile as solvent;

[0051] 1.3) Preparation of blank matrix mixed standard working solution: The sample was processed to obtain a blank matrix solution, and the mixed standard working solution was diluted stepwise with the blank matrix solution to a blank matrix mixed standard working solution of 0.0004, 0.0008, 0.0020, 0.0040, 0.0100, 0.0200, 0.0400, 0.0600, 0.0800, and 0.1000 μg / mL.

[0052] In order to achieve better results, the present invention optimized the extraction method of pesticide residues during the research and development stage:

[0053] The type of extraction reagent, type of extraction solvent, solid-liquid ratio, and ultrasonic time were selected as the optimization factors affecting the sample extraction method. The factor level table is shown in Table 1. L 16 (4 4 ) orthogonal table, scientifically optimize the sample extraction process, and characterize the experimental results of this group by the recovery rate of each pesticide. The results were subjected to intuitive analysis of orthogonal test and analysis of variance (ANOVA).

[0054] Table 1. Factor level table of orthogonal test of sample extraction based on QuEChERS

[0055]

[0056] Embodiment 3:

[0057] This embodiment is further optimized on the basis of any of the above embodiments, and the similarities with the above technical solutions are not repeated here. In order to better implement the method for determining 5 new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry according to the present invention, the following setting is particularly adopted: the step 2) is specifically:

[0058] 2.1) Weigh 2.00±0.02g of sample into a 50mL centrifuge tube (preferably a polypropylene centrifuge tube), add 5mL of extraction solvent (preferably an acetonitrile / acetone solution (1:1, V:V) containing 1% acetic acid), vortex for 10min, and sonicate for 10min;

[0059] 2.2) Then add an appropriate amount of extraction dispersant (preferably 5 g NaCl) into the centrifuge tube, vortex again for 10 min, and refrigerate centrifuge at 8000 rpm for 5 min;

[0060] 2.3) Transfer 1.0 mL of the upper organic phase to a dSPE mixture (preferably 120 mg PSA, 30 mg C 18 , 180 mg MgSO4) in a 15 mL centrifuge tube (preferably a polypropylene centrifuge tube), vortex for 3 min, centrifuge at 4000 r / min for 3 min, and take the supernatant and filter it through a 0.22 μm filter membrane.

[0061] Embodiment 4:

[0062] This embodiment is further optimized on the basis of any of the above embodiments, and the similarities with the above technical solutions are not repeated here. In order to better realize the method for determining 5 new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry described in the present invention, the following setting is particularly adopted: the sample in step 2.1) is a beef sample that is sealed and stored at -20°C after homogenization and then thawed before use; or it is a beef sample that is homogenized on site.

[0063] Embodiment 5:

[0064] This embodiment is further optimized on the basis of any of the above embodiments, and the similarities with the above technical solutions are not repeated here. In order to better realize the method for determining 5 new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry described in the present invention, the following setting is particularly adopted: the extraction solvent is acetonitrile or 1% acetic acid acetonitrile, acetonitrile / acetone (1:1, V:V) or acetonitrile / acetone solution containing 1% acetic acid (1:1, V:V); the appropriate amount of extraction dispersant is 5g NaCl or 4g MgSO4+1g NaCl or 6g MgSO4+1.5g NaOAc or 4g MgSO4+1g NaCl+1g Na3C6H5O7+0.5gC6H6O7Na2·1.5H2O or 6g MgSO4+1.5g CH3COONa or 4g MgSO4+1g NaCl+1g C6H5O7Na3+0.5gC6H6O7Na2·1.5H2O; the dSPE mixture uses 0, 30, 60, 90, 120 mg of PSA or 3, 5, 10, 15 mg of MWCNT, 30, 60, 90, 120 mg of C 18 and 30, 60, 90, 120, 180 mg of MgSO4; the centrifuge tube is a polypropylene centrifuge tube.

[0065] Embodiment 6:

[0066] This embodiment is further optimized on the basis of any of the above embodiments, and the similarities with the above technical solutions are not repeated here. In order to better realize the method of determining 5 new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry of the present invention, the following settings are particularly adopted: the extraction solvent is an acetonitrile / acetone solution (1:1, V:V) containing 1% acetic acid; the appropriate amount of extraction dispersant is 5g of NaCl; the dSPE mixture uses 120mg of PSA, 30mg of C 18 and 180 mg of MgSO4.

[0067] In order to achieve better technical effects, the present invention optimized some experimental conditions during the research and development stage:

[0068] 1. Sample extraction optimization:

[0069] The experiment selected different extraction conditions to analyze the spiked recoveries of 5 pesticides. 16 (4 4) Orthogonal test design, obtained orthogonal test grouping and results, the results are shown in Table 2. The obtained test results were imported into the SPSS data editor. For the multi-factor orthogonal table, the general linear model should be selected. In the main effect analysis, the average recovery rate was specified as the dependent variable, A, B, C, and D were fixed factors, and the sample pretreatment orthogonal test ANOVA results were obtained. The number of degrees of freedom in the table is the highest level number minus 1, the mean square is equal to the sum of squares divided by the degree of freedom, and the F value is the mean square of each factor divided by the error mean square. According to the significant result P value, it is judged whether the influence of each factor level on the spiked recovery rate of each pesticide in beef is significant.

[0070] Table 2 Grouping and results of orthogonal test for extraction of beef samples

[0071]

[0072]

[0073] Table 3 Analysis of variance of the recovery rate of each compound added in the orthogonal test

[0074]

[0075]

[0076] Table 4 Multiple comparisons of recovery rates at various levels of extraction solvent and solvent amount under extraction conditions

[0077]

[0078] Note: Different letters in the same column of the same factor indicate significant differences (P < 0.05).

[0079] The analysis results in Table 3 show that for the five compounds (pesticide standards), the extraction solvent and the liquid-to-solid ratio have significant effects on the recovery rate, while the ultrasonic time and the extraction dispersant have no significant effects on the recovery rate. Combined with Table 4, it can be seen that the extraction solvent has the largest range for the five compounds (pesticide standards), followed by the liquid-to-solid ratio, followed by the extraction dispersant, and the ultrasonic time has the smallest range, that is, the order of the influence of each factor on the recovery rate of each compound is: extraction solvent>liquid-to-solid ratio>extraction dispersant>ultrasonic time.

[0080] From the above analysis, it can be seen that the type of extraction solvent (extraction solvent) is the most important parameter affecting the sample pretreatment process. The effects of four different extraction solvents (extraction solvents) on the recovery rate of pesticides were compared: acetonitrile, 1% acetic acid acetonitrile, acetonitrile / acetone (1:1, V:V) and acetonitrile / acetone (1:1, V:V; 1% acetic acid). The most commonly used extraction solvent (extraction solvent) for detecting pesticide residues is acetonitrile. When extracting a large number of pesticides, the number of pesticides that can be extracted well is large and the extraction of pigments is small, but the extraction efficiency for some pesticides is limited. The results showed that the range of pesticide recovery obtained after adding sodium acetate to the extraction with pure acetonitrile was 75.25-86.92%, which was significantly higher than the recovery range of 53.40-71.30% in other groups. The recovery rate of pesticides extracted after adding acetic acid to acetonitrile was better than that of pure acetonitrile, but there were still cases where the recovery rate was as low as 60% or less, which made it difficult to meet the methodological indicators. When acetonitrile / acetone (1:1, V:V) is selected as (extraction solvent), the extraction effect is very good, and the recovery rate is 75.84% to 104.53%. Fat is the main matrix interference of beef. Acetone can extract fat, while acetonitrile is incompatible with animal fat. Therefore, when acetonitrile / acetone (1:1, V:V) is used, pesticides are extracted and fat is discarded. After adding 1% acetic acid to acetonitrile / acetone (1:1, V:V), the recovery rate of pesticides reaches the best, and the recovery rate of 5 pesticides is 81.37% to 113.42%. Therefore, acetonitrile / acetone (1:1, V:V) plus 1% acetic acid is selected as the preferred extraction solvent scheme of the present invention.

[0081] MgSO4 in the initial salt package (4g MgSO4+1g NaCl) promotes the salting out of acetonitrile and absorbs excess water. NaCl improves the efficiency of organic phase extraction by changing the polarity of the extraction solvent (extraction solvent); the combination of MgSO4 and NaCl can not only control the polarity of the extraction medium, but also improve the shape of the peak and reduce most polar interferences. Extraction salt package 1 (6g MgSO4+1.5g NaOAc) uses sodium acetate as a buffer solution, and citrate in extraction salt package 2 (4g MgSO4+1g NaCl+1gNa3C6H5O7+0.5gC6H6O7Na2·1.5H2O) provides a stable environment of pH 5-5.5, which is beneficial for the extraction of a large number of acidic pesticide compounds and acid-sensitive pesticide compounds. When NaCl is used alone, the addition amount reaches a certain level, and the aqueous phase and the organic phase can be completely separated. However, the results of variance analysis show that the type of extraction salt package has no significant effect on the recovery rate of the five pesticide residues. After multiple comparisons, the optimal extraction conditions for the five pesticides in beef were: acetonitrile / acetone (1:1, V:V) containing 1% acetic acid as the extraction solvent, a solid-liquid ratio of 1:2.5, an ultrasonic time of 10 min, and 5 g NaCl as the extraction dispersant.

[0082] 2. Sample purification optimization:

[0083] GC-MS / MS technology has been widely used to detect pesticides and fungicides in animal-derived foods. However, if the extracts of animal food samples are not thoroughly purified, the sensitivity of the analyte may be greatly reduced and the service life of the chromatographic column may be shortened. Therefore, before GC-MS / MS detection, sample pretreatment to remove non-target compounds that may affect the analyte signal is a key step in determining pesticides in complex sample matrices. By studying animal-derived food samples rich in matrices such as fat and protein. In particular, lipids, due to their high solubility in organic solvents, are easily dissolved in extraction solvents. Therefore, in the research and development stage, the supernatant obtained after pretreatment extraction is further purified by appropriate methods. On this basis, the present invention adopts dSPE method for purification of multiple pesticide multi-residue analysis. In order to study the retention performance and matrix effect of dSPE adsorbent content on the selected 5 pesticides, different dSPE adsorbent combinations (PSA, C 18 and MWCNT) to evaluate the recovery rate. In order to achieve better technical results, the adsorbent content used in dSPE was optimized during the research and development stage, and the matrix matching standard was used for quantification. The most suitable dSPE content combination for beef was selected based on the recovery results of each pesticide.

[0084] (1) Optimization of PSA content

[0085] PSA can remove a variety of organic acids, fatty acids, sugars, lipids and polar pigments through weak anion exchange, and can also adsorb polar pesticides. During purification, the addition amount of PSA was 0, 30, 60, 90, and 120 mg, and the optimal addition amount of PSA was determined based on the recovery rate of each pesticide. The results are shown in Figure 1 . With the increase of the amount of PSA added, the recovery rates of 2,6-diisopropylnaphthalene, benzathine, and Flupyrimin showed an upward trend, and the recovery rate reached the best at 120 mg. There was no significant difference in the recovery rate of fluazifop-cyclohexanilide at the dosage of 0-90 mg PSA, but it reached the best at 120 mg. The recovery rate of fluchlorfenvinphos was also good at 120 mg. Therefore, the addition amount of PSA was selected to be 120 mg, and its recovery rate was 92.97% to 106.85%, and the result was satisfactory. Figure 1 Different letters indicate significant differences among the same compounds in different columns (P < 0.05).

[0086] (2)C 18 Optimization of content

[0087] C 18 It has a strong affinity for non-polar interfering components such as sterols and long-chain fats, absorbs high lipid content and non-polar components, and is suitable for the purification of meat matrices. 30, 60, 90, and 120 mg of C18 The effect on the extraction efficiency was determined based on the recovery results of each pesticide. 18 The results show that ( Figure 2 ), with the increase of the added amount, the recovery rate of each pesticide basically showed a downward trend. This may be due to the fact that C 18 It generates strong van der Waals forces with the aromatic groups in the target compound. Excessive use will increase the adsorption of the target compound, reduce the recovery rate, and reduce the purification performance of the sample. Therefore, when purifying beef samples, C 18 The addition amount was selected as 30 mg, and the recovery rate ranged from 95.20% to 105.59%. Figure 2 Different letters indicate significant differences among the same compounds in different columns (P < 0.05).

[0088] (3) Optimization of MWCNT content

[0089] MWCNT has been used in the pretreatment analysis of complex matrices to adsorb interfering substances in fruits and vegetables due to its high specific surface area, good adsorption performance, and multilayer and hollow structure. In previous work, it was mainly used as a substitute for graphitized carbon black (GCB) to remove pigments. There are also studies that have conducted experiments to compare the cleaning performance of MWCNT and PSA on vegetables, and it is considered to combine QuEChERS sample preparation with MWCNT as a substitute for PSA. During purification, the amount of MWCNT added (i.e., 3, 5, 10, and 15 mg) and the optimal amount of PSA added (120 mg) were compared and optimized. The cleaning performance of MWCNT and PSA was judged based on the recovery rate of each pesticide. The results are shown in Figure 3 .exist Figure 3 Different letters indicate significant differences among the same compounds in different columns (P < 0.05).

[0090] Depend on Figure 3 It can be seen that after adding MWCNT to purify the beef sample, as the amount of MWCNT added increases, the recovery rates of the five pesticides are all above 90%, with no significant change. In contrast, the recovery rate after adding PSA is better than that after adding MWCNT for purification, indicating that MWCNT as a purification material has no optimization effect on the recovery rate of each pesticide in the beef sample. There is no significant difference in the chromatograms obtained after purification with MWCNT and PSA, so MWCNT is not used in purification, and PSA adsorbent is preferably used.

[0091] Embodiment 7:

[0092] This embodiment is further optimized on the basis of any of the above embodiments, and the similarities with the above technical solutions are not repeated here. In order to better implement the method of determining 5 new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry of the present invention, the following setting is particularly adopted: when performing GC-MS / MS analysis,

[0093] The chromatographic conditions are:

[0094] A SH-I-5Sil mass spectrometry capillary column (30m×0.25mm×0.25μm) was used; the injection volume was 1μL, non-split injection, and the carrier gas was helium; the column oven temperature program was as follows: the initial temperature was set at 70°C and maintained for 1.0min, then increased to 150°C at 30°C / min; increased to 280°C at a rate of 12°C / min; and finally increased to 290°C at a rate of 5°C / min and maintained for 1.0min; the flow rate was 1.6mL / min, the collision gas was argon, and the injection port temperature was 280°C;

[0095] Mass spectrometry conditions: ionization mode was electron impact (EI) source, detection voltage (relative to tuning result) was 0.3 Kv, ion source temperature was 230 °C, scanning mode was multiple reaction monitoring (MRM) mode, solvent delay time was 5.0 min, and total running time was 17.5 min.

[0096] In order to achieve better technical effects, the present invention optimized the mass spectrometry conditions during the research and development stage:

[0097] The GC-MS / MS detection conditions of five pesticides were optimized using the SH-I-5Sil MS separation column. A 10 μg / mL mixed standard solution of five pesticides was subjected to a Q3SCAN full scan, and the mass range was set to 50-550 m / z to obtain the parent ions of the five pesticides. Then, different collision-induced dissociation (CID) energies (3-45 eV) were used to perform product ion scanning to obtain the product ions corresponding to the parent ions. Two pairs of ions with relatively large abundance were selected, one pair as the quantitative ion pair, and the other pair as the qualitative ion pair. The MRM method was created by combining the retention time, qualitative ion pair, and optimal collision energy. The residence time of each substance was adjusted according to the peak width of the target compound, and the ion ratio was set to finally determine the MRM method. The total ion chromatogram detection diagram of GC-MS / MS detection of 1.0 μg / mL standard solution of five pesticide compounds is shown in the figure below. Figure 4 The optimal mass spectrometry parameters are shown in Table 5.

[0098] Table 5 Retention time and mass spectrometry conditions of five pesticides

[0099]

[0100]

[0101] In order to illustrate the technical effect of the invention, this method is verified:

[0102] 1. Verification of linearity, detection limit and quantification limit:

[0103] In order to evaluate the linear relationship between this method and the target compounds and reflect the matrix effect, a matrix-matched standard solution (blank matrix mixed standard working solution) was used for GC-MS / MS analysis at a mass concentration of 0.0004-0.1000 mg / L. The analysis results are shown in Table 6. The r of the compounds was between 0.9993 and 0.9999, showing a good linear relationship, which was in line with GB / T27404-2008 (r≥0.99). Therefore, this analytical method was confirmed to have moderate linear quantification. The quantitative limit of 4 compounds in this analytical method was 0.4 μg / kg, and the quantitative limit of flumethrin was 87.7 μg / kg, which was less than the MRLs value specified in Japan and met the detection requirements.

[0104] Table 6 Detection limit, quantification limit and linear relationship of 5 compounds in samples

[0105]

[0106] 2. Verification of accuracy and precision:

[0107] In order to verify the validity and reliability of the analytical method, its accuracy was determined by recovery rate, and its intra-day precision was determined by RSD. Spiked samples at three concentration levels were prepared, and each concentration level was repeated 5 times within 1 day, and the recovery rate and RSD were determined. The spiked concentration levels of the five compounds in the beef samples and the recovery and precision results are shown in Table 7. As shown in Table 7, the recovery rate of beef was 73.23% to 111.92%, achieving a satisfactory recovery rate, and the RSD was 1.09% to 11.55%. The RSD of all samples at three concentration levels met the requirements of GB / T 27404-2008 and did not exceed 20%. The results show that the method established by the present invention has high accuracy and reproducibility, and the method can be reliably applied to the analysis of five pesticides in beef. At present, there are few research articles on the detection methods of the five pesticides. Jiang Huatao et al. established an analytical method for the residue of fluazifop in brown rice (the limit of quantification is 4 μg / kg, RSD≤12%); LEE et al. established a GC-MS / MS analytical method for the determination of 2,6-diisopropylnaphthalene residues in agricultural products, and the limit of quantification of the method is 10 μg / kg. Therefore, the method of the present invention has higher sensitivity and good stability.

[0108] Table 7 Average recoveries and relative standard deviations of five compounds in beef samples (n=5)

[0109]

[0110] 3. Matrix effect verification:

[0111] To verify the applicability of this method to the target pesticides, beef samples were used for recovery determination. According to the standard GB / T 27404-2008 "Laboratory Quality Control Specifications for Physical and Chemical Testing of Foods", the multi-residue analysis method for beef was verified based on sensitivity, linearity, accuracy and precision. In order to determine the sensitivity of the analytical method, the signal-to-noise ratio (S / N) of 3 and 10 was used as the limit of detection (LOD) and limit of quantification (LOQ) values. Three different spike levels were used, with the spike concentrations of flumethrin being 100, 150 and 200 μg / kg, and the spike concentrations of other substances being 2, 5 and 10 μg / kg. Five parallels were made, and the average recovery and relative standard deviation (RSD) were calculated. The accuracy was verified by the recovery test. The recovery was satisfactory when it was between 60% and 120%, and the RSD of the recovery was satisfactory when it was less than 20%. In order to evaluate the analytical method considering matrix effects (ME), a matrix matching calibration method was established. The mass concentration range for calibration was 0.0004-0.1000 mg / L. The calibration curve and correlation coefficient r were calculated based on the peak area of ​​the matrix matching solution. The correlation coefficient should not be less than 0.99. Since the compounds extracted from each sample may produce matrix effects in the mass spectrometry-based analytical method, it is necessary to estimate the matrix effect of pesticides in the sample, which is expressed by the linear regression slope of the matrix matching calibration curve and the linear regression slope of the calibration curve of the standard solution without matrix, as shown in formula (1):

[0112]

[0113] |ME| values ​​of 0 to 20% are considered to be low or tolerable matrix effects, while |ME| values ​​of 20% to 50% are considered to be moderate matrix effects, and when |ME| values ​​are greater than 50%, they are considered to be strong matrix effects.

[0114] Animal-derived foods are very complex matrices containing proteins, lipids, saturated fatty acids, and monounsaturated fatty acids. Even if standard sample preparation protocols are followed and advanced instruments are used, the signal of the target compound may be enhanced or weakened due to the presence of coeluting substances after the sample analysis solution is injected into the instrument, resulting in matrix effects, which have a strong impact on the accuracy of the proposed method. The results of the beef matrix effect are shown in Table 7. The matrix effects of the five pesticides ranged from -22.09% to 78.67%. Among them, flumethrin showed matrix suppression, and the other four pesticides all showed ion enhancement. This matrix-induced signal (response) enhancement is a typical phenomenon in GC-MS / MS analysis, because matrix interference prevents the active surface sites on the injector bushing, column, or detector from being blocked, resulting in an overestimation of the analyte residue. The matrix effect value of 2,6-diisopropylnaphthalene was very high, reaching more than 50%, which was a strong matrix enhancement effect; the matrix effect values ​​of flumethrin, benzathine, flupyrimin and flupyrimidine were between 20% and 50%, which was a medium matrix effect. Since all interferences were medium and strong, in order to reduce the influence of matrix effect, the matrix matching standard curve was prepared with beef blank sample extract for quantitative analysis of the five pesticides.

[0115] During the experiment, the data were preferably analyzed using Shimadzu LabSolutions for the spectra of samples and standards, analyzed using IBM SPSS27.0.1 software, and tabulated and plotted using Origin 2022 software.

[0116] The invention is applied to the determination of actual samples:

[0117] The established method was applied to actual application scenarios to verify the effectiveness and applicability of the method in daily analysis. The established method was used to analyze 16 batches of samples purchased from the local market, including 4 batches of beef, fish, chicken, and eggs, and pesticide multi-residue detection was performed. No pesticide residues involved in this method were detected.

[0118] Therefore, the sample was extracted with acetonitrile / acetone (1:1, V:V) containing 1% acetic acid, then extracted with 5 g NaCl, and then with MgSO4, C 18 , PSA was cleaned up and analyzed by GC-MS / MS. The proposed analytical method was verified and optimized according to the Codex Alimentarius guidelines. In the analysis of 5 pesticides, the method pretreatment operation was simple and sensitive, and the recovery test also confirmed the high accuracy, precision and repeatability of the method, which can meet the limit requirements of Japan for these 5 new insecticides and fungicides.

[0119] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for determining five new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry, characterized in that: The steps include: 1) Preparation of standard solutions: prepare standard stock solution, mixed standard working solution and blank matrix mixed standard working solution respectively; 2) Sample pretreatment: Place the sample in a centrifuge tube, vortex and centrifuge with the extraction solvent and extraction dispersant, and continue to vortex and centrifuge the upper organic phase in the centrifuge tube containing the dSPE mixture to obtain the supernatant, which is filtered and set aside; 3) The filtered supernatant was subjected to GC-MS / MS analysis.

2. The method for determining five new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The step 1) comprises the following specific steps: 1.1) Preparation of standard stock solutions: Accurately pipette 1 mL of each pesticide standard and prepare a standard stock solution with a mass concentration of 10 mg / L or 100 mg / L using methanol, acetonitrile and acetone respectively; 1.2) Preparation of mixed standard working solution: Accurately pipette a certain amount of standard stock solution and prepare a mixed standard working solution with a mass concentration of 1 mg / L using acetonitrile as solvent; 1.3) Preparation of blank matrix mixed standard working solution: The sample was processed to obtain a blank matrix solution, and the mixed standard working solution was diluted stepwise with the blank matrix solution to a blank matrix mixed standard working solution of 0.0004, 0.0008, 0.0020, 0.0040, 0.0100, 0.0200, 0.0400, 0.0600, 0.0800, and 0.1000 μg / mL.

3. The method for determining five new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: The step 2) is specifically as follows: 2.1) Weigh 2.00±0.02g of sample into a 50mL centrifuge tube, add 5mL of extraction solvent, vortex for 10min, and sonicate for 10min; 2.2) Then add an appropriate amount of extraction dispersant to the centrifuge tube, vortex again for 10 minutes, and refrigerate centrifuge at 8000r / min for 5 minutes; 2.3) Transfer 1.0 mL of the upper organic phase to a 15 mL centrifuge tube containing the dSPE mixture, vortex for 3 min, centrifuge at 4000 rpm for 3 min, and filter the supernatant through a 0.22 μm filter membrane.

4. The method for determining five new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry according to claim 3, characterized in that: The sample in step 2.1) is a beef sample that is homogenized, sealed and stored at -20°C, and then thawed before use; or a beef sample that is homogenized on site.

5. The method for determining five new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry according to claim 3, characterized in that: The extraction solvent is acetonitrile or 1% acetic acid acetonitrile, acetonitrile / acetone (1:1, V:V) or acetonitrile / acetone solution containing 1% acetic acid (1:1, V:V); the appropriate amount of extraction dispersant is 5g of NaCl or 4g of MgSO4+1g of NaCl or 6g of MgSO4+1.5g of NaOAc or 4g of MgSO4+1g of NaCl+1g of Na3C6H5O7+0.5g of C6H6O7Na2·1.5H2O or 6g of MgSO4+1.5g of CH3COONa or 4g of MgSO4+1g of NaCl+1g of C6H5O7Na3+0.5g of C6H6O7Na2·1.5H2O; the dSPE mixture uses 0, 30, 60, 90, 120mg of PSA or 3, 5, 10, 15mg of MWCNT, 30, 60, 90, 120mg of C 18 and 30, 60, 90, 120, 180 mg of MgSO4; the centrifuge tube is a polypropylene centrifuge tube.

6. The method for determining five new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry according to claim 5, characterized in that: The extraction solvent is an acetonitrile / acetone solution (1:1, V:V) containing 1% acetic acid; the appropriate amount of extraction dispersant is 5g of NaCl; the dSPE mixture uses 120mg of PSA, 30mg of C 18 and 180 mg of MgSO4.

7. The method for determining five new pesticide residues in animal-derived foods by gas chromatography-tandem mass spectrometry according to claim 1, characterized in that: When performing GC-MS / MS analysis, The chromatographic conditions are: A SH-I-5Sil mass spectrometer capillary column was used; the injection volume was 1 μL, non-split injection, and the carrier gas was helium; The column oven temperature program was as follows: the initial temperature was set at 70°C and maintained for 1.0 min, then increased to 150°C at 30°C / min; increased to 280°C at a rate of 12°C / min; and finally increased to 290°C at a rate of 5°C / min and maintained for 1.0 min; the flow rate was 1.6 mL / min, the collision gas was argon, and the injection port temperature was 280°C; Mass spectrometry conditions: ionization mode was electron bombardment source, detection voltage was 0.3 Kv, ion source temperature was 230 °C, scanning mode was multiple reaction monitoring mode, solvent delay time was 5.0 min, and total running time was 17.5 min.

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